Enzyme immunoassay measurement of the urinary metabolites of thromboxane A 2 and prostacyclin

Abstract

We have used a recently developed enzyme immunoassay (EIA) method for measuring urinary concentrations of TXB 2, 6-keto PGF 1α, 2,3-dinor-TXB 2, 2,3-dinor-6-keto PGF 1α and 11-dehydro-TXB 2 using acetycholinesterase from Electrophorus Electricus coupled to TXB 2, 6-keto PGF 1α and 11-dehydro-TXB 2. Urinary PGl2 and TXA@ breakdown products and their metabolites were extracted from 3–40 ml of urine corresponding to 100 μmoles creatinine. Measurements were performed after Sep-Pak extraction and thin layer chromatography separation in a system that allows separation between dinor- and parent derivatives. Because of the relatively high cross reactivity (10–15%) of the anti-TXB 2 serum with 2,3-dinor TXB 2 and the anti-6-keto PGF 1α serum with 2,3-dinor-6-keto PGF 1α, measurements were done using 3 antisera (anti-TXB 2) and anti-6-keto PGF 1α diluted 1/50,000, anti 11-dehydro-TXB 2 diluted 1/200, 000). The reproducibility of the technique was assessed by measuring the same urine stored frozen in aliquots together with each series of samples (Coefficient of variation 6–12% (n = 20), depending on the compound). In addition, the use of a different solvent system for the thin layer chromatography did not affect the result although the migration of the compounds was modified significantly. Determination of the urinary excretion of TXB 2 and prostacyclin metabolites in 17 healthy individuals by this method provided results in agreement with those obtainedby other methodologies. In addition, comparisons made between EIA and gas chromatography/mass spectrometry analysis showed good correlation between the urinary metabolites as determined by each technique (r = 0.98).