Measurement of human venous plasma prostacyclin and metabolites by radioimmunoassay: a reappraisal.

Two radioimmunoassays, the first specific for 6-oxo-PGF1 alpha and the second for 6-oxo-PGF1 alpha, 13, 14-dihydro-6-oxo-PGF1 alpha, 13,14-dihydro-6, 15-dioxo-PGF1 alpha and 6-oxo-PGE1 are described. These radioimmunoassays were used to measure levels of immunoreactive 6-oxo-PGF1 alpha and alleged metabolites of prostacyclin in human venous plasma. Procedures for the direct measurement and extraction of plasma 6-oxo-PGF1 alpha are described and the limitations to which radioimmunoassay of plasma 6-oxo-PGF1 alpha is exposed are discussed. Direct measurement of plasma immunoreactive 6-oxo-PGF1 alpha gave maximal levels of 6.1 pg ml-1 and 4 pg ml-1 in the first and second radioimmunoassays respectively. The latter value reflected combined levels of less than 2 pg ml-1 of 6-oxo-PGF1 alpha and cross-reacting metabolites in venous blood. Extraction of human plasma gave 88.7 +/- 2.2% recovery (mean +/- S.E.M. n = 5 experiments) of [3H]-6-oxo-PGF1 alpha and 80 - 86% recovery of exogenous 6-oxo-PGF1 alpha as immunoreactive 6-oxo-PGF1 alpha. Basal plasma levels of extractable immunoreactive 6-oxo-PGF1 alpha were less than 2.5 pg ml-1. Prostacyclin incubated in vitro with blood was recovered in plasma as immunoreactive 6-oxo-PGF1 alpha and confirmed that conversion to metabolites that cross-reacted with the second antibody, in particular 6-oxo-PGE1, did not occur under experimental conditions. Extraction of [3H]-6-oxo-PGF1 alpha from acidified plasma with methanol resulted in formation of a prostanoid that had properties consistent with the methylated hemiketal isomer of 6-oxo-PGF1 alpha. Under radioimmunoassay conditions this prostanoid was less immunogenic than native [3H]-6-oxo-PGF1 alpha and [3H]-6-oxo-PGF1 alpha extracted from plasma using methyl formate. The low levels of plasma 6-oxo-PGF1 alpha which we report questions the validity of clinical studies that previously described altered levels of plasma 6-oxo-PGF1 alpha in pathophysiological conditions. These studies which were based upon measurement of plasma 6-oxo-PGF1 alpha by radioimmunoassay and GC/MS should now be re-evaluated. Preliminary results from this study have been reported elsewhere (1, 2).