Androstenedione Research Articles

Relationship of serum sex-steroid hormones and prostate volume in African American men.

Previous epidemiologic investigations of the associations of sex-steroid hormones and benign prostatic hyperplasia (BPH) have focused on predominately white populations. The objective of this study was to evaluate potential associations of body mass index (BMI), cigarette smoking, use of alcohol, and endogenous sex-steroid hormones with prostate volume in a population-based sample of African American (AA) men, ages 40-79 yr. A total of 369 AA men without clinical evidence of prostate cancer were identified in the Flint Men's Health Study by using a population-based sampling procedure. All subjects underwent a complete urologic evaluation that included prostate volume determination by transrectal ultrasonography and serum assays for androgens and estrogens. After age adjustment, BMI (weight (kg)/height (m)2) was positively correlated with increasing levels of androstanediol glucuronide (AG), estradiol (E2), estrone sulfate (E1S), and the ratios of E2:total testosterone (TT) and E2:free testosterone (FT); however, increasing BMI was negatively correlated with androstenedione (AD), FT, TT, and sex hormone-binding globulin (SHBG). Multivariable regression models demonstrated that prostate volume increased with age (P < 0.001) and BMI (P = 0.02) and decreased with increasing levels of SHBG (P = 0.01). Larger prostatic volumes were also marginally associated with increasing levels of TT (P = 0.058). Circulating serum levels of SHBG and endogenous sex-steroid hormones are correlated with prostate volume and potentially impact the natural history of BPH. However, longitudinal studies are needed to demonstrate the temporal relationships of hormones and growth factors in the pathogenesis of BPH in AA men.

Effect of phase composition on the whole-cell bioconversion of β-sitosterol in biphasic media

The multi-step bioconversion of β-sitosterol by Mycobacterium sp. NRRL B-3805 for the formation of androstenedione (AD) as major product, a key intermediate in the production of therapeutic steroids, is a well established industrial application of a biocatalytic process. In the present work, this sterol side-chain cleavage was selected as model system for a study of whole-cell biocatalyst operational stability in prolonged bioconversions in two-phase aqueous-organic media. The aim was to evaluate the effect of phase composition in the process yields by using four high logPoctanol phthalates as organic phases and phosphate buffer and nutritional media as aqueous phases. The effect of a non-ionic surfactant added to the aqueous phase was also studied. Results show no relation between bioconversion yields and solvent logPoctanol values, suggesting that, in the used range, solvent molecular structures and the resulting specific interactions with the cell envelope are determining for the biocatalyst behaviour. Different responses were obtained from the use of different aqueous media, the complex nutritional media leading to the highest conversion yields, while the lowest were observed with the mineral nutritional media. The effect of surfactant addition in the bioconversion yields was apparently not significant. In the systems with bis-(2-ethylhexyl) and bis-(3,5,5-trimethylhexyl) phthalates as the organic phases, the fed-batch operational mode allowed the maintenance of stable biocatalytic activity levels up to at least 6 days of operation, with high androstenedione yields.

Human 17βHSD type 2 reversibly interconverts androgens and estrogens at equal rates in vivo

Objective: The 17β-hydroxysteroid dehydrogenase enzymes (17βHSDs) are responsible for interconversion between the hydroxy- and ketosteroid pairs using the cofactors NAD(P)H/NAD(P)+. Thus, the 17βHSDs complete biosynthetic pathways to active hormones and regulate the intracellular concentrations of active and inactive forms of steroids. In vitro, these enzymes drive the reaction in either oxidative or reductive directions, depending on the pH and cofactors. However, in vivo, these enzymes appear to function unidirectionally, either as a reductase or as an oxidative dehydrogenase. When studied in transfected HEK-293 cells, 17βHSD type 2 converts estradiol (E2) almost completely to estrone (E1) but appears not to convert E2 to E1, suggesting that the 17βHSD type 2 functions exclusively as a steroid dehydrogenase in living cells. Likewise, 17βHSD type 2 also converts testosterone (T) almost completely to androstenedione (AD) without measurable activity in the reverse direction. Conversely, 17βHSDs types 1 and 3 principally reduce E1 to E2 and AD to T, respectively.

