J. Homoki

Thyroid Autoimmunity in Children and Adolescents with Type 1 Diabetes mellitus

Type 1 diabetes is often associated with additional autoimmune phenomena. However, data reported on the frequency of thyroid autoimmunity differ vastly. Therefore, the prevalence of thyroid autoantibodies was evaluated at a large pediatric diabetes center in Southern Germany. 2,305 determinations (TPO and TG, ELISA) were performed in 495 patients with type 1 diabetes (234 boys, 261 girls; age at last measurement: 15.4 ± 0.3 years, duration of diabetes 7.5 ± 0.2 years). The prevalence of elevated thyroid antibodies increased dramatically with age: from 3.7% in patients less than 5 years of age up to 25.3% in the age group 15–20 years (p < 0.0001). For children older than 10 years, girls were significantly more affected than boys (p < 0.0001). Thyroid autoimmunity tended to be more prevalent in the subgroup of patients with the HLA type DR3/DR4 compared to patients with other HLA types (p = 0.08). In children older than 10 years, basal TSH concentrations were significantly elevated in antibody-positive patients (p < 0.05). In conclusion, thyroid autoimmunity is prevalent in children and adolescents with type 1 diabetes. Adolescent girls and young women are especially affected. Yearly routine determinations of thyroid antibodies are therefore recommended.

17α-Hydroxyprogesterone, 4-Androstenedione, and Testosterone Profiled by Routine Stable Isotope Dilution/Gas Chromatography-Mass Spectrometry in Plasma of Children

ABSTRACT: Using stable isotope dilution/gas chromatography-mass spectrometry (ID/GC-MS), a physicochemical method, we have profiled the plasma steroids 17α-hydroxyprogesterone, 4-androstenedione, and testosterone in normal children of various age groups. Comparison of our values with those obtained by direct immunologic assays and those using an extraction or purification step showed that immunoassays in general overestimate steroid concentrations. This was especially true for plasma samples in the neonatal period and was most expressed for the concentrations of 17α-hydroxyprogesterone. Our study demonstrated the applicability of ID/GC-MS to routine clinical steroid analysis. The application of ID/GC-MS is recommended whenever problems from matrix effects or cross-reactivity are likely to arise or suspicious results by immunoassays need to be rechecked.

Androgen metabolism assessment by routine gas chromatography/mass spectrometry profiling of plasma steroids: part 1, unconjugated steroids

Using gas chromatography/mass spectrometry we have developed a method for the simultaneous determination of six plasma steroids: testosterone, 4-androstenedione, 17 alpha-hydroxyprogesterone, 5 alpha-androstane-3 alpha,17 beta-diol, 5 alpha-dihydrotestosterone, and dehydroepiandrosterone. For each analyte, a deuterium-labeled internal standard was used for quantification. Due to the high isotopic purity of our standards, no complex corrections for isotope contributions were necessary. The procedure provides a sensitive and specific technique with good accuracy and precision.

Suppression of Growth Hormone by Oral Glucose in the Evaluation of Tall Stature

Excess secretion of growth hormone is a rare diagnosis in children or adolescents with tall stature. An oral glucose tolerance test (OGT) with determination of growth hormone is generally recommended to exclude this disorder. In order to test the validity of this approach in pediatric subjects, OGT tests were performed in 126 tall subjects (age: 12.4 ± 1.8 years; height: 3.1 ± 0.8 SDS). Nonsuppression was present in 39 subjects, however, anthropometric analysis and follow-up excluded the diagnosis of eosinophilic pituitary adenoma in all patients. The lowest GH concentration was reached 90 min after ingestion of oral glucose, GH rose above baseline at 180 min. Plasma concentrations of glucose and insulin did not differ between suppressors and nonsuppressors. In conclusion, absent suppression of growth hormone by oral glucose is common in tall children and adolescents. The test is therefore not recommended as a general screening for excess growth hormone. Prolonging the test beyond 120 min does not increase the diagnostic value.

