Further evidence that there is more than one adrenal 21-hydroxylase system

The 21-hydroxylase activity of microsomes isolated from bovine adrenal cortex have been assayed using [21-3H]17-hydroxypregnenolone and [1,2-3H]17-hydroxyprogesterone as substrates. When the assays are performed in the presence of an NADH regenerating system, to inhibit steroid 3 beta-hydroxy isomerase-dehydrogenase activity, the microsomes oxidize the 3 beta-hydroxy-5-ene steroid at a rate of 0.37 nmol/min.nmol cytochrome P-450 and the 3-keto-4-ene steroid at a rate of 6.4 nmol/min.nmol. When the microsomes are solubilized with Triton CF-54 they lose the ability to oxidize the 3-hydroxy-5-ene steroid, while the specific activity of the microsomes for the 3-keto-4-ene steroid is enhanced 3-fold. In contrast, when the microsomes are solubilized with sodium cholate, their specific activity towards the 4-ene steroid is decreased by 50% while the specific activity for a low concentration of the 5-ene steroid, 1 microM, is unchanged. In addition, when the oxidations of the labeled steroids (at 1 microM) by the microsomes, are examined in the presence of unlabeled 17-hydroxyprogesterone (at 20 microM) the oxidation of the 3-keto-4-ene steroid is inhibited by 92% while the oxidation of the 3 beta-hydroxy-5-ene steroid is only inhibited by 20%. These results all suggest that there are at least two 21-hydroxylases in bovine adrenal tissue, one of which can utilize the 3-keto-4-ene steroids only, the other of which, in addition, can utilize the 3 beta-hydroxy-5-ene steroids as substrates.     

Urinary excretion of 5beta-pregnane-3alpha,20alpha,21-triol in human gestation

The steroids in urine from normal pregnant women have been studied. After extraction of conjugate steroids, solvolysis and enzymatic hydrolysis, the liberated steroids were separated by chromatography on Sephadex LH-20, and were analysed by gas-liquid chromatography and gas chromatography mass spectrometry. The following steroids were isolated and completely identified in the LH-20 fraction 7: 5beta-pregnane-3alpha,20alpha-diol, 5beta-pregnane-3alpha,17,20alpha-triol, 5beta-pregnane-3alpha,20alpha,21-triol and 5alpha-pregnane-3beta,16alpha,20alpha-triol. In addition, two metabolites tentatively identified as 5xi-pregnane-2xi,3xi,20xi-triol and 2xi,3xi,16xi-trihydroxy-5xi-pregnan-20-one, have not been reported as occcurring in urine from pregnant women. The 5beta-pregnane-3alpha,20alpha,21-triol was detected only in the third trimester of pregnancy and the urinary excretion values are between 320 and 650 microgram per 24 h. With the present data, it is not possible to establish the precursor(s) of this steroid. However, these results tentatively suggest that 5beta-pregnane-3alpha,20alpha,21-triol arises from foeto-placental unit.     

Serum 17-hydroxypregnenolone and 17-hydroxypregnenolone sulfate concentrations in patients with congenital adrenal hyperplasia due to 21-hydroxylase deficiency

Serum concentrations of 17-hydroxypregnenolone, 17-hydroxypregnenolone sulfate and 17-hydroxyprogesterone were measured simultaneously in patients with congenital adrenal hyperplasia due to 21-hydroxylase deficiency, using a combined radioimmunoassay method. All these precursor steroids were found to be markedly elevated in the sera of untreated patients with a salt-losing form of the disease, whereas, in untreated patients with a simple virilizing form, only the concentration of unconjugated steroids was increased and the 17-hydroxypregnenolone sulfate concentration remained within the normal range. Among the patients with a salt-losing form under maintenance therapy, these steroids were all still significantly increased in those on insufficient control, whereas only 17-hydroxyprogesterone was significantly but slightly increased in those on adequate control. Although the mechanism whereby the serum 17-hydroxypregnenolone sulfate concentration is not increased in the untreated simple virilizers is unknown, both a milder degree of 21-hydroxylase deficiency and a role of 17-hydroxypregnenolone sulfate in adrenal steroid production as a kind of supplier are suggested as possible explanations, especially in the neonatal period and early infancy. Thus, this study showed the serum concentrations of 17-hydroxypregnenolone and its sulfate together with 17-hydroxyprogesterone in patients with 21-hydroxylase deficiency in various conditions.     

