OBJECTIVE

The treatment goal in congenital adrenal hyperplasia (CAH) is to replace glucocorticoids while avoiding androgen excess and iatrogenic Cushing's syndrome. However, there is no consensus on how to monitor disease control. Our main objectives were to evaluate hormonal circadian rhythms and use these profiles to identify optimal monitoring times and novel disease biomarkers in CAH adults on intermediate- and long-acting glucocorticoids.

METHODS

This was an observational, cross-sectional study at the National Institutes of Health Clinical Center in 16 patients with classic CAH.

METHODS

Twenty-four-hour serum sampling for ACTH, 17-hydroxyprogesterone (<em>17OHP</em>), androstenedione (A4), androsterone, DHEA, testosterone, progesterone and 24-h urinary pdiol and 5β-pdiol was carried out. Bayesian spectral analysis and cosinor analysis were performed to detect circadian rhythmicity. The number of hours to minimal (TminAC) and maximal (TmaxAC) adrenocortical hormone levels after dose administration was calculated.

RESULTS

A significant rhythm was confirmed for ACTH (r(2), 0.95; P<0.001), <em>17OHP</em> (r(2), 0.70; P=0.003), androstenedione (r(2), 0.47; P=0.043), androsterone (r(2), 0.80; P<0.001), testosterone (r(2), 0.47; P=0.042) and progesterone (r(2), 0.64; P=0.006). The mean (s.d.) TminAC and TmaxAC for <em>17OHP</em> and A4 were: morning prednisone (4.3 (2.3) and 9.7 (3.5) h), evening prednisone (4.5 (2.0) and 10.3 (2.4) h), and daily dexamethasone (9.2 (3.5) and 16.4 (7.2) h). AUC0-24 h progesterone, androsterone and 24-h urine pdiol were significantly related to <em>17OHP</em>.

CONCLUSIONS

In CAH patients, adrenal androgens exhibit circadian rhythms influenced by glucocorticoid replacement. Measurement of adrenocortical hormones and interpretation of results should take into account the type of glucocorticoid and time of dose administration. Progesterone and backdoor metabolites may provide alternative disease biomarkers.

OBJECTIVE

To determine normal concentrations of 17alpha-hydroxyprogesterone (<em>17OHP</em>) for premature infants.

METHODS

<em>17OHP</em> was measured in 66 consecutive premature infants once a week during the first month, and once every two weeks thereafter, until the age of 3 months. The <em>17OHP</em> values in 100 full term healthy neonates on the third day of life served as controls. Blood was sampled on filter paper using a neonatal radioimmunoassay kit. Findings were correlated with gestational age, birthweight, mode of delivery, Apgar scores, presence of respiratory distress syndrome and intake of maternal steroids.

RESULTS

Mean <em>17OHP</em> was raised at 7 days of age (138.9, 46.3, 53.3, 29.9 nmol/l, respectively, for infants whose gestational age was under 29 weeks, 29 to 30 weeks, 31 to 32 weeks, and 33 weeks and above). It fell sharply in the first two weeks after which it gradually decreased further, reaching 32.7, 23.6, 16.9, and 13.0 nmol/l, respectively, by the age of 90 days. The mean (SEM) <em>17OHP</em> concentration in full term infants on day 3 of life was 17.8 (8.9) nmol/l. These values were independent of the presence and severity of respiratory distress syndrome and of prenatal maternal steroids.

CONCLUSIONS

The increased <em>17OHP</em> concentrations found at birth fell to those found in term infants during the first three months of life in infants over 31 weeks of gestation. Postconceptional age is the most important factor determining <em>17OHP</em> concentration.

Congenital adrenal hyperplasia (CAH) is a common disorder of impaired adrenal cortisol biosynthesis with associated androgen excess. The clinical presentation of 21-hydroxylase deficiency, the commonest cause of CAH, forms a spectrum and can be divided into classic and non-classic types. The former consists of life threatening salt wasting and non-life threatening simple virilizing phenotypes. Patients with the non-classic form are asymptomatic or have mild features of androgen excess. Most developed countries have newborn screening facilities for CAH. In the absence of newborn screening, the diagnosis of CAH may be missed or delayed. This can result in neonatal mortality in salt wasting forms and incorrect sex of rearing in females with simple virilizing form. The diagnosis is reached by demonstrating high serum 17-hydroxyprogesterone (<em>17OHP</em>) levels. Preterm birth and neonatal illness can cause physiological elevation of <em>17OHP</em>, thus complicating the diagnosis of CAH in the newborn period. Prenatal diagnosis and treatment with dexamethasone to prevent virilization of affected female fetuses is another area of controversy. The management of CAH is complicated by the need to use supraphysiologic doses of glucocorticoids to suppress adrenal androgen synthesis. In this review, the authors address pertinent issues related to the diagnosis and management of CAH in children.

