Formation of N ε-(Hexanonyl)lysine in Protein Exposed to Lipid Hydroperoxide

Abstract

The objectives of this study were to estimate the structure of the lipid hydroperoxide-modified lysine residue and to prove the presence of the adducts in vivo. The reaction of lipid hydroperoxide toward the lysine moiety was investigated employing N-benzoyl-glycyl-L-lysine (Bz-Gly-Lys) as a model compound of Lys residues in protein and 13-hydroperoxyoctadecadienoic acid (13-HPODE) as a model of the lipid hydroperoxides. One of the products, compound X, was isolated from the reaction mixture of 13-HPODE and Bz-Gly-Lys and was then identified as N-benzoyl-glycyl-N ⑀ -(hexanonyl)lysine. To prove the formation of N ⑀ -(hexanonyl)lysine, named HEL, in protein exposed to the lipid hydroperoxide, the antibody to the synthetic hexanonyl protein was prepared and then characterized in detail. Using the anti-HEL antibody, the presence of HEL in the lipid hydroperoxide-modified proteins and oxidized LDL was confirmed. Furthermore, the positive staining by anti-HEL antibody was observed in human atherosclerotic lesions using an immunohistochemical technique. The amide-type adduct may be a useful marker for the lipid hydroperoxide-derived modification of biomolecules.During lipid peroxidation, biomolecules such as proteins or aminolipids can be covalently modified by lipid decomposition products. For the case of aliphatic aldehydes (alkanals) such as 1-hexanal or 1-nonanal, the N ⑀ -amino groups of the lysine residues in protein can be modified through the formation of a Schiff base. ␣,␤-Unsaturated aldehydes (alkenals) such as acrolein or 4-hydroxy-2-nonenal react with lysine, cysteine, and histidine through a Michael-type addition (1, 2). On the other hand, lipid hydroperoxide might covalently react with protein without serious decomposition of its structure (3). Keto fatty acid (4), which is one of the products by lipoxygenase reaction, can also react with protein and amino acids as previously suggested (5-7). In addition, the pyrrole compounds from long chain epoxides and lysine were identified (8). However, the mechanism of lipid hydroperoxide-derived protein modification is not so clear.To estimate the structure after lipid hydroperoxide-derived lysine modification, the reaction of 13-hydroperoxyoctadecadienoic acid 1 with N-benzoyl-glycyl-L-lysine (BzGly-Lys) was investigated. In this study, a novel compound, N-benzoyl-glycyl-N ⑀ -(hexanonyl)lysine (named HEL), was identified as one of the lipid hydroperoxide-modified lysine residues. The formation of HEL in lipid hydroperoxide-modified proteins including oxidatively modified LDL was confirmed using the specific antibody to the HEL residue. In addition, the HEL moiety was detected in human atherosclerotic plaques by immunohistochemical approach. As far as we know, the formation of an amide-type adduct has not been previously reported. This novel adduct derived from lipid hydroperoxide may become an initial marker for the oxidative damage of biological molecules in vivo. EXPERIMENTAL PROCEDURESMaterials-The chemicals used were from the following sources. Bz-Gly-Lys an...

Ability of Orally Administered IFN-.ALPHA.4 to Inhibit Naturally Occurring Gingival Inflammation in Dogs

“…In the present study, we evaluated the levels of 8-OHdG [36] and HEL [20], which are both oxidative stress markers, in whole saliva as biomarkers in clinically healthy individuals (Group 1). Both 8-OHdG and HEL decreased significantly after LDOA of CaIFN-4 In Group 1, 4 of 5 dogs tested positive for occult blood, but this rate decreased during LDOA of CaIFN-4 (data not shown), possibly reflecting stress levels.…”

“…The saliva samples of the dogs in Group 1 were centrifuged at 10,000  g for 10 min, and the supernatant was used to determine 8-hydroxydeoxyguanosine (8-OHdG) and N-(Hexanoyl) Lysine (HEL) levels as oxidative stress biomarkers [36,20] with enzyme-linked immunosorbent assay (ELISA) kits (Japan Institute for the Control of Aging, Nikken SEIL Corporation, Fukuroi, Shizuoka, Japan).…”

Reduction of Cerebral Infarction in Stroke-Prone Spontaneously Hypertensive Rats by Statins Associated With Amelioration of Oxidative Stress

“…Immunoreactivities for N ⑀ -(hexanonyl)lysine (HEL), 4 4-hydroxynonenal (4-HNE), and 8-hydroxy-2Ј-deoxyguanosine (8-OHdG) in tMCAO models were detected using antibodies against HEL (1:500; MHL-020; JAICA, Shizuoka, Japan), 4-HNE (1:400; MHN-020; JAICA), and 8-OHdG (1:50; MOG-020; JAICA). 5 The numbers of positive cells for each marker in the 3 randomized 0.5 mm 2 areas in the ischemic core were counted.…”

Accumulation ofNε-(Hexanoyl)lysine, an Oxidative Stress Biomarker, in Rice Seeds during Storage

“…We have reported that HEL accumulates in animal tissue in which oxidative stress is induced. [7][8][9] In this study, we expected that biomolecules in which proteins and/or aminolipids are modified by lipid decomposition products might be a useful oxidative stress marker in rice damaged oxidatively, as well as in animal tissue. Hence, we investigated whether the reduction in germination ability resulted from oxidative stress during storage of rice at high temperatures, and whether HEL can be used effectively as an oxidative stress biomarker in rice.…”

“…6,20) We have identified N " -(hexanonyl)-lysine (HEL) as an adduct with an amide bond between the N " -amino group and lipid-derived part. 7) It has reported that the antibody against this adduct was useful as a biomarker of oxidative stress in animal tissue, 8,9) but it had not yet been reported whether HEL accumulates in plant tissue damaged by oxidative stress. As Fig.…”