A test for heterozygocity of 21-hydroxylase deficiency

Summary The urinary excretion of steroids was studied in 8 parents of children with congenital adrenal hyperplasia due to 21-hydroxylase deficiency of the simple virilizing and of the salt-losing type. Eight parents of normal children served as controls. 24-hour urines before and after the injection of 40 IU of ACTH were fractionated using gas liquid chromatography on glass capillary columns.Before stimulation no excretion of pregnanetriolone was detected in heterozygous and in normal parents. Following ACTH only heterozygotes showed an excretion of pregnanetriolone in the urine. This averaged 289 g per 24 h.Employing gas liquid chromatography on glass capillary columns heterozygous carriers of congenital adrenal hyperplasia due to 21-hydroxylase deficiency may reliably be detected by their increased urinary excretion of pregnanetriolone following ACTH.Supported by Deutsche Forschungsgemeinschaft, SFB 87, Project C₃.Presented in part at the Annual Meeting of the European Society for Paediatric Research, Budapest, August 21st–24th, 1975.     

Characterization of the major steroids present in amniotic fluid obtained between the 15th and 17th weeks of gestation

In pooled amniotic fluid obtained between the 15th and 17th weeks of gestation the concentration of free steroids and steroid glucuronides was found to be 40 micrograms/dl. The concentration of steroid monosulfates and disulfates was 19 micrograms/dl. About half of the characterized steroids are progesterone metabolites. The "fetal type" 3 beta-hydroxy-5-ene steroids were found exclusively in the sulfoconjugated form. Their concentration represents 20% of the total steroid content. The identification of two 15 beta-hydroxylated C21 steroids, 3 beta,15 beta,17 alpha-tridoxy-5-pregnen-20-one and 5-pregnene-3 beta,15 beta,17 alpha,20 alpha-tetrol isolated from mid-pregnancy amniotic fluid is reported here. Metabolites of cortisol and 17-deoxycorticosteroid metabolites had similar quantitative importance, 8.6 and 9.4%, respectively.     

Structural requirements for progesterone binding to the hepatic endoplasmic reticulum in the female rat

Microsomes isolated from the liver of the female rat specifically bind progesterone. The progesterone-microsomal complex shows highly specific characteristics. The binding is probably associated with the carbonyl groups at positions C-20 and C-3. Other steroids compete for microsomal binding sites less effectively. Competition for progesterone binding sites by other steroids in percentages: testosterone 33; testosterone propionate, 9; 17-methyltestosterone, 23.2; cortisol, 6.4; estradiol-17 beta, 1.8; 17 alpha-ethynyl estradiol, 4.7; mestranol, 1.0; norethynodrel, 4.5; ethisterone, 7.1; lynestrenol, 4.3; medroxyprogesterone, 23.3; medroxyprogesterone acetate, 15.2; 5 alpha-pregnane-3,20-dione, 47.6; 5 beta-pregnane-3,20-dione, 20.7; pregnenolone, 14.8; 6-methylpregnenolone, 1.2; 16 alpha-methylpregnenolone, 3.8%; 20 beta-hydroxy-4-pregnen-3-one, 2.8; 3 beta-hydroxy-5 alpha-pregnan-20-one, 5.2; 4-pregnene-3 beta, 20 beta-diol, 2.1; 11 alpha-hydroxyprogesterone 21.0; 16 alpha-hydroxyprogesterone, 7.9; 17-hydroxyprogesterone, 26.7; 16 alpha, 17-epoxyprogesterone, 2.7; 16 alpha-methylprogesterone, 3.8; 6-methylpregnenolone, 1.2; 16 alpha-methylpregnenolone, 3.8; promegestone, 27.0. 3 beta-Hydroxy-5 beta-pregnan-20-one, 3 alpha-hydroxy-5 beta-pregnan-20-one, 5-pregnene-3 beta,20 beta-diol, 5-pregnene-3 beta, 20 alpha-diol; 5 alpha-pregnane-3 beta, 20 beta-diol, 5 alpha-pregnane-3 beta, 20 alpha-diol, 5 beta-pregnane-3 alpha, 20 alpha-diol, 5 beta-pregnane-3 alpha, 20 alpha-diol diacetate, 5 beta-pregnane-3 alpha, 20 beta-diol, 3 alpha, 17-dihydroxy-5 beta-pregnan-20-one, 17-hydroxypregnenolone, 6-methyl-17-hydroxypregnenolone, pregnenolone-16 alpha-carbonitrile, dihydrotestosterone and cholesterol show no competition at all. The varying degree of competition by different steroids is unrelated to their lipid solubility.     

