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Technical information for oxidative stress biomarkers.  
In this web site, technical information about trouble shootings, application examples and list of reference papers are presented. If you have any questions or any comments, please feel free to contact us by e-mail.

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Type Product name / code Click below
DNA
oxidation		 8-OHdG Check ELISA (Code. KOG-200SE / KOG-HS10E) Tech Info Ref.
Anti 8-OHdG monoclonal antibody (N45.1) (Code: MOG-020P / MOG-100P) Tech Info Ref.
Anti Thymidine Glycol (TG) monoclonal antibody (Code: MTG-100P) Tech Info Ref.
Lipid
oxidation		 Hexanoyl-Lysine (HEL) ELISA kit (Code: KHL-700E) Tech Info Ref.
Anti Hexanoyl-Lysine (HEL) monoclonal antibody (Code: MHL-021P / MHL-020P) Tech Info Ref.
Anti 4-HNE monoclonal antibody (HNEJ2) (Code: MHN-020P / MHN-100P) Tech Info Ref.
Anti Acrolein (ACR) monoclonal antibody (5F6) (Code: MAR-020n / MAR-100n) Tech Info Ref.
Anti MDA monoclonal antibody (1F83) (Code: MMD-030n) Tech Info Ref.
Anti 4-hydroxy-2-hexenal (4-HHE) monoclonal antibody (Code: MHH-030n) Tech Info Ref.
Anti Crotonaldehyde (CRA) monoclonal antibody (Code: MCA-030n) Tech Info Ref.
Anti Methylglyoxal (MG) monoclonal antibody (Code: MMG-030n) Tech Info Ref.
Anti 7-ketocholesterol (7-KC) monoclonal antibody (Code: MKC-020n) Tech Info Ref.
Protein
oxidation		 Dityrosine (DT) ELISA (Code: KDT-010E) Tech Info Ref.
Anti Dityrosine (DT) monoclonal antibody (Code: MDT-020P) Tech Info Ref.
Anti Dibromo-Tyrosine (DiBrY) monoclonal antibody (Code: MBY-020P) Tech Info Ref.
Antioxidant
assay		 Total Antioxidant Assay(PAO) (Code: KPA-050) Tech Info Ref.
Traceelements Assay Fe / Iron Assay Kit (ferrozine) (Code: CFE-005) Tech Info Ref.
Fe / Iron Assay Kit (Nitroso-PSAP) (Code: CFE-010) Tech Info Ref.
UIBC Assay Kit (Bathophenanthroline) (Code: CBC-800) Tech Info Ref.
Cu / Copper Assay Kit (3,5-DiBr-PAESA) (Code: CCU-400) Tech Info Ref.
Zn / Zinc Assay Kit (5-Br-PAPS) (Code: CZN-001) Tech Info Ref.
Ca / Calsium Assay Kit (Chlorophosphonazo III) (Code: CCA-020) Tech Info Ref.
Ca / Calsium Assay Kit (o-Cresolphtalein-Complexone) (Code: CCA-030) Tech Info Ref.
Mg / Magnesium Assay Kit (Xyridil Blue-I) (Code: CMG-035) Tech Info Ref.
Anti 4-HNE monoclonal antibody
No. Title Content
1 Applicable species: This antibody can be applied to tissue samples from human, rabbit, rat and other animals, because lipid oxidation is species independent.
But if you are planning to apply to mouse tissue, blocking of internal mouse IgG will be needed before staining. For example, Vector M.O.M. Immunodetection kit (code.PK2200) may be useful for blocking of mouse IgG.
2 Immunohistochemistry: IHC protocol is available HERE.

4-HNE is generated during lipid peroxidation process, and may react with Lys / His / Arg residues in proteins to form stable adducts. Anti 4-HNE antibody (clone HNEJ2) reacts with mainly 4-HNE-His adducts. Cytoplasm may be stained by this antibody, but in some cases nuclear may be also stained. Recently, 4-HNE is reported to form DNA-adducts, and there may be some possibilities that this antibody would react with them.
3 Western blotting: This antibody can be applied to western blotting. Please try lower antibody concentration than that for immunohistochemistry. For example, 2.15 - 15 µg/mL. Multiple bands may be detected by western blotting because 4-HNE may react with various proteins. Molecular weight of 4-HNE itself is 156.2 Da.

[Ref.]
The monoclonal antibody specific for the 4-hydroxy-2-nonenal histidine adduct.
FEBS Lett. Vol.359, p189-191 (1995), S Toyokuni, et. al.

Differential Effects of High Glucose and Methylglyoxal on Viability and Polyol Metabolism in Immortalized Adult Mouse Schwann Cells.
Open Diabetes J. 1, p1-11(2008),K Sango, et. al.

Purified 4-HNE is available at Cayman Chemical.
4 Flowcytometry: In case if 4-HNE-modified proteins would exist on the cell surface, it may be possible to apply this antibody to flowcytometry. But we are very sorry we have no data about it.

