Carbon isotope ratios of endogenous steroids found in human serum-method development, validation, and reference population-derived thresholds.

In order to detect the misuse of testosterone (T), urinary steroid concentrations and concentration ratios are quantified and monitored in a longitudinal manner to enable the identification of samples exhibiting atypical test results. These suspicious samples are then forwarded to isotope ratio mass spectrometry (IRMS)-based methods for confirmation. Especially concentration ratios like T over epitestosterone (E) or 5α-androstanediol over E proved to be valuable markers. Unfortunately, depending on the UGT2B17 genotype and/or the gender of the athlete, these markers may fail to provide evidence for T administrations when focusing exclusively on urine samples. In recent years, the potential of plasma steroids has been investigated and were found to be suitable to detect T administrations especially in female volunteers. A current drawback of this approach is the missing possibility to confirm that elevated steroid concentrations are solely derived from an administration of T and cannot be attributed to confounding factors. Therefore, an IRMS method for plasma steroids was developed and validated taking into account the comparably limited sample volume. As endogenous reference compounds, unconjugated cholesterol and dehydroepiandrosterone sulfate were found suitable, while androsterone and epiandrosterone (both sulfo-conjugated) were chosen as target analytes. The developed method is based on multi-dimensional gas chromatography coupled to IRMS in order to optimize the overall assay sensitivity. The approach was validated, and a reference population encompassing n = 65 males and females was investigated to calculate population-based thresholds. As proof-of-concept, samples from volunteers receiving T replacement therapies and excretion study samples were investigated.

Simultaneous quantification of cholesterol sulfate, androgen sulfates, and progestagen sulfates in human serum by LC-MS/MS.

Steroids are primarily present in human fluids in their sulfated forms. Profiling of these compounds is important from both diagnostic and physiological points of view. Here, we present a novel method for the quantification of 11 intact steroid sulfates in human serum by LC-MS/MS. The compounds analyzed in our method, some of which are quantified for the first time in blood, include cholesterol sulfate, pregnenolone sulfate, 17-hydroxy-pregnenolone sulfate, 16-α-hydroxy-dehydroepiandrosterone sulfate, dehydroepiandrosterone sulfate, androstenediol sulfate, androsterone sulfate, epiandrosterone sulfate, testosterone sulfate, epitestosterone sulfate, and dihydrotestosterone sulfate. The assay was conceived to quantify sulfated steroids in a broad range of concentrations, requiring only 300 µl of serum. The method has been validated and its performance was studied at three quality controls, selected for each compound according to its physiological concentration. The assay showed good linearity (R(2) > 0.99) and recovery for all the compounds, with limits of quantification ranging between 1 and 80 ng/ml. Averaged intra-day and between-day precisions (coefficient of variation) and accuracies (relative errors) were below 10%. The method has been successfully applied to study the sulfated steroidome in diseases such as steroid sulfatase deficiency, proving its diagnostic value. This is, to our best knowledge, the most comprehensive method available for the quantification of sulfated steroids in human blood.

Resveratrol reduces the levels of circulating androgen precursors but has no effect on, testosterone, dihydrotestosterone, PSA levels or prostate volume. A 4-month randomised trial in middle-aged men.

BACKGROUND: Resveratrol is a naturally occurring polyphenol with purported inhibitory effects on prostate growth and cancer development. A number of studies have demonstrated that resveratrol reduces prostate growth in animal models and reduces prostate cell growth in vitro. Based on these pre-clinical findings, interest in resveratrol is increasing in relation to the management of benign prostate hyperplasia (BPH) and prostate cancer. So far, no human trials have evaluated the effects of resveratrol on circulating androgens, prostate size, or biochemical markers of prostate size. METHODS: In a randomized placebo controlled clinical study using two doses of resveratrol (150 mg or 1,000 mg resveratrol daily) for 4 months, we evaluated the effects on prostate size, prostate specific antigen (PSA) and sex steroid hormones in 66 middle-aged men suffering from the metabolic syndrome(MetS). RESULTS: At baseline, prostate size and PSA were positively correlated (R = 0.34, P < 0.007) as was prostate size and age (R = 0.37, P < 0.003). Prostate size did not correlate with testosterone, free testosterone, dihydrotestosterone (DHT), or any other androgen precursor at baseline. The highest dose of resveratrol lowered the serum level of androstenedione 24% (P = 0.052), dehydroepiandrosterone (DHEA) 41% (P < 0.01), and dehydroepiandrosterone-sulphate (DHEAS) 50% (p<0.001), compared to the control group. However, prostate size and levels of PSA, testosterone, free testosterone and DHT remained unchanged. CONCLUSION: In this population of middle-aged men suffering from MetS, high dose resveratrol (1,000 mg daily) administration for 4 months significantly lowered serum levels of the androgen precursors androstenedione, DHEA and DHEAS, whereas prostate size and circulating levels of PSA, testosterone, free testosterone, and dihydrotestosterone were unaffected. The present study suggests that resveratrol does not affect prostate volume in healthy middle-aged men as measured by PSA levels and CT acquired prostate volumes. Consequently, we find no support for the use of resveratrol in the treatment of benign prostate hyperplasia.

