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Purpose of Review: 21-Hydroxylase deficiency (21-OHD), the most common form of congenital adrenal hyperplasia, is an autosomal recessive disorder caused by pathogenic variants in CYP21A2 . Although this disorder has been known for several decades, many challenges related to its monitoring and treatment remain to be addressed. The present review is written to describe an overview of biochemical monitoring of this entity, with particular focus on overnight fasting urine pregnanetriol., Recent Findings: We have conducted a decade-long research project to investigate methods of monitoring 21-OHD in children. Our latest studies on this topic have recently been published. One is a review of methods for monitoring 21-OHD. The other was to demonstrate that measuring the first morning PT level may be more practical and useful for biochemical monitoring of 21-OHD. The first morning pregnanetriol (PT), which was previously reported to reflect a long-term auxological data during the prepubertal period, correlated more significantly than the other timing PT in this study, with 17-OHP, before the morning medication., Summary: In conclusion, although the optimal method of monitoring this disease is still uncertain, the use of overnight fasting urine pregnanetriol (P3) as a marker of 21-OHD is scientifically sound and may be clinically practical., (Copyright © 2024 Wolters Kluwer Health, Inc. All rights reserved.)

Background: Biochemically monitoring 21-hydroxylase deficiency (21-OHD) is challenging. Serum/blood 17-hydroxyprogesterone (17OHP) measurements are normally used for this purpose. Urinary pregnanetriol (PT), a urinary metabolite of 17OHP, may also be used. Based on auxological data, we previously reported that the optimal first morning PT value fell in the range of 2.2-3.3 mg/gCr (95% confidence interval of the mean) and 0.59-6.0 mg/gCr (10 th - 90 th percentile) for monitoring 21-OHD treatment. No report thus far has directly compared the first morning urinary PT value with the 17OHP value at various times during the day., Objective: To explore the correlation between the first morning urinary PT value before glucocorticoid administration and the serum/blood 17OHP value at three time points, namely, before and two and four hours after glucocorticoid administration., Design: This was a prospective study done at two children's hospitals., Methods: In total, 25 patients with 21-OHD aged 3-25 years were recruited. Their urinary PT levels and 17OHP levels were measured for three days within a total period of one week. The first morning PT value was collected on all three days. Dried blood spots and serum were used to measure 17OHP., Results: The range for the first morning PT value for all the samples (n=69) was 0.10-56.1 mg/gCr. A significant, positive correlation was found between the first morning PT and 17OHP values before medication (r=0.87, p<0.01), and weaker correlation was observed between the first morning PT and 17OHP values after medication., Conclusions: The first morning PT correlated more significantly with 17OHP before the morning medication. Measuring the first morning PT value may be more practical and useful for monitoring 21-OHD biochemically., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Itonaga, Izawa, Hamajima and Hasegawa.)

Rationale: Isotope ratio mass spectrometry (IRMS) is an analytical technique required by the World Antidoping Agency (WADA) before releasing of an adverse finding for the abuse of pseudoendogenous steroids (i.e. testosterone). For every single individual, the delta 13 C values (‰) of the selected target compounds (TCs, i.e. testosterone and/or its precursors/metabolites) are compared with those of endogenous reference compounds (ERCs). The aim of this work is to investigate the individual variation in the delta values of four different commonly used ERCs to establish the maximum acceptable variation, in order to detect potential outliers., Methods: Routine urine samples collected for antidoping purposes were submitted to IRMS confirmation. After a specific liquid chromatographic purification of the analytes of interest, the final extracts were analyzed by gas chromatography/combustion (GC/C)-IRMS. The selected ERCs monitored were pregnanediol, pregnanetriol, 11-keto-etiocholanolone and 11β-hydroxyandrosterone. The obtained 13 C delta values were statistically analyzed to evaluate their inter- and intra-individual distribution., Results: The delta values of the ERCs studied showed a normal distribution and no major differences among genders were observed. As expected, there are differences depending on the geographical origin of the samples, reflecting different dietary habits and food sources. The intra-individual dispersion, expressed as the standard deviation (SD) of the values of the studied ERCs, did not greatly exceed the instrumental error (0.5‰), demonstrating the good preservation of the delta values along the metabolic pathway., Conclusions: For the selected ERCs of non-sporting volunteers and the urinary specimens from more than 1000 sportsmen, we can propose a maximum SD of 0.54‰ and range of 1.2‰ for delta 13 C values as acceptance criteria to detect potential outliers. These cases can be caused by the external masking effect of the administration of a substance modifying the delta values or outliers due to unforeseen procedural artifacts., (© 2018 John Wiley & Sons, Ltd.)

Context: The classic androgen synthesis pathway proceeds via dehydroepiandrosterone, androstenedione, and testosterone to 5α-dihydrotestosterone. However, 5α-dihydrotestosterone synthesis can also be achieved by an alternative pathway originating from 17α-hydroxyprogesterone (17OHP), which accumulates in congenital adrenal hyperplasia (CAH). Similarly, recent work has highlighted androstenedione-derived 11-oxygenated 19-carbon steroids as active androgens, and in CAH, androstenedione is generated directly from 17OHP. The exact contribution of alternative pathway activity to androgen excess in CAH and its response to glucocorticoid (GC) therapy is unknown., Objective: We sought to quantify classic and alternative pathway-mediated androgen synthesis in CAH, their diurnal variation, and their response to conventional GC therapy and modified-release hydrocortisone., Methods: We used urinary steroid metabolome profiling by gas chromatography-mass spectrometry for 24-hour steroid excretion analysis, studying the impact of conventional GCs (hydrocortisone, prednisolone, and dexamethasone) in 55 adults with CAH and 60 controls. We studied diurnal variation in steroid excretion by comparing 8-hourly collections (23:00-7:00, 7:00-15:00, and 15:00-23:00) in 16 patients with CAH taking conventional GCs and during 6 months of treatment with modified-release hydrocortisone, Chronocort., Results: Patients with CAH taking conventional GCs showed low excretion of classic pathway androgen metabolites but excess excretion of the alternative pathway signature metabolites 3α,5α-17-hydroxypregnanolone and 11β-hydroxyandrosterone. Chronocort reduced 17OHP and alternative pathway metabolite excretion to near-normal levels more consistently than other GC preparations., Conclusions: Alternative pathway-mediated androgen synthesis significantly contributes to androgen excess in CAH. Chronocort therapy appears superior to conventional GC therapy in controlling androgen synthesis via alternative pathways through attenuation of their major substrate, 17OHP.

