Michaela F. Hartmann

Homozygous Mutation G539R in the Gene for P450 Oxidoreductase in a Family Previously Diagnosed as Having 17,20-Lyase Deficiency

CONTEXT Very few patients have been described with isolated 17,20-lyase deficiency who have had their mutations in P450c17 (17alpha-hydroxylase/17,20-lyase) proven by DNA sequencing and in vitro characterization of the mutations. Most patients with 17,20-lyase deficiency have mutations in the domain of P450c17 that interact with the electron-donating redox partner, P450 oxidoreductase (POR). OBJECTIVE Our objective was to clarify the genetic and functional basis of isolated 17,20-lyase deficiency in familial cases who were previously reported as having 17,20-lyase deficiency. PATIENTS Four undervirilized males of an extended Bedouin family were investigated. One of these has previously been reported to carry mutations in the CYP17A1 gene encoding P450c17 causing isolated 17,20-lyase deficiency. METHODS Serum hormones were evaluated before and after stimulation with ACTH. Urinary steroid metabolites were profiled by gas chromatography-mass spectrometry. Exons 1 and 8 of CYP17A1 previously reported to harbor mutations in one of these patients and all 15 coding exons of POR were sequenced. RESULTS Gas chromatography-mass spectrometry (GC-MS) urinary steroid profiling and serum steroid measurements showed combined deficiencies of 17,20-lyase and 21-hydroxylase. Sequencing of exons 1 and 8 of CYP17A1 in two different laboratories showed no mutations. Sequencing of POR showed that all four patients were homozygous for G539R, a previously studied mutation that retains 46% of normal capacity to support the 17alpha-hydroxylase activity but only 8% of the 17,20-lyase activity of P450c17. CONCLUSION POR deficiency can masquerade clinically as isolated 17,20-lyase deficiency.

Genotype-phenotype analysis in congenital adrenal hyperplasia due to P450 oxidoreductase deficiency

Context: P450 oxidoreductase deficiency (PORD) is a unique congenital adrenal hyperplasia variant that manifests with glucocorticoid deficiency, disordered sex development (DSD), and skeletal malformations. No comprehensive data on genotype-phenotype correlations in Caucasian patients are available. Objective: The objective of the study was to establish genotype-phenotype correlations in a large PORD cohort. Design: The design of the study was the clinical, biochemical, and genetic assessment including multiplex ligation-dependent probe amplification (MLPA) in 30 PORD patients from 11 countries. Results: We identified 23 P450 oxidoreductase (POR) mutations (14 novel) including an exonic deletion and a partial duplication detected by MLPA. Only 22% of unrelated patients carried homozygous POR mutations. p.A287P was the most common mutation (43% of unrelated alleles); no other hot spot was identified. Urinary steroid profiling showed characteristic PORD metabolomes with variable impairment of 17α-hydroxylase and 21-hydroxylase. Short cosyntropin testing revealed adrenal insufficiency in 89%. DSD was present in 15 of 18 46,XX and seven of 12 46,XY individuals. Homozygosity for p.A287P was invariably associated with 46,XX DSD but normal genitalia in 46,XY individuals. The majority of patients with mild to moderate skeletal malformations, assessed by a novel scoring system, were compound heterozygous for missense mutations, whereas nearly all patients with severe malformations carried a major loss-of-function defect on one of the affected alleles. Conclusions: We report clinical, biochemical, and genetic findings in a large PORD cohort and show that MLPA is a useful addition to POR mutation analysis. Homozygosity for the most frequent mutation in Caucasians, p.A287P, allows for prediction of genital phenotype and moderate malformations. Adrenal insufficiency is frequent, easily overlooked, but readily detected by cosyntropin testing.

Adrenarche and Bone Modeling and Remodeling at the Proximal Radius: Weak Androgens Make Stronger Cortical Bone in Healthy Children†

Adrenarche, the physiological increase in adrenal androgen secretion, may contribute to better bone status. Proximal radial bone and 24‐h urinary steroid hormones were analyzed cross‐sectionally in 205 healthy children and adolescents. Positive adrenarchal effects on radial diaphyseal bone were observed. Obviously, adrenarche is one determinant of bone mineral status in children.

