Jürgen Kratzsch

Serum Insulin-Like Growth Factor I Reference Values for an Automated Chemiluminescence Immunoassay System: Results from a Multicenter Study

Background: Analysis of insulin-like growth factor I in serum (S-IGF-I) is an integral component in the diagnosis of GH-related disorders and is going to be of interest in the diagnosis and follow-up of many disorders. The objective of the present study was to develop cross-sectional reference values for S-IGF-I measured by an automated chemiluminescence immunoassay (Nichols Advantage®). Methods: The study included samples from 3,961 healthy subjects (2,201 males, 1,760 females) aged 1 month to 88 years. Six laboratories were involved in this study and the samples were analyzed by one of seven automated immunoassay systems run in these laboratories. For data analysis, polynomial age and sex-specific models were fitted after transformation of S-IGF-I values. Results: The results show the well-known age dependency of S-IGF-I levels. At ages <20, higher S-IGF-I levels were seen in girls with an estimated mean peak of 410 µg/l at age 14 and an estimated mean peak of 382 µg/l at age 16 in boys. Thereafter, a rapid decrease was seen to approximately 25 years of age, followed by a slow age-dependent decrease. In adulthood, S-IGF-I in males were slightly, but significantly higher than in females. It could be shown that the mean values of some reference sample subgroups differed significantly from the total mean. However, the multicenter approach used in this study reduces the impact of systematic population, sample handling and laboratory differences on the calculated reference mean. Conclusion: The present study establishes age- and sex-specific reference values for a fully automated immunoassay system based on a large population of healthy subjects. The established reference values may be used for this immunoassay system in different laboratories provided that the systematic difference between systems is low.

Serum Vaspin Concentrations in Human Obesity and Type 2 Diabetes

OBJECTIVE— Vaspin was identified as an adipokine with insulin-sensitizing effects, which is predominantly secreted from visceral adipose tissue in a rat model of type 2 diabetes. We have recently shown that vaspin mRNA expression in adipose tissue is related to parameters of obesity and glucose metabolism. However, the regulation of vaspin serum concentrations in human obesity and type 2 diabetes is unknown. RESEARCH DESIGN AND METHODS— For the measurement of vaspin serum concentrations, we developed an enzyme-linked immunosorbent assay (ELISA). Using this ELISA, we assessed circulating vaspin in a cross-sectional study of 187 subjects with a wide range of obesity, body fat distribution, insulin sensitivity, and glucose tolerance and in 60 individuals with normal glucose tolerance (NGT), impaired glucose tolerance (IGT), or type 2 diabetes before and after a 4-week physical training program. RESULTS— Vaspin serum concentrations were significantly higher in female compared with male subjects. There was no difference in circulating vaspin between individuals with NGT and type 2 diabetes. In the normal glucose-tolerant group, circulating vaspin significantly correlated with BMI and insulin sensitivity. Moreover, physical training for 4 weeks resulted in significantly increased circulating vaspin levels. CONCLUSIONS— We found a sexual dimorphism in circulating vaspin. Elevated vaspin serum concentrations are associated with obesity and impaired insulin sensitivity, whereas type 2 diabetes seems to abrogate the correlation between increased circulating vaspin, higher body weight, and decreased insulin sensitivity. Low circulating vaspin correlates with a high fitness level, whereas physical training in untrained individuals causes increased vaspin serum concentrations.

Clinical aspects of obesity in childhood and adolescence

The level of fatness of a child at which morbidity acutely and/or later in life increases is determined on an acturial basis. Direct measurements of body fat content, e.g. hydrodensitometry, bioimpedance, or DEXA, are useful tools in scientific studies. However, body mass index (BMI) is easy to calculate and is generally accepted now to be used to define obesity in children and adolescents clinically. An increased risk of death from cardiovascular disease in adults has been found in subjects whose BMI had been greater than the 75th percentile as adolescents. Childhood obesity seems to substantially increase the risk of subsequent morbidity whether or not obesity persists into adulthood. The genetic basis of childhood obesity has been elucidated to some extent through the discovery of leptin, the ob gene product, and the increasing knowledge on the role of neuropeptides such as POMC, neuropeptide Y (NPY) and the melanocyte concentrating hormone receptors (for example, MC4R). Environmental/exogenous factors largely contribute to the development of a high degree of body fatness early in life. Twin studies suggest that approximately 50% of the tendency toward obesity is inherited. There are numerous disorders including a number of endocrine disorders (Cushings syndrome, hypothyroidism, etc.) and genetic syndromes (Prader‐Labhard–Willi syndrome, Bardet Biedl syndrome, etc.) that can present with obesity. A simple diagnostic algorithm allows for the differentiation between primary or secondary obesity. Among the most common sequelae of primary childhood obesity are hypertension, dyslipidemia, back pain and psychosocial problems. Therapeutic strategies include psychological and family therapy, lifestyle/behaviour modification and nutrition education. The role of regular exercise and exercise programmes is emphasized. Surgical procedures and drugs used in adult obesity are still not generally recommended in children and adolescents with obesity. As obesity is the most common chronic disorder in industrialized societies, its impact on individual lives as well as on health economics has to be recognized more widely. This review is aimed towards defining the clinical problem of childhood obesity on the basis of current knowledge and towards outlining future research areas in the field of energy homoeostasis and food intake in relation to child health. Finally, one should aim to increase public awareness of the ever increasing health burden and economic dimension of the childhood obesity epidemic that is present around the globe.

