The necessity of niacin in rats fed on a high protein diet

It is known that niacin itself is not necessary in rats when tryptophan is given in adequate amounts, because rats can biosynthesize niacin from tryptophan. In our experiment, young rats were fed on a 20%, 40%, 60%, or 70% casein diet with or without niacin. The rats fed on the 20%, 40%, and 60% casein diets did not require niacin for growth, but the rats fed on the 70% casein diet needed it. This phenomenon was attributed to the supposition that liver aminocarboxymuconate-semialdehyde decarboxylase activities increased according with the dietary casein levels. The conversion ratio of tryptophan-niacin in rats fed on the 70% casein diet became extremely low, and then the rats needed niacin.     

A Simple Bioassay for Chemicals Active to the Photosynthetic or Respiratory Systems of Plants

After male rats of the Sprague Dawley strain, 5 weeks old, were fed a 20% casein diet with or without 0.5% nicotinamide for 13 days, 180 mg/kg body weight of alloxan was injected in- traperitoneally into the rats. The rats were kept for 18 days with the same diet. The level of blood glucose was increased 6-fold in the group on a 20% casein diet by the injection of alloxan, while there was only a 2-foid increase in the group on a nicotinamide-containing diet and the decreased body weight was also lower in the group on the nicotinamide diet than the group on the casein diet. The body weight was indirectly related to the concentration of blood glucose. A marked increase was observed in the activities of tryptophan oxygenase, aminocarboxymuconate-semialdehyde decarboxylase, and nicotinamide methyltransferase upon the injection of alloxan with both diets; on the other hand, the activities of kynureninase and NAD⁺ synthetase were decreased by the injection of alloxan. The activity of kynurenine aminotransferase increased in the group on the 20% casein diet by the injection of alloxan, while in the group on the nicotinamide-containing diet its activity was not increased by the injection. These changes in the above enzyme activities mean that the conversion ratio from tryptophan to niacin is lower in the alloxan diabetic rat than normal rat. It was found that the activities of tryptophan oxygenase, aminocarboxymuconate-semialdehyde decarboxylase, and nicotinamide methyltransferase were directly related to the concentration of blood glucose, and that the activities of kynureninase and NAD⁺ synthetase were inversely related. There was no difference in the activities of 3-hydroxyanthranilic acid oxygenase and nicotinamide mononucleotide adenylyltransferase upon the injection of alloxan with both diets.     

Effects of fatty liver induced by niacin-free diet with orotic acid on the metabolism of tryptophan to niacin in rats

The effects of dietary orotic acid on the metabolism of tryptophan to niacin in weaning rats was investigated. The rats were fed with a niacin-free, 20% casein diet containing 0% (control diet) or 1% orotic acid diet (test diet) for 29 d. Retardation of growth, development of fatty liver, and enlargement of liver were observed in the test group in comparison with the control group. The concentrations of NAD and NADP in liver significantly decreased, while these in blood did not decrease compared to the control group. The formation of the upper metabolites of tryptophan to niacin such as anthranilic acid, kynurenic acid, and 3-hydroxyanthranilic acid were not affected, but the quinolinic acid and beyond, such as nicotinamide, N1-methylnicotinamide, N1-methyl-2-pyridone-5-carboxamide, and N1-methyl-4-pyridone-3-carboxamide, were significantly reduced by the administration of orotic acid. Therefore, the conversion ratio of tryptophan to niacin significantly decreased in the test group in comparison with the control group.     

Effect of Adding Branched-chain Amino Acids to a Nicotinic Acid-free,Low-protein Diet on the Conversion Ratio of Tryptophan to Nicotinamide in Rats

The effects of adding branched-chain amino acids to a nicotinic acid-free, low-protein diet on the conversion of tryptophan to nicotinamide were investigated in rats. The conversion ratio [urinary excretion of nicotinamide and its metabolites (μmol/day) × 100/tryptophan intake during urine collection (μmol/day)] was significantly lower in the groups fed with the 3% Leu-, Val-, or Ile-added diet than in the group fed with the control diet. Namely, the inhibition of this conversion was observed not only by the addition of Leu, but also by the addition of Val or Ile. The addition of Ile and/or Val to the Leu-added diet did not antagonize the Leu effect.     

