Helmuth G. Dörr

Automated, fast and sensitive quantification of 17α-hydroxy-progesterone, androstenedione and testosterone by tandem mass spectrometry with on-line extraction

Plasma 17 alpha-hydroxyprogesterone (17-OHP), androstenedione and testosterone measurements are important for the diagnosis and monitoring of hyperandrogenic disorders, most importantly for congenital adrenal hyperplasia (CAH) due to 21-hydroxylase deficiency. The reliability of immunoassays has proved questionable especially for newborns and children. In order to reduce the analytical interferences due to cross-reactivity or matrix effects, to improve accuracy and shorten the analysis time, we have developed a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method with atmospheric pressure chemical ionization (APCI) for simultaneous measurement. An on-line extraction cartridge with column-switching technique and liquid chromatography over a Chromolith RP 18 e column allow a rapid and easy quantification. The lowest limit of detection was 0.03-0.06 microg/L. Our method has proved linear up to 250 microg/L (r=0.999). Recoveries (S.D.) of 17-OHP, androstenedione and testosterone in plasma were 100% (5), 102% (2) and 92% (4), respectively. The regression equation for the LC-MS/MS (x) and immunoassay (y) methods for 17-OHP (excluding neonate samples) was y=1.942 x+0.255 nmol/L (r=0.695; n=97). In comparison to our values, the immunoassay generally overestimates steroid concentration. The regression equation for the LC-MS/MS (x) and immunoassay (y) methods for testosterone was y=0.963 x+0.035 nmol/L (r=0.955; n=107). Preliminary reference intervals for children were determined as a function of age and sex. The sensitivity and specificity of the LC-MS/MS method offer advantages over routine immunoassays due to the elimination of interferences especially for newborns, high throughput and short chromatographic run time.

Plasma Levels of Aldosterone, Corticosterone, 11-Deoxycorticosterone, Progesterone, 17-Hydroxyprogesterone, Cortisol, and Cortisone During Infancy and Childhood

Summary: Plasma aldosterone (A), corticosterone (B), deoxycorticosterone (DOC), progesterone (P), 17-hydroxyprogesterone (17-OHP), cortisol (F), and cortisone (E) were measured simultaneously by specific radioimmunoassays in small plasma samples obtained from 174 normal infants and children between 2 hr and 15 yr of age. The significantly elevated neonatal mean levels (ng/ml) of 2.5 (A), 4.1 (DOC), 53.0 (P), and 6.6 (17-OHP) dropped significantly during infancy reaching prepubertal levels between 3 months and 3 yr of age, with a transient, significant DOC increase between 1-7 yr. The glucocorticoids F and B declined significantly from means of 68 and 4.4 to 11.4 and 0.28 ng/ml, respectively, during the first weeks of life, then increased significantly reaching adult levels between 1-3 yr of age. Mean E fell progressively from 74 ng/ml after birth to 10 ng/ml during 1-5 yr (P << 0.0001), then slightly increased to adult levels. After age 7 yr, P and 17-OHP, in contrast to the other steroids, rose significantly in both boys and girls relative to pubertal development.The observed changes are thought to be due to (1) adaptation of the adrenal neocortex to extrauterine life after disruption of the fetoplacental unit, (2) a physiologic lack of corticosteroid binding globulin (CBG) during infancy due to maturation of hepatic CBG biosynthesis, (3) the functional immaturity of the infant kidney compensated by an increased activity of the renin-angiotensin-aldosterone system, and (4) gradually increasing gonadal secretion of progestins during puberty.Speculation: From birth to adulthood, marked evolutional changes were observed in the basal plasma concentrations of all physiologically important unconjugated corticosteroids and progestins in normal children. Detailed knowledge of the age-dependent normal plasma steroid pattern reflecting maturational processes of both the hypothalamo-adrenocortical and the hypothalamo-gonadal axis, of the renin-angiotensin-aldosterone system, and of hepatic and renal function, therefore, is a prerequisite for understanding pathologic conditions in pediatric endocrinology.

