Karin Weber

Significance of Basal IGF-I, IGFBP-3 and IGFBP-2 Measurements in the Diagnostics of Short Stature in Children

The role of IGF-I and IGFBP-3 measurements in the diagnostic work-up of short children is established but remains controversial. Little information exists on the value of IGFBP-2 measurements. Based on reference data established in 388 children we have reinvestigated the issue, using data from 392 short children who underwent the same diagnostic procedures between 1987 and 1998 (GHD, n = 187; non-GHD, n = 205, including patients with ISS, n = 76; IUGR, n = 46; and TS, n = 83). In comparing IGF-I, IGFBP-3 and IGFBP-2 serum levels of GHD and ISS children with reference data, we calculated the sensitivity, specificity, efficiency and positive predictive value for the diagnosis of GHD. The overall sensitivity of the parameters was high, the rank order being as follows: IGF-I >IGFBP-3 >IGFBP-2 (75, 67 and 62%, respectively). In contrast, the specificity was relatively low: IGFBP-3 >IGFBP-2 >IGF-I (50, 50 and 32%, respectively). The efficiency and positive predictive value of parameters was in the order of 40, 60 and 70–80%, respectively. In repeated measurements, the recorded basal levels of IGF-I and IGFBP-3 showed an overall narrow range of variation. We conclude that the determination of basal IGF parameters is, together with anthropometry and imaging techniques, an indispensable tool for differentiating between GHD and ISS; and that IGFBP-2 plays an additional role in this process.

Relevance of IGF-I, IGFBP-3, and IGFBP-2 Measurements during GH Treatment of GH-Deficient and Non-GH-Deficient Children and Adolescents

Background: Little information is available on the relevance of parameters representing the insulin-like growth factor (IGF) system with regard to growth hormone (GH) treatment during childhood. In adults, high IGF-I levels were found to be associated with side effects and long-term risks. Aim/Method: Our aim was to monitor the serum levels of IGF-I, IGF-binding protein (IGFBP) 3, and IGFBP-2 during long-term GH treatment of 156 patients with GH deficiency (GHD) and of 153 non-GHD patients. We determined the extent to which the IGF parameters exceed the normal ranges and identified those parameters which are predictive of 1st-year growth. Results: In prepubertal GHD children, the levels of IGF-I, IGFBP-3, and IGF-I/IGFBP-3 exceeded the 95th centile of the reference values for this age group in 2.3, 0.3, and 7.9% of the cases, respectively, whereas in prepubertal non-GHD children, the same parameters exceeded the 95th reference centile in 20.1, 3.5, and 32.2%, respectively. In pubertal GHD children IGF-I, IGFBP-3, and IGF-I/IGFBP-3 levels exceeded the 95th reference centile in 11.1, 1.5, and 15.4%, respectively. In pubertal non-GHD children, these levels also exceeded the 95th centile in 26.7, 7.0, and 41.4%, respectively. In both GHD and non-GHD groups, however, some patients had IGF parameters which were below the reference values. Our analysis showed that, in both groups, in addition to maximum GH, all IGF parameters (IGF-I, IGFBP-3, IGF-I/IGFBP-3 ratio, IGFBP-2 or derivatives) significantly extend the scope of a calculated model for predicting 1st-year height velocity. Conclusion: For reasons of safety and optimization of GH therapy, it is essential to follow up IGF-I, IGFBP-3, and IGFBP-2 levels regularly during childhood.

