Quantitation of substance-P and its metabolites in plasma and synovial fluid from patients with arthritis

Abstract

Substance-P (SP) and its metabolites, SP-(1-7) and SP-(5-11), were quantitated in arthritic synovial fluids and plasma using a validated procedure. This process involved collection into appropriate enzyme inhibitors, extraction with acid-acetone, high pressure liquid chromatography, and RIA using region-specific antisera. Our results demonstrate that the levels of authentic SP in these fluids are less than 3.5 pmol/L, which is 50- to 10,000-fold less than those previously reported by others. These discrepant findings were not attributable to degradation, because added SP was recovered in good yield, and the measured levels of the metabolites SP-(1-7) and SP-(5-11) were also extremely low. In search of an explanation, we noted that many of the earlier reports involved direct assay of these fluids (without extraction and chromatography). Further work indicated that proteolytic enzymes (e.g. protease 24.11) present in these unextracted fluids can give rise to artifactually high SP measurements. We conclude that if SP is released within the joint space and if it participates in the inflammatory reaction and/or healing process, it most likely does so in a local fashion, which would not involve its accumulation in synovial fluid or plasma.