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Context:The classic androgen synthesis pathway proceeds via dehydroepiandrosterone, androstenedione, and testosterone to 5α-dihydrotestosterone. However, 5α-dihydrotestosterone synthesis can also be achieved by an alternative pathway originating from 17α-hydroxyprogesterone (17OHP), which accumulates in congenital adrenal hyperplasia (CAH). Similarly, recent work has highlighted androstenedione-derived 11-oxygenated 19-carbon steroids as active androgens, and in CAH, androstenedione is generated directly from 17OHP. The exact contribution of alternative pathway activity to androgen excess in CAH and its response to glucocorticoid (GC) therapy is unknown.Objective:We sought to quantify classic and alternative pathway-mediated androgen synthesis in CAH, their diurnal variation, and their response to conventional GC therapy and modified-release hydrocortisone.Methods:We used urinary steroid metabolome profiling by gas chromatography–mass spectrometry for 24-hour steroid excretion analysis, studying the impact of conventional GCs (hydrocortisone, prednisolone, and dexamethasone) in 55 adults with CAH and 60 controls. We studied diurnal variation in steroid excretion by comparing 8-hourly collections (23:00–7:00, 7:00–15:00, and 15:00–23:00) in 16 patients with CAH taking conventional GCs and during 6 months of treatment with modified-release hydrocortisone, Chronocort.Results:Patients with CAH taking conventional GCs showed low excretion of classic pathway androgen metabolites but excess excretion of the alternative pathway signature metabolites 3α,5α-17-hydroxypregnanolone and 11β-hydroxyandrosterone. Chronocort reduced 17OHP and alternative pathway metabolite excretion to near-normal levels more consistently than other GC preparations.Conclusions:Alternative pathway-mediated androgen synthesis significantly contributes to androgen excess in CAH. Chronocort therapy appears superior to conventional GC therapy in controlling androgen synthesis via alternative pathways through attenuation of their major substrate, 17OHP.We studied diurnal urinary steroid excretion in glucocorticoid-treated patients with congenital adrenal hyperplasia and found increased alternative pathway androgen synthesis that was ameliorated by modified-release hydrocortisone.

The metabolomics of urinary steroids was studied by gas chromatography-mass spectrometry in 25 patients with Cushing’s syndrome without malignant potential and in 12 patients with malignant potential of adrenal neoplasms (Weiss score 1-3). Patients with adrenocortical adenoma ( N =24) constituted the control group. In patients with Cushing’s syndrome and malignant potential, increased urinary excretion of 16-oxo-androstendiol, tetrahydro-11-deoxycortisol, and 16-hydroxypregnendiol, which had 100% specificity and sensitivity >90% for the diagnosis of malignant potential. Additionally, non-classical 5-ene-pregnenes (16-OHpregnenolone, 21-OH-pregnenolone, 3β,16,20-pregnentriol, and 3β,17,20-pregnentriol) were identified. The revealed changes in the metabolomics of steroids can be early signs of malignant potential in patients with Cushing’s syndrome. In patients with malignant potential, three signs of reduced activity of 11β-hydroxysteroid dehydrogenase type 2 were detected and in patients without malignant potential, one sign was found. In patients with and without malignant potential, three signs increased activity of 5β-reductase were found.

We use gas chromatography–mass spectrometry (GC–MS) to determine the urine peak area ratio of tetrahydrocortisol (THF) to tetrahydrodeoxycortisol (THS) in spot urine samples of eight male volunteers after a single intramuscular injection of 100 mg hydrocortisone (HC) and after a single oral administration of 10 mg HC at six different post-treatment times over 24 h with 1 week between the two treatments. Control spot urine samples were also obtained from a group of 100 volunteers of each sex for GC–MS analysis. In addition, one female volunteer was collected for GC–MS and isotope ratio mass spectrometry (IRMS) analysis after a single oral administration of 40 mg HC and 40 mg cortisone (C) at 15 and 10 different post-treatment times over 30 h, respectively. IRMS analysis focused on the acetylated derivative of 11-keto-etiocholanolone (11KE) and 11β-hydroxy-etiocholanolone (11OHE) as target metabolites, and on androsterone (A) as an endogenous reference compound (ERC) for calculating the corresponding δ 13C (‰) depletion values. There was a small but significant sex-related difference for the THF/THS ratio in the control group with mean THF/THS ratio values of 10 and 13.5 for women and men, respectively. A cut-off value of 28 (mean + 2 S.D.) for the THF/THS ratio offered a narrow detection window with 39% of suspicious samples after HC-oral treatment, and a wide detection window with 94% of suspicious samples after HC-intramuscular administration in men. For the woman the same cut-off value offered a wide detection window after HC and C administration with 100% and 90% of suspicious samples, respectively. On the basis of a cut-off value of 3‰ for the δ 13C (‰) depletion, the exogenous origin was widely evidenced for at least one target compound in 93% and 80% of the HC and C samples, respectively. We conclude by discussing the predictive ability of the urine THF/THS ratio and its usefulness in pointing out suspicious samples resulting from the systemic administration of HC and C.

