Applications and limitations of measurement of 15-keto,13,14-dihydro prostaglandin E 2 in human blood by radioimmunoassay

Abstract

It has been anticipated that the inherent limitations of radioimmunoassays for prostaglandin E (PGE) would be obviated by assays for its major circulating metabolite, 15-keto, 13,14-dihydro PGE 2 (KH 2-PGE 2) which has a longer half-life in blood. We examined the effects of PGE 2 infusion and alterations in lipolysis in vivo , and of clotting, prolonged storage and hemolysis in vitro , on KH 2-PGE 2 immunoreactivity in unextracted human plasma and serum samples. Indeed KH 2-PGE 2 levels rose several hundred fold during infusions of PGE 2 at doses which cause little or no increment in peripheral PGE levels. During stimulation of lipolysis by infusions of epinephrine, apparent KH 2-PGE 2 levels rose five-fold. However, the dilution curve of plasma obtained during stimulation of lipolysis was not parallel to the standard curve; furthermore, apparent KH 2-PGE 2 levels were correlated strongly with free fatty acid (FFA) levels, suggesting that FFA's cross-reacted in the RIA weakly but significantly due to their very high molar concentration in blood. Clotting and prolonged storage of samples, but not hemolysis, also caused marked apparent increments in KH 2-PGE 2 levels. Competition curves using dilutions of such samples were again not parallel to the standard curves in plasma or buffer, but resembled dilution curves of samples containing high levels of FFA. These results suggest that handling of human blood samples for KH 2-PGE 2 measurement must be carefully standardized to avoid significant artifacts which presumably are due in part to fatty acids released from triglyceride stores in vivo or from disrupted membrane phospholipids in vitro . Unextracted plasma appears to be unsatisfactory for use in this RIA.