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Technical information for oxidative stress biomarkers.  
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Type Product name / code Click below
DNA
oxidation
8-OHdG Check ELISA (Code. KOG-200SE / KOG-200TE / KOG-HS10E) Tech Info References
Anti 8-OHdG monoclonal antibody (N45.1) (Code: MOG-020P / MOG-100P) Tech Info References
Anti Thymidine Glycol (TG) monoclonal antibody (Code: MTG-100P) Tech Info References
Lipid
oxidation
Hexanoyl-Lysine (HEL) ELISA kit (Code: KHL-700E) Tech Info References
Anti Hexanoyl-Lysine (HEL) monoclonal antibody (Code: MHL-021P / MHL-020P) Tech Info References
Anti 4-HNE monoclonal antibody (HNEJ2) (Code: MHN-020P / MHN-100P) Tech Info References
Anti Acrolein (ACR) monoclonal antibody (5F6) (Code: MAR-020n / MAR-100n) Tech Info References
Anti MDA monoclonal antibody (1F83) (Code: MMD-030n) Tech Info References
Anti 4-hydroxy-2-hexenal (4-HHE) monoclonal antibody (Code: MHH-030n) Tech Info References
Anti Crotonaldehyde (CRA) monoclonal antibody (Code: MCA-030n) Tech Info References
Anti Methylglyoxal (MG) monoclonal antibody (Code: MMG-030n) Tech Info References
Anti 7-ketocholesterol (7-KC) monoclonal antibody (Code: MKC-020n) Tech Info References
Protein
oxidation
Dityrosine (DT) ELISA (Code: KDT-010E) Tech Info References
Anti Dityrosine (DT) monoclonal antibody (Code: MDT-020P) Tech Info References
Anti Dibromo-Tyrosine (DiBrY) monoclonal antibody (Code: MBY-020P) Tech Info References
Antioxidant
assay
Test kit for Potential Antioxidant(PAO) (Code: KPA-050) Tech Info References
Traceelements Assay Fe / Iron Assay Kit (ferrozine) (Code: CFE-005) Tech Info References
Fe / Iron Assay Kit (Nitroso-PSAP) (Code: CFE-010) Tech Info References
UIBC Assay Kit (Bathophenanthroline) (Code: CBC-800) Tech Info References
Cu / Copper Assay Kit (3,5-DiBr-PAESA) (Code: CCU-400) Tech Info References
Zn / Zinc Assay Kit (5-Br-PAPS) (Code: CZN-001) Tech Info References
Ca / Calsium Assay Kit (Chlorophosphonazo III) (Code: CCA-020) Tech Info References
Ca / Calsium Assay Kit (o-Cresolphtalein-Complexone) (Code: CCA-030) Tech Info References
Mg / Magnesium Assay Kit (Xyridil Blue-I) (Code: CMG-035) Tech Info References
New/ Highly Sensitive 8-OHdG Check
No. Title Content
1 Animals applicable Q: What kind of animals is applicable to 8-OHdG ELISA?
A: Applicable to urine samples from human, mouse, rat, rabbit, dog, cattle, horse, etc. 8-OHdG Check ELISA kit can detect 8-OHdG in urine, serum, plasma, blood cells, tissue and cultured cells. There are two types of 8-OHdG ELISA products with different assay ranges. For urine samples, "New 8-OHdG Check" is suitable. For serum and tissue samples, we recommend to apply to "Highly Sensitive 8-OHdG Check" after pretreatment of samples.
The sample pretreatment procedure may be different depending on the type of samples. Please confirm the pretreatment procedure for your samples. If you are planning to measure 8-OHdG in urine samples, please remove cells and insluble materials from urine before application to ELISA.
2 Sample dilution Q: What should be used for sample dilution?
A: Phoshate buffered saline (PBS, pH7.4) is recommended. If urine sample is from healthy human, sample can be applied to "New 8-OHdG Check ELISA" as it is, but if urine sample is from patients, we recommend to dilute samples for 2 or 4 times with PBS.
3 Stability of urinary 8-OHdG Q: How urine samples should be stored?
A: 8-OHdG is known to chemically stable. Urine sample can be stored for 3 days at room temperature, for 7 days at 4 °C. But please take care for contamination of bacteria. If hydrochloric acid is used to prevent bacterial growth, please adjust pH before apply to ELISA.
Urine samples can be stored for longer period, for years at -80 °C. But please avoid repeated freeze / thaw cycle. When frozen urine sample is thaw, insolbule materials may be observed in some cases. Please remove insoluble materials by centrifuge before application to ELISA.