Hydrocortisone has long been the treatment of choice for congenital adrenal hyperplasia (CAH). However, treatment with this medication remains problematic. Patients with 21-hydroxylase deficiency CAH have significant diurnal variation in the secretion of 17-hydroxyprogesterone (<em>17OHP</em>). When considering treatment strategies, this variation must be considered along with the pharmacokinetic and pharmacodynamic properties of exogenous glucocorticoids. Orally administered hydrocortisone is highly bioavailable, but it has a short time to maximum concentration (T(max)) and half life (T(1/2)). While prednisone has a somewhat longer T(max) and T(1/2), they remain relatively short. There have been several studies of the pharmacodynamics of hydrocortisone. We present data indicating that the maximum effect of hydrocortisone in CAH patients is seen 3 hours after a morning dose. After an evening dose, suppression of adrenal hormones continues until approximately 0500 the next day. In both situations, however, there is a large degree of intersubject variability. These data are consistent with earlier published studies. Use of alternate specimen types, possibly in conjunction with delayed release hydrocortisone preparations under development, may allow the practitioner to design a medication regimen that provides improved control of androgen secretion. Whatever dosing strategy is used, clinical judgment is required to ensure the best outcome.

Clinical and hormonal changes in girls with precocious pubarche, a multifactorial entity, have not been thoroughly investigated.

OBJECTIVE

To describe anthropometric features and bone age in Brazilian girls with precocious pubarche, and to compare clinical and hormonal findings for patients with isolated precocious pubarche (IPP) or non-classical congenital adrenal hyperplasia (NC-CAH).

METHODS

Twenty-eight girls with precocious pubarche were consecutively seen at the Child and Pubertal Gynecologic Clinic and the Gynecological Endocrinology Unit of Hospital de Clinicas de Porto Alegre. Patients with central precocious puberty and pseudopuberty were excluded from the study. Diagnostic criteria: NC-CAH: 17-hydroxyprogesterone (<em>17OHP</em>) >12 ng/ml 60 min after 0.25 mg i.m. ACTH; IPP: hormonal levels compatible with adrenarche and normal adrenal function confirmed by ACTH testing. Anthropometric and hormonal features and bone age were assessed.

RESULTS

Six patients had NC-CAH, and 22 IPP. NC-CAH frequency was 21.42%. Weight and BMI were above 50th percentile except for patients <4 years; 46% of the girls were either overweight or obese. Clinical and hormonal features, except for response to ACTH testing, were similar in both groups; all patients were in or above the 50th percentile for height. Bone age was advanced in 43% of the patients. There was no statistical difference between the groups.

CONCLUSIONS

Slight acceleration of height and bone age, one of the clinical manifestations of precocious pubarche, is unlikely to be progressively severe.

Basal serum <em>17OHP</em> measurement remains the first screening step for nonclassic congenital adrenal hyperplasia (NCCAH) and the accuracy of the test is of high value. The aim of this study was to compare the accuracy of immunoassays to LC-MS/MS in the assessment of serum <em>17OHP</em> and androgens concentration in women with hyperandrogenism and controls. <em>17OHP</em>, total testosterone, androstendione and DHEA-S were measured in 39 women with clinically and/or biochemically evident hyperandrogenism and in 29 age-matched controls without clinical hyperandrogenism. <em>17OHP</em> and androgens were measured by immunoassays and by LC-MS/MS. In patients group median <em>17OHP</em> level measured by immunoassays was significantly higher compared to LC-MS/MS (5.49 nmol/l-ELISA NovaTec® and 3.57 nmol/l-ELISA DRG® versus 1.56 nmol/l-LC-MS/MS p < 0.0001) as well as in the control group (2.58 nmol/l-ELISA DRG® versus 1.14 nmol/l-LC-MS/MS p < 0.0001). Additional, unnecessary diagnostic procedures explaining elevated <em>17OHP</em> level were undertaken in 85% of patients when NovaTec® test was used, in 50% when ELISA DRG® and in none when LC-MS/MS method was applied. Total testosterone, androstendione and DHEA-S concentrations in the patients and the controls assessed by the immunoassays were also significantly higher compared to LC-MS/MS. LC-MS/MS is more reliable diagnostic tool in the measurement of serum <em>17OHP</em> and androgens concentrations compared to immunoassays in women with hyperandrogenism.

OBJECTIVE

To evaluate function of the hypothalamic-pituitary-adrenal (HPA) axis, adrenomedullary hormonal system (AMHS), and sympathetic noradrenergic system (SNS) in premenopausal women with systemic sclerosis (SSc).

METHODS

Insulin-induced hypoglycemia (0.1 IU/kg) was performed in 17 longterm, glucocorticoid-naive SSc patients with low disease activity and in 18 healthy women matched for age and body mass index (BMI). Concentrations of glucose, adrenocorticotrophic hormone (ACTH), cortisol, androstenedione (ASD), dehydroepiandrosterone (DHEA), dehydroepiandrosterone sulfate (DHEAS), 17a-hydroxyprogesterone (<em>17OHP</em>), epinephrine (EPI), norepinephrine (NE), interleukin 1ss (IL-1ss), IL-6, and tumor necrosis factor-a (TNF-a) were analyzed in plasma.

