Diagnosis of 21-hydroxylase deficiency by urinary metabolite ratios using gas chromatography-mass spectrometry analysis: Reference values for neonates and infants.

One major issue of newborn screening programs for 21-hydroxylase deficiency (21OHD) is the high rate of false-positive results, especially in preterm neonates. Urinary steroid metabolite analysis using gas chromatography-mass spectrometry (GC-MS) is suitable as a confirmatory diagnostic tool. The objective of this study was to analyze retrospectively diagnostic metabolite ratios in neonates and infants with and without 21OHD using GC-MS with emphasis on glucocorticoid metabolism, and to develop reference values for the steroid metabolite ratios for the diagnosis of 21OHD. We retrospectively analyzed urinary steroid hormone metabolites determined by GC-MS of 95 untreated neonates and infants with 21OHD (1-148 days), and 261 neonates and infants (100 preterms) without 21OHD (0-217 days). Metabolites of 17α-hydroxyprogesterone showed specificities below 98%, whereas the 21-deoxycortisol metabolite pregnanetriolone clearly separated 21OHD from non-21OHD subjects. The best diagnostic ratio for 21OHD was pregnanetriolone to 6α-hydroxy-tetrahydrocortisone. The lowest value of this ratio in the 21OHD group (0.47) was at least eight times higher than the highest values in the non-21OHD group (0.055). We have given appropriate reference values for steroid metabolite ratios in the largest 21OHD cohort so far described. Consideration of glucocorticoid metabolism, especially the use of typical neonatal 6α-hydroxylates metabolites, leads to improvement of diagnostic metabolite ratios.

The effects of sex, age and commensal way of life on levels of fecal glucocorticoid metabolites in spiny mice (Acomys cahirinus).

We studied levels of fecal glucocorticoid metabolites (GCM) in a social rodent - Egyptian spiny mouse. As breeding adults are socially dominant over subadults, and adolescent males are driven away by the dominant males, we addressed the question whether animals within extended families are stressed differently depending upon their social category. In addition, we evaluated whether there are differences between non-commensal (outdoor) and commensal (adapted to human settlements) populations. Concentrations of fecal GCM were assessed from samples collected in a special cage that allowed continuous individual sampling of undisturbed mice housed as a semi-natural social unit. First we performed an ACTH challenge test to validate two enzyme immunoassays (EIA): a 5alpha-pregnane-3beta,11beta,21-triol-20-one EIA and an 11-oxoetiocholanolone EIA to measure a group of fecal GCM in this species. Next we monitored concentrations of fecal GCM in 68 individuals belonging to 10 family groups and two populations. Commensal spiny mice showed higher fecal GCM levels than non-commensal ones. No effect of age (i.e., social dominance) and only a small effect of sex (in the commensal population only, with males exhibiting lower values) on fecal GCM levels were found. On the other hand, considerable variations in measured fecal GCM between family groups were revealed, indicating that the social settings of the particular group play an important role.

Maternal urinary steroid profiles in prenatal diagnosis of Smith-Lemli-Opitz syndrome: first patient series comparing biochemical and molecular studies.

Smith-Lemli-Opitz syndrome (SLOS) is an autosomal recessive disorder caused by reduced activity of 7-dehydrocholesterol (7DHC) reductase, resulting in a decreased level of cholesterol and increased concentrations of 7DHC and 8DHC in body fluids and tissues. Ten pregnancies at 25% risk of SLOS underwent prenatal testing. Diagnostic studies included DHCR7 mutation analysis in chorionic villus samples, amniotic fluid sterol analysis and serial measurements of oestriol (E3), pregnanetriol (PT), 7-dehydropregnanetriol (7DHPT) and 8-dehydroesteriol (8DHE3) concentrations in maternal urine samples obtained between 9 and 20 weeks of gestation. All tests were diagnostic and revealed nine unaffected foetuses (two normal homozygotes and seven DHCR7 heterozygotes) and one affected foetus. In the affected pregnancy, 7DHC and 8DHC in amniotic fluid were 9.87 and 3.7 microg/ml, respectively [reference range (RR) 0.0026 +/- 0.0015 microg/ml and not detectable, respectively] and maternal urinary steroid analyses showed increased ratios of 7DHPT/PT and 8DHE3/E3 of 0.74 and 1.7, respectively (RR 0-0.0147 and 0-0.019). In the heterozygous foetuses, 7DHPT/PT and 8DHE3/E3 ratios did not exceed those found in 48 normal controls. This is the first series of prenatal diagnostic testing for SLOS where non-invasive biochemical testing was performed in tandem with invasive diagnostic testing. We conclude that steroid measurements in maternal urine are a reliable means of prenatal diagnosis for SLOS.

