Valkonen M, Kuusi T
Department of Medicine, University of Helsinki, Finland.
J Lipid Res. 1997 Apr;38(4):823-33.
Antioxidants prevent modification of low density lipoprotein (LDL) by free radicals and possibly also atheroma formation. The capacity of human serum to resist attacks by free radicals is measured by the total peroxyl radical-trapping potential (TRAP). Its measurement has thus far required equipment not available in many clinical laboratories such as a thermostated oxygen electrode cell or a luminometer. To develop a simpler method we used a free radical probe, dichlorofluorescin-diacetate (DCFH-DA), described before in studies of respiratory burst in inflammatory cells. Its oxidation by radicals from thermal decomposition of 2,2'-diazobis(2-amidinopropane)dihydrochloride (AAPH) converts this compound to highly fluorescent dichlorofluorescein (DCF). The DCF also has absorbance at 504 nm thus enabling the determination of TRAP either fluorometrically or spectrophotometrically. Increasing the concentration of AAPH enables the measurement of DCF formation and its inhibition by serum samples at room temperature. The intra- and interassay coefficients of variation of this assay are 3.4% and 4.6%, respectively. The mean value for serum TRAP of healthy subjects is 1155 mumol/l (n = 38). The TRAP in human serum can be increased by adding various antioxidant substances to the assay in vitro or by dietary supplementation of healthy subjects with vitamin E in vivo (P < 0.025). An increase was also found in serum vitamin E levels (P < 0.0001) and in the length of the time human LDL is able to resist oxidation (P < 0.05). Thus the determination of TRAP by this method, which requires only commercially available chemicals, can be used for the evaluation of phenomena associated with lipid accumulation in human artery wall.
抗氧化剂可防止自由基对低密度脂蛋白（LDL）的修饰，也可能预防动脉粥样硬化的形成。人血清抵抗自由基攻击的能力通过总过氧自由基捕获能力（TRAP）来衡量。迄今为止，其测量需要许多临床实验室没有的设备，如恒温氧电极池或发光计。为开发一种更简单的方法，我们使用了一种自由基探针，二氯荧光素二乙酸酯（DCFH-DA），之前在炎症细胞呼吸爆发研究中已有描述。它被2,2'-偶氮二（2-脒基丙烷）二盐酸盐（AAPH）热分解产生的自由基氧化，将该化合物转化为高荧光的二氯荧光素（DCF）。DCF在504nm处也有吸光度，因此能够通过荧光法或分光光度法测定TRAP。增加AAPH的浓度可以在室温下测量DCF的形成及其被血清样品的抑制情况。该检测方法的批内和批间变异系数分别为3.4%和4.6%。健康受试者血清TRAP的平均值为1155μmol/l（n = 38）。通过在体外检测中添加各种抗氧化物质或在体内对健康受试者进行维生素E膳食补充，可以提高人血清中的TRAP（P < 0.025）。还发现血清维生素E水平有所升高（P < 0.0001），人LDL抵抗氧化的时间长度也有所增加（P < 0.05）。因此，通过这种仅需市售化学试剂的方法测定TRAP，可用于评估与人类动脉壁脂质积累相关的现象。
