Alexandra Keller

High-Sensitivity Chemiluminescence Immunoassays for Detection of Growth Hormone Doping in Sports

BACKGROUND Recombinant human growth hormone (rhGH) is abused in sports, but adequate routine doping tests are lacking. Analysis of serum hGH isoform composition has been shown to be effective in detecting rhGH doping. We developed and validated selective immunoassays for isoform analysis with potential utility for screening and confirmation in doping tests. METHODS Monoclonal antibodies with preference for pituitary hGH (phGH) or rhGH were used to establish 2 pairs of sandwich-type chemiluminescence assays with differential recognition of rhGH (recA and recB) and phGH (pitA and pitB). We analyzed specimens from volunteers before and after administration of rhGH and calculated ratios between the respective rec- and pit-assay results. RESULTS Functional sensitivities were <0.05 microg/L, with intra- and interassay imprecision < or =8.4% and < or =13.7%, respectively. In 2 independent cohorts of healthy subjects, rec/pit ratios (median range) were 0.84 (0.09-1.32)/0.81 (0.27-1.21) (recA/pitA) and 0.68 (0.08-1.20)/0.80 (0.25-1.36) (recB/pitB), with no sex difference. In 20 recreational athletes, ratios (median SD) increased after a single injection of rhGH, reaching 350% (73%) (recA/pitA) and 400% (93%) (recB/pitB) of baseline ratios. At a moderate dose (0.033 mg/kg), mean recA/pitA and recB/pitB ratios remained significantly increased for 18 h (men) and 26 h (women). After high-dose rhGH (0.083 mg/kg), mean rec/pit ratios remained increased for 32 h (recA/pitA) and 34 h (recB/pitB) in men and were still increased after 36 h in women. CONCLUSIONS Using sensitive chemiluminescence assays with preferential recognition of phGH or rhGH, detection of a single injection of rhGH was possible for up to 36 h.

Secular trends in body mass index in German children and adolescents: a cross-sectional data analysis via CrescNet between 1999 and 2006

To assess secular trends in alterations in body mass index (BMI) in German children and adolescents between 1999 and 2006, we performed an analysis using data from a computerized database (CrescNet) and focusing on the data ranges above the 97th percentile (P97) and below the median (P50). This cross-sectional assessment of BMI data used a total of 143 495 single values (73 290 males and 70 205 females aged 0.5-17.5 years) from screening and/or consulting visits at 1 of the 294 participating German pediatricians. Body mass index data were calculated from standardized measurements of body weight and height entered into the CrescNet database. Individual percentiles were estimated according to German reference data sets. Across all age groups, the respective mean value of children with BMI above P97 increased from 5.32% to 7.02% in boys and from 5.70% to 7.18% in girls between 1999 and 2006, whereas those below P50 decreased from 48.52% to 43.71% in boys and from 47.48% to 42.57% in girls. The proportions of obese children (above the 97th percentile) were significantly higher than estimated by German reference values throughout the study period. The significant increase in childhood obesity between 1999 and 2006 was more pronounced in boys compared to girls. In conclusion, the cross-sectional study performed at a large cohort of German children and adolescents reveals an alarming increase in the number of obese children and adolescents and an accompanied shift toward higher BMI values. As the number of children below the 50th centile decreases accordingly, the shift in the distribution panel of the German reference percentile curves affecting the whole population can be observed.

Effects of a 1‐year exercise and lifestyle intervention on irisin, adipokines, and inflammatory markers in obese children

Exercise improves weight status and metabolism. Irisin, a novel myokine, may be involved in the regulation of metabolic function. The effect of an exercise and dietary lifestyle intervention for 1‐year on irisin, adipokines (leptin, adiponectin, resistin) and inflammatory markers (C‐reactive protein (CRP), soluble tumor necrosis factor receptor II (sTNFR‐II) was evaluated, and predictors of irisin levels were characterized in obese children.

