Changes in Creatine and Urea Formation in Rats Fasted and Fed Low Protein Diets

Changes in the time course of the urinary excretion of creatinine, creatine and urea, and the activities of kidney transamidinase and liver urea-cycle enzymes were investigated in rats fasted and fed on a 10% casein diet and 10% casein diets supplemented with 10% glycine and/or 1.4% arginine.

The urinary total-creatinine of the fasted rats increased extremely during fasting for 7 days, while that of the animals given the 10% casein diet supplemented with glycine and arginine rose exceedingly on the 3rd day and thereafter no significant change was observed. Most of the increase of total-creatinine could be accounted for by the increase of creatine. The activity of kidney transamidinase in the fasted rats decreased in the 3rd day and thereafter kept nearly constant. The transamidinase activity of rats fed on the 10% casein diet after giving a protein-free diet for 5 days increased in the 3rd day. An inverse relation was observed between the urinary creatine and the transamidinase activity. The urinary urea increased in the rats fasted or fed on the 10% casein diets with the supplement of glycine and/or arginine. In fasting, the activities of liver urea-cycle enzymes, except arginase, had a tendency of increasing with the lapse of time. The arginase activity remained more or less constant. The reason of the extreme increase of urinary creatine during starvation was discussed.     

Intrauterine Malnutrition and the Brain: Effects on Enzymes and Free Amino Acids Related to Glutamate Metabolism

Abstract: The effect of feeding pregnant rats with wheat and Bengal gram (black chick pea) diets during the later part of pregnancy on brain growth, enzymes, and free amino acids of glutamate metabolism in 1-day-old rats was investigated. These diets did not induce growth dissociation, and the body and brain weights were equally affected. The concentrations of DNA, RNA, protein, and free α-amino nitrogen in brain decreased significantly and the activities of glutamine synthetase, glutamine transferase, glutaminase 1, glutaminase 11, and glutamate decarboxylase and the concentrations of free amino acids, glutamic acid, glutamine, alanine, and GABA were also decreased. The concentration of aspartic acid, however, was increased. Wheat and Bengal gram diets fortified with lysine and with methionine, cystine, and tryptophan respectively showed various beneficial effects on the changes observed in the brain. A 20% casein diet induced higher body and brain weights and better brain protein and free α-amino nitrogen concentrations than those observed on a 10% casein diet.     

Supplementation of orotic acid to the casein, but not to egg protein, soy protein, or wheat gluten diets, increases serum ornithine carbamoyltransferase activity

Effects of dietary supplementation of orotic acid to a diet containing the casein protein were compared with diets containing egg protein, soy protein, or wheat gluten on lipid levels in the liver and serum and activities of ornithine carbamoyltransferase (OCT) and alanine aminotransferase in the serum of rats. We found that supplementation of orotic acid to each diet increased the contents of the liver total lipids, triacylglycerol, and phospholipids compared with those not supplemented. The contents of liver total lipids, triacylglycerol, cholesterol, and phospholipids in rats fed the casein diet were significantly higher than those of rats fed the other three diets when orotic acid was supplemented. The levels of triacylglycerol, cholesterol, and phospholipids in the serum of rats fed the casein diet were markedly decreased by addition of orotic acid. The supplementation of orotic acid significantly increased the activities of both serum OCT and alanine aminotransferase in rats fed the casein diet, but not in rats fed the other diets. In conclusion, liver lipid accumulation induced by dietary orotic acid depends on the type of dietary protein. The enhancement of serum OCT activity may result from liver lipid accumulation in rats fed the casein diet supplemented with orotic acid, demonstrating hepatic damage.     