RESULTS

Basal plasma levels of cortisol, ASD, <em>17OHP</em>, DHEAS, IL-1ss, IL-6, and TNF-a were not significantly different in SSc compared to controls. Patients had higher basal ACTH (6.76 +/- 1.0 pmol/l in SSc vs 4.14 +/- 0.45 pmol/l in controls; p < 0.05), lower basal DHEA (9.02 +/- 1.64 nmol/l in SSc vs 17.0 +/- 2.8 nmol/l in controls; p < 0.05), and lower basal NE (1.61 +/- 0.26 nmol/l in SSc vs 2.57 +/- 0.38 nmol/l in controls; p < 0.05). Patients had comparable responses of glucose and ACTH to hypoglycemia. General linear model for repeated measurements, with BMI and age as covariates, revealed that the responses of <em>17OHP</em> (p < 0.05), ASD (p < 0.05), DHEA (p < 0.01), EPI (p < 0.001), and NE (p < 0.001) to hypoglycemia were lower in SSc compared to controls. Cortisol response to hypoglycemia tended to be lower in SSc patients (p = 0.06) compared to controls.

CONCLUSIONS

Our data indicate decreased adrenocortical and adrenomedullary functions in premenopausal women with SSc. Whether the observed changes in the neuroendocrine system are secondary to chronic disease deserves further investigation.

OBJECTIVE

To evaluate the function of the hypothalamic-pituitary-adrenal axis and sympathoadrenal system in premenopausal women with rheumatoid arthritis (RA).

METHODS

Insulin-induced hypoglycaemia (0.1 IU/kg) was produced in 15 glucocorticoid-naive patients with long term RA with low disease activity and in 14 healthy women matched for age and body mass index. Concentrations of glucose, adrenocorticotropic hormone (ACTH), cortisol, Delta4-androstenedione (ASD), dehydroepiandrosterone (DHEA), dehydroepiandrosterone sulphate (DHEAS), 17alpha-hydroxyprogesterone (<em>17OHP</em>), epinephrine (EPI), norepinephrine (NE), interleukin 6 (IL6), and tumour necrosis factor alpha (TNFalpha) were analysed in plasma.

RESULTS

Patients had comparable responses of glucose, cortisol, ACTH, ASD, and <em>17OHP</em> to hypoglycaemia, without any signs of hypothalamic insufficiency. Patients had lower basal DHEAS than controls (3.03 (0.37) micromol/l v 5.1 (0.9) micromol/l, respectively; p<0.05); borderline lower basal DHEA levels (p = 0.067); while the response of DHEA to hypoglycaemia was comparable to that of controls. Patients with RA had lower EPI (p = 0.005) and NE (p<0.001) responses to hypoglycaemia. TNFalpha and IL6 were higher (p<0.05) in patients with RA (TNFalpha 8 (2.8) pg/ml in RA v 1.1 (0.5) pg/ml in controls and IL6 15.1 (6.7) pg/ml v 1.4 (0.7) pg/ml).

CONCLUSIONS

Lower basal DHEAS levels, without concomitant differences or changes in DHEA, ASD, <em>17OHP</em>, and cortisol responses to hypoglycaemia in patients with RA, indicate an isolated decrease in adrenal androgen production. Significantly lower responses of EPI and NE to hypoglycaemia may suggest sympathoadrenal hyporeactivity in patients with RA.

BACKGROUND

Neonatal screening for congenital disorders like phenylketonuria (PKU), congenital hypothyroidism (CH) and congenital adrenal hyperplasia (CAH) is generally performed in dried blood spots on filter paper. The analytes of interest for testing for PKU, CH and CAH are phenylalanine, thyrotropin (TSH) and 17alpha-hydroxyprogesterone (<em>17OHP</em>), respectively. The International Society for Neonatal Screening (ISNS) decided to prepare a combined reference preparation for the three analytes on filter paper Schleicher & Schuell #903, Whatman BFC180 and Toyo Roshi 545. This 'First ISNS Reference Preparation for Neonatal Screening for TSH, phenylalanine and <em>17OHP</em> in blood spots' (1st ISNS-RPNS) has been prepared by the RIVM (Bilthoven).

METHODS

The number of filter paper cards prepared, each with two sets of six blood spot calibrators, was 480, 42 and 69 for Schleicher & Schuell #903, Whatman BFC180 and Toyo Roshi 545, respectively. The volume of blood dispensed was 50 microl. The range of concentrations for TSH was 1-121 mIU/L blood, for phenylalanine 65-865 micromol/L blood and for <em>17OHP</em> 2.2-302 nmol/L blood.

RESULTS

The linearity of the blood spot calibrators and the homogeneity of the batch (only tested for Schleicher & Schuell) were good. The differences between the three filter papers were small: i.e. the potency of the ISNS-RPNS on Whatman and Toyo Roshi in terms of Schleicher & Schuell was between 0.98 and 1.09 for the three analytes.

