Effect of dietary protein composition on placental protein, nucleic acid, free alpha amino N and enzymes in rats

Pregnant rats were fed six different diets from the first to the 15th, 17th or 19th day of pregnancy. Diets 1 to 5 contained the same amount of nitrogen (10% casein and unsupplemented or supplemented wheat or Bengalgram diets). Diet 6 contained 20% casein. Total placental protein, RNA, free alpha amino N contents and the activities of the enzymes arginase (EC 3.5.3.1), tyrosine amino transferase (TAT, EC 2.6.1.5) and lactate dehydrogenase (LDH, EC 1.1.1.27) were estimated. The fetal weight and placental weight, total placental protein, RNA and free alpha amino N and the activities of the enzymes increased with the gestational age, but the DNA content became constant after day 17 of gestation. The placental weight, protein, free alpha amino N and RNA contents were significantly reduced on wheat and Bengalgram diets as compared to 10% casein (control) diet. The low activities of arginase, TAT and LDH on these diets indicated impaired protein synthesis, as a result of reduction in the amino acid pool size. The fortification of wheat with lysine and Bengalgram with cystine, methionine and tryptophan showed significant improvement in the fetal weight and placental parameters. The values on the 20% casein diet were significantly higher than those observed on the 10% casein diet.     

Neither glutamine nor arginine supplementation of diets increase glutamine body stores in healthy growing rats

The aim of the work was to resolve whether glutamine and arginine supplemented diets affect plasma and tissue (muscle, liver and intestinal mucosa) glutamine concentrations, as well as glutaminase and glutamine synthetase specific activities. The trial was performed in growing rats fed 10% protein diets for 3 weeks. Protein sources were: whey proteins (W); whey proteins+free glutamine (WG); whey proteins+arginine (WA); and casein+wheat protein hydrolysate+acid whey (39:39:22), as source containing protein-bound glutamine (CGW). Rats fed the control diet (6.4% glutamine) (W) showed comparable glutamine body stores to those of rats fed the WG diet. In fact, glutamine sup- plementation down-regulated the hepatic glutamine synthetic capacity of growing rats (W/WG: 6.8+/-0.3 vs 6.0+/-0.2 nmol/min/mg protein). Arginine supplementation of the diet (up to 9% of the protein content) resulted in a decrease in plasma and tissue glutamine concentrations (W/WA: plasma, 1218+/-51 vs 1031+/-48 micromol/L; liver 7.5+/-0.4 vs 6.5+/-0.2 micromol/g; muscle: 5.7+/-0.2 vs 4.0+/-0.2 micromol/g). These data suggest that glutamine supplementation of the diet does not increase plasma and tissue glutamine concentrations in healthy growing rats, while the addition of arginine to the diet decreases glutamine body stores.     

The optimum dietary amino acid pattern for growing pigs. 1. Experiments by amino acid deletion

A series of four nitrogen-balance experiments was carried out with growing pigs to determine the optimum balance amongst the amino acids in the diet. The reduction in N retention when 20% of a single amino acid was removed from the diet was used to calculate a dietary amino acid pattern in which each amino acid would be equally limiting. A mixture of amino acids simulating the amino acid pattern of casein was used with the same efficiency as casein. From two successive deletion experiments an optimum balance amongst the essential amino acids was derived. Expressed relative to lysine = 100 this had threonine 72, valine 75, methionine + cystine 63, isoleucine 60, leucine 110, phenylalanine + tyrosine 120, tryptophan 18. No estimate was made for histidine. Essential amino acids in this pattern were mixed with non-essential amino acids in ratios of 36:64 up to 57:43. The highest efficiency of N retention was achieved with diets having a ratio of at least 45:55. This included (g/16 g N) lysine 6.5, threonine 4.7, valine 4.9, methionine + cystine 4.1, isoleucine 3.9, leucine 7.2, phenylalanine + tyrosine 7.8, tryptophan 12. The N of diets with this amino acid pattern was utilized significantly better than when the pattern proposed by the Agricultural Research Council (1981) was used. The flow of amino acids past the terminal ileum of pigs given the semi-synthetic diet with this amino acid pattern was no greater than that observed with protein-free diets. The proposed pattern thus describes the intrinsic requirements of the growing pig for absorbed amino acids.     

