Assessment of adrenal function at birth using adrenal glucocorticoid precursor to product ratios to predict short-term neonatal outcomes.

BACKGROUND: Most neonatal outcomes in neonates are related to normal adrenal gland function. Assessment of adrenal function in a sick preterm neonate remains a challenge, thus we hypothesized that adrenal steroid precursors to their product ratios have a direct relationship with neonatal outcomes. METHODS: We studied demographics of pregnancy and neonatal outcomes in 99 mother-infant pairs (24-41 weeks) and assayed 7 glucocorticoid precursors in the cortisol biosynthesis/degradation pathway. We correlated antenatal factors and short-term neonatal outcomes with these precursors and their ratios to assess maturity of individual enzymes. RESULTS: We found no correlation between cortisol levels with antenatal factors and outcomes. Antenatal steroid use impacted several cortisol precursors. 17-OH pregnenolone-to-cortisol ratio at birth was the best predictor of short-term neonatal outcomes, such as hypotension, RDS, IVH and PDA. A cord blood 17-OH pregnenolone:cortisol ratio of <0.21 predicts which neonate will have a normal outcome with a high sensitivity and specificity. CONCLUSIONS: Maternal factors and antenatal steroids impact neonatal adrenal function and leads to maturation of adrenal function. 17-OH pregnenolone:cortisol ratio and not cortisol is the best predictor of adrenal function. Adrenal function can be assessed by evaluating the profile of adrenal steroids.

Female and male serum reference intervals for challenging sex and precursor steroids by liquid chromatography - tandem mass spectrometry.

Measuring some sex and precursor steroids is still challenging even by liquid chromatography - tandem mass spectrometry (LC-MS/MS), and few normal values are available. We developed a LC-MS/MS method for estradiol, estrone, dihydrotestosterone and 17-hydroxypregnenolone measurement, compared it with direct immunoassays, and generated sex, age, menopausal and menstrual status specific reference intervals. Liquid-liquid extraction was optimized on 300 µL serum spiked with isotopic internal standards. A 2D-LC system allowed on-line purification and separation in 11 min run. Electrospray ionization was enhanced by ammonium fluoride. MS-detection was obtained by multiple reaction monitoring. Direct ECLIA for estradiol (n = 80) and RIA for estrone (n = 41) were compared with LC-MS/MS. Reference values were estimated in healthy, lean women in reproductive age (n = 118), menopausal women (n = 33) and men (n = 159). The assay showed satisfying imprecision, trueness, recovery and selectivity. Adequate functional sensitivity was achieved for measuring estrone (18.1 pmol/L) and 17-hydroxypregnenolone (117 pmol/L) in all subjects, and estradiol (35.9 pmol/L) and dihydrotestosterone (134 pmol/L) in women in reproductive age and men, but not in menopausal women. Compared with LC-MS/MS, immunoassays showed good agreement for estradiol but severe disagreement for estrone. Estrogens exhibited sex, menopausal and menstrual variations. Dihydrotestosterone and 17-hydroxypregnenolone depended on sex and menopause, the latter also declining with age in men. Strictly defined reference intervals in the adult female and male population were generated for challenging steroids such as estrogens, dihydrotestosterone and 17-hydroxypregnenolone by a novel LC-MS/MS method. Our achievement can be used to deepen the comprehension of several endocrine diseases.

Cortisol precursors in neonates with vasopressor-resistant hypotension in relationship to demographic characteristics.

OBJECTIVE: To correlate between cortisol precursors in neonates with vasopressor resistant hypotension and demographic characteristics. METHODS: We investigated 48 neonates with vasopressor-resistant hypotension. Gestation at birth ranged from 34 to 42 weeks and postnatal age from 4 to 14 days. Cortisol and precursor steroids were measured soon after the onset of volume expansion and inotropes for treatment of shock. Their concentrations were determined using liquid chromatography/mass spectrometry. RESULTS: In neonates with vasopressor-resistant hypotension, the serum levels of cortisol were within normal nonstress range. There was a strong negative linear association between postnatal age and dehydroepiandrosterone level (r = -0.50, p < .01), which decreased with neonatal age. In addition, there was a significant positive association between gestational age at birth and 17-hydroxy-pregnenolone (r = 0.33, p = .02). No further significant associations were evident between the neonatal weight, duration of gestation or gender and of the levels of cortisol or the other steroids (p > .05). The cause of therapy-resistant hypotension did not appear to influence the steroid levels. CONCLUSIONS: Cortisol stress response is absent in these severely ill late preterm and term infants. This may be due to inhibition of the distal pathway of cortisol synthesis.

