Leading the way in oxidative stress research: Genox Oxidative Stress
Hexanoyl-lysine adduct (HEL)
Biomarker for early stage of lipid oxidation

Oxidative damage of lipids caused by reactive oxygen species (ROS) play an important role in some diseases, lesion of cell functions and aging. Aldehydes such as malondi-aldehyde (MDA) and 4-hydroxy-2-nonenal (4-HNE) have been reported as one of the advanced lipid peroxidation products. But recently in the earlier stage of lipid peroxidation, 13-hydroperoxyoctadecanoic acid (13-HPODE) is found to be covalently bound to proteins1). Hexanoyl-Lysine adduct (HEL) is a novel lipid hydroperoxide-modified lysine residues. HEL is formed by oxidative modification by oxidized omega-6 fatty acids such as linoleic acid or arachidonic acid. HEL may be a useful biomarker for initial stage of lipid peroxidation.Monoclonal antibodies and ELISA kit have been developped, and HEL can be detected in oxidatively modified LDL, in human atherosclerotic lesions, human urine and serum. It is also reported that HEL is formed in rat muscle during exercise, and the formation is prohibited by antioxidants such as flavonoids.

Hexanoyl-Lys (HEL) ELISA kit

JaICA have developed HEL ELISA kit in collaboration with Dr. Toshihiko Osawa (Nagoya University) and Dr. Yoji Kato (Univerisity of Hyogo). This ELISA kit can be applied to urine, serum and cultured cells form human and animal.
Product name Code Assay range Application
Hexanoyl-Lys (HEL) ELISA KHL-700E 2-700 nmol/L Urine, serum, cultured cells and other biological materials
Related products:    Anti Hexanoyl-Lys monoclonal antibody   
FOR RESEARCH USE ONLY. Not for diagnostic, medical or other use.
Assay principle: Competitive ELISA (detection: 450 nm)
Specifity: Specific to N-epsilon-Hexanoyl-Lysine adduct
Measuring range: 2 - 700 nmol/L
Time to answer: Over night and 2 hours.
Format: 96 wells (54 samples in single assay)
Applications: Urine, serum and cultured cells from human and animals.
Storage: Store at 2 - 8°C (don't freeze).
Expiry: 2 years after the day of manufacturing.
Required but not provided: 50 µL micropipettor and pipette tips
8-channel (50-200 µL) micropipettor and tips
8 or 12-syncronous multichannel pipet and reagent tray for multichannel pipet.
4-7 °C incubator
Microtiter plate reader (measuring wavelength 450 nm)
Content of this kit
HEL-coated Microtiter Plate: 1 plate (96 wells)
Primary Antibody (ready to use): 1 vial
Secondary Antibody: 1 vial
Secondary Antibody Buffer: 1 vial
Chromogen (TMBZ solution): 1 vial
Chromogen Buffer: 1 vial
Washing Buffer (5X): 1 vial
Stop Solution: 1 vial
Standard solution (6 levels): 1 vial each
Plate seal: 2 sheets
Assay procedure
Sample pretreatment: Urine samples can be applied to ELISA without pretreatment. Serum, plasma, cultured cells and tissue homogenates should be digested by alpha-chymotrypsin for overnight at 37 °C.
Primary antibody: 50 µL of samples and 50 µL of primary antibody solutuion are poured into wells. Stand for over night at 4-7 °C.
Secondary antibody: 100 µL of HRP-conjugated anti mouse IgG. Incubate for 1 hour at room temperature.
Substrate reaction: 100 µL of TMBZ reagent. Incubate for 15 minutes at room temperature.
Reading: Add 100 µL of stop solution, and read absorbance at 450nm.
FOR RESEARCH USE ONLY. Not for diagnostic, medical or other use.
1) Yoji Kato, Yoko Mori, Yuko Makino, Yasujiro Morimitsu, Sadayuki Hiroi, Toshitsugu Ishikawa, Toshihiko Osawa, Formation of N epsilon-(hexanonyl) lysine in protein exposed to lipid hydroperoxide. J Biol Chem 274(29), p20406-20414 (1999)
