Steroid profiling for the diagnosis of congenital adrenal hyperplasia by microbore ultra-performance liquid chromatography-tandem mass spectrometry.

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.

Associations of residential greenness, ambient air pollution, biological sex, and glucocorticoids levels in rural China.

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 PM1, PM2.5, and PM10. A general linear regression model was performed to estimate the associations of NDVI500-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 NDVI500-m value in the total population was associated with a decrease in cortisol levels (ß = -0.063, 95 % confidence interval (CI): - 0.118, - 0.008), and 11-deoxycortisol levels (ß = -0.118, 95 % CI: -0.190, -0.047), as well as an increase in cortisone levels (ß = 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, PM2.5: P interaction=:0.018; PM10: 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.

A preliminary study on the relationship between environmental endocrine disruptors and precocious puberty in girls.

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.

Time-dependent association of glucocorticoids with adverse outcome in community-acquired pneumonia: a 6-year prospective cohort study.

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.

[Mass spectrometry for steroid assays]. / Dosages des stéroïdes par spectrométrie de masse.

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.

Open-sandwich enzyme immunoassay for one-step noncompetitive detection of corticosteroid 11-deoxycortisol.

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.

Determination of adrenal steroids by microfluidic chip using micellar electrokinetic chromatography.

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.

Newborn screening for congenital adrenal hyperplasia: additional steroid profile using liquid chromatography-tandem mass spectrometry.

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.

Identification of 17,20beta,21-trihydroxy-4-pregnen-3-one as an oocyte maturation-inducing steroid in black porgy, Acanthopagrus schlegeli.

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.

Analysis of 21-deoxycortisol, a marker of congenital adrenal hyperplasia, in blood by atmospheric pressure chemical ionization and electrospray ionization using multiple reaction monitoring.

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.

Delta sleep response to metyrapone in post-traumatic stress disorder.

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.

Plasma steroids in mature common dentex (Dentex dentex) stimulated with a gonadotropin-releasing hormone agonist.

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.

Prenatal dexamethasone treatment in pregnancies at risk for congenital adrenal hyperplasia due to 21-hydroxylase deficiency: effect on midgestational amniotic fluid steroid levels.

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.

[Analysis of corticoids in adrenal glands by high performance liquid chromatography (HPLC)].

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.

Bovine adrenal cytochrome P-450(11 beta)-mediated conversion of 11-deoxycortisol to 18- and 19-hydroxy derivatives; structural analysis by 1H-NMR.

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 steroid hormonal milieu of the undisturbed human fetus and mother at 16-20 weeks gestation.

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.

Identification of, and development of radioimmunoassays for 17 alpha, 21-dihydroxy-4-pregnene-3,20-dione and 3 alpha,17 alpha, 21-trihydroxy-5 beta-pregnan-20-one in the ovaries of mature plaice (Pleuronectes platessa).

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.