Development of Valid Methods to Measure Insulin-Like Growth Factors-I and -II in Bone Cell-Conditioned Medium*

Abstract

A variety of human cells and biological fluids have been shown to produce or contain insulin-like growth factor (IGF)-specific binding proteins (BPs). The existence of these BPs in serum and conditioned medium of cell and organ cultures has complicated radioligand assays for measurement of IGFs. Various strategies have been proposed to avoid interference of BPs with these assays, including acid-ethanol precipitation of BPs and acid-gel filtration. Many of these procedures are time consuming, exhibit low recoveries, and do not completely eliminate BP artifacts. In this study we have investigated interference of inhibitory IGF-BP (In-IGF-BP) purified from bone cell-conditioned medium in an IGF-II RRA and IGF-I RIA and developed methods to neutralize In-IGF-BP artifacts in IGF assays. In the IGF-II RRA, purified In-IGF-BP competed for [125I]IGF-II binding to H-35 cells in a dose-dependent manner and, thus, increased the apparent value for IGF-II in the medium. Fifty percent inhibition of [125I]IGF-II binding to H-35 cells was seen at 12.2 and 5.7 ng/ml unlabeled IGF-II and IN-IGF-BP, respectively. In-IGF-BP also competed for [125I]IGF-I in the IGF-I RIA; however, the interference was much less in the IGF-I RIA compared to the IGF-II RRA. Fifty percent displacement of [125I]IGF-I binding was seen at 0.25 and 5 ng/ml unlabeled IGF-I and In-IGF-BP, respectively. Our approach to eliminate BP artifacts was as follows. We knew that In-IGF-BP showed comparatively equal binding affinities with both IGF-I and IGF-II, and binding of these ligands to cell receptors (IGF-II) and antibodies (IGF-I) was very specific (2% and 0.5% cross-reactivity for IGF-I and IGF-II, respectively). Therefore, in the IGF-I RIA we blocked the In-IGF-BP artifacts by adding an excess of IGF-II, and in the IGF-II RRA we blocked the In-IGF-BP artifacts by adding an excess of IGF-I. By incubating purified In-IGF-BP with different amounts of IGF-I, we found that 30-min preincubation of 5 ng In-IGF-BP with 10 ng IGF-I completely blocked BP artifacts in the IGF-II RRA. Similarly, preincubation of In-IGF-BP with IGF-II blocked BP artifacts in the IGF-I RIA.(ABSTRACT TRUNCATED AT 250 WORDS)

Postnatal and Pubertal Skeletal Changes Contribute Predominantly to the Differences in Peak Bone Density Between C3H/HeJ and C57BL/6J Mice

“…The difference in cortical thickness between C3H and B6 mice appeared to be caused by significantly smaller endosteal circumference in C3H mice (approximately 20% less) compared with B6 mice. The increase in cortical thickness of approximately 25% during puberty (days [23][24][25][26][27][28][29][30][31][32][33][34][35] in both strains of mice appeared mainly to be caused by increased periosteal circumference, because the endosteal circumference did not show significant change during this period (Fig. 3).…”

“…In addition, serum levels of IGF-I and osteocalcin provided statistically significant contributions to the prediction of various bone parameters (Table 3). To further evaluate the relative contribution of the previously mentioned five independent variables to predict the gender differences in bone size, we performed multiple regression analyses using data from prepubertal (days 7-23), pubertal (days [23][24][25][26][27][28][29][30][31][32][33][34][35], and postpubertal (days 35-56) periods. We found that the relative contribution of gender to differences in periosteal circumference was much greater during the postpubertal period than the prepubertal period (␤-coefficient of 0.36 vs. 0.11).…”

Generalized osteoarthritis associated with increased insulin‐like growth factor types i and ii and transforming growth factor β in cortical bone from the iliac crest. possible mechanism of increased bone density and protection against osteoporosis

“…Louis Underwood and Judson Van Wyk, University of North Carolina, Chapel Hill), at a dilution of 1:6,000, as described previously (29). IGF-binding protein artifacts (30) in the IGF-I RIA or in the IGF-I1 radioreceptor assay (RRA; see below) were removed by preincubating samples with an excess of IGF-I1 or IGF-I, respectively, as described by Mohan et a1 (31). The cross-reactivity of IGF-I1 in the IGF-I RIA was <0.5%.…”

“…IGF-I1 concentrations were determined by RRA, using H-35 rat hepatoma cells (American Type Culture Collection, CRL 1548) as the receptor source (32). Human IGF-I1 purified from bone matrix (33) was used as standard and tracer, and the assay was performed as described elsewhere (31). The sensitivity of the RRA was -0.4-0.6 ng/well (4-12 ng/ml); the range of the assay was 8-250 ng/ml.…”

Ovariectomy selectively reduces the concentration of transforming growth factor beta in rat bone: implications for estrogen deficiency-associated bone loss.

“…(i) IGF-I was determined by RIA using recombinant human IGF-I (CIBAGeigy) as tracer and as standard and rabbit polyclonal antiserum UB 286 (kindly provided by Louis Underwood and Judson Van Wyk, University of North Carolina) at a dilution of 1:6000 as described (31). Artifacts known to be produced by the presence of IGF-binding proteins (32) in IGF-I RIA or in IGF-II radioreceptor assay (see below) were eliminated by preincubation of samples with excess IGF-I or IGF-II as described (33 (Table 1). At 9 weeks, 17fl-estradiol-treated rats had gained less weight than the untreated animals.…”