Initial genetic dissection of serum neuroactive steroids following chronic intermittent ethanol across BXD mouse strains.

Neuroactive steroids modulate alcohol's impact on brain function and behavior. Ethanol exposure alters neuroactive steroid levels in rats, humans, and some mouse strains. We conducted an exploratory analysis of the neuroactive steroids (3α,5α)-3-hydroxypregnan-20-one (3α,5α-THP), (3α,5α)-3,21-dihydroxypregnan-20-one (3α,5α-THDOC), and pregnenolone across 126-158 individuals and 19 fully inbred strains belonging to the BXD family, which were subjected to air exposure, or chronic intermittent ethanol (CIE) exposure. Neuroactive steroids were measured by gas chromatography-mass spectrometry in serum following five cycles of CIE or air exposure (CTL). Pregnenolone levels in CTLs range from 272 to 578 pg/mL (strain variation of 2.1 fold with p = 0.049 for strain main effect), with heritability of 0.20 ± 0.006 (SEM), whereas in CIE cases values range from 304 to 919 pg/mL (3.0-fold variation, p = 0.007), with heritability of 0.23 ± 0.005. 3α,5α-THP levels in CTLs range from 375 to 1055 pg/mL (2.8-fold variation, p = 0.0007), with heritability of 0.28 ± 0.01; in CIE cases they range from 460 to 1022 pg/mL (2.2-fold variation, p = 0.004), with heritability of 0.23 ± 0.005. 3α,5α-THDOC levels in CTLs range from 94 to 448 pg/mL (4.8-fold variation, p = 0.002), with heritability of 0.30 ± 0.01, whereas levels in CIE cases do not differ significantly. However, global averages across all BXD strains do not differ between CTL and CIE for any of the steroids. 3α,5α-THDOC levels were lower in females than males in both groups (CTL -53%, CIE -55%, p < 0.001). Suggestive quantitative trait loci are identified for pregnenolone and 3α,5α-THP levels. Genetic variation in 3α,5α-THP was not correlated with two-bottle choice ethanol consumption in CTL or CIE-exposed animals. However, individual variation in 3α,5α-THP correlated negatively with ethanol consumption in both groups. Moreover, strain variation in neuroactive steroid levels correlated with numerous behavioral phenotypes of anxiety sensitivity accessed in GeneNetwork, consistent with evidence that neuroactive steroids modulate anxiety-like behavior.

Age-dependent Increases in Adrenal Cytochrome b5 and Serum 5-Androstenediol-3-sulfate.

CONTEXT: Adrenal production of dehydroepiandrosterone sulfate (DHEA-S) increases throughout childhood owing to expansion of the zona reticularis (ZR). ZR features cells with a steroidogenic phenotype distinct from that of the adjacent zona fasciculata, with higher expression of cytochrome b5 type A (CYB5A) and steroid sulfotransferase type 2A1 but decreased 3ß-hydroxysteroid dehydrogenase type 2 (HSD3B2). In addition to DHEA-S, three adrenal Δ5-steroid sulfates could provide additional tools to define adrenal maturation. OBJECTIVE: This study sought to simultaneously measure serum levels of four adrenal Δ5-steroid sulfates, pregnenolone sulfate (Preg-S), 17α-hydroxypregnenolone sulfate (17OHPreg-S), DHEA-S, and 5-androstenediol-3-sulfate (Adiol-S) as a function of age and relate their production to the age-dependent adrenal localization of CYB5A. PARTICIPANTS AND METHODS: Δ5-steroid sulfates were quantified by liquid chromatography-tandem mass spectrometry in sera from 247 normal children (129 males,118 females) age 1.5-18 y and 42 adults (20 males, 22 females). Immunofluorescence localized HSD3B2 and CYB5A in normal adrenal glands from subjects age 2-35 y. Finally, HAC15 adrenocortical cells were transduced with lentiviral short hairpin RNA to suppress CYB5A expression. RESULTS: Of the Δ5-steroid sulfates quantified, DHEA-S was most abundant. Adiol-S increased in parallel with DHEA-S. Steroid ratios (17OHPreg-S/DHEA-S) suggested increases in 17,20-lyase activity during childhood. Immunofluorescence analysis showed age-related increases in ZR CYB5A immunoreactivity. Furthermore, silencing CYB5A in HAC15 adrenocortical cells significantly reduced DHEA-S and Adiol-S production. CONCLUSION: Adiol-S shows a similar age-related increase to that of DHEA-S. This likely results from the childhood expansion of CYB5A-expressing ZR, which enhances 17,20-lyase activity and the production of DHEA-S and Adiol-S.