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., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

In many species, males produce elaborate signals used by females in the evaluation of potential mates. Two urinary steroid epimers have now been shown to be components of a courtship display by male Mozambique tilapia that promotes female maturation., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Knowledge of the chemical identity and role of urinary pheromones in fish is scarce, yet it is necessary in order to understand the integration of multiple senses in adaptive responses and the evolution of chemical communication [1]. In nature, Mozambique tilapia (Oreochromis mossambicus) males form hierarchies, and females mate preferentially with dominant territorial males, which they visit in aggregations or leks [2]. Dominant males have thicker urinary bladder muscular walls than subordinates or females and store large volumes of urine, which they release at increased frequency in the presence of subordinate males or preovulatory, but not postspawned, females [3-5]. Females exposed to dominant-male urine augment their release of the oocyte maturation-inducing steroid 17α,20β-dihydroxypregn-4-en-3-one (17,20β-P) [6]. Here we isolate and identify a male Mozambique tilapia urinary sex pheromone as two epimeric (20α- and 20β-) pregnanetriol 3-glucuronates. We show that both males and females have high olfactory sensitivity to the two steroids, which cross-adapt upon stimulation. Females exposed to both steroids show a rapid, 10-fold increase in production of 17,20β-P. Thus, the identified urinary steroids prime the female endocrine system to accelerate oocyte maturation and possibly promote spawning synchrony. Tilapia are globally important as a food source but are also invasive species, with devastating impact on local freshwater ecosystems [7, 8]. Identifying the chemical cues that mediate reproduction may lead to the development of tools for population control [9-11]., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Objective: To characterize the urinary steroid metabolome of neonates and infants born either at term or preterm., Study Design: We retrospectively analyzed urinary steroid hormone metabolites determined by gas chromatography-mass spectrometry of 78 neonates and infants born at term and 83 neonates and infants born preterm (median 34 weeks of gestational age). The subjects' 11β-hydroxylase and 21-hydroxylase activities were assessed on the basis of urinary metabolite substrate-to-product ratios., Results: Preterm neonates and infants had elevated urinary concentrations of 17α-hydroxyprogesterone (17OHP) metabolites (P<.001) but lower urinary concentrations of the 21-deoxycortisol metabolite pregnanetriolone (PTO) (P<.01). One reason was lower 11β-hydroxylase activity in preterms. We could demonstrate a correlation between low 11β-hydroxylase activity and high urinary concentrations of 17OHP metabolites (r=0.51, P<.001) but low urinary concentrations of the 21-deoxycortisol metabolite PTO (r=-0.24, P=.03) in preterms., Conclusions: Low 11β-hydroxylase activity may explain increased 17OHP but decreased 21-deoxycortisol metabolite excretion in preterms. Our analysis clarifies, first, why preterms have higher 17OHP levels and thus higher rates of false-positive screening results for congenital adrenal hyperplasia than do term infants, and, second, why 21-deoxycortisol or its urinary metabolite PTO is more specific than 17OHP for the diagnosis of 21-hydroxylase deficiency., (Copyright © 2014 Elsevier Inc. All rights reserved.)

The steroidogenic enzyme 21-hydroxylase is necessary for the synthesis of both glucocorticoids and mineralocorticoids. 21-hydroxylase is a cytochrome P-450 enzyme and is encoded by the gene CYP21A2. Here we report a 68-year-old phenotypically 'male' but genetically female patient with 21-hydroxylase deficiency (21OHD) and the concomitant virilizing adrenocortical carcinoma. This patient grew up as a male and has not encountered any episodes of adrenal insufficiency without glucocorticoid replacement in his lifetime. A chromosome test at admission, however, identified the 46, XX karyotype, and serum 17-hydroxyprogesterone and urine pregnanetriolone and 11β-hydroxyandrostendione were all elevated, consistent with 21OHD. Moreover, serum testosterone was 1.90 ng/ml, much higher than the female standard levels, and serum cortisol was 5.7 µg/ml, slightly lower than standard levels. Genetic analysis identified the patient as a heterozygote of the two pathogenic mutations in the CYP21A2 gene: IVS2-13C(A)>G and R356W. Magnetic resonance imaging (MRI) revealed the presence of left adrenal tumor measuring 6 cm, which was subsequently diagnosed as adrenocortical carcinoma based on the criteria of Weiss. Immunohistochemical analysis of the tumor specimens revealed the expression of various enzymes involved in testosterone production, including 3β-hydroxysteroid dehydrogenase, 17α-hydroxylase/17,20-lyase, and 17β-hydroxysteroid dehydrogenase. Importantly, the expression of immunoreactive 21-hydroxylase was detected in these tumor cells. The levels of adrenal tumor-derived steroid metabolites were all markedly decreased following the surgery. This is the first report on a virilized 21OHD patient associated with the adrenocortical tumor that produces testosterone. Moreover, the concomitant adrenocortical tumor may ameliorate adrenocortical insufficiency by producing cortisol.

The urinary steroid profile has been used in clinical endocrinology for the early detection of enzyme deficiencies. In the field of doping, its evaluation in urine samples is used to diagnose the abuse of substances prohibited in sport. This profile is influenced by sex, age, exercise, diet, and ethnicity, among others; laboratories own reference ranges might compensate for ethnic differences among population and inter-laboratory biases. This paper shows the reference ranges obtained in the Antidoping Laboratory of Havana for the following steroid profile parameters: ten androgens (testosterone, epitestosterone, androsterone, etiocholanolone, 5α-androstan-3α,17β-diol, 5β-androstan-3α,17β-diol, dehydroepiandrosterone, epiandrosterone, 11β-hydroxyandrosterone and 11β-hydroxyetiocholanolone), three estrogens (estradiol, estriol and estrone), two pregnanes (pregnanediol and pregnanetriol) and two corticosteroids (cortisol and tetrahydrocortisol). The urine samples (male: n = 2454 and female: n = 1181) and data obtained are representative of population from Latin-American countries like Cuba, Venezuela, Mexico, Dominican Republic, Guatemala and Chile. Urine samples were prepared by solid-phase extraction followed by enzymatic hydrolysis and liquid-liquid extraction with an organic solvent in basic conditions. Trimethylsilyl derivatives were analyzed by gas chromatography coupled to mass spectrometry. Reference ranges were established for each sex, allowing the determination of abnormal profiles as a first diagnostic tool for the detection of the abuse of androgenic anabolic steroids. The comparison with the Caucasian population confirms that the urinary steroid profile is influenced by ethnicity., (Copyright © 2013 John Wiley & Sons, Ltd.)

