EXTRACTION AND QUANTITATION OF NEUROPEPTIDES IN BONE BY RADIOIMMUNOASSAY

The feasibility of extracting and quantifying neuropeptides in bone by radioimmunoassay was investigated in a study including 60 diaphyseal rat femora. Substance P, calcitonin gene-related peptide, neuropeptide Y and vasoactive intestinal polypeptide, previously identified in bon e by immunohistochemistry, were extracted from separate homogenates of bone, periosteum and bone marrow in a solution of 4% EDTA and 2 M ace tic acid. Measurable amounts of all four neuropeptides in bone, perios teum and bone marrow were obtained by radioimmunoassay in a reproducib le manner. The neuropeptide immunoreactivities were characterized by r everse-phase high performance liquid chromatography. Among the four ne uropeptides analyzed, neuropeptide Y consistently exhibited the highes t concentrations in the different tissues. Overall, cortical bone show ed the lowest neuropeptide concentrations. The concentration of vasoac tive intestinal polypeptide was higher in periosteum than in bone marr ow, whereas that of calcitonin gene-related peptide was uniform in the se tissues. The distributional differences observed in bone tissue may be explained by a variety of physiological roles attributed to neurop eptides such as regulation of nociception, vasoactivity, immune functi on and local bone metabolism. The described methodology offers a new m eans of investigating a neuropeptidergic involvement in various disord ers of the skeleton.