M.Clyde Otey

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. Mixtures 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%. Mixtures 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. V. Effects of combined administration of nonprotective compounds and subprotective levels of l-arginine.HCL on ammonia toxicity in rats

A level of l-arginine. HCl on the one hand, and of monosodium l-glutamate, l-glutamine, disodium α-ketoglutarate, monosodium l-aspartate, l-asparagine, disodium oxalacetate, l-alanine, sodium pyruvate, glucose, or sodium chloride on the other, neither of which on intraperitoneal injection will protect rats against an intraperitoneal injection of an LD99.9 dose of ammonium acetate, will however do so when employed together. Survival of all animals was observed when 1 mmole l-arginine. HCl/kg. body weight was mixed with 4 mmoles/kg. of one of the following: glutamate, ketoglutarate, and glucose. Aspartate and its derivatives were less effective in this connection, and replacement of the 1 mmole/kg. dose of l-arginine. HCl by an equivalent amount of l-ornithine. HCl led to no protection whatever against ammonia toxicity. Each of the effective components of the mixtures, such as 1 mmole/kg. of l-arginine. HCl and 4 mmoles/kg. of l-glutamate, reduces the blood NH3 N level of animals given a lethal dose of ammonium acetate to a significant extent, but only when employed together is the blood NH3 N reduced to the normal level with survival of the animals. It is suggested that the effective partners in such mixtures accomplish the ammonia detoxification by involving separate mechanisms.

Studies on polycysteine peptides and proteins. II. Apparent dissociation constants, and ultraviolet and infrared absorption spectra of isomeric cystinylcystine peptides

Abstract The apparent dissociation constants, and ultraviolet and infrared absorption spectra were determined for l -cystinyl- l -cystine prepared by oxidation of l -cysteinyl- l -cysteine at pH 6.5 and at pH 8.5. The p K 1 ′ and p K 2 ′ values, relating to the two carboxyl groups, differed between themselves, but were identical for the two l -cystinyl- l -cystine preparations. The p K 3 ′ and p K 4 ′ values, relating to the two amino groups, not only differed between themselves, but differed significantly in the two l -cystinyl- l -cystine preparations. These results were interpreted in part as indicating considerable interaction among the ionizing groups which would be consistent with a condensed or ring structure for these compounds as previously suggested. The ultraviolet absorption between 205 and 280 mμ and the infrared absorption between 2 and 15 μ were in each case identical for the two l -cystinyl- l -cystine preparations. Under the same conditions, the spectra in the ultraviolet region were identical for l -cystinyl- d -cystine and d -cystinyl- l -cystine, and so too were the spectra of these compounds in the infrared region. Both ultraviolet and infrared spectra were, however, different for the optically diastereoisomeric cystinylcystine peptides. Even more striking were the differences noted in the infrared absorption spectra of optically diastereoisomeric alanylalanine peptides. The cystinylcystine peptides possess bands at 3.0, 3.2, 6.0, and 6.6 μ. The infrared spectra of the diastereoisomeric cystinylcystine peptides differ most markedly toward the longer wavelengths of the region studied. This is true also when comparisons are made of the infrared spectra of l -cystine and l -homocystine. The characteristic 240–250-mμ band of cystine in the ultraviolet is much less distinct in peptides of cystine, due in part to the intense absorption of the peptide link which rises toward the shorter wavelengths. Calculations of the contribution of the peptide bond to the absorption of the peptides at 205 mμ suggested an unusually high order of magnitude for this value in peptides involving cystine. The physical criteria employed in the present study did not reveal the striking differences between l -cystinyl- l -cystine (6.5) and l -cystinyl- l -cystine (8.5) which were observed when such criteria as optical rotation, paper chromatography and the formation of derivatives were employed. Indeed, the identical spectra plus the equal susceptibility of the two l -cystinyl- l -cystines to peptidase action suggest that the two compounds are identical in certain respects, and, by previous criteria, quite different in others. Further studies on this problem are in progress.

Quantitative Nutritional and In Vivo Metabolic Studies with Water-Soluble, Chemically Defined Diets

Publisher Summary This chapter presents quantitative, nutritional, and in vivo metabolic studies with water-soluble and chemically defined diets. The resolution of racemic compounds, as initially developed by Louis Pasteur, consists of three independent methods: (1) mechanical separation of those crystals capable of showing hemihedrism; (2) employing an optically active acid or base, which can transform the racemate into a diastereomeric mixture wherefrom the more insoluble diastereomeric salt can separate; and (3) utilizing a biological agent that can preferentially metabolize one or the other isomeric form of the racemate. The enzymatic resolution procedures, developed by Jesse Philip Greenstein and his associates, are based primarily upon the L-directed asymmetric hydrolysis, either by hog renal acylase I or carboxypeptidase of a suitable N-acyl-DL-amino acid, or by hog renal amidase of a DL-amino acid amide. Such resolution procedures are employed to obtain the L-and D-antipodes of a large variety of α-amino acids on the large scale and permit the formulation of water-soluble, chemically defined diets composed of crystalline essential and nonessential L-amino acids, the requisite vitamins and salts, glucose, and ethyl linoleate or mazola oil as the essential fatty acid component.