EFFECTS OF DIETARY PROTEINS ON FETAL BRAIN PROTEIN AND GLUTAMIC ACID METABOLISM IN RAT

Abstract— The effects of feeding dietary wheat and Bengal gram proteins to pregnant rats on brain protein and glutamic acid metabolism in 15-, 17- and 19-day fetuses were investigated. Wheat and Bengal gram diets resulted in loss of brain weight with decreased DNA, RNA, protein, free x amino N and deficits in the activities of brain glutamine synthetase, glutaminase I. glutaminase II and glutamate decarboxylase at all the gestational ages studied without any change in glutamine transferase activity. The concentrations of the amino acids alanine, glutamic acid, glutamine and GABA were found to be significantly lower on wheat and Bengal gram diets than the control on a 10% casein diet. The wheat with lysine and Bengal gram with methionine, cystine and tryptophan resulted in similar mean values of all the characteristics studied to the mean values observed in rats on the control diet. However, glutaminase I activity remained significantly low on lysine fortified wheat diet, and aspartic acid content was found to increase on both fortified and unfortified wheat and Bengal gram diets. A 20% casein diet showed increased brain weight, DNA. RNA. protein and free x amino N concentrations as compared with the 10% casein diet, while the other parameters remained unchanged.     

Plasma arginine and ornithine levels and selected enzyme activities in uremic rats fed various amounts of arginine

Rats weighing 100 g were made chronically uremic by partial left renal artery ligation and contralateral nephrectomy. Rats with urea clearances below 0.30 ml/min and sham-operated controls were pair-fed arginine-free diets, diets containing normal amounts of arginine or diets with high levels of arginine. After 4 to 8 weeks, rats were killed and plasma levels of arginine, ornithine and lysine were measured. In addition, activities of various urea cycle enzymes in liver and kidney and renal transamidinase were determined. Plasma amino acid levels and enzyme activities of the urea cycle remained constant in control rats fed diets differing in arginine content. However, renal transamidinase activity was elevated in control rats fed arginine-free diets. In plasma of uremic as compared with control rats, arginine levels varied with the arginine intake, and lysine levels were elevated when arginine supplements were fed. With all diets, plasma ornithine remained constant in uremic rats at slightly but not significantly increased levels. Hepatic carbamoyl phosphate synthetase activity and renal arginine synthetase activity were reduced in uremic as compared to control rats. Renal transamidinase activity, expressed per g of kidney, was elevated in uremic rats with all diets except arginine-free. When amino acid diets were fed, hepatic arginase activity was higher in uremic rats and this increase was enhanced by arginine-free diets. Other enzyme activities in uremic rats were not affected by the amount of arginine in the diet.     

Rats fed prolonged high protein diets show an increase in nitrogen metabolism and liver megamitochondria

Rats were fed diets containing 20, 50 and 80% protein for 14 months. The urea excreted by the rats fed diets containing 50 and 80% protein when compared to rats fed diets containing 20% protein increased ca. 2- and 3-fold, respectively, in ca. 2 days; this increase was maintained essentially unchanged through the experimental period. The serum levels of urea increased 2.5- and 4-fold, respectively, in the first days and were also maintained during the experiment. Glutamate dehydrogenase activity of liver remained unchanged. The five urea cycle enzymes increased with respect to the control values. Orotic acid excretion increased as well as orotidylate decarboxylase and orotate phosphoribosyltransferase, but aspartate transcarbamylase did not. The key amino acids involved in the urea and pyrimidine pathways in liver were also measured; aspartic and glutamic acids and citrulline were increased, and ornithine and arginine did not change with the higher protein intake. In general, no differences were observed between animals fed 50 and 80% protein in their diets. Protein synthesis did not increase with the increase of protein content of the diet. Stereological analysis of ultrathin sections showed that the high protein diet induced a significant increment in the volumetric density, numerical density and size of hepatocyte mitochondria. Moreover, the presence of giant mitochondria, a hundred times larger than normal, was also observed in some periportal hepatocytes of rats fed the 80% protein diet.     

Comparative utilization of inorganic and organic compounds as sole nitrogen sources by the submergent duckweed,Lemna trisulca L.

The capability of the submergentLemna trisulca L. to utilize various inorganic and organic sources of nitrogen was studied using both non-axenic and axenic cultures. When doubling time for frond production was measured, the nitrogen sources in order of effectiveness were urea, aspartic acid, nitrate, glutamic acid, arginine, ammonium and casein hydrolysate. Nitrite supported a relatively rapid growth rate after an initial lag of 7 days. Other parameters of growth such as fresh or dry weight per frond or ohlorophyll content did not oorrelate well with rate of frond production. Casein hydrolysate and urea were found to elicit a morphology different from that seen in cultures containing the other nitrogen sources. These preferences for source of nitrogen were different than those known for the emergent species ofLemnaceae. The unique value ofL. trisulca as a subject for plant physiological research is discussed. This study also provides a possible explanation for the existence of nutritional niches existing in aquatio ecosystems containing several different species ofLemnaceae.     