Pharmacokinetics and pharmacodynamics of GH: dependence on route and dosage of administration

OBJECTIVE Pharmacokinetic and pharmacodynamic data after recombinant human GH (rhGH) administration in adults are scarce, but necessary to optimize replacement therapy and to detect doping. We examined pharmacokinetics, pharmacodynamics, and 20 kDa GH after injection of rhGH at different doses and routes of administration. DESIGN Open-label crossover study with single boluses of rhGH. METHODS Healthy trained subjects (10 males, 10 females) received bolus injections of rhGH on three occasions: 0.033 mg/kg s.c., 0.083 mg/kg s.c., and 0.033 mg/kg i.m. Concentrations of 22 and 20 kDa GH, IGF-I, and IGF-binding proteins (IGFBP)-3 were measured repeatedly before and up to 36 h after injection. RESULTS Serum GH maximal concentration (Cmax) and area under the time-concentration curve (AUC) were higher after i.m. than s.c. administration of 0.033 mg/kg (Cmax 35.5 and 12.0 microg/l; AUC 196.2 and 123.8). Cmax and AUC were higher in males than in females (P < 0.01) and pharmacodynamic changes were more pronounced. IGFBP-3 concentrations showed no dose dependency. In response to rhGH administration, 20 kDa GH decreased in females and remained suppressed for 14-18 h (low dose) and 30 h (high dose). In males, 20 kDa GH was undetectable at baseline and throughout the study. CONCLUSIONS After rhGH administration, pharmacokinetic parameters are mainly influenced by route of administration, whereas pharmacodynamic variables and 20 kDa GH concentrations are determined mainly by gender. These differences need to be considered for therapeutic use and for detection of rhGH doping.

Computer-guided, population-based screening system for growth disorders (CrescNet) and on-line generation of normative data for growth and development.

The mean age at which the diagnosis of growth disorders such as Turner’s syndrome, growth hormone (GH) deficiency or true GH-dependent gigantism is established is still rather late in many countries around the world. In addition, the question of secular trends in a given population and the rate at which childhood obesity is increasing in industrialized countries make it mandatory to establish a time-adapted system to develop percentiles for body height, weight and body mass index (BMI) and also to develop a screening system for growth disorders. In 1998 we established a network, now involving more than 160 paediatric practices in Germany and seven paediatric endocrinology departments. Paediatricians record heights, weights and growth velocities of all children in their care and systematically feed the data into the database at our centre usually by mailing formatted, structured data tickets. Data are then continuously analysed at the centre and the paediatricians in the network are informed immediately about their individual patients’ growth situations via phone or E-mail (feedback system). Regular annual conferences including structured reports, scientific presentations and discussion groups are organized for all participants at our centre. By May 2001, the data of 83,721 children and adolescents had been analysed. The mean values for height were 1–1.5 cm higher than the mean values in the German Synthetic Growth Curve, which serves as an internal standard. However, and most importantly, in comparison with the internal standard and historical normative data from Germany and Switzerland, there is a continuous increase in the 97th percentile for weight and BMI, while the third percentile remains unchanged. In addition, many children with short stature and tall stature due to a variety of endocrine disorders and genetic diseases which had not been diagnosed previously are now being identified. In conclusion, the databank allows for a continuous adaptation of normative curves based on a large number of children in a given population, i.e. eastern Germany. Secondly, the system allows for detection of pathological growth curves and is already serving to diagnose growth disorders in a defined population in a systematic way.

Comparison of adrenal function tests in children--the glucagon stimulation test allows the simultaneous assessment of adrenal function and growth hormone response in children.

The accurate assessment of adrenal function is necessary in many children with suspicion of pituitary insufficiency. The objective of this study was to evaluate the adrenal response during the glucagon stimulation test (GST) and its diagnostic utility in children. A total of 290 children, aged 10.1 +/- 5.0 years, were evaluated for adrenal function using the corticotrophin releasing hormone (CRH) test, the GST, and/or the insulin tolerance test (ITT). Glucagon stimulation provoked a substantial rise in cortisol and adrenocorticotropin (ACTH) that was independent of gender, age, or underlying growth hormone deficiency. There were no differences in peak cortisol levels in the GST compared to the CRH test in pair-wise intra-individual analyses in children with both tests performed within one year (615.4 +/- 30.5 vs 602.8 +/- 22.4 nmol/l, n=52). Similarly, there were no differences in the cortisol response between the ITT and CRH test. Peak cortisol levels in the CRH test correlated with the GST and the ITT. The magnitude of ACTH response, in contrast, was highest in the ITT with a 9.8-fold increase over baseline, while the increase in the GST (3.1-fold) and CRH test (1.6-fold) were more subtle. Since there is controversy concerning reliable cut-off values for adrenal function tests in children, we analyzed cut off levels in 186 children, including 26 children with adrenal insufficiency, using the CRH test. A peak cortisol level of 450 nmol/l provided the best balance of sensitivity (88.5%) and specificity (86.8%), while higher cut-off levels did not increase sensitivity but lost in specificity. In summary, the GST constitutes an1 equally sensitive test for the assessment of adrenal function in children that is not confounded by anthropometric parameters and is generally not accompanied by major side effects. It allows the simultaneous assessment of corticotroph and somatotroph function and may thus constitute a valuable alternative to the ITT.