Chemical synthesis of 7- and 8-dehydro derivatives of pregnane-3,17alpha,20-triols, potential steroid metabolites in Smith-Lemli-Opitz syndrome.

Pregnane-3,17 alpha,20-triols bearing unsaturation at delta(7), delta(8), delta(5,7), or delta(5,8) have been tentatively identified as steroid metabolites in Smith-Lemli-Opitz syndrome (SLOS). Starting with 17 alpha-hydroxypregnenolone diacetate, we have synthesized 13 unsaturated C(21) triols by four different routes in one to four steps. These multifunctional steroids were prepared by a series of regio- and stereoselective transformations chosen to minimize facile olefin isomerization and 17-deoxygenation. The results include a study of stereoselectivity in the reduction of 17 alpha-hydroxy-20-ketosteroids, an alternative method for reducing diethyl azodicarboxylate adducts of delta(5,7) steroids, and an efficient oxidation-isomerization of a delta(5,7) steroid using cholesterol oxidase. The 13 triols and their synthetic precursors were fully characterized by 1H and 13C nuclear magnetic resonance (NMR) spectroscopy. The NMR data, together with molecular modeling, indicated unanticipated conformational heterogeneity for two synthetic intermediates, 17 alpha-hydroxypregna-4,7-diene-3,20-dione and 17 alpha-hydroxy-5 beta-pregn-7-ene-3,20-dione. The unsaturated C(21) triols are useful as reference standards to study adrenal steroid production in SLOS and to develop methods for pre- and postnatal diagnosis of this congenital disorder.

In vitro biosynthesis of steroids, including 11-deoxycortisol and 5 alpha-pregnane-3 beta,7 alpha,17,20 beta-tetrol, by ovaries of the goldfish Carassius auratus during the stage of oocyte final maturation.

To examine the production of steroids with potential oocyte maturation-inducing or pheromonal activity in the goldfish (Carassius auratus) we have incubated mature ovaries of this species with 17-[3H]hydroxyprogesterone. The metabolites in the unconjugated, glucuronide, and sulfate fractions were identified by chromatography, microchemical reaction, and, in most cases, crystallization to constant specific activity. A major metabolite, present in all three fractions, was tentatively identified as 5 alpha-pregnane-3 beta,7 alpha,17,20 beta-tetrol. Although 17,20 beta-dihydroxy-4-pregnen-3-one (17,20 beta-P) was found in only low yield (as a sulfate), the presence of the tetrol indicates that it is synthesized in high yield but very rapidly metabolized. The relative proportions of 17,20 alpha-dihydroxy-4-pregnen-3-one (17,20 alpha-P), 11-deoxycortisol (17,21-dihydroxy-4-pregnen-3,20-dione) and 17,20 beta,21-trihydroxy-4-pregnen-3-one (17,20 beta,21-P) varied significantly between incubations and may be affected by the maturational state of the ovary or the method used to stimulate oocyte maturation. Testosterone was present predominantly as its glucuronide. Significant production of glucuronides and sulfates was observed in all incubations. Twenty-five to 30% of the radioactivity remained associated with the tissue, but the distribution of activity between the metabolites did not differ greatly from that found in the medium. These results indicate that 11-deoxycortisol and its 20 beta-reduced derivative (17,20 beta,21-P) may be significant in spawning female goldfish.

Synthesis of 20 alpha-hydroxylated steroids by ovaries of the dab (Limanda limanda).

