Alternative pathway androgen biosynthesis and human fetal female virilization.

Androgen biosynthesis in the human fetus proceeds through the adrenal sex steroid precursor dehydroepiandrosterone, which is converted to testosterone in the gonads, followed by further activation to 5α-dihydrotestosterone in genital skin, thereby facilitating male external genital differentiation. Congenital adrenal hyperplasia due to P450 oxidoreductase deficiency results in disrupted dehydroepiandrosterone biosynthesis, explaining undervirilization in affected boys. However, many affected girls are born virilized, despite low circulating androgens. We hypothesized that this is due to a prenatally active, alternative androgen biosynthesis pathway from 17α-hydroxyprogesterone to 5α-dihydrotestosterone, which bypasses dehydroepiandrosterone and testosterone, with increased activity in congenital adrenal hyperplasia variants associated with 17α-hydroxyprogesterone accumulation. Here we employ explant cultures of human fetal organs (adrenals, gonads, genital skin) from the major period of sexual differentiation and show that alternative pathway androgen biosynthesis is active in the fetus, as assessed by liquid chromatography-tandem mass spectrometry. We found androgen receptor expression in male and female genital skin using immunohistochemistry and demonstrated that both 5α-dihydrotestosterone and adrenal explant culture supernatant induce nuclear translocation of the androgen receptor in female genital skin primary cultures. Analyzing urinary steroid excretion by gas chromatography-mass spectrometry, we show that neonates with P450 oxidoreductase deficiency produce androgens through the alternative androgen pathway during the first weeks of life. We provide quantitative in vitro evidence that the corresponding P450 oxidoreductase mutations predominantly support alternative pathway androgen biosynthesis. These results indicate a key role of alternative pathway androgen biosynthesis in the prenatal virilization of girls affected by congenital adrenal hyperplasia due to P450 oxidoreductase deficiency.

Increased central adiposity and decreased subcutaneous adipose tissue 11ß-hydroxysteroid dehydrogenase type 1 are associated with deterioration in glucose tolerance-A longitudinal cohort study.

OBJECTIVE AND CONTEXT: Increasing adiposity, ageing and tissue-specific regeneration of cortisol through the activity of 11ß-hydroxysteroid dehydrogenase type 1 have been associated with deterioration in glucose tolerance. We undertook a longitudinal, prospective clinical study to determine if alterations in local glucocorticoid metabolism track with changes in glucose tolerance. DESIGN, PATIENTS, AND MEASUREMENTS: Sixty-five overweight/obese individuals (mean age 50.3 ± 7.3 years) underwent oral glucose tolerance testing, body composition assessment, subcutaneous adipose tissue biopsy and urinary steroid metabolite analysis annually for up to 5 years. Participants were categorized into those in whom glucose tolerance deteriorated ("deteriorators") or improved ("improvers"). RESULTS: Deteriorating glucose tolerance was associated with increasing total and trunk fat mass and increased subcutaneous adipose tissue expression of lipogenic genes. Subcutaneous adipose tissue 11ß-HSD1 gene expression decreased in deteriorators, and at study completion, it was highest in the improvers. There was a significant negative correlation between change in area under the curve glucose and 11ß-HSD1 expression. Global 11ß-HSD1 activity did not change and was not different between deteriorators and improvers at baseline or follow-up. CONCLUSION: Longitudinal deterioration in metabolic phenotype is not associated with increased 11ß-HSD1 activity, but decreased subcutaneous adipose tissue gene expression. These changes may represent a compensatory mechanism to decrease local glucocorticoid exposure in the face of an adverse metabolic phenotype.

Urine steroid metabolomics as a diagnostic tool in primary aldosteronism.

