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Nutritional Vitamin B6 Deficiency Impairs Lipid Me Tabolism and Leads

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Nutritional Vitamin B6 Deficiency Impairs Lipid Me Tabolism and Leads J Nutr Sci Vitaminol, 47, 306-310, 2001 Xanthurenic Acid Inhibits Metal Ion-Induced Lipid Peroxidation and Protects NADP-Isocitrate Dehydrogenase from Oxidative Inactivation Keiko MURAKAMI,Masae ITO and Masataka YOSHINO* Department of Biochemistry, Aichi Medical University School of Medicine,Nagakute, Aichi 480-1195, Japan (ReceivedJanuary 26, 2001) Summary Vitamin B6 deficiency increases the lipid peroxidation and the synthesis of xanthurenic acid from tryptophan. Antioxidant properties of xanthurenic acid were exam ined in relation to the coordination of transition metals. Xanthurenic acid inhibited the for mation of thiobarbituric acid-reactive substances as a marker of iron-mediated lipid peroxi dation and copper-dependent oxidation of low density lipoprotein. NADP-isocitrate dehydro genase (EC 1.1.1.42), a principal NADPH-generating enzyme for the antioxidant defense system, was inactivated by reduced iron and copper, and xanthurenic acid protected the en zyme from the Fe2+-mediated inactivation. Xanthurenic acid may participate in the en hanced regeneration of reduced glutathione by stimulating the NADPH supply. Xanthurenic acid further enhanced the autooxidation of Fe2+ ion. Other tryptophan metabolites such as kynurenic acid and various quinoline compounds did not inhibit the lipid peroxidation and the inactivation of NADP-isocitrate dehydrogenase, and they showed little or no effect on the Fe2+ autooxidation. The antioxidant properties of xanthurenic acid are related to the metal-chelating activity and probably to the enhanced oxidation of reduced transition met als as a prooxidant, and this action may be due to the electron deficient nature of this com pound. Key Words xanthurenic acid, antioxidant, LDL, NADP-isocitrate dehydrogenase, reactive oxygen species Nutritional vitamin B6 deficiency impairs lipid me Lipid peroxidation. Lipid peroxidation was deter tabolism and leads to lipid peroxidation by a decrease in mined as the formation of thiobarbituric acid-reactive some antioxidant factors such as vitamin E and reduced substances (7) by iron/ascorbate system with liver mi glutathione (1, 2). Vitamin B6 deficiency further causes crosomes (8). The reaction mixture of 1mL contained an increased excretion of xanthurenic acid, and the in 60mM Tris-HCl buffer (pH 7.5), 10ƒÊM FeCl3, 0.5mM creased xanthurenic acid level is an indicator of vitamin ascorbic acid, and 0.2mg microsomal fraction in the B6 deficiency (3). Many reports suggest that xan presence and absence of xanthurenic acid or kynurenic thurenic acid is closely related to the pathogenesis of acid. Lipid peroxides produced were determined after in several diseases (4-6), although the physiological role cubation for 10min (7). of this compound is unknown. In this paper we show Autooxidation of Fe2+ ion. The interaction of xan that xanthurenic acid acts as an antioxidant by inhibit thurenic acid and quinoline compounds with iron was ing lipid peroxidation and copper-dependent oxidation evaluated by the effect of these compounds on the rate of low-density lipoprotein. Xanthurenic acid also pro of autooxidation of Fe2+ ion, as described previously (8). tected NADP-isocitrate dehydrogenase (EC 1.1.1.42), The samples of 1mL contained 10mM Tris-HCl (pH the reduced NADP generating enzyme, from the iron 7. 1), 0.1mM FeSO4, and the additive. The reaction was and copper-mediated inactivation. The antioxidant ac started by the addition of FeSO4. Aliquots of 0.2mL tion of xanthurenic acid may counteract the peroxida were mixed with 0.1mL of 1mM bathophenanthroline tive damage because of vitamin B6 deficiency. disulfonate at appropriate intervals, and the absorbance at 540nm was measured. MATERIALS AND METHODS Inactivation of NADP isocitrate dehydrogenase. Materials. NADP isocitrate dehydrogenase was a Purified NADP isocitrate dehydrogenase of 1.5ƒÊg was product of Oriental Yeast Co. (Osaka, Japan). inactivated by preincubating for 2.5min with various Xanthurenic acid, kynurenic acid, anthranilic acid, concentrations of FeCl2 in 1mL of 40mM Tris-HCl quinolinic acid derivatives, human low-density lipopro buffer (pH 7.1) containing 0.25mM threo-Ds-isocitrate, tein (LDL), bathophenanthroline disulfonate, threo-Ds the substrate, and 2.5mM MgCl2 in the absence and isocitrate, and NADP were products of Sigma-Aldrich presence of various concentrations of xanthurenic acid -Japan (Tokyo, Japan). and its related compounds. Enzyme reaction was started by the addition of 0.25mM NADP to the mix * To whom correspondence should be addressed . ture, and changes in absorbance at 340nm were fol E-mail: [email protected] lowed (9). 