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Background: A rapid and accurate measurement approach for 17α-hydroxyprogesterone (17-OHP) and related steroids in amount/volume-limited clinic samples is of importance for precise newborn diagnosis of congenital adrenal hyperplasia (CAH) and its subtypes in clinic., Methods: Sixteen steroids (17-OHP, androstenedione, cortisol, tetrahydro-11-deoxycortisol, pregnenolone, progesterone, 11-deoxycorticosterone, corticosterone, 21-deoxycortisol, 11-deoxycortisol, dehydroepiandrosterone, testosterone, aldosterone, 17α-hydroxypregnenolone, dihydrotestosterone and 18-hydroxycorticosterone) were included in the panel of high-throughput microbore ultra-performance liquid chromatography-tandem mass spectrometry. Samples were collected from 126 normal subjects and 65 patients including different subtypes of CAH., Results: The method was validated with satisfactory analytical performance in linearity, repeatability, recovery and limit of detection. Reference intervals for 16 steroids were established by quantifying the level of steroids detected in normal infants. The applicability of the method was tested by differentiating steroid metabolic characteristics between normal infants and infants with CAH, as well as between infants with different CAH subtypes. The relevance of 17-OHP, 21-deoxycortisol, and 17-OHP/11-deoxycortisol for 21-hydroxylase deficiency screening was demonstrated. The level of 11-deoxycorticosterone, 11-deoxycortisol, progesterone and androstenedione can be used for the diagnosis of different rare subtypes of CAH., Conclusion: This study provides a strategy for highly efficient steroid analysis of amount/volume-limited clinic samples and holds great potential for clinical diagnosis of CAH., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Aims: To evaluate the associations between residential greenness and glucocorticoid levels and whether air pollutants and sex modify the relationship between greenness and glucocorticoid level in Chinese rural adults., Methods: We collected cross-sectional survey data from 6055 participants from the Henan Rural cohort. The three-year average residential greenness for participants was assessed using normalized difference vegetation index (NDVI) values from a satellite platform. Liquid chromatography-tandem mass spectrometry was employed to quantify the concentrations of glucocorticoids, which were measured by morning blood draw after at least 8 hr of fasting. A random forest model was employed to obtain the average concentrations of PM 1 , PM 2.5 , and PM 10 . A general linear regression model was performed to estimate the associations of NDVI 500-m values with cortisol, 11-deoxycortisol, and cortisone. Furthermore, interaction plots were used to present the interaction effects of particulate matter, sex, and green space on glucocorticoid levels., Results: After adjusting for multiple variables, an elevated average NDVI 500-m value in the total population was associated with a decrease in cortisol levels (β = -0.063, 95 % confidence interval (CI): - 0.118, - 0.008), and 11-deoxycortisol levels (β = -0.118, 95 % CI: -0.190, -0.047), as well as an increase in cortisone levels (β = 0.130, 95 % CI: 0.079, 0.181). By adding the interaction terms of air pollutants and residential greenness into the regression model, interaction effects between air pollutants and residential greenness were found (cortisol, PM 2.5 : P interaction=:0.018; PM 10 : P interaction=0.016; 11-deoxycortisol, all pollutants: P interaction< 0.001), suggesting that the protective effect of residential greenness on serum glucocorticoids disappeared accompanying with increased concentrations of particulate matter. Moreover, trends towards modification in the association between green space and glucocorticoid levels were also evident by sex, but these did not reach statistical significance (for all glucocorticoids: P interaction> 0.05)., Conclusion: Long-term exposure to green space was negatively correlated with cortisol and 11-deoxycortisol levels, and positively correlated with cortisone levels. There may be sex differences in these associations. Moreover, the protective effect of residential greenness on serum glucocorticoids was altered by high levels of particulate matter., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

Objectives: To explore the associations of environmental endocrine disruptors on precocious puberty in girls., Methods: This was a case-control study in which 30 girls with precocious puberty and 46 age- and race-matched prepubertal females were enrolled. The concentrations of 10 environment endocrine disruptors (bisphenol A, bisphenol B, butylparaben, propylparaben, ethvlparaben, methylparaben, mono-butyl phthalate, mono-2-ethylhexyl phthalate, monoethyl phthalate, and monomethyl phthalate) in urine and 10 steroid hormones (dihydrotestosterone, corticosterone, hydrocortisone, 11-deoxycortisol, 17α-hydroxy progesterone, 4-androstene-3,17-dione, estrone, deoxycorticosterone, pregnenolone, and dehydroepiandrosterone) in serum were detected with the liquid chromatography-mass spectrometry (LC-MS)., Results: According to the Mann-Whitney U test, urinary levels of bisphenol A, monobutyl phthalate, and monomethyl phthalate were significantly higher in the precocious group than in the prepubertal group, and blood levels of hydrocortisone, 11-deoxycortisol, corticosterone, deoxycorticosterone, and pregnenolone were significantly lower in the precocious group than in the prepubertal group (p<0.05, VIP>1)., Conclusions: Our findings confirm the association between phthalate exposure and the incidence of precocious puberty in girls. Control and reduction of children exposure to phthalate esters should be considered as a health priority., (© 2022 Fang Zhou et al., published by De Gruyter, Berlin/Boston.)

