Androstenedione Research Articles

Reference Intervals for Important Serum Sex Steroid Hormones, Prostate-Specific Antigen, Insulin-Like Growth Factor-1 and IGF Binding Protein-3 Concentration in a Normal Kuwaiti Adult Male Population

Objectives: The purpose of this study was to determine the age-specific reference ranges for some important male sex steroid hormones, prostate-specific antigen (PSA), insulin-like growth factor-1 (IGF-1), and IGF binding protein-3 (IGFBP-3), for the Kuwaiti population. Subjects and Methods: Blood samples were taken from 398 consenting, fasting, healthy Kuwaiti males aged 15–80 years between 8.00 a.m. and 12.00 noon. The serum concentrations of total testosterone (TT), dehydro-epiandrosterone sulfate (DHEAS), androstenedione (ADT), sex hormone binding globulin, luteinizing hormone (LH), follicle-stimulating hormone (FSH), prolactin, PSA, IGF-1 and IGFBP-3 were determined. A distribution curve was plotted and age-specific reference levels were determined for each analyte. The reference interval for parameters with a normal distribution (Gaussian) was mean ± 2 SD, while for the non-normal distribution (non-Gaussian), it was 2.5–97.5 percentile. The reference intervals for the analytes obtained from this study were compared with those suggested by the kit manufacturers and currently used by the Ministry of Health, Kuwait Laboratories (MOHKL). Results: Serum IGFBP-3 and ADT had normal distribution while other analytes had non-normal distribution. The reference intervals from this study, manufacturers kit and MOHKL were as follows: TT 3–31, 9–60, 8–35 nmol/l; DHEAS 0.9–11, 1.0–7.3, 2.2– 15.2 µmol/l; ADT 0.5–4.3, 0.8–2.8, 2.0–9.2 nmol/l; LH 1–11, 0.8–7.6, 0.4–5.7 mIU/l; FSH 0.5–11, 0.7–11.1, 1.1–13.5 mIU/l; prolactin 42–397, 53–360, 80–230 nmol/l; IGF-1 41–542, 78–956, 71–261 ng/ml; IGFBP-3 88– 2,090, 900–4,000, 900–4,000 ng/ml, and PSA 0–3.1, 0–4, 0–4 ng/ml, respectively. Conclusion: These data indicate that for Kuwaitis lower reference ranges must be used for serum TT, DHEAS, ADT, IGFBP-3 and PSA. There is no need to change the currently used reference interval for FSH whereas higher values must be used for LH, prolactin, and IGF-1.

Key Fertility Hormones Detected in Tears Correlate to Systemic Concentrations

Most of the observed associations of androgens and sex hormone-binding globulin (SHBG) with subclinical cardiovascular disease (CVD) stem from selected study samples with immunoassay-based hormone measurements. Thus, we used a large population-based sample with total testosterone (TT) and androstenedione (ASD) concentrations measured by liquid chromatography-tandem mass spectrometry.Data of 2140 individuals (mean age: 60,8 years) from the cohort Study of Health in Pomerania were assessed at baseline and 5-year follow-up.Multivariable regression models were implemented to assess cross-sectional and longitudinal associations of TT, free testosterone (fT), ASD, SHBG and dehydroepiandrosterone-sulphate (DHEAS) with measures of subclinical CVD including intima media thickness (IMT), carotid plaques, left ventricular mass (LVM), fractional shortening (FS), relative wall thickness (RWT), and left ventricular geometry.Cross-sectional analyses yielded an association of TT with IMT in women (β-coefficient per log unit increase: 0.02; 95% CI: 0.007; 0.45) and ASD with FS in both sexes (men: β-coefficient: −2.94; 95% CI: −4.75; −1.12; women: β-coefficient: 1.64; 95% CI: 0.55; 2.73). In longitudinal analyses, DHEAS was positively associated with FS change (β-coefficient: 2.34; 95% CI: -0.59; 4.08). In women, SHBG was positively associated with incident plaques (Q1 vs. Q3 (Ref.): β-coefficient: 1.35; 95% CI: 1.04; 1.74). In both sexes, longitudinal analyses showed no consistent association of TT with subclinical CVD.Despite several sex-specific associations of androgens and SHBG with subclinical CVD, the present representative study for the age group ≥45 years among men and women from the general population detected no consistent associations in longitudinal analyses.