Comparison of the effects of di(2-ethylhexyl)phthalate, a peroxisome proliferator, on the vitamin metabolism involved in the energy formation in rats fed with a casein or gluten diet

In order to find an alleviation method for the adverse effect of environmental endocrine disrupters, we studied the effects of the putative endocrine disrupter and peroxisome proliferator, di(2-ethylhexyl)phthalate (DEHP), on animal growth and vitamin metabolism. It is known that the effects of chemical compounds such as xenobiotics differ according to the dietary protein source. We compared the effects of dietary DEHP administration on rats fed with a diet containing milk casein or wheat gluten. The increased conversion ratio of tryptophan to nicotinamide by DEHP administration was significantly higher in the casein group than in the gluten group. We also investigated the effects of DEHP on the urinary excretion of other vitamins. DEHP administration resulted in decreased urinary excretion of vitamin B(1), vitamin B(2), and pantothenic acid.     

Identification of Arsenobetaine,a Water Soluble Organo-arsenic Compound in Muscle and Liver of a Shark,Prionace glaucus

After male rats of the Sprague Dawley strain, 5 weeks old, were fed a 20% casein diet for 12 days, 70 mg of streptozotocin/kg body weight (STZ group) or 70 mg of streptozotocin and 500 mg of nicotinamide/kg body weight (STZ-Nam group) was injected intraperitoneally into the rats. The rats were kept for 21 more days on the 20% casein diet and killed by decapitation. Urine was collected for the last 2 days. The level of blood glucose was 2-fold higher in the STZ group than in the STZ-Nam group. Urinary excretion of large amounts of glucose was observed only in the STZ group. Extremely reduction of urinary excretion of nicotinamide was observed in the STZ group, but, urinary excretion of N¹-methylnicotinamide (MNA) and N-¹-methyl-2-pyridone-5-carboxamide (2-py) was about the same in the two groups and that of N¹-methyl-4-pyridone-3-carboxamide (4-py) was higher in the STZ group than in the STZ-Nam group. The sum of urinary excretion of nicotinamide, MNA, 2-py, and 4-py was higher in the STZ group than in the STZ-Nam group. The levels of NAD in liver, pancreas, and blood in the STZ group tended to be higher, or rather not to decrease compared to the STZ-Nam group. For enzyme activities concerned with the tryptophan-NAD metabolism, a marked increase was observed in the activities of aminocarboxymuconate-semialdehyde decarboxylase, 3-hydroxyanthranilic acid oxygenase, and nicotinamide methyltransferase, on the other hand, the activity of NAD⁺ synthetase decreased in the STZ group compared to the STZ-Nam group. The activities of tryptophan oxygenase, kynureninase, NMN adenylyltransferase, and MNA oxidase were about the same in the two groups. These changes in the above enzyme activities mean that the conversion ratio from tryptophan to NAD is lower in the streptozotocin diabetic rats than normal rats, but the tryptophan metabolites such as NAD and 4-py were higher in the STZ group than in the STZ-Nam group. This might be due to the higher food intake and the lower body weight gain in the STZ group than in the STZ-Nam group. Similar phenomena have reported in alloxan diabetic rats.     

Lunatoic Acid A,a Morphogenic Substance Inducing Chlamydospore-like Cells in Some Fungi

The effect of the addition of 0.26 % free tryptophan (Trp) to a 20 % casein diet containing 6 mg of nicotinic acid per 100 g of diet on the ratio of N¹-methyl-2-pyridone-5-carboxamide (2-py) plus N¹-methyl-4-pyridone-3-carboxamide (4-py) to -methylnicotinamide (MNA) excretion was investigated in rats. The urinary excretion of MNA, 2-py and 4-py, respectively, increased statistically significantly with the feeding of a 0.26% Trp (the same as the content of the 20% casein diet) supplemented 20% casein diet, although it did not increase with the feeding of a 40% casein diet, compared with in the case of the 20 % casein diet [Agric. Biol. Chem., 52, 1765 (1988)]. So, the total urinary excretion of Nam and its metabolites was 1.8 times higher in the group fed the Trp supplemented diet than in the group fed the 20 % casein diet. However, the ratio of 2-py plus 4-py to MNA excretion was much lower in the group fed the Trp supplemented diet than in the group fed the 20 % casein diet (13.16 ± 3.75→5.49 ± 2.25). This decreased ratio was considered to be partially due to a decrease in the 4-py forming MNA oxidase, which decreased significantly with the feeding of the Trp supplemented diet. Furthermore, the metabolic fate of Trp was greatly affected by the form of Trp, free or bound.     

The urinary excretory ratio of nicotinamide catabolites was associated with the conversion ratio of tryptophan to nicotinamide in growing rats fed a niacin-free 20% casein diet

Weaning rats were fed a niacin-free 20% casein diet. Twenty-four-h-urine samples were collected, and nicotinamide and its catabolites were measured. A correlation was found between the urinary excretory ratio of nicotinamide catabolites (N(1)-methyl-2-pyridone-5-carboxamide + N(1)-methyl-4-pyridone-3-carboxamide)/N(1)-methylnicotinamide and the tryptophan-nicotinamide conversion ratio during growing period of the rats. This indicates the possibility that the conversion ratio can be deduced from the excretory ratio.     