Background: The hypothalamic-pituitary-adrenal stress axis plays a crucial role in community-acquired pneumonia (CAP), with high cortisol being associated with disease severity and corticosteroid treatment resulting in earlier time to recovery. Our aim in the present study was to compare different glucocorticoid hormones, including cortisol, 11-deoxycortisol, cortisone, and corticosterone, regarding their association with short- and long-term adverse outcomes in a well-defined CAP cohort., Methods: We prospectively followed 285 patients with CAP from a previous Swiss multicenter trial for a median of 6.1 years and measured different admission glucocorticoid serum levels by liquid chromatography coupled with tandem mass spectrometry. We used adjusted Cox regression models to investigate associations between admission hormone levels and all-cause mortality at different time points., Results: Mortality was 5.3% after 30 days and increased to 47.3% after 6 years. High admission cortisol was associated with adverse outcome after 30 days (adjusted OR 3.85, 95% CI 1.10-13.49, p = 0.035). In the long term (i.e.,), however, high admission cortisol was associated with better survival (adjusted HR after 3 years 0.53, 95% CI 0.32-0.89, p = 0.017; adjusted HR after 6 years 0.57, 95% CI 0.36-0.90, p = 0.015). Compared with 11-deoxycortisol, cortisone, and corticosterone, cortisol showed the highest association with mortality., Conclusions: Among different glucocorticoid hormones, cortisol showed the highest association with mortality in CAP. Whereas a more pronounced glucocorticoid stress response on hospital admission was associated with higher short-term adverse outcome, long-term outcome was favorable in these patients. These data should support the correct interpretation of glucocorticoid blood data.

Steroid hormone measurement, first developed with radioimmunoassay, is now becoming easier with the use of automated platforms of immunoassay. However, some hormones remain uneasily detectable because of their low blood concentration, their structural homology or the presence of interferences. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) can be considered as an alternative to immunoassays. This approach allows the simultaneous determination of several parameters thanks to its selectivity led by the detector mass spectrometer and the separate dimension of chromatography liquid. In addition, recourse to UHPLC (ultra high performance liquid chromatography) allows improving selectivity and sensitivity while limiting the samples volumes. The "ready-to-use" kits are now available and added to the "homemade" techniques developed by laboratories, thus giving opportunity for measurement of a wide steroid panel with only one sample. Finally, mass spectrometry methods, including a prior extraction step, allow the use of varied biological fluids (blood, urine, saliva…). Also, several clinical indications could gain from mass spectrometry, especially when hormone levels are low, when several steroids have to be identified, when the sample volume is low. However, this technology represents an important financial investment and in-depth staff training. In addition, some steroids are not easily quantifiable by mass spectrometry. It is likely by immunoassay and mass spectrometry, well-matched technologies, that we could answer the best to clinical questions about steroids.

A noncompetitive immunoassay has the potential for improved sensitivity and working range compared with corresponding competitive assays. However, monovalent antigens with less than 1000 in molecular weight are not susceptible to sandwich assays due to their small size. As a noncompetitive immunoassay that can be performed with a clone of an antibody, an open-sandwich immunoassay (OS-IA) based on the antigen-dependent stabilization of the antibody variable region (V(H) + V(L)) was applied to the quantification of 11-deoxycortisol (11-DC; M(r) 346.5), a corticosteroid serving as a diagnostic index for pituitary-adrenal function, as a model target hapten. By one step OS-IA detection of enzyme-labeled V(H) fragment bound to immobilized V(L) in the presence of sample in microplate wells, 11-DC was measured with a femtomolar detection limit and the working range was wider than that with corresponding competitive assay. In addition, the selectivity against analogues was found almost identical to that of conventional assays. The effect of the mutagenesis of a V(H) residue at the V(H)/V(L) interface to reduce background signal was also shown, implying the wider application of OS-IA in small molecule analyses.

This paper describes a sensitive and convenient method to separate progesterone, 17alpha-hydroxy progesterone, cortexolone, hydrocortisone and cortisone, all of which are steroids and have similar structures, using microfluidic chip-based technology with UV detection at 252 nm. We successfully obtained high-speed separation of the five steroids within 70 s in optimized microfluidic controls and micellar electrokinetic chromatography (MEKC) separation conditions. Fairly good linearity with correlation coefficient of over 0.98 from 10 or 20 to 100 mg/l steroid chemicals was obtained. The limits of detection obtained at a signal to noise ratio of 3 were from 3.89 to 7.80 mg/l. The values of the relative standard deviation (RSD) were 0.98-1.34% for repetitive injection (n = 12) and the intraday and interday RSDs were below 6%. The highly stable response reflected the feasibility of this method.