Background: The clinical differential diagnosis of classic 21-hydroxylase deficiency (C21OHD) and cytochrome P450 oxidoreductase deficiency (PORD) is sometimes difficult, because both deficiencies can have similar phenotypes and high blood concentrations of 17α-hydroxyprogesterone (17OHP). The objective of this study was to identify biochemical markers for the differential diagnosis of C21OHD, PORD, and transient hyper 17α-hydroxyprogesteronemia (TH17OHP) in Japanese newborns. We established a 2-step biochemical differential diagnosis of C21OHD and PORD., Methods: We recruited 29 infants with C21OHD, 9 with PORD, and 67 with TH17OHP, and 1341 control infants. All were Japanese and between 0 and 180 days old; none received glucocorticoid treatment before urine sampling. We measured urinary pregnanetriolone (Ptl), the cortisol metabolites 5α- and 5β-tetrahydrocortisone (sum of these metabolites termed THEs), and metabolites of 3 steroids, namely dehydroepiandrosterone, androstenedione (AD4), and 11β-hydroxyandrostenedione (11OHAD4) by GC-MS., Results: At a cutoff of 0.020, the ratio of Ptl to THEs differentiated C21OHD and PORD from TH17OHP and controls with no overlap. Among metabolites of DHEA, AD4, and 11OHAD4, only 11β-hydroxyandrosterone (11HA), a metabolite of 11OHAD4, showed no overlap between C21OHD and PORD at a cutoff of 0.35 mg/g creatinine., Conclusions: A specific cutoff for the ratio of Ptl to THEs can differentiate C21OHD and PORD from TH17OHP and controls. Additionally, the use of a specific cutoff of 11HA can distinguish between C21OHD and PORD.

In 1937 Butler and Marrian found large amounts of the steroid pregnanetriol in urine from a patient with the adrenogenital syndrome, a virilizing condition known to be caused by compromised adrenal secretion even in this pre-cortisol era. This introduced the concept of the study of altered excretion of metabolites as an in vivo tool for understanding sterol and steroid biosynthesis. This approach is still viable and has experienced renewed significance as the field of metabolomics. From the first cyclized sterol lanosterol to the most downstream product estradiol, there are probably greater than 30 steps. Based on a distinctive metabolome clinical disorders have now been attributed to about seven post-squalene cholesterol (C) biosynthetic steps and around 15 en-route to steroid hormones or needed for further metabolism of such hormones. Forty years ago it was widely perceived that the principal steroid biosynthetic defects were known but interest rekindled as novel metabolomes were documented. In his career this investigator has been involved in the study of many steroid disorders, the two most recent being P450 oxidoreductase deficiency and apparent cortisone reductase deficiency. These are of interest as they are due not to mutations in the primary catalytic enzymes of steroidogenesis but in ancillary enzymes needed for co-factor oxido-reduction A third focus of this researcher is Smith-Lemli-Opitz syndrome (SLOS), a cholesterol synthesis disorder caused by 7-dehydrocholesterol reductase mutations. The late George Schroepfer, in whose honor this article has been written, contributed greatly to defining the sterol metabolome of this condition. Defining the cause of clinically severe disorders can lead to improved treatment options. We are now involved in murine gene therapy studies for SLOS which, if successful could in the future offer an alternative therapy for this severe condition.

Background: Congenital adrenal hyperplasia is a group of disorders caused by defects in the adrenal steroidogenic pathways. In its most common form, 21-hydroxylase deficiency, patients develop varying degrees of glucocorticoid and mineralocorticoid deficiency as well as androgen excess. Therapy is guided by monitoring clinical parameters as well as adrenal hormone and metabolite concentrations., Content: We review the evidence for clinical and biochemical parameters used in monitoring therapy for congenital adrenal hyperplasia. We discuss the utility of 24-h urine collections for pregnanetriol and 17-ketosteroids as well as serum measurements of 17-hydroxyprogesterone, androstenedione, and testosterone. In addition, we examine the added value of daily hormonal profiles obtained from salivary or blood-spot samples and discuss the limitations of the various assays., Summary: Clinical parameters such as growth velocity and bone age remain the gold standard for monitoring the adequacy of therapy in congenital adrenal hyperplasia. The use of 24-h urine collections for pregnanetriol and 17-ketosteroid may offer an integrated view of adrenal hormone production but target concentrations must be better defined. Random serum hormone measurements are of little value and fluctuate with time of day and timing relative to glucocorticoid administration. Assays of daily hormonal profiles from saliva or blood spots offer a more detailed assessment of therapeutic control, although salivary assays have variable quality.

Objective: It has been shown that adiponectin serves as an insulin-sensitizing adipokine. Serum concentrations of adiponectin are low in children with obesity, and increase with fat mass loss, indicating that adiponectin can serve as a biomarker. Since the prevalence of overweight and obesity is increased in children with congenital adrenal hyperplasia (CAH), our study aimed to evaluate serum levels of adiponectin in a cohort of CAH children and adolescents, and their associations with clinical parameters such as chronological age (CA), body mass index (BMI), Tanner stage (TS), medication and metabolic control., Patients and Methods: We studied 51 patients, aged between 5.6 and 19.6 years (median 11.8; 30 females, 21 males), cross-sectionally. All patients had genetically confirmed CAH and received standard steroid substitution therapy. Adiponectin was measured by an enzyme linked immunoassay. Since BMI SDS of the CAH cohort were significantly higher compared to the reference population, we built matched pairs with healthy Caucasian subjects from a normal representative cohort for sex, Tanner stage, chronologic age and BMI., Results: Adiponectin concentrations were significantly higher in CAH patients (median 11 microg/L) compared to the matched controls (6.7 microg/L, p < 0.0001). Correlation analyses in CAH patients revealed a significant inverse relationship between adiponectin and CA, TS, BMI, serum DHEAS and serum testosterone, but no correlation with hydrocortisone and fludrocortisone dosage., Conclusion: Currently, the importance of the elevated adiponectin concentrations in CAH children for risk assessment is not clear. However, our data imply that besides adequate metabolic control of glucocorticoid substitution, a long-term follow-up of other metabolic markers of insulin resistance should be conducted in CAH patients.

