Blood and adipose tissue steroid metabolomics and mRNA expression of steroidogenic enzymes in periparturient dairy cows differing in body condition.

In high-yielding dairy cows, the rapidly increasing milk production after parturition can result in a negative nutrient balance, since feed intake is insufficient to cover the needs for lactation. Mobilizing body reserves, mainly adipose tissue (AT), might affect steroid metabolism. We hypothesized, that cows differing in the extent of periparturient lipomobilization, will have divergent steroid profiles measured in serum and subcutaneous (sc)AT by a targeted metabolomics approach and steroidogenic enzyme profiles in scAT and liver. Fifteen weeks antepartum, 38 multiparous Holstein cows were allocated to a high (HBCS) or normal body condition (NBCS) group fed differently until week 7 antepartum to either increase (HBCS BCS: 3.8 ± 0.1 and BFT: 2.0 ± 0.1 cm; mean ± SEM) or maintain BCS (NBCS BCS: 3.0 ± 0.1 and BFT: 0.9 ± 0.1 cm). Blood samples, liver, and scAT biopsies were collected at week -7, 1, 3, and 12 relative to parturition. Greater serum concentrations of progesterone, androsterone, and aldosterone in HBCS compared to NBCS cows after parturition, might be attributed to the increased mobilization of AT. Greater glucocorticoid concentrations in scAT after parturition in NBCS cows might either influence local lipogenesis by differentiation of preadipocytes into mature adipocytes and/or inflammatory response.

The influence of low-carbohydrate diets on the metabolic response to androgen-deprivation therapy in prostate cancer.

BACKGROUND: Prostate cancer (PC) is the second most lethal cancer for men. For metastatic PC, standard first-line treatment is androgen deprivation therapy (ADT). While effective, ADT has many metabolic side effects. Previously, we found in serum metabolome analysis that ADT reduced androsterone sulfate, 3-hydroxybutyric acid, acyl-carnitines but increased serum glucose. Since ADT reduced ketogenesis, we speculate that low-carbohydrate diets (LCD) may reverse many ADT-induced metabolic abnormalities in animals and humans. METHODS: In a multicenter trial of patients with PC initiating ADT randomized to no diet change (control) or LCD, we previously showed that LCD intervention led to significant weight loss, reduced fat mass, improved insulin resistance, and lipid profiles. To determine whether and how LCD affects ADT-induced metabolic changes, we analyzed serum metabolites after 3-, and 6-months of ADT on LCD versus control. RESULTS: We found androsterone sulfate was most consistently reduced by ADT and was slightly further reduced in the LCD arm. Contrastingly, LCD intervention increased 3-hydroxybutyric acid and various acyl-carnitines, counteracting their reduction during ADT. LCD also reversed the ADT-reduced lactic acid, alanine, and S-adenosyl methionine (SAM), elevating glycolysis metabolites and alanine. While the degree of androsterone reduction by ADT was strongly correlated with glucose and indole-3-carboxaldehyde, LCD disrupted such correlations. CONCLUSIONS: Together, LCD intervention significantly reversed many ADT-induced metabolic changes while slightly enhancing androgen reduction. Future research is needed to confirm these findings and determine whether LCD can mitigate ADT-linked comorbidities and possibly delaying disease progression by further lowering androgens.

Circulating androgens and postmenopausal ovarian cancer risk in the Women's Health Initiative Observational Study.

Our knowledge of epidemiologic risk factors for ovarian cancer supports a role for androgens in the pathogenesis of this disease; however, few studies have examined associations between circulating androgens and ovarian cancer risk. Using highly sensitive LC-MS/MS assays, we evaluated associations between pre-diagnostic serum levels of 12 androgens, including novel androgen metabolites that reflect androgen activity in tissues, and ovarian cancer risk among postmenopausal women in a nested case-control study in the Women's Health Initiative (WHI) Observational Study (OS). We frequency-matched 169 ovarian cancer cases to 410 controls from women enrolled in WHI-OS who were not using menopausal hormones at enrollment/blood draw. We estimated associations overall and by subtype (n = 102 serous/67 non-serous) using multivariable adjusted logistic regression. Androgen/androgen metabolite levels were not associated with overall ovarian cancer risk. In analyses by subtype, women with increased levels of androsterone-glucuronide (ADT-G) and total 5-α reduced glucuronide metabolites (markers of tissue-level androgenic activity) were at increased risk of developing non-serous ovarian cancer: ADT-G tertile (T)3 versus T1 odds ratio [OR] (95% confidence interval [CI]) 4.36 (1.68-11.32), p-heterogeneity 0.002; total glucuronide metabolites 3.63 (1.47-8.95), 0.002. Risk of developing serous tumors was unrelated to these markers. ADT-G and total glucuronide metabolites, better markers of tissue-level androgenic activity in women than testosterone, were associated with an increased risk of developing non-serous ovarian cancer. Our work demonstrates that sex steroid metabolism is important in the etiology of non-serous ovarian cancers and supports a heterogeneous hormonal etiology across histologic subtypes of ovarian cancer.