Leptin Levels in Boys with Pubertal Gynecomastia

It has been reported that there is a relationship between circulating leptin and sex steroid hormones and leptin is able to stimulate estrogen secretion by increasing aromatase activity in adipose stromal cells and breast tissue. Leptin receptors have been also shown in mammary epithelial cells and it has been suggested that leptin is involved in the control of the proliferation of both normal and malignant breast cells. To investigate circulating leptin levels in boys with pubertal gynecomastia. Twenty boys with pubertal gynecomastia who were in early puberty and had no obesity, and 20 healthy individuals matched for age, pubertal stage and body mass index (BMI) with the study group, were enrolled in the study. Body weight, height and left midarm circumference (MAC) and left arm triceps skinfold thickness (TSF) were measured and BMI was calculated. A fasting blood sample was collected and routine hormonal parameters including prolactin, beta-human chorionic gonadotropin (betaHCG), total and free testosterone, estradiol, luteinizing hormone (LH), follicle stimulating hormone (FSH), prolactin, androstenedione (AS) and dehydroepiandrosterone sulfate (DHEAS) levels were studied. Serum leptin levels were analyzed using radioimmunoassay. The mean ages of the study and control group were not different (13.9 +/- 0.89 and 14.2 +/- 0.66, respectively). No significant difference was found for BMI, MAC and TSF values between the two groups. There was no significant difference for hormonal parameters including FSH, LH, total and free testosterone, estradiol, AS, DHEAS and estradiol/total testosterone ratio between boys with pubertal gynecomastia and the controls. Serum leptin levels were found significantly higher in the study group compared with the healthy controls (5.58 +/- 0.81 and 2.39 +/- 0.29 ng/ml, respectively; p <0.001). No correlation could be determined between serum leptin levels and hormonal parameters. The presence of higher leptin levels in boys with pubertal gynecomastia indicates that leptin may be involved in the pathogenesis of pubertal gynecomastia. The role of circulating leptin in pubertal gynecomastia is probably related to increase in estrogen and/or estrogen/ androgen ratio by the stimulating effect of leptin on aromatase enzyme activity in both adipose and breast tissues, or a direct growth stimulating effect of leptin on mammary epithelial cells, or increase in sensitivity of breast epithelial cells to estrogen with inducing functional activation of estrogen receptors by leptin in breast tissue.

DDT increases hepatic testosterone metabolism in rats.

DDT and its metabolites are considered as endocrine disruptors able to promote hormone-dependent pathologies. We studied the effects of technical-grade DDT on hepatic testosterone metabolism and testosterone hydroxylase activity ratios in the rat. Male and female Wistar rats were treated by gavage with a single dose of technical-grade DDT (0, 0.1, 1, 10, and 100 mg/kg body weight) and killed 24 h later. Hepatic microsomes were incubated with [4-14C]-testosterone and the metabolites were separated by thin-layer chromatography and quantified by radio scanning. DDT increased testosterone biotransformation and modified the profile of metabolites produced in a sex-dependent manner. Males treated with a representative dose (10 mg/kg) produced relatively less androstenedione (AD), 2alpha-hydroxytestosterone (OHT), and 16alpha-OHT but higher 6beta-OHT whereas treated females produced less 7alpha-OHT and AD but higher 6beta-OHT and 6alpha-OHT than their respective controls. In both sexes DDT decreased the relative proportion of AD and increased that of 6beta-OHT suggesting that the androgen-saving pathway was affected. The testosterone 6alpha-/15alpha-OHT ratio, a proposed indicator of demasculinization, was increased in treated males. This effect was in agreement with the demasculinizing ability proposed for DDT. The effects on 6alpha-/16alpha-OHT and 6-dehydrotestosterone/16alpha-OHT ratios followed a similar tendency, with the ratio 6alpha-/16alpha-OHT being the most sensitive marker. Interestingly, these ratios were reduced in treated females suggesting that technical-grade DDT shifted testosterone hydroxylations toward a more masculine pattern. Thus, technical-grade DDT altered the hepatic sexual dimorphism in testosterone metabolism and decreased the metabolic differences between male and female rats.

