TRACER TECHN iOUES

1. H a HEMDEASON

The understanding of metabolic processes has advanced a t a fan tas t ic r a t e since the advent of t racer methods. isotopes of the elements of concern t o biochemists, scone crude t racer experiments were done. dichlorostearic acid by adding chlorine across the double bond. type of l abe l could only t race the general f a t e of the f a t t y acid molecule by measuring chlorine but could not lead t o information regarding the molecular basis fo r the metabolic processes i n which this f a t t y acid is involved.

Prior t o the discovery

Oleic acid, fo r example, was converted t o 9,lO- T h i s crude

The discovery by Hevesy i n 19U. of an unstable isotope of lead (Pb2l0) which had been assumed t o be contaminating radium, marked the begin- ning of tags or t racers within the nucleus of an atom. pe r t i e s of the tagged atom are not s ignif icant ly a l te red i n these isotopic t racers . nature a s mixtures of two or more isotopes, were exploited by separating the s table isotopes of these elements. from the predominaat isotope by one mass unit. While the permissible d i lu t ion of such i s a t o es i s f a r below tha t for radioactive isotopes, deuterium, (a2) and N9, detectable by mass spectrometry, were suff ic ient ly useful t o provide data which revolutionized our concepts of metabolism. Schoenheber and coworkers a t Columbia presented an hypothesis which s ta ted t h a t body constituents were in a dynamic s t a t e of equilibrium with each other and with the nutr ients provided t o the organism.

The chemical pro-

F i r s t elements such a s nitrogen and hydrogen, which occur in

The s tab le isotopes usually differ

With the production of unstable isotopes of the l i gh te r elements,

The necessity of having an accelerator or a neutron source t o

A t t ha t time, nuclear reactors began t o

labels fo r most of the elements involved i n biological processes became available. produce isotopes, limited the number of laboratories using radioisotopes unt i l t h e close of World War 11. produce the unstable isotopes of biologically important elements in s u f f i - c ien t amounts and a t a cost low enough t o bring these precious tagged atoms t o t h e a i d of s c i en t i s t s i n most laboratories in t h i s country. increased ava i lab i l i ty of radioisotopes, great improvement i n instrumenta- t i o n a l so came. ing various isotopes present i n many com ounds is on the market. the ease of measuring Isotopic carbon (Cf4), t h i s method has frequently re- placed more conventional methods of following the course of a reaction in the laboratory. The ava i lab i l i ty of most common nutr ients and metabolites labeled i n specif ic carbon atoms with $4 has a lso enhanced the usefulness of t h i s technique.

With t h i s

Today a vast array of mass produced instruments fo r measur- Because of

While the specif ic biochemical reaction can be studied without isotopically labeled substrate in v i t ro , the ro le of t h i s reaction i n the in t ac t organism can only be evaluated by in vivo experiments and usually this requires isotopic substrate. Sane examples of t h i s application of isotopes will be c i ted i n the remarks which follow. B many cases the understanding

206.

of the metabolism of a compound has came from enzyme experiments designed a f t e r isotopic studies i n t h e Intact organism had indicated the general nature of the metabolic processes involved.

I. GENERAL PRIJ!?CIPLFS - A, Chemical properties and hence the name we give an element a re

Chemical reactions are determined by the num3er of extranuclear electrons. largely independent of the mass of the nucleus.

B. The rays or par t ic les emitted by unstable isotopes a re (a) electrons, (b) positrons, ( c ) d -par t ic les or (d) Y -rays, each with i t s own specif ic properties.

C. The r a t e of decay of unstable isotopes i s independent O f temperature, pressure, etc., and of the chemical combination of the element. Thus t he half' l i fe of C14 is 5,700 years regardless of the conditions or the chemical form In which it is found.

D. The isotope must be incorporated i n t o the ccmpound which you wish t o study, usually in to a specific posit ion in the molecule.

E. There must be a sui table detecting system fo r measuring the

For radioiso- isotope concentrations under the conditions of the experiment. isotopes the maximum di lu t ion tolerance is about 100 fold. topes di lut ion of approximately 100,000 I s permissible. be determined on material i n the gaseous, l iquid or so l id s ta te .