Hormone mediated changes of brain glutamic acid system in amino acid imbalance

The effects of adrenal cortical hormone and thyroxine on brain glutamic acid, gamma-amino butyric acid (GABA) and glutamine were studied in rats fed on the amino acid imbalanced diet (8% casein diet supplemented with 0.3% L-threonine). The studies revealed that the decrease in brain glutamic acid and GABA levels in threonine imbalance was recovered by hydrocortisone supplementation. The increased level of brain glutamine in threonine imbalance could not, however, be reversed by hydrocortisone supplementation. Thyroxine supplementation was found to have no impact on any of the members of glutamic acid family in the brain of rats receiving the threonine-imbalanced diet. It was suggested that the decreased levels of brain glutamic acid and GABA in threonine imbalance were caused by diminished adrenal cortical function and the influence of adrenal cortical hormone could be suggested to reside at the level of formation of both glutamic acid and GABA.     

Bitterness Reduction of Citrus Fruits by β-Cyclodextrin

The levels of free amino acid, ammonia nitrogen and guanidino compounds were examined in renal failure rats induced by adenine. Among the essential amino acids in the serum, the marked reduction of lysine, valine, leucine and isoleucine was confirmed in the adenine-fed group as compared with the control group. Tyrosine and ornithine were also significantly reduced in the adenine-fed rats, while glycine, arginine and aspartic acid were significantly elevated. The urinary excretion of leucine, isoleucine and non-essential amino acids (glutamic acid, histidine, aspartic acid, citrulline, tyrosine, ornithine) was found to be high. On the other hand, adenine administered orally caused hyperammonemia. Furthermore, the results of the present study show that intake of adenine increased extraordinarily the level of guanidinosuccinic acid and methylguanidine in the serum, while the value of serum guanidinosuccinic acid and methylguanidine in rats fed on a control diet was not detectable.     

Effect of Individual Essential Amino Acid-Devoid Diets on the Activities of Liver Urea Cycle Enzymes in Rats

The activities of all urea cycle enzymes (carbamyl phosphate synthetase, ornithine trans- carbamylase, argininosuccinate synthetase, argininosuccinase and arginase) have been determined in the liver of rats forcibly fed diets lacking in individual essential amino acids from amino acid mixture simulating to a casein. In general, these enzyme activities (units/g liver and total units/body wt) in rats fed the single essential amino acid-devoid diet decreased as compared with those activities in animals fed complete diet, but their decreases were not as large as those observed in group of all amino acid-devoid diet. The degree of decrease in these enzyme activities differed somewhat from each other in individual enzymes and each essential amino acie-devoid groups. In contrast, in rats fed the arginine devoid diet, the activities (total units/body wt) of all enzymes expect the case of arginase increased more than those in the group of complete diet.     

Variations in the amino acid composition of cyanophycin in the cyanobacterium Synechocystis sp. PCC 6308 as a function of growth conditions