Background: Neonatal screening programs for congenital adrenal hyperplasia (21-CAH) using an immunoassay for 17alpha-hydroxyprogesterone (17-OHP) generate a high rate of positive results attributable to physiological reasons and to cross-reactions with steroids other than 17alpha-OHP, especially in preterm neonates and in critically ill newborns., Methods: To increase the specificity of the screening process, we applied a liquid chromatography-tandem mass spectrometry method quantifying 17alpha-OHP, 11-deoxycortisol, 21-deoxycortisol, cortisol, and androstenedione. The steroids were eluted in aqueous solution containing d8-17alpha-OHP and d2-cortisol and quantified in multiple reaction mode., Results: Detection limit was below 1 nmol/liter, and recovery ranged from 64% (androstenedione) to 83% (cortisol). Linearity was proven within a range of 5-100 nmol/liter (cortisol, 12.5-200 nmol/liter), and total run time was 6 min. Retrospective analysis of 6151 blood samples and 50 blood samples from newborns with clinically confirmed 21-CAH, as well as prospective analysis of 1609 samples of a total of 242,500 testing positive in our routine 17-OHP immunoassay, allowed clear distinction of affected and nonaffected newborns. High levels of 21-deoxycortisol were only found in children with 21-hydroxylase deficiency. Calculating the ratio of 17alpha-OHP to 21-deoxycortisol divided by cortisol further increased the sensitivity of the method., Conclusion: Our liquid chromatography-tandem mass spectrometry procedure as a second-tier test can be used to reduce false-positive results of standard 21-CAH screening. The short total run time of 6 min allows for immediate reanalysis of all immunoassay results above the cutoff.

The identity of the maturation-inducing steroid (MIS) in black porgy, Acanthopagrus schlegeli, a marine protandrous teleost, is unknown. Previous studies demonstrated that two teleost MISs, the progestins 17,20beta,21-trihydroxy-4-pregnen-3-one (20beta-S) and 17,20beta-dihydroxy-4-pregnen-3-one (DHP) can induce maturation of black porgy oocytes in vitro. The purpose of the present study was to identify the major progestin produced during oocyte maturation (OM) in black porgy and investigate whether its secretion increases during this process. Females were injected twice with a LHRH analog to induce OM. Ovarian follicles undergoing OM were incubated in vitro with tritiated [3H]pregnenolone precursor and the tritiated products were extracted, purified, and identified by HPLC, TLC, acetylation, and recrystallization. Significant amounts of tritiated products were biosynthesized from [3H]pregnenolone that co-migrated with 20beta-S but not with DHP on HPLC and TLC. Similar TLC profiles were obtained with the tritiated products isolated from the HPLC/TLC 20beta-S fraction and standard 20beta-S after the acetylation reaction. The identity of the tritiated products as 20beta-S was confirmed by recrystallization. 20beta-S had a slightly higher potency than DHP in the inducing in vitro final oocyte maturation. Plasma 20beta-S concentrations increased significantly during the oocyte maturation after injection with a LHRH analog. The present data suggest that 20beta-S is the MIS in black porgy.

Congenital adrenal hyperplasia (CAH) is an autosomal recessive disorder mainly caused by 21-hydroxylase deficit (21-OHD). Deletions or mutations of the CYP21 gene induce the impairment of glucocorticoid and mineralcorticoid synthesis. 17-Hydroxyprogesterone (17-OHP) is the hormonal marker in patients, but not in the heterozygous subjects. Excess 17-OHP is hydroxylated into 21-deoxycortisol (21-DF), and therefore 21-DF can be used as a specific marker for diagnosis of heterozygous individuals. We report an analytical method for analysis of 21-DF in blood samples using electrospray (ESI) and atmospheric pressure chemical ionization (APCI), showing that ESI is very sensitive for the analysis of this marker molecule. The multiple reaction monitoring (MRM) approach was used to increase the specificity and the sensitivity of the method., (Copyright 2003 John Wiley & Sons, Ltd.)

