BIOLOGICAL CHEMISTRY

DIVISION OF BIOLOGICAL CHEMISTRY

Insulin Acts Mainly by Promoting Transfer of Glucose into Cells

• Synthesis of glucuronidase in body is controlled by hormonal and genetic factors

• X-ray dose needed to inactivate papain pro­vides clue to molecular weight

• Radioactive needles containing germanium ox­ide suggested for use in brain studies

Τ NSULLN has been lound to accelerate the -*• rate of entry o f galactose and mannose into tissue cells, said Arne N . Wick of Scripps Metabolic: Clinic. This transfer into the cells cannot be the result of a physical process such as diffusion but must b e brought about by some definite chemical reaction. In this case, insulin is relatively specific, although not absolutely so, since it acts on galactose and mannose in addition to glucose, but fails to react with such closely related hexoses as sor­bitol, gluconic acid, and fructose.

Many investigators have assumed that insulin acts on the enzymes that deal with the oxidation and storage of glucose within the cells. This view implies that glucose passes readily into the cell, possibly by diffusion. Another concept of insulin ac­tion, which has recently been advanced, is that insulin regulates the transfer of glu­cose into the cell, Dr. Wick explained. This latter theory suggests that insulin does not actually affect the enzymes pres­ent in the cells.

There are two ways by which glucose might get int© the cell. One would be by a chemical process that insulin could ac­tivate. The other would be by the physical process of diffusion, and in this case in­sulin would act b y increasing the perme­ability of the cell membrane. This latter view has been advanced by Levine and coworkers, who have presented evidence to show that insulin increases the rate of entry of galactose into the cells. They have found that galactose is distributed in a volume equivalent to 4 0 % of the body weight when no insulin is used, and that this volume increases to 70% after insulin administration.

Dr. Wick and associates have studied this process with the aid of carbon-14-labeled hexoses i n eviscerated, nephrec-tomized rabbits. The labeled compounds have the advantage that they can be read­ily followed as they disappear from the blood stream. I n a typical procedure, galactose having a known radioactivity is injected intravenously into the animal. After 2 0 minutes are allowed for mixing in the body, the course of the plasma radioactivity is determined for several hours. If galactose penetrates the tissues

by the process of diffusion, its distribution can be calculated from the dilution of the labeled material in the plasma.

When insulin is not used, galactose is quicldy distributed in a volume equivalent to the extracellular space—that is, 20 to 257c of the body weight. On the other hand, when insulin is given, the rate of entry of the galactose into the cells is markedly accelerated. "We are forced to conclude," said Dr. Wick, "that galactose does not get into the cells b y diffusion but must be forced into the cells by some chemical process analogous to a pumping meciianism that is accelerated by insulin. We can conclude that insulin acts on glu­cose in the same way.*'

Sorbitol, gluconic acid, and fructose do not distribute beyond the extracellular spac«, with or without the presence of in­sulin. These three compounds have the same basic configuration as glucose, but apparently the aldehyde group on the 1-caxbon is all-important for cell perme­ability, h e said.

Protein Hydroly iates . The preparation of acid hydrolyzates of proteins without

significant loss of tryptophan was re­ported by Bruce E. Baker of McGill Uni­versity. The new procedure makes pos­sible the preparation o£ better-tasting therapeutic diets for use before and after surgery and for shortening convalescence from injury and disease. Concentrated nutrients are produced by mild chemical treatment that avoids the formation of bitter, unpleasant tastes and, at the same time, preserves the essential nutrients, tryptophan, which is easily decomposed by older strong-acid methods.

According to Dr. Baker, sulfurous acid was used to hydrolyze casein, egg albumin, lactalbumin, and blood fibrin to 24 to 37% of complete hydrolysis. Losses of tryptophan ranged from Ο to 3 % of the total. Use of 1 Ν sulfuric acid saturated with sulfur dioxide at 10° C. gave 58 to> 7 0 % hydrolysis, with losses of tryptophan» ranging from 5 to 10% of the total. The-sulfurous acid was removed from the-hydrolyzates by exaporation in vacuo,, while the strong mineral acid was re­moved by precipitation or by use of an< ion exchange resin. The final dried prod­uct contained less than 400 parts per mil­lion of sulfurous acid.