Gas chromatography-mass spectrometry studies of the nitrogen isotopic composition of the N-trifluoroacetyl n-butyl ester derivatives of the amino acids from isolated hydrolyzed cyanophycin from ¹⁵N-enriched cells led to two major findings: (1) the amino acid composition of this granular polypeptide, isolated using procedures optimized for extracting and purifying cyanophycin from cells in the stationary growth phase, varied with the culture growth condition; (2) the rate of incorporation of exogenous nitrate differed for each nitrogen atom of the amino acid constituents of cyanophycin or cyanophycin-like polypeptide. Arginine and aspartic acid were the principle components of cyanophycin isolated from exponentially growing cells and from light-limited stationary phase cells, with glutamic acid as an additional minor component. The cyanophycin-like polypeptide from nitrogen-limited cells contained only aspartic and glutamic acids, but no arginine. The glutamic acid content decreased and arginine content increased as nitrate was provided to nitrogen-limited cells. These cells rapidly incorporated nitrate at different rates at each cyanophycin nitrogen site: guanidino nitrogens of arginine>aspartic acid >-amino nitrogen of arginine>glutamic acid. Little media-derived nitrogen was incorporated into cyanophycin of exponentially growing cells during one cellular doubling time.Abbreviations asp-TAB, glu-TAB, arg-TAB N-Trifluoroacetyl n-butyl ester derivatives of aspartic acid, glutamic acid and arginine, respectively - CAP chloramphenicol - CF correction factor - TFAA Trifluoroacetic anhydride - MBTFA N-Methyl-bis-trifluoroacetamide     

Nitrogen source and mineral optimization enhance d-xylose conversion to ethanol by the yeast Pichia stipitis NRRL Y-7124

Nutrition-based strategies to optimize xylose to ethanol conversion by Pichia stipitis were identified in growing and stationary-phase cultures provided with a defined medium varied in nitrogen, vitamin, purine/pyrimidine, and mineral content via full or partial factorial designs. It is surprising to note that stationary-phase cultures were unable to ferment xylose (or glucose) to ethanol without the addition of a nitrogen source, such as amino acids. Ethanol accumulation increased with arginine, alanine, aspartic acid, glutamic acid, glycine, histidine, leucine, and tyrosine, but declined with isoleucine. Ethanol production from 150 g/l xylose was maximized (61±9 g/l) by providing C:N in the vicinity of ∼57–126:1 and optimizing the combination of urea and amino acids to supply 40–80 % nitrogen from urea and 60–20 % from amino acids (casamino acids supplemented with tryptophan and cysteine). When either urea or amino acids were used as sole nitrogen source, ethanol accumulation dropped to 11 or 24 g/l, respectively, from the maximum of 46 g/l for the optimal nitrogen combination. The interaction of minerals with amino acids and/or urea was key to optimizing ethanol production by cells in both growing and stationary-phase cultures. In nongrowing cultures supplied with nitrogen as amino acids, ethanol concentration increased from 24 to 54 g/l with the addition of an optimized mineral supplement of Fe, Mn, Mg, Ca, Zn, and others.The mention of trade names or commercial products in this article is solely for the purpose of providing specific information and does not imply recommendation or endorsement by the U.S. Department of Agriculture.     

54Mn absorption and excretion in rats fed soy protein and casein diets

Rats were fed diets containing either soy protein or casein and different levels of manganese, methionine, phytic acid, or arginine for 7 days and then fed test meals labeled with 2 microCi of 54Mn after an overnight fast. Retention of 54Mn in each rat was measured every other day for 21 days using a whole-body counter. Liver manganese was higher (P less than 0.0001) in soy protein-fed rats (8.8 micrograms/g) than in casein-fed rats (5.2 micrograms/g); manganese superoxide dismutase activity also was higher in soy protein-fed rats than in casein-fed rats (P less than 0.01). There was a significant interaction between manganese and protein which affected manganese absorption and biologic half-life of 54Mn. In a second experiment, rats fed soy protein-test meals retained more 54Mn (P less than 0.001) than casein-fed rats. Liver manganese (8.3 micrograms/g) in the soy protein group was also higher than that (5.7 micrograms/g) in the casein group (P less than 0.0001), but manganese superoxide dismutase activity was unaffected by protein. Supplementation with methionine increased 54Mn retention from both soy and casein diets (P less than 0.06); activity of manganese superoxide dismutase increased (P less than 0.05) but liver manganese did not change. The addition of arginine to casein diets had little effect on manganese bioavailability. Phytic acid affected neither manganese absorption nor biologic half-life in two experiments, but it depressed liver manganese in one experiment. These results suggest that neither arginine nor phytic acid was the component in soy protein which made manganese more available from soy protein diets than casein diets.     

