Milton Winitz

Studies on the metabolism of amino acids and related compounds in vivo. I. Toxicity of essential amino acids, individually and in mixtures, and the protective effect of l-arginine

The l- and d-forms of arginine. HCl, histidine. HCl, isoleucine, alloisoleucine, leucine, lysine. HCl, methionine, phenylalanine, threonine, allothreonine, tryptophan, and valine were administered intraperitoneally to rats, and the LD50, LD99.9, LD99.99 levels were evaluated with the aid of statistical computations. Among the l-amino acids, alloisoleucine was the least toxic and tryptophan the most toxic, whereas among the d-amino acids, although alloisoleucine was still the least toxic, arginine. HCl was the most toxic. With the exception of tryptophan whose l-isomer was about three times more toxic than the d-isomer, the differences in toxicity between the l- and d-isomers of the amino acids were of relatively small degree, the d-isomers at the LD50 level being generally less than or equal to the corresponding l-isomers in toxicity. n nMixtures of the ten essential l-amino acids possessed a toxicity considerably less than that calculated from the mean of the toxicities of the individual components. This was found to be due to the presence of l-arginine. HCl, for a mixture of nine l-amino acids from which l-arginine was excluded possessed a toxicity not far from that calculated from the mean of the toxicities of the individual components. This protective effect of l-arginine was further demonstrated by adding it to a lethal mixture of nine l-amino acids which caused a reduction in mortality from 100 to 24%. n nMixtures of the d-isomers of the ten essential amino acids possessed a toxicity less than that calculated from the mean of the toxicities of the individual components, but no one component of this mixture could be implicated in this general effect.

Studies on the metabolism of amino acids and related compounds in vivo. III. Prevention of ammonia toxicity by arginine and related compounds.

Abstract The LD50 and LD99.9 levels for ammonium acetate injected intraperitoneally in rats starved for 24 hr. have been determined. Injection of 2 mmoles/kg. body weight of l -arginine. HCl at 60 min. prior to an LD99.9 dose of the ammonium salt resulted in complete protection of the animals. A comparable degree of protection with l -citrulline and l -ornithine. HCl was achieved at a level of 8 mmoles/kg. l -Arginine methyl ester. HCl, neutralized to pH 7.0, conferred nearly complete protection at a level of 4 mmoles/kg. of body weight. Compounds which when injected 1 hr. prior to an LD99.9 dose of ammonium acetate protected some but not all of the animals included the d -isomers of arginine. HCl, citrulline, ornithine. HCl, and arginine methyl ester. HCl, as well as α-keto-δ-guanidovaleric acid (the α-keto acid analog of arginine) and α-acetyl- l -ornithine. Compounds which, under the same conditions, were completely nonprotective, included acetyl- l - and d -arginine, α-acetyl- d -ornithine, δ-acetyl- l -ornithine, and the l -forms of lysine, homocitrulline, and homoarginine. Liver slices prepared from animals injected 60 min. earlier with various protective compounds showed, when incubated with ammonium chloride, an accelerated consumption of ammonia and formation of urea, whereas with nonprotective compounds under the same conditons either a less strongly marked acceleration or no acceleration at all was observed. It would appear that the effect of the previously injected protective substances consisted at least in part in a mobilization and acceleration of the classic Krebs-Henseleit urea synthesis mechanism in the liver.

Quantitative nutritional studies with water-soluble, chemically defined diets. I. Growth, reproduction and lactation in rats.

Abstract Mixtures of purified l -amino acids, salts, vitamins (B and C), and d -glucose were prepared in 50% aqueous solution and offered, together with a separate supplement of fat-soluble vitamins in corn oil to weanling rats. The tyrosine and cystine components were present in the form of ethyl l -tyrosinate. HCl and ethyl l -cysteinate. HCl, respectively. Calcium and phosphorus were present in the form of the soluble calcium fructose 1,6-diphosphate, and iron appeared in the form of the complex salt, ferrous ammonium sulfate. Potassium and magnesium appeared as the respective gluconates. With these precautions to maintain solubility, the diet solutions were stable and water-clear almost indefinitely. Tests for the presence of microorganisms in diet solutions not sterilized but in which the amino acid and calcium fructose diphosphate components had been carefully crystallized prior to solution revealed complete sterility 0n standing at 37 ° for at least 4 weeks. No bulk was provided in these soluble diets, nor was a training period necessary for their ready imbibition. The intestinal contents were considerably reduced as compared with similar animals on a chow diet, and fecal excretion was scanty and infrequent. The teeth of these animals and the entire length of the gastrointestinal tract were free of any pathologic lesion, gross or microscopic. The diets contained roughly 20% amino acids and 80% glucose, equivalent per gram to 4 cal., or per milliter to 2 cal. The animals grew well at a rate of over 4 g. per day on a diet the “non-essential” amino acid ratios of which were modeled after that of casein. Lesser growth was obtained with diets modeled after other animal proteins, with diets containing fewer “non-essential” amino acids, and with diets modeled after casein but containing dl -amino acids, all however with the same or greater total nitrogen. At maturity the animals on a diet limited in “non-essential” amino acids were mated and produced satisfactory litters which were nursed to weaning. The young, which were smaller than normal, were divided into two groups, one fed on the same diet and the other on that with a full complement of “non-essential” amino acids. Animals on the latter diet recovered rapidly from their bad start and grew at a faster rate than did their litter mates. On mating within each group at the same time, the animals on the superior diet yielded satisfactory litters which were nursed to weaning while those on the inferior diet now showed no pregnancies at all.