Body Fat Mass Is Associated With Ratio of Steroid Metabolites Reflecting 17,20-Lyase Activity in Prepubertal Girls.

CONTEXT: Pediatric obesity has been related to hyperandrogenism and premature adrenarche in previous studies. However, little is known regarding the association between body fat mass and steroidogenic enzyme activities in children. OBJECTIVE: To examine whether body fat mass is associated with serum steroid profiles in girls. DESIGN, PARTICIPANTS, AND SETTING: We enrolled 242 girls (125 prepubertal, 117 pubertal; age, 7-13 years). Early morning blood samples were drawn at a university hospital to measure serum steroid profiles using gas chromatography-mass spectrometry, and steroidogenic enzyme activities were assessed from the ratios of steroid metabolites. MAIN OUTCOME MEASURES: We evaluated serum steroid profiles and estimated steroidogenic enzyme activities and their association with anthropometric indices and body composition. RESULTS: Prepubertal obese girls demonstrated significantly higher progestin, androgens (dehydroepiandrosterone [DHEA], androstenedione [A-dione], T, androsterone), and ratio of steroid metabolites reflecting 17,20-lyase activity [(DHEA + A-dione)/17-hydroxypregnenolone] compared with prepubertal controls. Pubertal obese girls demonstrated significantly higher serum T and androsterone than pubertal controls; however, serum steroid metabolite ratios reflecting steroidogenic enzyme activities did not significantly differ among obese and non-obese girls. Partial correlation analysis revealed that body fat mass was positively correlated with pregnenolone, DHEA, A-dione, T, androsterone, and ratio of (DHEA + A-dione)/17-hydroxypregnenolone in prepubertal girls only. Prepubertal girls with increased body fat mass had significantly higher serum DHEA and ratio of (DHEA + A-dione)/17-hydroxypregnenolone than controls. CONCLUSIONS: Increased androgen production in prepubertal obese girls could be at least partly due to increased body fat mass and 17,20-lyase activity.

A Comprehensive Evaluation of Steroid Metabolism in Women with Intrahepatic Cholestasis of Pregnancy.

Intrahepatic cholestasis of pregnancy (ICP) is a common liver disorder, mostly occurring in the third trimester. ICP is defined as an elevation of serum bile acids, typically accompanied by pruritus and elevated activities of liver aminotransferases. ICP is caused by impaired biliary lipid secretion, in which endogenous steroids may play a key role. Although ICP is benign for the pregnant woman, it may be harmful for the fetus. We evaluated the differences between maternal circulating steroids measured by RIA (17-hydroxypregnenolone and its sulfate, 17-hydroxyprogesterone, and cortisol) and GC-MS (additional steroids), hepatic aminotransferases and bilirubin in women with ICP (n = 15, total bile acids (TBA) >8 µM) and corresponding controls (n = 17). An age-adjusted linear model, receiver-operating characteristics (ROC), and multivariate regression (a method of orthogonal projections to latent structure, OPLS) were used for data evaluation. While aminotransferases, conjugates of pregnanediols, 17-hydroxypregnenolone and 5ß-androstane-3α,17ß-diol were higher in ICP patients, 20α-dihydropregnenolone, 16α-hydroxy-steroids, sulfated 17-oxo-C19-steroids, and 5ß-reduced steroids were lower. The OPLS model including steroids measured by GC-MS and RIA showed 93.3% sensitivity and 100% specificity, while the model including steroids measured by GC-MS in a single sample aliquot showed 93.3% sensitivity and 94.1% specificity. A composite index including ratios of sulfated 3α/ß-hydroxy-5α/ß-androstane-17-ones to conjugated 5α/ß-pregnane-3α/ß, 20α-diols discriminated with 93.3% specificity and 81.3% sensitivity (ROC analysis). These new data demonstrating altered steroidogenesis in ICP patients offer more detailed pathophysiological insights into the role of steroids in the development of ICP.