RACIAL DIFFERENCES IN PROSTATE ANDROGEN LEVELS IN MEN WITH CLINICALLY LOCALIZED PROSTATE CANCER

We sought to determine whether there are racial differences in androgenic stimulation within the prostate tissue microenvironment. Steroid hormones were extracted from snap frozen tissue obtained intraoperatively from radical prostatectomy specimens of 36 black and 59 white Americans. Testosterone, dihydrotestosterone (DHT), androstenedione (ASD), dehydroepiandrosterone, dehydroepiandrosterone sulfate, sex hormone-binding globulin (SHBG) and prostate specific antigen (PSA) were measured using radioimmunoassay. The Wilcoxon 2 group test was performed to compare clinical characteristics and tissue steroid levels between white and black Americans. Nonparametric rank ANOVA was used to consider race and other clinical factors in a multivariable way. Black and white American men were similar with respect to serum PSA, and pathological grade and stage. However, black men were younger (p = 0.01) and had a significantly higher body mass index (p = 0.02). Black and white men had similar testosterone and DHT. However, black men had higher ASD (p = 0.006) and SHBG (p = 0.009). Racial differences in ASD (p = 0.015) and SHBG (p = 0.008) persisted after controlling for age, body mass index, PSA, and pathological Gleason sum and stage. Tissue levels of testosterone and DHT did not differ by race. However, black men had higher tissue ASD and SHBG than white men. Higher tissue ASD did not result in a greater conversion of ASD to testosterone in the prostate of black men. Higher tissue SHBG may activate the androgen receptor through cyclic adenosine monophosphate dependent pathways.

Physiological and biochemical improvement of the enzyme side-chain degradation of cholesterol by Fusarium solani

The conversion of cholesterol (C) to androstenedione (AD) and androstadienedione (ADD) was influenced significantly by the composition of the cultivation medium. A medium composed of (g/l): glucose, 50; peptone, 20; corn steep solid, 3; supported relatively high AD and ADD outputs. C convertibility was further accelerated when the cultivation medium was adjusted with phosphate buffer with the pH range 5.5–7.38. Supplementation of the medium with 8-hydroxyquinoline or methanol prevented the degradation of the C nucleus with concomitant formation of good AD and ADD yields.

In vitro permeation studies comparing bovine nasal mucosa, porcine cornea and artificial membrane: androstenedione in microemulsions and their components

The components of a carrier formulation can interact with an added drug as well as with the membrane surface they were applied on. Therefore, they can influence permeability of the membrane and permeation of the drug. The particular membrane structure might lead to different drug permeation out of one and the same carrier formulation. In this study, in vitro permeability of androstenedione (AD) as a highly lipophilic substance was investigated in excised bovine nasal mucosa, porcine cornea and the artificial cellulose membrane Nephrophan ®. Two microemulsions (ME) with either the addition of the co-surfactants hydroxypropyl-γ-cyclodextrin (HP-γ-CD; ME-CD) or propylene glycol (PG, ME-PG) were tested in order to be used as carrier systems. Both MEs also consisted of 5% isopropyl myristate (IPM), 20% Cremophor ® EL (CrEL), and water. Buffer solution (EBS) with 0.0025% AD served as control solution and was furthermore compared to 0.0025% AD/buffer-solutions containing the ME components HP-γ-CD in different concentrations (0.012, 0.024, 9%) as well as 20% CrEL. The AD-permeation behaviour through the three tissues was differently influenced by the MEs. The apparent permeability coefficients ( P app) of nasal mucosa for both ME systems did not differ from the P app of the AD/buffer solution. In case of the other two barriers (cornea, Nephrophan ®), ME-PG as well as ME-CD provoked extended time lags for AD to permeate, so the P app could not be calculated or declined to zero. P app of AD/buffer solution without any additives resulted for cornea, nasal mucosa and Nephrophan ® in a ratio of 1:3:4. CrEL and 9% HP-γ-CD diminished the P app, except HP-γ-CD in molar AD/HP-γ-CD-ratios of 1:1 (0.012%) and 1:2 (0.024%). It seems that the composition of lipophilic and hydrophilic structures of the carrier systems or the additives had a higher impact on the P app of cornea than on the P app of the other tissues. Structure and character of the different membranes are considered to be mainly responsible for the differentiated permeation behaviour.

