Radioimmunoassay of 15-keto-13,14-dihydro-prostaglandin F2alpha: an index for inflammation via cyclooxygenase catalysed lipid peroxidation

Abstract

The metabolism of PGF2alpha in human and other species results initially in the formation of 15-keto-dihydro-PGF2alpha and later to several beta-oxidized metabolites, which are species-specific. Since the discovery of cyclooxygenase-2 (COX-2), the importance of measuring various arachidonic acid metabolites during inflammatory conditions is on focus. This study presents the development and validation of a new radioimmunoassay of 15-keto-dihydro-PGF2alpha as an index of lipid peroxidation via cyclooxygenase (COX-1 and COX-2) pathway. Furthermore, its application in endotoxin-induced acute inflammation in pigs is presented. An antibody was raised in rabbits by immunization with 15-keto-dihydro-PGF2alpha coupled to BSA at the carboxylic acid by 1,1'-carbonyldiimidazole method. The cross-reactivity of the antibody with PGF2alpha, 15-keto-PGF2alpha, PGE2, 15-keto-13,14-dihydro-PGE2, 8-iso-15-keto-13,14-dihydro-PGF2alpha, 11beta-PGF2alpha, 9beta-PGF2alpha, TXB2 and 8-iso-PGF3alpha was 0.02, 0.43, < 0.001, 0.5, 1.7, < 0.001, < 0.001, < 0.001, 0.01%, respectively. The intra-assay precision was 12.2% (CV) at the level of 64 pg/0.1 ml and 14.0% with 512 pg/0.1 ml in the human plasma. Similarly, intra-assay accuracy was 108.6% and 103.3% for the low and the high standards, respectively. The detection limit was about 45 pmol/L. 15-keto-dihydro-PGF2alpha levels in plasma from normal human volunteers were evaluated and found to correlate with the obtained values by GC-MS methods from other studies. The levels of 15-keto-dihydro-PGF2alpha in the plasma increased several-fold after endotoxin infusion (10 microg/kg/h over 6 h) to the pigs. Thus, this 1 5-keto-dihydro-PGF2alpha radioimmunoassay method is relevant to apply in inflammatory injury, and other physiological and pathophysiological studies, as an index of in vivo enzymatic lipid peroxidation.