The quantitation of 7beta-hydroxy-epiandrosterone in the plasma and seminal plasma of men with different degrees of fertility.

7beta-hydroxy-epiandrosterone (7beta-OH-EpiA) is an endogenous androgen metabolite that has been shown to exert neuroprotective, anti-inflammatory and anti-estrogenic effects. However, to the best of our knowledge no information is available about this androgen steroid in relation to sperm quality. We analyzed 7beta-OH-EpiA in plasma and seminal plasma using a newly developed isotope dilution ultra-high performance liquid chromatography - mass spectrometry method. Validation met the requirements of FDA guidelines. Levels of 7beta-OH-EpiA were measured in 191 men with different degrees of infertility. One-way analysis of variance followed by multiple comparison and correlation analysis adjusted for age, BMI and abstinence time were performed to evaluate the relationships between this steroid and sperm quality. Concentrations of 7beta-OH-EpiA in seminal plasma were significantly higher in severely infertile men in comparison with healthy men and slightly infertile men. The same trend was found when blood plasma was evaluated. Furthermore, plasma 7beta-OH-EpiA negatively correlated with sperm concentration (-0.215; p<0.01) and total count (-0.15; p<0.05). Seminal 7beta-OH-EpiA was negatively associated with motility (-0.26; p<0.01), progressively motile spermatozoa (-0.233; p<0.01) and nonprogressively motile spermatozoa (-0.188; p<0.05). 7beta-OH-EpiA is associated with lower sperm quality and deserves more research in that respect.

The effect of tea consumption on the steroid profile.

Green tea (GT), along with its flavonol epigallocatechin-3-gallate (EGCG), has shown to inhibit the UGT2B17 isoenzyme, which is highly involved in the glucuronidation of testosterone (T) and its metabolites. Since the steroid profile (SP) is composed of urinary concentrations of T and related metabolites excreted in both the free and the glucuronide fractions, GT consumption could alter the SP, leading to misunderstanding in doping controls. The aim of the present work was to study the effect of GT consumption on the SP. This study was performed with 29 male volunteers, which could be classified in 2 arms depending on their T/E values (0.12 ± 0.02, n = 12; 1.64 ± 0.90, n = 17). The clinical protocol was designed to evaluate the effect of GT administration on the SP biomarkers. Participants were asked to consume GT with a high content of EGCG for 7 days (5 GT beverages along the whole day for days 1-6 and 9 GT beverages on day 7, corresponding to 520 and 936 mg/day of EGCG, respectively). Urine samples were collected before and during GT consumption at different time periods. The SP was measured using gas chromatography-mass spectrometry. The excretion rates of the SP metabolites did not change after GT consumption. Moreover, the individual evaluation of the subject's steroidal biological passport resulted in normal sequences. The results obtained show that GT consumption does not distort the establishment of normal ranges of SP parameters. Therefore, GT consumption does not need to be considered a confounding factor in the SP evaluation.

Urine and Serum Sex Steroid Profile in Testosterone-Treated Transgender and Hypogonadal and Healthy Control Men.

