Sumitaka Saisho

Congenital adrenal hyperplasia due to 21-hydroxylase deficiency: Newborn screening and its relationship to the diagnosis and treatment of the disorder

Abstract Congenital adrenal hyperplasia (CAH) due to 21-hydroxylase deficiency can be detected by newborn screening. The screening alone in 29 programs from 13 countries resulted in the diagnosis of CAH in 1 2 of affected newborns and expedited the diagnosis in 1 3 of affected newborns clinically suspected to have CAH. The benefits of newborn screening for CAH were prevention of severe adrenal crisis, its sequela, incorrect male sex assignment of severely virilized female newborns, and progressive signs of androgen excess. Screening revealed a higher incidence of CAH worldwide (1:15000 live births) compared with the case survey incidence (1:32000 live births) The false-positive rate (usually found in low birth weight and premature infants) was acceptably low (0.01–0.5%) except for three programs (0.7–2.5%). The false-negative rate of CAH screening was negligible. Prenatal diagnosis of CAH is possible by HLA typing or 21-hydroxylase B gene analysis of cultured fetal cells from chorionic villus biopsy sampling in the first trimester and from amniotic cells or hormonal analysis of amniotic fluid in the second trimester. Prenatal treatment of CAH is possible via maternal dexamethasone therapy beginning early pregnancy. However, efficacy and side effects of maternal dexamethasone therapy require further investigation.

Sleep Disturbance in Children with Growth Hormone Deficiency

We examined the effects of growth hormone (GH) deficiency on sleep development by performing all-night polysomnography in three female children with GH deficiency (GHD). The percentage of REM sleep seemed to be reduced before the treatment in 2 cases, and human GH (hGH) compensation slightly increased it. Submental twitch movements (mTMs), i.e., body movements during sleep localized in the submental muscle and lasting less than 0.5 seconds, were commonly disturbed in the three patients. Rapid eye movements in REM sleep (REMs) were reduced before the therapy in one case, this decrease being reversed on hGH compensation. REMs also seemed to increase after hGH treatment in the other two cases. Dopamines and cholinergic muscarinic agonists can cause GH release, while mTMs and REMs might be related to dopaminergic and cholinergic systems in the human brain. It is intriguing that GHD, and the disturbance of mTMs and REMs coexisted in children with GHD. Since a relatively poor social outcome in patients with GHD has been reported, even after hGH compensation, it is important to monitor their neurological development by means of evaluation of their sleep disturbance.

Changes of Several Adrenal Δ4-Steroids Measured by HPLC-UV Spectrometry in Neonatal Patients with Congenital Adrenal Hyperplasia due to 21-Hydroxylase Deficiency

We have developed an easy and rapid method of reverse-phase high-performance liquid chromatography (HPLC)-UV spectrometry for measuring adrenal delta 4-steroids. Three female neonates with adrenal 21-hydroxylase deficiency (2 salt-losers and 1 simple virilizer), two of whom were recalled by neonatal mass-screening for congenital adrenal hyperplasia (CAH), were diagnosed using this method. Changes of several adrenal steroids were examined in these patients before and after treatment with hydrocortisone. Before treatment, the cortisone and cortisol peaks were very low and those of 17 alpha-hydroxyprogesterone (17-OHP) and 21-deoxycortisol (21-DOF) were high in all 3 patients (17-OHP: 79.9-997 nmol/l, 21-DOF: 83.7-324 nmol/l). The androstenedione peak was also high in 2 of them. A peak produced by 21-deoxycortisone, which is a product of oxidation of 21-DOF at the C-11 position, was also detected in all cases (14.5-297 nmol/l). After treatment, all of these abnormally elevated delta 4-steroids decreased or disappeared. This new method is thought to be valuable for the rapid diagnosis of CAH, and especially for use in neonatal mass-screening for CAH.