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List of Ref.:
1) S.Toyokuni, N.Miyake, H.Hiai, M.Hagiwara, S.Kawakishi, T.Osawa and K.Uchida:
The monclonal antibody specific for the 4-hydroxy-2-nonenal histidine adduct.
FEBS Lett. 359, p189-191 (1995)
2) T.Tanaka, Y.Nishiyama, K.Okada, K.Hirota, M.Matsui, J.Yodoi, H.Hiai, and S.Toyokuni:
Induction and nuclear translocation of thioredoxin by oxidative damage in the mouse kidney: independence of tubular necrosis and sulfhydryl depletion.
Lab. Invest. 77(2), p145-155 (1997)
3) Y.Hattori, C.Nishigori, T.Tanaka, K.Uchida, O.Nikaido, T.Osawa, H.Hiai, S.Imamura, and S.Toyokuni:
8-Hydroxy-2'deoxyguanosine is increased in epidermal cells of hairless mice after chronic ultraviolet B exposure.
J. Invest. Dermatol. 107, p733-737 (1997)
4) S.Toyokuni:
Reactive oxygen species-induced molecular damage and its application in pathology.
Pathol. Internat. 49, p91-102 (1999)
5) S.Kondo, S.Toyokuni, Y.Iwasa, T.Tanaka, H.Onodera, H.Hiai and M.Imamura:
Persistent oxidative stress in human colorectal carcinoma, but not in adenoma.
Free Rad. Biol. Med. 27, p401-410 (1999)
6) T.D. Oberley, S.Toyokuni, and L.I.Szweda:
Localization of hydroxynonenal protein adducts in normal human kidney and selected human kidney cancers.
Free Rad. Biol. Med. 27, p695-703 (1999)
7) Hiroko Kimura, Masahiro Mukaida, Kyoko Kuwabara, Teruyo Ito, Kimikazu Hashino, Koji Uchida, Kazuko Matsumoto and Ken-ichi Yoshida:
4-Hydroxynonenal modifies IgA in rat intestine after lipopolysaccharide injection.
Free Radical Biology and Medicine, 41(6), p973-978 (2006)
8) Zhang N, Komine-Kobayashi M, Tanaka R, Liu M, Mizuno Y, Urabe T:
Edaravone reduces early accumulation of oxidative products and sequential inflammatory responses after transient focal ischemia in mice brain.
Stroke. 36(10):p2220-2225(2005)
9) Fiorella Biasi, Barbara Vizio, Cinzia Mascia, Ezio Gaia, Neven Zarkovic, Elena Chiarpotto, Gabriella Leonarduzzi and Giuseppe Poli :
c-Jun N-terminal kinase upregulation as a key event in the proapoptotic interaction between transforming growth factor-β1 and 4-hydroxynonenal in colon mucosa
Free Radical Biology and Medicine 41(3), p443-454(2006)
10) Isabella Dalle-Donne, Marina Carini, Giulio Vistoli, Luca Gamberoni, Daniela Giustarini, Roberto Colombo, Roberto Maffei Facino, Ranieri Rossi, Aldo Milzani and Giancarlo Aldini:
Actin Cys374 as a nucleophilic target of α,β-unsaturated aldehydes
Free Radical Biology and Medicine 42(5), p583-598(2007)
11) Shintaro Kodai, Shigekazu Takemura, Yukiko Minamiyama, Seikan Hai, Satoshi Yamamoto, Shoji Kubo, Yasukazu Yoshida, Etsuo Niki, Shigeru Okada, Kazuhiro Hirohashi, Shigefumi Suehiro:
S-allyl cysteine prevents CCl4-induced acute liver injury in rats.
Free Radical Research 41(4), p489-497(2007)
12) Tai-Ho Hung, Jeremy N. Skepper, and Graham J. Burton:
In Vitro Ischemia-Reperfusion Injury in Term Human Placenta as a Model for Oxidative Stress in Pathological Pregnancies.
American Journal of Pathology 159(3), p1031-1043(2001)
Anti 4-HNE antibody is applied to immunohistochemistry and western blotting of human placenta.
13) Sango K, Yanagisawa H, Kato K, Kato N, Hirooka H, Watabe K:
Differential Effects of High Glucose and Methylglyoxal on Viability and Polyol Metabolism in Immortalized Adult Mouse Schwann Cells.
Open Diabetes J. 1, p1-11(2008)
Detection of 4-HNE by immunostaining and western blotting in murine cultured cells.
14) Wei-Yi Ong,Andrew M. Jenner,Ning Pan,Choon-Nam Ong,Barry Halliwell:
Elevated oxidative stress, iron accumulation around microvessels and increased 4-hydroxynonenal immunostaining in zone 1 of the liver acinus in hypercholesterolemic rabbits.
Free Radical Research 43(3), p241-249(2009)
Application to rabbit liver.

