Biochemical monitoring of 21-hydroxylase deficiency: a clinical utility of overnight fasting urine pregnanetriol.

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.

High-Resolution, Accurate-Mass (HRAM) Mass Spectrometry Urine Steroid Profiling in the Diagnosis of Adrenal Disorders.

BACKGROUND: Steroid profiling is a promising diagnostic tool with adrenal tumors, Cushing syndrome (CS), and disorders of steroidogenesis. Our objective was to develop a multiple-steroid assay using liquid-chromatography, high-resolution, accurate-mass mass spectrometry (HRAM LC-MS) and to validate the assay in patients with various adrenal disorders. METHODS: We collected 24-h urine samples from 114 controls and 71 patients with adrenal diseases. An HRAM LC-MS method was validated for quantitative analysis of 26 steroid metabolites in hydrolyzed urine samples. Differences in steroid excretion between patients were analyzed based on Z-score deviation from control reference intervals. RESULTS: Limits of quantification were 20 ng/mL. Dilution linearity ranged from 80% to 120% with means of 93% to 110% for all but 2 analytes. Intraassay and interassay imprecision ranged from 3% to 18% for all but 1 analyte. Control women had lower excretion of androgen and glucocorticoid precursors/metabolites than men (P < 0.001), but no difference in mineralocorticoids was seen (P = 0.06). Androgens decreased with age in both sexes (P < 0.001). Compared with patients with adrenocortical adenoma (ACA), patients with adrenocortical carcinoma (ACC) had 11 steroids with increased Z scores, especially tetrahydro-11-deoxycortisol (14 vs 0.5, P < 0.001), pregnanetriol (7.5 vs -0.4, P = 0.001), and 5-pregnenetriol (5.4 vs -0.4, P = 0.01). Steroid profiling also demonstrated metabolite abnormalities consistent with enzymatic defects in congenital adrenal hyperplasia and differences in pituitary vs adrenal CS. CONCLUSIONS: Our HRAM LC-MS assay successfully quantifies 26 steroids in urine. The statistically significant differences in steroid production of ACC vs ACA, adrenal vs pituitary CS, and in congenital adrenal hyperplasia should allow for improved diagnosis of patients with these diseases.

Bile Acid Synthesis Disorders in Arabs: A 10-year Screening Study.

OBJECTIVES: Early diagnosis of bile acid synthesis disorders (BASDs) is important because, untreated, these conditions can be fatal. Our objectives were to screen children with cholestasis or unexplained liver disease for BASD and in those with confirmed BASD to evaluate the effectiveness of cholic acid therapy. METHODS: A routine serum total bile acid measurement was performed on children with cholestasis, liver cirrhosis, and liver failure. Patients were screened for BASD by fast atom bombardment ionization-mass spectrometry (FAB-MS) analysis of urine, and molecular analysis confirmed diagnosis. Treatment response to oral cholic acid (10-15 mg/kg bw/day) was assessed from liver function tests and fat-soluble vitamin levels. FAB-MS analysis of urine was used to monitor compliance and biochemical response. RESULTS: Between 2007 and 2016, 626 patients were evaluated; 450 with infantile cholestasis. Fifteen cases of BASD were diagnosed: 12 presented with infantile cholestasis (2.7%, 7 boys), an 8-year-old boy presented with cirrhosis, and two 18-month-old boys presented with hepatomegaly and rickets. Eleven were caused by 3ß-hydroxy-Δ-C27-steroid oxidoreductase dehydrogenase deficiency, 3 from Δ-3-oxosteroid 5ß-reductase deficiency, and 1 had Zellweger spectrum disorder. In all but 1, serum total bile acids were normal or low. With cholic acid therapy, 10 are alive and healthy with their native liver. Liver failure developed in 3 infants despite therapy; 2 died and 1 underwent liver transplantation. CONCLUSIONS: BASDs are rare but treatable causes of metabolic liver disease in Saudi Arabia. BASD should be considered in infants with cholestasis and low or normal serum total bile acid concentrations.

Reduced activity of 11ß-hydroxylase accounts for elevated 17α-hydroxyprogesterone in preterms.

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' 11ß-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 11ß-hydroxylase activity in preterms. We could demonstrate a correlation between low 11ß-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 11ß-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.

