physical chemistry of living tissues' and life processes

November, 1932.

PHYSICAL CHEMISTRY OF LIVING TISSUES' AND

LIFE PROCESSES

R. BEUTNER.

PHYSICA·L CHEMISTRY OF LIVING TISSUES AND

LIFE PROCESSES

As Studied by Artificial Imitation of Their Single Phases

BY

R. BEUTNER, M.D., PH.D., Professor of Pharmacology.} School of Medicine,

Unt"verst"ty of LJout'sville

"Life in all its complexity seems to be no more than one of the innumerable .properties of the compounds of carbon."

LONDON BAILLI:BRE, TINDALL & COX

8 Henrietta Street, Covent Garden, W.C. 2 1933

B. L. No. 64.

Imperial Institute of Veterinary Research :i--olmratocy. 6 ,:1.' 0 (5 B E I II _l - t C-I ;~ ,~ r .

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ALL RIGHTS RESERVED, 1933

PRINTED IN AMERICA

DEDICATED

IN ADMIRATION AND GRATITUDE

TO

PROFESSOR JOHN J. ABEL

OF JOIINS HOPKINS

UNIVERSITY

PREFACE The writer is much indebted to numerous friends and colleagues

for valuable suggestions, particularly to Dr. H. G. Barbour of Yale University, without whose unceasing encouragement this work would not have been accomplished, to Dr. R. Chambers of New York University, Dr. Peyton Rous and Dr. D. D. van Slyke of the Rocke~ feller Institute, Dr. E. Gellhorn of the University of Oregon and Dr. H. Freundlich and Dr. F. Haber of Berlin for their expert advice; also to Dr. W. F. Hamilton of George Washington University and to Drs. S. I. Kornhauser, S. E. Johnson and G. E. Wakerlin of the University of Louisville; to Dr. Jos. Lomer of Fordh~m Hospital, New York City, and to many others.

After this text has become accessible by publication to a larger circle, the writer hopes that his readers will likewise let him know their opinions and criticisms, by writing to his address given below. For a further discussion of the problems in question, informal gatherings will be arranged yearly, if possible. All those who are inclined to participate will, please, address the author, School of Medicine, Louisville, Ky. .

The text of this book can be readily understoo! by the average medical student. A mathematical treatment of theories has been avoided in every case (except in the Appendix).

Lectures on these subjects have been delivered not only to medical students at Louisville, but calso, on invitation, at the Marine Biological Laboratory, Woods Hole, Mass.,.at Bellevue Medical College a.nd Washington Square College of New York University, and at the Medical Center of Columbia University, New York.

Finally, the writer begs leave to state that this volume contains reports on original investigations which' are described here for the first time.

R. BEuTNER.

November, 1932.

vii

CONTENTS

Introduction: Life as a Scientific Problem.............................. 1

The First Attempt at Approach: Membranes, Osmosis and Related Forces

The Gap between the Living and the Inanimate World; Artificial Struc-tures Produced by Osmotic Forces............ .............. .... .... 11

Brief Outline of the Most Elementary Laws of Physical Chemistry Relating to Osmotic Forces, Formation of Ions, etc... . . . . . . . . . . . . . . .. 23

Evidence to Indicate the Presence of Osmotic Pressure in Li'ving Tissue in Some Cases.-In Other Cases: A Flow of Fluids and Solutes is Described to Occur in Living Organisms Independent of, or Opposed to Osmotic Forces; Attempts at Explaining this Flow as a Result of Electrical Forces ......... , ....... , .... , , .... , .. , , .. , . , . . . . . . . . . . .. 35

The Importance of "Oceanic" Salt Mixtures for the Maintenance of Membrane Permeability and of "Life."-The Physicochemical Causes Underlying Permeability Changes as Studied on Emulsion Membranes. -Conclusions Concerning the Origin of Life on the Earth ....... ,... 72

The Formation of Stratified Structures as a Result of Diffusion in a Gel and of Reversible Chemical Precipitation Reactions Occurring Simul-taneously ...................... , .... , . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 99

The Second Attempt at Approach: Life Processes Related to Crystallization or Due to Surface Forces

Microscopic Structures Which Are Formed in Colloids, Even in Non-living Ones, .. , ... , ............. , ............... , . , ... , ... , ...... ,. 109

An Explanation of the Origin of Such Structures as the Result of Forces Similar to Those Acting in Crystallization.-The Tracing of Crystalline Units in Substances Which Have Been Produced by Vital Growth. -Surface Forces as a Result of Molecular Orientation; Adsorption in Surface Layers, .. , . , , ... , , .... , .............. , ..... , , . , ......... 119

Cell Respiration as a Chemical Reaction Occurring in Adsorbed Layers. -Vital Movement Due to Surface Forces; Vital Movement Due to Other Molecular Actions ........................ , .............. , ... 149

The Third Attempt at Approach: Electrical Currents in Tissues and Their Relation to Life Processes

The Nature of the Single Electric Potential Differences Which Occur in Living Tissue; The Electromotive Effect of Concentration as Demonstrating a Certain Resemblance to Metallic Electrodes of Living and of Artificial Membranes ......................... , ...... , . . . . . . . . . .. 189

ix

PREFACE

The Physicochemical Laws Which Elucidate This Similarity to Metals, Even Though the Membranes Are Second Class Conductors ......... 215

The Make-up of the Vital Battery System.-The Relation of Electromo-tive Forces in Tissue to Stainability and to Metabolism ............ 227

Stimulation as the Result of an Electric Polarization of the Phaseboundaries in Tissues, in Other Words, as a "Charging of the Vital Battery System."-Traveling Waves of Polarization on Passive Iron Wires and Their Resemblance to the Traveling Wave of Excitation in the Nerve. -Experiments Which Elucidate the Action of a "Pacemaker" as it Occurs in the Heart.-The "Salt Bridge" Experiment.-Conclusions Regarding the Theory of Narcosis .................................. 245

Outlook to Future Possibilities or Development

Artificial Parthenogenesis.-Mitogenetie Rays ......................... , 279

Concluding Remarks

Possible Application of Physicochemical Methods of Investigation to the Explanation of Cerebral Activity.-Possibilities of Development in a Remote Future ................................................. 292

Appendix

New Investigations on Membrane Equilibria ............................. 297

AUTHOR INDEX ......................................•........... , .... 325 SUBJECT INDEX. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 331

LIFE AS A SCIENTIFIC PROBLEM

THE ApPROACH BY MEANS OF SYNTHESIS

Since living organisms have been frequently compared to machines, it is important to understand to what extent such an analogy is useful. To take an example, we may compare a muscle and an electrical motor. Apparently we are as ignorant in regard to the muscle as a man would be in regard to the motor if he had no knowledge of physics, especially if he were quite ignorant of the nature and action of electrical currents and unable to investigate their law~. What could such a man do to understand the working of the motor?

He might first study the structure of the object by taking it apart and noting its parts and their mutual relations. This is what we call anatomy in the case of living tissues. We would hardly expect to reach an understanding of the working of the motor by this method alone. Another method could be that of chemical analysis and chemical identification of materials in it, corresponding to the physiological chemistry of tissues. But, if the nature of electrical insulation is not understood would it help to determine more precisely the chemical make-up of the insulating materials in the motor? Still another line of investigation-hardly more efficient-would be the study of the functions of the motor, for instance: how the velocity is affected by the load, or to study its latent period, etc. Similar observations on living organs are classified as physiological.

It is evident that anatomical, chemical or physiological descriptions are insufficient by themselves; the shape, the functions or the chemistry will not tell us why and how the motor works. Towards this end, it is necessary that we know the laws of electrodynamics. Do we knowanything as definite about muscular dynamics? We certainly do not, but the comparison with the motor can show us the cause of our ignorance. A profound knowledge can be acquired about a machine like the electromotor, not because we can describe it perfectly, but because electromotors can be made artificially, and with innumerable variations. The simplest experimental electromotor would consist of nothing but a magnetic needle and a wire passing over it, through

1

2 INTRODUCTION

which an electrical current flows. By experimenting with such simple devices we find the basis of electrodynamics and can analyze the action of a complicated motor. A complete understanding, for which we are ultimately striving, can be obtained only of things which we can produce artificially. Since it is impossible to synthesize a real muscle we should at least try to approach such a synthesis as nearly as possible. In general, we should study all artificial systems whicq bear even the remotest similarity to certain features of living organisms.

As is well known, the possibility of artificially producing life has attracted the human fancy from times immemorial, and given rise to many fantastic tales. In contrast to the skeptical attitude of most investigators, J. Loeb writes about this point:

"I can see no reason for the pessimistic assumption that the artificial trans~ formation of dead into living substance might not be accomplished some time. On the contrary I believe it will only help science if the solution of exactly this question should appeal to the younger biologists as an ideal problem of biology. The conservative members of the scientific profession will be, of course, inclined to issue the usual warning that the time has not yet arrived for this kind of a problem."

"I believe that the time for the solution of a problem is at hand if an investigator appears who has the courage to attack the solution, also the brain and the knowledge (and, possibly, the good luck) to carry it out successfully."l

It does not seem likely that the artificial transformation of dead into living substances can be accomplished suddenly by the work of a single genius. A task which embodies such difficulties can only be attacked tentatively after preparatory efforts continued through centuries.

Naturally it is quite impossible to state anything definite about the remote future of such a development. Unknown handicaps may arise to prevent the synthesis of real living organisms. Such an assumption seems certainly more cautious, at the present time, than extravagant claims about artificial life. We may take it, however, that artificial systems can be produced wMch show a resemblance to certain features of living organisms or life processes. No more than this can be achieved at present.

1 Translated from Loeb, "Dynamik der Lebenserscheinungen," 1906.

LIFE AS A SCIENTIFIC PROBLEM 3

PHYSICS AND BIOLOGY

Such an approach by means of synthesis, even though it is still handicapped, is an imperative need for scientific progress. It certainly is more important than the formal application of those physical laws which have been derived from inorganic systems that are profoundly different from living organisms. Such physical laws are applicable only on account of their very general character. As examples we may quote the laws of thermodynamics. Painstaking experiments have been undertaken to prove the identity of heat production in a flame and in the respiration of living organisms, the basis for calculating the heat being the amount of CO 2 produced in both cases. The first experiments of this kind date back to the time of Lavoisier (about 1780). From that time up to the present the technique has been much improved so that there can be no doubt now that foodstuffs, when oxidized, produce the same amount of heat as they would if burned in a bomb calorimeter, provided the end products are the same. Has this led to any progress in the explanation of the mechanism of oxidation which is the main supply of energy for most vital processes? This can hardly be expected. The agreement merely shows that the first law of thermodynamics, or the law of the conservation of energy, suffers no exception in biology. The second law of thermodynamics, which is concerned with the mutual transformation of heat into "free" or available energy, has also been applied to biology, but, this has just led to the negative and almost self-evident conclusion that a muscle cll-n not be regarded as a heat engine.2 The

2 This is shown by the high efficiency of muscular work. One-fifth of the chemical energy of the food is converted into mechanical work according to A. V.Hill. If an animal wereaheat engine it would operate between two temperatures. One of these would be the body temperature, viz., 38°C. or T 1 = 38° + 273° '" 311° (absol.). The other temperature would be that of combustion: T2. From the second law T2 can be calculated, since the "efficiency" or fraction of the heat of food transformed into work, which is one-fifth as stated, must equal

or 1 T2 - 311 ° 5 - 311 ,hence T2 = 373.3 •

The combustion temperature would be 373.3° (abso!.) or 100°C. It is inconceivable that this can occur in living tissues.

4 INTRODUCTION

third law of thermodynamics (Nernst) is hardly applicable at all. The application of thermodynamics to biology appeared as a scientific feat at the time in which life was considered to be entirely separate from the non-living world, subject to laws of its own. It was ·then a scientific merit to oppose this mysticism and to demonstrate the universal applicability of such elementary physical laws. Nevertheless, since no information about the mechanism of vital processes can be obtained by thermodynamics, no further development can be expected.

Another rather general application of physical chemistry has been made by determining the temperature variability of certain life processes, with a view of determining whether they are of a physical' or chemical type. When this application was first attempted by J. Loebs a long time ago, it was hailed by many as an important progress. This historical merit should not be questioned. Nevertheless, in the meantime, many may demand some more specific results before they are willing to concede that the application of physical chemistry offers to biology an appreciable advantage. The distinction between physical and chemical processes is, moreover, quite ambiguous.

Frequently attempts are undertaken at utilizing mathematics by deriving empirical mathematical expressions which describe quantitatively the course of certain vital functions-preferably by means of statistical methods. Such methods would be promising if the formulae, thus derived, were characteristic of one definite physical or chemical process exclusively. But, such a coincidence is hardly ever found.

It seems, therefore, that the secrets of life can hardly be revealed by an insistent application of mathematics, physics or chemistry without a definite aim. Biology has problems of its own. To solve these, we should rather search how physical or chemical experience might be suitably adapted. Now, for that end, an approach by means of synthesis is frequently the best method of orientation, since it is usually less difficult to determine the physical and chemical laws governing artificial models which imitate certain phases of life processes. These laws should then be applied to the living structures themselves in order to make sure that we are on the right track. This is the method attempted here. Vital functions are analyzed in each of the three "attempts at approach," described in this book, by means of the following steps:

3 See J. Loeb, "Dynamics of Living Matter," Chicago, 1906.


1. The finding of suitable artificial models which reproduce certain phenomena peculiar to living organisms.

2. The study of the physical laws of the model, the observation of similar physical actions in related models, hence a generalization of our physical knowledge of the entire field.

3. The application of these physical laws to biological problems. 4. Finally, on the basis of all the experience gained, an extension

of the experiments with models may be attempted.

VITALISM, THE PURPOSEFULNESS OF LIVING ORGANISMS

The search for the underlying causes of life processes has at all times excited intensive interest, but, has seemed to 'tax science beyond its capacity. Innumerable attempts to solve vital mysteries have been shipwrecked. Is it surprising, therefore, that many biologists follow a doctrine of negation which claims that vital forces should be forever irreproducible by artificial means? This is the essence of the so-called vitalism. Oftentimes its claim is summarized by the statement that physics and chemistry are inadequate for solving fundamental biological problems. This statement is undoubtedly correct if physics or chemistry at their pr,esent stage of development are meant, but, it should also be remembered that their present stage is certainly not their final one. Some forces, acting in living organisms, have been imitated artificially, although they were quite mysterious for a long time. Those which are still puzzling us now may be reproduced at some future time.

In former centuries, the propounding of vitalism was chiefly the privilege of philosophy which used dialectic means. In our days vitalism seems to be rather the result of experimental work, due to the fact that the qualified biologist tends to limit his observations to living tissue strictly. This tendency leads him to the impression that the properties of the living are unparalleled in the inanimate world,-in agreement with the age old creed of a supernatural force as the source of life. But we should realize that little is gained by discussing, over and over, the supposedly wonderful mechanisms of self-regulation if we neglect all studies which lead up to an analysis of their physical causes. Also, we cannot help but insist that perfect freedom of thought must be the basis of science. If we can free our

6 INTRODUCTION

minds from all prejudices we find no evidence in nature of anything like a purpose or an aim at creating life. In other words: just as mountains, rivers, and oceans are formed, so also arise living structures on the surface of the earth by the play of the forces of nature.

Since the term of vital purposiveness has been impressed very frequently upon our minds, it may be considered indispensable by some. But can this be looked upon as an objection to the . above statement? It is a definition of an inherent difficulty of the problem without containing any suggestion as to its underlying causes. The development of living organisms in nearly identical forms, generation after generation, their adaptation to the environment, their seeming skillfulness and resourcefulness in overcoming innumerable handicaps, are all a matter of the most common observation. We may explain all this as the result of "determinism," or of Aristoteles' "entelechia" or of Driesch's "psychoid" or of Uexkuell's "zielstrebigkeit." We may also use any other technical terms. Such explanations are equivalent to the dialectic explanations of ancient times which stated, e.g., that "a stone falls on account of gravity." Gravity implies the fact that the stone is heavy, hence this statement means no more than that the stone falls because it falls. Another explanation was "that opium produces sleep on account of its somniferent power," which means no more than that it is sleep producing. These are just a few examples out of those thousands of "philosophical" explanations which have been discussed. Such explanations have nothing in common with scientific methods in the modern sense (Liebig).4 The fact is that we hardly know anything about the causes of what is termed "determinism," etc. Our ignorance in this respect is so great that some may doubt whether our science will ever grow great enough to elucidate the underlying causes thoroughly. Even in this case we should not mask our ignorance with high sounding technical terms.

Also in connection with the problem of evolution, such descriptive terms as "struggle for existence" do not imply any explanation of the underlying cause. We should not be content with the se1£evident fact that only those organisms can persist which adapt themselves to changes in environment. The scientific problem is to determine the physical and chemical variations which occur in an organism following changes of environment.

• J. v. Liebig, "Chemische Briefe."


Above all, however, the problem of evolution demands an answer to the greatest of all questions: how life originated on this earth. It is by no means impossible to find at least a tentative answer to this question as a result of studies on the physicochemical nature of life processes as we shall see.

Vitalistic mysticism has penetrated also into biochemistry, as expressed by the belief in a peculiar living substance, the so-called protoplasm, to whose mystical functions life phenomena are attributed. Of course, the ancient belief, that all organic substances are produced by dint of the vital force, has died out long ago, since thousands of such substances are now'made synthetically. Yet, the very substrate of the living, the "protoplasm" is held by many to be beyond the reach of synthetic chemistry. About this point, the remarks of an up-to-date biochemist, Gow~and Hopkins,6 are noteworthy:

"We are ceasing to believe in all this (the hypothetical molecule of protoplasm). The term "protoplasm" itself, we think, has merely been taken over from a vague abstract terminology. The characteristic of a living unit -whether it be the cell or another system-is that it is heterogeneous. There is no such thing as living matter in a specific sense. The special attribute of such systems from a chemical standpoint is that these reactions are organized, not that the molecules concerned are fundamentally different in kind from those the chemists meet elsewhere. This concept of the physical basis of life is certainly more stimulating to chemical thought, and gives more justification for chemical methods of attack than the forever elusive protoplasmic molecule."

6 Gowland Hopkins, Lancet (1925) (Italics by writer).

THE FIRST ATTEMPT AT APPROACH

MEMBRANES, OSMOSIS AND RELATED FORCES

THE FIRST ATTEMPT AT APPROACH: MEMBRANES, OSMOSIS AND RELATED

FORCES

EXPERIMENTS WITH MODELS: ORGANIZED CHEMICAL REACTIONS AND THE ARTIFICIAL OSMOTIC STRUC

TURES, THEIR DEVELOPMENT AND TROPISMS

1. THE ARTIFICIAL GELATIN TANNATE CELL

To bridge the gap between inanimate and living matter is very difficult at the present state of our knowledge. As far as it is possible, nothing is as instructive as the .study of the artificial growths which originate from many simple chemical reactions if these are performed in an organized form, which is the special attribute of the more complicated reactions in living tissue according to G. Hopkins as stated. The technique of performing reactions in such an organized form is simple. As an example an experiment may be quoted, which was devised by Moritz Traube about sixty years ago.1 The reaction between gelatin and tannic acid was studied in this case.

A solution of gelatin was prepared and a drop taken out of this solution with a glass rod. The drop remained hanging at the end of the rod, and was exposed to the air for several hours. It was then dipped into a 5 per cent solution of tannic acid. In about ten minutes a thin irridescent solid film formed at the surface of the drop. The gelatin and the tannic acid had formed a membrane of precipitation at the common surface which was permeable to the water but impermeable for both gelatin and tannic acid and thus prevented any further reaction between the two.

Traube further points out that this phenomenon explains certain features of the growth of a cell. If sugar, salts or other soluble substances are added to the solution of gelatin the drop formed from it rapidly increases in size in the tannic acid. If, however, such substances are dissolved in the tannic acid solution the drop will shrink. This shows that the dissolved substances exert a pressure on the

1 Traube's outstanding publications are in: Zentralblatt fiir die medic. Wissenschaften, 1864 (p. 609) and 1866 (pp. 97 and 113); also Archiv fiir Physiology, 1867 (pp. 87 and 129); see also his "Gesammelte Abhandlungen."

11

12 FIRST ATTEMPT AT APPROACH

gelatin tannate membrane from the inside or from the outside according to whether their concentration is higher inside or outsider This is the osmotic pressure which as we shall see is also a factor in the fluid exchange of living cells.

2. STRUCTURES FROM A COPPERFERROCYANIDE PRECIPITATE; SILI

CATE STRUCTURES; INFLUENCE OF GRAVITY

If one of the substances which enter into the chemical reaction is quite soluble and if this is applied in solid form, the resulting membrane formation develops into an unusual shape. As an example we may study the well known reaction:

2CUS04 + K4Fe(CN)6 = Cu2Fe (CN)6 + 2K2S04 red precipitate

We apply the CUS04 as 1 per cent solution and drop a crystal of K.tFe(CN)6 into it. The first change observed is the formation of a delicate brown membrane of Cu 2Fe(CN)6 around the crystal. This membrane expands rapidly since the driving force is much greater in this case than in the case of the gelatin tannate cell. Inside of the Cu 2Fe(CN)6 envelope a saturated solution of K.tFe(CN)s is formed from the solid salt, making a 28 per cent solution. Since the CUS04 solution outside the envelope is no more than 1 per cent a great excess of pressure is acting inside. Moreover, this excess pressure acts for a considerable time since more salt goes into solution as more water is drawn into the envelope by its expansion. The result of this excess pressure is to cause a bulge in the envelope which then no longer maintains the simple spherical shape but assumes complicated forms resembling vegetable structures.

The experiment can also be performed in the reverse way, viz., by placing a CUS04 crystal into a dilute K.tFe(CN)s solution\_ Particularly fine looking structures may be obtained by using "seeds," formed of one part of cane sugar and two parts of CUS04 and placing these in the solution of K4Fe(CN)6 to which some gelatin has been added. Structures 30 to 40 cm. high grow from it, which show rhizomes, roots, twigs, ramified branches and leaf-like structures (St. Leduc).2 See Figure 1, opposite page 14.

2 For Leduc's experiments see, "Theorie Physicochemique de la Vie," Paris, 1910, and "La Biologie Synthetique," Paris, 1912; also "Solutions and

EXPERIMENTS WITH MODELS 13

In order to study the formation of the CU2Fe(CN)6 membrane somewhat more closely, we make use of the microscope. CuSO, and K4Fe(CN)6, both in solution, are brought in contact in the following manner (Quincke, 1902):3 capillary tubes of 1 mm. in diameter are filled with a CUS04 solution having a specific gravity of 1.019. Two of these tubes are placed on a microscope slide upon which a I\4Fe(CN)6 solution of specific gravity 1.025 is poured. A coverglass is placed on top. The CU2.Fe(CN)u formed at the contact of the two solutions appears to be almost liquid when it is formed or at least exceedingly flexible. It has the appearance of a circular section as observed under the microscope. A second later, however, it stiffens up and is broken at one place owing to the higher specific gravity of the I\4Fe(CN)6 solution. The K4Fe(CN)6 solution then streams into the CUS04 solution and forms delicate tubules of Cu2Fe(CN)6. This observation shows that the initial flexibility or fluidity of the precipitation membranes and their subsequent rigidity play an important role in the formation of the structures.

M any other chemica:! reactions can be used for producing similar structures. Any reaction is suitable if it results in a cohercmt precipitation of a colloidal nature. Examples of this kind are the reactions between silicate solutions (commercial waterglass) and solid salts of heavy metals, earths (lr alkaline earths such as the sulphates, chlorides, nitrates or acetates of copper, cobalt, nickel, iron, manganese, calcium also ammonium chloride; also the reaction between aqueou!'! solutions of calcium nitrate or calcium chloride and alkali carbonates, phosphates or hydroxides (Quincke and Leduc). Peculiar structures are obtained by the interaction of metallic mercury and 5 or 10 per cent chromic acid (L. Rhumbler, 1902),4 also by the interaction of metallic iron or other metals and ferro cyanide solutions

Life," in J. Alexander's "Colloid~l Chemistry," New York, 1928. Leduc's extensive theoretical considerations keep the actual description of the experimental detail of his work somewhat in the background. The writer of this book begs leave to state that he personally would be unable to present an account of Leduc's work-such as it appears on the following pages-from a study of his books alone. This was possible only through a visit to Leduc's laboratory at Nantes (in 1914), which enabled the writer to observe Leduc's extraordinary experimental skill.

3 Annalen der Physik, 7, 647. • Zeitschrift f. allg. Physiologie, 2, 290.


(R. S. Lillie., 1922).5 The foregoing reactions have been the ones chiefly used but certainly numberless others are still available.

The appearance of these artificial structures varies widely according to their mode of preparation. The addition of small amounts of substances which do not enter into the membrane forming reaction may materially alter the aspect of the growth.

Sometimes these vegetations exhibit different colorations which are arranged according to a definite pattern. This occurs particularly if cobalt salts are placed in a solution of sodium silicate. The structures developed consist of cobalt silicate which contains alternate pink and blue layers. Similar variations of color are shown by iron and manganese silicate structures. The different colorations point to the presence of somewhat different chemical reactions occurring in different parts of the structures.

The question arises whether the growth of the artificial structures, described so far, is the result of gravity, in other words, whether a precipitation is simply pulled upward by dint of its specific gravity which is lower than that of the solution. It can be shown that this is merely an accessory factor and by no means necessary for the formation of an artificial growth. Under certain conditions osmotic structures grow in all directions: upward, horizontally and downward. This is shown, for instance, by worm-lil:e structures developed from fused calcium chloride in saturated sodium carbonate as is illustrated in Figure 2. Frequently such structures rise and descend in several successive eurves (St. Leduc). Hence hydrostatic pressure due to a difference of specific gravity cannot exert an appreciable influence in these cases, although it may have some influence in others.

Further evidence to show that the osmotic pressure of the dissolving salt crystal causes such structures to grow is found by adding an excess of sugar to the solution. Since sugar exerts an osmotic pressure which counterbalances the osmotic pressure of the salt, dissolving inside of the newly fanned membrane, the growth of an artificial structure should be retarded or inhibited entirely by such an addition. Dr. S. H. Mann has proved this point in experiments performed (at the suggestion of the writer) at the Cleveland Clinic Foundation, as shown by his photographs, see Figure 3. If gravity were a factor

~ Scientific )'Ionthly, February, 1922.

Photographs Illustrating the Appearance and the Functions of Artificial Osmotic Structures

FJ(l. l. l'REl'IPITATIO?ll STRUCTURE DE,']';l.OPED FROM COPI'ERS"LI'HATE IN \ :-\OLrTION IIF POTA!';"; II ~I FE [( l{OeY A ;>i'IOJJ;

From ,stpphanc Leduc, "L}1 Biologic Synthctiquc." 1'1Iiliisher, A, Poinal, l'1lrll'., HH2.


FI(;, 2, \\'OIOI-I,IKI·: :-;Tal'I"rl'H~: OF CALCU ' M CAHBO~XJ'I';' (inowl:'\(J [~ .\LL DIlU;('1'IO~S

Photographs of a worm-likC' sLl'ucLIII'C' of I':!lciurn (':lrhonatC' growing from :l piece of fu"pd ('all'ium ('hloridC' in a "aturatl'd "Illution of sodium I'arhonatl', Tlli", :;lru('tun' I'I'OW,., upward!:! horizont;Jlly and downward, hC'[1I'(' indl'pcndent of gravit,y. Owinl' to the ditTerl'n('e of speeific' l'ravity, tilC' f'ltrU('lllrc hllPP(,lled tl) break ill thl' last pllrt of the initial ('urvutUl'e «('Ol/lp'I1'(' Ow first :lIlIl secolld "<'lIker in th!' illuHtration). The hori;"ontal purt has LllllH hl'('omp lH'nt llP\\:tfll. The wound then hralcli up an([ (.(J'()wth lI;lS ('ontinllC'tl p('r,;istently LloWl1wufll, a::; Hho\\11 Oil tIll' third photograph.

From Nt. L('(ilw, "Th('orir pitysi('ol'himiqllc dc' la \ ie," l'uhlish{'l', \, I'"inn(" I'ari s, IfllO

Without. ,jug"r

2 'C.U t-

... :t~. 01

With ,;ugur

FJ(;. ;{. [~1l1111T1()" nt' '!'H}; nE\'~:I.()I'~IE"'!' OF A, O"'~IOT«' HTRl'I'lTHI: In '1'111,;

\IIDITlo:\ ot \ :-IOl.lT):; \\'IIIClI f<; "EH'l's A" O",'\IOTIC COl':\'I'hUl'll]<;SSlTRl';

A compariKolI of LII!'HC' two photographs sh(Jws thc influC'ncC' of an ('ntir{'ly inert l:;uh"taTl('l', a,; ('.g., sugar. The growth ('casp,' if a ,;uffi('i(,Tlt1:v large tun,mut of ,",u).!;a\' i" \\thlcd, lll'uLral salt,; wm'k in tlw i'oam('. W\\y. Thi" ,;how,; tha t t h(' gl'OlI"t h a~ ~11f']1 i::> d u(' cnl irely lo o<lrnoLic fOf('e~,

Photographs III ustrating the Appearance and the Functions of Artificial Osmotic Structures

a b Fill , 4, S1'Il.IT1Tl{I';S WITH rh' !.",; .\T TilE E:\TD OF !-ir..;"I)I~R ~+n:~l';

The- Hie-nrier ::;terns of lhese "trU('tllreS d<'vclop rrom tlolid particleli of tal .'ium l'il]orid(' pluf'cd in a dilute solution cllntainin~ potassium HiH('atc (6 per ccnt of 11 :;ollltion of :33 0 B('), soclimn efl,rbonate (6 per cent of a Haturated Kollltion) and ;;odium biphosphcLtc (3 per rent of a f!n.tumted solution), After they have formed the solutiun is dillltE"d with water 100 tilDes, The greatC'T difference of osmotie preSHllfe thus produC'C'd, lead~ to the fOl'ln~Ltion of blllhs.

Figure 4[1 ShOWB these 'bulb:s in tra.nsJ)tJ.rent li~ht revcillill,:!; a mOre CO.lU pact ("('ntral po!,tion. Figure 4h show!'!, i!3tructures produced.in !.l. similar manner Ironl rni..l.ILgtl n('~ sait:!'! i nstearl of c..al{'ium

C'lllorid(·, "I'he h ulb~ turn dark due to oxidation of manJ!,anesE' hydroxid ... · i n tht~ uir.

F1G, ;). "ARTIFICIAT, ::\fGSHROO:"l!S"

Dcvl'loped from large pic,.-,es of fUSE'd ca]l'ium chloride immersed in 3. ('oU('entraterl solution of sodiuIll sili('ute, ;;odiuID C'urbollut(' unci ,,,odium pho,,phate. On top of tId;, solution water W!lS poured in such ,L wny as to avoid mixinll; wi til thc solutioll as far a.~ pos:<ihle, ThE' ":,;tem~" df'v{']opc(l in the ('on('cntl'!lt.cd Ho]ution, the "hoods" in the Hllpernat:tnt wlIter.

Both Fig, 4: :Hld Fig , 0) ure taken frOhl SL Leduc HLa Biologh-" ~yr!thcti<111C'," Pari::;;, 1!J12.


FIG. l:-i. GALVANOTROPIC Bg:-WIr\G (n' AN O,;l\10TH.: STItUCTtTU],; OF COI'I')'][\

FE RltO('Y A NIDE

The stnwtun' h!uJ first df'veloped up\yard. After ('.lIlTf'nt WitS sent through the solution it, hent, tU\\'~\l'd the negative pole.

(See text on pl\i>:e lU)

+

Fw. !). GALYA~()1'llOl'l(' lh::\,Dlr\G TO\\'Aun,; 'l'lIE XEGA'l'rVB POLt] 01> 'l'IlE

~L"NI Aun:M A~D TE!\ITACLE OJ,' 1\fEl)USA POLYOH.CHj~

From J. Loeb, "Dynamik der L('benscr~cheinungen." Publisher, J. A. 1hz-th, Leipzig, lOOn.

(~oo text on page 1\))


accounting for the growth of these structures one should expect that addition of sugar would rather promote the growth on account of increased specific gravity of the solution, which would tend to promote the upward floating of the precipitate. This, however, is not the case.

Osmotic structures can alBa be grown outside of solutions, e.g., from a piece of fused calcium chloride immersed in a solution of carbonates and phosphates (St. Leduc). That part of the osmotic structure which stands out in the air frequently exhibits a special development. The top surrounds itself with an opaque crown containing a cavity into which the fluid rises which is absorbed from the base.

3. THE GENERATION OF COMPLICATED STRUCTURES BY MORE

ARTIFICIAL METHODS

Structures with so-called "end organs" are prepared according to Leduc as follows. Small pieces of fused CaCl 2 are dropped into a solution of silicates, carbonates and phosphates. When the slender rectilinear structure8, which are seen to develop, have attained a COn

siderable height the solution is diluted down to one-hundredth of its original salt content. Owing to the increased difference of osmotic pressure inside and outside, the structure bulges and end organs are formed. If the growth of the structure has not advanced far, the shape of the end organs becomes cuneiform or pearshaped. (See Figure 4.) In a similar manner other forms have been obtained, such as colored bulbs ("end organs" according to Leduc) on white stems, or white bulbs on dark stems, also bulbs containing granules and a differently colored center.

Osmotic structures in the shape of mushrooms are obtained by pouring water over a concentrated solution in which osmotic structures are developing, taking care that the water does not entirely mix with the underlying concentrated solution, see Figure 5 (Leduc).

The resemblance of these structures to real mushrooms is so great that botanists have mistaken them for real ones, according to Leduc. The stems of these osmotic mushrooms are formed by bundles of fine hollow fibers. The upper surfaces of the hoods are smooth or


covered with fine scales, while their lower surfaces present traces of vertical lamellae. Sometimes these lamellae are intersected by concentric lines.

Natural mushrooms are built up in a somewhat similar manner, since their stems are also made up of fibers which are bundled together and continue to extend into the hood. But, it must be added that the mode of origin of such artificial products is vastly different

\ t , I I I

f I I

I

FIG. 6. MICROSCOPIC ApPEARANCE OF OSMOTIC STRUCTURES, GROWING IN DIFFERENT

MEDIA

Magnified 25 times From St. Leduc, Theorie Physicochimique

de la Vie. Publisher, A. Poinat, Paris, 1910.

FIG. 7. REOPAX NODULOSA

For comparison with Figure 6

from that of natural mushrooms since, manifestly, Ilatural mushrooms do not develop in two different media. Moreover, their hood is usually formed before the stem has grown up. It would be indispensable, therefore, to devise further' experiments with models in order to understand this feature of vital growth.


Nevertheless, it is perhaps possible to evaluate the experiments described as being the first step toward a future development which may lead to an understanding of vital growth as a result of definite forces. The nature and working of these forces cannot be recognized by studying living matter functionally, chemically or by its shape. In order to understand it, a new physics must be created which tends to develop into a synthetic biology. The experiments described may be regarded as the first tentative step towards this remote ideal.

The membranes covering the various gross structures which were described are anything but homogeneous. They are cellular, although the "cells" which compose them are not nearly so complex as those in living tissue. St. Leduc has brought this out by a large number of photographs some of which are reproduced here. The last of these structures resembles in a striking manner one of polythalamic foraminiferae, Reophax Nodulosa (see Figure 6, compare Figure 7). The study of artificial microscopic structures involves so many details that a special section of this book is devoted to it.

4. TROPISMS MANIFESTED BY THE ARTIFICIAL STRUCTURES

The artificial osmotic structures have not merely a certain morphological resemblance to plants, but also exhibit the simplest functions manifested by living organisms, viz., tropisms, such as a phototropism, an example of which is the well known growth of plant stems toward the light. This is designated more precisely as "positive phototropism" while "negative phototropism" would be the, rather rare, direction of growth away from the light. This process is to some extent dependent on laws similar to those which govern the flying of insects toward the light-such actions constitute one of the most elementary reactions which living tissue may exhibit. They are closely related to the reflexes which are the components of the activity of the nervous system of higher animals.

A "reflex" is defined as a reaction of an organism caused by an external stimulus. Such terms as tropism are preferably used if the resultant reaction is a coordinated movement of the plant or animal as a whole, such as the flying of insects toward the light, or the upward movement of young caterpillars (which is called geotropism). Quanti-


tative experiments show that these movements of insects and other animals are in no way dependent on anything like will or intention. They occur in a perfectly machine-like manner. The animal through a photo-tropic stimulus orients itself toward the source of light because, as long as it is not oriented, one of the sides' of its body is stimulated more powerfully by the light rays. This stimulation results in asymmetrical reflexes on either side of the animal and consequently in a rotation until the axis is parallel to the light rays. If two sources of light of equal size and intensity are present the animal can be shown to move in a middle line. The machine-like character of tropisms is, moreover, emphasized by the' fact that they are not an exclusive function of nervous tissue since even plants show them although they have no nerves. All that is required to bring about a reflex or tropism is physiological "conductivity" and "irritability/'S

Certain artificial precipitation structures grow toward the light, hence are positively phototropic like plants and possess a certain "irritability." To observe this, a 5 per cent solution of CaCl2 is poured into a square glass container. A crystal of sodium carbonate is dropped into it, and sunlight or artificial light is allowed to shine on one side of the container. Simultaneously the same experiment is performed in a container which is covered up entirely with a tin cap. Delicate tubes of calcium carbonate grow up in both containers. In the illuminated container they creep up on the illuminated side only or they grow up a few millimeters and then bend toward the light. In the shaded container the calcium carbonate tubes creep up on both sides or grow up straight (G. Quincke, 1902).7 The physical cause of this bending is probably due to a lower viscosity of the CaCOa precipitation on the illuminated side possibly on account of the heat brought about by irradiation. A similar effect upon artificial growth can be demonstrated by means of Roentgen rays. Similar effects may be the cause of biological phototropism.

Electrical currents influence the growth and the movements of plants or animals, which is termed galvanotropism or galvanotaxis. The regeneration of new polyps from the cut stems of the hydroid Obelia may be controlled experimentally by weak electric currents

6 See J. Loeb, "Forced Movements, Tropisms and Animal Conduct," New York, ·1918.

7 Annalen der Physik, 7,647.


passing lengthwise through the stem. The formation of hydranths is promoted at the cut end facing the anode pole, and inhibited a.t the cathode pole (E. J. Lund, and collaborators, 1921, 1922 ff).8 The same can be shown for plant cells (J. C. Bose, 1918)9 and for eggs of Fucus (E. J. Lund).l° In most cases the direction of growth is the same, being toward the negatively charged portion or toward the anode. On the other hand one can show that growing parts of plants or animals, like the tip of stems, are electrically negative, thus forming a negative pole. This shows that electrical currents of a direction suitable for promoting growth are present in tissue even in the absence of a current sent in from the outside. Hence, electric currents seem to playa role in the growth of living tissue normally, being produced by the tissue itself (E. J. Lund).

At the suggestion of the writer, Dr. S. H. Mann has performed experiments at the Cleveland Clinic which show that copperferrocyanide structures grow likewise toward the negative pole, like hydranths (see Figure 8, opposite page 15). .

These experiments were performed in a square glass container; the copper electrodes were 4 cm. wide, 10 cm. high and 9 cm. apart. The intensity of current passing was about 10 milliamperes. Since the solution itself contains CUS04 no disturbing gas bubbles develop at the electrodes. Discharge of gas must be avoided, of course, since this interferes mechanically with the development of the structures.

Galvanotropism is also exhibited by certain living organisms which are not in an active process of growth, as for instance, by the bending of the tentacles and manubrium of medusae (see Fig. 9). It is possible that also in artificial structures the galvanotropic bending is independent of the process of growth.

In the experiments, previously described, performed by Dr. Mann, the physical cause of this growth toward the negative pole is probably the positive charge of the copperferrocyanide (cataphoretic migration). Similar actions may be the cause' of biological galvanotropism.

Another type of tropism is due to gravity, being known as geotropism, examples of which are the upward growth of stems (negative geotropism) or the downward growth of root (positive geo-

• 8 See a number of articles in the Journ. Exper. Zoology, volumes 36, 37, 39, and 41.

9 Proceed. Roy. Soc., B 90, 364. 10 Botanical Gazette, 78, 288.


tropism). Numerous examples of the structures described demonstrate that both types of geotropism can also be reproduced artificially.

5. EVALUATION OF THE RESULTS OBTAINED ON ARTIFICIAL OSMOTIC

STRUCTURES

None of these artificial structures is a living entity. They merely bear resemblances to certain features of living organisms as described, but the more closely we study such artificial structures in all their diversity, the more we are led to believe in transitions. Some of the artificial structures described so far, are imperfect, but in order to properly evaluate the results obtained we should also realize that very little laboratory work has been done in this particular line. As yet the facilities of modern laboratories have not been applied to studies of this kind and only sporadic attempts of quite an amateurish character have been undertaken,Il

In the artificial structures described so far, organized chemical reactions occur which are totally different in type from the reactions in living tissue. Most of these structures are altogether devoid of

11 To corroborate this statement it may be said that St. Leduc is a medical practitioner at Nantes, France. In past years he has held the chair of physics at the medical school in his home town, but he has never had up-to-date physical laboratory equipment. It is also well known that Moritz Traube who initiated the whole development about seventy years ago, was never able to gain sufficient recognition for his epoch making work to enter the academic profession. The qualified chemists declared his work on osmosis to be outside the realm of "pure" chemistry. Biologists refused cooperation on account of Traube's incompetency in the exclusively descriptive biology of those days. The result of this situation was that this man who presented to science the most far-reaching discovery had to become a wine merchant in a small German town where he pursued his research in a primitive laboratory at his home after the close of business.

Up to the present time these experiments with models have been neglected. The general conception is perhaps most clearly expressed by H. Bechhold in his book "Die Kolloide in der Biologie .... " (5 ed. Dresden and Leipzig, 1929). Bechhold believes that the striking similarity of artificial structures to natural ones is a disadvantage for scientific research since there also exist. considerable differences. An observer with less prejudice would rather believe that such a similarity in some and dissimilarity in other respects would meet the needs of scientific research by furnishing suitable points of attack.


organic substances. Nevertheless, it is a point of interest to compare these inorganic structures to living organisms in order to see to what extent the features of life can be found in chemically dissimilar systems. The reaction which occurs in the inorganic structures begins with the dissolving of a solid salt. This leads to membrane formation. The artificial structure develops as long as the salt is in the process of dissolving and the membrane forming reaction continues. When all the salt is dissolved and transformed "life" is over; the structure is "dead," using such expressions metaphorically.

Life, in the usual sense of the word, is, of course, also by no means a mere shape or structure but always an active process, a continuous chemical change, although the chemical reactions are totally different. There is no dissolving of a salt, but enzymatic reactions take place instead. A living organism is dead when the enzymes or the substances with which these react are exhausted, removed, disorganized; or when the chemical reactions are interfered with for any other reason.

In spite of this immense difference between the chemical reactions in an inorganic artificial structure and in a living organism, some truly noteworthy s~'milarities have been found since the chemical reactions are arranged ~'n an organized form in both cases. The resemblance £s by no means of an accidental or superficial nature, since the same kind of forces is responsible for the growth in both artificial and living structures,. as will be shown in subsequent chapters.

The question arises: is it reasonable to suppose that no other reactions than simple inorganic precipitations, on the one hand, or unexplored enzymatic reactions, on the other, can be arranged under conditions of organization? Should we not conclude that many of the millions of known chemical reactions can also be found occurring as organized ones if the research be only continued? This line of thought leads us to the conclusion that a whole world of artificial structures is possible. Of these, we have just become acquainted with the very simplest ones through the work of some amateurs.

By further continuing this line of .research, the inorganic structures which have been described can manifestly never be developed to living organisms or anything near it. But we may hope that future discoveries might lead to the production of more perfect artificial structures by performing enzymatic reactions in an "organized" manner. Even such more highly developed artificial structures


would probably not yet be "living," but, they would resemble living organisms to such an extent that we would no longer believe in the sharp boundary line which now seems to separate the living and the inanimate nature. The living organisms would then appear to us merely as limiting cases which occur in nature owing to peculiar coincidences which are now called self-preservation and reproduction. It may take ages for such a possibility to become real. Difficulties of an entirely unforseen nature may handicap further progress in this line. Even if the difficulties are greater than now appraised, the student of nature perceives the new outlook as a true revelation. It inspires him with new hope for the further development of science.

DERIVATION OF PHYSICOCHEMICAL LAWS: BRIEF OUTLINE OF THE LAWS OF OSMOSIS, ELECTROLYTIC

DISSOCIATION AND MEMBRANE EQUILIBRIUM

The fundamental problem which the study of the artificial structures presents is the nature of the peculiar driving force acting in them, the so-called osmotic force,' manifestly this is also one of the forces which playa role in living organisms although it may not be the most important one.

"Force" is the name for the "cause of changes." The causes of changes, due to osmosis, have been thoroughly investigated by the science of physical chemistry, of which a few of the most important principles are briefly summarized on the following pages. For more detailed information the reader is referred to textbooks of Physical Chemistry.l

1. DETERMINATION OF THE MAGNITUDE OF OSMOTIC PRESSURE

When salt, sugar or any other soluble substance passes into solution, a dissipation takes place similar to the one occurring in evaporation. Consequently we may speak of a "solution pressure" which drives the soluble salt into solution, just as we speak of a vapor pressure in vaporization. Or we may define a "diffusion pressure," which drives the highly concentrated solution, present in the immediate vicinity of the solid substance, further and further into the surrounding water. Such definitions as these are, of course, useless as long as no methods are available for measuring that "solution

1 The most exhaustive textbook of physical chemistry is W. N ernst's "Theoretical Chemistry," English translation by L. W. Codd (MacMillan Company, New York). Special treatises which are,adapted to the use of biologists are: Alexander Findlay's "Physical Chemistry for Students of Medicine" (1924), published by Longmans Green & Company, and M. Steel's "Physical Chemistry and Biophysics," 1928 (J. Wiley and Son). The more recently developed theory of membrane equilibria and the osmotic action of colloids is reviewed briefly in the appendix of this book. It may be added that certain criticisms of the physicochemical theory of osmotic pressure by J. B. Haldane (Silliman Lecture, 1922, p. 175, and other publications) refer to certain special points which are not described here. The generally acknowledged basic principles, briefly outlined below, cannot be questioned by this or any other critic.

23


pressure" or "diffusion pressure." This is difficult as long as they do not exert any direct mechanical effect.

The conditions are different if a solid substance goes in solution not into straight water but into a solution of another substance which produces a precipitation membrane with it, as for example, K4Fe(CN)6 going in solution into a dilute CUS04 solution. The boundary up to which ICtFe(CN)6 has gone into solution is now marked by a precipitation membrane and methods can be devised for measuring the solution pressure since it acts on that membrane. It is customary to speak, in this case, of "osmotic pressure" rather than of "solution pressure" although the phenomena are essentially similar. The measurement is done by counterbalancing osmotic pressure by a hydrostatic pressure.

It is necessary, for the purpose of such a measurement, to produce a membrane which is held in place rigidly. This is done by allowing the precipitation to take place inside of a cup of porous clay. For this end a CUS04 solution is poured into a moistened porous cup and, at the same time, this cup, with the solution in it, is immersed in a &Fe(CN)6 solution. The two solutions diffuse into the clay from opposite sides and produce a precipitation of semi-permeable CU2-Fe(CN)& in the inner layers of the clay where they meet. The cup is then rinsed and filled with the solution, the osmotic pressure of which is to be measured. The measurement is done by means of a mercury manometer, or a pressure gauge. Such an arrangement is known as an osmometer. The hydrostatic pressure must act, in this case on the solution so as to counteract the streaming of water into it. (See Fig. 10.)

In such an apparatus we observe that a 1 per cent solution of cane sugar requires 53.5 cm. of mercury to counterbalance its osmotic pressure; in other words, water will be driven into the porous cup and the level of the mercury will rise until it reaches 53.5 cm. (this would equal 7.57 meters or 24.8 feet of water column). Further measurements show that a 2 per cent sugar solution exhibits an osmotic pressure of 101.6 cm. of mercury which is slightly less than twice as high as the 1 per cent solution; a 4 per cent solution exhibits 203.4 cm.

The amazing magnitude of osmotic pressure was first discovered in 1877 by Pfeffer,2 a German botanist, by observ.ations on plants which perform

2 Pfeffer, "Osmotische Untersuchungen" 1877.

DERIVATION OF PHYSICOCHEMICAL LAWS 25

certain movements when touched, like Mimosa pudica, Cynara scolymus, or Centaurea jacea. Certain elongated cells of these plants lose their turgor after a stimulus because their osmotic pressure drops. From the pull required to stretch the filament of Cynara scolymus-after it has relaxed following a

The semi-permeable membrane is embedded in clay, and held in place rigidly hence the osmotic pressure can work only in the direction indicated against the manometer. The solution, the osmotic pressure of which is to be measured, is contained in the porous cup and in the wide tube attached to it.

«irteflon of !/Y«ro.5f"l/t 1 I're.ssurc •

a/redion Dr OSl11ol/, I'ressure . .

FIG. 10. PFEFFERS OSMOMETER

touch-one can conclude that the inside pressure which keeps the cells stretched when irritation is absent must be about 2-4 atmospheres, or 150 to 300 cm. of mercury. Pfeffer showed that such a high pressure can actually be measured in an osmometer of the type already described.


Great technical difficulties are involved in this measurement. In spite of the large force of osmotic pressure the movements of the fluid are very slow since the internal friction prevents rapid movements. It takes months before the final reading of a manometer is reached. The slightest leakage spoils the experiment. In order to check Pfeffer's original measurements, Morse and his collaborators3 have worked with an extremely elaborate osmomrter taking care to eliminate all sources of error or disturbance.

The osmotic pressure is found to increase if the temperature is increased. This increase amounts to 2 ~ 3 of its value at O°C. for each rise of temperature of 1°. This is striking because gas pressure also increases with the temperature at the same rate if a gas is heated in a closed vessel.

We let this close agreement induce us to investigate whether there are further similarities between gas pressure and osmotic pressure. Let us assume that cane sugar was a gas at ordinary temperature and let us suppose that this gas was compressed to the same volume which the cane sugar occupies in a 1 per cent solution. A physicist can well perform a calculation based on such an unreal assumption, for the gas laws are known in all details. He will tell us that this pressure will equal 53.4 cm. of mercury. This is in close agreement with the osmotic pressure which has been found experimentally, viz., 53.5 cm.

From this and many other similar agreements we may conclude that the osmotic pressure· of a dissolved substance is the same pressure which this substance would produce if it could be transformed into a gas without change of volume and temperature (Vant Hoff, 1887).4 This may also be expressed by stating that dissolved substances are in a state or condition resembling that of gases. The process of solution resembles that of volatilization. On account of this resemblance, osmotic pressure should depend on molecular weight as does gas pressure. This is shown more definitely by means of quantitative measurements, preferably by the well known indirect methods which will now be briefly described.

On account of the difficulties involved in direct measurements of osmotic pressure, indirect methods are used, preferably those based on determinations of freezing-point lowering. This lowering is pro-

3 Am. Chern. Journ., 26, 80, 1901. 4 Zeitschrift physik. Chemie, 1, 488~ .


portional to the osmotic pressure of the dissolved substance for the following reasons. Ice contains no dissolved substance. Consequently, when ice freezes out, the solution becomes more concentrated, hence the osmotic pressure is raised. This rise requires work to be done, and hence, it is more difficult to freeze a solution. In fact the higher the osmotic pressure of its solute the greater is the difficulty of freezing it. All solutions having the same osmotic pressure, hence containing the same number of molecules, have the same freezing point, irrespective of the size of the molecules. A solution which contains 1 gram molecule5 per 1000 cc. -a so-called molecular or 1 M solution freezes at -1.85°C. Thus, for instance a .6 per cent solution of urea freezes at -1.85° since 60 is the molecular weight of urea.

The freezing point method is most extensively used for determinations of the osmotic pressure of biological fluids of any kind. The apparatus of Beckmann which is usually employed for this end is described in all textbooks of physical chemistry.

2. OSMOTIC PRESSURE OF ELECTROLYTES, DISSOCIATION OF WEAK

ACIDS

All those aqueous solutions, which conduct the electrical current, lower the freezing point more than calculated from the molecular weight of the dissolved substances in them, hence these substances exert a greater osmotic pressure. Such substances are the so-called electrolytes, such as salts, acids or bases. Their molecules act as though they were split. into two or more parts-as shown by their greater osmotic action. Their split products, termed ions, carry electrical charges, as elucidated by electrolysis, where the "ions" are discharged at the electrodes and where their chemical nature is revealed; thus NaCI is split into Na+ and Cl-, Na2SO, into 2 Na+ and SO,--. Most salts and the so-called·strong acids and bases are split into ions almost completely at any concentration (S. Arrhenius, 1887}.6

In contrast to this, numerous organic acids and other acids which manifest weak acid properties are split up to a slight degree only, as shown by their low electrical conductivity and by their more nearly

6 A "gram molecule" means as many grams of the substance in question as its molecular weight.

6 Zeitschrift physik. Chemie, 1, 631.


normal freezing-point lowering. Moreover, the degree of splittingas calculated from the electric conductivity-varies enormously for these weak acids or bases. For instance, one-half of the "weak" acetic acid is split in extremely dilute solutions, viz., in solutions containing less than 0.00005 M. In aIM solution, however, no more than about 3 ~ 0 is split. The degree of splitting is always very much larger in more dilute solutions. This variable splitting depends on a chemical equilibrium established between the split and the non-split fraction. The result of this variability is that relatively more hydrogen ions are split off in dilute solutions; in other words, the hydrogen ion concentration varies only slightly with the concentration, in solutions of weak acids.

In a solution containing a salt of the weak acid and the acid itself, such as e. g. sodium acetate and acetic acid, a still greater invariability of this kind is found. The hydrogen ion concentration is practically independent of the concentration; in other words, it remains unchanged if the solution is diluted with water. Such solutions are the so-called buffer solutions. The majority of body fluids are of such a composition. The hydrogen ion concentration of such a "buffer" solution depends on the ratio of the weak acid to the salt of that acid.

In any solution, the hydrogen ion concentration is a measure of the actual acidity present. Consequently buffer solutions have a constant acidity, viz., one which remains unaffected by dilution, or is changed by further addition of an acid only if the ratio of the free weak acid to salt of the weak acid is altered materially thereby.

The amount of alkali or acid required to reach the neutral point (or to change the hydrogen ion concentration) does not depend on the above ratio but on the absolute' concentration of the weak acid or salt present. It is independent, therefore, of the H ion concentration and cannot be used to measure the acidity, in the case of buffer solutions.

The presence of buffer solutions in living organisms is the chief cause of the maintenance of a constant hydrogen ion concentration (or near neutrality) in them. In body fluids the most important weak acid is H 2CO a, its salt is NaHCO a; the ratio between the two determines the hydrogen ion concentration. The NaHCO a present chiefly determines the so-called "alkaline" reserve (L. J. Henderson, 1908).7 None of these rules holds if a strong free acid is present; in

7 Am. Journ, Physiol., 8, 274.


this case the hydrogen ion concentration depends largely on the dilution. This is true, for instance for the gastric juice.

The hydrogen ion concentration of a buffer solution, containing equivalent amounts of a weak acid and its salt, is equal to the "affinity constant" of that acid. This constant determines the acidifying power of the acid in question and is usually calculated from the equilibrium of dissociation of the acid (for details of this calculation see textbooks of physical chemistry). To give numerical examples, we may quote the affinity constant of the very weak carbonic acid, which is 0.000016; for the stronger lactic acid' it equals 0.00014; for the still stronger tartaric acid, 0.001. In general the hydrogen ion concentration (or acidity) of a buffer solution equals the affinity constant of the acid contained in it, multiplied by the ratio of acid: salt.

lt is customary to designate the hydrogen ion concentration of a buffer solution by its negative logarithm which is termed pH. For instance, if the H+ concentration, always expressed in grams per 1000 cc., equals 1/10 = 10-1, pH equals 1.

If it is 1/100 = 10-2, pH = 2 If it is 1/1000 = 10-3, pH = 3, etc. lt is important to remember that at the neutral point the hydrogen

ion concentration equals 10-7 = 1:10 million, hence pH = 7. The neutral point is defined by the presence of equal amounts

of H+ ions-which denote acidity, and OH- ions-which dissociate from bases and characterize alkalinity. Hence, at the neutral point, the OH- concentration also equals 10-7•

Alkaline solutions should be characterized by their OH- concentrations. It is possible and customary, however, to quote hydrogen ion concentrations, or pH values, even for alkaline solutions since the product of OH- and H+ concentrations must be constant and equal to 10-14

according to well known physicochemical laws. Hence a hydrogen ion concentration smaller than 10-7 corresponds to any OH- concentration larger than 10-7, in other words pH values larger than 7 and ranging up to 14 denote alkaline solutions.

To quote numerical examples:

pH = 12, means that the H ion concentration equals 10-12, hence the OHconcentration 10-' since 10-12 • 10-' = 10-14 as postulated; 10-' would correspond about to a O.01M NaOH solution.

pH = 10 would correspond to a OH- concentration of 10- 4 as found in a nearly 0.0001 M NaOH solution.


It should be remembered that, in general, the smaller the pH the more acid the solution; the larger the pH, the more alkaline it is.

Determinations of pH can be made without a detailed chemical analysis of all their constituents. The simplest method is based on a colorimetric comparison of the unknown solution with a standard mixture of known pH, following the addition of an indicator. The indicators are weak colored acids which form salts of a different color, as e.g., litmus which is a pink acid forming blue salts (in alkaline solution).

At the turning point of the indicator the acid and its differently colored salts are present in equal amounts. On either side of this turning point lies a zone of transitional coloration where, for instance, pink and blue litmus are present in a variable ratio, producing purple or violet shades. Within a range of 2 pH the transitional coloration is distinctly to be recognized and can be used for pH determinations since each coloration corresponds to a definite pH, hence two solutions giving the same color, have the same pH. A large number of indicators having turning points at any desired pH are available.

More accurate are the electrometric methods of pH determination which are based upon the principles of concentration cells (see below, page 217).8

3. MEMBRANE EQUILIBRIUM AND OSMOTIC PRESSURE OF ELECTRI

CALLY CHARGED COLLOIDS AS THE CAUSE OF COLLOIDAL

SWELLING

Besides the osmotic forces which are the cause of water exchange in the tissues, a different type of action exists, namely, colloidal swelling. Even without any visible membrane, proteins or other high molecular substances which are constituents of living tissue, can take up water or give it up, depending upon the conditions. Such high molecular substances are known as "colloidal" or glue-like substances, more particularly they are the so-called "hydrophilic colloids. "

Up to a short time ago, it was believed that osmotic and colloidal

8 For further details on pH determination, see Mansfield Clark's "Determination of Hydrogen Ions," 3d ed., Balt~more, 1928.


swelling had nothing to do with each other. More recently the presence of forces similar to oEmotic forces has been traced in colloids, and it has been possible to explain colloidal swelling, partly at least, as a result of these forces.9 This can be shown by studies of the osmotic pressure, which solutions of high molecular substances, like soaps or proteins, exert on a membrane impermeable to them but permeable to salt. These investigations which comprise the theory of membrane equilibria (Donnan equilibrium) and other subjects are explained in detail in the Appendix.

The most important principles of this theory may be briefly summarized as follows. The osmotic pressure of a colloidal solution is relatively small and can easily be counterbalanced by a water column of convenient height. It is thus always measured directly in heights of water column and not by the freezing point method. Measurements, done in this way, show however, that this osmotic pressure is not a definite entity, but varies in an uncontrollable fashion. This leads to the conclusion that colloidal molecules are not uniform. They consist of aggregates (or so-called "micellae") the size of which varies within wide limits. The osmotic pressure of colloids, as such, does not afford interest for investigations. However, its variations are an object of research.

Special interest is attached to its variations following the addition of acids and salts, such as HCI and NaC!. These variations, which are very large, are due to an interaction of electrical forces since HCI forms colloidal salts with many colloids, such as gelatin or other proteins. The colloidal ions which dissociate from this salt attract, by dint of their multiple charges, a large number of monovalent ions and prevent them from passing the membrane. Held within the membrane by electrical attraction, these small otherwise penetrating ions have ar;tually become non-penetrating and increase the osmotic pressure of the colloid enormously. An effect of this type accounts for the increase of the osmotic pressure of gelatin following addition of HC!. Gelatin combines with HCI to form gelatin chloride which dissociates into gelatin ion and chlorine ions. Each gelatin molecule gives rise to about one gelatin ion, but, to each of these correspond many chlorine ions, on the average 50 to 100. All these are held within the shell and thus increase the osmotic pressure.

9 See J. Loeb, "Proteins and Colloidal Behavior," New York, 1922. This book also gives further references.


If NaCI is also present this salt is displaced by the heavy colloidal ions. If equilibrium is attained, NaCI must stay largely outside of the membrane, viz., in the protein free solution. Hence it exerts an osmotic counterpressure wh1'ch manifests itelf by a drop of the observed osmotic pressure of the colloid. Such a counterpressure is also exerted by HCI, if more is added than corresponds to the binding capacity of gelatin. Thus, while moderate additions of HCI must increase the osmotic pressure of gelatin, an excess of HCI must decrease this pressure. This is verified by experimental observations (J. Loeb).9

The osmotic pressure of the colloid cannot decrease to zero, however, even if an excess of NaCI is added. Its lowest value is identical with the osmotic pressure which the colloid exerts at its iso-electric point. The iso-electric point which is characterized by a more or less definite pH (for gelatin pH = .. P) lies in the transition between the more alkaline solutions in which gelatin combines with bases-thus acting as an acid-and the more acid solutions where gelatin combines with acids,-thus acting as a base. The iso-electric point of a colloidal protein can be determined by combining the protein with stainable or colored ions, according to Loeb. (Compare Figs. 11 and 12 and legends.)

Another peculiarity of these membrane equilibria is the unequal distribution of ions on either side of the membrane. Gelatin HCI has a higher CI, but a lower H ion concentration than the gelatin free HCI solution in equilibrium with it. In other words, an acid protein solution is less acid than a straight acid solution on the other side of the membrane in equilibrium with it. On the alkaline side of the iso-electric point, the reverse is true, the protein solution is less alkaline (or more acid) than the protein free solution on the other side of the membrane.

These results and many others have been confirmed both by theory and experiment. In regard to the theory one of the most recent contributions has been added as late as 1930 by the writer, who showed how the valence of the gelatin ion can be calculated from the depressing effect of salt addition on the osmotic pressure. lO

10 A complete account of this theory has not been published; for a preliminary communication see: Proceed. Soc. Exper. Biology and Med., 27, 692, 1930, for a detailed account see the Appendix.


FIG. 11. THE COMBINATION OF CATIONS (OR BASES) WITH PROTEINS ON THE ALKALINE SIDE OF THE ISOELECTRIC POINT

Proof that cations combine with proteins only on the alkaline side of the isoelectric point. Powdered gelatin brought to different pH was treated in a dark room with M/64 AgNOa and then washed with cold water to remove the silver not in combination with gelatin. The gelatin was liquefied, brought to a 1 per cent solution, and the pH was determined. The solutions were then poured into test tubes and exposed to light. In about half an hour the gelatin of pH > 4.7 was dark while the gelatin of pH 4.7 or less remained permanently clear though exposed to light for over a year. The pH of each gelatin solution is marked at the head of each-test tube. (From Loeb's "Proteins and Colloidal Behavior.")

FIG. 12. THE COMBINATION OF ANIONS (OR ACIDS) WITH PROTEINS ON THE ACID SIDE OF THE ISO-ELECTRIC POINT

Proof that anions combine with proteins only on the acid side of the isoelectric point. Doses of powdered gelatin solutions of different pH were treated with M/128 K4Fe(CN)& and then washed with cold water. All the samples of gelatin solution of pH < 4.7 turned blue (through the formation of some ferric salt), while all the gelatin solutions of pH 4.7 or above remained colorless.


This has been possible by a further development of Donnan's theory which considered nothing but monovalent colloidal ions. According to this rather too simple assumption, the osmotic pressure should never be depressed by NaCI to less than one-half of its original value, while the experiment shows a very much larger decrease in many cases. Theoretical considerations show that the osmotic pressure can be lowered by an excess of salt to one-half for

1 monovalent ions, to one-third for bivalent ions, hence to -- for n-valent

n+1 ions. Since the osmotic pressure of gelatin can be lowered to about one sixtysixth, as calculated from Loeb's measurements, the gelatin ion has a valence of 67,11 just as the Na ion has one, the Ca ion two, the Al ion three valences.

As a consequence of these conditions, the osmotic ,pressure of a gelatin solution

(1) is a minimum at the iso-electric point. (2) it first increases and then decreases on gradual addition of

HCI. (3) it decreases on addition of N aCI to the same minimum as at

the iso-electric point. Further experiments show that the swelling of solid gelatin granules

is subject to the same rules, and so is the variation of the viscosity of gelatin solutions. Consequently both solid gelatin granules, as well as gelatin solutions, behave as though they consisted of innumerable bags made of a semi-permeable shell and each containing a colloidal solution. The higher the osmotic pressure of the solution inside the bag, the more it enlarges. This would account for the swelling as well as for the increase of viscosity. This theory of membrane equilibrium is not applicable, however, to explain the swelling of gelatin in every case. Gelatin swells also in various neutral salt solutions (salts of the "Hofmeister" series). For an explanation of these effects, see Northrup and Kunitz.12 (For details concerning these ·relationships see Appendix.)

11 This is true only for the gelatin sample chosen for those determinations. Since gelatin is not a definite chemical entity, somewhat varying results may be expected in other cases.

12 Journ. Physical Chern., 36, 102.

APPLICATION OF PHYSICOCHEMICAL LAWS: TRACING OSMOTIC FORCES IN THE LIVING TISSUE. LIMITATIONS OF THE OSMOTIC THEORY. THE SEARCH FOR OTHER FORCES WHICH MAY SHIFT FLUIDS IN TISSUES

The starting point for our first attempt at approach has been the empirical investigation of artificial structures which exhibit striking similarities to certain elementary life phenomena. This has induced us to review the well known physicochemical rules which elucidate the nature of the driving force in these structures. In pursuing the physicochemical line of research, the investigators have been led further and further into innumerable details of a strictly physical character so that finally they have lost sight entirely of the starting point, viz., the search for the nature of some of the driviryJ forces in life processes. Gowland Hopkins describes the development of chemistry by stating:! "The hand and thought of the qualified chemist has become divorced from the living plant and alJimal." Certainly, the same is true of the physicists and physical chemists who have investigated osmotic pressure, electrolytic dissociation and all the multitude of related pherwme"!_a.

Whatever usefulness we may ascribe to purely physicochemical re-" search we should realize that these studies are naturally insufficient by themselves to approach ow: goal, the exploration of life processes. In' this respect we may compare the physicochemical studies to the athletic exercises which a mountain climber performs before an ascent. Even though they may be indispensable he cannot reach the peak by doing them exclusively.

1. THE OSMOTIC PRESSURE OF THE BLOOD AND OTHER BODY FLUIDS

The composition of the blood fluid or plasma is constant in all higher animals. In mammals it contains about 9.8 per cent of dissolved substances of which roughly 9 per cent are proteins and 0.8 to 0.9 per cent are salts. Of all salts present sodium salts amount to more than 95 per cent. In spite of their greater concentration the proteins exert almost no osmotic pressure as compared to that of

1 Lancet, 1925.

35


the salts, since their average molecular weight is more than two hundred times higher than that of the salts.

All vertebrate animals and many invertebrates have a constant osmotic pressure in spite of the large amount of substances which are constantly being introduced with the food. The excess of dissolved substances is excreted by the liver and the kidney. Consequently the osmotic pressure of the urine frequently changes, varying between 12 to 26 atmospheres, for the same individual within 24 hours. Blood in the portal vein which is loaded with food stuffs may contain up to 10 per cent more dissolved substances than the rest of the blood, as measured by its freezing point (Fano and Bottazzi, 1896).2

As the study of artificial structures has shown, osmotic pressure is likely to be connected somehow with the process of growth, although it is by no means its sole cause. The constancy of osmotic pressure in animals seems to be related to the more regular type of growth which they exhibit as contrasted to that of plants. The higher the animal stands in the evolutionary series the more perfectly developed are its mechanisms for keeping the osmotic pressure constant. The lowest types of animal life which are found abundantly in the ocean have no such regulatory mechanisms; their osmotic pressure is kept constant by the perfusion of their entire body with sea water. Land plants, on the other hand, have no osmotic regulation (deVries, 1884).3 Their osmotic pressure varies within the widest limits, roughly between 3 and 30 atmospheres according to temperature, humidity, season, age and species of plants. In desert plants it ranges up to 100 atmospheres I

The lowest types of animal life are likewise devoid of regulatory mechanisms for keeping their osmotic pressure constant. Their life processes are, however, not compatible with such changes to the same extent as in plants. This is probably the reason why an immensely larger number and variety of low forms of animal life are met with in the ocean. o The following figures may serve to illustrate these facts. (Bottazzi, 1897).4

2 Archiv. Ital. Biologo, 26, 45. 3 Jahrbuch wiss. Botanik, 14, 427. 4 Quoted from an article of F. Botazzi's in "Physik. Chemie und Medizin,"

1907.

APPLICATION OF PHYSICOCHEMICAL LAWS 37

The freezing point of the blood of man is -0.56°C., hence the molecular content of blood is ~:: ~ = 0.303. (This would correspond to 1.78 per cent NaCI if NaCI was not dissociated into ions; taking into account ionization, it corresponds to 0.9 per cent NaC!.)

The corresponding osmotic pressure at body temperature (37.5°) equals 22.4 (1 + "2"h·37.5)·0.303 = 7.72 atmospheres.

The blood of other mammals and of reptiles contains a greater quantity of dissolved substances, the osmotic pressure amounting to about 9 atmospheres. Most amphibians have a lower osmotic pressure; viz., 4 to 5 atmospheres, corresponding to a freezing point of -0.42°C.

In marine fish we find in general that, the higher the fish stands in the phylogenetic series, the lower the osmotic pressure. The blood of ganoides, the highest type of fish, has a freezing point of :'_0.76°C. close to that of whales which have -0. 7°C.; in teleosts it is -0.76° to -1.04°C. In selachiae, however, it is _2.2° to -2.4°C., practically equal to that of the ocean which is -2.3°C. on the average. These fish, then, have not developed any osmotic regulation. The same is true for all invertebrates of the ocean, like cephalopodes, crustaceae, worms, echinoderms and coelenterates. Those invertebrates, however, which live outside the ocean have a certain osmoregulation; their osmotic pressure being nearly within the range of that of vertebrates. The blood of freshwater fish has a freezing point of -0.45° to -0.69°C.

2. OSMOTIC EFFECTS IN CELLS OF PLANTS AND ANIMALS

The osmotic properties of cells or tissues are tested by placing theIl! in salt solutions of higher or lower concentrations than the natura~ body fluid. If membranes, impermeable to salts, are in the tissue, the altered conditions thus produced will lead to an intake-or a giving off of fluid, due to the play of osmotic forces. In certain plant cells, as for instance in the epidermal cells of Tradescantia Discolor, a fluid exchange IS directly visible. In this case the cellulose frame, in which the cell proper is encased, remains unchanged, but, the protoplasmic body, contained in it, shrinks in concentrated salt solutions, as indicated diagrammatically in Figure 13. Evidently the protoplasmic body is surrounded by a membrane permeable to water and less permeable to salt and other dissolved substances like sugar (deVries, 1884,5 and Overton, 1895).6

With red blood cells osmotic effects are readily demonstrable; in

5 Jahrbuch wiss. Botanik, 14, 427. 6 Vierteljahrsschrift der naturforsch. Ges. in Zurich, 40, 1.


hypertonic salt solutions7 the cells shrink while in hypotonic solutions they either swell or burst, if the solution is sufficiently diluted. For blood corpuscles of cattle, for instance, the dilution must be carried from the normal level of i molecular down to about n molecular, if the majority of the red blood cells is to be broken up or "hemolyzed." Such osmotic experiments are easily performed by mixing one or two drops of defibrinated blood with a few cubic centimeters of a saline solution and observing the highest concentration at which a distinct hemolysis occurs. Or, if it is desired to observe volume changes of the cells due to osmotic fluid exchange, the mixture con-

1 Original aspect of cell

2 Aspect of cell in !1 concentrated

solution FIG. 13. DIAGRAM ILLUSTRATING THE EFFECT OF OSMOSIS ON TRADESCANTIA

CELLS

taining the cells is filled in a graded capillary, a so-called hemotacrit, and centrifuged after sufficient standing. The total volume of the sedimented cells is -then compared to that of normal whole blood.

The result of such experiments both on plant and animal (blood) cells is, in general, in agreement with the rules of osmotic pressure. If a variety of salts is tested on red blood cells of cattle, for instance, by determining the highest concentration at which hemolysis occurs, £t is observed that the amount of salt invariably corresponds to its molecular weight. Thus a 0.56 per cent NaCI is just sufficiently diluted for

? Solutions which cause neither shrinkage nor swelling are termed "isotonic." More dilute solutions are termed "hypotonic;" those more concentra ted "hypertonic."


hemolysis, or a 100 per cent KNO s solution, or a 1.58 per cent NaI solution, or a 1.66 per cent KI solution. All these solutions are nearly -lIT molecular, since the molecular weight of NaCI is 56, of KNO a 101, of NaI 155, and KI 166 (Hamburger, 1890).8 Even approximate determinations of molecular weight are possible by such biological tests.

Not only red blood cells but also numerous other body cells can be shown to exhibit osmotic properties, in other words to act as though composed of bags permeable to water, less permeable to salts, sugars, or other dissolved substances. This holds particularly for freshly excised skeletal muscle, also for~kidney, liver and other parenchymatous organs. Skeletal muscle of frog, for instance, keeps its weight constant in 0.7 per cent NaCI for many hours, it swells in less concentrated and shrinks in more concentrated solution. The weight is also kept up in isotonic NaBr, NaNO~, LiCI, or cane sugar solutions· (0. Nasse, 1869;9 Overton, 1902).10 But not all other salt solutions, which are osmotically active on blood cells, can be used. Many salts such as sulphates, iodides, calcium or potassium salts produce secondary variations of the muscle membrane. The use of muscles of warm blooded animals in such experiments involves great difficulties since decomposition sets in rapidly.

It should be noted, however, that smooth muscle is not subject to osmotic actions of the same kind as skeletal muscle. It seems that it does not contain such semi-permeable membranes but acts rather like a colloid such as gelatin (E. B. Meigs, 1912).u

If either hypertonic or hypotonic solutions are introduced into the veins of a living animal the salt concentration is altered temporarily only; restitution of the normal concentration occurs within a few minutes (Sollmann, 1901).12

3. THE OSMOTIC ACTION OF LIPOLYTICS (OVERTON, 1895)13

The osmotic pressure of any dissolved substance can be measured by its freezing point lowering or by other suitable methods, but, this

8 Zeitschr. fUr physik Chemie, 6, 319. 9 Pfliigers Archiv., 2, 67.

10 Pfliigers Archiv., 92, 115. n Journ. Exper. Zoology, 13, 497. 12 Arch. exper. Path. Pharm., 46, 1. 13 Vierteljahrss~rift d. naturforsch. Ges., Ziirich, 40, 159.


pressure can only become effective in or on a given tissue provided· that this tissue contains membranes which retain the dissolved substance. A penetrating substance cannot exert an osmotic pressure. We know of a large number of substances which are not withheld by any membrane in tissue; they penet.rate even more rapidly than water, hence never cause shrinking of cells, even in the highest concentrations.

Such substances are: all the monovalent alcohols and their ethers, all water-soluble aldehydes, aldoximes, ketoximes, nitriles, hydrocarbons and halogenated hydrocarbons-as far as they exhibit some solubility in water, as e.g., chloroform-neutral esters of organic and inorganic acids, and finally many organic acids and bases, including G0 2, NHa and boric acid. All these substances are soluble in oil, hence are usually termed lipolytics. In general, tissues of plants as well as of animals behave alike; lipolytics penetrate rapidly in all of them.

To demonstrate the penetration of these substances, they may be added to an isotonic NaGl solution into which a muscle is placed. The weight of the muscle will remain unchanged as though NaGl alone was in the solution. For example, a sartorius, the weight of which has remained constant in 0.7 per cent NaGI solution, does not change its weight in a solution of 0.7 per cent NaGl + 5 per cent methyl alcohol, although this solution has the osmotic pressure of a 5.2 per cent N aCl solution. If the alcohol is added to a hypotonic solution swelling occurs in spite of the addition.