The follicular hormonal profile in low-responder patients undergoing unstimulated cycles: Is it hypoandrogenic?

STUDY QUESTION: What is the final hormonal milieu of pre-ovulatory follicles of low-responder (LR) patients undergoing unstimulated cycles? SUMMARY ANSWER: Neither androgen secretion nor LH was impaired in pre-ovulatory follicles of LR women. WHAT IS KNOWN ALREADY: Therapies currently used to improve ovarian response in LR women have an impact on the final hormonal follicular milieu, and these changes are believed to be partially responsible for determining the success rate in these women. Surprisingly, as far as we know, there is no report of the final hormonal profile of LR women undergoing unstimulated cycles or evidence that follicular androgen secretion in LR women is impaired. STUDY DESIGN, SIZE AND DURATION: A prospective case-control study including 94 women, 36 normal controls and 58 LR patients (19 Young ≤ 35 years LR and 39 Aged >35 years LR) from 2009 to 2011. PARTICIPANTS/MATERIALS, SETTING AND METHODS: Fifty-eight LR women were divided into two groups: Young LR (age ≤ 35; n = 19) and Aged LR (ALR; age >35; n = 39). The control group (group C) comprised 36 egg donors undergoing an unstimulated cycle in our IVF unit. Serum and follicular fluid hormonal concentrations for estradiol (E2), progesterone, testosterone and androstendione were measured. The spindle parameters of metaphase II oocytes generated from these groups were also analysed. MAIN RESULTS AND THE ROLE OF CHANCE: Pre-ovulatory follicles from LR patients had similar androgenic and LH concentrations to those observed in the control group. However, higher intrafollicular concentrations of FSH and progesterone were observed in ALR. Moreover, no differences were found for the spindle evaluation of oocytes between groups by the Oosight technology. LIMITATIONS, REASONS FOR CAUTION: The controls were younger and had a lower BMI than the LR women. The sample size available restricted statistical power. WIDER IMPLICATIONS OF THE FINDINGS: This study suggests that the problem with LR women is not the final pre-ovulatory follicular androgen concentration since this is similar to normal responders, but in the ability to respond to controlled ovarian stimulation protocols. Therefore, efforts should be focused on long-interval androgen priming to potentially increase the recruitment of small antral follicles rather than increasing the intraovarian androgen levels within the current cycle. STUDY FUNDING/COMPETING INTEREST: The present project has been supported by the R+D programme from the Generalitat Valenciana (Regional Valencian Government) IMPIVA MIDTF/2010/95. The authors have no conflict of interest to declare.

Hyperandrogenism sensitizes mononuclear cells to promote glucose-induced inflammation in lean reproductive-age women.

Hyperandrogenism and chronic low-grade inflammation are related in polycystic ovary syndrome (PCOS), but it is unknown whether hyperandrogenemia can activate inflammation. We determined the effect of oral androgen administration on fasting and glucose-stimulated nuclear factor-κB (NF-κB) activation and expression and related markers of inflammation in mononuclear cells (MNC) of lean reproductive-age women. Sixteen lean, ovulatory reproductive-age women were treated with 130 mg of DHEA or placebo (n = 8 each) for 5 days in a randomized, controlled, double-blind fashion. Nuclear activation of NF-κB, p65 and p105 NF-κB subunit RNA, TNFα and IL-1ß mRNA, and NF-κB p65 and inhibitory-κB (IκB) protein were quantified from MNC obtained while fasting and 2 h after glucose ingestion, before and after DHEA or placebo administration. Before treatment, subjects receiving DHEA or placebo exhibited no differences in androgens or any inflammatory markers while fasting and after glucose ingestion. Compared with placebo, DHEA administration raised levels of testosterone, androstenedione, and DHEA-S, increased the percent change in fasting and glucose-challenged activated NF-κB, p65, p105, TNFα, and IL-1ß RNA and p65 protein, and decreased the percent change in fasting and glucose-challenged IκB protein. We conclude that elevation of circulating androgens to the range observed in PCOS upregulates the NF-κB inflammation pathway in lean reproductive-age women. Thus, hyperandrogenemia activates and sensitizes MNC to glucose in this population.

