Search

In spite of over 40 years of clinical use of 6-mercaptopurine, many aspects of complex pharmacology and metabolism of this drug remain unclear. It is thought that 6-mercaptopurine is oxidized to 6-thiouric acid through 6-thioxanthine or 8-oxo-6-mercaptopurine by one of two molybdenum hydroxylases, xanthine oxidase (XO), however, the role of other molybdenum hydroxylase, aldehyde oxidase (AO), in the oxidation of 6-mercaptopurine and possible interactions of AO substrates and inhibitors has not been investigated in more details. In the present study, the role of AO and XO in the oxidation of 6- mercaptopurine has been investigated. 6-mercaptopurine was incubated with bovine milk xanthine oxidase or partially purified guinea pig liver molybdenum hydroxylase fractions in the absence and presence of XO and AO inhibitor/substrates, and the reactions were monitored by spectrophotometric and HPLC methods. According to the results obtained from the inhibition studies, it is more likely that 6- mercaptopurine is oxidized to 6-thiouric acid via 6-thioxanthine rather than 8-oxo-6-mercaptopurine. The first step which is the rate limiting step is catalyzed solely by XO, whereas both XO and AO are involved in the oxidation of 6-thioxanthine to 6-thiouric acid.

Partially purified aldehyde oxidase (EC 1.2.3.1) has been prepared from human, rabbit, guinea pig, and baboon liver by heat treatment and precipitation with ammonium sulfate. The interaction of 35 substituted quinazolines and phthalazines with human liver enzyme has been studied using a spectrophotometric assay. Fifteen quinazoline and 14 phthalazine derivatives were found to be substrates for human liver aldehyde oxidase with Km values ranging from 5 to 500 microM. The substrate specificity of the quinazolines toward rabbit, guinea pig, and baboon liver aldehyde oxidase has also been investigated; the reaction of substituted phthalazines with mammalian liver enzyme has been reported previously (Beedham et al., 1990, Biochem. Pharmacol. 39, 1213-1221). Oxidation products of 2-substituted (4-substituted) quinazolines with rabbit liver aldehyde oxidase were identified by MS as 4-oxo (2-oxo)-quinazolines, respectively. In all cases, unsubstituted compounds gave the highest oxidation rates and the presence of lipophilic substituents presumably facilitated hydrophobic binding to the enzymes. However, there were marked differences in substrate specificity between human liver aldehyde oxidase and hepatic enzyme from rabbit, guinea pig, and baboon with the size of substrate being the differentiating factor. The molecular sizes of the substrates, estimated using calculated molar refractivities, ranked the size of the binding site of aldehyde oxidase in the order rabbit < guinea pig < baboon < man. Isoelectric points of the different aldehyde oxidase isozymes ranged from pH 5.10 for rabbit to 6.40 for the human liver isozyme. These results indicate that rabbit liver aldehyde oxidase shows marked differences from the human liver enzyme in its handling of quinazoline and phthalazine substrates.

Molybdenum hydroxylase activity in guinea pig liver has been compared with that of marker enzymes in mitochondria (succinate dehydrogenase), microsomes (glucose-6-phosphatase) and cytosol (lactate dehydrogenase). Aldehyde oxidase activity was highest in the cytosol, with about 10-fold activity of xanthine oxidase. Significant molybdenum hydroxylase activity was found in mitochondria with minimal levels in microsomes. Mitochondrial and cytosolic aldehyde oxidase varied in substrate specificity and electrophoretic mobility with two major bands in each fraction, one of which was common to cytosol and mitochondria.