Naoki Ozawa

Determination of linezolid in plasma by reversed-phase high-performance liquid chromatography

An HPLC-UV method was developed for assay of linezolid in dog, rat, mouse, and rabbit plasma. Linezolid and the internal standard were extracted on a solid phase cartridge (SPE) and separated on a reversed-phase column (C8, 4.6x150 mm, 5 microm) with 20% acetonitrile in water as mobile phase. The SPE quantitatively recovered linezolid and the internal standard from plasma samples. The chromatographic peak height ratio or peak area ratio based on UV absorbency at 251 nm was used for quantitative analysis. The assay procedures were simple and the assay was specific and had adequate precision and accuracy. Calibration standards with concentrations over the range of 0.01 20 microg/ml were validated for routine sample analysis to support the pharmacokinetic and toxicology studies with linezolid in dog, rat, mouse, and rabbit. Analysis of quality control samples showed the coefficients of variation were usually <10% and the measured and theoretical concentrations differed by <10% in most assays. Linezolid in the plasma samples was stable when stored at below -20 degrees C for at least 63 days, at room temperature (22-23 degrees C) for up to 24 h, and after three freeze-thaw cycles. This HPLC method has been successfully used in multiple laboratories to assay plasma samples from pharmacokinetic and toxicology studies with linezolid in the animal species.

Studies on metabolism and toxicity of styrene--VI. Regioselectivity in glutathione S-conjugation and hydrolysis of racemic, R- and S-phenyloxiranes in rat liver.

Rat liver cytosol converted phenyloxirane enantiomers regioselectively to glutathione S-conjugates. R-(+)-Phenyloxirane was converted to S-(1-phenyl-2-hydroxyethyl)glutathione (conjugate 1) and S-(2-phenyl-2-hydroxyethyl)glutathione (conjugate 2) (ratio 6.1:1), and S-(-)-phenyloxirane to conjugates 1 and 2 (ratio 1:32). Racemic phenyloxirane was converted to conjugates 1 and 2 (ratio 1.8:1). The conjugates were separated by HPLC on an octadecylsilicone column and identified with synthetic specimens whose structures were assigned by 13C NMR spectrometry. R-(+)-, S-(-)- and racemic phenyloxiranes were hydrolyzed to R-(-)-, S-(+)- and racemic phenylethanediols by microsomal epoxide hydrolase without inversion of absolute configurations of their benzylic carbons. R-(+)-Phenyloxirane had much smaller Km and Vmax than the S-(-)-oxirane did. The R-(+)-oxirane potentially inhibited the microsomal hydrolysis of the S-(-)-oxirane and was preferentially hydrolyzed when the racemic oxirane was used as the substrate. Microsomal monooxygenase oxidized styrene to R-(+)- and S-(-)-phenyloxiranes (ratio 1.3:1), and the ratio was little changed by the pretreatment of the animal with phenobarbital, 3-methylcholanthrene and polychlorinated biphenyls.

Subunit Ya-specific Glutathione Peroxidase Activity toward Cholesterol 7-Hydroperoxides of Glutathione S-Transferases in Cytosols from Rat Liver and Skin

Dermal 7α- and 7β-hydroperoxycholest-5-en-3β-ols (cholesterol 7α- and 7β-hydroperoxides), regarded as good aging markers in the rat (Ozawa, N., Yamazaki, S., Chiba, K., Aoyama, H., Tomisawa, H., Tateishi, M., and Watabe, T. (1991) Biochem. Biophys. Res. Commun. 178, 242-247), were reduced in the presence of glutathione (GSH) with concomitant formation of GSSG by cytosol from rat liver in which no detectable level of the hydroperoxides had been demonstrated to occur. The GSH peroxidase (GSH Px) activity toward the toxic steroid hydroperoxides was exerted to almost the same extent by both Alpha-class GSH S-transferases (GSTs), Ya-Ya and Ya-Yc, and by selenium-containing GSH Px (Se-GSH Px) in rat liver cytosol. None of three Mu-class GSTs, Yb1-Yb1, Yb1-Yb2, and Yb2-Yb2, and a Theta-class GST, Yrs-Yrs, from rat liver and a Pi-class GST, Yp-Yp, from rat kidney showed any appreciable GSH Px activity toward the hydroperoxides. The subunit Ya-bearing GSTs and Se-GSH Px purified from rat liver cytosol showed marked differences in apparent specific activity toward the cholesterol hydroperoxides (GSTs Ya-Ya > Ya-Yc ≫ Se-GSH Px). However, a kinetic study indicated that Se-GSH Px had a higher affinity for steroid hydroperoxides than did the GSTs, so that Se-GSH Px could catalyze the reduction of lower concentrations of cholesterol 7-hydroperoxides with approximately equal Vmax/Km values to those by the GSTs. Rat skin had no GST bearing the subunit Ya but contained only a very low concentration of Se-GSH Px, possibly resulting in the accumulation of cholesterol 7-hydroperoxides in the skin but not in the liver. From rat skin cytosol, GSTs Yc-Yc, Yb1-Yb1, Yb1-Yb2, Yb2-Yb2, and Yp-Yp were isolated, purified to homogeneity, and identified with the corresponding GSTs from liver and kidney. The GSTs accounted for 0.23% of total skin cytosolic protein, and the most abundant isoform of skin GSTs was Yb2-Yb2, followed by Yc-Yc, Yp-Yp, Yb1-Yb1, and Yb1-Yb2 in decreasing order.