306 Xanthurenic Acid as Antioxidant 307 Oxidation of human low-density lipoprotein. The oxi dependent LDL oxidation by prolonging lag phase and dation of LDL was determined as described previously by slightly suppressing propagation rate. 8-Hydroxy (10). LDL was diluted to the concentration of 60ƒÊg/mL quinoline caused a complete inhibition of LDL oxida with 10mM potassium phosphate buffer (pH 7.4) con tion, but quinaldic acid showed no effect on the oxida taining 1.5ƒÊM EDTA and 0.15M NaCl. LDL oxidation tion (Fig. 2, 3). was performed at 37•Ž in 1mL of the phosphate buffer NADP-isocitrate dehydrogenase, the principal (pH 7.4) containing 2ƒÊM CuSO4, 5ƒÊM xanthurenic NADPH generating enzyme, was irreversibly inacti acid or quinoline compounds, and 0.15M NaCl. The vated by Fe2+ ion, and the concentration of ferrous ion progress of oxidation was monitored spectrophotomet required for the enzyme inactivation was about 3 to rically (Shimadzu 1600, Tokyo, Japan, equipped with a 4ƒÊM (Fig. 4). Fe3+ ion did not inactivate the enzyme Peltier-thermostatted six-cell holder) by the formation (data not shown). The addition of xanthurenic acid of conjugated dienes at 234nm (10). completely protected the enzyme from the ferrous Spectrophotometric analysis of the interaction of xan ion-dependent inactivation. 8-Hydroxyquinoline also thurenic acid with iron. Stock solutions of xanthurenic acid were diluted to 20 or 25ƒÊM solutions in 10mM Tris-HCl buffer (pH 7.4). Absorption spectra were recorded from 300 to 600nm. Scans were started by the addition of 0.1mM FeSO4 and repeated at intervals of 1 min. The spectra of FeCl3 and FeSO4 solution were also recorded. RESULTS We examined the effect of xanthurenic acid and kynurenic acid as principal tryptophan metabolites on the microsomal lipid peroxidation. The incubation of microsomes with iron and ascorbate produced thiobar bituric acid-reactive substances as a marker of lipid per oxidation. The antioxidant effect was determined as the inhibition of the formation of the thiobarbituric acid-re Fig. 2. Effect of xanthurenic acid and its related com active substances by quinoline compounds. The con pounds on the copper-dependent oxidation of low density lipoprotein. LDL was diluted to a concentra centrations of xanthurenic acid required for 50% inhi tion of 60ƒÊg/mL, with 10mM potassium phosphate bition of the peroxidation were about 30ƒÊM, but buffer (pH 7.4) containing 1.5ƒÊM EDTA and 0.15M kynurenic acid showed no inhibitory effect (Fig. 1). NaCl, and LDL oxidation was started at 37•Ž by the The addition of 2ƒÊM copper caused the oxidation of addition of 2ƒÊM CuSO4 in the absence and presence of LDL with the concomitant formation of conjugated 5ƒÊM xanthurenic acid or quinoline compounds. The dienes (Fig. 2). Xanthurenic acid inhibited copper progress of oxidation was monitored spectrophoto metrically by means of a thermostatic control (37•Ž) and an automatically exchangeable six-position cu vette holder operating at 234nm (10). Curve 1, no addition; curve 2, xanthurenic acid added; curve 3, quinaldic acid added; curve 4, 8-hydroxyquinoline added. Fig. 1. Effect of xanthurenic acid and kynurenic acid on the iron-mediated lipid peroxidation of rat liver microsomes. Lipid peroxidation was induced by 10ƒÊM FeCl3, 0.5mM ascorbic acid, and 0.2mg microsomal fraction (8) in the presence and absence of xan thurenic acid or kynurenic acid. The mixture was in cubated at 37•Ž for 10min, and the reaction was stopped by the addition of 100% trichloroacetic acid. Lipid peroxides produced were expressed as the thio barbituric acid-reactive substances (7). •œ, xan thurenic acid; •¢, kynurenic acid. Fig. 3. Structure of quinoline compounds. 308 MURAKAMI K et al. showed a complete protection of the enzyme, but compound required for the 50% protection of the kynurenic acid and quinaldic acid showed no protective enzyme was 80 to 90ƒÊM (Fig. 5). The inactivation by effect on the enzyme (Fig. 4). We further examined the reduced copper of NADP-isocitrate dehydrogenase was effect of the increasing concentration of xanthurenic also protected by xanthurenic acid (Fig. 5). acid on the iron-mediated inactivation of NADP-iso The effect of xanthurenic acid and its related citrate dehydrogenase, and the concentration of the compounds on the rate of Fe2+ autooxidation was examined. After incubation of FeSO4 with quinoline compounds or tryptophan metabolites, Fe2+ ion was determined by bathophenanthroline disulfonate. Xanthurenic acid increased the rate of Fe2+ oxidation markedly, and 8-hydroxyquinoline showed a large stim Fig. 4. Inactivation of NADP-isocitrate dehydrogenase by Fe2+ ion in the presence of xanthurenic acid and its related compounds. NADP-isocitrate dehydrogenase was incubated for 2.5min with a mixture containing various concentrations of FeCl2, 0.25mM threo-Ds isocitrate, 2.5mM MgCl2, and 40mM Tris-HCl buffer Fig. 5. Effect of increasing concentrations of xan thurenic acid on the Feet and Cut-mediated inactiva (pH 7.1) in the absence and presence of xanthurenic acid and its related compounds. Activity was deter tion of NADP-isocitrate dehydrogenase. The enzyme mined by the addition of 0.25mM NADP. •ž, no addi was inactivated by iron or copper of 10ƒÊM, and the tion; • , 0.1mM xanthurenic acid added; •›, 0.1mM 8 conditions were similar to those in the legend to Fig. 4. •Ÿ hydroxyquinoline added; •~, 0.1mM quinaldic acid , Inactivation by 10ƒÊM FeC12; •¢, Inactivation by added; •£, 0.1mM kynurenic acid added. 10ƒÊM CuSO4 plus 0.5mM ascorbate.
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