Stability of urinary thromboxane A2 metabolites and adaptation of the extraction method to small urine volume

Background: Thromboxane (TX) A2 is a pro-thrombotic prostanoid synthesized by activated platelets, biotransformed into 11-dehydro-TXB 2, measurable in urines. Eleven-dehydro-TXB2 excretion is increased in high risk cardiovascular diseases; however, this cardiovascular biomarker awaits validation in large trials. The need of large urine volume (8-10 mL) and the unknown stability of 11-dehydro-TXB2 in urine after collection might limit its implementation. Methods: We scaled the original method for urine extraction and 11-dehydro-TXB2 measurement down to 1 mL, and assessed its stability at 4°C or 25°C up to 6 days after collection. The sensitivity of the 1 mL procedure was also tested in aspirin-treated patients with low 11-dehydro-TXB2 excretion Results: The 1 mL adapted method was highly correlated with the original assay (rho = 0.98, p < 0.001, n = 33). Both methods showed similar recoveries in samples spiked with exogenous 11-dehydro-TXB2. Urinary 11-dehydro-TXB 2 values in samples immediately frozen were comparable and highly correlated to values in samples at 4°C (day 6: rho = 0.99, p > 0.001, n = 8) or 25°C (day 6: rho = 0.98, p < 0.001, n = 23) up to 6 days in controls and patients. Conclusions: Eleven-dehydro-TXB2 can be measured in small urine volumes and is relatively stable for a few days after collection, even at 25°C. These data allow the validation of this non-invasive cardiovascular biomarker in large studies.