Oleuropein supplementation increases urinary noradrenaline and testicular testosterone levels and decreases plasma corticosterone level in rats fed high-protein diet

The effects of oleuropein, a phenolic compound in extra virgin olive oil, on protein metabolism were investigated by measuring testicular testosterone and plasma corticosterone levels in rats fed diets with different protein levels. In Experiment 1, rats were fed experimental diets with different protein levels (40, 25 and 10 g/100 g casein) with or without 0.1 g/100 g oleuropein. After 28 days of feeding, the testosterone level in the testis was significantly higher and the plasma corticosterone level was significantly lower in rats fed the 40% casein diet with oleuropein than in those fed the same diet without oleuropein. The urinary noradrenaline level, nitrogen balance and hepatic arginase activity were significantly higher in rats fed the 40% casein diet with oleuropein supplementation than in those fed the 40% casein diet without oleuropein supplementation. In Experiment 2, the effects of oleuropein aglycone (a major phenolic compound in extra virgin olive oil and the absorbed form of oleuropein ingested in the gastrointestinal tracts) on the secretion of luteinizing hormone (LH) from the pituitary gland, which regulates testosterone production in the testis, were investigated in anesthetized rats. Plasma LH level increased dose dependently after the administration of oleuropein aglycone (P<.001, r= 0.691). These findings suggest that dietary supplementation with 0.1 g/100 g oleuropein alters the levels of hormones associated with protein anabolism by increasing urinary noradrenaline and testicular testosterone levels and decreasing plasma corticosterone level in rats fed a high-protein diet.     

The Urinary Excretory Ratio of Nicotinamide Catabolites Was Associated with the Conversion Ratio of Tryptophan to Nicotinamide in Growing Rats Fed a Niacin-Free 20% Casein Diet

Weaning rats were fed a niacin-free 20% casein diet. Twenty-four-h-urine samples were collected, and nicotinamide and its catabolites were measured. A correlation was found between the urinary excretory ratio of nicotinamide catabolites (N ¹-methyl-2-pyridone-5-carboxamide + N ¹-methyl-4-pyridone-3-carboxamide)/N ¹-methylnicotinamide and the tryptophan-nicotinamide conversion ratio during growing period of the rats. This indicates the possibility that the conversion ratio can be deduced from the excretory ratio.     

Effect of phytate in soy protein on the serum and liver cholesterol levels and liver fatty acid profile in rats

Dietary soy protein, in comparison with casein, generally lowers the serum cholesterol concentration in rats fed on a cholesterol-enriched diet, while mixed results were observed in rats fed on a diet free of cholesterol. Soy protein also suppresses the conversion of linoleic acid to arachidonic acid in the rat liver. The present study examines whether phytate, a minor component of a soy protein isolate, is responsible for these beneficial effects of soy protein. Weanling male rats were fed for 4 weeks on a purified diet containing a 20% level of protein (either casein (CAS), soy protein (SOY), phytate-depleted SOY (PDSOY) or phytate-replenished PDSOY (PRSOY)) and cholesterol (0 or 0.5%). The dietary protein source and phytate level only affected the serum and liver cholesterol concentrations when the animals were fed on the cholesterol-enriched diet, being significantly lower in those rats fed on the SOY and PRSOY diets than in those fed on the CAS diet, while the concentrations in the rats fed on the PDSOY diet were intermediate. When the animals were fed on the cholesterol-free diet, the ratio of (20:3n-6 + 20:4n-6)/18:2n-6 in liver phosphatidylcholine, a delta6 desaturation index, was significantly lower in the SOY diet group than in the CAS, PDSOY and PRSOY diet groups. Dietary cholesterol significantly depressed the ratio, but neither depletion nor replenishment of phytate affected the ratio. These results suggest that phytate in soy protein played a limited role in the cholesterol-lowering effect of soy protein and was not involved in the metabolism of linoleic acid.     