Leucocytes are a major source of circulating nicotinamide phosphoribosyltransferase (NAMPT)/pre-B cell colony (PBEF)/visfatin linking obesity and inflammation in humans.

Aims/hypothesisNicotinamide phosphoribosyltransferase (NAMPT) is a multifunctional protein potentially involved in obesity and glucose metabolism. We systematically studied the association between circulating NAMPT, obesity, interventions and glucose metabolism and investigated potential underlying inflammatory mechanisms.MethodsFasting morning NAMPT serum levels were measured in cohorts of lean vs obese children, cohorts of intervention by lifestyle, exercise and bariatric surgery, and during an OGTT. In addition, mRNA expression, protein production and enzymatic activity of NAMPT were assessed from isolated leucocytes and subpopulations.ResultsCirculating NAMPT was significantly elevated in obese compared with lean children and declined after obesity interventions concomitantly with the decline in BMI, high-sensitivity C-reactive protein (hsCrP) and leucocyte counts. Circulating NAMPT significantly correlated with glucose metabolism and cardiovascular variables in univariate analyses, but only the association with glucose response during an OGTT was independent from BMI. We therefore assessed the NAMPT dynamic following an oral glucose load and found a significant decline of NAMPT levels to 77.0 ± 0.1% as a function of time, and insulin-to-glucose ratio during an OGTT in obese insulin-resistant adolescents. Circulating NAMPT was, however, most strongly associated with leucocyte counts (r = 0.46, p < 0.001). The leucocyte count itself determined significantly and independently from BMI insulin resistance in multiple regression analyses. We systematically evaluated NAMPT expression among several tissues and found that NAMPT was predominantly expressed in leucocytes. In subsequent analyses of leucocyte subpopulations, we identified higher NAMPT protein concentrations in lysates of granulocytes and monocytes compared with lymphocytes, whereas granulocytes secreted highest amounts of NAMPT protein into cell culture supernatant fractions. We confirmed nicotinamide mononucleotide enzymatic activity of NAMPT in all lysates and supernatant fractions. In monocytes, NAMPT release was significantly stimulated by lipopolysaccharide (LPS) exposure.ConclusionsLeucocytes are a major source of enzymatically active NAMPT, which may serve as a biomarker or even mediator linking obesity, inflammation and insulin resistance.

New predictors of the metabolic syndrome in children: Role of adipocytokines

There is ample discussion of the relevance of the metabolic syndrome, the definition criteria, and predictive power. Nevertheless, along with the increasing prevalence of childhood obesity, the prevalence of the metabolic syndrome in obese children is reported at 30%, irrespective of the definition applied. Because children are otherwise relatively free of co-morbidities, they constitute an interesting population in which to study the sequence of events of obesity-related pathology. The adipocytokines appear to be important in this respect. Leptin was initially suggested as a promising “antiobesity” hormone. New concepts indicate that, in humans, leptin and its soluble receptor may be more important in states of energy deficiency rather than a predictor of the metabolic syndrome. Adiponectin, on the other hand, is not only related to obesity and insulin resistance, but appears to be the strongest predictor for metabolic syndrome, even in children. In newborns and infants, both adipocytokines occur in high concentrations, even though this cannot completely explain the increased risk for ensuing metabolic disease later in life. Finally, low-grade systemic inflammation may underlie the clustering of metabolic risk factors, but their role in children remains to be specified. Overall factors from the adipose tissue may constitute not only markers but also mediators of metabolic sequelae of obesity.