Therapeutic implications of vitamin D and calcium in overweight women with polycystic ovary syndrome.

OBJECTIVE: To assess effects of vitamin D and Calcium (Ca) on hormonal and metabolic milieu of polycystic ovary syndrome (PCOS). DESIGN: Single arm open label trial. METHODS: Twelve overweight and vitamin D deficient women with PCOS underwent a 2 hour oral glucose tolerance testing at baseline and following 3-month supplementation with vitamin D (daily dose of 3533 IU, increased to 8533 IU after the first five participants) and 530 mg elemental Ca daily. MAIN OUTCOME MEASURES: Blood pressure (BP), plasma glucose, insulin, total testosterone (T) androstenedione (A), sex hormone binding globulin, lifestyle parameters were assessed at baseline and following 3-month intervention. Insulin resistance (IR) and area under the curve for glucose and insulin were computed; paired analyses were conducted. RESULTS: Improved serum 25OHD (p < 0.001) and reductions in total T (p = 0.036) and A (p = 0.090) levels were noted following 3-month supplementation, compared to baseline. Significant lowering in BP parameters was seen in participants with baseline BP ≥ 120/80 mmHg (n = 8) and in those with baseline serum 25OHD ≤20 ng/ml (n = 9). Parameters of glucose homeostasis and IR remained unchanged (p > 0.05). CONCLUSIONS: Androgen and BP profiles improved followed three month intervention, suggesting therapeutic implications of vitamin D and Ca in overweight and vitamin D deficient women with PCOS.

Androgens and sexual dysfunction in naturally and surgically menopausal women.

AIM: To evaluate the influence of surgical versus natural menopause on sexual dysfunction, and the role of androgens in that context. MATERIAL & METHODS: Sexual functioning and androgen levels were studied in 35 surgically and 83 naturally menopausal women. Sexual dysfunction was defined as a total score of less than 23 on the Female Sexual Function Index. RESULTS: Sexual dysfunction was significantly more common in surgically than in naturally menopausal women (65.7% vs 44.6%; P = 0.036). The mean total testosterone level in women with sexual dysfunction was significantly lower than that in their counterparts (49.3 ± 21.0 vs 58.8 ± 23.6 ng/mL; P = 0.022). On the other hand, androgen levels did not differ significantly between surgically and naturally menopausal women. Androstenedione (B = 2.253; P = 0.039) and dehydroepiandrosterone sulfate levels (B = 0.222; P < 0.001), and time from menopause (B = -0.064; P = 0.040) were found to be independent determinants of total testosterone levels. While the duration of menopause was significantly longer in surgically menopausal women, this co-factor was not an independent predictor of sexual dysfunction. Logistic regression analysis proved mode of menopause and total testosterone levels to be the only two independent determinants of sexual dysfunction. CONCLUSIONS: Our findings suggest that while surgical menopause is detrimental to sexual functioning by itself, lower testosterone levels are predictive of sexual dysfunction, especially in naturally menopausal women.

Anthropometric, clinical and laboratory comparison of four phenotypes of polycystic ovary syndrome based on Rotterdam criteria.