Determination of dehydroepiandrosterone sulfate in human plasma by gas chromatography/mass spectrometry using a deuterated internal standard: a method suitable for routine clinical use.

A capillary gas chromatographic-mass spectrometric method for the determination of dehydroepiandrosterone sulfate (DHEA-S) in human plasma is described. [7,7-2H2]DHEA-S is used as internal standard. After desulfation by methanolysis, heptafluorobutyryl derivatives are prepared, and selected ion monitoring of characteristic fragment ions--m/z 270 for DHEA and m/z 272 for [7,7-2H2]DHEA--carried out. Requiring small amounts of plasma, the rapid, convenient work-up and the application of bench-top GC/MS instrumentation proved our method to be suited for routine clinical use in adults and children. The method needs no complex corrections for isotope contributions and provides good accuracy and precision. Comparative values of samples assayed by our GC/MS procedure and by a direct RIA indicated that the RIA overestimates the true concentration.

Severe hypoaldosteronism due to corticosterone methyl oxidase type II deficiency in two boys: metabolic and gas chromatography-mass spectrometry studies

Infection-triggered, life-threatening salt-loss and hyperkalaemia developed in two male infants with wasting, inappropriately low plasma aldosterone concentrations and elevated plasma renin activity. The presumptive diagnosis of a defective terminal step in aldosterone biosynthesis was made by the presence of large amounts of 11-dehydrotetrahydrocorticosterone and its 18-hydroxylated metabolite (18-OH-THA), free 18-hydroxycorticosterone (18-OH-B) and 18-hydroxytetra-hydrocorticosterone in the urine of both patients. The diagnosis of corticosterone methyl oxidase type II (CMO II) deficiency was confirmed by an elevated urinary 18-OH-THA to tetrahydroaldosterone ratio in one boy and by an elevated plasma 18-OH-B to aldosterone ratio in the other boy. Unknown steroids responsible for the salt-loss were not identified. Sodium supplementation but not short-term high dose oral 9α-fluorcortisol (FF) normalized the hyponatraemia in one patient, in whom sodium (Na+)/potassium (K+) co-transport was decreased. Both patients eventually received long-term FF treatment to prevent impairment of longitudinal growth caused by chronic salt-loss. The diagnosis of CMO II deficiency should always be confirmed by elevated precursor-product ratios in urine or plasma, using radioimmunoassays with prior chromatographic separation. Metabolic studies as the short-term response of serum Na+ to high dose FF may not be helpful in differentiating aldosterone biosynthetic defects from endorgan resistance to mineralocorticoids.

Urinary excretion of 17-hydroxypregnanolones in patients with different forms of congenital adrenal hyperplasia due to steroid 21-hydroxylase deficiency

To improve diagnostic criteria in different (classical salt-wasting (SW), classical simple virilizing (SV) and non classical late onset (LO)) forms of congential adrenal hyperplasia (CAH) due to steroid 21-hydroxylase deficiency, we investigated the urinary excretion of 17-hydroxypregnanolones (17OH-PO(5β) and (5α), 15β-hydroxypregnanolone(15βOH-PO), pregnanetriol(PT) and 11-oxo-pregnanetriol (11-O-PT) compared to hydrocortisone metabolities During the 1st month of life newborn infants with CAH-SW excreted from barely detectable to very large amounts of 17OH-PO(5β), 15βOH-PO and PT, and, in 12 of 14 cases, also 11-O-PT in their urines. From the 1st to the 28th day of life, cortisol metabolites were virtually absent in urines of CAH-SW infants. This was in contrast of 36 healthy newborn infants. We measured the excretion of 17OH-PO(5α) in children with CAH of whom 19 patients with CAH-SV had a median 17OH-PO(5α) excretion of 1110 μg/day (range: 152–5515). In 21patients with CAH-LO, median excretion of 17OH-PO(5α) was 294μg/day (range: 66–1273). Besides the conventional metabolites of 17-hydroxyprogesterone (17OH-PO(5β), PT and 11-O-PT),no 17OH-PO(5α) was detected in the urines of 14 patients with precocious pubarche, in 14 patients with virilization of unknown origin and in 94 healthy children of comparable age. The ratio of 17OH-PO(5α) to tetrahydrocortisone (THE) discriminated between CAH-SV and CAH-LO from the 1st to the 18th year of age. The determination of urinary 17OH-PO(5α) is an excellent diagnostic method in CAH-SV as well as CAH-LO.

