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Objective: Determination of insulin-like growth factor (IGF)-I is now a routine adjunct to multiple research and clinical investigations. Evidence has associated higher IGF-I levels with various human pathologies, but the reported associations have not been invariably confirmed. We examined the potential for post-sampling proteolysis and evaluated the impact of such events on IGF-I immunoassays. Design and methods: We compared IGF-I in different sets of fresh and frozen old samples using four different and commonly used immunoassays. The potential for post-sampling proteolysis was further examined by assaying fresh samples stored for 4 weeks at various temperatures in the absence or presence of protease inhibitors. Results: IGF-I levels in fresh serum samples from adult males, females, and pregnant subjects by all methods were similar and were highly correlated (r = 0.85–0.97). The same was true for levels in frozen (∼2 years at −80°C) samples from diabetic patients, which are reportedly associated with enhanced proteolytic activity. In contrast, in another set of frozen adult male and female samples (∼8 years at −20°C), the inter-method median IGF-I levels varied by ∼3- to 4-fold and the values poorly correlated. Similar variability in the inter-method response was also observed when IGF-I in the replicates of fresh samples stored at 4°C for 4 weeks was measured. However, the 4°C storage effect could be completely blocked by the addition of protease inhibitors, allowing for all assays to detect 92–101% of the expected mean levels. Conclusions: The data indicate susceptibility of IGF-I to significant post-sampling proteolysis and suggest the importance of immunoassays for the intact molecule. Immunoassays that lack specificity for intact IGF-I may mask the potential pathophysiological effects of proteolysis and generate misleading results, particularly in studies involving inappropriately stored and/or proteolyzed samples. In such cases, underestimation of the in vivo levels by the intact assays would occur, but the findings of low IGF-I levels may be indicative of questionable sample quality.

Summary objective Age-related decline in IGF-I and gonadal hormones have been postulated to play an important role in the pathogenesis of age-related bone loss in men. In this cross-sectional study, the relation between serum IGF-I and gonadal hormones with bone mineral density (BMD) was examined in community-dwelling men. design and subjects Serum IGF-I, testosterone and BMD were examined in 61 community-dwelling men over the age of 27, who were randomly selected from the Calgary cohort of 1000 subjects in the Canadian Multicentre Osteoporosis Study. In the present study, IGF-I, serum testosterone, SHBG, free androgen index (FAI), parathyroid hormone (PTH), 25-hydroxy-vitamin D [25(OH)D] and other markers of bone turnover were measured. BMD was measured at the spine and hip (HOLOGIC 4500). Simple linear regression was used to assess the linear relation between IGF-I, testosterone, BMD and other biochemical markers of bone metabolism and potential confounding variables and subsequent multivariate regression models were constructed separately for each BMD measurement to assess the importance of IGF-I and testosterone in the presence of potential confounding variables. results Serum IGF-I, FAI and SHBG significantly decreased as a function of age, whereas serum levels of PTH increased. Only 25(OH)D, total testosterone and FAI were positively associated with serum IGF-I after adjusting for age and BMI. Multiple linear regression models revealed that IGF-I was a significant predictor of BMD at the total hip, femoral neck and femoral trochanter neck (P ≤ 0·001). In contrast, the FAI was a significant predictor of BMD at the lumbar spine and wards area (P ≤ 0·011), and SHBG was a significant predictor at the total hip and femoral trochanter (P ≤ 0·045). conclusion These data support the hypothesis that the age-related decline in bone mass in men is associated with declining levels of IGF-I and testosterone.