Obesity Among Children and Adolescents With Classic Congenital Adrenal Hyperplasia Due to 21-Hydroxylase Deficiency

OBJECTIVES. Congenital adrenal hyperplasia (CAH) due to 21-hydroxylase deficiency is the most common inherited disorder of adrenal steroid biosynthesis. Patients with the classic form of CAH show androgen excess, with or without salt wasting. There are few studies reporting on higher rates of overweight and obesity among children with CAH. In addition to its role in the regulation of energy balance, leptin is involved in various endocrine and metabolic pathways. In this context, elevated serum leptin levels were reported recently for patients with CAH and were thought to be involved in the development of obesity among these patients. Therefore, the aim of this study was to analyze BMI values, compared with population-based references, for children and adolescents with CAH. Possible contributing factors, such as glucocorticoid therapy, skeletal maturation, birth weight and length, and parental BMI, were correlated with current BMI SD scores (SDS). In addition, the implications of serum leptin levels, corrected for BMI, gender, and Tanner stage, were investigated. METHODS. We performed a cross-sectional retrospective study of 89 children and adolescents with cah (48 female and 41 male subjects; age: 0.2-17.9 years) who presented in our outpatient department during 1 year. All individuals had classic cah, confirmed with molecular genetic analyses, and received substitution therapy (glucocorticoids and mineralocorticoids, if necessary). The quality of therapy was monitored in follow-up visits every 3 to 6 months, on the basis of clinical presentation and laboratory measurement findings according to current guidelines. We grouped the patients into salt wasting and simple virilizing groups, as well as according to current metabolic control. Leptin levels were measured with a commercial radioimmunoassay and calculated as sds. For statistical analyses, standard parametric and nonparametric methods were used. RESULTS. The chronologic ages of the children with CAH were between 0.20 and 17.9 years (mean ± SD: 8.9 ± 4.6 years). The BMI SDS of the whole group ranged from −2.7 to 4.3 (mean ± SD: 0.88 ± 1.3) and was significantly elevated above 0. Fifteen subjects (16.8%) had BMI SDS of >2.0, which indicated a significantly greater frequency of obesity among patients with CAH than expected for the normal population (expected: 2.27%). There was no significant difference in age and BMI between genders and clinical forms (salt wasting versus simple virilizing). BMI SDS was correlated positively with chronologic age. The BMI SDS did not differ significantly between children receiving hydrocortisone, prednisone, or dexamethasone. Hydrocortisone dosages (including equivalent dosages of prednisone and dexamethasone) ranged from 6.2 to 30.1 mg/m2 body surface area (mean ± SD: 14.7 ± 4.8 mg/m2 body surface area). Hydrocortisone dosages were correlated positively with BMI SDS. The relative risk of having a BMI SDS of >2.0 was not significantly elevated among children with prednisone/dexamethasone medication, compared with those with hydrocortisone treatment. In contrast to this, fludrocortisone dosage was not correlated with BMI SDS. Bone age delay, as calculated from the difference of bone age and chronologic age, ranged from −2.9 years to 5.6 years (mean ± SD: 1.11 ± 1.8 years) and was significantly elevated; it was correlated positively with BMI SDS. The BMI of parents ranged from 17.8 to 39.0 kg/m2 (median: 24.2 kg/m2). Median BMI values did not differ significantly between fathers and mothers. The relative risk for obesity among our children (BMI SDS of >2.0) was significantly elevated for children with obese parents, compared with those with nonobese parents (relative risk: 4.86). There was no significant correlation of birth length, birth weight, or gestational age with BMI SDS. Serum leptin values ranged from 0.10 to 32 μg/L (median: 4.4 μg/L); they were correlated positively with BMI SDS, chronologic age, and Tanner stage. After transformation into leptin concentration SDS values, the median SDS of 0.42 (range: −5.4 to 3.1) did not differ significantly from 0. CONCLUSIONS. Children and adolescents with CAH have a higher risk of obesity. Glucocorticoid dosage, chronologic age, advanced bone age maturation, and parental obesity contributed to elevated BMI SDS, whereas birth weight and length, serum leptin levels, used glucocorticoid, and fludrocortisone dosage were not associated with obesity. Therefore, children with CAH who become obese should be tightly monitored and should participate concurrently in weight management programs that include obese family members.

