Atsuko Noda

Spin trapping of a free radical intermediate formed during microsomal metabolism of hydrazine

A radical formed during oxidative metabolism of hydrazine in rat liver microsomes was spin-trapped with alpha-phenyl-t-butylnitrone. The trapped species was identified as hydrazine radical by comparison of its ESR parameters and mass spectrum with those of the adduct formed during CuCl2 catalyzed oxidation of hydrazine. The requirement for oxygen and NADPH in the microsomal oxidation and the occurrence of a typical binding spectrum by difference spectroscopy suggest the involvement of the participation of the cytochrome P-450 enzyme system in the formation of hydrazine radical which must be a precursor of diimide during microsomal oxidation of hydrazine.

Simultaneous determination of antiepileptic drugs and their metabolites, including chiral compounds, via β-cyclodextrin inclusion complexes by a column-switching chromatographic technique☆

Using beta-cyclodextrin as a mobile-phase additive, a column-switching chromatographic system equipped with two 25-mm short ODS cartridge columns and two UV detectors was successfully employed for the simultaneous determination of some antiepileptic drugs and their metabolites, including chiral compounds, in human serum. The compounds examined were phenobarbital, zonisamide, phenytoin and its metabolites, S- and R-5-(p-hydroxyphenyl)-5-phenylhydantoin, R- and S-mephobarbital, carbamazepine and its main metabolites, 10,11-dihydro-10-11-epoxycarbamazepine and trans-10,11-dihydroxy-10,11-dihydrocarbamazepine (as a racemate), and allobarbital (as an internal standard).

Hydrazine radical formation catalyzed by rat microsomal NADPH-cytochrome P-450 reductase

Using NADPH-cytochrome P-450 reductase purified from rat liver microsomes, the oxidation of hydrazine to its radical was proved to proceed smoothly. The catalytic effect of NADPH cytochrome P-450 on the radical formation in the hepatic microsomes obtained from phenobarbital-pretreated rats was also supported by the fact that Hz radical formation was stimulated by flavin adenin dinucleotide or methyl viologen and markedly inhibited by superoxide dismutase, however, carbon monoxide showed no effect. Expectedly, anti-NADPH-cytochrome P-450 IgG decreased the radical formation. The present study provides the first evidence for the NADPH-cytochrome P-450 reductase catalyzed oxidation of hydrazine to its radical in the presence of O2 and NADPH.

Chiral separation of barbiturates and hydantoins by reversed-phase high-performance liquid chromatography using a 25 or 50 mm short ODS cartridge column via β-cyclodextrin inclusion complexes

A high-performance liquid chromatographic method on a 25 or 50 mm short ODS cartridge column has been developed for the resolution of the enantiomers of some optically active barbiturates and hydantoins in human serum. beta-Cyclodextrin was used in the mobile phase. This method also seems to be an easy and effective way to test whether beta-cyclodextrin would be a useful chiral discriminator for a particular racemate.

Effects of rifampicin and phenobarbital on the fate of isoniazid and hydrazine in vivo in rats

After the intraperitoneal (i.p.) administration of isoniazid (INH) to male Wistar rats, the liver and plasma levels of hydrazine (Hz) and acetylhydrazine (AcHz), which are hazardous metabolites of INH and well known as mutagens, carcinogens and hepatotoxins, were determined by gas chromatography-mass spectrometry (GC-MS). The levels of Hz in rifampicin (RMP)- or phenobarbital (PB)-pretreated groups were lower than those in the control group, while the amount of AcHz was scarcely altered. In each of the pretreated groups a pronounced increase in the oxidative elimination rate of Hz was observed. These results are of important toxicological significance in INH therapy with RMP, since an active intermediate of Hz seems to be a hepatotoxin.

Relationship between oxidative metabolites of hydrazine and hydrazine-induced mutagenicity

Hydrazine (Hz) mutagenicity was observed in a test using Escherichia coli B/r strain, WP2 uvrA and was enhanced by the addition of rat liver microsomal fraction containing a generating system, while the enhanced mutagenicity was diminished by the addition of metyrapone to the microsome-free levels. On the other hand, an NADPH-dependent difference spectrum of the metabolic intermediate of Hz-complex, characterized by a maximum level of 448 nm, was also inhibited by metyrapone. The results show that the oxidative intermediates, which are diimide and its precursor, hydrazine free radical [Biochem. Biophys. Res. Commun., 133 (1986) 1086], are responsible not only for hepatotoxicity but also for the enhancement of genotoxicity or mutagenicity.