CONCLUSIONS

The 1st ISNS-RPNS for TSH, phenylalanine and <em>17OHP</em> can be said to be suitable as formal reference preparation and as a source for (re)calibrating kit calibrators.

Immunoassays of steroid hormones are still used in the diagnosis and monitoring of patients with congenital adrenal hyperplasia. However, cross-reactivity between steroids can give rise to falsely elevated steroid levels. Here we compare the use of immunoassays and liquid chromatography-tandem mass spectrometry (LC-MS/MS) in the monitoring of patients with classic 21-hydroxylase deficiency (21OHD). Steroid profiles in different mutation groups (genotypes) were also compared. Fifty-five patients with classic 21OHD (38 women) were studied. Blood samples were collected in the morning after an overnight medication fast. LC-MS/MS and immunoassays were employed to assay 17-hydroxyprogesterone (<em>17OHP</em>), testosterone and androstenedione. In addition, 21-deoxycortisol (21DF), 11-deoxycortisol (11DF), corticosterone, deoxycorticosterone, cortisone and cortisol were analyzed by LC-MS/MS. Testosterone, androstenedione and <em>17OHP</em> levels were consistently lower (by about 30-50%) when measured by LC-MS/MS compared with immunoassays, with exception of testosterone in men. There was a significant correlation between 21DF and <em>17OHP</em> (r=0.87, p<0.001), but three patients had undetectable 21DF. Subjects with no enzyme activity had significantly lower mean 11DF concentrations than subjects with residual activity. The use of LC-MS/MS gives a more specific view of adrenal steroid levels in 21OHD compared with immunoassays, which seem to considerably overestimate the levels of <em>17OHP</em> and androstenedione. Falsely elevated levels of <em>17OHP</em> and androstenedione could lead to overtreatment with glucocorticoids.

OBJECTIVE

(1) Characterize serum (S) and urinary (U) steroid metabolites in complete CYP17 deficiency (cCYP17D); (2) analyze the relative 17α-hydroxylase (17OH) and 17,20-lyase (17,20L) activities in vivo; and (3) comparedata from the two most prevalent mutations in Brazil.

METHODS

20 genotyped cCYP17D patients from a previously reported cohort were homozygous for W406R or R362C; 11 controls were CYP17 wild types (WT). WT and cCYP17D patients had S and U samples drawn to measure: cortisol (F), corticosterone (B), deoxycorticosterone (DOC), 18OH-B, 18OH-DOC, and <em>17OHP</em>; and tetrahydro (TH)-B, THA, THDOC, THF+5α-THF, TH-cortisone, androsterone, etiocholanolone, 5-pregnenediol, 17OH-pregnenolone and pregnanetriol.

RESULTS

Compared to WT, cCYP17D patients had marked elevations of B, DOC, 18OH-B and 18OH-DOC, whereas <em>17OHP</em>, F and adrenal androgens (AA) were reduced; U steroids parallel S findings. Metabolite ratios revealed that both 17OH and 17,20L activities were impaired in cCYP17D. There were nodifferences between W406R andR362C mutations.

CONCLUSIONS

cCYP17D patients show parallel overproduction/overexcretion of 17-deoxysteroids, and marked reduction of F and AA. In addition to 17OH, 17,20-L activity was also impaired in cCYP17D. W406 and R362C mutations disclose similar Sand U patterns.

The concentration of 17-OH-progesterone (17-OHP) was measured retrospectively in a second-trimester amniotic-fluid sample obtained from a mother who had an infant with congenital adrenal hyperplasia (CAH) due to 21-hydroxylase deficiency. The concentration was more than three times the mean amniotic-fluid-<em>17OHP</em> concentration determined in pregnancies of comparable gestational age with normal outcome. In four further pregnancies tested, where the parents were heterozygous for CAH, amniotic-fluid concentrations of 17-OHP were normal. To date, three of the mothers have delivered normal infants. CAH can be detected in early pregnancy by specific radioimmunoassay techniques for steroid-hormone analysis in amniotic fluid. This antenatal test could be useful in those cases in which parents do not wish to risk having affected offspring.

A neonatal screening for both 21-hydroxylase and 11-beta-hydroxylase deficiencies, responsible for congenital adrenal hyperplasia (CAH), has been conducted in Campania Region, Southern Italy. In 4380 neonates, aged 2-10 days, capillary blood from a heel prick was collected on microfilter paper, and 17-alpha-hydroxyprogesterone (<em>17OHP</em>) measured by radioimmunoassay (RIA) using a highly specific antibody (Ab A). In addition, in 295 of these samples, both <em>17OHP</em> and 11-deoxycortisol (S) were measured using an anti-deoxycortisol antibody (Ab B) cross-reacting with <em>17OHP</em> 100%. All results were compared with plasma <em>17OHP</em> and S levels in 21 patients with diagnosed 21-hydroxylase deficiency and in 5 healthy volunteers on metyrapone test used for blocking the 11-beta-hydroxylase activity. CAH due to 21-hydroxylase deficiency was diagnosed in a female newborn. The assay, based on the antibody reacting with both <em>17OHP</em> and S, is particularly suitable for wide-scale screening programs enabling the simultaneous detection of two congenital enzyme defects.