Blood Lipids. Because of the presence of polar groups which are soluble in water and nonpolar groups which remain on the surface, lipids are able to form mono-molecular films on water surfaces. The areas of these films vary from about 4 to 9 square centimeters per microgram of sample, depending on the lipid, the tem­perature, and the pressure on the film. In these measurements, a pressure is used such that a minimum change in area occurs for a given change in pressure. A pressure of 22 dynes per centimeter is generally satisfactory. The lipids are separated by chromatography, using latex-coated filter paper. The lipids that remain on the paper are removed by extraction with ethyl al-

Befo>re Division of Biological Chemistry, William E. McKee ( right ) of Chaney Chemi­cal Laboratories answers question from audience after presenting paper on estrone and estradiol. Listening is Otto Schales of Tulane, chairman of afternoon program

1285 / U L U M h 3 Ί , N U . 1 3 » » M A K C H 3 Ο, 1 V 5 3

123rd NATIONAL ACS MEETING

H. Bayard Milne of State College of Washington follows progress of enzyme reaction. Above the constant-temperature bath is an automatic sample changer for exposure to x-rays

Standing in front of birefringence apparatus are (left to r ight): G. F. Hanna, J. T . Yang, and J. F . Foster of I o w a State College, authors of paper presented at meeting of Biochemical Division

cohol. This technique permits the use of small amounts of blood serum and pro­vides a rapid method for measuring small quantities of lipids present.

Barbiturates in Blood. Measurement of the increase in optical density in the ultra­violet range of the spectrum when solu­tions of barbiturates are made alkaline has been used in several recently reported methods for the quantitative determina­tion of barbiturates. In the technique out­lined by Bernard F. McKenzie of the Mayo Foundation, 2 milliliters of oxalated blood is extracted with peroxide-free ether for 45 minutes in an all-glass apparatus. The extract is evaporated to dryness and the residue taken up in 3 milliliters of 95% ethyl alcohol containing 0.2 milliliters of acetate buffer at pH 4.65.

The optical density of this solution, in comparison with that of the alcohol-buffer mixture as a blank, is measured over the range of 220 to 330 ηΐμ by means of a Beckman spectrophotometer. A 0.2 milli­liter portion of borate buffer of strength sufficient to increase the pH to 9.5 is then added to each absorption cell and the reading repeated over the same range. The increase in optical density at 240 τημ is proportional to the content of barbi­turate. Individual barbiturates cannot be determined by this method since the ab­sorption curves of the various barbiturates are similar.

• Synthesis of Glucuronidase

The significantly greater concentration of ^-glucuronidase in tumor tissue, as compared to normal tissue, has aroused considerable speculation as to the possible role of this enzyme in cancer. According to a report b y William H. Fishman of Tufts College medical school, various hor­mones have a marked effect on the concen­tration of /3-glucuronidase in specific or­gans of the body. For example, testosterone is able to increase the concentration of glu­

curonidase some 5 to 15 times in the kid­ney, while producing no such effect in the liver. On the other hand, stilbesterol greatly increases the concentration of glu­curonidase in the liver, but not in the kidney. It has been known for some time, said Dr. Fishman, that certain strains of mice possess tissue ^-glucuronidase activ­ity which, in the liver and kidney, i s about one tenth that found in the majority of other inbred mice. It can be concluded, he said, that the synthesis o f glucuronidase in the body is controlled by both hor­monal and genetic factors.

Estrone and Estradiol. The determina­tion of urinary estrone and estradiol was discussed by William E . McKee of Chaney Chemical Laboratories. T h e urinary estro­gen conjugates are hydrolyzed b y auto-claving in the presence of 1.6 Ν sulfuric acid. An extract that is free of most of the usual red and purple pigments is ob­tained by adjusting the urine to p H 9 be­fore extraction with benzene. The solvent layer i s purified by sodium carbonate and sulfuric acid washes. The phenolic frac­tion is extracted -with 1 Ν sodium hyrox-ide, and the pH is then reduced to 11.0 with sodium bicarbonate. Shaking the bicarbonate layer with benzene serves to reextract the estrogens. Final purification is carried out by chromatography on 1 gram of alumina.