The penetrating power can also be traced by direct chemical methods, since lipolytic substances can be shown to accumulate in cells or tissues. In order to demonstrate this, red biood cells or muscles are shaken with an isotonic NaCl solution containing an alcohol or other lipolytics. By determining the freezing point of this solution .before and after shaking with the blood cells or muscle, an accumulation of a part of the alcohol in these cells can be demonstrated (Hedin, 1897).14

Oil solubility, per se, is not as important for penetration as the ratio of the solubilities in oil and in water, or the distribution of the substance in question between oil and water. This ratio is determined by shaking a weighed amount of the lipolytic substance with measured volumes of oil and water. The oily phase and the aqu{lous phase are then

14 Pfluger's Archiv., 68, 229; see also 144, 469 (1912) (Warburg).


separated and each of them is analyzed. In this way the distribution between water and oil is determined for a number of lipolytic substances. Almost invariably it is found that the more the lipolytic accumulates in the oil, the more rapidly it will penetrate tissue of any type (Overton, 1895).15

For a number of substances comparison has been made (by Overton) between the distribution in water and oil on the one hand and the power of penetration through tissue membranes on the other hand. Not only the lipolytics, named above, were used in these experiments, but also bivalent alcohols, and ami des of monovalent acids. These dissolved substances accumulate only slightly in oil, being more soluble in water. Yet they have a slight oil solubility, and hence the osmosis which they produce is of a short duration only. In the beginning these dissolved substances are entirely outside of the membrane, although water passes through the cell membrane at once, and osmosis sets in immediately. If the concentration outside is higher than normal the cell will shrink. Later, however, the dissolved substance also passes the membrane and no further shrinking· occurs. As the process continues, more and more of the dissolved substance passes into the cells, until finally the direction of the osmotic forces is reversed (Overton, 1902).16

Such experiments may be performed, for instance, with glycol, a bivalent alcohol having a low oil solubility. Further experiments have been made with substances like glycerol, which is a trivalent alcohol, and with a variety of sugars and urea. The latter are insoluble in oil. Accordingly they were found not to penetrate tissues, but to cause osmosis, like the majority of salts. Glycerol still has a slight penetrating power being on the borderline of the lipolytics (Overton, 1902).16

Experiments on a variety of physiological objects have been performed to test the penetration of lipolytics. All of them have confirmed the parallelism to oil-solubility in general. Summarizing, we may accept Overton's rule of the parallelism of cell penetration and oilsolubility of a water-soluble substance as an approximate biological law. The exceptions from this law relate to rather special cases only (see below, pages 43-46).17

15 Vierteljahrschrift d. naturforsch. Ges., Ziirich, 40, 1. 16 Pfliigers Archiv, 92, 115. 17 It may be added that swelling or shrinking is not the exclusive criterion


It has been found, furthermore, that substances which are oil-soluble and penetrate tissues have the power of narcotizing animals. To a certain degree oil-solubility and narcotic power have been found to run parallel, although further complications cause deviations from this simple rule (see below, page 273).

4. LIPOIDS AS CONSTITUENTS OF TISSUE MEMBRANES

The parallelism of oil-solubility and penetration into tissue seems to indicate that the semi-permeable membrane consists of some sort of water immiscible substance resembling fats, although it need not be a genuine fat. It may also be a special kind of protein with properties similar to those of fats. These fat-like substances take up lipolytics from the aqueous solutions because lipolytics are better soluble in the membrane itself than in water. After this, the lipolytics pass from the membrane into the interior of the cell.

The essential feature of osmosis is, of course, the rapid penetration of water through the semi-permeable membranes. It should be emphasized that most fat-like substances also dissolve water. Hence the lipoid theory is also capable of accounting for the penetration of water,18 Even genuine fats dissolve water and allow it to penetrate, as the following experiment shows. Finely ground N aCI is .thoroughly mixed with olive oil so as to form a stable emulsion. If droplets of this oil-salt mixture are placed in water they swell rapidly. A closer examination shows that vacuoles filled with saline solution are being formed within them. This can only be due to a penetration of water through the oil. The water then dissolves the salt to form a hypertonic solution and attracts more water through the oil. In this. w::)'y ia,t membranes can give rise to jlJst that type of osmosis which we observe in tissue. Tissue membranes are, of course,

in all cases. In the case of certain plant cells, for example, color changes are observed if acids or bases penetrate. Strong acids or bases like HCI, H2S04 or NaOH fail to bring about a change, since they do not penetrate. Weak acids or bases like CH.C02H, CO2, NH., etc., react rapidly since they penetrate on account of their oil-solubility. Many other special methods have been devised. (See E. Gellhorn's "Permeabilitiits Problem," Berlin, 1929.)

18 This important point has been doubted by some writers, notably by R. Hober; compare his handbook of "Physikal. Chemje cler Zelle," 5th ed., 1924, p.502.


far more delicate than the rather heavy oil layers used in this experiment, and hence let water pass more rapidly (Butschli, 1892).19 Moreover, the fat-like substances, contained in tissues, e.g., lecithin, dissolve more water than a genuine fat of the kind used in this experiment. Hence, the water permeability of the cell membrane could be easily accounted for, even if it contained no protein.

These statements concerning the.nature of the cell membrane are, of course, quite indefinite. We ought to be more specific and attempt to give its exact make-up. This is not yet possible, but experiments with models can be performed by preparing artificial membranes and then testing their permeabilities. Thus, to put the entire problem on a broader basis, not only lipoidal membranes but also membranes of collodion, proteins, or other materials have been investigated.

The results of such experiments have furnished an additional proof, although indirect, for the lipoid theory since artificially made membranes of gelatin and collodion exhibit a permeability of an entirely different type than tissue membranes. These membranes allow all substances of a low molecular weight to pass while high molecular substances are retained. Lipoid solubility plays no role for these artificial membranes, very much in contrast to most tissue membranes. We may describe these artificial membranes as sieves. Their semipermeability is due to the size of their pores which are insufficient for the large colloidal particles to pass. Such membranes consist of collodion or gelatin. By suitably modifying the mode of their preparation, their pores can 'be made smaller so that not only colloidal particles but, even larger molecules, such as those of cane sugar (molecular weight 342) are retained. Urea, however (molecular weight 60) will pass slowly, and ammonia (molecular weight 17) passes rapidly (R. qollander, 1926 and 1927).20 Such artificial membranes are impermeable to alkaloidal bases, to amylalcohol, to

19 "Untersuchungen tiber mikroscop. Schaume," Leipzig, 1928. 20 Societas Scientiarum Fennica Comment at Biolog., II, 6; also Proto-

plasma, 3, 213. , In order to prepare such a sieve membrane a solution of collodion is poured on

a mercury surface and allowed to dry completely. It is then pasted over the end of an open glass tube. A similar membrane is obtained by impregnating paper shells with a 30 per cent gelatin .solution and hardening them with formaldehyde, when dry. . .


valerianic acid and to many other high molecular lipolytics which penetrate into tissues rapidly.21

On the other hand, artificial membranes containing lipoids, like lecithin and cholesterol, have been prepared and these were found to exhibit a permeability similar to tissue membranes in that alkaloidal bases did penetrate (Thieulin, 1920).22 Even by merely impregnating a collodion membrane with an ether extract of muscle which contains various lipoids, the permeability is changed very considerably. In contrast to the permeability of a dried collodion membrane, this artificial lipoidal membrane has a greater permeability for the lipolytics. Butyric acid penetrates through it more rapidly than acetic acid which in turn is more penetrating than pumic acid. In the order given these acids exhibit a decreasing lipoid solubility. Through a dried collodion membrane, however, butyric acid has the lowest permeability, formic acid the highest corresponding to the molecular weight. All these experiments certainly lend support to the idea that in most cases, the cell membrane consists of lipoids.

5. THE THEORY OF SURFACE ACTION (ADSORPTION) (J. TRAUBE,

1904)23

Extensive discussions-which have obscured the issue-have arisen about the relation of surface actions to cell permeability. It has been observed that many rapidly penetrating lip olytics , like alcohols l

also lower the surface tension of water. Now it is well known that a dissolved substance which lowers the surface tension of a solution

21 Membranes with a.permeability like molecular sieves have been found so far in a few very special organisms only, as for instance, in sulphur bacteria, Beggiatoa Mirabilis. (See Ruhland and Hoffmann, Archiv fUr wissensch. Botanik, 1, 1, 1925.) Most other cell membranes in plants and animals exhibit the type of permeability depending on oil-solubility, as stated. It is noteworthy that the precipitation membrane of copper ferrocyanid, mentioned above, acts as a kind of a molecular sieve differing markedly from cell membranes in this regard. (See Collander, Kolloidchem. Beihefte, 19, 72; 20, 273 (1924).) Concerning the permeability of red blood cells to substances which are not lipoid soluble, Mond and Hoffmann (Pfliigers Arch., 219, 467 (1928» find a predominating influence of molecular size. Thus, e.g., urea passes easier than methylated or ethylated urea. .'

22 Comptes rendus Soc. Biolog., 83, 1347. 23 Pfliigers Archiv., 106, 540; 123, 419; 1~0, 123; 163, 282.


accumulates at the surface. Hence, the conclusion was drawn that lipolytics accumulate at the surface of membranes and are held there by forces of adsorption without penetrating through the membranes at all.

In order to prove this theory, J. Traube has measured the surface tension by counting the number of droplets formed by a solution of the lipolytic in question. Usually more drops are counted than for the same volume of pure water, which indicates a lowering of surface tension. This lowering is to be matched'with the penetration of the lipolytic substance in question into tissues, but, this has not been measured directly by Traube. Instead he uses for comparison the narcotic power of the substance, as determined from the minimal amount required for immobilizing small fish or tadpoles. This is parallel to the penetration, as stated.

When. performing measurements of this kind with substances of a homologous series like methyl alcohol, ethyl alcohol, or with similar other homologous substances, Traube observes indeed a parallel course of surface tension lowering and of narcotic power. However, the analogy fails completely if surface effects and penetration of substances are compared, such as chloroform, CHCl 3 24 and ether, C 2H 6·0· C 2Ho. Ether lowers the surface tension considerably, chloroform has hardly any effect at all, under the same conditions. Yet chloroform penetrates far more rapidly into tissue and its narcotising power is about three to five times that of ether. If the surface theory were correct, chloroform should be about as inactive for narcosis as water. Numerous other examples of this. type can be given.

An agreement can hardly be expected since the surface tension is measured at the surface of aqueous solution and air, while it ought to be observed at the junction of aqueous solutions with either lipoids or proteins. This, of course; would have a more direct bearing on the problem of penetration into tissues.

J. Traube has finally (1924) performed such measutements in his later investigations.2• A direct measurement of surface action is impossible, in this case, but, the adsorption can be measured which is parallel to the lowering of surface tension. Measurenfents were performed by pouring a solution of homologous alcohols on a gelatin jelly in a Petri dish with or without addition of lecithin, and observing the adsorption of this alcohol by the jelly in a given time. The result was that a greater "adsorption:' for the higher homologues occun;ed in such mixtures exclusively, which contained lipoids. In this way the supposed contradiction turned out to be a new proof for the lipoid solubility theory, since a penetration into the lipoid certainly occurs .

• 24 CHCl 3 solutions show a certain surface tension lowering in an atmosphere

of CHCl 3 but it is not nearly large enoug~. (See Traube, Pfluger's Archiv., 218, 749, 1928.) The parallelism between oil-solubility and penetration into tissues is far more satisfactory. Exceptions are found, in this case too, but these can be reasonably accounted for. (See below, page 46.)

25 Biochem. Zeitschrift, 163, 335, 358; 167, 371, 377, 385, 477.


6. FURTHER UNJUSTIFIED OBJECTIONS AGAINST THE LIPOID THEORY:

OLIVE OIL AND DIAMYLAMINE AND FATTY ACID AS A

MODEL OF COMPARISON

The lipoid theory of cell permeability has been subjected to'many further criticisms. Various special observations have been interpreted as contradictory to the theory.

Certain dyes, for instance, were found to stain tissue, which demonstrates penetration into or through the semi-permeable membrane, but, these dyes fail to penetrate into olive oil. This was considered as contradicting the lipoid theory until it was found that th€lse dyes do penetrate and dissolve in olive oil if an oil-soluble base (viz., aiamylamine) and an oil-soluble higher fatty acid are added to the oil (Nirenstein, 1920).26 In order to test this, the stainability of paramecia by 100 different dyes was compared with the distribution of these same dyes between water and olive oil. It was found that certain basic dyes like methylene green or thionine, stain paramecia well, but fail to penetrate into the oil. However, they do penetrate into the oil after adding oleic acid to it, the reason being apparently that the ·basic dye combines with oleic acid to form an oil-soluble oleate. Acid dyes on the other hand require the addition of I1n oilsoluble base (diamylamine) in order to become oil-soluble. Olive oil, with both oleic acid and amyl amine added to it, was found to dissolve all dyes which stain the paramecia (Nirenstein).26 The conclusion is that the lipoid which forms the cell membrane should also contain both a fat soluble base and a fat soluble acid. This is likely since lecithin contains both fatty acids and such bases as choline or neurine in its molecule. Since it decomposes easily, it resembles the mixture which reproduces the permeability of tissue membranes in many cases.

This result shows how the perfection of experiments with models serves to elucidate further the nature of the lipoid membrane. Dlive oil with the addition of an amine and a fatty acid is a better model, but, of course, even this latter mixture does not hold good in all.cases. Certain dyes were found which fail to penetrate into muscle tissue, while they dissolve in that oil mixture, and again other dyes which show the opposite deviation. Manifestly such discrepancies can

26 Pfliigers Archiv., 179, 233.


only stimulate further research in order to find better models. If a mixture of olive oil and amine and fatty acid is unsatisfactory, another fatty mixture may be found which will agree with the tissue membrane more satisfactorily. It would be erroneous to believe that all oils behave like olive oil in all details, or that all bases behave like diamylamine. Also the semi-permeable membranes do not consist of genuine fats, but, of lipoids like lecithin and cholestero1.27

7. THE ORIGIN OF THE CELL MEMBRANE

While the exact composition of the semi-permeable membranes in tissUfl awaits further investigation, it seems important to know to what extent living tissue resembles those artificial osmotic structures tlie growth of which is similar to vital growth in some respects as we have seen. If the semi-permeable membrane in those artificial osmotic structures is broken it will form anew. This new formation will take place as long as there is any salt left which is not yet dissolved. Is there any evidence to show that the lipoid membrane of tissues regenerates in a similar manner?

MaIW cells fail to regenerate their membrane if it is broken. If the membrane of red blood cells, for instance, is pierced by a very fine needle, using a micromanipulator, the hemoglobin flows out, no regeneration takes place. A cell of this type would correspond to one of those artificial structures the active growth of which has ceased, since no membrane forming material is left for regeneration.

There are, however, other types of living tissues in which the new formation of a membranous envelope can be readily observed. This is seen, for instance, by microscopic observation of the root hairs of Hydrocharis, a water plant of the order of Helol)iae. If these delicate root hairs are placed into a stained solution and pressed by the coverglass the "protoplasmic" content 'Will flow out, forming droplets some of which enclose the solution like vacuoles. Each single droplet can now be seen to exhibit all the osmotic properties of whole cells. Each little lump of protoplasm surrounds itself automatically with

27 Compare Coli ander, Jahrbuch f. wissensch. Botanik, 60, 354, 1921. Ruhland, same journal, 51, 376, 1912. Kiister, same journal, 50, 261, 1911. Molfendorf, Ergebnisse der Physiologie, 1920.


a precipitation membrane and exhibits osmotic shrinking or swelling (Kuhne, 1864, Nageli, Pfeffer, 1877).28 A new formation of a visible membrane can also be observed around the protoplasmic exudate, obtained from cells of low marine animals such as Arbacia (Seifriz, 1921).29

This shows that in some living substances, just as in some artificial osmotic structures, the semi-permeable membrane is continually regenerated through the contact of cell content and the solution on the outside. Manifestly this new formation of 'the membrane is a factor in the growth of cells.

The all important question would be to determine precisely the nature of this precipitation reaction. Some progress towards the solution of this difficult question has been achieved recently through the discovery that a part of the entire lipoidal content of tissue exists in it in water soluble form since it can be extracted by means of water from ulant or animal tissue (Hansteen Cranner, 1919;&0 V. Grafe, 1925).31

According to Hansteen Cranner, these water soluble lip aids are best obtained by extracting fresh sugar beets, but they are found also in other plant and animal tissue, as for instance, in muscle. These lipoids are extremely unstable being precipitated from their aqueous solutions by the addition of salts and likewise by the addition of such fat solvents as ether and alcohol. For this reason, water, and not fat solvents like ether, must be used if undecomposed water soluble lipoids are to be obtained.

If the assumption is justified that similar water,soluble lipoids are contained within the cells, a semi-permeable membrane might be formed by the interaction of these lipoids within and of the salts outside of the cells. In agreement with this assumption would be the observation of Hansteen Cranner that calcium salts hlwe a, more powerful precipitating influence on the water soluble lipoids than other salts such as sodium salts. This hypothesis is also supported by the finding that calcium salts tend to m~e tissues more imperme-

28 Kuhne, "Untersuchungen uber Protoplasma," Leipzig, 1864. Pfeffer, "Osmotische Untersuchungen, Studien zur Zellmechanik," Leipzig,

1877. Nageli, "Pfianzenphysiolog. Untersuchungen," Zurich. 29 Annales of Botany, 36, 260 (1921). 30 Biochem. Zeitschr., 169, 444 and 449; 162, 366; 17~, 266; 177, 16. 31 Berichte der Deutschen botan. Ges., 37, 380.


able, as shown by the decrease of electric conductivity and by the impaired penetration of various substances. (See below, page 83.) But, additional evidence would be needed to corroborate this hypothesis. Perhaps some evidence for this assumption is obtained by Heilbrunn's observation32 that calcium salts and lipolytics, which also precipitate lipoids as stated, tend to produce a precipitation membrane around protoplasmic exudates, as observed on exudates from crushed Arbacia eggs and other cells. An important physical property of the dissolved lipoids. is their power to lower the surface tension of water, hence they accumulate at the surface. This would not only facilitate the formation of a membrane, but would perhaps result in producing it even in the absence of a chemical reaction.

The nature of the membrane forming reaction in living cells can thus be explained, at least tentatively. The membrane forming reactions, whatever their nature may be, should be looked upon as one of the most important life processes. The cell membrane is not to be regarded as a kind of a protecting envelope inside of which life proper takes place, it is rather the essence of life, or a part of it, in itself. It is only natural, therefore, that the permeability is subject to change from a great variety of conditions, such as irradiation, heat, electric current, countless chemical agents, but, above all, it is changed by life processes themselves. In general, stimulation 1'ncreases the permeability of contractile tissue (Verzar, 1925 on frog's muscle,33 Lange and Simon on Chromodoris, 1922).34 The same is true for fertilization which may be looked upon as a process of stimulation35 (McClendon, 1910, and others).36

8. TISSUE MEMBRANES IMPERMEABLE TO COLLOIDS; THE OSMOTIC

ACTIONS OF BLOOD COLLOIDS IN THE CAPILLARIES

While the membranes, considered so far, are impermeable to most salts, we will now describe the actions of a vastly different type of

32 Heilbrunn, Archiv. fiir exper. Zellforschung, 4, 246. 33 Pfliiger's Archiv., 207, 192. 34 Zeitschrift f. physiolog. Chemie, 120, 1. 35 In certain cases a decrease of permeability seems to occur, e.g., in the

skin following electric stimulation. (Gildemeister, 1920, Pfluger's Archiv, 200,278.)

36 Science, 32, 317.


membranes, viz., of those through which simple salts can pass freely, but not colloids, such as the dissolved proteins or other high molecular compounds. The membranes which form the walls of the blood capillaries, connecting arteries and veins, have such properties. They allow the NaCI of the blood to pass freely. Hence no osmotic pressure is exerted by this salt on the capillaries, just the blood proteins are retained. These exert an osmotic pressure although a very small one. While the osmotic pressure of the N aCl in the blood amounts to 6000 mm. of mercury column, that of the proteins is no more than 25 mm., on account of the immense size of their molecular units, in spite of their higher total concentration.

In spite of this comparatively diminutive magnitude the osmotic pressure of the blood colloids is important, since it keeps the blood from passing through the wall of the capillaries into the interstitial spaces. The capillaries have the thinnest walls of all parts of the vascular system. A considerable mechanical pressure, chiefly due to the pumping action of the heart, is resting upon them. Under the influence of this pressure the blood fluid would leak through the capillaries if it was not kept back by the osmotic pressure of the proteins which, tending to draw in water, acts in the opposite direction (E. H. Starling, 1895)_37 Starling injected isotonic saline into a dog's tissues and perfused its blood vessel with serum. The saline was absorbed into the perfusing serum in the vessels, but no absorption occurred if the perfusing fluid itself was saline.

The osmotic pressure of blood colloids, viz., 25 mm. of mercury, under normal conditions, lies between the hydrostatic blood pressure in the arteries, 50 to 80 mm., and that in the veins which is less than 10 mm. A part of the total difference of the pressure between arteries and veins is lost in the narrow -lumen of the arterioles, but another portion is used to drive the blood through the capillaries. In that portion of the capillaries which is nearer to the arterial side the hydrostatic pressure exceeds the osmotic pressure and the fluid will be forced out through the walls of the capillaries into the connective tissue spaces. As the blood streams through the narrow lumen of the capillaries, the hydrostatic pressure drops until in the neighborhood of the veins it becomes smaller than the osmotic pressure. Hence in this portion of the capillary

37 Journ. Physiology, 19, 312.


tract water will be drawn into the capillaries from the connective tissue spaces (Schade, 1924).38

The initial loss and the later gain of fluid nearly compensate each other in some cases, in others the initial loss is greater, the excess is drained off by the lymph vessels. Direct measurements of the pressure in the capillaries of frog's mesentery have recently been possible by means of microscopic glass tubes introduced with micromanipulators into the capillaries. The pressure was found to differ from moment to moment, since the capillaries also collapse and dilate alternately.39 Nevertheless, the average pressure was found to be higher than the osmotic pressure of the blood colloids at the arterial end of the capillaries, and lower at the venous end (E. M. Landis, 1929).40 Experiments with models have given further evidence. Capillaries have been constructed of silk impregnated with collodion, and suspended in a saline solution (representing the'lymph fluid). If these capillaries were perfused with saline under pressure, fluid was found to leak out all along their course. But, if they were perfused with a protein solution, fluid was observed to leak out in the initial part only where the hydrostatic pressure exceeded the osmotic pr~ssure, while at the other end fluid was drawn in (R. Schade, 1928).41

An additional factor is the osmotic pressure exerted by the proteins of the lymph or tissue fluid. This pressure naturally also counteracts the osmotic pressure of the blood colloids by tending to draw water from the blood stream. The tissue fluid or lymph is not free from protein -even under normal conditions.42 The protein content of the lymph varies widely, in the liver lymph it is particularly high. Del Baere (1931)43 believes that this is the result of the low hydrostatic pressure in the liver capillaries. It seems therefore, that, in the liver, the osmotic pressure of the blood colloids is equilibrated to a greater extent by the osmotic pressure of the lymph colloids and to a lesser extent by hydrostatic pressure. '

38 Zeitschrift Klin. Med. 100, 402. 39 This has been described previously by Krogh. 40 Am. Journ. Physiology, 76, 548; 82, 217. 41 Zeitschrift Klin. Med., 108, 581. Refractometric measurements served

to observe the dilution or concentration of protein, in other words, the inward or outward water shifting -along these artificial capillaries.

42 Drinker, Journ. Physiology, 94, 40; 97, 32. 43 Nederlandsche Tijdschrift voor Geneeskundf', 76, 1540, 1737.


9. ApPLICATION TO THE THEORY OF EDEMA

While thus osmotic and mechanical pressure compensate each other, under normal conditions, a disturbance of this equilibrium may lead to an outflow of fluid from the blood into the interstitial spaces. This is one of the common causes of swelling or edema of the tissues. Disproportionate .size of either the osmotic or the mechanical pressure may be the cause. If the hydrostatic pressure rises above the normal level, water will be squeezed out into the interstitial spaces even though the osmotic pressure of the colloids remains normal. This factor in edema of cardiac origin was understood long before any other cause of edema was known. The increased pressure in the capillaries is usually due to an obstruction of the flow in the veins, the blood is backed up, accumulation of metabolites due to impairment of the circulation in turn affects certain brain centers which accelerate the heart, thus leading to a still higher hydrostatic pressure favoring edema.

The equilibration of hydrostatic and osmotic pressure can also become disturbed from the other side if the hydrostatic pressure remains normal, but the osmotic pressure of the blood proteins falls. This leads .likewise to a leakage of fluid into the interstitial spaces. Such a fall of osmotic pressure of proteins must occur, of course, if the supply of proteins is insufficient as in protein starvation or in starvation in general. Shortage of osmotically active blood proteins occurs also in so-called parenchymatous nephritis or lipoid nephrosis, as pointed out by Epstein (1914).44 The kidney, losing its normal impermeability, lets the blood proteins passisometimes as much as 50 per cent-and edema follows. In fact, the osmotic pressure of the blood of nephrotic patients is diminished as can be observed in an osmometer made of cellophane which is permeable to salts (Govaerts, 1926).45 Thls pressure was found to decrease to 12 mm. of mercury in some nephrotic cases, viz., to less than one-half of the normal pressure of 25 mm. However, in cases of cardiac edema with increased hydrostatic blood pressure no decrease in the osmotic pressure of blood colloids was found, as is to be expected.

Further support for this explanation is obtained by dialyzing blood

44 Journ. Exper. Med., 20, 334. 45 Presse medicale, No. 26.


serum of nephrotic patients through highly permeable collodion shells. These shells are prepared by adding water to the ether alcohol solution of the nitrocellulose from which they are made j they are 80 permeable that some of the proteins of normal blood serum will pass. However, from the blood serum of a patient with nephrosis no proteins will pass through them evidently because of the filtering off of the most diffusible proteins through the damaged and hence more permeable kidney (Del Baere, 1931).46 Accordingly the serum of nephrotic patients contains relatively more globulins.

Cases are known also, in which the edematous fluid contains particularly abundant protein due to an abnormally high permeability of capillary walls. As already explained this protein on the outside of the membrane exerts an osmotic counterpressure. Consequently the hydrostatic pressure preponderates and edema follows. According to Schade (1927)47 such an edema "rich. in protein" occurs particularly in glomerular nephritis. It tends to be localized since the increased membrane permeability is not necessarily present all over the body.

The different causes of edema frequently overlap. Thus, increased hydrostatic pressure in "cardiac" edema makes the capillaries "leaky" and hence diminishes the osmotic pressure, adding another cause for edema. Also lack of oxygen makes the capillaries "leaky" according to Krogh.48

"There is furthermore to be considered the fact that fluids can leave the blood by two paths, either into the tissue spaces or through the kidneys into the urine. The balanced forces include unknown factors which divide the fluid leaving the blood stream between these two outlets."49 A patient with nephritic edema "without change in either plasma protein content or in blood pressure, may suddenly cease to store edema and begin to excrete his accumulated fluids at a surprising rate"-as case observations show.

46 Nederlandsche Tijdschrift voor Geneeskunde, 76, 3694. 47 Ergebnisse der Medizin, 32, 425. 48 A. Krogh, "Anatomy and Physiology of the Capillaries," Yale Univ.

Press, 1922. 49 Quoted from van Slyke's "Factors Affecting the Distribution of Water

and Electrolytes," Philadelphia, 1925.


10. OSMOTIC AND NON-OSMOTIC FACTORS IN KIDNEY FUNCTION

A mutual compensation of osmotic and mechanical pressure can be shown to exist also in the kidneys-which, as is well known, are the great eliminating organs of the body. In this respect the formation of urine may be compared to the formation of edematous fluid although it is considerably different in other regards. As explained, an initial loss of fluid and a subsequent gain of fluid occurs in the capillaries of the body. In an analogo)ls manner there is an initial loss of fluid in the kidney capillaries, vi~., in those nearer to the arterial side, and which still have a high mechanical pressure. These are the glomerular capillaries which lie inv.aginated in Bowman's capsule. In their further course, the capillaries coalesce into one narrow vessel which emerges from Bowman's capsule. On account of its narrow lumen, the mechanical blood preflsure decreases probably below the osmotic pressure of the proteins. The narrow vessel subsequently breaks up agai; to form a second capillary network, around the convoluted tubules through which the urine is ,drained off. Just as in the capillaries, the greatly reduced mechanical ,pressure favors a gain of fluid by the blood from the initially formed glomerular urine, this being usually called re-absorption. The conditions are such that not all of the urine is re-absorbed, the excess is drained through the kidney pelvis. But, it must be added that osmotic and mechanical forces cannot accoun~ for all details of kidney function. The available evidence which shows that such forces act in some ca!les may be summarized as follows. In the glomerular capillaries the filtration of urine due to the excess of the mechanical pressure over the osmotic pressure seems to be fairly well established. Thus, whenever the blood pressure falls below the osmotic pressure, for instance following hemorrhage, urine secretion is found to be arrested immediately, as is to be expected according to the theory. On the other hand, a decrease of the osmotic pressure of the blood proteins, as it occurs after the injection of saline, results in an increased flow of urine.

The partial retention of NaCI in the blood may be perhaps explained on the basis of this theory, as the result of a loose combination of NaCl with globulin (which is one of the soluble proteins of blood serum). According to Milroy and Donegan/o a mixture of globulin and NaCl, dialyzed against pure water in a collodion shell,

50 Quoted according to D. Burn's "Biophysics," London, 1921.


exhibits a considerable retention of Na81 inside the shell, while NaCI alone passes the collodion more rapidly.

It is more difficult to account for the rapid excretion of such a salt as Na 2S04, CaCl2 or other so-called "diuretics." These have been found to be excreted preferably even if the blood pressure is lowered (Cushny). This may be ~xplained to some extent by assuming that Na 2S04 or CaC12 tends to render the kidney membranes more permeable. An effect of this kind has been demonstrated in model experiments on soap cups by M. H. Fischer.51 These cups were made of soft soap jelly which contained a large amount of water. CaCh ~olutions or some other diuretics were found to filter more rapidly through such a cup, evidently o~ account of a dehydration of the soap cup. In contact with eaCh, calcium soaps are formed in the substance of the filtering cup. These soaps hold less water; the soap particles thus shrink leaving channels between them. The soap cups may therefore be considered as useful models which illustrate the possible influence of physico-chemical alterations of living membranes, while e;xperiments with such unchangeable membranes as collodion films are rather misleading in that respect. In fact, CaCh or Na ZS04 passes through collodion slower than NaCl, while through the kidney, and also through the soap cup model, CaCl2 passes more rapidly. However, a direct evidence is still missing to show whether or not Fischer's soap cups are actually models of certain kidney membranes.

The theory of glomerular filtration has frequently been doubted, for instance, by Heidenhain, 1874,52 on the basis of his observation that urine secretion was arrested by compression of the renal veins although the pressure is increased thereby. This objection is unjustified because a stagnation of blood occurs if the veins are clamped. A. N. Richards pumped blood through a rabbit's kidney by means of a special pump which delivered a constant stream independent of the existing pressure; partial obstruction of the renal veins was then found to increase the flow of urine.53 A. N. Richards also produced evidence in favor of glomerular filtration in the frog, by collecting "glomerular urine" by means 'of micro pipettes and showing that it

HM. H. Fischer, "Edema and Nephritis," New York, 1915. 5~ Arch. mikr. Anat., 10, 1 53 Journ. Pharmacology, 8, 407.


was an "ultrafiltrate" of the frog's blood.54 Under the conditions of Richards' experiments the presence of osmotic forces seems well established in the glomeruli. The conclusion is that the influence of such simple forces extends much further than has been assumed formerly. Nevertheless this evidence does not prove that osmotic and hydrostatic forces are the only possible causes for movement of fluid in the glomeruli. This is shown by R. R. Bensley's65 experiments which demonstrate the inadequacy of the filtration theory even for the glomerulus under certain conditions.

In the re-absorption in the tubules, actions are observed which certainly cannot be explained by the osmotic or "filtration" theory. This is more than a fluid intake due to diminished mechanical pressure in contrast to the actions occurring in the body capillaries. Highly specific actions occur. Water, salts and sugars are re-absorbed in such adequate amounts as are required to maintain the composition of the blood constant. N a 2SO 4 and other diuretics are not re-absorbed at all which also seems to account for their diuretic action to some extent. Moreover, as shown by A. N. Richards,56 "chlorides disappear from the fluid within the tubules even when the fluid with which the tubules are filled has exactly the same composition as that which flows through the capillaries surrounding them." This finding points to the presence of driving forces which can move fluid even in the absence of osmotic forces since the fluid on either side of the membrane has the same composition in Richards' experiment. As the following pages will show, fluid movement independent of osmosis is by no means an entirely mysterious process, but also accessible to a physico-chemical analysis, at least to some extent.

11. FLUID MOVEMENT OPPOSED TO OSMOTIC FORCES

We have attempted, so far, to reach an understanding of the underlying causes of the shifting of fluids in 'living organisms on the basis of the osmotic theory. Even the seemingly complex changes in the kidney can be explained in this way to some extent. But, as already stated, it is not likely that osmotic and hydrostatic forces suffice to explain

64 Beaumont Foundation Lectures, Series No.8, 1929. 65 Am. Journ. of Anatomy, 47, 241, 1930. 66 Beaumont Foundation Lectures, Series No.8, 1929.


the re-absorption in the kidney. Moreover, in many organs of the body, fluid movements independent of or opposed to osmotic forces can be demonstrated directly, as e.g., in the intestine in the following manner. The small intestine of a rabbit, killed with a half digested meal in its lower digestive tract, is excised and split lengthwise. This is tied over one end of a glass tube which is open at both ends; the serosa being on the upper surface, the mucosa on the lower one. N aCI solution is then poured into the tube and the whole tube is immersed in NaCI solution so that NaCI is on either side. The NaCI solution will then slowly rise in the glass tube.

The same kind of experiment can be performed with many other physiological membranes, e.g., with the Skin of a frog. Fluid will usually be driven from the outside to the inside. The height to which the fluid rises depends particularly on the condition of the animal from which the skin is taken. If it is taken from lively and healthy animals and used immediately after excision, the rise is larger than if it is taken from morbid ones. In some cases the isolated frog's skin seems to give rise to a flow of fluid in the opposite direction, viz., from the inside to the outside. The nature of the solution in contact with the skin has a marked influence on the magnitude and direction of the flow. The greatest rise of fluid, in the direction to the inside, is observed if the skin flaps of healthy living animals left in situ, are used so as to preserve their nerve and blood supply (E. Gicklhorn, 1927).67 It is apparent that osmotic forces cannot be responsible for this flow of fluid, as the concentration of NaCl, and hence the osmotic pressure, is equal on both sides of the gut or the frog's skin.

Manifestly the same type of flow prevails in the living animal. Fluid is constantly 5hifted from the intestine into the abdominal veins. Through the frog's skin water passes into the animal, to be thrown out again through the urinary and intestinal tract. The intake through the skin is the only one since these animals usually do not drink water. Such fluid movements are known in physiology as resorption. Hence the fluid driving forces have been termed "resorptive forces." But such a ,descriptive term bears no relation to any possible underlying cause of the phenomenon.

67 See R. Keller, "Der Elektrische Faktor des Wassertransport," Ergebnisse der Physiologie, 30, 294 (1930); Krogh, in his collected papers, 10, 127-46 (1931), points out that these permeability effects are small. To render them more certain, additional observations would be desirable.


Such an unexplained biological fact requires new experiments to elucidate its nature. The crucial point is the question whether movement of fluid without or even opposed to osmotic forces can also be observed in artificial systems composed of inanimate matter. It has been known long ago that this is the case. Dutrochet found in 182558 that water diffuses from a dilute solution of oxalic acid through a (dead) pig's bladder into straight water. Later a similar "negative osmosis" was observed with a porcelain filter (F. E. Bartwell, 1916).59 Manifestly these observations bear a remote relationship only to the intestinal resorption. But, since better models are not known as yet, the only possible approach seems to be a physicochemical research tending to elucidate the nature of such forces which apparently are thoroughly different from the osmotic ones. With a certain knowledge of this point, the question might be discussed, whether the existence of this new type of forces seems possible in living organisms.

12. ELECTRICAL FORCES WHICH CAUSE WATER MOVEMENT

Anti-osmotic fluid movement of the type described can be understood by studying the movements of fluids under the influence of electrical forces, the so-called phenomena of endosmosis.

In order to understand the underlying principle, let·us first consider what occurs when oil droplets or electrically charged particles are in the pathway of an electric current. Due to electrical attraction the particle will move to the negative pole if it has a positive charge or to the positive pole if it has a negative charge. Another experiment may be performed as follows: A porous cup stands in a solution which produces an electrical charge on the cup. If an electrical current is sent through the cup in the manner shown in the diagram (Fig. 14a), a force will be exerted on the charged particles of which the porous cup is composed, in the same way as in the previous

68 Ann. Chim. et phys., 40, 337 (1835). 69 Journ. Am. Chern. Soc., 38, 1029 (1916). A similar flow of fluid has been observed in 1932 by W. J. V. Osterhout and

W. M. Stanley (Proceed. Soc. Exper. BioI. and Med., 29, 577), who described "models consisting of a non-aqueous layer separating two aqueous solutions, the inner being more acid than the outer." In this case, "osmotic pressure and Ie-concentration become greater inside than outside." This model was found to resemble, in this and other respects, plant cells, particularly those of Valonia.


experiment, but, since the porous cup is held rigidly in place, the fluid will move in the opposite direction. Hence it will rise within the cup and drop outside of it if the electric current flows in the direction indicated in the figure.

In the type of experiment described, the porous cup, acting as a membrane, acquires its electrical charge through mere contact with the solution, while an electrical current is sent through the membrane from the outside. It is possible, however, to modify the experimental conditions in such a manner that this flow of current is also produced from within the solution. In this case an electrical,' or "endosmotic" flu~·d transport must manifestly occur without the necessity of sending in an electrical current from an outside source. Evidence for this possibility is found through observations on the flow of water through collodion &hells impregnated with gelatin (J. Loeb, 1920).60 These shells are filled with a salt solution of varying composition and immersed in pure water. Just as in osmosis, water will now be drawn into the shells, yet, this effect cannot be due to osmotic forces since the salt solutions used are so dilute-viz., less than 0.002 M-that their osmotic pressure is too weak to draw fluid through the membrane. This is shown by the observation that no fluid movement occurs at all, if an equally concentrated sugar solution is in the shell. Yet, as measurements show, the flow of water into the salt solutions in those shells is quite appreciable. In fact, a distinct pressure can be measured if a rising tube is attached to each shell. To quote the result of a few tests: within twenty minutes the level of the fluid rose to

80 mm. if a l>h- M NaCl solution was in the shell 155 mm. if a dlr M Na2SO. sQlution was in the shell 385 mm. if a dlr M Na,Fe(CN) 6 solution was in·the shell

(all these solutions were alkalinized, pH = 11, compare diagram Figure 14b). These figures show the very much greater effect of salts of high valent anions.

Comparative experiments were then performed, in order to demonstrate the existence of electrical forces as causes of fluid movement in these cases, by determining the electric charge of the collodion shells. For this end three shells of the same kind were again filled with the same salt solutions, and were immersed not in water but in the same salt solutions which were inside. As is to be expected, no flow was

60 Journ. Gen. Physiol., 2, 387.


observed now. It was possible, however, to produce a flow by sending an electric current through the system from the outside. If the negative pole was in the shell, the direction of flow was such as to raise the level inside, compare diagram Figure 14a. The rise was found to depend on the nature of the salt, increasing with higher valence of the anions in exactly the same manner as in the previously described experiments where water was outside and no current sent

a r to Battery

a. Salt solution inside and outside the shell; fluid moves due to cur

rent sent in from the outside.

b

b. Salt solution inside, water outside; fluid moves due to current

generated by potential difference across the membrane. No outside source of cur

rent needed. FIG. 14. DIAGRAM TO ILLUSTRATE How ELECTRICAL ENDOSMOSIS MAY OCCUR

IN THE ABSENCE OF CURRENT SENT IN FROM THE OUTSIDE

through the system. With the same current intenSity the level was observed to i'ise within 15 minutes as follows:

3.5 mm. when lfh molecular NaCI was added 10.0 mm. when lfh- molecular Na 2SO 4 was added 22.0 mm. when lfh- mblecular Na.Fe(CN) 6 was added

There can be no doubt about the cause of the action of the salts in this case. The membrane must be negatively charged according to the direction of the flow observed and this charge increases with the valence of the negative ion, as is shown by the increase of the flow.


These experiments lead to the conclusion that in the first set of experiments, the membrane is also negatively charged, to an increasing degree for the higher valent anions. In addit~on to the charge of the membrane, a potential difference across the membrane arises, if water is outside, and this leads to a flow of current even if no current is sent in from the outside, resulting in a flow of water into the shell, as observed (J. Loeb).60

Numerous other experiments of this kind have been described by J. Loeb.61 In some of them a quantitative proof for the existence of electrical forces as the cause of the flow through the membranes has been offered by direct measurement of the electrical magnitudes concerned and comparison of their variation to that of the fluid flow. 62 A parallel course of the electrical magnitudes and the flow of water was found indicating that electrical forces are the ~ause of fluid movement in these cases.

Evidence of the same kind is also available to indicate that the anti-osmotic fluid transport in the old experiment of Dutrochet on the pig's bladder and in the experiments of Bartell is due to such electrical actions.

As already stated, two independent electrical magnitudes are concerned, either one of them was measured separately by Loeb, viz., (1) the charge of the membrane-without which any electrical fluid movement would manifestly be impossible-and (2) the potential across the membrane-which produces an electrical current that acts as "a substitute" for an external current as explained.

Apparently no current could flow if the membrane was entirely homogeneous. This, however, is not likely to be the case, since many observations show that these membranes have pores. This means that a potential difference across the membrane exists at one place, while there is none in the immediate vicinity where a pore perforates the membrane. Hence a local electrical circuit arises

61 Journ. Gen. Physiol., 4, 463 (1922). 62 Compare also the important investigations by SoUner and GroUman,

Zeitschrift f. Electrochemie, 38, 274 (1932), in which the influence of these two independent electric magnitudes is clearly demonstrated as follows: two membranes are used, one of which has charged pores, but no electromotive force across the membrane and hence no current flowing through the pores. A second cup containing a membrane gives rise to an electromotive force across the membrane and is connected with the first one in such a way as to drive a current through the pores of the first membrane. This current gives rise to a flow of fluid in some cases in a direction opposite to the osmotic flow. The connection is made by means of an agar bridge without using metallic wires.


in such a manner that the current passes through the pore at one place and returns through the substance of the membrane, or possibly through the surface layer. Thus the current required for the transport is generated within the system. The intensity of current is proportional to the potential difference "across the membrane" which generates the current, if other conditions are kept constant such as the width of the pores. It is justified, therefore, to substitute measurements of the potential difference for measurements of current, although the potential difference as such can never move fluid.

13. ELECTRICAL FLUID TRANSPORT BY INHOMOGENEOUS MEM

BRANES; IRRECIPROCAL PERMEABILITY

While thus an electrical fluid transport seems fairly well established for certain artificial membranes, it remains doubtful how a similar evidence can be presented for living tissue membranes. In the experiments, described so far, an electrical transport of fluid has been observed in systems in which there are two different solutions on either side of the membrane. In the case of the frog's skin, the same saline solution is originally in contact with the skin on either side. However, the solution outside soon takes up products from the acid secreting glands while that of the inner surface takes up tissue products of a widely different pH.53 Consequently the solutions on either side of the skin would soon become different from each other and potential differences might be generated in the same manner as described before, compare Figure 14.

Whether such an asymmetry of the solutions on either side is possible also in the case of the rabbit's intestine (page 57) or in the case of kidney tubules (page 56) remains doubtful. However, electrophysiological investigations show that electrical potential differences may also be. produced by a membrane which is in contact with two identical solutions on both sides, if this membrane is built up of different layers. (See below, page 228.) Currents generated by such a potential difference may be the cause of fluid movement which occurs through the gut or the tubules even if pure NaCI solution is on both sides. In fact it has been known for a long time that such membranes as the frog's skin gives rise to electric currents. As early as 1857, Du Bois Reymond in his classical "investigations on animal electricity" described the electric polarity of the frog's skin. Accord-

63 The writer is indebted to Dr. J. J. Abel for this valuable suggestion.


ing to E. F. Adolph, 1925,68 it seems that this polarity is in such a direction as to give rise to an electric current capable of transporting fluid as actually observed. For, if we assume that the pores carry a negative charge-as do the majority of pores in membranes-and since the outer surface is negative, water should move from the inside to the outside. In that specimen of isolated frog's skin with which Adolph58 experimented such an (inverse) flow was actually observed. Whether this explanation is applicable to frog's skin in situ or to other membranes remains to be investigated.

It would be possible to find additional evidence for this explanation if a movement of fluid was known to occur also through artificial membranes, independent of osmotic forces. This has not been possible so far, but, such inhomogeneous membranes have been found to exhibit another property which is probably related to the electric fluid transport, viz., "irreciprocal permeability."

"Irreciprocal permeability" means that the solution or in some cases the water or a certain dissolved substance passes more rapidly in one direction than in the opposite one. Artificial membranes with such properties have been .prepared, by coating a parchment membrane-on the one side exclusively-with chromated protein or in another experiment with collodion (H. J. Hamburger, 1904).64 Under the influence of slight osmotic differences such an asymmetrical membrane allows water to pass more rapidly from the parchment to the protein than vice versa. Pepsin passes easier in the opposite direction. That irreciprocal permeability is related to the electrical fluid transport, already described, is indicated by the fact that membranes like the gut exhibit it distinctly. For instance, if a loop of the rabbit's intestine is filled with Ringer solution, the N aCI rapidly passes out into the blood vessels of the intestinal walls, however, if the loop is filled with isotonic glucose solution, none or very little NaCl will pass from the blood into the gut (0. Cohnheim, 1899).65 This is to be expected on account of the fluid streaming through the intestinal wall from the inside to the outside. Sodium chloride is carried along by the streaming fluid into the blood vessels, but, naturally it cannot move up against the stream (even though it diffuses from a NaCI solution into straight water or glucose solution if both fluids are at rest).

64 Biochem. Zeitschrift, 11, 443. 65 Zeitschrift f. Biologie, 37, 443.


On account of the ext~emely low velocity of diffusion an irreciprocal permeability must manifest itself even if the electrical fluid transport cannot be observed directly, the force being too small to raise the fluid column noticeably on one side. Any preferred permeability through a membrane in one direction may be taken to indicate the presence of circulating electric currents which would be capable of actually transporting fluid if their intensity was not too low. Thus irreciprocal permeability is found to persist even after death when the fluid transport has ceased to be perceptible. Moreover, electrical currents, passing through electrically charged capillary spaces, are known to transport dissolved substances, some of them in a direction opposite to water. This would account for the irreciprocal permeability of dissolved substances.66 The explanation for irreciprocal permeability, given here, does not exclude however, the possibility of other causes of a different nature.

14. ELECTRICAL FORCES AS A FACTOR CAUSING THE RISE OF SAP

The following experiment seems to indicate that electrical forceb playa role in the rise of the sap of plants, although there may be also other influences, such as the evaporation of water from the leaves.

Seedlings of corn (Zea Mays) are used for this experiment. The root of the plant is partially smeared over with cocoa butter, leaving a flmall zone uncovered, in each test a different one, in order to investigate the penetration of water and salts from the soil (St. Popescu, 1926).67 As the result of extensive experiments of this kind a definite zone of the root has been found to be exclusively responsible for the intake of fluid from the soil. This active zone is the same in all experiments, viz., the one in which the root exhibits the greatest longitudinal growth, the so-called "stretching zone" (Strekkungs zone").

6~ Also an active movement of dissolved substances can be observed in living tissues. A very striking example is the piling up of certain dyes in the lumen of kidney tubules, which are grown in a culture. This has been quite recently discovered by Dr. R. Chambers. This result has not bel;Jn published as yet. The author is much indebted to Dr. Chambers for a communication about these important findings.

67 Bulletin Agriculture, Bukarest, 1926.


Other investigators have measured the electrical potential differences of this root (Lund and Kenyon, 1927, Pekarek in Keller's laboratory, 1930)68 by attaching one electrode permanently to some upper part while the second electrode was gradually moved along the root measuring the potential difference against the fixed electrode at points of regular distance. In this way it was found that the "stretching zone," through which fluid is taken in, exhibits outstanding electromotive forces which are distinctly different from those of the rest of the root or whole plant, viz., a markedly negative potential. This points to the possibility that electrical forces may influence the rise of the sap.

This type of fluid transport is somewhat different from the transport through the frog's skin or the gut, in that capillary spaces extend through the entire length of the tissue. We may assume that electrical currents passing along these capillaries are the cause of the fluid movements. It seems· likely that such an electrical fluid transport through extensive capillary spaces is not limited to plants, but, occurs likewise in the animal body. In many cases the movement of blood or lymph through capillaries cannot be understood as a result of simple mechanical forces such as blood pressure. It would be worth while to investigate to what extent these types of fluid movement are influenced by electrical forces.

Attempts have been made to determine this electrical factor of the water transport through observation of the migration of certain dyes (R. Keller, 1927).69 Experiments with aqueous solutions of various

68 Journ. Exper. Zoology', 48, 333, 1927; also 61, 309, 1928, by M. Gordon. 69 The studies of the transport of fluid by electrical forces are presented in

the scientific literature in a confusing manner. The older physiological investigators merely observed anti-osmotic streaming which they called resorption, without considering that the same occurs in inorganic systems. (See R. Heidenhain, Pflugers Archiv. 66, 579, 1894. Voit and Bauer, Zeitschrift f. Biologie, 6, 536, 1869.)

The early work of Dutrochet, Annal. de chim. et phys., 60, 337 (1835) and Graham, Phil. Trans. Roy. Soc., London, 1«, 177 (1854) demonstrated the existence of anti-osmotic flow, likewise Flusin's experiments on water imbibing membranes, Ann. de chim. et phys., 13, 480 (1908). Some of these early workers had already been led to assume the electrical forces as the cause of anti-osmotic flow, but the electrical mechanism was elucidated some time later only by F. E. Bartell, H. Freundlich and J. Loeb. See E. Bartell and co-worker, Journ. Am. Chern. Society, 36, 444 (1914); 38, 1029, 1036 (1916); 44,289. H.


dyes show that these are transported in an electrical field, some of them in the same direction as water. This method is handicapped, however, by the variability of the migration of the dyes, which depends on pH changes, addition of colloids, or other conditions.

Summarizing we may state that unusual difficulties still handicap the investigation of the details of the electrical forces which cause fluid movements in tissues. Nevertheless, the facts presented here, show that there are such forces and some observations at least indicate their occurrence in tissues.69

15. ApPLICATION OF THE THEORY OF "MEMBRANE EQUILIBRIUM" TO

TISSUES; MEMBRANES PERMEABLE TO NEGATIVE IONS ONLY

The cause of fluid movement in tissues may be traced either to osmotic forces or electrical forces, according to the description given so far. Now there is another different mode of action in which elec-

Freundlich and co-workers, Kolloid Zeitschrift, 18, 11 (1916). J. Loeb, Journ. Gen. Physiology, 1 and 2, several articles.

The merit of pointing out the necessity of actually circulating electric current belongs to Freundlich, subsequently Bartell has utilized this idea.

The early papers of J. Loeb are chiefly descriptive such terms being used as "the water acts as though it carried a positive or negative charge." These papers should not be quoted without considering Loeb's later results. E. Wertheimer, however, seems to know the early paper of Loeb only. He likewise fails to consider the entire work of Freundlich and Bartell. (Pfliiger's Archiv., 199, 383; 200, 82, 354; 201, 488, 591 (1923); 203, 542; 206, 16~ (1924); 207, 254; 208, 669; 209,494; 210,527 (1925); 211,255; 213, 735 (1926).)

Wertheimer's experiments are chiefly modifications of the well known observations of irreciprocal permeability. The physical mechanism of the actions is not considered. Loeb, Freundlich and Bartell on the other hand while dealing with the phenomena of anomalous osmosis do not yet investigate how this may explain irreciprocal permeability.

R. Keller has attempted to solve these problems by studying the migration of dyes and vital staining in relation to electric potential differences and transport of fluid caused by electric current. [See Ergebnisse der Physiologie, 30, 3!J4; "Methoden der Bio-electrostatik" Handbuch der biolog. Arbeits methoden, V, 2, 1189 (1928).1 His statement that water is sucked into negative membranes is obviously no complete presentation of the underlying electrical mechanism. Yet these inadequacies are merely temporary. It seems likely that Keller's work particularly will lead to new important developments in this line.


trical and osmotic forces cooperate in a peculiar manner, viz., the so-called "membrane equilibrium" in which smaller ions attracted by larger ones increase the osmotic pressure, as already described for gelatin solutions. (See page 31.) A priori it would seem likely that actions of this type occur in tissues. In fact, it can be shown that the osmotic forces acting in red blood cells are re-inforced by electrical ones in such a manner, because the membrane around the blood cells is impermeable to cations such as Na+, K+, but, permeable to anions like HCO a-, CI-, S04--. This is shown by the higher K+ content inside the blood cells and the higher N a + content of the plasma surrounding them, between which no exchange occurs. But, anions are exchanged; for instance, if blood cells are placed in a Na 2S04 solution the SO 4 -- can be found to be exchanged for other anions in the cell (v!1n Slyke, 1925).70

There is, however, one anion in the blood cell which does not penetrate: viz., that of the high molecular hemoglobin. This is particularly important for the establishment of a membrane equilibrium, (in other words, for the re-inforcement of osmotic forces by electrical forces). Hemoglobin acts as a polyvalent acid. It has so many valences, that, under certain conditions existing in the blood, it can combine with as much as half of the total alkali in the blood cells. This leaves only the other half to combine with CI- and HCO a- (van Slyke, 1925).70 Applying the rules of membrane equilibria explained before, we should expect that the concentration of the CI- and HCOa- ions in the cell is lower than in the surrounding plasma and that the concentration of H ions in the cell is larger than outside. Chemical analyses have shown that this conclusion is indeed correct.

The base binding power of hemoglobin is still more increased following the loose binding of oxygen which changes hemoglobin to oxyhemoglobin when in contact with air. Since oxyhemoglobin has stronger acidic properties it drives out some of the carbonic acid of the bicarbonate in the cell, the cell membrane being freely permeable to both O2 and CO 2• This occurs in the lungs. In the tissues, however, the reverse process occurs: oxyhemoglobin gives up the loosely bound oxygen to reducing substances in the tissues. Thereby some of the alkali of the blood is liberated since the weaker acid, hemoglobin, is now present. In the tissue capillaries this alkali combines with CO 2 formed through

70 Van Slyke "Factors Affecting the Distribution of Electrolytes," Philadelphia, 1925.


respiration.71 "The alkali is thus setjree at the exact place and moment where it is needed to combine with CO 2 entering the blood from the tissues" (van Slyke, 1925).

This variation in base binding must also influence the osmotic pressure within the blood cells. Since, following oxygenation each single polyvalent colloidal hemoglobin ion can replace a large number of monovalent HCO a- and CI- ions, the osmotic pressure inside the blood cell must necessarily decrease. Such a replacement is the only shifting which can possibly occur; one may think of an entrance of N a + ions to neutralize the additional base binding power of oxyhemoglobin but this is impossible since Na+ ions cannot penetrate as stated. On account of this decrease of osmotic pressure, oxygenation should cause shrinking, while manifestly deoxygenation must act in the opposite sense and likewise acidification since this brings hemoglobin nearer to its iso-electric point. _ All these theoretical conclusions have been fully ven'fied. The entire exchange of salt and water to or from the blood cells following oxygenation can thus be forecast by the theory of joint osmotic and electrical actions ("theory of membrane equilibrium") on the basis of the two experimental facts described:

(1) the preferential permeability of the cell membrane to anions; (2) the large base binding of hemoglobin which is further increased by

oxygenation. (See diagram, Figure 15.) For further details on these membrane equilibria, see van Slyke's book mentioned before.70)

Red blood cells are not the only body cells which swell following deoxygenation. Lack of oxygen is known to be the cause of swelling or edema, also for many other tissue cells. These do not contain hemoglobin, but one may perhaps assume that the base binding of other proteins is raised- by oxygenation since it is a common experience in chemistry that many oxidized substances are more acidic than those containing less oxygen.

It should be emphasized that such a "membrane equilibrium," which has been described here as a "joint electric and osmotic action," is thoroughly different from the action described before as "electrical fluid transport" in which nothing but electrical forces shift fluids. The marked distinction between these two actions will be better

71 These reversible shiftings may also be described as occurring through a transfer of ions, viz., a shifting of HOO a- and 01- ions from the cells to the plasma outside.

APPLICATION OF PHYSICOCHEMICAL LAWS

--T--~

I I : I I I I I I

ell I NCI+ I :o'Iiefl1 1

I I I I I I

I I [.__-----f I

I : I I

.1 _ _J

Cells Serum f\eductd

69

FIG. 15. DIAGRAM INDICATING THE DISTRIBUTION OF IONS BETWEEN BLOOD CELLS AND SERUM ACCORDING TO VAN SLYKE

The shaded portion reJ>resents the base binding power of hemoglobin (Hb) or of serum protein (P). However, the osmotic pressure of these proteins is practically zero. Osmotic pressure is exerted merely by the ions represented by non-shaded areas. Following oxygenation, the base binding power of hemoglobin increases leading to a displacement of CI- ions. But the large polyvalent oxyhemoglobin ions do not make up for the loss of osmotic pressure caused by loss of CI- ions. Hence, the osmotic pressure in the corpuscles drops and they shrink, giving off water to the serum The serum IS thus diluted as shown by the diagram, which indicates a lower salt content of the Berum following oxygenation.


understood after a more detailed study of membrane equilibria which is presented in the Appendix.

16. COLLOIDAL SWELLING AS DISTINGUISHED FROM OSMOTIC SWELL

ING; SUMMARIZING STATEMENT CONCERNING NON

OSMOTIC SHIFTING OF FLUID

Still another force, responsible for the shifting of fluid in or into tissues, is the swelling or shrinking of colloidal substances. This occurs even when membranes of any description are absent. In these cases, water is attracted or given off largely due to forces of chemical attraction. The nature of these actions is little understood in detail. To some extent membrane equilibria may be regarded as the cause of this "colloidal swelling" even though no visible membranes are present, as already described, see page 34. Concerning the nature of the methods for tracing colloidal swelling in tissues by means of the Hofmeister series, the reader is again referred to the Appendix (see Appendix, page 321).

A factor of considerable importance for the swelling of colloidal proteins is the pH of the surrounding fluid. If the pH of this fluid is shifted so as to approach the iso-electric point of the protein in question, swelling will be counteracted. With the exception of the globulins, most tissue proteins have an iso-electric point of pH = 6 to 7. They are, therefore, on the alkaline side of their iso-electric point in the blood and a small shift to the acid side brings them nearer to their iso-electric point which means that swelling is counteracted. A marked acidification would, of course, shjft the pH of the fluid away from the iso-electric point of the tissue proteins and hence cause swelling (sec page 34). However, this can rarely occur, -even under pathological conditions, on account of the buffer substances present in tissues (see also page 76ff.). A colloidal swelling due to a piling up of acid seems, therefore, practically impossible in tissues.

The influence of acidification or alkalinization upon the swelling of tissue colloids is rather complicated and in some cases may exhibit antagonistic effects. Thus the fibrillary substance of connective tissue swells considerably in 0.01 molecular alkali, but swells little in 0.01 molecular acid. The collagenous fibres exhibit the opposite phenomenon. This shows that different constituents of the same tissue exhibit antagonistic swelling, probably on account of a


difference in their isoelectric points. The tissue containing both types of fibers shows a slight swelling only, due to the counteracting action of its constituents (H. Schade, 1924).72

The most important facts described on the last pages may be condensed into the statement that we know of three different actions, at Zeast, which may result in a shifting of fluid, if the simple osmotic forces play no role. These three actions are caused by:

(a) electrical forces, resembling endosmosis in the manner described; (b) membrane equilibria, also described here as "joint osmotic and

electric forces;" (c) the swelling or shrinking of colloids "!lithout visible membranes. Numerous details concerning the mode of action of these different

forces still await investigation. Also their incidence or their importance for fluid shifting or swelling in every particular case is not known in all details, but, evidence is available to show that such forces can act in living tissues. Hence there is no reason to describe all functions which cannot be accounted for by osmotic forces as vital, or to label them as unexpla1·nable resorbing or secreting forces.

72 Kolloid Zeitschrift, 35, 306.

APPLICATION OF PHYSICO-CHEMICAL LAWS, CONTINUED: OCEANIC SALT MIXTURES INDISPENSABLE TO LIFE. INFLUENCE OF THESE MIXTURES ON MEMBRANE PERMEABILITY AND THE PHYSICAL CAUSES THEREOF, AS STUDIED ON EMULSION MEMBRANES. THE ORIGIN OF LIFE ON THE EARTH

1. THE RINGER SOLUTION

The salts contained in blood serum do not consist of sodium salts exclusively. Traces of calcium salts and potassium salts are present and these are indispensable to life.

The average composition of the salts contained in human blood is the following. All sodium salts combined amount to about 7.9 per cent (of which 5.3 per cent is NaCI, 2.3 per cent NaHC0 3 and about 0.3 per cent sodium sulphate and phosphate). There is 0.01 per cent CaCh and 0.008 per cent KCl. Slight amounts of magnesium salts are also present. On the basis of such observations the well known Ringer solution1 has been devised as the most suitable salin(l solution for perfusing organs, for rinsing exposed organs or wounds, and for transfusions. Outside of varying amounts of bicarbonates, it contains about 0.85 per cent NaCI for all warm blooded animals or mcn+ 0.02 per cent Kel + 0.02 per cent CaCho

These three salts, combined in the approximate proportion of 2 parts of CaCh and 2 parts of KCI for every 100 parts of NaCI, are more favorable than pure salts for the development and maintenance of any form of life, not only for that of vertebrates. Even plants require a similar mixture for growth. Roots of wheat, e.g., require NaCI + KCI + CaCl2 for growth, see Figure 16. In a solution containing NaCI + CaCh, growth is retarded to slight extent only. In pure NaCI or CaCh solution growth is entirely handicapped as the figure shows distinctly (Osterhout).2

Most striking is the fact, that ocean water contains N aCl, CaCl 2 and KCl in the same proportion as blood although the total salt concentration of the ocean is four times higher than that of the blood, averaging 3.5

1 Journ. Physiology, 16, 1, 1894. Other similar solutions are those of Tyrode and of Locke.

2 Journ. BioI. Chern., 1, 303.

72

per cent. CA. B. Macallum, 1903).3 The KCI and CaCb content of the ocean for each 100 parts of NaCI is found to be about: 3.0 parts of KCI and 4.5 parts of CaCI2, while in Ringer solution suitable for frog's organs, it is 3.3 parts of KCI and the same amount of CaCl2.4

This relationship seems to indicate that all life has evolved from the ocean. All living organs of animals must be bathed in some kind of ocean water to keep them alive. Those of the lowest forms of animal life are directly perfused with the ocean water itself as these living forms of the ocean have no body fluid of their own. The organs of more highly developed animals are bathed in a 4 to 5 times dilute ocean water. By means of excretory organs this compara-

in 0.12 mol. NaCI + inO.12 mol. NaCl + in 0.12 mol. in 0.12 mol. 0.0026 mol. KCl + 0.0012 mol CaCb CaCl2 NaCI 0.0012 mol. CaCI2•

FIG. 16. GROWTH OF THE ROOTS OF WHEAT IN SALT SOLUTIONS OF VARYING COMPOSITION UNDER OTHERWISE IDENTICAL CONDITIONS

tively lower concentration of the body fluid is kept constant. It seems also possible to account for the higher salt content of the ocean. We have to consider that the salt which is now dissolved in the ocean has been extracted from the surface layers of the earth by the rivers. This process of extraction is still carried on at present. Consequently at former geological periods the salt concentration of the ocean must have been much lower. At that early time in the earth's l],istory, the first animals left the ocean and adapted themselves to conditions on land. They have preserved the low salt concentration of that early ocean life ever since.

3 Journ. Physiology, 29, 213. 4 Besides, this ocean water contains considerable amounts of magnesium

salts which, however, do not seem to be quite as important for the maintenance of life as N aCI, KCI and CaCI 2•


Surprising observations are made if the action of salt combinations is studied on those animals or plants which are surrounded by such a dense skin that none or little water penetrates and hence no osmosis occurs. We find that in such cases pure NaCl solutions, even though they are isotonic, are much more harmful, than distilled water (J. Loeb).5

For example, a small marine fish, Fundulus Heteroclitus, can live in distilled water, as well as in ocean water, or in any mixture of the two. Also its eggs develop in distilled water. Yet, the fish dies rapidly in a pure N aCl solution of the same concentration as ocean water. N aCl without the other salts is therefore poisonous. The point of attack for this poisonous action is probably located in the gills. Pure N aCl also checks the development of the eggs of this fish. Similar experiments can be performed on certain plants. An alga, Vaucheria, develops equally well in distilled water and in a solution of NaC} + CaC1 2• In pure NaCI, or in pure CaCl 2 solution, no germination takes place (Osterhout).6

2. THE MECHANISMS WHICH KEEP THE Ca CONTENT OF BODY FLUIDS

CONSTANT; THE CONSTANCY OF pH AND THE ALKALINE

RESERVE IN THE BLOOD

The oceanic salt combination of N aCl, CaC12 and KCl in the approximate ratio 100:2:2 is essential likewise for more highly developed animals. But in their body fluids the constant rati9 of NaCI, CaC12

and KCI can only be maintained if conditions of equilibrium of some sort are established. If this were not the case, variations would seem unavoidable since varying amounts of all salts are introduced into the animal body continuously. For this reason; an -intravenous injection even of large amounts of pure NaCI solution fails to produce any poisonous symptoms. Probably those small amounts of calcium and potassium salts which are needed are set free from other tissues. Large depots of both are available in the body: calcium salts in the bones and potassium salts from the red blood cells and other body cells which contain more potassium than sodium salts.

6 Am. Journ. Physiology, 6, 411,1902; Biochem. Z., 2, 83 (1906); also numerous later publications in this journal.

6 University of California Publications Botany, 2, 317 (1907); also Botanical Gazette, 42, 127, and 44, 259 (1907); 64, 532 (1912).


Little is known so far about the nature of the equilibria which automatically keep the salt concentrations of the body fluid constant. Nothing at all is known in this respect about the potassium salt concentration. Concerning the NaCI concentration, we know that complete excretion is prevented by the kidneys (see page 54). Concerning the mechanism on which the constancy of the calcium concentration depends, we know that it bears some relation to the parathyroid glands. This gland produces a hormone which prevents complete excretion of calcium salts. In the absence of this hormone, calcium salts decrease rapidly in the blood and the poisonous actions of a pure salt solution or of salt mixtures, which are different from the natural ones, manifest themselves on the most sensitive tissue of the body, viz., the neuro-muscular system, leading to tetany. Almost one-half of the total calcium content of the blood is kept in solution by the action of the parathyroid hormone, the mechanism of this action being unknown. This fraction is also most effective physiologically, viz., by preventing tetany (van Slyke and his collaborators, Hastings and Sendroy, 1927).6a

Another fraction, viz., about 30 to 40 per cent of the total calcium of the blood, is bound to the dissolved proteins which have acid properties as described before. The remainder of the total calcium content of the blood, which amounts from 20 to 30 per cent, is "held in solution by forces which ordinarily govern solubility in solutions" (van Slyke). The amount of this fraction depends on the pH of the blood, for the following reasons. The blood is slightly alkaline having a pH of 7.3 to 7.4. Among other bicarbonates it contains calcium bicarbonate, which is partly split into insoluble calcium carbonate and free carbonic acid. Now it is well known that insoluble CaCOa is dissolved by any acid even by the weak carbonic acid. Consequently, the calcium content of the blood depends primarily on its acidity. The more acid is present, the more CaCOa is dissolved, as long as a sufficient amount of undissolved CaCOa is present.

The conditions existing in the blood are described more accurately by stating that a chemical equilibrium exists between dissolved calcium bicarbonate on the one hand and insoluble calcium carbonate and carbonic acid on the other. By application of the well known physico-chemical laws of chemical equilibrium we find that the cal-

6. Quoted according to Peters and van Slyke, "Quantitative Clinical Chemistry," Baltimore, 1931, Vol. I, page 810.


cium concentration in the blood must be proportional to the HT ion concentration (acidity) and inversely proportional to the NaHCO a content (alkaline reserve). Consequently both magnitudes must be constant if the calcium concentration in the blood is to stay at a constant level. In fact, we find that the pH of the blood is very constant, varying within the narrow range of pH 7.3 to 7.Jf corresponding to CH

0.44 . 10-7 This constancy is of importance not only for the calcium concentration but also for enzymatic chemt'cal reactions which are the essence of life processes. 7

The constancy of pH in the blood fluid depends in the first place on the weak acids and their salts, contained in the blood, which act as "buffer" substances (see above, page 28). Carbonic acid, C02, or in the hydrated form in solution H 2CO a, is the most important weak acid present. Just as in all buffer solutions the hydrogen ion concentration is proportional to the ratio of free acid to its salt; hence, in the blood plasma, to the ratio: H 2COa to NaHCOa.

Under pathological conditions the NaHCOa in the blood may become reduced, due to the production of an excess of non-volatile or "fixed" acids which set free H 2COa (or CO2) from NaHCOa. This leads to an increase in the ratio H 2COa:NaHCO a, and consequently to an increase of the hydrogen ion concentration. However, the respiratory center is promptly stimulated by such a change. Increased ventilation of the lungs through more intense respiration follows and a portion of the H 2CO a in the blood is eliminated as CO 2 until the ratio H 2COa:NaHCOa is back again to its normal value. Such a condition is known as compensated acidosis (V3Jl Slyke and collaborators). 8

However, if the respiratory center fails to respond and consequently CO 2 or other acids pile up in the blood while·the' NaHCO a concentration of the blood is diminished, only then does a considerable acidification occur. Such a condition, known as uncompensated acidosis, is not compatible with life for a considerable time. Even in diabetes when overwhehning amounts of acids are produced the pH never falls below 7.0. The acidotic condition chiefly consists in a

1 Straub and Meyer, Deutsch. Archlv. f. Klin. Med., 129, 54 (1919). Michaeli's "Hydrogen Ion Concentration," Baltimore, 1926. Donegan and Persons, Journ. Physiology, 52, 315 (1919).

8 See a number of articles in the Journ. Biological Chern., 1917, and the following years, particularly vol. 41, 567 (1920).

APPLICATION OF PHYSICO.CHEMICAL LAWS 77

diminution of the alkaline reserve, except in the last stages of severe diabetes which are associated with deep coma (van Slyke and collaborators, 1917). Elimination of acid is not limited to CO2 ex.piration through the'ungs. Non-volatile or fixed acids are excreted as such by the kidney. Eventually alkali is secreted if an excess of this is present in the blood. Consequently the pH value of the urine varies within wide limits.

NaHCO a is not the only buffer substance which tends to maintain neutrality in spite of all the acid products of oxidation. The dissolved proteins of the blood as well as the tissue proteins neutralize acids and likewise dissolved buffer substances which are contained within the body cells. Red blood cells, e.g., contain alkaline potassium phosphate and dissolved hemoglobin, a protein. The potential alkali reserve of the body as a whole, as represented by all these conditions, is far greater than the NaHCO a content of the blood (van Slyke).8 Ocean water, although poor in buffer substances, is also slightly alkaline, pH equalling 7.8 to 8.3 (S¢rensen and Palitsch, 1910).9 Saline solutions, used for perfusion, likewise contain alkaline buffer salts.

As stated before, the third calcium fraction of the blood depends not only on the pH value but also on the N aH CO 3 content which is a part of the "alkaline reserve." For maintaining a constant calcium level it is equally important, therefore, that the alkaline reserve of the blood should be constant. In fact, it is nearly constant under normal conditions. It is most conveniently measured by liberating its total CO 2 content by the addition of an excess of H 2S04 and measuring the CO 2 in a gas burette ("van Slyke apparatus"). The volume of CO 2,

thus liberated from 100 cc. of blood plasma, ranges from 50 to 65 cc. This indicates the degree of constancy of the alkaline reserve.

For determining the pH value of the blood in vitro, anyone of the well known methods is available. For whole blood, the electrometric method is exclusively available. Care must be taken, however, not to lose a part of the dissolved CO 2 through volatilization. Methods, worked out recently, allow us to determine pH directly in the streaming blood by inserting a Pt wire, coated with Mn02 electrolytically, int6the blood stream; such a wire functions as a H2 electrode.l° For measuring pH in subcutaneous tissue a regular H2

9 Biochem. Zeitschrift, 24, 387. 10 R. Gesell and Hertzmann, 1925, Proc. Soc. Exper. Biolog. and Medicine,

20,298.


electrode (using H2 gas) has been adapted by perforating the skin and introducing a glass capillary with a Pt wire in it, through which H2 gas + CO 2 (40 mm.) is conducted.11 This method, in contrast to the one described before, aims at determining pH in tissue. This is found to be 7.09 to 7.29, viz., more acid than blood. Following exhaustion by muscular exercise, this drops to about 6.6.

A series of splendid investigations performed by Peyton Rous and his associates have shown that healthy tissues are always relatively acid as compared with the blood. Their reaction can be rendered directly visible by injection of indicators into living animals.12 The indicators are not injurious to the health of the animal and those of the phthalein series yield reliable color phenomena under body conditions. Connective tissue was found to have a pH of less than 7.2, most parenchymatous tissues less than 6.6. In general the organs showing a marked acidity are such as have a greater metabolic activity. Vigorous skin grafts are acid, weak ones alkaline. Local constriction of blood vessels from any cause was found to raise tissue acidity. (Rous calls this "outlying acidosis.") The local tissue acidity thus developing is independent of the pH of the blood. When enough concentrated sodium carbonate was given to a mouse to render the blood more alkaline while producing anhydremia, the "shaved body surface instead of participating in the alkalinity became more acid" (P. Rous). The acidity of parenchymatous tissue (liver, kidney) is probably the result of a membrane equilibrium which leads to a higher acidity inside the cells, but, there are certainly other factors involved which are less completely understood.

3. DETOXIFICATION OF N aCI BY HEAVY METAL SALTS; THE MELTZER

NARCOSIS

After this review of the equilibrium conditions which contribute toward the maintenance of a constant calcium: level in the blood of higher animals, we will now continue the description of such antag-

11 Schade, Neukirch and Halpert, 1921, Zeitschrift f. d. gesamte experim. Mediz., 24, 11.

12 See a number of articles by P. Rous and collaborators, Journ. Exper. Med. vol. 41-49, (1925-8). C. Voegtlin and his associates have recently performed more accurate determinations of tissue pH by means of a glass electrode (according to a private communication).


onistic salt actions which are investigated chiefly on aquatic animals or plants. Numerous experiments show that, for a detoxification of a pure NaCI solution, an addition of CaCl 2 alone in the optimal proportion is almost, although not quite, as efficient as an addition of both CaCl 2 and KCI. This is demonstrated, e.g., by the experiments on wheat seedlings by Osterhout, already mentioned, compare Figure 16, as well as by numerous observations on animal organs. Further experiments which were performed on a fish, Fundulus Heteroclitus, show that in the place of CaCb, other similar salts may be used, such as MgCl 2 0r SrCI 2, with the same effects (J. Loeb, 1902)Y

Even such poisonous salts of heavy metals as ZnCl 2, NiCl 2, BaCl2,

and M nCl2 can be used to render pure N aClless harmful in experiments on fish. Naturally in these cases the detoxifying action is impaired, since such salts are poisonous themselves. Yet, even a mixture of NaCI with some uranium chloride is slightly less poisonous to Fundulus than pure N aCI. This observation would seem almost unbelievable, since uranium chloride is known in pharmacology as a strong protoplasmic poison, yet a detoxification can be demonstrated by unmistakable experiments on fertilized eggs of Fundulus.

The percentage of eggs which have developed in a given mixture of NaCI and any heavy metal gives an approximate measure of the toxicity of that mixture (J. Loeb),13 For the mObt efficient mutual detoxification, the two antagonistic salts must be present in a definite ratio. This optimal ratio was found to vary for the different salts used. For the highly poisonous salts of heavy metals the percentage of developing fish eggs is, of course, relatively small, even if the optimal mixture is used. Further experiments have shown that trivalent salts, such as AICb, also detoxify pure NaCI, mere traces of AICl a or FeCls sufficing in these cases. In the absence of CaCb pure N aCI solutions can also be detoxified by KCI or other monovalent salts, pure CaCl 2 can be detoxified by MgCI2, NaCI or other salts. To summarize the results obtained with fish or fish eggs: almost any mixture of two or more salts, if combined in the most favorable proportions, is less poisonous than a solution of one salt alone.

Such salt mixtures which contain poisonous salts cannot be used, however, without discrimination for mutual detoxification on any type of tissue. The more specialized organs of vertebrates are in general more sensitive. Not so many substitutes for the natural combinatwn are

13 Pflligers Archiv., 88, 68, and 93, 246.


capable of maintaining a normal condition in these cases. For instance. the fibrillations of a frog's muscle in pure N aCl are counteracted by CaCl 2,

also by SrCl 2, but not by MgCl 2• An isolated heart will beat in NaCI + eaCb, or in NaCI + SrCl2 solutions but not in NaCI + MgCh. An isolated frog's kidney becomes permeable to sugar when perfused with pure NaCI solution; with the addition of traces of CaCI2, sugar is retained, but an addition of MgCh has no such effect (Hamburger and AloUS).14 Also heavy metal salts fail to detoxify like CaCb in anyone of these cases.

In these more specialized organs, magnesium salts not only fail to detoxify NaCI; on the contrary, large amounts of magnesium salts added to NaCI are poisonous themselves and can be detoxified by calcium salts, the antagonists of NaCl. The action of magnesium salts resembles, therefore, in this case, rather that of monovalent salts like N aCl.

Special interest is attached to the mutual detoxification of MgCh and CaCb since these salts playa role in the so-called Meltzer narcosis. To understand the nature of this narcosis, it is important above all to know that a large addition of CaCl2 is needed for detoxification of pure MgCh. This is very much in contrast to the above mentioned fact that N aCI can be detoxified by slight additions of CaCI 2• The most favorable combination is that of 2 MgCb and 1 CaCh in equivalents. Meltzer and Auer16 have fpund that rabbits or other animals become paralyzed and narcotized by the injection of a large dose of magnesium salts into their veins. In this case, we are not dealing with the deleterious action of a pure solution, of course, since the natural salts of the blood are also present. But, the natural saline combination is so much altered, that the most sensitive tissues of the body'become deranged. These sensitive tissues certainly are in the central nervous system, hence narcosis results. In this case the antagonism of calcium salts can be demonstrated, as shown by Meltzer, since an animal narcotized by MgS04 or MgCI2, promptly awakens, when CaCh is injected. If CaCl 2 is injected before or simultaneously with the magnesium salt, no narcosis occurs. The conclusion is, that the excess of the magnesium salt narcotizes since the amount of calcium salt naturally present in the blood is insufficient to

14 Biochem. Zeitschrift., 94, 129 (1919). 15 Am. Journ. Phsyiology, 14, 16, 16, and Proceed. Soc. Exper. Biol., 46,

11, 13, 1905 to 1907, several articles.


counteract it. The narcotizing action of magnesium salts can also be overcome by injections of sodium, potassium or lithium salts, but much larger amounts are needed as we might expect according to the experiments on fish described above (Hirschfelder, 1929).16

4. INCREASE OF PERMEABILITY IN PURE SOLUTIONS AS THE CAUSE

OF THEIR TOXICITY

The cause of the poisonous action of pure salt solutions is the increase of permeability which these solutions prodUce in certain tissue membranes. Salt combinations of definite proportions are required to maintain a normal condition of cell impermeability. The natural salt combinations are best adapted to this purpose.

