Development and validation of a radioimmunoassay for prostaglandin E2 metabolite levels in plasma

Abstract

Measurements of primary prostaglandins (PGs) in plasma are unreliable, and determinations of their metabolite levels are to be preferred. The principal circulating metabolite of PGE2 [13,14-dihydro-15-keto-PGE2 (PGEM)] is itself unstable however, and this has hindered development of PGE2 metabolite assays. Alkalinization of plasma uniformly converts PGEM and its degradation products to a stable bicyclo compound 11-deoxy-13,14-dihydro-15-keto-11 beta, 16 epsilon-cyclo PGE2 (bicyclo-PGEM). Measurement of bicyclo-PGEM, therefore, enables the PGEM instability problem to be circumvented. This paper describes a specific and sensitive RIA for bicyclo-PGEM in plasma. The assay uses a rabbit antiserum against bicyclo-PGEM and a radiolabel produced by alkalinization of tritiated PGEM. The assay is direct, employing neither chromatographic nor extraction steps. The least detectable mass of bicyclo-PGEM is 1 pg, and the mean mass of added bicyclo-PGEM required to displace zero point binding by 50% is 15 pg. Cross-reactivities of the antiserum against prostanoids are less than 1%. The assay is both accurate and reproducible. Intra- and interassay coefficients of variation are 9.8% and 15.3%, respectively. Plasma concentrations of bicyclo-PGEM in man, rhesus monkey, sheep, and rabbit are reported. This assay allows for reliable measurements of plasma PGE2 metabolites and opens the way for in vivo studies on the significance of PGE2 in health and disease.