Juilee Rege

Liquid Chromatography–Tandem Mass Spectrometry Analysis of Human Adrenal Vein 19-Carbon Steroids Before and After ACTH Stimulation

CONTEXT A broad analysis of adrenal gland-derived 19-carbon (C19) steroids has not been reported. This is the first study that uses liquid chromatography-tandem mass spectrometry to quantify 9 C19 steroids (androgens and their precursors), estrone, and estradiol in the adrenal vein (AV) of women, before and after ACTH stimulation. OBJECTIVE The objective of this study was to define the adrenal androgen metabolome in women before and after ACTH infusion. DESIGN This was a retrospective study. PATIENTS Seven women, aged 50.4 ± 5.4 years, with suspected diagnosis of an adrenal aldosterone-producing adenoma were included in the study. METHODS AV and iliac serum samples were collected before and after administration of ACTH (15 minutes). AV samples were analyzed using for concentrations of 9 unconjugated C19 steroids, estrone, and estradiol. Dehydroepiandrosterone sulfate (DHEA-S) was quantified by radioimmunoassay. RESULTS AV levels of DHEA-S were the highest among the steroids measured. The most abundant unconjugated C19 steroids in AV were 11β-hydroxyandrostenedione (11OHA), dehydroepiandrosterone (DHEA), and androstenedione (A4). ACTH significantly increased the adrenal output of 9 of the 12 steroids that were measured. ACTH increased the mean AV concentration of DHEA-S by 5-fold, DHEA by 21-fold, A4 by 7-fold, and 11OHA by 5-fold. 11β-Hydroxytestosterone and testosterone were found to be potent androgen receptor agonists when tested with an androgen-responsive cell reporter model. CONCLUSION The current study indicates that the adrenal gland secretes primarily 3 weak androgens, namely DHEA, 11OHA, and A4. Active androgens, including testosterone and 11β-hydroxytestosterone, are also produced but to a lesser degree.

The steroid metabolome of adrenarche

Adrenarche is an endocrine developmental process whereby humans and select nonhuman primates increase adrenal output of a series of steroids, especially DHEA and DHEAS. The timing of adrenarche varies among primates, but in humans serum levels of DHEAS are seen to increase at around 6 years of age. This phenomenon corresponds with the development and expansion of the zona reticularis of the adrenal gland. The physiological phenomena that trigger the onset of adrenarche are still unknown; however, the biochemical pathways leading to this event have been elucidated in detail. There are numerous reviews examining the process of adrenarche, most of which have focused on the changes within the adrenal as well as the phenotypic results of adrenarche. This article reviews the recent and past studies that show the breadth of changes in the circulating steroid metabolome that occur during the process of adrenarche.

Profiles of 21-Carbon Steroids in 21-hydroxylase Deficiency.

CONTEXT Marked elevations of 17-hydroxyprogesterone (17OHP) are characteristic of classic 21-hydroxylase deficiency (21OHD). Testing of 17OHP provides the basis for 21OHD diagnosis, although it suffers from several pitfalls. False-positive or false-negative results and poor discrimination of nonclassic 21OHD from carriers limit the utility of serum 17OHP and necessitate dynamic testing after cosyntropin stimulation when values are indeterminate. OBJECTIVE The objective was to provide a detailed characterization of 21-carbon (C21) steroids in classic 21OHD, which might identify other candidate steroids that could be employed for the diagnosis of 21OHD. SETTING AND PARTICIPANTS Patients (11 women, 10 men) with classic 21OHD and 21 sex- and age-matched controls seen in a tertiary referral center were studied. METHODS C21 steroids in the peripheral sera from all subjects, as well as in media from cultured testicular adrenal rest tumor (TART) cells and normal adrenal (NA) cells, were analyzed using liquid chromatography/tandem mass spectrometry (10 steroids). Additionally, the dynamics of C21 steroid metabolism in TART and NA cells were assessed with radiotracer studies. RESULTS Five C21 steroids were significantly higher in 21OHD patients: 17OHP (67-fold; P < .01), 21-deoxycortisol (21dF; 35-fold; P < .01), 16α-hydroxyprogesterone (16OHP; 28-fold; P < .01), progesterone (2-fold; P < .01), and 11β-hydroxyprogesterone (11OHP; not detected in controls; P < .01). The same steroids were the highest in media from TART cells relative to the NA cells: 11OHP, 58- to 65-fold; 21dF, 30- to 41-fold; 17OHP, 9-fold; progesterone, 9- to 12-fold; and 16OHP, 7-fold. CONCLUSION Measurement of 16OHP and 11OHP along with 17OHP and 21dF by liquid chromatography/tandem mass spectrometry might comprise a biomarker panel to accurately diagnose all forms of 21OHD.

