Microbial pathways leading to steroidal malodour in the axilla

Odorous steroids, specifically the 16-androstenes, 5alpha-androstenol and 5alpha-androstenone, are widely accepted as being contributors to underarm odour, but the precursors and pathways to these odorous steroids were unclear. This study demonstrated that the axillary microflora could only generate odorous 16-androstenes from precursors that already contain the C16 double bond, such as 5,16-androstadien-3-ol and 4,16-androstadien-3-one. In incubations containing 5,16-androstadien-3-ol, mixed populations of Corynebacterium spp., isolated from the axilla, could generate many different 16-androstene metabolites, several of which were odorous. Isolation of individual Corynebacterium strains, followed by pure culture incubations with 5,16-androstadien-3-ol, revealed organisms capable of efficient, rapid reactions. However, no single isolate could carry out a full complement of the observed biotransformations. 16-Androstene metabolites were identified by gas chromatography-mass spectrometry (GC-MS), either by comparison with known standards, or by prediction from molecular ion and fragmentation patterns. Based on detection of these metabolites, a metabolic map for axillary corynebacterial 16-androstene biotransformations was proposed, detailing potential enzyme activities. In summary, the formerly implicated 4,16-androstadien-3-one, 5alpha-androstenone and 5alpha-androstenol were detected, along with previously unreported hydroxy- and keto-substituted 16-androstenes, 16-androstatrienones and 16-androstatrienols. Additionally, many other metabolites with steroidal fragmentation patterns were present, but have remained unidentified.A key observation was that very low prevalences of microorganisms capable of biotransforming 16-androstenes were present on skin. For example, from a panel of 21 individuals, only 4 of 18 mixed populations of corynebacteria, and only 4 of 45 Corynebacterium isolates, could biotransform 5,16-androstadien-3-ol.This study has increased understanding of the metabolic pathways involved in steroidal malodour formation, and has demonstrated that the biotransformations are more complex than previously anticipated. However, it is clear that further research is required, both to assess the level of contribution of 16-androstenes to underarm odour, and to further elucidate the pathways and odour molecules formed by corynebacteria.     

The development and application of a radioimmunoassay for 5α-androst-16-en-3α-ol in plasma

At radioimmunoassay (RIA) for 5α-androst-16-en-3α-ol in human and porcine plasma has been developed. Antibodies were produced in rabbits immunized against 5α-androst-16-en-3-(O-car☐ymethyl) oxime conjugated to BSA. The antiserum cross-reacted with other 16-androstenes as follows: 5α-androst-16-en-3α-ol, 42%; 4,16-androstadien-3-one, 23.8% 5α-androst-16-en-3β-ol, 16.1% and 5,16-androstadien-3β-ol, 1.19%.Although the method gave an acceptable standard curve for 5α-androst-16-en-3-one (10–1000 pg), it was not possible to separate by t.l.c. an unknown contaminant in plasma which interfered with the RIA of the steroid. However, by exploiting the high (42%) cross-reaction with 5α-androst-16-en-3α-ol, a RIA has been developed involving a thin layer chromatographic step. Regression analysis of the data in an accuracy study gave the equation y = 0.986 x + 151.5, withr = 0.999, while the coefficients of variation of replicate assays of plasma 5α-androst-16-en-3α-ol were in the range 9.5–15.6%.The mean values for plasma 5α-androst-16-en-3α-ol in 31 healthy men and 16 healthy women were 3.08 (range 0.3–14.9) and 0.66 (range 0–2.4) ng/ml, respectively. In boars, sows and castrated male pigs, the corresponding mean values were 30.7 (range 10.9–58.9), 0.24 (range 0.08–0.77) and 0.27 (range 0.06–0.51) ng/ml, respectively.     

