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.     

Inhibitory effect of platelet-activating factor (PAF) on luteinizing hormone-releasing hormone and somatostatin release from rat median eminence in vitro correlated with the characterization of specific PAF receptor sites in rat hypothalamus

Platelet-activating factor (PAF) exhibits a wide range of biological activities, including the stimulation of secretory processes in various cell types. However, little is known regarding its possible influence on the release of brain neuropeptides. In the present study we have examined the effect of PAF on the release of three hypothalamic releasing hormones in adult male rats, and have characterized the presence of specific PAF binding sites in rat hypothalamic membranes. PAF decreased LHRH and somatostatin (SRIF) release from the median eminence with a maximal inhibition at 10(-14) M for both neuropeptides, whereas GRF release was not significantly altered. Moreover, PAF strongly counteracted the Ca2+ ionophore A 23187-stimulated release of LHRH and SRIF from median eminence and medial basal hypothalamus (greater than 50% inhibition). These results suggest an involvement of Ca2+ dependent events in PAF action. This inhibitory effect was specifically exerted at a hypothalamic site because PAF failed to depress LH and GH release from the anterior pituitary. A specific, reversible and saturable binding of [3H]PAF to membrane preparations of rat hypothalamus was demonstrated and two classes of binding sites were characterized. The affinity (KD) of each binding class was 2.14 +/- 0.32 nM and 61.63 +/- 16.4 nM, respectively, and the corresponding maximal number of each binding class was 25.41 +/- 3.2 fmol/mg protein and 146.2 +/- 47.5 fmol/mg protein. In the same conditions no specific binding was observed using rat pituitary membranes. The specificity of PAF analogs for these binding sites was well correlated to their relative effectiveness in altering LHRH and SRIF release (order of potency: L-652,731, kadsurenone greater than BN 52021 greater than Lyso-PAF). These data suggest that the binding sites identified in the hypothalamus have the characteristics expected of a specific PAF receptor and that PAF effect on neuropeptides release is a receptor-mediated process.