Metyrapone blocks cortisol synthesis, which results in the stimulation of hypothalamic cortiocotropin-releasing factor (CRF) and a reduction in delta sleep. We examined the effect of metyrapone administration on endocrine and sleep measures in male subjects with and without chronic PTSD. We hypothesized that metyrapone would result in a decrease in delta sleep and that the magnitude of this decrease would be correlated with the endocrine response. Finally, we utilized the delta sleep response to metyrapone as an indirect measure of hypothalamic CRF activity and hypothesized that PTSD subjects would have decreased delta sleep at baseline and a greater decrease in delta sleep induced by metyrapone. Three nights of polysomnography were obtained in 24 male subjects with combat-related PTSD and 18 male combat-exposed normal controls. On day 3, metyrapone was administered during normal waking hours until habitual sleep onset preceding night 3. Endocrine responses to metyrapone were measured in plasma obtained the morning following sleep recordings, the day before and after administration. Repeated measures ANOVAs were conducted to compare the endocrine and sleep response to metyrapone in PTSD and controls. PTSD subjects had significantly less delta sleep as indexed by stages 3 and 4, and total delta integrated amplitude prior to metyrapone administration. There were no differences in premetyrapone cortisol or ACTH levels in PTSD vs controls. PTSD subjects had a significantly decreased ACTH response to metyrapone compared to controls. Metyrapone caused an increase in awakenings and a marked decrease in quantitative measures of delta sleep that was significantly greater in controls compared to PTSD. The decline in delta sleep was significantly associated with the magnitude of increase in both 11-deoxycortisol and ACTH. The results suggest that the delta sleep response to metyrapone is a measure of the brain response to increases in hypothalamic CRF. These data also suggest that the ACTH and sleep EEG response to hypothalamic CRF is decreased in PTSD.

The aim of this study was to identify the major C21 steroids produced in vivo during artificially induced final oocyte maturation and spawning in female common dentex (Dentex dentex). During the spawning season, mature females were treated with a gonadotropin-releasing hormone agonist (GnRHa)-loaded delivery system, with or without pimozide (given as a single dose at the beginning of the experiment). Blood samples were collected at various intervals during the experiment and were assayed for GnRHa, 17,20beta-dihydroxy-4-pregnen-3-one (17,20beta-P), and 17,20beta,21-trihydroxy-4-pregnen-3-one (17,20beta,21-P). A higher percentage of ovulated females was observed in GnRHa-implanted fish, which produced over 10 times more eggs than controls. Relative fecundity was highest in the GnRHa + pimozide group and lowest in controls. The viability of naturally released eggs was low (2 to 15%) in all groups. Plasma concentrations of 17,20beta-P in GnRHa-implanted fish did not increase, but those in control fish decreased, such that there was a significant difference between control and treated fish between 2 and 10 days after treatment. In another experiment, ovulating common dentex were injected intramuscularly with a single dose of 50 microg kg(-1) of GnRHa in saline and were sampled for blood at 0, 3, 6, 12, and 24 h postinjection. A single water sample was taken from the tanks at 9 h postinjection, the tanks having been emptied and refilled at 6 h. Measurements were made of plasma and water concentrations of free and conjugated 17,20beta-P, 17,20beta,21-P, 17beta-oestradiol (E2), and GnRHa (plasma only). The GnRHa injection increased plasma levels of all steroids, with free 17,20beta-P reaching maximal levels within 3 h. GnRHa treatment also increased the amounts of free and conjugated steroids released into the water between 6 and 9 h.

The HPLC system was used to separate and measure 10 kinds of corticoids in adrenal tissues. Calibration curves were drawn as straight lines that ranged from 1.25 to 20ng, or 1.25 to 200ng by peak area calculated with the chromatointegrator. The samples for the assay were extracted from homogenized tissues and treated with methanol to remove non-steroidal contaminants which may interfere with the ultraviolet absorption monitor. The recovery rate during the assay procedure was calculated using testosterone as the internal standard, because testosterone was not detected in any adrenal tissue examined in the present study. Contents of corticoids were measured in normal adrenal glands obtained during radical nephrectomy for renal cancer and in functioning adrenal adenomas. Steroid levels in the adrenal glands and tumors have been measured by radioimmunoassay until now, and the data obtained in the present study were compared with those in previous reports. Main steroids in normal adrenals were cortisol (F) and corticosterone (B), and there were certain amounts of 11-deoxycortisol (S), 11-deoxycorticosterone (DOC) and precursor steroids. 11 beta-hydroxy-androstenedione was the main androgen in the adrenal gland. Mineralocorticoids other than B and DOC were very low in the normal adrenals. There was a certain balance between the production of cortisol and corticosterone in normal adrenals. In functioning adenomas, the levels of F, B and aldosterone, and F to B ratios (F/B) varied according to their biological features. Although with the HPLC system it was possible to obtain the production balance of each steroid clearly in the chromatogram, we could not detect the delta 5-3 hydroxysteroids such as pregnenolone and dehydroepiandrosterone using the ultraviolet absorption monitor.