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Hexanoyl-Lys (HEL) ELISA kit
No. Title Contents
1 Animals HEL ELISA kit is expected to be applied to human, rabbit, rat and other experimental animals, because lipid oxidation is common to many species. Until today, human urine, dog urine and cat urine are tested.
We would like to share any information about oxidative stress research with researchers, and would like to contribute to your research. If you have any experiences or any ideas for HEL assay, please don't hesitate to submit to our web site (sales@jaica.com).
2 Application to human urine: Dilution (x 4) with PBS (pH7.4) is recommended. Concentraion of HEL in normal human urine is expected to be:
Mean 105.4 +/- 67.0 nmol/L.
Mean 85.1 +/- 61.8 pmol/mg creatinine.
Mean 77.2 +/- 54.9 pg/hour kg body weight.
3 Application to human serum: Sample pretreatment by protease is needed to detect HEL in serum samples.
[Sample pretreatment procedure]
1) Mix 1 volume of serum with 1 volume of PBS (pH7.4).
2) Prepare enzyme solution (14 mg/mL of alpha-chymotrypsin in PBS (pH7.4)).
3) Add 60 µL of enzyme solution to 300 µL of diluted serum.
4) Incubate at 37 degree C overnight (10-18 hours).
5) Apply the reaction mixture to ultrafilter with molecular cut-off 10kDa to remove protease and high molecular weight substances. For example, Microcon YM-10 (Millipore, USA) is suitable.
6) Collect the filtrate and apply to HEL ELISA.
[Assay example]
HEL concentration in normal human serum: 7.96 ± 4.32 nmol/L.
4 Application to animal samples: 1) Dog urine:
x 4 dilution by PBS is recommended. Assay example 14-58 nmol/L.
2) Cat urine:
x 10 dilution by PBS is recommended. Assay example 108-298 nmol/L.
3) Mouse urine:
x 20 dilution by PBS is recommended. 4) Rat serum:
The same protorol as that for human serum. Assay example 25.8 nmol/L.
5 Detection of HEL in LDL: HEL can be detected in oxidized LDL. Sample pretreatment is needed.
[Sample pretreatment procedure]
1) Dialyze purified LDL or oxidized LDL to PBS(pH7.4).
2) Add 100 µL of Trypsin (40 mg/mL in PBS) and 2 µL of CaCl2solution (100 mM) to 100 µL of samples.
3) Incubate at 37 °C overnight (10-18 hours).
4) Apply the reaction mixture to ultrafilter with molecular cut-off 10kDa to remove protease and high molecular weight substances. For example, Microcon YM-10 (Millipore, USA) is suitable.
5) Collect the filtrate and apply to HEL ELISA.
[Assay example]
HEL level in native human LDL=13.6 nmol/L, oxidized human LDL=363.6 nmol/L
6 Application to tissue samples: HEL is expected to be detected by follwoing procedure.
[Sample pretreatment procedure]
1) Prepare tissue homogenate in PBS containing 0.1% BHT (butylated hydroxytoluene).
2）Centrifuge, and collect the supernatant.
3) Prepare enzyme solution (14 mg/mL of alpha-chymotrypsin in PBS (pH7.4)).
4) Add 60 µL of enzyme solution to 300 µL of the supernatant.
5) Incubate at 37 °C overnight (10-18 hours).
6) Apply the reaction mixture to ultrafilter with molecular cut-off 10kDa to remove protease and high molecular weight substances. For example, Microcon YM-10 (Millipore, USA) is suitable.
7) Collect the filtrate and apply to HEL ELISA.
NOTE) BHT may be difficult to dissolve in PBS. Please prepare BHT / ethanol solution, and add BHT solution to PBS just before start preparing homogenate.
7 Application to cultured cells: HEL can be detected in cultured cells by follwoing procedure.
[Sample pretreatment procedure]
1) Culture the cells to reach confluent in 6 wells plate.
2) Exchange the medium to serum free.
3) Wash the cells by PBS for 3 times.
4) Scrape the cells in 600 µL of PBS (pH7.4).
5) Add 0.1% BHT (butylated hydroxytoluene), and prepare cell lysate by sonication.
6) Centrifuge, and collect the supernatant.
7) Add 60 µL of enzyme solution to 300 µL of the supernatant.
8) Incubate at 37 °C overnight (10-18 hours).
9) Apply the reaction mixture to ultrafilter with molecular cut-off 10kDa to remove protease and high molecular weight substances. For example, Microcon YM-10 (Millipore, USA) is suitable.
10) Collect the filtrate and apply to HEL ELISA.
8 How to prepare standard curve: Any algorithm function can be used alternatively to calculate the results if it is an approximate semi-logarithm curve (Absorbance=Antilogarithm, Concentration=Logarithm). Especially since recent micro plate readers have useful functions to draw the standard curves, you can use it. The semi-logarithm line graph is also enough to use as an approximate function.
9 Partial use of the ELISA kit: The ELISA kit is made up for 8 wells * 12 columns, therefore can be divided accordingly to meet the number of samples being measured. Please be aware that a new calibration curve is necessary for each measurement of samples. For a calibration curve 6 levels * 3=18 wells are needed. Almost all samples remain stable when stored at lower than -20°C. Therefore more samples can be measured when you test all of them at the same time.
10 Reference wavelength: 600 - 650 nm would be used as reference wavelength.
11 alpha-chymotrypsin : Alpha-chymotrypsin is available from Sigma-Aldrich.
Code C-4129, a-chymotrypsin from bovine pancreas. type II, 250 mg, 40 units/mg protein
12 Ultrafilter device: Millipore Microcon® YM-10 has been discontinued.
Nanosep® centrifugal device (MW cut off: 10KDa, Code. OD010C34) from Pall corporation may be useful for sample pretreatment.