Thyroid function in term newborn infants with congenital goiter

Eighty-four term newborn infants without goiter and 45 newborn infants with congenital goiter were studied with regard to thyroid function. The radiologic development of the femoral and tibial epiphyses was evaluated in those with goiter. Fifty-eight percent of the patients had retarded bone age, markedly elevated TSH levels, elevated TBI, decreased total T4I, and decreased PBI values. Forty-two percent of newborn infants with congenital goiter had a normal bone age, normal values for TSH, PBI, and total T4I, and elevated values for TBI. It is concluded that the 58% of the newborn infants with congenital goiter had subtle hypothyroidism. They require substitution therapy with thyroid hormones in order to avoid possible retardation of normal brain development. Patients with congenital goiter who have no biochemical evidence of hypothyroidism should also be treated with thyroid hormones to achieve rapid regression of goiter.

Insulin Resistance (HOMA) in Relation to Plasma Cortisol, IGF-I and IGFBP-3

Objective: To investigate the possible contribution of plasma cortisol and growth hormone (GH) as reflected by insulin-like growth factor-I (IGF-I)/insulin-like growth factor-binding protein-3 (IGFBP-3) on insulin action in short-statured children. Methods:In this study, insulin resistance (HOMA) was determined in 34 normal short-statured (age 9.4 ± 3.5 years) and in 19 GH-deficient children (age 10.4 ± 2.2 years). HOMA was examined in relation to fasting plasma cortisol, IGF-I, IGFBP-3 and in addition to birthweight and body mass index (BMI). Results: Birthweight was not correlated to insulin resistance. In GH-deficient children, BMI was significantly augmented and was associated with HOMA (p < 0.02). In both groups of patients, fasting plasma cortisol was related to HOMA (normal: r = 0.295, p < 0.05, GH-deficient: r = 0.495, p < 0.02). Only in normal short-statured children IGF-I (r = 0.338, p < 0.03) and IGFBP-3 (r = 0.493, p < 0.002) were associated with insulin resistance. Conclusion: The results indicated that at a young age cortisol contributed to insulin resistance in short-statured children. In normal short-statured children HOMA was associated with IGF-I and IGFBP-3. Possibly GH, a known cause of insulin resistance, contributed to HOMA as IGF-I and IGFBP-3 do not mediate insulin resistance but reflect growth hormone secretion. The results in GH-deficient children supported this conclusion as in the absence of GH insulin resistance was not associated with IGF-I/IGFBP-3.

Determination of 17α-hydroxypregnenolone in human plasma by routine isotope dilution mass spectrometry using benchtop gas chromatography-mass selective detection

A first assay based on stable isotope dilution/gas chromatography-mass spectrometry has been developed for plasma 17alpha-hydroxypregnenolone, the leading marker of 3beta-hydroxysteroid dehydrogenase deficiency. Equilibration of plasma with internal standard was followed by solid phase extraction. After clean up using Sephadex LH-20 mini columns, heptafluorobutyrates were prepared as derivatives. Quantification was achieved by selected ion monitoring of m/z 467.0 (analyte) and m/z 471.0 (internal standard). 0.030 pmol of 17alpha-hydroxypregnenolone gave a signal to noise ratio of 3.7. Calibration plot was linear. Spiking experiments showed good accuracy with relative errors < 3.7%. Intraassay precision CV was 8.3% and interassay precision CV was 5.6%. Requiring small amounts of plasma, the rapid, convenient work up and the application of bench top GC/MS instrumentation proved our method suitable for routine clinical use in adults and children.