Evidence for gonadotrophin secretory and steroidogenic abnormalities in brothers of women with polycystic ovary syndrome.

STUDY QUESTION: Are there abnormalities in gonadotrophin secretion, adrenal steroidogenesis and/or testicular steroidogenesis in brothers of women with polycystic ovary syndrome (PCOS)? SUMMARY ANSWER: Brothers of women with PCOS have increased gonadotrophin responses to gonadotrophin releasing hormone (GnRH) agonist stimulation and alterations in adrenal and gonadal steroidogenesis. WHAT IS KNOWN ALREADY: PCOS is a complex genetic disease. Male as well as female first-degree relatives have reproductive features of the syndrome. We previously reported that brothers of affected women have elevated circulating dehydroepiandrosterone sulfate levels. STUDY DESIGN, SIZE, DURATION: This was a case-control study performed in 29 non-Hispanic white brothers of 22 women with PCOS and 18 control men. PARTICIPANTS/MATERIALS, SETTING, METHODS: PCOS brothers and control men were of comparable age, weight and ethnicity. Adrenocorticotrophic hormone (ACTH) and GnRH agonist stimulation tests were performed. Gonadotrophin responses to GnRH agonist as well as changes in precursor-product steroid pairs (delta, Δ) across steroidogenic pathways in response to ACTH and GnRH agonist were examined. MAIN RESULTS AND THE ROLE OF CHANCE: Basal total (T) levels did not differ, but dehydroepiandrosterone (DHEA) levels (0.13 ± 0.08 brothers versus 0.22 ± 0.09 controls, nmol/l, P = 0.03) were lower in brothers compared with control men. ACTH-stimulated Δ17-hydroxypregnenolone (17Preg)/Δ17-hydroxyprogesterone (17Prog) (7.8 ± 24.2 brothers versus 18.9 ± 21.3 controls, P = 0.04) and ΔDHEA/Δandrostenedione (AD) (0.10 ± 0.05 brothers versus 0.14 ± 0.08 controls, P = 0.04) were lower in brothers than in the controls. GnRH agonist-stimulated Δ17Prog/ΔAD (0.28 ± 8.47 brothers versus 4.79 ± 10.28 controls, P = 0.003) was decreased and luteinizing hormone (38.6 ± 20.6 brothers versus 26.0 ± 9.8 controls, IU/l, P = 0.02), follicle-stimulating hormone (10.2 ± 7.5 brothers versus 4.8 ± 4.1 controls, IU/l P = 0.002), AD (1.7 ± 1.4 brothers versus 0.9 ± 1.5 controls, nmol/l, P = 0.02) and ΔAD/ΔT (0.16 ± 0.14 brothers versus 0.08 ± 0.12 controls, P = 0.005) responses were increased in brothers compared with controls. LIMITATIONS, REASONS FOR CAUTION: The modest sample size may have limited our ability to observe other possible differences in steroidogenesis between PCOS brothers and control men. WIDER IMPLICATIONS OF THE FINDINGS: Decreased ACTH-stimulated Δ17Preg/Δ17Prog and ΔDHEA/ΔAD responses suggested increased adrenal 3ß-hydroxysteroid dehydrogenase activity in the brothers. Decreased Δ17Prog/ΔAD and increased ΔAD/ΔT responses to GnRH agonist stimulation suggested increased gonadal 17,20-lyase and decreased gonadal 17ß-hydroxysteroid dehydrogenase activity in the brothers. Increased LH and FSH responses to GnRH agonist stimulation suggested neuroendocrine alterations in the regulation of gonadotrophin secretion similar to those in their proband sisters. These changes in PCOS brothers may reflect the impact of PCOS susceptibility genes and/or programming effects of the intrauterine environment. STUDY FUNDING/COMPETING INTERESTS: This research was supported by P50 HD044405 (A.D.), K12 HD055884 (L.C.T.), U54 HD034449 (A.D., R.S.L.) from the National Institute of Child Health and Development. Some hormone assays were performed at the University of Virginia Center for Research in Reproduction Ligand Assay and Analysis Core that is supported by U54 HD28934 from the Eunice Kennedy Shriver National Institute of Child Health and Human Development. Partial support for some of the clinical studies was provided by UL1 RR025741 and UL1 TR000150 (Northwestern University Clinical and Translational Sciences Institute) from the National Center for Research Resources, National Institutes of Health, which is now the National Center for Advancing Translational Sciences. The authors have no conflict of interest to declare.