Transcriptome profiling reveals differentially expressed transcripts between the human adrenal zona fasciculata and zona reticularis.

CONTEXT The human adrenal zona fasciculata (ZF) and zona reticularis (ZR) are responsible for the production of cortisol and 19-carbon steroids (often called adrenal androgens), respectively. However, the gene profiles and exact molecular mechanisms leading to the functional phenotype of the ZF and ZR are still not clearly defined. In the present study, we identified the transcripts that are differentially expressed in the ZF and ZR. OBJECTIVE The objective of the study was to compare the transcriptome profiles of ZF and ZR. DESIGN AND METHODS ZF and ZR were microdissected from 10 human adrenals. Total RNA was extracted from 10 ZF/ZR pairs and hybridized to Illumina microarray chips. The 10 most differentially expressed transcripts were studied with quantitative RT-PCR (qPCR). Immunohistochemistry was also performed on four zone-specific genes. RESULTS Microarray results demonstrated that only 347 transcripts of the 47 231 were significantly different by 2-fold or greater in the ZF and ZR. ZF had 195 transcripts with 2-fold or greater increase compared with its paired ZR, whereas ZR was found to have 152 transcripts with 2-fold or greater higher expression than in ZF. Microarray and qPCR analysis of transcripts encoding steroidogenic enzymes (n = 10) demonstrated that only 3β-hydroxysteroid dehydrogenase, steroid sulfotransferase, type 5 17β-hydroxysteroid dehydrogenase, and cytochrome b5 were significantly different. Immunohistochemistry and qPCR studies confirmed that the ZF had an increased expression of lymphoid enhancer-binding factor 1 and nephroblastoma overexpressed, whereas ZR showed an increased expression of solute carrier family 27 (fatty acid transporter) (SLC27A2), member 2 and TSPAN12 (tetraspanin 12) CONCLUSION: Microarray revealed several novel candidate genes for elucidating the molecular mechanisms governing the ZF and ZR, thereby increasing our understanding of the functional zonation of these two adrenocortical zones.

Clinical, Biochemical, and Molecular Characterization of Macronodular Adrenocortical Hyperplasia of the Zona Reticularis: A New Syndrome

CONTEXT Macronodular adrenocortical hyperplasia classically presents with progressive hypercortisolemia and Cushing syndrome. We describe a 29-yr-old man with massive macronodular adrenocortical hyperplasia without hypercortisolemia but rather markedly elevated and nonsuppressible production of dehydroepiandrosterone (DHEA) and its sulfate (DHEAS). OBJECTIVE To characterize the clinical and molecular features of this case and to determine whether the tissue biochemically resembles the zona reticularis or fetal adrenal. SETTING University clinic, hospital, and laboratories. DESIGN Static and dynamic blood and urine testing were performed preoperatively. Tissue was studied by light microscopy, immunoblot, RNA microarray, and enzyme assay. PARTICIPANT A 29-yr-old man with incidentally discovered bilateral adrenal enlargement. INTERVENTION Bilateral adrenalectomy. MAIN OUTCOME MEASURES Molecular studies compared with control samples. RESULTS Hypercortisolism and 21-hydroxylase deficiency were excluded. DHEA, DHEAS, and 17-hydroxypregnenolone were markedly elevated and did not suppress with dexamethasone 2 mg/d for 4 d. Homogenates of the adrenals demonstrated high 17-hydroxylase, good 17,20-lyase, and low or absent 21-hydroxylase and 3β-hydroxysteroid dehydrogenase activities. Immunoblots confirmed robust expression of cytochrome P450c17 and AKR1C3 but not P450c21. Microarray analysis demonstrated high CYP11A1 and CYP17A1 expression but low or absent HSD3B1, HSD3B2, and CYP21A2 expression. Expression of mRNA for cytochrome b(5) (CYB5A) and AKR1C3, markers of the zona reticularis, were markedly elevated. CONCLUSION This is the first case of macronodular hyperplasia of the adrenal zona reticularis confirmed with studies of enzyme activity, mRNA expression, and protein identification. We speculate that this condition can be clinically silent in men but might cause severe hyperandrogenemia in women.