Colon Cancer in a 14-Year-Old Female With Turner Syndrome

Abstract A 14-year-old female with Turner syndrome (karyotype 45,X) had a history of abdominal pain with distention, constipation, and fever. She was first operated on for the suspicion of appendicitis, failed to improve, and was later hospitalized for further investigation and treatment. Studies demonstrated an obstructing tumor of the transverse colon, and an emergency laparotomy was performed. The final diagnosis was a signet-ring cell carcinoma of the colon with diffuse peritoneal dissemination and metastasis to paracolic lymph nodes. On the basis of this case, we report the association of Turner syndrome with malignancies and also some aspects of colon cancer in childhood.

Neonatal mass screening for congenital adrenal hyperplasia in Tokyo

Abstract Nation-wide neonatal mass screening for congenital adrenal hyperplasia (CAH) due to 21-hydroxylase deficiency (21-OHD) has been performed in neonates in Japan since January 1989. In Tokyo, between April 1989 and March 1991, 190,094 newborns were screened, and 12 cases of CAH were detected. There were 2 cases with the simple virilizing (SV) type, and 10 of the salt-losing (SL) type. The incidence of CAH was I in 15,841 live births, which is two- to threefold higher than previously found in case-assessment studies in this country. The ratio of the SL to the SV type was also higher than previously reported, and the patients clinical symptoms were less marked than previously reported. The usefulness of mass screening for CAH is strongly recommended, especially for salvaging those with the severe type of 21-OHD, and detecting symptomatic patients with the SV type.

A highly specific heterologous enzyme immunoassay for 5α-androstane-3α, 17β-diol 17-glucuronide (androstanediol-17G) and developmental patterns of urinary androstanediol-17G excretions

We established a highly specific enzyme immunoassay (EIA) for 5 alpha-androstane-3 alpha, 17 beta-diol 17-glucuronide (androstanediol-17G). Rabbit antisera raised against 5 alpha-androstane-3 alpha, 11 alpha, 17 beta-triol 17-glucuronide 11-glutaryl bovine serum albumin and a heterologous tracer of androstanediol-17G conjugated with horseradish peroxidase at the glucuronic acid group were used. The EIA showed excellent specificity: there were no remarkable cross-reactivities with related androgens. The assay range for urine samples was 0.3-30 ng/ml. Recoveries of standards added to samples were 100-108%. Intra-assay and inter-assay coefficients of variation were 2.9-4.4% and 5.7-7.9%, respectively. The EIA was applied to urine samples of 407 males and 322 females to determine developmental patterns and normal ranges of androstanediol-17G excretions in 11 age groups (0 y, 1 y, 2-3 y, 4-5 y, 6-7 y, 8-9 y, 10-11 y, 12-13 y, 14-15 y, 16-17 y, and over 18 y). Urinary androstanediol-17G/creatinine (androstanediol-17G/Cre) ratios in both sexes were high in infancy, tended to decrease during childhood, and began to increase near adolescence. While androstanediol-17G/Cre ratio in girls increased at 8-9 y and reached a plateau during adolescence, that in boys increased at 10-11 y and continued to increase throughout adolescence. Androstanediol-17G/Cre ratios in girls were higher than those in boys at 6-7 y (P < 0.05) and at 8-9 y (P < 0.01). Androstanediol-17G/Cre ratios in boys were higher than those in girls at 12-13 y and at older ages (P < 0.01). These developmental patterns are parallel to age-related changes in androgenicity and serum androstanediol-17G, suggesting that urinary androstanediol-17G/Cre ratio could be a good marker for androgenicity in childhood.

Enzyme-linked immunosorbent assay for determining urinary pregnanetriol-3α-glucuronide