Numerous observations on a variety of plants and arumals can be quoted to support this statement. In the roots of certain plants the increase of permeability of the skin, which is acquired in N aCI or other pure solutions, becomes directly visible owing to their slimy gelatinous appearance. Similar changes are visible in tile membranes of various animals (R. S. Lillie)P Even more striking are observations on those eggs of fish, which are covered by a dense membrane, impermeable to both salts and water, as stated above (J. Loeb, 1912).18 This dense skin also keeps intact and impermeable if the eggs are transferred to a highly concentrated mixed salt solution, viz., a NaCl solution with a small addition of CaCl 2 and KCl, all salts being 3 or 4 times more concentrated than in ocean water. Such a solution has a higher specific gravity than the eggs. Hence the eggs will float in it, and they will keep floating for several days since no osmosis occurs in spite of the excessive osmotic pressure of the solution. The membrane remains impermeable to water. However, in a pure NaCI solution of equal

16 Journ. Pharmacology, 37, 399. 17 Am. Journ. Physiology, 6,6,10,17, several articles. Decreased permea

bility following addition of CaCl 2 can. be demonstrated also by a diminished velocity of staining as a result of a slower penetration of dyes into cells, E. Gellhorn, Protoplasma, 12, 66; 14, 28, 1931. The increase of permeability of a muscle after stimulation can be compensated by CaCl 2 addition. Hence in a Ringer solution with a higher CaCl 2 content muscles fatigue later than in a normal Ringer solution, after tetanic stimulation. E. Gellhorn, Biolog. Bulletin, 60, 382, 1931.

18 Biochem. Zeitschrift, 47, 127 (1912).


concentration and specific gravity the eggs float for a short time only, the membrane becomes permeable to water and osmosis draws water from the eggs on account of the high concentration of the surrounding solution. Consequently the specific gravity of the eggs increases and they sink. The same occurs in concentrated CaClz solution or in other pur~ salt solutions. These experiments have but one meaning, since death or life is not a criterion entering into discussion in this case. The essential change is the increase in permeability of the egg membrane, which allows osmotic forces to draw water out of the egg. Also in a highly concentrated N aCI solution with a slight addition of CaCh, or with the addition of SrCl 2 or BaCl2 solution, the impermeability of the membrane is maintained and hence the eggs will float longer than in pure NaCl. Additions of salts of heavy metals, however, were found to have no such action in this case.

Another method for demonstrating the increased permeability in pure salt solutions is based on observations of the hemolysis of red blood cells (S. M. Neuschloss, 1920).19 Pure salt solutions hemolyze more readily and likewise all those mixtures which have a composition different in proportion from the natural one. (These we might call "non-physiological" ones.) In order to demonstrate this more rapid hemolysis, slightly hypotonic mixtures are used, viz., 0.112 molecular solutions. The difference of osmotic pressure inside and outside the cell is so small in these cases that no 'hemolysis occurs in Ringer solutions. However in a non-physiological mixture some cells will break up. Neuschloss has experimented also with mixtures of NaCI and CaClz in varying proportions, all of which have a total concentration of 0.112M. All of them hemolyze slightly except the mixture of these two salts in the proportion of 20: 1 which is about the "physiological" ratio.

5. THE Loss OF IMPERMEABILITY IN PURE NaCI AND PURE CaCIz SOLUTIONS AS BEING DUE TO DIFFERENT CAUSES

That the normal impermeability of the membrane is kept up by CaCI2 addition, as demonstrated by the experiments described, is not surprising, if we remember that calcium salts precipitate soaps

19 Pfliigers Archiv., 181, 40.


or water soluble lipoids most powerfully and accelerate the new formation of a membrane around protoplasmic effusions (see above, page 49). In pure NaCI solution the membrane evidently tends to dissolve. Probably the small addition of CaCb required keeps up the normal degree of impermeability by its dehydrating effect, causing a moderate shrinking. However, if more CaCl 2 is added, the tissue membranes may become too much dehydrated and may shrink to such an extent that fissures arise in them. It seems, therefore, that the loss of permeability in pure NaCl and in pure CaCl 2 solution is due to different causes.

These assumptions are supported by observations of the electric conductivity of plant tissues ·in pure salt solutions and in mixed solutions. Intact tissue offers a considerable resistance to the passage of the electric current as long as its membranes have their normal condition of impermeability. When impermeability is lost in pure NaCI, the resistance drops. In pure CaCl 2 solution, however, there is at first an increase of electric resistance owing to dehydration of the colloids followed later by a drop in resistance probably on account of the formation of fissures. This has been demonstrated on plants, for instance, on the leaves of a marine alga, Laminaria (W. J. V. Osterhout, 1912).20 100 to 200 discs of uniform size are cut from these leaves and pressed together tightly. In sea water their resistance is about three times higher than in pure NaCI of equal concentration. In pure CaCl 2 solution t,he resistance is, at first, still higher than in sea water. Within a few hours it drops to the same level as in NaCI or lower, probably on account of fissures. The same course is observed in solutions of other bivalent salts or heavy metal salts.

A definite condition of hydrqtion, an intermediate one between too high a hydration and complete dehydration seems, therefore, to be essential for maintaining the normal membrane impermeability. This is indicated likewise by observations regarding another type of equilibration, viz., that of acids and salts. Free acids of any description are poisonous to nearly all tissues. The addition of neutral salts like NaCI or CaCI 2, detoxifies considerably although such salts do not neutralize the free acids in the least, of course. Now, there can be no doubt about the underlying cause of poisoning and detoxification in this case. It is well known that gelatin and other proteins swell in acid, which would thus tend to increase the permeability of a protein mem-

20 Science, 35, 112.


brane. It is also well known that the addition of salt to the acid leads to shrinkage. The detoxification of acids by salt addition is thus easily explained, but, is it true, in this case also, that a moderate degree of hydration serves best for detoxification? The total amount of shrinkage produced by salt addition may be very large, as we have seen before. But, since extreme dehydration is likewise unfavorable we should expect that too high a salt addition to acid should rather increase the toxicity. In fact, systematic experiments (by Loeb and Wasteneys, 1911)21 have clearly demonstrated that this conclusion is correct.

If small fish (Fundulus Heteroclitus) are poisoned by placing them in a dilute solution of acid, the addition of a definite amount of salt was found to be most favorable for keeping the fish alive for the longest period. This occurred, for instance, if t molecular N aCI was to be added to a 0.0006 molecular solution of acid (viz., HNO s). With either larger or smaller additions of NaCl the fish was observed to die sooner.

6. THE SURFACE TENSION AND AGGLUTINATION OF LIPOIDS AND

PROTEINS IN PHYSIOLOGICAL SALT MIXTURES (NEUSCHLOSS, 1920)

It may seem from the experiments described that the antagonistic action of NaCl and CaCl2 is the result of their influence on the hydration of tissue proteins. It can be shown, however, that the hydration of lecithin, a typical lipoid, is also influenced in a similar manner by such salt mixtures as NaCI + CaCl2 in the physiological ratio, but not by pure NaCI solutions or, "non-physiological" mixtures. This influence is far more specific than the rather indefinite action on protein hydration.

To demonstrate this specific influence, emulsions of lecithin are used (S. M. Neuschloss, 1920).22 As is well known, lecithin, although water insoluble, forms finely divided suspensions in water which have a lower surface tension than water, like soap or protein solutions. This lowering of surface tension can be measured in the usual manner by counting the number of drops from a given pipette (stalagmom-

21 Biochem. Zeitschrift, 33, 489. 22 Pflugers Archiv., 181, 17, 45.

APPLICATION OF PHYSI(jOCHEMICAL LAWS 85

eter). The lowering is quite considerable: a decrease to 75 per cent of the surface tension of water may be observed. By the addition of salts like N aCI, KCI or CaC1 2 the surface tension of a lecithin emulsion is again raised; in other words, it is brought back to 80 or 90 per cent of that of pure water. This action may be explained by a dehydration or rather an agglutination produced by the salts. Salts act in this case in a similar manner as on other colloids, causing the finely divided lecithin particles to flock together to larger aggregates which fail to lower §urface tension. This salt action is nearly the same whether much or little salt is added, at least within the limits of 1 to -.to molecular, but it occurs after the addition of pure salts only and not after the addition of those mixtures which have an outstanding physiological action like Ringer solution or N aCI + CaCl2 mixtures in the ratio 20: 1. These mixtures, no matter what their concentration may be, fail to agglutinate lecithin, the surface tension stays almost as low as in pure water. In other words, these special mixtures act like water, while pure salt solutions or all "non-physiological" mixtures act in the opposite way. M anifestly this corresponds exactly to the observations on living organisms: Fundulus lives in distilled water, and in ocean water, but dies in pure NaCl solution or in "non-physiological" combinations.

As explained above, N aCl and CaC1 2 seem to act on biological membranes in opposite directions; this is not the case in the experiments on lecithin. Both pure CaC1 2 and NaCl raise the surface tension alike. But the striking feature is, that the ratio of the salts, in that mixture, in which lecithin has the lowest surface tension, agrees very closely with the ratio of the optimal mutual detoxification as tested on fish and other animals or plants.

In order to determine the ratio of N aCI and CaCI 2, or of other salts, at which the surface tension of lecithin is lowest, it was necessary, of course, to perform systematic measureme'nts varying the ratio of the salts and determining the surface tension in every case. Both pure N aCI and pure CaCl 2 were found to raise the surface tension most, mixtures to a lesser extent, the less so the more they resembled the optimal mixture.

For instance, in a NaCI + CaCb mixture 10:1 or 50:1, the surface tension would be already markedly low, viz., about 80 to 82 per cent of that of water, although not as low as in the optimal 20: 1 mixture, where it amounts to about 75 per cent just as in a lecithin emulsion in straight water. For a N aCI + CaCl 2

+ KCI mixture, the optimal ratio, as found by systematic testing, was 50: 1: 1, and this is even more efficient than NaCl + CaCl 2 (20:1) just as a complete


Ringer solution detoxifies better than NaCl + CaCl 2 in biolt;Jgical tests. The ratio 50: 1: 1 very nearly corresponds to a Ringer solution since, for frogs, for instance, the standard composition is in the ratio of 60:1.2:1 (Neuschloss).22

... In the absence of CaC12 or other bivalent salts, (NaCl and KCl

also antagonize each other in regard to the surface ten!,!ion of lecithin just as they show a mutual detoxification in mixtures aa shown by the greater resistance of red blood cells. As in the case of blood cells, the optimal ratio is 20: 1.

The maximal mutual detoxification of CaCh and MgCl,2 solution occurs in mixtures containing 2MgC12 to lCaCh as statep above. It would seem to be almost too much to expect that with a m~ture of these salts in this ratio the surface tension measurements shou~d also yield identical results. Yet this is very nearly what takes place. In fact the largest number of drops were obtained in this instance from a mixture of equal' parts of MgC12 and CaCh (Neuschlo~s).22

The least agglutinating mixtures of the other salts investigated were found to be mixtures in the ratio 20: 1 for KCl and CaCh or for N aCI and MgCh and mixtures in the ratio 100: 1 for N aCl and AICIa. Such mixtures are likewise least toxic for most organisms.

In general, the investigations carried out so far show, that' all salt mixtures in those proportions which are least toxic for living organisms, are also least agglutinating for lecithin (Neuschloss).22 A similar antagonistic action of salts mixed in the biological ratio has also been Qbserved by a totally different technique on an enzyme of protein nature, viz., invertase. As is well known invertase is a colloidal enzyme, prepared from yeast, which decomposes cane sugar into glucose and fructose. Like lecithin it is insoluble in water, out forms emulsions. The degree of dispersion, in other words, the number of colloidal particles per unit volume, influences the degree of the enzymatic action. The more finely invertase is dispersed, the more cane &ugar will be decomposed.

Neuschloss22 has measured by polarimetric methods the amountlof cane sugar which is decomposed within 18 hours in solutions all fof which contain 10 per cent cane sugar and the same amount of invertase in the beginning. Under these conditions, 77 per cent of the cane sugar is decomposed in an aqueous llolution to which no salts are added. After addition to the above mixture of! molecular NaCI, 52 per cent of the cane sugar was decomposed, after the addition of l molecular CaC1 2, 7 per cent. With mixtures of these two salts at

.A!PPLICATION OF PHYSICOCHEMICAL LAWS 87

the same total concentration (t molecular) a 13 per cent decomposition was observed if equal amounts of N aCI and CaCl2 were added. A 71 per cent decomposition was observed if CaCb and N aCI either 10: 1 or 50: 1 were aqded, but 76 per cent decomposition, which is practically the same ,as in pure water, if these salts were added in the ratio 20: 1. T~us, in this case also, the mixture of NaCl and CaCl 2 20: 1 acts like pure! water. Analogous experiments have been performed with other sf1lt mixtures (Neuschloss).22

Further experiments have shown that pure invertase-prepared according to Willstae.tter-fails to show the antagonistic salt effects described (Hober and Schurmeyer,1926).23 In fact, its enzymatic activity is not influenced by any salt adqition at all, not even by pure salts. Howe':er, if globulin is again added to the solution of the purified enzyme, this mixture will exhibit an enzymatic activity which can be influenced by salt additions in the same manner as was observ.ed for the non-purified enzyme.

Manifestly the peculiar salt actions are not due to the enzyme invertase as such, but to protein impurities which are adsorbed by the enzyme. In the place of globulin, we may likewise add lecithin to invertase to observe the same dependence on salt addition and the equilibration of the preferred mixtures. However, gelatin or albumin cannot be substituted.

7. THE ACTION OF SALT MIXTURES AT OIL-WATER INTERFACES; ANTAGONISTIC EMULSIFICATION (G. H. A. CLOWES, 1916)24

The observations on biological salt actions and on agglutination of lipoids and proteins lead to the conclusion that agglutination increases membrane permeability, both effects being produced by pure salt solutions. The mechanism of this process remains to be investigated. Our knowledge regarding the state of hydration of the membrane and the relation of hydration to permeability remains hypothetical as long as we cannot produce an artificial membrane, made of well known substances, which maintains its impermeability exclusively in a solution mixed in the physiological ratio, such as a Ringer solution or in other "equilibrated" mixtures, and becomes permeable in pure salt solutions. This goal has been reached through the experiments of

23 Journ. Gen. Physiology, 8,265. Antagonistic salt actions can be demonstrated in the precipitation of gelatin by alcohol (W. O. Fenn, Journ. BioI. Chem., 34, 141, 1918), but the similarity to biological antagonism is not as striking as in experiments on lecithin and invertase.

24 Journ. Physical Chem., 20, 407 (1916).


Clowes which are based on his observation of the antagonistic effect of NaCl and CaCl 2 on the interfacial tension between dilute alkali and olive oil. This tension is increased by N aCl and decreased by CaCl 2

showing that there is an opposite action of these two salts, in this case, as on biological membranes.

The method of measuring interfacial tension is the same as that used for determining surface tension of a fluid in air, viz., .to count the

NaOH .s/trrtj/tt NaO/I"4tJt:J15111d Nab/lftJ,IS'IM/ Na{/. Ca(/.t I-qIl(J/5CaCM.

.3()() a'rcp$ . The number of drops in the diagram is about one-tenth of those actually observed.

FIG. 17. DIAGRAM TO DEMONSTRATE THE ANTAGONISM AND MUTUAL EQUILIBRATION OF NaC! ANT) CaC!. ON THE SURFACE TENSION OF NaOH

SOLUTION IN OLIVE OIL

Addition ofNaC! increases the number of drops, decreases the surface tension. Addition of CaC!. decreases the number of drops, increases the surface tension.

number of drops from a given volume. Figure 17 indicates diagrammatically how the method is operated: aqueous solutions of NaOH with or without an addition of either NaCl, or of CaCb, or of both salts, in the concentrations indicated on the diagram are contained in pipettes. The tips of the pipettes are submersed in olive oil. One counts the number of drops which are formed as the pipettes empty through the oily layers. Both the increase In the number of drops


following NaCI addition and the decrease following CaCl 2 addition are exceedingly large as represented diagrammatically in Figure 17. "But if NaCl and CaCl 2 are employed together at concentrations of 0.15 molecular of NaCl and 0.0015 molecular CaCl 2, the number corresponds closely with that given by the original NaOH" (G. H. A. Clowes).24 (Compare diagram, Figure] 7.)

This shows that the opposite actions of NaCI and CaCl2 compensate each other when these two salts are present in the ratio 100: 1. Nearly the same ratio is found if the total salt concentration is varied, although at high concentration the ratio equals ,100: 1.6. Manifestly this approaches the ratio of mutual detoxification of salts' as closely as can be reasonably expected. ~

Now, the cause of the surface tension changes in these experiments can be traced with a fair degree of certainty. As explained on subsequent pages (page 143), a lowering of surface tension is due to the formation of a surface film of oriented molecules. In the above described experiments the film probably consists of sodium oleate formed by interaction of NaOH and oleic acid. If CaCl2 is added, sodium oleate of which the surface film consists, is partly transformed into calcium oleate, while after addition of NaCI the film will contain more sodium oleate. These two soaps exhibit different solubilities: sodium oleate is chiefly water soluble, calcium oleate chiefly oil soluble. The result is a different curvature of the surface film (W. D. Bancroft, 1913)25 as indicated in Figure 18. The sodium oleate film being more water soluble, tends to spread the watery phase around the oil, thus producing oil globules floating in water (see Figure 18a). Calcium oleate promotes a curvature of film in the opposite direction (see Figure 18b), leading to the formation of water globules floating in o_il. The sodium oleate surface film tends to lower the interfacial tension between water and oil and hence leads to the formation of numerous small droplets of water, while the calcium oleate film has the opposite effect.

Both films lead to the formation of stable emulsions by enveloping the particles and keeping them from fusing together. However, on account of their different interfacial tensions these emulsions are of a different nature, viz.; (1) oil suspended in water in the case of a sodium oleate surface film, produced by addition of NaCI; such emul-

25 Journ. Physical Chern., 17,501 (1913).


sions being analogous to cream; (2) water suspended in oil, for a fat soluble calcium oleate film-produced by the addition of CaCh; such emulsions being analogous to butter.

Contrary to the view expressed by Bancroft, these adsorbed surface films are not to be considered as monomolecular. Curvatures of the kind described can be produced only by films consisting of layers which are several molecules thick, as pointed out by N. K. Adams, in his book "Physics and Chemistry of Surfaces," New York and Oxford, 1930.

"Oil in water" "Water in oil" Film consists chiefly of water-sol- Film consists chiefly of oil-soluble

uble Na oleate. More room in water Ca oleate. More room in oil on on surface! Leads to "oil in water" surface! Leads to "water in oil" emulsion. emulsion. FIG. 18. CURVATURE OF SODIUM OLEATE AND OF CALCIUM OLEATE SURFACE

FILMS AT A WATER-OIL INTERFACE '.

Evidently in between these two types of emulsions there must be a transitional condition where both "oil in water" and "water in oil" emulsions exist next to each other. In order to observe this transition, increasing amounts of CaCl2 are gradually added to an "oil in water" emulsion. The results of such an experiment are shown in Figure 19. In this case NaOH and CaCh were chosen as the two antagonistic agents, in the place of NaCl and CaC1 2• NaOH, as such, acts like NaCl, but much more powerfully, by building up a sodium oleate surface film, hence increasing the number of drops in the stalagmometer experiment described above. The result is that a double sided emulsion of "oil in water" and "water in oil" occurs only at one definite ratio of the two antagonistic agents. This ratio

Icc fa Ita OUf 0.25 0.5 0.75 1.0 cc~ CaCt2

2ccPJRaOU+ 0.25 0.6 0.75 I. 0 cc;g Caele

8ccf/JnaOH+ 0.25 0.5 0.76 1.0 cc m CaC/z

4ufVlaOHf- 0.25 0.5 /.0 ce f9 CaClz.

FIG. 19 To observe the opposing effect of NaOH and CaCI: on emulsification, 10 cc.

of olive oil + oleic acid were used, and 10 cc. of varying aqueous solutions. The oil was colored red with Sudan II~J consequently it appears black in the illustration. The additions to the Huia were 1 cc. of n molecular NaOH for all bottles in the first horizontal row of the illustration, 2 cc. in the second row

l 3 cc. in the third row and 4 cc. in the fourth row. Besides these additions al bottles in the first vertical row received an addition of 0.25 cc .• \ molecular CaCh, those in the second row, 0.5 cc. n molecular CaC!.. Those in the third row, 0.75 cc. and in the fourth row; 1.0 cc. In every instance the total volume of tne aqueous Huid was made up to 10 cc. "Emulsification was then effected by means of a mechanical shaker supplemented with shaking by hand." (G. H. A. Clowes.)

91


is 4: 1 in the present instance, but it need not be of the same magnitude in every case. The amount of oleic acid, added to the olive oil, and other conditions influence the equilibrium. In agreement with this is the biological finding that the optimal ratio of mutual detoxification is different for different tissues.

The chlorides of calcium and sodium are not the only salts which exhibit this type of antagonism. All the salt combinations, which have been tested in regard to their mutual detoxification, may be substituted for the NaCI + CaCl 2 mixtures in order to observe antagonistic emulsification. If this is done, "electrolytes are found to be divided roughly into two main groups, (1) salts of the monovalent cations, Li, Na, K, etc., which raise the number of drops and favor the formation of "oil in water" emulsions and (2) salts of the di- and trivalent cations, Ca, Sr, Ba, Fe, Al and of other heavy metals which tend to diminish the number of drops and favor "water in oil" systems" (G. H. A. Clowes).24 These two classes correspond to the two classes of salts with antagonistic effects in detoxification as shown above.

In biological experiments, certain substances like magnesium salts have been found to be capable of functioning under varying conditions in one or the other class. In physicochemic!11 experiments of the kind described, magnesium salts also function in"either direction depending on the conditions. "If soap is employed as the dispersing agent in making up a drop system, MgCl 2 functions like CaCh "If however, NaOH is employed as the dispersing agent and soap is formed by interaction between the fatty acid in the oil phase and the NaOH, MgCl 2 functions in an entirely different manner, exerting a dispersing effect similar to but considerably stronger, than that of NaCl, which may b!') counteracted 'by CaCl 2 in ratios 1 to 2 Mg to 1 of Ca" (see above, page 80). "In one case soap is present in the system prior to the introduction of MgCI 2 ; in the other case MgCb has had an opportunity of reacting with NaOH with the formation of colloidal aggregates of Mg(OH)2 prior to the production of soap" (G. H. A. Clowes).24

It may be added that a reversal of emulsion of the kind described above can be brought about by numerous other substances particularly by narcotics; for details see H. Freundlich's Kapillarchemie 4. ed. Leipzig 1930/32.

The results described may be condensed into the statement that the antagonistic influence or'salt mixtures on interfacial tension re-


sembles their antagonistic actions on tissues. But, even more has been achieved as already mentioned. Clowes has obtained an insight into the mechanism which causes these puzzling. salt actions by producing artificial membranes, which show the same variations of permeability as tissue membranes. Such a membrane consists of a reversible emulsion. If N aCl is added, water will be tM outer phase, hence aqueous solutions and water soluble substances will readily pass. One should expect therefore that the permeability is increased just as in tissue membranes. At the same time the electrical conductivity should be almost as good as that of the solution without oil in it, since the electric current can pass through the solution around the oil droplets. However, if by a sufficient addition of CaCI 2, the oil is made the outer phase, the coherent layer of oil formed will arrest water soluble substances and block the current since olive oil is practica1ly an insulator. In other words, CaCh should be expected to counteract both permeability and conductivity, as in tissues. Such results have indeed been obtained by Clowes. The parallelism to the permeability variations in tissue is truly remarkable. His own description may be inserted here.26

"Artificial emulsion membranes suitable for electrical conductivity and permeability experiments may be prepared by interposing layers of filter paper saturated with an emulsion of oil in soap between supporting sections of rubber tubing in a glass U-tube of the type commonly employed for electrical conductivity determinations. A thicker layer or film of emulsion is generally preferable and may be prepared by introducing into the U-tube a section of rubber tubing of any desired length which is then filled with emulsion. Retaining layers of filter paper above and below are held in position by additional supporting sections of tightly fitting rubber tubing."

"Emulsion membranes of this type when exposed to the influence of various antagonistic electrolytes exhibit variations in electrical conductivity and permeability corresponding almost exactly with those observed by Osterhout in the case of Laminaria under similar working conditions. For example, 0.52 M NaCI causes a rapid rise in the conductivity of a saturated filter-paper membrane until the level of the environing solution is almost reached, while 0.278 M CaCl 2

causes first a considerable fall in conductivity which is followed sub-

2. From the Proceedings of the Society for Experimental Biology and Medicine, 15, 107 (1918).


sequently by a rise to approximately the same level as in~the case of NaCI."

"The most remarkable paralleling of Osterhout's results may be obtained by exposing emulsion layers or films to brief alternating treatments with NaCI and CaCI 2• As in the case of Laminaria, al ternating variations in conductivity within comparatively wide limits may be obtained without any apparent injury to the membrane which may subsequently be returned to sea-water or a balanced solution of NaCI and CaCI. and exposed to a similar treatment the next day. However, just as in the case of Laminaria, too prolonged an exposure to either NaCI or CaCh may cause changes in electrical conductivity and permeability beyond the critical point at which recovery is still possible and the membranes subsequently exhibit extremely erratic results or fail entirely to respond to treatment."

"That electrical conductivity experiments on membranes of this type or living tissues afford an index of their permeability to water and water-borne substances was demonstrated by paralleling the above experiments in the following manner: Layers of emulsion supported between filter paper and rubber tubing were introduced into a series of long glass tubes and the speed with which distilled water, sea-water and solutions of NaCI and CaCI. and a balanced mixture of N aCI and CaCl 2 flowed through the membrane was determined by measuring the fall of the fluid in each tube at given time intervals. The distilled water, sea-water and suitably balanced mixtures of NaCl and CaCl 2 flowed through the membranes at approximately equal speed while NaCI flowed through the membrane at a vastly greater speed and CaCI. at a somewhat reduced speed. The relative speeds of flow of various solutions were found to correspond to an extraordinary degree with data previously accumulated by the writer by means of the capillary pipette" (see above, page 88) (G. H. A. Clowes).26

From these important experiments of Clowes we may conclude that one single assumption suffices to explain all the various phenomena ex~ hibited by equilibrated salt mixtures, viz. that, in those equilibrated solu~ tions only, a "double sided emulsification" occurs as described. This is the pre-requisite condition for maintaining the membranes of living tissues in their normal impermeability, and hence for the maintenance of life. Under these conditions only, lipoids are as finely divided as in water as shown by the experiments of N euschloss. In short, the most intimate mutual impregnation of watery and fat-like (or protein-like) phases occurs at this point of equilibration only. We have thus found a new lead to understanding the basis of life phenomena.

To quote Clowes: "Variations of plasma membrane permeability are attributable to the action of electrolytes and metabolic products on delicately balanced soap films and emulsion systems."


8. THE ORIGIN OF LIFE ON THE EARTH AND OTHER PLANETS

It is worth while to consider even a small contribution to the solution of this dominant problem. Any explanation which we may attempt to give must naturally be hypothetical at the present state of knowledge. The similarity of the saline components in the animal blood and in the ocean has lead us to the conclusion that life has originated from the ocean. The same conclusion has been reacl!ed independently by studies on geological evolution. As stated, proteins and lipoids can be finely dispersed and brought to form a double sided emulsion in the physiological salt mixtures only, the composition of which resembles that of the ocean. This dispersion may be assumed to be one of the prerequisite conditions for the origin of life. Hence the lipoidal constituents of living tissue seem to be peculiarly adapted to the saline composition of the terrestrian ocean.

On the other hand, this saline composition of the ocean can hardly be due to anything but the total amounts of all water extractable salts on the surface of the earth. Ever since the earth had cooled sufficiently for water vapour to condense, the soluble substances began to go into solution. This solution, viz., the ocean, became homogeneous throughout, by the action of winds and tides. If upon other planets, the soluble salts were present in other proportions, another form of life would evolve from such oceans. Even the chemical organic compounds of living forms on those planets would be different from those of terrestrian life, since on those planets lipoids of the earth would show no do.uble sided emulsification in the ocean.

The physico-chemical investigations thus lead to the idea that life has been entirely adapted to the terrestrian conditions from its very beginning, and probably has arisen from the earth. This conception is somewhat contradictory to a theory widely accepted formerly, the so-called theory of panspermia according to which life should be propagated from germ cells scattered through the universe. The beginning of life and evolution w~s supposed to depend on the condition that such germ cells were dropped on a planet like the earth where conditions were favorable for germination. Since such germ cells were supposed to be carried by radiation pressure27 from planet to

27 This radiation pressure has also been demonstrated experimentally by a movement of dust particles in a vacuum following radiation from one side as shown by Nichols and Hull. For details see S. Arrhenius, "Worlds in the Making."


planet and from solar system to solar system, life was considered to be eternal like time and matter; it should never have been generated anew. This theory, popularized by extensive physical discussions by such an authority as S. Arrhenius, has been invented on account of the widely accepted doctrine that life can never arise from inanimate matter. As is well known this doctrine dates back to the famous experiments by which Pasteur exposed the delusion of the old time "life creators," viz., those careless experimenters who drew conclusions from observations on nutritive media, handled without adequate control of asepsis. The reason given for the impossibility of spontaneous generation was simply that so far nobody had ever observed it. But, such a negative inductive proof cannot be convincing!

Considering the great uncertainty prevailing about this problem it is worth while to recall the varying opinions about another fundamental question, viz., the transformation of one chemical element into another. During the sixteenth, seventeenth and eighteenth centuries such a transformation, especially that of other metals into gold, was believed to be possible, since fraudulent reports about alJeged results were widely circulating. This delusion was unmasked at last when scientific chemistry developed early in the 19tp. century. At the end of the nineteenth century, the indivisibility of the elements was better proven than is now the case for the impossibility of spontaneous generation. For nearly a century hundreds of able chemists had investigated indefatigably every possibility of transforming elements, without success. Hence they deemed it justified to announce that such a transformation would never be accomplished. Yet a very short period of time later, new investigations definitely showed that elements can be transformed by radioactivity.

Considering this experience, the impossibility of spontaneous generation seems to be hardly more than a creed based on doubtful authority. To prove it, we should exclude by experimentation all possibilities of generating artificially anything remotely resembling a living organism. Manifestly this has never been undertaken.

Nothing definite is known so far about experimental methods leading to spontaneous generation. One possibility may be suggested which is based upon the assumption that the very first forms of life to arise on the earth were of the size of molecules or molecular aggregates. ]If any may object to the idea that a single molecule can be a living or-


ganism since this would lead to the rather odd conclusion that a chemically pure substance may consist eventually of a multitude of living beings.

Further objections have been raised against this concept by some who assume that a living thing proper must have "organs" of some sort or, at least, a plasma membrane around it. It will be remembered that formerly the requirements for a real living organsim were set considerably higher yet: the great Liebig, whom J. J. Abel28 deservedly honors as "the most notable among the modern founders of biological chemistry since Lavoisier," held that yeast was not an organism but just an enzyme. The motive for this assumption was that the too simple make-up of yeast cells did not conform with Liebig's rather fastidious ideas of what a living organism should be! At present, scientists have become more unpretending in that respect, but perhaps it is permissible to state that not all of them are sufficiently unassuming as yet. The fact is that the smallest living things known, viz., filtrable submicroscopic viruses, are actually hardly above the size of molecular aggregates as is shown by an estimation of their size through the method of gold impregnation of Bechhold and Villa (1926).29 Such filtrable viruses are, for instance, the active virus of rabies, of vaccine, of smallpox, of bird pest or of plant mosaics. It is only because of their pathogenic power that we have some knowledge of these living organisms.

There is positively no reason to deny that a single molecule or a molecular aggregate, a micella, has all the properties of a living organism if it is an enzyme which has the power of producing in a naturally occurring environment chemical reactions which lead to its own multiplication. This would mean that such an enzyme propagates itself in the same manner as a living organism. Evidently it would be really alive in the common sense of this word.

Furthermore, if it is true that living organisms can develop from lower to higher forms, there is no reason why the monomolecular forms should be excluded from this evolution. It would seem possible, therefore, that the monomolecular forms, viz. self reproducing enzymes, stand at the very beginning of the evolutionary scale. The hypothesis would be that they have been formed somehow by a process of molecular aggregation in an unknown manner.

28 See J. J. Abel's "Willard Gibbs Lecture," Science, 66, 307, 337. 29 Zeitschrift f. Hygiene, 106, 601. The method consists in the deposition

of gold on every particle that is present.


In order to understand the origin of life, it would be necessary, therefore, to synthesize enzymes which reproduce themselves in a natural environment and to study their actions. At present synthetic chemistry is not nearly sufficiently developed to venture the synthesis of such compounds, but who can claim that it will never develop that far? If a self-regenerating enzyme of such a kind could be made it would certainly ·be a carbonic compound. Life, therefore, in spite of all its complexity, seems to be no more than one of the innumerable properties of the compounds of carbon. Thus we realize that our knowledge of organic chemistry is just in its infancy. As is well known, the chemistry of carbon compounds has proved to be many times more complicated than the entire chemistry of all other compounds. 150,000 carbon compounds have been registered in scientific literature as compared with 30,000 known compounds of all other elements. Who will deny that further unknown properties rest in the carbon atom so as to enable it to cause spontaneous generation in the manner suggested?

Some modern biologists, who have renounced the viewpoints of the panspermic theory, still believe that a spontaneous generation of submicroscopic organisms could have occurred at the early time of terrestrian existence only. There seems to be no strict reason for this belief.

EXTENSION OF EXPERIMENTS WITH MODELS: THE LIESEGANG RINGS AS A RESULT OF REVERSIBLE CHEMICAL REACTIONS UNDER CONDITIONS OF ORGANIZATION. AN EXPLANATION FOR NEUROBIOTAXIS

1. THE ADVANTAGE OF PRECIPITATION MEMBRANES OVER FIXED

MEMBRANES IN MODEL EXPERIMENTS

In order to elucidate the functions of tissue membranes, we have at first described the origin and growth of precipitation membranes from solutions, these processes being similar in some respects to the origin and growth of membranes in tissues. Subsequently, however, in order to analyze the various properties of tissue membranes, growing membranes proved unsuitable on account of their variability. It was necessary to substitute for them fixed membranes such as:

Precipitation membranes firmly embedded in porous clay for observing the laws of osmotic pressure;

Collodion membranes for studying the complicated ionic membrane equilibria; (compare also the Appendix)

Emulsion membranes (Clowes) for understanding the cause of antagonistic salt actions.

Each of these membranes is prepared in a definite manner for investigating one definite feature of tissue membranes. We should realize that the comparison with such "fixed membranes" is necessarily limited since variability is the most characteristic peculiarity of tissue membranes.

This variability can be imitated only by experiments on membranes which are being generated from solutions. Such an experimentation is truly a source of wealth for imitating vital phenomena which might appear inaccessible to artificial reproduction at first sight. An example of such' an unforeseen achievement is the artificial reproduction of stratified structures by means of diffusion in gels.

Stratified structures· are frequently met with in living tissue. The deposition of calcium salts around the blood vessels of the bones, running through the Haversian canals shows distinctly stratified layers. Other stratifications are found in plants; the configurations on the wings of butterflies are further examples.

99

100 FIRST ATTEMPT AT A,PPROACH

R. E. Liesegang first observed and described artificial stratified strudures in 1896. 1 This discovery will be looked upon as a milestone of pmfl'Te8S 2/ synthetic biology will be generally recognized as a science -as we hope it will be some time. Previous to Liesegang, to explain 8tl'atijicat1'ons, assumptions were involved like a periodic supply of one or both components, ar a periodicity of e.rternal conditions like temperature. None of these assumptions is needed; striking periodic stratifications can be formed when all the factors involved remain constant or, at any rate, do 1Wt undergo periodic var-latiolls, synchronous with the formation of the strata.

Licsegang rings are obtained by a simple diffusion of a solution into a gel (jelly) which contains a precipitating agent. As an example we may mention the diffusion of an AgNO a solution into a gel containing bichromate. Since bichromate precipitates soluble silver salts, a dark red precipitation occurs in the gelatin. This takes the configuration of beautiful concentric ring[o; as shown in Figure 20. The experiment can also be performed in such a manner that the bichromate is poured into a test tube. When this has set to a gel, silver nitrate is poured on top. After several days parallel striations appear in the gelatin.

Similar bands or rings can be obtained with numerous other precipitations. Thus FeCla may be added to the gelatin, and a solution of potassium ferrocyaniae pJacea in the center. Blue rings of ferriferrocyanide or Prussian Blue will then be formed.. Also the simple crystallization of certain substances occurs in parallel stratifi~ations. This is true, e.g., for tolunitril (E. Schubert, 1924).2 The well known stratifications of starch granules arc flimilar.

Furthermore, striation occurs in the abRence of gelatin in layers of fluid held by capillary forces within nmrow spaces. If a 10 per cent AgNO a solution is held between two microscope slides at a distance of about 2J.1. from each other and if NaCl is allowed to diffuse into this layer, silver chloride is precipitated at regular intervals forming parallel striations (T. Brodersen, 1924).3 Spiral shaped

1 NaturwisB. Wochenschr., 11, 353. 2 Koll. Zeitschrift, 35, 210; compare also II. Kagi, Koll. Zeitschr., 33, 284;

Zacharias, Koll. Zeitschr., 84, 37 (rhythmic sedimentation). 3 Koll. Zcitschrift, 35, 21; compare also A. Janek, Koll. Zeitschrift, 33, 86

(1923) (rhythmically striated precipitation membranes on the free !lurface).

Photographs Illustrating the Formation of Structures Through Diffusion In Gels

,. .. ~. , .

,

\

•

FlU. 20. LIESEGA:"lG RIXGS

A .it'lly or the following composition is prepared: 50 ('(', 10 pt'r (,Pllt geblin HO In tio rJ + 20 ('(', 10 per cen t gelatose tloj ution + 00 f'C, water + ;~ d rOj)iI HI I'('r' ('('11 t mnrnonillm hidu'omaLc solution + 5 drop" 5 per ccut citrif' :t('id solutioll' Lot" of' 10 (T, each of this mixture ,ue poured on a gln"~ platE' !lnd allowe(l to I-\p,t tu :'l !,;c1, After setting, 4 rl1:npR d " 25 \'(,,1' n'nt "i!ver uitl'atl' !Solution are plaeed in the center of the !!;elatin. The preparaLion i~ then Ret Il~idc in B. cltirk plae", ~ nd well pmtec ted agt,inst e vapo mti Oil, A riel' 2 to 4 daSH cone-eniric rings have formed as shown on photo,

XllLurul size, (Reproduction of photogmph taken by Dr, H, II, MaUll)

• 'rue gelatin \1sr-u in this m\xtuTI:: Bl'lO'uld hr- p\lre, viz" iahly [,I'll from soluble eonstituents, To purify it, it iR wushe[1 with [,.,ld w;,t,PT H'IH,'atedly, before dissolving it in warm water. "(~f'l:d()s('" "oJllt,i,m ii< a gdatin solution heated in an autoehn'e for :oe\'erul huur::;, It hap]H'TlH th'lt. Llie ('Iwaller brands of (,(Hnmt'ITial gplutin :-\ometimps eunt:"lin gelatoHl' and sometime:,; alRo aeiu and, in this Cllse, they ('un he uspu without ;<llI"h ail_litioll [or the experiments, ,\ llnilwT prcsr-riptinn of L i.C"PJ!;fLn1!, if' t hI' f oU'n, in I!; : ~\)!T, H} per l"eDt gcia tin holutirm + 50 ee, ,Yater + 14 drops ]0 Pl't" Cl'nt umml)nium bir'hromatc 'Hllution + 11 drops citric acid solution; spread un plate and pro('eell us before,

Photographs Illustrating the Formation of Structures Through Diffusion ill Gels

FIG. 22. D~;VI';L()rMENT GHO\'lTII 01.' Two ZONE" OF PRECIl'lTATIO'l WHICH AHJ<; Apl'B.OXl~tA.TED

Thi:'l phenom(,I1Un expluins n('urobiotaxis. A(·(·urt.lin~ to Liesel!;:lllg; from Annalen der PhYHik, 1906. Sep. text on ploijl;C 1Q5.

};XPERIMENTS WITI[ MODJ<;LS 101

striations can also be obtained under certain conditions.5 These are just a few examples which may serve to illustmte the great variety of morphological phenomena of the Liesegang type.

2. LIESEGA G RINGS AS A REsrLT OF INCOMPLETE HEVl<;USIBLI<; RE

ACTIONS LEADING TO A MUTUAL INTERFJ<JHENCE 01" THREE

DIFFUSIO. WAV1<;8 (Wo. OSTWALD, 1926)6

However, not every chemical precipitation reaction is equally well suited for the production of Licscgang rings. The precipitation of BaSO 4 hardly ever occur' in rhythmic hands, just one continuous layer of BaS04 is formed between the Ba salt on the one side and any sulphate on the other. \Vith AgCI precipitations, it seems very difficult to obtain "rings" although it can be accomplished under certain conditions. Yet other reactions lead eaRily to "ring" formation. A property which discriminates between the two types of reactions, is the degree of complet.eness or the limitation up to which they progress. "Complete" reactions do not form rings, incomplete renctions do. This empirical relation-which is well established by numerous experiments- forms the basis of the so-called theory of diiIusion waves (Wo. Ostwald, 1926).6 Although this theory is directly applicable to chemical reactions only, it affords the best physico-chemical explanation for the origin of the ring>; as far as we know at present.

In fact, the Btl.SO 4 precipitation, which !!,ives no "rill!!,""" is a !!,ood

example of a "complete" reaction. If equivalent amounts of Na2S04 and BaC1 2 are ffilxed in solution they arc transformed completely into insoluble BaS04; S04- - and Ba++ cannot exist together to any appr('ciable amount due to the extremely low solubility or the insolubility of BaS04.

As an example of an incomplete reaction, on the other hand, we may mention the well known precipitation of magnesium chloride by an aqueous solution of ammonia. In fact this reaction gives rise to beautiful "rings." The precipitate formed is, of course, mllgnesium

i ~ee Hatschek, KQll. Zeitschr., ~7, 225 (19'22). Stuckert, KQB. Zeitschr., 31, 2.1R (1926).

• Kolloid. Zeitscbr., 36, 38l.


hydroxide, but a considerable portion of the MgCh remains unchanged even if an equivalent amount of NH3 or more of it is added. The reaction is incomplete to a particularly great extent if NH 4Cl is added. No precipitate is formed at all if sufficient amounts of NH 4Cl are added. 7

Since NH 4ci is a product of the reaction itself, it is easy to understand that its formation and subsequent accumulation in certain zones are responsible for a disappearance of the precipitation in these zones. In other words, by the dissolving power of this reaction product (NH 4Cl) clear zones are formed between the zones of precipitate which thus appear as "rings."

To describe this mechanism of "ring" formation more completely, it may be interpreted as a mutual interference of the diffusion of three soluble components: MgCl 2, NH 4CI and NHs, acting against each other, as illustrated by the accompanying diagrams. (See Fig. 21.)

The deeper the diffusion of NH 3 penetrates into the jelly, the more dilute it becomes, consequently it takes more time for NHs in the lower layers to overtake the NH 4Cl diffusion which originates from the precipitation band. We should expect, therefore, from the theory that the more distant bands of precipitation have an ever increasing distance between them; the clear zones in deeper layers should be more extended. This conclusion is quite generally verified in agreement with the theory.

The theory agrees well with experimental observations also in other regards. Thus, the formation of the second and all the following rings should be delayed if some NH 4Cl is added to the gelatin while its MgCb content is kept constant. With t~is addition it should take a longer time for the NH s to predominate over the NH 4Cl (which counteracts precipitation) since some NH4Cl is present in the gel everywhere. Consequently the clear layers should become

7 This prevention of the Mg(OH) 2 precipitation is by no means due to a compound formed, but, is the typical result of the existence of a chemical equilibrium. As is known in Physical Chemistry, the law of chemical equilib-. rium states that the product of the concentrations of the substances entering into the reversible reaction, divided by the product of concentration of the reaction products is constant. If one of the reaction products as e.g., NH.CI is present in a high concentration, the reaction must proceed in the opposite direction in order to maintain the above mentioned constancy, in other words, MgCl 2 and NH 3 must form from Mg(OH) 2 and NH .CI, which means a dissolution of the precipitate.

EXPERIMENTS WITH ·MODELS 103

broader. Experiments show that this is the case. With a very high NH4CI addition they naturally fail to form entirely.

It is evident from all the observations described that incompleteness or reversibility of the reaction is the condition which inhibits and

Initial state tater state

3.

~I 0..

·'~I·· . ''0 ;r "', t

MgC1Z

I

a b

FIG. 21. DIAGRAM ILLUSTRATING Woo OSTWALD'S THEORY OF "DIFFUSION RlNGS"

a. In the beginning of the experiment a highly concentrated solution, viz., 12 molecular solution of NHa diffuses agajnst a 1 molecular solution of MgCb.

b. As the diffusion proceeds all the MgCl2 in the "plug" or first ring has been transformed into Mg(OH).; no further Mg(OH)2 and consequently no further NH,CI can be formed here. However, the supply of NH. is far from being exhausted. NH. continues to diffuse, it now penetrates unchanged through the "plug" and penetrates further through the clear layer under it, the diffusion of NH,Cllags behind-since there is no further supply ~ thus NHs finally reaches a still lower layer where it hits upon sufficient l\lgCI. with little or no NH,CI-so that a second zone of precipitation occurs. Mg(OH). is formed and along with it diffusible NH,Cl. From now on the game repeats itself; alternating clear and precipitation layers are continuously being formed. A rhythm is established.

104 'FIRST ATTEMPT AT APPROACH

interrupts the formation of a continuous precipitate. Now we only need to substitute NaOR or KOR for NHa if we want to precipitate Mg(OHh from MgCh solution completely since Mg(ORh does not dissolve in an excess of KCI or NaCI, formed in this reaction. Consequently we should expect that if NaOH is substituted for NRa on top of the gelatin containing MgCh no ring formation should occur. This conclusion from the theory h!1s also been verified.

Since Liesegang ring formation may occur also as a result of merely physical reactions, such as crystallization, as stated, it is difficult to see which kind of a dissolving substance can be formed in this case. Adsorption of some sort may account for the formation of the clear spaces. These cases are difficult to explain at present.

The merit of Ostwald's theory is to have selected those cases in which an explanation is possible and to have elaborated a complete experimental proof for them. The occurrence of Liesegang rings from such physical reactions can hardly be quoted as an argument against this theory.

3. EXPERIMENTS ON DIFFUSION AND PRECIPITATION IN GELS WHICH

EXPLAIN NEUROBIOTAXIS

All the similarities of artificial structures and certain features of living organisms, which have been enumerated so far, mlty appear rather too general to some. It may be worth while, therefore, to describe here a highly specialized feature of living growth, the nature of which has become well understood through further studies on the diffusion in gels. This phenomenon is the so-called neurobiotaxis which was first described by C. V. A. Kappers in 1908.8 It was known that nerve cells preferably send out dendrites following stimulation or injury. Kappers observed that if two nerve cells are stimulated or injured which are in close approximation, the dendrites grow from one cell to the other.

The growth of simple inorganic precipitations in gelatin exhibits quite a similar tendency, as was demonstrated by Liesegang9 in the following manner. If a drop of AgNO a solution is placed upon a gelatin jelly containing N aCI, a disc shaped zone of white opaque Agel develops which gradually enlarges as the silver salt continues to diffuse into the gelatin. If two drops of AgNO a solution are placed

8 Folia Neuro Biologica, vol. 1. 9 Annalen d. Physik, 19, 395 (1906); 32, 1095 (1910).


on the gelatin they grow towards each other-a phenomenon which is quite analogous to "neurobiotaxis." (See Fig. 22, opposite page 101.)

The reason for this development is easily understood: Through the formation of AgCI, CI- ions disappear from the NaCI gelatin in the vicinity of the Ag salt zone. These disappearing Cl ions are replaced by others from the more remote zones of the gelatin. If two drops of AgN03 solution are near each other, Cl ions are used up on both sides of the zone between the drops. Hence the Cl- ion concentration in the intermediate zone is reduced more rapidly, and the AgN03 has to advance further in this-zone in order to produce a AgCl precipitate.

Probably neurobiotaxis is due to similar causes, viz., to an exhaustion of membrane building substances between the two nerve cells. This is a striking example demonstrating the usefulness of experiments with simple models for analyzing the nature of a seemingly difficult biological observation.

THE SECOND ATTEMPT AT APPROACH

LIFE PROCESSES RELATED TO CRYSTALLIZATION OR DUE TO SURFACE FORCES

THE SECOND ATTEMPT AT APPROACH: LIFE PROCESSES RELATED TO CRYSTALLIZA

TION OR DUE TO SURFACE FORCES

EXPERIMENTS WITH MODELS: MICROSCOPIC STRUCTURES PRODUCED BY COLLOIDS IN VITRO

As is well known, the cell is one of the most generally observed microscopic structures in living tissue. A somewhat enthusiastic belief has prevailed that the study of the structure of this morphological unit would go far in revealing the nature of life processes. This belief has led to the collection of an immense amount of descriptive statements. But, morphological studies alone can hardly reveal the nature of vital processes in the cell. The true value of all the labor invested in the science of cell study (cytology) will be better understood in the remote future after a similar microscopic morphology has been found in artificially produced systems. Little is known of such possibilities at present. Many may doubt whether anything worth while will ever be accomplished along this line. Nevertheless, we can attempt to trace the beginning of ajuture development towards this remote ideal.

1. SIMPLE STRUCTURES ARISING IN ALBUMOSE SOLUTIONS FOLLOWING

PRECIPITATION

Most microscopic structures which compose animal organisms consist of protein next to lipoids. Hence they must be formed somehow from dissolved protein Of' from protein or lipoid forming materials. The question prt:}sents itself as to whether or not structures of some sort can be formed if dissolved protein or similar substances are precipitated in a suitable manner. Indeed numerous experiments have shown the possibility of producing primitive artificial microstructures of various types in this way. This is demonstrated, by the following experiment (Alfred Fischer, 1899).1 A square area is

1 As described in his book "Fixierung, Farbung und Bau des Protoplasma," Jena, 1899. Fischer's experimental work chiefly aimed at demonstrating that many structures in fixed microscopic preparations were artefacts, viz., similar to the artificial structures obtained from solutions of proteins or proteoses. He also demonstrated the stainability of these artificial structures.

109

110 SECOND ATTEMPT AT APPROACH

walled off on a microscope slide by means of vaseline. A glass capillary open at both ends is inserted so as to pass through the vaseline wall. This square area is then entirely filled with 3 per cent albumose solution and a cover glass placed on the top in such a way as to exclude all air bubbles. This solution is placed under the low power of the microscope. A drop of "fixing" or precipitating solution, as for instance, an aqueous 0.2 per cent picric acid is then allowed to run into the albumose solution. A shell shaped zone of precipitation is thus formed. In this precipitation a radial striation appears within about ten minutes, as shown in Figure 23. Not only radial striation but also a cellular or honey-comb structure is formed in such a precipitation of albumose or other protein split products. With a slowly acting precipitant the radial striation usually develops first, later a honeycomb structure appears allover the albumose solution and grows until it covers up the striation.

FIG. 23. SUBSEQUENT STAGES OF THE DEVELOPMENT OF STRUCTURES ARISING IN A PROTEOSE SOLUTION PRECIPITATED BY FIXING AGENTS

The figure presents subsequent stages of the same precipitated mass developing at the tip of the capillary.

Honeycomb structures can also be obtained from genuine proteins such as a 10 per cent gelatin jelly or concentrated egg albumin if milder or dilute precipitating agents are used. Thus chromic acid must be diluted down to about 0.5 per cent when acting on 10 per cent gelatin, while a 5 per cent chromic acid produces nothing but shapeless masses. In contrast to the forms produced from split products (proteoses) concentrated proteins usually-yield honeycomb structures directly without an initial formation of a radial striation. Moreover, a structure is formed in gelatin merely by allowing it to set to a geJ.2 Also such colloids as gums, starch, other proteins, or

2 The question whether structures pre-exist in a gelatin gel or whether they are formed by the precipitating agent in every case, has been debated on the ground of ultramicroscopic observations, but, it is doubtful whether this


various rosins, or colloidal silicic acid, when precipitated either by slow evaporation from their solutions or suitably mild reagents, will form such structures. Such structures have been demonstrated chiefly through the work of Butschli,3 who has been successful in the difficult application of microphotography although many of these objects are on the very limit of visibility.' (see Fig. 24). The medium size of the single meshes can be estimated at about 0.7 JL (= 0.0007 mm.). The size of these structures is therefore very much smaller than those described by Alfred Fischer.

Under the influence of mechanical forces definite arrangements arise in the uniform pattern of gel structures described. If gelatin gel is stretched the structural elements will arrange themselves in a linear fashion (see Fig. 25). In this way a seemingly fibrillar structure arises, the fibrils being arranged in parallel order in the direction of the stress. Considering this observation, the striated structure of tendons and aponeuroses appears as a result of the stretching to which these structures are subjected, while formerly it has been regarded by some as a purposeful arrangement tending to increase the tensile strength. Incidentally we may add that the formation of cancellous tissue in the bones-along the stress lines-should be looked upon from a similar viewpoint, although in this case a compression occurs.5

method is more reliable than Blitschli's microscopic studies which lead to the assumption of pre-existing structures.

3 O. Blitschli, "Untersuchungen liber Strukturen," Leipzig, 1898. • Biitschli's microphotographs have been published in a separate "Atlas"

zu den Untersuchungen liber Strukturen, Leipzig, 1898. 5 In the bones, the osseous material is not distributed evenly but collected

in a net work of thin osseous sheets, the so-called cancellous tissue. The arrangement of this net work coincides exactly with the lines of greatest compression under the given load or the so-called stress lines in the bone.

A story is told of an engineer who happened to see a drawing of this cancellous tissue in the femur. He was at once impressed with the resemblance of these lines to stress lines. Later he made a sketch of the landmarks of the femur and calculated the stress lines in it according to mechanical principles taking into account the incidence of the load according to the anatomical relations. In other words, he devised an internal structure, resembling a crane, suitable to meet the given stresses. On comparing this constructed system of lines and planes with the distribution. of the cancellous tissues in bones a complete agreement was found.

Subsequently extensive investigations have been made upon the finer archi-


One should expect that a radial striation appears in a gelatin gel if the ::;tretching force acts in a radiating fashion. Indeed this occurs if small air bubbles are includ('d in the coagulated gelatin, since on cooling down the air contracts, constricting the layers around it (see Fig. 26).

2. COXFIGURATIONS RESE 1SLY TGTH~ KARYOKINETIC FIGUHES

None of the artificial forms, dCficribed so far, can be matchpd with the diversity and complexity of microt-;copic structures observed in most living ti8sues. Yet a further development of such studies leads to the production of configurations which refiemble certain features of karyokinetic figures of cell division, at kaRt in part. Rew light is thus thrown upon this most important. cyi ological phenomenon.

As is well known, the mitotic or karyokinetic cell division is the prevailing "type although not the exclusive one. The unique picture presented by the dividing nucleus is met wit h in plants and animals alike. :Many attempts have been made to determine the physical nature of the forc s by which these striking intracellular movements and configurations arise. Mechanical, electrical or magnetic forces have been assumed. As shown by cataphoretic measurements, the chromatic substance, which is the chief constituent of the nucleus, has a negative electrical charge. The cytopInsmic colloids, on the other hand, arc found to have a positive charge (ll. S. Lillie, 1903).(> This seems to indicate that forces of electrical attraction and repulsion arc acting in karyokinesis. In order to eluciuate 1 he action of !;lIch attracting and repelling forces, R. S. Lillie constructed models of floating magnet. which were acted upon by a magnetic field. 7 In this manner it was possible to produce movements of the magnets in such a manner t,hat they took up positions giving the whole arrangement a remote rcsemblance to karyokinetic figures. But this

tccture of the hone. It has heen shown that thc struct\lre is perfectly in accordance with mechanical principles involved in the. trellS and strain to which the bone is sU\'jectcd. See J. Wolf, "Gesetz der Trlln~formation der Knochen," Berlin, 1892. J. C. Koch, "Laws of Bone Architecture," Am. Journ. Anatomy, :U, 117, 1917.

6 Biolog. BulL, 8, 19:1 . 7 Am. Journ. Physiol., 8, 4, (1905).

Microscopic and Macroscopic Structures Which Are Formed in Colloids without the Intervention of Osmotic Forces

)0'1('>, 24 FlO. 25

1"1(1, 24, HO]'.;E)('OM B HTltITTlIHf: IN I<;"AJ'ORATED SHfJr.r • .'$'

AC'('D!'dinl!; to HlLtsehli, "Atlas xu den 1llltersu('hUllgen iilwl' HtrllktnT<'Il," Leip"i/.!:, LHH5 (Table X, ::I), lfi I.!; 11 ifi catioll 1380,

Aftpl' evaporl1tion 30 pel' cl"nt aleohol \\':\., added. This ('auser( :1 sl"w Pl'(,('\ p~la tin\1 le,,,linj!; to the fOTm'ltiol1 <l r t h" Kt ru<'tUTC, 8i mi ltu s I.UC'llll"('f< ,',Ill

\Jf' nhtailll'fl from proteins or "thel' ('ol.loid".

FlO , 2;3. 1'.~I~AI.LEL HTRIA'l'ION 1N A HTI'lI';'I'(,111'~D TnREAD OF 50 1" ';1'( CI.;,'1 '1' UJ'; J.ATIN

:\ I if' 1"0 photograph ll('eonling to Bli tSI' 11 I i (Atlas, XV, 10). 1\ I agn i fic LLtion 7$0, T1H' )!;elntin ,~'lLS dl'i<ed, then imh\bed wiLh Wllt('l' ,,[I,d tl'Ctlted with Q,a per ('('nt

"hl'omie :I<'i<l i a lengthwise and [l, f;laTLtinl!; ""O,;:;WI>;(' Htriation is vi"ihlc.

FHi,27

FIG. 26. R .\O IAL ~'!'JUATION AROUND AN AlIt Humn,'('; IN A THH'K GELATlN C1GI,

.\IirroJlhf)lol!:r~'11h :lc'('ording to BiHschli (At.lafl, 1, 9) , ]\Iag;nincation 179. The I!:eltttin \\':l~ c;pread un " eover 11;1ass and treaLed with 0.:1 per pent ehromi,' "C' irl ,

(0'[(1, 27, Two A .ln HIII<IILI'~" 1V1'['HJ:' l'll('f'toH,'OP]( ' l)["'[,AN('['~ I'IUlLH"'I:\G A

"'HPINDLE';- BET1-VEEX ~rHe."f

A('['mdin):( "D Bi'tl,:;I' !lli (ALia,"", I, 7). )'bgnifi<'\\tinn 4.;",,0 . l' l'odu['ed in ('OIwr'ntT':,t",1 gelatin coagulated "y n,:l IlI'r I'('nl l'hromi(' n<'id,

The: hla"k ,.,,,,,t,,,, ""'1'(' in the' photngraphi<" pLttle'.

Fi~n ... (':s 24, 2.3, ~w :LIHl ~7 ar',' n."p .... ndllr·{'-d ... ,-jth ~p{'{"l~l p("rrlll:«"';'II~n (If I h,., [.(,1'-:-1 of thp bt.> P rOrl·S!-l.( ll· J.;_ut::-wl.li.

Microscopic and Macroscopic Structures Which Are Formed in Colloids without the Intervention of OsmotiC Forces

L Uradual llevelopmf'l1t of a r:tdinl st,-i:Ltion

2. Replacement or r,"dinl striation h.v 11 honeycomb like' configuration

YI(~. 28. COXFJ(H RAT10IVS AI!lSlNG CONSEC'I-TTV1<;l"Y IN A DIT"UTE DI{OP OF Is!"." INK PLAC"f]O IX A CONt:ENTHATli)i) HOLUTWN 0]<' [(X0 3

'\('l'or<lil1l1: tn LeduC"s tei'lllliqllC'; from an artiel~ b~' R. Beutner and \[. 1:'111""<', Zpit"phnft [til' cxpC'rirnent('lle \[e'dizin , Volf<. ::\2, 90 (192:1). Hep!"odllr'N.I "itb permi~si[JIl of.T. Hprinl(<'r, Bpl'lin


a

Hpiremc formed f !"om nucl('rtr

gr:h1llde,;

b

Chromosomes coller>ting in cqua

t!II'c,,1 pl:tte

t'

ChrmnmHlme dr:J.wn to ()pposite ('eotrosOInes

,[

Formation of two new lluelci

1"1 G. 2f1. CONSE('llTI VE ~TAGES 01" EA!! l'OK1."'i'ETH' FrGU ln~S DIA(;HA~DI ~TH' \i.["y

A"COROI"Q TO ~1]('I{(,s('nl'lC ORHER.YATIO~"

a

Formation of "pirenw

b

P:HLide" "olle('(,lJlg in E'qlluto

ria.! pInto

('

1':1I'tielc8 drlL" 11 to oppm;itc

POI('H

II

Forma t iOll of t"·,, drops (' [Irrespn 11 d

ing to two nudei

Two drop" of enn('entnlteu >mlt solutions lin eithet, sidC' of ,L drop of India illk in dilutc :salt O\olutiolL. Tlw figUIP shows the eon~('"utivn stagf''' \\' l1i('h develop :lutomati(':dl~',

fr'l(;, 30. CON"BCL'TI\,I~ STAGE>; ()~. D~~V~: I"OPIl1EN'l' OF RTH1'('TFln;" RE"t;\IRr.I"G TIlE CHHUMATH' FIGl ' ln~ OF KAIl.YOh.INE"I" !:<'OllMEll PHO\I [-';1)1 \ INK

From AtE'phune L('duc, "La Biologic SynthetiqIJC." 1)llhli~hl'r, A, Poinat, Paris, 1 !)12,

:-..; atuml siz('


:Fffl. 31. FIRl<.:R"; GROW r~G FRO!>! LIPOID

Origina\ pbotl)~l·aflh. l'\l1:;,gn ificCltion 1: 500. Til(' lipoid,.; llxIc'd in this ('R'P rue ether extracts of whole hrain, ,;tninc,l with

rhrHlamine. (Rpprocluction of photogra.ph trckclI by Dr. S H. l\Ianll.)

{;-':cC t(>--"'::f on p;_tge 117)


resem blancc was satisfactory in some respects only, in other respects it was quite unsatisfactory. No more than some incidental features of karyokinetic figures can thus be imitated.

It seems desirable to search for a model which imitates more closely the conditions existing in a cell, preferably by means of microscopic structures, such as those photographed by Biitschli. This has been possible to a limited ex.'tent only; an imitation is possible so far merely for the so-called karyokinetic "asters" and "spindles" (see Fig. 29). Radial striation around an air bubble in gelatin, as already described (Fig. 26), may be compared to an "aster." If two air bubbles happen to be within microscopic distance of each other, the two sets of radial striations around them interfere with each other and in this manner produce a "spindle" (sec Fig. 27). But, "spindle" and" aster" seem to be merely incidental, since the aster fails to appear in many dividing plant and animal cells. The more important part of karyokinesis is the so-called "chromatic figure" which includes all the varyin~ configurations which the chromatin material of the nucleus undergoes during cell division.

As is well known, the first of these changes is that the nuclear membrane disappears, and that the chromatin granules of the nucleus arrange themselves in the form of a long coiled band, the so-called spireme (see Fig. 29a). This has been described in some cases. The spireme thread then breaks up into a number of short lengths, known as the chromosomes (see Fig. 2gb). Meanwhile the "spindle" between the centrosomcs has been well developed, and the chromosomes "go on the spindle" grouping themselves in a so-called equatorial plate across its widest part (see Fig. 29c). Each chromosome separates lengthwise into two parts. Each of the two groups of split chromosomes migrates finally to opposite poles of the spindle to form two new nuclei (see Fig. 29d). (Turn back for Fig. 29.)

It would seem desirable to imitate this chromatic figure also by microscopic figures of the kind already described. So far this has not yet been done, but, we know of a macroscopic technique by which similar movements and configurations can be produced in an aqueous solution (St. LedUC). S This macroscopic technique makes use of the spontaneous grouping of diminutive soot particles which are contained

. in so-called "India ink," a colloidal suspension of soot. The simplest experiment of this kind consists in placing a drop of India ink

8 "La Biologic Synthetique," Paris, 1912.


upon a thin stratum of a concentrated salt solution (preferably a solution of potassium nitrate).9 The drop of ink appears as a compact disk, at first. A few minutes later it spreads out, while salt ;;olution streams into the drop. The soot particles arrange themselves automatically so as to form a radial striation, resembling an "aster." After about ten minutes this "aster" gradually disappears, and another configuration sets in; viz., the formation of a honeycomb (see Fig. 28). This arrangement does not last much longer than the previous stages, the final condition being a diffuse distribution of the soot particles over a wider area. This sequence of rapidly changing primitive forms corresponds to the microscopic pictures observed by Alfred Fischer in precipitated albumoses.

The macroscopic technique is, of course, simpler and permits one to recognize the physical causes of the phenomena to some extent. Experiments have shown that in the place of India ink various other colloidal solutions or fine suspensions can be used to obtain such structures, as e.g., milk, blood, colloidal dye, colloidal silver or other metals, etc. However, neither colored solutions, e.g., potassium permanganate, nor coarse suspensions, e.g., mastix, are adaptable to this purpose (Beutner and Busse, 1922).10 Consequently the phenomena cannot be explained as the results of diffusion only-as attempted by Leduc. Particles must be present, the size of which must not exceed a certain magnitude since coarse suspensions show no configurations.

It seems that a slow aggregation of the colloidal particles occurs, just as a slow precipitation is essential for microscopic structures as already stated. Fluid streaming plays a rOle also. Further experiments have shown that the chemical nature of the solution used is by no means unimportant. Thus, e. g., sodium salts and chlorides do not yield configurations as good as potassium nitrate (R. Beutner and M. Busse). This seems to be an indication that molecular forces also playa role in the formation of these configurations.

Now in order to produce configurations similar to those of karyokinesis, by this technique, the following procedure has been described by St. Leduc.8 A dilute potassium nitrate solution is spread in a thin layer on a glass plate which floats on mercury, as described above. A drop of India ink which also contains potassium nitrate at a slightly lower concentration is placed on this layer. Owing to the slight difference of concentration aster formation fails to occur. On either side of this drop,

9 The solution may be spread out on a petri dish; in order to protect the dish against the disturbing effects of shaking, it must be kept floating on mercury.

10 Zeitschrift fur die gesamte experim. Medizin, 32, 90.


two other drops are then placed which· contain a considerably higher concentration of salt and very little India ink. When this has been done, the following phenomena can be observed to develop automatically.

The black particles of soot, at first arrange themselves as striations, taking the appearance of a smooth band or "spireme" (see Fig. 30a). This "spireme" breaks up into shorter lengths which collect in the equatorial plane or move towards the two concentrated spots taking the appearance of a fan (see Fig. 30b) similarly to the "chromosomes." The particles collected in the equatorial plane are then pulled toward the two concentrated spots in a fashion similar to the chromosomes in their movement toward the centrosomes -(see Fig. 30c). If this last movement is completed, the black particles collect entirely in the two concentrated spots and merge, thus taking the appearance of two nuclei (see Fig. 30d) (Leduc). 8 (Turn back for Figs. 30a, b, c, d.)

A n approximate imitation of the chromatic figure of karyokinesis is obtained by this experiment as an entirely automatic process. If its mechanism is similar to that acting in karyokinesis, we may assume that the cause of cell division is the formation of two spots of a concentration different from that of the rest of the cell perhaps through the action of enzymes. If two such spots of different molecular pressure arise anywhere in the cell a karyokinetic movement of chromosomes should occur as the chromosomes are water insoluble like the soot particles with which the above described experiment is performed. In this way it is possible to throw some light on the dynamics of karyokinesis. Considering that the simple "asters" and "spindles" can be imitated both by a microscopic and a macroscopic technique, it ought to be possible to find also a microscopic technique which allows one to imitate the "chromatic" configurations.

3. VARIOUS MICROSCOPIC CONFIGURATIONS IN GELS

At the free margin of microscopic .honeycomb structures we may observe a differentiation resembling epithelium (0. Blitschli).u An artificial cellulose membrane, e.g., forms by evaporating a solution of cellulose in copperoxide ammonia on a water bath. This membrane, on cross section and after suitable staining, shows a fine "epi-

11 "Untersuchungen iiber Strukturen," Leipzig, 1898.


theloid" differentiation of even greater intricacy. The outermost layer of the membrane is formed by a dense highly refracting edge which resembles a pellicle. Parallel to this lie three alveolar layers, each consisting of a single layer of meshes, the outermost of which is most distinct. The appearance of these structures changes to some extent with the staining method used. The structure of this artificial cellulose membrane is similar to that of cellulose membranes of plants, according to Btitschli.u It is likely that these marginal structures are produced by the one sided pull of surface forces acting at the free margin.

A greater variety of forms is observed if crystallization takes place in the honeycomb structures formed from colloidal substances. As will be shown later, the amorphous state is not peculiar to any definite type of substances. Consequently not only regular colloids such as proteins, are capable of producing honeycomb structures, but eventually such low molecular substances as salts and sugars which are seen usually in the form of well defined crystals (Btitschli).ll Evidently low molecular substances like salts, exist in an amorphous state under certain conditions.12 A kind of transition to the regular crystallization frequently occurs in these "pseudo-colloids." The honeycomb structure which was originally lacking any further organization, begins to form stratifications under the influence of crystallization, or parallel or radial striations, as though mechanical forces were at play, but the picture is much more diversified. Fibrillary structures, sometimes with many contours, ramifications, leaflike structures, oval shapes, a seemingly endless variety of forms can be seen. Many of these forms rather resemble inorganic crytsals but they give us the impression that here we have encountered a force by which we might produce forms of a truly vital complexity provided that the details of its mode of action were better understood.

Among these various crystalline forms are also spherical ones, socalled "globulites"-to be prepared preferably by slow evaporation of a solution of arrow root starch, according to Btitschli. These globulites have been compared to cells, some of them show even a remote resemblance to ·nucleated cells since there is a central portion, the ·honeycomb structure of which stands out against the more striated

12 V. Weimarn emphasizes that any substance before crystallization occurs exists in the colloidal state (see page 135).


peripheral portion. But, it is hardly possible to draw conclusions as to the make-up of living cells from this remote similarity.

It may be added that the artificial production of structures resembling real living cells has been attempted also by various other methods, but so far this great problem remains largely unsolved. The so-called <lartificial" or <lsynthetic" cells, described in the literature, are, in most cases, either droplets of fats, oils or other water immiscible substances or they are mechanically produced fragments obtained by pulverizing protein and allowing it to swell in acid. (Compare A. L. Herrera, 1910-20;13 E. Newton Harvey, 1912/4 G. W. Crile and collaborators, 1931, and some others.!")

4. FILAMENTS DEVELOPING FROM LIPOIDS

Peculiar fibrillar structures develop from certain lipoids, for instance, from the soft substance (myelin sheath) which encloses the axis cylinder of nerves-after it has been killed and fixed in formalin (R. Virchow, 1854).16 Similar filaments grow from lipoids extracted from the brain (see Figure 31), or from lecithin obtained from other sources, also from various other substances, as for instance, soap, cresol, or ammonium oleate (0. Lehmann, 190817). (Fig.Sl is opposite to page 113.)

As seen from the microphotograph (Fig. 31) the growing fibrils show a double contour. It is seen also that some of them may detach to form floating globules. Sometimes they twist around each other exhibiting diversified configurations. In other cases, their tips appear to rotate.

The physical nature of these myelin filaments is quite different from those similar looking ones which are formed by osmotic forces as already described. The myelin filaments are not tubules con-

13 See article on Plasmogeny in Alexander's "Colloidal Chemistry." Some of these "cells" are oil drops performing "ameboid" movements as described on page 169 ff.

14 Science, 36, 930. These "cells" are lecithin drops coated with protein. They imitate certain features of sea urchin eggs.

15 Protoplasma, 1932. These "cells" are different from those described previously on account of enzymatic properties. They exhibit a "model respiration," as explained on pages 178-180.

16 Virchow's Archiv., 6, 571. 17 Ergebnisse der Physiologie, 16, 255.


taining an aqueous phase separated from the outer solution by a membrane. On the contrary, they consist of lipoidal matter throughout, being entirely immiscible in the surrounding aqueous medium, as demonstrated by microdissection. I8 Consequently osmotic forces cannot be the cause of the growth of these filaments. This is also demonstrated by the fact that they grow equally well in a concentrated sugar solution where osmotic structures fail to develop as already mentioned (see above .page 14).

The myelin filaments do not develop, however, in concentrated solutions of certain salts; this is not due to an osmotic influence, but probably to the lowering of the electric potential difference or charge of the lipoid. a It has been found that a lipoid exhibits growth only in those solutions in which it carries a charge as measured by migration of lipoid droplets in an electric field. Addition of salt depresses this charge, thus prevents both emulsification of the lipoids and the development of filaments.

Other factors responsible for this growth are forces of molecular attraction and molecular orientation, as is demonstrated by the double refraction of the more transparent outer layer which surrounds the filaments (0. Lehmann).2o This clear outer layer probably consists of symmetrically grouped lipoid molecules while the central portion is ordinary lipoid; viz., with the molecules arranged at random (see below, page 139).

18 The writer wishes to acknowledge with appreciation that Dr. R. Chambers, who performed microdissection on myelin filaments, informed him of this finding.

1~ Private communication from Dr. M. Telkes of Cleveland. 20 Ergebnisse der Physiologie, 16, 255.

DERIVATION OF PHYSICOCHEMICAL LAWS AND SOME APPLICATIONS: THE CRYSTALLINE CHARACTER OF COLLOIDS AS THE CAUSE OF THEIR FORM PRODUCING POWER; SURFACE FORCES AND ADSORPTION AS THE RESULT OF MOLECULAR ORIENTATION

1. THE MICELLAR THEORY (NAEGELI, 1858)

A problem of great biological importance is the cause of the inherent form producing power of colloids as manifested in living organisms. We have seen that even inanimate colloidal matter tends to produce primitive structures. Can we analyze the physical causes for this tendency? Can we form at least a vague conception of the mechanism by which such formative forces may give rise to the complicated forms that we find in the living world?

A priori, it would seem that a relation of some sort must exist between the growth of a crystal and that of a living thing. The determination of the nature of this relation ought to be looked upon as one of the greatest tasks. For a long time, however, research towards this end has been handicapped by the pre-conceived idea that only such substances were crystalline in which crystals were directly visible. This erroneous view was abandoned in the last decade only, although Carl Naegeli pointed out as early as 18581 that many colloids, particularly some of the constituents of living matter, are probably built up of diminutive crystalline units, the so-called micellae. . (These micellae may also be looked upon as molecular aggregates.) To quote one of Naegeli's earliest statements which outlines his "micellar theory:" "An organized substance which is permeable to water is somehow analogous to a crystal as far as its internal structure is concerned. Its smallest particles (micellae) are surrounded by water. . . . . "2

Naegeli defines as "organized" substances various materials which

I Carl Naegeli, "Die Starke Korner," .Zurich, 1858. 2 The existence of aqueous layers around the micellae was assumed by N ae

geli because some specimens of starch, when saturated with water, take up less, others more water. Almost any saturation point is possible. The assumption that water is contained in starch as a solid solution is, therefore, impossible. For further evidence see Appendix, page 299.

119


are produced by vital growth, particularly cellulose fibers or other fibers of any description, also starch globules, or a variety of membranes or of granules.

According to this concept an aqueous fluid is contained in the interstices between the crystalline submicrosopic "micellae." This aqueous fluid is probably a solution containing dissolved substances from which the micellae might grow by taking up dissolved molecules just as sugar crystals take up sugar molecules from a solution of sugar. The growth of a crystal and of a micellar substance-and possibly also that of living tissue-would be, therefore, essentially similar in type; a difference existing in that micellar substances or living tissues do not grow by apposition but by interposition. The crystalline surfaces would be scattered over an immense area on the surface of diminutive crystals, viz., the micellae. All the material which is added to the growing substance would have to penetrate into the intermicellar spaces first. In addition chemical reactions may occur at the micellar surface since they occur at the cell surface (see page 152).

Some indication for the crystalline character of the micellae has been found by optical methods. Substances like cellulose fibers, starch or cell membranes of plants or animals exhibit a double refraction (Naegeli, 1862).3 As is well known, double refraction, or optical anisotropy, occurs in the limespar, for instance, owing to the presence of elongated crystalline parts held in equi-ciistant parallel position by the molecular forces of attraction which build up the crystal. The conclusion, therefore, seems to be that the micellae of cellulose or starch are likewise crystalline and anisotropic.

Double refraction may also arise under the action of mechanical stress in substances which certainly are amorphous, such as glass. However, the double refraction of cellulose fibers or membranes is entirely different in nature. It cannot possibly be the direct effect of internal tension since it remains almost unaffected by mechanical stress. Evidently the anisotropic crystalline micellae are freely movable against each other, being held together merely by the water layers between them. Any stress exerted upon the substance as a whole merely leads to a mutual displacement of the single micellae against each other, but hardly to a distortion of the micellae themselves.