Effects of keratinase supplementation of corn-soybean meal based diets on apparent ileal amino acid digestibility in growing pigs and serum amino acids, cytokines, immunoglobulin levels and loin muscle area in nursery pigs

Two experiments were conducted to evaluate effects of keratinase for growing and nursery pigs. In Exp. 1, six pigs (32.3 +/- 2.8 kg body weight), fitted with a simple T-cannula at the distal ileum, were assigned to one of two 3 x 3 Latin squares involving three periods and three diets including a basal diet and the same diets supplemented with 0, 0.05 or 0.1% keratinase. Dietary keratinase supplementation increased the apparent ileal digestibility of crude protein (CP), arginine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, threonine, tryptophan, alanine, glutamic acid and proline (p < 0.05). Digestibility coefficients did not differ between pigs fed 0.05 and 0.1% keratinase. In Exp. 2, 24 piglets weaned at 30 +/- 2 d of age were used in a 2 x 2 factorial design experiment with two CP concentrations (19 vs. 22%) and two levels of keratinase supplementation (0 vs. 0.05%). Keratinase supplementation increased (p < 0.05) average daily gain, serum arginine concentration and loin muscle area but decreased (p < 0.05) serum interleukin-10 concentrations. The reduction in dietary CP level decreased (p < 0.05) serum urea nitrogen concentrations, isoleucine, serine and proline concentrations, but increased serum arginine concentrations. Few interactions between keratinase supplementation and dietary CP concentration were observed. This study indicated that dietary keratinase supplementation improved apparent ileal amino acid digestibility for growing pigs and had a positive effect on weight gain, immune response and loin muscle area for nursery pigs.     

Effect of urea and amino acids of the ornithine cycle on the biomass accumulation and amino acids synthesis by Candida guilliermondii]

When assimilating urea, arginine, ornithine and citrulline as the sole source of nitrogen, C. guilliermondii shows a higher economic coefficient of biomass accumulation (54.2, 59.7, 40.6% respectively) as compared with ammonium sulphate whose coefficient is 35.6%. Nitrogen sources exert a significant influence on the content of essential amino acids in the alcohol soluble fraction of cell biomass. For instance, urea and arginine are responsible for the accumulation of ornithine (220 and 480 mug/100 mg abs. dry weight), arginine (470 and 587 mug), aspartic acid (220 mug), glutamic acid (520 and 444 mug), alanine (460 and 500 mug), whereas ammonium sulphate provides an accumulation of serine--52 mug, glycine--57 mug, gamma-aminobutyric acid--480 mug, phenyl alanine--96 mug and leucine--96 mug.     

Analysis of regulatory factors for urea synthesis by isolated perfused rat liver. II. Comparison of urea synthesis in livers of rats subjected to different dietary conditions.

Capacities for urea synthesis and amino acid patterns in the perfused livers isolated from rats fed low and high-protein diets were compared. Urea formation with amjonium chlorode as the nitrogen source in perfused livers isolated from rats fed on a 70% casein diet was rapid and the efficiency of conversion of ammonia to urea was 97.9%. However, that in livers isolated from rats fed on a 5% casein diet was much slower and the efficiency of conversion of ammonia to urea was only 36.1%. The ratios of the rate of urea formation from ammonium chloride to activity of ornithine transcarbamylase [EC 2.1.3.3.] in the perfused livers of rats fed on 5 and 70% casein diets were calculated. The ratio of the former condition was much lower than that of the latter. The ratios reached nearly the same level by the addition of ornithine and N-acetylglutamate, the addition of which to the perfusate caused marked elevation of the ratios in both cases. In the perfused livers from rats fed on a 5% casein diet a considerable portion of the ammonia added to the perfusate was fixed into an amino ro an amide group of amino acids such as alamin, aspartate, and glutamine. On the other hand, in the perfused livers from rats fed on a 70% casein diet most of the ammonia added was converted to urea. The regulation of urea synthesis and the relation between anabolism and catabolism of amino acids in rat livers subjected to different dietary conditions were compared.     

