Katsumi Shibata

Role of gustation in the recognition of oleate and triolein in anosmic rats.

Recent studies suggest a chemical perception of dietary fat in the oral cavity. To examine the role of gustation for the recognition of oleate and triolein, very short-term (5-min), two-bottle preference tests were conducted in anosmic rats. To minimize the effects of olfaction, texture and postingestive effects, rats were rendered anosmic with intranasal zinc sulfate, test substances were suspended in 0.3% xanthan gum solution and test fluids were offered for 5 min. Rats preferred oleate fluid but not triolein fluid to the control of 0.3% xanthan gum solution. The preference threshold for oleate in the rat oral cavity was between 0.2% and 0.5%. In the two-bottle preference tests between oleate and triolein, rats preferred oleate fluid to triolein fluid, showing discrimination of oleate and triolein. The results suggest that rat recognizes oleate by a gustatory cue and that fatty acid but not triglyceride is important for gustatory recognition of fat.

Products of the Colonic Microbiota Mediate the Effects of Diet on Colon Cancer Risk

It is estimated that most colon cancers can be attributed to dietary causes. We have hypothesized that diet influences the health of the colonic mucosa through interaction with the microbiota and that it is the milieu interior that regulates mucosal proliferation and therefore cancer risk. To validate this further, we compared colonic contents from healthy 50- to 65-y-old people from populations with high and low risk, specifically low risk Native Africans (cancer incidence <1:100,000; n = 17), high risk African Americans (risk 65:100,000; n = 17), and Caucasian Americans (risk 50:100,000; n = 18). Americans typically consume a high-animal protein and -fat diet, whereas Africans consume a staple diet of maize meal, rich in resistant starch and low in animal products. Following overnight fasting, rapid colonic evacuation was performed with 2 L polyethylene glycol. Total colonic evacuants were analyzed for SCFA, vitamins, nitrogen, and minerals. Total SCFA and butyrate were significantly higher in Native Africans than in both American groups. Colonic folate and biotin content, measured by Lactobacillus rhamnoses and Lactobacillus plantarum ATCC 8014 bioassay, respectively, exceeded normal daily dietary intakes. Compared with Africans, calcium and iron contents were significantly higher in Caucasian Americans and zinc content was significantly higher in African Americans, but nitrogen content did not differ among the 3 groups. In conclusion, the results support our hypothesis that the microbiota mediates the effect diet has on colon cancer risk by their generation of butyrate, folate, and biotin, molecules known to play a key role in the regulation of epithelial proliferation.

Simultaneous micro-determination of nicotinamide and its major metabolites, N1-methyl-2-pyridone-5-carboxamide and N1-methyl-4-pyridone-3-carboxamide, by high-performance liquid chromatography

A simultaneous micro-determination of nicotinamide and its major metabolites, N1-methyl-2-pyridone-5-carboxamide (2-py) and N1-methyl-4-pyridone-3-carboxamide (4-py) by high-performance liquid chromatography is described. The method employs a 7-ODS-L (250 mm X 4.6 mm I.D., particle size 7 microns) column eluted with 10 mM potassium dihydrogenphosphate-acetonitrile (96:4, v/v; pH adjusted to 3.0 by the addition of concentrated phosphoric acid) at a flow-rate of 1.0 ml/min. The UV detector was set at 260 nm. The detection limits for nicotinamide, 2-py and 4-py were 10 pmol (1.22 ng), 2 pmol (304 pg) and 2 pmol (304 pg), respectively, at a signal-to-noise ratio 5:1. Isonicotinamide was used as an internal standard. The technique was applied to the analysis of rat and human urines. The total analysis time was ca. 15 min.

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…

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 α-amino-β-carboxymuconate-η-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.

Simultaneous High-performance Liquid Chromatographic Measurement of Xanthurenic Acid and 3-Hydroxyanthranilic Acid in Urine

absorptivity of 35,000 M- 1 . cm -1 at 240 nm in water. The calibration curve for XA was linear in the range of 1O-1000pmol per injection, with a correlation coefficient of 0.999. The relative standard deviation of the peak area ofXA (100 pmol per injection; five injections) was ca. 1 %, the detection limit being 10 pmol (2052 pg) at a signal-to-noise ratio of 5: 1. The concentration of 3-HA was calculated by using the value for molar absorptivity of 3860 M - 1 . cm - 1 at 340 nm. The calibration curve for 3-HA was linear in the range of 0.2-500 pmol per injection. The relative standard deviation of the peak area of 3-HA (10pmol per injection; five injections) was less than 1 %, the detection limit being 0.2 pmol (30.62 pg) at a signal-to-noise ratio of 5 : 1. To check the validity of this method, a urine sample was measured five times, the resulting coefficient of variation being l.9%. In order to determine the recovery of XA from rat urine, O.l ml of 0.164mM XA (dissolved in water) was added to a mixture of 0.3 ml of diluted rat urine and 0.7 ml of 0.5 M sodium citrate-citrate buffer (pH 3.0). The recovery was 98.9%. The recovery of 3-HA from rat urine was also satisfactory as described in ref. 4. Under the described HPLC conditions, XA and 3-HA were eluted at ca. 6.8 min and 9.8 min, respectively. The elution times for the other Trp metabolites were as follows (detecting at 240nm): 3-hydroxykynurenine, 3.5 min; 5-hydroxytryptophan, 4.3 min; serotonin, 5.l min; kynurenine, 5.6 min; 5-hydroxyindole-3-acetic acid, 5.7min; Trp, 9.1 min; kynurenic acid, 9.8min; and anthranilic acid, 34min. Although kynurenic acid was co-eluted with 3-HA, it did not interfere with the measurement of 3-HA since kynurenic acid is amperometrically inert. A chromatogram of the extract of rat urine is shown in Fig. 1. XA and 3-HA in the sample were characterized on the basis of their retention times, and by coinjecting the respective reference compounds. The total HPLC analysis time was ca. 40 min.

