122nd NATIONAL ACS MEETING

DIVISION OF BIOLOGICAL CHEMISTRY

Researchers Explore Metabolic Fate of Antituberculosis Drug

• Conversion of carotene to vitamin A in di­abetic rats is only one fourth that in normal rats

• Technique developed for preparation of crystalline rennin from commercial cheese rennet

• Inactivation of enzyme solutions can be pre­vented by addition of sucrose before freezing

ALTHOUGH the metabolism of pyridine-**- 3-carboxyl compounds has been in­vestigated repeatedly, there is little in­formation available on the metabolism of organic acid hydrazides. The marked anti-tubercular activity of isonicotinic acid hy-drazide makes a knowledge of its meta­bolic fate particularly important, said Jacques M. Kelly, Raymond B. Poet, and Lois H. Chesner of E. R. Squibb & Sons.

In their tests on dogs, 20 to 50% of the drug, administered orally in doses of 5 and 20 mg./kg., is excreted unchanged in the first 24 hours. About 1 to 5% of the drug is excreted in the second 24-hour period. Isonicotinic acid has been isolated as a principal metabolite of isonicotinic acid hydrazide, and about 20 to 40% of the drug is excreted as such. The oral admin­istration of 10 mg./kg. of isonicotinic acid to a dog results in the excretion of about 70% of the acid. The use of paper chromatography has suggested the pos­sible presence of small amounts of W-methylisonicotinic acid hydrazide in the urine, but this compound could not be iso­lated in sufficient quantity to establish its identity.

No metabolites could be detected that had the properties of synthetic N'-methyl-isonicotinamide or isonicotinamide. Like­wise, free hydrazine could not be detected in either the plasma or urine at any time after dosage.

In vivo experiments showed that the orally administered drug is uniformly dis­tributed in the tissues and apparently is not accumulated as shown by the analy­sis of dog tissues following chronic admin­istration. In vitro experiments with rat tissue homogenates at 37° C. for 24 hours indicated that the drug is destroyed in only small amounts by liver, lung, brain, kid­ney, heart, and plasma. However, slightly hemolyzed blood rapidly destroys the com­pound at, room temperature.

Virus Attachment. The identification of the specific groups of atoms by which viruses attach themselves to host cells in the initial stage of virus invasion was an· nounced by L. J. Tolmach and T. T. Puck of the University of Colorado. Earlier re­search by Dr. Puck indicated that the first

step in a virus invasion is the establish­ment of bonds of electrical attraction be­tween virus and host cell. With this theory as a basis, he and coworkers rea­soned that there are probably only six cnemical groups in the virus' protein struc­ture that are capable of participating in such bond formation.

Viruses tagged with radioactive phos­phorus were treated with reagents known to block specific functional groups. The ability of such modified viruses to combine with Escherichia coli host cells was then tested. It was found that the carboxyl group was necessary for the attachment of both T l and T2 type viruses. The T l virus also required the presence of amino groups. The four chemical groups also present in the cells but not essential for the initial attachment of the viruses were the substituted amino group, the phos­phoric acid group, the sulfhydryl group, and the phenolic hydroxyl group.

The attachment of labeled T2 to host cells was also studied as a function of pH. As was expected from the proposed

The Division of Biological Chemistry heard three papers on the effect of tumors on normal tissue metabolism by Irving R. Sherman and Emily F . Chaffee of Yale XJiaiversity

theory, the virus-cell binding displays a pH maximum. The rate of attachment Is diffusion-limited at pH's between 5Λ) and 8.5 and falls off rapidly to zero on either side of this pH range.

• Diabetes and Vi tamin A

Discovery of a chae to "the nature of premature aging in diabetes patients was reported by Albert E. Sobel and Abraham Rosenberg of the Jewish Hospital of Brooklyn. It has been estimated that the arteries of diabetics age three times as fast as those of normal individuals. Fatty substances are believed to play an impor­tant role in this process, which is; char­acterized by hardening of the arteries.

In laboratory tests, young rats were placed on a vitamin. A-fre e diet. When signs of vitamin A deficiency weare ob­served, each litter of animals was ddvided. One half was made diabetic by tSie use of alloxan, while both groups of rats re­ceived a standard dose of carotene- Tests showed that diabetic rats htad storesd only one fourth as much vitamin A as haud their normal litter-mates. This -difference was observed in each of 20 litters cororprising a total of over 100 animals. Wheal vita­min A was fed to the rats instead o>f caro­tene, practically the same amount o f vita­min A was found in the "body stores of both the diabetic and normal animals. This indicated that there was neither im­paired absorption nor destruction of vita­min A in the diabetic animals.