Physiological and biochemical improvement of the enzyme side-chain degradation of cholesterol by Fusarium solani

The conversion of cholesterol (C) to androstenedione (AD) and androstadienedione (ADD) was influenced significantly by the composition of the cultivation medium. A medium composed of (g/l): glucose, 50; peptone, 20; corn steep solid, 3; supported relatively high AD and ADD outputs. C convertibility was further accelerated when the cultivation medium was adjusted with phosphate buffer with the pH range 5.5–7.38. Supplementation of the medium with 8-hydroxyquinoline or methanol prevented the degradation of the C nucleus with concomitant formation of good AD and ADD yields.

Study of key operational parameters for the side-chain cleavage of sitosterol by free mycobacterial cells in Bis-(2-ethylhexyl) phthalate

The selective side-chain cleavage of β-sitosterol by free cells of Mycobacterium sp. NRRL B-3805 is a well-established multi-enzymatic process for the production of the pharmaceutical steroid precursors androstenedione (AD) and androstadienedione (ADD). In this study, bis(2-ethylhexyl) phthalate (BEHP) was used as a reaction medium for carrying out the process with freely suspended cells. The work aimed to show that microbial sitosterol side-chain cleavage is possible in this essentially mono-phasic organic medium, provided that some important parameters are adequately controlled. The effects of the biocatalyst/substrate mass ratio, system aeration rate and minimum buffer addition to the organic medium on the product yield and the reaction rate were thus evaluated.

Postmenopausal serum androstenedione levels are associated with the calcitonin receptor gene polymorphism T1377c. A pilot study.

We tested the hypothesis that homeostasis of sex-steroids is related to the calcitonin receptor (CALCR) genotype. To determine the CALCR genotype PCR amplification followed by digestion with Alul restriction enzyme were carried out according to Nakamura et al. Indeed, a single nucleotide difference at position 1377 of cDNA generates two alleles (CC genotype or TT genotype). Serum estradiol, testosterone and their precursors androstendione (AD) and DHEA levels were estimated in a cohort of 113 postmenopausal women. While serum DHEA levels did not differ between the individual allele combinations, AD levels as well as AD/DHEA ratio were higher in carriers of TC and CC genotypes than those with TT genotype (p<0.05 and p<0.02, respectively, ANCOVA). We postulate that the 3beta-hydroxysteroid dehydrogenase activity is associated with C allele at least in C19 steroids. The data correspond with the functionality of CALCR.

Low calcium intake is associated with decreased adrenal androgens and reduced bone age in premenarcheal girls in the last pubertal stages.

In 50 premenarcheal girls selected from the lowest and highest end of the calcium-intake distribution of a large population sample, we evaluated bone mineral density (BMD), together with the following hormonal-metabolic parameters: androstenedione (ASD), dehydroepiandrosterone (DHEA), dehydroepiandrosterone sulfate (DHEAS), testosterone (T), estradiol (E2), the apparent free fractions of T (AFTC) and E2 (AFEC), osteocalcin (OC), parathyroid hormone (PTH), and 25-hydroxyvitamin D. Dietary calcium was assessed by 3-day food records, and BMD was measured at ultradistal (ud) and proximal (pr) radial sites, using dual-energy X-ray absorptiometry. Calcium intake, which was below the recommended levels set for the Italian population and below the recommended daily allowance (RDA) in both subgroups of girls, did not show any apparent relationship with ud- and pr- BMD. However, despite the similar chronological age of the two premenarcheal groups, in the low-calcium consumers, we found lower bone age, delayed pubertal development, and lower circulating adrenal androgens. Of interest, in girls who had a low calcium intake, PTH levels were significantly higher. In all premenarcheals, we observed that DHEA, T, and AFTC were positively correlated with bone age and with bone density at both radial sites. Even though bone density at the two radial sites did not show any apparent relationship to calcium consumption, the increased mean PTH in the girls with low calcium intake seems to underscore the hormonal attempt in maintaining calcium homeostasis. In conclusion, low calcium intake and reduced levels of adrenal androgens, leading to decreased bone age and delayed pubertal development, indicate a link between calcium intake, the hormonal milieu, and skeletal maturation.

