J Nutr Sci Vitaminol, 60, 334–339, 2014 Large Amounts of Picolinic Acid Are Lethal but Small Amounts Increase the Conversion of Tryptophan-Nicotinamide in Rats Katsumi SHIBATA and Tsutomu FUKUWATARI Department of Nutrition, School of Human Cultures, The University of Shiga Prefecture, Shiga 522–8533, Japan (Received May 13, 2014) Summary Picolinic acid (PiA) is an endogenous metabolite of tryptophan that has been reported to possess a wide range of physiological actions. We investigated the effects of dietary PiA on the metabolism of tryptophan to nicotinamide in growing rats. Feeding an ordinary diet containing 1% PiA to growing rats (6 wk) caused death within a few days. Toxicity of PiA was higher than that of analogs such as nicotinic acid and quinolinic acid. Feeding an ordinary diet containing 0.05% and 0.1% PiA did not elicit decreased intake of food or loss in body weight. PiA did not affect the in vitro liver activities of quinolinic acid phosphoribosyltransferase or a-amino-b-carboxymuconate-e-semialdehyde decarbox- ylase (ACMSDase, a Zn-dependent enzyme). Concentrations of NAD and NADP in the liver and blood were not affected by PiA. PiA administration did not affect tryptophan metabo- lites such as anthranilic acid, kynurenic acid, and xanthurenic acid. However, quinolinic acid and subsequent metabolites such as nicotinamide and its catabolites were increased by administration of a diet containing 0.05% PiA but not by a 0.1% PiA diet. These results suggest that the in vivo activity of ACMSDase is controlled by the Zn level. Therefore, a small amount of PiA has a beneficial effect for conversion of tryptophan to nicotinamide, but an excessive amount of PiA can be very toxic. Key Words picolinic acid, tryptophan, nicotinamide, toxicity, rat Picolinic acid (PiA) is a metabolite of the complete As in the case of QA, excessive amounts of nicotinic degradation pathway of tryptophan (Trp) (see, Fig. 1), acid, Nam, N1-methylnicotinic acid, and N1-methylnic- which has been reported to possess a wide range of otinamide (MNA) affect food intake and body-weight physiological actions (e.g., neuroprotective (1, 2) and gain only slightly (10). Administration of nicotinic acid immunological (3) effects) and zinc (Zn) metabolism (4). and Nam elicit a much greater increase in Nam catabo- PiA formation is controlled by the activities of two lites such as MNA, N1-methyl-2-pyridone-5-carbox- enzymes: a-amino-b-carboxymuconate-e-semialdehyde amide (2-Py) and N1-methyl-4-pyridone-3-carboxamide decarboxylase (ACMSDase) and a-aminomuconate-e- (4-Py). However, administration of MNA and N1-meth- semialdehyde dehydrogenase (AMSDHase). ACMSDase ylnicotinic acid do not produce increases in Nam catab- catalyzes the reaction of a-amino-b-carboxymuconate- olites (10). Whether dietary PiA affects the metabolism e-semialdehyde (ACMS) to a-aminomuconate-e-semial- of Trp to Nam is unknown. We investigated the effects dehyde (AMS) and AMSDHase the reaction of AMS to of dietary PiA on the growth, food intake, and metabo- 2-aminoadipic acid in the presence of NAD(P)H. lism of Trp to Nam in growing rats. PiA is created by non-enzymatic means from AMS. MATERIALS AND METHODS Quinolinic acid (QA) is produced non-enzymatically from ACMS, which is metabolized to nicotinic acid Chemicals. Vitamin-free milk casein, sucrose, and mononucleotide by QA phosphoribosyltransferase L-methionine were obtained from Wako Pure Chemical (QPRTase) in the presence of 5-phosphoribosyl 1-pyro- Industries, Ltd. (Osaka, Japan). Corn oil was purchased phosphate (PRPP). QA is the direct precursor of NAD in from Ajinomoto (Tokyo, Japan). Gelatinized cornstarch, the tryptophan (Trp)-nicotinamide (Nam) biosynthetic a mineral mixture (11), and a vitamin mixture (11) pathway, but dietary QA is not incorporated efficiently were purchased from Oriental Yeast Co., Ltd. (Tokyo, into NAD molecules (5). The low replacement efficiency Japan). PiA, anthranilic acid (AnA), QA and Nam were of QA for NAD is considered to result from inefficient obtained from Wako Pure Chemical Industries. MNA penetration of QA into cells (6–9). chloride, xanthurenic acid (XA), kynurenic acid (KA), The toxicity of QA is reported to be low. For exam- and 3-hydroxyanthranilic acid (3-HA) were purchased ple, feeding a diet containing 0.5% QA to weaning rats from Tokyo Chemical Industries (Tokyo, Japan). Com- affects food intake and body-weight gain slightly com- pounds 2-Py and 4-Py were synthesized according to pared with those of rats fed a non-QA control diet (10). the method of Pullman and Colowick (12) and that of Shibata et al. (13), respectively. All other chemicals were E-mail: [email protected] of the highest purity available from commercial sources. 