Primary aldosteronism (PA) causes 5-10% of hypertension cases, but only a minority of patients are currently diagnosed and treated because of a complex, stepwise, and partly invasive workup. We tested the performance of urine steroid metabolomics, the computational analysis of 24-hour urine steroid metabolome data by machine learning, for the identification and subtyping of PA. Mass spectrometry-based multi-steroid profiling was used to quantify the excretion of 34 steroid metabolites in 24-hour urine samples from 158 adults with PA (88 with unilateral PA [UPA] due to aldosterone-producing adenomas [APAs]; 70 with bilateral PA [BPA]) and 65 sex- and age-matched healthy controls. All APAs were resected and underwent targeted gene sequencing to detect somatic mutations associated with UPA. Patients with PA had increased urinary metabolite excretion of mineralocorticoids, glucocorticoids, and glucocorticoid precursors. Urine steroid metabolomics identified patients with PA with high accuracy, both when applied to all 34 or only the three most discriminative steroid metabolites (average areas under the receiver-operating characteristics curve [AUCs-ROC] 0.95-0.97). Whilst machine learning was suboptimal in differentiating UPA from BPA (average AUCs-ROC 0.65-0.73), it readily identified APA cases harbouring somatic KCNJ5 mutations (average AUCs-ROC 0.79-85). These patients showed a distinctly increased urine excretion of the hybrid steroid 18-hydroxycortisol and its metabolite 18-oxo-tetrahydrocortisol, the latter identified by machine learning as by far the most discriminative steroid. In conclusion, urine steroid metabolomics is a non-invasive candidate test for the accurate identification of PA cases and KCNJ5-mutated APAs.

Mutations in the genes encoding 11beta-hydroxysteroid dehydrogenase type 1 and hexose-6-phosphate dehydrogenase interact to cause cortisone reductase deficiency.

In cortisone reductase deficiency (CRD), activation of cortisone to cortisol does not occur, resulting in adrenocorticotropin-mediated androgen excess and a phenotype resembling polycystic ovary syndrome (PCOS; refs. 1,2). This suggests a defect in the gene HSD11B1 encoding 11beta-hydroxysteroid dehydrogenase type 1 (11beta-HSD1), a primary regulator of tissue-specific glucocorticoid bioavailability. We identified intronic mutations in HSD11B1 that resulted in reduced gene transcription in three individuals with CRD. In vivo, 11beta-HSD1 catalyzes the reduction of cortisone to cortisol whereas purified enzyme acts as a dehydrogenase converting cortisol to cortisone. Oxo-reductase activity can be regained using a NADPH-regeneration system and the cytosolic enzyme glucose-6-phosphate dehydrogenase. But the catalytic domain of 11beta-HSD1 faces into the lumen of the endoplasmic reticulum (ER; ref. 6). We hypothesized that endolumenal hexose-6-phosphate dehydrogenase (H6PDH) regenerates NADPH in the ER, thereby influencing directionality of 11beta-HSD1 activity. Mutations in exon 5 of H6PD in individuals with CRD attenuated or abolished H6PDH activity. These individuals have mutations in both HSD11B1 and H6PD in a triallelic digenic model of inheritance, resulting in low 11beta-HSD1 expression and ER NADPH generation with loss of 11beta-HSD1 oxo-reductase activity. CRD defines a new ER-specific redox potential and establishes H6PDH as a potential factor in the pathogenesis of PCOS.

Analysis of bioactive oxysterols in newborn mouse brain by LC/MS.

Unesterified cholesterol is a major component of plasma membranes. In the brain of the adult, it is mostly found in myelin sheaths, where it plays a major architectural role. In the newborn mouse, little myelination of neurons has occurred, and much of this sterol comprises a metabolically active pool. In the current study, we have accessed this metabolically active pool and, using LC/MS, have identified cholesterol precursors and metabolites. Although desmosterol and 24S-hydroxycholesterol represent the major precursor and metabolite, respectively, other steroids, including the oxysterols 22-oxocholesterol, 22R-hydroxycholesterol, 20R,22R-dihydroxycholesterol, and the C(21)-neurosteroid progesterone, were identified. 24S,25-epoxycholesterol formed in parallel to cholesterol was also found to be a major sterol in newborn brain. Like 24S- and 22R-hydroxycholesterols, and also desmosterol, 24S,25-epoxycholesterol is a ligand to the liver X receptors, which are expressed in brain. The desmosterol metabolites (24Z),26-, (24E),26-, and 7α-hydroxydesmosterol were identified in brain for the first time.