Longitudinal Analysis of Maternal Serum Leptin Levels during Pregnancy, at Birth and Up To Six Weeks after Birth: Relation to Body Mass Index, Skinfolds, Sex Steroids and Umbilical Cord Blood Leptin Levels

Leptin is an important regulator of body fat mass and energy expenditure during adult life. The mechanisms by which maternal and fetal weight are regulated during pregnancy are poorly understood. In order to gain more insight into a potential role of leptin during gestation, a prospective, longitudinal study was carried out to measure leptin concentrations in maternal serum of 29 healthy women during pregnancy up to 6 weeks after birth and also in umbilical cord blood of their newborns. Leptin concentrations were measured using a specific RIA. In addition, estradiol, testosterone, and sex hormone binding globulin were determined using commercially available RIAs. The mothers’ skinfolds were determined at four sites using a Holtain caliper.Leptin levels increased continuously during pregnancy and reached 25.8 ± 14.7 ng/ml at 38–40 weeks. At birth, leptin concentrations were 23.5 ± 15.4 ng/ml. Three days after delivery a significant decrease of leptin levels to 10.6 ± 6.0 ng/ml was observed. Six weeks after birth the leptin concentration in maternal serum was 13.8 ± 8.6 ng/ml. At birth, maternal serum levels were significantly higher than levels in cord blood and did not correlate with leptin levels in cord blood or neonatal weight. Furthermore, leptin levels did not correlate with maternal sex steroids and sex hormone binding globulin levels. At 6–8 weeks of pregnancy, maternal leptin serum levels correlated significantly with BMI (r = 0.81). The correlation coefficients (leptin vs. BMI) dropped with increasing gestational age and at birth only a poor correlation persisted (r = 0.50). Six weeks after birth there was again a high correlation between leptin levels in maternal serum and BMI (r = 0.76). Subscapular skinfold thickness was correlated to leptin concentrations in maternal serum during the whole period of the investigation.In conclusion, maternal leptin levels continuously increased from 6–8 weeks up to 38–40 weeks of pregnancy. Maternal leptin levels decreased dramatically after birth. Six weeks after delivery, leptin levels were comparable to the values measured at the beginning of pregnancy. We hypothesize that leptin might play an important role during pregnancy and fetal development.

Total and High–Molecular Weight Adiponectin in Relation to Metabolic Variables at Baseline and in Response to an Exercise Treatment Program: Comparative evaluation of three assays

OBJECTIVE—Adiponectin, an adipocyte-secreted hormone, circulates in the serum in several multimeric forms. Compared with total adiponectin, high–molecular weight (HMW) adiponectin has been suggested to be a better predictor of metabolic parameters and insulin sensitivity in humans. Our objective was to compare total adiponectin with HMW adiponectin as predictors of metabolic variables and insulin sensitivity at both baseline and after an exercise intervention. RESEARCH DESIGN AND METHODS—We obtained blood samples from 60 men and women with normal glucose tolerance (n = 20), impaired glucose tolerance (IGT) (n = 20), or type 2 diabetes (n = 20) at baseline and after 4 weeks of training to measure metabolic variables. Using commercially available assays, we measured plasma total adiponectin using LINCO, Mediagnost, and ALPCO assays and HMW adiponectin using an ALPCO assay. RESULTS—HMW adiponectin and total adiponectin (ALPCO) had similar ability to predict the presence of insulin resistance. Total adiponectin, as measured by radioimmunoassay (LINCO) and enzyme-linked immunosorbent assay (ELISA) (Mediagnost), correlated most strongly with measures of insulin sensitivity (P < 0.01) and lipid profile (P < 0.01) at baseline, showed greater improvements of adiponectin levels (P < 0.001), was more closely associated with improvements of lipid measures with exercise training (P < 0.01), and more accurately predicted insulin resistance and IGT in comparison with total adiponectin or HMW measured with the ALPCO ELISA. CONCLUSIONS—These results do not support the superiority of HMW over total adiponectin (measured using currently available assays) in assessing metabolic variables at baseline or in response to physical training. Moreover, there are significant differences in the ability of commercially available assays for total adiponectin to predict metabolic variables.

