Ramesha C S, Taylor L A
Department of Inflammation Biology, Syntex Research, Palo Alto, California 94303.
Anal Biochem. 1991 Jan;192(1):173-80. doi: 10.1016/0003-2697(91)90203-6.
a simple gas chromatographic method for the assay of phospholipase A2 (PLA2) has been described in which arachidonic acid released from endogenous phospholipid pools is measured following its extraction and derivatization to pentafluorobenzyl esters. Using this assay, PLA2 activities in control and calcium ionophore-stimulated human neutrophils, as well as in control, thrombin, and calcium ionophore stimulated human platelets, have been measured. These values are compared with those obtained by monitoring the release of radioactivity from [3H]- or [14C]arachidonic acid prelabeled cells. While the radiometric assay measures only the release of exogenously incorporated radioactive arachidonic acid, the gas chromatographic assay measures arachidonic acid released from all the endogenous pools. Thus, the apparent increase in PLA2 activity in stimulated cells measured by the gas chromatographic assay is four- to fivefold higher than that by the radiometric assay. Inclusion of fatty acid free bovine serum albumin in the reaction buffer significantly increases the amount of arachidonic acid that is measured by gas chromatography. The gas chromatographic method has also been successfully utilized for measuring PLA2 activity in cell-free preparations derived from physically disrupted human neutrophils.
已描述了一种用于测定磷脂酶A2（PLA2）的简单气相色谱方法，其中，在内源性磷脂池中释放的花生四烯酸经提取并衍生化为五氟苄酯后进行测量。使用该测定法，已测量了对照和钙离子载体刺激的人中性粒细胞以及对照、凝血酶和钙离子载体刺激的人血小板中的PLA2活性。将这些值与通过监测来自[3H]-或[14C]花生四烯酸预标记细胞的放射性释放所获得的值进行比较。虽然放射性测定法仅测量外源掺入的放射性花生四烯酸的释放，但气相色谱测定法测量从所有内源性池中释放的花生四烯酸。因此，通过气相色谱测定法测量的刺激细胞中PLA2活性的表观增加比放射性测定法高四至五倍。在反应缓冲液中加入无脂肪酸的牛血清白蛋白可显著增加气相色谱法测量的花生四烯酸量。气相色谱法也已成功用于测量源自物理破坏的人中性粒细胞的无细胞制剂中的PLA2活性。