Premature pubarche in girls is associated with functional adrenal but not ovarian hyperandrogenism

Objective: We hypothesized that there would be evidence of functional ovarian hyperandrogenism in girls with premature pubarche (PP) at diagnosis. Methods: White girls <8 years of age and black girls <6 years with PP (n = 15) were studied. Prepubertal girls (n = 13; 5.3-10.9 years) and early pubertal girls (n = 8) served as control subjects. The biochemical marker for functional ovarian hyperandrogenism was the 17-hydroxyprogesterone (17-OHP), androstenedione (AD), and estradiol (E2) response to subcutaneous leuprolide during adrenal suppression with dexamethasone. This was studied in girls with PP and in control subjects. Results: ACTH stimulated 17-hydroxypregnenolone (17-OH Preg), dehydroepiandrosterone (DHEA), and AD levels, and 17-OH Preg:17-OHP and DHEA:AD ratios were significantly higher in girls with PP than in prepubertal control subjects (n = 18) (P ≤.003). The ovarian response to leuprolide stimulation was comparable in girls with PP and prepubertal control subjects, but the response in prepubertal study subjects was significantly lower than in pubertal control subjects (P =.016 for Δ17-OHP, P =.001 for ΔAD, and P =.026 for ΔE2). Conclusions: Contrary to the hypothesis, PP in girls was not associated with prepubertal evidence of ovarian hyperandrogenism but was associated with functional adrenal hyperandrogenism. (J Pediatr 2002;141:91-8)

Biosynthesis of estradiol-17beta in the ovarian follicles of the red seabream Pagrus major during vitellogenesis

: The red seabream Pagrus major is a useful experimental fish for studying the endocrine control of oogenesis in teleosts. This study investigated the steroidogenic pathway for estradiol-17β (E2) biosynthesis in the ovarian follicles of red seabream. Intact follicles were isolated during vitellogenesis and incubated in vitro with different radiolabeled steroid precursors. When 17-hydroxyprogesterone (17-P), dehydroepiandrosterone (DHEA), or androstenedione (AD) were used as precursors, both testosterone (T) and estrone (E1) were synthesized by follicles, leading to estradiol-17β (E2) production. Serum steroid levels measured by enzyme-linked immunosorbent assay showed that T, E1, and E2 were present in the circulation at levels ranging from 1 ng/mL to 2 ng/mL throughout the day during the spawning season. In vitro conversion of E1 into E2, however, was 15.8-fold greater than T conversion into E2, suggesting that E2 is synthesized mainly via E1 rather than T. The results showed that E2 was synthesized from pregnenolone via 17-hydroxypregnenolone, DHEA, AD, and E1. Thus, the study demonstrated the complete steroidogenic E2 synthesis pathway in the ovarian follicles of red seabream, and revealed that E1 is the major precursor of E2.

Aromatization of 16α-hydroxyandrostenedione by human placental microsomes: effect of preincubation with suicide substrates of androstenedione aromatization

Estrogen synthase (aromatase) catalyzes the aromatization of androstenedione (AD) as well as 16α-hydroxyandrostenedione (16α-OHAD) leading to estrone and estriol, respectively. We found that several steroid analogs including 4-hydroxyandrostenedione ( 1), 6-oxoandrostenedione (6-oxoAD, 2) and its 19-hydroxy analog ( 3), 10β-acetoxyestr-5-ene-7,17-dione ( 4), androst-5-ene-4,7,17-trione ( 5), and 17α-ethynyl-19-norteststerone ( 6), which are known suicide inactivators of AD aromatization, are not effective in inactivating 16α-OHAD aromatization in a time-dependent manner. The compounds were tested with the use of human placental microsomes and 1β-tritiated-16α-OHAD as the substrate. The results of the tritium water method of 16α-OHAD aromatization was confirmed by the gas chromatography–mass spectrometry (GC–MS) method of estriol formation. The 1β-tritiated-AD was used to measure AD aromatization as a positive control for these experiments. The compounds were tested at concentrations up to 40-fold higher than the K i ’s determined for inhibition of AD aromatization. These studies suggest that differences exist in the binding site structures responsible for aromatization of 16α-OHAD and AD.

Effects of endogenous steroids on CYP3A4-mediated drug metabolism by human liver microsomes.

