D. B. Gower

Comparison of 16-Androstene steroid concentrations in sterile apocrine sweat and axillary secretions: Interconversions of 16-Androstenes by the axillary microflora—a mechanism for axillary odour production in man?

The concentrations of five 16-androstene steroids were determined, by a GC-MS method, in freshly-produced apocrine sweat (adrenaline-induced), in 8 men and 2 women. The ranges of concentrations (nmol/microliter) in apocrine sweat were: 5 alpha-androst-16-en-3-one (5 alpha-A), 0.1-2.0 and 4,16-androstadien-3-one (androstadienone), 0-1.9, 5,16-Androstadien-3 beta-ol (androstadienol) was also found in 5 of the subjects (range 0.05-1.05). 5 alpha-Androst-16-en-3 alpha- or 3 beta-ols [3 alpha (beta)-androstenols] were only found in small amounts (< 0.1 nmol/microliters) in a few subjects. In the second study, prior to apocrine sweat collection (adrenaline injection), the axillary skin of 6 of the male subjects was washed with diethyl ether on an adjacent site of the axillary vault. The concentrations of 16-androstenes were compared in the ethereal extracts and apocrine sweat. The former contained detectable levels (pmol/cm2) of androstadienone (17.9 +/- 2.4), 3 alpha-androstenol (6.9 +/- 3.7), 3 beta-androstenol (1.8 +/- 1.0) and androstadienol (1.9 +/- 0.5) (means +/- SEM) in all 6 subjects. All but 1 subject also had 5 alpha-androstenone, the mean value for the others being 2.5 +/- 0.6. The axillary skin levels of 3 alpha- and 3 beta-androstenols, androstadienol and, in 3 subjects, androstadienone exceeded those in the apocrine sweat obtained from the same subjects, whereas levels of 5 alpha-androstenone in the skin extracts were all lower than in apocrine sweat samples, when related to the corresponding areas of skin sampled. The metabolism of 16-androstenes was studied in vitro in the presence of two aerobic coryneform bacteria, previously shown to metabolize testosterone as well as being capable of producing odour from extracts of axillary sweat in an odour-generation test. Although both coryneforms caused complex metabolic reactions and were capable of oxidation or reduction at C-3 and C-4, the overall direction favoured reduction. For example, large quantities of the more odorous 5 alpha-androstenone and 3 alpha-androstenol were formed from androstadienol and androstadienone. In contrast, strains of corynebacteria, unable to produce odour and incapable of metabolizing testosterone, were also unable to metabolize 16-androstenes.(ABSTRACT TRUNCATED AT 400 WORDS)

IN-VITRO AND IN-VIVO STUDIES OF HUMAN AXILLARY ODOUR AND THE CUTANEOUS MICROFLORA

The axillary microflora of 34 male subjects were studied in relation to their underarm odour intensity. The predominant groups of micro‐organisms were aerobic coryneforms, Micrococcaceae and propionibacteria. There was no competition for habitat between these groups (Fishers exact test P <0·05). There was an association between the population density of aerobic coryneforms and the intensity of odour (Spearman P = 0.001). Dominance of aerobic coryneforms within the axillary microflora was associated with high odour intensity (x2, P= 0.005). An in‐vitro odour model was developed using a diethyl ether extract of axillary skin incubated with test bacteria. Underarm odour was produced exclusively by aerobic coryneform bacteria. Of aerobic coryneforms, 71.4% were odour producers and these were identified as Corynebacterium xerosis.