In a Swiss screening programme for detection of congenital adrenal hyperplasia (CAH), 27 of over 120,000 newborns examined from 1992 to 1994 were further studied because of persistingly high 17 alpha hydroxyprogesterone (<em>17OHP</em>). Out of 27, 11 were later confirmed to have CAH by specific gas chromatography of urinary steroids and ACTH test at age 3-4 months. Of 27, 11 were born at term (7 confirmed 21-hydroxylase deficiency, one 11 beta-hydroxylase deficiency). Out of 27, 16 were preterm newborns. Of them, only 2 were confirmed to have CAH (one 21-, one 11 beta-hydroxylase deficiency). In 3 cases with high <em>17OHP</em>, but later not confirmed CAH, what appeared to be a pregnanetriolone peak in the gas chromatograms was shown to be 3 beta, 15 beta, 17 alpha-pregnenetriol. This compound may be misleading in confirming the diagnosis of CAH. 15 beta-Hydroxylated compounds occur in fetuses, neonates, and amniotic fluid. Since human tissues do not have 15 beta-hydroxylating capacity, their origin is unclear. However, since some bacteria (Bacillus megatherium) and mycelial fungi (fusaria) are known to hydroxylate steroids in position 15 beta, it is likely that this compound is formed by micro-organisms in the enterohepatic circulation of newborns or their mothers.

CONCLUSIONS

For the confirmation of the diagnosis of CAH in cases suspected by screening, later ACTH stimulation and specific steroid analysis are necessary.

BACKGROUND

Midtrimester ultrasound is a valuable method for identifying asymptomatic women at risk for spontaneous preterm delivery (PTD). However, response to various treatments (cerclage, progestogen) has been variable in the clinical setting. It remains unclear how other biomarkers may be used to guide intervention strategies.

OBJECTIVE

We applied an amniotic fluid inflammatory scoring system to determine if the degree of inflammation is associated with intervention efficacy in patients with midtrimester short cervix.

METHODS

Women carrying a singleton fetus between 16-24 weeks' gestation with a short cervix (≤25 mm) on transvaginal ultrasound underwent amniocentesis and were assigned to McDonald cerclage, no cerclage, or weekly 17-alpha hydroxyprogesterone caproate (<em>17OHP</em>-C). Our previously described inflammatory risk score (comprised of 14 inflammatory markers) was used to classify patients as high (score ≥8) or low (score <8) risk for inflammation. Gestational age at delivery was compared for each intervention and risk score status. Risk of delivering as a function of the remaining gestation was evaluated using modified Cox proportional hazards models with incorporation of methods to account for both left and right truncation bias.

RESULTS

Ninety patients were included: 24 were in the nonintervention control group, 51 received cerclage, and 15 received <em>17OHP</em>-C. Inflammation status at time of sampling influenced the efficacy of the treatment (P < .001). Compared to the nonintervention control group, in patients with low inflammation (score < 8), both cerclage (adjusted hazard ratio [HR], 2.86; 95% confidence interval [CI], 1.28-6.37) and <em>17OHP</em>-C (HR, 3.11; 95% CI, 1.04-9.30) were associated with increased hazard of PTD. In contrast, in patients with high inflammation (score ≥8) both cerclage (HR, 0.22; 95% CI, 0.08-0.65) and <em>17OHP</em>-C (HR, 0.20; 95% CI, 0.05-0.81) were associated with lower hazard of delivering preterm.

CONCLUSIONS

Cerclage placement or administration of <em>17OHP</em>-C therapy for midtrimester short cervix for PTD prevention appears beneficial only in the subset of patients with high inflammation. Knowledge of the amniotic fluid inflammatory status may aid in guiding the appropriate therapy for women presenting with midtrimester short cervix who are at increased risk of PTD.

