Context:Androgen excess is a defining feature of polycystic ovary syndrome (PCOS), but the exact origin of hyperandrogenemia remains a matter of debate. Recent studies have highlighted the importance of the 11-oxygenated C19 steroid pathway to androgen metabolism in humans. In this study, we analyzed the contribution of 11-oxygenated androgens to androgen excess in women with PCOS.Methods:One hundred fourteen women with PCOS and 49 healthy control subjects underwent measurement of serum androgens by liquid chromatography-tandem mass spectrometry. Twenty-four–hour urinary androgen excretion was analyzed by gas chromatography-mass spectrometry. Fasting plasma insulin and glucose were measured for homeostatic model assessment of insulin resistance. Baseline demographic data, including body mass index, were recorded.Results:As expected, serum concentrations of the classic androgens testosterone (P < 0.001), androstenedione (P < 0.001), and dehydroepiandrosterone (P < 0.01) were significantly increased in PCOS. Mirroring this, serum 11-oxygenated androgens 11β-hydroxyandrostenedione, 11-ketoandrostenedione, 11β-hydroxytestosterone, and 11-ketotestosterone were significantly higher in PCOS than in control subjects, as was the urinary 11-oxygenated androgen metabolite 11β-hydroxyandrosterone. The proportionate contribution of 11-oxygenated to total serum androgens was significantly higher in patients with PCOS compared with control subjects [53.0% (interquartile range, 48.7 to 60.3) vs 44.0% (interquartile range, 32.9 to 54.9); P < 0.0001]. Obese (n = 51) and nonobese (n = 63) patients with PCOS had significantly increased 11-oxygenated androgens. Serum 11β-hydroxyandrostenedione and 11-ketoandrostenedione correlated significantly with markers of insulin resistance.Conclusions:We show that 11-oxygenated androgens represent the majority of circulating androgens in women with PCOS, with close correlation to markers of metabolic risk.

Summary Background Cross-sectional imaging regularly results in incidental discovery of adrenal tumours, requiring exclusion of adrenocortical carcinoma (ACC). However, differentiation is hampered by poor specificity of imaging characteristics. We aimed to validate a urine steroid metabolomics approach, using steroid profiling as the diagnostic basis for ACC. Methods We did a prospective multicentre study in adult participants (age ≥18 years) with newly diagnosed adrenal masses. We assessed the accuracy of diagnostic imaging strategies based on maximum tumour diameter (≥4 cm vs <4 cm), imaging characteristics (positive vs negative), and urine steroid metabolomics (low, medium, or high risk of ACC), separately and in combination, using a reference standard of histopathology and follow-up investigations. With respect to imaging characteristics, we also assessed the diagnostic utility of increasing the unenhanced CT tumour attenuation threshold from the recommended 10 Hounsfield units (HU) to 20 HU. Findings Of 2169 participants recruited between Jan 17, 2011, and July 15, 2016, we included 2017 from 14 specialist centres in 11 countries in the final analysis. 98 (4·9%) had histopathologically or clinically and biochemically confirmed ACC. Tumours with diameters of 4 cm or larger were identified in 488 participants (24·2%), including 96 of the 98 with ACC (positive predictive value [PPV] 19·7%, 95% CI 16·2–23·5). For imaging characteristics, increasing the unenhanced CT tumour attenuation threshold to 20 HU from the recommended 10 HU increased specificity for ACC (80·0% [95% CI 77·9–82·0] vs 64·0% [61·4–66.4]) while maintaining sensitivity (99·0% [94·4–100·0] vs 100·0% [96·3–100·0]; PPV 19·7%, 16·3–23·5). A urine steroid metabolomics result indicating high risk of ACC had a PPV of 34·6% (95% CI 28·6–41·0). When the three tests were combined, in the order of tumour diameter, positive imaging characteristics, and urine steroid metabolomics, 106 (5·3%) participants had the result maximum tumour diameter of 4 cm or larger, positive imaging characteristics (with the 20 HU cutoff), and urine steroid metabolomics indicating high risk of ACC, for which the PPV was 76·4% (95% CI 67·2–84·1). 70 (3·5%) were classified as being at moderate risk of ACC and 1841 (91·3%) at low risk (negative predictive value 99·7%, 99·4–100·0). Interpretation An unenhanced CT tumour attenuation cutoff of 20 HU should replace that of 10 HU for exclusion of ACC. A triple test strategy of tumour diameter, imaging characteristics, and urine steroid metabolomics improves detection of ACC, which could shorten time to surgery for patients with ACC and help to avoid unnecessary surgery in patients with benign tumours. Funding European Commission, UK Medical Research Council, Wellcome Trust, and UK National ...