For stable

Radioisotopes can

A l i s t of the isotopes most frequently used t o study biochemical processes is

Element and Mass Number

(de u t e r i um)

( t r it i u n )

H2

E3

c13

Cl4

N E 018

Na22 NaZ4 P32

s35

shown in Table 1; -

Type of

TABLE I SOME CHARACTEKETICS OFTHE ISUI'OFES USED I N STUDYIYYG LIVING PROCESSES

Half- Comments or Energy of Radiation

L i f e Part i c l e s Y -Rays - Radiation

None ----- 0.02% i n Nature

B- 12.1 yr. 0.017 mev

None ----- 1.1% i n Nature

B - 5,700 yr. 0.154 mev ----- None

None ----- 0.38% i n Nature 0.20% i n Nature

af, 7 2.6 yr. 0.58 mev B- 14.8 hr. 1.4 mev 8- 14.3 days 1.7 mev

8- 87.1 days 0.17 mev

1.3 mev 1.38, 2.7 mev

Element and Mass Number

K 4 O

K4'

~ a 4 5

Fe5'

c 060 zn65 ~ r 9 0 1131

Type of

Radiation

8- 8-

8-

8- B-, Y Y 0-

8-, Y

207

TABLE I (Continued)

Half- L i f e

8 -

4.5 x 10 yr. 12.4 hr,

180 days

45.1 days

5.3 yr.

250 days

19.9 yr4

8 days

Comments or Energy of Radiation

Part i c l e s Y -Rays

1.3 - 1.9 mev 1.46 mev 2.0 mev (25$) 3.6 mev (75$)

0.26 mev (50%) 0.46 mev (50%)

0.257, 0.460 mev 0.31 rnev 1.16, 1.32 mev

-I_

1.120 mev

0.61 mev

0.6 mev 0.080 - 0.722 mev

Tracer techniques as the name implies permits following the f a t e of atoms or molecules by the mass or radioactivity of the isotopes. of t he great sens i t iv i ty of the methods of measuring radioisotopes they a re much preferred when available. separation from natural sources the radioisotopes a re prepared with a re- actor or an accelerator, The simple procedure of tracing an element such a s sodium or iron while useful, does not present t h e elegant power of t r ac - ing a specif ic carbon or hydrogen atom through the complexities of metabolic reactions. Examples of the various types of application or' isotopes t o physiology and biochemistry w i l l be presented, attempting t o classify these applicstions.

Because

Whereas s table isotopes a re prepared by

11. APPLICATICNS OF ISCrrOPES TO THE STUDY OF LIVING PROCESe

A. Requirements and Limitations of the Tracer TechEique

The t racer technique impinges upon biology chiefly a t t h e bio- chemical and physiological level. area of intermediary metabolism, an understanding of which is essent ia l t o the development of a l l areas of biological science. t racers has made it possible t o follow the wanderings of a specif ic atomic grouping wlthin the organism.

Its greatest contribution has been i n the

The advent of isotopic

Before an isotope can be used as a t racer , one must make cer ta in tha t the i n i t i a l concentration of isotope i s high enough t o to le ra te the d i lu t ion which occurs during metabolism and s t i l l remain detectable i n the products separated a t the close of the experiment. condition can usually be met, but f o r s table isotopes, it often presents a problem. The labeled atom must remain attached t o the molecule or portion of a molecule during the metabolic processes under study. The presence of the isotope must not affect the metabolic process.

For radioisotopes, t h i s

This becomes of some

208

importance fo r isotopes of hydrogen where "isotope effects" a re observed. The ha l f - l i f e of the radioisotope must be long enough t o permit the experi- ment t o be done. Unstable isotopes of oqgen and nitrogen have half- l ives too short f o r most purposes, but fortunately the s tab le isotopes $8 and N15 a re available in nature and a re separated and used fo r t r ace r experiments,

B. Physiological Applications of Isotopes

1. Permeability, Transport, Cellular Uptake, and Distribution

Before the use of isotopes, physiological processes were often studied using "unphysio3.ogical concentrations" of various rner;a'-lolites , Radioactive metabolites can often be used a t normal or less < h m normal physiological concentrations t o measure permeability under conditions where there i s no net t ransfer of a metabolite. back and fo r th across a c e l l membrane can be measured coiive~Lcni;ly using Na24, Transgort of compounds in to axones, across the in t e s t i r31 wall and placenta and aptake of compounds by bac te r i a l c e l l s have a l l been studied effectively using isotopes. The kinet ics of such processes can be readily evaluated by frequent analysis of the medium fo r t he isotopic t racer .