Growth-promoting activity of pyrazinoic acid,a putative active compound of antituberculosis drug pyrazinamide,in niacin-deficient rats through the inhibition of ACMSD activity

We have recently reported that the antituberculosis drug, pyrazinamide (PZA), caused a significant increase in the conversion ratio of tryptophan to niacin in rats. In the present work, we investigated whether or not pyrazinoic acid (POA), a putative metabolite of PZA, increased the conversion ratio of tryptophan to niacin. Weaning rats were fed with a niacin-free and tryptophan-limited diet (negative control diet), or with the negative control diet supplemented with 0.003% nicotinic acid (positive control diet) or 1% POA (test diet) for 27 days. The growth rate was almost same between the groups fed on the positive control diet and the test diet. Dietary POA significantly increased the conversion ratio of tryptophan to niacin. Although POA did not directly inhibit the activity of alpha-amino-beta-carboxymuconate-epsilon-semialdehyde decarboxylase (ACMSD), the rate-limiting enzyme in the tryptophan-niacin pathway, liver ACMSD activity was only not detected in the test diet group. These results suggest that a derivative of POA metabolized by rats inhibited the ACMSD activity.     

Elucidation of the toxic mechanism of the plasticizers,phthalic acid esters,putative endocrine disrupters: effects of dietary di(2-ethylhexyl)phthalate on the metabolism of tryptophan to niacin in rats

We have previously reported that the administration of a large amount of di(n-butyl)phthalate (DBP) increased the conversion ratio of tryptophan to niacin in rats. In the present experiment, the effect of di(2-ethylhexyl)phthalate (DEHP) on the conversion ratio and how altering the conversion ratio of tryptophan to niacin depended on the concentration of DEHP were investigated to elucidate the toxic mechanism of phthalic acid esters (PhE). Rats were fed with a diet containing 0%, 0.01%, 0.05%, 0.1%, 0.5%, 1.0%, or 3.0% DEHP for 21 days. To assess the conversion ratio of tryptophan to niacin, urine samples were collected at the last day of the experiment and measured for metabolites on the tryptophan-niacin pathway. The conversion ratio increased with increasing dietary concentration of DEHP above 0.05%; the conversion ratio was about 2% in the control group, whereas it was 28% in the 3.0% DEHP group. It is suggested that the inhibition of alpha-amino-beta-carboxymuconate-epsilon-semialdehyde decarboxylase (ACMSD) by DEHP or its metabolites caused this increase in the conversion ratio. We conclude that PhE such as DEHP and DBP disturbed the tryptophan-niacin metabolism.     

Effects of dietary di(2-ethylhexyl)phthalate, a putative endocrine disrupter, on enzyme activities involved in the metabolism of tryptophan to niacin in rats

We have reported that the conversion ratio of tryptophan to niacin increased with increasing dietary concentration of di(2-ethylhexyl)phthalate (DEHP); the conversion ratio was about 2.0% in the control rat, which increased by about 30% in the rat fed with 3.0% DEHP diet. In this study, we investigated whether this abnormal increase in the conversion ratio by DEHP occurred through the alteration of the enzyme activities involved in the metabolism of tryptophan to niacin. Rats were fed with a diet containing 0%, 0.1%, 0.5%, or 1.0% DEHP for 21 days. The nine kinds of enzyme activities involved in the biosynthesis and catabolism in the liver and kidney were measured. Based on previous findings that the formation of quinolinic acid and its' metabolites significantly increased with DEHP administration, we proposed that the activity of 2-amino-3-carboxymuconate-6-semialdehyde decarboxylase would be inhibited by DEHP intake. However, we found that the activities in the liver and kidney did not decrease in the rat fed with DEHP-containing diet. We discuss the discrepancy between the metabolite results and the enzyme activities.     

Effects of excess nicotinamide administration on the urinary excretion of nicotinamide N-oxide and nicotinuric acid by rats

We investigated a useful chemical index for an excessive nicotinamide intake and how this excessive nicotinamide intake affects the tryptophan-nicotinamide metabolism in rats. Weaning rats were fed on a tryptophan-limited and nicotinic acid-free diet containing no, 0.003%, 0.1%, 0.2%, or 0.3% nicotinamide for 21 days. Urine samples were collected on the last day and analyzed the intermediates and metabolites on the tryptophan-nicotinamide pathway. Nicotinamide N-oxide, nicotinic acid and nicotinuric acid, metabolites of nicotinamide, were detected when nicotinamide at more than 0.1% had been taken. An intake of nicotinamide of more than 0.1% increased the urinary excretion of quinolinic acid, an intermediate on the pathway. Nicotinamide N-oxide and nicotinuric acid increased with increasing dietary concentration of nicotinamide. These results show that the measurements of nicotinamide N-oxide and nicotinuric acid in urine would be useful indices for an excessive nicotinamide intake.     

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.     