Sulfur amino acid requirements of the growing rat fed eight percent dietary protein

Methionine and methionine plus cystine requirements of the growing rat fed equivalent to 8% dietary protein were determined. Diets formulated to be adequate in all nutrients except sulfur amino acids (SAA) and providing 4% protein from ANRC casein and equivalent to 4% casein protein from amino acids (excluding methionine and cystine) were supplemented with graded levels of L-methionine. The diet providing 0.34% total SAA (0.32% L-methionine + 0.02% L-cystine) gave the best feed/gain ratio, relative net protein ratio (RNPR), liver protein utilization (LPU) and plasma amino acid acid parameters. When similar ANRC casein + crystalline amino acid diets providing 0.44% total SAA but varying in ratio (by weight) by methionine/cystine were fed to rats, optimal feed/gain ratio, RNPR and plasma amino acid parameters were obtained when cystine replaced 33 to 60% of dietary L-methionine. Inclusion of L-cystine at the expense of L-methionine in these 8% protein diets improved overall rat performance and utilization of dietary methionine. Supplementation of the casein + amino acid basal diet with methionine sulfoxide, methionine sulfone (oxidized forms of methionine) or cysteic acid (oxidized form of cysteine/cystine) to provide 0.34% total SAA, indicated that relative to methionine (100), methionine sulfoxide was completely available and methionine sulfone (68) and cysteic acid (55) were less available. Correction for methionine and cystine digestibilities in ANRC casein suggests that the SAA requirements for the growing rat are 0.33% of diet or 4.1% of dietary protein when 8% protein diets are fed.     

Non-essential nitrogen and protein utilization in the growing rat

Three series of nitrogen-balance experiments were carried out on growing rats fed on purified isonitrogenous diets (16 g N/kg) to study the importance of non-essential N and the essential:total N (E:T) ratio for attaining maximum N balance (NB) and biological value (BV) of protein. Minimum dietary levels of asparagine, proline and glutamic acid required for maximum NB and BV were estimated to be 1.0, 2.0 and 5.0 g/kg respectively. In an essential amino acid-based diet, the levels of individual amino acids were successively reduced to 110% of the requirement. Reducing the level of arginine, lysine or methionine + cystine resulted in a significant increase in NB and BV while the response of rats given the isoleucine-reduced diet significantly decreased. Addition of asparagine, proline and glutamic acid in the estimated minimum amounts to an essential amino acid-based diet resulted in a significant increase in NB and BV. A further significant increase was found when the levels of arginine, lysine and methionine + cystine in the diet were reduced to 110% of the requirement. The performance of rats fed on the latter diet was similar to that of rats given a diet with the optimum E:T ratio. It is concluded that the optimum protein utilization may be influenced by the presence of some non-essential amino acids and by the surplus of some essential amino acids rather than by the E:T ratio per se.     

The quality and quantity of dietary protein affect brain protein synthesis in rats.

The influence of the amino acid supply in diets with different quality and quantity of protein on the rate of protein synthesis in the brain was investigated. Amino acid concentrations in serum and brain altered in accordance with the amino acid levels of the diets, with the exception of some amino acids such as aspartic acid, glutamic acid, glycine and threonine. When rats were fed various levels of dietary casein (0, 5 and 20%), the aggregation of ribosomes increased and the fractional rate of protein synthesis tended to increase with the increase in dietary protein. When rats were fed a 20% casein diet, greater aggregation of brain ribosomes and protein synthesis rate were observed compared with those in rats fed 20% wheat gluten or gelatin diets. The RNA activity was related to the degree of the aggregation of brain ribosomes and the fractional rate of protein synthesis.     

Effect of dietary methionine and arginine on uric acid excretion of cocks fed a protein-free diet.

Cocks were fed a protein-free diet supplemented with methionine plus arginine or glutamic acid for 25 days to investigate the effect of these amino acids on fecal and urinary nitrogen excretion. Addition of either methionine plus arginine or glutamic acid did not change the fecal nitrogen excretion. Methionine plus arginine supplementation reduced the urniary nitrogen excretion compared to the protein-free diet, whereas glutamic acid supplementation increased it. The reduced urinary nitrogen excretion resulting from supplementation with methionine plus arginine was mostly accounted for by a reduction in uric acid excretion. In the methionine plus arginine group, free amino acid analysis showed that free glutamine and glutamic acid content significantly decreased in liver while no differences were found in plasma. Since glutamine may play an important role in the formation of uric acid for chickens, the reduced amount of free glutamine and glutamic acid in the liver of cocks fed the diet suplemented with methionine plus arginine might account for the reduced excretion of uric acid, and therefore for the nitrogen sparing action of methionine plus arginine in chickens fed a protein-free diet.     