Studies on the metabolism of amino acids and related compounds in vivo. IV. Blood ammonia and urea levels following intraperitoneal administration of amino acids and ammonium acetate, and the effect of arginine thereon

Blood ammonia and urea levels were determined in rats which had been injected intraperitoneally (a) with LD99.9 ammonium acetate alone and with a prior injection of l-arginine. HCl, l-ornithine. HCl, or l-citrulline; (b) with LD99.9 levels of l- or d-amino acids; (c) with LD99.9 levels of l-amino acids together with l-arginine · HCl; and (d) with an LD99.9 level of a mixture of nine essential l-amino acids in the presence and absence of l-arginine. HCl. n1. n(a). Injection of an LD99.9 dose of ammonium acetate was followed by a rapid rise in blood ammonia, a moderate increase in blood urea, and death of the animals; in the presence of protective levels of arginine, ornithine, or citrulline, the rise in blood ammonia was quickly checked and the values of this component rapidly fell to normal levels as the urea markedly increased, and the animals survived. n n2. n(b). Animals injected with LD99.9 levels of l- and d-amino acids died with elevated blood ammonia, and with elevated blood urea except for l-valine, and l- and d-histidine. HCl which evoked normal blood urea levels. n n3. n(c). Addition of l-arginine. HCl to each of the l-amino acids, resulted in survival of the threonine-injected rats, a drop in the blood ammonia levels of rats given isoleucine, leucine, threonine, lysine, and tryptophan, with no change in the blood ammonia levels of rats given methionine, valine, histidine. HCl, and phenylalanine; the blood urea levels in animals injected with isoleucine, leucine, threonine, and phenylalanine appreciably increased, whereas with the five other amino acids no change in urea levels was noted. n n4. n(d). Animals given the LD99.9 mixture of nine essential l-amino acids died with a moderately elevated blood ammonia and a slightly subnormal blood urea level. In the presence of a relatively low level of l-arginine. HCl the animals survived with a normal level of blood ammonia and an elevated urea level. In the presence of a much higher level of l-arginine. HCl, the animals died with elevated blood ammonia and urea levels, the innate toxic effect of so much arginine outweighing its ordinary protective effect.

Quantitative nutritional studies with water-soluble, chemically defined diets. VIII. The forced feeding of diets each lacking in one essential amino acid

Abstract Male Sprague-Dawley rats weighing 130–140 g. were force-fed water soluble, chemically defined diets, either complete or free of one essential amino acid, and the subsequent changes in weight were determined over a period of time. Diets free of histidine, tryptophan, lysine, phenylalanine, and threonine caused the animals to lose between 0.2 and 1.0 g./day over an 11-day period. Diets free of methionine, leucine, and isoleucine caused the animals to lose, over the same period, 1–2 g. weight/day. Most of the animals on these diets survived, and all survivors gained in weight when force-fed the complete diet. However, all the animals on the valine-free diet rapidly sickened and failed to survive beyond 9 days on this diet. Rats bearing the rapidly growing Walker tumor were subjected to the deficient diets, and all gained in weight by an amount nearly equivalent to the weight of the tumor, the carcasses losing weight to about the same extent as their normal controls on the same diets. The rates of tumor growth varied according to the essential amino acid deleted from the diet. On the lysine-free diet the tumor growth rate was the same as that on the complete diet, and on diets lacking phenylalanine, histidine, or tryptophan the tumor growth rate was only slightly slowed. The tumor growth rate, however, was considerably slowed when the animals were force-fed diets lacking in methionine, isoleucine, or valine. Normal animals force-fed diets free of lysine, tryptophan, or isoleucine were in negative nitrogen balance. The balance values became more positive in similar animals bearing the Walker tumor, the nitrogen loss from the carcass being nearly equal to that gained by the tumor on lysine- and tryptophan-free diets. On the isoleucine-free diet a considerable proportion of the nitrogen lost by the carcass was not picked up in the tumor.