Steroid profiling in the study of rat testicular steroidogenesis

Twenty authentic steroids, derivatized as O-methyl oximes (MO), trimethylsilyl (TMS) ethers or as MO-TMS ethers have been subjected to capillary gas chromatography using two different columns. Virtually all of the steroid derivatives have been resolved, one difficult pair to separate being 5,16-androstadien-3 beta-ol and 5 alpha-androst-16-en-3 beta-ol on the non-selective phase OV-1. Where syn and anti forms of MO derivatives arose, these were also resolved under the conditions utilised. This technique of 'steroid profiling' has been applied to the separation and quantification of metabolites of pregnenolone which were formed during incubations of the microsomal and cytosolic fractions from rat testes. The majority of the metabolites were found in the microsomal incubation. These compounds included some odorous 16-androstenes as well as other C21 and C19 steroids, the formation of which was consistent with the 5-ene and 4-ene pathways of testosterone biosynthesis being operative. In addition, evidence was obtained for 16 alpha-hydroxylation of C21 steroids. Very much less metabolic activity was found in the cytosolic fraction of rat testes. Metabolic pathways have been proposed which both confirm and extend earlier work. We conclude that the rat testis can only form some of the odorous, possibly pheromonal, 16-androstenes and that these are quantitatively less important than in the porcine testis.     

Studies on the metabolism of 5α-androst-16-en-3-one in boar testis in vivo

1. [5α-³H]5α-Androst-16-en-3-one (5α-androstenone) was infused at a constant rate for 180min into the spermatic artery of a sexually mature boar. Samples of spermatic-venous blood were collected at 1min intervals for the first 10min of the infusion and thereafter at 15min intervals for the first hour, then at 64, 125, 155 and 172min. After infusion, the testis was removed and immediately cooled to −196°C. 2. From both the testicular tissue and the spermatic-venous plasma, endogenous and ³H-labelled androst-16-enes were isolated, characterized and quantitatively determined and their specific radioactivity was calculated. 3. The specific radioactivities of 5α-androstenore, 5α-androst-16-en-3α-ol and 5α-androst-16-en-3β-ol (an-α and an-β) in testicular tissue were different from those in the spermatic-venous plasma, suggesting that these compounds may be present in more than one compartment of the testis and differentially secreted into the spermatic-venous blood. 4. The ratios of the specific radioactivities of an-α and an-β to their respective sulphate conjugates in the testicular tissue were less than the ratios of the same compounds in the spermatic-venous plasma. 5. The patterns of secretion of these labelled compounds in the spermatic-venous blood during the period of infusion were demonstrated. 6. The urine that accumulated during the infusion was analysed and found to contain ³H-labelled an-β, conjugated as both glucuronide and sulphate, the specific radioactivities of which were determined. Little or no androst-16-enes occurred as free steroids. 7. The presence of an-β glucuronide in the urine is discussed.     

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.     

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-3 beta-ol, 4,16-androstadien-3-one, 5 alpha-androst-16-en-3-one and 5 alpha-androst-16-en-3 alpha- and 3 beta-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-androstene-3,17-dione) were 50-70 times lower than in the adult animal. The kinetic parameters and cofactor preference of the 3 alpha- and 3 beta-hydroxysteroid dehydrogenases were determined in the cytosolic, microsomal and mitochondrial fractions of neonatal porcine testes.     

Detection of methandienone (methandrostenolone) and metabolites in horse urine by gas chromatography—mass spectrometry

The metabolic transformation of methandienone (I) in the horse was investigated. After administration of a commercial drug preparation to a female horse (0.5 mg/kg), urine samples were collected up to 96 h and processed without enzymic hydrolysis. Extraction was performed by a series of solid—liquid and liquid—liquid extractions, thus avoiding laborious purification techniques. For analysis by gas chromatography—mass spectrometry, the extracts were trimethylsilylated. Besides the parent compound I and its C-17 epimer II, three monohydroxylated metabolites were identified: 6β-hydroxymethandienone (III), its C-17 epimer (IV) and 16β-hydroxy-methandienone (V). In addition, three isomers of 6β,16-dihydroxymethandienone (VIa–c) were discovered. Apparently, reduction of the δ⁴ double bond of 16β-hyroxymethandienone (V) in the horse yields 16β,17β-dihydroxy-17α-methyl-5β-androst-1-en-3-one (VII). Reduction of the isomers VIa–c results in the corresponding 6β,16,17-trihydroxy-17-methyl-5β-androst-1-en-3-ones (VIIIa–c). The data presented here suggest that screening for the isomers of VI and VIII, applying the selected-ion monitoring technique, will be the most successful way of proving methandienone administration to a horse.     