Dehydroepiandrosterone metabolism by 3beta-hydroxysteroid dehydrogenase/Delta5-Delta4 isomerase in adult zebra finch brain: sex difference and rapid effect of stress.

Dehydroepiandrosterone (DHEA) is a precursor to sex steroids such as androstenedione (AE), testosterone (T), and estrogens. DHEA has potent effects on brain and behavior, although the mechanisms remain unclear. One possible mechanism of action is that DHEA is converted within the brain to sex steroids. 3beta-Hydroxysteroid dehydrogenase/Delta5-Delta4 isomerase (3beta-HSD) catalyzes the conversion of DHEA to AE. AE can then be converted to T and estrogen within the brain. We test the hypothesis that 3beta-HSD is expressed in the adult brain in a region- and sex-specific manner using the zebra finch (Taeniopygia guttata), a songbird with robust sex differences in song behavior and telencephalic song nuclei. In zebra finch brain, DHEA is converted by 3beta-HSD to AE and subsequently to estrogens and 5alpha- and 5beta-reduced androgens. 3beta-HSD activity is highest in the diencephalon and telencephalon. In animals killed within 2-3 min of disturbance, baseline 3beta-HSD activity in portions of the telencephalon is higher in females than males. Acute restraint stress (10 min) decreases 3beta-HSD activity in females but not in males, and in stressed animals, telencephalic 3beta-HSD activity is greater in males than in females. Thus, the baseline sex difference is rapidly reversed by stress. To our knowledge, this is the first demonstration of 1) brain region differences in DHEA metabolism by 3beta-HSD, 2) rapid modulation of 3beta-HSD activity, and 3) sex differences in brain 3beta-HSD and regulation by stress. Songbirds are good animal models for studying the regulation and functions of DHEA and neurosteroids in the nervous system.

In Vitro Effects of γ-Hexachlorocyclohexane on Biosynthesis and Metabolism of Steroids in Goldflsh Carassius auratus (L.)

Testicular and ovarian fragments of Carassius auratus , taken during the reproductively active prespawning phase (June) of its annual reproductive cycle, were incubated with different concentrations (0 mg/ml,0.001 mg/ml, lppm;0.01 mg/ml, 10ppm; and 0.02 mg/ml, 20 ppm) of γ-hexachlorocyclohexane (γ-HCH) in the presence of either exogenous precursor [ 3 H]-17-hydroxyprogesterone ([ 3 H]-17-P 96.5kBq, 13.04ng/Incubation) or carp hypophyseal homogenate (chh, 100 μγg/incubation). The free (unconjugated) and conjugated metabolites (glucuronides and sulfates) of [ 3 H]-17-P [androstenedione (AD), androstenetrione, 17-hydroxyprogeste-rone, testosterone (T), 11- deoxycortisol (S), 17, 20α-dihydroxy-4-pregnen-3-one (17,20αP), 17,20β-dihydroxy-4-pregnen-3-one (17, 20βP), 7α-pregnanetetrols (7α-P), and other polar metabolites] were separated by thin layer chromatography and high performance liquid chromatography. The endogenous production of unconjugated (free) steroids T, 17,20βP, S and 11-ketotestosterone (11-KT) in response to γ-HCH were measured by radioimmunoassay. Among the in vitro metabolism of [ 3 ]-17-P, in males; free steroids of AD, T, 17,20aP, S and polar free steroids were increased with the decreased yield of 11-KT. Percentage yield of testosterone glucuronide (TG) was increased with highly significant decreased yields of polar glucuronide steroids. The sulfate steroids of 17, 20αP, 17, 20βP, S and 11-KT remain unchanged. In females, the decreased percentage of yield of AD and S, and elevated T were noticed. The yield of TG was increased with decreased yield of 7α-P glucuronides. The percentage yield of AD sulfate and sulfate steroids of 17, 20αP, 17, 20βP and S were noted to be increased, but the yield for S sulfate was very high. Endogenous production of T was increased in both sexes in the presence of γ-HCH, but 11-KT in male and S in female were depressed. 17,20βP was stimulated at some concentrations in both sexes but levels were very low. Results indicate that γ-HCH in vitro perturbed the steroids biosynthesis during this phase thereby affecting reproductive physiology.