Markers of myocardial injury have been vital in the assessment of patients with coronary heart disease. Pregnancy associated plasma protein A (PAPP)-A is an insulin-like growth factor (IGF) binding protein (IGFBP)-4 protease and a potential early indicator of unstable angina. We developed an ultrasensitive enzyme-linked immunosorbent assay (ELISA) for PAPP-A and measured serum PAPP-A in patients with biochemical evidence of acute coronary syndrome.Method development was based on pair-wise evaluation of a panel of antibodies and determination of PAPP-A specificity and sensitivity relative to those of a conventional method. Association of PAPP-A with myocardial damage was assessed in serum samples classified based on serum creatine kinase (CK)-MB or cardiac troponin-T levels.Serum PAPP-A was significantly higher in samples with elevated CK-MB or troponin-T than in samples with normal CK-MB (p0.001). Marker-association studies showed strong correlation between PAPP-A and troponin-T (r = 0.59, p0.001) in a subset of troponin-T positive samples. Indications for both parallel as well as divergence in the expression of PAPP-A and troponin-T was also evident when serial timed samples available from a number of patients were analyzed.The data are consistent with the conclusion that expression of PAPP-A is enhanced in patients with biochemical evidence of acute coronary syndrome and suggest strongly that demonstration of PAPP-A association with other cardiac markers might be influenced by their relative release dynamics (timing and duration). The availability of the ultrasensitive PAPP-A ELISA should facilitate systematic investigations of PAPP-A expression in this and other pathophysiological conditions that might involve altered expression of the IGF/PAPP-A system.

Insulin-like growth factor-I (IGF-I) has mitogenic and anti-apoptotic effects on breast cancer cells. Epidemiologic studies have shown that high plasma levels of IGF-I and low levels of IGF binding protein (BP)-3 are associated with increased risk of breast cancer in premenopausal women. The actions of IGF-I are mediated through the IGF-I receptor (IGF-IR) and are regulated by IGFBPs. In circulation, most of the IGF-I binds to IGFBP-3, and binding of IGF-I to IGFBP-3 inhibits the actions of IGF-I. Since free IGF-I, which does not bind to IGFBPs, can readily cross the endothelial barrier to interact with IGF-IR, circulating free IGF-I is thought to be more relevant to the biologic activity of IGF-I. To examine the association of free IGF-I with breast cancer, we compared free IGF-I levels between 40 newly diagnosed breast cancer patients and 40 age- and race-matched healthy controls. Plasma levels of free IGF-I, total IGF-I and IGF-II, as well as total, intact and fragment IGFBP-3, were measured using commercial immunoassay kits. The association between IGF-I and breast cancer was examined using the conditional logistic regression analysis. Analysis of correlation (Spearman) showed that free IGF-I was correlated with total IGF-I and IGFBP-3 but not with IGF-II. The odds ratios for breast cancer patients having high plasma IGF-I (≥median) after adjusting for menopausal status and IGFBP-3 were 2.00 (p < = 0.376) for total IGF-I and 6.31 (p < = 0.047) for free IGF-I. A high ratio of IGF-I to IGFBP-3 was also associated with breast cancer (p < 0.05). No association was found for IGF-II, nor for total, intact and fragment IGFBP-3. The findings of this study suggest that measuring free IGF-I in circulation is more useful than measuring total IGF-I with respect to evaluation of an association between IGF-I and breast cancer risk. © 2001 Wiley-Liss, Inc.

Measurements of insulin-like growth factor I(IGF-I), IGF-binding protein-3 (IGFBP-3), and the acid-labile subunit (ALS) are important in assessing the GH-IGF axis. As nearly all IGF-I, IGFBP-3, and ALS circulate in a GH-dependent ternary protein complex, direct determination of the complex may be of significant analytical and clinical importance. We evaluated a panel of monoclonal antibodies (mAb) to human IGFBP-3 and classified them into four groups (G-1 to G-4). G-1 antibodies recognized epitopes that mapped at or near IGFBP-3 ligand (IGF)-binding site. This region overlapped with the G-2 defined region, which, in turn, overlapped with G-3 epitopes defined by one antibody (mAb 3). Only G-1 and G-3 antibodies paired without interference. mAb 9 recognized a conformational epitope (G-4), and mAb 10 was nonreactive. In pairwise mixed antibody evaluation, mAbs in G-2 and G-3 showed simultaneous binding to serum IGFBP-3 complexes in combination with an anti-IGF-I or an anti-ALS antibody. On this basis, two novel enzyme-linked immunosorbent assays (ELISAs) involving IGFBP-3/IGF-I (ELISA-1) and IGFBP-3/ALS (ELISA-2) recognition partners were developed, both demonstrating acceptable analytical performance characteristics. IGFBP-3 complexes measured by ELISA-1 and -2 in samples from normal individuals and subjects with GH deficiency, acromegaly, and GH receptor deficiency more tightly correlated with IGF-I, IGFBP-3, and ALS than IGF-II. ELISA-1 determinations were comparatively more age dependent and, in comparison to ELISA-2, showed better discriminations among the various sample groups, particularly among GH receptor deficiency, normal, and GH deficiency subjects. The development of IGFBP-3 complex ELISAs may simplify diagnostic applications and facilitate investigations of the physiological relevance of the ternary complex formation.