In order to document testicular 17beta-reduction deficiency (17RD) and to search for additional metabolic aberrations possibly associated with this disorder, the metabolism of 14C-labeled pregnenolone (delta5P), 17-HYDROXYPROGESTERONE (<em>17OHP</em>), dehydroepiandrosterone (DHEA), androstenedione (A), testosterone (T) and estrone (E1) was studied in testicular minces from a 46-year-old male pseudohermaphrodite (MPH) with highly elevated testicular A and minimal T secretion but normal extragonadal conversion of A to T. Testicular minces from a 20-year-old MPH with apparently normal testicular T biosynthesis served as a control. The results of this investigation show that the 17RD testes metabolized delta5P along delta5- and delta4- pathways but, in contrast to the control, converted more <em>17OHP</em>, metabolizing it predominantly to A rather than T, failed to reduce DHEA to androst-5-ene-3beta,17beta-diol, metabolized DHEA very efficiently to A and produced little T, and converted only minimal quantities of A and E1 to their 17beta-reduced counterparts. 17beta-Reduction increased slightly but was far from being restored to control levels upon addition of NADH plus NADPH. However, oxidation of T to A by 17RD testicular minces, with and without additional NAD plus NADP, was comparable to that by the control. These results document 17RD for A, DHEA and E1 and suggest that the lack of elevated <em>17OHP</em> and DHEA secretion by the 17RD testes was due to increased 17, 20-lyase and perhaps elevated 3beta-hydroxysteroid dehydrogenase and/or isomerase activity. The observation that 17beta-reduction was only slightly increased upon addition of NADH plus NADPH, but that oxidation of T to A was normal, is consistent with the assumption that more than one 17beta-hydroxysteroid dehydrogenase may be involved in testicular 17beta-reduction and/or 17-oxidation, and that the 17RD testes studied either lacked the enzyme which acts predominantly as 17beta-reductase or that the affinity of this 17beta-reductase for reduced cofactor(s) and/or substrates was abnormal.

We describe a direct, solid-phase time-resolved fluoroimmunoassay (TRFIA) for measuring 17 alpha-hydroxyprogesterone (<em>17OHP</em>) in serum and blood spots on filter paper. We used <em>17OHP</em>-3-carboxymethyloxime (<em>17OHP</em>3CMO) coupled to polylysine as the label, which enabled incorporation of up to 34 atoms of europium per molecule of <em>17OHP</em>, for a very high specific activity. The assay is based on competition between labeled <em>17OHP</em>3CMO and <em>17OHP</em> in blood specimens for polyclonal rabbit anti-<em>17OHP</em> antibodies. The antibody-label complex is separated by binding to anti-rabbit antibodies coated onto microtiter strips. The assay buffer contains danazol to displace <em>17OHP</em> from steroid-binding proteins in serum. For serum samples, the assay is accomplished in 1 h of incubation at room temperature. The blood spot assay with filter paper discs involves incubation overnight at 4 degrees C. Results for both types of specimens from the same subjects correlated well. The lowest measurable concentrations of <em>17OHP</em> (nmol/L) were 0.10 (3 SD) and 0.75 (3 SD) for serum and dried blood on filter paper, respectively. Intra- and interassay CVs were about 5-15% for both types of samples.

BACKGROUND

Although much is known about the increased levels of the 21-hydroxylase substrates 17-hydroxyprogesterone (<em>17OHP</em>) and 21-deoxycortisol (21DF) - the biochemical markers of all forms of 21-hydroxylase deficiency (21OHD), only limited information is available on the zona fasciculata (ZF) products distal to the enzymatic block: 11-deoxycortisol (S), 11-deoxycorticosterone (DOC), and corticosterone (B).

OBJECTIVE

To investigate whether basal and post-ACTH levels of S, DOC, and B and the 21-hydroxylase precursor-to-product ratios determined by tandem mass spectrometry preceded by high-performance liquid chromatography separation (liquid chromatography-tandem mass spectrometry) could disclose distinct profiles in genotypically confirmed classic (no.=14) and non-classic (NC) (no.=18) patients, heterozygote carriers (no.=61) and wildtypes (WT) (no.=27) for 21OHD.

RESULTS

Salt wasting (SW) and simple virilizing (SV) had higher basal levels of DOC with no further increase in response to ACTH. Stimulated DOC was similar in 21OHD patients and carriers but was reduced as compared to WT. ACTH-stimulated B increased gradually from SW and SV through WT. The post-ACTH 21DF/B ratio was able to detect 92% of the carriers among WT. All NC patients could be detected by post-ACTH <em>17OHP</em>/DOC and 21DF/B, with no overlap with 21OHD carriers.

CONCLUSIONS

Although 21-hydroxylase is a key enzymatic step in both 17-hydroxy and 17-deoxy pathways of ZF, the reaction is mostly affected in the latter pathway, leading to a significant impairment of B production, which may further characterize the 21OHD subtypes. Also, the precursor-to-product ratios, particularly 21DF/B, can demonstrate the distinctive outline of 21OHD subtypes, including carriers and normal subjects.