AIM: To compare all phenotypes of polycystic ovary syndrome (PCOS) for anthropometrical, hormonal, and metabolic differences according to Rotterdam criteria. MATERIAL & METHODS: Women with PCOS (Rotterdam definition; n = 127) and women without PCOS used as controls (n = 44). There were four phenotypes of PCOS: 56/127 (44.09%) of the patients were polycystic ovaries on ultrasound (PCO) + oligo or anovulation (O) + biochemical and/or clinical hyperandrogenism (HA), 29/127 (22.84%) were HA+O, 24/127 (18.90%) were PCO+O and 18/127 (14.17%) were HA+PCO. And 42/127 (33.07%) patients represented the new phenotypes (PCO+O and HA+PCO). Phenotypical, hormonal and metabolic parameters were compared between the groups. RESULTS: Waist-to-hip ratio, luteinizing hormone-to-follicle stimulating hormone ratio, testosterone, androstenedione, dehydroepiandrostenedione were lower in women with PCO+O and controls than the other three phenotypes. The highest score regarding hirsutismus was found in the HA+O phenotype and the lowest score in the PCO+O phenotype. Prevalence of metabolic syndrome and degree of insulin resistance in PCO+O phenotype was closer to control subjects than the other three phenotypes. CONCLUSIONS: Anthropometrical, hormonal, and metabolic differences suggest that PCO+O phenotype is closer to control group than the other PCOS phenotypes.

Effects of a monophasic combined oral contraceptive containing nomegestrol acetate and 17ß-oestradiol in comparison to one containing levonorgestrel and ethinylestradiol on markers of endocrine function.

OBJECTIVES: To compare the effects of two monophasic combined oral contraceptives, containing either nomegestrol acetate/17ß-oestradiol (NOMAC/E2) or levonorgestrel/ ethinylestradiol (LNG/EE) on endocrine function, androgens, and sex hormone-binding globulin (SHBG). METHODS: Randomised, open-label, multi-centre trial involving 121 healthy women, aged 18-50 years old. Participants received NOMAC/E2 (2.5 mg/1.5 mg) in a 24/4-day regimen (n=60) or LNG/EE (150 µg/30 µg) in a 21/7-day regimen (n=61) for six cycles. The primary outcome was the change from baseline to cycle 6 in markers of adrenal and thyroid function, androgens, and SHBG. RESULTS: Total cortisol, corticosteroid-binding globulin (CBG), and thyroxine-binding globulin (TBG) increased from baseline in both groups, with significantly greater increases in the LNG/EE group. No relevant changes from baseline or differences between the groups were observed for thyroid-stimulating hormone (TSH) and free thyroxine (T4). Androgens and androgen precursors decreased from baseline in both groups, with significantly greater decreases in the LNG/EE group (except for free testosterone). A greater increase in SHBG was observed with NOMAC/E2 than with LNG/EE. CONCLUSIONS: NOMAC/E2 has significantly less influence on markers of adrenal and thyroid function and androgens than LNG/EE. The clinical relevance of these findings requires further study.

Determination of anabolic steroids in dried blood using microsampling and gas chromatography-tandem mass spectrometry: Application to a testosterone gel administration study.

Anabolic androgenic steroids (AAS) have been the most commonly abused substances taken by not only professional sportsmen but also recreational bodybuilders. The detection of micro-dose testosterone (T) misuse is particularly challenging as it possesses pseudo-endogenous origin and is sometimes impossible to be identified in urine samples. Dried blood (DB) obtained by finger pricking has been proven to be an alternative matrix for better correlating to physiological responses. Moreover, the introduction of the volumetric absorptive microsampling (VAMS) technology allows overcoming some major limitations of spotting blood onto a filter paper card. In this work, a fast and sensitive GC-MS/MS method was developed and validated for the quantification of AAS in DB collected by means of VAMS. T and the eight top abused synthetic AAS, namely nandrolone, boldenone, mesterolone, drostanolone, metenolone, metandienone, oxandrolone, and dehydrochloromethyl T were selected as the target analytes. The method based on VAMS exhibited good precision, accuracy as well as stability, and superior extraction recoveries over the punched DB spots reported in the literature. The chromatographic separation was achieved within 6.4 min and the detection limit is as little as 50 fg (i.e. able to detect 0.10 ng mL-1 in 20 µL of DB). Confirmed by forty real blood samples, the Deming regression and Bland-Altman analysis revealed that the VAMS DB could be employed for quantifying blood T level in agreement with using the serum specimen. The feasibility of the method was then successfully proven by the analysis of samples collected from a three-arm T administration trial. Our results highlighted that DB total T was a sensitive indicator for identifying transdermal micro-dosing of T. In the groups of receiving T gel administration, T concentrations could rise up to ten times higher than the baseline at 9 h after the application. As a future step, this approach is being expanded to a large cohort screening of bodybuilders at gym and ultimately may allow universal applications on monitoring sports drug misuse.