The broad phenotypic spectrum of 17α-hydroxylase/17,20-lyase (CYP17A1) deficiency: a case series.

CONTEXT: 17α-Hydroxylase/17,20-lyase deficiency (17OHD) caused by mutations in the CYP17A1 gene is a rare form of congenital adrenal hyperplasia typically characterised by cortisol deficiency, mineralocorticoid excess and sex steroid deficiency. OBJECTIVE: To examine the phenotypic spectrum of 17OHD by clinical and biochemical assessment and corresponding in silico and in vitro functional analysis. DESIGN: Case series. PATIENTS AND RESULTS: We assessed eight patients with 17OHD, including four with extreme 17OHD phenotypes: two siblings presented with failure to thrive in early infancy and two with isolated sex steroid deficiency and normal cortisol reserve. Diagnosis was established by mass spectrometry-based urinary steroid profiling and confirmed by genetic CYP17A1 analysis, revealing homozygous and compound heterozygous sequence variants. We found novel (p.Gly111Val, p.Ala398Glu, p.Ile371Thr) and previously described sequence variants (p.Pro409Leu, p.Arg347His, p.Gly436Arg, p.Phe53/54del, p.Tyr60IlefsLys88X). In vitro functional studies employing an overexpression system in HEK293 cells showed that 17,20-lyase activity was invariably decreased while mutant 17α-hydroxylase activity retained up to 14% of WT activity in the two patients with intact cortisol reserve. A ratio of urinary corticosterone over cortisol metabolites reflective of 17α-hydroxylase activity correlated well with clinical phenotype severity. CONCLUSION: Our findings illustrate the broad phenotypic spectrum of 17OHD. Isolated sex steroid deficiency with normal stimulated cortisol has not been reported before. Attenuation of 17α-hydroxylase activity is readily detected by urinary steroid profiling and predicts phenotype severity. SIGNIFICANCE STATEMENT: Here we report, supported by careful phenotyping, genotyping and functional analysis, a prismatic case series of patients with congenital adrenal hyperplasia due to 17α-hydroxylase (CYP17A1) deficiency (17OHD). These range in severity from the abolition of function, presenting in early infancy, and unusually mild with isolated sex steroid deficiency but normal ACTH-stimulated cortisol in adult patients. These findings will guide improved diagnostic detection of CYP17A1 deficiency.

Prevalence of steroid sulfatase deficiency in California according to race and ethnicity.

OBJECTIVE: Estimate steroid sulfatase deficiency (STSD) prevalence among California's racial/ethnic groups using data from a previous study focused on prenatal detection of Smith-Lemli-Opitz syndrome (SLOS). SLOS and STSD both have low maternal serum unconjugated estriol (uE3) levels. METHODS: Prevalence was estimated using three steps: listing clinically identified cases; modeling STSD frequency at three uE3 intervals using diagnostic urine steroid measurements; applying this model to determine frequency in pregnancies not providing urine. RESULTS: Overall, 2151 of 777 088 pregnancies (0.28%) were screen positive; 1379 of these were explained and excluded. Fifty-four cases were diagnosed clinically among 707 remaining pregnancies with a male fetus. Urine steroid testing identified 74 additional STSD cases: 66 (89.2%) at uE3 values < 0.15 MoM, 8 (10.8%) at 0.15-0.20 MoM, and 0 (0%) at > 0.20 MoM. Modeling estimated 107.5 STSD cases among 370 pregnancies without urine samples. In males, STSD prevalence was highest among non-Hispanic Whites (1:1230) compared to Hispanics (1:1620) and Asians (1:1790), but differences were not significant. No STSD pregnancies were found among 65 screen positive Black women. CONCLUSION: The overall prevalence estimate of 1:1500 males is consistent with published estimates and is reasonable for counseling, except among Black pregnancies where no reliable estimate could be made.