We developed a specific, simple, and rapid RIA for the direct quantification of estrone sulfate (E1S) and established its performance characteristics. The assay has a dynamic range of 0.05–90 μg/L with a detection limit of 0.009 μg/L. Intraassay CVs were 9.2%, 4.5%, and 4.6% at 0.35, 9.0, and 60 μg/L, respectively. Interassay CVs were 8.8%, 5.1%, and 5.5% at 0.076, 0.5, and 12 μg/L, respectively. Linearity of dilution studies showed values of 80–105% of expected, and recovery of E1S added to serum samples ranged from 82% to 102%. Cross-reactivities with structurally related estrogens were

Insulin-like growth factor (IGF) I and IGF binding proteins (IGFBPs) modulate metabolic activity and tissue repair and are influenced by nutritional status. IGF-I circulates in free, ternary [IGF-I + IGFBP-3 + acid labile subunit (ALS)], and binary (IGF-I + IGFBP) molecular complexes, and the relative proportions regulate IGF-I extravascular shifting and bioavailability. This study examined the hypothesis that sustained physical activity and sleep deprivation superimposed on a short-term energy deficit would alter the IGFBP concentrations and alter the proportions of IGF-I circulating in ternary vs. binary molecular complexes. Components of the IGF-I system (total and free IGF-I; IGFBP-1, -3, and ALS; nonternary IGF-I and IGFBP-3), biomarkers of metabolic and nutritional status (transferrin, ferritin, prealbumin, glucose, free fatty acids, glycerol, beta-hydroxybutyrate), and body composition were measured in 12 men (22 +/- 3 yr, 87 +/- 8 kg, 183 +/- 7 cm, 20 +/- 5% body fat) on days 1, 3, and 4 during a control and experimental (Exp) period. During Exp, subjects performed prolonged work (energy expenditure of approximately 4500 kcal/day) with caloric (1600 kcal/day) and sleep (6.2 h total) restriction. IGF-I and IGFBP-3 were measured by immunoassay before and after immunoaffinity depletion of ALS-based complexes (i.e., ternary complex removal). Exp produced losses in body mass (-3.0%), lowered total IGF-I (-24%), free IGF-I (-42%), IGFBP-3 (-6%), nonternary IGF-I (-27%), and IGFBP-3 (-16%), and increased IGFBP-1 (256%). No Exp effects were observed for ALS. No changes were observed in the proportion of IGF-I circulating in free ( approximately 1.2%), ternary ( approximately 87.4%), or nonternary ( approximately 11.4%) molecular complexes. During Exp, glucose concentrations were lower on day 3, but days 1 and 4 were statistically similar. In conclusion, during a short-term energy deficit in young, healthy men, 1). IGF-I system components differentially respond (both in direction and magnitude) to a given metabolic perturbation and 2). the relative proportion of IGF-I sequestered in ternary vs. nonternary molecular complexes appears to be well maintained.

