Kumlin M, Granström E
Prostaglandins. 1986 Nov;32(5):741-67. doi: 10.1016/0090-6980(86)90196-6.
A radioimmunoassay was developed for 11-dehydro-TXB2, a prominent metabolite of TXB2 in blood and urine of several species. In order to reliably assay 11-dehydro-TXB2, its chemical stability as well as its chromatographic properties were first examined. Since dehydrogenation at C-11 converts the thromboxane ring into the delta-lactone form of a dicarboxylic acid, which can also occur in an open form, the analysis of 11-dehydro-TXB2 may be somewhat complicated. In some chromatographic systems, the compound thus migrated with pronounced tailing, and during extraction using the common Sep-Pak procedure the two forms were partially separated. The lactone as well as the open form could be conclusively identified using mass spectrometry. The equilibrium between the two forms of 11-dehydro-TXB2 was studied in buffers of different pH and in plasma. Higher pH favoured hydrolysis into the acyclic structure. The lactonization and hydrolysis processes were also shown to be time and temperature dependent. Two different antiplasms, raised in rabbits against conjugates of 11-dehydro-TXB2 with bovine serum albumin, displayed somewhat different properties in their recognition of the two forms of 11-dehydro-TXB2. A radioimmunoassay employing these antibodies was developed. The labeled antigen was prepared by incubation of 3H-TXB2 with rabbit lung supernatant. The limit of detection was 1.5 pg. For validation of the assay, analysis of blood and urinary samples, obtained after injection of TXB2 to a human volunteer, was done. The values obtained were compatible with previous isotope studies. Results from an inhibition experiment with rabbit lung incubated in the presence or absence of indomethacin further supported the identity of the assayed substance.
已开发出一种针对11 - 脱氢 - TXB2的放射免疫分析法，11 - 脱氢 - TXB2是几种物种血液和尿液中TXB2的一种主要代谢产物。为了可靠地检测11 - 脱氢 - TXB2，首先检测了其化学稳定性及其色谱性质。由于C - 11位的脱氢作用将血栓烷环转化为二羧酸的δ - 内酯形式，该形式也可以以开放形式存在，因此11 - 脱氢 - TXB2的分析可能会有些复杂。在一些色谱系统中，该化合物迁移时拖尾明显，并且在使用常规Sep - Pak方法提取过程中，两种形式会部分分离。使用质谱法可以明确鉴定内酯形式以及开放形式。在不同pH的缓冲液和血浆中研究了11 - 脱氢 - TXB2两种形式之间的平衡。较高的pH有利于水解为无环结构。内酯化和水解过程也显示出与时间和温度有关。用兔制备的两种不同的抗血清，针对11 - 脱氢 - TXB2与牛血清白蛋白的偶联物，在识别11 - 脱氢 - TXB2的两种形式时表现出略有不同的性质。开发了一种使用这些抗体的放射免疫分析法。标记抗原通过将3H - TXB2与兔肺上清液孵育制备。检测限为1.5 pg。为了验证该分析方法，对向一名人类志愿者注射TXB2后获得的血液和尿液样本进行了分析。得到的值与先前的同位素研究结果相符。在有或没有吲哚美辛存在的情况下孵育兔肺的抑制实验结果进一步支持了所检测物质的同一性。