In the present study, we investigated the effects of 14 endogenous steroids on the CYP3A4-mediated drug metabolism by human liver microsomes in vitro. Nevirapine (NVP) 2-, 12-hydroxylations, carbamazepine (CBZ) 10,11-epoxidation, triazolam (TZM) 1'-, 4-hydroxylations, erythromycin (EM) N-demethylation, and 2-sulphamoylacetylphenol (SMAP) formation from zonisamide (ZNS) were investigated. The activities of the NVP 2-, 12-hydroxylations, the CBZ 10,11-epoxidation, and the TZM 4-hydroxylation were activated by endogenous androgens, such as androstenedione (AND), testosterone, and dehydroepiandrosterone. However, these androgens inhibited EM N-demethylation, TZM 1'-hydroxylation, and SMAP formation. To understand the mechanisms of these effects of androgens on CYP3A4 activities, we performed a kinetic analysis of the metabolism of CBZ and ZNS in the presence or absence of AND using the modified two-site equation model. The addition of AND to the reaction mixture caused a drastic increase in the activity of CBZ 10,11-epoxidase, especially at a low substrate concentration, and resulted in a change in the kinetics from the sigmoid to Michaelis-Menten type. On the other hand, the metabolism of ZNS was strongly inhibited by AND, although no allosteric change was observed in this case. These data demonstrate that endogenous steroids, especially androgens, strongly affect CYP3A4-mediated drug metabolism in vitro. The postulated mechanisms of the interactions between AND and CBZ or ZNS are discussed.

High performance liquid chromatographic separation of steroids from ovarian follicles of fresh water perch Anabas testudineus: identification and characterization of the maturation-inducing hormone.

Accurate separation and identification of steroids from the postvitellogenic ovarian follicles of Indian climbing perch Anabas testudineus was performed by high-performance liquid chromatography (HPLC) and specific radioimmunoassay (RIA). The steroids from such follicles, incubated in Cortland's saline with or without homologous fish pituitary extract (FPE), were extracted with dichloromethane and separated on a micro Bondapak C(18) column. Identification of the HPLC fractions was further confirmed by thin layer chromatography. As HPLC peaks for 17 alpha, 20 beta-dihydroxy-4-pregnen-3-one (DHP) and testosterone (T) were close, clear separation of these steroids and accurate measurement of their quantities was achieved by RIA of HPLC fractions using specific antibodies. Altogether, nine eluted fractions in the FPE-untreated and ten in FPE-treated samples were obtained. Of these, six were identified as: 5 beta-pregnan-3 alpha,17 alpha,20 beta-triol (5 beta-3 alpha,17 alpha,20 beta-P); DHP; T; 17 alpha-hydroxyprogesterone (17 alpha-P(4)); progesterone (P(4)); and androstenedione (AD). Three fractions from untreated and four from FPE-treated samples, however, remained unidentified. Of all the HPLC fractions examined for their relative maturational inducing (MI) potency on full grown (postvitellogenic) ovarian follicles of perch, the fraction identified as DHP was found to be the most effective inducer of germinal vesicle breakdown (GVBD) both at low and high concentrations. Fractions identified as 5 beta-3 alpha, 17 alpha, 20 beta-P and 17alpha-P(4) could induce only 32% and 20% GVBD at their highest concentration, while none of the unidentified fractions showed any MI activity. FPE caused increased production of DHP, testosterone, and 5 beta-3 alpha, 17 alpha, 20 beta-P. The qualitative differences between the fractions obtained from FPE-treated samples and those from FPE-untreated samples were only the appearance of a new polar metabolite of unknown function. The present study showed that, as a single steroid, DHP was the most potent MIH for A. testudineus.

Inadequately low serum levels of steroid hormones in relation to interleukin-6 and tumor necrosis factor in untreated patients with early rheumatoid arthritis and reactive arthritis.