Idiopathic intracranial hypertension (IIH) is a condition of unknown etiology, characterized by elevated intracranial pressure frequently manifesting with chronic headaches and visual loss. Similar to polycystic ovary syndrome (PCOS), IIH predominantly affects obese women of reproductive age. In this study, we comprehensively examined the systemic and cerebrospinal fluid (CSF) androgen metabolome in women with IIH in comparison with sex-, BMI-, and age-matched control groups with either simple obesity or PCOS (i.e., obesity and androgen excess). Women with IIH showed a pattern of androgen excess distinct to that observed in PCOS and simple obesity, with increased serum testosterone and increased CSF testosterone and androstenedione. Human choroid plexus expressed the androgen receptor, alongside the androgen-activating enzyme aldoketoreductase type 1C3. We show that in a rat choroid plexus cell line, testosterone significantly enhanced the activity of Na + /K + -ATPase, a surrogate of CSF secretion. We demonstrate that IIH patients have a unique signature of androgen excess and provide evidence that androgens can modulate CSF secretion via the choroid plexus. These findings implicate androgen excess as a potential causal driver and therapeutic target in IIH.

Treatment options for idiopathic intracranial hypertension are limited. The enzyme 11β-hydroxysteroid dehydrogenase type 1 has been implicated in regulating cerebrospinal fluid secretion, and its activity is associated with alterations in intracranial pressure in idiopathic intracranial hypertension. We assessed therapeutic efficacy, safety and tolerability and investigated indicators of in vivo efficacy of the 11β-hydroxysteroid dehydrogenase type 1 inhibitor AZD4017 compared with placebo in idiopathic intracranial hypertension. A multicenter, UK, 16-week phase II randomized, double-blind, placebo-controlled trial of 12-week treatment with AZD4017 or placebo was conducted. Women aged 18–55 years with active idiopathic intracranial hypertension (&gt;25 cmH2O lumbar puncture opening pressure and active papilledema) were included. Participants received 400 mg of oral AZD4017 twice daily compared with matching placebo over 12 weeks. The outcome measures were initial efficacy, safety and tolerability. The primary clinical outcome was lumbar puncture opening pressure at 12 weeks analysed by intention-to-treat. Secondary clinical outcomes were symptoms, visual function, papilledema, headache and anthropometric measures. In vivo efficacy was evaluated in the central nervous system and systemically. A total of 31 subjects [mean age 31.2 (SD = 6.9) years and body mass index 39.2 (SD = 12.6) kg/m2] were randomized to AZD4017 (n = 17) or placebo (n = 14). At 12 weeks, lumbar puncture pressure was lower in the AZD4017 group (29.7 cmH2O) compared with placebo (31.3 cmH2O), but the difference between groups was not statistically significant (mean difference: −2.8, 95% confidence interval: −7.1 to 1.5; P = 0.2). An exploratory analysis assessing mean change in lumbar puncture pressure within each group found a significant decrease in the AZD4017 group [mean change: −4.3 cmH2O (SD = 5.7); P = 0.009] but not in the placebo group [mean change: −0.3 cmH2O (SD = 5.9); P = 0.8]. AZD4017 was safe, with no withdrawals related to adverse effects. Nine transient drug-related adverse events were reported. One serious adverse event occurred in the placebo group (deterioration requiring shunt surgery). In vivo biomarkers of 11β-hydroxysteroid dehydrogenase type 1 activity (urinary glucocorticoid metabolites, hepatic prednisolone generation, serum and cerebrospinal fluid cortisol:cortisone ratios) demonstrated significant enzyme inhibition with the reduction in serum cortisol:cortisone ratio correlating significantly with reduction in lumbar puncture pressure (P = 0.005, R = 0.70). This is the first phase II randomized controlled trial in idiopathic intracranial hypertension evaluating a novel therapeutic target. AZD4017 was safe and well tolerated and inhibited 11β-hydroxysteroid dehydrogenase type 1 activity in vivo. Reduction in serum cortisol:cortisone correlated with decreased intracranial pressure. Possible clinical benefits were noted in this small cohort. A longer, larger study would now be of interest.