The skin microflora and the formation of human axillary odour

We have examined the relationship between human axillary skin microflora and underarm odour (UAO), in particular, the ability of cutaneous bacteria to transform steroids. A study was made of bacterial population density and odour intensity of the axillae of 34 normal male subjects. There was a statistically significant association between population density of aerobic coryneform bacteria and UAO intensity. No associations could be found between population densities of staphylococci, micrococci or propionibacteria and UAO intensity. An in vitro model for formation of UAO was developed, and used to test individual bacterial isolates. Only aerobic coryneforms could produce axillary odour in vitro, most notably C. xerosis. Many aerobic coryneforms could transform testosterone, the principal metabolites being 5α‐ and 5β‐DHT, androstenedione, and 5α‐ and 5β‐androstanedione. UAO positive coryneforms were more metabolically active than UAO negative bacteria. Micrococci also transformed testosterone to androstenedione, whilst staphylococci and propionibacteria could not metabolize it. A hypothesis for the role of aerobic coryneforms in the formation of human axillary odour is discussed.

The use of human gingival fibroblasts in culture for studying the effects of phenytoin on testosterone metabolism.

In initial experiments, monolayer cultures of human gingival fibroblasts from healthy male and female subjects were incubated for various time intervals with [4-14C]-testosterone. This was rapidly taken up by the cells to reach 1.8 fmol/50,000 cells by 2 h. At 6, 12 and 24 h, the values were considerably lower (0.1-0.2 fmol/50,000 cells). In order to maintain a sufficient intracellular concentration of testosterone, unlabelled testosterone was incubated in the presence of [14C]-testosterone. This gave optimum yields of metabolites, which were separated by thin-layer chromatography and provisionally identified by comparison of their mobilities with those of authentic steroids. Final characterization of 5 alpha-dihydrotestosterone was achieved by combined capillary gas chromatography-mass spectrometry. The metabolites of testosterone were 5 alpha-dihydrotestosterone (5 alpha-DHT), 4-androstenedione, 5 alpha-androstanedione and 5 alpha-androstanediols, but the quantities formed varied with different cell lines. A similar pattern of metabolites was noted for minced human gingival tissue. Low concentrations of phenytoin generally increased the production of 5 alpha-DHT and 4-androstendione but there were marked variations between individual cell lines with regard to the magnitude of stimulation. Higher concentrations of phenytoin generally caused inhibition of steroid formation but the concentration required for this again varied with different cell lines. Thus human gingival fibroblasts in culture provide a suitable model for the study of testosterone metabolism and of the effects of drugs such as phenytoin. Variation in these effects may be reflected in individual susceptibility to phenytoin-induced gingival overgrowth.

GC-MS studies of 16-androstenes and other C19 steroids in human semen

Human semen was examined for the presence of 16-androstenols, 16-androstenones and androgens. Extracts were analysed by gas chromatography-mass spectrometry after derivatization of steroids under study. In a qualitative study, 5 alpha-androst-16-en-3 alpha- and 3 beta-ols, 5,16-androstadien-3 beta-ol and 5 alpha-androstan-3 beta-ol were detected in a semen pool A. Hydroxyl groups were converted to tert-butyldimethylsilyl ethers, the ions selected for monitoring being [M-57]+, consistent with loss of the tert-butyl group. For a more detailed quantitative study, a second semen pool B was used. In this case, all hydroxyl groups were converted to trimethylsilyl ethers, while oxo groups were not derivatized. As with semen pool A, separation of steroids was achieved using capillary gas chromatography with appropriate temperature programming. Quantification was carried out by mass spectrometry using selected ion monitoring of two significant ions and appropriate internal standards. The following steroids were identified at the concentrations indicated: 5 alpha-androst-16-en-3 alpha- and 3 beta-ols and 5,16-androstadien-3 beta-ol (concentration range, 0.5-0.7 ng/ml). 5 alpha-Androst-16-en-3-one and 4,16-androstadien-3-one were also present at levels of 0.7-0.9 ng/ml. Two androgens, testosterone and 5 alpha-dihydrotestosterone were found at concentrations of 0.5 and 0.3 ng/ml, respectively. These data, showing the presence of 16-androstenes and androgens in human semen, appear to be consistent with testicular formation of these steroids. The possible significance of the odorous 16-androstenes is discussed.