According to the recent paper, urine samples are stable for at least 2 years if stored at below -80 °C.
Y Matsumoto, et.al., J Occup Health 50, p366-372 (2008)
4 Type of samples applicable Q: What kind of samples are applied to 8-OHdG ELISA?
A: In addition to urine samples, serum / plasma, ascite samples, outer liquid of artificial dialysis and saliva samples are known to be applied to 8-OHdG ELISA. Pretreatment is required for these samples. DNA samples isolated from blood cells, sperm, organs and cultured cells can be applied to 8-OHdG ELISA.

[Assay example for saliva]
New biomarker evidence of oxidative DNA damage in whole saliva from clinically healthy and periodontally diseased individuals. M Takane, et. al., J Periodontol.73(5), p551-554 (2002)
5 8-OHdG concentration
in normal human urine:
A) Mean concentration: 13.6 ng/mL (250 males) and 10.3 ng/mL (250 females)
B) 8-OHdG extretion ratio: 8.6 ng/kg/h (250 males and 250 females)
C) Creatinin correction: 8.4 ng/mg CRE (250 males and 250 females)
6 Urine sampling and correction: 8-OHdG concentraion in random urine is not stable, depending on individual variation, the time in the day of sample collection, sex and some other factors. To obtain the best result, lease take care for sampling and correction methods.

About creatinine correction:
Creatinie correction is known to be a good method to obtain stable data. But it is not suitable for some researchers who are interested in the effect of exercise to oxidative stress. That's because creatinine concentration will be increased by exercise.

1) Effect of repeated exercise on urinary 8-hydroxy-deoxyguanosine excretion in humans. Free Radic Res. 1997 Jun;26(6):507-14. Okamura K, et. al.
In this paper, effects of running are assessed using 8-OHdG excretion per day. A significant increase of urinary 8-OHdG is observed (from 265.7+/-75.5 to 335.6+/-107.4 pmol/ kg/ day).

2) Habitual long-distance running does not enhance urinary excretion of 8-hydroxydeoxyguanosine. Eur J Appl Physiol Occup Physiol. 1997;75(5):p467-469. Pilger A,et. al.
In this paper, effects of running are assessed by urinary 8-OHdG with creatinine correction. Both 8-OHdG and creatinine concentration increased. But the effect of running to 8-OHdG level is not significant (0.12 - 6.45 µmol/mol CRE).
7 Trouble shootings: Please clice HERE to see technical tips for New 8-OHdG Check ELISA kit.
8 Serum / plasma samples: To detect 8-OHdG in serum and plasma samples, pretreatment is required. Please remove proteins by ultrafiltration, and apply to "Highly Sensitive 8-OHdG Check". For more information, click HERE.
9 Blood collection tubes: Blood collection tubes are commercially available. For example, NIPRO code.30-122 is suitable. Please note that serum 8-OHdG concentration may be increased if whole blood is placed for hours at room temperature. We recommend to start centrifuge in 20 minutes after blood collection. Serum samples must be stored below -20 °C.
10 Human serum 8-OHdG: 8-OHdG concentration in male serum is higher than that of female. Serum 8-OHdG is higher in smokers than in non-somokers.
Reference:The relationship between smoking habits and serum levels of 8-OHdG, oxidized LDL antibodies, Mn-SOD and carotenoids in rural Japanese residents., J Epidemiol 2003;13(1):p29-37.Suzuki K, et.al.
11 8-OHdG in tissue samples: After the hydrolysis of DNA extracted from tissues or cells, 8-OHdG levels can be measured using "Highly Sensitive 8-OHdG Check".
Reference:Coexposure to benzo[a]pyrene plus ultraviolet A induces 8-oxo-7,8-dihydro-2'-deoxyguanosine formation in human skin fibroblasts: preventive effects of anti-oxidant agents. Environmental Toxicology and Pharmacology 12, p37-42, 2002
12 Extraction of DNA: In order to measure 8-OHdG in tissue samples or cultured cells, DNA must be extracted by NaI method. NaI-based kits for DNA extractions are commercially available. For example,
DNA extractor® TIS kit (WAKO Chemicals, code. 296-67701) may be useful.

