Alfred E. Harper

Branched-chain amino acid oxidation by isolated rat tissue preparations.

Branched-chain amino acid transaminase activity, branced-chain alpha-keto acid dehydrogenase activity, and leucine oxidation were measured in homogenates and slices of several rat tissues. Transaminase activity was highest in heart, while dehydrogenase activity was highest in liver. Leucine oxidation in isolated tissues may be limited by either transaminase or dehydrogenase activity depending upon the relative activities of these two enzymes in the tissue. The results suggest that, as the load of branched-chain amino acids increases, the liver may become an increasingly important site for the degradation of branched-chain alpha-keto acids.

BALANCE AND IMBALANCE OF AMINO ACIDS

Recognition of the importance of the proportions of individual amino acids in dietary proteins can be traced to the classic investigations of Willcock and Hopkind and of Osborne and Mende12 about fifty years ago. These pioneer studies, which gave rise to the concept of dispensable and indispensable amino acids, indicated clearly that the nutritive value of a protein was related to its amino acid composition. These observations led to extensive investigations by many nutritionists, culminating in 1935 in the isolation of threonine by McCoy, Meyer, and This discovery, together with the previously accumulated knowledge, made it possible to estimate quantitatively the amino acid requirements of several species and, as methods of determining the amino acid composition of proteins were improved, to relate differences in the nutritive value of proteins to differences in their content of indispensable amino acids4, These developments paved the way for detailed studies of interrelationships among amino acids, and between amino acids and other dietary components, thus leading to an awareness of the importance of amino acid balance and imbalance in nutrition.

Valine metabolism in vivo: Effects of high dietary levels of leucine and isoleucine

The short-term effects of feeding rats high levels of L-leucine or L-isoleucine on valine metabolism in vivo have been investigated. Consumption of a low-protein diet containing an additional 5% of leucine resulted in depression within one hour of the plasma concentrations of isoleucine, valine, alpha-keto-beta-methylvalerate, and alpha-ketoisovalerate. Concurrently with these changes in blood branched-chain amino acids and branched-chain ketoacids was a rapid increase (51%) in whole-body L-[1-14C]-valine oxidation. Studies with intragastrically administered leucine solutions indicated that the depressions in blood concentrations of valine occurred over the same time period as the stimulation in valine oxidation. In contrast, consumption of a low-protein diet containing an additional 5% of isoleucine had no significant effect on the plasma concentrations of leucine, valine, and alpha-ketoisocaproate; a significant (P less than 0.01) depression in the plasma concentration of alpha-ketoisovalerate was observed three hours after the diet containing excess isoleucine had been consumed. In contrast to the results obtained with excess leucine, consumption of excess isoleucine had no significant effect on the rate of valine oxidation in vivo. As part of an effort to explain the leucine-induced depletion of plasma valine and stimulation of valine oxidation, liver and muscle branched-chain aminotransferase and liver branched-chain ketoacid dehydrogenase activities were measured. Consumption of excess leucine had no significant effect on either muscle or liver aminotransferase activities, but was associated with a greater than two-fold increase in hepatic dehydrogenase activity.(ABSTRACT TRUNCATED AT 250 WORDS)

Gas-liquid chromatography of α-keto acids: Quantification of the branched-chain α-keto acids from physiological sources

Abstract Gas-liquid chromatography of several α-keto acids as their O -trimethylsilyl-quinoxalinol derivatives is described. Application of the method to quantification of branched-chain α-keto acids from several physiological sources is emphasized. The α-keto acids are partially purified by deproteinization with acetone, cation-exchange chromatography on Dowex 50W-X8 (H + ), and conversion to quinoxalinol derivatives by reaction with o -phenylenediamine in acid solution. Prior to gas-liquid chromatography, the quinoxalinols are trimethylsilylated with N,O -bis(trimethylsilyl)trifluoroacetamide in pyridine.

Influence of dietary protein level on protein self-selection and plasma and brain amino acid concentrations☆

Control of protein intake was studied in young rats that were allowed to choose between either protein-free and 55% casein diets or 15% and 55% casein diets. Animals on the protein-free vs. 55% casein regimen exhibited a lower weight gain, a lower cumulative energy intake and a greater cumulative total protein intake during the 13-day study compared to rats selecting between 15% and 55% casein. The daily average proportion of total food selected as casein by animals choosing between protein-free and 55% casein diets increased from 15% to 38% during the course of the study. In contrast, rats choosing between 15% and 55% casein chose 18-22% of total food as protein throughout the entire study. Long-term protein intake or protein selection did not correlate significantly with whole-brain contents of 5-HT or 5-HIAA. Our results suggest that protein intake is not regulated at a constant proportion of total calories, but is controlled between a minimum level that will support rapid growth and a maximum that, if exceeded, would require the animal to undergo substantial metabolic adaptation. The mechanism controlling protein selection may involve diet-induced changes in the brain content of total free indispensable amino acids.

Protein and energy consumption, plasma amino acid ratios, and brain neurotransmitter concentrations.