Objective: To examine the hypothesis that 12 weeks of resistance training would alter circulating concentrations of IGF-I system components in end-stage renal disease (ESRD) patients. Design: Ten ESRD patients underwent 12 weeks of resistance training after a 6 week control period and had morning fasted blood drawn on four occasions (weeks – 6, 0, 6, 12). Immunoassays were performed for serum total and free IGF-I, IGF binding proteins (IGFBPs) 2 and 3, and the acid labile subunit (ALS). Immunoaffinity depletion of ALS-based complexes allowed measurement of non-ternary (i.e., binary) IGF-I and IGFBP-3. Results: Significant improvements in strength and functional performance were observed. All IGF-I measures were stable during the control period and no changes were observed for the first 6 weeks of resistance training. At week 12, total IGF-I (−15.4 ± 28.9%), ternary IGF-I (−16.4 ± 36.7%), and the IGF-I/IGFBP-3 ratio had significantly (P⩽0.05) declined from week 0 values. No changes were observed for free IGF-I, IGFBPs 2 and 3, or the acid labile subunit. The proportion of IGF-I in ternary (∼76.3 ± 6.8%), non-ternary (∼22.5 ± 6.6%), and free (∼1.2 ± 0.5%) forms remained constant throughout the training. Conclusions: 12 weeks of resistance training in ESRD patients induced a decline in total IGF-I, but did not alter the proportion of IGF-I circulating in free, ternary or non-ternary molecular complexes. The decline in IGF-I occurs in the presence of positive training adaptations on physical performance and we conclude that this response pattern appears to be reflective of favorable neuromuscular anabolic adaptations.

In view of evidence indicating significant involvement of the insulin-like growth factor (IGF) system in the pathogenesis of prostate cancer, we measured serum IGF-I and IGF-binding protein-3 (IGFBP-3) in men with benign prostatic hyperplasia (BPH; n = 75) or prostatic carcinoma (CaP; n = 84). The age-matched patient populations were selected to have circulating prostate-specific antigen (PSA), the most reliable predictor of CaP, in the overlapping diagnostic gray zone range of approximately 4--10 microg/L. Of particular interest was investigation of intact, fragment, and total IGFBP-3 levels in relation to PSA, which is also a well established IGFBP-3 protease. Among the key findings were significantly higher IGF-I and intact IGFBP-3 levels in CaP vs. BPH (P0.001), whereas changes in fragment and total IGFBP-3 were statistically insignificant. As expected, total PSA levels were similar in the two groups of patients (P = 0.173), whereas free PSA levels were significantly lower in those with CaP (P0.001). IGF-I and IGFBP-3 (intact and total) correlated significantly (P = 0.024 to0.001) and inversely (r = -0.26 to -0.35) with free PSA in BPH, but not in CaP, and no correlations were found in comparisons involving total PSA. Statistical analysis of the various markers and their combinations indicated enhanced performance of IGF-I/free PSA [receiver operating characteristics area under the curve (AUC) = 0.728] and intact IGFBP-3/free PSA (AUC = 0.737) ratios in discriminating between BPH and CaP compared with the currently used free/total PSA ratio (AUC = 0.689). Multivariate logistic regression models confirmed the observed relationships and identified IGF-I/free PSA and intact IGFBP-3/free PSA as independent factors in predicting the presence of CaP. We conclude that increases in IGF-I and intact IGFBP-3 levels are positively associated with the presence of CaP in this group of patients with low to moderately elevated PSA, and that their measurements in relation to PSA may help improve diagnostic discrimination between BPH and prostate cancer.

Posttranslational modifications, particularly proteolysis, may play a significant role in the regulation of insulin-like growth factor (IGF)-binding protein-3 (IGFBP-3) physiology, and thus, measurement of modified variants of IGFBP-3 and/or their combination ratios may have important research and diagnostic relevance. Based on evaluation of a panel of monoclonal and polyclonal IGFBP-3 antibodies, we constructed three new enzyme-linked immunosorbent assays (ELISAs) using a common capture and polyclonal (ELISA-3) or monoclonal (ELISA-1 and -2) detection antibodies and evaluated them in a two-step colorimetric procedure. Evaluation of ELISA-1-3 demonstrated detection limit, dynamic range, overall precision, and recovery of the added IGFBP-3 to be generally less than 0.04 microg/L, 2-100 microg/L, less than 10%, and 91-113%, respectively. IGF-I and -II, and IGFBP-1, -2, -4, -5, and -6 did not interfere. In normal adult sera (n = 26), seminal plasma (n = 14), pregnancy sera (n = 30), and amniotic fluid (n = 30), ELISA-1-3 detected significantly different IGFBP-3 levels (by up to 6-fold, on the average), whereas levels in seminal plasma determined by ELISA-1 were undetectable. Comparison of the values obtained vs. corresponding levels by an established method (Diagnostic Systems Laboratories, Inc., active IGFBP-3 ELISA) were similarly sample dependent and, on the average, varied by up to 19-fold. Only ELISA-3 compared well with the Diagnostic Systems Laboratories, Inc., IGFBP-3 ELISA when samples from normal adults were analyzed. The observed variability could not be totally explained by 50% lower reactivity of ELISA-1-3 for glycosylated IGFBP-3 vs. the nonglycosylated form, and changes in phosphorylation had no effect on immunoreactivity. Evaluation of IGFBP-3 after proteolysis by seminal plasma, plasmin, or thrombin suggested recognition of intact IGFBP-3 by ELISA-1, whereas ELISA-3 appeared to measure intact and proteolyzed IGFBP-3 (total IGFBP-3) with similar potency. In contrast, levels determined by ELISA-2 increased severalfold, indicating preferential recognition of IGFBP-3 fragments. We propose that immunoassay capable of differential determination of IGFBP-3 variants may help better define the physiological importance and potential clinical value of IGFBP-3 measurements.