Urine steroid metabolomics for the differential diagnosis of adrenal incidentalomas in the EURINE-ACT study: a prospective test validation study.

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 Institute for Health Research, US National Institutes of Health, the Claire Khan Trust Fund at University Hospitals Birmingham Charities, and the Mayo Clinic Foundation for Medical Education and Research.

Maternal urine and serum steroid measurements to identify steroid sulfatase deficiency (STSD) in second trimester pregnancies.

OBJECTIVE: To document the performance of second trimester maternal urine and serum steroid measurements for detecting fetal steroid sulfatase deficiency (STSD). METHODS: We studied detection rate and false positive rate (DR, FPR) of analytes in maternal urine [combinations of 16alpha-OH-dehydroepiandrosterone sulfate (16alpha-OH-DHEAS), 11beta-hydroxyandrosterone, total estriol] and serum [combinations of 16alpha-OH-DHEAS, 11beta-hydroxyandrosterone, total estriol, unconjugated estriol (uE3)]. Samples were obtained from pregnancies which were screen positive for Smith-Lemli-Opitz syndrome (SLOS). RESULTS: Among 1 079 301 pregnancies, 3083 (0.29%) were screen positive for SLOS. Urine and/or serum samples were available from 917 viable pregnancies with known gender. We assigned likelihood ratios (LRs) to steroid measurements from male fetuses with known STSD and unaffected female fetuses. An LR > or = 100 was present in urine from 84 of 86 STSD pregnancies (98% DR, 95% CI 92-99), along with 0 of 198 pregnancies with normal female fetuses (0.0% FPR, CI 0-1.9). LRs were > or = 100 in 4 of 129 female fetuses with major abnormalities (3% FPR). In maternal serum, steroid measurements performed less effectively, achieving a 71% DR for STSD at a 1.6% FPR. CONCLUSION: Maternal urine steroid measurements are effective for detecting STSD, including those with point mutations and those with full deletions.

Human steroid biosynthesis, metabolism and excretion are differentially reflected by serum and urine steroid metabolomes: A comprehensive review.

Advances in technology have allowed for the sensitive, specific, and simultaneous quantitative profiling of steroid precursors, bioactive steroids and inactive metabolites, facilitating comprehensive characterization of the serum and urine steroid metabolomes. The quantification of steroid panels is therefore gaining favor over quantification of single marker metabolites in the clinical and research laboratories. However, although the biochemical pathways for the biosynthesis and metabolism of steroid hormones are now well defined, a gulf still exists between this knowledge and its application to the measured steroid profiles. In this review, we present an overview of steroid hormone biosynthesis and metabolism by the liver and peripheral tissues, specifically highlighting the pathways linking and differentiating the serum and urine steroid metabolomes. A brief overview of the methodology used in steroid profiling is also provided.

Steroid Metabolome Analysis in Disorders of Adrenal Steroid Biosynthesis and Metabolism.

