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Differential distribution of insulin‐like growth factors and their binding proteins within bone: Relationship to bone mineral density

To evaluate the possibility that insulin‐like growth factors (IGFs) and their binding proteins (BPs) in bone play a role in regulating cortical bone formation in growing animals, we compared changes in IGF and IGF BP levels with changes in bone mineral density (BMD) at three different regions (proximal, middle, and distal) along the rabbit femoral shaft. BMD measured by dual‐energy x‐ray absorptiometry decreased progressively from proximal to distal regions of the shaft, from 0.449 ± 0.005 to 0.354 ± 0.002 g/cm2 (mean ± SEM; n = 9), respectively; total protein concentrations also decreased toward the distal region. We extracted the IGFs and their BPs from bone by demineralization in 10% EDTA and 4 M guanidine‐HCl (pH 4.5). The IGFs were then separated from their BPs by size exclusion HPLC. The pH of the extraction buffer profoundly influenced the recoveries of the IGFs and, to a lesser extent, the total protein; at least 100% more IGFs were recovered at acid (4.5) pH than at neutral (7.5) or basic (10.5) pH. The levels of IGF‐I decreased markedly from proximal to distal regions, from 273 ± 27 to 100 ± 38 ng human IGF‐I equivalent/g bone (or 103 ± 10 to 52 ± 11 ng human IGF‐I equivalent/mg protein), respectively. IGF‐II was uniformly distributed (385 ± 17 ng human IGF‐II equivalent/g bone; mean of all three regions). Levels of the predominant 28–32 kD IGF BP doublet increased by about 100% from proximal to distal segments, regardless of whether the data were expressed per unit mass or protein. Thus, the differential distribution of bone‐associated IGF‐I paralleled that of BMD and total protein, whereas levels of the 28–32 kD bone IGF BP(s) were inversely related to cortical bone density.