Simultaneous quantification of five odorous steroids (16-androstenes) in the axillary hair of men

Five 16-androstenes have been simultaneously quantified in extracts of the axillary hair of men (age range 18-40 years) using combined capillary gas chromatography-mass spectrometry, with specific ion monitoring. Quantities found (pmol/mg.hair, with approximate 24-h totals in parentheses) were: 5 alpha-androst-16-en-3-one, 0-15 (0-433); 4, 16-androstadien-3-one, 0-143 (0-4103); 5,16-androstadien-3 beta-ol, 0-3.5 (0-728); 5 alpha-androst-16-en-3 alpha-ol, 0-17 (0-1752) and 5 alpha-androst-16-en-3 beta-ol, 0-4 (0-416). There were no significant relationships with age of the subjects for any of the steroids measured but significant relationships were found between the amounts of the two ketones and between 5 alpha-androst-16-en-3 alpha- and 3 beta-ols. These findings may indicate the existence of a pathway of metabolism in axillary bacteria in which 4,16-androstadien-3-one is reduced to 5 alpha-androst-16-en-3-one and thence to the 3 alpha- and 3 beta-alcohols. The data are discussed in the context of axillary odour because of the low olfactory thresholds of several of the 16-androstenes measured and because of the relatively large quantities found in some subjects.     

4-Methyl-3-oxo-4-aza-5α-androst-1-ene-17β-N-aryl-carboxamides: an approach to combined androgen blockade [5α-reductase inhibition with androgen receptor binding in vitro]

4-Aza-5α-androstan-3-one 17β-(N-substituted carboxamides) are potent human type 2 5α-reductase (5aR) inhibitors with generally poor binding to the human androgen receptor (hAR). When the 17-amide N-substituent included an aromatic residue, potent dual inhibitors of both type 1 and 2 5aR are produced, but hAR binding remained poor. Tertiary-substituted-17-amides have reduced inhibition of both 5aR isozymes. The addition of an N⁴-methyl substitutent to the A-ring profoundly increased hAR affinity and the addition of unsaturation to the A-ring (Δ¹) modestly augmented hAR binding. The unsubstituted carbanilides in the Δ¹-N⁴-methyl series show some selectivity for type 1 5aR over the type 2 isozyme, whereas addition of aryl substituents, particularly at the 2-position, increased type 2 5aR binding to provide dual inhibitors with excellent hAR binding, e.g. N-(2-chlorophenyl)-3-oxo-4-methyl-4-aza-5α-androst-1-ene-17β-carboxamide (9c). Compounds of this type exhibit low nanomolar IC₅₀s for both human 5aR isozymes as well as the human androgen receptor. Kinetic analysis confirms that the prototype 9c displays reversible, competitive inhibition of both human isozymes of 5aR with K_i values of less than 10 nM. Furthermore, this compound binds to the androgen receptor with an IC₅₀ equal to 8 nM. Compounds in this series are projected to be powerful antagonists of testosterone and dihydrotestosterone action in vivo, with potential utility in the treatment of prostatic carcinoma (PC).     

Sulphoconjugation of steroids in porcine liver: Partial purification of the cytosolic sulphotransferases for pregnenolone and 5α-androst-16-en-3β-ol

Steroid sulphotransferase activities for 5α-androst-16-en-3β-ol and pregnenolone in porcine liver cytosol have been assayed using 3′-phosphoadenosine-5′-phospho[³⁵S]sulphate as sulphate donor. 5α-Androst-16-en-3β-ol sulphotransferase activity was obtained from porcine liver cytosol by gel filtration chromatography; activity was linear with time up to about 5 min., the optimum pH was near 8.0 and optimum temperature 37°C. Pregnenolone sulphotransferase activity was partially purified from porcine liver cytosol using DEAE-cellulose chromatography with an ionic gradient of KCl. This enzyme activity was linear with time up to 10 min and had optimum pH and temperature of 8.0 and 37°C, respectively.     