Capillary gas chromatography with chemical ionization negative ion mass spectrometry in the identification of odorous steroids formed in metabolic studies of the sulphates of androsterone, DHA and 5α-androst-16-en-3β-ol with human axillary bacterial isolates

The products of metabolism of the sulphates (0.5 micromol/l) of androsterone, dehydroepiandrosterone (DHA) and 5alpha-androst-16-en-3beta-ol have been investigated after incubation with 72 h cultures of human axillary 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 testosterone 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, 5alpha-androstan-3beta-ol and 5alpha-androstan-3-one (50 ng each), extracted and purified metabolites were subjected to combined gas chromatography-mass spectrometry with specific ion monitoring. Steroidal ketones were derivatized as their O-pentafluorobenzyl oximes; steroidal alcohols (only androst-16-enols in this study) were derivatized as their tert-butyldimethylsilyl ethers. Analysis was achieved by negative ion chemical ionization mass spectrometry for the pentafluorobenzyl oximes at [M-20]- and electron impact positive ion mass spectrometry for the tert-butyldimethylsilyl ethers at [M-57]+. The incubation broth contained two compounds which had gas chromatographic and mass spectrometric properties identical to those of DHA and 4-androstenedione. It was not possible, therefore, to show unequivocally that DHA sulphate (DHAS) was converted microbially into DHA, although this is implied by the finding of small quantities of testosterone and 5alpha-dihydrotestosterone in incubations with F1. With androsterone S, no free androsterone was recorded and only very small (5 pg or less) amounts of testosterone. Two odorous steroids, androsta-4,16-dien-3-one and 5alpha-androst-2-en-17-one (Steroid I) were formed (mean quantities 40 and 45 pg, respectively). The sulphate of 5alpha-androst-16-en-3beta-ol was metabolized with F1 into large quantities of the odorous steroids, 5alpha-androst-16-en-3-one and Steroid I. In addition, much smaller quantities 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 free steroid together with large amounts of Steroid I. In incubations of DHAS and androsterone S with F1, no 16-unsaturated steroids were formed, although 5alpha-androst-16-en-3beta-yl S was de-sulphated and the free steroid further metabolized. No evidence was obtained for androst-16-ene metabolism in incubations with F46. In incubations with S. hominis/epidermidis (IIR3), androsterone S was converted into androsterone and, in high yield, to Steroid I plus some 5alpha-androst-16-en-3-one. Both DHAS and androsterone S were converted into androst-16-enols. Sulphatase activity was also manifested when 5alpha-androst-16-en-3beta-yl S was utilized as substrate with IIR3, large quantities of Steroid I and 5alpha-androst-16-en-3-one being formed, together with further metabolism of androst-16-enes. In view of the fact that both DHAS and androsterone S occur in apocrine sweat, the metabolism of these endogenous substrates by human axillary bacteria to several odorous steroids may have important implications in the context of human odour formation.

Chromatographic separation of C19-16-dehydro-steroids

Abstract The chromatography of a closely related series of 16-dehydro-steroids and their acetates on thin layer plates and silicic acid-impregnated paper is described. Fairly rapid and easy separations of most of the compounds studied can be achieved although the separation of androst-16-en-3β-ol and androsta-5, 16-dien-3β-ol is only obtained by an overrunning technique. Silicic acid-impregnated paper appears to be free from disadvantages associated with papers impregnated with other stationary phases such as phenylcellosolve, liquid paraffin and kerosene. The use of a number of colour reagents is described, including phosphomolybdic acid, phosphotungstic acid, resorcylaldehyde—sulphuric acid, the Allen reagent and uranyl nitrate in sulphuric acid. Some of the steroids give specific colours which may be useful in their identification.