3ß-hydroxysteroid dehydrogenase type II deficiency on newborn screening test.

3ß-hydroxysteroid dehydrogenase II (3ß-HSD) deficiency represents a rare CAH variant. Newborns affected with its classic form have salt wasting in early infancy and genital ambiguity in both sexes. High levels of 17-hydroxypregnenolone (Δ517OHP) are characteristic, but extra-adrenal conversion to 17-hydroxyprogesterone (17OHP) may lead to positive results on newborn screening tests. Filter paper 17OHP on newborn screening test was performed by immunofluorometric assay, and serum determinations of 17OHP and Δ517OHP, by radioimmunoassay. A 46,XY infant with genital ambiguity and adrenal crisis at three months of age presented a positive result on newborn screening for CAH. Serum determinations of 17OHP and Δ517OHP were elevated, and a high Δ517OHP/cortisol relation was compatible with the diagnosis of 3ß-HSD deficiency. Molecular analysis of the HSD3B2 gene from the affected case revealed the presence of the homozygous p.P222Q mutation, whereas his parents were heterozygous for it. We present the first report of 3ß-HSD type II deficiency genotype-proven detected at the Newborn Screening Program in Brazil. The case described herein corroborates the strong genotype-phenotype correlation associated with the HSD3B2 p.P222Q mutation, which leads to a classic salt-wasting 3ß-HSD deficiency. Further evaluation of 17OHP assays used in newborn screening tests would aid in determining their reproducibility, as well as the potential significance of moderately elevated 17OHP levels as an early indicator to the diagnosis of other forms of classic CAH, beyond 21-hydroxylase deficiency.

Adrenal enzyme impairment in neonates and adolescents treated with ritonavir and protease inhibitors for HIV exposure or infection.

BACKGROUND: Human deficiency virus (HIV) protease inhibitors (PIs) are widely used drugs whose effects are pharmacologically enhanced by ritonavir, a potent cytochrome P450 inhibitor. We reported previously that prophylactic postnatal ritonavir-PI therapy in HIV-exposed neonates was associated with increases in plasma 17-hydroxyprogesterone (17-OHP) and dehydroepiandrosterone sulfate (DHEA-S). AIMS: To further investigate adrenal function in neonates and adolescents given ritonavir-PI. METHODS: Adrenal function was assessed prospectively in 3 HIV-exposed neonates given short-term prophylactic treatment and 3 HIV-infected adolescents given long-term treatment. Plasma cortisol, 17-OHP, 17-OH-pregnenolone, DHEA-S, and androstenedione were measured before and after ACTH administration. RESULTS: None of the patients had clinical signs of adrenal dysfunction. The only neonate exposed to ritonavir-PI in utero had up to 3-fold increases in plasma 17-OHP. Increases in 17-OH-pregnenolone of up to 3.1-fold were noted in 4 of the 6 patients, and all 6 patients had elevations in DHEA-S (up to 20.4-fold increase) and/or DHEA (up to 4.7-fold) and/or androstenedione (up to 5.2-fold). All these parameters improved after treatment completion. CONCLUSION: Neonates and adolescents given ritonavir-PI exhibit a similar adrenal dysfunction profile consistent with an impact on multiple adrenal enzymes. These abnormalities require evaluation, given the potentially long exposure times.

The environmental regulation of maturation in goldfish, Carassius auratus: effects of various LED light spectra.

