Kato Y, Makino Y, Osawa T
Department of Applied Biological Sciences, Nagoya University, Japan.
J Lipid Res. 1997 Jul;38(7):1334-46.
Lipid hydroperoxide may react with protein or amino phospholipid without secondary decomposition. We prepared a polyclonal antibody to lipid hydroperoxide-modified proteins using 13S-hydroperoxy-9Z, 11E-octadecadienoic acid-modified keyhole limpet hemocyanin (13-HPODE-KLH) as immunogen. The antibody recognized 13-HPODE-modified bovine serum albumin (BSA), but not aldehyde-modified proteins, such as malondialdehyde-modified BSA. The antibody also recognized adducts derived from 13-HPODE and 13S-hydroperoxy-9Z, 11E, 15Z-octadecatrienoic acid (13-HPOTRE(alpha)). The oxidized alpha-linolenic acid- and linoleate-protein adducts were recognized by the antibody. Oxidized phospholipid-protein adducts were scarcely recognized by the antibody. However, when ester bonds of phospholipids containing linoleic acid were hydrolyzed by alkaline treatment, the cross-reactivities appeared. The result suggests that a phospholipid hydroperoxide can react with a protein directly or indirectly, and a carboxyl terminal (COOH) of the lipid in an adduct was needed as an epitope. Oxidized LDL (ox-LDL) was prepared by the incubation of LDL with copper ion or 2,2'-azobis(2-amidinopropane)dihydrochloride (AAPH), and the formation of lipid hydroperoxide-modified apolipoprotein was confirmed using the antibody. A slight immunoreactivity was observed in ox-LDL without alkaline treatment. When the ox-LDL was treated with alkali to hydrolyze the ester bonds of the lipid, enhanced antigenicity appeared with time-dependency. The results suggest that lipid hydroperoxide-modified apolipoprotein was formed during the oxidation of LDL.
脂质氢过氧化物可能与蛋白质或氨基磷脂发生反应而不发生二次分解。我们以13S-氢过氧基-9Z,11E-十八碳二烯酸修饰的钥孔血蓝蛋白（13-HPODE-KLH）作为免疫原，制备了针对脂质氢过氧化物修饰蛋白的多克隆抗体。该抗体识别13-HPODE修饰的牛血清白蛋白（BSA），但不识别醛修饰的蛋白，如丙二醛修饰的BSA。该抗体还识别由13-HPODE和13S-氢过氧基-9Z,11E,15Z-十八碳三烯酸（13-HPOTRE(α)）衍生的加合物。氧化的α-亚麻酸和亚油酸-蛋白质加合物可被该抗体识别。氧化的磷脂-蛋白质加合物几乎不被该抗体识别。然而，当含亚油酸的磷脂的酯键经碱性处理水解后，交叉反应性出现。结果表明，磷脂氢过氧化物可直接或间接与蛋白质反应，加合物中脂质的羧基末端（COOH）作为表位是必需的。通过将低密度脂蛋白（LDL）与铜离子或2,2'-偶氮二（2-脒基丙烷）二盐酸盐（AAPH）孵育制备氧化型LDL（ox-LDL），并使用该抗体确认脂质氢过氧化物修饰的载脂蛋白的形成。未经碱性处理的ox-LDL中观察到轻微的免疫反应性。当ox-LDL用碱处理以水解脂质的酯键时，抗原性随时间依赖性增强。结果表明，在LDL氧化过程中形成了脂质氢过氧化物修饰的载脂蛋白。