For example, t he passage of Na+

2. B t r a c e l l u l a r and Extracellular Space Determination by Isotope Dilution

Frequently the f lu id volume, or space available for solution of' electrolytes , both inside and outside the c e l l s of an organism must be measured. can be a noma1 coapnent of the space being measured, HgO and $0 have been used. be used. recently P32 d i -is opopylshosphorof luor ida t e, which combines with plasma proteins and with ce l lu la r components of blood, have been used, space of the brain has been measured using S35-sulfate. Cardiac output and the pat tern of blood flow through the heart a r e examples of similar applications of isotopes in the c l i n i c a l f ield,

Isotopes a r e useful fo r such measurements because the indicator

For extra-cel lular space Na+, C1' and Br' may For t o t a l body water,

For plasma volume, serum albumin, iodinated with IU1, and more

The water

3. Hormone Studies

Because of the minute quantit ies required, hormones have been d i f f i c u l t t o study. Without isotopes, the ana ly t ica l methods demanded t h a t massive doses of hormone be given. whether the findings were related t o the t rue metabolic f a t e of the hormone i n question. investigated using isotopical ly labeled hormone a t physiological concentra- t ions. (1) who observed tha t hexoesterol was concentrated more in the reproductive organs than i n any other organ except the kidney, in 5.5 hours af'ter being given a t a l eve l of 1 %/kg of body weight.

This l e f t considerable doubt a s t o

The selective uptake of hormones by the ta rge t organ can be

A good example of such experiments has been presented by Glascock

Iodine-131 has been very useful i n labeling the thyroid hormone and related compounds a t a specif ic ac t iv i ty suff ic ient ly high for experi- ments in the whole animal using near physiological concentrations of the hormone.

209 . 4. Mineral Metabolism

The metabolism of t race minerals, a s i n the case of hormones, can be studied most readily a s radioisotopes, t o make the t r ace amounts sus- ceptible t o analyt ical attack. the r a t e of i ron absorption and incorporation in to heme. Isotopic studies have shuwn t h a t red blood c e l l s l i v e about four months and p l a t e l e t s about lweek, studies of these t race metals (l), C a g and $2 have been extensively used t o study the deposition and mobilization of bone ash.

Fe55 and FeS9 have been used t o es tabl ish

Isotopes of cobalt and copper have been u s e f u l i n metabolism

C Biochemical Applications

The e a r l i e s t experiments with isotopes revolutionized the exis t ing views regarding the general nature of metabolic processes. The par t i t ioning between exogenous and endogenous metabolism, a concept developed by Folin on the basis of the e f fec t of dietary components on the concentration urinary constituents, was abandoned when the deuterium and NE experiments of Schoenheimer and coworkers demonstrated the dynamic nature of metabolic processes. The more excit ing contributions with isotopes have been those i n which en t i r e metabolic sequences and cycles have come t o l igh t .

A number of examples of these contributions w i l l be presented Where appropriate, I w i l l draw these examples from my own area of briefly.

research.

1. Isotope Dilution

A fundamental concept, essent ia l t o the understanding of many applications of isotopes, is the isotope d i lu t ion principle. Analytically, t h i s method is precise t o about I$. It depends upon the f ac t t ha t when a

is added t o a complex mixture containing an unknown amount (xz) of S, the specif ic ac t iv i ty (A1) of S w i l l be reduced t o a new figure (%) t o t he degree tha t the radioactive S i s di luted with unlabeled S. The following equation expresses t h i s relationship:

knuwn quantity (xl) of a substance, S , labeled with an isotope, e.g,, c14,

A1 Xl + Since A and xl a r e known, i f a small sample of S from the mixed,

labeled and unlabeled S can be isolated and i ts specif ic ac t iv i ty , AZ, determined t h i s equation can be solved f o r *, the quantity desired. variations in the application of this principle a re used. a highly labeled compound is present i n a biological material, it can be recovered and the t o t a l quantity of isotope ascertained by adding unlabeled compound (cold car r ie r ) in a known amount and isolat ing a portion of the compound. If a micro-assay is available fo r the substance, the specif ic ac t iv i ty of the labeled compound ar i s ing from a metabolic process can be calculated .