To compare levels of steroid hormones in relation to cytokines and to study levels of cortisol or dehydroepiandrosterone (DHEA) in relation to other adrenal hormones in untreated patients with early rheumatoid arthritis (RA) and reactive arthritis (ReA) compared with healthy controls. In a retrospective study with 34 RA patients, 46 ReA patients, and 112 healthy subjects, we measured serum levels of interleukin-6 (IL-6), tumor necrosis factor (TNF), adrenocorticotropic hormone (ACTH), cortisol, 17-hydroxyprogesterone (17-OH-progesterone), androstenedione (ASD), DHEA, and DHEA sulfate (DHEAS). RA patients had higher serum levels of IL-6, TNF, cortisol, and DHEA compared with ReA patients and healthy subjects, but no difference was noticed with respect to ACTH and DHEAS. However, in RA and ReA patients compared with healthy subjects, levels of ACTH, cortisol, ASD, DHEAS, and 17-OH-progesterone were markedly lower in relation to levels of IL-6 and TNF. Furthermore, the number of swollen joints correlated inversely with the ratio of serum cortisol to serum IL-6 in RA (R(Rank) = -0.582, P = 0.001) and, to a lesser extent, in ReA (R(Rank) = -0.417, P = 0.011). In RA patients, the mean grip strength of both hands was positively correlated with the ratio of serum cortisol to serum IL-6 (R(Rank) = 0.472, P = 0.010). Furthermore, in these untreated patients with RA and ReA, there was a relative decrease in the secretion of 17-OH-progesterone, ASD, and DHEAS in relation to DHEA and cortisol. This indicates a relative predominance of the nonsulfated DHEA and cortisol in relation to all other measured adrenal steroid hormones in the early stages of these inflammatory diseases. This study indicates that levels of ACTH and cortisol are relatively low in relation to levels of IL-6 and TNF in untreated patients with early RA and ReA compared with healthy subjects. The study further demonstrates that there is a relative increase of DHEA and cortisol in relation to other adrenal hormones, such as DHEAS. This study emphasizes that adrenal steroid secretion is inadequately low in relation to inflammation. Although changes in hormone levels are similar in RA and ReA, alteration of steroidogenesis is more pronounced in RA patients than in ReA patients.

Dehydroepiandrosterone status in postmenopausal women is determined by the gene for the vitamin D receptor.

Data documenting the indirect interaction of vitamin D and bone metabolism via hormonal systems are rare. The authors analysed the predictive role of the vitamin D receptor (VDR) gene for circulating sex steroids and their precursors in postmenopausal women. Using the PCR technique, the polymorphic FokI, ApaI, TaqI and BsmI sites of the VDR gene were determined in relation to serum dehydroepiandrosterone sulphate (DHEAS), androstenedione (AD), testosterone, and estradiol levels. After adjustment to body mass and years since menopause, circulating DHEAS was higher in the Ff genotype than in ff (p < 0.001) and FF genotypes (p < 0.05, ANCOVA followed by least significant difference multiple comparison tests). The Ff genotype also contributed to the highest BMD at the hip (p < 0.01 as compared to ff genotype) and at the spine (p < 0.05). No significant associations were found between ApaI, TaqI and BsmI polymorphisms and serum DHEAS or between FokI, ApaI, TaqI or BsmI and serum androstenedione, testosterone or estradiol. The study shows that the VDR gene predicts synthesis and/or metabolism of sexual steroid preursor DHEA in parallel with bone mineral density (BMD). The results indicate that DHEA production and bone mass share a common genetic control through VDR.

Production, release and olfactory detection of sex steroids by the tench (Tinca tinca L.).

The present study is concerned with pheromone communication in tench (Tinca tinca L.), establishing firstly whether males have a high olfactory sensitivity to some typical teleost sex steroids and prostaglandins; and secondly whether males and females might be able to synthesise and release some of these steroids into the water. The C21 steroid, 17,20β-dihydroxy-4-pregnen-3-one (17,20β-P) was found to give large electro-olfactogram responses with an estimated threshold of detection of 10 −12 M. The male tench were equally sensitive to glucuronidated 17,20β-P (10 −11.6 M) but 100 times less sensitive to sulphated 17,20β-P (11 −9.7 M). Preliminary data from cross-adaptation studies suggest that both the free and conjugated forms are detected by the same olfactory receptor(s). Male tench also had high olfactory sensitivity to prostaglandin F2α (PGF2α) and 15-keto PGF2α (11 −11.5 and 10 −11.4 M). They were relatively insensitive, however, to testosterone (T), androstenedione (AD), 11-ketotestosterone (11-KT), 17β-oestradiol (E2), 17,20β,21-trihydroxy-4-pregnen-3-one (17,20β,21-P) and 17,20α-dihydroxy-4-pregnen-3one (17,20α-P). Radioimmunoassays were used to measure the steroids in plasma and water and all samples were processed for the measurement of free, sulphated and glucuronidated fractions. In females, free 17,20β-P, 17,20α-P, free and glucuronidated T, and AD in plasma showed the largest increases in response to injection with mammalian gonadotropin-releasing hormone analogue (GnRHa) or Ovaprim (a mixture of GnRHa and a dopamine inhibitor). Free 17,20β-P was released into the water at the greatest rate. Plasma concentrations of the two conjugated forms of 17,20β-P were also elevated 18 h after the administration of GnRHa, but not by as much as the free steroid. In males, AD and 11-KT showed the greatest increase in response to GnRHa and were moreover released into the water at a higher rate in the treated group than in the control. The data support a possible pheromonal role for free and glucuronidated 17,20β-P.