Menstrual cycle-related changes in circulating androgens in healthy women with self-reported normal sexual function.

INTRODUCTION: There is currently neither a clinically useful, reliable and inexpensive assay to measure circulating levels of free testosterone (T) in the range observed in women, nor is there agreement on the serum free T threshold defining hypoandrogenism that is associated with female-impaired sexual function. AIM: Following the Clinical and Laboratory Standards Institute guidelines, we generated clinically applicable ranges for circulating androgens during specific phases of the menstrual cycle in a convenience sample of 120 reproductive-aged, regularly cycling healthy European Caucasian women with self-reported normal sexual function. METHODS: All participants were asked to complete a semistructured interview and fill out a set of validated questionnaires, including the Female Sexual Function Index, the Female Sexual Distress Scale, and the 21-item Beck's Inventory for Depression. Between 8 am and 10 am, a venous blood sample was drawn from each participant during the midfollicular (day 5 to 8), the ovulatory (day 13 to 15), and the midluteal phase (day 19 to 22) of the same menstrual cycle. MAIN OUTCOME MEASURES: Serum levels of total and free testosterone, Delta(4)-androstenedione, dehydroepiandrosterone sulphate and sex hormone-binding globulin during the midfollicular, ovulatory and midluteal phase of the same menstrual cycle. RESULTS: Total and free T levels showed significant fluctuations, peaking during the ovulatory phase. No significant variation during the menstrual cycle were observed for Delta(4)-androstenedione and dehydroepiandrosterone sulphate. Despite the careful selection of participants that yielded an homogeneous group of women without sexual disorders, we observed a wide range of distribution for each of the circulating androgens measured in this study. CONCLUSIONS: This report provides clinically applicable ranges for androgens throughout the menstrual cycle in reproductive-aged, regularly cycling, young healthy Caucasian European women with self-reported normal sexual function.

A dose-response study of testosterone on sexual dysfunction and features of the metabolic syndrome using testosterone gel and parenteral testosterone undecanoate.

The objective of this study was to observe the dose-response effects of testosterone (T) treatment on symptoms of sexual dysfunction and the metabolic syndrome. Two cohorts of elderly men with late-onset hypogonadism were followed over 9 months. Group 1, consisting of 28 men (mean age, 61 years; mean T level, 2.07 +/- 0.50 ng/mL), received long-acting T undecanoate (TU; 1000 mg); group 2, composed of 27 men (mean age, 60 years; mean T level, 2.24 +/- 0.41 ng/mL), received T gel (50 mg/day) for 9 months. In patients treated with T gel, plasma T levels rose from 2.24 +/- 0.41 to 2.95 +/- 0.52 (statistically significant) at 3 months, 3.49 +/- 0.89 (statistically significant) at 6 months, and 3.80 +/- 0.73 ng/mL at 9 months (T level at 6 months was compared with T level at 3 months). With TU, plasma T levels rose from 2.08 +/- 0.56 to 4.81 +/- 0.83 (statistically significant) at 3 months, 5.29 +/- 0.91 at 6 months, and 5.40 +/- 0.77 ng/mL at 9 months. With TU, the plasma T levels were statistically significantly higher than with T gel With TU, there was a greater improvement in sexual symptoms and in symptoms of the metabolic syndrome. With both treatments, changes in waist circumference correlated with changes in total, low-density, and high-density lipoprotein cholesterol. Parameters of safety were not different between the 2 treatments. T administration had a beneficial effect on sexual dysfunction and symptoms of the metabolic syndrome in elderly men. The higher plasma levels of T generated with TU than with T gel were clearly more effective, indicating that there is a T dose-effect relationship.

Validation of commercial ELISAs for quantifying anabolic growth factors and cytokines in canine ACD-A anticoagulated plasma.