Many When a t race of

210.

2. Precursor-Product Relaticnship

One of the simplest questions often asked i s whether compound A i s converted t o compound B. t i v e resu l t s a re needed.

To answer t h i s question, frequently only quali ta- For example: the conversion of phenylalanine t o

tyrosine (2) . *

CHz -C H -C OOH

and tryptophan t o N1-methyl nicotinamide (3).

C% -CH -COOH NH2 ____.___p

H

Cil

WCONHz 0 CH3

I n cases where isotope from A i s found i n substance B, but with SO much a s t o cast doubt on the directness of t h e relationship, other approaches w i l l be needed t o es tabl ish a precursor-product relationship, type of application are: cholesterol from acetate and isopentanyl pyrophosphate, (2) C% f ixat ion in to cer ta in simple molecules during photosynthesis, (3) the formation of porphyrin from succinate and glycine, (4) the formation of purines from COz, formate, glycine, aspar ta te N and glutamine N.

Examples of t h i s (1) the formation of the s teroid nucleus of

3. Method of Isotope Competition

Th i s method i s applicable specif ical ly t o bac ter ia l systems. If a metabolic sequence, represented by A --> B 4 C ----3D, exis t s i n a c e l l then isotopically labeled A w i l l lead t o labeling of D. unlabeled B or C would then be expected t o suppress the labeling of D.

Addition of

4. Metabolite Gverloadin(S

Ln animals many metabolic processes cannot be studied with isotopes because the end product of one sequence of reactions (C) becomes the reactant of another sequence of reactions:

A- B - C-Cl- Cz-C3 + co2* If it i s suspected tha t "C" i s involved i n the conversion of "A" t o CO hypothesis might be tested by administering a small dose of isotopic&

the

211.

labeled "A" and simultaneously a dose of "C" of such magnitude tha t it appears in the urine. frcm "A''b it should be labeled and the l abe l should be present in the urinary C" which i s isolated, A posit ive result implicates "C" a s an intermediate i n the degradation of "A"; but a negative r e su l t does not eliminate "C" a s an intermediate.

If the exogenous "C" equilibrates with the "C" formed

5. Labeling Patterns i n Tissue Components

When any compound, labeled i n a specif ic carbon atom is degraded through simple 2, 3 and 4 carbon compounds, these small molecules a re labeled in a single carbon atom and lead t o character is t ic labeling pat terns i n the glucose of l i v e r and muscle glycogen, i n f a t t y acids and/or i n non- essent ia l amino acids. of degradation products a re the amino acids alanine, aspar t ic acid, glutamic acid, glycine and ser ine, pyruvate or acetate give character is t ic labeling patterns especially i n glutamic acid and alanine. pound of in te res t , uniquely labeled, is fed or injected in to an animal and the animal is sacr i f iced about twelve hours l a t e r . The l i v e r glycogen and l i ve r or carcass protein a re isolated, hydrolyzed and t h e fragments de- graded, carbon by carbon, t o C02 which i s examined quantitatively fo r C 1 4 ac t iv i ty .

I11 (4) it is evident t h a t the labeling pat tern is Glutamfp and alanine from l i v e r and carcass of a r a t receiving tryptophan-7a-C i s essent ia l ly t h e same a s tha t observed when a ~ e t a t e - 1 - C ~ ~ i s administered (5). the pat tern i n these two amino acids i s similar t o tha t from a ~ e t a t e - 2 4 ~ 4 when t ryp t0phan-5 -C~~ was given (6).

Perhaps the most useful indicator of the ident i ty

Compounds leading t o labeling in any posit ion i n

The experiment i s thus very simple. The com-

Table I1 i l l u s t r a t e s the label in pat tern i n the most indicative amino acids when acetate-l-C1*, acetate-2-C f4 are fed t o r a t s . From Table

Likewise

These r e su l t s c lear ly indicate t h a t

C-5 of tryptophan > C-2 of acetate

C-7a o f tryptophan 3 C - 1 of acetate

6 Trapping Techniques

A small molecule suspected t o be a product of degradation might ex is t i n the metabolic pool i n quantit ies too small t o detect and it might not be excreted in the urine normally. If a sample of the fragment can be withdrawn from the pool i n to the urine by detoxification of some foreign compound, it can be degraded and its labeling pat tern determined. Acetate (7) and glycine a re good examples of molecules which can be trapped i n t h i s way, glycine a s hippuric acid and acetate a s an acetylated amine,

When t r y p t ~ p h a n - C ~ ~ was injected in to the r a t together with cyclohexylalanine, t he acetylcyclohewlalanine was excreted. from the urine, hydrolyzed and the resul t ing acetate degraded stepwise t o reveal a labeling pat tern in acetate consistent with t h e amino acid labeling pattern. Table IV shows the r e su l t s obtained (6, 8) .