Production of androstenedione using mutants ofMycobacterium sp

Mycobacterium sp VKM Ac-1815D strain was able to cleave sterol side chain giving androstenedione (AD) as a major product with a molar yield of 63–68%. Clones having altered resistance to antibacterial agents were selected. After treatment with ethyl methane sulfonate or mitomycin C, mutants were obtained that retained the ability to produce AD from sitosterol with molar yields of 70–75%. A mutant strain able to effectively reduce 3,17-diketosteroids at C-17 was selected. The 17β-hydroxy steroid dehydrogenase activity of its crude extract was twice as high as that found for the parent organism. The approach offers the possibility of obtaining improved and labelled biocatalysts for AD or testosterone production from sterols. © 2002 Society of Chemical Industry

Dehydroepiandrosterone in Songbird Plasma: Seasonal Regulation and Relationship to Territorial Aggression

Many male animals are territorial in the breeding season, when plasma testosterone (T) levels are high, and nonterritorial in the nonbreeding season, when plasma T levels are basal. In contrast to this common pattern, male song sparrows (Melospiza melodia morphna) are territorial year-round, except briefly during molt. Song sparrows are highly aggressive in the nonbreeding season (autumn and winter), even though plasma T, 5α-dihydrotestosterone, androstenedione (AE), and 17β-estradiol levels are undetectable (≤0.1 ng/ml). Castration has no effect on nonbreeding territoriality. However, aromatase inhibitors decrease aggression in the nonbreeding season, indicating a role for estrogens in winter. The androgenic substrate for brain aromatase in winter is unclear, because plasma T and AE levels are basal. Here, we measured plasma levels of dehydroepiandrosterone (DHEA). DHEA is a precursor to AE and T, and the avian brain can convert DHEA into sex steroids. In nonbreeding male song sparrows, plasma levels of DHEA were detectable and several times higher than plasma AE and T levels. Plasma DHEA levels were similar in the breeding and nonbreeding seasons, but significantly lower during molt, which parallels seasonal changes in male aggression. Adrenal glands and testes from nonbreeding males had high concentrations of DHEA, suggesting that both tissues may secrete DHEA. However, stress and gonadotropin-releasing hormone (GnRH) did not increase plasma DHEA in nonbreeding birds. We hypothesize that in the nonbreeding season, circulating DHEA, possibly of adrenal origin, is converted into active sex steroids by steroidogenic enzymes in the brain. This mechanism would create high local levels of sex steroids in the brain to support winter aggression.

Ontogeny of steroid metabolizing enzymes in rat oligodendrocytes

Rat oligodendroglial cells were isolated from newborn and developing brains and used immediately after, for quantification of steroid metabolizing activities. Oligodendrocytes (Ol) and their progenitor cells were incubated with [ 14C] testosterone, [ 14C] progesterone, [ 14C] pregnenolone or [ 14C] dehydroepiandrosterone (DHEA). Oligodendrocytes and their progenitor cells expressed different steroid metabolizing enzymes. The main activities were 5α reduction of testosterone and progesterone and 3β hydroxy steroid dehydrogenase–isomerase which transformed pregnenolone into progesterone and DHEA into Δ4 androstenedione. 5α reductase activity increased in male and female rats in parallel with testosterone or progesterone. Contrary to this, 3β hydroxysteroid dehydrogenase-isomerase activity was found to be high in the young rat and to decrease when testosterone and progesterone plasma concentration increased.

