Cloning and sequencing of the gene encoding the mouse vitamin D receptor.

The mouse vitamin D receptor (VDR)-encoding cDNA was cloned and the coding regions were sequenced. Comparison of the amino-acid sequence to that of humans and rats revealed high homology in the DNA- and ligand-binding domains. Divergence appeared in the internal region between the two domains.

Expression of the putative membrane fatty acid transporter (FAT) in taste buds of the circumvallate papillae in rats.

The putative membrane fatty acid transporter (FAT) protein and its mRNA, originally expressed in adipose tissue, were found in the tongue of rats. Northern blot analysis showed a significant expression of FAT mRNA in the epithelial layer of circumvallate papillae. Immunohistochemical staining revealed that immunoreactivity for FAT is specifically localized in the apical part of taste bud cells, possibly gustatory cells, in the circumvallate papillae.

The orosensory recognition of long-chain fatty acids in rats.

To determine the selectivity of long-chain fatty acid (LCFA) in the oral cavity, short-term (5 min) two-bottle tests were conducted in rats. Fifteen male Wistar rats were given oleic acid, linoleic acid, linolenic acid, and their derivatives. All compounds used were 99% pure. The concentration of test fluids was made 1% in 0.3% xanthan gum to minimize postingestive and textural effects. The rats preferred LCFA fluids to the control of 0.3% xanthan gum solution. The preference order of LCFA was linolenic acid > linoleic acid > oleic acid. Four LCFA derivatives (methyl oleate, oleyl alcohol, methyl linoleate, and linolyl alcohol), triolein, and capric acid were not preferred to LCFA, but LCFA derivatives were preferred to the control of xanthan gum solution. These studies suggest that rats select LCFA from olfactory or gustatory cues that are related to both the carbon chain and carboxylate group.

Increased conversion ratio of tryptophan to niacin by dietary di-n-butylphthalate.

We reported that the growth promoting activity of di-n-butylphthalate (DBP) was observed when rats were fed with a niacin-free and tryptophan-limiting diet (Shibata et al.. 1982. J Nutr Sci Vitaminol 28, 173-177). The present experiment was performed to investigate whether this phenomenon is attributable to the increase in the conversion ratio of tryptophan to niacin. The weaning rats were fed with a 10% (low protein diet) or 20% protein (conventional protein diet) diet with or without adding 1% DBP. The conversion ratio of tryptophan to niacin was significantly higher in the DBP group than in the control group; for 10% casein diets, it increased two-fold and for 20% casein diet, about five-fold. From these results, the previous finding is possibly explained by DBP increasing the conversion ratio of tryptophan to niacin.

Elevation of blood NAD level after moderate exercise in young women and mice.

We previously reported that the blood NAD levels are decreased by severe exercise, and administration of nicotinamide, a precursor of NAD, improves the endurance capacity of mice. In the present study, we determined whether moderate exercise changes the blood NAD levels in humans and mice. College female students exercised moderately with bike-ergometers. The blood NAD levels elevated after moderate exercise. Mice were forced to swim in a running water pool for 5 min as a moderate exercise, 15 min as a strong exercise, and until exhaustion as a severe exercise (average swimming time was 28.7 min). A 5 min swim gave a result similar to that of moderate exercise by human subjects. However, the blood NAD levels decreased after all-out exercise. The changes in whole blood tryptophan (a precursor of pyridine nucleotides) levels were similar to that in NAD. The glucose levels in whole blood and the non-esterified fatty acid levels in serum decreased according to exercising time. These data are the first demonstration of moderate exercise raising the blood NAD levels in human and mice. Elevation of the blood NAD levels may reflect changes in niacin metabolism that occur in response to exercise.

[Effect of feeding with a poisonous mushroom Clitocybe acromelalga on the metabolism of tryptophan-niacin in rats].

The poisonous mushroom Clitocybe acromelalga contains clitidine, which resembles nicotinic acid mononucleotide, and 4-amino-pyridine-2,3-dicarboxylic acid, which resembles quinolinic acid. Both are important intermediates in the tryptophan-niacin pathway. Therefore, we investigated the effect of feeding a niacin-free and tryptophan-limited diet containing the toadstool Clitocybe acromelalga on the metabolism of tryptophan to niacin in rats. The toadstool diet was fed to the rats for only one day (this day was designated day 0). Urinary excretion of intermediates in the tryptophan-niacin pathway, such as anthranilic acid, kynurenic acid, xanthurenic acid, 3-hydroxyanthranilic acid, quinolinic acid, nicotinamide, N1-methylnicotinamide, N1-methyl-2-pyridone-5-carboxamide, and N1-methyl-4-pyridone-3-carboxamide, was higher in the toadstool group than in the control on day 0-day 1 and day 1-day 2. The blood levels of tryptophan and NAD on day 1 were also higher in the toadstool group. Accordingly, intake of Clitocybe acromelalga appeared to increase the conversion of tryptophan to niacin.

