Ralph H. Evans

Rat hepatic cytochrome P-450 isozyme specificity for the metabolism of the steroid sulfate, 5α-androstane-3α,17β-diol-3,17-disulfate

Abstract Nine rat hepatic microsomal cytochromes P -450 ( P -450a- P -450i) have been purified to electrophoretic homogeneity and assayed as potential catalysts of 15β-hydroxylation of 5α-androstane-3α,17β-diol-3,17-disulfate in a reconstituted system containing NADPH-cytochrome c reductase and dilauroylphosphatidylcholine. Of the nine isozymes, only cytochrome P -450i, which is present in adult female but not adult male rats, catalyzes the hydroxylation of 5α-androstane-3α,17β-diol-3,17-disulfate. The reaction has an absolute requirement for cytochrome P -450i, NADPH-cytochrome c reductase, and NADPH, as well as a marked dependence on dilauroylphosphatidylcholine. Under optimal conditions, the K m ,app was 69 μ m , and the V max was 7.8 nmol min −1 nmol cytochrome P -450i −1 . The affinity of cytochrome P -450i for the substrate, as expressed by the apparent spectral dissociation constant ( K s app ), was 20 μ m . This female-specific isozyme had very low catalytic activity toward testosterone, and the metabolite profile from testosterone was distinct compared to the profiles obtained with the other eight isozymes. The results with androstane disulfate indicated that cytochrome P -450i is responsible for the sex-specific microsomal 15β-hydroxylase system in adult female rat liver, originally described by Gustafsson and Ingelman-Sundberg [(1974) J. Biol. Chem. 249: 1940–1945].

Preparative HPLC for the facile isolation of drug glucuronide conjugates from crude extracts of urine

Abstract Reverse-phase preparative HPLC has been used to advantage for the isolation of crystalline quantities of drug glucuronide conjugates from crude urinary extracts. Following chronic administration of large doses of diazepam, levorphanol and hydroxyethylflurazepam to dogs, urine was collected and the water soluble drug conjugates adsorbed on a column of Amberlite XAD-2 resin. In each instance elution with methanol and solvent evaporation yielded a crude oil (approx. 3 g) which was chromatographed in one portion on either PrepPAK 500 C18 (Waters) or Magnum 40 ODS-3 (Whatman) columns using aqueous methanol solvent systems. A greater than 90% purification was achieved in this single initial chromatographic step. Employing a combination of subsequent semi-preparative HPLC steps on either C18 or silica gel columns, milligram quantities of the glucuronides of oxazepam, levorphanol and hydroxyethylflurazepam were isolated. The isolation procedures provide a general approach for obtaining milligram quantiti...

X-Ray structure of Ro 21-6150, a polyether antibiotic related to Dianemycin

The constitution and absolute configuration of the polyether antibiotic Ro 21-6150 have been established by X-ray crystallographic analysis of the silver salt (C47H77O13Ag).

The structure determination of a novel C 17 metabolite from streptomyces X-537 using Eu (DPM) 3

Die Struktur destrans-6-[2-(1-hydroxybutyl)-4-tolyl]-hex-5-enamids (I), eines neuen mikrobiellen Metaboliten, wurde durch Protonenresonanz-Spektroskopie unter Verwendung des Verschiebungsreagenz Eu (DPM)3 bestimmt.