Platelet-rich plasma has been studied extensively in dogs, but validation of enzyme-linked immunosorbent assays (ELISAs) for quantifying anabolic growth factors and inflammatory cytokines in canine plasma prepared with citrate-based anticoagulants is not available. We performed a validation of commercial ELISAs for transforming growth factor-beta 1 (TGF-ß1), platelet-derived growth factor-BB (PDGF-BB), vascular endothelial growth factor (VEGF), tumor necrosis factor-alpha (TNF-α), and interleukin-1 beta (IL-1ß) for use with canine plasma prepared with acid-citrate-dextrose, solution A (ACD-A). Platelet-poor plasma (PPP) anticoagulated with ACD-A as well as PPP anticoagulated with ACD-A and spiked with the relevant canine recombinant proteins were evaluated with each ELISA to calculate the efficiency of spike recovery. Replicates of the spiked PPP were also assessed in 2 additional assays to quantify intra-assay and interassay precision. The efficiency of spike recovery was within 75-125% of the expected concentration for the TGF-ß1, PDGF-BB, and VEGF ELISAs. The intra- and interassay variability were <25% for the TGF-ß1, PDGF-BB, VEGF, and TNF-α ELISAs. The TGF-ß1, PDGF-BB, and VEGF ELISAs demonstrate acceptable efficiency of spike recovery and intra- and interassay variability, whereas the TNF-α and IL-1ß ELISAs did not meet industry standards of performance with ACD-A anticoagulated canine plasma.

Demographic, lifestyle, and other factors in relation to antimüllerian hormone levels in mostly late premenopausal women.

OBJECTIVE: To identify reproductive, lifestyle, hormonal, and other correlates of circulating antimüllerian hormone (AMH) concentrations in mostly late premenopausal women. DESIGN: Cross-sectional study. SETTING: Not applicable. PATIENT(S): A total of 671 premenopausal women not known to have cancer. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): Concentrations of AMH were measured in a single laboratory using the picoAMH ELISA. Multivariable-adjusted median (and interquartile range) AMH concentrations were calculated using quantile regression for several potential correlates. RESULT(S): Older women had significantly lower AMH concentrations (≥40 [n = 444] vs. <35 years [n = 64], multivariable-adjusted median 0.73 ng/mL vs. 2.52 ng/mL). Concentrations of AMH were also significantly lower among women with earlier age at menarche (<12 [n = 96] vs. ≥14 years [n = 200]: 0.90 ng/mL vs. 1.12 ng/mL) and among current users of oral contraceptives (n = 27) compared with never or former users (n = 468) (0.36 ng/mL vs. 1.15 ng/mL). Race, body mass index, education, height, smoking status, parity, and menstrual cycle phase were not significantly associated with AMH concentrations. There were no significant associations between AMH concentrations and androgen or sex hormone-binding globulin concentrations or with factors related to blood collection (e.g., sample type, time, season, and year of blood collection). CONCLUSION(S): Among premenopausal women, lower AMH concentrations are associated with older age, a younger age at menarche, and currently using oral contraceptives, suggesting these factors are related to a lower number or decreased secretory activity of ovarian follicles.

Understanding alterations on blood and biochemical parameters in athletes that use dietary supplements, steroids and illicit drugs.

In recent years it was verified there are an alarming growing number of teenagers and young adults using a combination of dietary supplements (DS) anabolic androgenic steroids (AAS) and drugs of abuse. This practice is used to improve physical fitness and appearance, may cause serious side effects. This article shows the alterations in the hematological and renal function parameters associate with these substances in 40 athletes. This research involved three steps: 1-the administration of a self-completion questionnaire ; 2-the assessment of hematological and biochemical parameters of renal function and; 3-toxicological urinalysis. Hematological and biochemical tests were conducted in an accredited laboratory and the toxicological urinalysis was validated in our laboratory using liquid-liquid extraction (LLE) and gas chromatography-mass spectrometry (GC-MS). The testosterone levels in the participants who consumed steroids increased 20-60% and alterations in serum creatinine, urea and uric reached values of up to 1.9; 60.6 and 7.5mg/dL, respectively. The toxicological urinalysis supports self-reports confirming the use of AAS and recreational drugs, putting at risk the health of those athletes increasing the chances of kidney diseases.

Testicular vs adrenal sources of hydroxy-androgens in prostate cancer.