[Establishment of animal model for elucidating the mechanism of intoxication by the poisonous mushroom Clitocybe acromelalga].

Dietary intake of a poisonous mushroom, Clitocybe acromelalga, causes acromelalgia. The symptom continues for over a month. Some papers reported that treatment with nicotinic acid is effective. We have established an animal model to elucidate the mechanism of toxicity of the poisonous mushroom Clitocybe acromelalga. Diet containing Clitocybe acromelalga was fed to niacin-deficient rats for 24 hours (designated as day 0). The food intake decreased to about one-half compared with that of day before, and body weight loss was noted. Although the diet was returned to the control diet on day 1, the food intake did not recover until day 7, and body weight gain was not seen until day 6. A severe symptom resembling acromelalgia in humans started to appear on day 3. This is the first report of an animal model for the intoxication of Clitocybe acromelalga in humans. Since no similar symptom resembling human intoxication was seen in a previous rodent study, the niacin-free/tryptophan-limited diet used in the present study may have contributed to the result.

Twenty-four-hour urinary water-soluble vitamin levels correlate with their intakes in free-living Japanese university students.

BACKGROUND/OBJECTIVES: We examined the association between 24-h urinary excretion of water-soluble vitamin levels and their intakes in free-living Japanese university students. The design used was cross-sectional study. SUBJECTS/METHODS: A total of 216 healthy, free-living male and female Japanese university students aged 18-27 years voluntarily participated in this study, of which 156 students were eligible for this assessment. All foods consumed for 4 consecutive days were recorded accurately by a weighed food record method. A 24-h urine sample was collected on the fourth day, and the urinary levels of water-soluble vitamins were measured. RESULTS: Each urinary water-soluble vitamin level, except for vitamin B(12), was correlated positively with its mean intake in the recent 2-4 days (vitamin B(1): r=0.42, P<0.001; vitamin B(2): r=0.43, P<0.001; vitamin B(6): r=0.40, P<0.001; vitamin B(12): r=0.06, P=0.493; niacin: r=0.35, P<0.001; niacin equivalents: r=0.33, P<0.001; pantothenic acid: r=0.47, P<0.001; folate: r=0.27, P=0.001; vitamin C: r=0.44, P<0.001). Mean estimated water-soluble vitamin intakes calculated from urinary levels and recovery rates showed 91-101% of their 3-day mean intakes, except for vitamin B(12) (61%). CONCLUSIONS: These results showed that urinary water-soluble vitamin levels, except for vitamin B(12), reflect their recent intakes in free-living Japanese university students, and could be used as a potential biomarker to estimate mean vitamin intake.

Reversed-phase high-performance liquid chromatography of nicotinic acid mononucleotide for measurement of quinolinate phosphoribosyltransferase.

A system has been developed for the determination of quinolinate phosphoribosyltransferase (QPRT) activity in liver and kidney homogenates using HPLC. A product, nicotinic acid mononucleotide (NaMN), is separated by reversed-phase chromatography (a Tosoh ODS 80TS was used as an analytical column) using a mixture of 10 mM KH2PO4-K2HPO4 buffer (pH 7.0) containing 1.48 g/l tetra-n-butylammonium bromide-acetonitrile (9:1, v/v) as a mobile phase. The flow-rate was 1.0 ml/min, the detection wavelength was 265 nm. The column temperature was maintained at 40 degrees C. Under these conditions, NaMN was eluted at about 8.1 min. Sample preparation was very straightforward. The reaction mixture of QPRT assay was stopped by immersing the tube into a boiling water bath, the resulting supernatant was filtered, and the filtrate was directly injected into a HPLC system. The total HPLC analysis time was approximately 20 min.

Effects of dietary pyrazinamide, an antituberculosis agent, on the metabolism of tryptophan to niacin and of tryptophan to serotonin in rats.

The effects of pyrazinamide on the metabolism of tryptophan to niacin and of tryptophan to serotonin were investigated to elucidate the mechanism for pyrazinamide action against tuberculosis. Weanling rats were fed with a diet with or without 0.25% pyrazinamide for 61 days. Urine samples were periodically collected for measuring the tryptophan metabolites. The administration of pyrazinamide significantly increased the metabolites, 3-hydroxyanthranilic acid and beyond, especially quinolinic acid, nicotinamide, N'-methylnicotinamide, and N1-methyl-4-pyridone-3-carboxamide, and therefore significantly increased the conversion ratio of tryptophan to niacin and the blood NAD level . However, no difference in the upper metabolites of the tryptophan to niacin pathway such as anthranilic acid, kynurenic acid and xanthurenic acid was apparent between the two groups. No difference in the concentrations of trytptophan and serotonin in the blood were apparent either. It is suggested from these results that the action of pyrazinamide against tuberculosis is linked to the increase in turnover of NAD and to the increased content of NAD in the host cells.