Normal Values of Circulating Insulin-Like Growth Factor-I Bioactivity in the Healthy Population: Comparison with Five Widely Used IGF-I Immunoassays

BACKGROUND IGF-I immunoassays are primarily used to estimate IGF-I bioactivity. Recently an IGF-I-specific kinase receptor activation assay (KIRA) has been developed as an alternative method. However, no normative values have been established for the IGF-I KIRA. OBJECTIVE The objective of the study was to establish normative values for the IGF-I KIRA in healthy adults. DESIGN This was a cross-sectional study in healthy nonfasting blood donors. STUDY PARTICIPANTS Participants included 426 healthy individuals (310 males, 116 females; age range 18-79 yr). MAIN OUTCOME MEASURES IGF-I bioactivity determined by the KIRA was measured. Results were compared with total IGF-I, measured by five different IGF-I immunoassays. RESULTS Mean (+/- sd) IGF-I bioactivity was 423 (+/- 131) pmol/liter and decreased with age (beta = -3.4 pmol/liter.yr, P < 0.001). In subjects younger than 55 yr, mean IGF-I bioactivity was significantly higher in women than men. Above this age this relationship was inverse, suggesting a drop in IGF-I bioactivity after menopause. This drop was not reflected in total IGF-I levels. IGF-I bioactivity was significantly related to total IGF-I (r(s) varied between 0.46 and 0.52; P < 0.001). CONCLUSIONS We established age-specific normative values for the IGF-I KIRA. We observed a significant drop in IGF-I bioactivity in women between 50 and 60 yr, which was not perceived by IGF-I immunoassays. The IGF-I KIRA, when compared with IGF-I immunoassays, theoretically has the advantage that it measures net effects of IGF-binding proteins on IGF-I receptor activation. However, it has to be proven whether information obtained by the IGF-I KIRA is clinically more relevant than measurements obtained by IGF-I immunoassays.

Rational Approach to the Diagnosis of Severe Growth Hormone Deficiency in the Newborn

CONTEXT Severe congenital GH deficiency (GHD) of the newborn is a rare disease, which can cause life-threatening hypoglycemias beginning in the first week of life. Reviews and consensus papers on the diagnosis of GHD repeatedly state the lack of a practical evidence-based approach to the diagnosis of GHD in the newborn. OBJECTIVE Here we provide for the first time sound reference values and a diagnostic cutoff for the GH levels in newborns at the age between d 3 and 5. DESIGN, SETTING, AND PATIENTS GH was measured in the eluate from 314 filter papers of the newborn screening test performed in our university hospital by using a highly sensitive human GH-ELISA. Reference data are compared with measurements from nine newborns with very high likelihood of having severe GHD, and cutoffs for the diagnostic work-up are defined. RESULTS In the presence of clinical evidence, the diagnosis of neonatal GHD can be confirmed during the first week of life by a single randomly taken GH level less than 7 microg/liter with 100% sensitivity and 98% specificity on the basis of our assay method. GH content in newborn screening cards stored for almost 3 yr were not different from the content found in recently used screening cards indicating high immunological stability of GH over time. Therefore, the diagnostic approach can use stored screening cards. In addition, we observed a clear gender dichotomy in respect to GH, with healthy female newborns having significantly higher GH levels than males. Cigarette smoking during pregnancy was associated with higher, transient tachypnea of the newborn with lower GH levels. CONCLUSIONS We provide the first rational approach to the diagnosis of severe GHD in the newborn and evidence for gender dichotomy of the neonatal GH axis.

Reference levels of insulin-like growth factor I in the serum of healthy adults: comparison of four immunoassays.

Abstract The measurement of insulin-like growth factor-I (IGF-I) has become an essential tool for diagnosing growth hormone deficiency and acromegaly, as well as for monitoring the efficacy of treatment in these disorders. The latter aspect gains significance in the light of epidemiological studies which indicate a relationship between IGF-I levels and the incidence of certain malignancies. We aimed to evaluate the performance of widely implemented IGF-I assays by testing four representative, commercially available immunoassays. Thus, four parallel determinations of the IGF-I levels of 427 healthy blood donors aged between 18 and 79 years were conducted. Apart from divergent performance criteria, the assays also differed systematically. These differences were, however, linear and of lower magnitude among the lower ranges. We conclude that despite the wide variance among commercially available IGF-I assays, which principally involve assayspecific normative data, each of the implemented assays was robust and thus an appropriate tool in the diagnostic workup of growth hormone deficiency in adult life, when IGF-I levels are low. Clin Chem Lab Med 2003; 41(10):13291334