Steroid biosynthesis and metabolism are reflected by the serum steroid metabolome and, in even more detail, by the 24-hour urine steroid metabolome, which can provide unique insights into alterations of steroid flow and output indicative of underlying conditions. Mass spectrometry-based steroid metabolome profiling has allowed for the identification of unique multisteroid signatures associated with disorders of steroid biosynthesis and metabolism that can be used for personalized approaches to diagnosis, differential diagnosis, and prognostic prediction. Additionally, steroid metabolome analysis has been used successfully as a discovery tool, for the identification of novel steroidogenic disorders and pathways as well as revealing insights into the pathophysiology of adrenal disease. Increased availability and technological advances in mass spectrometry-based methodologies have refocused attention on steroid metabolome profiling and facilitated the development of high-throughput steroid profiling methods soon to reach clinical practice. Furthermore, steroid metabolomics, the combination of mass spectrometry-based steroid analysis with machine learning-based approaches, has facilitated the development of powerful customized diagnostic approaches. In this review, we provide a comprehensive up-to-date overview of the utility of steroid metabolome analysis for the diagnosis and management of inborn disorders of steroidogenesis and autonomous adrenal steroid excess in the context of adrenal tumors.

Steroid biomarkers and genetic studies reveal inactivating mutations in hexose-6-phosphate dehydrogenase in patients with cortisone reductase deficiency.

CONTEXT: Cortisone reductase deficiency (CRD) is characterized by a failure to regenerate cortisol from cortisone via 11beta-hydroxysteroid dehydrogenase type 1 (11beta-HSD1), resulting in increased cortisol clearance, activation of the hypothalamic-pituitary-axis (HPA) and ACTH-mediated adrenal androgen excess. 11beta-HSD1 oxoreductase activity requires the reduced nicotinamide adenine dinucleotide phosphate-generating enzyme hexose-6-phosphate dehydrogenase (H6PDH) within the endoplasmic reticulum. CRD manifests with hyperandrogenism resulting in hirsutism, oligo-amenorrhea, and infertility in females and premature pseudopuberty in males. Recent association studies have failed to corroborate findings that polymorphisms in the genes encoding H6PDH (R453Q) and 11beta-HSD1 (Intron 3 inserted adenine) interact to cause CRD. OBJECTIVE: Our objective was to reevaluate the genetics and steroid biochemistry of patients with CRD. DESIGN: We analyzed 24-h urine collection for steroid biomarkers by gas chromatography/mass spectrometry and sequenced the HSD11B1 and H6PD genes in our CRD cohort. PATIENTS: Patients included four cases presenting with hyperandrogenism and biochemical features clearly indicative of CRD. RESULTS: Gas chromatography/mass spectrometry identified steroid biomarkers that correlated with CRD in each case. Three cases were identified as homozygous (R109AfsX3, Y316X, and G359D) and one case identified as compound heterozygous (c.960G-->A and D620fsX3) for mutations in H6PD. No mutations affecting enzyme activity were identified in the HSD11B1 gene. Expression and activity assays demonstrate loss of function for all reported H6PDH mutations. CONCLUSIONS: CRD is caused by inactivating mutations in the H6PD gene, rendering the 11beta-HSD1 enzyme unable to operate as an oxoreductase, preventing local glucocorticoid regeneration. These data highlight the importance of the redox control of cortisol metabolism and the 11beta-HSD1-H6PDH pathway in regulating hypothalamic-pituitary-adrenal axis activity.

Human Delta4-3-oxosteroid 5beta-reductase (AKR1D1) deficiency and steroid metabolism.

Conclusive in vivo evidence regarding the enzyme responsible for steroid hormone 5beta-reduction has not been obtained, although studies have suggested it may be the same enzyme as that utilized for cholic acid and chenodeoxycholic bile-acid synthesis. We have recorded the steroid metabolome of a patient with a defect in the "bile-acid" 5beta-reductase (AKR1D1) and from this confirm that this enzyme is additionally responsible for steroid hormone metabolism. The 13-year old patient has been investigated since infancy because of a cholestasis phenotype caused by bile-acid insufficiency. Several years ago it was shown that she had a 662C>T missense mutation in AKR1D1 causing a Pro198Leu substitution. It was found that the patient had an almost total absence of 5beta-reduced metabolites of corticosteroids and severely reduced production of 5beta-reduced metabolites of other steroids. The patient is healthy in spite of her earlier hepatic failure and is on no treatment. All her vital signs were normal, as were results of many biochemical analyses. She had normal pubertal changes and experiences regular menstrual cycles. There was no evidence for any clinical condition that could be attributed to attenuated ability to metabolize steroids in normal fashion. Both parents were heterozygous for the mutation but the steroid excretion was entirely normal, although an older female sibling showed definitive evidence for attenuated 5beta-reduction of cortisol. A younger brother had a normal steroid metabolome. The sibling genotypes were not available.

