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.

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.

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.

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.

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.

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.

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.

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.

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.

Longitudinal changes in glucocorticoid metabolism are associated with later development of adverse metabolic phenotype.

OBJECTIVE: Dysregulation of enzymes that control local tissue steroid metabolism has been implicated in the pathogenesis of obesity and insulin resistance; however, longitudinal changes in glucocorticoid metabolism have not been investigated. This study was performed to evaluate the role of glucocorticoid metabolism in the development of insulin resistance and obesity and to identify biomarkers for future development of metabolic disease. DESIGN: This was a prospective longitudinal observation study conducted over 5 years. METHODS: A 24-h collection was used to serially analyze urinary glucocorticoid and mineralocorticoid metabolites in 57 obese and overweight patients with no prior diagnosis of diabetes mellitus, recruited from the community. RESULTS: Baseline higher 5α-reductase (5αR) activity, but not 11ß-hydroxysteroid dehydrogenase type 1 activity, was predictive of increased fasting insulin at final visit (11.4 compared with 7.4 mU/l in subjects with lower 5αR activity, P<0.05), area under the curve insulin response to oral glucose tolerance test (176.7 compared with 89.1 mU/l.h, P<0.01), and homeostasis model assessment (HOMA2-IR; 1.3 compared with 0.8, P<0.01). Higher total glucocorticoid production was associated with abnormal glucose tolerance and increased BMI. During this study, systolic blood pressure increased (equivalent to ∼1 mmHg/year), as did plasma sodium levels; this evidence of increased mineralocorticoid activity was associated with increased aldosterone metabolites and decreased 11ß-hydroxysteroid dehydrogenase type 2 activity. CONCLUSIONS: Increased 5αR activity and glucocorticoid secretion rate over time are linked with the development of metabolic disease, and may represent targets for therapeutic intervention, which merits further study.

Diminished 11ß-hydroxysteroid dehydrogenase type 2 activity is associated with decreased weight and weight gain across the first year of life.

CONTEXT: Low birth weight is associated with adverse metabolic outcome in adulthood. Exposure to glucocorticoid (GC) excess in utero is associated with decreased birth weight, but the prospective longitudinal relationship between GC metabolism and growth has not been examined. OBJECTIVE: We have hypothesized that changes in GC metabolism leading to increased availability may impair growth. DESIGN: This was a prospective, longitudinal study with clinical measurements and 24-hour urinary steroid metabolite analysis at 1, 4, 12, 26, and 52 weeks after delivery in mothers and their babies. SETTING: The study was conducted with observations and samples collected in the volunteers' own homes. PARTICIPANTS: Healthy mothers and newborn babies/infants participated in the study. INTERVENTIONS: There were no interventions. MAIN OUTCOME MEASURES: Urinary steroid metabolite excretion quantified by gas chromatography/mass spectroscopy across the first year of life in relation to change in weight was measured. RESULTS: The total production of the GC metabolites quantified increased across the first year of life. Markers of 11ß-hydroxysteroid dehydrogenase type 1 activity increased from the age of 3 months as did those of 5α-reductase activity. After correcting for confounding variables, low markers of 11ß-hydroxysteroid dehydrogenase type 2 activity was associated with reduced absolute weight and decreased weight gain over the first year of life. In the mothers, 5α-reductase activity was low at birth and progressively increased to normal over the first 6 months postpartum. CONCLUSIONS: Increased GC exposure as a consequence of reduced 11ß-hydroxysteroid dehydrogenase type 2 activity is likely to be a critical determinant of growth in early life. This not only highlights the central role of GCs and their metabolism, but also emphasizes the need for detailed longitudinal analyses.

Remission of hypertension and electrolyte abnormalities following renal transplantation in a patient with apparent mineralocorticoid excess well documented throughout childhood.

Apparent mineralocorticoid excess syndrome (AME) is an autosomal recessive genetic disorder caused by a deficiency in the enzyme 11ß-hydroxysteroid dehydrogenase type 2 (11ß-HSD). We report a 36-year-old male who was hypertensive from birth and was diagnosed with AME at 8 years of age. There was continuous documentation of his hypertension and hypokalemic alkalosis throughout childhood, during which spironolactone and supplemental potassium were administered. At 33 years of age, the patient received a renal transplant, and following this the AME appears to have been cured clinically with remission of his low renin hypertension and hypokalemic alkalosis despite termination of treatment with spironolactone and potassium supplements.

Analysis by liquid chromatography-mass spectrometry of sterols and oxysterols in brain of the newborn Dhcr7(Δ3-5/T93M) mouse: a model of Smith-Lemli-Opitz syndrome.

In this study the sterol and oxysterol profile of newborn brain from the Dhcr7(Δ3-5/T93M) mouse model of Smith-Lemli-Opitz syndrome (SLOS) has been investigated. This is a viable mouse model which is compound heterozygous containing one null allele and one T93M mutation on Dhcr7. We find the SLOS mouse has reduced levels of cholesterol and desmosterol and increased levels of 7- and 8-dehydrocholesterol and of 7- and 8-dehydrodesmosterol in brain compared to the wild type. The profile of enzymatically formed oxysterols in the SLOS mouse resembles that in the wild type but the level of 24S-hydroxycholesterol, the dominating cholesterol metabolite, is reduced in a similar proportion to that of cholesterol. A number of oxysterols abundant in the SLOS mouse are probably derived from 7-dehydrocholesterol, however, the mechanism of their formation is unclear.