Axillary 5α-androst-16-en-3-one,cholesterol and squalene in men; Preliminary evidence for 5α-androst-16-en-3-one being a product of bacterial action

Twenty-four hour axillary levels of the odorous steroid 5α-androst-16-en-3-one, have been measured in six men by radioimmunoassay. Initially, no control of bacterial activity was made and conditions attempting a normal axillary environment were maintained. The level of 5α-androst-16-en-3-one was significantly higher (P = 0.028) in one axilla (“superior”) than the other (“inferior”) and levels showed considerable variation both between and within individuals. This difference between axillae was also observed for cholesterol (P = 0.0013) but not for squalene (P = 0.18). This suggests that the presence of 5α-androst-16-en-3-one in the axilla does not correlate with sebum secretion. The effect of a general germicidal agent was tested by shaving and applying Povidone-iodine to the “superior” axilla whilst treating the “inferior” axilla as a control. A highly significant drop in the level of 5α-androst-16-en-one in the “superior” axilla below that in the control axilla was obtained (P = 0.000014, double tailed, as calculated using the Fisher Exact test). Squalene and cholesterol were measured in an attempt to monitor glandular activity and their levels were not significantly affected by Povidone-iodine. It is likely, therefore, that the production of 5α-androst-16-en-3-one is from metabolism of a precursor in the axillae by skin micro-organisms.     

The role of porcine cytochrome b5A and cytochrome b5B in the regulation of cytochrome P45017A1 activities

Male pigs are routinely castrated to prevent the accumulation of testicular 16-androstene steroids, in particular 5α-androst-16-en-3-one (5α-androstenone), which contribute to an off-odour and off-flavour known as boar taint. Cytochrome P450C17 (CYP17A1) catalyses the key regulatory step in the formation of the 16-androstene steroids from pregnenolone by the andien-β synthase reaction or the synthesis of the glucocorticoid and sex steroids via 17α-hydroxylase and C17,20 lyase pathways respectively. We have expressed CYP17A1, along with cytochrome P450 reductase (POR), cytochrome b5 reductase (CYB5R3) and cytochrome b5 (CYB5) in HEK-293FT cells to investigate the importance of the two forms of porcine CYB5, CYB5A and CYB5B, in both the andien-β synthase as well as the 17α-hydroxylase and C17,20 lyase reactions. Increasing the ratio of CYB5A to CYP17A1 caused a decrease in 17α-hydroxylase (p < 0.013), a transient increase in C17,20 lyase, and an increase in andien-β synthase activity (p < 0.0001). Increasing the ratio of CYB5B to CYP17A1 also decreased 17α-hydroxylase, but did not affect the andien-β synthase activity; however, the C17,20 lyase, was significantly increased. These results demonstrate the differential effects of two forms of CYB5 on the three activities of porcine CYP17A1 and show that CYB5B does not stimulate the andien-β synthase activity of CYP17A1.     

Effect of sexual stimulation on concentrations of 5α-androstenone and testosterone in the peripheral plasma of boars reared individually

Six Yorkshire boars were reared from 107 days of age in individual pens. No female pigs were housed in the same building. When the boars were 200 days old, sows in oestrus were introduced to the pens of five boars and remained with the boars for 2 days. No oestrous sow was introduced to the pen with the sixth boar. Plasma 5α-androstenone and testosterone concentrations were low between 107 and 200 days of age in all boars. The maximum mean concentrations of these two steroids during this period were 6.18 ± 0.72 and 3.04 ± 1.02 ng/ml, respectively. Plasma 5α-androstenone concentrations increased with advancing age (P < 0.01). A similar trend was not seen for plasma testosterone concentrations. Plasma concentrations of 5α-androstenone and testosterone increased by 247 ± 27% (P < 0.02) and 1212 ± 204% (P < 0.001), respectively, in the samples drawn 24 h after the introduction of the sexually receptive sows. The maximal mean concentrations recorded following sexual stimulation were 12.90 ± 1.80 and 17.51 ± 1.96 ng/ml for 5α-androstenone and testosterone, respectively. The control boar also showed increases in plasma 5α-androstenone (221%) and testosterone (751%) concentrations in the same period, probably in response to auditory and olfactory stimuli originating in the pens nearby with introduced oestrous sows.     