References:
ML Hamilton, et.al. A reliable assessment of 8-oxo-2-deoxyguanosine levels in nuclear and mitochondrial DNA using the sodium iodide method to isolate DNA.
Nucleic Acid Res. 29(10), p2117-2126(2001)

HJ Helbock, et. al. DNA oxidation matters: The HPLV-electrochemical detection assay of 8-oxo-deoxyguanosine and 8-oxo-guanine.
Proc Natl Acad Sci USA 95,p288-293(1998)
13 The sample volume required to
measure 8-OHdG in tissue or cells:
Q: What is the required sample volume to measure 8-OHdG in tissue or cells?
A: This depends on the experiment, however, usually 8-OHdG exists between 10-6 and 10-5 per dG in healthy human tissue. 8-OHdG can be measured between 0.125-10ng/mL using the "Highly Sensitive 8-OHdG Check", therefore in order to detect 8-OHdG certainly we recommend prepareing 200 µg/135µL of DNA.
14 Nuclease P1 reagent: You may be able to find nuclease P1 enzyme from local agency for biochemical reagents. In addition, nuclease P1 is available at our web site.

Code: OPNUCP1, Nuclease P1 from Penicillium citrinum: 25,000 JPY + freight.
15 About Argon substitution: Q: What does "Argon substitution for detection of 8-OHdG in tissue DNA" mean?
A: Argon substitution is to replace air (containing oxygen) with argon gas. That's because purified DNA may be oxidized in the presence of oxygen. To prevent the possibility of oxidization during sample preparation, we recommend performing "argon substitution" for reagents and samples. Argon gas is gently blown into the test tube containing the sample solution or reagents. However if the experiment will be carried out immediately, argon substitution will not be necessary.
Reference:CG Fraga, et. al.: Oxidative damage to DNA during aging: 8-hydroxy-2'-deoxyguanosine in rat organ DNA and urine. Proc Natl Acad Sci USA 87, p4533-4537, 1990.
16 Pretreatment of tissue samples:
(What is Chelex® treatment?)
It is reported that DNA can be oxidized by the Fenton reaction in the presence of trace divalent metal ions. Divalent metal ions can be removed by Chelex® resin (Chelex® is a registered trademark of BioRad Laboratories). For reliable results, Chelex® -treated water should be used to detect 8-OHdG in DNA samples.
17 Can't find Ultrafree-MC: As far as we know Ultrafree-MC (Millipore corporation, USA, catalog #UFC3LGC) is out of production. As an alternative, Microcon YM-10 (Millipore, catalog#42407) can be used.
18 Anatlytic testing service: JaICA is a registered medical testing institute in Japan, and is able to accept your urine and serum samples.
Testing service is also available at Genox corporation (Baltimore, USA).
Please take care that the testing service is for research use only. Not applicable for medical, diagnostic or other use.
19 Number of samples measured: Q:How many samples can be measured using 1 kit?
A:If experiment will be conducted in N=3, 18 samples can be measured (total 54 assays). One ELISA kit contains a 96-wells micro titer plate.
An example for plate layout.
In some cases, at the top and bottom row of wells, error of variance from the ¡Èedge effect¡É can be observed. Although all wells are prepared uniformly to obtain reliable data, it is recommended not to use wells in top and bottom rows. 6 levels by 3 wells (total 18 wells) will be used for standards. As a result, 54 wells can be used for samples.
20 How to calculate the results: Q: What algorithms are suitable for the standard curve?
A: 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.