Liquid chromatography-tandem mass spectrometry analysis of human adrenal vein corticosteroids before and after adrenocorticotropic hormone stimulation.

Context  Although steroid hormones produced by the adrenal gland play critical roles in human physiology, a detailed quantitative analysis of the steroid products has not been reported. The current study uses a single methodology (liquid chromatography–tandem mass spectrometry, LC‐MS/MS) to quantify ten corticosteroids in adrenal vein (AV) samples pre‐ and post‐adrenocorticotropic hormone (ACTH) stimulation.

Cytochrome b5 Activates the 17,20-Lyase Activity of Human Cytochrome P450 17A1 by Increasing the Coupling of NADPH Consumption to Androgen Production

Human cytochrome P450 17A1 is required for all androgen biosynthesis and is the target of abiraterone, a drug used widely to treat advanced prostate cancer. P450 17A1 catalyzes both 17-hydroxylation and subsequent 17,20-lyase reactions with pregnenolone, progesterone, and allopregnanolone. The presence of cytochrome b5 (b5) markedly stimulates the 17,20-lyase reaction, with little effect on 17-hydroxylation; however, the mechanism of this b5 effect is not known. We determined the influence of b5 on coupling efficiency-defined as the ratio of product formation to NADPH consumption-in a reconstituted system using these 3 pairs of substrates for the 2 reactions. Rates of NADPH consumption ranged from 4 to 13 nmol/min/nmol P450 with wild-type P450 17A1. For the 17-hydroxylase reaction, progesterone oxidation was the most tightly coupled (∼50%) and negligibly changed upon addition of b5. Rates of NADPH consumption were similar for the 17-hydroxylase and corresponding 17,20-lyase reactions for each steroid series, and b5 only slightly increased NADPH consumption. For the 17,20-lyase reactions, b5 markedly increased product formation and coupling in parallel with all substrates, from 6% to 44% with the major substrate 17-hydroxypregnenolone. For the naturally occurring P450 17A1 mutations E305G and R347H, which impair 17,20-lyase activity, b5 failed to rescue the poor coupling with 17-hydroxypregnenolone (2-4%). When the conserved active-site threonine was mutated to alanine (T306A), both the activity and coupling were markedly decreased with all substrates. We conclude that b5 stimulation of the 17,20-lyase reaction primarily derives from more efficient use of NADPH for product formation rather than side products.

Bone Morphogenetic Protein-4 (BMP4): A Paracrine Regulator of Human Adrenal C19 Steroid Synthesis.

Bone morphogenetic proteins (BMPs) comprise one of the largest subgroups in the TGF-β ligand superfamily. We have identified a functional BMP system equipped with the ligand (BMP4), receptors (BMP type II receptor, BMP type IA receptor, also called ALK3) and the signaling proteins, namely the mothers against decapentaplegic homologs 1, 4, and 5 in the human adrenal gland and the human adrenocortical cell line H295R. Microarray, quantitative RT-PCR, and immunohistochemistry confirmed that BMP4 expression was highest in the adrenal zona glomerulosa followed by the zona fasciculata and zona reticularis. Treatment of H295R cells with BMP4 caused phosphorylation of the mothers against decapentaplegic and a profound decrease in synthesis of the C19 steroids dehydroepiandrosterone (DHEA), DHEA sulfate, and androstenedione. Administration of BMP4 to cultures of H295R cells also caused a profound decrease in the mRNA and protein levels of 17α-hydroxylase/17,20-lyase (CYP17A1 and P450c17, respectively) but no significant effect on the mRNA levels of cholesterol side-chain cleavage cytochrome P450 (CYP11A1) or type 2 3β-hydroxysteroid dehydrogenase (HSD3B2). Furthermore, Noggin (a BMP inhibitor) was able to reverse the negative effects of BMP4 with respect to both CYP17A1 transcription and DHEA secretion in the H295R cell line. Collectively the present data suggest that BMP4 is an autocrine/paracrine negative regulator of C19 steroid synthesis in the human adrenal and works by suppressing P450c17.