In glucocorticoid-remediable aldosteronism (GRA), there is a large interfamily variation of phenotype. We report three subjects with GRA in a single family (parents, two brothers and two sisters), of whom only one (proband) displayed classical features of the mineralocorticoid excess. The proband was a man found to be hypertensive and hypokalaemic at the age of 24 years. Plasma renin activity was suppressed and plasma aldosterone was repeatedly elevated. Blood pressure and aldosterone levels normalized within 5 days of dexamethasone therapy. The presence of a chimaeric CYP11B1 / CYP11B2 gene was demonstrated by long-PCR and Southern blotting (crossover site at the end of intron 3) in the proband, in the younger sister (sibling 1) and in the father. In these patients, sequencing of the chimaeric portion of CYP11B1 did not reveal any mutation, while sequencing of the chimaeric portion of CYP11B2 showed a V386A polymorphism in exon 7, known to cause only a minimal impairment of enzymatic activity. Sibling 1 was normotensive, normokalaemic and had normal PRA and aldosterone. The father had normal blood pressure and potassium, low-normal PRA and normal aldosterone. All three subjects had elevated levels of urinary 18-hydroxycortisol and 18-oxocortisol. Baseline 11-deoxycorticosterone (DOC), corticosterone (B) and aldosterone were high in the proband and normal in the father and sibling 1; 11-deoxycortisol (S) and cortisol (F) were normal. ACTH induced a normal increase of B, DOC, S and F, and an excessive aldosterone increase in all three patients. Abnormalities in the chimaeric portions of CYB11B1 or CYP11B2 genes did not account for the phenotypic disparity of the different members in a single GRA family. Altered regulation of the chimaeric gene may be responsible for differences in its activity.

Introduction: LY3045697 is a potent and selective aldosterone synthase (CYP11B2) inhibitor that was developed as a safer alternative to mineralocorticoid receptor antagonists. Effects of LY3045697 on aldosterone and cortisol synthesis, as well as potassium ion homeostasis, were evaluated in two clinical studies in healthy subjects. Materials and methods: Two incomplete, placebo-controlled crossover-design clinical studies examined safety, pharmacodynamics, and pharmacokinetics under single and repeated dose conditions in healthy subjects. Pharmacodynamics was assessed following oral potassium challenge and intravenous adrenocorticotropic hormone procedures with spironolactone 25 mg/d as an active comparator. Results: A total of 51 subjects participated in the two studies, which included 38 males and 13 females (of non-childbearing potential), from 18–65 years old. LY3045697 caused rapid dose and concentration-dependent unstimulated plasma aldosterone concentration reduction seen as early as 4 h after the first dose at dose levels as low as 1 mg, and reaching near complete suppression at high doses. The potency (IC50) decreased significantly upon multiple dosing. After eight days of dosing, post-adrenocorticotropic hormone challenge plasma aldosterone concentration increase was dose-dependently blunted by LY3045697 with high potency with a dose as low as 0.1 mg resulting in substantial effect, and with an overall IC50 of 0.38 ng/ml. Minor reductions in cortisol were observed only at the top dose of 300 mg. LY3045697 is generally safe and tolerated, and exhibits linear pharmacokinetics. Conclusions: LY3045697 is a potent and highly selective aldosterone synthase inhibitor with selectivity for CYP11B2, offering a substantial potential advantage over previous aldosterone synthase inhibitors evaluated in the clinic.

A six-month-old 46,XY infant with a female phenotype and ambiguous genitalia was evaluated for male pseudohermaphroditism. The principal findings were (1) low basal plasma levels of all measured C 19 steroids and their sulfates, which were unchanged or only minimally increased after stimulation with human chorionic gonadotropin or ACTH, (2) no urinary metabolites of C 19 11-deoxy steroids, and decreased amounts of C 19 11-oxo steroids, (3) normal basal plasma cortisol levels and normal urinary excretion of cortisol metabolites, (4) high plasma corticosterone and deoxycorticosterone levels and elevated urinary excretion of their metabolites, (5) high plasma progesterone and pregnenolone levels and increased urinary excretion of pregnanediol and pregnenediol, (6) high plasma 17α-hydroxyprogesterone and 21-deoxycortisol levels and increased urinary excretion of pregnanetriol, 17α-hydroxypregnanolone, and pregnenetriolone, (7) high plasma and urinary levels of 5-pregnene-3β,20α-diol sulfate, (8) low plasma levels of 21-hydroxypregnenolone and 5-pregnene-3β,17α,20α-triol sulfate, (9) high plasma ACTH levels, and (10) suppression of the high plasma steroid levels by dexamethasone. The unusual pattern of plasma and urinary steroids indicated that this child had multiple abnormalities of steroid-biosynthetic microsomal mixed-function oxidases — 21-hydroxylase, 17α-hydroxylase, and 17,20 desmolase. The deficit in the activities of the first two enzymes resulted in decreased cortisol synthesis with subsequent increased ACTH secretion and adrenocortical hyperplasia. The male pseudohermaphroditism resulted from deficient testosterone synthesis due to deficiency of 17α-hydroxylase and 17,20 desmolase. The mother and two sisters of the affected child had evidence of mild 17α-hydroxylase deficiency. (N Engl J Med 1985; 313:1182–91.) FIVE enzymatic reactions are involved in the synthesis of testosterone from cholesterol, and five distinct genetic defects of impaired testosterone biosynthesis that result in incomplete masculinization of the male fetus have been described. 1 Cortisol synthesis is dependent on 20,22 desmolase (Fig. 1, Site I), 5-ene-3β-hydroxysteroid dehydrogenase, 3-oxo-4,5 isomerase (II) and 17α-hydroxylase (VI), and 21-hydroxylase (III) and 11β-hydroxylase (IV). Except for the enzyme activities at Sites III and IV, these biosynthetic reactions and 17,20 desmolase (VII) and 17β-hydroxysteroid dehydrogenase (VIII) are required for testosterone biosynthesis. We present studies of a 46,XY child with male pseudohermaphroditism due to deficient testosterone synthesis. The . . .