Enzyme immunoassay measurement of the urinary metabolites of thromboxane A2 and prostacyclin.
We have used a recently developed enzyme immunoassay (EIA) method for measuring urinary concentrations of TXB2, 6-keto PGF1 alpha, 2,3-dinor-TXB2, 2,3-dinor-6-keto PGF1 alpha and 11-dehydro-TXB2 using acetylcholinesterase from Electrophorus Electricus coupled to TXB2, 6-keto PGF1 alpha and 11-dehydro-TXB2. Urinary PGI2 and TXA2 breakdown products and their metabolites were extracted from 3-40 ml of urine corresponding to 100 mumoles 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-TXB2 serum with 2,3-dinor TXB2 and the anti-6-keto PGF1 alpha serum with 2,3-dinor-6-keto PGF1 alpha, measurements were done using 3 antisera (anti-TXB2 and anti-6-keto PGF1 alpha diluted 1/50,000, anti 11-dehydro-TXB2 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 results although the migration of the compounds was modified significantly. Determination of the urinary excretion of TXB2 and prostacyclin metabolites in 17 healthy individuals by this method provided results in agreement with those obtained by 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).
Lellouche
F
F
U 150 INSERM, Hôpital Lariboisière, Paris, France.
Fradin
A
A
Fitzgerald
G
G
Maclouf
J
J
D000328:Adult; D002855:Chromatography, Thin Layer; D011464:Epoprostenol / Q000652:urine*; D005260:Female; D008401:Gas Chromatography-Mass Spectrometry / Q000379:methods; D006801:Humans; D007124:Immunoenzyme Techniques; D008297:Male; D012016:Reference Values; D015203:Reproducibility of Results; D013928:Thromboxane A2 / Q000652:urine*
D013928:Thromboxane A2; D011464:Epoprostenol