It was isolated

212.

7. Evaluation of the Extent t o Which Alternate Pathways Operate

Glucose metabolism i s recognized t o proceed by two pathways; glycolysis t o 2 molecules of pyruvate and via the pentose pathway by which C4, C 5 and c6 also give rise t o pyruvate while C1 yields CO2. findine of C14 from C - 1 of glucose i n lac ta te re f lec ts glycolysis and a comparison of the labeling i n lac ta te from C - 1 of glucose i n one experiment and C - 6 of glucose i n another experiment has been used t o determine the extent t o which glucose metabolism in the bovine, corneal epithelium proceeds by glycolysis (9), Another method proposed by Blmenthal e t a l e (1O)is based on the f a i lu re of C-1 of glucose t o labe l acetate when the pentose pathway prevails.

Thus, the

8 0 Reaction Mechanisms

Both b organic chemistry and biochemistry, isotopes present a powerful t oo l for determining the mechanism by which a reaction proceeds, Perhaps the best knom example of the solution of a reaction mechanism problem with isotopes was the elucidation of the stereospecific addition to, and removal of hydrogen frcm, carbon-4 of the pyridine nucleus of nicotina- mide-adenine dinucleotide (NAD) (11)

H H H

1 R -P

I R-P

Time w i l l not permit the detailed discussion of these experiments, but it was shown t ha t most NAD requiring enzymes a re specific for one of the spat ia l ly d is t inc t hydrogen atoms and tha t the atom is transferred direct ly (not via a proton i n the medium) t o the reduced product.

Another aspect of the mechanism of reactions brought t o l i gh t by isotope experiments was the concept of a meso-carbon atom such as tha t found i n glycerol or c i t r i c acid: (12, 13)

c % a

I I*

HOCH

CHZ OH

The carbon atom marked * organic chemists. These

a -C -a type structure. A b

d

(2% -COOH I I * I

HO-C -C OOH

C% -COOH

has been considered a s a syaetric carbon atom by molecules have a plane of symetry f o r they a re

closer examination shows tha t they a re in r ea l i t y

213

b

d al-C-aZ type molecules, where a 1 and a2 a re re la ted t o each other as a re

our r igh t and l e f t hands, It i s not surprising i n retrospect t ha t an enzyme or some other optically act ive reagent should react a t a different r a t e with a 1 than with a2. Thus the two "a" groups are d i f fe ren t t o the enzyme and only one group reacts a s hers been clear ly established with specif ical ly labeled c i t r a t e and glycerol.

Oxygen-18 has been used t o oxidize various substrates and a number of oxygenases shown t o incorporate molecular oxygen not oxygen from water i n to the product.

9. Isotopes in Enzyme Assays

Rapid methods of following an enzyme catalyzed reaction a re often based upon the use of radioisotopes, For example, D r . Ghblson and coworkers (6) have been studying the conversion of quinolinic acid t o nicot inic acid rnonoucleotA.de by following the release of C1402 from the 2-carboxyl group of the substrate

\ -t co2 R i b os e -P

When isotope methods can be develoved, they a re frequently more precise and rapid because of the ease of estimating radioactivity.

We have f e l t t ha t isotopic techniques a re ju s t one more t o o l which should be understood and used by a l l who work in biochemistry, students i n laboratory courses receive sane experience with C 1 4 and our graduate courses provide repeated contact with t h i s technique.

All of our

Th i s hasty exposure has been intended only t o t e l l you of some poss ib i l i t i e s and limitations of the isotopic methods i n solving biochemical problems. properties of matter. For s t a t i c or re la t ive ly s table s i tuat ions, t h e application of isotopes i s res t r ic ted largely t o their ana ly t ica l use. should l i ke t o close by discussing briefly a use of isotopes which has been the subject of discussions i n these conferences i n past years. t o discuss t h i s application because many of you a re working act ively i n t h i s f i e l d , of l i v e animals or cuts of meat.

