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.

A 68-year-old phenotypically male patient with 21-hydroxylase deficiency and concomitant adrenocortical neoplasm producing testosterone and cortisol.

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 11ß-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 3ß-hydroxysteroid dehydrogenase, 17α-hydroxylase/17,20-lyase, and 17ß-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 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.

Detection of late onset steroid 21-hydroxylase deficiency by capillary gas chromatographic profiling of urinary steroids in children and adolescents.

Patients suffering from late onset 21-hydroxylase deficiency (LO-CAH) excreted only slightly higher amounts of 17-hydroxypregnanolone (17-OH-PO), pregnanetriol (PT) and 11-oxo-pregnanetriol (11-O-PT) than age-matched healthy controls. To discriminate between LO-CAH and virilization of unknown origin and precocious pubarche, we calculated the following ratios: (1) pregnanetriol to tetrahydrocortisone (PT/THE), (2) the sum of 17-OH-PO, PT and 11-O-PT (OHP-M) to the sum of THE, tetrahydrocortisol (THF) and allotetrahydrocortisol (a-THF) (C-M) and (3) 11-O-PT to C-M. The following patients were studied: 9 patients with congenital adrenal hyperplasia due to 21-hydroxylase deficiency-non-salt losing (CAH-NSL), never treated; 8 patients with CAH (NSL/SL: 3/5) off treatment; 10 patients with LO-CAH; 11 patients with virilization of unknown origin (prepubertal/pubertal: 5/6) and 9 patients with precocious pubarche. Healthy individuals and obligatory heterozygote carriers of comparable ages served as controls. LO-CAH showed increased ratios (median (range] of PT/THE: 2.27, (1.15-9.09), OHP-M/C-M: 2.30, (1.24-8.15), and 11-O-PT/C-M: 0.24, (0.13-1.23) compared to healthy individuals and heterozygous carriers: PT/THE 0.28, (0.03-0.57), OHP-M/C-M 0.23, (0.06-0.46) and 11-O-PT/C-M less than 0.01, (less than 0.01-0.06), respectively. The calculation of ratios, rather than absolute amounts seems to allow the detection of LO-CAH in a single spontaneously voided urine specimen. The clinical and measurable hormonal manifestations of LO-CAH occur at the same time.

[Reevaluation of recalled infants by neonatal mass screening for congenital adrenal hyperplasia due to 21-hydroxylase deficiency. Diagnostic value of pregnanetriolone in a single urine specimen using glass capillary gas chromatography].

To establish a detailed reevaluation system for infants who were recalled by a neonatal mass screening for congenital adrenal hyperplasia (CAH) due to 21-hydroxylase deficiency, pregnanetriol (PT) and pregnanetriolone (PTL) in a single urine specimen combined with plasma 17 alpha-hydroxyprogesterone (17-OHP) and 21-deoxycortisol (21-DOF) were determined by a simple method using glass capillary gas chromatography. A pilot study of neonatal mass screening for CAH with a determination of "disc 17-OHP" value in dried blood on filter paper was carried out in Western Shizuoka Prefecture. During the study period (32 months), 37472 neonates were determined by mass screening, and 362 neonates proved to be abnormal candidates who needed further evaluations. From out of these candidates, 262 neonates responded with recall and were studied. Amongst these 262 neonates, 241 neonates visited directly our outpatient clinic at Hamamatsu University Hospital. The reevaluation conducted at our clinic included a physical examination, detailed family history, measurement of serum electrolytes, disc 17-OHP, plasma 17-OHP and 21-DOF values, and PT and PTL in a single urine specimen. Consequently, 3 neonates appeared to be patients with CAH. Two of them were the salt-losing type and the other was the simple virilizing type. The rest of the candidates who received reevaluation were finally decided to be healthy neonates, indicating false positivity by mass screening. Compared to the candidates who showed false positivity in the mass screening, the CAH patients had an apparently high urinary PT and PTL titer of ten or one hundred fold. Additionally, despite corticosteroid treatment in one case, significantly elevated levels of PT and PTL were detected. To assay PTL was a more reliable parameter for the detection of CAH and for following up the candidates because PTL was not detectable in 63.3% of the false positive cases, suggesting that PTL was less likely to indicate false positive cases. PTL was detected at more than 0.01 microgram/ml urine in 19.4% of false positive cases, however, no case showed further elevation of PTL during the follow up period. In all false positive cases, PTL was not detectable until the age of six months. Despite problems to be resolved, determination of urinary PTL titer is valuable for the detection of CAH patients. In addition, urinary PTL could be a good parameter for the further follow up of false positive cases in neonatal mass screening.

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.

[Estimations of pregnanetriolone, pregnanetetrol, delta 5-pregnenetriol by spectrophotometry and by gas liquid chromatography (author's transl)]. / Dosages de prégnanetriolone, prégnanetétrol, delta 5-prégnènetriol par spectrophotométrie et par chromatographie gaz-liquide.

The author describes two methods of simultaneous estimation of pregnanetriolone, pregnanetetrol and delta 5-pregnenetriol. These estimations may be carried out at the same time as those of pregnanediol and pregnanetriol. The first method is based on spectrophotometry of derivatives obtained with 80 % sulphuric acid. The second demands gas chromatography. It permitted us to verify the specificity of the first. Its specificity was also verified by coupling with mass spectrometry. These estimations are used in the diagnosis of deficiencies of 21 hydroxylase and 3-beta hydroxysteroid oxidoreductase from the adrenal cortex. The values frequently obtained for delta 5-pregnenetriol are given.

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

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