Novel H6PDH mutations in two girls with premature adrenarche: 'apparent' and 'true' CRD can be differentiated by urinary steroid profiling.

CONTEXT: Inactivating mutations in the enzyme hexose-6-phosphate dehydrogenase (H6PDH, encoded by H6PD) cause apparent cortisone reductase deficiency (ACRD). H6PDH generates cofactor NADPH for 11ß-hydroxysteroid dehydrogenase type 1 (11ß-HSD1, encoded by HSD11B1) oxo-reductase activity, converting cortisone to cortisol. Inactivating mutations in HSD11B1 cause true cortisone reductase deficiency (CRD). Both ACRD and CRD present with hypothalamic-pituitary-adrenal (HPA) axis activation and adrenal hyperandrogenism. OBJECTIVE: To describe the clinical, biochemical and molecular characteristics of two additional female children with ACRD and to illustrate the diagnostic value of urinary steroid profiling in identifying and differentiating a total of six ACRD and four CRD cases. DESIGN: Clinical, biochemical and genetic assessment of two female patients presenting during childhood. In addition, results of urinary steroid profiling in a total of ten ACRD/CRD patients were compared to identify distinguishing characteristics. RESULTS: Case 1 was compound heterozygous for R109AfsX3 and a novel P146L missense mutation in H6PD. Case 2 was compound heterozygous for novel nonsense mutations Q325X and Y446X in H6PD. Mutant expression studies confirmed loss of H6PDH activity in both cases. Urinary steroid metabolite profiling by gas chromatography/mass spectrometry suggested ACRD in both cases. In addition, we were able to establish a steroid metabolite signature differentiating ACRD and CRD, providing a basis for genetic diagnosis and future individualised management. CONCLUSIONS: Steroid profile analysis of a 24-h urine collection provides a diagnostic method for discriminating between ACRD and CRD. This will provide a useful tool in stratifying unresolved adrenal hyperandrogenism in children with premature adrenarche and adult females with polycystic ovary syndrome (PCOS).

Characterization of two novel mutations in the glucocorticoid receptor gene in patients with primary cortisol resistance.

OBJECTIVE: Primary glucocorticoid resistance is characterized by decreased sensitivity to cortisol signalling. We have performed genetic analysis of the glucocorticoid receptor (GR) gene in 12 unrelated patients with primary cortisol resistance as defined by a pathological dexamethasone suppression test. METHODS: Exon specific polymerase chain reaction amplification of the GR gene and sequencing of each exon was carried out. The two mutations were characterized in vitro in terms of glucocorticoid driven reporter gene activity in a transient transfection assay and in a ligand binding assay. Molecular modelling of the R477H mutant was performed based on the X-ray structure of the GR-DNA binding domain. RESULTS: Two novel mutations in the GR gene were found: R477H in the DNA-binding domain which is the first reported mutation in that region of the human GR gene and G679S in the ligand binding domain. The R477H mutation showed no transactivating capacity, whereas the G679S mutation had reduced transactivation capacity compared to the wild-type (wt) GR. When tested for ligand binding capacity, the G679S mutation had 50% binding affinity compared to the wt GR. The effect of the point mutation R477H was deduced by a comparison between the wt structure and the model of the mutant. The wt GR has direct and water mediated contact with the phosphate groups of the glucocorticoid responsive element (GRE) whereas, in the model, the mutation R477H has no contact with the GRE. The G679S mutation is located on the surface of the ligand binding domain, at a distance from the steroid-binding site. A previously reported polymorphism, AAT to AAC at amino acid position 766, was found in four of the patients. CONCLUSIONS: In two of 12 patients with clinical glucocorticoid resistance, mutant forms of GR could be found. The glucocorticoid resistance in vivo in these two patients corresponds to impaired function of the two mutated GR forms in two in vitro assays. The relevance of the conservative polymorphism for the glucocorticoid insensitivity noted in these patients remains to be clarified.

Increased 6-sulfatoxymelatonin excretion in women with polycystic ovary syndrome.

OBJECTIVE: To determine melatonin production in hyperandrogenic women. DESIGN: Controlled prospective study. SETTING: Outpatients in an academic medical center. PATIENT(S): Twenty-two women with polycystic ovary syndrome (PCOS), 20 women with idiopathic hirsutism, and 15 age-matched individuals who had similar body mass indexes as controls. INTERVENTION(S): Fasting blood samples and 24-hour urinary samples were obtained from all participants. MAIN OUTCOME MEASURE(S): All participants provided serum samples for the measurement of LH, FSH, testosterone, E(2), DHEAS, 17 alpha-hydroxyprogesterone (17-OHP), and insulin levels, as well as urinary 6-sulfatoxymelatonin (aMT6s). RESULT(S): Women with PCOS had higher aMT6s, testosterone, LH/FSH ratio, and insulin values than either women with idiopathic hirsutism or control women. Testosterone inversely correlated with aMT6s in PCOS. Regression analysis revealed that only testosterone was an important determinant of aMT6s in PCOS. CONCLUSION(S): Women with PCOS have increased melatonin production.