Efficiency of D-tryptophan as Niacin in rats.

L-tryptophan is a very important precursor of niacin in mammals. Food preparation in which proteins are exposed to an alkali and/or high temperature for a long period generate appreciable amounts of D-amino acids from racemization. The efficiency of D-tryptophan as niacin was thus investigated by using weanling rats. The availability of D-tryptophan was almost the same as that in L-tryptophan as the precursor of niacin and was 1/6 as active as niacin.

Effect of dietary histidine content on the change in content of skin urocanic acid isomers in hairless mice irradiated with ultraviolet B.

Hairless mice were fed with a 10% amino acid mixture diet (control diet, 0.42% histidine content), the control diet without histidine (histidine-free diet), or the control diet rich in histidine (histidine-rich diet; histidine content, 4.2%) for 32 days. They were irradiated with UV light of 312 nm for 30 min, and skin samples were periodically taken for measuring the urocanic acid isomers. Total urocanic acid isomers were decreased by UV irradiation in all the three groups, the recovery being the fastest in the histidine-rich group. The percentage increase in cis-urocanic acid/total urocanic acid was quickly increased by UVB irradiation. The recovery of the ratio was slightly higher in the histidine-rich group, although the total urocanic acid level was higher in the histidine-rich group than in the others. Therefore, the absolute cis-urocanic acid content in the skin was almost the same among the three groups. These results indicate that the increased histidine intake strengthened UVB protection without any decrease in immune suppression.

Synthesis and characterization of the sweetness-suppressing polypeptide gurmarin and ent-gurmarin.

The sweetness-suppressing polypeptide gurmarin isolated from the leaves of Gymnema sylvestre consists of 35 amino acid residues including three intramolecular disulfide bonds. Herein, the total chemical synthesis of gurmarin was performed by the stepwise fluoren-9-ylmethoxy-carbonyl solid-phase method, the yield of reduced gurmarin being 1.9% based on the starting amino acid resin. Disulfide formation was carried out in the presence of a redox system of reduced glutathione/oxidized glutathione to give gurmarin in a yield of 35.5%. The product was identical to natural gurmarin by analytical reverse phase high performance liquid chromatography (RP-HPLC), mass spectroscopy (MS), and peptide mapping, and suppressed the responses to sucrose, D-glucose, and L-glucose in a rat. The D enantiomer (all D-amino acid gurmarin) was also synthesized, and was shown to be the mirror image of gurmarin. Interestingly, the D enantiomer (ent-gurmarin) also suppressed the responses to sucrose, D-glucose, and L-glucose in a rat.

Changes of pyridine nucleotide levels during adipocyte differentiation of mouse 3T3-L1 cells.

The levels of NAD and NADP were measured in 3T3-L1 cells during a differentiation from preadipocytes to adipocytes. The cells were grown in the ordinary medium and differentiated in the medium by adding dexamethasone, 1-methyl-3-isobutylxanthine, and insulin for 2 days, and then they were grown in the medium by adding only insulin for another 8 days to accumulate fat. The levels of cellular NAD and NADP increased abruptly with days after differentiation, and the levels of NAD and NADP reached maximum at day 7, and at day 10 the values were decreased compared with the maximum values. These results suggest that expression of the pyridine nucleotide biosynthesis genes is induced in the differentiation process.

Influence of adenine-induced renal failure on tryptophan-niacin metabolism in rats.

To discover the role of the kidney in tryptophan degradation, especially tryptophan to niacin, rat kidneys were injured by feeding a diet containing a large amount of adenine. The kidney contains very high activity of aminocarboxymuconate-semialdehyde decarboxylase (ACMSD), which leads tryptophan into the glutaric acid pathway and then the TCA cycle, but not to the niacin pathway. On the other hand, kidneys contain significant activity of quinolinate phosphoribosyltransferase (QPRT), which leads tryptophan into the niacin pathway. The ACMSD activity in kidneys were significantly lower in the adenine group than in the control group, while the QPRT activity was almost the same, however, the formations of niacin and its compounds such as N1-methylnicotinamide and its pyridones did not increase, and therefore, the conversion ratio of tryptophan to niacin was lower in the adenine group than in the control group. The contents of NAD and NADP in liver, kidney, and blood were also lower in the adenine group. The decreased levels of niacin and the related compounds were consistent with the changes in the enzyme activities involved in the tryptophan-niacin metabolism in liver. It was concluded from these results that the conversion of tryptophan to niacin is due to only the liver enzymes and that the role of the kidney would be extremely low.