If you don't have appropriate software, please feel free to send us your law data by e-mail entitled "Calibration of ELISA kit". We will convert your raw data to 8-OHdG concentration.
21 Partial use of the ELISA kit: Q: How many times can the ELISA kit be partially used?
A: 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.
22 Assay example for ovarian endometrioma: To detect 8-OHdG in ovarian endometrioma solution, please remove viscous components by centrifuge or filtration, and subsequently remove proteins by ultrafilration as the same protocol as that for serum samples.
Ovarian endometrioma: 0.58 ng/mL 8-OHdG
Serous Cystadenoma: 0.06 ng/mL 8-OHdG
Mucinous cystadenoma: 8-OHdG not detected.
Reference: S Fujii, 31th meeting of Japan BioIron Sociery, Abstruct in Japanese p9-10 (2007)
23 Which micro plate reader to be used? Any micro plate reader with 450nm filter may be suitable.
24 Protocol to hydrolyse DNA using DNase I: Protocols are suggested in following papers:
Measurement of 8-hydroxy-2'-deoxyguanosine in DNA by high-performance liquid chromatography-mass spectrometry: comparison with measurement by gas chromatography-mass spectrometry.Nucleic Acids Res 29(3) E12(2001).Dizdaroglu M, Jaruga P, Rodriguez H

Increased mitochondrial DNA damage and down-regulation of DNA repair enzymes in aged rodent retinal pigment epithelium and choroid. Mol Vis 14 p644-651(2008)Wang AL, Lukas TJ, Yuan M, Neufeld AH.
25 Reference wavelenghth: 600 - 650 nm would be useful.
26 Feature of our 8-OHdG ELISA: The monoclonal antibody (clone N45.1) which is used for 8-OHdG Check ELISAs is highly specific to 8-OHdG. It is reported that cross reactivity to 8-OHdG analogues (guanosine(G),7-methyl-G, 6-SH-G, 8-Bromo-G, dA, dC, dT, dI, dU, dG, O6-methyl-dG, 8-OHdA, guanine(Gua),O6-methyl-Gua, 8-OH-Gua, creatinine, 8-sulfhydryl-G, 8-OH-G) is less than 1% in comparison with that to 8-OHdG.
[Reference] S.Toyokuni, et.al.: Quantitative immunohistochemical determination of 8-hydroxy-2'deoxyguanosine by a monoclonal antibody N45.1: Its application to ferric nitrilotriacetate-induced renal carcinogenesis model. Lab. Invest. 76(3), p365-374 (1997)
27 Creatinine determination: Adjustment by creatinine concentration is useful for assessment of 8-OHdG in urine samples. Creatinine assay kits are commercially available.
LabAssay™ Creatinin, Code. 290-65901, WAKO Chemical Co.Ltd.
This kit can be applied to human and animal urine.

Ask a question about 8-OHdG ELISA by e-mail.