Exercise Training Attenuates MuRF-1 Expression in the Skeletal Muscle of Patients With Chronic Heart Failure Independent of Age The Randomized Leipzig Exercise Intervention in Chronic Heart Failure and Aging Catabolism Study

Background— Muscle wasting occurs in both chronic heart failure (CHF) and normal aging and contributes to exercise intolerance and increased morbidity/mortality. However, the molecular mechanisms of muscle atrophy in CHF and their interaction with aging are still largely unknown. We therefore measured the activation of the ubiquitin-proteasome system and the lysosomal pathway of intracellular proteolysis in muscle biopsies of CHF patients and healthy controls in two age strata and assessed the age-dependent effects of a 4-week endurance training program on the catabolic-anabolic balance. Methods and Results— Sixty CHF patients (30 patients aged ⩽55 years, mean age 46±5 years; 30 patients aged ≥65 years, mean age 72±5 years) and 60 healthy controls (30 subjects aged ⩽55 years, mean age 50±5 years; 30 subjects aged ≥65 years, mean age 72±4 years) were randomized to 4 weeks of supervised endurance training or to a control group. Before and after the intervention, vastus lateralis muscle biopsies were obtained. The expressions of cathepsin-L and the muscle-specific E3 ligases MuRF-1 and MAFbx were measured by real-time polymerase chain reaction and confirmed by Western blot. At baseline, MuRF-1 expression was significantly higher in CHF patients versus healthy controls (mRNA: 624±59 versus 401±25 relative units; P=0.007). After 4 weeks of exercise training, MuRF-1 mRNA expression was reduced by −32.8% (P=0.02) in CHF patients aged ⩽55 years and by −37.0% (P<0.05) in CHF patients aged ≥65 years. Conclusions— MuRF-1, a component of the ubiquitin-proteasome system involved in muscle proteolysis, is increased in the skeletal muscle of patients with heart failure. Exercise training results in reduced MuRF-1 levels, suggesting that it blocks ubiquitin-proteasome system activation and does so in both younger and older CHF patients. Clinical Trial Registration— URL: http://www.clinicaltrials.gov. Unique identifier: NCT00176319.

Nicotinamide phosphoribosyltransferase (NAMPT/PBEF/visfatin) is constitutively released from human hepatocytes

Circulating NAMPT (PBEF/visfatin) has pleiotropic functions and is secreted from adipocytes. Since it is doubtful whether serum levels can be explained by secretion from adipocytes alone, we asked whether hepatocytes are also able to liberate NAMPT. Using HepG2 cells and primary rat and human hepatocytes, release of NAMPT into the cell culture supernatant was found to occur constitutively in a time-dependent manner. In primary human hepatocytes, secretion within 24h was far higher than the cellular content, but was neither influenced by inhibitors of secretion nor by glucose, insulin or TNFalpha. As determined by size exclusion chromatography, HepG2 lysates and supernatants primarily contained the dimeric form of NAMPT which exhibited similar in vitro specific enzymatic activity. In primary human hepatocytes, secreted NAMPT was less active. Our results demonstrate that human hepatocytes are a potential source of circulating NAMPT.

Rapid quantification of steroid patterns in human serum by on-line solid phase extraction combined with liquid chromatography-triple quadrupole linear ion trap mass spectrometry.

BACKGROUND The determination of steroids is important for the diagnosis and monitoring of endocrine diseases and infertility workup. We developed a rapid and reliable mass spectrometric method for the simultaneous quantification of steroid patterns in human serum. METHODS An on-line solid phase extraction (SPE)-liquid chromatography-triple quadrupole linear ion trap (LC-QTrap) method utilizing atmospheric pressure chemical ionization was developed. Following protein precipitation of 100 microL serum, on-line SPE and chromatographic separation was performed for 13 steroids in 1.8 min. Analytes were confirmed by the characteristic fragment patterns. RESULTS The total run time of the method was 4 min. Detection limits ranged between 0.02 microg/L (testosterone) and 9 microg/L (dehydroepiandrosterone sulfate). The method was linear up to 7000 microg/L for dehydroepiandrosterone sulfate, 500 microg/L for cortisol, 125 microg/L for 11-deoxycortisol, and 25 microg/L for aldosterone, 17-hydroxyprogesterone, progesterone, testosterone, androstenedione and beta-estradiol, respectively. Accuracy ranged between 80 and 114%. Between-day variance at three different concentration levels was <15%. Excellent correlations with immunoassays were observed for testosterone, cortisol and beta-estradiol with Pearsons correlation coefficient r=0.967, 0.963, and 0.998, respectively. CONCLUSION The novel on-line SPE-LC-MS/QTrap platform offers a very fast, reliable, and sensitive quantification of steroid patterns and fulfils the quality criteria for routine laboratory application.