Aldosterone-Receptor Deficiency in Pseudohypoaldosteronism

Pseudohypoaldosteronism, a syndrome characterized by salt wasting and failure to thrive, usually presents in infancy as high urinary levels of sodium despite hyponatremia, hyperkalemia, hyperreninemia, and elevated aldosterone levels. We have investigated this syndrome for the possibility of abnormal Type I or mineralocorticoid-like receptors, which have intrinsic steroid specificity indistinguishable from that of renal mineralocorticoid receptors and are found in many tissues and cells, including mononuclear leukocytes. We have studied three patients with pseudohypoaldosteronism: the 28-year-old index case in Melbourne (Patient 1) and two siblings in Munich, eight and two years of age (Patients 2 and 3); clinically, Patient 3 had a less severe case than his sister. Percoll-separated control monocytes bound [3H]aldosterone with high affinity (Kd approximately 3 nM) and limited capacity (150 to 600 sites per cell). On repeated examination, no [3H]aldosterone binding was found in monocytes from Patients 1 and 2; in Patient 3, the levels were 62 sites per cell, more than 2 S.D. below those of the control. Levels in the parents of the Munich patients (first cousins) were normal. It appears that pseudohypoaldosteronism is caused by a Type I receptor defect, that the defect may be complete or partial, that transmission may be autosomal recessive, and that the study of patients with pseudohypoaldosteronism may indicate physiologic roles for Type I receptors in nonepithelial tissues.

Etomidate: A selective adrenocortical 11β-hydroxylase inhibitor

SummaryTo investigate the adrenocortical suppression caused by the anesthetic etomidate, plasma levels of progesterone (P), 17-hydroxyprogesterone (17-OHP), 11-deoxycorticosterone (DOC), corticosterone (B), aldosterone (Aldo), 11-deoxycortisol (S), cortisol (F), and cortisone (E) were measured simultaneously before and after a short-term ACTH stimulation test in a 6.5-year-old boy whose convulsions could be kept under control only with constant etomidate infusions. During etomidate therapy, plasma levels of DOC and S were extremely elevated, the progestins P and 17-OHP were slightly elevated, whereas B and Aldo were in the lower normal range, and F and E were markedly decreased. A short-term ACTH stimulation test during etomidate infusion gave a blunted response of B, Aldo, F and E, whereas the level of DOC remained high and S even further increased. P and 17-OHP showed a positive response to ACTH. The ratios of B/DOC and F/S, which reflect adrenocortical 11β-hydroxylase activity, were extremely decreased during etomidate and did not change after ACTH stimulation. In contrast, the ratios of DOC/P and S/17-OHP, which relect 21-hydroxylase activity, were elevated and remained elevated after ACTH stimulation. After discontinuation of etomidate therapy, all the baseline steroid levels were somewhat elevated, but responded normally to ACTH. These results demonstrate that etomidate causes a specific and reversible blockade of the 11β-hydroxylation of adrenal steroid synthesis.

Does an altered leptin axis play a role in obesity among children and adolescents with classical congenital adrenal hyperplasia due to 21-hydroxylase deficiency?

OBJECTIVEnCongenital adrenal hyperplasia (CAH) patients are at a higher risk to develop obesity. The role of leptin in CAH is still controversial. Our study aimed to evaluate serum levels of leptin, the soluble leptin receptor (sOB-R), and the sOB-R: leptin molar ratios in a cohort of CAH children and adolescents, and their associations with clinical and metabolic parameters.nnnMETHODSnWe studied 51 CAH patients, aged 5.6-19.6 years (median 11.8, n=30 females) cross-sectionally. All patients had genetically proven CAH and received standard steroid substitution therapy. Blood specimens were taken after overnight fasting between 0800 and 1000 h. For the analyses of leptin and sOB-R, matched pairs were built with healthy Caucasian patients for sex, Tanner stage (TS), chronologic age (CA), and body mass index (BMI).nnnRESULTSnBMI and SDS were significantly elevated compared with the reference population. Leptin levels were not different between matched pairs, whereas sOB-R levels were significantly lower in CAH. Consequently, the sOB-R: leptin molar ratios were significantly decreased in CAH. Correlation analyses in CAH patients revealed significant relationship between leptin and CA, TS, BMI, and homeostasis model assessment of insulin resistance. Similar results were obtained for the matched control group. For sOB-R, we found no significant correlation for CA, TS, or BMI in CAH, but we did in the controls. There were significant correlations for androgens within the CAH group. Additional analyses revealed no correlation with steroid medication or metabolic control.nnnCONCLUSIONSnOur data show that an altered leptin axis with normal serum leptin concentrations but decreased sOB-R serum levels may contribute to the increased risk of overweight and obesity in CAH.