Side-chain metabolism of propranolol: involvement of monoamine oxidase and aldehyde reductase in the metabolism of N-desisopropylpropranolol to propranolol glycol in rat liver.

The further metabolism of N-desisopropylpropranolol (NDP), a side-chain metabolite of propranolol (PL), was investigated in isolated rat hepatocytes. Propranolol glycol (PGL) was generated from NDP as a major metabolite. Naphtetrazole (NTE), a potent inhibitor of monoamine oxidase (MAO), significantly retarded the disappearance of NDP from the incubation medium, suggesting the involvement of MAO in the deamination of NDP to an aldehyde intermediate. In a reaction mixture of rat liver mitochondria and cytosol with NADPH, phenobarbital, a specific inhibitor of aldehyde reductase, and 4-nitrobenzaldehyde (4-NBA), a substrate inhibitor of aldehyde reductase, decreased the formation of PGL from NDP. 4-NBA was a competitive inhibitor of the enzyme responsible for the PGL formation. The optimal pH for the formation of PGL from NDP in the reaction mixture was approximately 8.0. Based on these results, we propose the possibility that, in the rat liver, MAO catalyzes the oxidative deamination of NDP to an aldehyde intermediate and the formed aldehyde intermediate is subsequently reduced to PGL by aldehyde reductase. Furthermore, the enantioselective metabolism of NDP to PGL was examined. In isolated rat hepatocytes, the amount of PGL formed from S-NDP [S(-)-form of NDP] was larger than that of PGL formed from R-NDP [R(+)-form of NDP].

High-performance liquid chromatographic method for direct separation of 5-(p-hydroxyphenyl)-5-phenylhydantoin enantiomers using a chiral tris(4-methylbenzoate) column

After simple purification of the incubation mixture of phenytoin in isolated rat hepatocytes, 5-(p-hydroxyphenyl)-5-phenylhydantoin (p-HPPH), which formed as a major metabolite, was readily resolved to each enantiomer by direct high-performance liquid chromatography on a cellulose tris(4-methylbenzoate) column, with a mobile phase of ethanol-water. It was also observed that the formation of S-(-)-p-HPPH was dominant, and the S/R ratio was 11.5.

Studies of isoniazid metabolism in isolated rat hepatocytes by mass fragmentography.

Isoniazid metabolism in isolated rat hepatocytes was studied by mass fragmentography using single ion monitoring. Isoniazid and its metabolites were determined as the trimethylsilylated derivatives of acetylisoniazid and diacetylhydrazine and of the benzaldehyde hydrazones of isoniazid and acetylhydrazine. Deuterated analogues served as internal standards. Hydrazine was quantitated as benzalazine using 15N-labeled hydrazine as an internal standard. The method is well suited for the microanalysis of isoniazid metabolism, from which it was clarified that the greater part of hydrazine, a hazardous metabolite of isoniazid, was formed through the direct hydrolysis of isoniazid itself as expected.

Chiral separation of 10,11-dihydro-10,11-trans-dihydroxycarbamazepine, a metabolite of carbamazepine with two asymmetric carbons, in human serum

Chiral separation of 10, 11-dihydro-10, 11-trans-dihydroxycarbamazepine (CBZ-diol), a metabolite of carbamazepine (CBZ) with two asymmetric carbons, in serum taken from epileptic patients receiving CBZ alone for a long period, was performed by high-performance liquid chromatography using a polysaccharide stationary phase with n-hexane-ethanol (75:25, v/v) as the mobile phase. The enantiomeric ratio (S,S-/R,R-CBZ-diol) was 10.74 +/- 1.13 (mean +/- S.D.), which could demonstrate the presence of the stereospecificity in the hydrolysis of 10, 11-dihydro-10, 11-epoxycarbamazepine (CBZ-epoxide) to CBZ-diol and/or in the conversion of CBZ-diol to some metabolite such as 9-hydroxymethyl-10-carbamoylacridan. This is the first paper to report the determination of each enantiomer and the enantiomeric ratio of CBZ-diol in serum of epileptic patients who received CBZ.