Abstract The hypothesis that long-term protein and energy intakes are controlled, respectively, by brain concentrations of serotonin (5HT) and the catecholamines, dopamine (DA) and norepinephrine (NE) has been proposed by Anderson and associates [2,5]. This control is assumed to be mediated through the influence of diet composition on the availability of the amino acid precursors of the neurotransmitters to the brain. The hypothesis was tested in experiments in which plasma amino acid concentrations and brain neurotransmitters were measured in young rats allowed to choose between two isocaloric diets differing in protein content (15% vs 55% casein or 15% vs 55% wheat gluten) and containing additional amounts of certain amino acids. No consistent correlation was observed between protein consumption and either the plasma ratio of tryptophan to neutral amino acid (Trp/NAA) concentrations or brain 5HT concentration. Similarly, no correlation was observed between energy intake and either the plasma ratios of Tyr/Phe and Tyr/NAA concentrations or the brain concentrations of NE and DA. The most apparent association observed was a strong correlation between total protein consumption and the sum of plasma branched-chain amino acid concentrations. Under the conditions of this experiment, feeding behavior seemed to be associated positively with the needs of the animal for nutrients and negatively with ingestion of an excess of amino acids generally rather than with plasma amino acid concentration ratios and brain neurotransmitter levels.

Transport of threonine and tryptophan by rat brain slices: relation to other amino acids at concentrations found in plasma.

Threonine content of brain decreases in young rats fed a threonine‐limiting, low protein diet containing a supplement of small neutral amino acids (serine, glycine and alanine), which are competitors of threonine transport in other systems (Tewset al., 1977). Threonine transport by brain slices was inhibited more by a complex amino acid mixture resembling plasma from rats fed the small neutral amino acid supplement than by mixtures resembling plasma from control rats or from rats fed a supplement of large neutral amino acids. Greater inhibition was seen with mixtures containing only the small neutral amino acids than with mixtures containing only large neutral amino acids. On an equimolar basis, serine and alanine were the most inhibitory; large neutrals were moderately so; and glycine and lysine were without effect. Threonine transport was also strongly inhibited by α‐amino‐n‐butyric acid and homoserine, less so by α‐aminoisobutyric acid, and not at all by GABA.

Transport of nonmetabolizable amino acids in rat liver slices

Abstract Transport of α-amino [ i -14C]isobutyric acid (AIB) by rat liver slices against a concentration gradient has been demonstrated; uptake was improved by including a preincubation step. Similarities to other systems included linearity of uptake over an extended period of time, as well as indications of saturability of the system with increasing concentrations of substrate. The transport of AIB was inhibited by anoxia and by 2,4-dinitrophenol, while glucose was without effect; inhibition also occurred in the presence of ouabain. No evidence for the active transport of AIB was seen when Na+ was totally replaced in the medium. The removal of extracellular K+ or Ca2+ markedly decreased the transport of AIB, although some uptake against a concentration gradient still occurred. The replacement of Mg2+ had little or no effect on the gradients achieved. Uptake was lower in a Krebs-Ringer phosphate than in a Krebs-Ringer bicarbonate buffer. The Na+ and K+ concentrations of slices incubated in the presence of AIB were similar to those of fresh liver. Cycloleucine was also transported against a concentration gradient but to a lesser extent than AIB.

L-Tryptophan injection fails to alter nutrient selection by rats ☆

Nutritional studies on rats given a choice between two diets differing in protein content have led to the proposal that brain 5-HT content regulates protein intake [2]. Pharmacologic studies under similar conditions of dietary self-selection suggest that brain 5-HT controls carbohydrate intake [41]. We tested the effect of elevating brain 5-HT via tryptophan injection (100 mg/kg) on short-term food intake and selection by rats choosing between two diets differing in protein and carbohydrate content. Under these conditions neither total food intake nor protein and carbohydrate selection were affected despite increases of 50% in brain concentrations of 5-HT and 5-HIAA. The effect of Trp administration was selective to serotonin metabolism as brain concentrations of NE, DA and DOPAC were not affected. These results suggest that alterations in brain 5-HT content which may occur following meal ingestion may not be of physiological importance in regulating nutrient intake and selection.

Metabolism and transport of γ-carboxyglutamic acid

Abstract γ-Carboxyglutamic acid residues have been shown to be present in prothrombin, the other vitamin K-dependent clotting factors, and more recently in bone and kidney proteins. This amino acid is formed by a posttranslational vitamin K-dependent carboxylation of glutamyl residues in polypeptide precursors of these proteins. It has now been demonstrated that this amino acid, either in the free or peptide-bound form, it not metabolically degraded by the rat, but is quantitatively excreted in the urine. In nephrectomized rats, the tissue concentration of intravenously administered γ-carboxyglutamic acid is increased, but there is still no evidence of any oxidative metabolism of this amino acid. This amino acid is transported by kidney slices against a concentration gradient, but does not accumulate in liver, intestinal or brain tissues. Prelimininary data suggest that γ-carboxyglutamic acid may be concentrated by a carrier system from the utilized by other amino acids.