Comparison of sensitivity between gas chromatography–low-resolution mass spectrometry and gas chromatography–high-resolution mass spectrometry for determining metandienone metabolites in urine

In doping control laboratories the misuse of anabolic androgenic steroids is commonly investigated in urine by gas chromatography–low-resolution mass spectrometry with selected ion monitoring (GC–LRMS–SIM). By using high-resolution mass spectrometry (HRMS) detection sensitivity is improved due to reduction of biological background. In our study HRMS and LRMS methods were compared to each other. Two different sets were measured both with HRMS and LRMS. In the first set metandienone (I) metabolites 17α-methyl-5β-androstan-3α,17β-diol (II), 17-epimetandienone (III), 17β-methyl-5β-androst-1-ene-3α,17α-diol (IV) and 6β-hydroxymetandienone (V) were spiked in urine extract prepared by solid-phase extraction, hydrolysis with β-glucuronidase from Escherichia coli and liquid–liquid extraction. In the second set the metabolites were first spiked in blank urine samples of four male persons before pretreatment. Concentration range of the spiked metabolites was 0.1–10 ng/ml in both sets. With HRMS (resolution of 5000) detection limits were 2–10 times lower than with LRMS. However, also with the HRMS method the biological background hampered detection and compounds from matrix were coeluted with some metabolites. For this reason the S/N values of the metabolites spiked had to be first compared to S/N values of coeluted matrix compounds to get any idea of detection limits. At trace concentrations selective isolation procedures should be implemented in order to confirm a positive result. The results suggest that metandienone misuse can be detected by HRMS for a prolonged period after stopping the intake of metandienone.     

Purification of 5β-reductase from hepatic cytosol fraction of chicken

From the cytosol fraction (supernatant fluid at 105,000 g) of chicken liver, 4-en-3-oxosteroid 5β-reductase (EC 1.3.1.23) was purified by ammonium sulfate precipitation, followed by Butyl Toyopearl, DEAE-Sepharose, Sephadex G-75 and hydroxylapatite column chromatographies. The enzyme activity was quantitated from amount of the 5β-reduced metabolites derived from [4-¹⁴C]testosterone. During the purification procedures, 17β-hydroxysteroid dehydrogenase which was present in the cytosol fraction was separated from 5β-reductase fraction by the Butyl Toyopearl column chromatography. By the DEAE-Sepharose column chromatography, 3α- and 3β-hydroxysteroid dehydrogenases were able to be removed from 5β-reductase fraction. The final enzyme preparation was apparently homogenous on SDS-polyacrylamide gel electrophoresis. Purification was about 13,600-fold from the hepatic cytosol. The molecular weight of this enzyme was estimated as 37,000 Da by SDS-polyacrylamide gel electrophoresis and also by Sephadex G-75 gel filtration. For 5β-reduction of 4-en-3-oxosteroids, such as testosterone, androstenedione and progesterone, NADPH was specifically required as cofactor. K_m of 5β-reductase for NADPH was estimated as 4.22 × 10⁻⁶M and for testosterone, 4.60 × 10⁻⁶M. The optimum pH of this enzyme ranged from pH 5.0 to 6.5 and other enzymic properties of the 5β-reductase were examined.     