Effect of hyperandrogenism and menstrual cycle abnormalities on bone mass and bone turnover in young women.

OBJECTIVE: Prolonged period of amenorrhoea are regarded as a risk factor for the appearance of osteoporosis. Amenorrhoea is a feature of different pathological conditions with heterogeneous endocrine profiles. We evaluated bone mineral metabolism in patients with polycystic ovary syndrome (PCOS), hypothalamic amenorrhoea and idiopathic hirsutism in order to establish the relative importance for the maintenance of normal bone mass of ovulatory cycles and androgen and oestrogen production. PATIENTS AND MEASUREMENTS: Bone mineral density (BMD), bone turnover markers and endocrine profile were evaluated in 51 patients with PCOS, 24 patients with idiopathic hirsutism, 26 patients with hypothalamic amenorrhoea and 35 healthy women. Body mass index (BMI) ranged between 20.1 and 31.0 kg/m2, and age from 17 to 33 years. Thirty-eight of the PCOS patients were amenorrhoeic (< 4 menstrual cycles/year). RESULTS: Spine and femoral BMD were significantly decreased and bone markers (serum osteocalcin, and urinary excretion of free deoxypyridinoline, cross-linked N-telopeptide and hydroxyproline) significantly increased in the patients with hypothalamic amenorrhoea, when compared to control subjects and the other two patient groups. In the sub-group of PCOS patients with amenorrhoea, spine and femoral neck BMD was significantly lower than in patients with idiopathic hirsutism and the non-amenorrhoeic PCOS patients. In all PCOS patients, spine and neck BMD were positively correlated (P < 0.05) with serum androstenedione and free testosterone levels. CONCLUSIONS: The results of this study suggest that in patients with polycystic ovary syndrome the deleterious effect on bone of amenorrhoea is balanced by androgen overproduction.

Serum and urinary steroids in girls with precocious pubarche and/or hirsutism due to mild 3-beta-hydroxysteroid dehydrogenase deficiency.

To obtain data on the correlation of serum and urinary steroids in nonclassical 3 beta-hydroxysteroid dehydrogenase (3 beta-HSD) deficiency, 9 girls with precocious pubarche and 33 adolescent girls with mild to severe hirsutism were studied. Urinary steroid profiles were analyzed by capillary gas chromatography. Serum 17-OH-pregnenolone (17-OHPreg) and 17-OH-progesterone (17-OHP) were determined by RIA after column-chromatographic separation. One out of 9 girls with precocious pubarche and 4/33 girls with hirsutism had elevated ratios of 17-OHPreg to 17-OHP after ACTH stimulation in serum and elevated urinary excretion of 5-ene steroids under basal conditions. These patients were defined to have decreased adrenal 3 beta-HSD activity. Basal and ACTH-stimulated serum 17-OHPreg levels in patients with mild 3 beta-HSD deficiency overlapped those of healthy controls and peripubertally virilized female patients without enzyme deficiency. Post-ACTH 17-OHPreg/17-OHP ratios in serum discriminated patients with and without 3 beta-HSD deficiency using a cutoff value of 13 instead of mean + 2 SD for age-related control values (6.7 and 11.6 for girls with Tanner stage II-III and IV-V, respectively). Sums of urinary 5-ene steroids in patients with 3 beta-HSD deficiency overlapped those in patients without enzyme deficiency. Results showed that an abnormal post-ACTH serum 17-OHPreg/17-OHP ratio may not be associated with elevated urinary 5-ene steroid excretion, and vica versa. In conclusion, patients with simultaneous elevation of post-ACTH serum 17-OHPreg/17-OHP ratio and basal urinary 5-ene steroid excretion are supposed to have mild 3 beta-HSD deficiency.

Assessment of 5 alpha-reductase activity in hirsute women: comparison of serum androstanediol glucuronide with urinary androsterone and aetiocholanolone excretion.