334 Picolinic Acid and Tryptophan Metabolism 335 Fig. 1. Metabolic pathway of Trp. ACMS, a-amino-b-carboxymuconate-e-semialdehyde; AMS, a-aminomuconate-e-semi- aldehyde; NMN, nicotinamide mononucleotide. Table 1. Compositions of diets. 20% Casein diet 10.05% PiA 10.10% PiA 11.0% PiA g/kg diet Vitamin-free casein 200 200 200 200 L-Methionine 2 2 2 2 Gelatinized-cornstarch 464 463.5 463 454 Sucrose 224 224 224 224 Corn oil 50 50 50 50 Mineral mixture1 50 50 50 50 Vitamin mixture (NiA-free)1 10 10 10 10 PiA 0 0.5 1 10 1 Shibata K, Matsuo H. 1989. Effect of supplementing low protein diets with the limiting amino acids on the excretion of N1-methylnicotinamide and its pyridones in rats. J Nutr 119, 896–901. Animals and treatment. The care and treatment of and allowed to drink tap water ad libitum for 21 d; this experimental animals conformed to guidelines set by group was the control group (Table 1). The other groups The University of Shiga Prefecture (Shiga, Japan). The (n55) were fed the same diets with PiA addition (Table animal room was maintained at temperature of 22˚C 1) and allowed to drink tap water ad libitum for 21 d. and with 60% humidity. A 12-h light–dark cycle was The concentration of dietary PiA was selected from the also in operation (18:00–06:00). data about the toxicity of quinolinic acid (10) and nico- Six-week-old male Wistar rats obtained from CLEA tinic acid (10, 14). Urine samples (24 h; 09:00–09:00) Japan, Inc. (Tokyo, Japan) were housed individually in were collected at day 21 in amber bottles containing metabolic cages (CT-10; CLEA Japan). Rats were divided 1 mL of 1 mol/L HCl. Acidified urine samples were into four groups at 09:00, which was designated as stored at 225˚C until needed. After the last urine sam- the “zero time” of the experiment elapsed time. One ples had been collected, the rats were killed. Blood from group (n55) was fed a conventional 20% casein diet the carotid artery and the livers were removed, and were 336 SHIBATA K and FUKUWATARI T Fig. 2. Effects of dietary PiA on body-weight changes (A) and food intake (B) in rats. Values are mean6SE for 5 rats. Table 2. Effects of PiA on body weight, food intake, liver weight, NAD concentration and NADP concentration, and liver activities of QPRTase and ACMSDase in rats on day 21. 20% Casein diet 10.05% PiA 10.10% PiA Initial body weight, g 133.861.33 133.760.15 133.460.90 Final body weight, g 273.966.0 274.966.5 268.067.6 Body-weight gain, g/21 d 140.165.9 141.264.2 134.665.7 Total food intake, g/21 d 399610.8 392610.5 397613.6 Liver weight, g 12.2960.34 12.0760.48 11.9360.51 Liver NAD, nmol/g 826642.4 810633.7 824650.1 Liver NADP, nmol/g 43267.8 448637.5 437632.2 Blood NAD, nmol/mL 82.861.7 79.261.4 84.963.0 Blood NADP, nmol/mL 13.560.5 13.860.5 14.760.4 Liver QPRTase, nmol/h/g 1.1860.05 1.1560.04 1.1960.04 Liver ACMSDase, nmol/h/g 0.7460.11 0.9760.16 0.8660.13 Values are means6SE for 5 rats. Significant differences were not observed between values in rows. used for measurements of NAD and NADP as well as the Statistical analyses. Values are the mean6SE. Sig- enzyme activities of QPRTase and ACMSDase. nificance was determined by one-way ANOVA, followed Enzyme assays. The liver was dissected, and one by Tukey’s multiple-comparison test; p,0.05 was con- portion homogenized immediately with a Teflon-glass sidered significant. Prism version 5.0 (Graph Pad, San homogenizer in 5 volumes of cold 50 mmol/L KH2PO4– Diego, CA) was used for all analyses. K HPO buffer at pH 7.0. The resulting homogenate 2 4 RESULTS was used as an enzyme source. The methods of measuring of QPRTase (nicotinate Effects of a high-PiA diet on body weight and food intake nucleotide: pyrophosphate phosphoribosyltransferase, Body weights of rats fed the 1% PiA diet decreased by EC 2.4.2.19) (15) and ACMSDase (EC 4.1.1.45) (16) 1.9 g on day 1, and by 20 g on day 3. Three of the five have been reported previously. rats died on day 6, one rat on day 8, and one rat on day Measurement of Trp metabolites. Amounts of Trp 9. Rats ate 10 g of the 1% PiA diet on day 1, and 3 g/d metabolites such as AnA (17), KA (18), XA (19), 3-HA during days 2 to 5, but ate little after day 5. (19), QA (20), Nam (13), MNA (21), 2-Py (13), and Effects of low PiA on the metabolism of Trp to Nam 4-Py (13) in urine were measured by high-performance Feeding a diet containing low amounts of PiA (0.05% liquid chromatography.