Placental 11β-HSD2 Gene Expression at Birth Is Inversely Correlated With Growth Velocity in the First Year of Life After Intrauterine Growth Restriction

Intrauterine growth restriction (IUGR) is associated with an increased risk for short stature and diseases in adulthood thought to be inflicted by fetal programming. We hypothesized that placental endocrine systems involved in perinatal growth might also play a role in postnatal growth after IUGR. In a prospective controlled multicenter study, placental gene expression of IGF-binding protein-1 (IGFBP-1), leptin and 11β-hydroxysteroid dehydrogenase type 2 (11β-HSD2) were measured in 14 IUGR infants and 15 children born appropriate for gestational age (AGA) proven by serial ultrasound examinations. Postnatally, IUGR infants experienced a significantly higher growth velocity than AGA neonates (at 1 y: p = 0.001). Gene expression of 11β-HSD2 at birth correlated positively with birth length (r = 0.55, p = 0.04) and inversely with growth velocity in the first year of life (r = −0.69, p = 0.01) in the IUGR, but not in the AGA group. There was no correlation between gene expression of placental IGFBP-1, leptin and birth weight, length and growth velocity during the first year of life. AGA infants showed significantly higher concentrations of cortisone in venous cord blood after birth (p = 0.02) as a surrogate of a higher 11β-HSD2 activity in the fetoplacental unit. In conclusion, placental 11β-HSD2 gene expression might predict postnatal growth in IUGR.

Growth hormone therapy and the risk of tumor recurrence after brain tumor treatment in children.

ABSTRACT To assess the effect of human growth hormone (hGH) therapy and other factors on tumor recurrence after treatment of pediatric brain tumors (BTs), we retrospectively analyzed data from 108 craniopharyngioma, medulloblastoma, and ependymoma patients. Risk factors were identified using multifactorial univariate regression analysis. Recurrences occurred in 41 and second malignant neoplasms in 4 patients. There were significant correlations for completeness of tumor removal and recurrence-free survival (RFS). 13/44 hGH-treated and 28/59 non-hGH-treated children relapsed. This difference was found only for medulloblastomas and accounted for by higher rates of incomplete tumor removal in non-hGH patients. Craniopharyngioma recurrence correlated only with RFS. Malignant BT recurrence correlated with completeness of tumor removal, chemotherapy, and RFS. 4 children developed SMNs, 3/4 after hGH therapy. Our regression model yielded accurate within-sample prediction of recurrence for 90% of the study population. We conclude that hGH therapy after treatment of pediatric BTs does not increase tumor recurrence risk.

Evidence for change of 11β-hydroxysteroid dehydrogenase activity during infancy and childhood

The conversion of cortisol (F) to cortisone (E) is catalyzed by 11β-hydroxysteroid dehydrogenase type 2. The present study was designed to investigate the changes of F and E plasma concentration as an indirect measurement of 11β-hydroxysteroid dehydrogenase activity in infancy and childhood. Plasma samples were obtained from 262 healthy children and adolescents aged 1 d to 18 y. Plasma F and E were measured, using specific radioimmunoassays after extraction and automated Sephadex LH 20 chromatography. The F/E ratio was calculated to assess 11β-hydroxysteroid dehydrogenase activity. During the first year of life, plasma F levels rose significantly (r2 = 0,24;p = 0.01), and thereafter no further increase was seen until adulthood (r2 = 0.01;p = 0.86). In contrast, plasma E significantly decreased during the first year of life (r2 = –0.35;p < 0.001) and stayed unchanged thereafter (r2 = 0.02;p = 0.81). As a consequence, the F/E ratio rose significantly during the first year (r2 = 0.67;p < 0.001) but did not change afterward (r2 = 0.001;p = 0.99). During the first year of life, there is a change from the predominance of E, with low mineralocorticoid receptor affinity, to F, with high mineralocorticoid receptor affinity. This shift corresponds to the declining plasma concentrations of aldosterone during infancy. The changes may indicate a not yet recognized, significant change of 11β-hydroxysteroid dehydrogenase isoenzyme activity or alterations in the secretion of F and E, which may be of relevance for the development of arterial blood pressure in infancy.

Different relationships between the first 2 years on growth hormone treatment and the d3‐growth hormone receptor polymorphism in short small‐for‐gestational‐age (SGA) children

Backgroundu2002 There has been controversy in recent years on whether the d3 polymorphism of the GH receptor is associated with a better growth response to GH in idiopathic short children born small for gestational age (SGA).