Tsao Francis H C, Shanmuganayagam Dhanansayan, Zachman Derek K, Khosravi Mehdi, Folts John D, Meyer Keith C
Department of Medicine, Section of Pulmonary and Critical Care Medicine, University of Wisconsin Medical School, Madison, WI 53792, USA.
Clin Chim Acta. 2007 Apr;379(1-2):119-26. doi: 10.1016/j.cca.2006.12.023. Epub 2007 Jan 12.
Calcium-dependent secretory phospholipase A(2)-IIA (sPLA(2)-IIA) in the circulation is a marker of inflammation, associated with acute and chronic disease processes. We describe a quick, sensitive and reliable microplate continuous fluorescence assay for determining sPLA(2) activity in serum.
Liposomes composed of a fluorescent probe and varying amounts of L-alpha-phosphatidylglycerol (PG) and 1,2-dioleoyl-L-alpha-phosphatidylcholine (DOPC) were used as substrates to determine the optimal protocol for sPLA(2) activity determination without interference from serum albumin and lipoproteins.
Hydrolysis of the labeled substrate by sPLA(2)-IIA, characterized by increase in fluorescence intensity (FI) and confirmed by end-product analysis, occurred in a time-, calcium-, and protein-dependent manner. Liposomes containing 100% PG were most suitable for measurement of sPLA(2) activity without interference from serum components; LDL produced a Ca(2+)-independent increase in FI when liposomes containing DOPC were used. The assay determined that sPLA(2) activity in serum spiked with sPLA(2)-IIA and illustrated that endogenous sPLA(2) activity was markedly higher in sera from patients with sepsis than in healthy subjects. Intra-assay and inter-assay CVs were in the ranges of 1.6-8.8% and 3.0-11.5%, respectively.
The described method has potential for rapid and sensitive screening of sPLA(2) activity in both clinical and research settings.
循环中的钙依赖性分泌型磷脂酶A2-IIA（sPLA2-IIA）是一种炎症标志物，与急性和慢性疾病过程相关。我们描述了一种用于测定血清中sPLA2活性的快速、灵敏且可靠的微孔板连续荧光测定法。
由荧光探针以及不同量的L-α-磷脂酰甘油（PG）和1,2-二油酰基-L-α-磷脂酰胆碱（DOPC）组成的脂质体用作底物，以确定在不受血清白蛋白和脂蛋白干扰的情况下测定sPLA2活性的最佳方案。
sPLA2-IIA对标记底物的水解以荧光强度（FI）增加为特征，并通过终产物分析得到证实，其发生具有时间、钙和蛋白质依赖性。含有100%PG的脂质体最适合用于测量sPLA2活性，且不受血清成分干扰；当使用含有DOPC的脂质体时，低密度脂蛋白（LDL）使FI出现不依赖于Ca2+的增加。该测定法确定了添加sPLA2-IIA的血清中的sPLA2活性，并表明脓毒症患者血清中的内源性sPLA2活性明显高于健康受试者。批内和批间变异系数（CV）分别在1.6 - 8.8%和3.0 - 11.5%范围内。
所描述的方法在临床和研究环境中都具有快速、灵敏地筛选sPLA2活性的潜力。