Background: The impact of testosterone (T) treatment on antidoping detection tests in female-to-male (F2M) transgender men is unknown. We investigated urine and serum sex steroid and luteinizing hormone (LH) profiles in T-treated F2M men to determine whether and, if so, how they differed from hypogonadal and healthy control men. Method: Healthy transgender (n = 23) and hypogonadal (n = 24) men aged 18 to 50 years treated with 1000 mg injectable T undecanoate provided trough urine and blood samples and an additional earlier postinjection sample (n = 21). Healthy control men (n = 20) provided a single blood and urine sample. Steroids were measured by mass spectrometry-based methods in urine and serum, LH by immunoassay, and uridine 5'-diphospho-glucuronosyltransferase 2B17 genotype by polymerase chain reaction. Results: Urine LH, human chorionic gonadotropin, T, epitestosterone (EpiT), androsterone (A), etiocholanolone (Etio), A/Etio ratio, dehydroepiandrosterone (DHEA), dihydrotestosterone (DHT), and 5α,3α- and 5ß,3α-androstanediols did not differ between groups or by time since last T injection. Urine T/EpiT ratio was <4 in all controls and 12/68 (18%) samples from T-treated men, but there was no difference between T-treated groups. Serum estradiol, estrone, and DHEA were higher in transgender men, and serum T and DHT were higher in earlier compared with trough blood samples, but serum LH, follicle-stimulating hormone, and 3α- and 3ß,5α-diols did not differ between groups. Conclusion: Urine antidoping detection tests in T-treated transgender men can be interpreted like those of T-treated hypogonadal men and are unaffected by time since last T dose. Serum steroids are more sensitive to detect exogenous T administration early but not later after the last T dose.

Androst-5-ene-3ß,7α/ß,17ß-triols, their plasma levels and dependence on the hypothalamic-pituitary-adrenal axis.

Androst-5-ene-triols are metabolites of dehydroepiandrosterone, the most abundant steroid hormone in human circulation. Many observations in rodents have demonstrated the anti-inflammatory and immune modulating activity of 7ß-hydroxy-androst-5-enes, and on the basis of these experiments androst-5-ene-3ß,7ß,17ß-triol is considered as a potential agent in the treatment of autoimmune diseases. In contrast to the fairly abundant information on the levels and effects of androst-5-ene-triols in experimental animals and of their the pharmacological perspective, little is known about androst-5-ene-3ß,7α/ß,17ß-triols circulating in human blood, their regulation by the hypothalamo-pituitary-adrenal axis, or their daily concentration variability. Here we provide some data on androst-5-ene-3ß,7α/ß,17ß-triol concentrations under various conditions in men and women.

Concentration range of serum sex steroids in normal postmenopausal women and those with diagnosis of vulvovaginal atrophy.

OBJECTIVE: The aim of the study was to determine the range of serum sex-related steroids in normal postmenopausal women and in women of the same age with a diagnosis of vulvovaginal atrophy (VVA). METHODS: Validated mass spectrometry-based assays coupled to gas or liquid chromatography were used over a 10-year period for steroid measurements. Serum samples were obtained in up to 1,512 women aged 55 to 65 years. RESULTS: Serum estrone sulfate (E1S) and androsterone glucuronide (ADT-G), the main metabolites of estrogens and androgens, respectively, were 16.9% (P = 0.005) and 16.1% (P = 0.001) higher in women not diagnosed with moderate/severe VVA than those diagnosed with VVA. Serum estrone (E1) was 14.5% (P < 0.0001) higher in women with no diagnosis of VVA, whereas the other steroids did not show meaningful differences. The limited biological significance of serum estradiol (E2) and testosterone is supported by the lack of statistical significance in the serum concentrations of these two steroids between the two groups. Most importantly, for the women without a diagnosis of VVA, the normal upper limit (95 centile) of serum E2 was 9.15 pg/mL (n = 364) and 10.7 pg/mL (n = 67) for a weighted average of 9.99 pg E2/mL. A limit of 10 pg E2/mL has recently been found by two other laboratories. When comparing 50- to 59-year-old and 70- to 79-year-old women, serum E2, E1S, ADT-G, and DHEA were, respectively, 24.4%, 22.6%, 27.0%, and 85.9% higher in the younger group. CONCLUSIONS: Somewhat higher values, namely, 16.9% and 16.1%, are observed in the serum concentrations of the estrogen (E1S) and androgen (ADT-G) metabolites in normal compared with women with a diagnosis of VVA. Such data indicating a lower estrogenic and androgenic global exposure in women diagnosed with VVA offers an opportunity for the local intravaginal administration of DHEA to replace the deficiency in endogenous DHEA.

Simultaneous profiles of sulfonated androgens, sulfonated estrogens and sulfonated progestogens in postpubertal boars (sus scrofa domestica) measured by LC-MS/MS.