Quantitative nutritional studies with water-soluble, chemically defined diets. III. Individual amino acids as sources of “non-essential” nitrogen

Abstract The basal diet described earlier, and consisting of the ten “essential” amino acids to 9.5 g. total N, B-vitamins, ascorbic acid, salts, and glucose was dissolved in water to 50% concentration and offered to weanling male rats. Over a 21-day period there was an average weight gain of 9 g. or about 0.45 g. per day per animal. Various amino acids and other nitrogenous compounds were individually added to the basal diet at the expense of an equal amount of the glucose component, and the resulting diets, also in 50% aqueous solution, were likewise offered ad libitum to weanling rats. These supplemented diets were all isonitrogenous, i.e., either 25.2 or 22.0 g. total N/kg. The diets providing the best growth, about 3 g. per day, were those in which l -alanine, ammonium l -glutamate, l -glutamine, ammonium l -aspartate, and l -proline were individually furnished as the sole source of “non-essential” nitrogen. The additions of the d -isomers of alanine and of arginine to the basal diet accelerated growth but not to the same extent as that produced by the corresponding l -isomers when also employed singly. Ammonium acetate proved more effective in promoting growth than either urea or glycine, while l -serine, l -hydroxyproline, and l -cysteine, at the levels used (15.7 g. total N/kg. diet) proved to be toxic to the animals.

Studies on the metabolism of amino acids and related compounds in vivo. VI. Free amino acid levels in the tissues of rats protected against ammonia toxicity.

Abstract The free amino acids present in ethanolic extracts of homogenates of various tissues of the normal rat, and of rats treated with an LD 99.9 dose of ammonium acetate preceded or not by protective doses of l -arginine. HCl, were determined by quantitative paper chromatography. The most striking observations made were ( a ) a very considerable increase of liver aspartic acid in animals treated with ammonia alone, and of aspartic acid, glutamic acid, and urea in the livers of animals treated with ammonia and protective arginine, some 15 min. after injection of the ammonia, with no great change in glutamine levels at any time; ( b ) moderate increases of glutamine in the muscle extract of animals given ammonia alone, and of glutamine and urea in extracts of this tissue in animals given both ammonia and protective arginine; and ( c ) considerable increases of glutamine in extracts of brain and of testis with no change in urea levels at any time. It is concluded that ammonia is dealt with in the liver by the Krebs-Henseleit-Ratner cycle, and in brain and testis through the synthesis of glutamine.

A synthesis and resolution of DL-serine.

Abstract A single-step synthesis of dl -serine from the interaction of formaldehyde and glycine in an alkaline aqueous solution containing catalytic amounts of copper sulfate is described. The reaction proceeds in addition with the formation of a second as yet unidentified ninhydrin-reactive substance with an empirical formula of C 4 H 9 NO 4 . Separation of the two products from each other and from any unreacted glycine is readily achieved with the aid of a Dowex 50 column. The elemental analyses, chromatographic behavior, and infrared absorption spectrum of the serine ultimately secured were identical with those shown by an authentic sample of dl -serine. Additionally, a resolution of dl -serine as its acetyl derivative via the l -directed asymmetric hydrolytic action of hog renal acylase I and a racemization of optically active serine by treatment with acetic anhydride in aqueous pyridine are presented.

ϵ-Lysine acylase

Abstract An enzyme system in rat kidney has been observed which hydrolyzes ϵ-acetyl- l -lysine, and which, through two or three purification steps, has been concentrated in activity approximately 100 times. The enzyme is more highly active in phosphate than in other buffer mixtures studied, possesses a pH optimum at 7.0–7.2, and is not appreciably affected by most bivalent cations. It is somewhat more active toward ϵ-chloroacetyl- l -lysine than toward ϵ-acetyl- l -lysine and completely or nearly completely inactive toward ϵ-acetyl- d -lysine, ϵ-carbobenzoxy- l -lysine, biocytin, and δ-chloroacetyl- l -ornithine. The enzyme is seemingly active toward α,ϵ-dichloroacetyl- l -lysine, but because of a trace of acylase I in the preparation this observation is not altogether certain. Because of its apparent specificity requirements, the enzyme has been tentatively given the designation of “ϵ-lysine acylase.” Growth studies on rats fed a nearly chemically defined diet including all essential l -amino acids except l -lysine were performed. Supplementation of this diet with ϵ-acetyl- l -lysine led to growth, in accord with the earlier observations of Neuberger and Sanger, and explicable now in part at least in terms of the presence of the ϵ-lysine acylase in the tissues of the rat. Supplementation of the diet with acetyl- d -lysine led to loss of weight which was distinctly less than when the diet was supplemented with d -lysine, and the possibility of a slow inversion of the former compound to the corresponding l -isomer was expressed.

Quantitative nutritional studies with water-soluble, chemically defined diets. VII. nitrogen balance in normal and tumor-bearing rats following forced feeding

Abstract Water-soluble, chemically defined diets were prepared and force-fed to normal rats in amounts predetermined to produce no appreciable loss or gain of weight. When the Walker tumor was implanted in such animals and allowed to grow, and the forced feeding continued, the tumor-bearing animals gained greatly in weight, this gain being due practically entirely to the tumor. Although the carcass weight did not change appreciably, there was a loss of nitrogen which could only be accounted for by translocation of nitrogen from the carcass to the tumor. Otherwise adequate, nitrogen-free, water-soluble diets when force-fed produced nearly equal losses in body weight of normal rats and of the carcasses of tumor-bearing animals, and nearly equal excretion of nitrogen; the continued growth of the tumors in such animals together with the loss of nitrogen from the carcasses suggests that the tumor growth was based on nitrogen translocated from the carcass.