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

Abstract

Evidence has been obtained for the presence of a total spectrum of sulfate-conjugated metabolites of cortisol in human plasma: 19 monosulfates, two disulfates and six glucuronosulfates. Two hours and 15 min following i.v. administration of a tracer dose of [4- 14C]-cortisol to 11 normal subjects, large samples of heparinized blood were obtained. From the separated, deproteinized and defatted plasma, all conjugated steroids were extracted by means of an XAD-2 amberlite column. Mono- and di-sulfate conjugated steroids were then separated from other free and conjugated metabolites of cortisol by means of high voltage paper electrophoresis. Individual monosulfate conjugated metabolites and glucuronosulfates were subsequently separated (as seven sub-groups) by paper chromatography. These conjugates were eluted and subjected to cleavage by solvolysis (monosulfates and disulfates) or consecutive solvolysis and β-glucuronidase hydrolysis (glucuronosulfates). The liberated steroid moieties were separated by multiple successive paper chromatographies, and identified by reverse isotope dilution technique. The individual steroids were then quantitated. Sulfate-to-steroid molar ratios were also determined on major conjugates, prior to their cleavage. The steroid metabolites found in plasma are identical with those isolated by us previously from human urine. The total sulfate fraction constituted 5.3 ± 1.9% of all conjugated metabolites of cortisol in plasma. Of those, 76% were monosulfates, 19% were glucuronosulfates, and 5% were disulfates. A large proportion of monosulfates consisted of steroids conjugated at C-21 (27%), which contrasts with glucuronide conjugates. The steroids found in largest concentrations were: 20β-cortolone-3-sulfate (17%), 5α-tetrahydrocortisol-3-sulfate (14%), 6β-hydroxycortisol-21-sulfate (9%), cortisol-21-sulfate (8%), tetrahydrocortisol-3-sulfate (7%), and 20β-dihydrocortisol-21 -sulfate (6%). From the correlation of plasma and urinary concentration of individual steroid sulfates, the following conclusions are drawn: (1) sulfoconjugation is a consistent, though quantitatively minor, pathway in the peripheral metabolism of cortisol; (2) C-21 sulfates of 4-ene-3-oxo-steroids are formed (and excreted) considerably faster than C-3 sulfates, the formation of which requires a prior reduction of ring-A; (3) C-3 sulfates of steroids with dihydroxyacetone side-chain are formed (and excreted) more slowly than the corresponding sulfates of steroids with glycerol side-chain, ergo, the reduction of 20-ketone appears to facilitate sulfoconjugation.