While there have been a number of studies on the effects of photoperiod and duration of light and dark exposure, much less information is available on the importance of light intensity. This study investigated the effects of exposure of goldfish, Carassius auratus exposed to white fluorescent bulbs, and red (peak at 630nm), and green (530nm) light emitting diodes (LEDs) at approximately 0.9W/m(2) (12-h light:12-h dark) for four months on a number of hormones of the hypothalamus-pituitary-gonad (HPG) axis, in vivo and in vitro. We investigated the effects of native GnRH molecules (gonadotropin-releasing hormones; salmon GnRH, sGnRH; and chicken GnRH-II, cGnRH-II), gonadotropin hormones (GTHα; follicle-stimulating hormone, FSH-ß; luteinizing hormone, LH-ß2), kisspeptin 1 (Kiss1) and G protein-coupled receptor 54 (GPR54) mRNA levels. Furthermore, we measured LH and 17α-hydroxypregnenolone levels in plasma and we performed gonad histological observations. GnRHs, Kiss1, GPR54 and GTH mRNA and plasma LH and 17α-hydroxypregnenolone levels in the in vivo and in vitro groups exposed to green LEDs were significantly higher than the other groups. Histological analysis revealed the presence of oocytes in the yolk stage in fish exposed to green light. These results suggest that green wavelengths regulate the HPG axis and enhance sexual maturation in goldfish.

Kisspeptin regulates the hypothalamus-pituitary-gonad axis gene expression during sexual maturation in the cinnamon clownfish, Amphiprion melanopus.

Kisspeptins (Kiss) have been recognized as potent regulators of reproduction in teleosts, and Kiss is suggested to be a key regulator of the hypothalamus-pituitary-gonad axis (HPG). However, its regulatory role on reproduction in fish remains unclear. Therefore, to investigate the role of Kiss on fish reproduction, this study aimed to test differences in the hormones of the HPG axis, Kiss as neuropeptides, and sex steroids on the sexual maturation of paired cinnamon clownfish, Amphiprion melanopus, following treatment with Kiss. We investigated the actions of sex maturation hormones, including HPG axis hormones and sex steroid hormones, such as gonadotropin-releasing hormones, gonadotropin hormones (GTHs), GTH receptors, estrogen receptors, and vitellogenin in the pituitary, gonads, and liver following treatment with Kiss. The expression levels of HPG axis genes increased after the Kiss injection. In addition, the levels of plasma 17α-hydroxypregnenolone, estradiol-17ß, and 11-ketotestosterone increased. These results support the hypothesis that Kiss play important roles in the regulation of the HPG axis and are most likely involved in gonadal development and sexual maturation in cinnamon clownfish.

Ashwagandha root in the treatment of non-classical adrenal hyperplasia.

Congenital adrenal hyperplasia (CAH) is a well-characterised family of disorders of the adrenal cortices, resulting in varying degrees of cortisol, aldosterone and androgen deficiency or androgen excess, depending on the enzyme(s) affected and the degree of quantitative or functional enzyme deficit. Withania somnifera (WS), commonly known as Ashwagandha, is a medicinal plant that has been employed for centuries in ayurvedic medicine. Preclinical studies have shown that WS increases circulating cortisol levels and improves insulin sensitivity. We report the case of a 57-year-old woman with non-classical adrenal hyperplasia due to both 3-ß-ol dehydrogenase deficiency and aldosterone synthase deficiency who was self-treated with WS for 6 months. After 6 months of treatment her serum 18-OH-hydroxycorticoserone, 17-OH-pregnenolone, corticosterone and 11-deoxycortisol decreased by 31%, 66%, 69% and 55%, respectively. The biochemical improvement was accompanied by a noticeable reduction in scalp hair loss.

Differences in stimulated androgen levels in black and white obese adolescent females.

STUDY OBJECTIVE: Little is known about racial differences in androgen levels among obese children. The objective of this pilot study was to compare basal and stimulated androgen levels in a cross-sectional sample of obese black and white pubertal females. STUDY DESIGN, SETTING, AND PARTICIPANTS: This was cross-sectional study of obese (body mass index ≥ 95th percentile) but otherwise healthy female adolescents (10 black and 12 white; age range 8.8-13.9 y) who underwent adrenocorticotropic hormone stimulation testing at an academic medical center as part of a protocol for the study of obesity-related conditions. MAIN OUTCOME MEASURES: Basal and stimulated androgen levels. RESULTS: White and black participants were similar with regard to pubertal stage, body mass index, percentage body fat, and fasting glucose and insulin levels. Black girls had lower stimulated levels of 17-hydroxyprogesterone, and the differences between basal and stimulated levels of 17-hydroxyprogesterone and androstenedione were lower in black girls. Body mass index was negatively correlated with stimulated cortisol in blacks only (r = -0.69, P = .03). CONCLUSION: There appear to be race-related differences in stimulated androgen levels in obese adolescent females. These differences deserve further study, as measurements of androgen levels are commonly used in clinical practice and research.