Sulfonated steroids (s-St) have been usually regarded as inactive metabolites but are progressively considered as precursors for the intra-tissue formation of bioactive steroids. Moreover, independent effects without preceding removal of the sulfate group have been observed. We use the porcine testicular-epididymal compartment as a model to investigate the still largely unknown s-St physiology as the boar exhibits an intriguingly broad s-St spectrum predominantly originating from the testis. The application of LC-MS/MS in steroidomics enables the determination of unconjugated and intact sulfonated steroids with currently highest specificity and good sensitivity, allowing the concurrent measuring of numerous analytes in larger quantities of samples. Profiles (6h, 20min intervals) were generated for sulfonated 5-androstene-3ß,17ß-diol (Adiol-S), androsterone (A-S), dehydroepiandrosterone (DHEA-S), epiandrosterone (EA-S), epitestosterone (ET-S), estrone (E1-S), estradiol-17ß (E2-S), pregnenolone (P5-S), 17αOH-pregnenolone (OHP5-S) and unconjugated testosterone (T) in four unstimulated and four hCG-stimulated boars. Moreover, concentrations were measured in individual samples collected from testicular afferent and efferent blood to differentiate between testicular vs. extratesticular origin. Highest concentrations were found for EA-S, followed by ET-S, Adiol-S and DHEA-S, which mostly exceeded the levels of E1-S and A-S. Lowest concentrations were obtained for E2-S, P5-S and OHP5-S. The analytical profile also included sulfonated T, 5α-dihydrotestosterone and cholesterol. However, their concentrations were below the limit of quantification. Profiles of quantifiable s-St were consistent with a wave-like pattern associated with T pulses. In postpartal females (n=5) concentrations of all analytes assessed were undetectable, suggesting that in pigs the adrenals are not a quantitatively significant source of s-St.

Neurosteroid Levels in Patients With Bipolar Disorder and a History of Cannabis Use Disorders.

PURPOSE/BACKGROUND: In animal models, levels of the neurosteroid pregnenolone increase after tetrahydrocannabinol (THC) administration and pregnenolone appears to attenuate the brain effects of THC. Given these interactions between pregnenolone and THC, we evaluated baseline neurosteroid levels in participants with a history of a cannabis use disorders (CUDs). METHODS/PROCEDURES: Bipolar depressed participants were enrolled in a randomized placebo-controlled clinical trial to evaluate the efficacy of add-on pregnenolone for depression and before receiving pregnenolone or placebo. Baseline serum levels of neurosteroids (pregnenolone, allopregnanolone, pregnanolone, and androsterone) were analyzed in 53 participants with highly sensitive and specific gas chromatography/mass spectrometry. Current, active substance use disorders, or a positive baseline urine drug screen, were exclusionary. Participants were classified by past cannabis abuse or dependence diagnosis using the structured clinical interview for Diagnostic and Statistical Manual of Mental Disorders, Fourth Edition. Data were analyzed by independent t tests for separate neurosteroids. FINDINGS/RESULTS: Participants with a history of CUD had higher serum pregnanolone, lower allopregnanolone, a higher pregnanolone to allopregnanolone ratio, and a lower pregnenolone to pregnanolone ratio compared with those without a history of cannabis use. Similar findings were not observed based on a history of other substance use disorders with the exception of lower allopregnanolone in those with opioid use disorders. Notably, the majority of those with an opioid use disorder also had a CUD (75%). IMPLICATIONS/CONCLUSIONS: These findings potentially suggest either enduring changes in neurosteroids in people with past CUDs or represent a vulnerability marker for a CUD.

Androsterone glucuronide to dehydroepiandrosterone sulphate ratio is discriminatory for obese Caucasian women with polycystic ovary syndrome.

BACKGROUND: Androsterone glucuronide (ADTG) concentrations have been suggested as a marker of the effects of androgens at the target tissue level. As the mechanism for hyperandrogenemia in obese and nonobese polycystic ovary syndrome (PCOS) may differ, this study compared the different androgen parameters in non-obese compared to obese women with PCOS, and in normal subjects. METHODS: Eleven non-obese and 14 obese women with PCOS were recruited and compared to 11 control women without PCOS. Total testosterone, dehydroepiandrosterone sulphate (DHEAS), ADTG, and androstenedione were analysed using gold standard tandem mass spectrometry, and the free androgen index (FAI) was calculated. RESULTS: Total testosterone, ADTG and androstendione levels did not differ between non-obese (body mass index (BMI) ≤25 kg/m2) and obese PCOS (BMI >25 kg/m2) but all were significantly higher than for controls (p < 0.01). The ADTG to DHEAS ratio was significantly elevated 39 ± 6 (p < 0.01) in obese PCOS in comparison to non-obese PCOS and controls (28 ± 5 and 29 ± 4, respectively). The free androgen index (FAI) and insulin resistance (HOMA-IR) were significantly higher in obese PCOS compared to non-obese PCOS and controls (p < 0.01). DHEAS was significantly higher in the non-obese versus obese PCOS (p < 0.01). All androgen parameters were significantly lower and sex hormone binding globulin (SHBG) significantly higher in normal subjects compared to those with obese and non-obese PCOS. CONCLUSIONS: The ADTG:DHEAS ratio was significantly elevated in obese PCOS compared to non-obese PCOS and controls suggesting that this may be a novel biomarker discriminatory for obese PCOS subjects, perhaps being driven by higher hepatic 5α reductase activity increasing ADTG formation in these women.