Occurrence of cholesterol 7α- and 7β-hydroperoxides in rat skin as aging markers

Abstract Evidence for presence of cholesterol 7α- and 7β-hydroperoxides in rat skin was presented for the first time. The 7-hydroperoxides in rat skin were reduced with sodium borohydride and trimethylsilylated for identification with the authentic compounds by gas chromatography/mass spectrometry. A content of cholesterol 7-hydroperoxides in rat skin, determined by high performance liquid chromatography with a chemiluminescence detector, highly correlated with the age of rats ( r = 0.874; between 1 and 45 weeks old), indicating that cholesterol 7α- and 7β-hydroperoxides were good markers for aging.

Photogeneration of 3β-hydroxy-5α-cholest-6-ene-5-hydroperoxide in rat skin: evidence for occurrence of singlet oxygen in vivo

We identified singlet oxygen adduct of cholesterol, 3beta-hydroxy-5alpha-cholest-6-ene-5-hydroperoxide (5alpha-OOH), in skin of rats pretreated with oral doses of pheophorbide a and subsequent visible irradiation, that have been known to induce photosensitive diseases in animals and humans. In a living animal body, this is the first demonstration of presence of 5alpha-OOH, that is known to be formed exclusively by reaction in vitro between singlet oxygen and cholesterol. By the quantitative determination with high performance liquid chromatography equipped with a chemiluminescence detector, we observed time-dependent increase in concentrations of 5alpha-OOH in skin of rats pretreated with oral doses of pheophorbide a and subsequent visible irradiation, suggesting the occurrence of a labile activated oxygen species, singlet oxygen, in this system.

Differences in liver homogenates from Donryu, Fischer, Sprague-Dawley and Wistar strains of rat in the drug-metabolizing enzyme assay and the salmonella/hepatic S9 activation test

Comparison studies for detecting differences between liver microsome and S9 preparations from 4 strains (Donryu, Fischer, Sprague-Dawley, Wistar) of young male rats were carried out with pretreatment of the animals by inducers such as PCBs and PB plus 5,6-BF. Each microsome fraction was assayed for the enzymic activity of metabolism of model substrates such as aniline, benzophetamine, BP, DMN and 7-ethoxycoumarin. The hepatic S9 sample was also compared, as regards its metabolizing ability to activate 9 pre-mutagens (2AA, AAF, o-AAT, BP, DAB, DMBA, DMN, m-PDA, quinoline) to directly acting mutagens in the Salmonella/hepatic S9 activation test by using TA98, TA100 and TA1537 strains with or without cytochrome P450 inhibitors (SKF-525A, metyrapone, 7,8-benzo-flavone). In the enzymic assay with PCBs-induced microsomes, BP hydroxylation a strain-specific difference: the microsomes from Fischer and Wistar rats were more effective for metabolizing BP than those from the other strains of rat. The effect of induction by BP plus 5,6-BF for Fischer rats showed relatively higher enzymic activity in the same induction group. Other microsomes prepared from rats with and without induction by PB plus, 5,6-BF did not show a clear-cut strain dependency in the enzymic activities assayed. In the mutation experiments with hepatic S9 samples, the examination of DAB and quinoline revealed a marked strain difference when S9 samples prepared from PCBs-pretreated and PB-plus-5,6-BF-induced rats were used: the S9 sample from Fischer rats was available for activating the two pre-mutagens to directly acting mutagens. No marked difference in the metabolic activation of the remaining 7-pre-mutagens was observed on other S9 preparations. In examinations of mutagenicity activities with the use of three inhibitors, the two S9 preparations made with the two induction methods showed inhibition profiles closely similar to each other. However, there were minor differences in the profiles by these inhibitors. From these findings it was concluded that Fischer rat-liver S9 is useful for detecting mutagens in the metabolic activation test, when induction by PB plus 5,6-BF was used in the Ames Salmonella test.

Hepatic microsomal cholesterol epoxide hydrolase: selective inhibition by detergents and separation from xenobiotic epoxide hydrolase.

Cholesterol epoxide hydrolase (CEHase) activities of mouse liver microsomes were completely inhibited by Emulgen 108 and Lubrol PX and approximately half by cholate, Triton WR-1339, and Tween 80 when preincubated with 0.1% of these detergents, while their xenobiotic epoxide hydrolase (XEHase) activities were markedly enhanced under the same preincubation conditions. The CEHase and XEHase activities were separately eluted from a gel column applied with mouse liver microsomes which were solubilized in a phosphate buffer containing cholate, glycerol, urea, dithiothreitol, and EDTA.