Prenatal diagnosis of congenital adrenal hyperplasia (CAH) due to 21-hydroxylase deficiency by amniotic fluid (AF) steroid analysis is not possible in those cases in which prenatal dexamethasone (DEX) therapy is initiated to prevent virilization of female CAH fetuses because AF steroid levels are suppressed if DEX therapy is continued beyond amniocentesis (AC). In order to use AF steroids for prenatal diagnosis, it is necessary to discontinue DEX therapy for 5 days before AC. To study the effects of this interruption on AF steroid levels, we measured levels of aldosterone, corticosterone, 11-deoxycorticosterone, progesterone, 17-hydroxyprogesterone (170HP), 11-deoxycortisol, cortisol, and cortisone as well as androstenedione (delta 4-A) in AF samples (16-18 weeks) obtained from 25 pregnancies at risk for CAH treated with Dex (daily dosage: 1.0-1.5 mg). The prenatal diagnosis of 14 normal fetuses and 11 affected CAH fetuses was postnatally confirmed in all cases. Additionally, steroid levels were measured in AF samples (16-18 weeks) from 8 untreated CAH fetuses and in 19 AF samples (weeks 16-20) obtained in normal pregnancies. In 17/19 prenatally diagnosed CAH fetuses, the affected sibs had the salt wasting (SW)-form, in 2 cases the simple virilizing (SV)-form. All steroids were measured by RIA after extraction and Sephadex LH-20 chromatography. AF levels of aldosterone, corticosterone, deoxycorticosterone, progesterone, cortisol, cortisone, and 11-deoxycortisol were not different between CAH fetuses, prenatally DEX-treated normal fetuses and untreated controls. The 170HP-levels of the CAH-SW-fetuses (range: 19.9-59.8 mmol/L) were clearly above the normal range (3.74-11.6), but normal in the SV-fetuses (10.9, 11.5), whereas delta-4 A-levels (normal range: 0.87-5.13 mmol/L) were elevated both in the SW-(range: 6.53-37.6) and the SV-form (9.37,6.25) of CAH. 170HP and delta-4 A levels of prenatally DEX-treated pregnancies with normal fetuses were not different from levels found in normal pregnancies. Mean 170HP and delta-4 A AF steroid levels of prenatally DEX-treated CAH-pregnancies were slightly lower (NS) than levels of untreated CAH-pregnancies (170HP: 30.5 vs. 40.7; delta-4 A: 15.8 vs. 21.1). 170HP levels are elevated in the SW-form of CAH, but not in the SV-form. However, with the combination of 170HP and delta-4 A levels it is possible to diagnose prenatally both forms. There is no rebound phenomenon of AF steroid levels if DEX therapy is interrupted 5 days before amniocentesis.

Incubation of 11-deoxycortisol with a cytochrome P-450(11 beta)-reconstituted system yielded, in addition to cortisol, several new steroid products. In this study, structures of the three steroid products were elucidated. Retention time of the first product (Peak 2 substance) coincided with that of authentic 18-hydroxycortisol on reverse phase HPLC. To further confirm the chemical identity of this product, the purified sample was subjected to 1H-NMR analysis. The spectrum was essentially identical to that of 18-hydroxycortisol. The retention time of the second product (Peak 3 substance) did not coincide with those of commonly occurring steroids. The one- and two-dimension 1H-NMR spectra provided strong evidence for its structure of 19-hydroxy-11-deoxycortisol. The retention time of the third product (Peak 4 substance) did not coincide with those of commonly occurring steroids. The 1H-NMR spectrum showed the presence of signals of 19-CH3 and 18-CH2 protons. There was also evidence that this product is not hydroxylated at the 11-position. Further analysis of the COSY spectra identified its structure as 18-hydroxy-11-deoxycortisol. From these results, we conclude that bovine P-450(11 beta) can catalyze the hydroxylation of 11-deoxycortisol at 11 beta-, 18- and 19-positions and produce cortisol, 18-hydroxy-11-deoxycortisol, 18-hydroxycortisol and 19-hydroxy-11-deoxycortisol.

The interrelations of steroid hormone levels in plasma and amniotic fluid from mothers and their undisturbed fetuses at early midgestation of human pregnancy have not been defined previously. We, therefore, studied 12 healthy mothers and their fetuses undergoing termination of pregnancy for social reasons at 16-20 weeks gestation. Fetal arterial and venous blood was obtained by direct vessel puncture through a fetoscope in the conscious sedated mothers immediately before termination of pregnancy. Simultaneously, maternal peripheral venous blood and amniotic fluid were collected. Aldosterone (Aldo), corticosterone (B), 11-deoxycorticosterone, progesterone (P), 17-hydroxyprogesterone (17OHP), 11-deoxycortisol, cortisol (F), and cortisone were simultaneously determined by specific RIA after extraction and chromatography. Positive fetal arterio-venous differences were found for F, B, and Aldo, whereas arteriovenous differences were negative for P and 17OHP. In amniotic fluid, six of the eight corticosteroids showed significantly lower levels during fetoscopy than during routine amniocentesis, as reported previously using the same analytical methods. The present study demonstrates that the undisturbed human fetus at 16-20 weeks gestation actively secretes the most important gluco- and mineralocorticoids, F, B, and Aldo, independent of the mother. P and 17OHP were shown to be primarily derived from placental production and supplied to the fetus as a source of F and Aldo biosynthesis. The fetoscopy procedure with premedication seemed to give rise to less stress to the fetus than routine amniocentesis without sedation. Fetoscopy is, therefore, an ideal method to study feto-maternal steroid interrelations in human pregnancy.