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List of references:
1) T.Osawa, A.Yoshida, S.Kawakishi, Y.Yamashita, and H.Ochi:
Protective role of dietary anti-oxidants in oxidative stress.
Oxidative Stress and Aging (Esd:R.G.Cutler, L.Packer, Bertram and A.Mori) p367-377, Birkhser Verlag Basel, Switzerland (1995)
2) Kasai H:
Analysis of a form of oxidative DNA damage, 8-hydroxy-2'-deoxyguanosine, as a marker of cellular oxidative stress during carcinogenesis.
Mutat Res. 387(3):147-63.(1997)
3) J.Leinonen, T.Lehtimi, S.Toyokuni, K.Okada, T.Tanaka, H.Hiai, H.Ochi, P.Laippala, V.Rantalaiho, O.Wirta, A.Pasternack, and H.Alho:
New biomarker evidence of oxidative DNA damage in patients with non-insulin-dependent diabetes mellitus.
FEBS Lett. 417, p150-152 (1997)
4) M.Erhola, S.Toyokuni, K.Okada, T.Tanaka, H.Hiai, H.Ochi, K.Uchida, T.Osawa, M.M.Nieminen, H.Alho, and P.K-Lehtinen:
Biomarker evidence of DNA oxidation in lung cancer patients: association of urinary 8-hydroxy-2' deoxyguanosine excretion with radiotherapy, chemotherapy, and response to treatment.
FEBS Lett. 409, p287-291 (1997)
5) H.Tsuboi, K.Kouda, H.Takeuchi, M.Takigawa, Y.Masamoto, M.Takeuchi, and H.Ochi:
8-Hydroxydeoxyguanosine in urine as an index of oxidative damage to DNA in the evaluation of atopic dermatitis.
Br. J. Dermatol. 138, p1033-1035 (1998)
6) Y.Miyake, K.Yamamoto, N.Tsujihara, and T.Osawa:
Protective effects of lemon flavonoids on oxidative stress in diabetic rats.
Lipids 33(7), p689-695 (1998)
7) M-H.Kang, M.Naito, N.Tsujihara, and T.Osawa:
Sesamolin inhibits lipid peroxidation in rat liver and kidney.
J. Nutr. 128, p1018-1022 (1998)
8) S.S.Kantha, S.Wada, H.Tanaka, M.Takeuchi, S.Watabe, and H.Ochi:
Carnosine sustains the retention of cell morphology in continuous fibroblast culture subjected to nutritional insult.
Biochem. Biophys. Res. Commun. 223, p278-282 (1996)
9) S.S.Kantha, S.Wada, M.Takeuchi, S.Watabe, and H.Ochi:
A sensitive method to screen for hydroxyl radical scavenging activity in natural food extracts using competitive inhibition ELISA for 8-hydroxydeoxyguanosine.
Biotechnol. Techniques 10(12), p933-936 (1996)
10) T.Arimoto, T.Yoshikawa, H.Takano and M.Kohno:
Generation of rective oxygen species and 8-hydroxy-2'-deoxyguanosine formation from diesel exhaust particles components in L1210 cells.
Japanese J. Pharmacol. 80, p49-54 (1999)
11) M.D.Evans, M.S.Cooke, I.D.Podmore, Q.Zheng, K.E.Herbert, and J.Lunec:
Discrepancies in the measurement of UVC-induced 8-oxo-2'-deoxyguanosine: Implications for the analysis of oxidative DNA damage.
Biochem. Biophys. Res. Commun. 259, p374-378 (1999)
ELISA results show high correlation between those of HPLC-ECD when 8-OHdG in DNA samples are assessed.
12) M.S.Cooke, M.D.Evans, I.D.Podmore, K.E.Herbert, N.Mistry, P.Mistry, P.T.Hickenbotham, A.Hussieni, H.R.Griffiths, and J.Lunec:
Novel repair action of vitamin C upon in vivo oxidative DNA damage.
FEBS Lett. 363, p363-367 (1998)
13) M.S.Cooke, M.D.Evans, K.E.Herbert, and J.Lunec:
Urinary 8-oxo-2'-deoxyguanosine - source, significance and supplements.
Free Rad. Res. 32, p381-397 (2000)
14) T. Shimoike, T. Inoguchi, F. Umeda, H. Nawata, K. Kawano and H. Ochi:
The meaning of serum levels of advanced glycosylation end products in diabetic nephropathy.
Metabolism 49(8), p1030-1035 (2000)
15) W. Y. Fan, K. Ogusu, K. Kouda, H. Nakamura, T. Satoh, H. Ochi and H. Takeuchi:
Reduced oxidative DNA damage by vegetable juice intake: A controlled trial.
J. Physiol. Anthropol. 19(6), p287-289 (2000)
16) H.Ochi, M.Hashimoto, J.Kurashige:
Assessment of functional tea in human using oxidative stress profile technique.
Proceedings of 2001 International Conference on O-Cha (tea) Culture and Science (2001)