The next series of tests was undertaken to determine whether iit diabetic rats there is reduced conversion of carotene to vitamin A in the wall of the iratestine. Experiments showed that the noramal in­testine produced six; times as much vita­min A from tbe same amoomt of carotene as did the diabetic intestine. "These studies carry the clear implications," tbe

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BIOLOGICAL CHEMISTRY

report stated, "that the diabetic rat must receive some source of pre-formed vita­min A, such as fish oils, rather than the usual carotene sources, such as vegetables."

Germicidal Soaps. Previous studies on 2,2'-methylenebis 3,4,6-trichlorophenol have shown that this compound, unlike most phenol derivatives, does not lose its germicidal activity in soap, stated Max E. Chiddix of General Aniline and Film Corp. During a study of the properties of such compounds, a number of substituted 2,2'-methylenebis phenols were prepared and tested for germicidal activity in the pres­ence of soap. Although halogen and nitro substituents were more effective germi-cidally than methyl and phenyl groups, there was little correlation of the germi­cidal power and the position or frequency of these groups.

However, a good correlation was found between germicidal activity in the pres­ence of soap and the acidity of each of the two phenolic hydroxyl groups. A chelated structure that previously has been proposed for other methylenebis phenols may account for the difference in the acid­ity of these groups, Dr. Chiddix stated. The high acidity of the first phenol group increases the rate of absorption of the compound by bacteria. The low acidity of the second group enables it to take part in the toxic reaction as an unneutralized phenolic group, in spite of the high alka­linity of the soap.

Blood Preservation. Although plasma may be stored safely for long periods of time, whole human blood is considered suitable for transfusion purposes only within the first three weeks after collec­tion. During the storage of human blood, a gradual change takes place in the red cells, so that they do not survive after entering the circulatory system of the re­cipient. Enzymatic processes, it is be­lieved, may take place during storage that

may cause an increase in the fragility of the outer covering of the red cells.

With this in vitro fragility test as a rough measure of red-cell deterioration during storage, the effect of enzyme in­hibitors on the rate of development of os­motic fragility was investigated. Various inhibitors of lipases, proteases, and pepti­dases were tested. The most promising re­sults were obtained with 10-(2-dimethyl-amino-isopropyl)phcnothiazinc, which was first introduced as an antihistamine.

Blood from 10 different donors showed, on exposure to 0.6r/r sodium chloride solu­tion after 21 days storage, ο to 23% hemolysis and, after 42 days. 20 to 36% hemolysis. However, when the optimum amount of the phenothiazine derivative (0.4 millimole per liter) was added to the same blood samples, hemolysis was re­duced to 0.4 to 4.1'ν after 21 days and to 0.6 to 4.5% after 42 days on exposure to 0.6% sodium chloride solution.

ί» Crystalline Rennin A method of preparing crystalline

rennin from commercial cheese rennet was described by \V. M. Connors, R. \V. De Baun, and R. A. Sullivan of National Dairy Research Laboratories. The over­all yield was about 5r/<, and an eleetro-phoretic analysis revealed the presence of some three components. The major com­ponent (75*% ) contained both the milk-clotting and proteolytic activities, while the minor components were inactive. The presence of tyrosine in the molecule was indicated by ultraviolet absorption spec­tra.

Crystalline and crude rennins are af­fected in the same way by changes in pH, temperature, and concentration of sodium ion, calcium ion, and substrate. Of these factors, pH is quantitatively the most im­portant. Crystalline rennin exhibits a pH

Jacques M. Kelly (left), Lois H. Chesner, and Raymond B. Poet of E. R. Squibb & Sons coauthored a report on the metabolism of isonicotinic acid hydrazidc

optimum for proteolysis against hemo­globin at pH 3.9, which shifts to pH 3 in the presence of 1% sodium chloride.

Tumors. The effect of tumors on normal-tissue metabolism in tumor-bearing rats was the subject of three papers by Irving R. Sherman and Emily P. Chaffee of Yale University. They found that, although in normal rats citrate was consumed in the customary manner, it exhibited a positive inhibitory action in tumor-bearing rats. Oxygen uptake in the presence of suc­cinate was lowered considerably, and the utilization of glucose was slightly de­pressed.