DHEA and bone metabolism

A strong positive correlation of serum dehydroepiandrosterone sulfate (DHEA-S) and estrone (E1) with bone mineral density (BMD) in postmenopausal women but no correlation between serum estradiol (E2) and BMD in the same group suggest that circulating adrenal androgen may be converted to estrogen in peripheral tissues including osteoblast and may contribute to BMD maintenance. Actually, in cultured human osteoblast cells, DHEA can be converted to androstenedione and then androstenedione to estrone through the apparent aromatase activity. In human bone cells, intracrine mechanism through aromatase activity may contribute to the local production of estrogens, thus leading to protective effect against osteoporosis especially after menopause.

Probing the active site of aromatase with 2-methyl-substituted androstenedione analogs

To gain insight into the spatial nature of the androstenedione (AD) binding (active) site of aromatase in relation to the catalytic function of the enzyme, we synthesized 2,2-dimethylAD ( 4), 2β- and 2α-methylADs ( 5 and 6), 19-oxygenated derivatives of compounds 4 and 6, and 2-methyleneAD ( 17), and we then tested their inhibitory activity as well as their aromatase reaction (aromatization for 2-methyl and 2-methylene analogs or 19-oxygenation for 2,2-dimethyl steroids) with human placental aromatase. 2-Methyl and 2-methylene steroids 5, 6, and 17 were good competitive inhibitors of aromatase ( K i=22–68 nM), but less effective compared to the 2,2-dimethyl analog 4 ( K i=8.8 nM), indicating that a combination of 2β- and 2α-methyl moieties is essential for the formation of a thermodynamically stable inhibitor–aromatase complex. A series of 2α-methyl steroids were good substrates for aromatase, whereas 2β-methyl steroid 5 was an extremely poor substrate, and a series of 2,2-dimethyl steroids did not serve as substrate, suggesting that a 2β-methyl moiety of the 2,2-dimethyl and 2β-methyl steroids would prevent the aromatase reaction probably due to steric hindrance in each case. The 2-methylene compound 17 was also aromatized to produce 2-methylestrogen with a low conversion rate where the 1,4-diene structure may have been created before the C 10–C 19 bond cleavage. Kinetic analysis of the aromatization of androgens revealed that a good substrate was not essentially a good inhibitor for aromatase.

Prepubertal administration of estradiol valerate disrupts cyclicity and leads to cystic ovarian morphology during adult life in the rat: role of sympathetic innervation.

Administration of estradiol valerate (EV) to adult rats leads to anovulation and cystic ovarian morphology. Sympathetic ovarian nerve denervation (SONX) overcomes this disruption. In this study, we determined whether EV administration to juvenile rats prevents achievement of reproductive competence, disrupts cyclicity, and whether this programming is facilitated via activation of the sympathetic nerve input to the ovary. Prepubertal rats were administered 2 mg EV in corn oil or corn oil alone. One half of the animals from each group underwent SONX on d 71 of life. Rats were euthanized on d 91 for determination of serum gonadotropins, progesterone, Delta4 androstenedione, and estradiol concentrations, ovarian norepinephrine (NE), and 3beta-hydroxysteroid dehydrogenase (3beta-HSD) activities and ovarian dynamics. Results revealed that EV administration during juvenile period advanced pubertal onset, suppressed circulating LH, FSH, and Delta4 androstenedione, increased ovarian NE, estradiol, and 3beta-HSD activities, disrupted ovarian dynamics evidenced as absent corpus luteum and presence of ovarian cysts and culminated in anovulation. SONX restored cyclicity in these animals, normalized LH, estradiol, ovarian 3beta-HSD activities, and ovarian dynamics as evidenced by the disappearance of ovarian cysts and appearance of corpus luteum and restored corpus luteum function. These findings provide evidence that EV exposure during juvenile life leads to long-lasting deleterious reproductive consequences via activation of the sympathetic ovarian nerve.