A Metabolome-Wide Study of Dry Eye Disease Reveals Serum Androgens as Biomarkers.

PURPOSE: To test the association between serum metabolites and dry eye disease (DED) using a hypothesis-free metabolomics approach. DESIGN: Cross-sectional association study. PARTICIPANTS: A total of 2819 subjects from the population-representative TwinsUK cohort in the United Kingdom, with a mean age of 57 years (range, 17-82 years). METHODS: We tested associations between 222 known serum metabolites and DED. All subjects underwent nontargeted metabolomic analysis of plasma samples using gas and liquid chromatography in combination with mass spectrometry (Metabolon Inc., Durham, NC). Dry eye disease was defined from the validated Short Questionnaire for Dry Eye Syndrome (SQDES) as a previous diagnosis of DED by a clinician or "often" or "constant" symptoms of dryness and irritation. Analyses were performed with linear mixed effect models that included age, BMI, and sex as covariates, corrected for multiple testing. MAIN OUTCOME MEASURES: Primary outcome was DED as defined by the SQDES, and secondary outcomes were symptom score of DED and a clinical diagnosis of DED. RESULTS: Prevalence of DED as defined by the SQDES was 15.5% (n = 436). A strong and metabolome-wide significant association with DED was found with decreased levels of the metabolites androsterone sulfate (P = 0.00030) and epiandrosterone sulfate (P = 0.00036). Three other metabolites involved in androgen metabolism, 4-androsten-3beta,17beta-diol disulfate 1 and 2, and dehydroepiandrosterone sulfate, were the next most strongly associated of the 222 metabolites, but did not reach metabolome-wide significance. Dryness and irritation symptoms, as opposed to a clinical diagnosis, were particularly strongly associated with decreased androgen steroid metabolites, with all reaching metabolome-wide significance (androsterone sulfate, P = 0.000000029; epiandrosterone sulfate, P = 0.0000040; 4-androsten-3beta,17beta-diol disulfate 1, P = 0.000016; 4-androsten-3beta,17beta-diol disulfate 2, P = 0.000064; and dehydroepiandrosterone sulfate, P = 0.00011). Of these 5 androgens, epiandrosterone sulfate (P = 0.0076) was most associated with 2-year incidence of clinician-diagnosed DED. In addition, we found decreased glycerophosphocholines to be associated with DED, although not at metabolome-wide significance. CONCLUSIONS: This hypothesis-free metabolomic approach found decreased serum androgens to be highly associated with DED and adds important evidence to the growing body of research that links androgens to ocular surface disease and DED.

A sensitive and accurate LC-MS/MS assay with the derivatization of 1-Amino-4-methylpiperazine applied to serum allopregnanolone, pregnenolone and androsterone in pre- and postmenopausal women.

The concentrations of allopregnanolone (Allopreg), pregnenolone (Preg) and androsterone (ADT) are very low in the circulation, especially in postmenopausal women, resulting in a considerable challenge for their accurate measurements in serum or plasma. In this report, a sensitive and reliable LC-MS/MS assay method has been developed using a simple sample preparation and the 1-Amino-4-methylpiperazine (AMP) derivatization procedure. A 5pg/ml (0.1pg on column) of low limit of quantitation has been achieved for Allopreg, Preg and ADT, with a sensitivity comparable to data obtained with the commercial reagent. The major benefit of this reagent is to limit the matrix effect since the excess amount of reagent can be removed during the reaction. Multiple reaction monitoring (MRM) from the derivatization of AMP not only increases the detection of these compounds but also provides a good resolution for Allopreg, Preg and ADT from interferences, especially for Allopreg from its isomers. Within the calibration range of 5pg/ml to 2000pg/ml, a good linearity was obtained with R>0.99 where the weighing factor is 1/X. Bias and coefficients of variance are within 15% for all QC levels. The matrix effect has been evaluated, well meeting the acceptance criteria according to the FDA guidelines. With this method, the concentrations of Allopreg, Preg and ADT in postmenopausal serum are in the range of 6.4-53.6pg/ml, 16.2-68.0pg/ml and 23.9-114.0pg/ml, respectively, while the ranges in premenopausal serum are 8.2-701.5pg/ml, 31.2-135.2pg/ml and 47.8-310.0pg/ml, respectively.