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List of Ref.:
1) Y. Kato, Y. Mori, Y. Makino, Y. Morimitsu, S. Hiroi, T. Ishikawa, T. Osawa.:
Formation of N-ipsyron-(hexanonyl) lysine in protein exposed to lipid hydroperoxide.
J. Biol. Chem. 274(29) p20406-20414 (1999)
2) Y. Kato, T. 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
3) Y. Kato, Y. Miyake, K. Yamamoto, Y. Shimomura, H. Ochi, Y. Mori, T. Osawa:
Preparation of a monoclonal antibody to Nε-(hexanonyl) lysine: applycation 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)
4) M. Naito, Wu X, H. Nomura, M. Kodama, Y. Kato, Y. Kato, T. Osawa:
The protective effects of tetrahydrocurcumin on oxidative stress in cholesterol-fed rabbits.
J Atheroscler Thromb. 9(5), p243-250 (2002)
5) H. Arai, Y. Kato, K. Fukunaga, S. Mohri, and K. Nakamura:
Formation of Nepsilon-(hexanonyl)lysine in oxidized human very-low density lipoprotein.
J. Electrophoresis 48, p37-40 (2004)
6) Y. Kato, A. Yoshida, M. Naito, Y. Kawai, K. Tsuji, M. Kitamura, N. Kitamoto, and T. Osawa:
Identification and Quantification of N-epsilon-(Hexanoyl)lysine in human urine by liquid chromatography/tandem mass spectrometry.
Free Radic. Bio.l Med. 37(11), p1864-1874 (2004)
7) T. Osawa, Y. Kato:
Protective role of antioxidative food factors in oxidative stress caused by hyperglycemia.
Ann N Y Acad Sci. 1043, p440-451 (2005)
8) K. Minato, M. Gono,H. Yamaguchi, Y. Kato, T. Osawa:
Accumulation of Nepsilon-(Hexanoyl)lysine, an oxidative stress biomarker, in rice seeds during storage.
Biosci Biotechnol Biochem. 69(9), p1806-1810 (2005)
Rice is reported to be oxidized during storage, to form HEL.
9) 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):252-60.(2006)
HEL can be detected in saliva samples from patients with Sjogren's syndrome.
10) 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.
11) 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.
12) 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.
13) 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.
14) 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)p245-251(2008)
Urinary HEL level was reduced at 3 and 6 months after the initial treatment with etanercept.
15) 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.
16) Tomofuji T, Sanbe T, Ekuni D, Azuma T, Irie K, Maruyama T, Tamaki N, Yamamoto T: Oxidative damage of rat liver induced by ligature-induced periodontitis and chronic ethanol consumption. Arch Oral Biol. 53(12),p1113-1118 (2008)
Periodontitis and chronic ethanol consumption in rat is reported to cause oxidative stress.
17) Ekuni D, Tomofuji T, Sanbe T, Irie K, Azuma T, Maruyama T, Tamaki N, Murakami J, Kokeguchi S, Yamamoto T: Periodontitis-induced lipid peroxidation in rat descending aorta is involved in the initiation of atherosclerosis. J Periodontal Res. Feb 6 (2009)
In rat periodontitis model, HEL is reported to increase.
18) Sanbe T, Tomofuji T, Ekuni D, Azuma T, Irie K, Tamaki N, Yamamoto T, Morita M: Vitamin C intake inhibits serum lipid peroxidation and osteoclast differentiation on alveolar bone in rats fed on a high-cholesterol diet. Arch Oral Biol. 54(3),p235-240(2009)
Rat serum HEL(lipid oxidation) is reported to increase when fed high cholesterol diet. It is also reported that oral administration of vitamin C decreases serum HEL concentration.
19) Higuchi A, Takahashi K, Hirashima M, Kawakita T, Tsubota K: Selenoprotein P Controls Oxidative Stress in Cornea. PLoS ONE 5(3) e9911 (2010)
In rat dry-eye model, HEL (lipid oxidation) is reported to increase from 3.7 pmol/mg protein to 5.4 pmol/mg protein. In this paper essential role of selenoprotein P in the defense of corneas from oxidative stress.
20) Hiroshi Izuta, Nozomu Matsunaga, Masamitsu Shimazawa, Tetsuya Sugiyama, Tsunehiko Ikeda, Hideaki Hara:
Proliferative diabetic retinopathy and relations among antioxidant activity, oxidative stress, and VEGF in the vitreous body.
Molecular Vision 16, p130-136 (2010)