Urinary steroidomic profiles by LC-MS/MS to monitor classic 21-Hydroxylase deficiency.

21-hydroxylase deficiency, the most common enzyme defect associated with congenital adrenal hyperplasia (CAH) is characterized by an impairment of both aldosterone and cortisol biosynthesis. Close clinical and biological monitoring of Hydrocortisone (HC) and 9α-Fludrocortisone (FDR) replacement therapies is required to achieve an optimal treatment. As frequent and repeated reassessments of plasma steroids, 17-hydroxyprogesterone (17-OHP), androstenedione (Δ4-A) and testosterone (TESTO) is needed in childhood, urine steroid profiling could represent an interesting non-invasive alternative. We developed and validated a LC-MS/MS method for the measurement of 23-urinary mineralocorticoids, glucocorticoids and adrenal androgens. The usefulness of steroid profiling was investigated on single 08h00 am-collected spot urine for discriminating between 61 CAH patients and their age- and sex-matched controls. CAH patients were split into two groups according to their 08h00 am-plasma concentrations of 17-OHP: below (controlled patients, n = 26) and above 20 ng/mL (uncontrolled patients, n = 35). The lower limit of quantification and the wide analytical range allows to assay both free and total concentrations of the main urinary adreno-corticoids and their tetra-hydrometabolites. Extraction recoveries higher than 75% and intra-assay precision below 20% were found for most steroids. Urinary steroids upstream of the 21-hydroxylase defect were higher in uncontrolled CAH patients. Among CAH patients, plasma and urinary 17-OHP were closely correlated. As compared to controls, steroids downstream of the enzyme defect collapsed in CAH patients. This fall was more pronounced in controlled than in uncontrolled patients. Androgens (Δ4-A, TESTO and the sum etiocholanolone + androsterone) accumulated in uncontrolled CAH patients. A strong relationship was observed between plasma and urinary levels of androstenedione. Daily doses and urinary excretion of both FDR and HC were similar in both CAH groups. Urinary FDR was inversely related to the sodium-to-potassium ratio in urine. A partial least squares discriminant analysis model allowed to classify the patient's classes unaffected, controlled and un-controlled CAH patients based on urinary steroidomic profiles. Our LC-MS/MS method successfully established steroid profiling in urine and represents a useful and non-invasive tool for discriminating CAH patients according to treatment efficiency.

Urinary GC-MS steroid metabotyping in treated children with congenital adrenal hyperplasia.

BACKGROUND: Treatment of children with classic congenital adrenal hyperplasia (CAH) is a difficult balance between hypercortisolism and hyperandrogenism. Biochemical monitoring of treatment is not well defined. OBJECTIVE: Cluster analysis of the urinary steroid metabolome obtained by targeted gas chromatography-mass spectrometry (GC-MS) for treatment monitoring of children with CAH. METHODS: We evaluated 24-h urinary steroid metabolome analyses of 109 prepubertal children aged 7.0 ±â€¯1.6 years with classic CAH due to 21-hydroxylase deficiency treated with hydrocortisone and fludrocortisone. 24-h urinary steroid metabolite excretions were transformed into CAH-specific z-scores. Subjects were divided into groups (metabotypes) by k-means clustering algorithm. Urinary steroid metabolome and clinical data of patients of each metabotype were analyzed. RESULTS: Four unique metabotypes were generated. Metabotype 1 (N = 21 (19%)) revealed adequate metabolic control with low cortisol metabolites (mean: -0.57z) and suppressed androgen and 17α-hydroxyprogesterone (17OHP) metabolites (-0.79z). Metabotype 2 (N = 23 (21%)) showed overtreatment consisting of a constellation of elevated urinary cortisol metabolites (0.62z) and low metabolites of androgens and 17OHP (-0.75z). Metabotype 3 (N = 32 (29%)) demonstrated undertreated patients with low cortisol metabolites (-0.69z) and elevated metabolites of androgens and 17OHP (0.50z). Metabotype 4 (N = 33 (30%)) presented patients with treatment failure reflected by unsuppressed androgen- and 17OHP metabolites (0.71z) despite elevated urinary cortisol metabolites (0.39z). CONCLUSION: Metabotyping, which means grouping metabolically similar individuals, helps to monitor treatment of children with CAH using GC-MS urinary steroid metabolome analysis. This method allows classification in adequately-, over-, or undertreated children as well as identification of patients with treatment failure.