It is manifestly unsuitable f o r problems not involving dynamic

I

I hes i ta te

I refer t o the use of K4O measurements f o r estimating the composition

D. K40 Methods fo r Carcass Composition

There i s great need fo r a method which would permit one t o ascertain the body composition of a meat animal a t intervals during the growth period without sacr i f ic ing the animal. Even a rapid measure of the mass of muscle i n a cut of meat would be useful. Body composition of human

214

subjects and experimental animals has concerned the physiologist f o r over 100 years and a great deal of e f for t has been devoted t o t h i s problem recently. Skin fold thickness WId height-weight data, used t o assess the nutr i t ional s ta tus of human populations, i s of no value for other species. More precise methods a re needed if the energy or protein stored during a period of metabolic study is t o be determined without replicating the animals so tha t samples can be taken a t the desired intervals. Even if a sample of the population can be sacrificed, the methods available are not ideal, chiefly because of the d i f f icu l ty of obtaining a representative small sample of a carcass or a large cut of meat.

Potassium analysis by the usual means as an indication of the mass of muscle would not be considered seriously when samples of the tissue are available for l i p id or protein determination, The recognition of K40, aY-ray emitter, a s a normal constituent of natural potassium and the development of very sensit ive and ef f ic ien t l iquid sc in t i l l a t i on methods of detecting radiation suggested tha t whole animals or cuts of meat could be analyzed for K 4 0 which r e f l ec t s the potassium content, which i n turn re f lec ts the lean body mass.

The use of K U measurements t o assess the composition of l i ve animals, hcluding man, a s w e l l as cuts of meat has been described by many workers since the measurement of potassium i n the body by i ts radioactivity was first described (15). A brief review of t h i s work up t o 1961 was pub- lished by Anderson and Langham (16), and the problem was discussed by Andrews and Christian (17) a t the Mexico City Conference l a t e in 1961. method agrees well with other methods of determining lean body weights of man (18) .

The

Jh 1959, the value of 73 milliequivalents of potassium per kg of lean body weight was adopted in the place of a value of 63 used i n the ea r l i e r studies. Anderson and coworkers a t Los Alamos have worked on t h i s method fo r approximately t en years, It has been applied t o l i ve hogs (19), t o hams (20), and t o l i ve sheep (21) . Because the resul ts indicated tha t the errors of prediction of body composition were too hiGh for satisfactory use in most animal breeding programs, the K40 method was checked against the flame photcmetry method by Kirton and Pearson (22). was a significant correlation between carcass composition of lambs and the K content a s measured by flame photometry, but not when estimated by K40 con- tent . On ground lamb and g~ound pork the K40 measurements agreed with the flame photometry method, though the latter method gave values for K which were much more closely re- la ted t o $ water, $ fa t , and $ protein. This relationship was closer f o r pork than lamb. They concluded tha t "A degree of precision a t l ea s t com- parable t o t h a t obtained by flame photosnetry is needed for a non-destructive method (such a s e) before the accuracy i s great enough t o be useful."

They observed tha t there

Gn separable lean and f a t the two methods agreed.

E. Some Recent Developments

Isotopes can be expected t o make many more important contributions t o the field of biological sciences. Three exciting developments of recent years w i l l be mentioned briefly here. The development of density gradient centrifugation has made it possible t o separate macromolecules containing

215

heavier isotopes from t h e i r normal counterparts. bonucleic acid frcm bac ter ia l c e l l s grown with a N15 nitrogen source can be separated from N14 DNA using a cesium chloride solution a s a medium (23). This technique makes it possible t o t race DNA O r a m each parent of bac te r ia l c e l l s among progeny molecules.

For example, deoxyri-

A similar application of radioisotopes is exemplified in the use of t r i t i a t e d thymidine t o locate within chromosomes the DNA formed during a given period ju s t a f t e r administration of the labeled thymidine (24). T r i t i u m has a d i s t inc t advantage in such experiments i n t ha t i t s B' i s so low i n energy tha t the s i l ve r grains i n a photographic emulsion activated by tritium l i e clustered within one micron of the labeled locus.