17-Hydroxylase/C17,20-lyase (CYP17) is not the enzyme responsible for side-chain cleavage of cortisol and its metabolites.

The question addressed in this study was the nature of the enzyme required to remove the side-chain of 17-hydroxycorticosteroids, leading in the case of cortisol to the excretion of 11beta-hydroxyandrosterone, 11-oxo-androsterone and the corresponding etiocholanolones. We questioned whether it could be CYP17, the 17-hydroxylase/17,20-lyase utilized in androgen synthesis. The conversion of exogenous cortisol to C(19) steroids in patients with complete 17-hydroxylase deficiency (17HD) was studied rationalizing that if CYP17 was involved no C(19) steroids would be formed. The urinary excretion of the four 11-oxy-C(19) steroids as well as many of the major C(21) cortisol metabolites were measured by GC/MS. Our results showed that the conversion of cortisol to C(19) steroids was normal in 17HD indicating that a currently unidentified enzyme must be responsible for this transformation. A secondary goal was to determine to what extent 11-oxy-C(19) steroids were metabolites of cortisol or adrenal synthesized 11beta-hydroxyandrostenedione. Since cortisol-treated 17HD patients cannot produce androstenedione, all C(19) 11-oxy-metabolites excreted must be derived from exogenous cortisol. The extent to which 17HD patients have lower relative excretion of C(19) steroids should reflect the absence of 11beta-hydroxyandrostenedione metabolites. Our results showed almost all of 11-oxo-etiocholanolone and 11beta-hydroxyetiocholanolone were cortisol metabolites, but in contrast the excretion of 11beta-hydroxyandrosterone was less than 10% that of normal individuals, indicating that in excess of 90% must be a metabolite of 11beta-hydroxyandrostenedione.

Differential inhibition of CYP17A1 and CYP21A2 activities by the P450 oxidoreductase mutant A287P.

P450 oxidoreductase (POR) has a pivotal role in facilitating electron transfer from nicotinamide adenine dinucleotide phosphate to microsomal cytochrome P450 (CYP) enzymes, including the steroidogenic enzymes CYP17A1 and CYP21A2. Mutations in POR have been shown recently to cause congenital adrenal hyperplasia with apparent combined CYP17A1 and CYP21A2 deficiency that comprises a variable clinical phenotype, including glucocorticoid deficiency, ambiguous genitalia, and craniofacial malformations. To dissect structure-function relationships potentially explaining this phenotypic diversity, we investigated whether specific POR mutations have differential effects on CYP17A1 and CYP21A2. We compared the impact of missense mutations encoding for single amino acid changes in three distinct regions of the POR molecule: 1), Y181D and H628P close to the central electron transfer area, 2) S244C located within the hinge close to the flavin adenine dinucleotide and flavin mononucleotide domains of POR, and 3) A287P that is clearly distant from the two other regions. Functional analysis using a yeast microsomal assay with coexpression of human CYP17A1 or CYP21A2 with wild-type or mutant human POR revealed equivalent decreases in CYP17A1 and CYP21A2 activities by Y181D, H628P, and S244C. In contrast, A287P had a differential inhibitory effect, with decreased catalytic efficiency (Vmax/Km) for CYP17A1, whereas CYP21A2 retained near normal activity. In vivo analysis of urinary steroid excretion by gas chromatography/mass spectrometry in 11 patients with POR mutations showed that A287P homozygous patients had the highest corticosterone/cortisol metabolite ratios, further indicative of preferential inhibition of CYP17A1. These findings provide novel mechanistic insights into the redox regulation of human steroidogenesis. Differential interaction of POR with electron-accepting CYP enzymes may explain the phenotypic variability in POR deficiency, with additional implications for hepatic drug metabolism by POR-dependant CYP enzymes.