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Anti Hexanoyl-Lys (HEL) antibody
No. Title Content
1 Applicable species: This antibody can be applied to tissue samples from human, rabbit, rat and other animals, because lipid oxidation is species independent.
But if you are planning to apply to mouse tissue, blocking of internal mouse IgG will be needed before staining. For example, Vector M.O.M. Immunodetection kit (code.PK2200) may be useful for blocking of mouse IgG.
2 Immunohistochemistry: Protocol is available HERE.

Application of anti HEL antibody to HUVECs
Maeda R, et.al.:Hypertens Res 31(1)p141-151(2008)
3 Western blottings: Anti HEL antibody can be applied to western blottings. Multiple bands will be detected because HEL adducts can be formed in any proteins which have lysine residues.

[Reference]: H. Arai, Y. Kato, K. Fukunaga, S. Mohri, and K. Nakamura: Formation of Nepsilon-(hexanonyl)lysine in oxidized human very-low density lipoprotein. J. Electrophoresis 48, p37-40 (2004)
4 Rat kidney: Tissue: Rat kidney cisplatin treated.
Primary antibody: x 75 dilution
Antigen retrieval: none
Secondary antibody: Histofine® SimpleStain Rat(M), Nichirei corporation
[Reference]: Sugiyama A, et. al.; J Vet Med Sci. 73(6), p821-826 (2011)

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List of Ref.:
1) Y. Kato, Y. Mori, Y. Makino, Y. Morimitsu, S. Hiroi, T. Ishikawa, T. Osawa.:
Formation of N-epsilon-(hexanonyl) lysine in protein exposed to lipid hydroperoxide.
J. Biol. Chem. 274(29) p20406-20414 (1999)
2) Y. Kato, Y. Miyake, K. Yamamoto, Y. Shimomura, H. Ochi, Y. Mori, T. Osawa:
Preparation of a monoclonal antibody to N-epsilon-(hexanonyl) lysine: applycation 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)
3) H. Arai, Y. Kato, K. Fukunaga, S. Mohri, and K. Nakamura:
Formation of N-epsilon-(hexanonyl)lysine in oxidized human very-low density lipoprotein.
J. Electrophoresis 48, p37-40 (2004)
4) Tomaru M, Takano H, Inoue K, Yanagisawa R, Osakabe N, Yasuda A, Shimada A, Kato Y, Uematsu H:
Pulmonary exposure to diesel exhaust particles enhances fatty change of the liver in obese diabetic mice.
Int J Mol Med. 19(1):17-22 (2007)
5) Fukuchi Y, Miura Y, Nabeno Y, Kato Y, Osawa T, Naito M:
Immunohistochemical detection of oxidative stress biomarkers, dityrosine and N(epsilon)-(hexanoyl)lysine, and C-reactive protein in rabbit atherosclerotic lesions. J Atheroscler Thromb 15(4)p185-192(2008)
HEL was detected in rabbit atherosclerotic lesions.
6) Maeda R, Noiri E, Isobe H, Homma T, Tanaka T, Negishi K, Doi K, Fujita T, Nakamura E:
A water-soluble fullerene vesicle alleviates angiotensin II-induced oxidative stress in human umbilical venous endothelial cells. Hypertens Res 31(1)p141-151(2008)
Application to cultured cells (HUVECs).
7) Sango K, Yanagisawa H, Kato K, Kato N, Hirooka H, Watabe K: Differential Effects of High Glucose and Methylglyoxal on Viability and Polyol Metabolism in Immortalized Adult Mouse Schwann Cells. Open Diabetes J. 1, p1-11(2008)

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Anti Dibromotyrosine (DiBrY) antibody
No. Title Content
1 Applicable species: This antibody can be applied to tissue samples from human, rabbit, rat and other animals, because lipid oxidation is species independent.
But if you are planning to apply to mouse tissue, blocking of internal mouse IgG will be needed before staining. For example, Vector M.O.M. Immunodetection kit (code.PK2200) may be useful for blocking of mouse IgG.