Ovine steroid 17alpha-hydroxylase cytochrome P450: characteristics of the hydroxylase and lyase activities of the adrenal cortex enzyme

The steroid 17-hydroxylase cytochrome P450 (CYP17) found in mammalian adrenal and gonadal tissues typically exhibits not only steroid 17-hydroxylase activity but also C-17,20-lyase activity. These two reactions, catalyzed by CYP17, allow for the biosynthesis of the glucocorticoids in the adrenal cortex, as a result of the 17-hydroxylase activity, and for the biosynthesis of androgenic C(19) steroids in the adrenal cortex and gonads as a result of the additional lyase activity. A major difference between species with regard to adrenal steroidogenesis resides in the lyase activity of CYP17 toward the hydroxylated intermediates and in the fact that the secretion of C(19) steroids takes place, in some species, exclusively in the gonads. Ovine CYP17 expressed in HEK 293 cells converts progesterone to 17-hydroxyprogesterone and pregnenolone to dehydroepiandrosterone via 17-hydroxypregnenolone. In ovine adrenal microsomes, minimal if any lyase activity was observed toward either progesterone or pregnenolone. Others have demonstrated the involvement of cytochrome b(5) in the augmentation of CYP17 lyase activity. Although the presence of cytochrome b(5) in ovine adrenocortical microsomes was established, ovine adrenal microsomes did not convert pregnenolone or 17-hydroxypregnenolone to dehydroepiandrosterone. Furthermore the addition of purified ovine cytochrome b(5) to ovine adrenal microsomes did not promote lyase activity. We conclude that, in the ovine adrenal cortex, factors other than cytochrome b(5) influence the lyase activity of ovine CYP17.     

Phospholipases modulate the rat testicular androgen biosynthetic pathway in vitro

The role of membrane phospholipids in testicular androgen biosynthesis was investigated by monitoring the effects of phospholipase treatments on the activities of the steroid transforming enzymes. Androgen biosynthesis in untreated rat testicular microsomes was examined by monitoring the temporal appearance of pregnenolone metabolites and was found to proceed through the 4-ene route. When phospholipase A2 was included, the 5-ene steroids 17-hydroxypregnenolone and dehydroepiandrosterone (DHEA) were formed in greater quantities, and the production of 4-ene steroids was reduced indicating that the conversion of 5-ene steroids to the 4-ene configuration was inhibited by phospholipase A2 treatment. Phospholipase C, in addition to inhibiting this step, also inhibited the conversion of C21 steroids to C19 steroids. When the enzymatic steps were measured individually, phospholipase A2 inhibited 3 beta-hydroxysteroid dehydrogenase-isomerase (3 beta-HSD-Isomerase) with an ED50 of 73 mU/ml but had no effect on the activities of 17-hydroxylase, C-17, 20 lyase, or 17 beta-hydroxysteroid dehydrogenase (17 beta-HSD). However, though phospholipase C treatment inhibited 3 beta-HSD-Isomerase, it caused less inhibition (the ED50 value was 149 mU/ml). Furthermore, 17-hydroxylase and C-17, 20 lyase activities were also inhibited by phospholipase C treatment (ED50 values were 410 and 343 mU/ml, respectively), but no effect on 17 beta-HSD was observed. The differences in the apparent phospholipid requirements of the steroidogenic enzymes provides the possibility that the metabolic fate of pregnenolone may be regulated by changes in the phospholipid composition of the microenvironment.     

