Tagari P, Callaghan D H, Black C, Yerge J A
Dept of Pharmacology, Merck Frosst Centre for Therapeutic Research, Pointe Claire, Quebec, Canada.
Prostaglandins. 1994 Apr;47(4):293-306. doi: 10.1016/0090-6980(94)90024-8.
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 B2, in the urine of dogs, a species commonly used for functional studies of thromboxane pharmacology. The beta-metabolite 2,3-dinor TXB2 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 TXB2/mg creatinine (range 624-4493 pg/mg). Thromboxane immunoreactivity co-eluting with synthetic 2,3-dinor TXB2 was also identified by HPLC-RIA and similarly determined (2585 +/- 276 pg/mg creatinine). Exogenous 2,3-dinor TXB2 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 micrograms/kg/min) dose-dependently inhibited 2,3-dinor TXB2 excretion measured by IA/GC-MS, with maximal inhibition (83.0 +/- 4.2%) being achieved after 6h (25 micrograms/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.
免疫亲和萃取/气相色谱-质谱联用（IA/GC-MS）法和高效液相色谱-放射免疫分析法（HPLC-RIA）被开发用于分析犬尿中一种主要的人类尿血栓素代谢物——2,3-二去甲血栓素B2，犬常用于血栓素药理学的功能研究。通过IA/GC-MS在混合的正常犬尿中明确鉴定出β-代谢物2,3-二去甲TXB2，并在6只麻醉犬身上于7小时内测定其排泄量为2001±132 pg 2,3-二去甲TXB2/毫克肌酐（范围为624 - 4493 pg/毫克）。通过HPLC-RIA也鉴定出与合成的2,3-二去甲TXB2共洗脱的血栓素免疫反应性，并进行了类似测定（2585±276 pg/毫克肌酐）。两种方法在很宽的浓度范围（50 - 5000 pg/mL）内都能定量回收外源性2,3-二去甲TXB2，不过与HPLC-RIA（添加量与回收量；m = 0.89，r = 0.89）相比，IA/GC-MS的精度更高（添加量与回收量；m = 1.05，r = 0.99）。在麻醉犬中通过输注给予环氧化酶抑制剂吲哚美辛（2.5、8和25微克/千克/分钟），剂量依赖性地抑制了通过IA/GC-MS测定的2,3-二去甲TXB2排泄，在6小时后（25微克/千克/分钟）达到最大抑制（83.0±4.2%）。通过HPLC-RIA也得到了类似结果，药物处理后样品中两种方法之间的相关性为0.88（斜率 = 0.9）。这些数据表明犬体内血栓素的代谢和排泄情况与人相似，并为血栓素生物合成抑制剂的非侵入性体内评估提供了一个有用的动物模型。