Abstract Introduction: A highly sensitive and specific enzyme-linked immunosorbent assay (ELISA) was developed to determine the urinary concentrations of pregnanetriol-3α-glucuronide (Pt-3-G) for the diagnosis and confirmation of 21-hydroxylase deficiency (21-OHD) congenital adrenal hyperplasia (CAH). Method: The immunogenic complex was synthesized by an active ester method by coupling bovine serum albumin to the carboxyl group of the glucuronic acid moiety. Enzyme-labeled Pt-3-G was synthesized by coupling horseradish peroxidase to the same carboxyl group. Results: There was good correlation between the values obtained for Pt-3-G by this ELISA method and conventional capillary gas-chromatography (cGC). In normal 5-day-old infants the Pt-3-G levels were 4.9 ± 4.8 ng/ml (0.027 ± 0.014 mg/g creatinine), but in infants with CAH were 2473 ng/ml (age 3 days), 423 ng/ml (age 25 days) and 76 ng/ml (age 49 days) (3.6, 1.5 and 0.9 mg/g creatinine, respectively). Discussion: This ELISA method is simple and sufficiently specific to be performed without prior extraction or hydrolysis and sensitive enough to measure the low levels of Pt-3-G found in neonatal urine. The method was 2000-times more sensitive than cGC, requiring only 51 μ1 of urine and having a sensitivity of 10 fg/ml as compared to the requirement of 5 ml of urine and a sensitivity of 20 pg/ml for cGC. Thus, this ELISA method for Pt-3-G is useful in differentiating infants with 21-OHD from normal infants.

血中17α-hydroxypregnenolone, 17α-hydroxypregnenolone sulfateおよび17α-hydroxyprogesteroneの同時測定法

It is well recognized that in the fetal adrenal cortex, 3 beta-hydroxysteroid dehydrogenase (3 beta-HSD) activity is lower and in fetal tissues, steroid sulfokinase (SK) is higher than in the adult. In order to clarify a part of the development processes of the adrenal cortex or steroidgenesis in humans, a combined radioimmunoassay (RIA) method to estimate serum 17 alpha-hydroxypregnenolone (17-OH-delta 5 P), 17 alpha-hydroxypregnenolone sulfate (17-OH-delta 5 P-S) and 17 alpha-hydroxyprogesterone (17-OH-delta 4P) was devised. The method consisted of the following procedures: 1) diethyl ether extraction and chromatographic separation of unconjugated steroids (17-OH-delta 5P and 17-OH-delta 4P), 2) enzymatic hydrolysis of 17-OH-delta 5P-S using the residue of diethyl ether extraction for a material, 3) diethyl ether extraction and chromatographic purification of hydrolyzed 17-OH-delta 5P-S, and 4) RIAs for 17-OH-delta 5-P to estimate 17-OH-delta 5P and 17-OH-delta 5P-S concentration, and for 17-OH-delta 4P. Extracted 17-OH-delta 5P was well separated from 17-OH-delta 4P by Sephadex LH-20 microcolumn chromatography, using a benzene/methanol = 95/5 (v/v) solvent as a mobile phase. Several procedures for hydrolysis or solvolysis of 17-OH-delta 5P-S were compared using available tritiated delta 5-3 beta-hydroxysteroids including dehydroepiandrosterone (DHA), DHA sulfate (DHA-S) and 17-OH-delta 5P, and it was found that the most suitable method was an enzymatic hydrolysis by arylsulfatase from Helix Pomatia in an appropriate condition in which the percent hydrolysis was 92.9 +/- 1.2 (mean +/- SEM)%. The final percent recoveries were 88.7 +/- 1.2% in 17-OH-delta 4P, 90.7 +/- 1.4% in 17-OH-delta 5P and 78.1 +/- 2.1% in 17-OH-delta 5P-S, respectively. A suitable antiserum and its final dilution titer for RIA of 17-OH-delta 5P (hydrolyzed 17-OH-delta 5P-S also) was 1:12,000 dilution of anti-17-OH-delta 5P-3-succinate-BSA serum. An anti-7-oxo-17-OH-delta 5P-7-carboxymethyloxime-BSA serum was considered to be unsuitable for the measurement of hydrolyzed 17-OH-delta 5-P-S, presumably because of a significant cross-reactivity with a large amount of unknown steroid sulfates simultaneously hydrolyzed.(ABSTRACT TRUNCATED AT 400 WORDS)

Urinary pregnanetriol-3-glucuronide excretion in neonates and the use of urinary pregnanetriol-3-glucuronide/creatinine ratio in differentiating 21-hydroxylase deficiency