3 Sitzungsberichte Akad. der Wissenschaften, Miinchen, 4, 290.


From these observations the existence of crystalline units as constituents of organized substances appears rather likely. Thus, one of the artificially erected walls of separation between living and dead matter seems to fall to the ground. An antiquated teaching in biological sciences has been, however, that the growth of a crystal, as a typical inorganic process, bears no similarity to the growth of living things, this latter being due to obscure "vital" forces. It is only natural, therefore, that every possible effort has been made to disprove the arguments which indicate the crystallinity of products formed by vital growth. Every possible effort was made to prove that double refraction can also be produced by amorphous particles under the given conditions. .

In fact another type of double refraction can occur in a non-crystalline substance if this is built up of regularly striated layers and if these layers have a different index of refraction, or di-electric constant, like water and oil. The same occurs if elongated particles, such as the micellae, are arranged in a regular fashion with the intermicellar fluid between them. If the double refraction of cellulose fibers, membranes or other "organized" material was due to an effect of this type exclusively it could be explained by a difference of the refractive indices of the substance of the micellae and the fluid between them, but the micellae themselves need not be anisotropic crystals.

It can be shown, however, that this type of double refraction cannot be the exclusive cause of the double refraction of "organized" material. If it were, double refraction should disappear if the aqueous fluid between the micellae is replaced by another fluid which has the same index of refraction as the substance of the micella itself. This, however, is not the case, as is demonstrated by imbibing organized substances with suitable fluids other than water, having the same refractive index as the fiber. Experiments of this type have been performed with cellulose or nitrocellulose (Ambronn, 1916),4 with fibroin of silk, with the double refracting substance of smooth muscle, or with various cell membranes. The result was that the larger portion of the double refraction observed-which for cellulose, for instance, is seven times as large as for quartz-can be due only to the crystallinity of the micellae themselves.5

4 Kolloid Zeitschrift, 18, 90 and 273, 20, 173. 6 In some cases, the double refraction due to a striation acts in a direction


Thus we see that systematic investigations, stimulated by criticism, have served to corroborate the micellar theory. All this was insufficient, however, to overcome at once the prejudice of certain biologists who were unwilling to admit that crystallization had any relation to vital growth. New objections were raised in attempts to explain double refraction as the result of stress in the process of growth (Ebner, 1882),6 A weary polemic arose by which the erroneous impression was produced that the micellar theory was essentially unsound. Even though Ebner's sophisticated conclusions were disproved later CR. Ambronn) 7 the micellar theory fell into oblivion for half a century. The details of this discussion need not be reviewed since a definite proof for the micellar theory has been offered finally by means of the diffraction of Roentgen rays.

2. ROENTGEN RAY DIFFRACTION AS A MEANS OF PROVING THE

MICELLAR THEORY

As is well known, the study of Roentgen ray diffraction, instituted by v. Laue in 1912,8 has established a new experimental basis for the study of the crystalline state. Ever since this discovery, the tracing of Roentgen ray diffraction is considered as indispensable for a definite demonstration of the existence of crystalline units. The final proof for the micellar theory has been possible only by the application of this method. In fact a diffraction of Roentgen rays on fibrous or organized substances has been observed by methods briefly outlined on the following pages. The micellar theory has thus become an accepted idea.

The diffraction of Roentgen rays is based on the foll.owing well understood facts. Visible light is deflected when passing through fine openings. Owing to the mutual interference of the light rays,

opposite to the crystalline double refraction. In this case, the total double refraction observed will be increased by suitable imbibition. In some cases also-while acting in either direction-it is larget than the douhle refraction due to crystals.

6 "Untersuchungen liber Anisotropie," Leipzig, 1882. 7 Ann. der Physik, 38, 159. B For a summarizing review see Laue's report in Jahrbuch f. Radioakt. und

Elektronik, 11, 308 (1914). The discovery of Roentgen ray diffraction was made in collaboration with Knipping and Friederich.


which pass through narrow openings, alternating dark and light zones appear, as well as a spectrum, since light rays of varying wave lengths are deflected to varying degrees. Roentgen rays have a wave length of about 1 O.~ 0 0 or less of that of visible light. Manifestly they would require an opening which is 1 O.~ 0 0 of the width of a slit suitable for diffracting ordinary light, or rather, a grating containing such slits. It is very difficult to produce artificial openings of such diminutive size with sufficient accuracy, but, nature itself presents us with a grating in space, capable of deflecting Roentgen rays. These are the crystals. A crystal consists of a network of single atoms held in place rigidly by molecular forces, while empty spaces are between the atoms. The width of these spaces is from 1 to -t-u- J.lJ.I, which is just about the right size for a deflection of Roentgen rays. Therefore, when a ray of Roentgen light passes through a crystal, a number of symmetrical light spots and dark places should originate which are distributed symmetrically around the main ray in the center owing to mutual interference of the deflected Roentgen light. Laue and his collaborators have shown by experiments that this actually occurs.

A difference exists between any artificially made lattice used for diffraction of ordinary light and the natural lattice of crystals in that the former extend in one plane only, the latter, however, represent lattices in space. A lattice in space could be obtained by placing a large number of punctiform lattices behind each other, in such a manner that their distance equals the latticular constant. Manifestly an artificial product of such a type is technically impossible. The crystals, however, furnish lattices of such spacing. The diffracting action of a lattice in space is naturally more complicated than that of a plane lattice. For a complete mathematical analysis of this action, the reader is referred to textbooks of physics. It may be stated that in Laue's experiment, Roentgen rays of various wave lengths are sent into the crystal; the single diffracted rays, however, which pass it, are light of one particular wave length each. This is to be expected according'to the optical theory.

A disadvantage of this original method is, however, that it requires a crystal of sufficient size to be manipulated; it cannot be used for tracing diminutive crystalline units, such as the micellae. For this end another method has been devised in which the Roentgen rays do not pass through the crystals but are reflected from the surface. Roentgen rays of one particular wave length must be used in this case (Debye and Scherrer, 1916).9

9 Physik. Zeitschrift, 17, 277. Strictly monochromatic Roentgen ray light which would be desirable for these experiments can hardly be obtained. A


The following consideration may help to explain how a Roentgen ray of one particular wave length is reflected from the surface of a crystal. The approaching ray will first strike the uppermost layer of the crystal (see diagram, Fig. 32) from where a part of it is reflected under the same angle. Another part passes on until it hits the second layer from where again a part is reflected. Still another part is reflected from the third layer, etc., although the lower layers evidently participate less and less in the reflection. The reflected light, therefore, consists of at least two distinct parallel rays, which travel at such short distances that their waves interfere with each

FIG. 32. DIAGRAM ILLUSTRATING THE MUTUAL INTERFERENCE OF RAYS REFLECTED FROM NEIGHBORING CRYSTALLINE PLANES

This diagram shows that the difference in the pathway of the rays reflected from the first and the second crystalline plane equals 2d·sin a where d is the distance of the two IJlanes, and a the angle under which the approaching ray strikes the plane. Extinction occurs whenever this difference of pathway equals the wave length. Since this wave length is known and the angle a can be measured, the distance between the crystalline planes can be calculated.

other. Both rays travel over different paths. If the difference of their paths amounts to one, two or more entire wave lengths exactly, the light waves of these two rays will become superimposed upon each other, and hence the two rays will become summated. Under all other conditions, however, the waves will interfere wIth each other

Roentgen ray light of a limited range of waves is characteristic of the metal used as anti-cathode. To obtain these characteristic rays the voltage in the tube must exceed a certain limit, for instance, for copper as anti-cathode, in order to obtain the typical "K-lines" the voltage has to exceed 10,000 volts. (See Scherrer in Zsigmondy's "Colloid Chemistry," 3rd edition, Leipzig, 1920, p. 387ff).


which,leads to an extinction of the ray. The difference of the pathways of the two rays manifestly depends upon the angle between the ray and the crystalline planes as well as upon the distance between the two planes (see Fig. 32).

Now the reflection method is operated in such a manner that a powder of the crystalline substance in question, pressed into a cylindrical body, 1 cm. long and 1 mm. wide, is used (Debye and Scherrer).9 This cylinder is placed in the center of a circular photographic film as shown in Figure 33. A Roentgen ray passes in the direction of the arrow; ,a part of it is reflected by those crystalline surfaces which are in a suitable position. yielding a cone of light which produces a round black zone when hitting the film. (One cone of light only is shown on the diagram in order to simplify the picture; in reality a number of cones are formed in most cases.)

Fro. 33. DIAGRAM ILLUSTRATING THE FORMATION OF A CONE OF RAYS REFLECTED FROM CRYSTALLINE FRAGMENTS A~RANGED AT RANDOM

Technique of Debye and Scherrer. Reflection can occur unde'r certain angles only. Under other angles the rays are extinguished by interference.

The fragments of crystals which reflect the rays present a multitude of surfaces arranged in all possible directions. Some of them are in a suitable direction for the two reflected Roentgen rays from neighbouring planes to become superimposed. Hence in this particular direction one cone of reflected light will be formed. Planes oriented under another angle will extinguish the rays by interference, hence in this direction no reflection will occur. The appearance of distinct circular bands is, therefore, the criterion for the existence of crystalline particles. Even the minutest crystalline fragments can be traced and determined in that way. It can be proven, e.g., that the particles contained in a colloidal gold solution, the size of which is about 2 to 5fJ.J.L,


consist of gold crystals. These particles consist of a limited number of gold atoms only; yet even these few atoms are held rigidly at distances as in the case of crystals. However, if a fluid or an amorphous substance is used in the device no bands at all appear, but, just a m;tiform darkening of the film or sometimes very broad indefinite bands .

(

" -..

........

/ .. ,_--" FIG. 34. EXAMPLE OF INTERFERENCE BANDS OBTAINED FROM A MICELLAJt

SUBSTANCE

\ " ! i ~

{: , j I .

FIG. 35. EXAMPLE OF INTERFERENCE BANDS OBTAINED FROM A COARSE CRYSTALLINE POWDER

• l) ) FIG. 36. EXAMPLE OF INTERFERENCE BANDS OBTAINED FROM A FINE CRYSTAL-

LINE POWDER ..

By an application of the method described to such structures as cellulose fibers or other fibrous material, crystalline units are demonstrated through the appearance of definite bands (see Fig. 34). It is seen, how-


ever, that no continuous bands appear, but, rather interrupted lines resembling dots at some places. In order to understand this feature, we sh'ould compare the bands obtained from fine and from coarse powder. If a micro-crystalline powder is used in the above described meth9d of Debye and Scherrer, the bands appear as perfectly smooth distinct lines. If the powder is coarser, the bands not only become less dIstinct but also acquire an irregular punctiform appearance (see Figs. 35 and 36). The reason for this change is apparently as follows:! In the fine powder, the individual crystals are small. Hence so mJtny of them are present that a sufficiently large number of them are found in every possible place all around the compressed powder, in sJlch a way as to allow the formation of smooth lines. In a coarser powder, the number of individual crystals being smaller, suitably oriented planes are missing in certain places. Consequently, the cone of diffracted light fails to appear in some locations.

In the case of elongated regularly arranged crystallites, such as are found in micellar substances, the punctiform character of the bands is still more pronounced. Moreover, it is symmetrical since numerous orientations are now missing in a definitely arranged fashion (M. Polanyi, 1927).10 (See Fig. 34.)

3. THE MOLECULAR MAKE-UP OF MICELLAR SUBSTANCES, TAKING

AS AN EXAMPLE CELLULOSE FIBERS; THE DEFINITION OF

"MAIN VALENCE CHAINS"l1

The Roentgen ray analysis of crystalline chemical compounds leads to a determination of the location of the single atoms in the lattice. Concerning the qua:r;ttitative details of this analysis the reader is referred to textbooks of physical chemistry. Suffice it to say that a crystal of such a simple compound, as NaCI, consists of a cubic grating in which Na+ and CI- ions occupy alternate positions. In this case, the individuality of the molecule disappears; it is impossible to determine, for a given Na+ ion to which of all the surrounding CI- ions it belongs. The entire NaCI crystal may be regarded as one giant

10 For a summary of this work see Zeitschrift d. Vereins Deutscher Ingen., 71,565.

11 See K. H. Meyer and H. Mark, "Aufbau der hochpolymeren N aturstofie." Leipzig (1920).


molecule. In the crystals of more complicated compounds, however, particularly in all crystals of organic compounds, the entire molecule enters into the lattice as a part of it. In other words, these organic molecules pass unchanged from the solution into the crystal where they are held in the rigid lattice. Weak forces of secondary valence hold the molecules of organic compounds together, which accounts for their softness and their low melting point. An analysis of the crystals of organic compounds has led to a determination of the position not only of the molecules but of the single atoms as well. From an extensive series of such measurements, pursued systematically, the conclusion can be drawn that the atoms invariably occupy a definite space whatever their position may be. If the atoms are pictured as spheres, the diameter of a carbon atom in aliphatic compounds would be about 0.1551L1L; the diameter of a =C=O bond would be O.llJj/J; that of =C-C1 0.19JjJj.

In the same manner the molecular arrangement of submicroscopic crystals, such as the micellae, can be analyzed. Difficulties arise in this case on account of the large size of the molecular units from which micellar substances are composed, but, by application of the experience, gained from studies on lower molecular substances, and by further chemical and spectroscopic studies, it is possible to form a conception concerning the atomic make-up of certain micellae. An investigation of this kind is best started with a micellar compound of comparatively simple chemical composition, as an example of which we may select: cellulose. Chemical analysis has shown that cellulose is built up from cellobiose, a disaccharide. This in turn is built up of two glucose residues, which are rings of five carbon atoms and one oxygen atom.. The molecular make-up of glucose is shown in Figure 37, which shows the five carbon ring. In cellobiose such a ring is joined to another ring of the same kind which is rotated through 180°. The bond between these two rings is an oxygen bridge between the first and the fourth carbon atom. The molecular model of cellobiose is, therefore, such as shown in Figure 38. The model shows that the length of its molecule amounts to l.031-1/J. Chemically cellobiose is a glucose-/3-glucoside, which is quite similar to maltose or glucose-a-glucoside (Meyer and Mark).ll

It remains to determine how these cellobiose or sugar molecules are linked up to form cellulose. If we resort to Roentgen ray analysis to solve this question the result is rather puzzling since there is nothing


eli

o ::: 011

FIG. 37. THE MAKE-UP OF A GLUCOSE MOLECULE ACCORDING TO K. H. MEYERll

8=CH ~=Obridge

O=OH

FIG. 38. THE MAKE-UP OF A CELLOBIOSE MOLECULE ACCORDING TO K. H. MEYERll

The dimensions are given in Angstrom units (1 Angst. unit = 1~ f.l.JJ.)

to indicate, according to the appearance of the diffraction bands, that larger molecular units are present. Roentgen ray analysis shows that cellulose contains periodically repeated identical units, but, the size of these units is not larger than that of a single sugar molecule. In


fact it is found by various methods to amount to 1.02 to 1.03pp. In other words, it agrees within the experimental errors with the space occupied by the sugar molecule (cellobiose) from which cellulose is built up. Yet, according to all our chemical and physical knowledge of such a substance as cellulose, it must be built of larger molecular aggregates (considering the splitting by Hel, the water-insolubility of cellulose and other properties).

The Roentgen ray finding in the case of fibrous substances of a protein character, e.g., silk fibroin, is still more difficult to explain since the unit determined is so small in this case that it offers room for no more than 2 to 3 amino acids while more should be in it according to the chemical findings.

The cube indicates the unit shown by X-ray. The main valence chains extend beyond this.

This symbol represents a cellobiose molecule which is the elementary component of the main valence chain of cellulose.

FIG. 39. THE MAKE-UP OF A BUNDLE OF MAIN VALENCE CHAINS OF CELLULOSE, ACCORDING TO K. H. MEYERlI

It is likely, therefore, that the units, indicated by Roentgen ray diffraction, are/no more than a small portion of the large size molecular units existing in cellulose. According to K. H. Meyer,ll the assumption is justified that in cellulose these smaller units, which are the cellobiose radicals, are bound together by oxygen bridges in one line, thus forming the so-called "main valence chain" which extends in the direction of the fiber as indicated diagramatically in Figure 39. This assumption is made in order to account for the tensile strength of


the fiber. The main valences, arranged in line in the direction of the fiber, would be almost untearable. However, vertically to the fiber, in which direction it can be torn much more easily, weak or side valences are assumed (see diagram, Fig. 39).

An entire chain, held together by main valence bonds, would correspond to a molecule chemically. However, the length of the main valence is assumed not to be uniform even if it is composed of one type of units only, as in the case of cellulose, since the micellae which compose a definite substance are likewise never uniform, as stated above. The average length of tl}ese main valence chains may be about ten times the length of cellobiose, viz., about 10j.t,u.

The expression "molecule" is usually defined as the particle which exerts an osmotic pressure in solution. In the case of high molecular compounds the osmotically acting particles are the micellae which are still larger than the main valence chain. It is advisable, therefore, to discard entirely the confusing expression of "molecule" in the case of high molecular compounds (K. H. Meyer).ll

It should be borne in mind that neither the micellae nor the main valence chains are uniform in length.

FIG. 40. VISUALIZATION OF THE GROUPING OF THE MAIN VALENCE CHAINS IN THE MICELLAE

The main valence chains are supposed to extend in the direction of the cellulose fiber, but they cannot extend clear through the fiber to any considerable extent, since the primary unit of the fiber is the micella which is surrounded by an aqueous fluid for the reasons described above. The main valenc~ chain can extend, therefore, merely within one micella. Fifty to one hundred closely packed main valence chains are bundled together to form a micella, which in the case of cellulose is 15 to 50j.tj.t long .and from 2 to 5j.tj.t thick. A diagrammatic visualization of the structure of the cellulose fiber would be, therefore, such as is given in Figure 40 (K. H. Meyer).ll


4. MAIN VALENCE CHAINS IN MICELLAR STRUCTURES OF PROTEIN

NATURE

Cellulose is built up of one type of comparatively simple units: the glucose radicals. The combination of chemical and physical methods of investigation has led in this case to rather definite assumptions. There are, however, numerous other micellar or fibrous materials which also exhibit Roentgen ray diffraction: hairs, tendons, the ligamentum nuchae and various other ligaments, certain muscles, the fibers of silk, chitin (viz., the substance which makes up the shell of arthropods and mushrooms), furthermore stretched rubber, and other materials.

The micellae of these substances probably contain main valence chains, similar in type to those in cel1ulose, but different chemically in that most of them are proteins. They are built up therefore from amino acid radicals as units in the place of glucose radicals. Since the chemistry of proteins is less completely understood, we cannot form quite as definite conceptions in these cases. Silk is the best investigated example of a fib,rous substance with main valence chains of protein character. It consists of an amorphous ground substance into which crystalline components are embedded. The crystalline part amounts to 50 per cent of the whole and contains micella~ which are made up of bundles of long polypeptid chains (K. H. Meyer). The amino acids in these chains are alanine and glYCine, the uniting bridge between the single links of the chain is:-CO-NH-. This bridge takes the place of the oxygen bridge of cellulose. As stated. the main valence chains are never of uniform length, even if they are composed of identical links, as in cellulose. However, if the links are of varying nature, as in silk fibroin, then the main valence chains differ from each other not only in size but also in composition. It is useless, therefore, to search for a "chemical formula of silk fibroin" or any other protein or to attempt to determine its "molecular" size from the percentage of that split product which is contained in it to the least amount. Uniform or "chemically pure" proteins are practi.cally impossible (K. H. Meyer).l1

According to K. H. Meyer's calculations, the main valence chain in silk is 100 times longer than it is thick. It would represent a diminutive thread, probably very flexible since it is made up entirely of flexible single bonds and without rings, such as cellulose. It would


be almost untearable on account of the firm cohesion of the main valence bonds, but capable of undergoing innumerable chemical changes through reactions of its side chains. With many reactions the main chain remains intact. 12 The main valence chains of other proteins are probably similar.

The substance of living matter is chiefly composed of such units as these or others which are less completely known. It seems that science has made the first step, in our day, towards the realization of an old dream: viz., to determine how the complicated form of living organisms is built up from atoms linked together according to a definite order so as to form macroscopic structures. But, of course, it is hardly more than a first step. We cannot determine yet the detailed make-up of the different main valence chains of proteins. How these chains combine to micellae and the micellae in turn to cell-like structures, are problems which are little understood at present.

The Roentgen ray method is certainly very useful, as we have seen, to demonstrate regular molecular arrangements where other methods fail. Peculiarly enough, however, certain proteins like egg albumin, hemoglobin and others which can be obtained as distinct large size crystals, fail to produce bands in Roentgen light, showing that certain regular arrangements of molecules or main valence chains must be possible which fail to diffract Roentgen rays. It would, therefore, appear that the method is of limited application.

The cause of this failure is not completely understood. It may be stated, however, that protein crystals differ from crystals of lower molecular substances in that they are capable of swelling or shrinking. Simultaneously marked changes of the crystallographic characteristics occur, viz., the angles between the planes are continuously changed according to the water content. Hence these crystals are built up differently than ordinary crystals, probably from micellae with aqueous intermicellar spaces between them, capable of taking up water which leads to a distortion of the crystal.

12 Out of the numerous other substances of which the main valence chain has been studied, rubber is of interest. Roentgen ray diffraction bands appear only when rubber is stretched. K. H. Meyer assumes that in this case, the main valence chains coil up like a spiral which should account for the elasticity. A theory of muscular contraction is based on a similar spiral coiling up of main valence chains, but, this still needs further corroboration.


5. CONDITIONS WHICH DETERMINE THE SIZE OF CRYSTALS OR THE

FORMATION OF MIC:ELLAE

To throw further light upon the relationship between growth and crystallization, it is useful to determine the conditions under which the formation of crystalline micellae or crystals in general may occur. Evidently the time element is important, A cellulose or a silk fiber grows slowly. One molecule after the other is added, sufficient time being granted for each molecule to occupy its position in an orderly arranged main valence chain. Now, it is well known that artificial fibers can be produced, as for instance, those of artificial silk or "rayon," but, in this case the process is much cruder. It consists of a rapid coagulation of a nitrocellulose solution, the fiber being produced by pressing the nitrocellulose solution through narrow openings into the coagulating fluid. Evidently no time is available in such a process for the micellae to take up oriented positions. In fact we find no indication of a regular arrangement; no Roentgen ray bands appear in the light passing through such artificial fibers.

Moreover, it is quite a general experience that crystals grow the larger, the more time is allowed for the transition from the fluid to the solid state. If this transition is enforced with extreme rapidity no crystals appear at all. Even such a readily crystallizable substance as sodium chloride can be obtained as an amorphous mass, if the formation of solid NaCI is induced with extreme rapidity. This can be done, for instance, by mixing solutions of NaOH and of HCI, both being dissolved in a mixture of amylalcohol and ether. NaCI is practically insoluble in amylalcohol + ether. It is formed from the soluble NaOH and HCl immediately following the mixing and precipitated as a white cheesy mass resembling the AgCl precipitate from soluble silver salts and chlorides (P. P. v. Weimarn).13 This rapidly precipitated, amorphous looking N aCI has all the properties of the amorphous or "colloidal" precipitations well known in inorganic chemistry. The "crystalline" or the amorphous character is, therefore, not a specific property of a given substance, but, depends on the rapidity of formation of that substance under the existing circumstances. By suitably varying the conditions, any substance can be obtained either in crystalloid or in amorphous form (P. P. von Weimarn).13 This has been demonstrated for about. 200 substances.

13 P. P. von Weimarn, "Grundzuge der Dispersoid Chemie," Dresden, 1911.


As an outstanding example we may quote von Weimarn's experiments on BaSO. which can be precipitated by mixing solutions of sulphates and of barium salts in any desired form. The form in which BaSO. appears depends on the concentration of the sulphate and of the barium salt solutions which are mixed, to form BaSO., and hence on the velocity of precipitation. The more dilute these solutions, the more time is allowed for the formation of BaSO., and hence the larger the crystals. At extremely high concentrations -viz., 7 times molecular-an amorphous jelly is formed but no crystals not even submicroscopic ones. By mixing sulphate and barium salt solutions at a suitable dilation, viz., at a concentration of about m7J to lO.~OO molecular, BaSO. can also be obtained in the form of microscopic crystals.

It is noteworthy that these diminutive crystals may form a honey comb structure resembling that obtained from gelatin or other colloids, as already described (see Fig. 41). This seems to indicate that simple visible structures are built up from small crystals or probably from micellae.

FIG. 41. ALVEOLAR STRUCTURE FORMED FROM DIMINUTIVE BaSO. CRYSTALS, ACCORDING TO P. P. V. WEIMARN12

This is a secondary structure resembling Biitschli's gelatin structures

The rule that any substance can be obtained in crystalline or in amorphous form suffers no exception, not even for such material as gelatin, the prototype of colloids. Von Weimarn has obtained diminutive crystals by mixing a dilute gelatin solution with an excess of alcohol. The crystals, thus obtained, are barely visible under the high power of the microscope. These experiments have led v. Weimarnl3 to the above mentioned assumption that any substance, before crystallizing, passes through an amorphous or as he terms it, "colloidal" state although for readily soluble substances this condition is so transitory that it cannot be observed.

That the appearance of crystallinity depends on the velocity of the formation of a substance, can be demonstrated also by Roentgen ray analysis. Amorphous precipitations which have formed rapidly show no bands, e.g., amorphous beryllium hydroxide, if precipitated by NH 3• But the same hydroxide, if precipitated slowly by heating a soluble beryllium salt with NH 3, shows distinct bands.u

14 Quoted according to H. Freundlich, "KapiIIarchemie," 2nd ed., p. 457.


6. FLUID CRYSTALS: DOUBLE REFRACTION IN STREAMING AND

RESTING COLLOIDAL SOLUTIONS; SPONTANEOUS FORMATION

OF STRUCTURES IN COLLOIDAL SOLUTIONS

All the experiments described tend to demonstrate transitions between crystallinity and amorphous state. Transitions exist likewise between the fluid and the crystalline state: the so-called "liquid crystals." The existence of these seems plausible if we realize that the hardness of crystals can vary within the widest limits. Crystals of certain soaps, such as ammonium oleate, are so soft that they can actually flow together. Nevertheless, they do not form round drops under the influence of surface tension, but appear as distinct crystalline needles. Moreover, in polarized light their crystalline nature is revealed by optical anisotropy.

However, another type of crystalline fluid has been observed which forms perfectly rounded droplets and yet shows signs of optical anisotropy. A substance of this type is p-azoxyanisol; this forms solid crystals at room temperature which melt at 116° to form a fluid which appears entirely turbid and cannot be cleared by filtration or sedimentation. Upon further heating, however, this fluid becomes clear at 135.2°. Another substance of this type is the ethylester of p-azoxybromocinnamonic acid. It forms a turbid fluid within the range of 139° to 247°. Many other substances exhibit phenomena of this kind. On examining droplets of these turbid fluids (within the temperature ranges given) in polarized light, they present different pictures when viewed from different angles, in spite of their completely round shape (see Fig. 42) (0. Lehmann, 1890).15

In order to explain this, the following assumption is made: while in ordinary fluids the molecules are arranged at random, they are arranged in -these peculiar turbid fluids in circular layers around a central axis as indicated diagrammatically in Figure 43. Within these layers the molecules are freely movable, and hence the mechanical properties are those of a fluid. But, to account for their double refraction, we assume that the molecules are oriented III one direction in a manner comparable to lead pencils which are packed in a box in such a way that their tips point either to the left or to the right (F. Haber, 1926).158 Thus, this fluid crystalline condition represents

15 "Neue Welt der fiiissigen Krystalle," Leipzig, IMl. 15a Naturwissenschaften, 14, 849.


a transition between crystallinity and fluidity. Hence the term "mesomorphous" (middle shaped) has been proposed for it.

A. Appearance of drop of p-azoxyanizol viewed in polarized light in the

direction of the optical axis

B. Appearance of drop of p·azoxyanizol in'polarized light vertically to the

direction of the axis

FIG. 42. ApPEARANCE OF LIQUID CRYSTALS AND THEIR MOLECULAR MAKE-UP; ACCORDING TO O. LEHMANN

A. Grouping around the optical axis B. Grouping in a plane passing through the optical axis

FIG. 43. DIAGRAMS TO ILLUSTRATE THE HYPOTHETICAL GROUPING OF THE MOLECULAR AGGREGATES

The particles of some 'colloidal solutions are also anisotropic crystalline aggregates but they fail to show double refraction since they move around freely in the solution hence are arranged at random. One should expect, however, that if such a solution streams in a narrow tube some elongated particles will be forced to take up parallel positions. Hence the fluid should be rendered doubly refractive by streaming. As a matter of fact, double refraction can be observed in certain colloidal solutions, on al~owing them to stream. One such solution is that of vanadin pentoxyde (Freundlich and Disselhorst, 1915).16

Double refraction also persists for some time after the streaming has ceased in such a solution. Moreover, in certain concentrated

16 Physik. Zeitschrift. 16, 413.


colloidal solutions, as for instance, colloidal solutions of certain dyes such as benzopurpurin and chrysophenin, double refraction appears spontaneously in the following manner. When a hot 2 per cent solution of either one of these dyes is allowed to cool down, it will set to a jelly. On microscopic observation, this jelly is seen to contain a large number of leaf or spindle shaped bodies which exhibit double refraction and resemble microscopic crystals at first glance. However, a closer examination by more refined optical methods shows that they are not crystals, but, that they are built up of colloidal particles of the dye, which are arranged in oriented layers parallel to the longitudinal axis with aqueous layers between them, and hence similar to micellae (R. Zocher, 1925).17 These microscopic bodies are also somewhat similar to the liquid crystals, but are different from them on account of the aqueous fluid contained between the oriented particles while, in molten azoxyanisol between 116° and 135°C., the "intermicellar" fluid is the molten azoxyanisol itself.

Similar aggregates of oriented colloidal particles are formed also from a colloidal solution of iron hydroxide. In this case they settle to the bottom, forming there an optically anisotropic jelly (H. Zocher)_17 The colloidal solution of iron hydroxide contains particles which are disc shaped and tend to arrange themselves one upon another, like coins in a coin roll. The formation of aggregates of such a "coin roll" type can be observed according to Zocherl7 for instance by the following method. An ordinary colloidal solution of iron hydroxide is mixed with some of its optically anisotropic sediment. This mixture shows no double refraction at first. Soon, however, a process remotely resembling crystallization sets in, leading to the formation of "coin roll" aggregates. These structures have been investigated by means of optical methods (R. Zocher).l7 On account of their brilliant green opalescence they must contain particles the distance of which in the "coin roll" arrangement equals a multiple of one-half of the wave length of green light. This shows that these structures consist of particles which are held by forces of molecular attraction at equal distances, with water layers between them. In other words, they have just that type of organization, which is characteristic of "organized" substances, according to the micellar theory. This is also confirmed by other optical methods, worked out

17 Zeitschrift anorg. Chemie., 147, 93. "Freiwillige ("spontaneous") Strukturbildung in Solen."


.by Zocher, all the details of which cannot be described here. The formation of such "micellar" structures from colloidal solutions in vitro is an observation of considerable biological interest.

It seems likely that also many of the other primitive structures which arise in colloids, as already described, are formed by molecular forces in a similar manner. For the myelin filaments developing from lipoids this assumption is supported by the observation that their clear peripheral layer shows double refraction (0. Lehmann, 1908).18 It also seems probable that many structures in vivo are thus formed. Asfar as this is true, we may state that the living forms wtth their definite shape may be looked upon as products of a process which resembles crystallization, yet does not lead to the formation of ordinary crystals but to micellar aggregates as described.

7. POLAR ADSORPTION AS DUE TO CHEMICAL ATTRACTION

At the junction of two immiscible substances certain molecules are held in oriented positions by forces of chemical attraction similar to those acting in crystalline or micellar compounds. This chemical attraction by surfaces is known as adsorption. Since living tissue is inhomogeneous throughout, adsorption is liable to occur on many of its diversified surfaces or "phase junctions." The physicochemical rules governing adsorption are, therefore, bound to be of some biological significance.

In order to observe adsorption, a powdered solid substance, as for instance charcoal, may be shaken with a solution. The well known observation is then made that almost all dissolved substances are taken out of solution by the charcoal-some of them completely, as for instance dyes, colloids and "surface active" substances, others to a slight extent, such as ordinary salts or sugars. The carbon particles take up the dissolved molecules and hold them at their surface; there is no appreciable penetration into the carbon. This is indicated by the observations on "adsorptive replacement," which show that an adsorbed substance will be crowded out by another one, particularly by one which is better adsorbed. Striking experiments demonstrating adsorptive replacement can be performed with dyes. Almost any dye is adsorbed by charcoal (a process which is frequently used

18 Ergebnisse der Physiologie, 16, 255.


for decolorizing sugar syrup or other solutions). In many cases the adsorbed dye is held so fast by the charcoal that no visible trace is given off to the water. However, if the charcoal holding the dye is shaken with a dilute solution of any substance which lowers surface tension, the adsorbed dye is liberated again and the water appears stained. The feature of adsorptive replacement, as demonstrated by such tests, is the small amount of the surface active substance capable of liberating the adsorbed dye. This would not be possible in the case of absorption by a solvent in bulk.

That the attraction of the molecules to the adsorbing surface is due to chemical forces is difficult to prove in the case of charcoal since many diversified substances are adsorbed (although we may attempt to account for this adsorption on the basis of the well known fact that the carbon atom can enter into a chemical combination with almost any other atom, even those with antagonistic properties like H+ and Cl-). In general, chemical attraction is, of course, specific. Hence, in order to show that adsorption is caused by chemical affinity, we should trace a specific action for adsorption, at least in some cases. In order to obtain clear cut results, it is desirable to use, instead of charcoal, well defined, chemically known adsorbing powders and to. investigate their power of adsorption for various dissolved substances. This has been done by H. Bechhold (1929),19 who selected the following four bicyclic compounds:

1. Naphthalene ....... . 2. ~-N aphtho!.. ........ .

3. ~-Naphthylamine .... . 4. Amido naphthol. ... , .

C1oH 7NH2

ClOH6NH20H

A neutral hydrocarbon A substance with an acidic group

ing A substance with a basic grouping An amphoteric substance with

both a basic and an acidic grouping

Each of these insoluble substances, in the form of a finely divided powder, was shaken with aqueous solutions of various basic or acid dyes, for several minutes, then filtered and washed with water until the filtrate became colorless. An appreciable adsorption of dye, as seen from the coloration of the powder after washing, was then observed in those cases only where a chemical comb,ination was pos-

19 H. Bechhold, "Kolloide in der Biologie," 5th edition, Leipzig, 1929.


sible. Thus naphthalene failed to adsorb any dye, since after washing, it became nearly colorless. I3-Naphthol, an acidic adsorbent, appeared intensively colored after shaking with methylene blue, carbolfuchsin and crystal-violet, all of which are basic dyes. Acidic dyes were not adsorbed. The reverse was observed for napbthylaminc, a base, which adsorbed acidic dyes like eosin, aurantia or picric acid, but none of the basic dyes named. Finally amidonaphthol was seen to take up both basic and acidic dyes since it has both acidic and basic groupings.

It is manifest that in these experiments chemical combinations are the underlying cause of adsorption: the acidic substance adsorbs basic dyes, the basic substance acidic dyes from the solution. If naphthol or naphthylamine were used in liquid form (viz., above their melting point) they would take up the same dyes, however, by absorption with their bulk and not by surface adsorption. A solid powder, however, can bind only by means of the molecules in its outermost surface. Nevertheless the kind of combination is the same in both instances, viz., a chemical combination. The assumption seems justified that chemical surface reactions are the main factor causing adsorption also in those rather obscure cases as in adsorption on charcoal-although other unknown possibilities are difficult to exclude.

8. ApOLAR ADSORPTION AND SURFACE TENSION EFFECTS

The type of adsorption just described depends on mutual binding of acidic and basic groupings at surfaces. This is called "polar adsorption" as distinguished from apolar adsorption which depends on chemical or physical surface attraction of a different type, although in this case the same parallelism of adsorption and absorption can be observed. As an example of apolar adsorption we may mention the adsorption of higher alcohols to carbon (or a neutral adsorbent like napththalene). The higher alcohols are more oil-soluble and are accordingly the better adsorbed the longer their carbonic chains. Carbon or other neutral adsorbents act in this respect like a neutral oil; they attract the carbonic chains of the alcohols-the more powerfully the longer they are. A massive layer of oil, when agitated with a watery solution of an alcohol, would likewise attract the carbonic chains, but in this case the effect will gradually extend through the


entire bulk of the oil, while in adsorbing charcoal it remains limited to the surface.

Now, an apolar adsorption of this type occurs likewise at the free surface of a solution. This can be demonstrated by the fact that froth contains much higher amounts of an adsorb able substance than the bulk of the fluid. For instance, if a rapid stream of air is blown through an aqueous solution of amylalcohol and the froth thus formed carried over into another large container, where it is allowed to settle, the fluid in this second vessel will contain an excess of as much as 5 per cent of this alcohol (Benson, 1903).20 The higher alcohols are, in this case also, adsorbed more powerfully and completely.

This adsorption at the free surface of a fluid lowers markedly its surface tension, the degree of lowering being parallel to the adsorbability. Thus the higher homologues decrease surface tension much more than the lower ones. This decrease is so much larger, that for the same lowering of surface tension no more than one-third of a substance with one additional CRa group is needed as compared with the action of the preceding substance in the series. Measurements on homologous fatty acids may be quoted as an example: a 0.25 molecular solution of acetic acid, CHaC0 2H, will lower the surface tension of water from the normal level of 74 to about 69 dynes per centimeter. To bring about the same lowering no more than about 0.08 molecular propionic acid, C2H 5C0 2H, is needed, or as little as 0.02 to 0.03 molecular butyric acid, CaIl 7C0 2H (Rule of Traube 1891).21

As already mentioned surface tension can be measured at the free surface of a liquid against air by counting the number of drops formed from a given volume. Upon emerging froin the tip of the pipette (stalagmometer) each droplet grows as long as the weight is carried by the surface tension which acts like a rubber envelope around the drop. Since the weight grows more rapidly than the surface, the imaginary sheath around the drop will finally burst. Hence the smaller the drop the smaller has been the contracting tension.

Another method based on the same principle consists in measuring the force required to pull off a ring floating on the surface. This method offers the advantage of leaving the surface of the fluid to be mEl_asured undisturbed. It is thus possible to measure surface tension wbile the fluid is resting. But this method is not considered reliable by some writers and would seem to require further modification (see page 144). For further-modifications of the methods for surface tension measurement, see textbooks of physics.

20 Journal physic. Chem., 7, 532. 21 Liebig's Ann., 265, 27.


In general we find that adsorption of the apolar type and the concomitant lowering of surface tension are mamjested by those substances particularly which contain in their molecules groupings which tend to produce antagonistic solubility. Examples of such compounds are the higher alcohols. The OR grouping in their molecules tends to produce water solubility, the carbonic chains have the opposite effect. Other compounds with such antagonistic groupings are, for instance, the soaps in which the -C0 2Na grouping attracts water, the carbonic chains acting in the opposite sense. In fact soaps lower surface tension markedly.

Groupings which tend to produce water solubility may be assumed to be attracted by water. Thus for instance, in soap the -C0 2Na groupings are attracted by water while the carbonic chains are repelled by it. Hence, if soap molecules are adsorbed to the water surface, they must manifestly take up oriented positions due to attraction of one part of their molecules by the water and the repulsion of the other. This effect may be described as ';wlubility directed in space." It seems that the formation oj layers of oriented molecules is the cause of the lowering of surface tension. The oriented molecules cannot be equally well attracted to each other as water molecules, and hence the surface is contracted with less energy than a pure water surface.

Evidence for the existence of such an orientation of molecules can be obtained in superficial layers of fats or fatty acids if diminutive amounts of such substances are spread out on a water surface over the largest area which they can possibly cover (Langmuir, 1917).22 Oleic acid might be used for such experiments, also solid fatty acids, or various fats, or higher non-volatile alcohols. Anyone of these substances is dissolved in a suitable volatile fat solvent such as benzene. A droplet of this solution is then placed upon a water surface from which all dust particles have been carefully removed by wiping over it. In order to determine the maximal spreading, the water surface offered to the fat must be, of course, larger than the one which the fat can possibly cover. It is possible to determine to what extent the fat has spread by sprinkling talcum powder on the surface and gently blowing over it. Talcum will stick to the' surface which is covered with fat, even though it may be a monomolecular fat layer, but not to an absolutely clean water surface.

The thickness of the thinnest layer can thus be determined from the amount of fat applied and the area covered. For oleic acid, for

22 Journ. Am. Chern. Soc., 39, 1848.


instance, the result is 1.12,u,u. That this is actually the lengtp of the oleic acid molecule is demonstrated by the good agreement of this figure with that obtained through the work on Roentgen ray diffraction (see above, page 128). Langmuir has performed such experiments with a number of saturated higher fatty acids having carbonic chains of varying lengths. The thickness of the thinnest layers was then found to vary according to the length of their carbonic chains as is to be expected for monomolecular layers. Langmuir's assumption is that in these monomolecular layers the fatty acid molecules are held in a parallel position with their carboxyl groups attached to the water and the carbonic chains standing up vertically to the surface. This assumption is supported by the fact that paraffine hydrocarbons, which have no water attracting groups, cannot be spread out to monomolecular layers at all. They contract on the water surface to much thicker layers.

The attraction of a part of the molecule, by chemical affinity, to the water surface seems indispensable, therefore, for the existence of such monomolecular layers.23 In this respect these layers resemble adsorbed layers for which chemical attraction also plays an important role, as we have seen. This leads to the assumption that oriented molecules may also be contained in adsorbed layers. Certain experiments described by Lecomte du Nouy24 can be regarded as furnishing some evidence for this assumption although his experimental methods have been criticized by other observers.25 Du Nouy's experiments probably contain some element of truth, even for a highly critical student who may not feel inclined to accept all of his (du Nouy's) statements.

Du Nouy measures the surface tension of soap or protein solutions, after these solutions have been at rest for at least two hours. He uses the method of the adhering ring, already described, which however, his critics do not consider as quite reliable, possibly because of du Nouy's imperfect description of his method.

In one series of experiments the surface tension of extremely dilute solutions such as sodium oleate solutions 1: 700,000 to 1: 800,000 was measured. In spite of the great "surface activity" of sodium oleate,

23 Langmuir has offered much additional evidence for this assumption which cannot be described here.

%( P. Lecomte du Nouy, "Surface Equilibria of Colloids," New York, 1926. 25 Compare, e.g., J. M. Johlin, Journ. Gen. Physiol., 11, 301, 1928.


such solutions have almost the same surface tension a.s pure water, on account of their extreme dilution, as one might expect. Now, du Nouy24 describes the striking observation that at one definite concentration within the range mentioned, viz., at 1: 750,000, sodium oleate exhibits a marked lowering of surface tension. This is described to occur at 1:750,000 only, but not at 1:740,000 or 1:760,000, or other higher or lower concentrations.

This lowering of the surface tension of an extremely dilute solution at one concentration exclusively is explained by the assumption that the total number of molecules just suffices to form a monomolecular layer. "This is obviously only possible at one concentration, the other conditions being constant" (du Nouy).24 At higher concentrations, e.g., 1: 740,000, "the number of the molecules being too large, there is some crowding in places." . At lower concentrations, e.g., 1: 760,000, "on the contrary, the number of molecules is too small to cover the whole surface and yet keep their orientation. Channels are formed between the large molecular aggregates floating like ice on the lakes during a thaw. An absolute minimum of surface tension can exist only when all the molecules are in contact with one another and identically oriented. The surface of the liquid is then covered by a solid and homogeneous film" (du Nouy).24

Apparently the formation of a stable monomolecular layer does not depend on the concentlation of the colloid in the solution, but rather on the total amount present in relation to the surface to which it is adsorbed. This surface is both the free surface of the fluid and the surface in contact with the glass container. Consequently, we should expect that the same amount of the same sodium oleate dilution should show another minimum of the final surface tension in a container presenting a larger (or smaller) surface. In agreement with these considerations du N ouy24 described that the surface tension is considerably changed by placing glass beads in the container-all other conditions remaining unchanged. The typical minimal surface tension was then no longer at the same dilution but at another dilution which was considerably higher, since more sodium oleate was required for a monomolecular layer over this greater surface. Furthermore, on scrutinizing the surface tension of sodium oleate solution at other concentrations, du N ouy observed an outstanding lowering of surface tension also at a dilution of 1: 1,220,000 and at 1: 1,390,000, using in each case the same amount of solution (2 cc.) in the same container. Three different kinds of monomolecular layers of sodium oleate seem to be possible, therefore: the first minimum is due to the formation of a layer with vertical orientation of the molecules; the second, with their horizontal orientation, and the third, with a rotation of 90° of these molecules around their horizontal axis. Thus du NOUy24 is led to a calculation of the three dimensions of


space of the sodium oleate molecule. This molecule is calculated to be 1.21'1' long, O.75Jl.Ji. thick and O.65Jl.Jl. wide. The order of magnitude of these figures, agrees well with that of Langmuir's figures for oleic acid.

The validity of all these findings is questioned by the opponents of du Nouy who were unable to repeat the experimental observations produced by the alleged monomolecular layers. Du N ouy himself admits that he had to perform 8800 measurements in order to determine the described minima of surface tension. This shows that the formation of a monomolecular layer does not occur invariably at the given concentration and in the given container as described. It seems to be necessary to fulfill certain additional conditions, but the nature of these has not been distinctly recognized as yet. Hence a repetition of du Nouy's24 experiments has not been possible and criticism has been aroused. The conclusion is that layers oj oriented molecules may exist in adsorbed surface layers under certain conditions, but these conditions are not understood in all their detail, as yet. It also seems likely that the adsorbed layers at the junction of solid substances contain oriented molecules although no direct evidence is possible.

9. THE MORPHOGENETIC INFLUENCE OF ADSORPTION

The aggregation of crystals in the process of crystallization and the size of the crystals formed is markedly influenced if an adsorbed surface layer of a protein, soap or another colloid is allowed to settle on the growing crystals. Thus, for instance, a simple salt like NaCI, when crystallizing from about a 2 to 5 per cent gelatin solution, forms crystalline aggregates resembling leaf-like structures, as shown in Figure 44, while N aCl usually forms compact aggregates of larger crystals when crystallizing from a pure aqueous solution. As we have seen, living cells and other tissue constituents are likewise the product of an aggregation of diminutive crystals, viz., of micellae. It seems likely, therefore, that the morphology in vivo depends on adsorbed surface layers to a considerable extent. The resemblance to plant forms of the aggregate of NaCl crystals in gelatin seems certainly suggestive of such a possibility.

The dissolved colloids probably influence the aggregation of crystals by their adsorption to the surface of the growing crystal. In


Natural eize

The aggregate of NaCI crystals takes a leaf-like appearance

FIG. 44. SODIUM CHLORIDE CRYSTALLIZING FROM A COLLOIDAL SOLUTION

From Stephane Leduc, "La Biologie Synthetique." Publisher, A. Poinat, Parisr 1912.

Na Oleate added 1: 220000 1:221000 1:222000

The greatest spreading of the crystals occurs at that concentration at which sodium oleate forms a monomolecular layer.

Natural.ize

FIG. 45. SODIUM CHLORIDE CRYSTALLIZING FROM SLOWLY EVAPORATING SOLUTIONS, ON WATCHGLASSES, TO WHICH DIMINUTIVE TRACES OE' SODIUM

OLEATE HAVE BEEN ADDED

Photograph by Du Nouy. From du Nouy, "Surface Equilibria of Colloids" (Table XVII, p. 153). Publisher, Chemical Catalog Company, New York, 1926.


this way, they block the surface. Growth can only occur through the meshes or diminutive pores of .the colloidal envelope. This gives rise to numerous elongated crystals giving the whole a pinnate aspect Another factor to be considered is the adsorption on the free surface of the solution. A certain number of NaCl molecules are adsorbed on the colloidal molecules and follow them to the surface. Consequently small crystals are spread all over the surface and give rise to the peculiar aspect of the crystalline aggregate resembling plant forms. If diminutive amounts of an adsorbable substance are added to a solution in which crystals grow, we should expect that solutions with a monomolecular layer should have an outstanding power of spreading out crystallizing salts in the manner described above for more concentrated colloidal solutions. The illustration reproduced in Figure 45, shows that this is the case (from du Nouy).24 Sodium oleate in concentrations of 1 :220,-000, 1 :221,000 and 1 :222,000 in 0.9 per cent NaCl was allowed to evaporate on watch glasses. The monomolecular layer of oriented molecules occurred in this case at 1 :221,000. It is seen that the NaCl crystals are spread out more at this concentration than at the neighboring concentrations. These experiments indicate that an adsorbed layer of colloids is a morphogenetic factor.

It may be added that adsorption plays a r61e even in the delicate and obscure alterations of serum proteins following immunization (according to duNouy).24 The surface tension of blood serum was found to decrease slowly to a final value when left undisturbed. 26 However, if an antigen, viz., any foreign protein, is injected into an animal (rabbit) the surface tension of its serum decreases within two hours to a much greater degree than normal rabbit's serum. This is the only physical or chemical method, known so far, by which normal and immune serum can be discriminated. Manifestly the proteins of the immune serum are different from those of the normal serum although no chemical differentiation is possible. Yet, the adsorbed surface layers are built up differently, as shown, by surface tension measurements.

26 This occurs also for solutions of soap or other colloids as described.

APPLICATION OF PHYSICOCHEMICAL LAWS AND FURTHER EXPERIMENTS WITH MODELS: CELL RESPIRATION AS A CHEMICAL REACTION OCCURRING IN ADSORBED LAYERS; MOVEMENT DUE TO SURFACE FORCES AND OTHER MOLECULAR ACTIONS

1. WARBURG'S EXPERIMENTS ON RESPIRATWN ~

Adsorption plays an eminent part in the chemical reactions occurring in living tissue, particularly in respiration. The so-called cell respiration or combustion of organic matter to CO 2 and other substances, as it occurs in living tissue, is the most outstanding of all biochemical reactions. Itfurnishes heat and locomotor power for the living organism and probably some other forms of energy which are required. Ina general way, a living thing may be compared to a coal burning heat engine. H owever, in spite of numerous investigations, little was understood about the underlying mechanism until Warburg began a detailed study of the conditions governing respiration. In the development of his research he discovered artificial models which exhibit a respiration subject to the same conditions, depending on adsorption. Finally-by an ingeniou8 application of the most accurate physical methods, Warburg was able to identify in detail the p~ysical and chemical properties of the very ferment which controls respiration.

Even more important than W arburg' s findings are the results of Keilin, St. Gyorgy, and some other investigators who have revealed the chemical nature of the great variety of other reducible substances which playa r6le in cell respiration next to the respiratory ferment proper (compare pages 164 and 165). U1J,fortunately a detailed description of all this work cannot be given here, due to lack of space.

By cell respiration is meant the oxygen intake and the CO 2 output of living tissues; this is not only exhibited by most plants and animals but also by isolated animal organs, by cut off parts of plants, such as leaves and fruits, and likewise by tissue juices, extracts and cell suspensions. In physiology the term respiration is frequently used in a different sense, viz., as designating the mechanical intake of oxygen into the lungs. Nor is the loose combination of oxygen with hemoglobin a respiration proper in the sense defined above. This is just a

149


transporting of oxygen from the lungs to the body tissues where oxygen is again liberated to be used for combustion.

For a determination of cell respiration the following technique has been elaborated (Warburg, 1914).1 Three containers are used each of which has the peculiar shape shown in Figure 46. The total content of each of them, viz., fluid + gaseous space, is 15 cc. Each contains 1.5 cc. of respiring fluid in the

The flask is placed in a water bath and shaken

FIG. 46. ApPARATUS FOR· THE DETERMINATION OF CELL RESPIRATION

space c. In the first container the appendix a is empty; the central part b

contains 5 per cent KOH. In the second and in the third container, the appendix a contains an acid, -qsually 0.5 cc. 20 pet cent phosphoric acid, while the center b is empty.

Each container is then connected to a simple U-tube containing a fluid, serving as a manometer. The tubes are placed in II. water bath and shaken.

1 Zeitschrift pp.ysiol. Chemie, 92, 231.


The manometers are provided with a stop cock, d, opening to the outside atmosphere; this is kept open for the first ten minutes, viz., until each of the three vessels has equalized its temperature to that of the water bath. After closing the stop cocks the !lcid is poured over from a into c in the third container at once; CO 2 a priori dissolved in the fluid is thus liberated and can be determined from the small positive pressure produced on the manometer. The first and the second containers are kept in the water bath while shaken; respiration proceeds in each of them, leading to O2 consumption and CO 2 production. In the first container, due to the KOH in the center, CO 2 is absorbed, hence a negative pressure arises which increases gradually as the respiration progresses. In the second container the acid in a is poured over into c, at the end

of the experiment, thus liberating all the CO 2 produced into the gaseous space. The difference of the pressures in the first and the second container apparently corresponds to the CO 2 produced by respiration, if the CO 2 present a priori is subtracted as determined in the third container.2

2 The Redox Indicators. For an approximate estimation of the O2 consumption of living cells, the so-called redox indicators are also available. These substances are dyes which can be more or less easily reduced to colorless leucocompounds; they can be arranged in a series according to the readiness with which they are reducible; this "readiness" is accurately defined because the reduction and the oxidation are reversible for these "redox" dyes. This means that a given mixed solution of an oxidized dye and its leucocompound is in equilibrium with a definite O 2 concentration.

A certain low O 2 concentration corresponds also to a condition in which practically all of the dye is reduced so that the solution turns colorless; but for each redox indicator such a decoloration corresponds to a different O2 concentration. This makes possible an approximate estimation of the O2 consump-. tion by respiring cells. In a suspension of respiring cells the O2 concentration is, of course, decreased by the O 2 consumption due to respiration; on the other hand more O 2 diffuses in from the air.

Although these oxygen concentrations are exceedingly low, they can be determined by an electrometric method. The following is a list of some redox indicators giving the potential difference which characterizes them; this being measured against an H2 electrode in normal HCl, at 30°C.

volt Chloro indophenol (most easily reduced) . . . . . . . . . . . . . . . . . . .. +0.233 Indophenol. ............................................... +0.228 Methyl indophenol ........................................ +0.195 Methylene blue .................•.......................... +0.011 Indigo tetrasulfonate ................................ , ..... -0.046 Indigo disulfonate ...................................... , .. -0.125 Indigo monosulfonate ......................... , ............ -0.156 Neutral red (reduced with greatest difficulty) ........... about 0.3

To give an example of the application of these indicators: in a suspension of washed yeast cells methylene blue is not appreciably reduced,-even if


2. RESPIRATION AS A SURFACE REACTION; THE INHIBITION BY NAR

COTICS AND 'BY HeN

By means of this technique, the importance of cellular constituents for respiration can be demonstrated as follows. Red blood cells of. birds are used because they exhibit a distinct respiration.3 They are suspended in Ringer solution which is frozen rapidly. The delicate cell membranes are thus torn up. Upon thawing, a hemoglobin solution is obtained in which the disrupted membranous constituents are suspended. By centrifuging, the fluid can be divided into a transparent supernatant portion and a turbid portion containing the cell fragments. On measuring the respiration, the lower portion only is found to respire, showing that respiration depends entirely on solid cell constituents (0. Warburg, 1914).4

In order to demonstrate the importance of adsorption to solid cell constituents, the inhibition of respiration is investigated. Like all other life processes, respiration is interfered with and eventually arrested by narcotics which penetrate through membranes, according to their fat solubility (see above, page 40 ff.). Since absorption and adsorption are due to somewhat similar causes as demonstrated by Bechhold's experiments (page 140) the adsorbability is in general parallel to the oil solubility. Hence, if respiration is a surface reaction we should expect that more fat soluble compounds, with longer carbonic chains, interfere with the respiration to a greater extent. This is indeed found to be the case (Warburg, 1912),5 as for instance, in experiments with homologous substitution products of urethane:

the air is excluded; hence this dye preserves its blue color even though more powerful reducing agents thaI). yeast cells decolorize it.

Indophenol or dichloro-indophenol is reduced and decolorized more easily than methylene blue. In fact it is found that yeast cells decolorize them and maintain them colorless even if a vigorous stream of air is passed through the suspension. In most cell suspensions only the first, second, third or fourth indicators are reduced and decolorized. Following micro injection into cells (Amoeba) they are all reduced except the last one named (Cohen, Chambers and Reznikoff, 1928, Journ. Gen. Physiology, 11, 585).

8 Mammalian red blood cells have no respiration, viz., consume no oxygen, probably because they have no nucleus; following the addition of certain tissue extracts a respiration appears (Michaelis and. Salomon. 1929).

4 Zeitschrift physiol. Chemie, 92, 231. 6 Pfliigers Archiv, 144, 465.


NH2-COOC 2H6. Urethane is only weakly narcotic, but, by substitution of longer carbonic chains for the C 2H 5 group, more and more powerful narcotics are produced. Accordingly the inhibition of respiration was found to increase with the length of the carbonic chain of the narcotic added. This Wl;tS tested for various bacteria, yeast cells, sea urchin eggs, suspended liver cells, and other cells (Warburg, 1912).5

As already mentioned compounds with longer carbonic chains produce a more powerful adsorptive replacement, the rate of variation being the same as in respiratory inhibition. The analogy seems to indicate that the inhibition by narcotics is also an effect of adsorptive replacement. The combustible substances like sugar and aminoacids are replaced on the surface by inert and non-combustible narcotics. Consequently respiration is inhibited. By a more elaborate method a quantitative agreement between the inhibiting power of a narcotic and its absorption (or adsorption) by the cells has been demonstrated (Warburg).6

It must be emphasized, however, that the inhibition of respiration by these narcotics is partial only, and in some cases, very slight. To give an example: the respiration of fertilized eggs of echinoderms and other low animals is not arrested by amounts of narcotics great enough to stop completely the progress of the segmentation. Hence the inhibition of oxidation cannot be regarded as the cause of narcosis. .

In striking contrast to the inhibitory effects of narcotics is the much more powerful inhibition of such respiratory poisons as HCN, H 28, CO, N0 2 and some others. HeN is adsorbed to about the same degree as the weakest narcotics known. As estimated from the degree of adsorption a gram molecular HCN solution should just interfere slightly with the respiration. The experiment shows, however, that a 1 o.~ 0 0 molecular HeN interferes noticeably with respiration; HCN inhibits 10,000 times more than would correspond to its adsorption.

This powerful action is likely to be due to a specific chemical interaction with the iron compounds embedded on the cell surface which are responsible for respiration. In fact, it is a general chemical experience that HCN reacts with iron salts, forming complex compounds -in which iron is so firmly bound that no electrolytic dissociation occurs. Since diminutive traces of HeN suffice to inhibit respiration, it

B Warburg, "Katalytische Wirkungen der lebenden Substanz," Berlin, 1928.


appears likely that just a small fraction of the cell surface is covered with this active Fe. Thus, while the non-specific narcotics inhibit respiration by blocking the entire cell surface, HCN blocks only a small fraction consisting of iron compounds, by chemical interaction, 7

according to Warburg.

3. THE OXIDATION OF AMINO ACIDS ON CHARCOAL AS A MODEL OF

CELL RESPIRATION

It would be important to know whether any artificially made substances can be found which induce a respiration in a manner similar to cells, viz., a slow combustion of combustible substances in solution at ordinary temperature. This problem has been solved by O. Warburg in the following manner. By heating organic matter, preferably blood, to red heat, a charcoal is obtained which in contact with certain combustible substances in aqueous solution burns these to CO 2,

using the dissolved air as a source of oxygen. The iron contained in organic matter is not lost by charring. It is still effective for respiration, although somewhat altered in form. The oxidizable substances used -in these model experiments are amino acids, such as cystin, leucin, and tyrosin, or oxalic acid. All of these substances are fairly stable in aqueous solution in the presence of air. However, if blQod charcoal is added and the mixture agitated in the presence of air a combustion of the amino acids to CO 2, NHa (and H 2S04 in the case of cystin) takes place. These end products of combustion are the same as they occur in natural cell respiration. The velocity is also about the same. Charcoal in contact with a 0.0017 molecular cystin solution at 40°C. consumes about as much oxygen as the mammalian liver in vivo-viz., 84 cU.mm. O2 in twenty-one minutes in one given experiment (Warburg and Negelein).8 It is justifiable therefore to

7 All the statements, made so far, apply to the combustion of sugars and protein split products in living tissue. In regard to the oxidation of fats, entirely different conditions seem to prevail. For example, the oxidation of linseed oil is catalyzed by hemoglobin and other Fe containing compounds but this is not inhibited by HCN. For a more detailed discussion see Robinson, Biochem. Journ., 18, 255, (1924); also Kuhn and Meyer, Zeitschrift physiol. Chemie, 185, 193 (1929).



speak of a "respiration" or rather "model respiration" in the case of the charcoal. 9

The inhibition of the model respiration on charcoal occurs under the same conditions as on living cells, for instance, by the addition of narcotics. On comparing the inhibiting power of homologous narcotics, the same sequence is obtained as in the inhibition of living cell respiration. In this case it can be shown ·by quantitative measurements that adsorptive replacement accounts for the inhibition of respiration: "If we determine for various narcotics the adsorptive replacement on the one hand and the respiratory inhibition on the other, a satisfactory agreement is found" (Warburg, 1921).10

Hydrocyanic acid and its salts exert a specific inhibiting action on the model respiration of charcoal just as on cell respiration. The adsorption of HeN by charcoal is so slight that it cannot nearly account for its powerful inhibition of respiration. For an adsorptive replacement of 0.03 millimoles cystin from the surface of 1 gram of charcoal, a molecular solution of HeN (2.7 per cent) would be necessary. Yet for a 60 per cent ·inhibition of respiration a 1 0 ~ 0 0

molecular HeN solution is sufficient (Warburg, 1921). In the same manner HeN inhibits the oxidation of other amino acids. This small amount of HeN is of the same order of magnitude as the iron contained in the charcoal, suggesting that HeN blocks just the iron on the charcoal surface-occupying a small fraction only of the total surface, just as on living cells.

All the results described may be condensed into the statement that the similarity oj cell respiration and charcoal respiration points to an identical mechanism in both cases. Since charcoal respiration can be demonstrated to be a surJace reaction by quantitative measurement, the same should hold Jor tissue respiration.

Furthermore, the experiments with charcoal are accessible to a closer analysis. The "model respiration" of various types of charcoal, differing according to origin and composition, can be tested by the methods described, in order to determine the nature of the active

9 Other biologically important substances, particularly sugars, fatty acids and lactic acid are not oxidized by charcoal which shows that the model respiration is not identical with the respiration of living cells in every respect.



constituent on the surface of the adsorbing charcoal. Among the different kinds of charcoal, thus investigated by Warburg, we want to mention in particular one which is prepared by charring pure sugar while sodium silicate is added.ll This charcoal adsorbs amino acids as does blood charcoal, but, it does not bum them, since it is free from iron.12 One may expect that this inactive silicated charcoal should be activated by the addition of iron in some form. However, the addition of FeCl a or other soluble iron salts to the mixture of amino acids and suspended charcoal has no such effect. Likewise, the addition of iron salts in the process of making the charcoal from sugar by heating shows no influence. It takes something more to activate the silicated charcoal. Besides iron some nitrogen containing compound 'must be added while the charcoal is prepared, preferably hematin or hemoglobin. A small addition of this substance suffices to yield a charcoal with a considerable respiration, capable of inhibition by HeN. The most active charcoal is that obtained from pure hemin in a solution of amino acids. It has a respiration which is ten times as large as the respiration of commercial blood charcoal. Charcoal with a similarly large respiratory effect can also be obtained from other iron and nitrogen containing compounds as, e.g., from nitrogeneous organic dyes which contain traces of iron as impurities. "

4. THE INHIBITION OF RESPIRATION BY CO AND THE EFFECT OF

LIGHT ON THIS INHIBITION

Iron, bound to nitrogen and adsorbed to the surface, is the active catalytic agent of a charcoal with a respiratory activity as described. Further experiments indicate that the natural respiratory ferment likewise contains its iron bound to some other constituent since the addition of iron salts usually fails to increase cell respiration except in the case of a few special cells, as for instance, starfish eggs.1a Prob-

11 Warburg and Brefeld, Biochem. Zeitschrift, 146, 461. 12 Warburg has shown that there is also another type of charcoal which is free

from iron and yet shows "respiration" due to self oxidation. This coal is prepared from sugar without silicate or by extracting blood charcoal with HOI.

13 Addition of Fe up to 0.1 per cent of the weight of the eggs increases the respiration, further addition has no effect; F,e is added as ferro ammonium sulphate.


ably these eggs are about the only cells which contain an excess of that other constituent, but, its nature has remained undecided for a long time. The recent ingenious experiments of Warburg (1928) on the inhibition of respiration by CO and the influence of light upon it have nearly solved this question. His investigations have opened a new way of determining the nature of the ferment itself in the living cells as follows :14 CO is one of the substances with a specific inhibiting action on respiration just as HCN. It inhibits although it is only slightly adsorbed. Like HCN, it combines with the'iron of the ferment although the type of combination is different from that of HCN.15 However, this respiratory inhibition occurs in the dark only. If light shines on the CO gas mixture the respiration is inhibited to a lesser degree or not at all. It seems, therefore, that light breaks up the compound of the respiratory ferment and CO, allowing oxygen to combine with the ferment as before.

"This peculiar action of CO on cell respiration was first discovered in experiments with yeast. Later bacteria, plant seeds, liver, chorion, retina, embryos of chick and rat, tumors of rat, blood leukocytes and platelets were investigated. A~l of these different cells behaved in about the same manner towards CO" (Warburg, 1929).16

The action of monochromatic light of different wave lengths was th81u investigated and it was found that the different wave lengths, although all of them are active, show a different degree of activity. The ultraviolet line of 283JLJL wave length and the blue line of 436JLJL were found to have an outstanding action in preventing respiratory inhibition by CO, exceeding about ten to thirty times that of the other lines. Now, it is known that a photochemical action can come about only if light

14 See Warburg and Negelein, Biochem. Zeitschrift, 193, 334, 339. 15 Nevertheless the combination of the ferment with CO is not nearly so

strong as the combination of hemoglobin with CO. A few tenths of a percent of CO in the air suffice to form' a stable CO-hemoglobin and hence to check any combination of oxygen with hemoglobin so that the O 2 carrying capacity of the blood is completely abolished. In order to bring about a similar but much smaller effect on the respiratory ferment, viz., to diminish respiration, a gas mixture containing CO of nearly atmospheric pressure must be used. The respiration of cells in a gas mixture of 95 per cent CO + 5 per cent O2 is compared to that in a mixture of 95 per cent N 2 + 5 per cent O 2•

The cells in the CO mixture will then respire only about half as much as those in the other mixture.

16 Zeitschrift f. Electrochem., 35, 928.


is absorbed. If one wave length acts more than another it can do so only because of its higher absorption by the compound of CO and respiratory ferment. Bya comparison of photochemical activity we can obtain, therefore, a relative absorption spectrum of the CO compound of the ferment. This absorption spectrum exhibits a striking similarity to the spectra of natural (red) pigments, such as hemoglobin, which are accessible to direct determination. The conclusion is that the respiratory ferment must have a red color similar to that of hemoglobin,l7

However, the productiveness of the method is not yet exhausted. It can be further improved so as to determine the absolute spectrum also. Without giving the mathematical details the principle of the methods may be outlined as follows (Warburg, 1928).18 Let us suppose again that we deal with cells, the respiration of which is inhibited by CO. Following irradiation, the respiration does not rise to its maximal value at once, but, within a definite short time only, depending on the depth of the color of the ferment. Now, we can measure the time which it takes for a given light intensity to split up the CO ferment compound to a certain degree, say 50 per cent. From this time and from the magnitude of the acting light intensity the absolute coefficient of absorption of the ferment can be calculated because the more the .pigment absorbs light the faster the CO groups are split off as a result of the absorption.

"Perhaps the most brilliant part of Warburg's investigations consists in the overcoming of the serious difficulties involved in the measurements of the velocity constant of the reaction for the respiratory ferment. It is not feasible to measure accurately the rate at which respiration changes as CO groups are split off when the carbon monoxide derivation of the ferment is exposed to a definite intensity

17 Additional evidence in favor of the soundness of the procedure for obtaining the relative spectrum is given by experiments with known substances. The oxidation of cystein is catalyzed by nicotine hemochromogen and this catalysis is inhibited by carbon monoxide (Krebs, 1928). The spectrum of nicotine hemochromogen can therefore be determined in precisely the same manner as was the spectrum of the respiratory ferment by studying the effect of light on the CO inhibition. In this case, however, the spectrum may also be ascertained by direct physical measurements of light absorption. The spectra determined by the two independent methods agree perfectly (Warburg and Negelein, 1928, Biochem. Zeitschrift, 200, 414). .

18 Biochem. Zeitschrift, 202, 202.


of light. But, the difficult problem of measuring the changing respiration of yeast in a constant light has been reduced to the simpler problem of measuring the constant respiration of yeast in a changing light. When yeast whose respiration is inhibited by CO is exposed to light the rate of respiration rises until it reaches the value of the constant respiration in light. When the light is turned off the rate of respiration drops until it reaches the value of the constant respiration in the dark. The total respiration of the yeast in the whole period of oscillation of the light clearly depends on the value of the constant respiration in the dark and in the light (both of which can be measured separately) and on the absolute rates at which the CO groups combine with and become detached from the ferment molecule. Other things being equal the total respiration in a period of oscillation depends on the velocity constant of the reaction. The remarkable fact which has been demonstrated theoretically and experimentally is that when the light is made to oscillate faster and faster, a point is reached where the respiration is sensibly constant and not influenced by further increases in the rate of oscillation, and yet is still a function of the velocity constant of the reaction. It is from the value of this constant respiration in rapidly oscillating light that the velocity constant of the reaction and hence absolute absorption coefficients are obtained."19

For the most active wave length, 436,u,u, a value of 3.6 X 108 sq. cm. per gram atom was found by this method. This means that if a solution could be made, containing the ferment to an amount equivalent to one gram atom of iron per liter, a layer of two millionths of a centimeter would cut down the intensity of light of 436,u,u wave length to the extent of 50 per cent. The light absorbing power of the respiratory ferment is enormously large, therefore, comparable to the most intensive dyes known.

The fact that the ferment is nevertheless invisible can be accounted for by its extreme dilution in the tissue which is about one part of ferment to ten million parts ot cells. The concentration of the ferment can be estimated from the amount of CO adsorbed in the complete inhibition of respiration. This is so small that it cannot be measured exactly, but, it can be estimated to be smaller than 4 X 10-7 grams per gram of yeast. On the other hand, there must be at least so much of the ferment that every oxygen molecule on col-

19 Quoted from Anson and Mirsky, Physiol. Reviews, 10, 506 (1930).


liding with the ferment is consumed. On this basis we can estimate that the ferment must be larger than 3 X 10-8 grams per gram of yeast; 1: 10,000,000 is near the probable average. The concentration of the ferment in cells is, therefore, so small that a combination must occur whenever an oxygen molecule strikes a ferment molecule (Warburg).

5. THE RESPIRATORY FERMENT AS A HEMIN COMPOUND

The methods, briefly outlined above, make it possible to determine the absolute absorption spectrum of the respiratory ferment by observing the effect of light of different wave lengths. Warburg and his collaborators have worked out this problem very thoroughly by studying the effect of thirty-one definite wave lengths isolated from the light of mercury vapor lamps, from Pirani lamps, and from different carbon arcs by means of color filters. The result is represented in Figure 47a as a curve which exhibits a certain similarity to the well known absorption spectrum of hemoglobin, as mentioned already, see Figure 47b.

Hemoglobin is a derivative of hemin which belongs to the so-called porphyrrines. This class of substances has been elucidat_ed recently by the splendid syntheses achieved by Hans Fischer.20 All porphyrrines are built up of four pyrrol nuclei with varying side chains, such as vinyl-, methyl- groups, or various acidic groupings. Iron may enter into anyone of these compounds. Moreover, they combine with proteins as is the case with hemoglobin, for instance. Hemoglobin is one of the "red porphyrrines." This group includes also mesohemin and deuterohemin, which are closely related to hemoglobin, and coprohemin, the iron compound of coproporphyrrin (H. Fischer). All these red compounds have a similar absorption spectrum: the main or ,),-band being at 420JiJi and the a-band at 570JiJi (see Fig. 47b). For the respiratory ferment the main band is at 433JiJi and the aband at 590JiM (see Fig. 47a). The agreement is therefore, no more than an approximate one, indicating that the respiratory ferment, although similar, is still somewhat different from the red porphyrrines. For such a comparison of absorption spectra the absolute magnitude of the single bands is of little importance, since this depends on the

20 See the extensive pUblications of H. Fischer and his co-workers in Liebig's Annalen, 446, and following volumes, (1926-1928).


addition of salts, on the nature of the solvent used and other incidental factors. Merely the general order of magnitude of the bands should be considered, and this is the same for the hemins and the respiratory ferment. Little importance is also to be attributed to the 0- and E-bands (see diagrams), since these depend on the nature of the protein component.

= o .= a+ ..... o

® 3.11 "'" o:l

~ 2,6 tlIl

t 2,2 0.. £

S '"' 0< '"

FIG. 47a. ABSOLUTE ABSORPTION SPECTRUM OF THE RESPIRATORY FERMENT COMBINED WITH CO·, AS DETERMINED INDIRECTLY, ACCORDING TO WARBURG

From Warburg's "Nobel-Vortrag," Zeitschrift fur angewandte Chemie, 45, 1, 1932.

The results described so far have been condensed by Warburg, in 1929,21 into the following statement.

"Thus we see where the tasks of the future lie. Even though we know to which class of substances the respiratory ferment belongs, the details of its chemical constitution remain to be determined. The problem is to find out, by comparison of the absorption spectra, which one of the spectra of the innumerable hemin derivatives is not merely similar to but identical with the spectrum of the respiratory ferment."

21 Zeitschrift f. Electrochem., 35, 928.


In the meantime, Warburg and his collaborators have worked at the solution of this clearly outlined problem. A large number of hemin derivatives were prepared and the wave length of their a- and '}'-bands determined. Finally in 1931, a compound was found with aand '}'-bands, identical, within experimental errors, with the bands determined indirectly for the respiratory ferment. This compound is

3.8

260 - JlfO

I'"-"J If.ZfJ

.s '" at 570 flf'-

FIG. 47b. ABSOLUTE ABSORPTION SPECTRUM OF CO HEMOGLOBIN DETERMINED DIRECTLY

From Warburg's liN obel-Vortrag," Zeitschrift fiir angewandte Chemie, 45, 1, 1932.


spirographis hemoglobin, -y-band at 433f..Lf..L and a-band at 594f..Lf..L. (Respiratory ferment -y at 430f..Lf..L, a at 590f..Lf..L.)22 (See Fig. 47c.)

Spirographis hemoglobin is, therefore, very closely related to the respiratory ferment or possibly even identical with it. It exhibits interesting chemical properties: by oxidation it is transformed into a green dye which resembles chlorophyl, the green pigment of leaves; by reduction t is changed into a genuine red hemin derivative. In this respect spirographis hemoglobin resembles the phaeohemins, prepared by H. Fischer, which have also a similar spectrum. Moreover, the degree of oxidation of the side chains of these phaeohemins,

d 0

.!::: .... 0

8 0

a'"at¥3,sp,P ..., oj

F. 2,& .,0 oj .... ll.O .... 2,Z <l)

0..

8 1,8 '" 0-oo ...,- J." d <l)

'Z !El 1,0 <l) 0

'" d .S ..., 0.. ....

q2 0 00

.D < 26D -.3¥o 4oZO 4110 4B1J 610 i - l,"p]

FIG. 47c. ABSOLUTE ABSORPTION SPECTRUM OF SPIROGRAPHIS HEMOGLOBIN,22 COMBINED WITH CO, PREPARED BY WARBURG

From Warburg's "Nobel Vortrag," Zeitschrft fUr angewandte Chern ie, 45, 1, 1932.

22 This substance was obtained by linking together globin, the protein contained in hemoglobin with a hemin, obtained from the blood pigment, chlorocruorin, of the worm Spirographis; see Warburg's Nobel-prize lecture published in Zeitschrift fUr angewandte Chemie, 45, (1932). Compare also Biochern. Zeitschr. 227, 177, 184; 235, 240, 1930-31.


as well as of spirographis hemoglobin, is intermediate between that of chlorophyl and the red hemins.

This intermediate position of spirographis hemoglobin and the phaeohemins suggests the possibility that "both the red blood pigment and the green pigment of leaves have been formed from the respiratory ferment, the blood pigment by reduction, the leaf pigment by oxidation. For, it is.manifest that the respiratory ferment has been present prior to hemoglobin and chlorophyl" (Warburg).

Thus we see that the ingenious mode of attack worked out by Warburg makes use both of physical and chemical methods: Physical methods are necessary to determine the bands of the ferment. Organic chemistry is needed to identify these bands. The procedure, as a whole, resembles therefore, the spectral analysis of celestial bodies. In fact, the substance of the ferment, even though it is so near, is equally inaccessible as the substance of the stars (quoted according to Anson and Mirsky).