The three steroidal components of the goldfish preovulatory pheromone signal evoke different behaviors in males

The goldfish sex pheromone system is the best understood among the teleost fishes. Pheromones in this species are unspecialized hormonal products, which are released in ratios that vary with reproductive status. This study examined behavioral responses of male goldfish to three steroidal components of the female preovulatory pheromone: 17,20β-dihydroxy-4-pregnen-3-one (1720βP); 17,20β-dihydroxy-4-pregnen-3-one-20-sulfate (1720βP-S); and androstenedione (AD). Males were observed during exposure to nanomolar concentrations of each steroid over a 2-h period. We observed chasing, nudging (courtship behaviors) and pushing (an aggressive behavior). Each steroid elicited a different set of behaviors. 1720βP, which is released by ovulatory females, elicited a low level of chasing and nudging that persisted throughout the experiment. Exposure to 1720βP-S, which is released primarily by ovulatory females, triggered a large increase in nudging and chasing that lasted for only 5 min. In contrast, AD, which is released by females early in the ovulatory cycle and by mature males, elicited increases in aggressive behavior. 1720βP and 1720βP-S both caused increases in GtH-II release while AD did not. These results demonstrate that goldfish can discriminate components found in the female pheromone blend, suggesting that goldfish, and likely other fish species, may employ blends of hormonal products as pheromones.

Dynamics of the development of multiple follicles during ovarian stimulation for in vitro fertilization using recombinant follicle-stimulating hormone (Puregon) and various doses of the gonadotropin-releasing hormone antagonist ganirelix (Orgalutran/Antagon)

Objective: To investigate relations between dose of GnRH antagonist and follicular phase characteristics. Design: Randomized controlled multicenter trial. Setting: Tertiary referral fertility centers. Patient(s): Three hundred and twenty-nine IVF patients. Intervention(s): Ovarian stimulation for IVF with recombinant FSH starting on cycle day 2. From cycle day 7 onwards, cotreatment was provided with 0.0625, 0.125, 0.25, 0.5, 1.0, or 2.0 mg/d GnRH antagonist. Main Outcome Measure(s): Number of follicles, total follicular surface area, gonadotropin, and serum steroid concentrations. Result(s): In 311 patients, similar follicular growth was observed in all treatment groups. FSH levels increased during the follicular phase. Late follicular phase LH, androstenedione (AD), and E 2 levels showed a GnRH antagonist dose-related decrease ( P<0.05). Late follicular phase E 2 levels correlated with total follicular surface area, AD, LH, and FSH (all P<0.001). Increasing GnRH antagonist doses exhibited additional suppressive action on E 2 levels. Conclusion(s): Follicular growth was unaffected by the dose of GnRH antagonist. A rise in follicular phase FSH serum concentrations during the follicular phase, largely related to exogenous FSH, enabled ongoing follicular growth in all treatment groups. The effect of GnRH antagonist on late follicular phase E 2 levels could not be exclusively attributed to suppression of LH.

Androgens and the Role of Female “Hyperaggressiveness” in Spotted Hyenas (Crocuta crocuta)

It has been suggested that female dominance in spotted hyenas evolved due to selection for “hyperaggressive” androgenized females. According to this view, virilized external genitalia of female hyenas developed as a byproduct of selection for “androgen-facilitated social dominance.” The evidence that female hyenas have higher androgen levels than males or other female mammals is inconclusive. We compared concentrations of testosterone (T), 5α-dihydrotestosterone (DHT), and androstenedione (AE) from a population in the Serengeti. Females had significantly lower T and DHT levels than predispersal and postdispersal males. AE levels did not significantly differ between females and postdispersal males, but were significantly lower in predispersal males. Our results, and those from studies that have demonstrated that male and female hyenas experience similar levels of maternal androgens during fetal development, provide little support for a theory of selection for female dominance and hyperaggressiveness through enhanced secretion of androgens. Our data are consistent with an alternative view that female virilization is a byproduct of selection for precocial aggressive cubs of both sexes. According to this view, high investment in lactation favored selection for accelerated fetal development, high neonatal aggression, and facultative siblicide.