The effect of graded levels of dietary casein, with or without methionine supplementation, on glutathione concentration in unstressed and endotoxin-treated rats

Glutathione (GSH) concentration was measured in rats fed either graded levels of dietary casein (experiment 1; 180 g, 120 g, 80 g, or 60 g protein/kg diet) or graded levels of dietary casein, supplemented with methionine to equalize dietary sulfur amino acid content to that seen in an 180 g/kg casein diet supplemented with 0.3 g L-methionine/kg diet (experiment 2; 180 g protein +0.3 g L-methionine, 80 g protein +6.70 g L-methionine, or 60 g protein +7.45 g L-methionine/kg diet). Rats were given an inflammatory challenge by intraperitoneal injection of endotoxin (lipopolysaccharide from Escherichia coli), and were compared with ad libitum and pair-fed controls. Glutathione concentration in various organs (liver, lung, spleen, and thymus) decreased in animals fed the low-protein diets (80 g or 60 g/kg diet). Addition of the sulfur amino acid, methionine, to the low-protein diets restored glutathione concentrations in animals fed ad libitum and prevented the fall in GSH concentration, which occurred in lung, spleen, and thymus in response to the endotoxin. Despite the similarity in the amount of sulfur amino acid consumed between the groups fed the 180 g protein +0.3 g L-methionine and the 60 g protein +7.45 g L-methionine/kg diet, in experiment 2, hepatic GSH concentration significantly increased in the latter group, in animals fed ad libitum and in the endotoxin-treated animals, but not in the pair-fed controls.     

Methionine and arginine supplementation of casein-based high fat diets: Effects on rat growth

The effects of methionine and arginine supplementation on protein adequacy of two commercial caseins (ANRC and U.S. Biochemicals) in diets containing 20% fat were investigated in weanling rats. They were fed basal 18% protein (20% casein) diets and basal diets supplemented with 0.25% L-methionine, 0.35% L-arginine or methionine + arginine for a period of four weeks. The unsupplemented basal diets contained 1.38–1.41 mg of methionine + cystine and 1.46–1.49 mg of arginine per kilocalorie and were similar in nutrient composition to those utilized for evaluation of dietary oils. The methionine and arginine supplemented diets contained 1.92–1.96 mg of methionine + cystine and 2.23±2.27 mg of arginine per kilocalorie. Dietary supplementation with methionine, arginine or methionine + arginine had no significant (P>0.05) effects on food consumption; weight gain; protein efficiency ratio; net protein ratio; liver protein utilization; or liver, heart and kidney weight in rats. These results suggest that all amino acid requirements for rat growth are met by 20% unsupplemented casein of the types tested here.     

Sulfur-containing amino acid requirements of growing dogs

Three experiments were undertaken to establish the total sulfur-containing amino acid requirement of growing dogs. In experiment 1, six Labradors failed to grow normally when fed a soy isolate diet of 0.28% methionine with 0.18% cystine but grow well when the food was supplemented to either 0.57% or 0.74% methionine. In the next experiment, 12 Labradors and 21 beagles were fed the soy isolate diet containing either 0.39%, 0.57% or 0.74% methionine in the presence of 0.15% cystine for 12 weeks. Dogs fed 0.39% methionine had significantly lower body weights, nitrogen retentions, food intakes and feed efficiencies than their littermates fed the two higher levels. This diet provided 468 kcal metabolizable energy (ME) per 100 g, therefore a level of 116 mg total sulfur-containing amino acids (TSAA)/100 kcal ME was not adequate for growth. The lowest level found to be adequate was 0.57% methionine, or 154 mg TSAA/100 kcal ME, which is similar to the requirement of other young omnivores. A final experiment with a free amino acid diet indicated that a level of 117 mg TSAA/100 kcal ME while inadequate for Labradors, may be sufficient for some beagles, highlighting differences between intact protein and free amino acid diets and suggesting possible breed differences.     