Long-term anti-tumor necrosis factor antibody therapy in rheumatoid arthritis patients sensitizes the pituitary gland and favors adrenal androgen secretion.

New insights into the role of tumor necrosis factor (TNF) in the pathogenesis of rheumatoid arthritis (RA) have expanded our understanding about the possible mechanisms by which anti-TNF antibody therapy reduces local synovial inflammation. Beyond local effects, anti-TNF treatment may modulate systemic antiinflammatory pathways such as the hypothalamic-pituitary-adrenal (HPA) axis. This longitudinal anti-TNF therapy study was designed to assess these effects in RA patients. RA patients were given 5 infusions of anti-TNF at weeks 0, 2, 6, 10, and 14, with followup observation until week 16. We measured serum levels of interleukin-6 (IL-6), adrenocorticotropic hormone (ACTH), 17-hydroxyprogesterone (17[OH]progesterone), cortisol, cortisone, androstenedione (ASD), dehydroepiandrosterone (DHEA), and DHEA sulfate in 19 RA patients. Upon treatment with anti-TNF, we observed a fast decrease in the levels of serum IL-6, particularly in RA patients who did not receive parallel prednisolone treatment (P = 0.043). In these RA patients who had not received prednisolone, the mean serum ACTH levels sharply increased after every injection of anti-TNF, which indicates a sensitization of the pituitary gland (not observed for the adrenal gland). During treatment, the ratio of serum cortisol to serum ACTH decreased, which also indicates a sensitization of the pituitary gland (P < 0.001), and which was paralleled by constant cortisol secretion. The adrenal androgen ASD significantly increased relative to its precursor 17(OH)progesterone (P = 0.013) and relative to cortisol (P = 0.009), which indicates a normalization of adrenal androgen production. The comparison of patients previously treated with prednisolone and those without previous prednisolone revealed marked differences in the central and adrenal level of this endocrine axis during long-term anti-TNF therapy. Long-term therapy with anti-TNF sensitizes the pituitary gland and improves adrenal androgen secretion in patients who have not previously received prednisolone treatment. These changes are indicative of normalization of the HPA axis and must therefore be considered as evidence of an additional antiinflammatory influence of anti-TNF treatment in patients with RA.

DETERMINANTS OF BONE TURNOVER IN YOUNG WOMEN

Skeletal integrity is related to the balance between bone formation and bone resorption, which is mediated by factors including genetics, hormones, loading, and calcium supply. The extent to which these determinants of bone environment influence markers of bone turnover remains unclear. PURPOSE This investigation was conducted to evaluate the importance of 17β-HSD1 genotype, estradiol (E2), androstenedione (AD), testosterone (T), insulin-like growth factor-I (IGF-I), physical activity, and calcium intake to concentrations of markers of bone formation (osteocalcin, OC) and resorption (cross-laps, LAPS) in college-aged young women. METHODS Fasting serum was obtained from 32 women aged 21.6 ± 3.4 years (X ±SD) for the determination of OC, E2, AD, T, and IGF-I on day 11 ± of an ovulatory menstrual cycle. OC and IGF-I concentrations were determined by immunoradiometric assay, while E2, AD, and T were determined by radio-immuno assay. 17β-HSD1 genotype was determined by PCR as A/A, A/G, or G/G. LAPS were determined from urine by enzyme-linked immunosorbent assay. Physical activity and dietary calcium intake were derived from questionnaires. Stepwise linear regression analysis determined the proportion of the variance in OC and LAPS accounted for by the variables. Significance was set at p < 0.05. RESULTS For osteocalcin, E2 accounted for 18.8% of the variance (p = 0.012), with physical activity accounting for an additional 16.3% (p = 0.011). The variance in LAPS was explained by IGF-I concentrations (11.4%, p = 0.044) and calcium intake (11.1%, p = 0.039). None of the other variables were significant predictors of either marker. CONCLUSION It is evident from the results that diverse factors are important in determining rates of bone formation and resorption in young women. These results underscore the complex roles of estrogen, physical activity, IGF-I and calcium in the maintenance of skeletal integrity. Supported by USC James H. Zumberge Faculty Research and Innovation Fund, USC MFWA Research Fund, and BCRP 1KB-0060

Kinetic analysis of reversible inhibition of 16alpha-hydroxyandrostenedione aromatization in human placental microsomes by suicide substrates of androstenedione aromatization.