The liquid chromatography-tandem mass spectrometry (LC-MS/MS) is an increasingly common tool in the clinical laboratory. Established applications include routine assays for detecting inborn errors of metabolism and for monitoring therapeutic drugs and steroids. Steroid profiling is a very effective method for distinguishing almost all steroid related disorders. It allows accurate diagnosis and is very useful in many clinical situations. Most methods for the determination of steroid hormones are based on immunoassays, which are rapid and easy to perform. However, the reliability of steroid immunoassays has been shown to be doubtful because of the lack of specificity and of matrix effects. Immunological methods, especially direct assays, often overestimate true steroid values. This is of particular importance in the newborn period and in early infancy. Problems with steroid immunoassays have further been reported for female patients or when analysing different media, e.g. saliva. Patient follow-up over time or between laboratories, as well as longitudinal studies are extremely difficult. In contrast to immunoassays, which allow the measurement of only a single steroid at a time, LC-MS/MS has the advantage that a wide spectrum of steroid hormones can be measured simultaneously. The applicability for clinical samples and problems in pediatric endocrinology will be discussed.

Objective: Congenital adrenal hyperplasia (CAH) patients are at a higher risk to develop obesity. The role of leptin in CAH is still controversial. Our study aimed to evaluate serum levels of leptin, the soluble leptin receptor (sOB-R), and the sOB-R: leptin molar ratios in a cohort of CAH children and adolescents, and their associations with clinical and metabolic parameters., Methods: We studied 51 CAH patients, aged 5.6-19.6 years (median 11.8, n=30 females) cross-sectionally. All patients had genetically proven CAH and received standard steroid substitution therapy. Blood specimens were taken after overnight fasting between 0800 and 1000 h. For the analyses of leptin and sOB-R, matched pairs were built with healthy Caucasian patients for sex, Tanner stage (TS), chronologic age (CA), and body mass index (BMI)., Results: BMI and SDS were significantly elevated compared with the reference population. Leptin levels were not different between matched pairs, whereas sOB-R levels were significantly lower in CAH. Consequently, the sOB-R: leptin molar ratios were significantly decreased in CAH. Correlation analyses in CAH patients revealed significant relationship between leptin and CA, TS, BMI, and homeostasis model assessment of insulin resistance. Similar results were obtained for the matched control group. For sOB-R, we found no significant correlation for CA, TS, or BMI in CAH, but we did in the controls. There were significant correlations for androgens within the CAH group. Additional analyses revealed no correlation with steroid medication or metabolic control., Conclusions: Our data show that an altered leptin axis with normal serum leptin concentrations but decreased sOB-R serum levels may contribute to the increased risk of overweight and obesity in CAH.

In a large multi-center trial involving prenatal screening for Smith-Lemli-Opitz syndrome (SLOS), we evaluated maternal urine and serum steroid analysis as a non-invasive diagnostic alternative to amniotic fluid sterol analysis. Candidate steroid ratios included: 7-dehydropregnanetriol/pregnanetriol (7-PT/PT), 8-dehydropregnanetriol/PT (8-PT/PT), the sum of these two (7 + 8-PT/PT), and dehydroestriol/estriol (DHE3/E3). Results are presented from 19 SLOS pregnancies, and 732 reference pregnancies that were screen positive for SLOS but negative on testing in amniotic fluid. Steroid ratios are expressed as multiples of the 75th centile (MoS), rather than multiples of the median, as most reference measurements were undetectable. All four urine ratios were available in 12 SLOS pregnancies; the median 7-PT/PT MoS was 94, with no overlap between affected and reference pregnancies in the second trimester. The separation between these groups increased by 27% per week. The other three ratios performed similarly in urine, with (7 + 8)-PT/PT ratios being marginally superior, due to fewer high reference outliers. All four steroid ratios in urine were diagnostic for SLOS between 14 and 22 weeks' gestation. In six SLOS pregnancies in which all serum analytes were measured, the median 7-PT/PT MoS was 71, and there was slight overlap in the second trimester. The separation increased by 28% per week. Steroid ratios in serum were less definitive than in urine but might be useful in certain circumstances, at 14 weeks gestation or later. Urine testing performance prior to 14 weeks gestation appears promising, but reference data are sparse., ((c) 2007 Wiley-Liss, Inc.)

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.

Aim: To establish a method to determine the isotope ratios of 13C to 12C of dehydroepiandrosterone and its metabolites in urine, for detecting the source of dehydroepiandrosterone or its metabolites., Methods: Preliminary separation of endogenous anabolic androgenic steroids could be achieved using solid phase extraction, enzymolysis and thin layer chromatography. The source of dehydroepiandrosterone and other endogenous anabolic androgenic steroids could be detected by their delta values with gas chromat ography-combustion-isotope ratio mass spectrometry., Results: The 5 values of some metabolites of dehydroepiandrosterone reduced after the administration of dehydroepiandrosterone preparation. In these cases the data indicated that exogenous anabolic androgenic steroids were administrated., Conclusion: The source of dehydroepiandrosterone or its metabolites in urine could be detected by measuring their delta values with this method.

Elevated blood 17alpha-hydroxyprogesterone (17OHP) level, although widely used for the screening of classical 21-hydroxylase deficiency (21OHD) in neonates, has frequently been found in some neonates without classical 21OHD, particularly preterm neonates. We studied the diagnostic value of the metabolite of 21-deoxycortisol (pregnanetriolone, Ptl) and the metabolite of 17OHP (pregnanetriol, PT) in identifying 21OHD in term and preterm neonates with elevated blood 17OHP on the newborn screening. Spot urine samples from 59 classical 21OHD neonates (50 term, 9 preterm), 83 neonates without 21OHD having transiently elevated blood 17OHP (non-21OHD) (49 term, 34 preterm), and 62 control term neonates were studied using gas chromatography/mass spectrometry in selected ion monitoring analysis for Ptl, PT, 5beta-tetrahydrocortisone (betaTHE), and 5alpha-tetrahydrocortisone (alphaTHE). Ptl and Ptl/(betaTHE+alphaTHE) showed no overlap between 21OHD and non-21OHD, and 21OHD and controls, respectively (Ptl was 0.46-124 mg/g creatinine in 21OHD term, 0.80-26.9 mg/g creatinine in 21OHD preterm, < or = 0.08 mg/g creatinine in non-21OHD term, < or =0.06 mg/g creatinine in non-21OHD preterm, and < or = 0.07 mg/g creatinine in controls). PT and PT/(betaTHE+alphaTHE) showed significant overlap between 21OHD and non-21OHD. The above data indicate that spot urine Ptl is a highly specific marker of 21OHD with a cutoff value of 0.1 mg/g creatinine, yielding an unambiguous separation between 21OHD and non-21OHD in term and preterm neonates.