Carotid Intima-Media Thickness Is Associated with Increased Androgens in Adolescents and Young Adults with Classical Congenital Adrenal Hyperplasia.

BACKGROUND/AIMS: Youth with congenital adrenal hyperplasia (CAH) due to 21-hydroxylase deficiency develop cardiovascular disease (CVD) risk factors of obesity and hypertension. Carotid intima-media thickness (CIMT), a marker of CVD risk, is increased in CAH young adults. We examined CIMT and its relationship with androgens and obesity in adolescents/young adults with CAH. METHODS: Twenty CAH subjects (age 16 ± 3.3 years, 50% female) and 20 matched controls were studied cross-sectionally. Eight additional obese patients with CAH were included in within-group comparisons. CIMT by high-resolution ultrasound, androgens, anthropometry, bone age (BA), and metabolic/inflammatory markers were assessed. RESULTS: Within the CAH group, CIMT correlated with 17-hydroxyprogesterone (r = 0.48, p < 0.05) and androstenedione (r = 0.46, p < 0.05), and was greater in obese subjects. CIMT was greater in CAH males than females, but similar among CAH females with advanced BA, CAH males with normal BA, and control males. There was no difference in CIMT between CAH and controls, although high-density lipoprotein was inversely correlated with CIMT in both groups. CONCLUSION: CIMT is associated with increased androgens in CAH adolescents and young adults, with loss of sex differences in CAH females with excess androgen exposure. Our findings highlight the importance of hormonal control for CVD prevention in CAH.

An improved micro-method for the measurement of steroid profiles by APPI-LC-MS/MS and its use in assessing diurnal effects on steroid concentrations and optimizing the diagnosis and treatment of adrenal insufficiency and CAH.

Our goals were to (1) develop an improved micro-method usable for neonates for steroid profile measurements and a method to measure androsterone, a key steroid in the recently described androgen backdoor pathway together, with dehydroepiandrosterone and (2) to assess if dehydroepiandrosterone diurnal concentration fluctuations exist potentially necessitating strict adherence to time of blood sample draw and requirement of separate time-dependent reference intervals. Liquid chromatography-tandem mass spectrometry was performed with an atmospheric pressure photoionization source [1]. For each sample 50µL (100µL for the backdoor pathway) of serum was deproteinized by adding 75µL (150µL for the backdoor pathway) of acetonitrile containing the internal standards. After centrifugation, 75µL (150µL for the backdoor pathway) of supernatant was diluted with 250µL of water and injected onto a Poroshell 120 EC-C8 column (SB-C8 column for the backdoor pathway). Within-run coefficients of variation ranged from 2.4 to 10.4% and between-day coefficients of variation from 2.9 to 11.2%. Comparison studies yielded correlation coefficient between 0.97 and 1.00 with recoveries of 90% or greater. Our methods analyze a 9 steroid profile and an additional 2 steroid profile (backdoor pathway) with minimal sample volume (usable in neonates optimizing early diagnosis of endocrinopathies and genetic diseases). Low limits of quantitation make these methods ideal for steroid measurement in women and prepubertal children. As diurnal variations of dehydroepiandrosterone and other steroids [2] concentrations are clinically significant we recommend that separate reference intervals be developed for 8 am, 8 pm, and midnight sample draws. The use of this approach in improving the diagnosis of patients with adrenal insufficiency and congenital adrenal hyperplasia is discussed.

5α-Reductase Inhibition Prevents the Luteal Phase Increase in Plasma Allopregnanolone Levels and Mitigates Symptoms in Women with Premenstrual Dysphoric Disorder.