Unconjugated and sulfoconjugated steroids in plasma and zones of the adrenal cortex of the guinea pig

The following steroids were measured in their unconjugated and sulfoconjugated forms in plasma and in the outer and inner zones of the adrenal cortex of the guinea pig: pregnenolone, 17-hydroxypregnenolone, 21-hydroxypregnenolone, dehydroepiandrosterone and deoxycorticosterone. In plasma, pregnenolone and 21-hydroxypregnenolone were the predominant unconjugated steroids with concentrations 10-30 times higher than the other three steroids. Among the sulfoconjugated steroids, pregnenolone sulfate had a concentration 25-50 times higher than the other sulfoconjugates. For each steroid except 21-hydroxypregnenolone the sulfoconjugated form was present in a concentration 2-7 times higher than the unconjugated form. In the adrenal cortex, the content of 21-hydroxypregnenolone was significantly higher in the outer zone than in the inner zone and was present in amounts 3-100 times greater than the other unconjugated steroids in the outer zone. On the other hand, the content of pregnenolone was significantly greater in the inner zone than the outer zone, and was present in amounts 3-80 times greater than the other unconjugated steroids in the inner zone. With the exception of 21-hydroxypregnenolone and deoxycorticosterone, the steroid sulfoconjugates were significantly higher in the inner cortical zone. As in plasma, pregnenolone sulfate was the most abundant sulfoconjugated steroid. This report also describes preliminary studies concerning sulfurylated hydroxyl groups in different positions of 21-hydroxypregnenolone. The sulfoconjugate was prepared by using partially purified steroid sulfotransferase from the guinea pig adrenal. The results obtained indicated that of the total 21-hydroxypregnenolone conjugate formed, approximately 40% was the 21-sulfate and 20% the 3-sulfate, whereas 40% was non-hydrolyzable with the techniques used and was not further characterized.     

Isolation of urinary C-20 alpha- and C-20 beta-hydroxy-C21-steroid metabolites in cases of congenital adrenal hyperplasia, postpubertal virilizing syndrome and polycystic ovary syndrome.

The proportion of 20 alpha/20 beta-epimers of the urinary C21-steroid metabolites was estimated in normal volunteers and in patients with congenital adrenal hyperplasia (CAH), postpubertal virilizing syndrome (PVS) and polycystic ovary syndrome (POS). In the normal individuals 20 alpha- and 20 beta-epimers of 5 beta-pregnane-3 alpha, 17 alpha, 20-triol (pregnanetriol) and 5-pregnene-3 beta, 17 alpha, 20-triol (5-pregnenetriol) were measured. In the other case 20 alpha- and 20 beta-epimers of the characteristic, representative metabolites were also investigated. In 26 of 27 normal persons and in all the patients with normotensive CAH, PVS and POS the 20 beta-epimer comprised 0-10% of the total pregnanetriol. The 20 beta-epimer of 5-pregnenetriol was found in only one normal case (6% of the total). The percentage of 20 beta-epimers of 3 alpha, 17 alpha, 20-trihydroxy-5 beta-pregnan-11-one, 5 beta-pregnane-3 alpha, 11 beta, 17 alpha, 20-tetrol (in the normotensive CAH, PVS and POS) and 5 alpha- or 5 beta-pregnane-3 alpha, 17 alpha, 20, 21-tetrol (in the hypertensive CAH) varied from nil to 76%. The effect of functional groups and the stereochemistry of the molecule on the direction of C-20-keto group reduction is discussed; the existence of additional factors determining this reduction in certain pathological conditions is suggested.     