Adrenal steroid profiling, including 17α-OH progesterone (<em>17OHP</em>), 11-deoxycortisol (S), Δ4-androstenedione (Δ4-A) and cortisol (F) in blood spots by tandem mass spectrometry, is used for newborn screening to detect congenital adrenal hyperplasia (CAH). Pre-analytical sample processing is critical for assay specificity and accuracy; however, it is laborious and time-consuming. This study describes the development and validation of a new Liquid Chromatography-Tandem Mass Spectrometry (LC-MS/MS) method for the simultaneous quantification of five steroids: <em>17OHP</em>, S, Δ4-A, F and cortisone (E) in blood spots from newborns. Whole blood was eluted from a 5.00 mm dried blood spot by an aqueous solution containing the deuterium-labeled internal standards d8-<em>17OHP</em> and d4-cortisol. The steroids extracted from blood spot into aqueous solution were subsequently purified via Extelut mini NT1 column using diethylether. The extracts were evaporated and quantified using LC-MS/MS. The detection limit was 0.25 ng/mL for <em>17OHP</em> and S, 0.4 ng/mL for Δ4-A and 0.5 ng/mL for F and E. The limit of quantification was 0.5 ng/mL for <em>17OHP</em>, S and Δ4-A and 1 ng/mL for F and E. Precision for <em>17OHP</em>, S, Δ4-A at concentrations of 0.5, 2, and 8 ng/mL (n=5) in fortified steroid free serum samples was 1.3-3.5% (intra-assay CV) and 7-14.8% (inter-assay CV). Precision for F and E at concentrations of 5 and 20 ng/mL was 1.5-4.8% (intra-assay, CV%) and 6-15% (inter-assay, CV%). Accuracy was calculated at concentrations of 0.5, 2, and 8 ng/mL for <em>17OHP</em>, S and Δ4-A and ranged from -0.3 to 0.2%, while for F and E it ranged from -3.2 to 0.2%. Relative recoveries at concentration 2 ng/mL and 8 ng/mL for <em>17OHP</em>, S, Δ4-A and at 5 ng/mL and 20 ng/mL for F and E ranged from 55% to 80%. Reference intervals were estimated for all steroids in newborns (on day 3). The steroid profile assay herein described is sensitive, specific and accurate and involves a simple pre-analytical sample manipulation; it is therefore suitable for routine analysis and provides data for samples within normal range as well as those with elevated levels. For the first time to our knowledge, cortisone levels are reported in dried blood spots from newborns.

BACKGROUND

Men with Klinefelter syndrome (KS) show hypergonadotropic hypogonadism, but the pathogenesis of hypotestosteronemia remains unclear. Testicular steroidogenesis in KS men was evaluated over three decades ago after human chorionic gonadotropin (hCG) stimulation, but inconclusive results were obtained. Intriguingly, some recent studies show increased intratesticular testosterone concentrations in men with KS.

OBJECTIVE

To analyze serum steroid profile, as a proxy of testicular steroidogenesis, after hCG stimulation in KS compared with control men.

METHODS

A prospective, longitudinal, case-control, clinical trial.

METHODS

Thirteen KS patients (36±9 years) not receiving testosterone (TS) replacement therapy and 12 eugonadic controls (32±8 years) were enrolled. Serum steroids were measured by liquid chromatography-tandem mass spectrometry (LC-MS/MS) at baseline and for five consecutive days after intramuscular injection of 5000IU hCG.

RESULTS

Progesterone (P), 17-hydroxyprogesterone (<em>17OHP</em>), TS, and estradiol (E2) showed a significant increase (P<0.001) after hCG stimulation in both groups. On the contrary, androstenedione (AS) and dehydroepiandrosterone did not increase after hCG stimulation. The <em>17OHP</em>/P ratio increased in both groups (P<0.001), the TS/AS ratio (17β-hydroxysteroid dehydrogenase type 3 (17βHSD3) activity) did not increase after hCG in any group, and the E2/TS ratio (aromatase activity) increased significantly in both groups (P=0.009 in KS and P<0.001 in controls). Luteinizing hormone decreased after hCG in both groups (P=0.014 in KS and P<0.001 in controls), whereas follicle-stimulating hormone decreased only in control men (P<0.001).

CONCLUSIONS

This study demonstrates for the first time using LC-MS/MS that Leydig cells of KS men are able to respond to hCG stimulation and that the first steps of steroidogenesis are fully functional. However, the TS production in KS men is impaired, possibly related to reduced hydroxysteroid deydrogenase activity due to an unfavorable intratesticular metabolic state.

OBJECTIVE

To evaluate adrenal steroid hormone secretion in response to corticotropin-releasing factor (CRF) or to adrenocorticotropin hormone in women with hypothalamic amenorrhea.

METHODS

Controlled clinical study.

METHODS

Department of Reproductive Medicine and Child Development, Section of Gynecology and Obstetrics, University of Pisa, Italy.

METHODS

Fifteen women with hypothalamic amenorrhea were enrolled in the study. Eight normal cycling women were used as control group.

METHODS

Blood samples were collected before and after an injection of ovine CRF (0.1 microg/kg iv bolus) or after synthetic ACTH (0.25 mg iv).

METHODS

Plasma levels of ACTH, 17-hydroxypregnenolone (<em>17OHP</em>e), progesterone (P), dehydroepiandrosterone (DHEA), 17-hydroxyprogesterone (<em>17OHP</em>), cortisol (F), 11-deoxycortisol (S) and androstenedione (A).