OBJECTIVE: Recent evidence suggests that androstanediol glucuronide (AG), a metabolite of dihydrotestosterone (DHT) formed in skin, is frequently elevated in hirsute women, presumably reflecting enhanced 5 alpha-reductase activity. An alternative method of demonstrating 5 alpha-reductase activity is the androsterone (A)/aetiocholanolone (E) ratio in urine. A and E are the 5 alpha- and 5 beta-reduced metabolites, respectively, of androstenedione, which is the principal metabolite of dehydroepiandrosterone (D). Although serum AG and the urinary A/E ratio have both been considered valid methods for assessing 5 alpha-reductase activity, the two have not been previously compared in hirsute women. The present study was undertaken to assess 5 alpha-reductase activity in hirsute patients as determined by these two different methods. PATIENTS AND MEASUREMENTS: We surveyed 47 untreated women (ages 17-33) with various degrees of hirsutism. Serum testosterone, bioavailable testosterone, dehydroepiandrosterone sulphate, and AG were determined. Additionally, A, E and D were measured in 24-hour collections of urine. RESULTS: For the 47 women, 37 had elevated blood levels of AG (17.4 +/- 2.2, mean +/- SEM; normal < 8 nmol/l), but only 18 of these had an increased urinary A/E ratio (> 1.5). All but one of the remainder had elevated urinary and/or serum androgen levels. Overall, no significant correlation between AG and A/E was observed. There was a highly significant correlation between AG in serum and A in urine (r = 0.82, P < 0.001). AG was also positively related to dehydroepiandrosterone sulphate (r = 0.64; P < 0.005), bioavailable testosterone (r = 0.6; P < 0.001), aetiocholanolone (r = 0.58; P < 0.001) and total testosterone (r = 0.52; P < 0.01). In contrast, A/E was not significantly related to androgen production. CONCLUSIONS: There is a poor correlation between AG and the A/E ratio in hirsute women. Although AG may be raised by increased 5 alpha-reductase activity, it is probably also affected by the presence of elevated androgens regardless of 5 alpha-reductase activity.

[Potential side-effects of oral contraception: hirsutism and hypertension]. / Potenciální vedlejsí úcinky perorální antikoncepce: hirzutismus a hypertenze.

Since we observed the development of hirsutism and hypertension in a series of middle-aged women using peroral contraceptive drugs, we tried to ascertain whether there is any correlation between body mass index, blood pressure and urinary elimination of a series of steroid hormones. Although most correlations tested were statistically insignificant, we found a statistically significant correlation between the systolic blood pressure and the sum of 11 beta-hydroxyandrosterone + oxoetiocholanolone + 11 beta-hydroxyetiocholanolone urinary excretion, as assessed by thin-layer chromatography of urinary 17-oxosteroids, using dansylhydrazine as a prelabelling reagent.

Case report: acromegaly and Cushing's disease in a patient with synchronous pituitary adenomas.

A 40-year-old white woman presented with hirsutism, amenorrhea, generalized fatigue, diffuse weight gain, acral changes, and coarsened facial features. Physical examination revealed mild diastolic hypertension, acromegalic features, hirsutism, and seborrhea. The growth hormone concentration was elevated and did not suppress after glucose administration. Urinary free cortisol excretion was increased and was not suppressed during a 2 mg low-dose dexamethasone suppression test. Magnetic resonance imaging of the sella demonstrated a 1.3 x 1.2 x 0.8 cm pituitary adenoma. Trans-sphenoidal resection was performed, and portions of the resected tumor were analyzed by routine pathologic methods. Histopathologic and immunohistochemical findings indicated discrete growth hormone- and adrenocorticotropic hormone-producing pituitary adenomas. Coexisting acromegaly and Cushing's syndrome due to pituitary neoplasia was previously reported in two patients. However, to the authors' knowledge, this represents the first description of a patient with acromegaly and Cushing's disease resulting from discrete synchronous adenomas of the pituitary gland as defined by modern histopathologic techniques.

A chemiluminescent immunoassay for the direct measurement of urinary 5 alpha-androstane-3 alpha, 17 beta-diol-glucuronide in urine.

We described a chemiluminescent immunoassay (CIA) for 5 alpha-androstane-3 alpha, 17 beta-diol-glucuronide (3 alpha-diol-G) in human diluted urine. This method allowed the direct measurement in 1 microliter of urine avoiding the hydrolysis and extraction steps for sample pretreatment commonly used in routine methods. The hapten 3 alpha-diol-G was synthesized by a Koenigs-Knorr reaction. The immunogenic complex, 3 alpha-diol-G conjugated to bovine serum albumin (BSA), was employed to induce the formation of specific antibodies in New Zealand rabbits. In addition, the required chemiluminescent (CL) tracer was prepared. The characteristics of the antibody was determined as regard to specificity and sensitivity and the precision of the assay methods established. In 22 hirsute women affected by polycystic ovarian syndrome we found 3 alpha-diol-G values significantly (p less than 0.01) higher (146.28 +/- 73.77 micrograms/g of creatinine; mean +/- SD) than those observed in normal women (72.1 +/- 32.58 micrograms/g of creatinine; mean +/- SD).

