Hofmann U, Seefried S, Meese C O, Mettang T, Hübel E, Kuhlmann U
Dr. Margarete Fischer-Bosch-Institut für Klinische Pharmakologie, Stuttgart, Federal Republic of Germany.
Anal Biochem. 1990 Sep;189(2):244-8. doi: 10.1016/0003-2697(90)90115-p.
Thromboxane A2, the predominant product of arachidonic acid metabolism in the blood platelet, is a potent vasoconstrictor and platelet agonist. During its biosynthesis from cyclic endoperoxide, 12(S)-hydroxy-5Z,8E,10E-heptadecatrienoic acid (HHT) is formed in equal amounts. The further metabolism of HHT, catalyzed by 15-hydroxyprostaglandin dehydrogenase, leads to 12-oxo-5Z,8E,10E-heptadecatrienoic acid (Oxo-HT). Sample workup procedures are described which allow for the sensitive and reproducible determination of these two arachidonic acid metabolites in human plasma by gas chromatography-mass spectrometry in the presence of deuterated analogues as internal standards. HHT is derivatized to the pentafluorobenzyl ester tert-butyldimethylsilyl ether. In order to enable quantification of low concentrations of about 10 pg/ml in nonstimulated human plasma, the samples have to be purified by HPLC. Oxo-HT is derivatized to the pentafluorobenzyl ester, which is purified by HPLC, and then derivatized to the trimethylsilyloxime. The method allows quantification of Oxo-HT in concentrations down to 10 pg/ml plasma. The reported methods have been used to measure HHT and Oxo-HT in stimulated platelet rich plasma and to quantify HHT in nonstimulated plasma. Determination of endogenous levels of these two arachidonic acid metabolites may give new insights into the overall biosynthesis of thromboxane A2 in man.
血栓素A2是血小板中花生四烯酸代谢的主要产物，是一种强效血管收缩剂和血小板激动剂。在其从环内过氧化物生物合成过程中，等量生成12(S)-羟基-5Z,8E,10E-十七碳三烯酸（HHT）。由15-羟基前列腺素脱氢酶催化的HHT进一步代谢导致生成12-氧代-5Z,8E,10E-十七碳三烯酸（氧代-HHT）。本文描述了样品处理程序，该程序可在氘代类似物作为内标的情况下，通过气相色谱-质谱法灵敏且可重复地测定人血浆中这两种花生四烯酸代谢物。HHT被衍生化为五氟苄基酯叔丁基二甲基甲硅烷基醚。为了能够定量非刺激人血浆中约10 pg/ml的低浓度，样品必须通过高效液相色谱法进行纯化。氧代-HHT被衍生化为五氟苄基酯，通过高效液相色谱法纯化，然后衍生化为三甲基硅烷肟。该方法可定量血浆中低至10 pg/ml的氧代-HHT。所报道的方法已用于测量富血小板血浆中刺激后的HHT和氧代-HHT，并定量非刺激血浆中的HHT。测定这两种花生四烯酸代谢物的内源性水平可能会为人类血栓素A2的整体生物合成提供新的见解。
