Measurement of canine urinary thromboxanes by GC-MC and HPLC-RIA

Abstract

Immunoaffinity extraction/gas chromatography-mass spectrometry (IA/GC-MS) and high-performance liquid chromatography-radioimmunoassay (HPLC-RIA) methods were developed to analyse a major human urinary thromboxane metabolite 2,3-dinor thromboxane B 2, in the urine of dogs, a species commonly used for functional studies of thromboxane pharmacology. The β-metabolite 2,3-dinor TXB 2 was unequivocally identified in pooled normal canine urine by IA/GC-MS, and its excretion measured in 6 anesthetized dogs over a 7h period as 2001 ± 132 pg 2,3-dinor TXB 2/mg creatinine (range 624–4493 pg/mg). Thromboxane immunoreactivity co-eluting with synthetic 2,3-dinor TXB 2 was also identified by HPLC-RIA and similarly determined (2585 ± 276 pg/mg creatinine). Exogenous 2,3-dinor TXB 2 could be quantitatively recovered by both methodologies over a wide range of concentrations (50–5000 pg/mL), although with better precision by IA/GC-MS (added vs recovered; m = 1.05, r = 0.99) compared with HPLC-RIA (added vs recovered; m = 0.89, r = 0.89). The cyclooxygenase inhibitor indomethacin given by infusion in anaesthetized dogs (2.5, 8 and 25 μg/kg/min) dose-dependently inhibited 2,3-dinor TXB 2 excretion measured by IA/GC-MS, with maximal inhibition (83.0 ± 4.2%) being achieved after 6 h (25 μg/kg/min). Similar results were obtained by HPLC-RIA, with a correlation of 0.88 (slope = 0.9) between the methodologies in samples after drug treatment. These data suggest that the profile of metabolism and excretion of thromboxanes in dogs resembles that of man, and provide a useful animal model for the non-invasive in vivo assessment of inhibitors of thromboxane biosynthesis.