Objective: To clarify the pathogenesis of transient hyper-17alpha-hydroxyprogesteronemia, we initiated a laboratory investigation in a pre-term infant with persistently high serum 17alpha-hydroxyprogesterone (17-OHP) until 2 months of age., Methods: Serum 17-OHP level was measured by high-performance liquid chromatography and radioimmunoassay, and gene analysis of CYP21A2 (21-hydroxylase) was performed., Result: Serum 17-OHP level on the 29th day of life was 25.4 ng/ml, and the urinary steroid profile showed low pregnanetriolone. Gene analysis of 21-hydroxylase disclosed no mutation, and 17-OHP normalized by 3 months of age without specific treatment., Conclusion: Transient elevations in 17-OHP, which do not appear related to cross-reactions with products of a residual fetal adrenal cortex, may occur in the first few months of life., (Copyright 2004 S. Karger AG, Basel)

Gas chromatographic/mass spectrometric (GC/MS) analysis of maternal urine and serum steroids from 13 pregnancies at 25% risk for Smith-Lemli-Opitz syndrome (SLOS) was undertaken. All patients were between 12 and 31 weeks' gestational age. From dehydrocholesterol/cholesterol ratios determined in amniotic fluid and chorionic villus cells, five patients were shown to carry SLOS affected fetuses and eight patients were negative for the condition. Because it had previously been shown that dehydro-oestriol and dehydropregnanetriol were novel steroids produced in SLOS, these compounds were measured in the serum and urine samples of the 13 mothers. All five urine samples from SLOS affected pregnancies had high levels of both dehydrosteroid metabolites, which were below the detection limit in the non-affected pregnancies. The ratios of dehydro-oestriol/oestriol (DHE(3)/E(3)) were between 0.073 and 1.42 for the affected patients and less than 0.01 for unaffected patients. Corresponding values for dehydropregnanetriol/pregnanetriol (DHPT/PT) were 0.037-1.02 for affected and less than 0.01 for unaffected. In the positive serum sample available for analysis, the DHE(3)/E(3) ratio was 0.20 [unaffected (n=5), <0.014]. It is proposed that the measurement of DHE(3) and DHPT in maternal urine and serum may allow non-invasive antenatal diagnosis of SLOS., (Copyright 2001 John Wiley & Sons, Ltd.)

Air-borne volatile substances have been demonstrated to signal oestrus, induce ovulation and synchronize ovarian activity in different mammals. An oestrous-related pheromone of the female Asian elephant (Elephas maximus) is known to induce behavioural responses in elephant bulls. Additional data revealed that timing of oestrus in females with close social relationships tends to be synchronized. Therefore, urine from female Asian elephants might be expected to contain luteal phase-dependent volatile substances, which may function as additional chemical signals in this species. The aim of the present study was to identify such compounds and to investigate their pattern of excretion throughout the ovarian cycle. Urine samples were collected three times a week during the follicular phase and one to three times a week during the luteal phase from five adult female Asian elephants from a total of 13 non-conception cycles and one conception cycle, including the first 72 weeks of pregnancy. A simple headspace solid-phase microextraction method has been developed for quantification of urinary volatile substances and analysis was performed by gas chromatography. The comparison of urine collected during the follicular and the luteal phase indicated the presence of two luteal phase-dependent substances. Mass spectrometry was used to identify one substance as 5alpha-androst-2-en-17-one and a second substance as the corresponding alcoholic compound 5alpha-androst-2-en-17beta-ol. The 5alpha-androst-2-en-17beta-ol and -17-one profiles reflected cyclic ovarian activity with clear (10-20-fold) luteal phase increases. Furthermore, measurements of both compounds were correlated positively with the concentration of urinary pregnanetriol and indicated cycle duration (15.1 +/- 1.2 weeks) similar to that obtained from pregnanetriol measurements (15.2 +/- 1.6 weeks). The results demonstrate the presence of two luteal phase-specific steroidal volatile compounds in elephant urine. One of the substances, 5alpha-androst-2-en-17-one, has been demonstrated in human axillary bacterial isolates. The measurement of both volatile substances in elephant urine can be used for rapid detection of the stage of the ovarian cycle, as the analysis can be completed within 2 h.

Vocal disturbances in women with congenital adrenal hyperplasia and androgen excess should be extremely rare today since effective substitution with glucocorticoids is available. We present a 17 year-old female with congenital adrenal hyperplasia due to 21-hydroxylase deficiency and severe virilization because of long-term insufficient therapy. Laboratory data showed elevated serum levels of testosterone, 17-hydroxyprogesterone, plasma ACTH and a high excretion of urinary pregnanetriol. The phoniatric aspect showed a masculine voice. We discuss the different effects of androgens on the pubertal larynx and various hormonal disturbances that may cause voice changes as well as therapeutic options of voice therapy. From the pediatric point of view it might be important to perform a phoniatric examination in girls with congenital adrenal hyperplasia during puberty in order to monitor androgen effects.

The urinary steroid hormone metabolites and the ratio of pregnenetriol (delta 5P3) to pregnanetriol (P3) as indicators of 3 beta HSD activity in the urine of healthy young female were measured by means of capillary gas chromatography. All of the subjects have finished the normal pubertal development, and their adrenal steroid hormone secretion had reached to the stable state. We analyzed the diurnal variation, fluctuation during menstrual cycle and seasonal variation of delta 5P3/P3. We found that the hormone excretion in the urine of the morning during the follicular phase of menstrual cycle was relatively stable, and that the ratio of delta 5P3/P3 correlated highly with that in the total daily urine. In the seasonal variation, the urinary delta 5P3/P3 ratio in the subjects of high urinary DHEA group was relatively high, and that of the low DHEA group was low. Although the difference of delta 5P3/P3 ratio of the both groups was small, but statistically significant. Individual difference in the delta 5P3/P3 ratio was relatively small in comparison with that of the urinary DHEA excretion. About 5% of the all subjects showed marked high value of delta 5P3/P3 ratio. About 80% of the high urinary excretion group showed higher value than the average delta 5P3/P3 ratio. These findings suggest that the normal young female subjects were divided into several groups with regard to the urinary DHEA excretion pattern and delta 5P3/P3 ratio in the urine. Both of them may be a specific individual marker.