Fractionation of urinary 17-ketosteroids by gas chromatography: a neglected procedure in the assessment of hyperandrogenicity in the hirsute female.

17-Ketosteroids were determined by gas chromatography in twenty four-hour urine samples from 62 hirsute females. The method permitted the determination of androsterone (A), aetiocholanolone (E) and dehydroepiandrosterone (D). Elevated concentrations of one or more of these metabolites were detected in 81% of the samples. Two main patterns of hyperandrogenicity were observed: 1) Hyper A + E (27%) and 2) Hyper A (26%). Elevated AD, AED, D, ED or E were less common, but in total these patterns comprised another 28%. The plasma testosterone and total urinary 17-ketosteroid concentrations were elevated in only 21% and 23% of the samples, respectively. Thirty two out of 33 patients with elevated urine metabolites showed significant suppression following dexamethasone administration (2 mg/day during 6 days). Thus, dexamethasone suppressable hyperandrogenicity was predominant in this group of hirsute females. Elevations of urinary androsterone and aetiocholanolone are probably contingent on the relative activities of 5 alpha- and 5 beta-reductases) in the presence of increased androstenedione secretion. Elevations of urinary dehydroepiandrosterone suggest decreased adrenal cortical 3 beta-hydroxysteroid dehydrogenase) activity. Thus, fractionation of urinary 17-ketosteroids seems to be an effective test in the evaluation of hirsutism.

Urinary 5 alpha-androstane-3 alpha,17 beta-diol levels in normal and hirsute women: discriminating power and relation to other urinary steroids.

The urinary levels of seven steroids, 5 alpha-androstane-3 alpha,17 beta-diol, 5 beta-androstane-3 alpha,17 beta-diol, androsterone, etiocholanolone, tetrahydrocortisone, tetrahydrocortisol and allotetrahydrocortisol were measured in both normal (n = 18) and hirsute (n = 24) women. The results confirmed 5 alpha-androstane-3 alpha,17 beta-diol as the most significant steroid with respect to discrimination between hirsute and normal subjects. Investigation of the inter-steroid relationships, using multivariate techniques established that the mode of steroid metabolism was different between the two groups. Whereas in normal women the strong correlation amongst all the androgen metabolites inferred a predominant hepatic route to 5 alpha-androstane-3 alpha,17 beta-diol formation, the same analogy was not applicable to the hirsute subjects. Excellent agreement was found for the predicted vs actual excretion of 5 alpha-androstane-3 alpha,17 beta-diol in normal women, based on a regression model involving the six other steroids as independent variables. When the same model was used for estimation of 5 alpha-androstane-3 alpha,17 beta-diol levels in thirteen hirsute subjects, misclassified as "normal", 50% gave values which were considerably less than actually measured. It is suggested that this discrepancy, with respect to these hirsute subjects is a reflection of extrahepatic production of 5 alpha-androstane-3 alpha,17 beta-diol due to increased 5 alpha-reductase activity.

Effect of a melatonin-free extract from bovine pineal on plasma and urinary steroids and on serum prolactin in hirsute amenorrheic adolescents.

Basal and post-suppressive dexamethasone (dxm) levels of some urinary androgen metabolites, plasma testosterone (T), 17 hydroxyprogesterone (17 OHP) and of basal serum prolactin were determined in 34 hirsute amenorrheic adolescents aged 13-17 in whom a five day course of bovine pineal extract (4 ml a day i.m.) was instituted. No convincing effect of pineal extract administration on plasma T and 17 OHP and on serum prolactin was detectable in most of pineal-treated patients. Suppressibility to dxm of plasma T and 17 OHP was not a relevant index of both the origin of androgens or the clinical course of the patients.

A solid phase chemiluminescent immunoassay (LIA) for testosterone glicuronide in diluted urines.

A chemiluminescent immunoassay (LIA) method in solid phase for the measurement of testosterone 17 beta-D-glicuronide (TG) in diluted urine is described, which utilizes as tracer a TG-isoluminol conjugate (TG-ABEI). An IgG fraction of antiserum of TG-BSA, has been passively adsorbed to the walls of polystyrene tubes. After the binding reaction the coated tubes were washed with buffer and the measure of chemiluminescence reaction was performed at high pH. The assay was validated in terms of specificity, accuracy, sensitivity and precision. The values obtained by chemiluminescence immunoassay were compared with that obtained by the RIA method, and the two methods agreed well (r = 0.95, n = 28). The assay method offers the advantage of speed and does not involve the use of radioisotopes or of a centrifugation step. Preliminary results show that the mean 24 h urinary TG excretion in a group of hirsute women is higher than in the control group, and decreases after suppression with dexamethasone for 1 month of therapy.