Prenatal diagnosis of congenital adrenal hyperplasia caused by P450 oxidoreductase deficiency.

CONTEXT: Mutations in the electron donor enzyme P450 oxidoreductase (POR) result in congenital adrenal hyperplasia with apparent combined 17α-hydroxylase/17,20 lyase and 21-hydroxylase deficiencies, also termed P450 oxidoreductase deficiency (PORD). Major clinical features present in PORD are disordered sex development in affected individuals of both sexes, glucocorticoid deficiency, and multiple skeletal malformations. OBJECTIVE: The objective of the study was to establish a noninvasive approach to prenatal diagnosis of PORD including assessment of malformation severity to facilitate optimized prenatal diagnosis and timely treatment. DESIGN: We analyzed 20 pregnancies with children homozygous or compound heterozygous for disease-causing POR mutations and 1 pregnancy with a child carrying a heterozygous POR mutation by recording clinical and biochemical presentations and fetal ultrasound findings. In 4 of the pregnancies (3 homozygous and 1 heterozygous for disease-causing POR mutations), prenatal analysis of steroid metabolite excretion in maternal urine was carried out by gas chromatography/mass spectrometry during gestational weeks 11-23. RESULTS: Pregnancy complications in our cohort included maternal virilization (6 of 20) with onset in the second trimester. Seven pregnant women presented with low unconjugated estriol at prenatal screening (triple or quadruple antenatal screening test). Overt dysmorphic features were noted in 19 of the 20 babies at birth but observed in only 5 by prenatal ultrasound. These 5 had the most severe malformation phenotypes and poor outcome, whereas the other babies showed normal development. Steroid profiling of maternal urine revealed significantly increased steroids of fetal origin, namely the pregnenolone metabolite epiallopregnanediol and the androgen metabolite androsterone, with concomitant low values for estriol. Diagnostic steroid ratios conclusively indicated PORD as early as gestational week 12. In the heterozygous pregnancy, steroid ratios were only slightly elevated and estriol excretion was normal. CONCLUSION: Prenatal diagnosis in PORD is readily established via urinary steroid metabolite analysis of maternal urine. Visible malformations at prenatal ultrasound predict a severe malformation phenotype.

Mitotane therapy in adrenocortical cancer induces CYP3A4 and inhibits 5α-reductase, explaining the need for personalized glucocorticoid and androgen replacement.

CONTEXT: Mitotane [1-(2-chlorophenyl)-1-(4-chlorophenyl)-2,2-dichloroethane] is the first-line treatment for metastatic adrenocortical carcinoma (ACC) and is also regularly used in the adjuvant setting after presumed complete removal of the primary tumor. Mitotane is considered an adrenolytic substance, but there is limited information on distinct effects on steroidogenesis. However, adrenal insufficiency and male hypogonadism are widely recognized side effects of mitotane treatment. OBJECTIVE: Our objective was to define the impact of mitotane treatment on in vivo steroidogenesis in patients with ACC. SETTING AND DESIGN: At seven European specialist referral centers for adrenal tumors, we analyzed 24-h urine samples (n = 127) collected from patients with ACC before and during mitotane therapy in the adjuvant setting (n = 23) or for metastatic ACC (n = 104). Urinary steroid metabolite excretion was profiled by gas chromatography/mass spectrometry in comparison with healthy controls (n = 88). RESULTS: We found a sharp increase in the excretion of 6ß-hydroxycortisol over cortisol (P < 0.001), indicative of a strong induction of the major drug-metabolizing enzyme cytochrome P450 3A4. The contribution of 6ß-hydroxycortisol to total glucocorticoid metabolites increased from 2% (median, interquartile range 1-4%) to 56% (39-71%) during mitotane treatment. Furthermore, we documented strong inhibition of systemic 5α-reductase activity, indicated by a significant decrease in 5α-reduced steroids, including 5α-tetrahydrocortisol, 5α-tetrahydrocorticosterone, and androsterone (all P < 0.001). The degree of inhibition was similar to that in patients with inactivating 5α-reductase type 2 mutations (n = 23) and patients receiving finasteride (n = 5), but cluster analysis of steroid data revealed a pattern of inhibition distinct from these two groups. Longitudinal data showed rapid onset and long-lasting duration of the observed effects. CONCLUSIONS: Cytochrome P450 3A4 induction by mitotane results in rapid inactivation of more than 50% of administered hydrocortisone, explaining the need for doubling hydrocortisone replacement in mitotane-treated patients. Strong inhibition of 5α-reductase activity is in line with the clinical observation of relative inefficiency of testosterone replacement in mitotane-treated men, calling for replacement by 5α-reduced androgens.

A SULT2A1 genetic variant identified by GWAS as associated with low serum DHEAS does not impact on the actual DHEA/DHEAS ratio.