Steroid abnormalities and the developing brain: declarative memory for emotionally arousing and neutral material in children with congenital adrenal hyperplasia.

Steroid hormones modulate memory in animals and human adults. Little is known on the developmental effects of these hormones on the neural networks underlying memory. Using Congenital adrenal hyperplasia (CAH) as a naturalistic model of early steroid abnormalities, this study examines the consequences of CAH on memory and its neural correlates for emotionally arousing and neutral material in children. Seventeen patients with CAH and 17 age- and sex-matched healthy children (ages 12-14 years) completed the study. Subjects were presented positive, negative and neutral pictures. Memory recall occurred about 30min after viewing the pictures. Children with CAH showed memory deficits for negative pictures compared to healthy children (p<0.01). There were no group differences on memory performance for either positive or neutral pictures (p>0.1). In patients, 24h urinary-free cortisol levels (reflecting glucocorticoid replacement therapy) and testosterone levels were not associated with memory performance. These findings suggest that early steroid imbalances affect memory for negative material in children with CAH. Such memory impairments may result from abnormal brain organization and function following hormonal dysfunction during critical periods of development.

Androgen excess is due to elevated 11-oxygenated androgens in treated children with congenital adrenal hyperplasia.

Adrenal androgen excess is the hallmark of classic congenital adrenal hyperplasia (CAH) due to 21-hydroxylase deficiency. Recently, 11-oxygenated C19 steroids, a class of highly active adrenal-derived androgens, have been described in patients with CAH. The aim of our study was to elucidate the significance of 11-oxygenated androgens in children with CAH. We retrospectively analysed 190 daily urinary excretion rates of glucocorticoid-, 17α-hydroxyprogesterone (17OHP)-, and androgen metabolites determined by gas chromatography-mass spectrometry of 99 children aged 3.0-10.9 years with classic CAH on hydrocortisone and fludrocortisone treatment. Daily urinary steroid metabolite excretions were transformed into z-scores using references of healthy children. Androgen metabolite z-scores were separately calculated for androsterone (AN), the major urinary metabolite of androstenedione (A4), testosterone and 5α-dihydrotestosterone, for urinary metabolites of dehydroepiandrosterone (DHEA), and for 11ß-hydroxyandrosterone (11OHAN), the major urinary metabolite of adrenal-derived 11-oxygenated androgens. Multivariate regression analysis was applied to analyse the precursors of 11OHAN synthesis. 11OHAN, cortisol-, and 17OHP metabolite z-scores were elevated in treated children with CAH, whereas AN- and DHEA metabolite z-scores were normalized or suppressed. Multivariate regression analysis revealed that 11OHAN excretion was strongest associated with 21-deoxycortisol (ß = 0.379; P =.0006), followed by A4 (ß = 0.280; P = .0008)) and 17OHP (ß = 0.243; P = .04) metabolite excretion. Androgen excess in treated children with CAH is solely due to elevated 11-oxygenated androgens that derive in addition to the known conversion from A4 also by direct conversion from 21-deoxycortisol. 11-Oxygenated androgens may represent better biomarkers of adrenal androgen status and treatment response than conventional androgens.

Urinary excretion of pregnanetriol and 5 -pregnenetriol in two forms of congenital adrenal hyperplasia.

Although congenital adrenal hyperplasia due to 3beta-hydroxysteroid dehydrogenase deficiency generally reveals a predominance of Delta(5)-3beta-hydroxysteroids, on occasion substantial quantities of pregnanetriol have been found as well. It appears that the latter steroid more often occurs in the subjects who have survived beyond infancy. The use of the measurement of pregnanetriol alone may therefore not be relied upon as a sole determinant of the specific form of defective steroidal biogenesis. It is more characteristic of the 21-hydroxylase deficiency. However when both Delta(5)-pregnenetriol and pregnanetriol are measured the ratio of the former to the latter is always considerably below 1.0 in 21-hydroxylase deficiency and always above 1.0 in 3beta-hydroxysteroid dehydrogenase. Furthermore, 11-ketopregnanetriol has been found only in the urine of subjects with the 21-hydroxylase deficiency. Thus, these two forms of defective steroidal biogenesis may be distinguished by the measurement of these three urinary steroidal metabolites.