Adrenocorticotropin Acutely Regulates Pregnenolone Sulfate Production by the Human Adrenal In Vivo and In Vitro

Background Dehydroepiandrosterone sulfate (DHEAS) is the most abundant steroid in human circulation, and adrenocorticotropic hormone (ACTH) is considered the major regulator of its synthesis. Pregnenolone sulfate (PregS) and 5-androstenediol-3-sulfate (AdiolS) have recently emerged as biomarkers of adrenal disorders. Objective To define the relative human adrenal production of Δ5-steroid sulfates under basal and cosyntropin-stimulated conditions. Methods Liquid chromatography-tandem mass spectrometry was used to quantify three unconjugated and four sulfated Δ5-steroids in (1) paired adrenal vein (AV) and mixed venous serum samples (21 patients) and (2) cultured human adrenal cells both before and after cosyntropin stimulation, (3) microdissected zona fasciculata (ZF) and zona reticularis (ZR) from five human adrenal glands, and (4) a reconstituted in vitro human 17α-hydroxylase/17,20-lyase/(P450 17A1) system. Results Of the steroid sulfates, PregS had the greatest increase after cosyntropin stimulation in the AV (32-fold), whereas DHEAS responded modestly (1.8-fold). PregS attained concentrations comparable to those of DHEAS in the AV after cosyntropin stimulation (AV DHEAS/PregS, 24 and 1.3 before and after cosyntropin, respectively). In cultured adrenal cells, PregS demonstrated the sharpest response to cosyntropin, whereas DHEAS responded only modestly (21-fold vs 1.8-fold higher compared with unstimulated cells at 3 hours, respectively). Steroid analyses in isolated ZF and ZR showed similar amounts of PregS and 17α-hydroxypregnenolone in both zones, whereas DHEAS and AdiolS were higher in ZR (P < 0.05). Conclusion Our studies demonstrated that unlike DHEAS, PregS displayed a prominent acute response to cosyntropin. PregS could be used to interrogate the acute adrenal response to ACTH stimulation and as a biomarker in various adrenal disorders.

11-ketotestosterone is the dominant circulating bioactive androgen during normal and premature adrenarche.

Context Adrenarche refers to the rise of dehydroepiandrosterone sulfate (DHEA-S) associated with the development of a functional adrenal zona reticularis. Clinical features of adrenarche include onset of body odor, axillary hair, and pubic hair, which reflect increased androgen action. An early rise in adrenal androgens, or premature adrenarche (PremA), is a risk factor for adverse metabolic profiles in adolescence and adulthood. The bioactive androgens associated with adrenarche and PremA remain poorly understood. The adrenal gland is a potential source of testosterone (T) and the 11-oxygenated derivatives 11β-hydroxytestosterone (11OHT) and 11-ketotestosterone (11KT). Objective The objective of this study was to characterize the adrenal androgen biome contributing to adrenarche and PremA. Participants and Methods With the use of mass spectrometry, 19 steroids including the 11-oxygenated derivatives of T were measured in sera obtained from girls with PremA (n = 37; 4 to 7 years) and age-matched girls (n = 83; 4 to 10 years). Results In reference population girls, dehydroepiandrosterone, DHEA-S, androstenediol-3-sulfate, T, and 11KT all increased at the onset of adrenarche (6 to 8 years) and beyond (9 to 10 years) (P < 0.05 vs younger subjects 4 to 5 years). T, 11OHT, and 11KT were further elevated in PremA vs age-matched girls (P < 0.001). Circulating concentrations of 11KT during adrenarche and PremA exceeded those of T and 11OHT (11KT > T ≥ 11OHT). Androgen receptor activity and nuclear translocation studies demonstrated that 11KT is a potent androgen similar to T. Conclusions Our findings suggest that 11KT is the dominant bioactive androgen in children during adrenarche and PremA. Its androgenic capacity suggests that it may be responsible for the phenotypic changes seen in these phenomena.