The optimum dietary amino acid pattern for growing pigs. 2. Requirements for maintenance and for tissue protein accretion

Experiments were made to estimate separately the amino acid requirements of growing pigs for maintenance and for protein accretion. The relationship between nitrogen retention and amino acid intake was estimated for each essential amino acid (except histidine) by giving, at rates of N intake of 0.25 and 2.0 g/kg body-weight (W)0.75 per d, diets in which one amino acid was made specifically deficient. From the regression coefficients it was calculated that, for the accretion of 1 g body protein, the dietary amino acid requirements were (mg) threonine 47, valine 53, methionine + cystine 36, methionine 19, isoleucine 43, leucine 78, phenylalanine + tyrosine 84, phenylalanine 41, lysine 68 and tryptophan 12. The daily amino acid requirements for N equilibrium were also estimated. From the relationship between N retention and amino acid intake the daily amino acid requirements for N equilibrium were estimated to be (mg/kg W0.75 per d) threonine 53, valine 20, methionine + cystine 49, methionine 9, isoleucine 16, leucine 23, phenylalanine + tyrosine 37, phenylalanine 18, lysine 36 and tryptophan 11. It was estimated that both for maintenance and for protein accretion tyrosine could provide close to half the total phenylalanine + tyrosine needs. Cystine could supply close to half the total sulphur amino acid needs for protein accretion but 0.8 of the needs for maintenance.     

Effects of p-aminobenzoic acid, methionine, threonine and protein levels on susceptibility of mice to Plasmodium berghei

The effects of dietary p-aminobenzoic acid (PABA), protein, methionine and threonine on Plasmodium berghei infection in mice were investigated. Animals were fed diets containing 12 or 20% casein supplemented with PABA (0 or 2 mg/kg diet), methionine (0 or 15 mg/g casein) and threonine (0, 7.5, 27.5 or 47.5 mg/g casein). Percent mortality was lower in rats fed diets without PABA than in those fed diets containing PABA. All further experiments were conducted without supplemental PABA. While the mean day of death was greater in the groups fed 12% casein, percent mortality in these groups was nearly twofold higher than in the groups fed 20% casein. The presence or absence of 15 mg methionine per gram casein had no effect on percent mortality, mean day of death, or percent parasitemia, regardless of dietary casein level. Supplementation of threonine at any level to the 12% casein diet with supplemental methionine resulted in mortality rates similar to those from animals fed the 20% casein diets, but percent mortality was not altered by threonine in the absence of supplemental methionine. The mean day of death of animals fed 20% casein increased with increments of added threonine. Methionine had no influence on this phenomenon. It is concluded that the quantity of dietary protein and the quality of its amino acid composition can have profound effects on the susceptibility of mice to malaria.     

Dietary branched-chain amino acids and protein selection by rats

Consumption by rats of high protein diets is associated with elevated plasma and brain concentrations of branched-chain amino acids (BCAA). We examined the possibility that changes in BCAA concentrations in blood and brain might serve as modulators of protein consumption. After young rats had adjusted to selecting between a 10% or 25% casein diet and a 50% casein diet, a mixture of BCAA was included in the diet containing the lower amount of protein (10% + BCAA, 25% + BCAA). Supplementation of the 10% or 25% casein diets with BCAA and subsequent elevation of BCAA concentrations in plasma and brain were associated with increased selection of protein in rats given the 10% + BCAA/50% casein diet choice, but not in rats offered the 25% + BCAA/50% casein diets. When no alternative diet was available, addition of BCAA to a 15% casein diet depressed food intake, and rats given a choice between a 15% casein diet with or without added BCAA selected almost exclusively the diet without added BCAA. Although BCAA concentrations were high in plasma and brain in all experiments, concentrations of methionine, tyrosine, phenylalanine, tryptophan and histidine were low in brain in experiments in which rats altered their diet or protein selection after BCAA addition. High concentrations of BCAA in plasma and brain were not consistently associated with changes in protein selection.     