Ovaries from a female plaice (Pleuronectes platessa) that had been injected with human chorionic gonadotrophin were incubated in vitro with 17 alpha-hydroxy[1,2,6,7-3H]progesterone. The major steroids produced by the ovaries were tentatively identified as 17 alpha,21-dihydroxy-4-pregnene-3,20di-one (11-deoxycortisol; 17,21-P), 17 alpha,21-dihydroxy-5 beta-pregnane-3,20-dione (3 alpha, 17,21-P-5 beta). A high proportion of these steroids was found in a conjugated form (sulphates or glucuronides). Radioimmunoassays were developed for 11-deoxycortisol and for 3 alpha,17,21-P-5 beta and were applied to fractions of mature male and female plaice plasmas and plaice ovarian incubates that had been separated on thin-layer chromatography. The presence of all three steroids, in vivo and in vitro, was confirmed. Particularly high amounts of conjugated 3 alpha,17,21-P-5 beta were found in the plasma of mature females (200-400 ng ml-1). The 3 alpha,17,21-P-5 beta radioimmunoassay also identified 3 alpha,17 alpha-dihydroxy-5 beta-pregnane-20-dione in all three fluids, despite the fact that this steroid was not among the radioactive incubation products of the ovary. These findings are compared with those from another flatfish, the dab (Limanda limanda), where the major gonadal steroids have been shown to be 17 alpha,20 alpha-dihydroxy-4-pregnen-3-one and its 5 beta-pregnane (3-keto and 3 beta-hydroxyl) metabolites.

A high-performance liquid chromatographic method is described for the determination of seven steroids in adrenocortical extracts showing a delta 4-3-ketonic conjugated system. The seven steroids (cortisol, cortisone, 11-dehydrocorticosterone, corticosterone, 11-deoxycortisol, aldosterone and 11-deoxycorticosterone) were separated with a chloroform-methanol gradient on a 5-micron silica column and with a water-acetonitrile gradient on a 10-micron RP-8 column. Effluents were monitored by UV absorption at 242 nm. Quantitative analysis was performed by comparing peak areas, which are proportional to the amounts of the individual substances (external standard method). The method is rapid, sensitive, easy to perform and reproducible.

We developed polarization fluoroimmunoassays for 11-deoxycortisol in serum and saliva. To avoid interfering factors, the steroid was initially extracted from the biological fluids with dichloromethane. Assays could then be completed without any further separation procedures or need to correct for blank signals. The serum assay was suitable for following the response to the metyrapone test and results correlated acceptably with those by an established, specific, direct 125I-radioimmunoassay. The method is not sufficiently sensitive to detect 11-deoxycortisol in normal saliva, but greatly increased concentrations were found in post-metyrapone saliva and results agreed well with those by the radioimmunoassay as modified for salivary assay. Salivary 11-deoxycortisol assay would provide a convenient means of monitoring results of the metyrapone test.

Radioimmunoassays, combined with thin-layer chromatography, have been used to test for the presence of 17 alpha,20 beta-dihydroxy-4-pregnen-3-one, 17 alpha,20 beta,21-trihy-droxy-4-pregnen-3-one, and 20 beta-hydroxy-4-pregnen-3-one, and for the presence of their 5-pregnene and 5 alpha-pregnane analogs, in the blood and ovarian incubation media of maturing/ovulating female rainbow trout (Salmo gairdneri). All of these steroids have previously been shown to be more or less equipotent in in vitro oocyte final maturation assays. Only two steroids were found: 17 alpha,20 beta-dihydroxy-4-pregnen-3-one, the presence of which has already been firmly established, and 17 alpha,20 beta, 21-trihydroxy-4-pregnen-3-one, which has not been previously identified in rainbow trout. The amounts of 17 alpha,20 beta, 21-trihydroxy-4-pregnen-3-one found in vivo and in vitro were, however, only 1-1.5% of those of 17 alpha,20 beta-dihydroxy-4-pregnen-3-one, and therefore seem unlikely to play a significant role in the induction of oocyte final maturation.