Measuring IGF-I, IGFBP-2 and IGFBP-3 from dried blood spots on filter paper is not only practical but also reliable

OBJECTIVE The aim of this study is to adapt measurements of IGF-I, IGFBP-2 and IGFBP-3 to dried blood filter disk assays. METHODS Measurements of the three analytes in serum samples and in the corresponding blood spotted onto filter paper were compared by applying standard radioimmunoassays. RESULTS In paired experiments, the quantity of all antigens measured on dried filter spots showed an excellent correlation to that in serum (R>0.88) and in addition this correlation was independent of the whole blood hematocrit value. Recovery of IGF-I, IGFBP-2 and IGFBP-3 in experiments using recombinant standards mixed with whole blood was well correlated with the recovery of the plasma fraction on the filter paper. All of the blood spot assays showed a inter- and intra-assay variation of less than 10% and the blood spots were stable over a period of more than 5 months stored at -20 degrees C. CONCLUSIONS Taken together, these data clearly demonstrate that such a filter paper assay is a reliable procedure to monitor changes of IGF-I, IGFBP-2 and IGFBP-3 content in blood. The obvious advantages of this methods concerning storage, handling and shipping of blood probes helps to solve the logistics of centralised measurements of these three analytes.

Human growth hormone measurement by means of a sensitive ELISA of whole blood spots on filter paper.

BACKGROUND Measurements of human growth hormone (hGH) are a prerequisite for identifying a deficiency or excess. Our study is the first to investigate the reliability of a very sensitive assay for the quantification of GH in dried blood spots on filter paper. OBJECTIVE Validation of a commercially-available enzyme-linked immunoassay (ELISA) for measuring hGH from filter paper samples of dried blood. METHODS We used an assay system (ELISA, E022, Mediagnost) based on polyclonal rabbit antibodies. Its suitability is ascribable to its very high sensitivity (1.6 ng/L) and virtual absence of interfering factors, excepting for a cross-reactivity with high pegvisomant concentrations. RESULTS hGH was found to be stable in dried blood spots on filter paper (No. 903, Whatman) over eight days at 37 degrees C. Extraction of hGH from filter paper, in comparison to EDTA plasma, was 107% (SD 8.1%; n=6) over a range from 2.4 to 34.5 microg/L. Linear regression analysis (n=119) showed a correlation of R(2)=0.97 for the hGH concentration in serum and on filter paper samples. CONCLUSION Our findings demonstrate the reliability of measurements of hGH in dried blood spots on filter paper. The advantages of this method are the low sample volume and the easy transport, storage, and handling of samples. This method contributes to the standardisation of diagnostics pertaining to abnormal hGH secretion as it facilitates the comparison of decisive measurements.

Secretion of Noncomplexed ‘Big’ (10–18 kD) Forms of Insulin-Like Growth Factor-II by 12 Soft Tissue Sarcoma Cell Lines

The paraneoplastic production of pro-insulin-like growth factor-II (IGF-II) forms causes tumour hypoglycaemias and presumably also has an effect on tumour cell growth. We investigated the molecular weights of IGF-II forms and their ability to form complexes with IGF binding proteins (IGFBPs) in conditioned culture media (CM) from 12 paediatric soft tissue sarcoma (STS) cell lines and from two healthy fibroblast lines. Untreated CM were separated by size exclusion chromatography using biocompatible HPLC. Subsequently, IGF-II, IGFBP-2 and IGFBP-3 were determined in the HPLC fractions by specific RIAs. In the CM, IGF-II concentrations between 0.5 and 8.6 ng/106 cells were measured but no IGF-I was detectable. Parallel to this investigation, a high IGF-II mRNA level averaging 44.4 ± 29.7% was measured by semi-quantitative RT-PCR. The STS cell lines secreted a higher proportion of big-IGF-II forms reaching 10–18 kD (10–33% of the total IGF-II secreted) compared to the healthy fibroblasts (2.5–5%). At the same time, the proportion of IGF-II bound with IGFBP in complexes of 35– 70 kD and 150 kD was reduced by up to 85% in CM from tumour cells. The tumour cell lines apparently secrete a different spectrum of IGF-II forms than healthy fibroblasts. The reduced ability to form complexes with IGFBP and the higher molecular weight of the IGF-II forms produced by the tumour cells indicate that these forms could in fact be the known tumour-associated pro-IGF-II forms. Due to these characteristics, the big-IGF-II forms probably have an altered biological effect on the tumour cells when compared to IGF-II.