6. PREVIOUS ATTEMPTS TO SOLVE TliE PROBLEM OF RESPIRATION;

"0XIDASES," GLUTATHIONE, H ACCEPTORS

The great problem of organic respiration has always attracted interest. Every conceivable attempt to solve it has been undertaken prior to Warburg's discoveries of recent years. Numerous self oxidizing or respiring extracts have been prepared from plant or animal tissue. As a rule the respiration of such extracts is unstable. While it usually decreases spontaneously, its dependence on poisons is quite variable. HeN may inhibit or fail to inhibit or, at times, even stimulate oxidations. The chemical composition of these unstable extracts or oxidases, as they are called, is likewise indefinite. Some of their constituents are probably not preformed in the tissue but are rather products of decay. Such an indefinite chemical composition seems to be characteristic also of D. Keilin's23 so-called cytochromes, which are probably identical with McMann's histohemins. They are traced in tissues and tissue extracts directly by means of the spectroscope. Their spectrum is similar to that of the respiratory ferment indicating that the "cytochromes" are probably hemin derivatives. Some of these cytochromes are capable of alternating reduction and

23 D. Keilin, Proceed. Roy. Soc., B, 48, 312; 69, 129; 64, 206; 104, 206 (1929); 106, 418 (1931).


oxidation and of transmission of oxygen to benzidine, guaiacum or similar other substances in the presence of H 20 2 (Keilin).

The mere fact that cytochrome is directly visible by means of the spectroscope pleads against its identity with the respiratory ferment. This ferment is present in too small a concentration to permit of direct spectroscopic determinations; consequently, it cannot be identical with cytochrome (Warburg). Keilin holds the opposite view, but it is difficult to see which one of his numerous substances should be regarded as the respiratory ferment. For, as Warburg has shown, in contrast to the variability of the oxidases, the respiration of living cells is found to be unchanged under all conditions, being marked by CO or HeN inhibition, as described. Even if partly inhibited, the remaining respiration appears to be governed by the same rules. It seems, therefore, that there is just one respiratory ferment even though many oxidizable substances have been found in tissues. Many substances in tissues are also capable of transmitting oxygen to others. As Keilin states (1929) :23

"Oxidase in the living respiring cell represents only one link in the chain, forming the complicated respiratory mechanism, iii. which several other respiratory ferments and systems are involved and inter-connected."

He classifies these systems by stating that cytochrome acts as a carrier between:

(1) the dehydrases activating the hydrogen of organic molecules, (2) the "oxidase/! activating oxygen.

Perhaps the opposing views of Warburg and Keilin can be reconciled by the assumption that the respiratory ferment is formed from the cytochromes in the tissues. But, this remains to be investigated. More recently, viz., 1931, Keilin23 described that the cytochrome from yeast and the oxidase from heart muscle, when mixed, act in a similar manner as the respiratory catalyst; perhaps the respiratory ferment is formed in this complex mixture.

Nevertheless for a complete combustion the respiratory ferment seems indispensable. This indispensability has been demonstrated by W arburg, to some extent, for cystein which is the self-oxidizing constituent of glutathione. Glutathione, a protein isolated from tissues, oxidizes readily in alkaline solution by taking up oxygen from the air, while in acid solutions the reverse change occurs, the oxidation product giving up oxygen to be transformed again into glutathione. On account of this reversible change, glutathione can transmit oxygen


and hence it was held to be the respiratory ferment. Warburg,24 however, showed that cystein oxidizes only if it contains iron as an impurity. It seems likely, therefore, that also the oxidation of glutathione depends on traces of iron, probably in the form of the respiratory ferment-which is invariably in it as an impurity. This point, as well as the detailed mechanism of respiration, still awaits further investigation.

Numerous theories based on organic chemistry have been advocated to explain respiration. One of these is Wieland's theory of hydrogen activation which leads to the assumption of the existence of hydrogen activating ferments. Such ferments would be different, of course, from Warburg's respiratory ferment. Their existence has been demonstrated in vitro, but not yet with equal certainty in tissues.25

In the case of charcoal the chemical mechanism of "respiration" has been elucidated by the experiments of Negelein, who showed that charcoal oxidizes in the same manner as does H 20 2• All those amino acids which are slowly oxidized by H 20 2 are also slowly burned up in respiration on charcoa1.26 If the same finding could be made for cell respiration it would point to an oxygen activation by the respiratory ferment, but, there may be other ferments which activate hydrogen.

7. FERMENTATION AS A SURFACE REACTION; GLYCOLYSIS AND TUMOR

GROWTH

Further investigations have shown that also other reactions in living tissues are surface reactions, particularly the various fermentations (Warburg and collaborators, 1925-1927). As is well known, alcoholic fermentation, e.g., is an anerobic reaction, no oxygen being consumed yet CO 2 is produced through an intramolecular shifting of oxygen. Glucose is split in such a way as to yield a substance of a lower oxygen content, viz., alcohol and a substance of higher oxygen content, viz., CO 2• Fermentation is inhibited by the same poisons

24 See Warburg and Sakuma, PfHigers Archiv, 200, 203 (1923). 25 See Wieland, Ergeb. der Physiol., 20, 477, 1922; also Warburg, Biochem.

Zeitschrift, 142, 518 (1923). 26 Biochem. Zeitschrift, 142, 493 (1923).


as respiration: viz., by HCN, CO or H 2S, although not to the same extent in the case of HCN where a concentration 1000 times greater is needed. It is inhibited also by chemically indifferent narcotics which act by covering up the entire adsorbing surface. The nature of the ferment which brings about these reactions is not so thoroughly understood as the respiratory ferment. It seems likely that it is related to the respiratory ferment. As first shown by Buchner, fermentation occurs not only with living yeast cells, but likewise, with the juice pressed out of yeast. This is not in contradiction with the "surface theory" since the pressed juice still contains colloidal particles which adsorb.27

Respiration and fermentation, or speaking generally, aerobic and anerobic reactions, occur in most tissues simultaneously. A preponderance of one type over the other seems to be a factor influencing the production of certain pathological conditions. Warburg has shown that in cancer cells the anerobic reactions predominate.28

The cancer cells resemble yeast to some degree on account of their tendency to ferment sugar anerobically, although lactic acid is formed in this case instead of alcohol. This anerobic formation of lactic acid from sugar is called "glycolysis." Tumor cells show also a certain respiration, but, while normal body cells live by dint of respiration and perish in the absence of oxygen, the tumor cells have the possibility of persisting in the absence of oxygen by dint of such a "glycolysis." This is one factor accounting for malignant prolifera-· tion.

In normal cells, hardly any lactic acid appears. It is formed as an intermediary product, but, burned up in respiration. In dying or damaged cells respiration becomes inadequate to dispose of the entire lactic acid. In tumor cells, lactic acid formation has become permanent and is another outstanding factor accounting for the invasion of the malignant cells into healthy tissue through dissolution of the neighboring cells. The optimal conditions of tumor glycolysis have been determined by Warburg to be as follows: a sugar concentration of 0.15 per cent, a bicarbonate concentration of 0.025 molecular and a pH of 7.5S. Acidification of the tissues depresses the pH value and thus creates conditions less favorable for tumor glycolysis and hence for malignant proliferation. In fact, a certain inhibition of

27 Warburg, Biochem. Zeitschrift, 189, 1967 (1927). 28 Warburg, Biochem. Zeitschrift, 189, 354, 1967 (1927).


tumor growth has been observed if the tissues were acidified by inflammation or starvation.29

Experiments described by Hecht and Eichholtz (1929)30 seem to indicate that the glycolysis of tumor tissue may be due to a copper compound of unknown nature, just as the respiration of normal cells is due to an iron compound. Hecht and Eichholtz found that alanine, glycine, ethylene diamine Hel, or certain other substances inhibit the glycolysis of the tumor cell without a considerable inhibition of the respiration which occurs in tumor cells simultaneously with the glycolysis as stated. The only other chemical property, common to all these substances, is their tendency to form complex compounds with eu salts, just as HeN forms complex compounds with Fe salts. This is shown not only by chemical tests but also by experiments which were performed on mice, poisoned by an intravenous injection of at least .1 mgm. of copper salt per gram of body weight. All those substances which inhibit tumor glycolysis were found to inhibit this poisonous action of a coppersalt on intravenous injection. This detoxification seems also to be due to the formation of complex compounds with the injected coppersalts. No other poisonous heavy metal salt can be detoxified by all these special compounds (Hecht and Eichholtz). These experiments were verified independently by E. Krah (1930)31 who discovered certain additional compounds which inhibit glycolysis, detoxify coppersalts on intra venous injection and hence form complex compounds with them; By an analogous experimental method Krah was also led to the assumption that the inhibition of glycolysis by respiration, viz., the so-called reaction of Pasteur, is due to a ferment containing iron.

8. MOVEMENT OF LIVING ORGANISMS DUE TO SURFACE TENSION

CHANGES, AMEBOID MOVEMENTS

Respiration and other chemical reactions in living tissue furnish not merely heat but also locomotor power. How motion can arise

29 For further information compare Warburg, "Stoffwechsel der Tumoren," Berlin, 1926.

30 Biochem. Zeitschrift, 206, 282. 31 Biochem. Zeitschrift, 219, 432; see also page 444 in the same volume where

Krah shows that glucuronic acid promotes the reaction of Pasteur.


from chemical reactions in living organisms, viz., without the aid of such cumbrous mechanisms as a steam engine or an explosion motor, is a fundamental biological problem. A possible solution may be attempted by considering that most, or possibly all, chemical reactions in tissues are "surface reactions," as we have seen. In other words, they are chemical changes in layers of oriented molecules, and consequently entail changes of surface tension leading to motion.

The so-called ameboid movement of the low forms of life is amenable to this explanation. The most conspicuous and essential feature of this ameboid movement is the streaming of the. endoplasm. No continued locomotion can be observed unless accompanied by this streaming, the main endoplasmic streams being invariably in the direction in which the Amoeba is moving. At a certain part of the surface the protoplasm frequently exhibits a particularly intensive streaming, which leads to the formation of processes, the socalled pseudopodia. The mass in the center of these processes flows rapidly forward. It then turns back at the periphery, but flows at a much slower rate. Due to the greater rapidity of the forward movement the pseudopodium extends CA. A. Schaeffer).32

Ameboid movements can be imitated by placing oil droplets in water and then adding substances which lower the surface tension, for instance, in the following manner. "A drop of clove oil is mounted on an ordinary glass slide in a mixture of three parts of glycerin with one part of 96 per cent alcohol and covered with a cover glass. The clove oil and the alcohol are miscible, so that a little alcohol is continually passing into the drop of clove oil, a little of the clove oil into the alcohol." The. glycerin acts simply as a neutral medium to prevent a too rapid interaction of the clove oil and the alcohol. "Such a drop of clove oil will change its form, send out 'pseudopodia' and creep about much as 'amoeba 'does: At first it maybe circular, then a long projection will be sent out on the one side, the entire drop may elongate and progress as a whole in that direction. Currents may be formed within it, 'pseudopodia' may extend in several directions at once; at times, the drop may divide-as also happens in Amoeba. Altogether, the drop of clove oil imitates with some degree of closeness the behavior of Amoeba" CR. S. Jennings, 1902).33

"That a local chemical change taking place within the Amoeba

32 A. A. Schaeffer, "Ameboid Movement" Princeton Press, 1920. 33 Journ. Microscopy, 5, 1597 (1902).


(producing thus a new chemical substance in a certain area) might cause the formation of a pseudopodium, and movement in a certain direction, may be illustrated by introducing some chemically different substance into a certain region of the drop of clove oil. A very satisfactory method is to inject a little 70 per cent alcohol into the drop near one side. Take up a small drop of the alcohol in a pipette drawn to a very fine point; introduce this beneath the coverglass and into the drop, and press out a very little of the alcohol into

Projections, resembling pseudopodia are formed' from the oil drop by a localized lowering of surface tension in the same manner as in Amoeba, viz., by a more rapid forward streaming in the center. FIG. 48. DIAGRAM ILLUSTRATING AMEBOID MOVEMENT AND ·FORMATION OF

PSEUDOPODIA OF AN OIL DROP

the drop, removing the pipette at once. If this is skilfully done, and not too much alcohol is added, the drop will at once send out a 'pseudopodium' on the side nearest which the alcohol was introduced, . and often follows this up by moving in that direction. Of course, if the alcohol (or any substance having less surface tension than the clove oil) could have been produced through a chemical change within the clove oil, the resulting movement would be the same" CR. S. Jennings).33

Similar experiments can also be performed by means of emulsions of potassium carbonate solution in olive oil (Biitschli, 1892).34 In

34 Btitschli, "Untersuchungen tiber mikroscop. Schaume," Leipzig, 1892.


this case a localized lowering of surface tension occurs on account of saponification of the olive oil by potassium carbonate in the droplets of carbonate contained in the oil. If the soap, thus formed, comes to the surface of the oil drop at one place, surface tension is lowered and a "pseudopodium" formed. This effect can be demonstrated also by bringing a drop of olive oil between pure water and soap solution which border on each other. The oil drop then forms a process which

o

o FIG. 49. VARIOUS "AMEBOID" FORMS PRODUCED BY OIL DROPS

extends into the soap solution. Manifestly this process is formed due to the lowering of surface tension of the oil in the soap solution (Stempell and Koch).35 Hence, also in the experiments described above, an augmentation of the surface due to a local decrease of its tension is the cause of the formation of so-called "pseudopodia." Moreover, the processes projecting from the oil drop are formed by a more rapid forward streaming in their center and a slower back flow at the periphery, just as in the case of Amoeba (compare Fig. 48).

Locomotion of the Amoeba is brought about by the extension of the pseudopodium. Thereafter the tip attaches itself to the plane on which the Amoeba is creeping. Surface tension increases again and the pseudopodium contracts carrying the whole amoeba along. This type of locomotion can be imitated by means of a drop of chloroform placed upon a shellac coated plate under water.

35 "Elemente der Tierphysiologie," Jena, 1916.


Many different species of Amoeba are known. One of their marks of distinction is the form of their pseudopodia. The processes formed from oil drops exhibit an analogous variety of forms. Experiments with drops of oil, as described, using different kinds of oil and different concentrations of alkali added, show that almost any kind of "pseudopodia" can be produced if the composition of the oil or the alkali is varied (see Fig. 49).

9. FOOD INTAKE OF AMOEBA, ARTIFICIAL DIFFLUGIA SHELLS, PUL

SATING MECHANISMS AS EFFECTS OF SURFACE TENSION

"One of the most striking phenomena in the behavior of Amoeba is its power of selecting substances which shall serve as food. Amoeba takes its food simply by sending out pseudopodia, flowing around and enveloping small bodies. But it by no means takes these at random; sand, decayed plant tissue, bits of wood, dirt, etc., are as a rule rejected, while small living plant and animal cells, diatoms, infusoria, are enveloped, carried away and digested. It thus shows a distinct choice in the substances which it takes into itself; and the power of choice has often been considered evidence of a rather highly developed mind" (H. S. Jennings, 1902).33

"Before accepting this conclusion for Amoeba, it will be wise to test this matter of the power of choice for other fluids. A drop of chloroform is a good subject for experimentation. With a medicine dropper a drop of chloroform may be placed in the bottom of a watch-glass of water, and then with fine tweezers we may offer it various substances to test its power of choice. The whole proceeding may seem at first thought very absurd, but the results are striking. "

"We may first offer the drop of chloroform a fragment of glass; this is held with the tweezers against the surface of the drop. It is not accepted. We push the glass against the drop, but the latter withdraws its surface from it as far as possible. We force the bit of glass into the drop of chloroform and let go of it. It is at once thrown out with energy. We try a small piece of wood in the same way; it is rejected as decidedly as was the glass. We may now try a hard piece of gum shellac. This is accepted eagerly, one had almost said.


Hardly has an angle of the piece of shellac touched the surface of the drop, when the latter literally reaches out, envelops the shellac and draws it into itself. If we take hold of the piece of shellac again with the forceps and draw it away, the chloroform drop stretches out after it, and lets go of it only with the greatest apparent reluctance. If allowed to retain the bit of shellac, it proceeds slowly to dissolve it, -just as the Amoeba .proceeds to digest the substance which it has taken within itself. A second and a third piece of shellac will be accepted with the same avidity as the first" (Jennings).33

"Other substances may be offered to the chloroform drop. Glass, sand, dirt, wood, grass, gum arabic, and chlorate of potash, for examples, are rejected; shellac, paraffin, styrax, hard Canada balsam, and various other substances are accepted."

"It thus appears that a drop of chloroform exercises choice in determining what substances shall be taken into itself, fully as decidedly as Amoeba does. The same is true of other fluids, of whatever sort. We must then throw out completely the power of choice of food as any test of mental power or even of life. Amoeba merely shares this power with all other masses of fluid. It is a suggestive fact, and one which has possibly a deep significance, that the chloroform drop (or other fluids) tends to take into itself especially such substances as will dissolve within it, or have a chemical affinity for it, just as Amoeba tends to take within itself substances which it can digest" (Jennings). 33

"The method by which Amoeba takes a small particle of food is very similar to that by which the chloroform drop takes within itself a bit of gum shellac. The protoplasm simply flows over and envelops the food particle. But at times the problem presented to the Amoeba if food is to be obtained is much more difficult. Sometimes the food available is in the form of a long thread of Alga, many times the length of the Amoeba. How is such an awkward piece of material to be managed? There seems to be only two possibilities for getting such a long thread into a short Amoeba. One is to cut it into lengths, the other to coil it up. Amoeba has no teeth for cutting up the thread, so it adopts the plan of coiling it. Individuals engaged in this process are sometimes found among the specimens studied in the laboratory."

"The Amoeba first settles itself upon the filament somewhere in its length, and envelopes a portion of it. It stretches out a slight


distance along the thread, then bends over, of course bending the filament at the same time. The bending is continued until there is a loop formed within the Amoeba. The animal now continues to stretch out along the two ends and to bend them over, till the loop

FIG. 50. INGESTION OF A THREAD-LIKE ALGA BY AMOEBA

FIG. 51. COILING Up OF A THREAD OF SHELLAC BY A DROP OF CHLOROFORM UNDER WATER

According to L. Rhumbler, 1898. The thread of shellac is drawn into the drop in a similar manner as the Alga into the Amoeba.

is doubled, tripled, and a coil is in process of formation. This is continued until the entire filament is rolled up into a neat little coil within the Amoeba, where it is digested" (see Fig. 50) (Jennings).33

"What are we to say to such a clever solution of a somewhat difficult problem as we have here? Must we not admit to Amoeba the


power of grasping a situation and intelligently adapting means to end or overcome difficulties-qualities which we are accustomed to consider as characteristic of minds in a high degree of development?"

"It will be well in this case, as in others, to test inorganic fluids before deciding what is to be thought of this matter. Suppose we present a similar problem to our chloroform drop; how will it meet the situation ?"

"We may try the experiment as before, with a drop of chloroform at the bottom of a watch-glass of water. As the chloroform drop accepts hard shellac, we may present it with a bit of shellac drawn out into a long, fine thread, of length many times the diameter of the chloroform drop."

"The chloroform envelops the filament in some portion of its length, just as Amoeba did. Then it stretches out in both directions along the thread, exactly as was done by Amoeba. (This is most striking when a drop of chloroform floating on the surface of the water is used, though the experiment is otherwise much more difficult to perform under these circumstances.) Thereupon the thread bends, exactly as with Amoeba. The process now continues, precisely parallel with what occurs in the case of Amoeba and the alga thread, until the shellac thread is coiled up within the chloroform drop, like a filament of Oscillaria within an Amoeba" (H. S. Jennings)33 (See Fig. 51).

If the Amoeba feeds on a digestib-le object containing indigestible' particles inside, these latter will be thrown out as soon as all the digestible matter has been dissolved. This process, termed "defecation," can be imitated if a drop of chloroform is brought in contact with a tiny thread of glass coated with shellac. The thread is drawn into the chloroform, the shellac coating dissolved and the bare glass rod thrown .out (Rhumbler).36

"As is well known, DijJlugia, one of the close relatives of Amoeba, lives in a shell formed of sand grains, diatom shells, and other small particles cemented together. These particles are fitted together accurately in a single layer, so that no crevices can be discovered. How are these delicate houses built? It would seem that the process must require much care, skill, and intelligence, to select the proper pieces and put them together with such nicety that the shell is but a single layer thick, and yet no gaps are left."

36 Quoted from L. Rhumbler "Nachahmung von Lebensvorgiingen", in Handbuch der biologischen Arbeitsmethoden, Abt. V, 1921.


"But the drop of chloroform is not to be outdone, and under the proper conditions will produce a shell not inferior to that of Difllugia. This may be shown very simply. Chloroform is rubbed up with fragments of glass in a mortar until the glass is reduced to the finest dust. Then with a pipette drawn out to a small point drops of this mixture of chloroform and glass dust are injected into water. At once the grains of glass come to the surface of the drops so formed and arrange themselves there in a single layer, without chinks or crevices exactly as in the shell of Difllugia. The chloroform drop is covered with a shell of a delicacy and beauty equal to that of Diffiugia, and almost indistinguishable in texture from it. Some of these artificial shells, if unexpectedly found with the microscope, would certainly be taken for those of Difllugia" (see Fig. 52). "In place of chloroform, linseed oil or other oils may be used. They must be injected into 70 per cent alcohol, since the oil would float upon water. The process is exactly the same as when chloroform is used" (H. S. Jennings, 1902).33 All of this does not account, however, for the specific form of Difllugia.

FIG. 52. ARTIFICIAL "DIFFLUGIA SHELLS" AROUND A CHLOROFORM DROP

Magnified 1:100. Formed by shaking a drop of chloroform under water with glass fragments.

Scientific literature contains descriptions of numerous other artificial mechanisms which--{)perated by dint of surface tension-ex-


hibit some resemblance to movements of certain living forms. Thus, for instance, chemotropic movement can be imitated by the extension of oil drops towards an object which will be taken up in the manner just described (this extension occurring, of course, before the object comes in contact with the drop). The effect can readily be explained as the result of a dissolution and diffusion of traces of the substance in question leading to a one-sided lowering of surface tension and consequently to a movement of the drop towards the source of the diffusing solute.

Automatic pulsating mechanisms operated by surface forces have attracted attention, particularly Ostwalds' so-called "electric heart" which is set up as follows: A globule of mercury about 2 cm. in diameter is placed on a watch glass almost filled with 10 to 15 per cent H 2S04 ; enough K 2Cr207 (potassium bichromate) is added to render the sulphuric acid light yellow in color. A clean sewing needle, held in place by a cork, is placed in a diagonal position in such a manner that the point of the needle just touches the margin of the mercury globule. At the moment of contact with the needle the globule flattens out. This breaks the contact with the needle, consequently the mercury becomes spherical-a result of increased surface tensionand so again makes contact. This must lead to another contraction. The rhythmic pulsation of such a mercury droplet resembles cardiac pulsations to a striking degree.

10. MOVEMENTS OF ARTIFICIAL SYSTEMS WITH A RESPIRATION I

To summarize, we may state that a conspicuous resemblance of the movements of Amoeba and oil drops can be demonstrated in a variety of cases. However, the artificiality of the technique applied must be emphasized. The formation of a localized extension of the oil drop was brought about by the addition of a "surface active" substance from one side, leading to an uneven tension and formation of processes. Granting that the pseudopodia of Amoeba are likewise the result of an uneven surface tension, it seems likely that additional factors must be considered as causes of surface tension variation, e.g., the respiration which is located at the cell surface or at the surface of granules. We may assume that this surface reaction is also subject to variations and that certain intermediate products of com-


bustion are "surface active."a7 In this way local variations of surface tension would occur, leading to formation of pseudopodia. A part of the energy produced by respiration would thus be transformed directly into locomotion.

The question arises whether it is possible to find water immiscible substances with a surface tension which depends on respiration. From Warburg's charcoal experiments we have learned that respiration is not the exclusive property of living cells. Should it not be possible to find also a fluid which shows a "model respiration" like charcoal in order to investigate whether spontaneous ameboid movements depending on respiration occur in this case?

In fact, freshly extracted brain lipoids suspended in physiological saline solution containing phosphates have such properties according to Crile and Telkes.38 As already described, this lipoid forms so-called "myelin filaments" (see Fig. 31) which pinch off to form globules floating in the solution. Also a large number of smaller droplets are formed, as is revealed by a closer inspection. The suspended lipoid droplets, thus formed, show a respiration analogous to that of cells, viz., a consumption of oxygen and an output of CO 2 and other end products, quite like living cells or like charcoal. Small doses of HeN or somewhat larger doses of narcotics arrest this respiration in the same manner as the regular cell respiration. A further'similarity to cell or charcoal respiration is the following. By the addition of soluble proteoses, proteins, or sugars ~he CO 2 output and the oxygen consumption are found to increase. This.seems to indicate that the combustible material is not taken from the lipoid itself-or at least not entirely from the lipoid. Soluble substances are. burned in solution in this case as in cell respiration. These observations have been verified by several observers, who have used the well kpown method of Warburg, already described (see page 150).38

37 This means that they lower the surface tension. 38 Protoplasma, 16, 337, 1932. According to a private communication Dr.

D. R. McCullough of Cleveland has verified these findings. The lipoids used in these experiments contain unsaturated fatty acid radicals,

which are probably responsible for the respiration. It is well known that unsaturated fats, e.g., linseed oil, undergo a self-oxidation in the air which resembles a respiration since CO 2 is put out. At the same time the oil is condensed to a high molecular compound, this being the well-known process of drying of linseed oil. A similar building up of high molecular compounds occurs probably also in living tissue simultaneously with respiration apd actually caused by it.


For a qualitative demonstration the well known redox indicators may be used as follows (R. Beutner, 1931).39 Control experiments were first performed in which a trace of the reducible dye, chloroindo-phenol, was added to a suspension of brain lipoids in saline. No I,~duction or decoloration was found to occur in this case evidently since the oxygen consumption of the lipoids themselves is slight if not zero. The same was observed if this redox indicator was added to a solution containing proteoses extracted from brain with a boiling alkaline solution, but without lipoids. However, reduction and decoloration did occur if chloro-indo-phenol was added to the mixture containing both proteoses and suspended lipoid. That the oxygen consumption, thus indicated, is the result of a real respiration similar to that of living cells is indicated by the possibility of inhibiting it with narcotics. This was observed if ether was added to the mixture of proteoses and lipoids. It was then seen that indophenol is no longer reduced by the lipoids and proteoses, showing that ether inhibits this "model respiration" just as it inhibits the respiration of living cells (R. Beutner, 1931). Further experiments would be desirable, in order to decide whether or not this type of respiration agrees with cell respiration in every respect. As already mentioned a large number of smaller oil globules are contained in the solution. Probably these globules take part also in the "respiration," but another part of the respiration may occur at the surface of the large globules.

The term "autosynthetic cells," which G. W. eriIe uses for these respiring • ,1 globules, does not indicate any further similarity to living cells, except the concomitance of surface reactions and surface changes as described. The rentral portion of the "cells" represents an aqueous phase.

A variety of such respiring lipoidal globules is known. Some of thrm when placed in an alkaline solution (pH 10) show a distinct ameboid movement. Processes resembling pseudopodia are thrown out or retracted alternately, as shown in Figure 53. Probably the formation of those somewhat thinner myeloid filaments from brain lipoid::; as described above (see Fig. 31) is of the same nature; these thinner filaments form in a more nearly neutral medium. It will be remembered that also the processes formed from non-respiring oil drops may show a variety of forms (see Fig. 49), depending on the

89 Unpublished observations.


composition of the medium. The interdependence of the respiration of these lipoidal globules and formation of "ameboid" processes from them seems to be indicated also by the observation that respiratory poisons like HeN or narcotics interfere with both Simultaneously. Here then is a case of ameboid movement in an artificial system which is not caused by surface tension changes produced by the addition of a substance like alcohol from the outside but due to surface reactions, viz., the burning of oxidizable substances at the surface just as it is known to occur at the surface of living cells,

,':.:', I' '., : '"

\'. \\ 0 . ., \'. "''0 \ . '" , '. '

o o

o

\ '. , . "

o FIG. 53. DIAGRAMMATIC VIEW OF A LIPOIDAL MASS (ETHER EXTRACr OF BRAIN)

WHICH PERFORMS AMEBOID MOVEMENTS AND SHOWS A RESPIRATION, PARTLY SIlIIlLAR TO THAT OF LIVING CELLS

The lipoid is 8uspend-ed in an alkaline saline. As indicated by dotted lines the processes of this lipoidal mass slowly expand and later retract, Addition of cyanide or narcotics arrests the "respiration" and results in a complete retraction of aU of the processes, the mass taking up a spherical form. In respect to its shape and movements this lipoidal mass resembles an Amoeba to some extent.

11. TRANSIENT AND PERMANENT PROTOPLASMIC RIGIDITY

Another peculiarity of ameboid movement has remained unexplained so far: the pseudopodia of an Amoeba ~xhibit a certain rigid-


ity while they form. The living protoplasm alternately liquefies and solidifies, in contrast to the extension of an oil drop which, of course, remains fluid throughout. Variations of surface tension are manifestly insufficient to explain this transient rigidity, but, gels (jellies) of iron hydroxide exhibit likewise a transition from rigidity to fluidity. These gels turn completely fluid following shaking. Upon standing they revert again to the "gel" state. (H. Freundlich, 1928).40 Freundlich calls this a "tixotropic" change, (from tixis = touch). These changes depend also on the pH of the solution. With decreasing pH, "gel" formation takes a much longer time. For instance while at pH 3.9 82 seconds were needed, in one particular case, it took 9000 seconds at pH 3.1. In other words, a slight acidification counteracts gel formation markedly. Also an addition of amino acids has a marked influence. In this case, gel formation is retarded, even though such an addition of amino acids raises the pH -which per se would accelerate gel formation. The same phenomenon is exhibited by numerous other substances, such as jellies of aluminium hydroxide, certain organic colloids, dilute gelatin, under certain conditions. In the case of aluminium hydroxide Freundlich has shown that this so-called "tixotropic" behavior is related to the streaming double refraction of this colloidal solution.

According to these observations it would seem likely that the disappearance of protoplasmic rigidity in Amoeba is due to some sort of motion of the protoplasm which is equivalent to shaking. We may also assume that it depends on pH changes, or on the formation of amino acids from the proteins through the action of enzymesin the same manner as demonstrated by Freundlich for iron hydroxide gels or other gels.

In this connection some interesting observations of O. Lehmann on liquid crystals may be mentioned, even though they bear a remote relation to the problem of protoplasmic rigidity, since they occur at elevated temperature only.

According to O. Lehmann41 one pf those organic compounds with

40 Koll. Zeitschr., 46, 289. 4J O. Lehmann, "Die Lehre von den fiiissigen Krystallen und ihre Beziehung

zu den Problemen der Biologie," Ergebnisse der Physiologie, 16, 255-504 (1908); see also "Fliissige Kristalle und die Theorien des Lebens," by the same author, Leipzig, 1906.


an elongated molecule is used (see above, page 136) as e.g., p-azoxybromocinnamic acid ethylester between 139° and 247°C. A trace of a suitable solvent like kerosene must be added to obtain motility. After melting, the mass is allowed to cool down slowly while it is under a microscope. As crystallization from the molten mass proceeds the round liquid crystals are usually seen to increase in size, as a result of cooling. Eventually, however, a different type of growth

FIG. 54. "SNAKE"-LIKE PROCESSES DEVELOPING FROM A LIQUID CRYSTAL WHILE IT COOLS DOWN ON A MIC·ROSCOPIC SLIDE

Redrawn from O. Lehmann's "Flussige Kristalle und Theorien des Lebens," Leipzig, 1906.

occurs. A sort of a bud is formed upon a round liquid crystal and this bud develops into a "snake" of uniform thickness, shooting out with great velocity, shifting and turning around like a worm, performing violent movements. These "snakes" frequently grow to considerable length so as to cover the entire field of vision (see Fig. 54). They disappear rapidly by contracting to round crystals ..

These movements seem to be caused by a rapid alignment of the elongated molecules of the azoxybromocinnamic ethylester. The


molecules somehow slide upon each other and arrange themselves in a consecutive order thus leading to the formation of a protruding filament as described. Forces of this kind may play a part in the formation of the pseudopods of Amoeba and likewise in the formation of the fibers which develop from lipoids (see above, page 117) since the distinctly visible membrane of these fibers shows characteristics of liquid crystallinity, viz., double refraction, etc. (0. Lehmann).4'

FIG. 55a. EXPERIMENTS OF AMPHIBIAN EGGS. DEMONSTRATION OF THE RIGIDITY OF LIVING PROTOPLASM

8 n II

---=~I

FIG. 55b. EXPERIMENTS OF AMPHIBIAN EGGS. DEMONSTRATION OF THE Loss OF RIGIDITY AFTER DEATH

Moreover, a comparison of protoplasmic masses to oil drops is insufficient since living protoplasm exhibits a certain permanent rigidity. This can be demonstrated by dividing a blastula or gastrula of a developing amphibian egg into the single cells of which it consists, by careful removal of the egg membrane. The isolated cells are left floating under a thin layer of water or saline at the bottom of a Petri dish where they adhere due to their stickiness (Fig. 55a). Upon gently rocking the dish they roll slightly back and forth like little solid balls without an alteration of shape. But the shape is maintained merely as lung as the cells are alive. After death they vary back and forth into oval shaped forms following the oscillating movements of the dish (see Fig. 55b).

Apparently, following death, the cell is transformed into a fluid. 42 To ac-

42 A similar difference exists between the rigidity of living and dead Amoeba and other cells. In general, living tissue is firmer than dead tissue although there are exceptions to this rule, e.g., the rigor mortis of muscle which, however, is transitory.


count for the rigidity of the living cell we should realize that protoplasm is probably an emulsion.43

Experiments can be described to show that certain emulsions have a considerable rigidity. Thus for instance, by shakinr 99 parts of mineral oil with one part of a concentrated soap solution, a semI-solid mass is obtained, the so-called Pickering's Emulsion, which can be cut with a knife. This rigidity can be accounted for by the formation of semi-solid adsorbed layers of soap around the oil droplets which are formed by the shaking. The micellae of soap are crowded in the adsorption layer so tightly that they are actually semisolid. Each oil droplet is thus encased in a solid envelope. Almost all of the soap is adsorbed to the oil surface. Moreover, there is so little fluid left between the drops that they cannot float in it, and hence the mass as a whole takes a semi-solid appearance. A similar rigidity is exhibited by foam of any kind, as is shown by the fact that foam cannot be made to flow like a fluid by blowing into it or by stirring it up.

12. THEORIES OF MUSCULAR CONTRACTION

It seems likely that forces of the kind described above (see page 169 ff.) are also responsible for muscular contractions but it is unknown as yet how their action may account in detail for all the known facts of the metabolism of muscle.

Theories of muscular contraction have been advanced so frequently that all of them have finally become discredited. Frequently osmotic actions have been looked upon as,the most plausible explanations. Numerous observations seem to point to' this possibility. On the other hand, the double refraction which all types of muscle exhibit and its variability with the contraction seem to indicate t~e presence of forces caused by molecular orientation. "At equal thickness the striated muscles-which have a greater double refraction-develop a larger force than the smooth muscles which are feebly double refracting." A relatively greater anisotropy is exhibited by many ciliated organs and these also develop an outstanding force when contracting. "In the physiological contraction of muscles, the contracting force and double refraction decrease, therefore, simultaneously" (Th. W. Engelmann, 1906).44

The tendency of modern research is to establish facts about the

43 It is probably an emulsion of both water in oil and oil: in water (see above page 90 ff.).

44 Sitzungsberichte der preussichen Akademie, 39, 694.


physical and chemical changes occurring during muscular contraction. At present these changes have not yet been determined definitely.

Formerly a decomposition of a glucose-phosphoric acid or "lactacidogen" into lactic acid was believed to be the primary change, with consequent oxidation of lactic acid and partial synthesis or condensation of lactacidogen after the contraction (A. V. Hill and Meyerhof, 1922).45 Recent investigators have questioned this hypothesis: in a muscle poisoned with bromoacetic acid no lactic acid at all is formed, yet it contracts. A splitting of phosphocreatine into phosphoric acid and creatin is observed, however, and this is assumed to be the cause of the contraction (Lundsgaard). Also an abundant NHs formation occurs (Embden, 1930).46

Even if the present discussions about these questions were settled and if the chemical changes in muscular contraction were better understood, the mode of action of the reaction products would still remain an open question.

45 A. V. Hill, Physio!. Reviews, 2, 310 (1922). O. Meyerhof, "Chemical Dynamics of Life Phenomena," Philadelphia and London, 1924.

4S Klinische Wochenschrift, 9, 1337 (review). A. V. Hill corroborates this by his recent finding that the total osmotic changes are greater than can be accounted for by the generation of lactic acid. (See A. V. Hill's "Adventures in Biophysics," Springfield and Baltimore, 1931.)

THE THIRD ATTEMPT AT APPROAOH

ELECTRICAL CURRENTS IN TISSUES AND THEIR RELATION TO LIFE

PROCESSES

THE THIRD ATTEMPT AT APPROACH: ELECTRICAL CURRENTS IN TISSUES AND

THEIR RELATION TO LIFE PROCESSES

EXPERIMENTS WITH MODELS: THE ELECTROMOTIVE EFFECT OF CONCENTRATION AND ITS ARTI

FICIAL IMITATION

The investigation of the origin of electric currents and potential differences in living tissues involves the most comprehensive problems in natural sciences, for biological, chemical and physical questions are all concerned in it. It has long been known that electric phenomena are associated with life; in fact it was by dint of this association that the existence of electric currents was first discovered (Galvani and Volta). (For further details see the historical review £nserted at page 212.)

As we shall see, the most exacting electrophysiological investigations have led to the result that the irritability of living tissue, its most "vital" property, is in reality an electrical polarization, which means an electrochemical process. The investigation of the electrical conditions prevailing in tissue may be looked upon, therefore, as one of the scientific approaches to the basic problems of physiology.

In the case of the electric fish, the production of electricity is most striking, potentials of several hundred volts being produced. Naturally this phenomenon has attracted the attention of many investigators, who have advanced various explanations. Most of these theories consider bioelectricity as so closely associated with life that an artificial reproduction of anything remotely resembling it is considered impossible. A conception of this kind is far from being true. It is not at all impossible to reproduce artificially many of the essential underlying conditions which are responsible for the generation of electric currents in tissues, and to observe how such artificial systems produce potential differences in a similar fashion. M amfestly no explanation of bioelectricity can be adequate unless it is based on an imitation of the phenomena. The true importance of physical chemistry for electrophysiology becomes

189

190 THIRD ATTEMPT AT APPROACH

manifest only by applying physical laws to those artificial systems which reproduce definite features of biological potential differences. In this way we may hope to develop electrophysiology to that high level of scientific accuracy which marks the development of modern physics and chemistry.

1. METHODS OF MEASURING ELECTROMOTIVE FORCES OF TISSUE

Living tissue produces electrical currents quite like a galvanic battery, e.g., a dry cell. As is well known, the current producing force of any battery-or the electromotive force, as it is usually called-can be measured in volts by various methods, e.g., through compensation by a known electromotive force or by means of suitable direct reading instruments. (For further details see textbooks of physics.) For tissues, sensitive measuring instruments are needed, since the electromotive forces produced are usually small and the internal resistance of the tissues are high. Hence the actual intensity of the current produced is very low.

Moreover, precautions are necessary to avoid disturbing influences from the metallic wires which connect the tissue to the measuring instrument. If we would touch two points of the tissue in question with the two terminal wires which lead to the measuring instrument, we would, in most cases, observe a small electromotive force, but a similar electromotive force may also be observed if we immerse the two wires in any salt solution, since the immersed ends of the wires are never entirely identical. One of them may have a diminutive amount of oxide on its surface while the other one has none. It is, therefore necessary that the wires from the measuring instrument should have interposed between them and the tissue a liquid conductor, preferably physiological saline, which produces no electromotive forces by the contact with the tissue. At the contact of the terminal wires and the solution, another liquid is interposed so as to avoid irregular electric variations at the metallic surface. A device, containing these solutions as well as the terminal metals, is called a non-polarizable electrode. Numerous designs of such electrodes have been prepared: Figure 56 shows one possible form in which the terminal wires are attached to pieces of zinc amalgam which is placed


in a zinc sulphate solution; this solution being in contact with physiological saline contained in the sponge attached below.1

If two such electrodes are brought into direct contact (by touching one sponge with the other) no electromotive force arises since the system is symmetrical. But, an electromotive force of 0.01 to 0.1 volt is observed if the longitudinal uninjured surface of the muscle is touched with one electrode and the cross section with the other (see Fig. 56). This electromotive force produces the so-called "current of injury." The direction of the current in the muscle is from the injured to the non-injured portion. In other words, the injured portion is on the negative side (see Fig. 56).

HIJS(!'t.e Two identical electrodes are in contact with different parts of tissues

FIG. 56. DIAGRAM OF A SET-UP FOR MEASURING THE INJURY CURRENT OF A MUSCLE

Such electromotive forces are observed not only with muscle, but with any kind of tissue and even with plants. Moreover, the same effect is produced, not only by cutting, but by most any other type of injury such as heating the tissue on one side, or poisoning it. To produce an electromotive force) it is sufficient if the tissue at the one

J Various forms of non-polarizable electrodes may be used, e.g., the calomel electrode (see Figs. 58 and 59). In this electrode terminal wires are connected to metallic mercury which is in contact with a KCI solution, saturated with calomel. In general, the metal of the electrode should be in contact with a solution of its own salt, for further details see textbooks on physical chemistry.


pole is approximately in the same condition as in life, while at the other it is damaged. Likewise, portions of tissue which are non-homogen..:. eous can produce a current even when there has been no injury. Thus, the root of a plant is negative relative to the leaf or stem; or the tendon is negative to the muscle. The order of magnitude of these electromotive forces varies greatly even in the same experimental object for unknown reasons. This inconstancy renders difficult the interpretation of the experimental work done in this line.

Since electrophysiological research has always aimed at finding a relation between life and electricity, special attention has been paid to another kind of electrophysiological phenomenon, the so-called action current: which arises in muscle or nerve if the tissue, although uninjured on both sides, is in a stimulated condition on the one side. The stimulated side is negative relative to the non-stimulated side. In other words, stimulation acts in the same way as an injury, but, this action is transitory, lasting usually no longer than a few thousandths or hundredths of a second. The time curye of this negative variation is most complicated. Since the time of discovery, it has been subjected to investigations of ever increasing accuracy. By increasing the velocity of movement of electrical instruments to the highest degree-first in the string galvanometer (Einthoven), more recently through the cathode ray oscillograph (Gasser and Erlanger)2-the time curve of the negative variation is now known in such detail as was never dreamed of formerly. Yet, all this investigation has naturally been unable to add any material information to the question of the origin of currents in living tissue.

2. THE IMPORTANCE. OF WATER IMMISCIBLE SUBSTANCES FOR BIOLOGICAL ELECTROMOTIVE FORCES

The question of what substances in the muscle or other tissues are concerned in the production of electromotive forces, has been taken up by the earliest investigators in this line (DuBois Reymond).8 In order to attack this problem, observations not only on the physio-

2 For literature see Harvey Lectures, Series XXII, 1926-7. 3 Emil duBois Reymond, "Untersuchungen liber tierische Elektrizitat,"

Berlin, 1848.


logical object but also model experiments with materials of known composition had to be made in order to carry conclusions over from the known to the unknown. At the time of DuBois Reymond and later, physics and chemistry failed completely to solve this problem. To be sure electrical currents can be obtained with Volta cells, currents which are even stronger than the biological currents, but here metals are the effective agent, and these are naturally lacking in tissues. Purely aqueous fluid cell systems, composed of salt solutions similar to those in tissues,jurnish no satisfactory explanation for the biological production of current. They fail to produce electromotive forces of approximately equal strength. One may consider, for instance, the possibility that in the muscle potential differences arise through the direct contact of an aqueous solution of KCI, as contained in the muscle fiber, and a solution of NaCl, as contained in the adjoining lymph. However, the average electromotive force of the muscle is at least ten times that of a potential difference at the contact of N aCI and KCl solutions; the maximal theoretical value for this potential difference being no more than 0.004 volt.4

It is only by interposing electrolytic conductors that are not soluble in water between the aqueous solutions in the electrolyte cells that electromotive forces can be produced in a similar order of magnitude to those which appear in living tissues. Therefore, in living tissues, it is not alone the aqueous solutions contained in them which are active in the production of electric potential differences but also the intermediate layers which are not soluble in water and which are usually called membranes.

One way to understand the mode of action of these membranes is to set up and to investigate cell systems, composed of aqueous solutions and non-aqueous electrolytic conductors which do not mix with water. These latter may be either precipitation membranes or water immiscible organic substances like oils. So far as the principle is concerned, it makes no difference whether oily layers or membranes are placed between aqueous solutions; the membranes can be looked upon as a special kind of "oil" which is generated by the solution itself. It also makes no difference whether the membrane is liquid or

4 This theoretical value, calculated from Nernst's theory, is usually not attained since the solutions begin to mix as soon as they come in contact with each other.


solid. Thus, for instance, if a non-aqueous electrolyte is inserted between a NaCl and KCl solution, electromotive forces are obtained which are similar to those of tissues. If a fluid layer of phenol or cresol or their substitutes is interposed between ,these solutions, an electromotive force of about 0.01 V9lt is produced. Various other non-aqueous fluids produce electromotive f9rces of 0.01 to 0.02 volt when inserted between KCI and NaCI solution.5 The mode of setting up such a battery system is illustrated by the accompanying diagram (see Fig. 57).

In spite of the exceedingly high electrical resistance of the nonaqueous central conductor, the el~ctromotive force of such a battery

Calomel eleetl'ode

I{C{

to Eledr~meter

calomel electrode

FIG. 57. DIAGRAMMATIC VIEW OF A SIMPLE BIPHASIC CELL

The non-aqueous conductor in the center is in contact with two different solutions. This accounts for the origin of an electromotive force; the electrodes are identical and do not give rise to electromotive forces.

system can be measured, if a sensitive electrostatic electrometer is used. However, not any central conductor can be used. Hydrocarbons, like paraffin, or regular fats are practically insulators and hence render measurements nearly impossible. On the other hand, most organic compounds which contain OH, N0 2, CO or COH groupings have a conductivity which is sufficient even though it is rath\lr small.

6 Also various systems containing membranes like parchment produce an electromotive effect of a similar magnitude.


3. THE MAXIMAL EFFECT OF CONCENTRATION

This shows that the electromotive force of such a biphasic cell approaches the order of magnitude of biological currents, but a comparison which is merely based on the order of magnitude is too vague, particularly on account of the inconstancy of the injury current, as f lready mentioned. This uncertainty is due to the fact that the asymmetry giving rise to an electromotive force is located in the tissue itself, where unknown and uncontrollable changes occur.

The following remarks may serve to make this point clearer. An asymmetry is the cause of any electromotive force. Thus a Volta cell, consisting of two different metals immersed in an acid, produces an electromotive force due to the asymmetry of metallic electrodes which, in this case also, are subjected to uncontrollable variations.

Each electromotive force in a large mass of tissue is composed of a multitude of single potential differences which are located in the tissue wherever differenentiated structures come in contact with each other. These potential differences compensate each other if -the connections are made from symmetrical points. If the tissue is injured, the internal potential differences no longer compensate each other; an electromotive force is manifested on the outside even though the origin of this electromotive force is in the tissue itself.

A case with which it is more suitable to start the investigation is found if a symmetrical physiological structure is chosen, which, however, is in contact with two different solutions on either side. The two solutions may contain the same salt, but, in different concentrations. This alone is sufficient to produce a current. The positive pole is usually found to be on the side of the more dilute solution as follows:

- electrode I concentrated I any symmetrical I dilute salt I electrode + salt solution piece of tissue solution

We may designate the electromotive force of such a system as the electromotive effect of concentration. Although the direction of this effect is usually the same, its order of magnitude varies considerably.

Large and regular variations of the electromotive force with the concentration are obtained by using uninjured parts of plants as central conductors in such a system. This is a well defined phenomenon which may serve as the starting point for a thorough investigation of the under-


lying cause of bioelectricity. (R. Beutner in Loeb's laboratory, 1912).6 For these experiments any part of a green plant with a hard smooth 'surface, covered by a cuticula, may be used, as for instance, leaves, fruits, etc., a suitable object being an apple.

The experimental arrangement is shown in Figure 58. The apple lies in a flat dish, solutions of varying concentrations are poured into this dish and a measurement of the electromotive force is made with each solution. Connections to the measuring instrument are made by means of two calomel electrodes in the manner shown in the diagram. If the variable solution is identical with the solution on

to Electrometer

Calomel electrode

FIG. 58. SET-UP FOR MEASURING THE ELECTROMOTIVE EFFECT OF CONCEN'l'RATION OF A FRUIT (ApPLE)

The peel is in contact with different solutions at its opposite ends from which connections are made to the measuring instrument by means of electrodes. The upper contact is made by means of a concentrated solution, which is not varied, while the solution at the lower contact-in the dish-is varied. Electromotive forces arise through the contact of the homogeneous peel with different solutions.

top of the apple (see diagram, Fig. 58), both being, e.g. saturated KCI solutions, the whole arrangement is apparently symmetrical and should have no electromotive force at all. By performing this measurement of a symmetrical arrangement as control, we can make sure whether the fruit used is electrically symmetrical. The usual finding is a small electromotive force, showing that it is practically impossible to find a biological subject which is entirely symmetrical. To avoid

6 See Loeb and Beutnerj Biochem. Zeitschrift, 41, 1 (1913); compare also the control experiments performed by A. Fujita twelve years 'later with the same result described in the same journal, 168, 11.


errors from this source, two subsequent measurements are always performed, one with a more concentrated, the other with a more dilute solution. The difference is the effect of concentration. In this manner, a variation of as much as -Dr volt or more is observed for a dilution from T!i to T1fo-o molecular N aCI. The effect is not identical for all fruits, yet far more easily reversible and reproducible than the effect of concentration in animal tissue.

Apart from its magnitude, an important feature of this effect of concentration is that it can be observed with almo~t any other salt and even with acids or bases. It is of the same direction and order of magnitude in every instance. Thus, substituting KCI for NaCI in the above measurements, the total variation for a change from To to T1fo-1I molecular amounts also to 0.10 to 0.11 volt. The same is observed for most other electrolytes,7 although the magnitude of the effect is somewhat lower for the salts of bivalent metals, like CaCl 2

(Loeb and Beutner, 1913).6 The addition of acid or of alkali influences the electromotive force

in the same direction as the addition of neutral salts, either acid or alkali acting in the same direction as a neutral salt. The acidity (pH value) as such, however, has no influence upon this kind of electromotive force. What counts is the total electrolyte content since all electrolytes act in the same direction. Hence the addition of small amounts of HCI or NaOH to a NaCI solution has no material effect even though the pH value is considerably varied thereby. In order to evaluate the importance of these observations we may compare them to the well known properties 'of metallic electrodes. If we use in the place of the fruit, a solid piece of metal, we would observe the same relation between concentration and po,tential difference, provided that the salt in the variable solution is a salt of the metal itself. One such system is the well known silver concentration cell of Nernst, viz.,

- Ag ) dilute AgNOa solution 1 concentrated AgNOa solution I Ag +

In order to compare this system to the one with a fruit, we have to change the arrangement of the constituents, viz.:

- concentrated AgNOa solution I Ag I dilute AgNOa solution +

7 It may be mentioned that the following salts of monovalent metals have been found to show the same effect of concentration as KCI or NaCl: NaBr, Na 2SO., LiCI, RbCI, KI, KSCN, KN0 3, also HCI and NaOH.


comparable to

- concentrated salt solution I fruit I dilute salt solution +

The essential similarity between these two systems is, (1) the identity of the direction of the electromotive force; (2) the nearly identical magnitude.

According to a well known formula derived by Nernst, the silver salt cell has an electromotive force of 0.058 log'cI/c2 volt at room temperature where CI and C2 are the two AgNO s concentrations. For a variation of concentration in the ratio 1: 100, as e.g., diluting from 0.1 to 0.001 M, the electromotive force should vary 0.058 log 100 = 0.116 volt which agrees with the value observed.

The essential difference between the Ag cell and the cell with a fruit in the center is that nothing but Ag ions act in this Ag cell while almost any ions may act in the latter case. The apple peel is a homogeneous, wax-like covering, consisting chemically of a mixture of fatty acids and higher alcohols, and hence is lipoidal in character. Cell membranes may have a different character and that has given rise to extensive discussions as to their nature. No such uncertainty prevails in this case. Little or no chemical change of this wax-like substance in contact with neutral salt solutions of varying concentration seems possible. In this respect the similarity to a metallic electrode is also striking; the silver metal, likewise remains chemically unchanged if it is in contact with a dilute or concentrated solution of AgNOs and yet the potential difference clianges considerably, depending on the concentration.

The variable potential difference appears, therefo~e, to be located at the very boundary of the solution. Consequently the electrical changes following variations of concentration should be expected to be instantaneous. Observations show that this is the case (Loeb and Beutner, 1913).6 If an apple is first placed in a 0.1 molecular solution of KCI for instance, as in the apparatus shown above (Fig. 58), an electromotive force of 0.03 volts is measured. On exchanging the -h molecular solution for a 0.0008 M solution of KCI, the electromotive force rises instantaneously to 0.10 volt. On applying again -h molecular KCI solution, the electromotive force immediately drops to 0.03 volt.

However, this reversibility is observed only if weak solutions are


used. For solutions more concentrated than one-half molecular, the reversibility is less perfect. MoreOiJer, at higher concentrations, the effect of concentration becomes smaller. Thus, for instance, while a five fold dilution from rtlf to 0.0008 M was found to change the electromotive force at the cuticula to the extent of 0.035 volt, a five fold

dilution from 2.5 to 0.5 M caused a change of no more than 0.02 volt. (In the range of the lower concentrations the electromotive variation is almost as large as calculated from Nernst's logarithmic formula, viz., 0.058 Ig 5 = 0.040 volt.) It appears, therefore, that a perfect reversibility is observed whenever the variability is maximal (or approaching the value calculated from the logarithmic formula).

The question arises whether the described electromotive phenomena are not due to osmosis. If this were the case, they should be influenced by the addition of such non-electrolytes as sugars, which do not penetrate cell membranes and hence cause osmosis. The experiment shows, however, the complete absence of any influence of this kind. The addition of sugar or any other non-penetrating nonelectrolyte has no influence. On the contrary, an influence is exercised by the addition of those non-electrolytes which penetrate membranes, and hence are devoid of osmotic action, viz., lipolytics. The osmotic and the electromotive actions of membranes are therefore properties which exclude each other (R. Beutner, 1913).8 Even the addition of an overwhelming excess of sugar to a dilute salt solution has no influence. If, e.g., a 0.01 M NaCI solution is in the dish of the apparatus in contact with a fruit, as much cane sugar can be added so as'to have a concentration of 0.5 M (about 17 per cent) without any noticeable change of the electromotive force. To illustrate the action of penetrating nonelectrolytes, we may consider the effect of an addition of alcohol, ether or other so-called lipolytics. The variation of the electromotive force caused by such additions is always to the negative side. For example, if 5 per cent ethyl-alcohol is added to a 0.001 M solution in contact with the apple the electromotive force changes to 0.022 volt; if 10 per cent is added, it changes to 0.007 volt and still more for higher additions. The side to which alcohol is added is on the negative pole.

8 For literature see R. Beutner "Entstehung elektrischer Strome in Geweben," Stuttgart, 1920.


4. THE ELECTROMOTIVE EFFECT OF THE CONCENTRATION IN ANIMAL

TISSUE

The electrical effects, which were described, would deserve little interest if they were the exclusive property of plant cuticula, but, this is not the case. Similar effects can be observed on animal organs, although technical difficulties interfere with the observations, for the following reasons. An excised organ like a nerve or muscle, has a moist surface. The superficial moist layers adhere and cannot readily be removed. Hence, the structures which give rise to electromotive effects of concentration are not well accessible in this case. In fact it is never definitely known exactly what solution is in contact with the muscle or nerve fiber. Moreover, a variable short circuit is formed by the moist surface and the interfibrillar fluid. For these reasons, the apple experiment described above cannot be duplicated with excised muscle or nerve, although observation shows that when these organs are immersed in a series of NaCI solutions of increasing dilution, successively, the electromotive variability has the same trend as on the cuticula. But, the variations are irregular. Also the magnitude is much smaller.

These difficulties can be avoided, to a certain degree, in measurements on nerves, if the excised nerves, viz., sciatic nerves of frogs, are first placed in an isotonic glucose solution for about. two hours. The fluid on the surface and between the fibers is thus replaced by an electrolyte free solution. A nerve thus prepared is immersed at one end in a more concentrated KCI solution, e.g., T~molecular, on the other end in a more dilute one, viz., Tio--U- molecular. An electromotive force of about 0.07 volt is then measured. 9 This indicates that the nerve fibers themselves show a considerable electromotive effect of concentration. The maximal effect for a ratio of concentration such as 1: 100 should be o.li volt. This is not attained entirely, but probably would be attained if the adhering salts could be removed from the fiber completely.

The internal structures of animal organs of vital importance seem to have, therefore, the same electromotive properties, resembling metallic

9 These results are quoted from H. Netter, see Pfliigers Archi v, 218, 310 (1927). 'Netter attempts to show that his findings substantiate the pore theory of Michaelis which he takes to be the best explanation for the elcetromotive effect of concentration. The insufficiency of the pQre theory from a physicochemical point of view is, however, apparent (see page 207, note).


electrodes as cuticula does, although they are not equally well accessible to observation. Incidentally it may be added that an electromotive

. effect of concentration can also be demonstrated at the external intact surface of the animal organism. A favorable object of demonstration iK the human skin, particularly of the finger or finger nail. The variation is about as large as on the nerve, viz., 0.06 volt for a variation of concentration in the ratio 1: 1000 (Loeb and Beutner, 1913).6

5. THE ARTIFICIAL IMITATION OF THE MAXIMAL EFFECT OF CONCENTRATION (R. Beutner,- 1912)10

A certain resemblance of many living tissue membranes to metallic electrodes is the main finding of the experiments described. The problem is to find the underlying physical cause of this electrode-like effect. Above all, it should be emphasized that these effects are by no means "vital." Dead tissue may also exhibit them. Thus, if the fruit or leaf is placed in ether for a day, the effect of concentration remains unchanged, even though this treatment has certainly annihilated all "life" properties. Manifestly, some substance contained in the outermost layer of the apple skin is responsible for the electrode-like action, no matter whether the plant is dead or alive.

As stated before, the plant cuticula consists of fatty acids and higher alcohols. If this chemical composition is the cause of the effect of concentration, an artificial mixture of similar substances should exhibit similar electromotive effects. Experiments show that this is the case. The electromotive effect of concentration can be reproduced artificially by a number of substances which are more or less chemically similar to the substance of cuticula, such as various esters, aldehydes or phenol derivatives, if a fat soluble acid is dissolved in these water-immiscible fluids. As an example we may quote, salicylic aldehyde which through self-oxidation in the air invariably contains considerable amounts of salicylic acid.

Thus the electromotive force of the system:

- electrode I 0.1 M KCI I salicylic I 0.0008 M KCI I electrode + aldehyde

10 Am. Journ. Physiology, 31, 343.


equals 0.10 volt (R. Beutner, 1913)11 which is at least equal to that observed when cuticula is used as a central conductor.

Solutions of higher fatty acids in a non-aqueous solvent like cresol or an aldehyde also exhibit the maximal effect of concentration. This mixture is manifestly similar to the composition of the plant cuticula since the higher alcohols, contained in the cuticula next to the fatty acids, are chemically similar to cresol (Loeb and Beutner, 1913).12

For the measurement of the electromotive forces of systems with such non-aqueous central conductors, an arrangement may be used

ID (/et;lrM,efer

FIG. 59. EXPERIMENTAL ARRANGEMENT FOR MEASURING THE ELECTROMOTIVE EFFECTS OCCURRING AT THE CONTACT OF A NON-AQUEOUS CONDUCTOR AND

AQUEOUS SOLUTION

The non-aqueous fluid in the U-tube attached to the electrode is in contact with saturated KCI on the one side and with a variable aqueou,s solution on the other just as the apple peel on Figure 58. By varying the concentration in the beaker a maximal electromotive effect of concentration can be observed for certain non-aqueous centra_l conductors. (R. Beutner, 1912.)

similar to the one shown in Figure .57, but, for consecutive measurements of a series of dilutions it is more advisable to use an apparatus as represented diagrammatically in Figure 59, where the cresol-oleic acid is contained in a U tube attached to a calomel electrode. This tube is immersed successively in a series of Kel solutions of increasing dilutions and the variation of the electromotive force of the system is

11 Zeitschrift Electrochem. 19, 45 and 319. With salicylic aldehyde a successful imitation of an electromotive effcct of concentration has been first achieved in the writer's own laboratory.



observed. In one series of measurements, for example, the following was measured (see table):

Difference of Compare this e.rn.f. using to the follow-

ing values guaiacol + obtained by oleic acid in using an apple center in center

volt oolt

Changing from 0.5 M to 0.1 M KCi.. ............... 0.028 0.020 Changing from 0.1 M to 0.02 M KCI. ....... , .... ' ... 0.028 0.028 Changing from 0.02 M to 0.004 M KCI. ............ 0.032 0.034

Total change for 0.5 M to 0.004 M KCl. .......... 0.088 0.083

By using the same method the following effects of concentration were observed with the following lipoidal extracts as central conductors, for a change of concentration from 110 to 12\0 molecular KOI:

volt Guaiacol extract from egg-yolk ........ , ........... , .......... 0.10 Guaiacol extract from muscle ................................. 0.09 Guaiacol extract from frog's skin ............................. 0.09 Guaiacol plus 10 per cent tri-olein ............................ 0.085

These substances show all the peculiarities of the effect of concentration as manifested by plants or nerves, in that various salts may be used, KCl and N aCZ among many others. Moreover, the gradual decrease of the effect of concentration at higher concentrations can be observed. In the same way as on tissues, non-electrolytes, like sugars, which do not penetrate, have only a slight effect in this case. Penetrating non-electrolytes, like alcohol, lower the electromotive force in the same way. (For instance, the addition of 10 per cent alcohol lowers the electromotive force of a system with salicylic aldehyde in 2

10 molecular KCl solution 0.01

volt, 20 per cent alcohol 0.02 volt.) Similar to the biological electromotive forces, the electromotive

forcE's of cell systems with these non-aqueous solvents are constant. They are also reversible under all those conditions in which reversibility had been found for plant cuticula as explained before, viz., for diluted aqueous solution. As already explained, the effect of concentration decreases or disappears entirely at the highest salt concentrations. This decrease can also be observed for many of the non-aqueous central conductors named.


It should be emphasized that a few selected substances only can produce electromotive forces of this magnitude following insertion between KCl solutions as stated. Guaiacol without oleic acid will produce no more than 0.03 volt (in the same direction) for a change from 1 ~ to 12

150 molecular-which, of course, is entirely too small. Other sub

stances which produce similar small effects are 0, m, or p cresol, phenol or other OR compounds, like the higher alcohols, also ketones like acetophenone or benzophenone, many esters like ethylacetate, ·butylacetate, aceto-acetic ester. Moreover, such small electromotive effects of concentration are not limited to water immiscible substances as middle conductors. It is well known that the insertion of any slightly acid aqueous solution between two Kel solutions of different concentration produces an electromotive force, also certain alkaline solutions, alkaline gelatin, agar, kaolin, membranes of paraffin or rubber. In fact it is almost more difficult to find a material which fails to exhibit any electromotive effect of concentration than one which does. It would be useless, therefore, if we tried to identify such small effects of concentration with similarly small and uncontrollable effects produced by tissues, such as excised muscle. Almost anything will work as a model to imitate such small effects, hence nothing but tM maximal effect of concentration deserves a detailed study, this being the only property which is exhibited by a somewhat limited number of substances.13

13 Confusion has arisen in the literature due to failure to consider these facts; small electromotive effects have been taken to be equivalent to the maximal effect of concentration, for instance by Loeb in his meritorious book on "Proteins and Colloidal Behavior," New York', 1922. According to Loeb's own experiments on gelatin, quoted in that book, the effect of concentration on gelatin amounts to no more than about one-fifth of that on plants and about one-third of that on nerves.

R. Rober and his pupils, Matsuo, Mond and Deutsch have likewise studied the effect of concentration on proteins. Their figures are still smaller than Loeb's figures. Nevertheless, Rober claims that protein membranes exclusively are useful models of bioelectric currents (Zeitschrift physik. Chemie, 110, 142 (1924)). Rober tries to support his protein theory by observations on the effects of various salts at equal concentration as described on page 209, all of which are small and atypical. These alleged protein effects can also be reproduced by starch paste, moist kaolin or agar, hence they are just "water effects" as Beutner and Menitoff have shown (Proceed. Soc. Exper. Biology and Med., 24, 462 (1927)).


6. THE INVERSE ELECTROMOTIVE EFFECT OF ORGANIC BASES, THE

DOUBLE-SIDED EFFECT OF GELATIN

Most of the non-aqueous fluids, by means of which the peculiarities of the biological electromotive effect of concentration have been reproduced, contain a water insoluble acid or have acid properties themselves. If acid properties are important, we should expect that a basic water immiscible fluid would exhibit electromotive changes in the opposite direction. In fact certain bases, like aniline or toluidine, do exhibit an effect of concentration in the opposite direction. This observation, even though it is of indirect physiological importance only, elucidates the nature of the phenomenon. Toluidine is particularly suitable for studying this phenomenon; using toluidine as a central conductor in the hook shaped tube in the apparatus (Fig. 59) we find a variation of 0.06 volt for a change of concentration from 110

molceular to a 12\ 0 molecular KCI, the latter being on the positive side, hence the effect of concentration is opposite to that on salicylic aldehyde. With all neutral salts such an inverse effect can be observed particularly well with KSCN. The latter substance shows a change of 0.1 volt for a variation of the concentration from /0 molecular to 12

100 molecular (R. Beutner, 1913).11

As already described gelatin or other proteins function as bases at the acid side of the iso-electric point or as acids on the alkaline side (see above, page 33). We should expect, therefore, that gelatin shows an effect of concentration like the cuticula on the alkaline side of the iso-electric point, or an effect like aniline on the acid side of the isoelectric point. This has been found to be the case, although the variations are small; there are effects of concentration in either direction but no maximal effects as already stated.14 Moreover, the potential difference of gelatin is affected by pH variations alone without variation of the total salt content.

Animal organs like the frog's skin which contain protein, exhibit similar electromotive actions as gelatin (Amberson and Klein, 1928).15 This agreement points to the possibility of investigating the chemical

I4 This point is demonstrated independently by measurements of J. Loeb in his book on "Colloidal Behavior" and by Mond, Pfliigers Archiv, 203, 247 (1924).

1& JOlITn. Gen. Physiology, 9, 823.


nature of tissue membranes in detail by means of their electromotive functions.

7. THE ELECTROMOTIVE EFFECT OF CONCENTRATION ON ARTIFICIAL PRECIPITATION MEMBRANES (R. Beutner, 1913)16

In tissues, electromotive forces are evidently not generated by such massive layers of non-aqueous substances as have been used in the

To Blectromet82" --------------~-=

FIG. 60. EXPERIMENTAL ARRANGEMENT FOR MEASU,RING THE MAXIMAL ELECTROMOTIVE EFFECT OF CONCENTRATION ON A PRECIPITATION MEMBRANE

OF COPPERFERROCYANIDE

(R. Beutner, 1913)

experiments described, but, by "membranes" which are generated from aqueous solutions in some way. It is of interest, therefore, to investigate the electromotive properties of artificial precipitation membranes, as for instance the well known membrane of copperferrocyanide. As the following experiments show, such a membrane exhibits under certain conditions the "electrode-like" electromotive properties of tissues, viz., the maximal effect of concentration. The measurements were performed by immersing an open tube containing a gelated (solidified) solution of K4Fe(CN) 6 Uo molecular), in a CUS04

16 Journ. Physical Chern., 17,472.


solution (210 molceular) as shown in Figure 60. When connected

to a measuring instrument, this system showed an electromotive force of as much as 1\ volt or more.

H the CuSO. solution in contact with the membrane is diluted with water, small and irregular electromotive changes are observed, on account of the excess osmotic force acting on the membrane from the inside and leading to gross, visible changes. To avoid such disturbances one may dilute the CuSO, solution with isotonic C\ molecular) glucose solution. But even then the variations are irregular since the membrane disintegrates if CUS04 which is one of the membrane forming constituents, is removed.

"Apparently it is essential to have a constant osmotic pressure and also a constant quantity of CUS04 in the solution, in order to keep up a membrane of constant composition and properties. It was found that, if these conditions were observed, the electromotive force underwent regular and reversible changes if an alk'ali salt was added to the solution in _the beaker. The direction of the change was such that with increasing concentration of the alkali salt the electromotive force became smaller, i.e., the solution in the beaker more negative. The effect is therefore of the same kind as observed with the potential differences on tissue" (R. Beutner, 1913).16

The average electromotive variation for a change of concentration of 1: 5 was found to be 0.040 volt. This is the maximal effect which can be expected, being equal to the variation on plant cuticula and on metallic electrodes. Moreover, it is remarkably constant and reversible. A similar effect of concentration can also be produced by sodium or ammonium salts as well as by acids or by other electrolyt,es.

8. THE MAXIMAL EFFECT OF CONCENTRATION ON COLLODION FILMS

(L. MICHAELIS, 1925)17

Also certain solid substances allow us to imitate the maximal effect uf concentration, viz., collodion in the form of a well dried film (L.

J7 L. Michaelis and his co-workers have explained their discovery of the effect of concentration on collodion_film with an improbable theory; viz., that the film should act exclusively by means of ultra-microscopic pores through which the cations can pass but not the anions. No substantial proof for this explanation is available yet; for further details see footnote to page 226.


Michaelis and Akiji Fujita, 1925).17 We have seen before that collodion films or "membranes" are permeable to all salts and impermeable to colloids only, but, such membranes are prepared without complete drying, the collodion being immersed in water while it still contains some ether and alcohol. A permeable membrane of this kind shows slight electromotive effects only. However, if the collodion film is thoroughly dried in the air before it comes in contact with water, an almost impermeable membrane is obtained. No NaCl or HCl will pass through such films. These dried films, which shrink considerably, exhibit distinctly a maximal effect of concentration. Measurements can be performed by letting the film dry in the shape of a cup. One of the solutions is poured into the cup, the other into a beaker in which the cup is immersed. By connecting electrodes to the inner and outer fluid the effect of concentration can be measured directly. For KCI solutions the following is found:

1 mol. KCI! film / T~ mol. KCI + .............. 0.033 volt - T~ mol. KCI! film / Tto mol. KCI + .............. 0.047 volt - Thmol. KCI! film / T-d'u-o mol. KCI + .............. 0.048 volt

These electromotive forces are quite constant. They are also reversible just as the electromotive forces of the systems with salicylic aldehyde or with cuticula, under certain conditions as explained before (see pages 198 ff. and 203).

Water immiscibility and acid properties have been found to be common to all non-aqueous fluids which imitate the maximal effect of concentration of the cuticula (see above, page 202 ff.). It is noteworthy that collodion has also acid properties, being an ester of a strong acid, HNO 3. Such a compound must necessarily have a certain base binding power, which can be demonstrated byexperiments in the following manner. If nitrocellulose is shaken with a NaCI solution, the solution turns acid, as can be shown by titration with NaOH. Consequently an acid, probably HCl, has been set free, due to the fact that nitrocellulose binds some NaOH (R. Beutner,1932).18 Control experiments have been performed by shaking collodion with pure water. In this case also, some acid was found in the water, but, considerably less than in NaCI solutions after shaking with collodion under comparable conditions.19 The

18 Communication before the XIV. Internat. Physiol. Congress at Rome. 19 Prior to the titration NaCI was added. The difference in acidity cannot

,be due, therefore, to a "salt error."


importance of these experiments for the theory is explained in the following ch~pter (see page 223). Incidentally it may be stated that the CU2Fe(CN)6 membrane has also a certain base binding power.

As shown before, non-aqueous bases produce an effect of concentration in the opposite direction. One should expect, therefore, that a collodion film in wh~ch a sufficient amount of a strong base is dissolved shows likewise such an inverse effect. In fact this is the case as shown by experiments of R. Mond20 on a collodion film to which rhodamine, a basic dye, was added.

9. THE ELECTROMOTIVE EFFECT OF EQUALLY CONCENTRATED SOLU

TIONS OF DIFFERENT SALTS21

Differences of electromotive force are produced on the cuticula of plants, or on other tissue membranes also by different salis at the same concentration. Thus, when using whole (uninjured) leaves or fruits, distinct and reversible variations of the electromotive force are found if a leaf, e.g., is first immersed in a /0 molecular solution of one salt and then in a -to molecular solution of another, using the same experimental arrangement as already described (see Fig. 59). In this way a solution of KCI is found to be negative relative to NaCI on an average of 0.02 volt; HCI again is negative to KCI. CaCI 2, BaCl2

or MgCl 2 are all positive to NaCI, averaging 0.03 to 0.04 volt. Various salts of the same metal, like N aCI, N aBr, N aSCN, produce about the same electromotive force. Similar effects can be observed on animal tissue. Such electromotive actions may also be observed with non-aqueous central conductors, e.g., by using the apparatus shown in Figure 59. If the U tube attached to the calomel electrode is immersed in equimolecular solutions of N aCl, KCI, CaCl 2 or another salt, one after the other, and the electromotive force is measured in each, the same variation is usually observed as with tissues as central conductors. However, this rule suffers numerous exceptions since certain salts produce different electromotive actions, depending on thfl type of central conductor with which they are combined.

As an example of such differential electromotive actions, we may

20 Pfliigers Archiv., 220, 194. 21 Compare R. Beutner, "Entstehung elektrischer Strome im lebenden

Gewebe," Stuttgart, 1920.


mention the effects produced by salts of organic acids 'pr bases. If cresol or other aromatic substances, or mixtures containing these, are used as central conductors, alkali salts of organic acids exhibit a high positive potential relative to NaCI, as is shown fQr instance, by measuring the system:

- 0.1 M NaCI solution/cresol/D.1 M sodium oleate solution + 1

e.m.f. found: 10 volt

On the other hand, chlorides or organic bases, such as aniline HCI or the hydrochlorides of substituted amines, impart an equally high negative potential (relative to most inorganic salts), as is shown by measuring the system:

+ 0.1 M NaCI solution/cresol/D.1 M aniline hydrochloride -

e.m.f. : 0.08 volt

In general, we find that salts with an organic constituent produce a h£gh electromotive force in battery systems with non-aqueous fluids as central conductors, this being in the positive or negative direction, according to whether the positive or negative ion of the respective srilt is an organic ion. However, this effect does not occur with all central conductors since tissue membranes, as well as most precipitation membranes or collodion films, show no such electromotive effect of organic salts.22

As will be explained on the following pages, the electromotive action of organic salts is the result of their solubility in the non-aqueous central conductor. The organic salts are well soluble in cresol or similar fluids and hence have an electromotive action, but they are evidently not equally soluble in the tissue membranes or in the artificial membranes named, and hence devoid of electromotive action on membranes.

As already described, a mixture of cresol and a fatty acid gives rise to a maximal effect of concentration as do tissue membranes. However, the electromotive action of organic salts is different since guaiacol, as well as cresol, give large electromotive variations when used as central conductors, while tissue membranes give no effect, as already stated. Apparently tissue membranes are built up from lipoids without a solvent like guaiacol which, is totally different from

22 See Beutner and Kanda, Zeitschrift fiir physik. Chemie, 107, (1928).


any tissue co:p.stituent. If pure lecithin is used as a central conductor, organic salt~ are found to have no effect, just as in tissue membranes (R. Beutner, 1927).23 The effect of concentration exhibited by pure lecithin is found to be not quite high enough to compare with the maximal e.ffect on plants. The addition of some solvent seems, therefore, ,to be essential in order to produce the maximal effect of concentration. Probably plant cuticula contains a higher alcohol, like cetylalcohol in the place of the cresol. A mixture of such an alcohol with fatty acids would resemble the cuticula both in regard to the effect of concentration and in regard to the absence of the effect of organic salts.24

23 Journ. Pharmacology, 32, lOl. 24 R. Hober and his pupils, Matsuo, Natannsen, Mond and Deutsch, have

used these findings to corroborate their assertion that only proteins furnish suitable models for bioelectricity; denying emphatically the importance of lipoids on account of the different action of many "organic" salts in "oil" cells; the term "oil" designating water immiscible solvents like guaiacol, etc. (see Zeitschrift fUr physikal Chemie, 110, 142; also several articles in Pfliigers Archiv, 13~, 203 and 209). Unfortunately Hober concludes that alllfpoids should act like these "oils." If he or his pupils had performed any experiments on a true lipoid, like lecithin, they would have convinced themselves that such a conclusion is impossible, since lecithin gives rise to the same positive direction of electromotive force as tissue membranes between KClf and propyl amine HCI soluti0l!s as stated.

HISTORICAL REVIEW

Electrophysiology started in 1786 with the famous discovery of Galvani (1737-1798) that a frog muscle twitches when touched with a Zn-Cu couple. He made the correct interpretation of this observation 't'n so far as he recognized the appearance of "electricity" in some form as ,the cause of the twitching of the muscle in his experiment, but, had the mistaken idea that the muscle itself, because of some inherent vital force, created this electricity. Volta's famous experiments proved the incorrectness of this hypothesis (Volta, 1745-1827). Since every combination of Zn and Cu, separated by an electrolyte, produces an electric current, it follows that in Galvani's experiments the frog's leg plays the role of the intermediate salt solution and does not create currents of its own account.

So a polemic marks the very first development of electrophysiology: Volta versus Galvani, and polemics have continued ever since that time. Volta's correct views were generally accepted, but, he himself fell into another error, similar to that of Galvani's, when he stubbornly defended as being the only possible explanation of his epoch-making experiments an entirely incorrect hypothesis: he asserted that the junction of the two metals-Cu and Zn-in the cell arrangement, was the exclusive location where electricity was produced and that living tissue never gives rise to electric currents. Because of the fame of its originator, this view had a great following for about half a century.

