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Titolo:
DEVELOPMENT OF A NON-HIGH PRESSURE LIQUID-CHROMATOGRAPHY ASSAY TO DETERMINE [C-14]CHLORZOXAZONE 6-HYDROXYLASE (CYP2E1) ACTIVITY IN HUMAN LIVER-MICROSOMES
Autore:
DRAPER AJ; MADAN A; LATHAM J; PARKINSON A;
Indirizzi:
UNIV KANSAS,MED CTR,DEPT PHARMACOL TOXICOL & THERAPEUT,CTR ENVIRONM &OCCUPAT HLTH KANSAS CITY KS 66160 UNIV KANSAS,MED CTR,DEPT PHARMACOL TOXICOL & THERAPEUT,CTR ENVIRONM &OCCUPAT HLTH KANSAS CITY KS 66160
Titolo Testata:
Drug metabolism and disposition
fascicolo: 4, volume: 26, anno: 1998,
pagine: 305 - 312
SICI:
0090-9556(1998)26:4<305:DOANPL>2.0.ZU;2-#
Fonte:
ISI
Lingua:
ENG
Soggetto:
CYTOCHROME P4502E1; CHLORZOXAZONE 6-HYDROXYLATION; ACETAMINOPHEN ACTIVATION; O-DEALKYLATION; HUMAN CYP1A2; IN-VITRO; METABOLISM; 2E1; OXIDATION; PROBE;
Tipo documento:
Article
Natura:
Periodico
Settore Disciplinare:
Science Citation Index Expanded
Citazioni:
42
Recensione:
Indirizzi per estratti:
Citazione:
A.J. Draper et al., "DEVELOPMENT OF A NON-HIGH PRESSURE LIQUID-CHROMATOGRAPHY ASSAY TO DETERMINE [C-14]CHLORZOXAZONE 6-HYDROXYLASE (CYP2E1) ACTIVITY IN HUMAN LIVER-MICROSOMES", Drug metabolism and disposition, 26(4), 1998, pp. 305-312

Abstract

The activity of liver microsomal CYP2E1 is commonly measured as the rate of 5-chloro-2-benzoxazolone (chlorzoxazone) 6-hydroxylation, whichrequires separation of 6-hydroxychlorzoxazone and chlorzoxazone by high pressure liquid chromatography (HPLC). In the present study, we describe a solvent extraction (non-HPLC) assay for measuring CYP2E1 activity, based on the B-hydroxylation of [C-14]chlorroxazone. When [C-14]chlorzoxazone was incubated with human or rat liver microsomes in the presence of NADPH, the major product formed was 6-[C-14]hydroxychlorzoxazone. Unreacted [C-14]chlorzoxarone was quantitatively extracted fromthe incubation mixture with dichloromethane under conditions that resulted in similar to 45% extraction of 6-[C-14]hydroxychlorzoxazone. The amount of 6-[C-14]hydroxychlorzoxazone remaining in the aqueous incubation mixture (similar to 55% of the total amount formed) was quantified by liquid scintillation spectrometry. The limit of detection for this assay was 100 pmol of 6-[C-14]hydroxychlorzoxazone. The solvent extraction procedure was validated by comparing the rates of formation of 6-[C-14]hydroxychlorzoxazone with those determined by HPLC under a variety of experimental conditions. The close correspondence between the two analytical methods suggests that the extraction procedure for measuring 6-[C-14]hydroxychlorzoxazone provides a simple, sensitive, andrapid alternative to the HPLC procedure for measuring CYP2E1 activity. In rats, the assay is not specific for CYP2E1 because CYP1A1 also catalyzes the 6-hydroxylation of chlorzoxazone. Recombinant human CYP1A1also catalyzed the 6-hydroxylation of chlorzoxazone (at 1/5 the rate of CYP2E1), although CYP1A1 is not expressed in human liver microsomes. The non-HPLC assay was used to investigate the postulated role of CYP1A2 in the 6-hydroxylation of chlorzoxazone by human liver microsomes. Recombinant CYP1A2 did not catalyze the 6-hydroxylation of chlorzoxazone, and studies with dimethoxyphenyl)methyl]-6,7-dimethoxyisoquinoline, which inhibits CYP1A2 but not CYP2E1, indicated that, in human liver microsomes, the 6-hydroxylation of chlorzoxazone is catalyzed by CYP2E1 with little or no contribution from CYP1A2 enzymes over a wide range of substrate concentrations.

ASDD Area Sistemi Dipartimentali e Documentali, Università di Bologna, Catalogo delle riviste ed altri periodici
Documento generato il 28/09/20 alle ore 22:09:53