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List of Ref.:
1) Wu W, Chen Y, d'Avignon A, Hazen SL.
3-Bromotyrosine and 3,5-dibromotyrosine are major products of protein oxidation by eosinophil peroxidase: potential markers for eosinophil-dependent tissue injury in vivo.
Biochemistry 38(12), p3538-3548(1999)
2) Kato Y, Kawai Y, Morinaga H, Kondo H, Dozaki N, Kitamoto N, Osawa T.
Immunogenicity of a brominated protein and successive establishment of a monoclonal antibody to dihalogenated tyrosine.
Free Radic Biol Med. 38(1), p24-31(2005)

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Anti Dityrosine (DT) antibody
No. Title Content
1 Applicable species: This antibody can be applied to tissue samples from human, rabbit, rat and other animals, because lipid oxidation is species independent.
But if you are planning to apply to mouse tissue, blocking of internal mouse IgG will be needed before staining. For example, Vector M.O.M. Immunodetection kit (code.PK2200) may be useful for blocking of mouse IgG.
2 Western blotting: Concentration of primary antibody would be 0.1 - 1.0 µg/mL.
Multiple bands or smear staining would be observed because varuous proteins may be oxidatively damaged, and cleavage, cross-links or aggregation are expected.
3 Preparation of positive control: Dityrosine-modified proteins can be prepared as follows:
Lens protein (For example, Sigma-Aldrich, code.C4163): 0.5 mg/mL
H2O2: 5 mmol/L
CuSO4: 0.05 mmol/L
Incubate for several hours at 37 °C in 0.1M phosphate buffer (pH7.4).
Terminate reaction by EDTA (0.1 mM at final concentration).
Dialyze by phosphate buffered saline.
4 Rat kidney: Tissue: 10% formalin fixed.
H2O2 pretreatment: 3%
Antigen retrieval: none
Primary antibody: x 100 dilution (1 µg/mL), overnight at 4 °C.
Amplification: SimpleStain® MAX-PO(M), Nichirei corporation. 30 min at R/T.
Enzyme / substrate: Peroxidase / DAB
[Reference]: Kimoto Y, et. al.; J Vet Med Sci. 73(3), p403-407 (2011)

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List of Ref.:
1) Kato Y, Wu X, Naito M, Nomura H, Kitamoto N, Osawa T.
Immunochemical detection of protein dityrosine in atherosclerotic lesion of apo-E-deficient mice using a novel monoclonal antibody.
Biochem Biophys Res Commun. 275(1), p11-15 (2000).
2) Kato Y, Kitamoto N, Kawai Y, Osawa T.
The hydrogen peroxide/copper ion system, but not other metal-catalyzed oxidation systems, produces protein-bound dityrosine.
Free Radic Biol Med. 2001 Sep 1;31(5):624-32.
3) Matsuzaki M, Hasegawa T, Takeda A, Kikuchi A, Furukawa K, Kato Y, Itoyama Y.
Histochemical features of stress-induced aggregates in alpha-synuclein overexpressing cells.
Brain Res. 1004(1-2),p83-90(2004)
4) Atwood CS, Perry G, Zeng H, Kato Y, Jones WD, Ling KQ, Huang X, Moir RD, Wang D, Sayre LM, Smith MA, Chen SG, Bush AI.
Copper mediates dityrosine cross-linking of Alzheimer's amyloid-beta.
Biochemistry. 43(2), p560-568 (2004)
5) Shamoto-Nagai M, Maruyama W, Kato Y, Isobe K, Tanaka M, Naoi M, Osawa T.
An inhibitor of mitochondrial complex I, rotenone, inactivates proteasome by oxidative modification and induces aggregation of oxidized proteins in SH-SY5Y cells.
J Neurosci Res. 74(4), p589-597 (2003)
6) Son TG, Zou Y, Yu BP, Lee J, Chung HY.
Aging effect on myeloperoxidase in rat kidney and its modulation by calorie restriction.
Free Radic Res. 39(3), p283-289 (2005)
7) Son TG, Zou Y, Jung KJ, Yu BP, Ishigami A, Maruyama N, Lee J.
SMP30 deficiency causes increased oxidative stress in brain.
Mech Ageing Dev. 127(5), p451-457 (2006)
8) Fukuchi Y, Miura Y, Nabeno Y, Kato Y, Osawa T, Naito M:
Immunohistochemical detection of oxidative stress biomarkers, dityrosine and N(epsilon)-(hexanoyl)lysine, and C-reactive protein in rabbit atherosclerotic lesions. J Atheroscler Thromb 15(4)p185-192(2008)
Application to rabbit tissue.
9) Kimoto Y, Sugiyama A, Nishinohara M, Asano A, Masuda A, Ochi T, Takeuchi T:
Expressions of protein oxidation markers, dityrosine and advanced oxidation protein products in Cisplatin-induced nephrotoxicity in rats. J Vet Med Sci. 73(3), p403-407 (2011)
Application to rat kidney Cisplatin-treated.
10) Sugiyama A, Fujita Y, Kobayashi T, Ryu M, Suzuki Y, Masuda A, Ochi T, Takeuchi T:
Effect of protein malnutrition on the skin epidermis of hairless mice. J Vet Med Sci. 73(6), p831-835 (2011)
Application to mouse skin.
11) Sun J, Sugiyama A, Masuda A, Ochi T, Takeuchi T:
Expressions of protein oxidation markers, dityrosine and advanced oxidation protein products in acetaminophen-induced liver injury in rats. J Vet Med Sci. 73(9), p1185-1190 (2011)
Application to rat liver acetaminophen-treated.
12) Dominik Levigne, Ali Modarressi, Karl-Heinz Krause, Brigitte Pittet-Cuenod
NADPH oxidase 4 deficiency leads to impaired wound repair and reduced dityrosine-crosslinking, but does not affect myofibroblast formation. Free Radical Biology and Medicine 96, p374-384(2016)