Testosterone metabolism by isolated human axillary corynebacterium spp.: A gaschromatographic mass-spectrometric study

Transformations of [4-¹⁴C]testosterone have been studied in Corynebacterium spp. isolated from the axillae of men. Metabolites have been separated by TLC and capillary gas chromatography and have been identified by gas chromatography-mass spectrometry (GC-MS). The introduction of a clean-up step using Florisil columns, prior to TLC, removed Tween-80 which co-extracted from the medium with the metabolites. This procedure greatly improved TLC resolution.Testosterone was converted enzymically to 5α- and 5β-DHT, identification being assisted by the inclusion of [3,4-¹³C]testosterone in some incubations. Other metabolites formed enzymically were 4-androstene-3,17-dione, 5β-androstane-3,17-dione, 3β-hydroxy-5β-androstan-17-one and 5β-androstane-3α.l7α-diol. Some spontaneous breakdown of [¹⁴C]testosterone occurred giving rise to 5α(β)-DHT, androstanediol and a monohydroxy-diketo-androstene, the latter being reduced enzymically to 2 monohydroxy-diketo-androstanes. Under the conditions used, no clear evidence has been obtained for the formation of 5α-androst-16-en-3-one, an odorous steroid that occurs in the axillae of men; the possible reasons why we were unable to prove the biosynthesis of this compound are discussed.     

Microbial transformation of isosteviol oxime and the inhibitory effects on NF-κB and AP-1 activation in LPS-stimulated macrophages

Microbial transformation of isosteviol oxime (ent-16-E-hydroxyiminobeyeran-19-oic acid) (2) with Aspergillus niger BCRC 32720 and Absidia pseudocylindrospora ATCC 24169 yielded several compounds. In addition to bioconverting the d-ring to lactone and lactam moieties, 4α-carboxy-13α-hydroxy-13,16-seco-ent-19-norbeyeran-16-oic acid 13,16-lactone (7) and 4α-carboxy-13α-amino-13,16-seco-ent-19-norbeyeran-16-oic acid 13,16-lactam (10), one known compound, ent-1β,7α-dihydroxy-16-oxo-beyeran-19-oic acid (6), and five new compounds, ent-7α-hydroxy-16-E-hydroxyiminobeyeran-19-oic acid (3), ent-1β,7α-dihydroxy-16-E-hydroxyiminobeyeran-19-oic acid (4), ent-1β-hydroxy-16-E-hydroxyiminobeyeran-19-oic acid (5), ent-8β-cyanomethyl-13-methyl-12-podocarpen-19-oic acid (8), and ent-8β-cyanomethyl-13-methyl-13-podocarpen-19-oic acid (9), were isolated from the microbial transformation of 2. Elucidation of the structures of these isolated compounds was primarily based on 1D and 2D NMR, and HRESIMS data, and 3–5 were further confirmed by X-ray crystallographic analyses. Additionally, the inhibitory effects of all of these compounds were evaluated on NF-κB and AP-1 activation in LPS-stimulated RAW 264.7 macrophages. Among the compounds tested, 5 and 10 significantly inhibited NF-κB activation, with 5 showing equal potency to dexamethasone; 3 and 6–9 significantly inhibited AP-1 activation, particularly 8, which showed more inhibitory activity than dexamethasone.     

Nor-sterols in Axinella proliferans, sponge from the Indian Ocean

An examination of the sterol mixture of the sponge Axinella proliferans collected in the Indian Ocean led to the isolation of nine A-nor-sterols, including two rare nor-sterols with a D-ring unsaturation. The known 3β-(hydroxymethyl)-A-nor-5α-cholest-15-ene has been identified by a comparison with mass spectrum and ¹H and ¹³C nuclear magnetic resonance (NMR) data of the sterol isolated from Homoaxinella trachys, a marine sponge collected in the Indian Ocean. A new sterol, 3β-(hydroxymethyl)-A-nor-5α-cholest-14-ene-16α-ol, has been identified by their mass and two-dimensional NMR spectra compared with those of the D-ring unsaturated sterol, 5α-cholest-14-ene-3β,16α-diol isolated from the Mediterannean sponge Topsentia aurantiaca.     