The chemistry of experimental chlo-roma (the green tumors of certain my­elogenous leukemias of man) was discussed by Julius Schultz, Harry Shay, and Margot Gruenstein of Temple University. They stated that the nature of the green pig­ment has long been a subject of contro­versy. In human chloroma, protoporphy­rin, protoporphyrin-protein complex, and choleglobin have a t various times been suggested as the green pigment.

Blood Clotting. A light-scattering tech­nique has been developed for the study of fibrinogen clotting, reported Robert F. Steiner of the Naval Medical Research In­stitute. During blood clotting, fibrinogen, an elongated protein molecule, is acted upon by thrombin to form a slightly al­tered molecule that is spontaneously poly­merized to form a three-dimensional lat­tice. When a small amount of thrombin is added to a fluid solution of fibrinogen, the latter is transformed into a rigid gel whose properties depend upon both the concentration of hydrogen ion and added electrolyte.

Light-scattering studies have shown that, after an induction period whose length depends upon the thrombin con­centration and the concentration of added electrolyte, the fibrinogen polymerizes to form very long, thread-like molecules about two or three fibrinogen molecules in thickness. At high concentrations of elec­trolyte, these molecules interact directly to form a three-dimensional network of thin strands, giving rise to the so-called fine clot which is transparent and readily rup­tured. The increase in particle size with time gradually levels off as the point of gelation is approached.

Hyaluronidase Inhibition. Research on a drug to stop the destructive action of hyaluronidase (an enzyme suspected of contributing to arthritis and to the weak­ening of blood vessels) was described by J. M. Boiler, Souren Avakian, Robert Preston, and Gustav J. Martin of National Drug Co. In animal experiments, phos-phorylated hesperidin has been found to have a powerful antihyaluronidase effect.

. Currently, the drug is undergoing pre­liminary tests on humans suffering from such diseases as osteoarthritis, rheumatoid arthritis, and bursitis, but no results have been announced as yet.

"There have been frequent reports in the medical literature," said Dr. Boiler, "that hyaluronidase is involved in certain

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122nd NATIONAL ACS MEETING

pathological conditions. For example, various types of arthritis are believed to be associated with abnormal hyaluroni-dase metabolism, and it has been reported that the beneficial action of cortisone in these conditions is connected with its abil­ity to inhibit the enzyme. Similarly, the effect of the vitamin Ρ group of com­pounds in maintaining the integrity of the blood vessels has been linked with their ability to inhibit hyaluronidase."

Citric Acid. The enzymatic synthesis of citric acid was discussed in a paper by Joseph R. Stern, Minor J. Coon, and Alice del Campillo of N e w York University. They indicated that, in the presence of oxalaeetate and condensing enzyme, acetyl coenzyme A is converted to citrate with the liberation of a stoichiometric quantity of sulfhydryl groups.

• Effects of Freezing If washed homogenates of rat liver are

quickly frozen and then thawed, their ability to oxidize ordinary substrates is lost or greatly impaired, reported Ε. Μ. Scott and coworkers at the Arctic Health Re­search. Center of Alaska. This inactiva-tion can be prevented b y the addition of sucrose before freezing. The effect of sucrose appears to be caused by a change in the size of the ice crystals formed. In-activation of enzymes b y freezing appears to be caused, in part, by dissociation of coenzymes and the consequent increased requirements for coenzymes. Tests indi­cate a complex destructive action of freez­ing on tissue at a subcellular level, in ad­dition to effects on the cell membranes.

Dextran Metabolism. The fate of dex-tran, a high-molecular-weight polysac­charide, is important because certain other materials (acacia, polyvinylpyrrolidone) have been shown to accumulate in the tis­sues, said Irving Gray of Brooke Army Hospital. Although no evidence for the in vitro metabolism of dextran has been shown, recent work, using starved dogs as test animals, has produced evidence for in vivo metabolism. This process has been studied with dextran labeled with car­bon-14. Glucose tagged with carbon-14 has been found in the urine of starved, phlorizinized dogs given labeled dextran. This material was isolated as the glucos-azone and characterized by elemental analysis.