Neoadjuvant androgen deprivation therapy (NADT) is one strategy for the treatment of early-stage prostate cancer; however, the long-term outcomes of NADT with radical prostatectomy including biochemical failure-free survival are not promising. One proposed mechanism is incomplete androgen ablation. In this study, we aimed to evaluate the efficiency of serum hydroxy-androgen suppression in patients with localized high-risk prostate cancer under NADT (leuprolide acetate plus abiraterone acetate and prednisone) and interrogate the primary sources of circulating hydroxy-androgens using our recently described stable isotope dilution liquid chromatography mass spectrometric method. For the first time, three androgen diols including 5-androstene-3ß,17ß-diol (5-adiol), 5α-androstane-3α,17ß-diol (3α-adiol), 5α-androstane-3ß,17ß-diol (3ß-adiol), the glucuronide or sulfate conjugate of 5-adiol and 3α-adiol were measured and observed to be dramatically reduced after NADT. By comparing patients that took leuprolide acetate alone vs leuprolide acetate plus abiraterone acetate and prednisone, we were able to distinguish the primary sources of these androgens and their conjugates as being of either testicular or adrenal in origin. We find that testosterone, 5α-dihydrotestosterone (DHT), 3α-adiol and 3ß-adiol were predominately of testicular origin. By contrast, dehydroepiandrosterone (DHEA), epi-androsterone (epi-AST) and their conjugates, 5-adiol sulfate and glucuronide were predominately of adrenal origin. Our findings also show that NADT failed to completely suppress DHEA-sulfate levels and that two unappreciated sources of intratumoral androgens that were not suppressed by leuprolide acetate alone were 5-adiol-sulfate and epi-AST-sulfate of adrenal origin.

Effect of the 5α-reductase enzyme inhibitor dutasteride in the brain of intact and parkinsonian mice.

Dutasteride is a 5alpha-reductase inhibitor in clinical use to treat endocrine conditions. The present study investigated the neuroprotective mechanisms of action of dutasteride in intact and 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-lesioned mice using a low dose of MPTP not affecting motor activity modeling early stages of Parkinson's disease (PD). We hypothesized that dutasteride neuroprotection is due to altered steroids levels. Dutasteride pre-treatment prevented loss of striatal dopamine (DA) and its metabolite DOPAC. Dutasteride decreased effects of MPTP on striatal dopamine transporter (DAT), vesicular monoamine transporter 2 (VMAT2) and D2 DA receptor specific binding while D1 receptor specific binding remained unchanged. Dutasteride enhanced DAT specific binding and the glycosylated form of DAT in intact mice. MPTP-lesioned mice had plasma and brain testosterone and dihydrotestosterone levels lower than control mice whereas progesterone and its metabolites (dihydroprogesterone, isopregnanolone and tetrahydroprogesterone) pathway showed increases. Dutasteride treatment by inhibiting transformation of progesterone and testosterone to its metabolites elevated plasma and brain concentrations of testosterone compared to MPTP mice and decreased DHT levels in intact mice. Plasma and brain estradiol levels were low and remained unchanged by MPTP and/or dutasteride treatment. Dutasteride treatment did not affect striatal phosphorylation of Akt and its downstream substrate GSK3ß as well as phosphorylation of ERK1/2 in intact and MPTP lesioned MPTP mice. Striatal glial fibrillary acidic protein (GFAP) levels were markedly elevated in MPTP compared to control mice and dutasteride reduced GFAP levels in MPTP mice. Treatment with dutasteride post-lesion left unchanged striatal DA levels. These results suggest dutasteride as promising drug for PD neuroprotection.

Relationship of androgens to insulin resistance and chronic inflammation in morbidly obese premenopausal women: studies before and after vertical banded gastroplasty.

BACKGROUND: Morbid obesity is associated with insulin resistance (IR), type 2 diabetes, lipid abnormalities, and hypertension. The association of obesity with increased androgen production and low concentrations of sex hormone-binding globulin (SHBG) in women has been demonstrated as well as a strong association of androgens with markers of inflammation such as high-sensitive C-reactive protein (hsCRP). Because weight loss results in a significant decrease in cardiovascular risk factors, IR and inflammation, we questioned a possible interrelationship between androgens, IR and inflammation in a prospective study with 43 morbidly obese female patients undergoing bariatric surgery. METHODS: SHBG, dehydroepiandrosterone-sulfate (DHEA-S), and insulin were measured by ELISA, cortisol by fluorescence polarization immunoassay, androstendione by RIA, and testosterone by electrochemiluminescence immunoassay. The free androgen index (FAI) was calculated as the total testosterone/SHBG ratio. High sensitivity assays were used to obtain concentrations of fasting hsCRP, Interleukin-6 (IL-6) and tumor necrosis factor-alpha (TNF-alpha). RESULTS: Weight loss resulted in a significant increase in SHBG (17+/-12 vs 70+/-30 nmol/l, P<0.0001). Serum concentrations of androstendione, total testosterone, DHEA-S and the FAI decreased significantly (2.05+/-0.78 vs 1.40+/-0.54 ng/ml, P<0.0001; 1.88+/-0.84 vs 1.16+/-0.57 nmol/L, P<0.0001; 1.72+/-0.86 vs 1.49+/-0.78 microg/ml, P<0.01; 0.15+/-0.10 vs 0.02+/-0.01, P<0.0001; respectively). Preoperatively and postoperatively, no association between androgens and IR, chronic subclinical inflammation and lipid parameters could be observed. CONCLUSION: Massive weight loss induced by bariatric surgery is associated with a significant reduction of androgen concentrations but not related to the concomitant decline of chronic inflammation, IR, glucose and lipid abnormalities in premenopausal morbidly obese women.