Modified-Release and Conventional Glucocorticoids and Diurnal Androgen Excretion in Congenital Adrenal Hyperplasia.

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 11ß-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.

The urinary steroidome of treated children with classic 21-hydroxylase deficiency.

Monitoring treatment of children with classic congenital adrenal hyperplasia (CAH) is difficult and biochemical targets are not well defined. We retrospectively analysed 576 daily urinary steroid hormone metabolite profiles determined by gas chromatography-mass spectrometry of 150 children aged 3.0-17.9 years with classic 21-hydroxylase deficiency (21-OHD) on hydrocortisone and fludrocortisone treatment. Daily urinary excretion of glucocorticoid-, 17α-hydroxyprogesterone (17-OHP)-, and androgen metabolites as well as growth and weight gain are presented. Children with classic CAH exhibited increased height velocity during prepubertal age, which was then followed by diminished growth velocity during pubertal age until final height was reached. Final height was clearly below the population mean. 11ß-Hydroxyandrosterone was the dominant urinary adrenal-derived androgen metabolite in CAH children. Adrenarche is blunted in children with CAH under hydrocortisone treatment and androgen metabolites except 11ß-hydroxyandrosterone were suppressed. Cortisol metabolite excretion reflected supraphysiological hydrocortisone treatment dosage, which resulted in higher body-mass-indices in children with CAH. Reference values of daily urinary steroid metabolite excretions of treated children with CAH allow the clinician to adequately classify the individual patient regarding the androgen-, 17-OHP-, and glucocorticoid status in the context of the underlying disorder. Additionally, urinary 21-OHD-specific reference ranges will be important for research studies in children with CAH.

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 utility of three different methods for measuring urinary 18-hydroxycortisol in the differential diagnosis of suspected primary hyperaldosteronism.

OBJECTIVE: Urine 18-hydroxycortisol (18-OHF) measurements are claimed to discriminate between primary hyperaldosteronism due to Conn's syndrome/adrenal adenoma or idiopathic bilateral adrenal hyperplasia (BAH), and also to identify cases of glucocorticoid-suppressible hyperaldosteronism (GSH). We have evaluated three urine 18-OHF methods using a panel of urine samples from patients with hypertension. DESIGN: Clinical methods comparative study. METHODS: Urine samples from patients with primary hyperaldosteronism due to either adenoma (n = 6), BAH (n = 6), GSH (n = 9), or essential hypertension (n = 38) were analysed without knowledge of the diagnosis using three different methods in different laboratories. These included 'in-house' radioimmunoassay (RIA), 'in-house' time-resolved fluorometric assay (DELFIA), and gas chromatography mass spectrometry (GC-MS). RESULTS: The three assays showed good correlation, but there were large bias differences: RIA bias was greater than DELFIA which was greater than GC-MS. Discrimination between adenoma and BAH patients was best for the DELFIA method, with no overlap between results for these two groups. All three methods gave significantly elevated results for the GSH group compared with the BAH and essential hypertension groups. No assay distinguished BAH from essential hypertension. CONCLUSION: Measurement of urine 18-OHF may be a useful additional test in the differential diagnosis of primary hyperaldosteronism. The clinical diagnostic value of urinary 18-OHF measurements is method-dependent with the DELFIA assay having the best discriminatory value.

Focused metabolomics using liquid chromatography/electrospray ionization tandem mass spectrometry for analysis of urinary conjugated cholesterol metabolites from patients with Niemann-Pick disease type C and 3ß-hydroxysteroid dehydrogenase deficiency.

