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Titolo:
DEVELOPMENT OF A NON-HIGH PRESSURE LIQUID-CHROMATOGRAPHY ASSAY TO DETERMINE TESTOSTERONE HYDROXYLASE (CYP3A) ACTIVITY IN HUMAN LIVER-MICROSOMES
Autore:
DRAPER AJ; MADAN A; SMITH K; 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: 299 - 304
SICI:
0090-9556(1998)26:4<299:DOANPL>2.0.ZU;2-6
Fonte:
ISI
Lingua:
ENG
Soggetto:
CYTOCHROME-P-450 ENZYMES; DRUG-METABOLISM; TERFENADINE; OXIDATION; RAT; IDENTIFICATION; CYCLOSPORINE; INHIBITION;
Tipo documento:
Article
Natura:
Periodico
Settore Disciplinare:
Science Citation Index Expanded
Citazioni:
24
Recensione:
Indirizzi per estratti:
Citazione:
A.J. Draper et al., "DEVELOPMENT OF A NON-HIGH PRESSURE LIQUID-CHROMATOGRAPHY ASSAY TO DETERMINE TESTOSTERONE HYDROXYLASE (CYP3A) ACTIVITY IN HUMAN LIVER-MICROSOMES", Drug metabolism and disposition, 26(4), 1998, pp. 299-304

Abstract

The major pathway of testosterone oxidation by human liver microsomesis the formation of 6 beta-hydroxytestosterone, which is catalyzed byCYP3A4/5 and which accounts for 75-80% of all metabolites formed. In the present study, we describe a non-high pressure liquid chromatography assay (HPLC) of CYP3A4/5 activity based on the release of tritium (with formation of tritiated water) upon incubation of [1,2,6,7-H-3]testosterone with human liver microsomes and NADPH. Unreacted testosterone and its metabolites were quantitatively extracted from the incubation mixture with activated charcoal under conditions that resulted in noextraction of tritiated water. The amount of tritiated water formed was quantified by liquid scintillation spectrometry and compared with the amount of hydroxylated testosterone metabolites formed, as determined by HPLC. Rates of tritium release from [1,2,6,7-H-3]testosterone paralleled rates of testosterone 6 beta-hydroxylation as a function of incubation time, the amount of microsomal protein, and the concentration of substrate (which yielded identical apparent K-m and V-max values), The sample-to-sample variation in tritium release from [1,2,6,7-H-3]testosterone with a panel of human liver microsomes was highly correlated with rates of testosterone 6 beta-hydroxylation and terfenadine metabolism, two commonly used markers of CYP3A activity. Several recombinant human P450 enzymes were incubated with [1,2,6,7-H-3]testosterone,and only cDNA-expressed CYP3A4 catalyzed a high rate of tritium release. The close agreement between the tritium-release assay and HPLC procedure for measuring testosterone oxidation indicates that tritium release from [1,2,6,7-H-3]testosterone provides a simple and rapid alternative to the HPLC procedure for measuring CYP3A4/5 activity in human liver microsomes. However, the tritium-release assay may have limited value in measuring CYP3A activity in liver microsomes from other species due to the presence of other P450 enzymes that can catalyze tritium release from [1,2,6,7-H-3]testosterone.

ASDD Area Sistemi Dipartimentali e Documentali, Università di Bologna, Catalogo delle riviste ed altri periodici
Documento generato il 28/09/20 alle ore 21:55:14