It w_as not until fifty years after Volta's discoveries that Du Bois Reymond (1818-96), stimulated by the observations of Mateucci, gave proof by means of sensitive galvanometers that organic tissues do call forth electric currents. Following this discovery an interest for electrophysiology flared up again, but the great expectations fostered by DuBois Reymond were soon disappointed. Being unable to find a rational explanation for the origin of electric currents in tissues, he adopted the theory of bipolar molecules, according to which the smallest particles of living tissue should be positively charged on one end and negatively on the other. These particles were supposed to be present in living tissues only, and arranged there in regular layers, parallel to each other with the posit~'ve or negative ends pointing ~'n the same direction and capable of

212

HISTORICAL REVIEW 213

rotation. This theory £s nothing more than a figurative representat£on of certain experimental observations. Surely countless other similarly figurative representations could be given.25 This one was chosen apparently because Du Bois Reymond strove to link up his theory with one of the most discussed physical theories of his time, viz., Weber's theory of molecular magnets. Such a tendency has always been, and is still now, the greatest damage to physiological research. Why should an up-to-date physical theory be applicable to those physiological problems which happen to be investigated within the same period of time?

Probably the "theory of bipolar molecules" would have gradually sunk into oblivion. However, before this was possible another polemic was started against Du Bois Reymond by his own former pupil, L. H ermann, who without consideration thoroughly condemned the conclusions of his former master.

In the broadest sense Du Bois Reymond's theory had postulated some structure, present in tissue a priori, as the cause of the current and this was certainly correct. Hermann, however, simply branded any such "pre-existence" theory as wrong. He postulated, in his "alteration theory," that living tissue should be devoid of all electric potential differences throughout and that potential diffprences should arise merely as a consequence of death, decay or degradation of life processes. This is contradicted by simple physical evidence which shows that electrical potential differences must exist at every boundary of differentiated structures. The potential differences existing in such a compound mixture as living tissue, cannot be zero in every case. Such a basic postulate as that of the alteration theory could be true only if living tissue were homogeneous throughout, just like a salt solution.

To prove the alleged basic idea that bioelectricity was produced exclusively by degrading changes, Hermann attempted to show that the injury current appeared within a definite time after inflicting the injury. This period of time was found to be extremely short, being about 10

100 of

a second, and yet it was interpreted as the time required for the cut surface of a muscle to pass from life into death! It has also been frequently maintained that the absence of an electric current observed on an entirely uninjured muscle should be a proof of the alteration theory. Yet the absence of a current between electrodes applied outside does not disprove the pre-existence of potential differences inside, these merely compensating each other.

25 Thus a similar figurative explanation has been given in terms of ions and semipermeable membrane corresponding to more modern theories.


It may almost seem as though the alteration theory has been adopted, because of its neglect of physics. No matter how irrational such an attitude may appear, we should remember .how many drawbacks had been experienced in electrophysiology . We should realize that such famous and meritorious investigators as Galvani, Volta and Du Bois Reymond had misused their authority for promulgating some impossible hypothesis. Out of the confusion thus produced, the general opinion finally arose that vital electricity defies all laws of physics. As a result of the prevailing uncertainty, personal views held sway. An author's cleverness in advertising his theory had great influence.

The more recent development of electrophysiology is characterized by the application of physical chemistry. In 1890, W. Ostwald, a well known writer on physicochemistry, advanced the hypothesis that membranes endowed with a differential permeability for positive and negative ions should be the peculiar cause of bioelectricity. This view gave rise to extensive discussions as to the location, the nature and the mode of action of those hypothetical membranes. Just as Du Bois Reymond wished, wdh Ms molecular theory, to introduce into physiology a special theory of his time, so Ostwald purposed later. It is very evident that he drew his inspiration for the theory of the selective permeability of ions from the well known ionic theory of Arrhem·us. As we shall see attempts have been made to support the theory of selective ionic mobility by more extensive experimentation (see page 226). But in spite of all these and other attempts, this. permeability theory has never been substantiated by definite evidence.

DERIVATION OF PHYSICOCHEMICAL LAWS: THE THEORY OF PHASE BOUNDARY POTENTIALSl

From the experiments just described, one gets the impression that the biological production of current is a process which may be fully explained in time on a physical basis. For we have succeeded so far in proving that cell systems composed of organic materials or "membranes" reproduce those typical "electrode-like" electromotive forces which characterize living tissue. Accordingly, the production of bioelectricity primarily appears to be a characteristic of living matter just like color, density, viscosity or other physical qualities. If we seek to connect the production of electric current with certain life processes the first requirement is to broaden our knowledge of the physical nature of elementary electrophysiological phenomena such as those described.

1. PRINCIPLES FOR THE CALCULATION OF THE ELECTROMOTIVE

FORCE OF CELL SYSTEMS IN GENERAL

Modem physics and physical chemistry have left a gap at this point which is of decisive significance for electrophysiology. To be sure, thorough investigations have been made of Volta cells-that is, of combinations of metals and aqueous solutions-and likewise of th. cells which contain exclusively aqueous solutions, but, the study of combinations of aqueous and water-immiscible fluids has hardly been deemed worthy of a consideration. Besides the empiric rules which we have established about these latter combinations in the preceding chapter, we should strive for a more fundamental explanation on the basis of the mechanical theory of heat as in the case of Volta cells.

To explain the origin of an electric current by the mechanical theory of heat is to determine the equivalent of work done. An electric current passing through water decomposes it into hydrogen and oxygen. The resultant mixture of hydrogen and oxygen represents a

1 Compare R. Beutner, "Entstehung elektrischer Stroeme in lebenden Geweben," Stuttgart, 1920.

215


certain amount of energy since it can be used to drive a gas engine, thus performing mechanical work. This amount of energy cannot arise from nothing; it must have been taken from the electric current that formed hydrogen and oxygen. Therefore, the current, in order to bring about the decomposition of the water, must overcome a counter force also called "polarization" which means an electromotive force acting in the opp_osite direction.

In this case the disturbing factor is that the decomposition products are gases and escape rapidly from the electrode. After the current is turned off the greater part of the counter electromotive force disappears rapidly. However, if we send a current from an outside source through a Daniel cell (in the direction indicated by the arrow):

»>----~

Cu I CuSO, solution I ZnSO. solution I Zn

the products of decomposition are identical with the material of which the electrodes consist. The change which is brought about is merely a quantitative one. The amount of the Zn present is increased at the expense of the ZnS04 solution, while the eu decreases to the advantage of the CUS04. . Therefore, in this case also, a current that is sent through the system, from a source outside of it, has to overcome a current-creating force or "electromotive force" which is opposed to it, a force which corresponds to this chemical decomposition. However, since a qualitative change of the composition of the system does not take pla_ce as a result of the passage of the current, this cqunter force must be present even when no current is sent through the system. If the two metals are united by a conducting wire, a current flows through the latter and the chemical change described takes place in consequence. Such a system that does not change its qualitative composition when the current is passed through it, is called a reversible galvanic element. Since, according to this conception, the current of the reversible element is the result of the energy of a chemical reaction, the electromotive force can be calculated from this energy.

Another similar but simpler application of thermodynamics is the calculation of the electromotive force of a concentration cell like

Ag I AgNOa solution concentrated I AgNOa solution dilute I Ag

~--


The underlying change which occurs through the passage of current, in this case, is merely the equalization of the two AgNO a solutions of different content and hence a physical reaction rather than a chemical one. If an electric current passes in the direction indicated by the arrow, the dilute solution becomes more concentrated since silver passes from the metallic electrode into the (dilute) solution. Conversely on the other electrode silver metal is precipitated out of the concentrated solution, electrolytically. The dilute solution, therefore, gains in silver salt, while the content of the concentrated solution decreases.

The amount of work which such an equalization of concentrations can accomplish can be calculated in the same way as the work obtained from the equalization of pressure of two gases which are under different pressure, for the same laws hold for dilute solutions and gases, and likewise for ions (see above, page 26). This consideration leads to a calculation of the electromotive force of this system from the ratio of the concentrations of the two solutions, the result being that the electromotive force when expressed in volts equals

0.058 log· Cl/C2

where c 1 and C2 are the two concentrations. The electrolytic changes which produce this force take place at the

two boundary surfaces of the electrodes and of the aqueous solutions. Thus it is established that the electromotive force is also localized at these two surfaces. The total electromotive force may be divided therefore into two partial forces or "potential differences" which are located at the electrodes. Each of these can be influenced only by the concentration of the one solution which comes in question. A single potential difference can be expressed therefore, as

0.058 log C • const.

Incidentally it may be added that the electromotive determination of hydrogen ion concentrations is performed by measurements of similar systems in which electrodes of platinum saturated with hydrogen are used. These electrodes function as though they consisted of metallic hydrogen; c 1 and c 2

arc, in this case, the H ion concentrations of two solutions. If one hydrogen ion concentration is known the other can be calculated from the electrometric measurement.


2. THE ELECTROMOTIVE PROPERTIES OF PHASE BOUNDARIES; THE

RELATION OF SALT DISTRIBUTION TO ELECTROMOTIVE

FORCES (R. BEUTNER, 1914)2

In the case of a battery system containing a water immiscible central conductor it is not quite so easy to determine the reaction which causes the flow of current. The changes which would occur with the passage of a current cannot be observed directly because it is impossible to drive an appreciable amount of current through such a system on account of its high resistance. One can show, however, that there is a similarity of cell systems with non-aqueous conductors to those with metallic electrodes. Consequently the passage of current would be connected with an equalization of concentrations also in the case of those systems which contain non-aqueous fluids as central conductors. The following general considerations may help to elucidate this poin·t.

A neatly defined phase boundary exists between a non-aqueous water-immiscible fluid, containing ions, and an aqueous solution, just as it does between a metal and a salt solution. With the passage of a current, ions pass from one fluid into the other thus leading to an equalization of the concentrations. In this manner opposing forces are called forth in a similar manner as on the metallic electrodes. The same effect is never produced when two miscible solutions of different concentrations are placed together in such a manner that two layers are formed, one above tpe other. In this case no sharp boundary appears. The forces of diffusion at once pave the way for an equalization of concentration without the cooperation of an electric current and a small fraction only, or nothing at all, of- the available energy is transformed into electric energy.

To prove more precisely the peculiar properties of phase boundaries let us consider theoretically a cell of the following composition.

± metal salt of the same metal salt of the same metal metal =r dissolved in water dissolved in a non

aqueous substance

(the two terminal metals being identical)

2 Zeitschrift fUr physikalische Chemie, 87, 385. See also Beutner, "Entstehung elektrischer Strome in Iebenden Geweben," Stuttgart, 1920.


The aqueous and the non-aqueous solutions of the salt are supposed to be in a state of equilibrium in this cell, that is, the distribution of the salts is that which is obtained after prolonged shaking. In this distribution the aqueous and the non-aqueous concentrations are not in the least similar. One might expect that this system produces a considerable electromotive force, if the partial electromotive forccs at the boundary of the metal are taken into consideration. Nevertheless, according to the principles of thermodynamics, this cell can posaess absolutely no electromotive force, for no process is possible which could accomplish work on account of the equilibration of the entire system.

The absence of current cannot be caused, however, by the equality of the potential differences at the electrodes, it can only be explained by the fact that the effect of the partial electromotive forces a"t the electrodes is counteracted by a third partial electromotive force which has its seat at the boundary surface of the aqueous and the non-aqueous solutions.

This third partial electromotive force must be equal and opposite to the two partial forces at the electrodes, viz., equal to

CI 0.058 log Cl constl - 0.058 log C2 const2 = 0.058 log - . constl at 200

C2

When this formula is employed its derivation should be taken into consideration; in the derivation c 1 and c 2 were taken as concentrations of the same ion, in the two phases. This cannot be changed, to mean total ionic concentrations. In order to apply the formula at all, the two bordering phases must have one or more ions in common.

The result of this derivation may be condensed into the following statement: From the ratio of the concentrations of the common ions in the two bordering phases, the partial electromotive force (or potential difference) at the phase boundary is calculated in exactly the same manner as the potential difference at a metallic electrode.

This result may be applied to determine the direction of the electromotive force of the following two systems (already mentioned).

+ 0.1 M Nnel solution ,I cresol 1 0.1 M aniline Hel - 0.08 volt

!l,nd

- 0.1 M Nael solution I cresol I 0.1 M Na oleate solution + 0.1 volt

As stated before the organic salts are more soluble in cresol, in other words, they penetrate into the cresol or most other organic


fluids to a greater degree than the purely inorganic salts. This can be proved by analytical tests, e.g., by shaking solutions of "organic" or "inorganic" chlorides with cresol and determining the chloride content left in aqueous solutions. It is found that much less chloride is left in the case of the organic chloride because this passes into the cresol.

Other more efficient methods make use of such physical properties, e.g. conductivity, as follows. As one might expect the electric conductivity of a non-aqueous fluid like cresol increases by shaking it with aqueous salt solutions due to the passage of salt and water into the cresol, but, the conductivity increase following the shaking with organic salts enormously exceeds that which is observed following shaking with inorganic salts (R. Beutner, 1914).2

To give an example: A sample of p-cresol was used which had a conductivity of 3.2 reciprocal megohms. After shaking with a fourfold amount of 1~ molecular NaCI the conductivity rose to 6.7. After shaking with KCI to 8 reciprocal megohms, after shaking with aniline HCP however, to about 500 reciprocal megohms and after shaking with lIT molecular sodium oleate the conductivity was 630 reciprocal meghoms.

To elucidate the importance of this finding, we may compare the well known concentration cells

- Ag Ag+ ions in water Ag + lower higher

concentration

with the system mentioned above, viz.: .

- aqueous NaCI I cresol I aqueous sodium oleat~ solution +

The "layer of cresol, adjacent to the NaCl solution, is poorer in Na salt, the layer in contact with the sodium oleate solution is richer in Na salt. The two aqueous solutions contain as nearly as possible, equal amounts of Na ions. The system is, therefore, in reality the following:

- aqueous Na solution Na ions in cresol aqueous Na solution + lower higher

concentration

3 Substitution products of aniline give rise to still higher conductivity values, e.g., dimethyl toluidine Hel to 7300 reciprocal megohms.


In c.nalogy to the Ag cell, the phase boundary between the water and cresol solutions produces electromotive actions similar to the phase boundary between Ag metal and the aqueous AgNO 3 solution. In other words: the two aqueous solutions may be compared to the Ag electrode, the two N a ion solutions in cresol to the two aqueous Ag+ solutions. A further f sential similarity of these systems is that both have a transitional layer in the center between the two solutions of different content. (In this respect they are very different from the currentless system as discussed above.) A suitable combination of phase boundaries and transitional layers is essential for systems of this type in order to produce a current. In fact both systems, the Ag cell and the cell with cresol, produce a current in the same direction. The process, causing the flow of current, is the equalization of different ionic concentrations, viz., of Ag+ ions in the first or Na+ ions in the second instance (R. Beutner).l

In contrast to this example, the opposite flow of current must arise whenever the common ion carries a negative charge, as for instance, in the system containing NaCI and aniline HCI solutions with cresol in the center. In this case, different Cl- ion concentrations arise in the cresol, as demonstrated by conductivity measurements. The negative Cl ions migrate in a direction opposite to that of the positive Na ions if a current is passing. Hence in this case an electromotive force in the opposite direction must arise in order to equalize the

. two different concentrations (R. Beutner, 1920)1 Our theoretical conclusions are summarized in the following

diagram:

NaCl sodium oleate ,---------------"

N + I t· cresol J N + 1 t· + - a aqueous so u lOn Na+ low Na+ high a aqueous so U lOn

direction of current in system capable of equalizing concentrations: ~

N aCI aniline hydrochloride r----------------,

+ CI- aqueous solution cresol J . CI- low Cl- high CI- aqueous solutlOn -

direction of current in system capable of equalizing concentrations: ~

In agreement with the theory, we find that the more penetrating organic chlorides have a negative potential relative to N aCl since the negative Cl-


ion is common to both. The more penetrating organic sodium salts however have a positive potential relative to NaCl since the Na+ ion is common in this case. The essential causative relation between electromotive forces and salt penetration into the non-aqueous phase is thus demonstrated.4

This theory is also applicable quantitatively. The salt penetration can be measured by the increase of conductivity of the non-aqueous phase following shaking with a given salt solution. From this increase, the variation of the electromotive force can be calculated by using the above given logarithmic formula (R. Beutner, 1914).2

Additional evidence for the correctness of the theory is obtained by observing the electromotive action of salt mixtures and the imperfect reversibility following contact of the second phase with solutions of highly penetrating salts. Highly penetrating salts should be expected to influence the electromotive force to a greater extent than slightly penetrating salts in mixtures containing equal fractions. This is verified by measuring the potential difference at the junction of a non-aqueous solvent and a mixed solution of NaCI and an organic salt. The result is that the organic salt determines the potential difference almost exclusively (R. Beutner, 1914).2

Furthermore the greater penetration of organic salts manifests itself by an irreversibility of their electromotive action. Once the central conductor has come into contact, for instance, with an aqueous solution of an aniline salt, a high CI- concentration is produced in the marginal layer of the central conductor. This high CI- concentration cannot be removed from the central conductor by simple contact with a NaCI solution. Hence the potential difference becomes irreversible. For the same reason concentrated salt solutions lead to irreversibility, as already mentioned.

3. EXPLANATION OF THE ELECTROMOTIVE EFFECTS OF CONCEN

TRATION BY THE THEORY OF PHASE BOUNDARY POTENTIAL

(R. BEUTNER, i920)1

The same theory is applicable to those systems which are set up by inserting a non-aqueous layer between two solutions of the same salt

4 This theory has been contested by E. Baur, but, this writer has renounced entirely his antagonistic explanation based on "adsorption potential" and adopted the views explained here [see Zeitschrif_t fUr physikal. Chemie, 92, 81 (1916)J. Compare this with Baur's statements in Zeitschrift fur Electrochemie, 32, 548 (1926).


but having different concentrations. ' In this case also, potential dzJferences at phase boundaries are at play. If the theory was applicable we should expect that, whenever an effect of concentration appears, the concentration of the common ion in the second phase should be independent of the aqueous concentration or, at least, nearly independent. This conclusion seems inevitable since the ratio of ionic concentrations in the two neighbouring phases determines the potential difference. If these two ionic concentrations underwent parallel variaations-as one might expect according to the law of partition-no electromotive effect should arise. However, the fact, which can be demonstrated by experiments, is that the ionic concentrations are not parallel. Thus, for instance, the N a+ concentration in salicylic aldehyde, after shaking it with NaCf solution of varying concentrations, does not vary parallel to the aqueous concentration. This is shown by measurements of the conductivity increase of salicylic aldehyde after shaking it with N ael solutions of varying concentrations. The conductivity increase due to penetration of N a+ ions from the water into the salicylic aldehyde is found not to vary at the same rate as the aqueous concentrations. The same holds for other substances which exhibit the maximal effect of concentration like mixtures of cresol and fatty acid. These experiments indicate the correctness of the theory of phase boundary potentials and also point to exceptions from the law of partition.

The question arises how it is possible that salicylic aldehyde or cresol + fatty acid mixtures, take up nearly the same amount of salt from high or low concentrations of NaGl. We are led to the assumption that a chemical reaction occurs between the non-aqueous acid (as e.g., salicylic or fatty acid) and the NaGI in the adjacent aqueous solution as follows:

HX + NaCI = NaX + HCI

HX denotes salicylic or fatty acid

The amount of soap (or sodium salicylate) formed would depend, according to this assumption, chiefly on the amount of fatty acid present and not on the NaGI concentration. Since NaX accumulates in the second phase, the amount of salt taken up by the non-aqueous solvent would be independent of the aqueous concentration.

(The same assumption may be e:1Cpressed by stating that HX, the fatty acid, exchanges H+ for Na + from NaCl, but that X- is not exchangeable for Cl-.)


The objection may be raised that the postulated chemical reaction is impossible, since HCI will decompose soap to NaCI and free fatty acid-as is well known. That soap can form fatty acid and NaCl, while HCI is liberated, appears contradictory to all chemical experiience, and yet, this reaction does occur, to a slight extent, if a water insoluble solvent is present, as can be demonstrated by tracing the free HCl formed. This surprising finding, which confirms the theory of phase boundary potentials, has first been demonstrated by G. H. A. Clowes5

who found that "emulsions of oil in water to which enough NaOH has been added to render them strongly pink to phenolphthalein may subsequently be decolorized by the addition of moderate amounts of NaCl." This discoloration can only be accounted for by the formation of HCI from NaCI and fatty acid. The formation of soap is thus demonstrated indirectly.

According to the theory, all those non-aqueous mixtures should liberate HCI from NaCI which show an effect of concentration like cuticula or salicylic aldehyde. For collodion films, which show also such an effect of concentration, this has already been described above (see page 208). On the other hand, all those non-aqueous fluids which show an inverse effect of concentration like aniline or toluidine should liberate NaOH from NaC}' Titration experiments performed by Dr. M. Caplan at the suggestion of the writer, have shown that this actually is the case. All conclus~'ons drawn from the theory can thus be verified by experiment. . There i8 hardly any doubt that an accumulation of Na ions, independent to some extent of the aqueous concentration, occurs in cuticula, salicylic aldehyde, collodion film and other 8ub8tances which are capable of producing a maximal electromotive effect of concentration.

Additional evidence, concerning reactions occurring at interphases, can be obtained by electromotive measurements on systems with a non-aqueous central conductor in which both a non-aqueous acid and a non-aqueous base are dissolved. As an example of such a system we may quote the following:6

5 See Journ. Physical Chern., 20, 407. Clowes uses olive oil plus oleic acid which probably would show an effect of concentration although this is difficult to observe due to the very high resistance of the oil.

6 Gelatin as a central conductor between acid and alkali acts similarly to this mixture of amine and fatty acid (see page 235, fig. 63) and likewise glass according to Haber and Klemensiewicz. Glass as a central conductor produces very large electromotive forces, acting like a hydrogen electrode.


- HCI fatty acid and water insoluble NaOH + aqueous solution amine dissolved in a non- aqueous solution

aqueous solvent

The nature of the chemical reactions at both interphases is evident in this casei HCI combines with a part of the amine, and NaOH with the fatty acid. The system is therefore transformed into the following:

Amine HClj fatty acid + amine j soap solution + in non-aqueous solvent

at this interphase positive amine ions are common to both phases, hence the potential difference shIfts to the positive Bide on diluting the HCI.

at this interphase negative fatty acid ions are common to both phases, hence the potential difference shifts to the negative side on diluting the NaOH.

The conclusion is that an increased HCI concentration leads to an increased negative potential and an increased NaOH concentration to a greater positive potential. This has been verified by experiment. (For details concerning these experiments the reader is referred to pUblications of the writer.)!

The results obtained may be summarized as follows: If two phases, which have no common ion, are bordering each other like

A+ C+

B- D-

in nOD. aqueous in solvent water

an exchange of A + against C+ or an exchange of B- against D- or both may occur. Which one of these exchanges takes place depends on whether the <lit AD or the salt BC has a higher solubility in the non-aqueous solvent.

In general, the rule holds that organic salts have a higher solubility in the orgaDj~ solvent than inorganic salts, and salts with two organic constituents more than salts with but one organic constituent. Consequently, organic ions are interchangeable but an inorganic ion and an organic ion are not equally well exchangeable. If both A + and B- are organic constituents, as usual, then the type of exchange depends on whether C + or D- are organic ions.

According to the type of exchange, the electromotive effect of concentration may occur in either direction as follows: the positive pole is on the side of the

'Zeitschrift f. Electrochemie, 19, 45 (1913). Compare also the writer's book on "The Origin of Currents in Tissues."


dilute solution if positive ions are exchanged, or the opposite side if negative ions are exchanged. If tbese rules are applied to the interphase of salicylic aldehyde (or cresol-fatty acid mixtures), and NaCI solutions it is seen that in this case also, the products of the reaction cannot be equally distributed between water and the non-aqueous solvent (viz., salicylic aldehyde). Potassium salicylate, a salt with one organic constituent, is more soluble in this aldehyde than the "strictly inorganic" HCI. Consequently in this case, as in the one already described, a chemical reaction leading to an exchange of cations takes place. Sodium salicylate or potassium salicylate accumulates in the aldehyde. This accumulation leads to the electrode-like effect of concentration (R. Beutner).7

4. SUMMARY OF THE THEORY OF PHASEBOUNDARY POTENTIALS

The theory of phase boundary potentials which has been briefly summarized here, indicates that a boundary between immiscible electrolytic conductors is nearly equivalent to a boundary between a metal and an electrolyte-as far as the production of electric currents is concerned. The relation between salt distribution and electromotive forces, as postulated by the theory, has been verified experimentally on those artificial systems with non-aqueous central conductors which imitate the electromotive properties of tissues. Indirect evidence is thus furnished to show that the same laws hold for tissue. The "electrode-like" function of tissue constituents is thus demonstrated by the application of thermodynamic laws, and may be considered as one of the best understood facts in electrophysiology. One may assert. without exaggeration that thermodynamic laws differ from all other theories in their infallibility; in spite of numerous doubts and recheckings every cha.nge in scientific views has furnished them with new substantiation. 8

8 The theory explained has been contested several times in attempts to explain the electromotive phenomena in question as a result of differential ionic mobility in the second phase. A notable exponent of this explanation is M. Cremer, who has lately changed his views and adopted the explanation given here; compare Cremer's views as expressed in 1906 (see Zeitschrift fiir Biologie, 47,1) with those expressed in 1926 (see Handbuch der normalen und pathologischen Physiologie, 8, II part). 'L. Michaelis still believes that the collodion film requires a special explanation (viz., by differential ionic mobility in the pores) although this film has electromotive actions quite similar to salicylic aldehyde (see several articles in Biochem. Zeitschrift, 158, 159, 161, 162, 164; and in Journal of General Physiology, 8, 10, 11, and 12). Recent experiments by the writer have shown that HCI is formed from nitrocellulose and NaGI in the Bame manner as from oleic acid and N aCl. This demonstrates the applicability of the phase boundary theory under the same conditions as for other central conductors and renders the theory of Michaelis untenable. (See above, page 208.)

APPLICATION OF·PHYSICOCHEMICAL LAWS: THE CAUSE OF ELECTRICAL CURRENTS CIRCULATING INSIDE OF LIVING TISSUES; THE RELATION OF ELECTROMOTIVE FORCES TO STAINABILITY

1. TYPES OF SYSTEMS WHICH CAN PRODUCE, THE INJURY CURRENT

The experience gained from investigations on battery systems with non-aqueous conductors shall now be applied to an old electrophysiological problem, viz., the source of currents circ)llating in and generated by living tissue, such as the so-called "injury current," or in general the internal "electromotive asymmetry" (see page 195). The possibility has already been mentioned that the injury current of a muscle might be the result of the interposition of a membrane between a fluid richer in potassium salts inside the fiber and another fluid outside, viz., lymph, richer in sodium salts. (See page 193, Fig. 57.)

Even though this source of electrical currents may be one factor, evidence is missing that the fiber content has really such electromotive properties as assumed, viz., to give rise to a negative potential in contact with the membrane. The theory is substantiated to some extent by the observation that a muscle, which has imbibed isotonic KCI solution, has no injury current. In this case, the interfibrillar NaCI is partly replaced by KCI, and the asymmetry, by which an electromotive force can be generated, disappears.

However, this explanation, even if correct for the muscle, cannot be applied to plant tissues as the following observation shows. An apple gives rise to an injury current like the muscle, a cut surface being negative, relative to an uninjured one, to the extent of 0.02 volt. But, this injury current cannot be accounted for by a negativating action of the sap. Even though the sap contains acids and potassium salts, it fails to show any negativity if brought in contact with the uninjured apple peel. The electromotive action of the sap is even slightly more positive than that of a T\ molecular KCI solution, it is equivalent to a 0\ molecular KCI solution. This can be demonstrated by placing a whole apple in the sap and measuring the electromotive force by using the apparatus shown in Figure 58 (page 196).

227


In order to make the evidence more conclusive, analogous experiments should be performed on single cells. The sap from a single cell should be applied to the outside of the cell and the electromotive force measured as described. It has actually been possible to perforIl,l such tests on Valonia Macrophysa since the individual cells of this plant reach a length of 2 inches or more (Osterhout).! These cells

. consist of a thin layer of protoplasm (containing numerous chloroplasts and nuclei) outside of which lies the cell wall, while the interior part of the cell consists of the very large central vacuole filled with cell sap. In order to study the electromotive action of the sap of Valonia, a capillary is inserted in this cell and the cell itself is immersed in Valonia cell sap. It is manifest that if the membranes were symmetrical an arrangement of this kind should have no electromotive force. Experiments show however, that this is not the case (Osterhout, Damon and Jacques, 1927).1 An electromotive force arises with sap on both sides, the capillary inserted in the cell being on the'positive side.2

The only possible conclusion from this finding is that there must be at least two different non-aqueous conductors at the inner and the outer layers, in the case of Valonia.

The following instructive experiment performed with an apple indicates, in this case also, the presence of at least two different nonaqueous conductors. If we press upon the surface of the apple with a finger and then lead off from the squeezed surface, we_lind that it is negative to a normal uninjured part. To exclude errors, the electromotive force is measured first between two identical spots on the surface of the fruit. Then, one spot is pressed with a finger and the experiment repeated. A considerable electromotive force, viz., 0.04 to 0.06 volt appears as a result of this pressure. The preSilure, therefore, produces an electromotive change in the same d'irection and of the same magnitude g,s cutting or peeling the apple would produce. Yet, the effect of the concentration of the pressed peel is as large as that of the normal peel, and has the same direction, as the following measurement shows. When the lead at the squeezed spot is varied from T~ to Th molecular KCI, the electromotive force varies 0,05 volt. It is then varied from Th to T-ho molecular KCI and the electromotive force again varies 0.05 volt, the lead at the other spot being constant.

1 Journ. Gen, Physiol., 11, 193; see also 13, 207. 2 This would be therefore an injury current in the inverse direction.


The peel, therefore, still exhibits the normal electromotive effect of concentration, showing that the upper layers have not been broken. They stay in the pathway of the current.

What is the nature of the variation which causes the negative potential at the squeezed spot? According to the conception suggested before, the following answer would seem possible. The internal and more delicate cell layers are torn up by the pressure. In this way the direct electrical contact of cuticula and the membraneous constituents of the pulp, parenchyma or rather collenchyma is broken. Thus sap flows out in between them. In other words, an aqueous solution, the sap, becomes inserted between the two kinds of non-aqueous conductors. Such an insertion must necessarily change the total electromotive force in such a way that the squeezed spot becomes negative (R. Beutner, 1920).3 In order to elucidate 'this statement, we may consider the effect of pressure upon an apple, cut in half, which, of course, produces a normal "current of injury." However, if the peel is squeezed, the electromotive force of the fragment nearly drops to zero due to a negativity arising therefrom in a direction opposite to the injury current and nearly equal to it. If we assume that the apple is built up from at least two constituents or "membranes" having different electromotive action, the system would be

before squeezing the peel:

+ saline I membrane I I membrane II I saline -

This system produces an injury current

after squeezing:

+ saline [ membrane I I sap I membrane II I saline -

1 2 3 4

The latter system has no electromotive force since the potential differences at 1 and 2 antagonize each other and likewise those at 3 and 4

(Compare figures 61, I and III, turn to next page.) By comparison to biphasic battery systems the effect of insertion

of sap may be elucidated as shown in diagrams, Figs. 61, II and IV. The two non-aqueous conductors may be phenol and ether or another

3 See R. Beutner, "Entstehung elektrischer Stroeme in lebenden Geweben," Stuttgart, 1920.

230

I.

ll. + o.zVOlt

THIRD ATTEMPT AT APPROACH

Diagrammatic presentation of a fragment of an apple which shows a normal current of injury. The cell membranes of the sarcocarp are uninjured and enclose the sap which is thus electromotively inactive. The inner and outer membranes are in direct contact.

Diagrammatic presentation of a battery system with two non-aqueous conductors both of which are in direct contact. This system represents the electrical condition of the apple fragment and explains the origin of the current of injury.

Diagrammatic presentation of the effect of squeezing the surface of the apple. The hard and elastic outer peel has not been brokenFbut, the electrical connection between the inner and outer membranes has been interrupted, allowing an effusion of cell sap in between ~he two layers.

Diagrammatic presentation of a model representing the apple fragment after squeezing. The electromotive force now has dropped to zero, just as the electromotive force of the fragment has been changed by the effusion following squeezing.

FIG. 61. DIAGRAMS TO ILLUSTRATE THE ELECTROMOTlVE EFF]lCTS WHICH RESULT FROM PRESSURE EXERTED ON THE ApPLE PEEL

From R. Beutner, "Entstehung elektrischer Stroeme in lebenden Geweben," Stuttgart, 1920.


pair of similar solvents. Experiments show that considerable electromotive forces may arise by direct contact of two solvents of this kind in a system such as indicated diagrammatically in Fig. 61, II. For instance, the system

+ aqueous saline I phenol I ether I aqueous solution -

has an electromotive force of about 0.2 volt. (Both phenol and ether are saturated with water.) For measuring the electromotive force of such a system, the apparatus shown in Fig. 62 may be used. To give another example, the system

+ aqueous saline I phenol I amylalcohol I aqueous saline

has an electromotive force of about 0.11 volt. Similar combinations might be set up from two different precipita

tion membranes or from two films consisting of different kinds of collodion. It seems that an unlimited variety of such systems is possible. It remains to be determined which one of these corresponds most closely to the system underlying the injury current.

By application of the theory of phase boundaries we can account for such electromotive forces by means of the experimental fact that Nael or other salts penetrate more into phenol than into ether (or amyl-alcohol). This must lead to a higher N a+ ion concentration in the marginal layer of the phenol, taking into account the additional fact that phenol has a slight electromotive effect of concentration and hence tends to accumulate Nil, + ions preferably. According to the principles already explained, such a preferential accumulation of Na + ions on one side of the central conductors must lead to an electromotive force in the observed direction.

However, the application of the theory is somewhat difficult in this case, due to the presence of two different non-aqueous substances. It is simpler to apply it to a system like

+ aqueous saline

non-aqueous solvent containing fatty

acid

non-aqueous solvent without fatty acid

aqueous -saline

using as solvent nitrobenzene, or cresol (saturated with water); in the place of a fatty acid such an organic acid as picric acid or benzoic acid or its derivatives might he used. Such systems have been found to produce an electromotive force of about 10 volt.


The solvent containing fatty acid takes up more sodium salt on account of the chemical reaction occurring at the interphase as shown before (see page 223). Consequently the system is really the following:

Na+ in aqueous I Na+ high Na+ low I Na+ in aqueous solution in non-aqueous solvent solution

This is equivalent to a system like:

+ Na salicylate solution I solvent I NaCI solution -

which has been mentioned before.

2. THE RELATION OF DIFFERENTIAL STAINABILITY TO ELECTROMO

TIVE FORCES (BEUTNER AND LOZNER, 1930)4

The lesson to be learned from these experiments is that electromotive forces may arise in tissues by contact of structures which have a different chemical composition. As is well known, the differentiation of tissue structures is usually investigated by means of their stainability, preferably by means of their differential stainability by basic and acid dyes. Thus, for instance, the nucleus of fixed white blood cells stains ·intensely with methylene blue, ,basic fuschsin, rhodamine or other basic dyes, but, remains more or less unstained when the cell is placed in eosin, erythrosin or other acid dyes. The cytoplasm of the white blood cell exhibits the opposite relationship, being more stainable by the acid dyes. Numerous other tissue structures exhibit likewise such a differential stainability.

In order to determine what electromotive forces can arise by the contact of differentially stainable structures, we may prepare mixtures from fats, fatty acids or proteins, which show also a differential stainability and we may use such mixtures to set up battery systems the electromotive forces of which are measured. For this end, the following experiments were carried out'(Beutner, Lozner and Caywood, 1930).4 Fat, preferably olive oil, or other neutral esters were used as central conductors. These were rendered "basophilic" by the additon of a large amount of a higher fatty acid, as for instance, oleic

, Protoplasma, 10, 1, 12, 52 145, 380.


acid. A corresponding acidophilic mixture was prepared by the addition to the olive oil of a fat soluble base, viz., amylamine or diamylamine in molecular concentration. A differential staining effect

S/7vel' elec/pode

I I

About o,l/IOlt

Silvsf' electrode

FIG. 62. DIAGRAM OF A SYSTEM CONSISTING OF AN ACIDOPHILIC AND A BASOPHILIC SUBSTANCE (NON-AQUEOUS FLUID) PRODUCING AN ELECTROMOTIVE

FORCE

One U-tube contains the acidophilic, the other the basophilic mixture, both being connected by an inverted U tube.

The other ends of the basophilic and the acidophilic mixtures are in contact with saline which is held in sponges, in this case, to prevent dropping down into the non-aqueous mixture. Two silver chloride electrodes are used to make connections to the measurin~ instrument. Each electrode consists of a silver wire, coated with AgCl, whiCh is inserted in a tube filled with gelated saline, having this shape: ~

Similar systems may account for electromotive forces occurring in tissues, in some cases.

From R. Beutner and Joseph Lozner, "The Relation of Life to Electricity." Part IV. Published in Protoplasma.


may be produced with such oil mixtures by shaking them with a few drops of a histological stain like Wright's stain which contains eosin and methylene blue. This will stain the fatty acid mixture blue by means of the basic methylene blue, while the amine containing mixture takes up the red eosin. These colors will show up clearly if, after the staining, the oil mixtures are washed with an excess of water, this procedure being analogous to the procedure in histological technique. Similar basophilic or acidophilic mixtures can be obtained from numerous other bases or acids, if these are dissolved in any suitable solvent.

The "acidophilic" and "basophilic" oil mixtures are then combined to form a kind of battery system, in the manner indicated in Figure 62. This system contains two mixtures of differential stainability between two saline solutions.5 These differentially stainable mixtures may be fats such as olive oil with an addition of a fatty acid on the one side and an amine on the other. Or various esters, alcohols, phenol derivatives, or other neutral solvents with the same additions of an amine or a fatty acid in every instance may be used instead of olive oil. These non-aqueous mixtures are always saturated with water. Preferably such neutral solvents are used which are not entirely insulating. Hence pure hydrocarbons would be less suitable. The electromotive force of a large number of such systems has been measured. The observation was made in nearly every instance that the "basophilic" mixture was on the positive pole, and the "acidophilic' mixture on the negative pole. The magnitude of the electromotive force of such systems was found on the average to be as high as that produced by tissues. In many cases it was even higher, suggesting that the measurable biological electromotive fprces are usually diminished by internal short circuits (Beutner and Lozner).4

From the theory explained before, this finding must be expected since phaseboundary potential differences are located between the fatty mixtures and the alkaline saline in contact with them. These potential differences must be differentiated in the direction actually observed since sodium oleate, oilsoluble, is formed on one side from oleic acid and the NaHCO. in the aqueous solution. This leads to an excess of Na ions on that side. On the other side the presence of amine has no such action. The amine, on the contrary, combines with any oil-soluble acid constituents which may be present, e.g., oleic

6 These solutions are alkalinized by the addition of sodium bicarbonate in order to imitate the conditions in tissues more satisfactorily.


acid from saponification of olive oil-and thus tends to diminish the concentration of the Na + ions in the olive oil. Consequently the cell arrangement, mentioned above, is really a concentration cell in regard to Na + ions:

+ Na+ in water I Na+ in oil I Na+ in oil I Na+ in water -about 0.1 M high (Na oleate) low (amine) about 0.1 M

According to the laws explained above, this system must have an electromotive force in the direction observed.

It is noteworthy that the "stainability" of a non-aqueous mixture, in the sense of the word used here, can also be influenced by the addi-

to electrode _____ _ _----- to electrode

+

J,asoph//ic .sIgMaN/it!

FIG. 63. DIAGRAM INDICATING THE ORIGIN OF AN ELECTROMOTIVE FORCE BY INTERPOSITION OF ANON-AQUEOUS CENTRAL CONDUCTOR BETWEEN

AQUEOUS ACID AND ALKALI

Gelatin may be used in the place of the fatty mixture, with the same result

tion of water-soluble acid or alkali, and by the same addition it is possible to influence the potential differences at the junction with an aqueous solution. This influence works in the opposite direction for both "stainability" and potential differences. Thus, an addition of HOl leads to "acidophilic staining," in the sense explained before, and to a more negative potential, while NaOH works in the opposite direction, as is to be expected. The acid or the alkali is added in this case to the


aqueous phase. The electrical effects are best observed by the insertion of a central conductor containing fatty acid and amine between two solutions of different pH, as indicated diagrammatically in Figure 63. According to the theory of phase boundary potentials this effect is to be expected, as already explained. The staining effects are self-evident, since HeI will extract basic dyes from fatty mixtures but not acidic dyes. NaOH has the opposite effect.

Since both electromotive forces and differential staining are influenced in opposite directions by water-soluble and by water-insoluble acids or bases, the general empirical rule that basophilic matter is on the positive pole and acidophilic matter on the negative pole, holds in every case for oil mixtures and for fixed cells no matter how the changes in stainability are brought about.

If we want to use these findings to explain ·the stainability of tissue, we have to realize that, for the experiments described so far, mixtures containing fats or fat solvents have been used. But, the tissue structures which give rise to differential stainability cannot consist entirely of fat, since differential stainability does not disappear when tissue sections have been subjected to the ordinary histological technique which consists of paraffin imbedding, extraction, with benzol, ether, alcohol, etc. However, the described relationship between potential differences and stainability is of such a general nature that it holds for proteins as well as for fats. This can be shown by measuring the system.

- aqueous acid J gelatin I aqueous alkali +

This system has an electromotive force similar to the system described above, which contained a fatty mixture between acid and alkali (Fig. 63). Also the stainability is influenced correspondingly. Gelatin on the alkaline side of the iso-electric point (pH = 4.7) stains with basic fuchsin, neutral red or other basic dyes (see page 33). In other words, alkali has rendered it basophilic. On the acid side of the iso-electric point, the reverse is observed, gelatin is preferably stained by acidic dyes, such as acid fuchsin. 6 Thus, in this case also, the basophilic mixture is on the positive pole.

S This was found a long time ago by Loeb. See Loeb, "Protein and Colloidal Behavior," New York, 1922.


Instead of adding aqueous acid or alkali to gelatin, one may think of setting up a battery system consisting of a protein containing more acid groupings on the one side, and another protein with a basic character on the other. Such a system should give rise to an electromotive force between two identical aqueous solutions. Although this experiment has not yet been performed, it seems probable that just such systems are found in the tissues, since the stainable constituents cannot be extracted by means of fat solvents, as explained.

The question arises whether this relationship between stainability and electric potential differences can also be verified in tissues. Measurements, performed at the suggestion of Keller by Gicklhorn and Umrath, 7 seem to show that acidophilic tissue is at the negative pole relative to basophilic tissue. These investigators have inserted micro electrodes into plant tissues and found that structures which stain more intensely with basic dyes are at the positive pole. However, there are certain exceptions to this rule. Sections of plant tissue were used for these experiments. These sections can hardly be considered as entirely living.

In living tissue, on the other hand, such a differential stainability cannot be readily observed because of the toxicity of most dyes and because of the inhibition of their diffusion by living membranes. There is some evidence for "acidophily" of the nucleus, since neutral red, a basic dye,-when injected into the nucleus according to R. Chambers' techniqueS-rapidly diffuses from the nucleus into the cytoplasm (1927).9

Chambers (1930)10 describes that one amphoteric dye, viz., methyl red, will enter into certain living cells such as Ameba, sea-urchin egg, or plant cells, provided that the environing medium is at pH 6, which means more acid than the cytoplasm of the cell. At pH 6 methyl red is still at the alkaline side of its isoelectric point, hence functions as an acid dye. From the cytoplasm, methyl red then diffuses into the nucleus. Since, according to Chambers, 1927,9 the

7 Protoplasma, 4, 228 (1928). 8 Bolle Lee, "Microtomist's Vademecum," Chap. 30, 9th edition, London,

1928. See article in McClung's "Microscopical Technique," Chap. II, New York, 1929.

9 Journ. Gen. Physiol., 10, 739. The author is much indebted to Dr. R. Chambers for bis expert advice concerning these questions.

10 Proceed. Soc. Exper. BioI. Med., 27, 809.


nucleus contains an alkaline aqueous solution, methyl red apparently tends to diffuse from a more acid into a less acid medium. This diffusion inside of purely aqueous media and not a true stainability of water-immiscible cell structures seems therefore to be the cause of "vital" coloration. It can also be understood that, under these conditions, the living cell nucleus throws out basic dyes as stated, while the nucleus of fixed leucocytes attracts them. Nevertheless, the presence of chemically differentiated structures seems indicated, in living cells also. The relation between coloration and electromotive forces is reversed in this case. The nucleus attracts acid dyes although it is on the positive pole.

Moreover, we should consider that the contact of structures exhibiting differential stainability constitutes one possible mode of origin of electric currents in tissues, but, by no means the only one. As we have already seen, considerable electromotive forces can arise also through the contact of two heterogeneous substances such as phenol and ether. In this case, no differential stainability is found since phenol takes up both acid and basic dyes considerably, while ether takes up both to a slight extent. There is also some evidence tending to show that the cuticula and the parenchyma which produce the injury current of the apple have no differential stainability, but, are two heterogeneous conductors com-parable to phenol and ether. The same may be true of other tissue structures.

All the essential findings concerning the bioelectric currents stated so far may be condensed into the following statement. It has been found before that the essential property of the potential differences which compose biological electromotive forces is their similartty to metals. (See pages 197ff,226.) . In agreement with this result the findings, described on the last pages, may be condensed into the statement that tissue structures or membranes are differentiated also electrically in a marmer comparable to the difference of metallic electrodes of which commercial batteries are composed. The essential feature of the "vital battery system" is that the current producing asymmetry is not limited to the aqueous components. In other words, it is not always a question of a homogeneous membrane separating two different solutions. On the contrary, it appears that the membrane itself may be heterogeneous and thus may produce a current in contact with one kind of an aqueous solution, resembling a lead accumulator which also contains one aqueous electrolyte (H2S04 )


but two different metal electrodes. We have seen that the electric properties of the different "electrode-like" membranes are accessible to investigation by means of studies on stainability.

3. THE RELATIONSHIP OF METABOLISM TO ELECTRIC CURRENTS IN

TISSUE

It has been known for a long time that a relationship exists between electric currents in tissues and metabolism. In many cases dying or degenerating tissue, having an impaired metabolism, has been found to be at the negative pole relative to healthy tissue having a normal metabolism. This general statement is, of course, too indefinite. Moreover, numerous exceptions to this general rule have been found. More definite experimental evidence about the relationship of electric currents and metabolism was established, recently only, by limiting the observations to one definite kind of metabolism which is accessible to quantitative measurement, viz., respiration (E. J. Lund and collaborators, 1927),u Increased respiration was found to shift the potential difference in tissues to the positive side. This points to a relationship between electric currents and metabolism in general since respira tion is a determining factor for almost any kind of metabolic change.

On a growing onion root tip, Lund has demonstrated this influence of' respiration upon electromotive forces. He uses sliding electrodes by which connections from any portion of the root can be made to a measuring instrument.12 A peculiar distribution of electric potentials can thus be traced. Taking the upper section of the growing tip as the arbitrary zero of potential, a region near the tip is found to be highly positive. In fact this is the region of the most active cell division, high protoplasmic content and most active respiration as evidenced by a most intensive reduction of methylene blue. This "positive" active zone extends about 0.5 cm. from the tip upwards and is followed by an inactive and electrically negative zone. This active zone is probably identical with the "stretching" zone already mentioned. (See page 64.)

11 Journ. Exper. Zoology, 48, 333. 12 Various types of arrangements can be used; for detailed description see

publications of E. J. Lund, Journ. Exper. Zoology.


In order to test the reduction of methylene blue, which occurs with varying velocity in different regions, the root tip is inserted in a glass capillary filled with methylene blue. The velocity of reduction of this dye, which is a redox "indicator" (see above, page 151) is parallel to the intensity of respiration. Comparative experiments show that the greatest intensity of methylene blue reduction and "positive" potentials are invariably associated at the same level (Lund and Kenyon).

In an even more striking manner, the interrelation of bio-electric currents and respiration can be demonstrated by the following method on frog's skin (Lund, 1928).13 An excised piece of frog's skin is in contact with Ringer solution both on the inside and outside. On account of the different composition of its inner and outer layers, the skin gives rise to a small electromotive force. This force can be shown to depend on the oxygen content of the Ringer solution, in other words, on the oxygen supplied to the skin. With abundant oxygen supply the electromotive force amounts to about 50 millivolts. If oxygen is withdrawn it drops to about 5 millivolts. If oxygen is again supplied, it rises to the former range, but decreases abruptly if the respiration is inhibited by KeN. "A continued production of electrical energy depends among other conditions on the availability of free oxygen" (Lund).

In, order to analyze the physical cause of this dependency, a relationship between oxidation and electromotive forces should be traced in artificial systems. At the suggestion of the writer, Dr. J. Lozner measured the electromotive forces of combinatiop.s of oxidized products against such non-oxidized products from which they might have been formed. For example:

heptylic alcohol ~ 'heptylic aldehyde ~ heptylic acid C6HISCH20H +0 C6H1SCOH +02 C6H13CO,H

or mixtures of these were measured against phenol, in the apparatus described above (see page 233)., Some of his findings were:

.aU

- saline / alcohol/alcohol + 50 per cent aldehyde I saline I + ... 0.02 - saline / alcohol/alcohol + 10 per cent acid / saline / + ... 0.04

These measurements show that with progressing oxidation the electromotive force becomes more positive on the oxidized side, just

13 Journ. Exper. Zoology, 61, 291.


as in Lund's experiments on onion roots, the more vigorously respiring portions are electrically more positive. In numerous other instances oxidation products can be shown to be "positive," especially in all those cases where a water-insoluble acid is formed by oxidation.

To evalute the significance of such experiments it is necessary to realize that in living tissue such oxidation products are constantly being formed. Hence the vital battery system is not fixed like the artificial system which was set up for the sake of investigation. In living tissue a continuous oxygen consumption and a shifting of the chemical composition occurs, or as Lund expresses it, a "flux equilibrium." To account for this peculiarity, it would seem necessary to assume that the end products of oxidation are devoid of electromotive activity, and that the electromotive action depends on certain intermediate products. In fact the end product is probably CO2

which is volatile, and hence without action.

However, not all products of oxidation have a relatively positive potential. Conforming to experiments already described a positive potential can be expected only if the product of oxidation is acidic and soluble in the second phase. The conclusion, therefore, would be that the oxidation due to respiration in tissues leads to intermediate products of such a kind.

Another aspect of the interrelation of bioelectricity and metabolism is the following. As already stated, respiration is a determining factor for most other metabolic changes chiefly on account of the simultaneous formation of high molecular compounds. While a portion of the organic matter is burned to CO2, another portion is condensed to unknown higher compounds. Such changes can also be observed in the auto-oxidation of an unsaturated fat, as for instance, linseed oil. On the other hand, in the absence of respiration in tissues, chemical decomposition seems to prevail. This would explain why more actively respiring tissues show a greater content of high molecular compounds of some kind as described by Lund. Consequently, the higher positive potential of respiring tissues is conditioned not merely by oxidized products but also by higher molecular compounds which are the indirect product of respiration. In order to elucidate the possible mechanism by which molecular size may influence electromotive forces, a series of artificial systems containing homologous fatty acid has been measured.u The apparatus used for such

14 Beutner and Lozner, Protoplasma, 12, 380 (1930).


measurements is similar to that shown in Figure 62. Figure 64 shows how the stainability was found to vary regularly along with the molecular weight. The electromotive forces vary concomitantly.

All these findings can be readily understood as the result of graded variations of oil and water-solubility according to the length of carbonic chains of the various homologous fatty acids. As stated before, HCI, which is quite watersoluble, and oleic acid, which is quite oil-soluble, influence both electromotive forces and stainability in opposite directions. The electrical and tinctorial properties of the lower fatty acids vary between these extremes.

Mixture. containing

Acetic Butyric Caproic Caprilic Capric Myristic Stearic Oleic Acid Acid Acid . Acid Acid Acid Acid Acid --- --- --- --- --- --- --- ---

Relative voltage measured ...... 0.0 +0.01 +0.04 +0.06 +0.10 +0.12 +0.15 +018

Each of the test t\lpes contains 3 cc. of a solution of 0.1 M amyl amine +0.1 M of any definite fatty acid, as indicated, in amylacetate; for staining, one drop of a one per cent solution of safranine is added, and each sample is then washed with a double volume of water. The washing water is !leparated and drained off, leaving the stained and washed amylacetate layer. The picture shows distinctly how the higher fatty acids in the amylacetate mixture tend to hold the dye more than the lower fatty acids. The electromotive forces were measured in an arrangement similar to that shown in Figure 62.

FIG. 64. STAINABILITY AND ELECTROMOTIVE FORCES OF MIXTURES CONTAINING HOMOLOGOUS FATTY ACIDS

From R. Beutner and Joseph Lozner, "The Relation of Life to Electricity." Part IV, published in Protoplasma, Gebr. Borntraeger, Leipzig, 1931.


For a biological application of these findings, we should remember the most widely accepted electro-physiological rule, viz., that normal or "healthy" tissue is electrically positive to degenerated or dead tissue.

If the process of death or dissolution is associated with a breaking down of higher molecular compounds, then, we should expect that in any artificial system-which suitably reproduces the conditions in vivo-the split products or the mixture containing them are electrically negative to the mixture containing the high molecular compounds from which the split products are formed. This is exactly what we have found; the lower fatty acids are negative to the higher ones. That the lower fatty acids are in fact split products of the higher ones in metabolic processes in the organism is also well established. Oxidation of a fatty acid always occurs at the second carbon atom of the C0 2H group. Thus, a series of homologous acids is formed similar to those used in our experiments described above. I5

Such a breaking down is not limited to fats. Water soluble low molecular substances are invariably formed at the expense of higher molecular ones, in decomposition of tissue, until it liquefies. For example, glycogen breaks down to lactacidogen and lactic acid; proteins to peptones, amino acids and urea. It remains for future investigations on other artificial models to study the electric effects of these decompositions.

A diminution of stainability following exhaustion and finally death of tissue is also well known. A well known example of this kind is the partial disappear-· ance of the so-called Nissl bodies, a type of stainable granules in nerve cells, after exhaustion. IS Accordingly, we have found that the split products (viz., the lower fatty acids) have a diminished stainability. This agreement may be

16 Such a gradual breaking down of fats or fatty acids by p-oxidation is well known and described in many text books of Physiological Chemistry, cf. e.g. A. P. Mathews, "Physiological Chemistry," 4th edition, 1925, p. 81ff. If the splitting is due to oxidases it is certainly of the type described; Knoop demonstrated the production of lower fatty acids by p-oxidation by perfusing isolated decomposing liver. Oxidation may also occur in the absence of oxygen if there are other hydrogen acceptors.

16 A complete review of the literature pertaining to the relation of stainability and functional activity is not possible here; see E. S. Cow dry (Science, 68, 138) who finds an outstanding stainability of the nucleus of tumor cells from rat sarcoma. Cowdry uses Feulgen's method of nuclear stainability in which thymo-nucleic acid "is· stained by means of fuchsin sulphurous acid. These experiments indicate that the tumor cells, corresponding to their abnormal power of growth, have an extraordinarily high content of a nucleic acid and consequently an abnormally large stainability.


taken to indicate that our artificial systems imitate one possible type of arrangement which produces electric currents in tissue.

The same conclusion is reached by studying the variation of the electric resistance of dying tissue. The electrical resistance of living tissue is almost entirely due to the membranes in it. If the tissue dies the membranes gradually dissolve, consequently the resistance drops. This is the usual occurrence after a sufficient time. A striking fact, however, is that in the beginning of degeneration the resistance of the tissue increases i e.g., in a freshly excised frog's muscle the resistance at first goes up before the opposite variation sets in. This initial rise of the resistance can only be explained as a result of the decomposition of higher fatty acids; or similar compounds. The higher fatty acids and bases which are dissolved in the membranes tend to lower its resistance. When the tissue dies these fatty acids are transformed into lower ones which are more water-soluble and so must pass from the membranes into the neighboring aqueous phase. The resistance of the membranes and consequently the resistance of the tissue as a whole is thus increased, at least temporarily. In fact it can be shown that solvent mixtures with lower fatty acids have a higher resistance than those with higher ones,17 after shaking with aqueous saline. This accounts for the initial increase of the electric resistance.

17 For detailed measurements see Beutner, Mann and Blanton, Protoplasma, 12,498.

APPLICATION OF PHYSICOCHEMICAL LAWS CONTINUED AND FURTHER EXPERIMENTS WITH MODELS: STIMULATION AS AN ELECTRICAL POLARIZATION OR "BATTERY CHARGE;" THE NATURE OF THE PROPAGATION OF ELECTRICAL DISTURBANCES IN TISSUES; THEORY OF NARCOSIS

As we have seen electromotive forces may arise in tissue anywhere, depending on its structure and chemical composition, on metabolic changes or on respiration. Frequently such electromotive forces give rise to circulating currents in tissue which in turn may either drive fluid through membranes, as explained above (see pages 58-60), or else they act as a determining factor in the process of growth as proven by Lund's experiments which demonstrate the importance of electric polarity in growing plants, etc. (see above, page 18). The most important electric effect, however, is that of stimulation. The irritability of living tissue, the conduction of the impulse and its effects are so closely linked with electrical currents and electrochemical alterations that one may well describe them as an electrochemical process.

1. THE LAWS OF ELECTRICAL STIMULATION, CHRONAXIE AND

RHEOBASE l

In order to understand the nature of irritability, it is first of all important to know its mode of action. By artificial means tissues can be stimulated either mechanically, chemically, or by heat, but all these actions can hardly be similar to a natural stimulation since this may occur at extremely short intervals-in some cases a hundred times per second-over considerable periods of time with no damage to the tissue. Just one artificial stimulation can be repeated at equally short intervals with the production of nearly identical results, viz., the stimulation by means of electric currents. This seems to indicate that natural stimuli are likewise electrical in nature. As a perceptible sign an electrical wave, the so-called negative variation, spreads along the nerve simultaneously with the excitation.

1 Louis Lapicque, "L'excitabilite en fonction du temps," Paris, Presses Universitaires, 1926.

245


Investigations on electrical stimulation of nerve and muscle date back about one hundred and fifty years to the time of Galvani. Strange though it may seem, not until quite recently has it been possible to determine accurately the laws governing electrical stimulation. A sound development has been handicapped for at least half a century by the exclusive application of the so~caned "law of DuBois Reymond," according to which the variation of intensity of an electric current should be the only cause of excitation. That this law is insufficient can be proven, for instance, by observations on so-called "Tesla" currents.2 These are alternating currents of exceedingly high frequency, viz., 100,000 to one million cycles per second, consequently the rate of variation is enormously large. Nevertheless, these currents fail to produce any stimulation even at the highest current intensity. It is possible to send Tesla currents which will light up a large incandescent lamp through the human body without the slightest sensation to the man who holds the lamp and conne~ts it to the current by his own body. In clinical work such currents are made use of for the so-called diathermic treatment, since they yield heat. A physical phenomenon is well known which depends on variation of current intensity, viz., induction. Tesla currents induce powerful currents in a secondary coil. Their physiological inertia)s in striking contrast to their power of induction. This shows that variations of current intensity cannot be the only factor which determines stimulation. The same is proven by expe~iments on condenser discharge, or by other tests.

It can be shown that an electrical current has to pass for a certain minimal time in order to stimulate. The minimal time required for stimulation is exceedingly short, for striated muscle averaging Ti-hor second. Special apparatus is necessary for determining this minimal time, asfor instance, the ballistic rheotome of Weiss, in which the making and the breaking of the stimulating current is effected by means of a bullet, shot from a gun. The current begins to flow through the nerve when a bullet breaks the nearest wire, and is interrupted when a second wire is broken (see diagram, Fig. 65). By means of such a device currents can be sent through the sciatic nerve of a frog which fail to stimulate not on account of an insufficient intensity but merely due to the exceedingly short time of their passage.

2 Formerly the physiological pecularities of Tesla currents were a perfect mystery; such utterly impossible explanations were discussed, as the assumption that these currents pass merely over the surface of the human body.


Sensory or motor nerves and the striated muscle in the higher animals, are the most rapidly functioning organs: this means that even short durations of currents suffice to stimulate. In order to find a threshold, we have to descend to such exceedingly short periods as those named. There are, however, other tissues which function much, more slowly, as for instance, smooth muscle. For these the minimal duration of current amounts to a whole second or several tenths of a second, depending on the conditions. Currents extending over such periods can be limited by much simpler devices, as for instance by a suitable pendulum, or by rotating devices.

4 volt 'battery

,:;....+-.... electrodes

FIG. 65. WIRING DIAGRAM OF THE BALLISTIC RHEOTOME OF WIESS (ACCORDING TO LAPICQUE)

The bullet makes the current flow through the nerve by breaking the first wire. The bullet interrupts the current flow through the nerve by breaking the second wire. If the two wires are about 5 em. apart, the time interval is Ti~Ti of a second.

The striated muscles of slowly moving animals, as for instance, those of the toad, are intermediate between the striated and smooth muscles of frogs. On the other hand, the movements of certain plants, as e.g., of spirogyra, are still slower than those of smooth muscle; the minimal duration amounts to about 5 to 20 seconds, enabling one to determine it by taking the time with a stop-watch. Thus a complete gradation exists. Tissues of animals or plants representing any degree of "velocity" can be found, the minimal time required to stimulate the "fastest" ones averages less than a thousandth of a second, while for the "slowest" ones it is about a hundred thousand times as long.


Parallel to the minimal time required for stimulation the rate of conduction of the muscles varies, being faster for striated than for smooth muscle and still slower for plants.

intensity o£

stimulating

current

intensity of

stimula ting

current

rheobase

I

thousandths of second Time required to attain stimulation of frog's gastrocnemius

rheobase

'" 8 1.2 10 hundredths of second

Time required to attain fltimulation of toad's gastrocnemius

FIG. 66. TIME RELATIONS FOR A QUICKER AND A SLOWER MUSCLE

If the duration of an indirect current is extended over its minimal duration, it produces no further stimulation as long as it flows continuously. If the current is interrupted aI;lother stimulation occurs, viz., the so-called "break stimulation."

As one might expect the minimal duration of current necessary


for stimulation decreases with the increased intensity of this current3

and it increases if the current intensity is lowered. However, in this latter case, a new observation is made. The current intensity cannot be lowered under a certain value, for sufficiently weak currents fail to stimulate no matter how long they are allowed to pass. This minimal current intensity can be easily determined by a single closure of circuit for a comparatively· long time, say several seconds, although for "fast" organs one hundredth of a second represents a sufficiently long time. This minimal current is called the rheobasic current or the "rheobase" (Lapicque).l By plotting the variability of the minimal time with the current intensity for organs of different "velocity," the general trend of the curves is invariably the same in spite of the great difference of the time values (see Fig. 66). This indicates that probably one common underlying change causes stimulation in every case.

In order to have a common standard of comparison for the different time axes, the minimal time for a current having twice the intensity of the "rheobasic" current is selected and this is called the "chronaxie" (Lapicque).l This time constant is the most important characteristic of excitability. The following values have been measured:

For sciatic and gastrocnemius of frog .......... , .... . For sciatic and gastrocnemius of toad ............... . For stomach of frog ................................. . For spirogyra ................................... about

second8

0.0003-0.0004 0.02

i 8

Since chronaxie and rheobase are independent of each other, the irritability of any tissue can be defined only by at least two independent entities. Failure to consider this fact has handicapped investigations on irritability up to the present time. Thus, e.g., a study of the influence of temperature has formerly yielded unexplainable contradictory results, in some cases the irritability seemed to increase, in other cases to'decrease depending on the method of observation. 4 Rational measurements show that a rise in temperature raises the rheobase and depresses the chronaxie (Lapicque).

3 To vary the intensity of current in a circuit, as for instance, the one shown on the diagram (Fig. 65) the voltage applied is varied by a so-called potential reducer which is a shunt, short circuiting the battery and allowing one to make connections at variable intersections to obtain any voltage desired smaller than that of the battery.

4 Gotch and Macdonald, e.g. (Journ. Physiol., 20, 283, 1896), found that the threshold decreases with rising temperature if induction shocks are used for stimulation, but the opposite findings were described by them when condenser discharges were used.


Moreover, such studies are important for the theory of drug actions. The action of curare, for instance, can be explained according to Lapicque, by the difference of its action on nerve and muscle. While the unpoisoned muscle and nerve both have an equal chronaxie after the application of curare, that of the muscle is increased enormously, while that of the nerve remains constant. The normally existing iso-chronism of muscle and nerve, therefore, disappears and this accounts for curarization. It is superfluous to assume the intermediation of a junctional substance between nerve and muscle (Lapicque).

So far we have assumed that the making of the current was instantaneous. If, on the contrary, the circuit of the stimulating current is closed gradually, for instance, by means of a shunt, the stimulating action is considerably diminished to a degree corresponding to the decrease of the rate with which the current is made. By a gradual increase of current intensity relatively strong currents can be passed through the nerve without stimulation. It is possible to "slip in" the current. Likewise a gradual decrease is not stimulating in contrast to an abrupt discontinuation. Hence the change of current intensity is likewise a factor-as duBois Reymond thought-although by no means the only one. The minimal rate of change necessary for stimulation varies inversely with the chronaxie. For this reason a slowly increasing and decreasing current may still stimulate smooth muscle.

2. ELECTRICAL POLARIZATION OR BATTERY CIlARGE AS THE CAUSE

OF STIMULATION (L. LAPICQUE, 1926)1

The more important conclusions, described thus far, may be condensed into one statement, namely, that a cert[!,in amount of electricity has to pass'before stimulatiop occurs and that its passage must occur within both maximum and minimum time limits. What kind of change can a definite amount of electricity bring about if it is forced through the tissue within an appropriate time? There must necessarily occur a change of the electromotive forces located at all the phase boundaries in the tissues. As we have seen, these phase boundaries act similarly to metallic electrodes as far as the production of electric currents is concerned. Now, we are led to assume that they are likewise similar to metals in regard to the ,passage of currents. On metallic electrodes the current brings about chemical reactions-termed electrolysis-and consequently electromotive variations (see above,


pages 215-217). These are usually called electric polarization. In the case of a commercial storage battery we speak of the "charge," effected by the current, which is no more than another name for the same action. In tissues, the same should occur. There should be chemical, or in other words, "electrolytic" changes at the "electrode-like" phase boundaries and these in turn should entail electromotive changes or electric polarization at membranes. This polarization may be assumed to be an important factor for stimulation.

FIG. 67. DIAGRAM OF ApPARATUS FOR STUDYING THEl TIlII:E RElLATION OF MElII:BRANEl POLARIZATION ACCORDING TO LAPICQUE

The question presents itself whether the relationship between time and intensity for a current polarizing a membrane is the same as that of a stimulating current. That typical curve, universally veri-fied for the time relation of stimulation, as described, should be found again for the polarization or "charge" of membranes. In order to prove this, the following experiments have been performed by Lapicque, using a precipitation membrane of CaHP04• A glass vessel is used as shown in Figure 67 which contains a molecular CaCb solution in the central chamber and a saturated Na2HP04 in the two lateral chambers. The two solutions when mixed form insoluble


calcium phosphate. In this apparatus they are separated by two porous films of defatted pigs bladder within which a calcium phosphate membrane will form. The terminal portions of the two lateral chambers are shaped as U tubes and two pair of electrodes are arranged, to make connections either to a battery which polarizes the two membranes or to a galvanometer which measures the effect produced by the current. These alternating contacts are made by shifting the free level of the fluid in the two terminal U-tubes with a suitable

rheobase

t/",e '10 .se,onas reyui,.ea t()

q#fifln ~iyel1 1'(J/tYrJ,url/pn

FIG. 68. TIME RELATION OF A CURRENT WHICH POLARIZES A MEMBRANE

device, not shown in the diagram. Since different electrodes are used for introducing the current and for measuring the polarization, a disturbing polarizing effect on the electrodes themselves will not be measured. Since the resistance of the whole system may be assumed to remain nearly constant, the current sent into it is practically proportional to the voltage applied.

Measurements were performed with this apparatus in such a way


that the intensity of the polarizing current was varied from one experiment to another. In each case its duration was so adjusted, following sufficient experimentation, that the deviation of a galvanometer which measured the polarization was always the same. In this way, a time intensity relation can be determined under conditions resembling the physiological experiment with the ballistic rheotome. For, if we assume that stimulation occurs whenever a certain polarization is attained, then the nerve-muscle preparation in the rheotome is really equivalent to the galvanometer in the present experiment. The preparation, as well as the galvanometer, both indicate the point at which a certain polarization is attained. The result found is that the time-intensity relation for the polarization is quite analogous to this relation for stimulation, as a graphic representation of Lapicque's findings show. (See curve, Fig. 68.) In this case, as for stimulation, the curve ends parallel to the time axis, in other words, if the polarizing current is below a certain minimum, the desired degree of polarization can never be attained just as a sub-minimal current can never stimulate. This analogy evidently favors our assumption that electrical polarization is one of the possible factors which determine stimulation.

3. HISTORICAL REMARKS, UNSATISFACTORY MATHEMATICAL

THEORIES

The entire physiological literature, from Galvani's time on, contains observations which, in the light of our present knowledge, point to the electrical character of stimulation, but, in the previous century the recognition of this fact was handicapped by the then altogether deficient knowledge of the role of membranes, polarization, etc. It remained for a famous physicist to first point out these relations, viz., W. Nernst in 1899.5

Lapicque correctly evaluates the merits as well as the drawbacks of Nernst's work as follows:6 "By one of those synthetic glances ('coups d'oeil') which precede and dominate every analysis and which characterize the genius, Nernst perceives that an electric polarization is at the base of the problem. As soon as this polarization is recognized

6 Nachrichten der Koniglichen Gesellschaft Gottingen, 1899, p. 104. 6 See Lapicque's book on excitability, p. 155.


to be the only possible mechanism for the exciting action of the current, the diminution of the efficiency of alternating currents with their frequency becomes evident. Since the physical effects of each wave are destroyed by an equal wave in the opposite direction, the physiological effect cannot be attained, not even by an intensive wave if this is rapidly followed by the opposite wave . .... "

"But," Lapicque continues, "almost all further developments (by Nernst) are subject to criticism." This is true particularly of Nernst's mathematical theory7 according to which the minimal time required for stimulation should be inversely proportional to the square of the intensity of the current; in other words, even the feeblest current should stimulate provided that the time be extended sufficiently. Yet, with the feeblest currents stimulation ~r occurs, as already stated.

Evidently Nernst's theoretical development has been based on too simple premises. He assumed that certain ions would be arrested at a membrane during the flow of current, thus creating an increased salt concentration. StimUlation should occur if a certain amount of salt had accumulated at the membrane, in spite of the counteraction of diffusion. That these assumptions are insufficient can be understood from the experimental investigations on phase poundary potentials described above. Even if no current is passing chemical reactions may occur at phase boundaries. How these are shifted by the passage of current is not even considered. Not even on a simple artificial phase boundary is it permissible to describe all the electrolytic changes which a current can bring about by a simple arrest of one kind of ions. How much less justified is it to use such premises in tissues! "Put in. this simplified form it would be an exception to the general rule of biological complexity if the polarization of nerve followed this rule exactly. Yet, a lack of exact fitting has prompted many physiologists to search for stimulating effects other than polarization" (G. H. Bishop).8

Still another deviation from Nernst's theory occurs in the case of very short time intervals,in that with increasing frequency, alternating currents become even less stimulating or polarizing than provided

7 For details of this theory see Nernst's publications; also Lapicque's book quoted before.

8 Am. Journ. Physiol., 84, 417.


for by the theory; but the same is true also for the polarization on metallic electrodes, as shown by P. Hoefer (1927).9

Since the theory is insufficient from a physical and even more so from a physiological point of view no mathematical treatment can make up for its deficiencies. Nevertheless, attempts have been undertaken to remedy the situation by cumbrous mathematics. A. V. Hill, for instance, has undertaken a mathematical attack on the problem.10 One formula may be quoted as an example of the complexity of his treatment.

c + rt (~ + b) + 2:... [b2 (~ _ '!_) _ a3J +?:... 4a3

• e -Dfp,t . cos _.,... b 2 2D 2 3 12 D .,..4 a

~= ----~----~--~~~--~~--~------------------

a r a3 r 4a3 -DEi't c + rt - - - . -- + - . - . e a'

2 2D 12 D .,..4

In general, the difficulties of applying mathematics to biological problems are manifestly very great as long as the cause of the actions is insufficiently understood. Mathematical theories should rather be first applied to the models of nervous conduction described below (e.g., the "iron wire models").

Lapicque has derived another formula-starting from energy considerations-which, although likewise complicated, seems to fit the experimental findings better than the previous theories. But, even thi(l formula can hardly clear up the underlying conditions. The starting point for any of these theories is the play of the counteracting forces-such as electric convection versus diffusion.

A better visualization of their mode of action is obtained from the hydraulic models devised by Lapicque (see his book on excitability), but it seems hardly worth while to describe this in detail since Lillie's model experiments have led much further (see below, pages 259-272).

4. POLAR PROPERTIES OF STIMULATION, ACTION OF CONSTANT

CURRENTS

Electrical polarization is a polar phenomenon as the name implies. In order to charge a battery it is essential to pass the current in the

9 Zeitschrift fur Physik, 45, 261. 10 Journ. Physiol., 40, 190 (1910).


right direction. Consequently the stimulating action of polarization should likewise be polar. It is definitely proven that this is the case since the nerve impulse which is started by the making of the current arises at the cathode,u

For a muscle nerve preparation this can be demonstrated by separating the anode and cathode as far as possible and recording the time interval between the making of the current and the muscle contraction. If the cathode is nearer to the muscle the time interval will be shorter than for reversed poles by a time equal to that necessary for a nerve impulse to travel the distance between the two poles. This shows that the stimulus originates at the cathode. This polarity is demonstrated also by studies on chronaxie as follows. In poisoned nerves the chronaxie is changed. If a section of a nerve is poisoned and one electrode placed in the non-poisoned part, the other in the poisoned area, one will invariably find a chronaxie value corresponding to the area around the cathode.

+

--_.------~------~------~------~----------nerve secondary

current primary current

secondary current

The arrow points indicate the positions of electrodes on the nerve

Cathodic polarization spreads this way+-

Anodic polarization spreads this way-+ -

FIG. 69. DIAGRAM ILLUSTRATING EXTRAPOLAR ELECTROTONIC CURRENT IN NERVE

If a current-which has caused stimulation at the beginning of its passage-is kept flowing through the nerve for several seconds or even minutes, no further stimulation occurs. However, during the flow of the current, some alteration persists which manifests itself by a rise or a decrease of excitability for new stimuli. It is an electrically produced change or "electrotonus." For moderate current intensity,

11 The reverse is true for the breaking stimulus, this being started at the anode. This is true for moderate current intensities only. For the more complicated changes occurring with stronger current, due to "electrotonus," the reader is referred to textbooks of physiology, see "Pfliigers Law."


an increase of irritability occurs at the cathode and a decrease at the anode.12

Direct electrical measurements show that this so-called "electrotonus" is associated with an electric polarization since the electromotive forces of the nerve are found to be different before and after passage of current. More important is the fact that this polarization extends along the nerve. As indicated diagrammatically in Figure 69, the cathodic polariaation extends along the nerve trunk on the one side, the anodic polarization on the other side, both decreasing according to the distance from the primary electrodes. Consequently, if any two

primary. cvrr~/l'i

trl111a'/c . 1'''It¥r/~#lIII1''

No spreading of polarization can occur if the axial wire is left off or if this wire is in direct contact with the poles of the battery.

FIG. 70. MODEL OFAxI'AL CONDUCTION (KERNLEITER MODELL HERRMANN) DEMONSTRATING THE SPREADING OF POLARIZATION

12 This is meant for "make shocks" only. The reverse is true for stimulation occurring at the breaking of current. Manifestly this "break shock" is due to 'the sudden disappearance of'the polarization or electrotonus, which constitutes an abrupt electromotive change. If the polarizing current is very strong, or if it is kept up for an exceedingly long time, other electrotonic changes occur which lead to a diminished irritability near the cathode while at the anode the resistance of the nerve rises so high as to block the passage of the current.

Failure to consider electrotonic variations has induced some recent investigators to doubt the constancy of the rheobase.


points in the extrapolar regions are connected to galvanometers, as indicated in the diagram, currents will be observed in the direction in which the primary current is sent through the nerve. The above described variations of excitability also extend into the extrapolar regions, indicating again the extension of polarization. The intensity of the extrapolar currents increases with the intensity of the polarizing current, when the entire length of nerve section is permeated by the primary current. 'The increase is less on the anodic side than on the cathodic side.

Such extrapolar currents can be imitated by a model consisting of a wire fastened in the axis of a glass tube filled with a salt solution (Herrmann's "Kernleiter Modell" or "model of axial conduction") (see diagram, Fig. 70). Lateral tubes attached at right angles allow for a passage of current through a section of the salt solution, for instance, through the center section as shown in the diagram. Secondary currents will then be seen to arise in both lateral extrapolar regions. Manifestly the analogy to the extrapolar "electrotonic" currents in nerve is satisfactory. In the model, the current passes in the center, partly through the salt solution and partly along the wire. Consequently it produces a polarization where it passes from the solution into the wire and vice versa, viz., an "anodic" one on the one side and a "cathodic" on the other. These polarizations spread along the wire into the extrapolar regions, just as a spreading of polarization or "electrotonus" occurs in nerve. The spreading of the electrical polarization of the nerve can be thus imitated artificially and understood. This is important since this polarization is probably the most essential part of the nerve impulse, as we have seen. But, of course, such a "spreading of the polarization cannot account lor the wave-like traveling of the excitation in nerve.