BACKGROUND: Various conjugated cholesterol metabolites are excreted in urine of the patients with metabolic abnormalities and hepatobiliary diseases. We aimed to examine the usefulness of precursor ion scan and neutral loss scan for the characterization of conjugated cholesterol metabolites in urine. METHODS: A mixture of authentic standards of conjugated cholesterol metabolites was used for investigating the performance of the present method. The urine of patients with Niemann-Pick diseases type C and 3ß-hydroxysteroid dehydrogenase deficiency were analysed by precursor ion scan of m/z 97, 74, and 124. RESULTS: A precursor ion scan of m/z 97 was effective for identifying conjugates with ester sulphates on hydroxyl groups whereas ester sulphates on phenolic alcohols were signalled by a neutral loss scan of 80 Da. Monosaccharide-conjugated cholesterol metabolites were signalled by a precursor ion scan of m/z 113. Although precursor ion scan of m/z 74 and 124 was effective for finding glycine- and taurine-conjugated metabolites, high intensity of product ions (m/z 74 and 124) disturbed measurement of other multiply conjugated metabolites. The urine samples contained many conjugated cholesterol metabolites, and there were several disease-specific intense peaks. We found several unknown intense peaks with three known peaks in urine of the Niemann-Pick type C patient. In the patient with 3ß-hydroxysteroid dehydrogenase deficiency, intense peaks that were tentatively identified as 5-cholenoic acid sulphates and their glycine and taurine conjugates were present. CONCLUSION: The method should lead to the discovery of new urinary biomarkers for these disturbances of cholesterol catabolism and transport.

Steroid excretion in urine during suppression and stimulation of adrenals in the 17 alpha-hydroxylase deficiency syndrome.

Twenty-three steroids were determined by capillary column gas chromatography in urine specimens from a patient with the 17 alpha-hydroxylase deficiency syndrome during dexamethasone suppression and ACTH stimulation tests. These steroids included pregnanediols and pregnenediols, 21-deoxy metabolites of corticosterone (e.g. 3 alpha, 20 alpha-dihydroxy-5 alpha-pregnan-11-one), and 21-hydroxy metabolites [e.g. allo-tetrahydrocorticosterone (3 alpha, 11 beta, 21-trihydroxy-5 alpha-pregnan-20-one)]. During dexamethasone suppression or ACTH stimulation, the excretion of all steroids decreased and increased, respectively, indicating the pituitary-dependent adrenal origin of each compound. The changes in excretion of 21-deoxy steroids responded slowly (compared with the 21-hydroxylated metabolites) to these maneuvers, thereby providing further evidence for extensive enterohepatic circulation of corticosterone metabolites. 21-Dehydroxylated corticosterone metabolites accounted for approximately 40% the total steroids excreted during the control period, and this percentage probably approximates the proportion of corticosterone excreted in bile.

The diagnosis of congenital adrenal hyperplasia in the newborn by gas chromatography/mass spectrometry analysis of random urine specimens.

Definitive neonatal diagnosis of congenital adrenal hyperplasia (CAH) is frequently complicated by normal 17-hydroxyprogesterone levels in 21-hydroxylase-deficient patients, residual maternal steroids, and other interfering substances in neonatal blood. In an effort to improve the diagnosis, we developed a gas chromatography/mass spectrometry method for simultaneous measurement of 15 urinary steroid metabolites as early as the first day of life. Furthermore, we developed 11 precursor/product ratios that diagnose and clearly differentiate the four enzymatic deficiencies that cause CAH. Random urine samples from 31 neonatal 21-hydroxylase-deficient patients and 59 age-matched normal newborns were used in the development. Additionally, samples from two 11 beta-hydroxylase-deficient patients and one patient each for 17 alpha-hydroxylase and 3 beta-hydroxysteroid dehydrogenase deficiencies were used. The throughput for one bench-top gas chromatography/mass spectrometry instrument is 20 samples per day. Thus, this method affords an accurate, rapid, noninvasive means for the differential diagnosis of CAH in the newborn period without the need for invasive testing and ACTH stimulation.

Clinical and biochemical variability of congenital adrenal hyperplasia due to 11 beta-hydroxylase deficiency. A study of 25 patients.

