Radioimmunoassay of 8-iso-prostaglandin F2alpha: an index for oxidative injury via free radical catalysed lipid peroxidation

Abstract

8-iso-prostaglandin F2alpha (8-iso-PGF2alpha), a major F2-isoprostane, is biosynthesized in vivo through nonenzymatic free radical-catalysed peroxidation of arachidonic acid. The levels of F2-isoprostanes both free in the circulation and esterified to the tissue phospholipids increase intensely in animal models of oxidant injury. This study presents the development and validation of a radioimmunoassay of 8-iso-PGF2alpha for the measurement of this substance in the body fluids. Furthermore, its application in normal human volunteers, a pharmacokinetic study performed in rabbits with 8-iso-PGF2alpha, and hepatic lipid peroxidation in rats is reported. An antibody was raised in rabbits by immunization with 8-iso-PGF2alpha coupled to BSA at the carboxylic acid by 1,1'-Carbonyldiimmidazole method. The cross-reactivity of the antibody with 8-iso-15-keto-13,14-dihydro-PGF2alpha, 8-iso-PGF2beta, PGF2alpha, 15-keto-PGF2alpha, 15-keto-13,14-dihydro-PGF2alpha,TXB2, 11beta-PGF2alpha, 9beta-PGF2alpha and 8-iso-PGF3alpha was 1.7, 9.8, 1.1, 0.01, 0.01, 0.1, 0.03, 1.8 and 0.6%, respectively. The intraassay precision was 14.5% (CV) at the level of 64 pg/0.1 ml and 12.2% with 512 pg/0.1 ml in the human plasma. Similarly, intra-assay accuracy was 95.6% and 101% for the low and the high standard, respectively. The detection limit was about 23 pmol/l. The appearance and disappearance of 8-iso-PGF2alpha in the blood and urine following intravenous administration of 8-iso-PGF2alpha in the rabbit was rapid. Free 8-iso-PGF2alpha levels in plasma and urine from normal human volunteers are evaluated and found to correlate with the obtained values by gas chromatography-mass spectrometry methods from other studies. The levels of free 8-iso-PGF2alpha in the plasma and urine increased 7- and 102-fold, respectively, after CCl4 administration to rats. Thus, this 8-iso PGF2alpha radioimmunoassay method is relevant to apply in the oxidative injury studies as an index of in vivo lipid peroxidation through free radical catalysis mechanism.