THE IDENTIFICATION OF NOVEL STEROID N-ACETYLGLUCOSAMINIDES IN THE URINE OF PREGNANT-WOMEN

A series of pregnanediols and pregnanetriols doubly conjugated with N- acetylglucosamine and glucuronic or sulfuric acid has been identified in urine from pregnant women. Steroid conjugates were separated by ion -exchange chromatography and the glucuronide and monosulfate fractions were analysed by fast atom bombardment mass spectrometry. After remov al of the acid moiety, the neutral steroids were isolated, derivatized , and analysed by gas chromatography-mass spectrometry (GC-MS). The an alyses revealed the presence of steroids conjugated with N-acetylhexos amine both in the glucuronide and the monosulfate fractions. Following enzyme hydrolysis, the sugar was identified by GC-MS as N-acetylgluco samine (GlcNAc). The major steroid conjugated with GlcNAc both in the glucuronide and monosulfate fractions was identified as 5 alpha-pregna ne-3 alpha,20 alpha-diol. 5 beta-Pregnane-3 alpha,20 alpha-diol was al so present as a GlcNAc conjugate in both fractions whereas a GlcNAc co njugate of 5 alpha-pregnane-3 beta,20 alpha-diol was only found in the sulfate fraction. 5 alpha-Pregnane-3 alpha,20 alpha,21-triol was a do uble conjugate with GlcNAc in the sulfate fraction whereas a pregnane- 2,3,20-triol was a double conjugate in the glucuronide fraction. The p ositions of conjugation were determined by collision-induced dissociat ion of the pseudomolecular anions produced by fast atom bombardment io nization. The sulfate and glucuronic acid moieties were located at C-3 and N-acetylglucosamine at C-20. An alternative localization of GlcNA c at C-21 of 5 alpha-pregnane-3 alpha,20 alpha,21-triol cannot be excl uded. Judging from the enzymatic hydrolysis of the conjugates, the sug ar was attached in beta-glycosidic Linkage. The mean excretion of N-ac etylglucosaminides of the pregnanediols and pregnanetriols was 32.2 mu mol/g creatinine (range 17.9-49.1 mu mol) in five healthy women in th e 38th-39th week of pregnancy. The mean excretion of 5 beta-pregnane-3 alpha,20 alpha-diol glucuronide in the same women was 71 mu mol/g cre atinine, (range 27-127 mu mol). This indicates that conjugation with N -acetylglucosamine constitutes a quantitatively important pathway of p rogesterone metabolism in human pregnancy. Copyright (C) 1996 Elsevier Science Ltd.