title: Liquid chromatographic determination of urinary 6beta-hydroxycortisol to assess cytochrome p-450 3A activity in HIV positive pregnant women.
abstract: Assessing the activity of CYP3A4 is important for predicting the pharmacokinetic behavior of protease inhibitors in HIV positive patients, especially in pregnant women. The endogenous hormonal ratio of 6beta-hydroxycortisol (beta-OHF) to cortisol (F) in the urine is an index for metabolic enzyme activity of cytochrome p-450 (CYP) 3A4. Because the ratio is a unique way to assess the enzyme activity without using any exogenous probes for this isozyme, it is practical for use in pregnant women. In this paper, we describe a method using high performance liquid chromatography (HPLC) for 6beta-OHF in urine from pregnant women to estimate the ratio of 6beta-OHF/F. Urinary 6beta-OHF was measured by using C18-cartridge solid phase extraction and isocratic HPLC. Aliquots (1 ml) of urine samples spiked with internal standard, 6beta-hydroxyprednisolone (6beta-OHPSL), were alkalinized with NaOH, then applied to C18-cartridges, which were washed with water and hexane and eluted with ethyl acetate. After the effluents were dried and reconstituted in 10% acetonitrile, the samples were analyzed by HPLC using an isocratic mobile phase (acetic acid/acetonitrile/50 mM potassium dihydrogenphosphate: 0.2/9/90.8; v/v) and ultraviolet detection at 245 nm. The recoveries of 6beta-OHF from C18 cartridges were 93.2 and 93.9% when the authentic 6beta-OHF was added to the urine sample at the concentration of 50 and 300 ng/ml, respectively. Intra- and inter-day variations estimated at concentrations of 113-674 ng/ml were 2.9-5.6 and 4.9-8.1%, respectively. The method was applied to morning urine samples collected from HIV-positive pregnant women managed with protease inhibitor containing anti-retroviral regimens.
authors.affiliation: Department of Clinical Pharmacology, School of Pharmacy, Tokyo University of Pharmacy and Life Science, Hachioji, Japan. masatoh@mail.hosp.tsukuba.ac.jp
authors.forename: M
authors.initials: M
authors.lastname: Homma
authors.forename: K
authors.initials: K
authors.lastname: Beckerman
authors.forename: S
authors.initials: S
authors.lastname: Hayashi
authors.forename: A L
authors.initials: AL
authors.lastname: Jayewardene
authors.forename: K
authors.initials: K
authors.lastname: Oka
authors.forename: J G
authors.initials: JG
authors.lastname: Gambertoglio
authors.forename: F T
authors.initials: FT
authors.lastname: Aweeka
mesh_terms: D000328:Adult; D019380:Anti-HIV Agents / Q000009:adverse effects; D001189:Aryl Hydrocarbon Hydroxylases; D002138:Calibration; D002851:Chromatography, High Pressure Liquid; D051544:Cytochrome P-450 CYP3A; D003577:Cytochrome P-450 Enzyme System / Q000378:metabolism*; D005260:Female; D006679:HIV Seropositivity / Q000201:enzymology / Q000378:metabolism* / Q000652:urine; D006801:Humans; D006854:Hydrocortisone / Q000031:analogs & derivatives* / Q000652:urine; D010089:Oxidoreductases, N-Demethylating / Q000378:metabolism*; D011247:Pregnancy
chemical_list: D019380:Anti-HIV Agents; C001380:6 beta-hydroxycortisol; D003577:Cytochrome P-450 Enzyme System; D001189:Aryl Hydrocarbon Hydroxylases; D051544:Cytochrome P-450 CYP3A; D010089:Oxidoreductases, N-Demethylating; D006854:Hydrocortisone