To gain insight into the catalytic function of aromatase and its substrate specificity, we studied reversible inhibition of 16alpha-hydroxyandrostenedione (16alpha-OHAD) aromatization in human placental microsomes by several suicide substrates of androstenedione (AD) aromatization, including 4-hydroxyAD (1), 6-oxoAD (2) and its 19-hydroxy analogue 3, androst-5-ene-4,7,17-trione (4), and 10beta-acetoxyandrost-5-en-7,17-dione (5) that, in contrast, do not cause a suicide inactivation of 16alpha-OHAD aromatization. All inhibitors examined blocked 16alpha-OHAD aromatization in a competitive manner with apparent K(i) values ranging from 0.50 to 980 nM. The relative K(i) values between inhibitors 1-5 obtained in the 16alpha-OHAD aromatization experiments were markedly different from those obtained in the AD aromatization experiments. The results predict that all inhibitors examined bind to the 16alpha-OHAD binding site in a manner that does not cause suicide inactivation of 16alpha-OHAD aromatization. These findings would be useful for understanding the active (binding) site structure as well as the catalytic function of aromatase.

Analysis of the binding energies of testosterone, 5alpha-dihydrotestosterone, androstenedione and dehydroepiandrosterone sulfate with an antitestosterone antibody.

Molecular dynamics simulations and molecular mechanics-Poisson-Boltzmann surface area (MM-PBSA) free energy calculations were used to study the binding of testosterone (TES), 5alpha-dihydrotestosterone (5ADHT), androstenedione (AND), and dehydroepiandrosterone sulfate (DHEAS) to the monoclonal antitestosterone antibody 3-C(4)F(5). The relative binding free energy of TES and AND was also calculated with free energy perturbation (FEP) simulations. The antibody 3-C(4)F(5) has a relatively high affinity (3 x 10(8) M(-1)) and on overall good binding profile for testosterone but its cross-reactivity with DHEAS has been the main reason for the failure to use this antibody in clinical immunoassays. The relative binding free energies obtained with the MM-PBSA method were 1.5 kcal/mol for 5ADHT, 3.8 kcal/mol for AND, and 4.3 kcal/mol for DHEAS, as compared to TES. When a water molecule of the ligand binding site, observed in the antibody-TES crystal structure, was explicitly included in MM-PBSA calculations, the relative binding energies were 3.4, 4.9, and 5.4 kcal/mol for 5ADHT, AND, and DHEAS, respectively. The calculated numbers are in correct order but larger than the corresponding experimental energies of 1.3, 1.5, and 2.6 kcal/mol, respectively. The fact that the MM-PBSA method reproduced the relative binding free energies of DHEAS, a steroid having a negatively charged sulfate group, and the neutrally charged TES, 5ADHT, and AND in satisfactory agreement with experiment shows the robustness of the method in predicting relative binding affinities. The 800-ps FEP simulations predicted that the antibody 3-C(4)F(5) binds TES 1.3 kcal/mol tighter than AND. Computational mutagenesis of selected amino acid residues of the ligand binding site revealed that the lower affinities of AND and DHEAS as compared to TES are due to a combined effect of several residues, each contributing a small fraction to the tighter binding of TES. An exception to this is Tyr99H, whose mutation to Ala lowered the binding of DHEAS 0.7 kcal/mol more than the binding of TES. This is probably due to the hydrogen bonding interaction formed between the OH group of Tyr99H and the sulfate group of DHEAS. Computational mutagensis data also showed that the affinity of the steroids to the antitestosterone antibody 3-C(4)F(5) would be enhanced if Trp47H were repositioned so that it would make more extensive contacts with the bound ligands. In addition, the binding of steroids to antitestosterone, antiprogesterone, and antiestradiol antibodies is discussed.