FinaUy, mass spectrometry i s finding new use in establishing the

compounds a re frequently volat i le , their separation by gas ident i ty of ccmpounds separated by the rapid, gas chromatographic technique. Since flavor chromatography has proved feasible , Drs. Waller and Mason in our laborato- ries have separated the vola t i le flavor components from peanuts i n t h i s way and a re examining the separated compounds by mass spectrometry.

Biemann (25) has described the use of mass spectrometry fo r establishing the sidered vo la t i l e enough, The substances are sublimed d i rec t ly in to t h e ionizing electron beam. quires no chemical pretreatment.

s t ructure of substances which were not previously con-

The method is very sensi t ive and rapid, and re-

The i l l u s t r a t ions given may serve i n a small way t o indicate the wide variety of applications of isotopes, atoms w i l l provide a better understanding of a dynamic process or if you need precise analyt ical data, you might f ind isotopes useful.

If t racing an atcm or group of

1. Glascock, R . F., Use of Radioisotopes in Animal Biology and the Medical Sciences, Vol. 2, pp. 49-67, Intern. Atcmic Energy Agency, Academic Press, New York, 1962,

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4. Gholson, R. K., D. R. Rao, L. M. Renderson,

H i l l , R. J., D. C. Hobbs, and R. E. Koeppe,

J. Biol. Chem., 2 3 , 179 (1958).

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R. J. H i l l and R. E. Koeppe,

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Masurat, J. HI and M. Helfant, Science, 122, 72 (1955).

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Vennesland Birgit , J. Cell. and Comp. Physiol., 47_, Suppl. 1, 201 (19563

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217.

TABLE I1

CCMPOUND

ADMINISTERED

LABELING PATTERN I N NON-ESSEXTIAL AMINO

ACIDS FRCM CEETAIN METABOLDES

CH$1400H

C1%,C OOH

CH3CL40C OOH

COMPOUND

ISOLATED

GLUTAMATE

ASPARTATE

ALANINE

SERINE

GLUTAMATE

ALANINE

GLUMMATE

ALANINE

PATTERN

c5 + c1 > 95%

~ 5 / ~ 1 = 2

c 1 + c4 > 95%

c2 = c3 c1 = 90 - 95$

c1 = c4 and

C1 = 9C$

218

DISTRIBUTION OF CL4 IN GLVTAMIC A C I D

AND ALANIIE FRGM DL-TRYFTOPHAN-C14*

GLUI'AMIC A C I D Tryptophan-7a -C14

Carcass Liver

Sa muc/mole 2847 16.9

24.3 15.9 Percent of t o t a l $4 in

C 1 c2 c3 c5

2 00 m 8

c4 74.1 8345

SA muc/mmole 4 e2 Percent of t o t a l ~ 1 4 in

94 6 C 1

c2 c3

Wean values for two animals

DEERJBUI'ION OF $4 FRCM POS~TTIONS 7a AND 5 OF

TKYPTOPHAN I N TRAPPED ACEX'ATE

Acetate

c -1

C -2

14 Trypt ophan-5 -C

Carcass Liver

23.8 60 04

1246 12.4 23 45 23 43 24.5 20.7 38.8 39 02 2 -3 2.4

7.7 20 03

22.7 20.4 38 e 5 39.6 38.6 39 m7

14 Tryptophan-7A-C14 Tryptophan-5-C

muc/mmole rnuc/mmole

22.1 25 .5

1.4 19.9

m 3 24.5

219

MR, PEARSON: It is almost nom, but we have time for jus t one or two questions -- we don% have very much time,

If there a re no questions now, i f you should have any l a t e r tha t you would personally like t o ask the speakers about, you may t a l k t o them individually. a l i t t l e while, anyway.

I am sure most of them will be available for

A t t h i s time I will turn the program back t o our Chairman, D r . Kemp,

CHAIRMAN KHP: Thank you, A I ,

Just one thing before we go. You see t h e ten names here, we have put them back out there for you t o see i f you don't remember them. put the name on a sheet of paper and pass them down, and, Harold Hedrick, if you w i l l pick them up on t h i s side, and I 'U ask Carroll Schoonover t o pick them up on the other side,

Please

We w i l l now recess fo r lunch, and reconvene here a t 1:15 sharp.

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