Dab (Limanda limanda) ovarian fragments were incubated in vitro with either [4,7-3H]pregnenolone or 17 alpha-hydroxy[1,2,6,7-3H]progesterone to investigate the pattern of steroidogenesis. A major enzyme found in the dab ovary was 20 alpha-hydroxysteroid dehydrogenase. Among the steroids that were tentatively identified in ovarian incubates were 17 alpha,20 alpha-dihydroxy-4-pregnen-3-one (17,20 alpha-P). 17 alpha,20 alpha-dihydroxy-5 beta-pregnan-3-one, 3 beta, 17 alpha,20 alpha-trihydroxy-5 beta-pregnane (3 beta,17,20 alpha-P-5 beta), and 3 alpha,17 alpha,20 alpha-trihydroxy-5 beta-pregnane. The presence of these steroids in plasma of mature female and male dabs was studied by radioimmunoassay. The antiserum was raised against 17,20 alpha-P. The 17,20 alpha-[3H]P label was produced by incubating place milt with 17 alpha-hydroxy [3H]progesterone. The radioimmunoassay was shown to have a high cross-reaction with the 5 beta-reduced analogues of 17,20 alpha-P and was therefore used, in conjunction with thin-layer chromatography, to measure the steroids. High concentrations of both 17,20 alpha-P and 3 beta,17,20 alpha-P-5 beta found in female and male dab plasma. The possible role of these steroids is discussed.

5 beta-pregnane-3 alpha,6 alpha,17 alpha,20 beta-tetrol and 5 beta-pregnane-3 alpha,6 alpha,17 alpha-triol-20-one: steroids of ovarian origin in the African catfish, Clarias gariepinus, during oocyte maturation.

At the stage of oocyte maturation, three very polar steroids were demonstrated by in vitro incubations with tritiated pregnenolone in the ovaries of the African catfish, Clarias gariepinus. Two of these compounds could be identified by chromatography, derivatization, and oxidation as 5 beta-pregnane-3 alpha,6 alpha,17 alpha,20 beta-tetrol and 5 beta-pregnane-3 alpha,6 alpha,-17 alpha-triol-20-one. Blood plasma analysis by means of gas chromatography followed by mass spectrometry confirmed the presence of these steroids with selected ion monitoring. The occurrence of the five most characteristic ions of each steroid was demonstrated at the proper retention times and with the correct abundance ratios. These very polar steroids, which were identified in vitro and in vivo, might have a function in maturation and ovulation induction.

Abnormal steroid excretion in gestational trophoblastic disease complicated by ovarian theca-lutein cysts.

Serum and urine steroids were examined in two subjects with trophoblastic disease accompanied by large ovarian theca-lutein cysts and compared with those from 10 patients with trophoblastic disease but without palpable cysts. In the patients without cysts normal values were obtained for serum oestradiol, progesterone, 17 alpha-hydroxyprogesterone and androstenedione, and for urinary total oestrogens, pregnanediol, pregnanetriol, and 17-oxosteroids. Nineteen urinary steroid metabolites, quantified by capillary gas-liquid chromatography, were either within reference limits or marginally raised. In several cases relatively minor increases in serum testosterone and cortisol and urinary free cortisol were observed. In contrast, the subjects with cysts showed pronounced excesses of androgen metabolites, 17 alpha-hydroxypregnenolone, pregnanediol, and pregnanetriol, and both exhibited a similar pattern of unusual additional metabolites. The profiles superficially resembled those seen in 21-hydroxylase deficiency adrenogenital syndrome, but there were important discrepancies reflecting known differences in ovarian and adrenal steroid metabolism. Chemotherapy led to decline of human chorionic gonadotrophin concentrations, regression of the cysts, and return to normal of the steroid profile. Excess steroids in the patients with cysts may have originated in the ovary rather than in the trophoblastic tissue.

Effect of hydrocortisone dose schedule on adrenal steroid secretion in congenital adrenal hyperplasia.

To explore the potential effect of dose schedule on the adrenal suppressive action of hydrocortisone in congenital adrenal hyperplasia, eight patients (six with 21-hydroxylase deficiency and two with 11-hydroxylase deficiency) were given five different dose schedules. Two of the schedules used single daily doses (morning or evening), two twice daily doses (two-thirds dose in the morning or evening), one and three equal doses at morning, noon, and night. Each dose schedule used the same total daily hydrocortisone dose (12.5 mg/m2/day), which is within the normal range of hydrocortisone production rate. Each schedule was given for 4 to 6 weeks. The different dose schedules caused the predicted alterations in the temporal pattern of adrenal steroid levels, with the greatest apparent suppression during the 2 to 4 hours after each dose. None of the schedules, however, caused significant differences in the mean 24-hour plasma concentration of 17-hydroxyprogesterone (21-hydroxylase deficiency) or 11-deoxycortisol (11-hydroxylase deficiency) or in the 24-hour urine pregnanetriol or 17-ketosteroid concentrations, either in the six patients undertreated at the dose of 12.5 mg/m2/day or in the two patients adequately treated. Nocturnal administration of all or a part of the daily dose did not improve adrenal suppression. These observations suggest that treatment of congenital adrenal hyperplasia with a once-a-day hydrocortisone dose schedule may be as effective as conventional multiple-dose schedules. Until this hypothesis has been tested by more extended clinical studies, however, we do not recommend a once-a-day schedule. Regardless of the dose schedule, the total daily hydrocortisone dose must be adjusted to achieve a normal rate of growth and bone age advancement.