Introduction: A number of neonatal patients with 21-hydroxylase deficiency (21-OHD) have been discovered since the initiation of a nationwide mass screening program for congenital adrenal hyperplasia (CAH) in Japan. At the same time more than 80% of the referred neonates were found to be free from 21-OHD after detailed examination. Therefore, a simple method for identifying 21-OHD among referred neonates from mass screening or a second screening method for referred neonates is required. Methods: In order to examine whether measurements of pregnanetriol-3-glucuronide (PT-3-G) in a single spot urine enable us to diagnose neonatal patients with 21-OHD. urinary PT-3-G concentrations were measured with our newly developed enzyme-linked immunosorbent assay method in 163 urine specimens from 107 infants and those from 21 referred neonates from a mass-screening program for CAH. Results: Median PT-3-G concentrations in 5-, 6-and 14-day-old infants were 11.5 nmol/l (range: 2.9–85.6, n = 35), 20.1 nmol/l (range: 5.4–89.1, n = 43) and 38.8 nmol 1 (range: 8.9–143.5, n = 34), respectively. Median values of the molar ratio of PT-3-G to excreted creatinine (PT-3-G, creatinine; × 105) were 0.82 (range: 0.23–2.95), 1.2 (range: 0.40–6.5) and 3.7 (range: 0.92–8.2). respectively. Both concentrations and ratios increased significantly with age (5 days vs. 6 days: P < 0.001 for the concentrations, P < 0.01 for the ratios; 6 days vs. 14 days: P < 0.01 for the concentrations, P < 0.001 for the ratios). Seven of the 21 neonates were diagnosed with 21-OHD, while the remaining 14 neonates did not have this disease. PT-3-G concentrations and the ratios in the 14 false-positive cases ranged from 21.2 to 909 nmol/l and 1.7 to 28.0. Those in the 7 diagnosed patients ranged from 1161 to 2814 nmol/l and 81.4 to 365, respectively. Discussion: Neither value overlapped between the patients and the false-positive cases. Therefore, measurement of urinary PT-3-G was a useful method of detecting neonates with 21-OHD among referred neonates. However, the ratios appeared to be more useful than concrete concentrations in distinguishing the neonates with 21-OHD.

Age-related Changes of Serum 17^|^alpha;-Hydroxypregnenolone and 17^|^alpha;-Hydroxypregnenolone Sulfate Concentrations in Infancy and Childhood

In order to clarify a part of the developmental processes of the human adrenal cortex or steroidgenesis in infancy and childhood, serum concentrations of 17 alpha-hydroxypregnenolone (17-OH-delta 5 P), 17 alpha-hydroxypregnenolone-3-sulfate (17-OH-delta 5 P-S) and 17 alpha-hydroxyprogesterone (17-OH-delta 4 P) were measured using a combined radioimmunoassay method previously reported, and age-related changes of these steroids were evaluated. In addition to the serum concentrations, a ratio of 17-OH-delta 5 P-S to 17-OH-delta 5 P (S/delta 5 P ratio) and that of 17-OH-delta 4 P to 17-OH-delta 5 P (delta 4 P/delta 5 P ratio) were also estimated as the indices of the activities of steroid sulfokinase (SK) and 3 beta-hydroxysteroid dehydrogenase (3 beta-HSD), respectively. Serum 17-OH-delta 5 P, 17-OH-delta 5 P-S and 17-OH-delta 4 P concentrations in umbilical cord blood were 13.5 +/- 6.02 (Mean +/- SD) ng/ml, 965 +/- 363 ng/ml and 46.3 +/- 21.1 ng/ml, respectively. These values decreased consecutively to the nadirs which were 0.47 +/- 0.16 ng/ml in subjects 1 to 2 years old, 1.26 +/- 0.82 ng/ml in subjects 3 to 6 years old and 0.17 +/- 0.007 ng/ml in subjects 3 to 4 months old, respectively, and they were followed by gradual increases up to the adult levels. S/delta 5 P ratio also showed the same profile.(ABSTRACT TRUNCATED AT 250 WORDS)