Sakai Kazuo, Kino Satoko, Masuda Aino, Takeuchi Masao, Ochi Tairin, Osredkar Josko, Rejc Barbara, Gersak Ksenija, Ramarathnam Narasimhan, Kato Yoji
Japan Institute for the Control of Aging (JaICA), Nikken SEIL Co., Ltd., 710-1 Haruoka, Fukuroi-shi, Shizuoka, 437-0122, Japan,
Subcell Biochem. 2014;77:61-72. doi: 10.1007/978-94-007-7920-4_5.
Published evidences indicate that reactive oxygen species (ROS) can induce lipid peroxidation, which plays important role in the pathophysiology of numerous diseases including atherosclerosis, diabetes, cancer and aging process. Monitoring of oxidative modification or oxidative damages of biomolecules may therefore be essential for the understanding of aging, and age-related diseases. N-epsilon-Hexanoyl-lysine (HEL) is a novel lipid peroxidation biomarker which is derived from the oxidation of omega-6 unsaturated fatty acid. In this chapter, development of HEL ELISA and its applications are reported. Assay range of HEL ELISA was 2-700 nmol/L, and showed good linearity and reproducibility. Accuracy of this assay was validated by recovery test and absorption test. HEL concentration in human urine was 22.9 ± 15.4 nmol/L and it was suggested that HEL exists as low molecular substances, in a free or in the peptide-attached form. In contrast with the urine sample, serum HEL was suggested to exist in the protein-attached form, and hydrolysis by protease might be essential for the accurate measurement of HEL in protein containing samples such as serum and cultured cells. By sample pretreatment with proteases, HEL was successfully detected in oxidized LDL, oxidized serum, and rat serum. In conclusion, HEL ELISA can be applied to measure urine, serum, and other biological samples independent of the animal species, and may be useful for the assessment of omega-6 PUFA oxidation in the living bodies.
已发表的证据表明，活性氧（ROS）可诱导脂质过氧化，这在包括动脉粥样硬化、糖尿病、癌症和衰老过程在内的多种疾病的病理生理学中发挥重要作用。因此，监测生物分子的氧化修饰或氧化损伤对于理解衰老及与年龄相关的疾病可能至关重要。N-ε-己酰赖氨酸（HEL）是一种新型脂质过氧化生物标志物，它源自ω-6不饱和脂肪酸的氧化。在本章中，报道了HEL酶联免疫吸附测定（ELISA）的开发及其应用。HEL ELISA的检测范围为2 - 700 nmol/L，具有良好的线性和重现性。该测定的准确性通过回收率测试和吸收测试进行了验证。人尿液中HEL的浓度为22.9±15.4 nmol/L，提示HEL以低分子物质形式存在，呈游离或与肽结合的形式。与尿液样本不同，血清中的HEL提示以与蛋白质结合的形式存在，对于准确测量血清和培养细胞等含蛋白质样本中的HEL，蛋白酶水解可能必不可少。通过用蛋白酶对样本进行预处理，在氧化型低密度脂蛋白（LDL）、氧化血清和大鼠血清中成功检测到了HEL。总之，HEL ELISA可用于测量尿液、血清及其他生物样本，与动物物种无关，可能有助于评估生物体内ω-6多不饱和脂肪酸（PUFA）的氧化情况。