INCREASES IN CHOLESTEROL 7-HYDROPEROXIDES IN LIPIDS OF HUMAN SKIN BY SUNLIGHT EXPOSURE

Free and ester forms of cholesterol 7alpha- and 7beta-hydroperoxides (Ch 7-OOHs) in skin lipids of humans were separated and determined by high performance liquid chromatography with a chemiluminescence detector. We first demonstrated the presence of Ch 7-OOHs in lipids of human skin. The levels of Ch 7-OOHs found in skin lipids of healthy Japanese volunteers (n = 5) ranged from 2.78 to 25.2 pmol/cm2 skin, indicating large inter-individual differences. However, the intra-individual differences of Ch 7-OOHs levels in skin lipids between right and left arms were less than 25% (-16.4% to 24.0%). Inter-day differences of Ch 7-OOHs in 5 subjects at 1 week interval were also small (-36.7% to 47.7%). Additionally, we investigated effects of sunlight exposure on the levels of Ch 7-OOHs in skin lipids of healthy Japanese volunteers (n = 24). The levels of Ch 7-OOHs in skin lipids significantly increased from 10.0+/-6.7 to 38.9+/-38.0 pmol/cm2 skin by sunlight exposure (10-40 mJ/cm2/min) for 3 h. Therefore, natural sunlight exposure causes lipid peroxidation in skin lipids of humans. These results suggest that the level of Ch 7-OOHs is a good marker for lipid peroxidation in human skin.

Hepatic microsomal conversion of pregnenolone to 3β,5,6β-trihydroxy-5α-pregnan-20-one via pregnenolone α- and β-epoxides☆

Abstract In the presence of ferrous ion, ADP, and an NADPH-generating system, [4-14C]pregnenolone was oxidized by bovine liver microsomes to its α-epoxide (5,6α-epoxy-3β-hydroxy-5α-pregnan-20-one), β-epoxide (5,6β-epoxy-3β-hydroxy-5β-pregnan-20-one), trihydroxypregnanone (3β,5,6β-trihydroxy-5β-pregnan-20-one) which were separated, isolated on an octadecylsilicone column in 70% aq. methanol by high performance liquid chromatography, identified with respective synthetic specimens by gas-liquid chromatography-mass spectrometry. The microsomal Δ5-oxidation products of pregnenolone were detected in trace yield either when EDTA was added to the incubation mixture or when ferrous ion was omitted from the mixture. The microsomal oxidation system generated malondialdehyde significantly. It, however, was retarded to a negligible extent either by the addition of EDTA or by the omission of ferrous ion. Therefore, the microsomal formation of the significant yields of Δ5-oxygenated pregnenolones was reasonably attributed to a reaction linked to microsomal lipid peroxidation. The ratio of pregnenolone α- to β-epoxides formed was 1:3. A comparable study carried out under the same conditions by using [4-14C]cholesterol as the substrate resulted in the similar Δ5-epoxidation with concomitant formation of cholestane-3β,5α,6β-triol; cholesterol α- and β-epoxides formed were in the ratio 1:4. Both pregnenolone α- and β-epoxides were hydrolyzed by the microsomes to trihydroxypregnanone as the sole metabolite at a relative rate of 0.6:1. A similar relative value was also obtained in the microsomal hydrolysis of cholesterol α- and β-epoxides to the cholestanetriol.

Studies on metabolism and toxicity of styrene IV. 1-Vinylbenzene 3,4-oxide, a potent mutagen formed as a possible intermediate in the metabolism in vivo of styrene to 4-vinylphenol

1-Vinylbenzene 3,4-oxide, a putative intermediate in the metabolism of styrene to 4-vinylphenol, was synthesized and examined for its obligatory intermediacy to the phenol, its physical properties, and its mutagenicity toward Salmonella typhimurium TA98 and TA100. The 3,4-oxide had a half-life of 4.3 sec at pH 7.4 in an aqueous solution, and yielded 4-vinylphenol quantitatively without concomitant formation of any trace amount of 3-vinylphenol. The 3,4-oxide had a potent mutagenicity toward the TA100 bacteria but not toward the TA98 strain, whereas it showed a potent cytotoxicity to both of them His+ revertant colonies induced by the 3,4-oxide were 7233/plate at a total dose of 1.0 micromole/plate when it was applied in a sequential manner to the bacterial suspension during the pre-incubation of the testing system. Under the same conditions, benzo[a]pyrene 4,5-oxide and phenyloxirane showed 1283 and 1657 of His+ revertant colonies/plate at 19 nmoles and 10 micromoles/plate, respectively, as the maximal activities. The isomeric arene oxide, 1-vinylbenzene 1,2-oxide, had a longer half-life (1.63 min) than the 3,4-oxide at pH 7.4 in aqueous solution and was specifically rearranged to 2-vinylphenol. The 1,2-oxide also showed more potent mutagenicity to the TA100 strain bacteria than phenyloxirane but weaker than the 3, 4-oxide. 4- and 2-vinylphenols were neither mutagenic nor cytotoxic to the bacteria at concentrations ranging up to 4 micromoles/plate.