Measurement of Insulin-Like Growth Factor-II in Physiological Fluids and Tissues. I. An Improved Extraction Procedure and Radioimmunoassay for Human and Rat Fluids*

Increased concentrations of insulin-like growth factor (IGF) system components have previously been observed in rheumatoid arthritis (RA) and osteoarthritis (OA); however, disruption of the IGF axis and the implications for the disease process remain largely unaddressed. This study was undertaken to characterise the IGF binding protein (IGFBP)-3 proteolysis and complex formation systems in synovial fluid and to investigate changes in these systems in arthritic disease, and their impact on the availability of IGF. Western blotting or autoradiography of SDS gels was used to visualise IGFBP-3 or its proteolysis. IGF-I and IGFBP-3 concentrations were determined by radioimmunoassays and acid-labile subunit (ALS) was measured by ELISA. A shift in distribution of IGFBP-3 and IGF-I in RA and OA synovial fluids (RASynF, OASynF) and an associated increase in ALS suggested the presence of 150 kDa ternary complexes. IGFBP-3 proteolysis was decreased in RASynF and OASynF, but was apparent in size-fractionated fluid and resembled serum activity. The presence of serum-like inhibitors of IGFBP-3 proteolysis in RASynF was also demonstrated by the ability of this fluid, and 150 kDa fractions from its size fractionation, to inhibit IGFBP-3 proteolysis in the other synovial fluid. A marked disruption in the IGF system was observed, as considerably more IGF-I was retained in ternary complexes. We also classified the IGFBP-3 proteolysis system in synovial fluid and found it to be disturbed in RASynF and OASynF. These changes may be caused by an increased flux of circulatory proteins into synovial fluid, resulting from an inflammation-induced increase in vascular permeability. The net result in RA and OA would be a decrease in IGF availability in arthritic joints, and therefore loss of a potential anabolic stimulus. This disruption to the IGF axis would influence disease progression in RA and OA.

Section: Distribution Of Igf-i In Column Fractionsmentioning

confidence: 99%

Alterations in insulin-like growth factor binding protein-3 proteolysis and complex formation in the arthritic joint

Whellams

Maile²,

Fernihough³

et al. 2000

“…A formic acid-acetone extraction procedure was used to separate IGFs from IGFBPs (Bowsher et al 1991). Briefly, 30 µl plasma were added into 15 µl 0·8 M formic acid-0·5% Tween 20.…”

Section: Radioimmunoassaymentioning

confidence: 99%

Alteration of IGF system gene expression during the postnatal development of pcd mice

Zhang

Ghetti²,

Yang

et al. 1999

IGF-I promotes growth during postnatal development via both endocrine and autocrine actions. In pcd mice (pcd/ pcd), we previously found that IGF-I mRNA expression was decreased in cerebellar Purkinje cells as they underwent apoptosis. To investigate the endocrine function of IGF-I, we examined hepatic IGF-I mRNA by Northern hybridization, circulating IGF-I peptide by radioimmunoassay, and circulating IGFBP by Western ligand blot in pcd mice. At postnatal days (D) 17 and 24, hepatic IGF-I mRNA and circulating IGF-I and IGF-II concentrations were normal in pcd mice. From D45, both hepatic IGF-I mRNA and circulating IGF-I concentrations decreased. The decrease in circulating IGF-I concentrations was accompanied by a simultaneous increase in circulating IGF-II concentrations in both the D45 and adult pcd mice. An early decrease in the circulating IGFBP-3 levels and an increase in the IGFBP-2 levels were observed at D17 and were followed by decreases in both IGFBPs at D45 and in the adult. Therefore, after the cerebellar neurodegeneration, there was an overall decrease in IGF-I gene expression in pcd mice. Our results suggest that the decrease in IGF-I gene expression may contribute to growth deficiency and multiple system degeneration in pcd mice.

“…Serum samples underwent formic acid-acetone extraction (Bowsher et al 1991) before assay to separate IGFs from their binding proteins. After overnight equilibration with antibody and [ 125 I]rhIGF, the bound and free fractions were separated using Sac-Cel (Immunodiagnostic Systems, Boldon, Tyne and Wear, UK) and compared with a standard curve produced by rhIGF standards (a gift from Pharmacia and Upjohn, Stockholm, Sweden).…”

Section: Assaysmentioning

confidence: 99%

Nutritional modulation of canine insulin-like growth factors and their binding proteins

Maxwell¹,

Butterwick²,

Yateman³

et al. 1998

The response of canine insulin-like growth factors (IGFs) and IGF-binding proteins (IGFBPs) to moderate nutritional restriction followed by refeeding has not previously been studied in detail. The purpose of these studies was to examine the effects of nutritional restriction on the IGF system of adult dogs. Normal serum IGF values were established after validation of heterologous RIAs for measuring canine IGFs-I and -II. Canine serum IGFBP profiles were examined by Western ligand blotting (WLB), using radiolabelled recombinant human (rh) IGF-I as the ligand, and were found to be similar to those of other species. IGF-I and IGFBP-3 concentrations correlated with body weight, thus reflecting breed size as previously shown, whereas IGF-II concentrations did not.IGFBP-2 serum concentrations and band intensity on WLB were increased compared with normal human serum IGFBP-2. Overnight fasting had no effect on IGF or IGFBP concentrations, including IGFBP-1, nor did refeeding. Prolonged restriction to 56% and then 42·5% of maintenance energy requirements for 2 weeks decreased IGF-I concentrations by 20·4% and 32·7% respectively. Feeding of the same diet ad libitum for 2 weeks normalised IGF-I concentrations. There were no changes in IGF-II or insulin levels. Serum IGFBP-2 concentrations increased with 56% restriction of maintenance energy (P=0·03). We conclude that serum IGF-I is potentially a useful marker of short-term change in nutritional status in the adult dog.