Determination of HEL (Hexanoyl-lysine adduct): a novel biomarker for omega-6 PUFA oxidation.

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.

Expressions of protein oxidation markers, dityrosine and advanced oxidation protein products in Cisplatin-induced nephrotoxicity in rats.

The purpose of this study was to evaluate whether dityrosine and advanced oxidation protein products (AOPP) reflect the severity of cisplatin-induced nephrotoxicity. Immunoexpression of dityrosine in kidneys and plasma AOPP concentration were examined up to day 4 post-cisplatin injection in rats. Cisplatin injection induced tubular injury on days 2-4 after injection and increased serum creatinine and BUN on days 3 and 4. On days 2-4, dityrosine was immunostained in the cytoplasm of damaged tubular cells, and their immunostaining intensity increased time-dependently. Plasma AOPP levels were significantly increased on days 3 and 4. These results suggest that expressions of dityrosine and AOPP were associated with the severity of renal injury and may be useful markers for the development of cisplatin-induced nephrotoxicity.

Expressions of protein oxidation markers, dityrosine and advanced oxidation protein products in acetaminophen-induced liver injury in rats.

The purpose of this study was to evaluate whether dityrosine and advanced oxidation protein products (AOPP) were useful as biomarkers for monitoring the development of acetaminophen-induced liver injury. Dityrosine immunoexpression in the liver along with plasma AOPP concentration was examined up to 24 hr post-acetaminophen injection in rats. The histopathological changes in the liver appeared 3 hr after acetaminophen injection and became exacerbated with time. The immunohistological expression of dityrosine was also first detected in the damaged hepatocytes 3 hr after the injection and became more accentuated at 6, 12 and 24 hr in accompanying with the elevation of plasma AOPP concentration. These results suggested that dityrosine and AOPP expressions might be useful biomarkers for monitoring the development of acetaminophen-induced liver injury.

Expressions of lipid oxidation markers, N(ε)-hexanoyl lysine and acrolein in cisplatin-induced nephrotoxicity in rats.

The purpose of this study was to evaluate whether N(ε)-hexanoyl lysine (N(ε)-HEL) and acrolein reflect the severity of cisplatin-induced nephrotoxicity. Immunoexpression of N(ε)-HEL and acrolein in kidneys and their urinary concentration were examined up to day 4 post-cisplatin injection in rats. Cisplatin-induced tubular injury was observed histopathologically on days 2-4 after injection and became more severe time-dependently. On days 2-4, N(ε)-HEL and acrolein were immunostained in the cytoplasm of damaged tubular cells. Their immunostaining intensity and urinary levels increased as tubular injury became more severe. These results suggest that expressions of N(ε)-HEL and acrolein were associated with the pathogenesis of cisplatin-induced nephrotoxicity.

Effect of protein malnutrition on the skin epidermis of hairless mice.

The purpose of this study was to evaluate the effect of protein malnutrition on the skin epidermis of mice. A low protein diet induced thinning of the skin epidermis, a decrease of cell proliferative activity in epidermal cells and a decrease of stratum corneum hydration. Dityrosine was immunostained in the cytoplasm of epidermal cells in the low protein diet group. Plasma advanced oxidation protein product (AOPP) levels were significantly more increased in the low protein diet group than in the control diet group. These results suggest that protein malnutrition adversely affects the structure and water barrier and reservoir functions of the skin epidermis, and these pathological changes are associated with the expressions of protein oxidation markers, dityrosine and AOPP.

Analysis of antioxidant activities in vegetable oils and fat soluble vitamins and biofactors by the PAO-SO method.

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.