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Dityrosine (DT) ELISA
No. Title Content
1 Stability of reagents: All components will be stable for one week after the package is opened.
2 Assay examples for serum/plasma: (No data)
3 Application to animal urine Mouse urine:
x 20 dilution by saline is recommended.

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List of Ref.:
1) Kato Y, Wu X, Naito M, Nomura H, Kitamoto N, Osawa T.
Immunochemical detection of protein dityrosine in atherosclerotic lesion of apo-E-deficient mice using a novel monoclonal antibody.
Biochem Biophys Res Commun. 275(1), p11-15 (2000).
2) Hattori Y, Mukaide T, Jiang L, Kotani T, Tsuda H, Mano Y, Sumigama S, Hirayama T, Nagasawa H, Kikkawa F, Toyokuni S:Catalytic ferrous iron in amniotic fluid as a predictive marker of human maternal-fetal disorders. J Clin Biochem Nutr 56(1),p57-63(2015) doi: 10.3164/jcbn.14-82

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Anti Thymidine Glycol(TG) antibody
No. Title Content
1    

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List of Ref.:
1) Serum antioxidant capacity and oxidative injury to pulmonary DNA in never-smokers with primary lung cancer.
Anticancer Res. 32(3),p1063-1067(2012)
Ito K, Yano T, Morodomi Y, Yoshida T, Kohno M, Haro A, Shikada Y, Okamoto T, Maruyama R, Maehara Y.
IHC application to lung tissues from lung cancer patients.

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Test kit for Potential antioxidant (PAO)
No. Title Content
1 Application to human serum. Q: How to store serum samples for PAO assay?
A: Please prepare fresh serum or fresh plasma. Please store frozen below -20 °C, because some antioxidants such as ascorbic acid, uric acid and coenzyme Q10 is unstable.
Q: Are plasma samples applicable for PAO assay?
A: Plasma samples with heparin can be applied, but EDTA-plasma can't.
According to our reearch, the mean value of antioxidant level measured by PAO assay is 1069 µmol/L.
2 Application to urine samples. PAO assay can be applied to urine samples. But please take care that is the concentration of uric acid is over 2 mmol/L, dilution by distilled water will be needed. The significance of antioxidants in urine is unknown.
3 Application to food samples: 1) Red wines: dilute x 8 by distilled water before assay (assay example, 45,479 µmol/L).
2) Japanese sake: dilute x1 to x8 by distilled water before assay (assay example, 18 to 211 µmol/L).
3) Black tea: dilute x 8 by distilled water before assay.
4) Coffee: dilute x 28 by distilled water before assay.
5) Green tea: dilute x 8 or more by distilled water before assay (assay example, 8,728 to 60,816 µmol/L)
4 Antioxidants detectable. PAO assay can detect at least follwoing substances:
ascorbic acid, trolox, vitamin E, glutathione, bililubin, uric acid, cathecin, beta-carotene and other polyphenols.
5 Preparation of standard. Q: Can't draw standard curve.
A: Uric acid powder is used as standard. Please take care to dissolve uric acid completely before use. The volume of distilled water used is different between vials. Please confirm the volume printed on the label of standard.
Q: How to store dissolved standard?
A: Standard solution (2mM uric acid) can be stored frozen below -20°C. Avoid freeze / thaw cycles.
Q: To calculate antioxidant capacity, why multiply 2189 to uric acid equivalent concentration?
A: To convert from uric acid equivalent to reducing power of copper ion.
6 Ovarian endometrioma: Antioxidant power (PAO) can be assessed in ovarian endometrioma.
Ovarian endometrioma: 1197 µmol/L
Serous Cystadenoma: 672 µmol/L
Mucinous cystadenoma: 424 µmol/L
Reference: S Fujii, 31th meeting of Japan BioIron Sociery, Abstruct in Japanese p9-10 (2007)
7 Reference wavelength: 620 - 660 nm would be useful for reference wavelength.

