Kayganich-Harrison K A, Murphy R C
National Jewish Center for Immunology and Respiratory Medicine, Denver, CO 80206.
Biol Mass Spectrom. 1994 Sep;23(9):562-71. doi: 10.1002/bms.1200230906.
The source of arachidonic acid metabolized to eicosanoids by 5-lipoxygenase was studied in a cultured neoplastic mast cell using a stable isotope tracer and tandem mass spectrometry strategy. Selected reaction monitoring and fast atom bombardment were used to analyze eight major arachidonate molecular species of glycerophosphocholine, nine major molecular species of glycerophosphoethanolamine, three major species of glycerophosphocholine, nine major molecular species of glycerophosphoethanolamine, three major species of glycerophosphoinositol, and three major glycerophosphoserine molecular species. Incubation of the mast cells with (2H8)arachidonic acid led to a time-dependent isotopic incorporation in each of these molecular species. Following stimulation with calcium ionophore A23187, the isotope incorporation of leukotriene B4 (LTB4) was found to be higher than that of the major arachidonate-containing glycerophospholipid molecular species. The isotope incorporation of LTB4 was similar to that found for free arachidonic acid present in the unstimulated cell. In order to prevent direct labeling of the intracellular, free arachidonic acid pool, (2H4)linoleic acid was added to the culture medium as a biochemical precursor of labeled arachidonic acid. There was a time-dependent increase of the specific incorporation of labeled arachidonic acid into each of the phospholipid molecular species of each lipid class after incubation with (2H4)linoleic acid. Importantly, (2H4)linoleic acid incubation also resulted in deuterium-labeled arachidonic acid in the free arachidonic acid, intracellular pool. The arachidonic acid isotopic incorporation in this pool very closely correlated with the isotopic incorporation of LTB4 (correlation coefficient 0.97) synthesized after A23187 stimulation, while the isotopic incorporation of the extracellularly released, not esterified arachidonic acid, after stimulation, did not.(ABSTRACT TRUNCATED AT 400 WORDS)
利用稳定同位素示踪剂和串联质谱分析法，在培养的肿瘤性肥大细胞中研究了经5-脂氧合酶代谢生成类二十烷酸的花生四烯酸来源。采用选择反应监测和快原子轰击法分析甘油磷酸胆碱的8种主要花生四烯酸分子种类、甘油磷酸乙醇胺的9种主要分子种类、甘油磷酸胆碱的3种主要种类、甘油磷酸乙醇胺的9种主要分子种类、甘油磷酸肌醇的3种主要种类以及甘油磷酸丝氨酸的3种主要分子种类。用(2H8)花生四烯酸孵育肥大细胞导致这些分子种类中的每一种都出现时间依赖性同位素掺入。在用钙离子载体A23187刺激后，发现白三烯B4（LTB4）的同位素掺入高于含花生四烯酸的主要甘油磷脂分子种类。LTB4的同位素掺入与未刺激细胞中存在的游离花生四烯酸相似。为了防止细胞内游离花生四烯酸池的直接标记，将(2H4)亚油酸作为标记花生四烯酸的生化前体添加到培养基中。用(2H4)亚油酸孵育后，标记花生四烯酸在各脂质类别的每种磷脂分子种类中的特异性掺入呈时间依赖性增加。重要的是，用(2H4)亚油酸孵育还导致游离花生四烯酸细胞内池中出现氘标记的花生四烯酸。该池中花生四烯酸的同位素掺入与A23187刺激后合成的LTB4的同位素掺入密切相关（相关系数0.97），而刺激后细胞外释放的未酯化花生四烯酸的同位素掺入则不相关。（摘要截短于400字）