5. TRAVELING WAVES OF POLARIZATION: TRANSMISSION OF ACTI

VATION IN PASSIVE METALS AS A MODEL OF NERVOUS

CONDUCTION (LILLIE, 1918)13

The automatic traveling of the wave of excitation is the most outstanding property of the nervous function. If stimulation is really due

13 Science 48, 51 (1918); 60, 2594 and 416 (1919). Journ. Gen. Physiol., 3, 107 and 129 (1920). Physiol. Revie}V, 2, 1 (1922).


to electrical polarization it should be possible also for polarization in inorganic systems to exhibit such a wave-like traveling, in some cases. But the mechanisms, discussed so far, fail to show such a feature. Some hitherto unconsidered electric mechanisms must be at play. As long as this additional influence was unknown the electrical theory did not find many proponents and really did not deserve them.

Through the work of R. S. Lillie13 in 1918 and since, this influence is now well understood. His ingenious model experiments have clarified our understanding of the processes underlying nervous activity to a degree hardly anticipated.14 The most important feature of his discoveries is the following: For reproducing traveling waves of electric polarization, a system consisting of two phases, viz., any metal and a salt solution, is insufficient. A third phase must be interposed between the two. This must be unstable and yet capable of regeneration.

A film of iron oxide on a steel wire has such properties. As is well known, steel dissolves easily in acids, even dilute or weak acids. Nevertheless, it fails to dissolve in concentrated nitric acid as it becomes covered with a thin invisible film of oxide. This so-called "passive" steel behaves like a noble metal in most respects, having a spotlessly brilliant aspect and being highly electro-positive in a Volt cell. In strong nitric acid (70 to 80 volumes per cent) this condition is maintained indefinitely. Dilute nitric acid, viz., of 35 volumes per cent or less, dissolves steel wire rapialy, but fails to dissolve passivated steel which has been previously immersed in the strong acid. However, the steel is not as well protected as in 70 or 80 volumes per cent acid, because if the film is broken at one place, it cannot be repaired. The "lesion" extends over the entire surface and continues unchecked until all the iron is dissolved. In somewhat stronger solutions, intermediary from 55 to 65 volumes per cent-"the reaction is temporary and the metal rett?rns spontaneously to the passive condition." If a passivated steel wire suspended in HNO a (55 to 65 volumes per cent) is touched at one end with a piece of ordinary active iron, or with zinc, the film is broken locally at first. ,This "lesion" becomes visible by a darkening of the formerly bright metallic surface and by effervescence, H 2 being developed. The original "lesion" then rapidly repairs itself,

14 Numerous speculations have been advanced in a futile attempt to explain the nervous conduction as a sequence of axial conduction (Hermann, Boruttau, McDonald and others). A mere enumeration of all these theories would be impossible here.


and the local effervescence disappears. But almost instantly the neighboring portions of the wire show effervescence and discoloration, and in turn revert to the passive state, and so on. A wave of temporary activation-visible through effervescence and darkening-thus sweeps over the sur/ace of a passive steel wire. This resembles a traveling nerve impulse in many respects (Lillie).13

Just as in nerve the wave of activation on the steel wire may be started chemically by touching with zinc or with another base metal, as stated, or else it may be initiated mechanically, by bending the wire or tapping it sharply with a glass rod; or it may be started electrically by a local electric circuit at one end of the wire. In the latter

case, the impulse starts at the cathode, as on the nerve. "Activation with the electric current is thus typically a polar phenomenon just as is the excitation of a nerve" (Lillie).13

Moreover, the velocity with which the local state of activity is propagated along the wire is of the same order of magnitude as that of the excitation wave in living tissue, varying between several centimeters or meters per second-depending on the type of wire used and other conditions; both tissues and wire have high temperature coefficients. The steel wire also exhibits an analogy to the so-called "all or none" behavior of irritable living elements. By whatever means the activation wave is excited, the whole wire is involved. As in the nerve "the character, intensity and duration of the reaction are independent of the activating agent" (Lillie) .13

6. PHYSICAL CAUSES OF ACTIVATIOJII AND TRANSMISSION IN THE

STEEL WIRE; THE RECOVERY OF TnANSMISSIVITY, INFLUENCE

OF ELECTRIC CONDUCTIVITY, END TO END TRANSMISSION

It is impossible to determine directly the conditions underlying the propagation of the excitation in nerve. In the passive steel wire,

, however, the cause of the transmission can be analyzed and since nerve and wire act similarly, we can develop a hypothesis, at least, about the essential nature of propagation in nerve. Evidently the comparison of the nerve to a metallic wire cannot be satisfactory in every respect. It would seem more desirable to match the nerve with a system containing membranes or non-aqueous substances with


"electrode-like" electromotive properties, as described before. At present we have to content ourselves with the iron wire model, but we can be satisfied since it exhibits a surprisingly large number of analogies.

Current in this direction builds up film or passivates ("anodic" or oxi

dizing action)

Current in this direction dissolves the film (cathodic or reducing

action)

FIG. 71. DIAGRAM TO ILLUSTRATE THE ACTION OF ELECTRICAL CURRENTS ON THE PASSIVATING FILM ON STEEL WIRE

In order to understand the propagation on the wire, we have to realize that a current passing from the wire to the solution passivates by oxidation while a current in the opposite direction dissolves the film, (see diagram Fig. 71). If the passive steel wire is touched


with a base metal like zinc there must necessarily arise a local circuit resembling a Volta cell since the passive iron is a nobler metal (compare diagram, Fig. 72). The direction of the circulating currents is such as to break up the passivating film on the steel wire near the zinc (compare Figs. 71 and 72). The steel near the zinc is thus activated.1s

After the zinc has been removed the now active spot on the steel wire forms a similar circuit with the still passive steel next to it, activating this in turn, etc. In this manner the effect spreads automatically by means of locally circulating currents (see Fig. 73).

__ ....I.N~it.J:.ic acid._

:. - ------

Nitric acid FIG. 72. DIAGRAM TO ILLUSTRATE How AN ELECTROCHEMICAL EFFECT STARTS

A WAVE OF ACTIVITY ON THE PASSIVE :IRON WIRE

Cathodic reduction causing obliteration of the passivating film, is thus the immediate ag;_ent responsible for the propagation.

A t the same time, that portion of the circulating current which flows in the opposite direction-viz., from the wire to the nitric acid-repassivates each previously active spot by "anodic" oxidation. It is important that this re-passivation is far more intensive than the passive condition existing previously, as is demonstrated by the fact that no transmission occurs in a steel wire immediately after a wave has passed. An analogous phenomenon is a constant feature of the excitation process in all

15 The passivity of iron is explained by some electro chemists without the assumption of a protecting film, but the majority of electrochemists still favor the view that such a film exists, although it is not directly visible.


irritable tissues, viz., the disappearance of irritability during a certain interval succeeding the response to stimulation, viz., the so-called "refractory period." It certainly is noteworthy that even such a rather special feature of nervous excitability finds its analogy in the conduction of waves on the steel wire. It seems likely that, in nerve too, the refractory period is the result of a too intensive repassivation of some sort.

Both in irritable tissue and in the steel wire, the power of transmission is gradually recovered, in the so-called "relative refractory period." "The return of complete transmissivity in 55 per cent nitric acid is in less than one-half a minute. In stronger acids it takes longer; viz., twenty minutes in 90 per cent acid" CLillie).13

lIricht surta c.

-----._--cliscolol'ation las discharge

After the wave ha. passed, it leaves be- Active area in hind it a much heavier film which which the cannot be dissolved by the return currenta

phase of the locally circulating tend to re-currents. Gradually thi. film build the

is thinned out by the acid film

Currents dissolve the film

here, thus Pi~~~t:t-wave of activity

Propagation travels from left to right

FIG, 73. DIAGRAM TO ILLUSTRATE THE MECHANISM OF PROPAGATION OF A TRAVELING WAVE OF POLARIZATION ON A PASSIVE STEEL WIRE

The essential part of this recovery process is the gradual thinning out of the newly deposited, too heavy, passivating film. Further thinning is prevented by the oxidizing action of the nitric acid. Thus, for the iron wire we can understand the cause of the refractory period: since the film is not yet sufficiently "thinned out," the circulating currents cannot reduce it. Consequently the wave works against a large resistance and eventually dies out. The conclusion would be that a reaction of some sort is also responsible for the refractory period in nerve, but, the nature of this reaction is little understood so far.


Locally circulating electric currents thus determine the propagation of the wave on the iron wire. The intensity of these currents depends on the electromotive force of the local circuit-which is constant, equalling 0.7 volt-and on the electrical resistance of the surrounding electrolyte. To observe the influence oj electrical resistance on transmission with passive wires, the wires may be enclosed in glass tubes of different diameter. The more the conducting path is narrowed by glass tubes, the more the speed of transmission is decreased, as one should expect.16 If we assume that nerve fibers are comparable to such enclosed wires, we should expect that the velocity of transmission depends mainly on the fiber size. Experiments by Gasser and Erlanger have shown that this is the caseY

"This, however, applies only to the continuous travel of activation along a steel wire in a tube which is insulated from the outside. If the ends of the wire project through a tube immersed in a larger volume of acid it may happen that the resistance between the two ends of the wire is less than that between the projecting end and a passive area situated a short distance inside the tube" (Lillie). In such a case if one end is activated the other becomes instantly active (see Fig. 74a). The same occurs if a bent passive wire, held in the air is dipped by its two ends into nitric acid (see diagram, Fig. 74b). No "wave" travels along the wire in this case. Activation is transferred from end to end instantaneously by direct. passage of the electric current.

"Some curious results follow froIl). these conditions. When a passive wire is enclosed in an interrupted acid-filled tube immersed completely in acid, and one end is activated, the transmission may be many times more rapid than along a wire in an insulated continuous tube of the same length. In such an experiment the activation effect appears to leap from one interrupted area or "internode" to

16 It is impossible to perform analogous experiments on the nerve since the single nerve fibers are of microscopic dimensions and cannot be enclosed in a capillary. If the well conducting Nael on the surface of the fiber is replaced by an isotonic sugar solution, the conduction of the impulse is retarded. Experiments demonstrating this retardation have been performed on nerves of cold blooded animals (Lillie, et al.). This effect has been ascribed to a diminution of the electric conductivity of the medium surrounding the fiber.

17 This finding was the result of an elaborate determination of the curve of the action potential in the nerve by means of the cathode ray oscillograph (see J. Erlanger's "Analysis of the Action Potential in Nerve," Harvey Lectures, Series XXII, 1926).


the next, and rapidly passes from mid to end of the wire. Each internodal passive area of the wire is the chief cathode for the anode at the neighboring activated internode; hence when any internodal area becomes active it constantly activates the next by distance action. As one might expect and as experimental observations prove, the speed of transmission in a wire enclosed by such an interrupted tube is more rapid than in a wire lying free in the acid" (Lillie, 1925).18

"Conditions analogous to those just described may enter in the case of nerve. For instance, in the most rapidly conducting proto-

a

---- -

In both cases, represented by the two diagrams above and below, the rule holds that, when A is activated, B becomes active instantaneously due to the flow of current without a traveling wave along the wire.

-

FIG. 74. "END TO END" TRANSMISSION

plasmic tracts, viz., the medullated nerves of vertebrates, the conducting element (axone) is enclosed in a medullary sheath which is constricted or interrupted at regular intervals. The medullated nerve transmits impulses at about ten times the velocity of the nonmedullated nerve in which-except for the absence of the segmented sheath-the structure is similar" (Lillie).18 The analogy of the medullated nerve to the wire in an interrupted glass tube seems interesting particularly since the electrical resistance at the internodes is lower.

18 Journ. Gen. Physiol., 7, 473, 1925.


Definite evidence is difficult to obtain, however, since all the nonmedullated nerves are much thinner than the medullated ones. Hence the difference in velocity of conduction may be also accounted for by the fiber size. Whether fiber size or internodes are more important in their influence on velocity remains to be determined by a quantitative method; but, we should remember that prior to the local circuit theory, the possible influence of the internodes was not even considered.

7. FURTHER ANOLOGIES OF THE InoN WInE MODEL AND NERVES:

RHYTHMIC PULSATIONS IN SOFT InON WInE; THEOllY OF

THE PACE MAKEn

Numerous cases of periodic movements of living organs are known such as ciliary waves and C9.rdiM contractions, in which rhythmical waves of excitation continually pnss over the excitable organs at short regular intervals. No ms.tt(lr how exclusively vital these waves may appear, they Can be imitated by a vnriety of artificial inorganic systems, such as "Ostwald's artificial heart of m(lrcury" (se(l page 177). Furthermore, rhythmical pulsations may 09(lUr in the Mse of simple chemical reactionB, I1B for instance, in the reaction betwMn Hg and aqueous H 202. A discharge of oxygen which alternately increases and decreases at regular intervaIB iB obBerved in this case. "A constant feature of each single reaction in ElUch rhythmical ;sequences is the formation of an insoluble reaotion product which covers the metallic surface as a film, thus limiting the reaction; a secondary reaction then removes the film and the reaotion Bots in anew" (Lillie) _Is

According t.o recent. investigntions by R. Willstaetter, which lutve not been published as yet, certain enzymes, particularly oxidaBeB, may alBa exhibit rhythmical chemical reactionB. Manife6tly thill obllcrvation would eeem t.o be of an outBtanding biological importance. (The writer is much indebtAd to ProfeBsor F. Haber for a communication regnrding this finding.)

S6ffJ.(; Il1'5M1m~!H:~ (Jf imll in nitdu {wid LWtJ' llkcwi.;!c cxhibl.b rhythm, Cut steel wires usually show only an irregular rhythm, if they show any at all. A rhythm in which all parts of a long wire are simultane-

19 Science, 67, 593; 69, 305. Journ. Gen. Physi'ol., 31, 1.


ausly active is found only in iron having special properties, the chief of which are rapid and complete transmission and a rapid rgturn of transmissivity after passivation. "Such properties are shown most completely in soft iron of low carbon content)) (Lillie] HJ2g] lQ!W),lu When 6hort pieces of soft iron wire] previously passivated] nre plnced in 60 to 70 per cent nitric acid and touched with !Zinc, mmfllly fl rhythmical reaction sets in at once. , At regular intervflls of one half to one second] the dark effervescent surface of the metal becomes momentarily bright and passiveY In 70 per cent RNO ~ theM passive periods are brief. As the strength of the acid is incrMMd up to 80 per cent, their duration becomes longer.

ltFurlher IJtudy IJhowed that an cIJIJcntia-l condition determining lhe peTlri,6t(;nt rhythm i6 the prGlJfJnce oj 1J0me Zoca-l a-rca- in which the reuelion oj the meta~ with the a-cirl il} contimwul}" (Lillie). Such a continually active region iB formed wherever the mcial is protected against renewal of the acid, for mBtance by meertlng it in a glass tube for a

1"10.70. RHYTIIMlG WhYlllIS IN 130FT hON Wm:tJe

length of 2 to 1: mm. (Boe Fig. 75) nnd onoe notiynting it by touching with llinc. The ncid in the tube iB Boon depleted by tho rell!Qtion. The region in the tube iB, therefore, oonBtantly activtl ll!nd BendB out waves of activation which paBS along the wire!O

"Such a continually active region may be compared with tho TliodaZ or pace making region of the heart or with the basal body (or blopharoplal5~) of a cilium; it exerts a constant activating influence to which the external passive part of the wire responds" (Lillie). Other conditions being equlll, the rilte of the rhythm on the wire in(Jrellses with the tempera" ture. "The correspondenM of the tempernturg cogfficignt with that of the physiological rhythms it;1 striking" (Lillic).l9 Furthermore] {laB in. the Cll!15e of the heart beat, the rhythm of pa66iYe iron is afleated Ly t!leELdG1tl ,{JuhrIuLiuu, LeIug IUCm&llEJ by cul,hodlo and decreased by anodic polarization." Before these striking model experi-

~o OYer the rest of the surface of the wire the acid (60 to 80 per cent) is permanently renewed by letting it stream.


ments were described by Lillie in 1928, no one ever ventured to offer a concise explanation for the controlling action of the pace maker of the heart.

Summarizing all the analogies between passive wire and nerve, Lillie's statement is certainly not exaggerated that "the resemblances between transmission in the "passive" wire and in nerve are numerous and undoubtedly are to be referred to the essential structural feature which the two systems have in common, namely, the presence of a chemically reactive thin surface film." It remains for further investigations to determine more closely the nature of this surface film in nerve.

8. DIRECT EVIDENCE FOR THE LOCAL CIRCUIT THEORY: THE "SALT

BRIDGE" EXPERIMENT (W. J. V. OSTERHOUT, 1930)21

It may seem doubtful whether the mechanism of the conducting processes is really identical in the nerve and in the iron wire model, in spite of the numerous analogies described. The nerve might contain components which produce effects of an entirely different nature, yet resemble traveling waves of polarization to such an extent that an identity of the underlying cause is simulated.

Moreover, a distinction exists between the iron wire and tlie nerve in that the locally circulating currents flow in the opposite direction,22 if we compare the wire with the nerve fiber. Even though it is of no importance which phase (the metal or the' acid) is compared with the living tissue, it would be desirable to demonstrate directly the physiological applicability of the local circuit theory.

In order to test the theory, we may try to explain the action of

21 Journ. Physiol., 13, 547 (in collaboration with S. E. Hill). 22 Dr. R. S. Lillie has kindly acknowledged, in a correspondence with the

author, the correctness of the statement that the-local currents in the nerve and in the wire model flow in opposite directions. In the passive wire, the active region has been described as being negative. By this statement is meant that the active portion is found negative if connections are made from the active and the passive part of the wire to an instrument by means of metallic connections. However, in the nerve the connections are made by means of non-polarizable electrodes. If non-polarizable electrodes are brought in contact with the active and passive portions of an iron wire, the active region is doubtlessly positive. In the nerve, however, the active region is negative under the same condition,


narcotics. Application of the experience, gained from the wire model, leads to the assumption that narcotics act by electromotive interference in the following manner. A narcotic, like alcohol or ether, will shift the potential difference at the junction of the nerve fiber and the surrounding lymph fluid to the negative side. This appears to be an experimentally established fact, since all narcotics, tested so far, produce an electromotive negativity on cuticula, lipoids and all other artificial substances which present phase junctions with electromotive properties similar to those tissues, as already described (see page 199). According to the local circuit theory this negativity would be the sole cause which accounts for the arrest of a traveling wave of excitation. For, propagation can occur only if the wave of negativity has in front of it a zone of positive potential, or at least, a zone less negative than the wave itself. But, if the potential difference in the narcotized area is as negative as the wave itself, no electromotive force will be left from which a local circuit may arise. Hence, the wave must die out as soon as it reaches that area. 23

The question arises as to whether this explanation is really acceptable. That the wave of excitation is arrested as soon as it reaches a narcotized area of the nerve is, of course, a well established fact, but, how can we demonstrate more conclusively, that electromotive actions are the determining factor? An answer to this question would be important since narcotic substances may produce an ahnost endless variety of changes on the nerve. Nearly all of these changes have been suggested as possible causes of the narcotic action. What convincing arguments can be quoted in favor of the electromotive properties?

If it is true that locally circulating currents are the cause of the propagating wave, it should be possible to make a short circuit around a negative or narcotiz(Jd area by means of a simple mechanical device in the following manner. We imagine that it is possible to isolate a single nerve fiber, covered by a moist layer of lymph or saline.

The assumption is that the potential at the boundary between fiber and lymph can undergo variations which are propagated in a wave-like manner as described. A certain zone of the fiber should be narcotized, hence marked by a more negative potential difference between lymph and fiber.

23 This is no contradiction to the previously observed fact that a moderate shift to the negative side or "catelectrotonus" may increase irritability, thus facilitating the progress of the wave under certain conditions.


Consequently the wave of excitation cannot reach another non-narcotized area beyond this "negative" zone. In order to test the local circuit theory, we now place across this "negative" zone, a "salt bridge," viz., a piece of cotton soaked in saline, touching the nerve at two non-narcotized points on either side of the negative zone (see diagram, Fig. 76) (as suggested by Osterhout). The local circuit theory would demand that, as soon as the wave of negativity reaches one end of the bridge, an electrical current should flow from the now negative end of the bridge to the positive area at the other end of the bridge and back through the nerve fiber. In other words, an electrical circuit through the bridge should take the place of a local circuit; thus a negative potential difference should arise at the other end of the bridge and this should enable the wave of excitation to travel on. This hypothetic process evidently resembles, to some extent, the "end to end" transmission on the steel wire. It is essential to note that the "salt bridge," as such, cannot propagate waves of

FIG. 76. HYPOTHETIC DIAGRAM TO ILLUSTRATE A "SALT BRIDGE" OVER A SINGLE NERVE FIBER .

excitation at all. Hence, it cannot be interpreted as a ramification of the nerve.

In order to support the local circuit theory of transmission, attempts should be made to demonstrate a "bridge effect" of this type on a nerve fiber. This. seems difficult since a single nerve fiber is hard to manipulate due to its diminutive size. Even a slender nerve represents a bundle of a large number of fibers. It is known, however, that a conduction of impulses is exhibited also by certain other tissues, even by those of plants. A convenient object for experimentation is the fresh water plant Nitella (Osterhout and collaborators, 1928).24 The single cells of this plant are several cm. long and contain conducting plasm. One such large cell, while comparable to a single microscopic nerve fiber, is readily accessible to experiments. The passing of waves of ex~itation can be observed by means of a

24 Journ. Gen. Physiol., 11, 391, 673; 12, Hi7.


suitable electrical instrument (string galvanometer). Like most plants Nitella hasa large chronaxie, and hence a low rate of conduction, viz., amounting to lor 2 cm. per second. The form and magnitude of the propagated disturbances resemble, in general, the action currents of nerve and muscle, although a complete identity can not be expected due to the size and different make-up of Nitella.25

The boundary between protoplasm and sap, or between protoplasm and cell wall, may be assumed to be the polarizable surface along which the waves of excitation spread. The potential difference at this interface is positive if the plant cell is in contact with tapwater

O.

KCl + CHC13

FIG. 77. DIAGRAM OF AN EXPERIMENTAL ARRANGEMENT OF PRODUCING A BLOCK ON A CONDUCTING CELL SURFACE OF NITELLA AND AnRANGING A SALT

BRIDGE ACROSS THE BLOCK, ACCORDING TO OSTERHOUT

or with a 0.001 molecular KCl solution, but, is shifted to the negative side by saturating the water (or the solution named) with chloroform. These electromotive variations are similar to those described for the cuticula of the apple (see page 196ff). Also RCI solution of a higher concentration than 0.01 molecular produces a negative potential. By local application of such negativating agents, a wave of excitation is started on the plant cell in a similar manner as the wave on the steel wire is started by the negativating zinc. Frequently, also, continuous

26 For details see papers by Osterhout and collaborators, Journ. Gen. PhysioI., 12, 167, 355; 13, 459 (1928-1930).


rhythmical sequences of waves arise from chloroform or concentrated KCl (Osterhout),24 resembling the rhythmical waves on soft iron wire.

Such rhythmical waves will likewise arise if a piece of cotton, saturated with chloroform, is put around the middle of the cell of Nitella, for the purpose of producing a blocking of the propagation. In order to eliminate these rhythmical waves, which are disturbing, several pieces of cotton, soaked with KCI solutions of different concentrations, may be arranged on either side of the chloroformed spot in a manner indicated diagrammatically in Figure 77. Through this arrangement a gentle gradient of potential on either side of the chloroformed spot is produced (Osterhout).24 As experiments have shown, this gentle gradient prevents the formation of rhythmical waves. Nevertheless the chloroformed spot acts as a "block," and arrests waves of excitation. However, if a salt bridge is put over this "block," as also indicated in Figure 77, the wave of excitation can be shown to travel across (Osterhout). 24

The salt bridge experiment can thus actually be performed, according to the local circuit theory. It seems impossible to explain this finding by the assumption of unknown forces of a different nature. The conclusion seems inevitable that the traveling wave of negative eZqr,;tric polarization is the dominating factor of the excitation wave and that the local circuit theory is essentially correct.

9. THEORIES OF NARCOSIS

According to the local circuit theory, we should expect that a negativating agent like a narcotic should be capable of excitation of a wave or of interfering with an already existing wave depending on the conditions. The experiments on Nitella have clearly demonstrated these antagonistic actions: a chloroformed spot may act as a center from which new impulses originate or it may arrest passing waves. Furthermore, the local circuit theory provides for an increased irritability if low narcotic concentrations are applied. A moderately negative potential difference should facilitate the negativating action of the local circuit and in this manner promote the propagation of the wave. This is also demonstrated by the fact that a moderate negative polarization or catelectrotonus of nerve increases


the irritability. It has been known for a long time that narcotics can produce all these effects, a stimulation, an increased irritability at low doses or, finally, an inhibition of irritability or narcosis proper. Each one of these single actions may occur. In fact many narcotics stimulate at first or increase irritability. This is demonstrated by a large number of observations.26 It has been found that even typical narcotics like ether and chloroform stimulate or increase irritability. Such actions occur even on single nerve fibers, on single muscles, or on single conducting protoplasmic tracts. Hence, it is difficult to account for them by the assumption that an inhibitory mechanism is narcotized. It does not seem likely that a single nerve tract contains such a complicated mechanism. The increased irritability or the stimulation must be considered, therefore, as an inherent property of the narcotics themselves. These actions may occur but they need not occur in every case, nor would the local circuit provide for their invariable occurrence. For, if a high concentration of the narcotic is applied, an intensive negativity, in other words, a narcosis proper, sets in immediately.

In the narcosis of an entire organism the conditions are manifestly very complicated. The narcotic acts on a number of nervous elements which are not alike, and, moreover, their functions are interrelated in a complicated manner so that, for instance, stimulation of one part entails depression of another or vice versa. To explain the detail of narcotic action on an entire organism seems impossible, therefore, particularly since the same narcotic does not necessarily produce the same electromotive variation on two different nerve fibers. Even two iron wires may act differently as we have seen. It seems plausible on account of all these complications that in the majority of cases one substance may be chiefly stimulating while another one is chiefly depressing. The general rule, however, verified by pharmacological observations, is that no drug in every case either stimulates or depresses exclusively. This is to be expected according to the local circuit theory.

As already stated, such non-electrolytes only can produce electromotive action, as penetrate into the non-aqueous phase (see page 199). It is manifest, therefore. that the electromotive action of

26 A collection of quotations from the literature demonstrating this point is found in: H. Winterstein, "Die N arkose," 2nd edition, Berlin, 1926, see pp. 13 to 20.


narcotics-and hence their narcotizing power-is due to their penetration into the membrane or to their ·'oil-solubility." This rule had been found empirically long before the causative relationship between penetration and narcotic power was understood, being the basis of the Meyer-Overton theory of narcosis (1901).27 At that time it was a notable achievement to point to the analogy of physiological and physical properties of the narcotic even though the nature of the relationship remained unexplained. The power of penetration of a given substance into tissues was estimated from its distribution between water and oil. Although this is rather a crude approximation, a fair agreement between penetration and narcotic power has been found in many cases. A complete agreement can hardly be expected -as explained before (see pages 40-46).

Following Meyer and Overton numerous other authors have advanced suggestions which attempted to correlate physical properties of a substance with its narcotic power. Each of these suggestions has subsequently received the name of a "theory of narcosis." There is J. Traube's adsorption theory of narcosis (1904);28 Claude Bernard's semi-coagulation theory of narcosis, revived by W. D. Bancroft (1931)29 which emphasizes the reversible coagulation of protein by the narcotic; Moore and Roaf's protein adsorption theory of narcosis (1906)30 emphasizing the adsorption of narcotics to dissolved proteins; Verworn's anoxemia theory of narcosisl1 which emphasizes -the interference of narcotics with cell respiration-and several other theories.

All these theories are empirical. None of them is quite satisfactory as there is never a complete parallelism between a 'single physical property of a substance and its narcotic power. None of them can offer such detailed evidence as Lillie's local circuit theory corroborated by the salt bridge experiment. Nevertheless, it would be erroneous to consider these theories as entirely unimportant. We should realize

27 Meyer, Archiv fur die experim. Pathologie und Pharmacologie 42, 109. Overton, "Studien uber Narkose," Jena, 1901.

28 Pflugers Archiv, 105, 541. 29 This theory was first propounded by Claude Bernard in 1869. Recently it

has been discussed again by Bancroft and Richter (Journ. Physical Chern., 35, 215 (1931).

30 Proceed. Roy. Soc., 73, 382; 77, 86. 81 Verworn, "Narkose," Jena, 1912. The objection to this theory is that

inhibition of respiration does not ental! narcosis in many cases.


that the local circuit theory, even though it is essentially correct, altogether fails to disclose the nature of ·the reactive film of the nerve. It seems that some of the conditions under which this film becomes inactivated, are intimated by those theories of narcosis.

Recently the coagulation theory has been discussed again since W. D. Bancroft has claimed that some narcotics coagulate an unstable colloidal solution of egg white or of other colloids, as well as the colloids in the nerve cell itself.32 Within certain limits this coagulation is reversible just as the narcosis itself. It seems possible that this theory describes a factor the importance of which for narcosis has not been considered sufficiently so far, although this is no more than one factor out of many others, and hardly the most outstanding one. We may assume that the reactive surface film of nerve depends for its existence on a delicately balanced colloidal equilibrium, such as is demonstrated by Clowes' emulsion system. Narcosis would result from a disturbance of this equilibrium. An example of this action is the Meltzer narcosis by magnesium salts as described above. Additional evidence for this explanation is found in the fact that oilsoluble narcotics exert an antagonistic action like calcium or magnesium salt, not only on the nerve, but also on Clowes' emulsion system, according to Clowes and to Lillie.33

10. THE OUTSTANDING ELECTROMOTIVE ACTION OF ALKALOIDS

(BEUTNER, 1927)34

A number of drugs, particularly alkaloids, stand out because of their "potency," which means that diminutive amounts bring about a physiological effect of some sort. The detailed mechanism of their action cannot be analyzed as yet, but, whatever its cause may be, if the local circuit theory is correct, these potent alkaloids should certainly likewise stand out by their electromotive action. In fact

32 Proceed. National Acad. Sciences, 17, 105, 186, 294. Bancroft's further experiments concerning antagonistic effects of peptizing and agglutinating drugs still await confirmation, also his theory described as "Colloidal Chemistry of Insanity."

33 Compare R. S. Lillie, Am. Journ. Physiol., 29, (1912), Journ. Exper. Zoology, 26, (1914); G. H. A. Clowes, Proceed. Soc. Exper. Biology and Medicine, 11, 8 (1913).

34 Journ. Pharmacology, 31, 305; 34, 29.


this is found to be true in some cases; alkaloidal salts like other salts of organic bases are negativating, however, to a much greater extent than the salts of other organic bases.

This negativating influence is particularly strong at a phase boundary, which is positive to NaCI solution, as it exists, e.g., at the junction of

nitrobenzene plus 10 per cent oleic acid

and

a NaCl solution which is slightly alkalinized by the addition of soap.

If traces of an alkaloidal salt are added to the alkaline N aCI solution, the potential difference changes considerably. Thus, for instance, an addition of strychnine, atropine, cocaine or pilocarpine hydrochloride in a concentration of 1: 1 million, lowers the potential difference about 3 to 5 millivolts. This extremely low concentration is in the same order of magnitude at which such potent drugs act in vivo. These electromotive effects cannot be explained by pH changes.

The electromotive action is not parallel to the general toxicity in every case, but, this can hardly be expected since a phase boundary at nitrobenzene cannot resemble those occurring in tissues in every respect. More satisfactory model systems might yet be found. Theoretically, it should be possible to explain specific drug actions on this basis by finding a number of different systems in each of which a single drug has an outstanding electromot~ve action.

Substances like ether, chloroform, amyl alcohol, veronal and antipyrin, which have no physiological action in alkaloidal doses, are devoid of electrical action at concentrations of 1: 1 million; but do act in the same manner as alkaloids at higher concentrations.

The greatest drop of potential,following the addition of an alkaloid, viz., pilocarpine, in a dilution 1: 1 million, was found if 0.02 per cent soap had been added to the NaCI solution, while the effect was smaller with both larger and smaller additions of soap. The physiological action of pilocarpine also increases following the addition of sodium oleate, and, in this case too, the greatest increase occurs within a definite range of soap concentration only.35 Manifestly this special feature can be accounted for by the electrical theory, but hardly by any other explanation.

35 Storm van Leeuwen, Journ. Pharmacology, 17, 1 and 21; 18, 257,271.

OUTLOOK TO FUTURE DEVELOPMENTS

CONCLUDING REMARKS

APPENDIX

OUTLOOK TO FUTURE DEVELOPMENTS

The "attempts at approach" as outlined in this volume should by no means be looked upon as the sole possibilities of attacking fundamental biological problems. It happens that in those three lines of investigation, knowledge has accumulated to such an extent that we can demonstrate the possibilitiy of imitating, and thus understanding, certain phases of life processes. Of course, no more than some of them have been analyzed as yet, but the systematic presentation of the results, attained so far, indicates the points of attack for future developments.

It is evident that a large number of additional attempts have to be made and developed for a more satisfactory exploration of life phenomena. Many potential developments of that kind have been initiated but not yet developed to a sufficient extent. Only two can be described here, viz., the experiments on artificial parthenogenesis and those dealing with mitogenetic rays. Still another mode of approach will pT:obably be based on studies on Mendelian inheritance when we have come to know more about the chemical and physical nature of the units (probably the genes) which determine dominant and recessive characteristics or the other conditions responsible for heredity.l

1. ARTIFICIAL PARTHENOGENESIS2

Hardly any other biological discovery has fascinated the investigator so much, and has attracted so much public attention as the achievement of artificial parthenogenesis. Under normal conditions following penetration of the spermatozoon, the eggs of marine animals can be seen to develop with the regularity of a clock work. At all times, biologists have been puzzled with the question as to how this initiation of the development through the sperm might be explained.

1 For the work, done so far in this line, see T. H. Morgan, "Experimental Embryology," New York, 1927. E. B. Wilson, "The Cell in Development and Heredity," New York, 1925.

2 Parthenogenesis means "virgin birthi" the term implies the artificial initiation of development of an unfertilized ovum (without sperm).

279

280 OUTLOOK TO FUTURE DEVELOPMENTS

Suddenly it seemed that a definite answer had been found when Jacques Loeb announced in 19043 that he had succeeded in obtaining normal larvae from uninsemina ted eggs of the sea urchin by a double treatment, viz., with lower fatty acids and, subsequently, with hypertonic salt solutions. Since normally the sea urchin eggs develop only if fertilized, it might have seemed to a superficial observer that a 50 per cent synthesis of a real living being was already accomplished. Of course, this is not by any means a reasonable aspect of Loeb's experiments since the egg is far more essential for the development of the organism than the sperm.

In order to evaluate the merits of Loeb's discovery, the following well known facts should be remembered: a natural parthenogenesis, that is, a development of uninseminated eggs without any artificial treatment, is an "old and familiar story to zoologists" (T. H. Morgan).1 Even Aristotle was aware of this fact since he states that "bees produce drones without copulation." Also ants, wasps and other hymenoptera, orthoptera, homoptera, octracoda and others show a natural parthenogenesis. It seems thus that, in general, eggs have a tendency to develop without the spermatozoon. In many cases they can develop without it; in other cases they cannot, but perhaps there is just a slight handicap of some sort which might be r~moved by artifiCial means. Starting from such hypotheses, experiments on artifiCial fertilization were undertaken by various workers a long time before Loeb entered the field, notably by T. H. Morgan4 who treated eggs of Arbacia and Annelids with sodium and magnesium salts and observed that astrospheres formed or that cle.avage was started. However, the cleavage did not seem to be ·the beginning of a development in these cases; it appeared to be a pathological phenomenon, a degeneration due to the poisonous action of the salts. On observing the degenerated products resulting from salt action on the delicate ova, the experimenters were led to the idea that any attempt at substituting artificial agents for such an exclusively vital agent as the fertilizing sperm would be doomed invariably to failure. Yet, this unexpected success was achieved by J. Loeb a few years later, after initial experiments in which he too had less satisfactory results. Whether his success was due to the freedom of his mind from prejudices or rather to his untiring and painstaking experimen-

3 University of California Publications, .I, 83, 89, 113. 4 Archiv f. Entwicklungsmechanik, 2 and 3, 1895-1896.

ARTIFICIAL PARTHENOGENESIS 281

tation and the greater accuracy of his technique, is hard to decide. At any rate the accomplishment was there: normal larvae of the sea urchin were obtained, nearly all eggs developed just as after natural fertilization. The larvae exhibited a good vitality by swimming to the top of the aquarium like naturally fertilized ones in contrast to the sickly individuals obtained in initial experiments which were unable to raise themselves that high. Such healthy larvae were now obtained from eggs which otherwise had needed the sperm for development. A substitute for the sperm had been actually found and it performed the function equally satisfactorily, in this case at least.

Although Loeb was decidedly opposed to exaggerated conclusions drawn from his work, he believed that definite evidence could be reached as to the nature of the fertilizing action of the spermatozoon. Even this, however, was very difficult to obtain. In the initial experiments which gave unsatisfactory results, the eggs had been treated with hypertonic saline alone. The treatment with fatty acids, introduced in 1904, resulted in the formation of the typical fertilization membrane which Loeb considered very essential. The eggs disintegrate after acid treatment alone. Loeb explains that membrane formation "starts the development of the egg, but, that it leaves it in a sickly condition, which causes it to disintegrate. In order to make such eggs normal, they must undergo a second treatment" which acts as a corrective to the cytolytic process produced by the acid. For this purpose not only hypertonic sea water was used but also sea water in which the oxidations of the egg were suppressed by cyanide. Moreover, instead of butyric acid or other fatty acids, almost any substance that causes hemolysis may be used such as ether, hydrocarbons, various esters, certain bases such as amines, saponin, bile salts, or blood of other animals. No matter by what means membrane formation is induced, it initiates development if supplemented by a "corrective" treatment.s The conclusion was drawn from all these experiments that the sperm acts likewise by carrying into the egg at least two agents, one of which is cytolytic and the other "corrective" in the manner explained.

However, in contrast to the experiments of Loeb, E. E. Just6 has found (in 1922) that normal top swimming larvae of the sea urchin can be

5 See J. Loeb's "Artificial Parthenogenesis and Fertilization," Chicago, 1913. 6 Biolog. Bulletin, 43, 384.


produced by the use of hypertonic salt solutions alone as successfully as by butyric acid followed by a "corrective" treatment. Success depends on the exact proportion of the salt added to the sea water and on the length of exposure. Eggs of different females may require slightly different treatment. Hence the optimal conditions have to be found for the eggs of each female separately. Under such most carefully controlled conditions, parthenogenesis can be obtained by a single treatment with equally satisfactory results as with the "double" treatment. The larvae behave in every respect -like those from normally fertilized eggs. It seems, that Loeb needed a "corrective" treatment in his experiments merely because the acid treatment had not been so carefully controlled as the hypertonic salt treatment of Just. Following Just's finding, it is difficult to maintain that the double treatment with acid and hypertonic solutions should be in any way specific or resembling the action of the spermatozoon, as Loeb believed.

The developmental action of the sperm is now as obscure as formerly. The problem seems as unsolvable as an equation with many unknowns. Even 1f one unknown is determined the equation becomes hardly more intelligible. Further discoveries would seem indispensable for approach-ing vital problems from this angle. '

Methods of artificial parthenogensis have heen worked out also for a number of other eggs; the treatment is different in every case, even for members of the same species.

Also unfertilized frog's eggs can be made to develop, but the treatment, in this case, consists merely in pricking with a glass needle .. Following this crude operation, an exceedingly small fraction of the eggs develop, the vast majority die. One could hardly consider this as a substitute for spermatozoal action. 7 Other eggs can be induced to develop by simple shaking or by irradiation or by electrical stimulation.

7 Compare J. Loeb's "Artificial Parthenogenesis and Fertilization," Chicago, 1913. Also T. H. Morgan's "Experimental Embryology," New York, 1927.

MITOGENETIC RAYS 283

2. THE MITOGENETIC RAYS8

Truth is stranger then fiction.

Alexander Gurwitsch, formerly professor of histology at Moscow, now at the Institute for Experimental Medicine at Leningrad, had studied for many years the incidence of cell division in tissues under various conditions. His systematic investigations led him to the assumption that the immediate impulse which causes a cell to divide is not generated within the cell, but comes from an outside source. In order to divide, the cell must have- manifestly reached a certain developmental stage, but, even if it is ready and prepared to divide, Gurwitsch believed it should take an impulse from the outside to set the division going. It seemed difficult, however, to find definite evidence for this assumption.

Finally, in 1923, Gurwitsch resorted to a daring experiment in order to substantiate his view. 9 He tested the possibility as to whether or not that extraneous impulse was transmitted to the cell in the form of a radiation. The outcome of this test, when performed under carefully controlled and favorable conditions, was successful in demonstrating the possibility that living tissue may send out rays which are capable of inciting cell division in another tissue near it.

The experiment is set up as follows: The tip of an actively growing root of the onion, Allium Lepum, is put in an open glass tube and brought close to a second root which is held in another glass tube in the manner shown in Figure 78. The first root serves for inducing the effect; the second, for observing the mitotic changes produced. Both roots are kept moist. The adjustment of their mutual position,the one vertical and the other nearly horizontal as indicated in the diagram-must be done with extreme care by means of a micrometer, in such a way that the projected axis of the first root strikes the second root in the median line. The whole set is held rigidly in position. Mter standing for two or three ,hours, sections of the second root are made. A regular sharply circumscribed preponderance of mitoses is then revealed in the center of the induced side of the root.

The same observation is made if a lamella of quartz is interposed between the two roots, or if the inducing root is completely encased in quartz, but the effect disappears if glass is interposed or if the quartz

8 A. Gurwitsch, "Die mitogenetische Strahlung," Berlin, 1932. 9 Roux's Archiv, 100, 11; see also 101, 53, 102, 68.


lamella is covered with a layer of gelatin. Moreover, the effect can be reflected by a mirror like ordinary light; the increase in mitoses appears exactly at that spot where the reflected light strikes the object. In such experiments pulp of onion roots may be used in place of a whole root to produce the same effect.

The description of this new biological phenomenon excited considerable interest and instigated numerous attempts at repeating the experiments. However, considerable difficulties were experienced since the effect is by no means readily observed. Numerous sources of error are present. It is not astonishing, therefore, that some experimenters have described failures of finding any mitogenetic radiation at all, using the onion root method. Most of those who have seen positive results have obtained these results only after initial negative observations.lo (For positive findings see particularly Zirpolo, 1930.)1l

However, in the further development of this research, Gurwitsch and his school have developed other more reliable and demonstrative methods which make use of yeast cultures as "biological detectors" for the mitogenetic radiation.l2 These methods are preferred since they are easier, and hence have facilitated the search for sources of radiation. They have been used not only by the Gurwitsch school but also independently by Reiter and Gaborl3 and W. W. Siebertl4 who report positive results.

One of the "yeast methods" is performed as {ollows:l5 A drop of

10 Outside of numerous papers by Gurwitsch and his school, see Schwarz, Biolog. Zentralblatt, 48, 202 (1928); Rossman, Roux's Archiv, -113,346, and 114, 583; Guttenberg, same journal, 113, 414, who report failures; see also Magrou and Clioucroun-who report both positive and negative findings, Compt. rend. Acad. Sci., 186, 802 and 188, 733, Journ. Mar. Biolog. Assn. Plymouth, 17, 65 (1928-1930). Further literature quotations are found in the book of Gurwitsch already quoted.

11 Bollet. della Societa dei Naturalisti in _Napoli 42, 169. 12 First described by M. Baron, 1930, in Planta, 30. 13 Reiter and Gabor, "Zellteilung und Strahlung," Berlin, 1928. 14 Zeitschrift f. Klinische Medizin, 109, 360; Biochem. Zeitschrift, 202, 115,

123. Compare also D. N. Borodin, Plant Physiology, 6, 119-129, 1930. 16 This description is from a personal letter of Gurwitsch, of April, 1932.

According to Borodin there are four different yeast methods used at present: (1) the yeast budding method which is most sensitive, (2) the drop culture method, (3) the planimetric method, and (4) the mizetocryt method, which is described above.


a fairly dense yeast culture, containing about 8000 yeast cells per cubic centimeter, is placed in a moist chamber covered with a thin quartz plate. On this plate is placed the "sender," viz., the material whose mitogenetic radiation is to be examined. An equal amount of the same yeast culture is kept in another moist chamber as a control, protected from all possible radiation. Mter four hours, equal measured amounts of the irradiated and of the control yeast are filled into two separate measuring tubes provided with ampules, and are centrifuged. If there is any radiation the total volume of

Onion root

showing

mitotio

effect

FIG. 78. EXPERIMENTAL ARRANGEMENT FOR TRACING MITOGENETIC RAYS IN THE ONION ROOT (ACCORDING TO GURWITCH)

the irradiated yeast cells is larger than that of the control. The excess corresponds to the intensity of the radiation.

Furthermore, the diffraction of the rays through quartz lenses and a quartz prism has been traced by means of yeast cultures by usin~ a type of arrangement as is shown diagrammatically in Figure 79. In this manner it is possible to determine the spectrum of a mitogenetic radiation. The spectra of radiations from different sources are by no means identical. They differ just as visible light may differ spectroscopically.


By making use of the yeast method "mitogenetic" rays have been found to be of wide, possibly even of universal occurrence (Gurwitsch).8 As examples, the following organisms or materials, which have been found to radiate, may be noted: different kznds of bacteria, yeast, animal eggs in various cleavage stages, plant seedlings, blood from warm or cold blooded animals, muscle tissue, nerves, ciliated epithelium, various parenchymatous organs and especially: neoplasms. A radiation can be traced as an instigator of cell division in many cases, perhaps invariably.

radiating object

yeas t c:ul turea

FIG. 79 DIFFRACTION OF MITOGENETIC RAYS THROUGH A QUAR:rZ PRISM

However, the source of the radiation is not to be sought in cell division, as is shown by the fact that tissues which are in active cell division, as for instance, the meristematic tissues of plants, ,do not give off radiation, but derive their mitogenetic impulse from other sources. The term: "mitogenetic rays" is, therefore, really incorrect.· The rays are not generated by mitosis, on the contrary, they are used to stimulate mitotic activity. In some tissues radiation is alternately emitted and consumed by mitosis, the radiation being premitotic in these cases (Gurwitsch).8 The origin of these rays is due to chemical reactions or metabolic processes. This is shown by the observation that some chemical reactions produce a radiation even in vitro, which may be demonstrated by the induction of increased cell division of yeast cultures if these are hit by the emitted rays. One of the first experimenters to observe such an effect was W. W. Siebert, 1928.16 He was led to this discovery by the following observation. The radiation of contracting muscle persists even after it has been macerated and transformed to

16 Biochem. Zeitschrift, 202, 115, 123.


pulp. However, a pulp made from a non-contracting muscle fails to radiate, but, it can be caused to do so by the addition of lactic acid in an atmosphere of oxygen. This seems to indicate that the source of the radiation is the combustion of lactic acid by the ferment of the muscle. Combustion occurs in muscle pulp or in actively contracting muscle where the lactic acid is generated by the contraction. Pulp made from resting muscle, however, being free from lactic acid, has no combustion, and hence fails to radiate (W. W. Siebert).16 Most of the other parenchymatous organs, investigated by Siebert, fail to radiate or radiate little, with the exception of bone marrow, which functions probably on account of the exceptionally powerful oxidations occurring in it. Further evidence is found in the observation that the blood of starved animals, which fails to radiate, begins to emit rays following the addition of glucose. But this effect may also be due to glycolysis (see below, page 289).

These findings led W. W. Siebert16 to test whether or not Warburg's respiring charcoal model would emit mitogenetic rays. This was indeed found to be the case. Charcoal added to an oxalic acid solution through which a stream of oxygen is conducted gives rise to a distinct mitogenetic radiation, as indicated by means of yeast cell growth. As we have seen, a respiration, viz., a combustion of oxalic acid, occurs In this solution (see above, page 154). That this respiration is the source of the radiation seems indicated from Siebert's observation that a slight addition of cyanide arrests the radiation, just as it arrests respiration. Also the radiation from bone marrow is arrested by cyanide. Some other oxidations were also found to emit radiation, viz., the oxidation of fructose in phosphate solution, the oxidation of pyrogallol by H 20 2 in the presence of oxidases, the oxidation of oxalic acid by permanganate and some other oxidations. The oxidizable substances in the circulating blood which emit radiations, seem to be chiefly the proteins (Gurwitsch).17

Moreover other types of chemical reactions can emit this radiation, particularly proteolytic reactions such as the digestion of protein by pepsin or pancreatin, and furthermore, glycolytic reactions. A radiation from glycolysis has been traced by the yeast methods as follows. Laked blood is an efficient source of mitogenetic radiation for about

17 All these and the following statements are quoted from A. Gurwitsch, "Die mitogenctische Strahlung," Berlin, 1932, where further references can be found.


the first ten to fifteen minutes. After this time the radiation ceases. But, if after more than one hour, glucose is added to the blood, the radiation sets in again. "No other source of radiation can be acting in this case but glycolysis" (Gurwitsch),17

From the investigations, carried out so far, it seems that the spectrum of a radiation depends chiefly on the type of the chemical reaction. Thus, the oxidations from which the radiation arises (investigated so far) generate a radiation of a wave length between 222 and 234pp. The proteolytic reactions emit two different types of wave lengths, viz., from 194 to 213J.1,u and from 220 to 242J.1J.1; the glycolytic reactions emit from 190 to 197J.LJ.I and from 217 to 218J.1J.L. These figures show that the glycolytic and the oxidative reactions send out radiations of quite different wave lengths (Gurwitsch),17

The aim of the research in this line would be to determine the spectral composition of the radiation which various organs emit. If these spectra could be identified with those of certain chemical reactions, a new method would be available for determining precisely the metabolic changes in vivo. But, so far, such an identification has been possible for a few single spectral lines only (except in the case of tumor tissue). For instance, the metabolic changes occurring in contracting muscle-which are nowadays in the focus of scientific interest-have been determined by this method to be oxidative and glycolytic reactions. But, there must also be additional reactions since the spectrum, emitted from muscle tissue, contains other rays, the chemical origin of which has not yet geen definitely determined. Nearly the same is true for isolated nerve fibers. During tetanic contraction, muscles emit considerably more radiation than at rest, also the spectral composition is materially altered by contraction.

Moreover, both nerve and muscle are stimulated by mitogenetic irradiation to emit a seconaary radiation. In this manner an induced radiation is propagated along the nerve fiber. Gurwitsch17 considers this propagation as an important contributory factor in nervous conduction. Such an induction of mitogenetic radiation qy a primary radiation can be observed also in many other instances, even in vitro. It can be shown in vitro that mitogenetic rays act as catalysts.

Next to contracting muscle and some other tissues, the circulating blood is probably the most important source of mitogenetic radiation in the animal organism. "Blood of healthy men and animals never


fails as a mitogenetic radiator." Therefore, "an uninterrupted mitogenetic field exists in all organs and tissues which are properly provided with blood" (Gurwitsch)_I7 In the blood just as in muscle, .both oxidative and glycolytic processes seem to be the source of the radiation. Glycolysis is indicated by the increase of radiation following addition of glucose to blood as stated. Evidence for oxidations is found by the observation that the addition of cyanide interferes with the emission of radiation to some extent, although it does not arrest it completely.

The radiation of the blood persists even under many pathological conditions. It is inhibited only in extreme starvation or in such grave diseases as pernicious anemia, leucemia and advanced septicemia or pyemia. While in most other diseases the radiation persists, the blood of cancer infected animals and of cancer patients has lost its mitogenetic radiation even in the early stages of this disease. Thus, in mice, inoculated with transplantable tumors, a diminution of blood radiation appears, even before the tumor becomes .palpable (Lydia Gurwitsch, 1929).18 It seems therefore, that this decrease of radiation, as an early symptom, is characteristic of malignancy particularly. If cancer blood is added to normal radiating blood in vitro, the radiation is arrested. Hence, it seems that cancer blood contains a poison which interferes with the metabolic processes which normally produce the radiation.

Another disturbance occurring in malignancy is the extraordinarily strong radiation arising from the tumor tissue itself. Under normal conditions, an actively growing section of tissues does not generate rays as stated, but, receives its mitogenetic impulses from an outside source. It is believed that development usually ceases when the growth extends outside of the promoting mitogenetic field. Cancer tissue, however, is both a strong source of mitogenetic radiation and at the same time is rich in mitotic cells. This is probably due to the fact that the malignant cells carry their mitogenetic impulses along with them while they grow (Gurwitsch)P

The source of the radiation in actively growing cancer cells is chiefly glycolysis, since excised portions of cancer tissue, which soon cease to radiate, begin to radiate again when they are bathed in a glucose solution. Also the spectrum of the radiation from active cancer is ex-



elusively that of a glycolytic reaction (190 to 197 1lP. and 217 to 218p.p.). This finding is completely in agreement with the result of Warburg's investigations according to which glycolysis is the chief metabolic reaction in cancer tissue. (See above, page 167.) Furthermore, another source of radiation often exists in tumors which contain necrotic tissue. The necrotic portion of the tumor tissue sends out a radiation which is due to proteolytic decomposition. In fact, the spectrum of the radiation from necrotic tumor tissue coincides completely with that generated by peptic digestion. The distinction of these two types of radiation can be traced quite definitely by means of spectral analysis. In these cases no spectral lines of unknown origin are found (Gurwitsch)Y

To summarize we may state that according to the investigations, available so far, a double disturbance exists in malignancy:

1) The mitogenetic radiation of the blood subsides., 2) Mitogenetic radiation is set up in the foci of cell proliferation

and persists in spite of the abundant cell multiplication, its source being a glycolysis in the actively growing portions and a proteolysis in the necrotic portions.

In view of the great theoretical and practical importance of the mitogenetic radiation it seems strange that its presence has escaped the attention of experimental biology for sU,ch a IQng ,time, although we have known long ago of a great variety of special biological light effects and light emissions, such as the CO2 assimilation. of -plants influenced by light and bio-Iuminescence of various types. Failure to observe mitogenetic radiation, prior to Gurwitsch, may be due to the extremely low intensity of these rays, this being their most outstanding physical property. In fact it is almost impossible, or at least very difficult, to trace this radiation by purely physical means such as the photographic plate. By the biological methods described the radiation can be traced merely because of the marked sensitivity of the growing cell. Frank and Rodionow19 (in Gurwitsch's laboratory) estimate the intensity of biological sources of radiation to be of the order of magnitude of 1000 light quanta per sq. cm. and second. It seems that a secondary radiation of yeast cells can be induced by a single light quantum and that mitosis can be set going by as little as about ten light quanta.

19 Naturwissenschaften, 1931.


Nearly all of the results described have been obtained by the Gur~ witsch school. It would be desirable that these observations should be controlled by a larger number of independent workers than those mentioned above. The considerable difficulties prevailing in this field may be illustrated by quoting a recently published criticism of Gurwitsch's work, by G. W. Taylor and E. N. Harvey20 (1931), who come to the conclusion that mitogenetic rays do not exist because of their inability to darken the photographic plate, even after 89 days. This inability was already known to Gurwitsch. It does not, of necessity, demonstrate that there are no rays. The rays are simply too weak and do not exceed the threshold necessary for photochemical action. Taylor and Harvey also emphasize the inadequacy of the onion root as a "detector" on account of a natural asymmetry of mitoses, but, do not mention all the results obtained by the yeast methods.

Attempts have been undertaken to demonstrate the existence of mitogenetic rays by purely physical methods, viz., by the so-called photoelectric effect. In order to produce this effect, the mitogenetic radiation is allowed to act on a photoelectric cell (W. B. Rajewsky, 1931).21 This cell in turn emits ions into the surrounding gas space, which is in a closed chamber, where the resulting ionization is traced by means of the discharge of a charged wire, according to the method of Geiger and Miiller.22 Definite effects have thus been demonstrated, but, as Gurwitsch17 remarks, it still remains doubtful whether the rays which are traced by this or by any other physical method are actually identical with those which promote growth. Morever, these physical methods are rather uncertain and still await furthet perfection.

20 BioI. BulL, 61, 280. 21 Article published in "Zehn Jahre Forschung auf dem physikalisch-medi

zinischen Grenzgebiet," edited by F. Dessauer, Berlin, 1931. 22 For details see Rajewsky's article just quoted.

CONCLUDING REMARKS

The great difficulties which handicap the investigation of elementary life phenomena have led to the assumption that a complicated play of inter-related physical forces is the essence of life processes. This is certainly correct, but, in addition, the experience gained from the work of Gurwitsch would lead to the conclusion that some of. these physical forces are as yet quite unknown. Further progress seems to depend on discoveries of such a type as that of Gurwitsch, viz., discoveries that are made incidentally to systematic investigations.

POSSIBLE ApPLICATION OF PHYSICOCHEMICAL METHODS OF IN

VESTIGATION TO THE EXPLANATION OF CEREBRAL ACTIVITY

The difficulties of such investigations are manifest. Nevertheless, progress would be possible if the importance of solving sU,ch fundamental problems were more widely appreciated. Unfortunately even the possibility of attacking truly vital problems by physicochemical methods is not recognized by many. This applies particularly to all the phenomena which are connected with mental activity. For ages the idea has been impressed upon us that the activity of the brain cannot be understood on a physical basis and this idea has been widely accepted.

A scientific analysis of brain functions has revealed the fact that their elementary components are reflexes. As is well known, a reflex starts with stimulation of a sensory nerve ending. The impulse travels to the central nervous system, passes from there to the motor nerves and terminates in a muscle fiber producing contraction, or in a gland producing secretion, or in another "effector" organ, producing its specific effect. Even in highly developed organisms such as the vertebrates the activity of the brain and the nervous system is chiefly influenced by such reflex actions, but it is complicated here by the presence of the residual effect of previous activity, the result of which is mentally an association or ,so-called "memory." The es-

292

CEREBRAL ACTIVITY 293

sential part of this association is the reproduction of the effect of stimuli which have attacked the organism at some former period. The release of this after-effect will occur as a result of a similar stimulus. An immense number of such associations are in existence, varying according to the size and development of the brain. As is well known Pawlow1 has successfully analyzed the mechanism of these residual effects or associated reflexes by ingenious experiments on dogs with salivary fistulas or various other fistulas of the gastrointestinal tract. He calls the associated reflexes "conditioned" reflexes.

These associations or so-called memory impressions occupy isolated positions in the brain of young individuals. Later ·inter-connecting tracts open up between them. By making use of these, a number of so-called "memory images" once associated with each other are called into play on each subsequent occasion. Thus, the odor of a rose will call forth its image, its name, but more than that, the visual image of persons or surroundings which were present on a former occasion when the image or odor of a rose impressed the brain. By the joint function of memory images, observation transforms itself into "perception." Consciousness, another psychological term, is also determined by associative memory. The "consciousness of self" is due to the fact that certain constituents of memory are constantly and more frequently produced than others. "The complex of these elements of memory is the 'ego' or the 'soul' or the 'personality' of the metaphysicians." Some of the constituents of this complex are: "the visual image of the body, certain sensations of touch repeated frequently, the sound of our own voice, certain interests and cares and other impressions" (E. Mach). 2

The question arises as to whether this description of cerebral activity might be further elucidated by the physicochemical methods of "approach by means of synthesis" as outlined here. Reflex actions depend primarily upon nervous conduction, the nature of which has become better understood through the local circuit theory and other theories as explained. Concerning the mechanism underlying association, we may assume that it depends either upon the growth of new nerve fibers or upon their capacity to make variable inter-related connections. It seems also that a functional development due to

11. P. Pawl ow, "Conditioned Reflexes," Oxford Un. Press, 1927. 2 Mach, "Beitrage zur Analyse der Empfindungen," Jena, 1886.

294 CONCLUDING REMA.RKS

progressing myelinization plays a role. Anyone of these possible types of functional development would lead to the formation of new nerve tracts. We may assume that the formation of new tracts by anyone qf these modes is influenced by stimuli.s

According to this concept, the development of "memory impressions," which constitute the basis of all training and education, would primarily depend upon the development of new functioning nerve tracts. Consequently in senility when development of any kind ceases, the formation of all new memory associations should likewise cease, while those nerve tracts which were previously formed still persist. These theoretical conclusions are certainly verified by experience in a striking manner since it is common knowledge that in old age mental activity is limited chiefly to the reiterations of impressions gained at a former period. It, would seem, therefore, that for a further elucidation of mental activity the physicochemical method outlined here might be helpful. We should not depend exclusively on anatomical methods in this respect, as is emphasized also by the following remark of J. Loeb: 4

"There is at present a tendency to consider the anatomical and histological investigation of the brain as the most promising line for the analysis of these functions. It seems to me that we can no more expect to unravel the dynamics of electricl)-l phenomena by counting and locating the telephone conhections in·a big city/'

POSSIBILITIES OF DEVELOPMENT IN A REMOTE FUTURE

Concerning the future developments of the methods of "approach by means of synthesis," we may ask the following question. If we imagine that at some future time our knowledge of vital processes were so far advanced that practically all of them were completely understood, would not such an achievement entail the possibility of

3 Perhaps this growth of nerve fibers bears a remote resemblance to the growth of fibers from lipoidal masses. (See page 117.) According to personal communications by Dr. M. Telkes, the growth of these lipoid fibers is influenced by electrical currents. It would be important to know whether or not the growth of nerve fibers is also influenced by electrical currents and whether it depends on the action currents produced by the traveling waves of electric polarization. '

4 J. Loeb, "Comparative Physiology· of the Brain," New York, 1900.

CEREBRAL ACTIVITY 295

producing one of the most elementary forms of life? This conclusion need not be necessarily correct since factors might be involved which are beyond' human control. In order to illustrate such a possibility, it may be)worth while to quote, as an example, the crystallization of diamond., As is well known, the diamond is a crystalline modification of carbon. Attempts to make it artificially have been successful to such an extent that diminutive crystalline fragments were obtained, but, valuable large and clear specimens have -not been made as yet. Comparison of the natural conditions to those in the experiments producing-, diminutive diamonds leads to the conclusion that the large specimens have formed in nature under immense pressure which cannot be produced by artificial means. Moreover, the crystallization must have taken several thousand years. Since these conditions cannot be produced in a laboratory, the making of large, clear diamonds has remained impossible. The same is true of other large crystals which occur in nature. Would it not seem possible, or even probable, that also the processes leading to the formation of primitive forms of life require millenniums or possibly millions of years, particularly since processes resembling crystallization form the basis of vital growth as we have seen?

Geology and palaeontology have taught us that the evolution of life: has taken millions of years. The postulate of synthesizing primitive life would involve the necessity of reproducing within days materials or conditions for which nature needed millions of years. This ha,s been possible in some cases, but exactly to what extent it is possible in this case, remains unknown.

However, even if the synthesis of living organisms should remain impossible, the imitation of the majority of the single phases of life processes will result in a control over man's own existence to an extent unknown at present. In order to evaluate the importance of this development, we should look upon it as an outstanding event in the history of the earth, or of mankind. Experience has shown that appreciable scientific progress can be made in a single century, which in terms of geological periods is hardly more than a moment. Scientific development of our day appears to us, therefore, as a turning point in the history of the earth, leading to the development of life thoroughly controlled by human knowledge.

APPENDIX NEW INYESTIGATIONS ON MEMBRANE EQUILIBRIAl

As briefly outlined above, a theory has been developed which explains the swelling of colloids in many cases, on the same basis as osmotic action. One may express this also by stating, as J. Loeb2 does, that swelling is explained as a result of an equilibrium condition of classical chemistry. A mQre detailed presentation of this theor:y will be given on the following pages. The writer begs leave to express his opin~on that a detailed study of this theory is suitable preferably for an advanced student. Some knowledge of physical chemistry seems indispensable for a study of this kind.

1. INTRODUCTION: ApPLICABILITY OF GAS LAWS TO COLLOIDAL SOLUTIONS

For an application of the classical theory it is first of all important to know to what extent the well known gas laws hold for colloidal solutions. In order to answer this question, it is necessary to resort to methods different from those applied to ordinary solutions. The best method is to study the variation of density with the height. In a gas such as the atmosphere of the earth, the density decreases with increasing height. This fact can be derived from the gas laws by taking into account the continual decrease of pressure with increasing altitude. The same decrease of density must occur in any solution with increase of height. If the gas laws are applicable to all colloidal solutions and even to suspensions, we should find the same type of decrease of density, in these cases too, although on an infinitely smaller scale since colloidal particles are enormously large. In fact this su.pposition has been verified even for rather coarse emulsions where we should least expect it. Hence any suspended particle, whatever its size, may be considered as a gas molecule. This amazing result .... has been demonstrated by preparing a colloidal solution containing particles of equal size, viz., a colloidal solution of mastix (J. Perrin, 1911}.3 Such an emulsion is prepared from an ordinary emulsion wi th unequal particles by repeated. sedimentation, usually in a centrifuge. Thus, for instance, starting with 1 kilogram of an ordinary mastix emulsion the largest particles are sedimented l)y centrifuging and the top layers removed. This process is repeated several hundred times until finally less than 1 gram of an emulsion containing uniform particles is obtained,'each particle having a diameter of 0.0005 mm. This uniform emulsion was observed under the ultramicroscope and the

1 This part consists largely of unpublished investigations. 2 Loeb, "Proteins and Theory of Colloidal Behavior," New York, 1922. 3 J. Perrin, "Les Atomes," 2nd edition, Paris, 1913.

297

298 APPENDIX

average number of particles at different levels was determined. Such an observation presents considerable difficulties since the particles are constantly moving around erratically (Brownian movement). Nevertheless by repeated counting, the number of particles was seen to decrease with increasing height in exactly the same manner as in the case of the gas molecules. Since the particles are of enormous size as compared to gas molecules the jlistance of level which brings about an appreciable diminution is of microscopic dimensions. The number of the suspended particles of 0.51' was found t.o decrease by 50 per cent at a level which was 0.005 mm. higher.

This decreas_e of the number of particles is to be compared to the decrease of density of the terrestrian atmosphere. In the lower layers of the atmosphere near the surface of the earth such determinations cannot be performed on account of the disturbing influence of the winds. However, in the higher altitudes, a gradual rarefaction of the air occurs quite regularly. It is a'different one for every single gaseous constituent of the air: the density of the heavier gases decreasing much more rapidly than that of the lighter ones. Consequently the lower layers contain chiefly oxygen and nitrogen, while in the highest layers such light gases as hydrogen and helium prevail. After eliminating by calculation the influence of temperature, it is found that oxygen lis diluted to one-half of its original density by each rise of 5 kilometers. However, in order to dilute helium to one-half, a rise of 40 kilometers is necessary. These two distances have the same relation as the molecular weight of these two gases, viz., 5:40 = 4:32.

The rise which is necessary for diluting oxygen to one-half is,. therefore, a billion times larger than that for diluting a uniform mastix emulsion containing particles of 0.5J,1 diameter. (For oxygen the distance of level amounts to 5 kilometers = 5,000,000 mm. While for the mastix particles it is 0.005 mm.)

If we attempt to apply the gas laws to the mastix emulsion, the decrease of density, or rather the decrease of the number of particles, may be explained in the same manner as that of a gas. In other words, each mastix· particle of O.5J,1 diameter may be looked upon as a giant gas molecule. The weight of a mastix particle should be_as. many times that of an oxygen molecule as corresponds to the relation of the differences of level over which dilution to one-half occurs. Since the weight of a mastix particle in solution is known, it ought to be possible, on the basis of this consideration, to calculate the weight of an oxygen molecule. The result of such a calculation, taking into account the necessary corrections, is that the weight of an oxygen molecule should be 45 . 10-U grams. This value agrees satisfactorily with the values found by entirely different methods (see handbooks of physics). The conclusion is that even visible particles distribute themselves as though thEty were very heavy gas molecules. Since the gradual dilution of the atmosphere with increasing altitude and increasing pressure depends >on the gas Jaws, these laws can also be applied to coarse emulsions. There is no doubt that they can be applied likewise to finer emulsions, in other words, to colloidal solutions. Incidentally it may be added th,l1t it is customary to designate as colloidal solutions, emulsions, the particle size of which ranges b!ltween 1 and 200ILIL.

APPENDIX 299

From an experimental viewpoint the application of gas laws tocolloidalsolutions seems quite promising, since the osmotic pressure is quite accessible to measurements preferably by direct methods. For low molecular substances the osmotic pressure is so large that its direct measurement offers the greatest difficulties. Being inversely proportional to the molecular weight, it comes down to magnitudes equivalent to a water column of a few inches, if very high molecular substances are used in dilute solutions, viz., not much more concentrated than about 5 per cent. Suitable impermeable membranes can readily be found, since the difference in size between colloidal particles and regular molecules is so immense that many sieves act as, membranes, such as parchment, pig's bladder, collodion or dried gelatin.

Nevertheless, an application of the gas laws for determinations of molecular weight of colloids from the osmotic pressure meets numerous handicaps. Just as in the:case of a mastix: emulsion, the size of the particles which are contained in any colloidal solution varies within the wide~t limits. This is proved particularly bY' observations of the diffusibility of colloids. It is found that a part of the dissolved colloid, such as protein, will diffuse through the membrane. The fraction, which diffuses, largely depends on the size of the pores of the membrane. The larger the pores the greater the number of colloidal particles that will pass. This can be observed even on such chemically pure solutions as those of starch. That portion of the colloidal solution which has passed through the membrane is different from the portion which has not passed and it can be shown that the difference consists in an accumulation of the larger particles in the solution inside.

Another observation which demonstrates the same point is the varying water imbibition of many solid colloidal materials. Starch shows preferably such a varying water imbibition. As pointed out by Naegeli, 1858,4 some specimens of starch, when saturated with water, take up less, others more water, and in between these two extremes a complete gradation exists. Almost any saturation point is possible. It would be difficult to account for this finding by a theory that water is contained in the starch as a solid solution. This would lead to the improbable conclusion that every type of starch should consist of a peculiar type of molecules, or, that an almost endless variety of different starch molecules should exist. It seems more reasonable to assume, as Naegeli did, that there' exists just one type of starch molecule, but, that aggregates of molecules or "micellae" of varying size, are formed, and that water should be held in the spaces between the micellae, being bound by chemical attraction to the surface of each micella, but without penetrating into the micella itself. In those types of starch which have a lower power of holding water the micellae are relatively large and hence the interstices occupy relatively less space, while in a. starch with a. high water holding power the interstices occupy more space,-in other words, the particles are smaller.

4 C. Naegeli, "Die Starke-Korner," 1858.

300 APPENDIX

2. THE PRINCIPLE OF THE SO-CALLED MEMBRANE EQUILIBRIUM (ACCORDING TO

F. G. DONNAN, 1911)5

It is evident that under these conditions determinations of osmotic pressure of colloids have but little importance. However, their variations under comparable conditions are of interest. Particularly large variations occur following the addition of salts and acids on account of the electrical charges of the molecular particles. Donnan's theory of membrane equilibrium attempts to account for these variations. In order to explain this theory we discuss first a special hypothetic case, viz., t}le variations of osmotic pressure of a mixture of soap and sodium chloride (NaCI). Soap, the sodium salt of fatty acids, may be supposed to consist of a freely diffusible positive ion: N a +, and a nondiffusible negative ion, viz., the fatty acid radical; this we may call R-. We assume that the osmotic pressure of such soap + N aCI mixtures is measured in a shell of parchment or collodion which is permeable not only to water but also to all other substances except colloids or colloidal ions like R-.

If no NaCl is present the osmotic pressure of the soap is due to all the Na + and all the R- ions present besides the undissociated remainder. Apparently the Na + ion is retained within the membrane by electrostatic attraction to the non-penetrating R- ion, even though it could pass through the membrane by itself. Without this attraction the soap solution as a whole, would acquire an electric charge which manifestly never occurs. Hence in any solution, the number of positive and negative particles must be equal. This is the "rule of electro-neutrality."

If N aCl is also present, this will distribute itself in both solutions, inside and outside the membrane, since it can pass freely. A superficiaz.. consideration may lead to the conclusion that, after a sufficient time, NaCI would be evenly distributed in both solutions, whether or not soap is present in the solution inside of the shell. This, however, is not true; the final distribution of NaCI is not equal on both sides. There must be less NaCl inside the shell if soap is present, since the non-diffusible soap prevents the invasion of the diffusible salt to a certain extent, due to the fact that soap also splits off Na + ions, and these repel a part of the Na + ions split off by NaCI, on acc~unt of a shifting of the chemical equilibrium. A determination of this repelling influence of soap is possible through the thermodynamic laws of chemical equilibrium. These laws show that the ratio of the concentration of Na +ions inside and outside the shell, mUltiplied by the ratio of the concentration of CI- ions inside and outside the shell, must be equal to one. In other words, these two ratios must be inversely proportional.

C'Na: C"Na = C"Cl: c'Cl

c' designating concentrations insid~; e" designating concentrations outside.

This distribution is reached after the diffusion has1asted sufficiently long to establish a stationary condition. This relation can be derived from the

Ii Zeitschrift f. Elektrochernie, 17, 572.

APPENDIX 301

thermodynamic law of partition according to which the distribution of each molecule between two phases in equilibrium must be constant. Hence the distribution of undissociated NaCI between water and the substance of the membrane is constant, or the concentration of undissociated NaCI in both aqueous solutions must be equal. Since the product of CNa' Celis proportional to CNapl in both solutions, we have:

C'Na'C'Cl = const·c"Na·C"Cl,

This is the formula given above. Another derivation as proposed by Donnan himself and quoted in his own

words is the following. "When this equilibrium is established the energy required to transport

reversibly and isothermally 1 gram molecule of Na from (2) to (1) equals the energy which can be gained by the corresponding reversible and isothermal transport of a gram molecule C1. In other words, we consider the following infinitely small isothermal and reversible change of the system:

:Zn mol N a + (2) --> (1) :Zn mol 01- (2) --> (1)

The energy which can be gained in this way (Le., the diminution of free energy) is zero, hence:

fNa+h fCI-h :Zn RT log fNa+J. +:Zn RT log [Cl-]l = 0

or

where the brackets signify molar concentrations."

It is interesting to apply this relationship to a case where the soap concentratfon is extremely high, as compared to the NaCl concentration, e.g., 1000 times higher. Whatever portion of NaCl diffuses, the ratio of the Na ions inside and outside the shell must be very high. The reverse relation must exist between the Cl ions inside and outside. The conclusion is that no more than diminutive traces of these ions can penetrate. This appears to be the only possibility of keeping the product of the two ratios of the two ions equal to one. Hence, an excess of the non-diffusible electrolyte displaces the diffusible one almost entirely to the outside.

It is necessary to derive a general law or formula which allows us to calculate the varying distribution of NaCI from the initial soap and NaCl concentration. Which fraction of NaCI is liable to penetrate the shell? The initial concentration of NaCI may be termed C2, that of soap Clo A fraction of NaCl equaling

302 APPENDIX

x has passed inside the membrane after a state of equilibrium has been established. The respective ionic concentrations are then

inside: Na+ ............ Cl + x CI-............ . x

outside: both Na + and Cl-............ Cz - x.

We have to determine the ratio of NaCI remaining to that penetrating into the shell, which is:

In order to do this, we apply the thermodynamic law mentioned that the ratio of the Na and CI inside to outside are inversely proportional and find:

Cl + X C2 - X --=

or

(Cl + x)x = (C2 - X)2

or

or

X C2 - 2x -=

or

Cz - 2x Cl --_-- =

adding 1 to each side of the equation, we obtain:

Cz - 2z + Z CI + C, =--

X C2

Cz - x = Cl + Cz

X Cz

or

remaining portion ot NaCl initial concentration of "soap" + NaCl penetrating portion of NaCl initial concentration of NaCI

APPENDIX 303

For a better understanding of this fundamental relation we will calculate the salt distribution in a few numerical cases. As one example let us suppose that in the initial state the same amount of soap is present inside the shell as NaCl outside. (We also assume that the soap, Na R, is totally dissociated into Na + and R- ions.) Hence

or

Cl+C2=2

Cl

According to the above given equation, the portion of NaCI remaining outside, must be twice as large as the penetrating portion, in this case; hence two-thirds of the NaCl stay outside while one-third penetrates. If twice as much NaCl is added, we find that two-fifths are inside and three-fifths outside. The excess on the outside is, therefore, one-fifth of the total NaCl present, or two-fifths of the amount of "soap" present. If ten times as much "salt" as "soap" is present the excess on the outside is -IT of the total amount of "salt" which amounts to as much as ~H of the "soap" content.

The most important question is the influence of this excess NaCI, on the osmotic pressure of the "soap." It is evident that the partial obstruction of the N aCl diffusion must influence the osmotic pressure of the "soap" materially. Since a greater portion of NaCI is kept outside, the excess must exert an osmotic counter-pressure. If a minimal amount of the NaCl is present this counter-pressure may be neglected on account of its low magnitude, even though all of the NaCl is kept outside. If more NaCl is present the counterpressure is considerable. For instance, in the case of equal amounts of "soap" and N aCl, the excess of N aCl amounts to one-third of the total "soap" content, hence the osmotic pressure of the "soap" is decreased by one-third. The actually observed osmotic pressure is two-thirds of its magnitude in the absence of salt. If twice as much NaCl is added, the excess outside amounts to twofifths of the "soap" as stated, and hence the counter-pressure takes away two-fifths of the original pressure leaving only three-fifths. If 10 times more NaCl than "soap" is present the excess of salt amounts to H of the "soap" content. Hence the counter-pressure of NaCl takes away H of the osmotic pressure of "soap." If 100 times more NaCl is present, !H of the "soap" pressure is counter balanced; if 1000 times more is present Hn is counterbalanced, etc. This shows that even with an enormous increase of the salt, the excess on the outside of the shell can never be more than one-half of the "soap." In other words, the osmotic pressure can decrease no more than to one-half of its original value by the action of the counter pressure of NaC!.

304 APPENDIX

3. THE INSUFFICIENCY OF DONNAN'S THEORY TO EXPLAIN QUANTITATIVELY

THE DEPRESSION OF THE OSMOTIC PRESSURE OF COLLOIDS BY SALTS

(R. BEUTNER, 1930)6

According to the theory discussed (Donnan, 1911), the depression of the osmotic pressure of colloidal electrolytes by the addition of a diffusible electrolyte should never amount to more than one-half of the original pressure no matter how great an excess of diffusible electrolyte is added. Experimental observation shows, however, that this is far from being true. The osmotic pressure is depressed in many cases to ,)" to .. \ of its original magnitude or even lower, by the addition of NaCI. This is clearly shown, by the measurements performed by Loeb on gelatin and other colloids.'