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List of Ref.:
1) Oxidative stress and its association with coronary artery disease and different atherogenic risk factors.
C VASSALLE, L PETROZZI , N BOTTO, MG ANDREASSI and GC ZUCCHELLI.
Journal of Internal Medicine, Vol.256, p308-315 (2004)
2) Vitamin E-coated dialyzers reduce oxidative stress related proteins and markers in hemodialysis ? a molecular biological approach.
LA Calo, A Naso, E Pagnin, PA Davis, M Castoro, R Corradin, P Riegler, C Cascone, W Huber and A Piccoli
Clinical Nephrology, Vol.62(5), p355-361 (2004)
3) Oxidative stress-related factors in Bartter's and Gitelman9s syndrome: relevance for angiotensin IIsignalling.
Calo LA, Pagnin E, Davis PA, Sartori M, Semplicini A.
Nephrol Dial Transplant ,Vol.18(8) p1518-25 (2003)
4) Effect of epoetin on HO-1 mRNA level and plasma antioxidants in hemodialysis patients.
Calo LA, Stanic L, Davis PA, Pagnin E, Munaretto G, Fusaro M, Landini S, Semplicini A, Piccoli A.
Int. J Clin. Ther, Vol.41(5), p187-92 (2003)
5) Restored Antioxidant Capacity Parallels the Immunologic and Virologic Improvement in Children with Perinatal Human Immunodeficiency Virus Infection Receiving Highly Active Antiretroviral Therapy.
M Martino, F Chiarelli, M Moriondo, M Torello, C Azzari, and L Galli
Clinical Immunology, Vol.100(1),p82-6 (2001)
6) Oxidative imbalance and cathepsin D changes as peripheral blood biomarkers of Alzheimer disease: A pilot study.
E Strafacea, P Matarresea, L Gambardella, R Vona, A Sgadari,MC Silveri, W Malorni
FEBS Letters 579, p2759-766(2005)
Serum antioxidant power is lower in patient with Alzheimer disease in comparison with that in control subjects.
7) Antioxidant capacity as a reliable marker of stress in dairy calves transported by road
P Pregel, E Bollo, FT Cannizzo, B Biolatti, E Contato, and PG Biolatti
Veterinary Record 156, p53-54 (2005)
8) Effetcts of the daily administration of a rehydrating supplement to trotter horses.
A Falaschini, G Marangoni, S Rizzi and MF Trombetta
J Equine Sci 16(1),p1-9 (2005)
Application to horse serum samples.
9) Effects of kakisu (persimmon vinegar) on plasma antioxidant power and urinary 8-isoprostane level. [Article in Japanese] Mure K, Takeshita T, Morioka I, Arita M
Nippon Eiseigaku Zasshi. 62(1),p32-38 (2007)
Randomized cross over study. Kakisu is one of the Japanese traditional vinegar made from persimmon, and contains various polyphenols and minerals. After drinking vinegar 20mL / day for 56 days, serum antioxidant power was significantly higher than control (1447 ± 36.3 µM vs 1315.9 ± 31.8 µM, p<0.001).
10) Antioxidant effects of flavonoids used as food additives (purple corn color, enzymatically modified isoquercitrin, and isoquercitrin) on liver carcinogenesis in a rat medium-term bioassay. Yokohira M, Yamakawa K, Saoo K, Matsuda Y, Hosokawa K, Hashimoto N, Kuno T, Imaida K J Food Sci. 73(7)C561-568(2008)
Application to rat serum. Serum antioxidant power from rats administrated flavonoids was significantly higher than that from control.
11) Contents of endometriotic cysts, especially the high concentration of free iron, are a possible cause of carcinogenesis in the cysts through the iron-induced persistent oxidative stress.
Yamaguchi K, Mandai M, Toyokuni S, Hamanishi J, Higuchi T, Takakura K, Fujii S.
Clin Cancer Res.14(1),p32-40(2008). doi: 10.1158/1078-0432.CCR-07-1614.
Assay example for total antioxidants in endometriotic cyst fluid. Samples were centrifuged at 10,000 x g for 60 minutes, and were treated with ultrafilter with molecular cut-off 10 kDa.
12) Proliferative diabetic retinopathy and relations among antioxidant activity, oxidative stress, and VEGF in the vitreous body.
Hiroshi Izuta, Nozomu Matsunaga, Masamitsu Shimazawa, Tetsuya Sugiyama, Tsunehiko Ikeda, Hideaki Hara:
Molecular Vision 16, p130-136 (2010)
Application to serum and vitreous body from diabetic patients. Serum antioxidant concentration from patients with proliferative diabetic retinopathy is 1177 µmol/L, and is higher than that from patients with macular hole(962 µmol/L). Antioxidants in vitreous body from patients with roliferative diabetic retinopathy (505 µmol/L) is higher than that from patients with macular hole(331 µmol/L).

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What is oxidative stress: FAQ
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