History and Clinical Findings: A 7.3-year-old boy was presented at out-patient clinic because of tallness and premature puberty. His height was 150.2cm (+ 4.74 standard deviation score for chronological age), body mass index 18.4 kg/m2, pubic hair stage 3 (after Tanner), testicular volume of 5.0 ml each. Bone age was accelerated by 6.5 years (SD -1.55 for height according to bone age). Expected final height was 166 cm, mean genetic target height 180 cm., Investigations: Basal serum concentration of 17-hydroxyprogesterone was 142.1 ng/ml (normal: < 1.9) and testosterone of 93 ng/dl (normal: < 11). 24-hour urine showed an increased excretion of pregnantriol of 8280 micrograms/d (normal < 500). Gonadotropine-releasing hormone test (GnRH), blood collected at 0 and 30 min, showed an increased rise of the serum LH concentration of 0.6 to 8.2 mU/ml (normal < 0.3 and < 3.6, respectively) and a normal FSH increase of 1.3 to 3.2 mU/ml (normal < 1.3 and < 4.0, respectively). The diagnosis of adrenogenital syndrome (AGS) with 21-hydroxylase defect was confirmed by molecular genetic testing., Treatment and Course: The boy was treated with hydrocortisone (average dose 18.3 mg/m2 body surface area). Because of the premature puberty and the poor growth endprognosis treatment with the GnRH agonist Decapeptyl Depot, 3.75 mg every 4 weeks i.m., was started., Conclusion: The correct diagnosis should have been made in the neonatal period on the basis of the family history (15-year-old brother with AGS) and at the latest on correct interpretation of the clinical signs during early childhood.

The effects of hypercortisolemia and ACTH on the metabolism of cortisol in congenital adrenal hyperplasia, Cushing's syndrome, and exogenous ACTH and cortisol administration were investigated by analysis of the respective urinary tetrahydro-metabolites of cortisol (THF and aTHF) and cortisone (THE) by capillary gas chromatography. The results for the patients with congenital adrenal hyperplasia establish that ACTH hypersecretion in the absence of an associated marked elevation of plasma cortisol does not cause inhibition of the 11beta-OHSD enzyme. In contrast elevated plasma cortisol levels (adrenal adenoma or intravenous cortisol administration) in the presence of suppressed ACTH secretion leads to significant inhibition of the peripheral conversion of cortisol to cortisone. The latter results are equivalent to the mode of cortisol metabolism noted during clinical states of ACTH hypersecretion and hypercortisolemia (Cushing's disease, ectopic ACTH syndrome and ACTH administration). The overall findings provide convincing evidence that ACTH hypersecretion is not associated with specific in vivo inhibition of 11beta-OHSD enzyme activity.

A series of pregnanediols and pregnanetriols doubly conjugated with N-acetylglucosamine and glucuronic or sulfuric acid has been identified in urine from pregnant women. Steroid conjugates were separated by ion-exchange chromatography and the glucuronide and monosulfate fractions were analysed by fast atom bombardment mass spectrometry. After removal of the acid moiety, the neutral steroids were isolated, derivatized, and analysed by gas chromatography-mass spectrometry (GC-MS). The analyses revealed the presence of steroids conjugated with N-acetylhexosamine both in the glucuronide and the monosulfate fractions. Following enzyme hydrolysis, the sugar was identified by GC-MS as N-acetylglucosamine (GlcNAc). The major steroid conjugated with GlcNAc both in the glucuronide and monosulfate fractions was identified as 5alpha-pregnane-3alpha,20alpha-diol. 5beta-Pregnane-3alpha,2Oalpha-diol was also present as a GlcNAc conjugate in both fractions whereas a GlcNAc conjugate of 5alpha-pregnane-3beta,20alpha-diol was only found in the sulfate fraction. 5alpha-Pregnane-3alpha,20alpha,21-triol was a double conjugate with GlcNAc in the sulfate fraction whereas a pregnane-2,3,20-triol was a double conjugate in the glucuronide fraction. The positions of conjugation were determined by collision-induced dissociation of the pseudomolecular anions produced by fast atom bombardment ionization. The sulfate and glucuronic acid moieties were located at C-3 and N-acetylglucosamine at C-20. An alternative localization of GlcNAc at C-21 of 5alpha-pregnane-3alpha,20alpha,21-triol cannot be excluded. Judging from the enzymatic hydrolysis of the conjugates, the sugar was attached in beta-glycosidic linkage. The mean excretion of N-acetylglucosaminides of the pregnanediols and pregnanetriols was 32.2 micromol/g creatinine (range 17.9-49.1 micromol) in five healthy women in the 38th-39th week of pregnancy. The mean excretion of 5beta-pregnane-3alpha,20alpha-diol glucuronide in the same women was 71 micromol/g creatinine, (range 27-127 micromol). This indicates that conjugation with N-acetylglucosamine constitutes a quantitatively important pathway of progesterone metabolism in human pregnancy.

Urinary concentrations of pregnanetriol-3-glucuronide (PT-3-G) were determined in 485 normal Japanese subjects (277 males and 208 females), aged 5 days to 20 years, using an enzyme-linked immunosorbent assay (ELISA). The usefulness of urinary PT-3-G concentrations before giving the morning dose of medications in monitoring the adequacy of glucocorticoid treatment was assessed in eight patients with 21-hydroxylase deficiency (21-OHD). The ratio of PT-3-G to excreted creatinine (PT-3-G/Cre ratio) increased significantly during the first month and did not change from age 1 month to 1 year of life. The ratio decreased to a nadir at age 3 or 4 years followed by continuous, significant increase until late adolescence. In the subjects treated with corticosteroids for 21-OHD, PT-3-G/Cre ratios at the 50th percentile or below suggested a risk of excessive treatment, as judged by the patients' growth. Measurement of the PT-3-G/Cre ratio enabled recognition of corticosteroid overtreatment, which was not demonstrated by determining the serum concentrations of 17 alpha-hydroxyprogesterone (17-OHP). On the other hand, ratios at the upper 95-99% tolerance limits seemed to be required for optimal control. The present study revealed the normal age-related changes in urinary excretion of PT-3-G and showed it to be a reliable marker for evaluating glucocorticoid treatment in young children with 21-OHD.