Manifestly such a deviation must be due to the fact that the premises of the theory are insufficient. It seems likely that this deficiency is due to the assumption that the colloidal molecular aggregates (micellae) carry a single electric charge. It seems more likely that high molecular colloidal ions carry multiple charges, probably about as many as the number of free acid or basic groups in their molecule. A protein molecule contains a large number of basic NH.or NH-groups. If HCI be added to a protein solution, many of these groups enter into a salt like combination with HCI which in turn dissociates into ions. The negative ions are, of course, the CI- ions. But the positive ions are the protein cations and manifestly as many CI- ions have to correspond to one single protein cation as the number of free basic groups in the protein molecule that have combined with HCI. The protein ion must carry about as many positive charges as the number of CI- ions, corresponding to it, or, else the solution would no longer be electroneutraI. .

The question arises how such a multiple valence of non-penetrating ions affects the membrane equilibrium. We may discuss a similar case, as above,

Before diffusion:

Concentration of N a.R inside: Cl

Concentration of N aCI outside: c,

the concentration of ions are as follows:

inside: Na+ .................. 2Cl

R--.................. Cl

outside: Na+ .................. c. Cl- .................. C2

After x gram molecules of N aCI have passed from the outside to the inside:

The con~centration of N aR inside remains unchanged.

N aCI has decreased outside to C2 - x N aCI has increased inside to x

hence the concentration of the ions are changed as follows:

inside: Na+ ............... 2Cl + x R--............... Cl

CI- .............•. x outside: Na+ ............... C2 - x

Cl-............... C2 - X

6 See preliminary pUblication in Proceed. Soc. Exper. Biology, 27, 692.

APPENDIX 305

with a non-penetrating sodium salt of a colloidal acid, but of a higher valence, such as 2, hence: R Na2. If the molecular concentration of this compound RNa 2 inside the shell is CI the N a + concentration must be 2 c I, assuming 100 per cent electrolytic dissociation. This would be the concentration inside before the equilibrium with the NaCI outside is established. Due to the diffusion of NaCI through the shell, an equilibrium is established and the Na + concentration inside increases from 2 CI to 2 CI + x. The change of concentration of the other ions is given in the table on page 304.

In general, if the valency of R equals n and if non-penetrating salts like R Nan are formed, the N a + concentration will be n C 1 + x, (and not c 1 + X

as in the case of a monovalent colloidal ion). If we repeat the derivation, described above, we have to substitute n c 1 for CI in every instance, and consequently arrive at the conclusion that the relation of the remaining portion of NaCI to that penetrating into the shell is not

but

This modified formula means that much more NaCI must remain outside the shell. Consequently a counterpressure which decreases the osmotic pressure of the colloid may rise to much higher levels, particularly if the N aCI concentration (c 2) exceeds the colloid concentration (c I) materially.

As an example, we may calculate the influence of valence, for a constant concentration of the colloid electrolyte R Nan, viz., c 1 = ,!o, while the NaCI concentration before the diffusion is the same in all cases, viz., c 2 = 1. We have calculated already above the lowering of the osmotic pressure for a monovalent colloid, viz., n = 1 and found it to equal !H = 49.8 per cent. A calculation performed in a similar manner for a bivalent colloid R Na2 shows that the osmotic pressure will drop 66.0 per cent; for R Naa it will drop 74.5 per cent; for R NalO the drop would be 86.1 per cent.

For c 1 = 17>'00, c 2 = 1 and n = 100, the decrease would go as far as 94.2 per cent; in other words, the osmotic pressure of the colloid would be reduced to 5.8 per cent of its original value! These figures clearly demonstrate the influence of the valence.

For higher valent colloid electrolytes the theory demands that the osmotic pressure should be counteracted far in excess of the 50 per cent limit. Since the experiments indicate such a large depression, we find our conclusion justified that the higher valence plays an important r{)le in these phenomena, as predicted by our extension of the Donnan theory. A polyvalent electrolyte displaces a far greater share of the NaGI to the outside by dint of its powerful multiple electric charges.

306 APPENDIX

4. THE DERIVATION OF A GENERAL FORMULA FOR THE MEMBRANE EQUILIBRIUM

OF POLYVALENT COLLOIDAL ELECTROLYTES (R. BEUTNER, 1930)

It seems desirable to express the result of this modified type of membrane equilibrium by means of a general formula. We have to solve the problem to what extent the osmotic pressure7 of a colloidal electrolyte of multiple valence (n) can be lowered by the addition of a salt like NaC!. The molecular concentration of the colloidal electrolyte may be expressed as C I. The assumption may be made that it is completely dissociated into ions. Hence each molecule of R Nan would produce one colloidal R ion and n Na + ions. The concentration of the colloidal ions would be therefore CI and that of the Na ions n·cI.

In the absence of any diffusible electrolyte the osmotically active concentration would be equal to that of the colloidal electrolyte C I plus the concentration of the Na + ions which are held within the cup, formed by the membrane, by electric attraction. Hence the total osmotically active concentration would becI(n+l).

The next problem is to express the counterpressure of the excess of diffusible ions if these are present. If the diffusible electrolyte is divided, after equilibrium is established, outside and inside the shell in the ratio a: b, then

a a + b ·c. is the concentration outside the shell.

b a + b ·c. is the concentration inside the shell.

a-b a + b ·c. is the excess of ions outside, which is responsible for the counter

pressure.

This last magnitude must now be expressed in terms of CI, c. and n. We have to eliminate a and b. This can be done if we remember that

remaining portion of N aCI a nCI + C2 =-=---

penetrating portion of NaCI b c.

as stated above (see page 305). Adding 1 to each side we get:

a nCI + c. -+1=--+1 b C2

or

a + b nCI + Co + C2 --=

b c.

or

(I)

7 It should again be emphasized that ~he osmotic pressure is measured in a shell permeable to all molecules or ions, except colloidal ones.

APPENDIX

On the other hand, we have also the reverse relation:

Adding 1 to each side we get:

C2 b:a= --

nc) + C2

a + b C2 + nc) + C2~ --=

a nc)+c2

a+ b nel + 2C2

By subtracting equation I from II we obtain:

a - b nCl + C2

a + b = nCl + 2C2

C2 nCl

nCl + 2C2 nCl + 2C2

307

(II)

SUbstituting this term, we find that the excess of NaCI outside, giving rise to counter pressure, is:

a - b nC'C2 -- 'C2= a+b nCl+2c2

Since each NaCl molecule may be supposed to be split into two ions the active osmotic concentration giving rise to counter pressure is twice as large, hence equals:

The relative lowering of the osmotic pressure must equal the ratio of

the osmotically active concentration of the colloidal electrolyte in the absence of N aCI which is:

to

the osmotically active concentration of NaCI giving rise to counter pressure which is:

2nCICt

no, + 202

The latter divided by the former is:

308 APPENDIX

this being the relative lowering mentioned. In order to find the maximal relative lowering of the osmotic pressure of the colloidal electrolyte by an excess of NaCI, we may assume that concentration of the colloid is negligible as compared to that of NaCl, hence c 1 = 0, also n Cl = O. The relative maximal lowering is then equal to

2nc2 n

(1 + n)2C2 n + 1

Thus we see that the final result of the theoretical calculation can be expressed in very simple terms: the maximal lowering depends exclusively on the valence of the colloidal electrolyte. It is independent of the concentrations both of the colloidal and the diffusible electrolyte.

For a monovalent colloidal electrolyte a fraction of the osmotic pressure equal

to __!!:_ = _1_ = lis taken away if anex~ess of the diffusible electrolyte is added. n+l 1+1

2 For bi-valent electrolyte this fraction amounts to 1 + 2 = ~, in other words the

pressure drops to ! of its original magnitude. For trivalent colloidal electrolytes the pressure can be dropped to t by an excess of diffusible salt; for a fourvalent to

. 1 t, or, in general, for an n-valent colloidal electrolyte to -- of its original mag

n+l nitude in the absence of diffusible salts.

This result can be verified also by a general derivation. Manifestly the fraction of the osmotic pressure which remains after maximal lowering by an

n 1 excess of NaCl amounts to 1 - -- or to --1'

n+ 1 n + The reader will be aware of the fact that for the derivation of this general

relation, certain simplifying assumptions have been unavoidable. In particular it has been necessary to assume that both the colloidal and the diffusible electrolyte should be completely dissociated into ions. While this assumption is not entirely correct the error thus introduced is probably slight. In the above given derivation we hav~ calculated the relation of the osmotic pressure of the colloidal electrolyte to the counter pressure of the excess N aCI . outside, from the respective ionic concentrations. If the assumption of complete ionic dissociation is inaccurate it seems likely that both the colloidal electrolyte and NaCI are dissociated to the same degree. Hence, the ratio of the two ionic concentrations, which affects the end result of the calculation would be nearly the same as given in the above derived formula.

From this theory the conclusion can be drawn that the depressing effect of an excess of NaCI on the osmotic pressure of a colloidal electrolyte always reaches a definite limit. The lowest limit to which the osmotic pressure can be depressed may be far below the maximal pressure for a polyvalent electrolyte, yet, it can never drop to zero. This postulate of the theory is well verified by J. Loeb's measurements2 which show that with large additions of NaCl the osmotic pressure decreases to a definite minimum, but never quite to zero.

APPENDIX 309

A depressing effect of a neutral diffusible salt, as discussed above, would occur not only in the case of "soap," R Nan, but also for a colloidal electrolyte consisting of a polyvalent cation and many monovalent diffusible anions. Consequently, it must be applicable likewise to an amphoteric colloid like gelatin. In this case the colloidal polyvalent ion is the cation on the acid side of the isoelectric point (due to the formation of gelatin chloride). On the alkaline side of the iso-electric point, however, sodium gelatin ate is formed. Hence, the colloidal gelatin ion is the anion. Concerning the method of determining the iso-electric point of gelatin by means of staining experiments, see Figures 11 and 12, also legends.

From all that we have stated, it is evident that the osmotic pressure of gelatin must depend on the pH of the solution. At the iso-electric point the osmotic pressure should be at a minimum. We should expect that at the iso-electric point, the osmotically active concentration should be equal to c 1 which is the (molecular) concentration of gelatin, since no gelatin chloride or Na gelatin ate is formed, hence no ions are present which can be held within the membrane by electric attraction.

As stated above, by addition of HCl, gelatin chloride is formed and, since n Cl- ions are present for each gelatin ion, the osmotically active concentration rises to c 1 (n + 1). By the addition of an excess of diffusible salt the osmotic

1 pressure of gelatin chloride is depressed to the -- part of its original value,

n+1 as we have seen. Hence what is left over is an active osmotic concentration

n+l -- 'c,= c, n+l

in other words the same osmotically active concentration which prevails at the iso-electric point. We see, therefore, that by the addition of an excess of N aCl the osmotic pressure of gelatin chloride can be lowered to the level of the osmotic pressure of iso-electric gelatin.

According to Loeb's measurements this conclusion is approximately correct: the osmotic pressure of a 1 'per cent gelatin at the iso-electric point is given by him as about 25 mm. water column, the osmotic pressure of gelatin chloride is depressed to a minimum of about 23 mm. by NaCl. A complete agreement can hardly.·.be expected since the molecular size of the gelatin micellae may be altered by ionization to some extent. Moreover, salt addition has some specific influence upon the osmotic pressure at the iso-electric point. This cannot be accounted for by the "classical theory," discussed so far, and points to other influences which will be described later.

5. CALCULATION OF THE VALENCE OF THE GELATIN ION (R. BEUTNER, 1930)6 ACCORDING TO LOEB's MEASUREMENTS OF 19222

It should be possible to calculate the valence of the gelatin ion from the relation of its maximal osmotic pressure to the minimal osmotic pressure which

310 APPENDIX

is observed following the addition of an excess of salt. However, the maximal osmotic pressure cannot be determined directly since even in the absence of NaCI or another salt, a gelatin chloride solution is never free from diffusible electrolyte. A so-called hydrolytic dissociation takes place. In other words: gelatin chloride splits up partly into gelatin and HCl. This leads to a chemical equilibrium in such a way that gelatin chloride and HCI are present in a definite proportion. For a calculation of the valence of the gelatin ion, it is not necessary to determine this proportion. All we need to know is that some free HCI must invariably be present. Consequently a membrane equilibrium will be established. HCl will diffuse through the semi-permeable shell of collodion which is used for measuring the osmotic pressure of the gelatin chloride. According to the rules of membrane equilibrium, as explained before, an equilibrium across this membrane can be established only if a higher HCI concentration exists outside on account of the gelatin chloride present inside which cannot penetrate. Consequently, the excess HCI outside, or rather the H+ and Cl- ions produced by its ionization, exert an osmotic counter pressure. The osmotic p1:essure, which is actually observed, is that of the gelatin chloride minus this counterpressure.

The magnitUde of the counterpressure, due to the excess !lCI outside, can be determined by means of hydrogen ion determinations, as follows. We can determine the H + ion concentration inside the shell in the gelatin solution, and outside in the acidulated water. As is to be expected, the latter has been found to be larger (Loeb).· In order to calculate from these H+ ion concentrations the excess of the osmotically active concentration outside over inside, we have to consider that one CI- ion exists for each H + ion; hence the difference of the H + ion Iloncentrations must be multiplied by two. "

In order to obtain the osmotic pressure in terms of the height of water column, we should consider that a gram molecular solution theoretically exerts a pressure of 22.4 atmospheres, or 22.4·760 mm. which equals 22.4·760·13.6 mm. of water. This holds good at O°C.; at room temperature 20°C. or 293 abso-

lute, the pressure is slightly higher, viz.: 22.4:760·13.6· 299 = 250,000 mm. 273 .

A 0.00001 molecular solution therefore, exerts a pressure of 2.5 mm. water column. Hence- the magnitude of the counterpressure is obtained by multiplying the difference of the H ion concentration inside and outside, which must be expressed in lOO!OOO molecular units, first with ~ and then with 2.5., or, on the whole, by multiplying it with §.

This mode of calculation may be applied to the measurements of osmotic pressure of acidified gelatin solutions in collodion bags, and pH determinations inside and outside the bag, performed by J. Loeb in 1922.2 In one instance the following was observed; a solution of 1 per cent gelatin was poured into a collodion shell provided with a tube to measure the osmotic pressure in mm. of water. Some HCI was added to the gelatin solution and to the water outside, and sufficient time allowed to establish an equilibrium. :The osmotic pressure was then observed to be 100 mm. of water. The hydrogen ion concentration inside was found to be 0.0001·2.7 M and outside 0.0001·7.2 M. The difference, 4.5 multiplied by 5 gives 22.5 mm. water as the magnitude of the counterpressure. This should be added to the actual osmotic pressure in order to obtain

APPENDIX 311

the sum total of the osmotic pressure due to gelatin and gelatin chloride, hence this sum amounts to 122.5 mm. water.

A series of further experiments of the same kind were performed with 1 per cent gelatin, adding more and more Hel, the results can be tabulated as follows:

I II III - -- --

Actual hydrogen ion concentration: OU~8ide~' in l(~O~OOO mol. u.nits ................ 16.6 363 549 InsIde, In ~ mol. unIts ........ ......... 4.9 93 14.1 Outside minus inside, in 100:000 mol. units .. Outside minus inside, in lOO~OOO moL units

11.7 27.0 40 8

times 5 (counterpressure) ................ 58.5 135 0 201 6 Observed osmotic pressure in millimeter

water column .................... ...... 202.0 322.0 375 0 Observed osmotic pressure in millimeter

water column pluB counterpressure . ..... 2605 4570 576.0

IV V VI VII -- - - -135.0 155 0 295 524 46.8 56.2 141 302 88 2 98 8 154 222

441.0 494.0 770 1110

443.0 4420 360 303

884.0 9360 1130 1413

VIII IX -- -

1000 1288 741 1023 259 265

1295 1325

198 162

1493 1487

X

190 162

5 2

283

141 5

11 o

152

The figures of the last line in this table represent the pressure of gelatin and gelatin chloride as calculated by adding to the observed pressure the (calculated) counterpressure. We see that with increasing addition of acid these values rise steadily. They seem to approach a maximum between 1500 and 1600 mm., probably at about 1530 mm. With increasing addition of acid the calculated osmotic pressure of gelatin chloride increases at first very markedly, until at a concentration of about 10~~~OO = rfTfTf molecular Hel, a pressure of nearly 1300 mm. of water is reached. On further addition of acid, the rise of osmotic pressure is slight: in a n\Tf molecular solution it is about 1460 mm., in a rtll"

molecular it is just very slightly larger since it still stays helow the 1500 mm. mark. According to the theory, such a course should be expected since increasing amounts of gelatin chloride must form as more acid is added. The rise of the pressure is, of course, exclusively due to the formation of gelatin chloride from gelatin, the osmotic pressure of the latter being quite negligible.

As explained above, the gelatin chloride is always partly split into gelatin and Hel, in such a way that a chemical equilibrium exists. This means that theoretically gelatin cannot be transformed completely into gelatin chloride. Nevertheless, if a great excess of Hel is added, it is practically completely transformed since, according to the laws of chemical equilibrium, the ratio:

Cgelatin • eiiel t = cons.

e gelatin chloride

Hence a high concentration of free Hel entails an extremely low concentration of free gelatin, which means that practically the entire gelatin is transformed into gelatin chloride. Apparently this is the case for (free) Hel concentrations of about l~~."Oooo = T~ll" molecules or higher.

Since tpe minimal pressure observed by adding to gelatin chloride an excess of Nael has been found to be about 23 mm.-which is the same as the osmotic pressure of iso-electric gelatin-we can now calculate the valence of the gelatin ion. As figured above, the maximal pressure of gelatin chloride, viz., 1530 mm., divided by the minimal pressure, 23 mm., equals n + 1, hence n + 1 =

312 APPENDIX

1530 23 = 66, or n = 65. The gelatin ion carries a charge 65 times. larger than a

H+ or N a+ ion, according to these calculations. Manifestly this can be only the average charge carried by gelatin ions. Since the size of the gelatin micellae is never uniform, as shown above, their charge must vary accordingly. Some carry a higher mUltiple charge, possibly up to 100 or higher, others have a lower charge. We have to content ourselves with an approximate estimate of the order of magnitude of this huge multiple electric ion.

It should also be pointed out that the result obtained holds only for that sample of gelatin which had been used in Loeb's experiments and if treated in exactly the same manner as he happened to treat it. The size of the particles and hence their charge depends on uncontrollable circumstances. Hence, if some one else repeats these experiments with another brand of gelatin, or, if he heats Or shakes the same gelatin, either more or less than Loeb in his experiments, when preparing solutions, the results may be different.

6. THE CALCULATION OF THE OBSERVED OSMOTIC PRESSURE OF ACID GELATIN

SOLUTIONS FROM THE DIFFERENCE OF ACIDITY INSIDE AND OUTSIDE

THE SHELL AT EQUILIBRIUM (LOEB, 1922)2

The variability oj the actually observed osmotic pressure oj the 1 per cent gelatin solution with increasing addition oj acid presents a picture entirely different from the calculated pressure oj gelatin chloride. We see that it first increases as more and more acid is added up to a maximum oj 443 mm. at a concentration of about 0.0015 mol. HCI. Upon further addition of HCl, it decreases almost as much as though N aCI was added. (See the line above the last one in the table, page 311.)

This peculiar influence of acid addition has puzzle.d many investigators. According to the theory explained, it seems easy to understand it. The actual osmotic pressure is the difference of two antagonistic pressures, in each instance, viz.: (1) the pressure of gelatin plus gelatin chloride, which increases as more acid is added and (2) the counterpressure of the excess HCI outside which also increases with the addition of the former. However, the increase of (1) is large at first, later reaches a maximum, while (2) keeps on increasing. Consequently, at low acid concentrations the count~rpressure is hardly felt at all and an addition of acid serves to raise the actual pressure. At higher concentrations the increase of (1) comes to a standstill, and the actual pressure drops since the counterpressure (2) grows steadily. (Compare table, page 311.)

On account of the extremely high average valence of the gelatin, viz., 60 to 70 times as high as the Cl- ion, the CI- ions alone are practically responsible for the rise of the osmotic pressure of gelatin plus gelatin chloride. The por~ tion due to gelatin as such, ionized or not ionized, can be neglected, since there is no more than one gelatin ion for every 60 or 70 CHons. We may state, therefore, that the actual osmotic pressure is practically due to all the diffusible ions, Cl- and H+, minus the same ions outside.

On the basis of this consideration, J. Loeb2 has been led to a calculation, different from the one described before. He calculates the osmotic pressure

APPENDIX 313

of gelatin chloride from the H + ion concentration difference inside minus outside and neglects the share due to the gelatin itself. Manifestly this neglect implies the assumption of a very high valence of the gelatin ions. Yet, it is not possible by this method to form a definite conception about the magnitude of this valence. Loeb's calculation may be explained, in a somewhat simplified manner, as follows.

In the solution outside, the same number of H + and Cl- ions must be present, since there are no other ions at all and positive and negative ones must be equal. Hence the osmotic pressure, neglecting the gelatin fraction, is equal to the concentration of the H + and Cl- inside minus twice the outside H + concentration. The Cl- concentration inside is, of course, far from being identical with the H + concentration inside since there are also the Cl- ions, dissociating from gelatin chloride. At this point, Donnan's theory can be applied. This states that the product of the Cl- and H + inside is equal to the same product outside, or, equal to the square of H + outside (since the concentration of Cl- and H + is identical here.) Hence the Cl- concentration inside equals the square of

the outside H+ concentration divided by the H+ concentration inside, or~ t

(if we call the outside H+ concentration 0 and the inside H+ concentration i). When this ratio is substituted for the Cl- concentration inside, the observed osmotic pressure neglecting the gelatin fraction should be

0 2 1 (0 - i)2 -;- + i - 2 0 = -; (02 + i2 - 2 io) = --.-1 1 t

This simple relation is true merely for gelatin plus HCI mixtures in the absence of NaC!. If NaCI is present, or for gelatin sulphuric acid mixtures, a more complicated formula is derived.

In other words, the square of the difference of the H + concentrations outside minus inside divided by the H+ concentration inside should represent the actual osmotically acitve concentration. If this concentration is expressed in 100~OOO molecular units and its numerical value multiplied with 2.5, the osmotic pressure of the acidified gelatin solution expressed in mm. water column should be obtained. This calculation has been carried out numerically for those ten cases, mentioned above, in which J. Loeb performed pH measurements inside and outside the osmotic bag. The results are as follows.

Case

I II III IV V VI VII VIn IX X -- -- -- -- ---- -- -- -- --

Calculated from dif-

}70 ference of H+ 196 296 416 436 422 410 227 115 115 concentration in-

side and outside

These calculated values should be compared to those actually observed which are as follows:

Observed values .... \202\32213751 44314421360 130211981162 IUO

314 APPENDIX

The agreement is far from being perfect since for the first five cases the calculated values are all smaller than the observed ones, while for the last five cases the reverse is true. If our tentative assumption of the constancy of the osmotic pressure, produced by ionogenic and non-ionogenic gelatin, was correct, we should expect that all observed values are somewhat larger than the calculated ones, since the calculated values leave out of consideration the "gelatin" fraction. The fact that for higher acidity the observed values are lower seems to indicate that for higher acidity the osmotic pressure of gelatin, ionized or not, decreases with increasing acidity. Within this range the gelatin micellae seem to be united into fewer and larger aggregates.

We see, however, that the calculated values rise, pass through a maximum and then decrease like the observed values. The maximum occurs nearly at the same acidity for both sets of values, viz., at or near the fifth numerical example in the table given above. Hence the result may be condensed into the statement that-in spite of the disagreement mentioned-the osmotic pressure of an acid(fied gelatin solution is almost parallel to the lIalues calculated/rom pH differences outside minus inside, according to the above derived theoretical formula.

As we have seen before, the addition of N aCI to a gelatin Hel solution ~nvariably decreases the osmotic pressure. In this case also the decrease of ,'Vsmotic pressure is associated with a decrease of the pH difference outside minus inside, as can be predicted by the theory (although by a more complicated calculation). The following examples may serve to show that this conclusion from the theory can also be verified by experiment; in one instance, the osmotic prcs'sure of salt free gelatin chloride was 425 mm. and the pH difference 0.53.

By the addition of N aNOa (~) the pressure dropped 215 mm. and the pH dif

ference to 0.19. Numerous other examples could be quoted to support the general statement that osmotic pressure and pH changes are parallel in this case. (See Loeb's book2 for further data.)

The unequal distribution of all diffusible ions, particularly of H + ions, gives rise to electric potential differences between the gelatin solution inside the shell and the aqueous solution outside. This is due to the difference of ionic concentrations on either side of the membrane. As we have seen, this difference is maintained even after equilibrium is established. It is possible, therefore, to apply the thermodynamic rules derived above (dee page 218), just as in the case of two phase boundaries which are in equilibrium. If c J be the H ion concentration in the gelatin solution and c 2 the one in the gelatin free solution, then a potential difference must exist across the membrane equal to

APPENDIX 315

Cl 0.058 log -. const. volts, at room temperature. This formula can be derived

C2

by assuming that a definite H ion concentration exists in the membrane itself, and that consequently two phase boundary potentials exist on either side of the membrane, the difference of which constitutes the potential difference across the membrane. If Cm be this H ion concentration in the membrane,

then the two phase boundary potentials must be 0.058 log ~. const. ' and 0.058 Cl

C log...!". const.". It is obvious that the difference of these two values equals

C2

that given above for the potential difference across the membrane, since const' . const· " may be considered as equal to const.

The correctness of this formula has been verified by numerous measurements of Loeb who believed he had found in this manncr a new evidence for the Donnan equilibrium.

It is obvious, however, that the existence of such a "membrane potential" is merely a thermodynamic consequence of the ionic inequality existing at equilibrium. "Membrane potentials" are virtually no more than differences of 'two "phase boundary potentials." The existence of these potential difference8 cannot be regarded, therefore, as an independent proof of the theory of membrane equilibrium, contrary to Loeb'8 statements which have been justly criticized byrA. V. Hill (1923).8

7. THE INFLUENCE OF THE VALENCE OF THE DIFFUSIBLE IONS (LOEB, 1922)2

Additional evidence for the existence of an ionic membrane equilibrium as tll,e cause of osmotic pressure is found by studying the influence of polyvalent diffusible acids like H2S04. In order to understand this influence, we may compare tbe effect of HOI and H 2S04 solutions of equal pH since it is evident tliat the H + ions exclusively are instrumental in bringing about a change of non-ionized gelatin into gelatin ions.

The addition, e.g., of 2 cc. of T~ molecular HOI to 100 cc. of 1 per cent gelatin w.ill produce nearly the same amount of ionized gelatin and the same degree of acidity as 1 cc. io molecular H 2S04, since H2S04 is dissociated into two H+ ions and one S04-- ion. On the other hand, 2 cc. of that HOI solution contain twice as many negative ions as 1 cc. H 2S04• 9 Since the number of negative ions which are held by the gelatin ions inside the shell is only half, the addition of H2S04 can produce only about half the rise of osmotic pressure which HOI produces. The following measurements (performed by Loeb) show that this

8 Proceed. Roy. Soc., Ser. A, 62, 703, also Journ. Gen. Physio!., 6, 91. 9 One-tenth molecular H 2SO, is usually called T"u normal H 2S04 counting

the H+ ions.

316 APPENDIX

is as nearly true as .one can reas.onably expect. One cubic centimeter .of a lu m.olecular H 2SO. was added t.o 100 cc. 1 per cent gelatin s.olutiDn; this mixture was p.oured int.o a cDllDdi.on bag which was immersed in water c.ontaining a little H 2SO.. The .osm.otic pressure was fDund t.o be 110 mm. while with 2 cc . .of HCI it waS 202 mm. (c.ompare figure qUDted abDve.on page 314). Up .on add· ing 4 cc . .of 10 mDlecular H 2S04 tD 100 cc. 1 per cent gelatin, the DsmDtic pressure was fDund to be 208 mm., while with HCl .of the same acidity it was abDut 440 mm. as qUDted ab.ove. These figures, 280 mm. fDr H 2S04 additi.on and 440 mm. f.or HCI additi.on, clearly demDnstrate the wide difference between the actiDn .of these tWD acids .on the .osmDtic pressure .of gelatin. This difference is in nD way related tD a weakness .of the acidity. It is exclusively due tD the fact that H2SO. is a bivalent strDng acid, in .other wDrds that it splits .off 2 H + iDns per mDlecule.

HgPO. seems tD be a trivalent acid and might be expected tD raise the .os· mDtic pressure still less than H 2S04• HDwever, the diss.ociatiDn .of its secDnd and third H + i.on is quite incDmplete. Hence H gPO 4 functi.ons as a mDn.obasic acid, disSDciating practically .only intD H2P04- and .one H + iDn. In fact ex· perimental Dbservati.on ShDWS that HsPO. raises the DsmDtic pressure .of gela. tin just as much as HCl. The highest pressure .obtainable is 436 mm. water at a pH = 3.24, while HCI at pH = 3.25 prDduces a pressure .of 442 mm. Tak· ing int.o cDnsideratiDn the experimental errDrs, these values may be c.onsidered as identical (LDeb).2

Acc.ording tD the relatiDn derived abDve, the DsmDtic pressure .of gelatin depends .on the pH difference inside and .outside the membrane. C.onsequently we shDuld expect that in the case .of gelatin sulphate the pH differences ShDUld be smaller, cDrrespDnding tD the I.ower DsmDtic pressure .of gelatin H 2S0 4. A detailed calculatiDn ShDWS that this difference must be tWD-thirds .. Df the dif· ference, prDduced by the additiDn .of HCI at the same pH. Experimental Db· servatiDns have verified these cDnclusiDns. As .one numerical example .out .of many .others, the fDII.owing may be qUDted: A bag was filled with a mixture.of 1 cc. y\ mDl. H 2S0 4 and 100 cc. 1 per cent gelatin' and plaped in acidulated water. After an equilibrium was established the pH inside was fDund tD be 4.34 while .outside it waS 3.99. The difference is 0.35. If HCl .of the same acidity was used a pH difference .of 0.53 was .observed, which is .one and one-half times greater than fDr H2SO. (L.oeb).2

On the whole, this agreement between observed osmotic preSl'lures and the ones expected from the theory of ionic equilibrium is such that there hardly remains room for doubt of the validity of the the6ry. In evaluating the degree of agreement of all these theoretical and observed figures the reader should bear in mind, (1) that a great accuracy of measurements is impossible on account of the indefinite and irregular composition of the colloids; (2) that the theory of membrane equilibria is evidently so complicated that simplifying assumptions cannot be missed, yet it is hardly possible that these simplifying assumptions should be entirely true. Considering all these handicaps the agreement is almost blJtter than expected.

APPENDIX 317

8. ApPLICATION OF THE THEORY: THE PARALLEL COURSE OF OSMOTIC PRESSURE

SWELLING AND VISCOSITY

The peculiar variations of the osmotic pressure of a gelatin solution have been studied in detail on the foregoing pages on account of their relation to colloidal swelling. As briefly mentioned before, the osmotic pressure of gelatin solutions is parallel to the swelling of solid gelatin granules. This has been demonstrated by Loeb10 in an extensive series of experiments which were carried out as follows. Gelatin granules of equal size were first brought to their iso-electric point by immersion in rh molecular acetic acid and then placed in acid solutions of different concentration. The degree of swelling was determined approximately by reading the volume occupied by the granules after they had been in contact with the solution in question for two hours.

It was found that gelatin swells more at pH values larger or smaller than pH = 4.7. Swelling is minimal at the iso-electric point pH = 4.7, iust as is the case for the osmotic pressure. By the addition of increasing amounts of HCl the swelling of the gelatin granules first rises to a maximal value, but, on further addition of acid, it decreases again, just as osmotic pressure decreases. Maximal swelling occurs at pH = 3.3, which is identical, within experimental errors, with that pH at which the osmotic pressure is at a maximum.

Salts like NaCl, KCI and CaC12 which depress the osmotic pressure of acidified gelatin are also found to depress the swelling of gelatin granules. The influence of valence is manifested in the same way. H2S0 4 which raises the osmotic pressure to one-half of that produced by HCI acts in the same way on swelling. Accordingly, Na2S04 or other sulphates have particularly powerful depressing actions on swelling just as they have on osmotic pressure, corresponding to the lesser effect of H2S04• It seems, therefore, that each gelatin granule swells or shrinks about as much as though it consisted of a semipermeable membraneous bag in which a gelatin solution is contained.

A property oj gelatin solutions which runs parallel to the osmotic pressure is their viscosity. This can be measured by observing the time of outflow of the solution through a narrow opening and then dividing this by the time of outflow of water through the same opening. If such measurements are performed for gelatin solutions to which varying amounts of acid have been added, the finding is that at the iso-electri.c point, the viscosity is at its lowest level. The gelatin is, therefore, more liquid, or takes less time to flow out, than at any other pH.

Furthermore, the viscosity of acid gelatin solutions varies with the pH in the same manner as does the swelling and the osmotic pressure. With increasing additions of HCI the gelatin becomes more and more viscous and at pH 3.2

10 Loeb, "Proteins and Colloidal Behavior." Prior to Loeb, H. R. Procter emphasized the relation of osmotic forces to swelling and developed a quantitative theory on the basis of Donnan's ideas. See Journ. Chem. Soc., 105, 313 (1914).

318 APPENDIX

the highest degree of viscosity is reached. Upon still further additions of acid, the viscosity decreases again. H 2SO. raises the viscosity to a lesser degree. Salts depress the viscosity in the same manner in which they act on swelling and osmotic pressure. Thus Na2SO. depresses more than NaCl. All these agreements lead us to conclude that similar underlying causes should be re· sponsible for all these properties of gelatin. Hence in regard to viscosity also, solid gelatin behaves as though it consisted of innumerable bags of a semi-permeable shell and containing a colloidal solution. The higher the osmotic pressure of the solution in the hypothetical bag, the more.it enlarges.

It remains to be seen whether such "bags" are really in existence. The observations, decribed so far, may also be explained by the assumption that no real osmotic actions occur, but, that forces of cohesion ("molecular forces") hold the water in the gelatin and that these simulate osmotic actions since they resemble them completely. In order to investigate this point, it is important that gelatin can be separated into two different components, without a chemical splitting, by a simple extraction with water (J. H. Northrop and M. Kunitz).l1 One of these constituents is quite soluble at all temperatures, it never sets to a gel. When dissolved, it produces a high osmotic pressure, but, a low viscosity. The other constituent is almost insoluble, its solutions were found to have a low osmotic pressure and a high viscosity. This latter constituent is really never truly dissolved to an appreciable extent. When whole gelatin goes into solution, the insoluble constituent is contained in the solution in the form of molecular aggregates or "micellae." That these micellae actually unite to form bags, which enclose within them the soluble and osmotically active constituents, is shown by the following findings. As shown before, the viscosity of a gelatin solution increases when HCl is added, manifestly due to a content of the "soluble constituent" in the "bags." This explanation involves the assumption that the solution outside is nearly free from this soluble gelatin. We have to assume that whole gelatin, before it is dissolved, contains the soluble constituent both inside and outside of the "micellar bags." When dissolving gelatin in an excess of water, the portion of soluble gelatin which is outside is diluted so much that the outside solution is practically free from gelatin. However, when gelatin is dissolved in a little water, both solutions, inside and outside, contain gelatin. HCI addition would then increase the osmotic pressure both inside and outside and hence the micellae would fail to swell. We should expect, therefore, that Hel addition increases the viscosity slightly or not at all in sufficiently concentrated solutions. This conclusion has been verified experimentally (Kunitz and Northrop).ll Thus, the viscosity of 0.5 per cent gelatin can be increased more than five-fold by Hel addition. The viscosity of a 10 per cent gelatin solution is hardly influenced at all.

Additional evidence for the existence of osmotic bags is found by measuring

11 A summary of this important work is found in Journ. Physical Chern., 36, 102 (1931).

APPENDIX 319

the double refraction of gelatin. The solid wall of the micellae is distended by Bwelling and compressed by shrinking. These mechanical stresses must lead to double refraction, which in fact can be demonstrated as expected (M. Kunitz).12 Further support for this explanation is found by studying the shrinking of iso-electric gelatin gel of less than 10 per cent concentration in distilled water or of dilute buffer solutions of the same pH as that of the isoelectric point of the gelatin used (M. Kunitz).12 It should be noted that these findings about gelatin do not apply to other proteins. In fact, Northrop and Kunitz show that egg albumin acts differently in many respects. The possibility remains that, for the swelling of these proteins, forces of cohesion play a more important role.

Ae we have Seen the osmotic pressure of an acidified gelatin solution is associated with differences of acidity between the gelatin solution inside the semi-permeable shell and the gelatin free solution outside. If it is true that osmotic forces regulate the variations of swelling of gelatin granules following acid or salt addition, we should expect that differences of acidity exist also between these granules and the surrounding aqueous solutions. Experiments have shown that this supposition is true (Loeb).' In this case also, the difference of acidity runs parallel to the degree of Bwelling just as it runs parallel to osmotio pressure. If salts are added the other diffusible ions are Ukewise unequally distributed.

Due to the unequal concentration of H ions (or other ions) in the gelatin and in the solution at equilibrium with the gelatin, electrical potential differences must arise. On account of the equilibrium which exists between gelatin and solution, the boundary between gelatin and solution has the character of a true phase boundary. The rules derived for phase boundary potentials (see page 218) are, therefore, applicable in this case also. Consequently, if Cl be the H ion concentration in the gelatin and C2 the H ion concentration in the solution, the potential difference between the two phases must equal 0.058

log.~ const. This conclusion can, therefore, be derived even without consider-C2

ing Donnan's membrane theory. Numerous measurements of Loeb have verified the correctness of this relation.

As stated above, proteins can combine with alkali on the alkaline side of the iso-electric point. In this way non-diffusible anions are formed, as in the case of "soap." A membrane equilibrium should be established if an alkaline protein is placed in a semi-permeable collodion bag which is immersed in alkaline water. With increasing addition of alkali the osmotic pressure of the protein should rise up to a certain point. This postulate of the theory has been verified by experiments to some extent, but the measurements vary rather irregulnrly.

In this instance also, osmotic pressure runs parallel to viscosity and swelling. All three properties show a similar initial rise and a subsequent fall at higher alkalinity. All are depressed by addition of salts, and, as we should expect

12 Journal Gen. Physiol., 12,289 (1928); 13, 565 (1930).

320 APPENDIX

from previous experience and theoretical considerations, polyvalent salts exert a much more powerful depressant action upon all these properties. In this case we should expect such an increased depression from poly~alent cations such as Ca++, Sr++, Mg++, and Th++++, in other words, from the salts of alkaline earths and possibly heavy metals. This conclusion has been verified by experiments. Hence the basic theoretical conclusion is certainly justified that osmotic pressure as well as swelling and viscosity are depressed particularly by polyvalent ions carrying an electric charge of a sign opposite to that of the colloidal ion. While on the acid side of the iso-electric point sulphates or sulfosalicylates are particularly depressing, Ca++ and Mg++ ions have a large depressing influence upon these properties on the alkaline side of the iso-electric point (J. Loeb).2 .

A parallel course of swelling and osmotic pressure is observed also in those cases in which swelling is brought about by neutral salts without the addition of acid. 13 As is well known, numerous salts such as iodides or sulfocyanides, promote swelling of gelatin without concomitant pH changes.13 These salts are known as "hydrating" ones, while other salts like sulphates, which have the opposite action are known to be "dehydrating." Systematic investigations of the hydrating or dehydrating effect of a number of salts have led to classifying them in the so-called "Hofmeister series" in the following order: alkali sulphates, tartrates, citrates, acetates, chlorides, bromides, nitrates, iodides and sulfocyanides.

In this series alkali sulphates have a dehydrating or shrinking effect, sulfocyanides favor swelling or have a hydrating effect. The other alkali salts exert an effect intermediary between these two extremes in the order given. The series is the so-called anionic Hofmeister series: there is also a cationic series which consists of the following salts.

CaCh, MgCh, LiCI, NaCI, KCI and NH,CI

CaCl2 in this case has a dehydrating influence; NIi. Cl fa~ors swelling. The order in which these salts act is not the same in all cases. For the an

ionic series, the -differences between actions of these salts are greater in acid solutions, for the cationic series, they are greater in alkaline solutions. Yet, such salt actions may be observed even at the iso-electric point.

When the concentration of one of these salts is changed, both osmotic pressure and swelling exhibit parallel variations. The degree of swelling can be calculated to a certain degree from the increase of osmotic pressure (J. H. Northrop, 1926)14 (as long as the elastic limit of the gelatin cells is not exceeded). Also the viscosity of gelatin exhibits parallel variations.

13 Considerable comment has been aroused by J. Loeb's statement that the actions of these salts should be explained entirely as a result of pH changes produced by them. All other experts have disagreed with Loeb on this point since there are no corresponding pH changes.

14 Journ. Gen. Physiol., 8, 317:

APPENDIX 321

Incidentally it may be added that the swelling of colloids also plays a role for the water exchange in tissues. For tracing this type of swelling in tissue, the salts of the Hofmeister series are used preferably. For instance, by immersing samples of smooth muscle in solutions of these salts, the degree of swelling is found to vary in the same manner as would be the case for pieces of gelatin. This shows that smooth muscle, in contrast to striated muscle, swells or shrinks like a colloid, and not like a red blood cell which is surrounded by a membrane at the surface.

To a certain degree the influence of the salts of the Hofmeister series on osmotic pressure and swelling can be explained by a chemical combination with the protein (gelatin) similar to the combination of acid and bases with gelatin. This chemical combination must lead to the formation of non-diffusing ions which attract other ions and this leads to the establishment of a membrane equilibrium, as described above. However, the increase of osmotic pressure cannot be calculated entirely from a membrane equilibrium in this case, the observed value is much greater than the one calculated from the excess of ions in the gelatin. Other factors have to be considered such as a splitting of molecular aggregates, since gelatin in the presence of N aSCN will pass through a membrane which is impermeable to gelatin in water (J. H. Northrop, 1926). Moreover, the addition of NaSCN to gelatin causes the same change of mutarotation as when the gelatin is transformed into a solution by heat. In the latter case, the effect can hardly be due to anything else but to a molecular splitting which leads to an increased osmotic pressure.

The nature of the "dehydrating" influence of Na2S04 and the "hydrating" influence of NaSCN at the other end of the "Hofmeister" series is further elucidated by the fact that Na2S0410wers the solubility in water of almost any substance. This is the well known "salting out" effect which is frequently used in organic chemistry. The reverse solubility, viz., that of water in organic substances is also lowered by Na2S0f. NaSCN has the opposite effect, it raises the solubility of almost any substance in water. The other salts occupy an intermediary position.

These examples show that the "classical" or thermodynamic theory, cannot explain the variations oj osmotic pressure oj a gelatin solution in every case. The same is proved by the simplejact that iso-electric gelatin shows a slight swelling although no unequal salt distribution can possibly occur. Many other limitations of the "classical" theory are known. Thus, acetic acid promotes the swelling of gelatin much more than expected from the pH changes caused, more than Hel, for instance (J. Loeb).2 This is probably due to a diminution of the cohesion of the gelatin.

9. THE WATER IMBIBITION OF THE ALKALI SALTS OF HOMOLOGOUS FATTY ACIDS

A drawback of the experiments with gelatin is the chemically indefinite character of this substance. Even the soluble and the insoluble constituents, isolated by Northrop and Kunitz, are probably not chemically pure substances.

322 APPENDIX

Instead of adding H2S04 or HCI to gelatin and studying osmosis and swelling of the complex mixture thus obtained, it would be desirable to use some protein chloride or sulphate in pure form; but, this is practically impossible. We may use, however, a different type of colloidal substances the chemical constitution of which is more perfectly understood, viz., soaps, the sodium salts of fatty acids. These can be readily prepared in pure form by mixing and heating equivalent amounts of NaOH, or other bases, with pure fatty acids. Soaps lend themselves to investigations which cannot be performed very well with proteins; viz., to observations on the effect of molecular size. The "osmotic" properties of proteins, such as swelling and viscosity, depend ultimately on their non-diffusibility and hence on the large size of the protein micellae. We know, however, that in living organisms, through the actions of enzymes, protein molecules may be either decomposed or built up. Hence enzymatic actions must affect the water holding power of tissues even if pH and salt content remain unchanged.

In order to study the influence of molecular size, we should use proteins of graded molecular size or of a homologous series. Such experiments would seem to be somewhat difficult on account of the insufficient development of protein chemistry. With soap, however, we can obtain such a series of gradedmolecular size by using the sodium salts of homologous fatty acids. Their swelling in water has been studied as follows (M. H. Fischer, 1922):111 Pure soaps were prepared by mixing equivalent amounts of N aOH and fatty acids and subsequent evaporation. Samples of one gram of solid soap, each, were then dissolved in varying amounts of warm .water and the solution cooled so that it would set to a gel. The largest amount of water which could be added to each soap without obtaining a fluid aqueous layer upon cooling was determined in each case by testing. The following sodium salts of fatty acids were used.

Molecular Water held in _Formula weight the form of gel

per gram

cc.

1. Sodium valerate ............. C4H gC02Na 124 0 2. Sodium capronate ........... CSHnC02Na 138 '0 3. Sodium caprylate ............ C 7H 16C02Na 166 1 4. Sodium caprate .............. C 9H 19C02Na 194 2.5 5. Sodium laurate .............. CllH"28C02Na 222 18 6. Sodium myristate ........... ClaH27C02N a 250 48 7. Sodium palmitate ............ C16H s1C02N a_ 278 72 8. Sodium margarate ........... C16H aaC02N a 292 80 9. Sodium stearate ............. C17H3SC02N a 306 88

10. Sodium arachidate ........... ClgH39C02Na 334 111

The increase of water holding power is seen to be very great for soaps with a molecular weight of more than 200, and almost zero below it, compare sodium valerate, capronate, caprylate, and caprate. From sodium~.laurate (molecular

"M. H. Fischer, "Seifen und Eiweisstoffe," Lj:lipzig, 1922.

APPENDIX 323

weight 222) on, the anion becomes more and more non-diffusible, hence water is held fast in amounts increasing with the molecular size.

The lower members of the series as far as sodium caprylate form clear solutions in water. They never set to a jelly. The high molecular sodium soaps can also be dissolved if a sufficient quantity of water is used, but these solutions contain submicroscopic colloidal particles which do not consist of solid soap only. They contain water somehow dissolved in them. These same particles can be brought closely together by using less water than required for a solution. The particles will then form a semi-solid mass or "gel."

This holding of water by soaps may be explained in a similar manner as the attraction of watcr by gelatin chloride or sodium gclatinate. The soap anions are not monovalent, as indicated by the chemical formula, but the higher fatty acid anions form large molecular aggregates with manifold charges, each of which attracts many Na + ions and these in turn hold water by molecular attraction. Even though no membrane may be present in this case, an equilibrium condition of the type described above becomes established. But this is possible only if the anion is non-diffusible as in the case of higher fatty acids. The lower fatty acids which diffuse freely cannot bind water.

10. FURTHER ApPLICATIONS OF THE THEORY OF MEMBRANE EQUILIBRIA: THE

WATER EXCHANGE OF THE RED BLOOD CELLS

A priori it would seem likely that membrane equilibria play an important rOle for the water exchange between body cells and surrounding body fluid. We know of one instance in which this influence has been demonstrated by quantitative measurements, viz., the red blood cells (see above, pages 67-68). As pointed out, the membrane which surrounds these cells, is impermeable to cations but permeable to anions, except to the colloidal hemoglobin anion. We have shown that the rules of membrane equilibria apply in this case, as is manifested by the higher concentration of diffusible anions outside the cell, or by the decrease of the osmotic pressure in the cell following oxygenation which causes an increase of the base binding of hemoglobin. It should be emphasized that this type of permeability is nevertheless quite different from that of a parchment or collodion shell as is used for measuring the osmotic pressure of gelatin solutions. In the case of erythrocytes the membrane is also impermeable to all cations. If the membrane of the red blood cells consisted of a material like parchment, which is permeable to all ions, except colloidal ions, then an increase of the base binding power of hemoglobin would lead to an intake of cations and consequently to swelling. In reality, however, a shrinking occurs.

It remains to explain the origin of the peculiar selective permeability of the red blood cells. We may consider it as a consequence of the amphoteric character of the lipoids or proteins which compose the cell membrane. On the acid side of the iso-electric point the substance of the membrane will combine with water soluble acids like Hel, hence it resembles an insoluble chloride. If this comes in contact with a soluble sulphate, it transforms itself into an insoluble sulphate which in turn in contact with the chloride solution inside gives off SO. in exchange. The seemingly selective anionic permeability is, there-

324 APPENDIX

fore, in reality a selective chemical reactivity, lipoid chloride as well as protein chloride can react with sulphate, but, of course, it cannot react with KCI or other chlorides (compare Loeb's experiments on gelatin). Hence no exchange of cations can occur.

If this is correct, we should expect that at the alkaline side of the iso-electric point, the reverse phenomenon exists, viz., a selective permeability for cations, since the cell membrane now consists of a sodium lipoate or sodium proteinate which can exchange its sodium for other metals, but, cannot react with other sodium salts, just as gelatin on the alkaline side of the iso-electric point fails to react with ferrocyanides, but, does react with silver salts (see above, page 33). If red blood cells of the pig are suspended in an alkalinized Na.SO. solution (pH == 10) for half an hour and analyzed, no sulphate is found in them, but, K ions are found in the supernatant fluid. This shows that at a pH larger than 8 the cells have become permeable to cations, and impermeable to anions which is just the reverse of their normal property (R. Mond, 1927).16

According to these experiments, the passage of a salt through a membrane occurs because of a chemical interaction. The same is true for other membranes, as for instance for the highly impermeable skin which surrounds the eggs of marine fish. If these eggs are immersed in a pure KCI solution, potassium salt will slowly penetrate and kill the embryo as is seen from the cessation of the heart beat. Dut if the eggs are first immersed :in water the penetration of potassium salts is retarded considerably (J. Loeb, 1916).u This is apparently caused by the extraction of salt from the membrane by the water. The membrane then chiefly consists of lipoids or proteins. Since the straight lipoids or proteins cannot react as readily with KCI, the penetration is retarded, showing that the penetration depends on the chemical reactivity of the membrane with the adjoining solution.

Another example is the penetration of potassium salts through the peel of an apple (L. Michaelis and A. Fujitan, 1925).18 If an apple is placed into a solution of NaCI, e.g., traces of potassium salt penetrate from the apple into this solution indicating a selective cationic permeability in this case, however, if the apple is placed into distilled water no KCI will diffuse out of it. Otherwise the apple peel is very little permeable to salts and water.1O

All this shows that a dependence of permeability on chemical interaction is a general property of membranes with widely different properties. Artificial membranes show this feature likewise (see above, page 208).

16 These experiments are quoted from R. Mond, Pflugers Archiv., 217, 618 (1927). Mond attempts to explain his interesting findings on the basis of the erroneous theory of Michaelis (see pages 207 and 226).

11 Journ. Biological Chern., 27, 339ff. 18 Biochem. Zeitschr, 161, 46. 19 The authors who have investigated these phenomena, J. Loeb and L. Mich

aelis, have different explanations. Loeb assumes a coagulation of membrane globulins by the water, tending to make the membrane denser. Michaelis' hypothesis is that of a selective ionic permeability. A difference between the experiments of Loeb and Michaelis is, of course, that in Loeb's experiments the water acts temporarily, yet the underlying cause is manifestly the same.

AUTHOR INDEX

A

Abel, J. J., 97 Adams, N. K., 90 Adolph, E. F., 63 Amberson and Klein, 205 Ambronn, H., 121, 122 Anson and Mirsky, 159, 164 Arrhenius, S., 27, 95 Auer and Meltzer, 80

B

Bancroft, W. D., 89, 275 and Richter, 274

Baron, M., 284 Bartell, E., 65

and Freundlich, 66 Bartwell, F. E., 58 Baur, E., 222

and Voit, 65 Bechhold, H., 20, 140

and Villa, 97 Benson, 142 Beutner, R., 32, 179, 199,201,202, 205,

206, 208, 209, 211, 215, 218, 220, 221, 225, 226, 229, 230, 275, 304, 306, 309

and Busse, 114 Blanton and Mann, 244 and Kanda, 210 and Loeb, 196, 197, 198, 201, 202 and Lozner, 232, 233, 234, 241, 242 Lozner and Caywood, 232 and Menitofi, 204

Bishop, G. H., 254 Blanton, Beutner and Mann, 244 Borodin, D. N., 284 Boruttau, 259 Bose, J. C., 19 Bottazzi, F., 36

and Fano, 36

Brefeld and Warburg, 156 Brodersen, T., 101 Burn, D., 54 Busse and Beutner, 114 BiitschIi, 0., 43, 111, 116, 170

C

Caywood, Beutner and Lozner, 232 Chambers, R., 64, 237

Cohen and Reznikoff, 152 Choucroun and Magrou, 284 Clark, Mansfield, 30 Clowes, G. H. A., 87, 89, 91, 92, 93, 94,

224,275 Cohen, Chambers and Reznikofi, 152 Cohnheim, 0., 63 Collander, R., 43, 44, 47 Cowdry, E. S., 243 Cranner, Hansteen, 48 Cremer, M., 226 Crile, G. W., 117

and Telkes, 178 Cushny,55

D

Damon, Osterhout and Jacques, 228 Debye and Scherrer, 123, 125 Del Baere, 51, 53 Deutsch, 204, 211 De Vries, 36, 37 Diesselhorst and Freundlich, 137 Donegan, F. G., 300

and Persons, 76 Drinker, 51 Du Nouy, Lecomte P., 144, 145, 146,

147, 148 Dutrochet, 58, 65

E

Ebner, 122 Eichholtz and Hecht, 168

325

326 AUTHOR INDEX

Embden,l85 Engelmann, Th. W., 184 Epstein, 52 Erlanger, J., 264

and Gasser, 192

F

Fano and Bottazzi, 36 Fenn, W.O., 87 Findlay, Alexander, 23 Fischer, Alfred, 109 Fischer, H., 160 Fischer, M. H., 55, 322 Flusin,65 Frank and Rodionow, 290 Freundlich, H., 66, 92, 135

and Bartell, 66 and Diesselhorst, 137

Fujita, A., 196 Fujita and Michaelis, 208, 324

G

Gabor and Reiter, 284 Galvani, 189, 212 Gasser and Erlanger, 192 Geiger and Muller, 291 Gellhorn, E., 42, 81 Gesell, R. and Hertzmann, 77 Gickelhorn, E., 57

and Umrath, 237 Gildemeister, 49 Gordon, M., 65 Gotch and Macdonald, 249 Govaerts, 52 Grafe, V., 48 Graham, Phil, 65 GroUman and Sollner, 61 Gurwitch, A., 283, 284, 285, 286, 287,

288, 289, 290 Gurwitch, Lydia, 289 Guttenberg, 284

H

Haber, F., 136 and Klemensiewicz, 224

Haldane, J. B., 23 Halpert, Schade and Neukirch, 78

Hamburger, H. J., 39, 63 and Alous, 80

Harvey, E. Newton, 117,291 Hastings, Sendroy and van Slyke, 75 Hatschek, 101 Hecht and Eichholtz, 168 Hedin, 40 Heidenhain, R, 55, 65 Heilbrunn, 49 Henderson, L. J., 28 Hermann, L., 259 Herrera, A. L., 117 Hertzmann and R. Gesell, 77 Hill, A. V., 3, 185, 255, 315 Hill, S. E., 268 Hill and Osterhout, 268 Hirschfelder, 81 Hober, R., 42, 204, 211

and Schurmeyer, 87 Hoefer, P., 255 Hoff, J. H. van't, 26 Hoffman and Mond, 44 Hoffmann and Ruhland, 44 Hopkins, Gowland, 7, 35 Hull and Nichols, 95

J

Jacques, Osterhout and Damon, 228 Janek, A., 101 Jennings, ~. S., 169, 170, 172, 173, 174,

175,176 Johlin, J. M., 144 Just, E. E., 281

K

Kagi, H., 100 Kanda and Beutner, 210 Kappers, C. V. A., 104 Keilin, D., 164, 165 Keller, R., 57, 65, 66 Kenyon and Lund, 65, 240 Klein and Amberson, 205 Klemensiewicz and Haber, 224 Knipping and Friederich, 122 Knoop, 243 Koch, J. C., 112 Koch and Stempell, 171

AUTHOR INDEX 327

Krah, E., 168 Krebs, 158 Krogh, A., 51, 53, 57 Kuhn and Meyer, 154 Kuhne, 48 Kunitz, M., 319

and Northrop, 34, 318 Kuster, 47

L

Landis, E. M., 51 Lange and Simon, 49 Langmuir, 143, 144 Lapicque, Louis, 245, 247, 249, 250,

253,254,255 Laue, v., 122 Leduc, St., 12, 15, 113, 115, 147 Lee, Bolle, 237 Leeuwen, Storm van, 276 Lehmann, 0., 117, 118, 136, 139, 181,

182, 183 Liebig, 6, 142 Liesegang, R. E., 100, 105 Lillie, R. S., 14, 81, 112, 258, 260, 264,

265,266,267, 268, 275 Loeb, J., 2, 4, 18, 31, 59, 61, 66, 74,

79, 81, 204, 205, 236, 280, 281, 282, 294, 297, 309, 310, 312, 313, 315, 316, 317, 320, 321, 324

and Beutner, 196, 197, 198, 201, 202, 242

and Wasteneys, 84 Lozner and Beutner, 232, 233, 234,

241 Beutner and Caywood, 232

Lund,_ E. J., 19, 239, 240 and Kenyon, 65, 240

M

Macallum, A. B., 73 Macdonald and Gotch, 249 Mach, E., 293 Magrou and Choucroun, 284 Mann, Beutner and Blanton 244 Mark and Meyer, 127, 128 '

Mathews, A. P., 243 Matsuo, 204, 211 McClendon, 49 McDonald, 259 Meigs, E. B., 39 Meltzer and Auer, 80 Menitoff and Beutner, 204 Meyer, K. H., 129, 130, 131, 132, 274

and Kuhn, 154 and Mark, 127, 128 and Straub, 76

Meyerhof, 0., 185 Michaelis, L., 207, 226, 324

and Fujita, 208, 324 and Solomon, 152

Milroy and Donegan, 54 Mirsky and Anson, '159, 164 Miillendorf, 47 Mond, R., 204, 209, 211, 324 Mond and Hoffmann, 44 Moore and Roaf, 274 Morgan, T. H., 279, 280, 282 Morse, 26 Muller and Geiger, 291

N

Naegeli, Carl, 48, 119, 120, 299 Nasse, 0., 39 Natannsen, 211 Negelein, 166

and Warburg, 154, 157, 158 Nernst, W., 23, 253, 254 Netter, H., 200 Neukircb, Schade and Halpert, 78 Neuschloss, S. M., 82, 84, 86, 87 Nichols and Hull, 95 Nirenstein, 46 Northrop, J. H., 320, 321

and Kunitz, 34, 318 Nouy, Lecomte P. de 144, 145, 146,

147, 148

o Osterhout, 58, 72, 74, 83, 270, 271, 272

Damon and Jacques, 228 and Hill, 268

328 AUTHOR INDEX

Ostwald, Wo., 101, 103 Ostwald, W., 214 Overton, 37, 39, 41

P

Palitsch and SS'lrensen, 77 Pawl ow, 293 Pfeffer, 24, 48 Perrin, J., 297 Persons and Donegan, 76 Peters and van Slyke, 75 Polanyi, M., 127 Popescu, St., 64 Proctor, H. R., 317

Q

Quincke, 13, 18

R

Rajewsky, W. B., 291 Reiter and Gabor, 284 Reymond, Emil du Bois, 192, 212 Reznikoff, Cohen and Chambers, 152 Rhumbler, L., 13, -175 Richards, A. N., 55, 56 Richter and Bancroft, 274 Roaf and Moore, 274 Robinson, 154 Rodionow and Frank, 290 Rossman, 284 Rous, Peyton, 78 Roux, 283 Ruhland,47

and Hoffman, 44

S

Sakuma and Warburg, 166 Salomon and Michaelis, 152 Schade, H., 51, 53, 71 Schade, Neukirch and Halpert, 78 Schaeffer, A. A., 169 Scherrer, 124 Scherrer and Debye, 123, 125 Schubert, E., 100 Schurmeyer and Hober, 87 Schwarz, 284 Seifriz, 48

Sendroy, Hastings and van Slyke, 75 Siebert, W. W., 284, 287 Simon and Lange, 49 Slyke, van, 53, 67, 68, 76, 77

and Peters, 75 Hastings and Sendroy, 75

Sollm::mn, 39 Sollner and GroUman, 61 SS'lrensen and Palitsch, 77 Stanley, W. M., 58 Starling, E. H., 50 Steel, M., 23 Stempell and Koch, 171 Storm, Leeuwen van, 276 Straub and Meyer, 76 Stuckert, 101

T

Taylor, G. W., 291 Telkes, M., 118, 294

and Crile, 178 Thieu1in, 44 Traube, J., 44, 45, 274 Traube, Moritz, 11, 20

U

Umrath and Gicklhorn, 237

V

Van Slyke, 53, 67, 68, 76, 77 and Peters, 75 Hastings and Sendroy, 75

Van't Hoff, J. H., 26 Verworn, 274 Verzar, 49 Villa and Bechhold, 97 Virchow, R., 117 Voit and Bauer, 65 Volta, 189, 212 Von Weimarn, P. P., 116, 134, 135

W

Warburg, 0., 150, 152, 153, 155, 156, 157, 158, 161, 162, 163, 166, 167, 168

and Brefeld, 156 and Negelein, 154, 157, 158 and Sakuma, 166

AUTHOR INDEX

Wasteneys and Loeb, 84 Weimarn, P. P. von, 116, 134, 135 Wertheimer, 66 Wieland, 166 Willstiitter, R., 266 Wilson, E. B., 279

Winterstein, H., 273 Wolf, J., 112

Zoe her, H., 138 Zirpolo, 284

z

329

SUBJECT INDEX

A

Absolute spectrum of the respiratory ferment, 158ff.

Absorption spectrum of the respiratory ferment, relative, 158

Achromatic figure of karyokinesis, 112

Acid propcrties of collodion, 208 Acidity, definition of, 28 Acidophilic oil mixtures, 234 Acidosis, 76

uncompensated, 76 "Action" current, 192 Adsorption, 139ff.

apolar, 141ff. in froth, 142 polar, 139ff. theory of narcosis, 272

Adsorptive replacement, 139ff., 153ff. Aerobic reactions, 167 Affinity constant, 29 After effect, cerebral, 293 Ag+ cell, 198 Agglutination of lipoids, 84ff. Alignment of the elongated mole-

culcs, 182 Alkaline Reserve, 28, 74ff. Alkaloids, electromotive action of,

275 "All or none" law, 260 "Alteration" theory, 213 Amoeba, food intake of, 172 Ameboid movements, 168ff. Amorphous NaCI, 134 Anerobic reactions, 167 Anionic permeability, 67, 323 Anoxemia theory of narcosis, 274 Antagonistic emulsification, 87 ff.

salt mixtures, 78 ff. Anti-osmotic fluid transport, 56ff. Apolar adsorption, 141

Application of thermodynamics, 3, 216ff.

Approach by means of synthesis, Iff. "Artificial cells" (alleged), 117

emulsion membranes, 93ff. fertilization, 280ff. mushrooms, 15 osmotic structures, llff. parthenogenesis, 279ff. systems with a respiration, 177ff.

"Asters" in karyok. etc., 113 Asymmetrical membranes, 63 Axial conduction, model of, 258 Azoxyanisol, 136

B

"Bags," osmotic in gelatin, 318 Ballistic rheotome, 246 Base binding of hemoglobin, 67, 68 Basophilic oil mixtures, 234 Bio-electric currents and metab-

olism, 239ff. currents and respiration, 239ff.

Biological detectors, 284 Biphasic battery systems, 194, 202,

230, 233, 235 Blood colloids, 49ff. Break stimulation, 248, 256 Brownian movement, 298 Buffer solutions, definition of, 28ff.

substances, 77

c Cancellous tissue, 111 Capillaries, 49ff. Cataphoresis, 59ff. Cathode ray oscillograph, 192 Cationic permeability, 324 Cells, artificial, alleged, 117

331

division, 112, 283 respiration, 149ff.

Cellobiose, 128

332 SUBJECT INDEX

Cellulose, molecular make-up of, 128ff.

Centrosomes, 113, 115 Cerebral activity, 292 Charcoal, silica ted, 156 Charge of chromatic substances, 112

of cytoplasmic colloids, 112 Chemotropic movement, 177 Choice, power of, 172 Chromatic figure, 113 Chromosomes, 113 Chronaxie, 245ff. Clove oil, ameboid movement of, 169 "Coin roll" arrangement, 138 Colloids, swelling of, 70ff. Colloidal electrolyte, 300 ff.

solutions and gas laws, 297 swelling, 30ff.

Concentration cell, 216 of the respiratory ferment, 160

Conditioned reflexes, 293 Conductivity of a non-aqueous fluid,

220 of plant tissues, 76

Consciousness, definition of, 293 Constancy of pH in tissue fluids, 74 Constituents of tissue membranes, 42 Coprohemin, 160 Copperferrocyanide structures, 12ff. "Corrective" treatment,r281 Counterpressure of NaCI, osmotic,

32,303 Cresol + fatty acid, 202 Crystalline fluid, 136 Crystals, liquid, 136, 181 Currentless cell, 218 Curvature of surface films, 89 Cystein, oxydation, 165 Cytochromes, 164ff. Cytology, 112

D

Debye and Scherrer, method of, 123 Dehydrases, -165 Detectors, biological, 284 Determinism, 6 Detoxification, 78ff.

of acids by salts, 83

Deuterohemin, 160 Diabetes, 76 Differential permeability, theory of.

214 staining, 232

Differentially stainable mixtures, 232 Diffiugia shells, 172 Diffraction of mitogenetic rays, 285

roentgen ray, 122, 133 Diffusion pressure, 24 Diuretics, 55, 56 Donnan's equilibrium 31, 300ff.

theory, insufficiency of, 304 Double refraction, 120ff.

refraction, streaming, 137, 181 sided ellluision, 90ff.

Dried collodion films, 208 Dubois Reymond, law of, 246, 250

E

Edema, 52ff. Effect of concentration, 189ff.

of concentration, imitation, 201ff. of concentration in animal tissue,

200 of concentration in nerve, 200 of concentration, maximal, 195ff.

"Ego," definition of, 293 Electric conductivity of plant tissues,

83 fish, 189

Electrical factor of water ttansport, 65 fluid transport, 58ff.

Electrode-like phase boundaries, 197ff., 251ff.

Electrolytic dissociation, definition of, 27

Electromotive action of alkaloids, 275

action of organic salts, 210 effect of precipitation membranes,

206 effect on collodion film, 207 effect of concentration, 1951'1'. effect of different salts, 209 effects, inverse, 205 forces of tissue, 190ff. forces and stainability, 227ff.

SUBJECT INDEX 333

Electrotonus, 256ff. "End to end" transmission, 260 Endomosis, 58f£. Endoplasmic streams, 169 "En telechia," 6 Enzymatic reactions, 21 Epitheloid differentiation, 116 Equilibrium, Donnan's, 31, 300ff. Evolution, 95ff.

F

Fats, oxidation of, 154 Fermentation, definition of, 166ff. Fiber size, influence of, 264 Fibrillary structures, 116 Fibrous material, 126 Filaments from lipoids, 117 Filtration theory, 55ff. Fixed acids, 76 Food intake of Amoeba, 172 Formula of Nernst, 198 Freezing point, determination in

blood, etc., 36ff. point method, 27

Fluid crystals, 136

G

Galvani's experiments, 212 Galvanotropism, 18 Gas laws and colloidal solutions, 297 Gelatin ions, valence of, 32, 309

tannate cell, Traube's, 11 Globulites, 116 Glomerular filtration, 54ff.

urine, 55 Glutathione, 165 Glycolysis, 167

of tumor tissue, 167 Gold impregnation, method of, 97 Gravity, influence of, 12 Guaiacol + oleic acid, 203

H acceptors, 164 Hematocrit, 38

H

Hemin compounds, 160

Hemoglobin, 160 base binding of, 67

Hemolysis, 38, 82 History of electrophysiology, 212 Hofmeister series, 320 Honeycomb, 110, 116, 135 Hydration of the membrane, 87ff. Hydraulic models, 255 Hydrogen ion concentration, defini-

tion of, 28ff. Hydrocharis, roothair of, 47 Hydrostatic 'pressure, 50ff. Hypertonic, 38 Hypotonic, 38

I

-Immune serum, 148 Impermeability, loss of in pure NaCl,

82ff. Increased permeability in pure solu

tions, 81ff. Indicators, 30 Influence of gravity, 12

of valence, 315 Ingestion by Amoeba, 174 Inhibition of respiration, 152ff.

of respiration by CO, 156 Injury current, 191, 227ff. Interfacial tension, 88ff. Internal asymmetry, electromotive,

195, 227ff. Internodes, 264 Ionic permeability, selective, 207,

226,324. Irreciprocal permeability, 62ff. Irritability of living tissue, 189ff. Iron wire model, 259ff. Iso-electric point, 32, 309ff. Isotonic, 38

K

Karyokinetic figures, 112 Kidney function, Mff.

L Lactacidogen, 185 Leaf-like structures, 116

334 SUBJECT INDEX

Least agglutinating mixtures, 86 Liesegang rings, 99ff. Light absorption by the respiratory

ferment, 159 Lipoid nephrosis, 52 Lipo!ytics, definition of, 39 Liquid crystals, 136, 181 Loca! circuit theory, 262ff., 268ff. Locomotion of Amoeba, 171 Loss of impermeability in pure NaC!,

82 Lowering of surface tension, 142ff. Lymph vessels, 51

M

Main valence chains, 130ff. Maximal effect of concentration,

195ff. Mechanical theory of heat, applied,

215ff. Meltzer narcosis, 80 Membrane equilibrium, 30, 66, 67,

297ff. forming reactions, 49 permeability, 72ff., 93ff. potentials, 314

'Membranes, semi-permeable, 37li. Memory images, theory of, 293 Mendelian inheritance, 279 Mesohemin, 160 "Mesomorphous," definition of, 137 Metabolism and biological currents,

239ff. of tumors, 166

Method of Debye and Scherer, 123 Micella, definition of, 119 Micellar substances, 127ff.

theory, 119ff. Microscopic structures, Willi. Mitogenetic radiation, secondary,

288 radiation, source of, 286 radiation, spectrum of, 288

Mitogenetic rays, 283ff. rays, diffraction of, 285

Mitosis, 112, 283ff. Model respiration, 154

Models of floating magnets, 112 Molecular make-up of cellulose, 128li.

sieves, 44 solution, 27 weight, 26 weight of colloids, 299

Monomolecular layers, 144 living forms, 97

Morphogenetic influence of adsorp-tion, 146

Movements, ameboid, 168ff. Multiple valence, 310 Muscular contraction, 184 Mutual detoxification, 85, 86 Myelin, filaments, 117, 118, 139, 178

N

Narcosis, adsorption theory of, 274 anoxemia theory of, 274 semi-coagulation theory of, 274 theories of, 272ff.

Narcotic power, 42 Narcotics, action of, analyzed, 269ft·. Natural parthenogenesis, 280ff. Negative osmosis, 58fI. Negativating action of the sap, 227 Nephritis, parenchymatous, 52 Nephrosis, lipoid, 52 Nernst's squarc root law, 254 Neurobiotaxis, 104 Neutral point, definition of, 29 Nicotine hemochromogen, 158 Non-aqueous central conductors,

194fI. Non-physiological mixtures, 85 Non-polarizable electrodes; 191

o Oceanic salt mixtures, 7'lff. Oil solubility, 40, 274

suspended in water, 89 -water interfaces, 87

Onion root method, 283 Optical anisotropy, 120 Organized chemical reactions, 11ff.

substances, 119ft'. Oriented molecules. 143ff.

SUBJECT INDEX 335

Origin of cell membranes, 47 of electric currents in tissues, 189 of life, 95

Osmometer, 25 Osmosis, negative, 58 Osmotic counterpressure, 32, 300ff.

fluid exchange, 37 pressure, 12, 23ff. pressure of the blood, 35ff. regulation, 36 structures, artificial, llff. structures outside of solutions, 15

Ostwald's electric heart, 177 "Outlying" acidosis, 78 Oxidases, 164, 165 Oxidation and potential difference,

240ff. of fats, 154

P

Pace maker, theory of the, 266ff. "Panspermia," theory of, 95 Parathyroid hormone, 75 Parenchymatous nephritis, 52 Parthenogenesis, artificial, 279ff.

natural, 280 Perception, 293 Permeability, 49ff., 72ff., 87ff.

irreciprocal, 63 in pure solutions, increased, 81ff.

"Personality," 293 pH,29 Phase boundary potentials, 218ff. Photoelectric cells, 291 Pickering's emulsion, 184 Polar adsorption, 139ff. Polarity of stimulation, 255 Polarization and stimulation, 250ff. Pore theory, 200, 207, 226 Porphyrrins, 160 Possibility of artificially producing

life, 2. Potent drugs, electromotive action

of,275 Potential difference and oxidation,

240ff. difference on apple, 196ff.

Power of choice, 172 "Pre-existence" theory, 213 Proteins, pure, 132 "Protoplasm," term of, 7 Protoplasmic rigidity, 180, 183 Pseudopodia, 169, 171 "Psychoid," 6 Pulsations, rhythmic, 266 Pure NaCl solutions, toxicity of, 74,

80. proteins, 132

R

Radial striations, 109 Radiation from tumor tissue, 289

of the blood, 289 pressure, 95

Re-absorption (in kidney), 56 Reaction at phase boundary, 223 Recovery of transmissivity, 260 Red blood cells, water exchange of,

68,323 "Redox" indicators, 151, 179 Reflex, 17, 292 Refractory period, 263 Relative absorption spectrum of the

respiratory ferment, 158 refractory period, 263

Re-passivation, 262 Replacement, adsorptive, 139ff., 153ff. Reproduction, 22 Respiration and biological currents.

239 on charcoal, 154, 155

Respiring lipoidal globules, 179 Respiratory center, 76

ferment, 149ff. ferment, absolute spectrum of, 158 ferment, concentration of the, 160 ferment, light absorption by the

157ff. Resorptive forces, 57ff. Retention of NaCl, 54 Reversible chemical reactions, 99 Reversibility of potential differences,

198 Rheobase, 245ff.

336 SUBJECT INDEX

Rigidity of foam, 184 of protoplasm, 180, 183

Ringer solution, 72ff. Rise of sap, 64 Roentgen ray diffraction, 122, 133

S

Salicylic aldehyde, 201 "Salt bridge" experiment, 268ff.

distribution and electromotive forces, 218ff'

mixtures, oceanic, 72 Secondary mitogenetic radiation, 288 Selective ionic permeability, 207,

226,324 Self-regenerating enzyme, 98 Self-preservation, 22 Semi-coagulation theory of narcosis,

272 Semi-permeable membranes, 37ff.,

48ff. Sieve membranes, 43 Silicate structures, 12 Silicated charcoal, 156 Silk fibroin, 132 Size of crystals, 134 Soap cups, as models, 55 Soaps, water imbibition of, 321 Solution pressure, 23 Solubility directed in space, 143 "Soul," definition of, 293 Source of mitogenetic radiation, 286 Spectrum of mitogenetic radiation,

288 "Spindles," 113, 115 Spireme, 113, 115 Spirographis hemoglobin, 163, 164 Spontaneous generation, 96 Stainability and electromotive forces,

227ff. of a non-aqueous mixture, 232ff. of paramecia, 46

Stalagmometer, 142 Stimulation and polarization, 250ff. Stratified structures, 100ff. Streaming double refraction, 137, 181

Stress lines in bones, 111 "Stretching" zone, 239 String galvanometer, -192 Structures, copper ferrocyanide, 12ff.

silicate, 12 Struggle for existence, 6 Submicroscopic viruses, 97 Surface action, theory of, 44

tension, determination of, 142 tension, lowering, 84ff., 141ff.

Swelling, colloidal, 30, 70, 317 of colloids, 30ff" 70.

Synthetic cells (alleged), 117 Synthesizing primitive life, 295

T

Temperature variability of life proc-esses, 4

Tesla currents, 246 Theories of narcosis, 272ff. Tixotropic change, 181 Toxicity of pure NaCI solutions, 74ff. Transmission in passive metals (steel

wire), 259ff. Traveling waves of polarization, 259ff. Traube's gelatin tannate cell, 11 Tropisms, 17 Tumor glycolysis, 167ff.

growth, 167 tissue, radiation from, 289

Tumors, metabolism of, 167

U

Uncompensated acidosis, 76 Unequal distribution of ions, 32, 300ff.

V

Valence chains, marn, 130ff. of gelatin ions, 32, 309ff. influence of, 315ff. multiple, 310ff.

Valonia, 228 Variations of the electromotive force

of cuticula, 195ff. Varying water imbibition, 299 Velocity -of stimulation, 249

SUBJECT INDEX 337

Viscosity, 317 Vital battery system, 227ff.

coloration, 238 purposiveness, 6

Vitalism, 5

W

Warburg's determination of cell respiration, 150

Water exchange of red blood cells, 68,323

imbibition, varying, of starch, 119, 299

imbibition of soaps, 321

immiscible substances, electrical action of, 192

soluble lipoids, 48 suspended in oil, 90

Wave of activation, 259ff. Waves of polarization, traveling,

259ff. Weak acids, 28

y

Yeast methods, 284

Z

"Zielstrebigkeit," 6

physical chemistry of living tissues' and life processes

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