Corticosteroids in human blood. VI. Isolation, characterization and quantitation of sulfate conjugated metabolites of cortisol in human plasma

Abstract A total spectrum of glucuronide-conjugated metabolites of cortisol has been isolated from human plasma, and their structure and quantitative relationship have been determined: 22 steroid monoglucuronides (MG), 7 steroid-diglucuronides (DG), and 6 steroid-sulfoglucuronides (GS). Large heparinized blood specimens (450–550 ml) were obtained from 15 normal subjects 2 1 4 –2 1 2 h after i.v. administration of a tracer dose of [4-14C]-cortisol. Plasma was deproteinized and defatted and the “free” steroids were extracted with ethyl acetate. All the conjugated steroid metabolites were then extracted by means of an Amberlite XAD-2 column and purified on a polyethyleneimine-impregnated cellulose column. The three groups of conjugates (MG, DG, GS) were then separated from each other, and from sulfate conjugates by means of high voltage paper electrophoresis. Individual MG conjugated steroid metabolites were then separated as conjugates by means of 15 consecutive paper chromatographies. Each steroid conjugate was subjected to the following characterization studies: (1) identification by reverse isotope dilution (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. Steroid DG and GS were also characterized by similar studies. The following steroids have been isolated and identified as MG: cortisol, cortisone, 20β-dihydrocortisol, 6β-hydroxy-20β-dihydrocortisol, tetrahydrocortisol, 5α-tetrahydrocortisol, tetrahydrocortisone, 5α-tetrahydrocortisone, cortol-20α, 5α-cortol-20α, cortol-20β, 5α-cortol-20β 60β-hydroxy-cortol-20β cortolone-20α, 5β-cortolone-20α, cortolone-20β, 5α-cortolone-20β, 11-hydroxyetiocholanolone, 11-hydroxyandrosterone, 11-oxoetiocholanolone, 11-oxoandrosterone. The following steroids have been isolated and identified as DG: cortol-20α, and -20β, cortolone-20α, and -20β tetrahydrocortisol, 5α-tetrahydrocortisol and tetrahydrocortisone. The following steroids have been isolated as GS: cortols, cortolones (individual isomers not separated), tetrahydrocortisol and tetrahydrocortisone. All ring-A reduced steroid MG were found to be conjugated at C-3, with the exception of tetrahydrocortisol which was also found as a C-21 conjugate. All 4-en-3-oxosteroids were conjugated at C-21. Steroid DG were conjugated at C-3 and C-21. Steroid GS were found to have glucuronide moiety at C-3 and sulfate moiety at C-21. All individual MG, DG and GS conjugates identified were identical with those previously isolated by us from urine. Most of these conjugated metabolites of cortisol have not been heretofore isolated from plasma as conjugates. Of all plasma metabolites of cortisol, total “free” metabolites constituted 47.5 ± 8.6%, and total conjugated metabolites 52.5 ± 9.8%. Of the latter, MG constituted 86.0 ± 2.7%, DG 1.8 ± 1.2%, and GS 1.3 ± 1.1%. The relative plasma concentrations of individual MG, DG and GS conjugates were found to be very similar to those of the urinary steroids. Only concentrations of C-21 MG of 4-en-3-oxosteroids were 2–3 times higher than those in urine, presumably due to the binding of these conjugates to transcortin. The quantitative relationships between concentrations of various individual conjugates suggest certain substrate predilections of steroid glucuronyl-transferases and sulfotransferases, which are discussed.