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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.

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.

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.

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.

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.