Conversion of β-sitosterol by Mycobacterium sp. NRRL B-3805 cells immobilized on Celite supports

Mycobacterium sp. NRRL B-3805 cells immobilized on Celite were effectively used for the selective side-chain cleavage of sitosterol to androstenedione (AD) in organic media (phthalate derivatives). Kinetic studies were performed with sitosterol concentrations up to 24 mM, with different Celite materials and particle sizes. Higher activity levels were observed when a larger pore size Celite was used as immobilization matrix. Substrate inhibition was observed for sitosterol concentrations above 6 mM. Toxicity effects were not apparently correlated with the high log P solvents (>9) here used as bioconversion media. The use of immobilized cells in repeated batch biotransformations did not prove effective, mainly due to biocatalyst desorption during the periodical, aqueous washing steps used for nutrient delivery to the cells.

Stimulation of pregnenolone metabolism and aromatase activity by luteinizing hormone in mouse uterus.

In vitro metabolism of pregnenolone (P5) as well as production of 17beta-estradiol (E2) were studied in uteri of untreated and luteinizing hormone (LH)-treated mice that had been ovariectomized (OVX) at late-diestrus stage. In the uteri of untreated mice, [H]pregnenolone was shown to be metabolized to Delta-components such as 17alpha-hydroxypregnenolone (17alpha-P5) and dehydroepiandrosterone (DHEA), whereas LH treatment resulted in significant increases in the formation of progesterone (P4), 17alpha-hydroxyprogesterone (17alpha-P4), androstenedione (AD) and testosterone (T). This was assessed by thin-layer chromatography (TLC) and high-performance liquid chromatography (HPLC). The content and release of P4 was shown to be stimulated by LH. Trilostane, an inhibitor of 3beta-hydroxysteroid dehydrogenase (3beta-HSD), inhibited LH-induced P4 synthesis and its release in a dose-dependent manner. A considerable increase in [H]estradiol formation from [H]testosterone was recorded in LH-stimulated uterine tissue as compared with the control, indicating the stimulatory effect of LH on aromatase activity. LH-stimulation in the synthesis of P4 and E2 in OVX mouse uteri was mimicked by dbcAMP (cell-permeable cAMP). Incubation with LH was shown to augment the conversion of P4 to various delta-3-oxosteroids. In vitro effects of LH on the synthesis and metabolism of P4, as well as on the stimulation of aromatase activity, were more pronounced in the uterine tissue of LH-primed OVX mice. Thus the results of the present study indicate that, under specific conditions, the uterus of the mouse behaves like steroidogenic tissue. Its prompt response to LH reveals the probable physiological relevance of the existence of LH receptors of high binding affinity in the uterine tissue of the mouse, as reported earlier.

Study of the microbial aggregation in mycobacterium using image analysis and electron microscopy

Cellular aggregation, which occurs in both prokaryotes and eukaryotes, is controlled by the hydrophobicity as well as the electrokinetic potential of the cell surface and substratum. It is known that the Mycobacterium genus form aggregates, but the influence of sugar on the cellular aggregation has not been reported for this genus. The mutant strain Mycobacterium sp. MB-3683 that transforms sterol to androstendione (AD), a steroidal precursor used by the pharmaceutical industries, was employed in this study. This strain was cultivated in a synthetic medium on three sugars (glycerol, glucose and fructose) at different concentrations, and at 144h microbial growth, cellular aggregation, hydrophobicity, lipid content, fatty acid composition, and width of cellular walls were measured. It was observed that at different sugar concentrations, similar growth and pH were obtained. However, in fructose, the aggregation level was significantly high, followed by glycerol and glucose (fructose < glycerol < glucose). These results were confirmed using electron microscopy and the aggregate area quantified by image analysis. Hydrophobicity was the highest in fructose and the lowest in glucose. The total lipids, in contrast to cellular hydrophobicity, were higher in glucose than glycerol. Although, the hydrophilic-lipophilic balance (HLB) of principal fatty acids isolated was similar regardless of sugar used. In glycerol and fructose, the paraffins were observed, which are responsible for the high cellular hydrophobicity detected above. The width of cell wall of the organisms grown on glucose and fructose was similar, but in glycerol the walls were very thin. There is a correspondence between cell wall width and lipid content.