The steroid metabolome in lamotrigine-treated women with epilepsy.

BACKGROUND: Epilepsy in women may be associated with reproductive disorders and alterations in serum steroid levels. Some steroids can be induced by epilepsy and/or treatment with antiepileptic drugs; however, there are still limited data available concerning this effect on the levels of other neuroactive steroid metabolites such as 3a-hydroxy-5a/b-reduced androstanes. AIM: To evaluate steroid alterations in women with epilepsy (WWE) on lamotrigine monotherapy. SUBJECTS AND METHODS: Eleven WWE and 11 age-matched healthy women underwent blood sampling in both phases of their menstrual cycles (MCs). The steroid metabolome, which included 30 unconjugated steroids, 17 steroid polar conjugates, gonadotropins, and sex hormone-binding globulin (SHBG), was measured using gas chromatography-mass spectrometry (GC-MS) and radioimmunoassay (RIA). RESULTS: WWE had lower cortisol levels (status p<0.001), but elevated levels of unconjugated 17-hydroxypregnenolone (status p<0.001). Progesterone was higher in the follicular menstrual phase (FP) in WWE than in the controls (status×menstrual phase p<0.05, Bonferroni multiple comparisons p<0.05), whereas 17-hydroxyprogesterone was higher in WWE in both menstrual phases (status p<0.001). The steroid conjugates were mostly elevated in WWE. The levels of 5α/ß-reduced androstanes in WWE that were significantly higher than the controls were etiocholanolone (status p<0.001), 5α-androstane-3α,17ß-diol (status p<0.001), and the 5α/ß-reduced androstane polar conjugates (status p<0.001). CONCLUSIONS: WWE showed a trend toward higher circulating 3α-hydroxy-5α/ß-reduced androstanes, increased activity of 17α-hydroxylase/17,20 lyase in the Δ(5)-steroid metabolic pathway, and increased levels of the steroid polar conjugates.

Serum concentrations of adrenal steroids and their precursors as a measure of maturity of adrenocortical function in very premature newborns.

BACKGROUND: Relative adrenocortical insufficiency is often seen in sick premature newborns. As the human fetal adrenal cortex does not express the 3ß-hydroxysteroid dehydrogenase (3ß-HSD) enzyme before about 23 weeks of gestation, we hypothesized that this enzymatic step may be rate limiting in cortisol synthesis in premature infants of less than 28 weeks postmenstrual age at birth. METHODS: We measured cord, first day (D0) and median fourth day (D4) serum 17-OH-pregnenolone (17-OHPreg), 17-OH-progesterone (17-OHProg), 11-deoxycortisol, cortisol (F) and dehydroepiandrosterone sulphate concentrations and calculated the substrate/product ratios in 67 infants with gestational age 23.6-33.1 weeks. RESULTS: The mean 17-OHPreg/17-OHProg ratio as a marker of 3ß-HSD activity did not differ between the gestational age groups (gestational age <28 vs. ≥28 weeks: 0.40 vs. 0.48, p = 0.52 for cord, 3.1 vs. 2.4, p = 0.25 for D0, and 1.6 vs. 1.9, p = 0.62 for D4). In addition, the 17-OHPreg/17-OHProg ratio did not differ between the infants in the lowest F tertile compared to those in the highest F tertile group, and the serum 17-OHPreg and 17-OHProg concentrations were parallel with the respective F concentrations. CONCLUSION: We did not find evidence of significant immaturity in adrenal 3ß-HSD activity in preterm infants between 24 and 28 weeks of gestation.

Simultaneous determination of 17alpha-hydroxypregnenolone and 17alpha-hydroxyprogesterone in dried blood spots from low birth weight infants using LC-MS/MS.