Effect of dietary protein and methionine on sulfite oxidase activity in rats

Sulfite oxidase catalyzes the oxidation of sulfite to sulfate. To investigate whether or not sulfite oxidase activity (EC 1.8.3.1) is regulated by the amount of sulfur from dietary protein or excess methionine, we fed rats diets containing 5, 10, 20 and 50% casein with or without excess methionine and measured sulfite oxidase activity in liver and intestinal mucosa. Hepatic sulfite oxidase activity was significantly lower in rats fed 5 or 10% casein diets and significantly higher in rats fed 50% casein than in rats fed the control diet containing 20% casein, but activity did not change in response to the addition of methionine at any level of protein. Sulfite oxidase activity in the intestinal mucosa was only 5% of that seen in liver and did not change in response to dietary protein or methionine. Activity did not change in rats fed low iron diets (5 mg Fe/kg diet) at any level of protein tested or in response to glycine. These results show that sulfite oxidase activity can adapt to different levels of dietary protein but is unaffected by the level of methionine, total amino nitrogen or iron in the diet.     

Induction of lysine imbalance in rats: relation to competition for lysine transport into the brain in vitro

The ability of amino acids inhibitory of lysine transport into brain slices to induce lysine imbalance was determined by feeding wheat gluten or casein diets with additions of such amino acids. Lysine transport was only moderately inhibited by amino acids; the most effective were basic amino acids or mixtures of indispensable (IAA) or branched chain amino acid (BCAA). Only mild depressions in growth and food intake occurred during a 10-day period when male, 60--65 g rats of the Sprague-Dawley strain were fed lysine-limiting, 18% wheat gluten diets with additions of these amino acids. The effects were prevented by added lysine. Rats allowed a choice between the lysine-imbalanced or non-protein diets selected the imbalanced, wheat gluten diets (in severe imbalances rats will choose the non-protein diet). Growth depression, prevented by added lysine, occurred in rats fed a 6% casein diet supplemented with IAA; individual amino acids were ineffective. Growth depressions also occurred when rats were fed a basal diet containing 6% case in + 5% of an equimolar mixture of nine IAA and supplemented with arginine or more IAA; BCAA were less effective. Additional lysine completely prevented the growth depressions, but growth of rats fed the diets containing arginine and BCAA was greater than that of those fed the extra IAA. It is difficult to induce a severe lysine imbalance; this is consistent with the failure of amino acids to cause under our conditions strong inhibition of lysine transport into brain.     

Comparative contributions of dietary protein quality and quantity to growth during gestation, lactation and postweaning in the rat

The growth promotion by feeding purified diets containing 25%, 12% or 7% of either casein or wheat gluten (W.G.) and the supplementation value of adding cystine to the low casein diets, and lysine or lysine plus threonine to the wheat gluten diets ascertained in rats by comparing the weight achieved at birth, after 3 weeks suckling or 4 weeks postweaning. Birth weight was depressed by 30% with 7% casein; by 20% with 7% W.G.; by 15% with 25% W.G. compared to controls and there was no change when lysine alone was added to 7% W.G. Addition of lysine plus threonine to 7% W.G., lysine to 25% W.G. and cystine to 7% casein increased birth weight. The weight gain of dams reflected birth weight of pups in most cases. Weaning weight was depressed by 60% with either 12% casein or 12% W.G., and by 40% with 25% W.G. compared to controls. Addition of cystine to 12% casein and lysine to 25% W.G. increased the weaning weight slightly. Addition of lysine, lysine plus threonine or lysine plus glutamic acid (G.A.) to 12% W.G. had no effect on weaning weight. Maternal weight loss was decreased by amino acid supplementation of the diet. Four-week postweaning body weight was equal to the control when cystine was added to 12% casein and lysine was added to 25% W.G. Addition of lysine to 12% W.G. improved the growth rate, and addition of lysine plus threonine further improved the growth rate. Food intake was not decreased by protein deficiency during gestation. Food intake was significantly lowered by protein deficiency during both lactation and postweaning, however, supplementation of amino acid partially restored food intake to normal levels.     