The mean control level of 11-deoxycortisol as determined by radioimmunoassay in eighty-one human amniotic fluid samples was 1.20 +/- 0.07 ng/ml. Markedly elevated levels were found at term in amniotic fluid of two pregnancies with fetuses affected with 11 beta-hydroxylase deficiency, congenital adrenal hyperplasia (135.0 and 64.0 ng/ml respectively) as well as in the maternal serum of one of these cases (28.0 ng/ml). It is suggested that the determination of 11-deoxycortisol in amniotic fluid be a prenatal diagnostic test for 11 beta-hydroxylase deficiency congenital adrenal hyperplasia.

Amniotic fluid levels of 21-deoxycortisol (21-DOF) and 17-hydroxyprogesterone (17-OHP) were measured in 49 pregnancies, including 31 pregnancies at risk for CAH. The results were compared with those obtained by HLA typing and linkage analysis to a HLA DNA probe. The mean amniotic fluid levels in the control pregnancies were 0.28 nmol/L for 21-DOF and 4.1 nmol/L for 17-OHP. The levels were similar in early and midpregnancy for 21-DOF (0.29 vs. 0.27 nmol/L) and 17-OHP (3.4 vs. 4.2 nmol/L). The amniotic fluid 21-DOF level was 1.75 nmol/L in affected pregnancies, significantly higher than in the control pregnancies (mean, 0.28 nmol/L). The mean amniotic fluid 17-OHP level in the affected pregnancies (30.5 nmol/L) also was significantly higher than that in the control pregnancies (4.10 nmol/L). Simultaneous measurement of 21-DOF and 17-OHP levels in amniotic fluid from 10-18 weeks of gestation can be used for early diagnosis of congenital adrenal hyperplasia.

In human urinary pH 1 extracts prepared for the aldosterone-18-glucuronide estimation, several other substances are present, crossreacting not only with aldosterone antisera, but also with various corticosteroid and tetrahydrocorticosteroid antisera. Aldosterone was measured before and after chromatographic purification. Further characterization of the non-aldosterone immunoreactive material was made by immunological analysis of paper chromatogram eluats. Pregnancy, and administration of ACTH, dexamethasone, and metopirone led to a change of excretion in the antigenic equivalents. A method for the separation of the antigenic material is described. For structural elucidation the gaschromatography-mass spectrometry (GC-MS) method was applied.

This study describes the identification of 17 alpha,20 beta, 21-trihydroxy-4-pregnen-3-one (20 beta-dihydro-11-deoxycortisol, 20 beta-S) as a major steroid product of the ovary of Atlantic croaker (Micropogonias undulatus) incubated in vitro. This is the first report which describes 17 alpha,20 beta,21-trihydroxy-4-pregnen-3-one as a major product of teleost steroidogenic tissue. The steroid was identified by a variety of methods, including HPLC, TLC, GC-MS, UV absorbance, and reactions with specific enzymes. Full grown oocytes, prior to final oocyte maturation (FOM), accumulated only small amounts of 20 beta-S. However, a substantial increase in 20 beta-S production occurred at the time of FOM. These results suggest that 20 beta-S is a maturational steroid in this species.

21-deoxycortisol is a steroid produced mainly by the adrenal gland. Its normal plasma baseline concentrations (0.03 to 0.30 n/ml) and its concentrations after tetracosactide injection (0.15 to 0.76 ng/ml) do not significantly vary with age, sex and phases of the menstrual cycle. 21-deoxycortisol was assayed in plasma by a specific radioimmunological method and its values were compared with those of 17-OH progesterone in heterozygous subjects with the classical and non-classical forms of 21-hydroxylase deficiency, and in the amniotic fluid of foetuses with this deficiency. Baseline concentrations of 21-deoxycortisol in the classical forms of 21-hydroxylase deficiency (n = 12; 55.36 to 186.6 ng/ml) and post-tetracosactide concentrations in non-classical late onset forms (n = 31; 4.04 to 47 ng/ml) were much higher than in normal subjects, thus making this steroid as sensitive as, or even more sensitive than 17-OH progesterone in diagnosing 21-hydroxylase deficiency. Post-tetracosactide assays of 21-deoxycortisol in 84 heterozygous subjects with 21-hydroxylase deficiency (0.70 to 5.40 ng/ml) enabled these subjects to be detected with a more than 90 percent sensitivity, which cannot be obtained with 17-OH progesterone assays. 21-deoxycortisol concentrations in the amniotic fluid of foetuses with 21-hydroxylase deficiency (n = 11; 0.391 to 0.930 ng/ml) were constantly superior to those observed in normal foetuses (n = 38; 0.034 to 0.221 ng/ml), so that the deficiency can be diagnosed with the steroid as easily as with 17-OH progesterone.