Cholesterol biosynthesis from birth to adulthood in a mouse model for 7-dehydrosterol reductase deficiency (Smith-Lemli-Opitz syndrome).

Smith-Lemli-Opitz syndrome (SLOS) is caused by deficiency in the terminal step of cholesterol biosynthesis, which is catalyzed by 7-dehydrocholesterol reductase (DHCR7). The disorder exhibits several phenotypic traits including dysmorphia and mental retardation with a broad range of severity. Pathogenesis of SLOS is complex due to multiple roles of cholesterol and may be further complicated by unknown effects of aberrant metabolites that arise when 7-dehydrocholesterol (7-DHC), the substrate for DHCR7, accumulates. A viable mouse model for SLOS has recently been developed, and here we characterize cholesterol metabolism in this model with emphasis on changes during the first few weeks of postnatal development. Cholesterol and 7-DHC were measured in "SLOS" mice and compared with measurements in normal mice. SLOS mice had measurable levels of 7-DHC at all ages tested (up to 1 year), while 7-DHC was below the threshold for detection in normal mice. In perinatal to weaning age SLOS mice, cholesterol and 7-DHC levels changed dramatically. Changes in brain and liver were independent; in brain cholesterol increased several fold while 7-DHC remained relatively constant, but in liver cholesterol first increased then decreased again while 7-DHC first decreased then increased. In older SLOS animals the ratio of 7-DHC/cholesterol, which is an index of biochemical severity, tended to approach, but not reach, normal. While these mice provide the best available genetic animal model for the study of SLOS pathogenesis and treatment, they probably will be most useful at early ages when the metabolic effects of the mutations are most dramatic. To correlate any experimental treatment with improved sterol metabolism will require age-matched controls. Finally, determining the mechanism by which these "SLOS" mice tend to normalize may provide insight into the future development of therapy.

Cortisol metabolism in the Bolivian squirrel monkey (Saimiri boliviensis boliviensis).

New World squirrel monkeys (Saimiri spp.) have high circulating cortisol levels but normal electrolytes and blood pressures. The goal of the present study was to gain insight into adaptive mechanisms used by Bolivian squirrel monkeys to minimize the effects of high cortisol on mineralocorticoid receptor (MR) activity and electrolyte and water balance. Aldosterone levels in serum from 10 squirrel monkeys were 17.7 +/- 3.4 ng/dl (normal range in humans, 4 to 31 ng/dl), suggesting that squirrel monkeys do not exhibit a compensatory increase in aldosterone. The squirrel monkey MR was cloned and expressed in COS-7 cells and found to have similar responsiveness to cortisol and aldosterone as human MR, suggesting that squirrel monkey MR is not inherently less responsive to cortisol. To determine whether altered metabolism of cortisol might contribute to MR protection in squirrel monkeys, serum and urinary cortisol and cortisone were measured, and a comprehensive urinary corticosteroid metabolite profile was performed in samples from anesthetized and awake squirrel monkeys. The levels of cortisone exceeded those of cortisol in serum and urine, suggesting increased peripheral 11beta-hydroxysteroid dehydrogenase 2 activity in squirrel monkeys. In addition, a significant fraction (approximately 20%) of total corticosteroids excreted in the urine of squirrel monkeys appeared as 6beta-hydroxycortisol, compared with that in man (1%). Therefore, changes in cortisol metabolism likely contribute to adaptive mechanisms used by Bolivian squirrel monkeys to minimize effects of high cortisol.

Evidence for Increased 5α-Reductase Activity During Early Childhood in Daughters of Women With Polycystic Ovary Syndrome.

