Short Communication ESTIMATION OF ALDEHYDE OXIDASE ACTIVITY IN VIVO FROM CONVERSION RATIO OF N-METHYLNICOTINAMIDE TO PYRIDONES, AND INTRASPECIES VARIATION OF THE ENZYME ACTIVITY IN RATS

The in vivo conversion ratio of N-methylnicotinamide (NMN) to N-methyl-2-pyridone-5-carboxamide (2-PY) and N-methyl-4pyridone-3-carboxamide (4-PY) as a parameter for the estimation of aldehyde oxidase level in rats was examined. NMN and its pyridones (2-PY and 4-PY) are usually detected in the urine of rats. When we measured the ratio of the amount of pyridones to the total amount of NMN and pyridones (RP value) in the urine of rats, marked intraspecies variations were observed. The variation in RP value among strains was closely related to the differences of liver aldehyde oxidase activity measured with NMN as a substrate. RP values after administration of NMN to different strains of rats confirmed the existence of strain differences of aldehyde oxidase activity in vivo. We demonstrated that measurements of NMN and its pyridones usually excreted in the urine can be used to predict the in vivo level of aldehyde oxidase. Aldehyde oxidase (EC 1.2.3.1), a cytosolic enzyme, contains FAD, molybdenum, and iron-sulfur centers, and is closely related to xanthine oxidase (Beedham, 1987). The two enzymes have a very close evolutionary relationship, based on the recent cloning of the genes, and they show a high degree of amino acid sequence homology (Calzi et al., 1995; Wright et al., 1999; Terao et al., 2000). They have been suggested to be relevant to the pathophysiology of a number of clinical disorders (Berger et al., 1995; Wright et al., 1995; Moriwaki et al., 1997). Aldehyde oxidase commonly exists in vertebrates. The enzyme in liver of various species catalyzes the oxidation of a number of aldehydes and nitrogenous heterocyclic xenobiotics, such as methotrexate and cyclophosphamide (Beedham, 1987), and also catalyzes the metabolism of physiological compounds such as retinaldehyde and monoamine neurotransmitters (Huang and Ichikawa, 1994). Moreover, the enzyme in the presence of its electron donor can mediate the reduction of a variety of compounds, such as sulfoxides, N-oxides, nitrosamines, hydroxamic acids, azo dyes, oximes, epoxides, aromatic nitro compounds, and 1,2-benzisoxazole derivatives (Sugihara et al., 1996). Recently, aldehyde oxidase homologs, which may exhibit different metabolic roles, were identified in mice (Garattini et al., 2003; Kurosaki et al., 2004; Vila et al., 2004). Marked interspecies variation of the enzyme activity in oxidative and reductive reactions was reported (Sugihara et al., 1996; Schofield et al., 2000). We found a significant variation of liver aldehyde oxidase activity in 12 strains of rats in an assay using benzaldehyde or methotrexate as a substrate (Sugihara et al., 1995; Kitamura et al., 1999). Variations of benzaldehyde oxidase in white people and Japanese have also been reported (Rodrigues, 1994; Sugihara et al., 1997). However, no report is available on the prediction of aldehyde oxidase levels in vivo. N-Methylnicotinamide (NMN), which is formed from nicotinamide by nicotinamide methyltransferase, is widely distributed in animals, like nicotinamide (Yan et al., 1997). NMN is neurotoxic, and is detoxified by oxidation to N-methyl-2-pyridone-5-carboxamide (2-PY) and N-methyl-4-pyridone-3-carboxamide (4-PY) (Fig. 1). The conversion of NMN to 2-PY and 4-PY has been reported to be catalyzed by aldehyde oxidase, but the activity shows marked strain differences in mice (Stanulovic and Chaykin, 1971a). In this study, we attempted to estimate the in vivo level of aldehyde oxidase in rats by using NMN as a substrate. The amounts of NMN and its pyridones excreted in the urine were measured by using high-performance liquid chromatography (HPLC). The conversion ratio of NMN to pyridones (RP value) in urine may be a useful parameter for noninvasive prediction of aldehyde oxidase activity in rats. We also observed considerable interstrain variations of RP value, which were correlated to those of liver aldehyde oxidase activity in rats. Materials and Methods Chemicals. NMN and N -methylnicotinamide were obtained from Tokyo Chemical Industry Co., Ltd. (Tokyo, Japan). 2-PY and 4-PY were prepared by the method of Shibata et al. (1988). Animals. Six different strains of male rats (6–7 weeks of age) were used. Slc:Wistar/ST and Slc:SD rats were obtained from Japan Slc, Inc. (Shizuoka, Japan); Jcl:SD from Clea Japan Inc. (Tokyo, Japan); Crj:SD from Charles River Japan, Inc. (Yokohama, Japan); and WKA/Sea and Sea:SD from Seiwa Experimental Animals, Ltd. (Fukuoka, Japan). Among these strains, Slc: Wistar/ST and WKA/Sea are Wistar strains, and Jcl:SD, Sea:SD, Crj:SD, and Slc:SD are Sprague-Dawley strains. WKA/Sea is inbred, and the others are from closed colonies. This work was supported by a Grant-in-Aid for Scientific Research on Priority Areas from the Japanese Ministry of Education, Science, Sports and Culture, and a Grant-in-Aid for Scientific Research from the Japan Society for the Promotion