Metabolic evidence for impaired 17α-hydroxylase activity in a kindred bearing the E305G mutation for isolate 17,20-lyase activity

CONTEXT The CYP17A1 gene encodes many enzymatic reactions including 17alpha-hydroxylase and 17,20-lyase activities. Mutations that selectively ablate the 17,20-lyase activity, causing isolated 17,20-lyase deficiency, are exceedingly rare and may belong to the rarest of all disorders of steroidogenesis. We have previously reported an E305G mutation in the active site of CYP17A1 that apparently causes isolated 17,20-lyase deficiency. Expression studies suggested intact 17alpha-hydroxylase activity which was at odds with subnormal tetracosactrin stimulated cortisol in the patients. OBJECTIVES To investigate the in vivo activity of the adrenal enzymes, we used the metabolomics approach with urinary steroid profiling by gas chromatography-mass spectrometry. PATIENTS Of the 11 subjects investigated, 6 patients in the kindred were found to be homozygous, 4 members were asymptomatic heterozygous, and 1 was homozygous for the wild-type allele. RESULTS In the homozygous patients for E305G, both serum and urinary steroids showed a severe lack of androgens (C(19)-steroids) pointing to the absence of 17,20-lyase activities. Furthermore, precursor/product ratios of urinary steroid metabolites characterizing 17alpha-hydroxylase activity showed variable decreases in 17alpha-hydroxylase activities. CONCLUSIONS The results confirm the complete absence of 17,20-lyase activity in vivo, as in the in vitro expression studies. On the other hand, in vivo 17alpha-hydroxylase activity was partially impaired. Thus, the in vivo metabolic data seem to be more sensitive than the expression study and suggests that this mutation also impairs 17alpha-hydroxylase activity.

Membrane Transporters for Sulfated Steroids in the Human Testis - Cellular Localization, Expression Pattern and Functional Analysis

Sulfated steroid hormones are commonly considered to be biologically inactive metabolites, but may be reactivated by the steroid sulfatase into biologically active free steroids, thereby having regulatory function via nuclear androgen and estrogen receptors which are widespread in the testis. However, a prerequisite for this mode of action would be a carrier-mediated import of the hydrophilic steroid sulfate molecules into specific target cells in reproductive tissues such as the testis. In the present study we detected predominant expression of the Sodium-dependent Organic Anion Transporter (SOAT), the Organic Anion Transporting Polypeptide 6A1, and the Organic Solute Carrier Partner 1 in human testis biopsies. All of these showed significantly lower or even absent mRNA expression in severe disorders of spermatogenesis (arrest at the level of spermatocytes or spermatogonia, Sertoli cell only syndrome). Only SOAT was significantly lower expressed in biopsies showing hypospermatogenesis. By use of immunohistochemistry SOAT was localized to germ cells at various stages in human testis biopsies showing normal spermatogenesis. SOAT immunoreactivity was detected in zygotene primary spermatocytes of stage V, pachytene spermatocytes of all stages (I–V), secondary spermatocytes of stage VI, and round spermatids (step 1 and step 2) in stages I and II. Furthermore, SOAT transport function for steroid sulfates was analyzed with a novel liquid chromatography tandem mass spectrometry procedure capable of profiling steroid sulfate molecules from cell lysates. With this technique, the cellular inward-directed SOAT transport was verified for the established substrates dehydroepiandrosterone sulfate and estrone-3-sulfate. Additionally, β-estradiol-3-sulfate and androstenediol-3-sulfate were identified as novel SOAT substrates.

Intravenous Lipid and Heparin Infusion-Induced Elevation in Free Fatty Acids and Triglycerides Modifies Circulating Androgen Levels in Women: A Randomized, Controlled Trial

