DEVELOPMENT OF HIGHLY SELECTIVE AND SENSITIVE DETERMINATION METHOD FOR LIPID PEROXIDES AND ITS APPLICATION TO BIOLOGICAL SAMPLES

采用化学发光（CL）法对脂质过氧化物进行了高选择性、高灵敏度的测定。为此，建立了一种具有在线光反应器和过氧草酸盐化学发光（PO-CL）检测的高效液相色谱（HPLC）系统。PO-CL反应需要过氧化氢、荧光团和二乙基草酸盐才能显示CL。在所提出的体系中，过氧化氢是由过氧化脂在紫外线照射下生成的，因此只有用荧光团标记的过氧化脂才会显示CL，这使得该方法具有很强的选择性。用油酸在亚甲基蓝存在下光敏氧化制得标准脂质过氧化物，然后用4,5-二苯基-2-（4-肼羰基）苯基咪唑（HCPI）或2-（2,3-萘酰亚胺）乙基三氟甲烷磺酸盐（NE-OTf）荧光标记（FL）对体系进行评价。结果表明，该方法对脂质过氧化物具有选择性，但灵敏度不如预期。这是由于紫外线照射过氧化氢生成过氧化氢的产率很低。因此，我们试图用芬顿反应代替紫外线照射来提高灵敏度。用芬顿反应观察到CL强度增加了15倍。通过优化芬顿反应，将进一步提高该灵敏度。在本研究中，我们选择具有过氧化氢和荧光团的分析物分子进行PO-CL检测。但是，荧光团可以用化学发光团代替。通过检查脂质过氧化物产生活性氧的反应，以进行有效的CL反应，将提高该方法的灵敏度。

Highly selective and sensitive determination of lipid peroxides was examined by the use of chemiluminescence (CL) detection. For this purpose, a high-performance liquid chromatographic (HPLC) system with on-line photoreactor and peroxyoxalate chemiluminescence (PO-CL) detection was developed. The PO-CL reaction requires hydrogen peroxide, fluorophore and diaryloxalate in order to show CL. In the proposed system, hydrogen peroxide is generated from lipid peroxide under UV radiation, and thus only the lipid peroxide labeled with fluorophore is expected to show CL, which makes the method very selective.The system was evaluated by using standard lipid peroxides, which were prepared by photosensitized oxidation of oleic acid in the presence of methylene blue and followed by fluorescence (FL) labeling with 4,5-diphenyl-2-(4-hydrazinocarbonyl)phenyl imidazole (HCPI) or 2-(2,3-naphthalimino)ethyltrifluoromethane sulfonate (NE-OTf). As a result, the method was found to be selective for lipid peroxides but was not as sensitive as expected. This was due to the low generation yield of hydrogen peroxide from hydroperoxide by UV irradiation. We therefore attempted to increase the sensitivity by using Fenton reaction instead of UV irradiation. Fifteen-fold increase in CL intensity was observed by using the Fenton reaction. This sensitivity will further be improved by optimizing the Fenton reaction.In this research, we selected the analyte molecule having hydroperoxide and fluorophore for PO-CL detection. However, the fluorophore will be able to replace with chemiluminophore. The sensitivity of the method will be improved by examining the reaction that generates reactive oxygen species from lipid peroxides for the efficient CL reaction.