Discriminatory inhibition of adrenocortical 17 alpha-hydroxylase activity by inhibitors of cholesterol side chain cleavage cytochrome P-450

Two inhibitors of the cholesterol side chain cleavage reaction were tested for their ability to inhibit bovine adrenocortical 17 alpha-hydroxylase and 21-hydroxylase activities. One inhibitor, 22-amino-23,24-bisnor-5-cholen-3 beta-ol (22-ABC), was found to be a potent inhibitor of 17 alpha-hydroxylation of either progesterone or pregnenolone but was inactive on 21-hydroxylase activity. 22-ABC was found to be a competitive inhibitor of 17 alpha-hydroxylase (cytochrome P-45017 alpha) activity, having an apparent inhibitor constant of 29 nM when using pregnenolone as the substrate. Spectral binding studies showed that 22-ABC produces a type II difference spectrum when added to a bovine adrenocortical microsomal preparation, due presumably to a coordination of its amine nitrogen atom to the heme-iron of cytochrome P-45017 alpha. The second cholesterol side chain cleavage inhibitor tested, (20R)-20-phenyl-5-pregnene-3 beta,20-diol (20-PPD), was found not to inhibit either the 21- or 17 alpha-hydroxylase activities. It is proposed that the phenyl group projecting from C-20 of 20-PPD prevents this steroid from binding to cytochrome P-45017 alpha. The discriminatory interaction of these two steroids with adrenocortical cytochromes P-450 provides some insight with respect to possible structural features of the active-site regions of these enzymes.     

Identification of 2 alpha-hydroxy-4-pregnene-3,20-dione in human pregnancy urine.

2 alpha-Hydroxyprogesterone (2 alpha-hydroxy-4-pregnene-3,20-dione) was identified in human late pregnancy urine by liquid-gel chromatography, GLC and GC-MS. In addition, the following 2-hydroxylated C21 steroids were found and identified as 2 zeta-hydroxy-5 zeta-pregnane-3,20-dione, 2 zeta,20 zeta-dihydroxy-4-pregnen-3-one, 2 alpha,3 alpha-dihydroxy-5 alpha- (and 5 beta)-pregnan-20-one, two isomers of pregnane-2,3,20-triol and 2 zeta,3 zeta,16 zeta-trihydroxy-5 zeta-pregnan-20-one.     

Formation of 5,16-androstadien-3 beta-ol from pregnenolone in human testicular microsomes

A microsomal fraction of testicular tissue from a patient with prostatic carcinoma was incubated with [4-14C]pregnenolone in the presence of an NADPH-generating system for different periods of time. The metabolites were separated by Sephadex LH-20 column chromatography and then identified by thin-layer chromatography, radio-gas chromatography, and crystallization studies. Pregnenolone was converted to a major metabolite, 5-androstene-3 beta,17 beta-diol via 17-hydroxypregnenolone and then dehydroepiandrosterone. Another major metabolite was 5,16-androstadien-3 beta-ol, which increased with the time of incubation and accumulated in the incubation medium. After 120 min of incubation, 34.6% of the precursor was converted to 5-androstene-3 beta,17 beta-diol and 15.1% to 5,16-androstadien-3 beta-ol. In addition to the above-mentioned steroids, 16 alpha-hydroxypregnenolone, 5-pregnene-3 beta,20 alpha-diol, and 5-androstene-3 beta,17 alpha-diol were identified as minor metabolites of pregnenolone. From these results it was concluded that human testicular microsomes possess enzymic activities for the synthesis of 5,16-androstadien-3 beta-ol, as well as androgens from pregnenolone.     

Urinary analysis of 16(5alpha)-androsten-3alpha-ol by gas chromatography/combustion/isotope ratio mass spectrometry: implications in anti-doping analysis