3alpha,5alpha-THP: a potential plasma neurosteroid biomarker in Alzheimer's disease and perhaps non-Alzheimer's dementia.

RATIONALE: A plasma biomarker for neurodegenerative disease is desirable because blood is relatively simple to obtain compared with other biological samples such as cerebrospinal fluid. Recent literature suggests that neurosteroid metabolism may be altered in Alzheimer's disease (AD). OBJECTIVES: We sought to measure the plasma levels of seven steroids to assess their potential as biomarkers for dementia and AD. METHODS: Steroids were measured using validated radioimmunoassay methods in AD (n=15), non-AD dementia (n=4), and control subjects (n=20). Demented subjects were in the mild-to-moderate stages of illness. Measurements were done blind to subject status in an independent laboratory. RESULTS: The notable finding was the significantly lower 5alpha-pregnan-3alpha-ol-20-one (3alpha,5alpha-THP) level in demented subjects compared with controls (25% decrease; p=0.004); 3alpha,5alpha-THP was the only one of the steroids demonstrating an effect of dementia. CONCLUSION: Lowered 3alpha,5alpha-THP levels appear promising as a biomarker in dementia, but further work is needed to establish the sensitivity and specificity of these findings in AD.

Open-label pilot study of testosterone patch therapy in men with opioid-induced androgen deficiency.

UNLABELLED: We conducted a 24-week open-label pilot study of testosterone (T) patch therapy in 23 men with opioid-induced androgen deficiency (OPIAD). The T dosage was 5 mg/day for the first 12 weeks and 7.5 mg/day for the second 12 weeks. Seven subjects discontinued prematurely: 4 for noncompliance, 2 for skin irritation and 1 for hepatitis C treatment. In the "completers" population (n = 16), mean (SD) free T levels (normal range 52 to 280 pg/mL) were 28.5 (18.6) pg/mL at baseline, 72.8 (29.6) pg/mL on 5 mg/day (P < .001 vs. baseline), and 120.2 (69.5) pg/mL on 7.5 mg/day (P < .001 vs. baseline and P < .01 vs. 5 mg/day). Total T, dihydrotestosterone, and estradiol showed parallel changes. Sex hormone-binding globulin levels were elevated at baseline and decreased modestly with treatment (P < .05 vs. baseline at 5 mg/day; P < .01 vs. baseline at 7.5 mg/day). Luteinizing hormone levels were in the low-normal range at baseline and suppressed markedly with treatment (P < .001 vs. baseline at both doses). Androgen deficiency symptoms (ADSQ), sexual function (Watts SFQ), mood (PGWB), depression (BDI-II), and hematocrit levels showed improvement during treatment, generally more so at the 7.5 mg/day dosage (P < .001 vs. baseline for most parameters). Pain scores (BPI-SF) decreased slightly on 7.5 mg/day (interference score: P < .05 vs. baseline and 5 mg/day); the use of opioids did not change appreciably. The testosterone patches were generally well tolerated. PERSPECTIVE: Long-acting opioid preparations suppress the hypothalamic-pituitary-gonadal axis in men and produce a symptomatic state of opioid-induced androgen deficiency (OPIAD). Testosterone patch therapy at a dose of 7.5 mg/day normalizes hormone levels and appears to improve a number of quality of life parameters (eg, sexual function, well-being, mood) in men with OPIAD.