Objectives: The study was designed to assess the reliability of measurement of 24-hour urinary 17 alpha-hydroxyprogesterone (17-OHP) by radio-immunoassay (RIA) as an alternative biochemical assessment for monitoring the treatment of congenital adrenal hyperplasia (CAH) due to 21-hydroxylase deficiency (21-OHD) and to assess the need for sample purification by column chromatography to improve assay specificity., Methodology: Morning serum 17-OHP was measured using RIA and 24-hour urinary pregnanetriol using gas chromatography. Twenty-four-hour urinary 17-OHP was measured in samples from 17 prepubertal patients with CAH due to 21-OHD, and 20 normal prepubertal children as controls. In 24 urine samples, RIA of 17-OHP was performed with and without column chromatography., Results: There was a good correlation between 24-hour urinary 17-OHP and 24-hour urinary pregnanetriol (r = 0.962, P < 0.01) and between 24-hour urinary 17-OHP and morning serum 17-OHP (r = 0.955, P < 0.01). There was no significant difference in the RIA of the urine samples with and without purification by column chromatography., Conclusions: The measurement of 24-hour urinary 17-OHP is a reliable alternative for the biochemical monitoring of 21-OHD, and RIA specificity is unaffected by omission of column chromatography.

The development of an enzymeimmunoassay for 5 beta-pregnanetriol and its use for non-invasive monitoring of reproductive cycles in Asian elephants is described. Gas chromatography-mass spectrometry (GCMS) and high performance liquid chromatography (HPLC) confirmed the presence of 5 beta-pregnane-3 alpha,17 alpha,20 alpha/beta-triols as the two most abundant urinary progesterone metabolites. The assay developed used the antiserum anti-5 beta-pregnane-17 alpha,20 alpha-diol-3 alpha-gamma l glucuronide but was designed to measure the free steroid in urine samples after hydrolysis and extraction. HPLC confirmed the presence of immunoreactive pregnanetriol in urine, but indicated that the measurement was nonspecific. Immunoreactive pregnanetriol concentrations were significantly correlated with the concentrations of both progesterone (r = 0.98, n = 269, P < 0.01) and 17 alpha-hydroxyprogesterone (r = 0.95, n = 205, P < 0.01), the metabolic precursor of pregnanetriol. The mean +/- SEM deviation of cycles as determined by measurements of plasma progesterone, 17 alpha-hydroxyprogesterone and urinary pregnanetriol, respectively, were 15.54 +/- 1.5 (n = 23, where n = number of cycles), 15.21 +/- 1.7 (n = 15) and 15.45 +/- 0.94 weeks (n = 20). These results demonstrate that it is possible to monitor ovarian function in Asian elephants by the measurement of urinary immunoreactive pregnanetriol concentrations.

We studied height velocity (HV), bone age progression (delta BA/delta CA), urinary pregnanetriol (PT) and plasma 17-hydroxyprogesterone (17-OH-P) during the first years of life in 12 patients with 21-hydroxylase deficiency, treated by cortisone acetate. In the well-controlled phases normal growth rate (SDS between -1 and +1), satisfactory bone age progression (delta BA/delta CA < or = 1) and no clinical sign of poor treatment were found; in the undertreatment phases enhanced growth rate, rapid bone age progression and, in some instances, signs of virilization were found; in the overtreatment phases, reduced growth rate was the only sign of poor treatment. Hormonal values were only weakly correlated to therapeutic control. Therefore, growth rate evaluation can represent the best method of monitoring treatment in very young patients with 21-hydroxylase deficiency.

The utility of urinary 17 alpha-hydroxyprogesterone (U-17-OHP) in the diagnosis and management of congenital adrenal hyperplasia (CAH) due to 21-hydroxylase deficiency (21-OHD) was evaluated in 16 patients with 21-OHD. The normal values for U-17-OHP in relation to age and other physiological conditions were investigated in 96 normal subjects and 7 low birth weight infants. Levels of U-17-OHP, serum 17-OHP (S-17-OHP) and urinary free cortisol (U-FC) were simultaneously determined using enzyme-linked immunosolvent assay (ELISA) combined with fractionation by high performance liquid chromatography (HPLC). Pregnanetriol (PT) levels in the same urine specimens were determined using glass capillary gas chromatography (GC). The results were as follows: 1) Normal subjects and low birth weight infants. A significant correlation between U-17-OHP excretions corrected for body surface area (BSA) in 2-h urine specimens and S-17-OHP concentrations at the midpoint of the urine sampling period was observed in normal subjects (r = 0.768, p less than 0.01). Circadian U-17-OHP excretion in 6 adult males was synchronous with that of U-FC. Age-related changes in actual U-17-OHP excretions (Mean +/- SD ng/day) were as follows: neonates: 31.9 +/- 10.3, children aged 2 to 4 years old: 29.1 +/- 14.5, 5 to 8 years old: 68.6 +/- 29.9, 9 to 11 years old: 151.3 +/- 50.0, 12 to 15 years old: 222.7 +/- 82.0, adult males: 400.1 +/- 62.5, adult females (luteal phase): 339.1 +/- 109.7, and adult females (follicular phase): 185.6 +/- 72.3, respectively. Correlation between U-17-OHP and PT excretions in 24-h urine specimens from normal subjects greater than 2 years of age was highly significant (r = 0.871, p less than 0.01). Although U-17-OHP values were measurable in neonates, those of PT were not detectable by GC in the same specimens because of low conversion of 17-OHP to PT. 2) Cases of 21-OHD. In 4 cases of 21-OHD diagnosed in the neonatal period (aged 11 to 15 days, all were of the salt-losing type), the U-17-OHP concentration in a single urine specimen was significantly higher than that of age-matched controls, whereas the PT concentration in one case was low and therefore had no diagnostic value. In 12 patients with 21-OHD receiving suppressive therapy, correlation between S-17-OHP concentrations and 24-h U-17-OHP excretions corrected for BSA was significant (r = 0.847, p less than 0.01).(ABSTRACT TRUNCATED AT 400 WORDS)