DHEA is the major precursor of human sex steroid synthesis and is inactivated via sulfonation to DHEAS. A previous genome-wide association study related the single nucleotide polymorphism (SNP) rs2637125, located near the coding region of DHEA sulfotransferase, SULT2A1, to serum DHEAS concentrations. However, the functional relevance of this SNP with regard to DHEA sulfonation is unknown. Using data from 3300 participants of the population-based cohort Study of Health in Pomerania, we identified 43 individuals being homozygote for the minor allele of the SNP rs2637125 (AA) and selected two sex- and age-matched individuals with AG and GG genotype (n=172) respectively. Steroid analysis including measurement of serum DHEA and DHEAS was carried out by liquid chromatography/mass spectrometry, employing steroid oxime analysis for enhancing the sensitivity of DHEA detection. We applied quantile regression models to compare median hormone levels across SULT2A1 genotypes. Median comparisons by SULT2A1 genotype (AA vs AG and GG genotypes respectively) showed no differences in the considered hormones including DHEAS, DHEA, androstenedione, as well as cortisol and cortisone concentrations. SULT2A1 genotype also had no effect on the DHEA/DHEAS ratio. Sex-stratified analyses, as well as alternative use of the SULT2A1 SNP rs182420, yielded similar negative results. Genetic variants of SULT2A1 do not appear to have an effect on individual DHEA and DHEAS concentrations or the DHEA/DHEAS ratio as a marker of DHEA sulfonation capacity.

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.

A missense mutation in the human cytochrome b5 gene causes 46,XY disorder of sex development due to true isolated 17,20 lyase deficiency.

CONTEXT: Isolated 17,20 lyase deficiency is commonly defined by apparently normal 17α-hydroxylase activity but severely reduced 17,20 lyase activity of the bifunctional enzyme cytochrome P450 (CYP) enzyme 17A1 (CYP17A1), resulting in sex steroid deficiency but normal glucocorticoid and mineralocorticoid reserve. Cytochrome b5 (CYB5A) is thought to selectively enhance 17,20 lyase activity by facilitating the allosteric interaction of CYP17A1 with its electron donor P450 oxidoreductase (POR). OBJECTIVE: We investigated a large consanguineous family including three siblings with 46,XY disorder of sex development (DSD) presenting with isolated 17,20 lyase deficiency. DESIGN: We investigated the clinical and biochemical phenotype, conducted genetic analyses, and functionally characterized the identified CYB5A mutation in cell-based CYP17A1 coexpression assays. RESULTS: All three siblings presented with 46,XY DSD, sex steroid deficiency, normal mineralocorticoids and glucocorticoids, and a urine steroid metabolome suggestive of isolated 17,20 lyase deficiency. CYP17A1 and POR sequences were normal, but we detected a homozygous CYB5A missense mutation (g.28,400A→T; p.H44L). Functional in vitro analysis revealed normal CYP17A1 17α-hydroxylase activity but severely impaired 17,20 lyase activity. In silico analysis suggested the disruption of CYB5A heme binding by p.H44L. CONCLUSION: We have identified the first human CYB5A missense mutation as the cause of isolated 17,20 lyase deficiency in three individuals with 46,XY DSD. Detailed review of previously reported cases with apparently isolated 17,20 lyase deficiency due to mutant CYP17A1 and POR reveals impaired 17α-hydroxylase activity as assessed by steroid metabolome analysis and short cosyntropin testing. This suggests that truly isolated 17,20 lyase deficiency is observed only in individuals with inactivating CYB5A mutations.

Role of a disordered steroid metabolome in the elucidation of sterol and steroid biosynthesis.

In 1937 Butler and Marrian found large amounts of the steroid pregnanetriol in urine from a patient with the adrenogenital syndrome, a virilizing condition known to be caused by compromised adrenal secretion even in this pre-cortisol era. This introduced the concept of the study of altered excretion of metabolites as an in vivo tool for understanding sterol and steroid biosynthesis. This approach is still viable and has experienced renewed significance as the field of metabolomics. From the first cyclized sterol lanosterol to the most downstream product estradiol, there are probably greater than 30 steps. Based on a distinctive metabolome clinical disorders have now been attributed to about seven post-squalene cholesterol (C) biosynthetic steps and around 15 en-route to steroid hormones or needed for further metabolism of such hormones. Forty years ago it was widely perceived that the principal steroid biosynthetic defects were known but interest rekindled as novel metabolomes were documented. In his career this investigator has been involved in the study of many steroid disorders, the two most recent being P450 oxidoreductase deficiency and apparent cortisone reductase deficiency. These are of interest as they are due not to mutations in the primary catalytic enzymes of steroidogenesis but in ancillary enzymes needed for co-factor oxido-reduction A third focus of this researcher is Smith-Lemli-Opitz syndrome (SLOS), a cholesterol synthesis disorder caused by 7-dehydrocholesterol reductase mutations. The late George Schroepfer, in whose honor this article has been written, contributed greatly to defining the sterol metabolome of this condition. Defining the cause of clinically severe disorders can lead to improved treatment options. We are now involved in murine gene therapy studies for SLOS which, if successful could in the future offer an alternative therapy for this severe condition.