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.
Accumulated evidences indicate that reactive oxygen species (ROS) are involved in the pathophysiology of aging process. Antioxidants are believed to play an important role in the defense system to counteract ROS in the body. While excess hydrophilic antioxidants can be excreted easily in urine, lipophilic antioxidants can penetrate into blood lipoproteins and cell membranes, and may maintain long and high bioavailability. These lipophilic antioxidants are thus expected to contribute greatly to the prevention of age-related diseases. Oils extracted from plant seeds are known to contain various lipophilic antioxidants such as vitamin E (alpha-tocopherol) and carotenoids. They are known to not only decrease serum low-density-lipoprotein (LDL) level, but also prevent oxidation of LDL. In addition to vitamin E (alpha-tocopherol) and carotenoids, other lipophilic antioxidants such as gamma-oryzanol and sesaminol (from sesamolin) are in rice bran and sesame, respectively. They are sometimes called "vitamin-like food factors" or "biofactors." Although there are several methods for measuring the total antioxidant activities for various plant extracts, most of these methods are designed for hydrophilic antioxidants, and not for lipophilic antioxidants present in various plant seed oils. In this report, we present an assay method for the total potency of antioxidants that are soluble in oil (PAO-SO) utilizing bathocuproine (BC) as a chromogen. BC-based antioxidant activity assay shows good linearity (r(2) = 0.9986), good reproducibility (CV < 10%), and good recovery (86-91%) when dl-alpha-tocopherol, for example, is added to sesame oil. Total antioxidant activity of rape-seed oil, olive oil, and sesame oil could also be successfully measured.
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