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 quantitation by radioimmunoassay was tested. The investigation, based on 25 adult Lewis rats, focused on substance P, calcitonin gene-related peptide, neuropeptide Y, and vasoactive intestinal polypeptide. The relative recovery of the peptides in different extraction media was assessed Both knee joints including the articulating epiphysis were dissected and cut into small pieces. The series was divided into five subgroups, 10 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 extraction media provided measurable concentrations, 2 M acetic acid in 4% EDTA was found to give the highest overall yield of the four neuropeptides analyzed. Reverse-phase HPLC confirmed that the immunoreactivities assessed by RIA corresponded to the four neuropeptides of interest. Experimental and clinical evidence suggest a neurogenic involvement in the pathophysiology of inflammatory joint disease, e.g., rheumatoid arthritis. The extraction procedure described offers a means of determining neuropeptide concentrations in joint tissue under normal and pathologic conditions by RIA.
Ahmed
M
M
Department of Orthopaedics, Karolinska Hospital, Stockholm, Sweden.
Bjurholm
A
A
Srinivasan
G R
GR
Theodorsson
E
E
Kreicbergs
A
A
D000818:Animals; D001169:Arthritis, Experimental / Q000378:metabolism; D015740:Calcitonin Gene-Related Peptide / Q000276:immunology / Q000302:isolation & purification; D002851:Chromatography, High Pressure Liquid; D005260:Female; D007719:Knee Joint / Q000737:chemistry*; D008297:Male; D009478:Neuropeptide Y / Q000276:immunology / Q000302:isolation & purification; D009479:Neuropeptides / Q000276:immunology / Q000302:isolation & purification*; D011863:Radioimmunoassay / Q000379:methods*; D051381:Rats; D013373:Substance P / Q000276:immunology / Q000302:isolation & purification; D014020:Tissue Extracts; D014660:Vasoactive Intestinal Peptide / Q000276:immunology / Q000302:isolation & purification
D009478:Neuropeptide Y; D009479:Neuropeptides; D014020:Tissue Extracts; D013373:Substance P; D014660:Vasoactive Intestinal Peptide; D015740:Calcitonin Gene-Related Peptide