Current understanding of vitamin D tends to be focussed on the measurement of the major circulating form 25‐hydroxyvitamin D3 (25OHD3) and its conversion to the active hormonal form, 1α,25‐dihydroxyvitamin D3 (1α,25(OH)2D3) via the enzyme 25‐hydroxyvitamin D‐1α‐hydroxylase (CYP27B1). However, whilst these metabolites form the endocrine backbone of vitamin D physiology, it is important to recognise that there are other metabolic and catabolic pathways that are now recognised as being crucially important to vitamin D function. These pathways include C3‐epimerization, CYP24A1 hydroxylase, CYP11A1 alternative metabolism of vitamin D3, and phase II metabolism. Endogenous metabolites beyond 25OHD3 are usually present at low endogenous levels and may only be functional in specific target tissues rather than in the general circulation. However, the technologies available to measure these metabolites have also improved, so that measurement of alternative vitamin D metabolic pathways may become more routine in the near future. The aim of this review is to provide a comprehensive overview of the various pathways of vitamin D metabolism, as well as describe the analytical techniques currently available to measure these vitamin D metabolites.

Recent studies suggest that vitamin D-deficiency is linked to increased risk of common human health problems. To define vitamin D ‘status’ most routine analytical methods quantify one particular vitamin D metabolite, 25-hydroxyvitamin D3 (25OHD3). However, vitamin D is characterized by complex metabolic pathways, and simultaneous measurement of multiple vitamin D metabolites may provide a more accurate interpretation of vitamin D status. To address this we developed a high-throughput liquid chromatography-tandem mass spectrometry (LC-MS/MS) method to analyse multiple vitamin D analytes, with particular emphasis on the separation of epimer metabolites. A supportive liquid-liquid extraction (SLE) and LC-MS/MS method was developed to quantify 10 vitamin D metabolites as well as separation of an interfering 7α-hydroxy-4-cholesten-3-one (7αC4) isobar (precursor of bile acid), and validated by analysis of human serum samples. In a cohort of 116 healthy subjects, circulating concentrations of 25-hydroxyvitamin D3 (25OHD3), 3-epi-25-hydroxyvitamin D3 (3-epi-25OHD3), 24,25-dihydroxyvitamin D3 (24R,25(OH)2D3), 1,25-dihydroxyvitamin D3 (1α,25(OH)2D3), and 25-hydroxyvitamin D2 (25OHD2) were quantifiable using 220 μl of serum, with 25OHD3 and 24R,25(OH)2D3 showing significant seasonal variations. This high-throughput LC-MS/MS method provides a novel strategy for assessing the impact of vitamin D on human health and disease.