BACKGROUND The polycystic ovarian syndrome (PCOS) is characterized by hyperandrogenism and associated with obesity and impaired glucose metabolism. Despite the high prevalence of PCOS and the considerable clinical impact, the precise interplay between metabolism and hyperandrogenemia is not entirely clear. OBJECTIVE The objective of the study was to analyze the effects of iv lipid and heparin infusion on circulating androgen levels in healthy women. DESIGN This was a randomized, controlled, crossover trial. SETTING The study was conducted at an endocrinology center. PATIENTS Patients included 12 healthy young women during the early follicular phase of two subsequent cycles. INTERVENTION After an overnight fast, a 20% lipid/heparin or a saline/heparin infusion was administered in random order for 330 min. MAIN OUTCOME MEASURES A detailed characterization of androgen metabolism was performed. RESULTS Elevations in free fatty acids and triglycerides, induced by lipid/heparin infusion, elevates the levels of androstenedione, dehydroepiandrosterone (DHEA), dehydroepiandrosterone sulfate (DHEAS), testosterone, 5alpha-dihydrotestosterone, estrone, and 17beta-estradiol. Urinary excretion of DHEA, DHEAS, 5-androstene-3beta,17beta-diol, and the sum of urinary excreted DHEA and its 16-hydroxylated downstream metabolites, 16alpha-hydroxy-DHEA and 5-androstene-3beta,16alpha,17beta-triol, were reduced. CONCLUSION The mechanism of iv lipid and heparin infusion-induced elevation of circulating androgens described here might contribute to the development of hyperandrogenism in women with PCOS and suggests that lowering of hyperlipidemia might be a potential therapeutic target in patients with PCOS to treat hyperandrogenemia.

Exaggerated adrenarche and altered cortisol metabolism in Type 1 diabetic children

Reported literature data strongly suggest that steroid metabolism is dysregulated in Type 1 diabetes mellitus. The aim of this study was to non-invasively examine the cortisol metabolism in children with Type 1 diabetes mellitus (T1DM) in detail and to test the hypothesis that adrenarche is affected under conventional intensive insulin therapy. In 24-h urine samples of 109 patients aged 4-18 years with T1DM of more than 1 year, steroids were profiled using gas chromatography-mass spectrometry. Additionally, urinary free cortisol (UFF) and cortisone (UFE) were quantified by RIA after extraction and chromatographic purification. Data on urinary steroids from 400 healthy controls served as reference values. Enzyme activities were assessed by established steroid metabolite ratios, e.g. 5alpha-reductase and 11beta-hydroxysteroid dehydrogenase Type 2 (11beta-HSD2) by 5alpha-tetrahydrocortisol/tetrahydrocortisol and UFE/UFF, respectively. Urinary markers of adrenarche, especially dehydroepiandrosterone and its direct metabolites were elevated in patients, as were urinary 6beta-hydroxycortisol, UFE, and 11beta-HSD2 activity. However, overall cortisol secretion, as reflected by the sum of major urinary cortisol metabolites, was mostly normal and activity of 5alpha-reductase clearly reduced. Our study provides evidence for an exaggerated adrenarche in T1DM children, which may help to understand reported sequelae in female patients like hyperandrogenic symptoms. The findings also suggest a reduced cortisol inactivation via 5alpha-reductase that is not compensated by a fall in cortisol secretion. Whether the elevated urinary 6beta-hydroxycortisol and cortisone excretion, observed in the patients, are also present in other forms of hypercortisolism and may thus serve as non-invasive clinical stress markers deserves further study.

Prepubertal Adrenarchal Androgens and Animal Protein Intake Independently and Differentially Influence Pubertal Timing

CONTEXT Whether adrenarche impacts on pubertal development is controversial. OBJECTIVE The objective of the study was to examine the associations of adrenal androgen (AA) secretion with early and late pubertal markers, independent of potential influences of dietary animal protein intake. DESIGN AND PARTICIPANTS This was a prospective cohort study of healthy free-living Caucasian children (n = 109) who provided both 24-h urine samples and 3-d weighed dietary records 1 and 2 yr before the biological age at take-off of the pubertal growth spurt (ATO). MEASUREMENTS Twenty-four-hour excretion rates of androgen (C19) metabolites quantified by gas chromatography-mass spectrometry were measured. MAIN OUTCOMES ATO, age at peak height velocity (APHV), age at menarche/voice break, duration of pubertal growth acceleration, and ages at Tanner stage 2 for breast (girls) and genital (boys) development (B2-G2) and pubic hair (PH2). RESULTS Higher adrenarchal C19 steroids predicted earlier ages at Tanner stage 2 for pubic hair (P < 0.0001) and B2-G2 (P = 0.009) as well as a shorter pubertal growth acceleration period (P = 0.001), independently of animal protein intake. Children with a higher AA secretion had a 1.5-yr earlier beginning of pubarche and a 0.8-yr earlier beginning of B2-G2 than those with a lower AA excretion. Furthermore, animal protein intake was independently negatively associated with ATO and APHV (P < 0.05 each) and tended to be negatively associated with age at menarche/voice break (P = 0.07). CONCLUSION A higher animal protein intake may be involved in an earlier attainment of ATO and APHV, whereas a more intensive adrenarchal process may precipitate a shorter pubertal growth spurt and a notably earlier onset of breast and genital development in girls and boys, respectively.