Impact of weight status on the onset and parameters of puberty: analysis of three representative cohorts from central Europe.

It is controversial whether obesity in children is associated with earlier onset of puberty and advanced appearance of distinct parameters of pubertal development. To investigate the impact of obesity on markers of the onset and parameters representing stages of puberty, we analyzed auxological parameters and secondary sex characteristics in three representative cohorts of Caucasian children. Body weight, height, peak height velocity and pubertal stages were evaluated in two recent German cohorts (CrescNet and Leipzig Schoolchildren), and a historical Swiss cohort. According to body mass index (BMI), children were classified into three weight groups of lean, overweight, or normal weight with limits defined below -1.28 and above +1.28 BMI SDS. Peak height velocity (PHV) occurred significantly later in lean compared to normal weight children in the CrescNet and Swiss cohort, while there was no difference between obese and normal weight children. There was a trend towards acceleration of parameters of puberty onset and progression in obese children in all three cohorts. Height SDS was significantly higher in obese children compared to normal weight peers, but after completion of pubertal development it was similar in adolescents. The impact of overweight on the acceleration of puberty seems to be slightly stronger in boys. Once girls have reached a critical weight for entering the process of maturation, further increase in body weight does not seem to advance the onset of puberty.

Auxological computer based network for early detection of disorders of growth and weight attainment.

INTRODUCTION For several years practising pediatricians and pediatric endocrinologists in Leipzig have been collaborating closely to achieve early detection of growth disorders. As a result of this collaboration a data-bank was established in September 1998 into which height and weight measurements were entered and related to age and gender. This well established network of pediatricians in practice, pediatric endocrinologists and the auxological data-bank functions well. METHODS All data are anonymized and continually monitored. By September 2000, the data for 60,984 children had been assessed. The data were evaluated using the German Synthethic Norm Curve for body height and German normal data for BMI. Pathology of growth dynamics was assumed when change in height SDS was greater than 0.5 height SDS/year. RESULTS Analysis showed that 2,216 children (3.6%) had a height greater than the 97th percentile and 2,775 children (4.5%) had a height less than the 3rd percentile of the normative reference. BMI above the 97th percentile was found in 7,687 children (12.6%) and below the 3rd percentile in 2,678 children (4.4%). When assessing growth development (n = 5,665), 194 (3.4%) showed acceleration and 155 (2.7%) showed deceleration of growth. CONCLUSION The computer-based monitoring system is useful to document short-term and long-term changes of growth and weight attainment in the general population. The presence of growth disorders and/or disturbed weight gain can be detected early in the individual child.

The one year exercise and lifestyle intervention program KLAKS: Effects on anthropometric parameters, cardiometabolic risk factors and glycemic control in childhood obesity

OBJECTIVE Regular physical exercise within structured lifestyle programs may improve weight status and minimize metabolic risk factors in childhood obesity. The aim of this study was to evaluate the effect of the one-year combined physical exercise/lifestyle program KLAKS on anthropometric and metabolic parameters and glycemic control in childhood obesity. MATERIALS AND METHODS 142 overweight/obese (BMI>90th percentile) candidates (7-18years) were enrolled, 115 participants completed the program. Anthropometrics and biochemical parameters were obtained at beginning and completion. An oral glucose tolerance test (OGTT) was performed in a subgroup of participants. Course of glucose and insulin levels within OGTT was correlated with several parameters and is reported here for those who completed the program. RESULTS The mean standard deviation scores (SDS) decreased significantly for BMI, waist circumference, waist-to-height ratio (WHtR) and percentage body fat (all p≤0.01). Improved metabolic risk markers included mean glucose levels within an OGTT at follow-up compared to baseline (p<0.0001) and HbA1c (p=0.05) as well as indications of improvement for gamma-glutamyl-transferase and free fatty acids. CONCLUSIONS The one-year combined exercise/lifestyle program KLAKS significantly improves markers of obesity and glycemic control. Impaired cardiometabolic risk markers, even subclinical, are also favorably influenced by program participation.

Effect of erythrocyte transfusion on longitudinal bone growth of premature infants assessed by mini-knemometry.

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