Changes in neurosteroid levels during the luteal phase of the menstrual cycle may precipitate affective symptoms. To test this hypothesis, we stabilized neurosteroid levels by administering the 5α-reductase inhibitor dutasteride to block conversion of progesterone to its neurosteroid metabolite allopregnanolone in women with premenstrual dysphoric disorder (PMDD) and in asymptomatic control women. Sixteen women with prospectively confirmed PMDD and 16 control women participated in one of two separate randomized, double-blind, placebo-controlled, cross-over trials, each lasting three menstrual cycles. After one menstrual cycle of single-blind placebo, participants were randomized to receive, for the next two menstrual cycles, either double-blind placebo or dutasteride (low-dose 0.5 mg/day in the first eight PMDD and eight control women or high-dose 2.5 mg/day in the second group of women). All women completed the daily rating form (DRF) and were evaluated in clinic during the follicular and luteal phases of each menstrual cycle. Main outcome measures were the DRF symptoms of irritability, sadness, and anxiety. Analyses were performed with SAS PROC MIXED. In the low-dose group, no significant effect of dutasteride on PMDD symptoms was observed compared with placebo (ie, symptom cyclicity maintained), and plasma allopregnanolone levels increased in women with PMDD from follicular to the luteal phases, suggesting the absence of effect of the low-dose dutasteride on 5α-reductase. In contrast, the high-dose group experienced a statistically significant reduction in several core PMDD symptoms (ie, irritability, sadness, anxiety, food cravings, and bloating) on dutasteride compared with placebo. Dutasteride had no effect on mood in controls. Stabilization of allopregnanolone levels from the follicular to the luteal phase of the menstrual cycle by blocking the conversion of progesterone to its 5α-reduced neurosteroid metabolite mitigates symptoms in PMDD. These data provide preliminary support for the pathophysiologic relevance of neurosteroids in this condition.

Hormonal circadian rhythms in patients with congenital adrenal hyperplasia: identifying optimal monitoring times and novel disease biomarkers.

OBJECTIVES: The treatment goal in congenital adrenal hyperplasia (CAH) is to replace glucocorticoids while avoiding androgen excess and iatrogenic Cushing's syndrome. However, there is no consensus on how to monitor disease control. Our main objectives were to evaluate hormonal circadian rhythms and use these profiles to identify optimal monitoring times and novel disease biomarkers in CAH adults on intermediate- and long-acting glucocorticoids. DESIGN: This was an observational, cross-sectional study at the National Institutes of Health Clinical Center in 16 patients with classic CAH. METHODS: Twenty-four-hour serum sampling for ACTH, 17-hydroxyprogesterone (17OHP), androstenedione (A4), androsterone, DHEA, testosterone, progesterone and 24-h urinary pdiol and 5ß-pdiol was carried out. Bayesian spectral analysis and cosinor analysis were performed to detect circadian rhythmicity. The number of hours to minimal (TminAC) and maximal (TmaxAC) adrenocortical hormone levels after dose administration was calculated. RESULTS: A significant rhythm was confirmed for ACTH (r(2), 0.95; P<0.001), 17OHP (r(2), 0.70; P=0.003), androstenedione (r(2), 0.47; P=0.043), androsterone (r(2), 0.80; P<0.001), testosterone (r(2), 0.47; P=0.042) and progesterone (r(2), 0.64; P=0.006). The mean (s.d.) TminAC and TmaxAC for 17OHP and A4 were: morning prednisone (4.3 (2.3) and 9.7 (3.5) h), evening prednisone (4.5 (2.0) and 10.3 (2.4) h), and daily dexamethasone (9.2 (3.5) and 16.4 (7.2) h). AUC0-24 h progesterone, androsterone and 24-h urine pdiol were significantly related to 17OHP. CONCLUSION: In CAH patients, adrenal androgens exhibit circadian rhythms influenced by glucocorticoid replacement. Measurement of adrenocortical hormones and interpretation of results should take into account the type of glucocorticoid and time of dose administration. Progesterone and backdoor metabolites may provide alternative disease biomarkers.

Measuring Faecal Epi-Androsterone as an Indicator of Gonadal Activity in Spotted Hyenas (Crocuta crocuta).