Relationship between amino acid composition of diet and plasma cholesterol level in growing rats fed a high cholesterol diet

The effects of dietary sulfur-containing amino acids and glycine on plasma cholesterol level were studied in rats fed amino acid mixture diets containing cholesterol. The relationship between the amino acid composition of dietary proteins and plasma cholesterol levels was also investigated in rats fed diets containing various kinds of protein such as casein, egg albumin, pork protein, fish protein, corn gluten, wheat gluten and soy protein. Feeding the amino acid mixture corresponding to casein led to an approximately two-fold level of plasma total cholesterol as compared with feeding the amino acid mixture corresponding to wheat gluten. It was possible to reduce the plasma cholesterol of rats fed the amino acid mixture of the casein type by increasing the proportion of cystine in the total sulfur amino acids. Inversely, the deprivation of cystine resulted in an enhancement of the plasma cholesterol of rats fed the gluten type amino acid mixture. Glycine had a tendency to resist increases in the plasma cholesterol level. A significant negative correlation was noted between plasma cholesterol levels and the content of cystine in intact dietary proteins. The results suggest that the differential effect of dietary proteins on plasma cholesterol level is mainly associated with sulfur-containing amino acids included in the protein, regardless of whether it is of animal or plant origin.     

Factors affecting dietary requirement and deficiency signs of L-tryptophan in rainbow trout

Two experiments were conducted to determine the concentration of dietary tryptophan needed for optimal growth and survival of fingerling rainbow trout (Salmo gairdneri), to characterize signs of tryptophan deficiency, and to ascertain the effects of niacin on deficiency signs. Test diets containing either hydrolyzed or intact casein were fed with graded levels of added L-tryptophan (0.0, 0.25, 0.50, 0.75 and 1.0 g/100 g protein). Maximum growth occurred at 0.75 and 0.50 g tryptophan in fish fed hydrolyzed and intact casein, respectively. Changes in mineral and amino acid mixtures in the intact casein diet yielded a cation-anion (Na + K - Cl) balance of +15.3 meq/100 g diet and pH of 6.44, compared with a balance of -21.3 and -25 meq, and pH 5.8 and 4.4 for unmodified hydrolyzed and intact casein diets, respectively. Analysis of growth by the polynomial regression function; Y = 0.16 + 0.429X - 0.26X2, where Y = mean daily gain in grams, and X = grams of added tryptophan/100 g protein, predicted that at least 0.58 g of tryptophan was needed for maximum weight gain. Analysis of apparent pathology-free fish by the function; Y = 34.33 + 147.33X -83.87X2, where Y = percentage with no signs of deficiency, and X = grams of added tryptophan, predicted that 0.63 g tryptophan/100 g protein was needed for optimum health scores. Deletion of supplemental niacin did not affect fish response to tryptophan.     

Amino acid requirements for growth of Nile tilapia

A series of feeding experiments was conducted in aquaria to determine the quantitative requirements of the 10 essential amino acids for growth of young Nile tilapia (Oreochromis niloticus). The test diets contained casein and gelatin supplemented by crystalline L-amino acids to provide an amino acid profile similar to 28% whole egg protein except for the test amino acid. Each set of test diets consisted of seven isonitrogenous diets containing varying levels of the amino acid to be tested. Weight gains analyzed by the broken line regression method indicated the following requirements as a percentage of the dietary protein: lysine, 5.12; arginine, 4.20; histidine, 1.72; valine, 2.80; leucine, 3.39; isoleucine, 3.11; threonine, 3.75; tryptophan, 1.00; methionine with cystine (0.54% of the protein), 3.21; and phenylalanine with tyrosine (1.79% of the protein), 5.54.     

Free amino acid levels during lactation in rats: effects of protein quality and protein quantity

Free amino acid levels were determined in serum, mammary gland, liver and muscle during lactation in rats fed diets varying in protein. Using a 3 X 2 factorial design, after conception female rats were fed purified diets varying in protein quality (wheat, wheat + lysine + threonine or casein + methionine) and in protein level (11.6% vs. 22.3%). On d 15 of lactation dams were decapitated; blood was collected, and the serum was quickly frozen. Samples of mammary gland, liver and calf muscle were also removed and quickly frozen. After deproteinization, free amino acids were determined on an LKB 4400 Amino Acid Analyzer. As the protein nutritional value of the diet increased, milk yield increased threefold and the level of free lysine in the mammary gland increased six- to eightfold. With protein quality or quantity improvement, the level of free methionine increased two- to threefold, and no consistent changes were seen in the levels of free threonine, aromatic and branched chain amino acids in mammary glands. Free lysine concentrations in mammary gland, liver and muscle were highly correlated with free lysine in serum (r2 = 0.94, 0.96, 0.93, respectively). Under the conditions of this experiment, a direct relationship between dietary lysine, free lysine in the mammary gland and milk secretion was observed.