GLC/MS identification of new pregnenolone metabolites in confluent embryonic rat fibroblast cultures.

Four new metabolites and their fatty acid derivatives have been identified as components of the steroid metabolic pathway in rat embryo fibroblasts grown during the confluent phase in the presence of [4-14C]-pregnenolone and [4-14C]-progesterone. These compounds are 3 alpha, 20 alpha and 3 beta, 20 alpha-dihydroxy-5 alpha-pregnanes, 4-pregnene-3 alpha, 20 alpha-diol and 5 alpha-pregnane-3 beta, 20 alpha, 21-triol. The labelled free and esterified steroids synthetized in the cells were released into the culture medium. The purpose of this study was to demonstrate that, besides t.l.c. enzymatic and chemical reactions, g.l.c./mass spectrometry coupling is a powerful tool for identifying the structures of sub-microgram amounts of radiolabelled steroids. The consequent metabolic features are discussed in terms of steroid cellular biosynthesis and kinetics.

A case of adrenogenital syndrome with aberrant 11beta-hydroxylation.

A 17 year old female patient with hypertension, amenorrhoea and hirsutism was found to have subnormal levels of plasma and urinary cortisol, significant plasma levels of Reichstein's compound S and 21-deoxycortisol, high urinary levels of THS and pregnanetriolone as well as elevated levels of plasma and urinary testosterone. Treatment with 0.5 mg/day of dexamethasone or 25 mg/day cortisone reduced her hypertension and restored her menstrual cycles, but also resulted in the development of moon face, body striae and a gain in weight. Lower doses of cortisone were without effect. The deficient cortisol production coupled with the presence of unusual intermediates such as Reichstein's compound S and 21-deoxycortisol can be explained by a shift in the substrate specificity of 11beta-hydroxylase from C-21-hydroxylated substrates (i.e. compound S) to C-21-deoxy substrates (i.e. 17-hydroxyprogesterone).

Adrenocortical hydroxylase deficiencies in acne vulgaris.

To investigate the possible occurrence of partial 11- or 21-hydroxylase deficiences in acne, an androgen-dependent disorder, 11 women with chronic nodulocystic acne were subjected to a 24-hr infusion of ACTH and their urine analyzed for tetrahydro S and pregnanetrio. The results obtained were compared to those found in 8 control women. Seven of the patients exhibited elevated excretion of either tetrahydro S or pregnanetriol, probably indicative of partial 11- or 21-hydroxylase deficiencies, respectively.

A compact perfusion system for studies on the control of metabolic processes in isolated organs from small animals.

A compact apparatus for perfusion of isolated organs from small animals is described. The system is based on a module principle making it useable both in low and high pressure perfusions. 40-80 ml of heparinized, diluted rat blood used as liver-perfusion medium, is recirculated in the system, which is temperature controlled by means of a water jacket. A new type of capillary membrane oxygenator is used for oxygenation. The system permits rapid measurements of perfusate flow, vascular resistance, bile production (when the isolated liver is used), urinary production (when the isolated kidney is used), gas tensions, oxygen consumption, pH and perfusion pressure. Descriptions of the techniques used for operation and perfusion of isolated livers and kidneys from rats are given. By the generally accepted criteria of viability the isolated rat liver remains almost normal for the test period of 3 h. Liver functions were normal as judged by formation of glucose, respiratory rate, urea and bile production and gross morphology. There was very little leakage of liver enzymes into the perfusate. The transformation and elimination of labelled corticosterone were studied to test the steroid metabolizing activity of the isolated rat liver preparation.