Identification of HEL, which is lysine adduct of 13-HPODE.
2) Yoji Kato, Toshihiko Osawa: Detection of lipid hydroperoxide-derived protein modification with polyclonal antibodies. Methods in Molecular Biology, vol. 186, Oxidative stress biomarkers and antioxidant protocols, D Armstrong, Ed., Human Press Inc., NJ, USA, p37-44
Immunohistochemical detection of hexanoyl-lysine adduct.
3) Kato Y, Miyake Y, Yamamoto K, Shimomura Y, Ochi H, Mori Y, Osawa T, Preparation of a monoclonal antibody to N(epsilon)-(Hexanonyl)lysine: application to the evaluation of protective effects of flavonoid supplementation against exercise-induced oxidative stress in rat skeletal muscle. Biochem Biophys Res Commun 274(2),p389-393(2000)
Development and characterization of anti HEL monoclonal antibody.
4) Ryo K, Yamada H, Nakagawa Y, Tai Y, Obara K, Inoue H, Mishima K, Saito I, Possible involvement of oxidative stress in salivary gland of patients with Sjogren's syndrome. Pathobiology 73(5), p252-260 (2006)
HEL can be detected in saliva samples from patients with Sjogren's syndrome.
5) Suzuki T, Kazui T, Yamamoto S, Washiyama N, Ohkura K, Ohishi K, Bashar AH, Yamashita K, Terada H, Suzuki K, Akuzawa S, Fujie M, Effect of prophylactically administered edaravone during antegrade cerebral perfusion in a canine model of old cerebral infarction. J Thorac Cardiovasc Surg 133(3),p710-716 (2007)
Application to canine serum samples.
6) Shimizu K, Ogawa F, Akiyama Y, Muroi E, Yoshizaki A, Iwata Y, Komura K, Bae S, Sato S, Increased Serum Levels of N(epsilon)-(hexanoyl)lysine, A New Marker of Oxidative Stress, in Systemic Sclerosis. J Rheumatol. 2008 Sep 1.
Serum HEL concentration from systemic sclerosis is higher than that from healthy subjects.
7) Tamura H, Takasaki A, Miwa I, Taniguchi K, Maekawa R, Asada H, Taketani T, Matsuoka A, Yamagata Y, Shimamura K, Morioka H, Ishikawa H, Reiter RJ, Sugino N, Oxidative stress impairs oocyte quality and melatonin protects oocytes from free radical damage and improves fertilization rate. J Pineal Res 44(3),p280-287(2008)
Intrafollicular concentration of HEL was significantly reduced by these antioxidant treatment.
8) Sakamoto Y, Ishikawa T, Kondo Y, Yamaguchi K, Fujisawa M, The assessment of oxidative stress in infertile patients with varicocele. BJU Int 101(12), p1547-1552 (2008)
Azoospermic and oligospermic patients had a significantly higher HEL concentration in seminal plasma.
9) Kageyama Y, Takahashi M, Nagafusa T, Torikai E, Nagano A, Etanercept reduces the oxidative stress marker levels in patients with rheumatoid arthritis. Rheumatol Int 28(3),pp245-251(2008)
Urinary HEL level was reduced at 3 and 6 months after the initial treatment with etanercept.
10) Rummenie VT, Matsumoto Y, Dogru M, Wang Y, Hu Y, Ward SK, Igarashi A, Wakamatsu T, Ibrahim O, Goto E, Luyten G, Inoue H, Saito I, Shimazaki J, Tsubota K, Tear cytokine and ocular surface alterations following brief passive cigarette smoke exposure. Cytokine 43(2),p200-208(2008)
HEL concentration in tear was increased by brief passive exposure to cigarette smoke.
11) K Sakai, S Kino, A Masuda, M Takeuchi, T Ochi, J Osredkar, B Rejc, K Gersak, N Ramarathnam, Y Kato;
Determination of HEL (Hexanoyl-Lysine Adduct): A Novel Biomarker for Omega-6 PUFA Oxidation.
Lipid Hydroperoxide-Derived Modification of Biomolecules: Subcellular Biochemistry 77,p61-72(2014)
An original paper for HEL ELISA development.
FOR RESEARCH USE ONLY. Not for diagnostic, medical or other use.