Fat+y Acid Oxidation. Fatty acid oxi­dation has been studied in fractions iso­lated centrifugally from homogenates of germinating peanut cotyledons. The frac­tions included the supernatant layer and microsomal particles that are sedimented b y centrifugation for one hour at 100,000 X gravity. The microsomal particles that oxidize both palmitic acid l-C-14 and palmitic acid 3-C-14 to labeled carbon dioxide require inorganic phosphate, co­enzyme 1, and coenzyme A for the oxida­tion. On the other hand, the supernatant fraction which produces carbon dioxide from palrnitate-l-C-14 only has no co-factor requirements.

Short-Chain Fatty Acids. Cell-free ex­tracts of the bacterium, Clostridium Kluy-veri, catalyze the synthesis of short-chain fatty acids from ethyl alcohol and ethyl acetate, said E. R. Stadtman of the Na­tional Institutes of Health in his Paul-Lewis Award address. These extracts also catalyze the oxidation o f fatty acids by molecular oxygen, he said. Detailed studies of this soluble enzyme system have led to a better understanding of the in­dividual reactions involved in fatty acid oxidation and synthesis.

In this system, ethyl alcohol is oxidized to acetaldehyde which, owing to the pres­ence of a coenzyme Α-dependent, D P N -specific aldehyde dehydrogenase, is further oxidized to acetyl coenzyme A. The acetyl coenzyme A thus formed assumes a key-role in the biosynthesis o f fatty acids, said Dr. Stadtman.

• Papain Inactivation

T h e inactivation of dry papain with 30,000-volt x-rays was described by H . Bayard Milne of State College of Wash­ington. The activity o f the papain was determined by means o f the reaction b e ­tween carboallyloxy-L-leucine and phenyl-hydrazine to form the phenylhydrazide. A plot of the log of the activity of papain against t ime of irradiation gives a straight line, in agreement with the target theory proposed by Lea .

T h e x-ray unit was calibrated b y deter­mining the inactivating x-ray does on a sample of catalase whose apparent cross section had been found by means of t h e deuteron cyclotron at Yale. On the basis of the x-ray dose required t o produce i n ­activation, the molecular weight of p a ­pain was calculated to he 28,000.

Ovalbumin and Plasma Albumin. It is n o w well known that both ovalbumin and bovine plasma albumin combine with ani­onic detergents. By equihbrium dialysis

1286 C H E M I C A L A N D E N G I N E E R I N G N E W S

CELLULOSE CHEMISTRY

Ellis V. Brown of Fordham, author of paper on thiamine labeled with radioac­tive sulfur. Fate of compound in rats was reported before Division o f Biological Chemistry

and electrophoretic studies, it has been shown that ovalbumin reacts with a cat-ionic detergent, Zephiron, in a similar manner—one cation being bound per car-boxylate group. According to Joseph F. Foster of Iowa State College, the reaction is accompanied by an increase in viscosity and in optical rotation, and b y the devel­opment of intense streaming birefringence. The product tends to aggregate* the rate of aggregation being at a minimum near pH 2.5 at low ionic strength.

Growth Hormone. T h e purity of the growth hormone, somatotropin, isolated from ox pituitary glands has recently been investigated b y chromatographic tech­niques and zone electrophoresis, accord­ing to a paper b y Choh Hao L i of the Uni­versity of California at Berkeley. The re­sults obtained thus far show no evidence of inhomogeneity. As previously reported, the hormone protein is denatured in an acetate solution at pH 4 , as indicated by the increase in its intrinsic viscosity and molecular weight. Ultracentrifugal stud­ies of the hormone in buffers at various pH indicate that the somatotropin mole­cule tends to aggregate or dissociate into different sizes. The molecular weight observed at pH 2.32 is 49 ,000 , but at p H 11.5 it decreases to 29,000.

Paper Chromatography. Previous stud­ies of mixtures of purified plasma albumin and *7-globulin suggested that these com­ponents might b e estimated quantitatively by use of two-dimensional paper chroma­tography. The report presented b y Henry Tauber of the University of North Caro­lina concerned the applicatioa of this method to serum specimens. It was found that paper chromatography, because of its simplicity and because it requires little special equipment, might b e of value in the detection of increases in t h e y-globmlin fractions of serum. At present, however, the method does not afford a precise quantitative indication of t h e y-globmlin content.