Short-term, increasing dietary protein and fat moderately affect energy expenditure, substrate oxidation and uncoupling protein gene expression in rats

Macronutrient composition of diets can influence body-weight development and energy balance. We studied the short-term effects of high-protein (HP) and/or high-fat (HF) diets on energy expenditure (EE) and uncoupling protein (UCP1-3) gene expression. Adult male rats were fed ad libitum with diets containing different protein-fat ratios: adequate protein-normal fat (AP-NF): 20% casein, 5% fat; adequate protein-high fat (AP-HF): 20% casein, 17% fat; high protein-normal fat (HP-NF): 60% casein, 5% fat; high protein-high fat (HP-HF): 60% casein, 17% fat. Wheat starch was used for adjustment of energy content. After 4 days, overnight EE and oxygen consumption, as measured by indirect calorimetry, were higher and body-weight gain was lower in rats fed with HP diets as compared with rats fed diets with adequate protein content (P<.05). Exchanging carbohydrates by protein increased fat oxidation in HF diet fed groups. The UCP1 mRNA expression in brown adipose tissue was not significantly different in HP diet fed groups as compared with AP diet fed groups. Expression of different homologues of UCPs positively correlated with nighttime oxygen consumption and EE. Moreover, dietary protein and fat distinctly influenced liver UCP2 and skeletal muscle UCP3 mRNA expressions. These findings demonstrated that a 4-day ad libitum high dietary protein exposure influences energy balance in rats. A function of UCPs in energy balance and dissipating food energy was suggested. Future experiments are focused on the regulation of UCP gene expression by dietary protein, which could be important for body-weight management.     

Effect of dietary protein, alfalfa, and zeolite on excretory patterns of 5',5',7',7'-[3H]zearalenone in rats

A series of experiments was conducted to determine how dietary protein, alfalfa, or zeolite influence the excretory patterns of zearalenone (Z), a uterotropic mycotoxin synthesized by Fusarium fungi. Rats were fed diets containing 16.3% casein, 40% casein, 11.2% casein + 25% alfalfa, or 25% casein + 25% alfalfa. Also fed were diets containing 0, 1, 2, or 5% anion exchange zeolite. Tracer doses of [3H]Z were administered either as a constituent of the diet or as a topical application on the skin at the base of the skull. When Z was administered orally, no differences were seen in the fraction of the dose excreted in urine or feces as a result of varying dietary levels of alfalfa and protein. Topical doses resulted in rats fed 25% casein + 25% alfalfa or 40% casein excreting more Z in urine than those fed 25% alfalfa or 16.3% casein. Fecal excretion of Z was greatest for rats fed 25% casein + 25% alfalfa whereas rats fed 40% casein excreted more fecal Z than those fed 16.3% casein. Feeding Z to rats receiving dietary zeolite resulted in a positive correlation between dietary zeolite and fecal excretion of Z but a negative correlation with urinary excretion of Z. Topical administration of Z produced a positive correlation between dietary zeolite and fecal Z excretion but no effect on urinary excretion. It may be concluded that protein and alfalfa treatments alleviate Z toxicosis through increased metabolism whereas zeolite binds Z in the digestive tract to prevent absorption.     

Soy protein diet ameliorates renal nitrotyrosine formation and chronic nephropathy induced by puromycin aminonucleoside

It has been shown that reactive oxygen species are involved in chronic puromycin aminonucleoside (PAN) induced nephrotic syndrome (NS) and that a 20% soy protein diet reduces renal damage in this experimental model. The purpose of the present work was to investigate if a 20% soy protein diet is able to modulate kidney nitrotyrosine formation and the activity of renal antioxidant enzymes (catalase, glutathione peroxidase, Cu,Zn- or Mn-superoxide dismutase) which could explain, at least in part, the protective effect of the soy protein diet in rats with chronic NS induced by PAN. Four groups of rats were studied: (1) Control rats fed 20% casein diet, (2) Nephrotic rats fed 20% casein diet, (3) Control rats fed 20% soy protein diet, and (4) Nephrotic rats fed 20% soy protein diet. Chronic NS was induced by repeated injections of PAN and rats were sacrificed at week nine. The soy protein diet ameliorated proteinuria, hypercholesterolemia, and the increase in serum creatinine and blood urea nitrogen observed in nephrotic rats fed 20% casein diet. Kidney nitrotyrosine formation increased in nephrotic rats fed 20% casein diet and this increase was ameliorated in nephrotic rats fed 20% soy protein diet. However, the soy protein diet was unable to modulate the antioxidant enzymes activities in control and nephrotic rats fed 20% soy protein diet. Food intake was similar in the two diet groups. The protective effect of a 20% soy protein diet on renal damage in chronic nephropathy induced by PAN was associated with the amelioration in the renal nitrotyrosine formation but not with the modulation of antioxidant enzymes.