Four post-menopausal women had Cushing's syndrome due to adrenal cortical carcinomas. Comprehensive analyses of blood and urinary steroids showed that although the steroid profiles differed between patients, the pattern in each patient remained almost constant as the disease progressed, or remitted due to therapy. Elevations of serum testosterone and oestradiol were commensurate with the extent of virilisation, and the urinary output of aldosterone was associated with the severity of hypertension. A new finding was that all had substantially increased urinary free deoxycorticosterone. Complete surgical removal of the primary tumours was impossible but when most of the tumour tissue was removed, full clinical and biochemical remissions were obtained for a short time in 2 patients. One patient obtained a clinical and biochemical remission from op'DDD. In another patient the drug caused reduction both in blood pressure and in urinary aldosterone excretion, but there were unpleasant side effects. A third patient could not tolerate op'DDD. Metyrapone therapy produced neither clinical nor biochemical improvement in 3 patients. The mean duration of survival was 17 months after the first symptoms and 10 months from the date of operation. Despite advances in drug therapy, adrenal cortical carcinoma remains a lethal disease. Biochemical screening of multiple steroids offers a means of early diagnosis and disease monitoring. Extensive surgical removal of the tumour offers the best chance of a clinical and biochemical remission.

We have studied plasma renin activity, the plasma aldosterone level and urinary metabolites of glucocorticoid precursors before and during a dexamethasone suppression test in three non-salt-losers with congenital adrenal hyperplasia due to 21-hydroxylase deficiency, who had never been treated with glucocorticoid. Plasma renin activity, the plasma aldosterone level and urinary pregnanetriol excretion were found to be abnormally elevated before dexamethasone administration. After 7 days' dexamethasone administration, plasma renin activity still remained high above the normal level, while the plasma aldosterone level as well as urinary 17KS and pregnanetriol excretion were lowered to the normal ranges. Several possible mechanisms for this discordant suppression of plasma renin activity and aldosterone level were discussed and the presence of mineralocorticoid resistance, which is not related to ACTH dependent aldosterone antagonists, was suggested in the case of these patients.

The effects of congenital adrenal hyperplasia on adult height and fertility were studied in 30 afflicted men. The patients' heights ranged from 150.0 to 178.6 cm (mean +/- 1 S.D. of 164.0 +/- 7.6), which is significantly lower than both the mean adult height for American men and that of the patients' parents (P less than 0.005). There was no correlation between adult height and the age at which therapy was begun, possibly because the patients treated before one year of age had the salt-losing form of the syndrome. Therapeutic compliance may also have been involved. Apparently normal fertility, indicated by paternity and normal sperm counts, was found in 18 out of 20 patients evaluated. This group included five untreated patients who were found to be fertile and to have normal plasma testosterone and gonadotropin but elevated androstenedione levels.

The authors determined the amounts of oestrone, oestradiol, oestriol, alpha and beta pregnanediol, and pregnanetriol in 24-hour urine of 11 women with epileptic seizures occurring two and one day before the onset of menstruation, and in 7 women with seizures not dependent on the menstrual cycle. Determinations of these hormons were done during three successive menstrual cycles on the 2nd and 1st day before the onset of bleeding. In both groups a significant decrease was observed in the urinary levels of oestradiol and oestriol as compared with normal values. On the other hand, the amount of excreted oestrone was only slightly lowered in relation to the amount of this hormone excreted by controls. A significant decrease in the amounts of alpha pregnanediol (the difference being statistically significant at p less than 0.01 level) was observed two days before menstruation. On the other hand, pregnanetriol was decreased only in women in whom the seizures showed no temporal correlation with the stage of the menstrual cycle. The authors suggest that the observed reduction in the metabolites of progesterone in both groups was associated with an increased seizure readiness of the brain on the days preceding menstrual bleeding.

The effects of angiotensin have been studied in four adult patients with the simple virilizing form of congenital adrenal hyperplasia due to 21-hydroxylase deficiency. They were treated with hydrocortisone (25 mg/day) throughout this investigation. Plasma ACTH was normal in three cases, and androstenedione was normal in all cases. However, urinary pregnanetriol, plasma 17-hydroxyprogesterone (17 OHP), aldosterone, and renin activity was increased. The patients were then submitted to three protocols: sodium depletion (10 meq Na/day) for 5 days, sodium repletion (200 meq Na/day) for 5 days, and angiotensin infusion (sufficient to maintain a pressor response) for 60 min. Urinary pregnanetriol, plasma 17 OHP, and androstenedione levels increased in all patients after sodium depletion and decreased after sodium repletion. Plasma ACTH levels were not modified by changes in the sodium balance. Furthermore, angiotensin infusion increased aldosterone and 17 OHP plasma concentrations without any change in the plasma ACTH level. This study shows the direct action of angiotensin on adrenal steroidogenesis, at least in 21-hydroxylase deficiency. It confirms that even in the simple virilizing form, combined treatment with glucocorticoids and mineralocorticoids helps to normalize plasma 17 OHP levels.

Forty-seven patients with gout, 28 of whom had not previously been treated with allopurinol, and 25 normal subjects, were examined for 24-h urinary excretion of the most important adrenal steroid derivatives. Results were submitted to statistical analysis and several variables have been taken in consideration. The untreated patients showed significantly higher values of uricemia, urinary uric acid, triglycerides, slightly higher values of androsterone, 11-oxo-androsterone + 11-oxo-etiocholanolone, dehydroepiandrosterone, and slightly lower values of 11-hydroxyandrosterone and pregnanetriol, in comparison to normal subjects. The different hormonal pattern seems to discriminate between patients with gout and normal subjects.

A component was observed in the steroid spectrum of the urine of salt-loosing children with C/21-hydroxylase deficiency, which was eluted from Sp 2100 stationary phase before pregnanetriol but, unlike pregnanetriol, exhibited heat and acid stability. This component was isolated by paper chromatography and identified as 3 alpha, 20 alpha-dihydroxy-5 beta-pregnan-11-one by GC-MS and further gas chromatographic analysis. The amount of the steroid was minimal in the urine of infants, while in children submitted to substitution corticoid therapy it showed an increasing tendency, especially during puberty. The maximal value of excretion, in one case, amounted to 17% relative to total steroids. In puberty a significant excretion of 11-keto-pregnanediol indicates that under the given conditions the 11 beta-hydroxylation of steroid intermediates in the adrenals may be considerable not only at the level of 11-hydroxy-progesterone but also at that of progesterone.