Elevation of cerebral levels of nonessential amino acids in vivo by administration of large doses

Taurine, aspartic acid, glutamic acid, glycine, and GABA were administered either intragastrically or in liquid diets to mice and rats. This resulted in a great increase in the plasma concentration of the administered amino acid, with plasma levels remaining elevated for several days.The prolonged increase in plasma levels resulted in significant increases in brain levels. Under these experimental conditions, taurine, aspartic acid, and glutamic acid were increased 30–60%; glycine and GABA 100%. During these experiments, plasma levels of taurine, aspartate, and glutamate were below brain levels; those of glycine and GABA were above.The findings show that even slowly penetrating amino acid levels can be increased in brain after parenteral administration of large doses.     

Effects of soy protein and isoflavone on hepatic fatty acid synthesis and oxidation and mRNA expression of uncoupling proteins and peroxisome proliferator-activated receptor gamma in adipose tissues of rats

Soy protein rich in isoflavones profoundly affects lipid metabolism in experimental animals. To distinguish the roles of the protein and isoflavone components of a soy protein preparation in regulating lipid metabolism, we compared the effects of diets containing methanol-washed soy protein low in isoflavone supplemented with a 0-, 0.5- and 4-g/kg isoflavone preparation on hepatic fatty acid metabolism and adipose tissue gene expression in rats. Diets containing soy protein irrespective of the isoflavone levels decreased the activities and mRNA expression of enzymes involved in hepatic fatty acid synthesis to similar levels. Methanol-washed soy protein compared to casein increased the mRNA expression of peroxisome proliferator-activated receptor (PPAR) alpha, and supplementing the soy protein diet with isoflavone further increased this parameter dose-dependently. However, methanol-washed soy protein compared to casein was totally ineffective in altering the activities and mRNA levels of enzymes involved in fatty acid oxidation. Supplementation of soy protein diets with isoflavone slightly increased these parameters. The mRNA level of uncoupling protein (UCP) 1 in brown adipose tissue was significantly increased and mRNA levels of UCP2 and 3, and PPARgamma2 tended to be higher in rats fed methanol-washed soy protein not supplemented with isoflavone than in the animals fed casein. Adding isoflavone to the soy protein diets dose-dependently increased these parameters. These results suggested that the protein rather than isoflavone component is primarily responsible for the physiological activity of soy protein rich in isoflavones in reducing hepatic lipogenesis. However, isoflavones may have a role in regulating heptic fatty acid oxidation and adipose tissue gene expression.     

Effects of Arginine and Methionine on the Growth Depression of Rats Fed Diets High in Glycine

In order to determine if the growth retardation by dietary exceess glycine could be prevented by the addition of arginine and/or methionine, weanling rats were fed a 25% casein diet (standard) or a 10% casein diet (low protein diet) with a supplement of several combinations of glycine, arginine, or methionine.

The changes in body weight, urinary creatinine, and kidney transamidinase activity were determined. The growth depression effect by excess glycine was prevented considerably in animals receiving standard diet and completely in animals receiving low protein diet by the addition of arginine and methionine to the high glycine diets.

The total urinary creatinine was increased by the supplement of both glycine and arginine, while the growth rate was not invariably raised and kidney transamidinase activity had a tendency to decrease.     

Nitrogen Metabolism of Lemna minor. I. Growth, Nitrogen Sources and Amino Acid Inhibition

Lemna minor grown in sterile culture on a minerals-sucrose medium can utilize as nitrogen source, in order of increasing growth rate: ammonia, nitrate, a mixture of glutamic and aspartic acids plus arginine, or a balanced mixture of amino acids (hydrolyzed casein). Maximum growth is found with nitrate plus hydrolyzed casein.Many synthetic mixtures of amino acids are unable to support growth. Many single amino acids are inhibitory, and when added (at 2 mm or less) to cultures, growing in the presence of nitrate, cause a decrease in growth rate or even death of the plants (e.g. with alanine, valine, methionine or leucine). Some of these inhibitory effects are also found when the amino acid is added to cultures growing on ammonia or hydrolyzed casein. Arginine was the only amino acid of those tested which gave a marked stimulation of growth when added to cultures growing with inorganic nitrogen.The rapid rate of growth, sterile nature of tissue, decreased biological variation of samples containing many plants and ability to utilize different culture media make this an attractive organism for studies on higher plant metabolism.