Corticosteroids (CS) are essential for fetal organ maturation; yet, knowledge of endogeneous CS and precursor levels throughout fetal life is limited. Therefore, unconjugated aldosterone (Aldo), corticosterone (B), 11-deoxycorticosterone (DOC), progesterone (P), 17 alpha-hydroxyprogesterone (17-OHP), 11-deoxycortisol (S), cortisol (F), and cortisone (E) were simultaneously determined by RIA after automated Sephadex LH-20 chromatography in 70 control samples of amniotic fluid (AF) obtained at all gestational ages between 14-42 weeks. Levels of the progestins P and 17-OHP slowly increased from means (+/- SE) of 14.7 +/- 2.8 and 1.63 +/- 0.21 ng/ml, respectively, in early gestation to maximum levels of 32.4 +/- 3.5 and 3.80 +/- 0.74 ng/ml at 36-38 weeks (P less than 0.005), then dropped significantly (P less than 0.01) to 19.2 +/- 2.2 and 1.58 +/- 0.22 ng/ml at term. All CS levels except E rose very markedly by 3- to 12-fold (P less than 0.0001) from the weeks 14-16 (DOC, 0.44 +/- 0.08; B, 1.49 +/- 0.23; Aldo, 0.043 +/- 0.012; S, 0.51 +/- 0.10; F, 5.96 +/- 0.93 ng/ml) until the 36-38th weeks (DOC, 3.50 +/- 0.66; B, 4.60 +/- 0.78; Aldo, 0.530 +/- 0.109; S, 6.00 +/- 0.75; F, 60.8 +/- 8.9 ng/ml). Term levels were significantly reduced (P less than 0.01) in the less active CS DOC (0.51 +/- 0.07 ng/ml), B (2.35 +/- 0.35 ng/ml), and S (1.14 +/- 0.14 ng/ml), whereas those of the biologically most potent CS Aldo and F declined less markedly (0.272 +/- 0.053 and 23.0 +/- 0.75 ng/ml, respectively, at 39-42 weeks). Levels of the inactive glucocorticoid E rose from 8.83 +/- 1.08 ng/ml at 14-16 weeks to 16.8 +/- 2.6 ng/ml at 31-35 weeks (P less than 0.01), then remained rather constant around 11.5 ng/ml until term. It is concluded that after the 25th week, large amounts of biologically active CS are available in AF which probably directly induce the final epithelial maturation of fetal lungs and intestinal tract.

Concentrations of unconjugated cortisol, 11-desoxycortisol, and 21-desoxycortisol were measured by radioimmunoassay in amniotic fluid throughout gestation. Cortisol levels rose from a median of 6.5 nanograms per milliliter prior to 20 weeks to 13.9 ng/ml at 28 to 37 weeks. Median levels of 11-desoxycortisol and 21-desoxycortisol were 2.6 ng/ml and 0.21 ng/ml, respectively, and did not change with advancing gestation. These normal values provide a basis for the application of assays of 11-desoxycortisol and 21-desoxycortisol in amniotic fluid in the prenatal diagnosis of congenital adrenal hyperplasia.

The sensitivities of monoclonal antibody-based enzyme immunoassays for 11-deoxycortisol using alkaline phosphatase (AP), horseradish peroxidase (HRP), beta-galactosidase (beta-GAL) and glucose oxidase (GOD) as labels were compared. The anti-11-deoxycortisol antibody used was that produced in ascites by inoculating antibody-secreting hybridoma cells into mice. Enzyme labeling of 11-deoxycortisol was carried out by the N-succinimidyl ester method. The activated ester of 4-(2-carboxyethylthio)-11-deoxycortisol was treated with each enzyme to give a homologous enzyme-labeled antigen. In the competitive immunoassay, the bound and free enzyme-labeled antigens were separated by a double antibody method and the enzymic activity of the immune precipitate was determined by colorimetric and fluorimetric methods. The AP activity was measured in three ways, using p-nitrophenyl phosphate, nicotinamide adenine dinucleotide phosphate (NADP), and 4-methylumbelliferyl phosphate as substrates. o-Nitrophenyl beta-D-galactopyranoside and 4-methylumbelliferyl beta-D-galactopyranoside were used for beta-GAL, and 3,3',5,5'-tetramethylbenzidine (TMB) and 3-(p-hydroxyphenyl)propionic acid (HPPA) for HRP. In the case of GOD, TMB and HPPA were used in combination with HRP. A dose-response curve with a high sensitivity was obtained in each 11-deoxycortisol assay system by the use of a minimum amount of the enzyme-labeled antigen at an appropriate dilution of monoclonal anti-11-deoxycortisol antibody (Ka = 2 x 10(10) M-1). The amounts of 11-deoxycortisol needed to displace 50% of the bound label ranged from 5 to 15 pg in the colorimetric methods, and 4-9 pg in the fluorimetric methods.(ABSTRACT TRUNCATED AT 250 WORDS)