CAPILLARY GAS-CHROMATOGRAPHY WITH CHEMICAL-IONIZATION NEGATIVE-ION MASS-SPECTROMETRY IN THE IDENTIFICATION OF ODOROUS STEROIDS FORMED IN METABOLIC STUDIES OF THE SULFATES OF ANDROSTERONE, DHA AND 5-ALPHA-ANDROST-16-EN-3-BETA-OL WITH HUMAN AXILLARY BACTERIAL ISOLATES

The products of metabolism of the sulphates (0.5 mu mol/1) of androste rone, dehydroepiandrosterone (DHA) and 5-alpha-androst-16-en-3-beta-ol have been investigated after incubation with 72 h cultures of human a xillary bacterial isolates for 3 days at 37 degrees C. The medium used , tryptone soya broth (TSB), contained yeast extract and Tween 80. The isolates used were Coryneform F1 (known previously to metabolize test osterone and to be involved in under-arm odour (UAO) production, i.e. UAO +ve), Coryneform F46 (inactive in both the testosterone metabolism and UAO tests, i.e. UAO -ve) and Staphylococcus hominis/epidermidis ( IIR3). Control incubations of TSB alone, TSB plus each of the steroid sulphates and TSB plus each of the bacterial isolates were also set up . After termination of reactions and addition of internal standards, 5 -alpha-androstan-3-beta-ol and 5-alpha-androstan-3-one (50 ng each), e xtracted and purified metabolites were subjected to combined gas chrom atography mass spectrometry with specific ion monitoring. Steroidal ke tones were derivatized as their O-pentafluorobenzyl oximes; steroidal alcohols (only androst-16-enols in this study) were derivatized as the ir tert-butyldimethylsilyl ethers. Analysis was achieved by negative i on chemical ionization mass spectrometry for the pentafluorobenzyl oxi mes at [M-20](-) and electron impact positive ion mass spectrometry fo r the tert-butyldimethylsilyl ethers at [M-57](+). The incubation brot h contained two compounds which had gas chromatographic and mass spect rometric properties identical to those of DHA and 4-androstenedione. I t was not possible, therefore, to show unequivocally that DHA sulphate (DHAS) was converted microbially into DHA, although this is implied b y the finding of small quantities of testosterone and Sa-dihydrotestos terone in incubations with F1. With androsterone S, no free androstero ne was recorded and only very small (5 pg or less) amounts of testoste rone. Two odorous steroids, androsta-4,16-dien-3-one and 5-alpha-andro st-2-en-17-one (Steroid I) were formed (mean quantities 40 and 45 pg, respectively). The sulphate of 5-alpha-androst-16-en-3-beta-ol was met abolized with F1 into large quantities of the odorous steroids, 5-alph a-androst-16-en-3-one and Steroid I. In addition, much smaller quantit ies of androsta-4,16-dien-3-one were formed. In contrast, incubations of DHAS with F46 resulted in no metabolites except, possibly, DHA, but the sulphate moiety of androsterone S was also cleaved to yield the f ree steroid together with large amounts of Steroid I. In incubations o f DHAS and androsterone S with F1, no 16-unsaturated steroids were for med, although 5-alpha-androst-16-en-3-beta-yl S was de-sulphated and t he free steroid further metabolized. No evidence was obtained for andr ost-16-ene metabolism in incubations with F46. In incubations with S. hominis/epidermidis (IIR3), androsterone S was converted into androste rone and, in high yield, to Steroid I plus some 5-alpha-androst-16-en- 3-one. Both DHAS and androsterone S were converted into androst-16-eno ls. Sulphatase activity was also manifested when 5-alpha-androst-16-en -3-beta-yl S was utilized as substrate with IIR3, large quantities of Steroid I and 5-alpha-androst-16-en-3-one being formed, together with further metabolism of androst-16-enes. In view of the fact that both D HAS and androsterone S occur in apocrine sweat, the metabolism of thes e endogenous substrates by human axillary bacteria to several odorous steroids may have important implications in the context of human odour formation. (C) 1997 Elsevier Science Ltd. All rights reserved.