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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.

Insulin-like growth factor-I (IGF-I) is sensitive to nutritional stress and is reduced in soldiers during stressful field training. Methods have recently been developed to measure IGF-I from filter paper blood spots. Filter paper has advantages over traditional blood sampling in that neither blood separation equipment nor refrigeration is necessary after sample collection. This study determined whether filter paper blood spots collected in a field environment could measure IGF-I and subsequent changes during military operational stress. Thirty-four Marines participating in an 8-day military field exercise characterized by near-continuous physical work (total daily energy expenditure 17-25 MJ/day) and underfeeding (dietary intake 7.0 MJ/day) had blood samples taken on day 0, day 4, and day 8. IGF-I was measured by filter paper blood spot assays from fingertip blood samples and by conventional methods using serum. Correlation and measurement agreement were assessed. Blood spot (Day 0 152 +/- 6 ng mL(-1)Day 4 111 +/- 6 ng mL(-1)Day 8 74 +/- 4 ng mL(-1)) and serum IGF-I (Day 0 412 +/- 10 ng mL(-1)Day 4 258 +/- 14 ng mL(-1)Day 8 203 +/- 13 ng mL(-1)) concentrations declined (p0.05) progressively over the 8-day exercise. Overall, the two methods significantly (p0.05) correlated (r = 0.92); however, the blood spot values were on average 61% lower than serum, but could be used to predict serum values ( +/- 10%). IGF-I is a biomarker of metabolic status. The filter paper blood spot method for IGF-I detected reductions accompanying nutritional stress and may be of potential value for characterizing the IGF-I response when conventional blood sampling methods are not feasible.

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.

Measurement of serum insulin-like growth factor I (IGF-I) is important in assessing the growth hormone/IGF axis. Competitive immunoassay methods for IGF-I are complicated by substantial interference from IGF-binding proteins (IGFBPs) due to the relatively low ratio of specific antibody to IGFBPs in the sample, even after standard acid-ethanol sample extraction. We report of development of a noncompetitive ELISA for human IGF-I that avoids this problem by utilizing excess amounts of capture and detection antibodies. Serum samples were prepared by using an abbreviated acid-ethanol extraction method. Neutralized supernatant was added to microwells coated with IGF-I capture antibody; horseradish peroxidase-labeled detection antibody was then added, incubated for 2 h, and then developed. Compared with the "gold standard" method of acid-column chromatography, the simplified acid-ethanol extraction yields a mean +/- SD recovery of 103% +/- 5.5% despite the presence of residual IGFBPs in the extracted sample. Comparisons with a centrifugal filtration sample extraction method are also shown. The ELISA is specific for IGF-I with an absolute sensitivity of 0.03 microgram/L and inter- and intraassay CVs of 3.9-8.8% and 2.6-6.7%, respectively. The availability of a rapid IGF-I ELISA combined with a simple and reliable sample preparation procedure should facilitate clinical and research studies of this important growth factor.

Development of an ELISA for phosphorylated isoform of IGFBP-1. Serine phosphorylation is an important regulator of IGFBP-1 bioactivity, but specific immunoassays for its measurement are currently lacking.Assay design was based on a novel approach of first capturing the phosphorylated and non-phosphorylated IGFBP-1 by an anti-IGFBP-1 antibody and then selectively detecting the phosphorylated form by an anti-phosphoserine antibody. Method development involved pair-wise evaluation of the candidate antibodies and determinations of analytical performance and specificity. Specificity was monitored by reactivity with dephosphorylated IGFBP-1, with antibodies against other phosphorylated residues that are not expressed, and by comparative analysis of sample containing different IGFBP-1 phosphorylation profile.Analytical evaluation demonstrated acceptable performance; detection limit 0.3 microg/L, dynamic range 1.56-100 microg/L; intra- and inter-assay CVs 2.1-8.6%; mean recovery (+/-SD) 97.8+/-9.2%, and mean recovery of sample dilution 93.4+/-6.0%. The phosphorylated and total IGFBP-1 medians in non-pregnant adult serum, which mostly contain the highly phosphorylated isoform, were 11.9 and 18.6 microg/L, respectively, and the sample values were tightly correlated (r=0.99). As expected, the corresponding medians in 1st trimester (17.4 and 63.0 microg/L) and 2nd trimester (30.9 and 75.8) samples with altered IGFBP-1 phosphorylation were significantly different (p0.001). Similarly, a fraction (1.29%) of total IGFBP-1 (13.3 mg/L) in amniotic fluids was found to be phosphorylated (0.172 mg/L). There was no reactivity with dephosphorylated IGFBP-1.The present ELISA is highly specific for the phosphorylated isoform of IGFBP-1 and its availability should help expedite further investigations of IGFBP-1 phosphorylation.

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.

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.

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.