In tests with mice, 0.2 ml. of a 6% solu­tion of dextran was injected intravenously. The biological half-life of the carbon-14 of the dextran, determined by following the total retained activity in the whole carcass, was found to be 6.1 days. The carcass was fractionated into fat, carbo­hydrate, and protein, and the activity in each fraction was determined. It was ίοιιικί that there was a rapid and large incorporation of activity into the amino acids, with a much slower and lesser in­corporation into the fat. The carbohy­drate activity fell rapidly at first and then approached the same rate of loss as the other constituents.

DIVISION OF CARBOHYDRATE CHEMISTRY

Sugars from Starch Hydrolysis Also Formed in Dextrose Polymers

• Inhibiting effect of water on the browning reaction demonstrated by model system

• Interfering substances in sugar beet refining shown to be galactinol and a new inositol

*-pHE presence of certain sugars in starch •*· hydrolyzatcs is no indication that they

existed in the original starch structure, an­nounced W. R. Fetzer of Clinton Foods, Inc. Speaking on the program of the Divi­sion of Carbohydrate (formerly Sugar) Chemistry, Dr. Fetzer reported investiga­tions showing that some of the sugars found in starch hydrolyzatcs are also pro­duced from pure dextrose by a treatment identical with that used for the hydrolysis of starch.

The polymerization of dextrose proceeds readily under proper conditions of con­centration and temperature and with amounts of acid so small that they are normally disregarded, said Dr. Fetzer. This polymerization or reversion must therefore be taken into account in inter­preting work with dextrose in aqueous acid systems. Others in Dr. Fetzer's laboratory, for instance, have obtained maltose in amounts of Ί0ΓΑ and gentio-biose in amounts of 5% from polymerized dextrose solutions. Both were identified as their octaacetates. Presumptive evi­dence indicates the presence of trel iose and isogentiobiose, he reported. The identification of maltose was confirmed in another laboratory in a sample whose origin was not revealed.

The composition of the equilibrium mix­ture that results from the extended acid heat treatment of cornstarch slurries varies with the solids concentration of the sirup at equilibrium, revealed Dr. Fetzer. The equilibrium can be shifted repeatedly from substantially "pure" dextrose to a complex mixture of monomeric and poly­merized dextrose by altering the concen­tration of the substrate; polymerization in­creases with the concentration of sugar solids. The rate at which equilibrium is reached for a given solids concentration is dependent on temperature and p l î .

Dr. Fetzcr's studies have also shown that changes in reducing power and in specific rotation of sugar solutions an» not necessarily related. In certain cas* acid-treatment may cause a large change in reducing power but very little change in rotation. In others the opposite may occur.

In some of the older literature, observed Dr. Fetzer, the effect of heat and acid on carbohydrate systems is reported either in terms of reducing power or in change in

rotation. His findings demonstrate that neither criterion alone is sufficient to war­rant specific conclusions as to the nature or extent of the change that has occurred.

The division's sessions opened with a memorial program celebrating the 100th anniversary of the birth of Emil Fischer and J. II. van't HofiF. Claude S. Hudson, National Institutes of Health, spoke on the work of both men, and H. O. L. Fischer, University of California, reminisced about his father, Emil Fischer.

Reducing Sugars. Two methods for de­termining minute quantities of reducing sugars have been developed by H. S. Isbell and J. D . Mover of the National Bureau of Standards. One depends on the amount of carbon-14 fixed in the cyano-hydrin reaction. This method is not specific but is extremely sensitive. The other method, based on an isotope dilution technique, provides a means for determin­ing specific sugars in complex mixtures. It depends on the formation of a radioac­tive derivative with separation by co-crystallization with the known carrier. The amount of parent substance in the unknown is calculated from the specific activity of the carefully purified product.

• Browning Reaction

A model system for studying the non-enzymatic browning reactions of sugars with amino acids was described by W. Ward Pigman of the University of Ala­bama Dental School and College of Medi­cine. Studying glucosylaniline. which is more labile than the glycosides, Dr. Pig-man found that water exerts an inhibiting effect on darkening in methanol solutions in the presence of hydrochloric acid; color develops strongly in anhydrous methanol.

In early stages of darkening (90 min­utes) the rate increases with the concen­tration of hydrogen chloride. When the ratio of hydrogen chloride to glucosylani­line is less than unity, there is an initial inhibitory period, but this stage disappears as the ratio goes above one. For systems containing up to 20% water, the molar ratio required to overcome the inhibition increases with the water content.

During later stages (2*1 hours) color formation reaches a maximum when the molar ratio is about unity. Atmospheric oxygen, cupric ion catalysis, and the

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