Enzyme immunoassays (EIA) that measure faecal testosterone metabolites (fTM) are useful tools to monitor gonadal activity. The aim of this study was to validate an "in-house" epiandrosterone EIA to monitor fTM in spotted hyenas. FTM were characterised in a male and a female hyena that each received an injection of 3H-testosterone. High-performance liquid chromatography (HPLC) analyses revealed a cluster of highly polar enzyme-hydrolysable hormone metabolite conjugates. We performed hydrolysis using ß-glucuronidase to deconjugate metabolites and improve sensitivity of the assay. Because ß-glucuronidase from Helix pomatia has been reported to bias testosterone measurements in some species, we compared the enzymatic activity of the commonly used ß-glucuronidase extracted from H. pomatia with the same enzyme from Escherichia coli. Our results showed that ß-glucuronidases from both sources produced similar results from spotted hyena faeces. We therefore hydrolysed samples with H. pomatia enzymes. HPLC analyses also demonstrated that following hydrolysis the epiandrosterone EIA measured significant amounts of immunoreactive metabolites corresponding to radiolabelled metabolites in both sexes. Additionally, HPLC and GC-MS analyses confirmed the presence of epiandrosterone in faeces of spotted hyenas. The biological relevance of the epiandrosterone EIA was validated by demonstrating (1) a significant increase in fTM levels in response to a testosterone injection within 16 h, (2) no biological responsiveness to an adrenocorticotropic hormone (ACTH) injection and (3) significant differences in fTM levels between juvenile males and adult immigrant males in a free-ranging wild population. Our results clearly demonstrate that the epiandrosterone EIA is a reliable non-invasive method to monitor gonadal activity in spotted hyenas.

DHEA metabolism to the neurosteroid androsterone: a possible mechanism of DHEA's antidepressant action.

BACKGROUND: Alterations in neurosteroid secretion have been implicated in the efficacy of antidepressants. In a previous study, the adrenal androgen DHEA, a precursor of the neurosteroid androsterone, produced antidepressant and libido-enhancing effects in patients with midlife depression. To investigate the mechanisms underlying DHEA's behavioral effects in this same patient group, we examined plasma levels of four additional neurosteroids implicated in the regulation of affective behavior. METHODS: Blood samples were assayed for neurosteroids in men (n = 13) and women (n = 10) with midlife depression who previously participated in a crossover study in which DHEA and placebo were administered for 6 weeks each. Depression severity was measured by the Center for Epidemiologic Studies Depression Scale (CES-D). Plasma levels of androsterone (ADT), allopregnanolone, pregnanolone, and pregnenolone were measured by GC-MS at baseline and week 6 of each treatment phase. Data were analyzed with repeated measures analysis of variance (ANOVA-R) and Bonferroni t tests. RESULTS: ADT levels (but not allopregnanolone, pregnanolone, and pregnenolone) increased after DHEA but not after placebo (F 2,42 = 3.3, p < 0.05). Post-DHEA ADT levels were higher in women than men [t 63 = 2.9, p < 0.05]. However, in both men and women who met criteria for clinical response on the CES-D, baseline ADT levels significantly increased post-DHEA, and the magnitude of the ADT increase post-DHEA treatment was similar in men and women. Consequently, it was the non-responders who accounted for the sex difference in post-DHEA plasma ADT levels, a difference that was driven by values in two women (the only female non-responders). CONCLUSIONS: The small sample size notwithstanding, these data emphasize the potential behavioral relevance of ADT in humans, which may include contribution to the antidepressant effects of DHEA.

A rapid and sensitive UPLC-MS/MS method for the simultaneous quantification of serum androsterone glucuronide, etiocholanolone glucuronide, and androstan-3α, 17ß diol 17-glucuronide in postmenopausal women.

Quantification of steroidal glucuronide conjugates by the indirect methods of immunoassay and GC-MS/MS may underestimate some conjugates since hydrolysis is needed in sample processing. In the present work, a sensitive and rapid liquid chromatography-tandem mass spectrometry (LC-MS/MS) method has been developed and validated for the simultaneous direct quantification of androsterone glucuronide, etiocholanolone glucuronide, and androstan-3α, 17ß diol 17-glucuronide in postmenopausal women's serum. The quantification limits are 0.1ng/mL for 3α-diol-17G and 4ng/mL for both ADT-G and Etio-G, respectively, with an extraction from 200µL serum while the total run time is less than 6min for all three glucuronides. In this method, solid phase extraction is used for sample preparation. The assay has been validated in compliance with EndoCeutics SOPs and FDA guidelines for bioanalytical method development and validation. The recovery of glucuronides in stripped serum is consistent with that in unstripped serum, where the average difference in stripped and unstripped is less than 10%. A linear regression model fits well the standard curves of all three compounds with R≥0.99 where the weighting factor is 1/X. Interday accuracy and CV for all levels of QCs are within the range of 15% in both stripped and unstripped serum while all calibration curves are within the range of 6% except for LLOQs, which are within the range of 9%. Other parameters have also been assessed such as selectivity, matrix, lipemic and hemolysis effects as well as stabilities in solution and matrix. Incurred sample reanalysis has been performed with a result of over 93% within 20% of the original values. This reliable, sensitive and fast method is ready for large-scale clinical sample assays.