Twenty five patients (10 males and 15 females) aged 0-23 yr with congenital adrenal hyperplasis due to 11 beta-hydroxylase deficiency were studied. They were divided into 13 classic (group A), and 12 mild (group B) patients. The patients of group A were diagnosed at a younger age and had more severe clinical symptoms (ambiguous genitalia in girls, pseudoprecocious puberty in boys). Two had neonatal salt wasting before treatment, and one gynecomastia. Seven had moderate to severe hypertension. Their mean 3 alpha,17,21-trihydroxy-5 beta-pregnan-20-one (THS) and 3 alpha, 21-dihydroxy-5 beta-pregnane-11,20-dione (THDOC) excretion was 14.2 +/- 4.1 and 7.2 +/- 4.2 mg/m2 . day, respectively. The patients of group B had mostly late onset of symptoms (hirsutism, amenorrhea in girls, pseudoprecocious puberty in boys, tall stature, and advanced bone age in both sexes). One boy had bilateral cryptorchidism. Four had moderate hypertension. In seven patients, THS (5.3 +/- 2.3 mg/m2 . day) and THDOC (3.9 +/- 0.5 mg/m2 . day) responded to ACTH. In five, only THS (4.3 +/- 1.1 mg/m2 . day) responded, but THDOC remained undetectable. It is concluded that the clinical and biochemical expression of 11 beta-hydroxylase deficiency is variable, that hypertension in not directly related to deoxycorticosterone, and that, regardless of the intensity of the defect, there are patients in whom the 11 beta-hydroxylation of 17 alpha-hydroxylated steroids only is impaired, and others in whom both the conversion of 17,20-dihydroxy-4-pregnene-3,20-dione and deoxycorticosterone are reduced.

11 Beta-hydroxylase deficiency congenital adrenal hyperplasia: update of prenatal diagnosis.

Hormonal measurements in maternal urine and amniotic fluid (AF) during pregnancy and/or at delivery correctly predicted the postnatal diagnosis of 11 beta-hydroxylase deficiency congenital adrenal hyperplasia (11 beta-OH deficiency CAH) in 7 fetuses at risk. In the 4 affected ones, maternal urinary tetrahydro-11-deoxycortisol (THS) excretion was high during the first trimester [0.3-2.2 mg/day (1.1-7.7 mumol/day)] and rose further during the third trimester [0.5-3.5 mg/day (1.8-12.3 mumol/day)] compared to urinary THS excretion in 20 normal pregnancies of the same gestational age (P less than 0.01). In 1 mother, dexamethasone administration (2 mg/day for 72 h) greatly reduced urinary THS excretion (and plasma steroid levels). Urinary THS excretion was low after delivery in these mothers, in normal pregnancies, and in parents of affected individuals [less than 0.05 mg/day (less than 0.08 mumol/day); P = NS]. However, 2 of the 3 heterozygous mothers who carried nonaffected fetuses excreted moderately increased amounts of THS during pregnancy, ranging from 0.15-0.26 mg/day (0.53-0.91 mumol/day), significantly higher than normal (P less than 0.01). Although urinary THS excretion in these mothers was similar to that in 2 mothers with affected fetuses early in pregnancy, urinary THS excretion was higher in mothers with affected compared to those with nonaffected fetuses after the first trimester (P less than 0.01). AF THS and 11-deoxycortisol concentrations were markedly elevated in pregnancies with affected fetuses (P less than 0.01), but normal in nonaffected ones. AF delta 4-androstenedione levels were high in 2 pregnancies and borderline elevated in a third. Although the AF tetrahydrocortisol and tetrahydrocortisone levels were always within the normal range, the AF THS to tetrahydrocortisol plus tetrahydrocortisone ratio was significantly elevated in all pregnancies with affected fetuses (2.8-5.5; P less than 0.01) and normal in nonaffected ones (0.48-1.2; P = NS) compared to that in 160 normal pregnancies [0.64 +/- 0.34 (+/- SD)]. AF 17-hydroxyprogesterone, testosterone, and 11-deoxycorticosterone levels were normal in all pregnancies. Maternal plasma 11-deoxycortisol and delta 4-androstenedione concentrations, determined sequentially throughout gestation, were variable and did not contribute to prenatal diagnosis. All affected infants were born hyperpigmented, 2 were large for gestational age, and the female was severely virilized. In the first week of life 2 males developed severe hypertension with seizures and adrenal insufficiency, respectively.(ABSTRACT TRUNCATED AT 400 WORDS)