To facilitate broader applications of insulin-like growth factor I (IGF-I) and IGF-binding protein-3 (IGFBP-3) analysis, we developed procedures for their measurements in extracts of whole blood dried on filter paper. A single 8-mm diameter filter paper disc containing about 13 microL blood was used. IGFBP-3 was efficiently extracted in a buffer within 1 h of incubation. IGF-I extraction involved incubation in buffer followed by acidification and neutralization steps. Blood spot assays showed intra- and interassay coefficients of variation (including interspot variations) of 5.4-16.7% for IGF-I and 6.6-11.7% for IGFBP-3; recoveries were 97 +/- 7.1% and 101 +/- 8.7%, respectively. Recoveries of IGF-I and IGFBP-3 in response to 4- to 8-fold variations in extraction buffer volume were 97 +/- 8.2% and 107 +/- 6.1%, respectively. Dried blood spot IGF-I and IGFBP-3 showed greater than 1-month stability at -20 C, 4 C, and room temperature and retained more than 65% of the immunoreactivity after approximately 1 month at 37 C. Both IGF-I and IGFBP-3 were contained within the plasma fraction of whole blood, and variations (mean +/- SD) in IGF-I (204 +/- 29 micrograms/L) and IGFBP-3 (4.4 +/- 0.48 mg/L) measured in extracts of dried blood spot with adjusted hematocrit of 0.2-0.62 were acceptable. IGF-I and IGFBP-3 in paired plasma and dried blood spot extracts of random samples (n = 46) showed excellent correlation (r0.94) with slopes of near unity. Compared to conventional methods, the filter paper procedures were equally effective in distinguishing IGF-I and IGFBP-3 levels in untreated GH receptor-deficient (n = 11) and age-matched normal controls (n = 16). We conclude that blood collected on filter paper is ideal for IGF-I and IGFBP-3 analysis and may find applications in pediatric and large scale infant screening programs.

Accurate measurement of insulin-like growth factor (IGF) binding protein-1 (IGFBP-1) is important for precise definition of its physiological roles and potential diagnostic values. Because altered phosphorylation results in altered IGFBP-1 immunoreactivity, current assays may significantly underestimate or fail to detect physiological changes in the IGFBP-1 concentrations. We developed three ELISAs (ELISA 1–3) using a common capture but three different detection antibodies. IGFBP-1 in serum, synovial fluid (SF), cerebrospinal fluid (CSF), and amniotic fluid (AF) were measured before and after treatment with alkaline phosphatase (ALP). Among the methods, only ELISA-1 was unaffected by IGFBP-1 phosphorylation and generated identical results before and after ALP treatment. The serum and SF values by ELISA-2 and -3 were lower by ∼4- to 10-fold, but increased after ALP treatment to within 66–98% of those by ELISA-1. The medians in AF, and to a lesser extent in CSF, by all methods were similar and did not change significantly after dephosphorylation. ELISA-1 showed excellent correlation with ELISA-2, ELISA-3, and a commercial IGFBP-1 IRMA only after ALP-treated samples were analyzed by the comparative methods. ELISA-1 is highly specific for IGFBP-1 and demonstrated acceptable analytical performance characteristics.