17alpha-hydroxypregnenolone (17OHPreg) has heretofore been considered to be the major cause of the false elevated 17alpha-hydroxyprogesterone (17OHP) value in the immunoassay-based newborn screening for congenital adrenal hyperplasia (CAH). To verify this point, we developed a liquid chromatography-tandem mass spectrometric (LC-MS/MS) method that enables the simultaneous quantification of 17OHPreg and 17OHP in the dried blood filter papers and measured their blood levels in infants, especially in infants with low birth weights. Steroids were extracted from the filter papers with methanol, purified using a Strata-X cartridge, derivatized with 2-hydrazinopyridine and subjected to LC-MS/MS. Validation tests proved that this method was specific and reproducible; endogenous steroids did not interfere with the quantifications, and the intra- and inter-assay coefficients of variation were below 5.2%. The limits of quantitation were 1.0 and 0.5 ng/mL for 17OHPreg and 17OHP, respectively, when 3 disks (3 mm in diameter) of the filter papers (corresponding to 8 microL of whole blood) were used. The blood 17OHPreg level was elevated in the very low birth weight (1000-1500 g) infants and extremely low birth weight (<1000 g) infants, compared to those in the normal birth weight (>2500 g) infants (P<0.05). However, the 17OHPreg concentration was not high enough to cause the false positive results in the enzyme immunoassay-based screening, and it was considered that the false positive results come from other endogenous components rather than 17OHPreg.

Evaluation of cortisol precursors for the diagnosis of pituitary-dependent hypercortisolism in dogs.

The serum concentrations of cortisol, 17alpha-hydroxypregnenolone, 17alpha-hydroxyprogesterone, 21-deoxycortisol and 11-deoxycortisol were measured in 19 healthy dogs, 15 dogs with pituitary-dependent hypercortisolism (pdh) and eight dogs with other diseases before and one hour after an injection of synthetic adrenocorticotrophic hormone (acth). At both times the dogs with pdh had significantly higher concentrations of cortisol, 17alpha-hydroxypregnenolone, 17alpha-hydroxyprogesterone and 21-deoxycortisol than the healthy dogs. Basal 11-deoxycortisol concentrations were also significantly higher in dogs with pdh compared with healthy dogs. When compared with the dogs with other diseases, the dogs with pdh had significantly higher basal and post-acth cortisol and basal 21-deoxycortisol, and significantly lower post-acth 11-deoxycortisol concentrations. The dogs with other diseases had significantly higher post-acth cortisol, 17alpha-hydroxyprogesterone and 11-deoxycortisol concentrations than the healthy dogs. In general, the post-acth concentrations of 17alpha-hydroxypregnenolone, 17alpha-hydroxyprogesterone, 11-deoxycortisol and 21-deoxycortisol were more variable than the post-acth concentrations of cortisol, resulting in large overlaps of the concentrations of these hormones between the three groups. A two-graph receiver operating characteristic (ROC) analysis was used to maximise the sensitivity and specificity of each hormone for diagnosing hypercortisolism; it showed that the post-acth concentration of cortisol had the highest sensitivity and specificity. The overlaps between the healthy dogs, the dogs with pdh and the dogs with other diseases suggested that the individual precursor hormones would not be useful as a screening test for hypercortisolism.

Determination of 17alpha-hydroxypregnenolone sulfate and its application in diagnostics.

New combined radioimmunoassay for determination of 17-hydroxypregnenolone sulfate (17-PregS) involving the hydrolysis of analyte by methanolysis was developed. 17-PregS, in addition to being secreted by the adrenals, is also formed by peripheral sulfoconjugation of 17-hydroxypregnenolone (17-Preg) or directly by hydroxylation of pregnenolone sulfate with 17alpha-hydroxylase/C17-20lyase. The measurement of 17-PregS can be used as a tool for detection of enzymatic deficiency particularly in pregnancy and for detection of congenital adrenal hyperplasia or gonadal dysfunction. The serum levels of 17-PregS, 17-Preg, dehydroepiandrosterone, dehydroepiandrosterone sulfate, pregnenolone and pregnenolone sulfate were measured in different age groups of human and in pregnant women respecting the age of gestation. The levels of 17-PregS are approximately three times higher than the levels of free 17-Preg in all subject groups. The levels of 17-PregS during pregnancy reached the local minimum in the 3rd month of gestation. The ratio of 17-PregS to free 17-Preg showed increasing profile during pregnancy with a maximum in the 8th month of gestation. These findings indicate that, the conversion of pregnenolone sulfate to 17-PregS is the major metabolic pathway for biosynthesis of 17-PregS.