Age-associated decline in muscle function represents a significant public health burden. Vitamin D-deficiency is also prevalent in aging subjects, and has been linked to loss of muscle mass and strength (sarcopenia), but the precise role of specific vitamin D metabolites in determining muscle phenotype and function is still unclear. To address this we quantified serum concentrations of multiple vitamin D metabolites, and assessed the impact of these metabolites on body composition/muscle function parameters, and muscle biopsy gene expression in a retrospective study of a cohort of healthy volunteers. Active serum 1,25-dihydroxyvitamin D3 (1α,25(OH)2D3), but not inactive 25-hydroxyvitamin D3 (25OHD3), correlated positively with measures of lower limb strength including power (rho = 0.42, p = 0.02), velocity (Vmax, rho = 0.40, p = 0.02) and jump height (rho = 0.36, p = 0.04). Lean mass correlated positively with 1α,25(OH)2D3 (rho = 0.47, p = 0.02), in women. Serum 25OHD3 and inactive 24,25-dihydroxyvitamin D3 (24,25(OH)2D3) had an inverse relationship with body fat (rho = -0.30, p = 0.02 and rho = -0.33, p = 0.01, respectively). Serum 25OHD3 and 24,25(OH)2D3 were also correlated with urinary steroid metabolites, suggesting a link with glucocorticoid metabolism. PCR array analysis of 92 muscle genes identified vitamin D receptor (VDR) mRNA in all muscle biopsies, with this expression being negatively correlated with serum 25OHD3, and Vmax, and positively correlated with fat mass. Of the other 91 muscle genes analysed by PCR array, 24 were positively correlated with 25OHD3, but only 4 were correlated with active 1α,25(OH)2D3. These data show that although 25OHD3 has potent actions on muscle gene expression, the circulating concentrations of this metabolite are more closely linked to body fat mass, suggesting that 25OHD3 can influence muscle function via indirect effects on adipose tissue. By contrast, serum 1α,25(OH)2D3 has limited effects on muscle gene expression, but is associated with increased muscle strength and lean mass in women. These pleiotropic effects of the vitamin D ‘metabolome’ on muscle function indicate that future supplementation studies should not be restricted to conventional analysis of the major circulating form of vitamin D, 25OHD3.

Introduction Epidemiology has linked preeclampsia (PET) to decreased maternal serum 25-hydroxyvitamin D3 (25(OH)D3). However, alterations in systemic and placental/decidual transport and metabolism of 25(OH)D3 during pregnancy suggest that other forms of vitamin D may also contribute to the pathophysiology of PET. Methods In a cross sectional analysis of normal pregnant women at 1st (n = 25) and 3rd trimester (n = 21), pregnant women with PET (n = 22), and non-pregnant female controls (n = 20) vitamin D metabolites were quantified in paired maternal serum, placental, and decidual tissue. Results Serum 25(OH)D3 was not significantly different in sera across all four groups. In normal 3rd trimester pregnant women serum active 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) was significantly higher than non-pregnant, normal 1st trimester pregnant, and PET women. Conversely, PET sera showed highest levels of the catabolites 3-epi-25(OH)D3 and 24,25-dihydroxyvitamin D3 (24,25(OH)2D3). Serum albumin was significantly lower in normal 3rd trimester pregnant women and PET relative to normal 1st trimester pregnant women, but there was no change in free/bioavailable 25(OH)D3. In PET placental tissue, 25(OH)D3 and 3-epi-25(OH)D3 were lower than normal 3rd trimester tissue, whilst placental 24,25(OH)2D3 was highest in PET. Tissue 1,25(OH)2D3 was detectable in 1st trimester decidua, which also showed 10-fold higher 25(OH)D3 relative to paired placentae. 3-epi-25(OH)D3 and 24,25(OH)2D3 were not different for decidua and placenta. In normal 3rd trimester pregnant women, total, free and bioavailable maternal 25(OH)D3 correlated with placental 25(OH)D3, but this was not conserved for PET. Discussion These data indicate that PET is associated with decreased activation, increased catabolism, and impaired placental uptake of 25(OH)D3.