We present a method for the analysis of urinary 16(5alpha)-androsten-3alpha-ol together with 5beta-pregnane-3alpha,20alpha-diol and four testosterone metabolites: androsterone (Andro), etiocholanolone (Etio), 5alpha-androstane-3alpha,17beta-diol (5alphaA), 5beta-androstane-3alpha,17beta-diol (5betaA) by means of gas chromatography/combustion/isotopic ratio mass spectrometry (GC/C/IRMS). The within-assay and between-assay precision S.D.s of the investigated steroids were lower than 0.3 and 0.6 per thousand, respectively. A comparative study on a population composed of 20 subjects has shown that the differences of the intra-individual delta(13)C-values for 16(5alpha)-androsten-3alpha-ol and 5beta-pregnane-3alpha,20alpha-diol are less than 0.9 per thousand. Thereafter, the method has been applied in the frame of an excretion study following oral ingestion of 50 mg DHEA initially and oral ingestion of 50mg pregnenolone 48 h later. Our findings show that administration of DHEA does not affect the isotopic ratio values of 16(5alpha)-androsten-3alpha-ol and 5beta-pregnane-3alpha,20alpha-diol, whereas the isotopic ratio values of 5beta-pregnane-3alpha,20alpha-diol vary by more 5 per thousand upon ingestion of pregnenolone. We have observed delta(13)C-value changes lower than 1 per thousand for 16(5alpha)-androsten-3alpha-ol, though pregnenolone is a precursor of the 16-ene steroids. In contrast to 5beta-pregnane-3alpha,20alpha-diol, the 16-ene steroid may be used as an endogenous reference compound when pregnenolone is administered.     

Intratesticular unconjugated steroids in elderly men

As an extension of our studies on the influence of age on testicular function and with the aim of detecting whether the decline in testosterone production by aged testes is accompanied by a block in the biosynthetic chain leading from cholesterol to testosterone, we determined in the testis of young and elderly men, who died suddenly either from a cardiac incident or from accident, intratesticular steroids: pregnenolone, 17 hydroxypregnenolone (3 beta, 17 alpha-dihydroxy-5-pregnen-20-one), dehydroepiandrosterone, androstenediol, (5-androsten-3 beta, 17 beta-diol), progesterone, 17 hydroxyprogesterone, androstenedione, 17 beta-estradiol as well as testosterone, dihydrotestosterone (5 alpha-androstan-17 beta-ol-3-one) and androstanediol (5 alpha androstane-3 alpha, 17 beta-diol). The intratesticular steroid pattern in elderly men was essentially characterized by a decrease of the 5-ene steroid concentration, whereas we did not observe a decrease in the 4-ene steroids, progesterone concentration being even significantly higher in the aged testes. There was no evidence for a decrease in either lyase or 17-hydroxylase activity. It is suggested that the steroid pattern as observed in the aged testes is the consequence of a decreased oxygen supply, due to a decreased testicular perfusion.     

Regulation of steroid and steroid sulfate production and aromatase activity in cultured human granulosa-luteal cells

The regulation of the production of steroids and steroid sulfates and the activity of aromatase in human luteinized granulosa cells were investigated. The cells were cultured for 48 h in the presence or absence of hCG and FSH. Basal production of pregnenolone (Pre, 0.3 +/- 0.03 ng/micrograms protein) and progesterone (P, 19.3 +/- 1.7 ng/micrograms protein) were high compared with that of other steroids beyond P in the steroidogenic pathway. The concentration of 17 alpha-hydroxyprogesterone (17-OHP) was lower 0.17 +/- 0.06 ng/micrograms and that of other steroids in the 4-ene and 5-ene pathways and steroid sulfates less than 0.05 ng/micrograms. Both hCG and FSH (100 ng/ml) stimulated the production of Pre and P 3- to 5-fold, but only minimal stimulation of other steroids and steroid sulfates was observed. Aromatase activity of granulosa-luteal cells was measured from the rate of formation of 3H2O from 1 beta-[3H]androstenedione (1 beta[3H]A) after exposing the cells to hCG, FSH or estradiol (E2) for 48 h. Basal aromatase activity was relatively low, but hCG and FSH stimulated aromatase 8- and 4-fold, respectively. The incubation of granulosa-luteal cells with E2 did not affect basal aromatase activity, but E2 augmented FSH-stimulated aromatase 1.4-fold (P less than 0.025). The results suggest that there is low 17 alpha-hydroxylase and steroid sulfokinase activity in human granulosa-luteal cells. Aromatase activity in these cells is regulated by both hCG and FSH, and intra-ovarian estrogens may regulate granulosa cell aromatase activity.     