Identification and Expression of a cDNA Encoding Human α-Amino-β-carboxymuconate-ε-semialdehyde Decarboxylase (ACMSD) A KEY ENZYME FOR THE TRYPTOPHAN-NIACINE PATHWAY AND “QUINOLINATE HYPOTHESIS”

Quinolinate (quinolinic acid) is a potent endogenous excitotoxin of neuronal cells. Elevation of quinolinate levels in the brain has been implicated in the pathogenesis of various neurodegenerative disorders, the so-called “quinolinate hypothesis.” Quinolinate is non-enzymatically derived from α-amino-β-carboxymuconate-ε-semialdehyde (ACMS). α-Amino-β-carboxymuconate-ε-semialdehyde decarboxylase (ACMSD) is the only known enzyme that can process ACMS to a benign catabolite and thus prevent the accumulation of quinolinate from ACMS. ACMSD seems to be regulated by nutritional and hormonal signals, but its molecular mechanism has, to date, been largely unknown. Utilizing partial amino acid sequences obtained from highly purified porcine kidney ACMSD, a cDNA encoding human ACMSD was cloned and characterized. The cDNA encodes a unique open reading frame of 336 amino acids and displays little homology to any known enzymes or motifs in mammalian databases, suggesting that ACMSD may contain a new kind of protein fold. Real-time PCR-based quantification of ACMSD revealed very low but significant levels of the expression in the brain. Brain ACMSD messages were down- and up-regulated in response to low protein diet and streptozocin-induced diabetes, respectively. The enzyme activities measured from partially purified brains were closely correlated with the changes in the message levels. Expression of quinolinate phosphoribosyltransferase (QPRT), another enzyme that catabolizes quinolinate, was also found in the brain. This suggests that a pathway does exist by which the levels of quinolinate in the brain are regulated. In this report, we address the molecular basis underlying quinolinate metabolism and the regulation of ACMSD expression.

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, N1-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.

Effects of UVA Irradiation on the Concentration of Folate in Human Blood

Although it is well known that ultraviolet A (UVA) irradiation destroys folate, no definite conclusion for the biological degradation has yet been drawn. In the present study, we determined the effects of UVA exposure on the blood folate concentration in vitro and in vivo. UVA irradiation reduced the synthesized folate pteroylmonoglutamic acid (PGA) content in the blood, but not 5-methyltetrahydrofolate, a major folate form in the blood stream. Exposure to sunlight also decreased the plasma folate concentration in human subjects who took PGA prior to the exposure, but not in subjects who did not take PGA. These results suggest that UVA exposure destroyed PGA but not 5-methyltetrahydrofolate in human blood in vivo.

The Niacin Required for Optimum Growth Can Be Synthesized from l-Tryptophan in Growing Mice Lacking Tryptophan-2,3-Dioxygenase

In mammals, nicotinamide (Nam) is biosynthesized from l-tryptophan (l-Trp). The enzymes involved in the initial step of the l-Trp→Nam pathway are l-Trp-2,3-dioxygenase (TDO) and indoleamine-2,3-dioxygenase (IDO). We aimed to determine whether tdo-knockout (tdo(-/-)) mice fed a diet without preformed niacin can synthesize enough Nam to sustain optimum growth. Wild-type (WT) and tdo(-/-) mice were fed a chemically defined 20% casein diet with or without preformed niacin (30 mg nicotinic acid/kg) for 28 d. Body weight, food intake, and liver NAD concentrations did not differ among the groups. In the groups of mice fed the niacin-free diet, urinary concentrations of the upstream metabolites kynurenine (320% increase, P < 0.0001), kynurenic acid (270% increase, P < 0.0001), xanthurenic acid (770% increase, P < 0.0001), and 3-hydroxyanthranilic acid (3-HA; 450% increase, P < 0.0001) were higher in the tdo(-/-) mice than in the WT mice, while urinary concentrations of the downstream metabolite quinolinic acid (QA; 50% less, P = 0.0010) and the sum of Nam and its catabolites (10% less, P < 0.0001) were lower in the tdo(-/-) mice than in the WT mice. These findings show that the kynurenine formed in extrahepatic tissues by IDO and subsequent enzymes can be metabolized up to 3-HA, but not into QA. However, the tdo(-/-) mice sustained optimum growth even when fed the niacin-free diet for 1 mo, suggesting they can synthesize the minimum necessary amount of Nam from l-Trp, because the liver can import blood kynurenine formed in extrahepatic tissues and metabolize it into Nam via NAD and the resulting Nam is then distributed back into extrahepatic tissues.

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.