^Germanium Oxide Carrier The use of germanium oxide as a car­

rier for radioactive materials was d e ­scribed b y H. C. Dudley and T. G. Mitchell of U. S. Naval Hospital, St. Albans, Ν . Υ. Germanium oxide has been shown to have little toxicity, while radio­active germanium delivers no significant radiation because of its low specific activ­ity and rapid elimination. Melts of ger­manium oxide, containing either 10% gold or 14.5% phosphorus, have been pre­pared at 1400° C. Threads have been withdrawn from the melts and formed into glass-like needles, 0.5 to 1.0 milli­meter in diameter.

These needles, once activated, have been implanted subcutaneously in rabbits. The germanium oxide is dissolved by the tis­sue within 10 days and is eliminated by way of the urine. The insoluble radio­active materials, however, remain in the tissue and produce localized radiation effects as a result of their beta emission.

These slowly soluble needles may b e used for the destruction of a small locus of tissue, as in the study of the function of specific areas of the brain. In the study of the effects of radiation on neoplasms, these radioactive needles may b e e m ­ployed in place of radium, cobalt-60, or radon seeds. An advantage is that the

γ -RAY diffraction, microscopy, elec--**• tron microscopy, the ultracentrifuge, and infra-red studies are among those b e ­ing made by cellulose chemists to unravel the secrets of the composition and struc­ture of cellulose and its derivatives. The complete story has not yet been unfolded but substantial progress is being made.

Symposia held by the Division of Cel­lulose Chemistry give a picture of recent progress made in the field. H. M. Spurlin, Hercules Powder Co., chairman of the symposium, stated that the meeting was oganized.to examine the validity and use­fulness of methods used in these studies. Molecular weight and molecular weight distribution papers were omitted as they were covered in a previous symposium. Dr. Spurlin noted, however, that the pres-

germanium oxide preparations d o not re­quire subsequent removal from the tissue.

Radioactive Thiamine. As part of a study of the metabolism of vitamin Bi, radioactive thiamine was syntbesized in the laboratory, said Ellis V. Brown of Fordham University. Dosages of radio­active thiamine varying from 5 0 to 10O micrograms were injected intramuscularly into thiamine deficient and sufficient young adult rats. Since the thiamine sufficient rats excreted a major portion of trie in­jected thiamine unchanged, studies were continued only on the deficient rats. A separation was made of the sulfur com­ponents of the urine and feces into in­organic sulfate, ethereal sulfate, and neu­tral sulfur fractions. Radioactivity meas­urements on these sulfur components in the form of barium sulfate precipitates were made using a Geiger-Muller counter.

Negligible amounts of radioactivity were detected in any of the sulfur fractions of the feces, said Dr. Brown. On the other hand, significant amounts of thiamine "were oxidized and found in the inorganic sul­fate fraction of the urine. When oxythia-mine was administered within 24 hours after the intramuscular injection of labeled thiamine, there was a decrease in the ex­cretion of radioactive sulfur as inorganic sulfate and an increase in the radioactivity of the neutral sulfur fraction.

ent symposium lacked papers dealing with such important subjects as optical rotation, application of physical methods to solid polymers, and adsorption chromatography.

One major problem has been to deter­mine the size and shape of cellulose mole­cules. Three different methods of ap­proaching this problem were described by Michael Wales, National Bureau of Stand­ards; Seymour Newman, Hercules Powder Co., and Paul Doty, Harvard University. All three authors noted that the cellulose molecules are outstanding among polymers because of their stiffness.

Dr. Wales directed his work t o sedimen­tation velocity and equilibrium studies us­ing the ultracentrifuge. Dr. Newman and Dr. D o t y approached the problem through light scattering studies.

DIVISION OF CELLULOSE CHEMISTRY

Usefulness of Tools for Cellulose Research Examined

• Research trends of five years ago revived, augmented by paper chromatography and radioisotopes

• Carboxymethylhydroxyethylcellulose shows promise for oil well drilling muds, may compete with CMC

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