Biosynthesis of androgens and pheromonal steroids in neonatal porcine testicular preparations

The biosynthesis of testosterone and 4‐androstene‐3,17‐dione and some 16‐androstenes has been studied in homogenates or subcellular fractions of testes from 3‐week‐old Landrace piglets. Pregnenolone was converted into 5,16‐androstadien‐3β‐ol, 4,16‐androstadien‐3‐one, 5α‐androst‐16‐en‐3‐one and 5α‐androst‐16‐en‐3α‐ and 3β‐ols, but the quantities were some 50 times less than those formed in the mature boar testis. Androgens were also formed in the microsomal fractions but the quantities of 4‐androstene‐3,17‐dione (from side‐chain cleavage of 17‐hydroxyprogesterone) and of testosterone (from reduction of 4‐andro stene‐3,17‐dione) were 50–70 times lower than in the adult animal. The kinetic parameters and cofactor preference of the 3α‐ and 3β‐hydroxysteroid dehydrogenases were determined in the cytosolic, microsomal and mitochondrial fractions of neonatal porcine testes.

Use of gas chromatographic-mass spectrometric techniques in studies of androst-16-ene and androgen biosynthesis in human testis; cytosolic specific binding of 5α-androst-16-en-3-one

Homogenates of histologically normal human testis from three men were incubated separately with pregnenolone, 16-dehydropregnenolone, 5alpha-pregnane-3,20-dione, 3beta-hydroxy-5alpha-pregnan-20-one and androsta-5,16-dien-3beta-ol (androstadienol) in the presence of NADPH in a study of androst-16-ene and androgen biosynthesis. After the addition of internal standards and initial extraction and purification, metabolites were identified using gas chromatography-mass spectrometry (GC-MS) and monitoring selectively for three principal ions in each case at the appropriate GC retention time. Quantification was achieved by comparison with calibration lines for authentic steroids, together with the appropriate internal standards, prepared by monitoring three ion fragments for each analyte. In all experiments, androstadienol was found to be the major androst-16-ene metabolite of pregnenolone (seven times the control, i.e. endogenous, quantity; 19.8 +/- 3 ng/100 mg homogenate protein, mean +/- SEM, n = 9). Pregnenolone was also converted to androsta-4,16-dien-3-one (androstadienone) with three times the endogenous quantity (44 +/- 10 ng/100 mg homogenate protein, mean +/- SEM, n = 9) being formed. The formation of testosterone occurred only in trace amounts in the incubations of testis taken from one man (a 69-yr-old) but appreciable yields (six times endogenous levels 90 +/- 7 ng/100 mg homogenate protein, mean +/- SEM, n = 9) were found with testes from two younger men. Only traces of 5alpha-dihydrotestosterone were detected. Using androstadienol as the substrate, androstadienone was shown to be the major metabolite (approximately 10 times greater than control incubations) together with 5alpha-androst-16-en-3alpha- and 3beta-ols at approximately twice the endogenous quantities (5 ng/100 mg homogenate protein). In some incubations with androstadienol, 5alpha-androst-16-en-3-one (5alpha-androstenone) was formed (32 +/- 1 ng/100 mg homogenate protein/h; mean +/- SEM, n = 3); surprisingly, no endogenous 5alpha-androstenone could be detected. No evidence was obtained for the production of testosterone or 5alpha-DHT from androstadienol. Using cytosolic fractions of human testis, specific (displaceable) binding of 5alpha-androstenone was determined, with binding sites of approximately 200 fmol/mg tissue and a Ka of approximately 8 nmol/l.

Identification of 16-dehydropregnenolone as an intermediate in 16-androstene biosynthesis in neonatal porcine testicular microsomes

The biosynthesis of 16‐androstenes has been studied in neonatal porcine testicular microsomes using 17‐hydroxypregnenolone and 16‐dehydropregnenolone, separately, as substrates. The metabolites formed after microsomal incubation with these substrates were purified, derivatized as O‐methyloxime‐trimethylsilyl ethers and analysed by capillary gas chromatography‐mass spectrometry. In the incubation of 17‐hydroxy‐pregnenolone with microsomes, 16‐dehydropregnenolone was identified as an intermediate in the biosynthesis of 16‐androstenes. Further microsomal incubation of 16‐dehydropregnenolone has established the intermediary role of this steroid in the production of 16‐androstenes.