CONTEXT: Polycystic ovary syndrome (PCOS) is a heritable, complex genetic disease. Animal models suggest that androgen exposure at critical developmental stages contributes to disease pathogenesis. We hypothesized that genetic variation resulting in increased androgen production produces the phenotypic features of PCOS by programming during critical developmental periods. Although we have not found evidence for increased in utero androgen levels in cord blood in the daughters of women with PCOS (PCOS-d), target tissue androgen production may be amplified by increased 5α-reductase activity analogous to findings in adult affected women. It is possible to noninvasively test this hypothesis by examining urinary steroid metabolites. OBJECTIVE: We performed this study to investigate whether PCOS-d have altered androgen metabolism during early childhood. DESIGN, SETTING, AND PARTICIPANTS: Twenty-one PCOS-d, 1-3 years old, and 36 control girls of comparable age were studied at an academic medical center. MAIN OUTCOME MEASURES: Urinary steroid metabolites were measured by gas chromatography/mass spectrometry. Twenty-four hour steroid excretion rates and precursor to product ratios suggestive of 5α-reductase and 11ß-hydroxysteroid dehydrogenase activities were calculated. RESULTS: Age did not differ but weight for length Z-scores were higher in PCOS-d compared to control girls (P = .02). PCOS-d had increased 5α-tetrahydrocortisol:tetrahydrocortisol ratios (P = .04), suggesting increased global 5α-reductase activity. There was no evidence for differences in 11ß-hydroxysteroid dehydrogenase activity. Steroid metabolite excretion was not correlated with weight. CONCLUSIONS: Our findings suggest that differences in androgen metabolism are present in early childhood in PCOS-d. Increased 5α-reductase activity could contribute to the development of PCOS by amplifying target tissue androgen action.

Steroid Sulfatase Deficiency and Androgen Activation Before and After Puberty.

CONTEXT: Steroid sulfatase (STS) cleaves the sulfate moiety off steroid sulfates, including dehydroepiandrosterone (DHEA) sulfate (DHEAS), the inactive sulfate ester of the adrenal androgen precursor DHEA. Deficient DHEA sulfation, the opposite enzymatic reaction to that catalyzed by STS, results in androgen excess by increased conversion of DHEA to active androgens. STS deficiency (STSD) due to deletions or inactivating mutations in the X-linked STS gene manifests with ichthyosis, but androgen synthesis and metabolism in STSD have not been studied in detail yet. PATIENTS AND METHODS: We carried out a cross-sectional study in 30 males with STSD (age 6-27 y; 13 prepubertal, 5 peripubertal, and 12 postpubertal) and 38 age-, sex-, and Tanner stage-matched healthy controls. Serum and 24-hour urine steroid metabolome analysis was performed by mass spectrometry and genetic analysis of the STS gene by multiplex ligation-dependent probe amplification and Sanger sequencing. RESULTS: Genetic analysis showed STS mutations in all patients, comprising 27 complete gene deletions, 1 intragenic deletion and 2 missense mutations. STSD patients had apparently normal pubertal development. Serum and 24-hour urinary DHEAS were increased in STSD, whereas serum DHEA and testosterone were decreased. However, total 24-hour urinary androgen excretion was similar to controls, with evidence of increased 5α-reductase activity in STSD. Prepubertal healthy controls showed a marked increase in the serum DHEA to DHEAS ratio that was absent in postpubertal controls and in STSD patients of any pubertal stage. CONCLUSIONS: In STSD patients, an increased 5α-reductase activity appears to compensate for a reduced rate of androgen generation by enhancing peripheral androgen activation in affected patients. In healthy controls, we discovered a prepubertal surge in the serum DHEA to DHEAS ratio that was absent in STSD, indicative of physiologically up-regulated STS activity before puberty. This may represent a fine tuning mechanism for tissue-specific androgen activation preparing for the major changes in androgen production during puberty.