Effects of Dehydroepiandrosterone Therapy on Pubic Hair Growth and Psychological Well-Being in Adolescent Girls and Young Women with Central Adrenal Insufficiency : A Double-Blind, Randomized, Placebo-Controlled Phase III Trial

CONTEXT AND OBJECTIVE The efficacy of oral dehydroepiandrosterone (DHEA) in the treatment of atrichia pubis and psychological distress in young females with central adrenal insufficiency is unknown. Our study aimed to evaluate this therapy. DESIGN AND PATIENTS A total of 23 young females (mean age 18 yr, range 13-25) was enrolled in a double-blind randomized placebo-controlled trial. Inclusion criteria were ACTH deficiency plus two or more additional pituitary deficiencies, serum DHEA less than 400 ng/ml, and pubertal stage more than B2. Exclusion criteria were cerebral radiation with more than 30 Gy, tumor remission less than 1 yr, amaurosis, hypothalamic obesity, psychiatric disorders, and unstable hormone medication. INTERVENTION Patients were randomized to placebo (n = 12) or 25 mg HPLC-purified DHEA/d (n = 11) orally for 12 months after stratification into a nontumor (n = 7) and a tumor group (n = 16). MAIN OUTCOME MEASURES Clinical scoring of pubic hair stage was performed at 0, 6, and 12 months (primary endpoint), and psychometrical evaluation (Symptom Check-List-90-R and the Centre for Epidemiological Studies-Depression Scale) at 0 and 12 months (secondary endpoint). Androgen levels and safety parameters were measured at 0, 6, and 12 months; 24-h androgen urinary excretion rates were calculated at 0 and 12 months. RESULTS In the placebo group, four patients dropped out because of recurrence of craniopharyngioma, manifestation of type 1 diabetes, and change of residence (n = 2); in the DHEA group, one patient dropped out because of recurrent anxiety attacks. DHEA substitution resulted in normalization of DHEA sulfate and androstanediol glucuronide morning serum levels 2 h after drug intake (P < 0.006), and of its 24 h urinary metabolite levels (P < 0.0001), placebo had no effect. Morning serum levels of androstenedione increased in the DHEA group (P < 0.02) but did not normalize. The DHEA group exhibited significant progress in pubic hair growth from Tanner stage I-III to II-V (mean: +1.5 stages), whereas the placebo group did not (relative risk 0.138; 95% confidence interval 0.021-0.914; P = 0.0046). Importantly, eight of the 10 Symptom Check-List-90-R scores, including those for depression, anxiety, and interpersonal sensitivity, and the global severity index improved in the DHEA group in comparison to the placebo group (P < 0.048). DHEA was well tolerated. CONCLUSIONS In adolescent girls with central adrenal insufficiency, daily replacement with 25 mg DHEA orally is beneficial: atrichia pubis vanishes, and psychological well-being improves significantly.

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

Steroids are primarily present in human fluids in their sulfated forms. Profiling of these compounds is important from both diagnostic and physiological points of view. Here, we present a novel method for the quantification of 11 intact steroid sulfates in human serum by LC-MS/MS. The compounds analyzed in our method, some of which are quantified for the first time in blood, include cholesterol sulfate, pregnenolone sulfate, 17-hydroxy-pregnenolone sulfate, 16-α-hydroxy-dehydroepiandrosterone sulfate, dehydroepiandrosterone sulfate, androstenediol sulfate, androsterone sulfate, epiandrosterone sulfate, testosterone sulfate, epitestosterone sulfate, and dihydrotestosterone sulfate. The assay was conceived to quantify sulfated steroids in a broad range of concentrations, requiring only 300 μl of serum. The method has been validated and its performance was studied at three quality controls, selected for each compound according to its physiological concentration. The assay showed good linearity (R2 > 0.99) and recovery for all the compounds, with limits of quantification ranging between 1 and 80 ng/ml. Averaged intra-day and between-day precisions (coefficient of variation) and accuracies (relative errors) were below 10%. The method has been successfully applied to study the sulfated steroidome in diseases such as steroid sulfatase deficiency, proving its diagnostic value. This is, to our best knowledge, the most comprehensive method available for the quantification of sulfated steroids in human blood.