RESULTS

Basal plasma concentrations of ACTH, cortisol, 11-deoxycortisol, DHEA and <em>17OHP</em>e were significantly higher in patients than in controls, whereas plasma levels of progesterone and 17-OHP were significantly lower in patients than in controls. In amenorrheic women the ratio of 17-OHPe/DHEA, of 17-OHPe/17-OHP and of 11-deoxycortisol/cortisol were significantly higher than in controls, while a significant reduction in the ratio of 17-OHP/androstenedione, of 17-OHP/11-deoxycortisol was obtained. In response to corticotropin-releasing factor test, plasma levels of ACTH, cortisol, 17-OHP, 11-deoxycortisol, DHEA and androstenedione were significantly lower in patients than in controls. In response to adrenocorticotropin hormone, plasma levels of 17-OHP, androstenedione and androstenedione/cortisol were significantly higher in patients than in controls.

CONCLUSIONS

Patients suffering for hypothalamic amenorrhea showed an increased activation of hypothalamus-pituitary-adrenal (HPA) axis, as shown by the higher basal levels and by augmented adrenal hormone response to corticotropin-releasing factor administration. These data suggest a possible derangement of adrenal androgen enzymatic pathway.

Aldosterone-producing adenomas (APAs) demonstrate exquisite sensitivity to endogenous ACTH. We previously showed an ACTH receptor overexpression in APAs compared with the other adrenal tumors. To evaluate the meaning of such findings, we investigated the response of aldosterone, cortisol, and 17OH progesterone (<em>17OHP</em>) to 1 microg ACTH in 42 patients with adrenocortical tumors (23 NHAs, 9 APAs, and 10 CPAs) and 10 normal subjects (C). All 52 subjects were responsive to ACTH, and hormone peak levels were reached at 30 min. The aldosterone peak level was significantly higher in APAs [mean +/- SEM: 84.3 +/- 13.1 ng/dl (2335.1 +/- 362.9 pmol/liter)] than in other tumors and control (C). Cortisol peak levels was higher in CPAs [37.1 +/- 3.9 microg/dl (1023.9 +/- 107.6 nmol/liter)] than in NHAs (P < 0.01), in C (P < 0.01) and in APAs (P = n.s.). <em>17OHP</em> peak levels were significantly higher in patients with adrenocortical tumors toward C. In summary: 1) low-dose ACTH induces an important stimulation in all tumors, suggesting preservation of high responsiveness to ACTH; 2) this is especially true for aldosterone in APA and could be of primary importance when performing diagnostic tests for hyperaldosteronism; and 3) <em>17OHP</em>-hyperresponsiveness to low-dose ACTH is the most common alteration both in functional and nonfunctional tumors.

Congenital adrenal hyperplasia due to 21-hydroxylase deficiency (21OHD-CAH) is an autosomal recessive disorder affecting steroidogenesis, due to mutations in CYP21A2 (6p21.3). 21OHD-CAH neonatal screening is based on 17-hydroxyprogesterone (<em>17OHP</em>) serum levels, showing high type I error rate and low sensitivity to mild CAH forms. Here, we used an epidemiological approach, which estimates the allelic frequency (q) of an autosomal recessive disorder using the proportion of homozygous patients, the mutational spectrum and the inbreeding coefficient in a sample of affected individuals. We applied this approach to 2 independent Italian cohorts of patients with both clinical and molecular diagnosis of 21OHD-CAH from mainland Italy (N = 240) and Sardinia (N = 53). We inferred q estimates of 2.87% and 1.83%, corresponding to a prevalence of 1/1214 and 1/2986, respectively. CYP21A2 mutational spectra were quite discrepant between the 2 cohorts, with V281L representing 74% of all the mutations detected in Sardinia vs 37% in mainland Italy. These findings provide an updated fine-grained picture of 21OHD-CAH genetic epidemiology in Italy and suggest the need for a screening approach suitable to the detection of the largest number of clinically significant forms of CAH.

Neonatal screening for congenital adrenal hyperplasia (CAH) is useful in diagnosing salt wasting form (SW). However, there are difficulties in interpreting positive results in asymptomatic newborns. The main objective is to analyze genotyping as a confirmatory test in children with neonatal positive results. Patients comprised 23 CAH children and 19 asymptomatic infants with persistently elevated 17-hydroxyprogesterone (<em>17OHP</em>) levels. CYP21A2 gene was sequenced and genotypes were grouped according to the enzymatic activity of the less severe allele: A1 null, A2 < 2%, B 3-7%, C>> 20%. Twenty-one children with neonatal symptoms and/or <em>17OHP</em> levels>> 80 ng/ml carried A genotypes, except two virilized girls (<em>17OHP</em> < 50 ng/ml) without CAH genotypes. Patients carrying SW genotypes (A1, A2) and low serum sodium levels presented with neonatal <em>17OHP</em>>> 200 ng/ml. Three asymptomatic boys carried simple virilizing genotypes (A2 and B): in two, the symptoms began at 18 months; another two asymptomatic boys had nonclassical genotypes (C). The remaining 14 patients did not present CAH genotypes, and their <em>17OHP</em> levels were normalized by 14 months of age. Molecular analysis is useful as a confirmatory test of CAH, mainly in boys. It can predict clinical course, identify false-positives and help distinguish between clinical forms of CAH.