EXTRACTION OF NEUROPEPTIDES FROM JOINT TISSUE FOR QUANTITATION BY RADIOIMMUNOASSAY - A STUDY IN THE RAT

The feasibility of extracting neuropeptides from rat knee joints for q uantitation by radioimmunoassay was tested. The investigation, based o n 25 adult Lewis rats, focused on substance P, calcitonin gene-related peptide, neuropeptide Y, and vasoactive intestinal polypeptide. The r elative recovery of the peptides in different extraction media was ass essed. Both knee joints including the articulating epiphysis were diss ected and cut into small pieces. The series was divided into five subg roups, 1;0 joints in each, for extraction in five different media: 1) 1 M acetic acid in 4% EDTA, 2) 2 M acetic acid in 4% EDTA, 3) neutral water in 4% EDTA, 4) 2 M acetic acid in 4% EDTA and 95% alcohol, and 5 ) 2 M acetic acid without EDTA. Measureable concentrations of the four neuropeptides were reproducibly assessed by RIA. Although all extract ion media provided measurable concentrations, 2 M acetic acid in 4% ED TA was found to give the highest overall yield of the four neuropeptid es analyzed. Reverse-phase HPLC confirmed that the immunoreactivities assessed by RIA corresponded to the four neuropeptides of interest. Ex perimental and clinical evidence suggest a neurogenic involvement in t he pathophysiology of inflammatory joint disease, e.g., rheumatoid art hritis. The extraction procedure described offers a means of determini ng neuropeptide concentrations in joint tissue under normal and pathol ogic conditions by RIA.