Relationship of Salivary and Plasma Cortisol Levels in Preterm Infants: Results of a Prospective Observational Study and Systematic Review of the Literature

Background and Objectives: (1) To investigate the relationship of salivary and plasma cortisol levels in preterm infants with a focus on the usability of salivary cortisol in diagnostic work-up of infants at risk of adrenal insufficiency. (2) To perform a systematic review addressing this question. Methods:Clinical study: We conducted a prospective observational single-center study in preterm infants. We analyzed plasma and saliva cortisol concentrations by enzyme immunoassay. Correlation analysis was used to determine the relation between salivary and plasma cortisol levels and the agreement of the measurement methods was analyzed according to Bland-Altman. Systematic review: A systematic literature search (PubMed and Embase) on the relationship of salivary and plasma cortisol levels in neonates was performed in November 2012. Results:Clinical study: We enrolled 58 preterm infants (median (interquartile range) gestational age at birth was 31.4 (28.1-32.7) weeks, birth weight 1,340 (974-1,745) g, respectively). Correlation analyses revealed a relationship of plasma cortisol and salivary cortisol levels. Rank correlation coefficient was 0.6. Estimating plasma cortisol levels based on measured salivary cortisol levels showed poor agreement of the two methods for determining plasma cortisol levels (direct and via salivary cortisol). Sensitivity and specificity of salivary cortisol for the detection of adrenal insufficiency were 0.66 and 0.62, respectively. Systematic review: Six studies in preterm infants and term neonates depicting the correlation of salivary and plasma cortisol were identified with a range of saliva-plasma correlation coefficients from 0.44 to 0.83. Conclusions: Substitution of plasma cortisol by salivary cortisol determination cannot be recommended in preterm infants because of unsatisfactory agreement between methods.

Investigation of myostatin serum levels before and after a 6-month lifestyle intervention program in obese children.

OBJECTIVE To investigate the relationship between myostatin serum levels and muscle mass, fat mass and HOMA before and after a 6-month lifestyle intervention program in obese children and adolescents. DESIGN A total of 57 overweight children and adolescents (female, n=27; age range, 6.0-16.1 years) were examined between 2007 and 2009. Mean BMI (±SD) was 31.1 (5.7) kg/m(2) corresponding to a mean BMI-SDS LMS of 2.2 (0.4). Muscle and fat mass were determined by means of DXA. Serum myostatin was measured by using a competitive ELISA. RESULTS [MEAN±SD]: After the 6-month intervention program, muscle mass (+2.1±2.7 kg, p<0.0001), and percentage myostatin serum levels (+23.7±26.7%, p<0.0001) were higher than before, whereas decreases in BMI (-0.4 kg/m(2)±1.5, p<0.0001), fat mass (-1.2±3.9 kg, p<0.0001), and HOMA insulin sensitivity index (-0.78±3.28 SD, p=0.0004) were observed. In 86% (n=49, p<0.0001) of all cases, the intervention program resulted in a higher level of myostatin. After lifestyle intervention, patients with the greatest increase of myostatin had a significantly lower increase of muscle mass (p=0.048) but did not differ for fat mass. There was no significant correlation between Myostatin and HOMA insulin sensitivity index before and after lifestyle intervention. CONCLUSION Both muscle mass and serum myostatin increased concordantly. Patients with the greatest rise of myostatin had a significantly lower increase of muscle mass suggesting a negative feedback loop between myostatin and muscle tissue. In our study, the change of myostatin serum levels was not associated with the amount of fat mass or HOMA insulin sensitivity index.