Steroids in newborns and infants identification by combined gas chromatography-mass spectrometry of the major steroids excreted by a newborn chimpanzee (Pan troglodytes)

The following steroids have been identified by combined gas chromatography-mass spectrometry in a urine specimen collected from a newborn chimpanzee; 5-androstene-3β, 17α-diol, 3β,16α (and 16β)-dihydroxy-5-androsten-17-one, 5-androstene-3β, 16α, 17β-triol, 5-androstene-3β, 16β, 17α-triol, 5-pregnene-3β, 20α-diol, 5-pregnene-3β, 20α, 21-triol, 3β,21-dihydroxy-5-pregnen-20-one, 3β, 16α-dihydroxy-5-pregnen-20-one, 5-Piegnene-3β, 16α,20α, 21-tetrol, 5-pregnene-3β,17α, 20ξ, 21-tetrol androstenetriolones and androstenetetrols.     

Capillary gas chromatography as a tool for characterization of urinary steroid excretion in patients with congenital adrenal hyperplasia

Urinary steroid excretion was studied by capillary gas chromatography in 23 patients with congenital adrenal hyperplasia. In 5 patients the estimated excretion rates of pregnanetriol were in or below the normal range and 7 patients presented supranormal excretion rates of tetrahydro-cortisone and/or other glucocorticoid metabolites. Deficiency of 21-hydroxylase was nevertheless demonstrated in each patient by an increased ratio of excreted precursors vs products of 21-hydroxylase, e.g. of pregnanetriol/tetrahydro-cortisone. Due to this relative deficiency of glucocorticoids the patients' steroid excretion was further characterized by a predominance of 5 alpha-hydrogenated C19O3 metabolites (11-keto-androsterone, 11-hydroxy-androsterone) over their 5 beta-hydrogenated homologues (11-keto-etiocholanolone, 11-hydroxy-etiocholanolone). An apparent preponderance in the excretion of pregnenetriol over that of pregnanetriol was found in 4 patients, but the presence of pregnenetriol was not confirmed by mass spectrometry following prepurification of the urine samples by thin-layer chromatography indicating interference of an unidentified steroid metabolite with the initial gas chromatographic analysis. The simultaneous determination of steroids serving as precursors or products of 21-hydroxylase by capillary gas chromatography helps to establish the diagnosis of 21-hydroxylase deficiency and to characterize the pattern of steroid excretion in this syndrome even in patients where the estimation of single urinary steroids may lead to erroneous conclusions.     

The measurement of 11 beta-hydroxy-4-pregnene-3,20-dione (21-deoxycorticosterone) by radioimmunoassay in human plasma

A specific radioimmunoassay (RIA) method is described for the determination of 21-deoxycorticosterone (21 DB) in human plasma. 21-Deoxycorticosterone-3-(O-carboxymethyl) oxime-bovine serum albumin conjugate was used to generate antisera in rabbits. Steroids which reacted significantly with the antisera were found to be progesterone, pregnenolone, corticosterone and 11-oxo progesterone. However, after extraction of plasma and column chromatography on Celite, all these steroids were separated from 21-deoxycorticosterone and consequently did not interfere with the radioimmunoassay. The intra- and interassays coefficients of variation were 8% and 11% respectively. Mean plasma 21-deoxycorticosterone level for healthy subjects was very low: 17.8 +/- 14.8 pmol/l (mean +/- SD) with no statistical difference between males and females. During the ACTH stimulation test, the 21-deoxycorticosterone levels of healthy subjects increased to 84.7 +/- 26.3 pmol/l (mean +/- SD) for males and 79.3 +/- 31.6 pmol/l (mean +/- SD) for females. Consequently high levels of plasma 21-deoxycorticosterone were found in treated patients suffering from congenital adrenal hyperplasia (CAH) with 21-hydroxylase deficiency, particularly in CAH salt-losers with high plasma renin activity (PRA), where the plasma level reached 40,545 pmol/l. Thus, 21-deoxycorticosterone may be a new marker for adrenal 21-hydroxylase deficiency.