Investigation of Testosterone,Androstenone, and Estradiol Metabolism in HepG2 Cells and Primary Culture Pig Hepatocytes and Their Effects on 17βHSD7 Gene Expression

Steroid metabolism is important in various species. The accumulation of androgen metabolite, androstenone, in pig adipose tissue is negatively associated with pork flavor, odour and makes the meat unfit for human consumption. The 17β-hydroxysteroid dehydrogenase type 7 (17βHSD7) expressed abundantly in porcine liver, and it was previously suggested to be associated with androstenone levels. Understanding the enzymes and metabolic pathways responsible for androstenone as well as other steroids metabolism is important for improving the meat quality. At the same time, metabolism of steroids is known to be species- and tissue-specific. Therefore it is important to investigate between-species variations in the hepatic steroid metabolism and to elucidate the role of 17βHSD7 in this process. Here we used an effective methodological approach, liquid chromatography coupled with mass spectrometry, to investigate species-specific metabolism of androstenone, testosterone and beta-estradiol in HepG2 cell line, and pig cultured hepatocytes. Species- and concentration-depended effect of steroids on 17βHSD7 gene expression was also investigated. It was demonstrated that the investigated steroids can regulate the 17βHSD7 gene expression in HepG2 and primary cultured porcine hepatocytes in a concentration-dependent and species-dependent pattern. Investigation of steroid metabolites demonstrated that androstenone formed a 3′-hydroxy compound 3β-hydroxy-5α-androst-16-ene. Testosterone was metabolized to 4-androstene-3,17-dione. Estrone was found as the metabolite for β-estradiol. Inhibition study with 17βHSD inhibitor apigenin showed that apigenin didn’t affect androstenone metabolism. Apigenin at high concentration (50 µM) tends to inhibit testosterone metabolism but this inhibition effect was negligible. Beta-estradiol metabolism was notably inhibited with apigenin at high concentration. The study also established that the level of testosterone and β-estradiol metabolites was markedly increased after co-incubation with high concentration of apigenin. This study established that 17βHSD7 is not the key enzyme responsible for androstenone and testosterone metabolism in porcine liver cells.     

Role of Neutral Metabolites in Microbial Conversion of 3β-Acetoxy-19-Hydroxycholest-5-Ene into Estrone

Biotransformation of 3β-acetoxy-19-hydroxycholest-5-ene (19-HCA, 6 g) by Moraxella sp. was studied. Estrone (712 mg) was the major metabolite formed. Minor metabolites identified were 5α-androst-1-en-19-ol-3,17-dione (33 mg), androst-4-en-19-ol-3,17-dione (58 mg), androst-4-en-9α,19-diol-3,17-dione (12 mg), and androstan-19-ol-3,17-dione (1 mg). Acidic metabolites were not formed. Time course experiments on the fermentation of 19-HCA indicated that androst-4-en-19-ol-3,17-dione was the major metabolite formed during the early stages of incubation. However, with continuing fermentation its level dropped, with a concomitant increase in estrone. Fermentation of 19-HCA in the presence of specific inhibitors or performing the fermentation for a shorter period (48 h) did not result in the formation of acidic metabolites. Resting-cell experiments carried out with 19-HCA (200 mg) in the presence of α,α′-bipyridyl led to the isolation of three additional metabolites, viz., cholestan-19-ol-3-one (2 mg), cholest-4-en-19-ol-3-one (10 mg), and cholest-5-en-3β,19-diol (12 mg). Similar results were also obtained when n-propanol was used instead of α,α′-bipyridyl. Resting cells grown on 19-HCA readily converted both 5α-androst-1-en-19-ol-3,17-dione and androst-4-en-19-ol-3,17-dione into estrone. Partially purified 1,2-dehydrogenase from steroid-induced Moraxella cells transformed androst-4-en-19-ol-3,17-dione into estrone and formaldehyde in the presence of phenazine methosulfate, an artificial electron acceptor. These results suggest that the degradation of the hydrocarbon side chain of 19-HCA does not proceed via C₂₂ phenolic acid intermediates and complete removal of the C₁₇ side chain takes place prior to the aromatization of the A ring in estrone. The mode of degradation of the sterol side chain appears to be through the fission of the C₁₇-C₂₀ bond. On the basis of these observations, a new pathway for the formation of estrone from 19-HCA in Moraxella sp. has been proposed.