Studies on steroid conjugates—VIII: Isolation and characterization of glucuronide-conjugated metabolites of cortisol in human urine

Abstract

A total spectrum of glucuronide-conjugated metabolites of cortisol has been isolated from human urine and characterized: 22 steroid monoglucuronides and 7 steroid diglucuronides. Following i.v. administration of a tracer dose of [4- 14C]-cortisol to 10 normal subjects, urine was collected for 2 consecutive 24-h periods. Free steroids were removed with ethyl acetate. All conjugated metabolites were extracted by means of an Amberlite XAD-2 column, and were further purified by chromatography on a polyethyleneimine-impregnated cellulose column. Groups of mono- and di-glucuronide conjugated steroids were then separated from each other, and from other groups of steroid conjugates by means of high voltage paper electrophoresis. Individual monoglucuronide-conjugated metabolites were then separated from each other by means of 15 consecutive paper chromatographies, and their homogeneity was ascertained. Each steroid conjugate was then subjected to the following characterization: (1) identification by RID of the steroid moiety released by β-glucuronidase hydrolysis; (2) identification of the glucuronide moiety; (3) determination of the steroid/glucuronide molar ratio; (4) determination of the site of conjugation. The following monoglucuronide-conjugated (-G) metabolites of cortisol were isolated: cortisol-21-G, cortisone-21-G, 20β-dihydrocortisol-21-G, tetrahydrocortisol-3-G, tetrahydrocortisol-21-G, 5α-tetrahydrocortisol-3-G,tetrahydrocortisone-3-G,5α-tetrahydrocortisone-3-G,cortol-20α-3-G, 5α-cortol-20α-3-G, cortol-20β-3-G, 5α-cortol-20β-3-G, cortolone-20α-3-G, 5α-cortolone-20α-3-G, cortolone-20β-3-G, 5α-cortolone-20β-3-G, ll-hydroxyaetiocholanolone-3-G,11-hydroxyandrosterone-3-G, 11-oxoaetiocholanolone-3-G, 11-oxoandrosterone-3-G. In addition, 6β-hydroxy-20β-dihydrocortisol and 6β-hydroxy-20β-cortol were also isolated as monoglucuronides, but their site of conjugation was not determined. The steroids identified as diglucuronide conjugates were: cortol-20α and −20β, cortolone-20α and −20β, tetrahydrocortisol, 5α-tetrahydrocortisol and tetrahydrocortisone. Most of these steroid metabolites have not been heretofore isolated and characterized as conjugates. Three of the isolated metabolites were not known to exist as glucuronide conjugates. The quantitation of individual steroid conjugates revealed interesting relationships between various metabolites, which are discussed.