17) T. Kuragano, T. Kuno, C. Yamamoto, Y. Nagura, S. Takahashi and K. Kanmatsuse:
Oxidative stress on DNA in chronic renal failure: the influence of different hemodialysis membranes.
J. Artif. Organs 4, p320-325 (2001)
18) M.S.Cooke, M.D.Evans, R.M.Burd, K.Patel, A.Barnard, J.Lunec and P.E.Hutchinson:
Induction and excretion of ultraviolet-induced 8-oxo-2'deoxyguanosine and thymine dimmers in vivo: implications for PUVA.
J. Invest. Dermatol. 116(2), p281-285 (2001)
19) M.S.Cooke, M.D.Evans and J.Lunec:
DNA repair: insights from urinary lesion analysis.
Free Rad. Res. 36(9), p929-932 (2002)
20) M.S.Cooke, M.D.Evans, N.Mistry and J.Lunec:
Role of dietary antioxidants in the prevention of in vivo oxidative DNA damage.
Nutr. Res. Rev. 15, p19-41 (2002)
21) T. Matsubasa, T. Uchino, S. Karashima, Y. Kondo, K. Maruyama, M. Tanimura and F. Endo:
Oxidative stress in very low birth weight infants as measured by urinary 8-OHdG.
Free Rad. Res. 36(2), p189-193 (2002)
22) Y.Ibuki, T.Warashina, T.Noro and R.Goto:
Coexposure to benzo[a]pyrene plus ultraviolet A induces 8-oxo-7,8-dihydro-2¡¦deoxyguanosine formation in human skin fibroblasts: preventive effects of anti-oxidant agents.
Environ. Toxicol. Pharmacol. 12, p37-42 (2002)
23) M.Kakimoto, T.Inoguchi, T.Sonta, H.Y.Yu, M.Imamura, T.Etoh, T.Hashimoto and H.Nawata:
Accumulation of 8-hydroxy-2¡¦deoxyguanosine and mitochondrial DNA deletion in kidney of diabetic rats.
Diabetes 51, p1588-1595 (2002)
24) Takane, Naoyuki Sugano, Hiroyasu Iwasaki, Yoshihiro Iwano, Noritaka Shimizu and Koichi Ito:
New biomarker evidence of oxidative DNA damage in whole saliva from clinically healthy and periodontally diseased individuals.
J.Periodontol 73(5), p551-554 (2002)
25) Kayoko Shimoi, Hiroshi Kasai, Naoko Yokota, Shinya Toyokuni and Naohide Kinae:
Comparison between high-performance liquid chromatography and enzyme-linked immunosorbent assay for the determination of 8-hydroxy-2' deoxyguanosine in human urine.
Cancer Epidemiol. Biomarkers & Prevention 11 p767-770 (2002)
26) Ha Won Kim, Akira Murakami, Marshall V Williams and Hajime Ohigashi:
Mutagenicity of reactive oxygen and nitrogen species as detected by co-culture of activated inflammatory leukocytes and AS52 cells.
Carcinogenesis 24(2), p235-241 (2003)
27) Nagayoshi Y, Kawano H, Hokamaki J, Miyamoto S, Kojima S, Shimomura H, Tsujita K, Sakamoto T, Yoshimura M, Ogawa H:
Urinary 8-hydroxy-2'-deoxyguanosine levels increase after reperfusion in acute myocardial infarction and may predict subsequent cardiac events.
Am J Cardiol. 95(4), p514-517 (2005)
28) Yutaka Otsuji, Eiji Kuwahara, Keiko Yuge, Goichi Yotsumoto, Takayuki Ueno, Kenichi Nakashiki, Shuichi Hamasaki, Sadatoshi Biro, Shinichi Minagoe, Robert A. Levine, Ryuzo Sakata, and Chuwa Tei:
Relation of Aneurysmectomy in Patients With Advanced Left Ventricular Remodeling to Postoperative Left Ventricular Filling Pressure, Redilatation With Ischemic Mitral Regurgitation.
Am J Cardiol 95, p517-521 (2005)
29) Someya T, Kaneko K, Yamada T, Yamashiro Y.:
Effect of a novel free radical scavenger, edaravone, on puromycin aminonucleoside induced nephrosis in rats.
Pediatr Nephrol.20(10):1430-1434(2005)
30) Watanabe E, Matsuda N, Shiga T, Kajimoto K, Ajiro Y, Kawarai H, Kasanuki H, Kawana M:
Significance of 8-hydroxy-2'-deoxyguanosine levels in patients with idiopathic dilated cardiomyopathy.
J Card Fail. 2006 Sep;12(7):527-32. (Journal of cardiac failure)
31) Morillas-Ruiz J, Zafrilla P, Almar M, Cuevas MJ, Lopez FJ, Abellan P, Villegas JA, Gonzalez-Gallego J:
The effects of an antioxidant-supplemented beverage on exercise-induced oxidative stress: results from a placebo-controlled double-blind study in cyclists
Eur J Appl Physiol. 2005 Aug 31:1-7
32) Shiihara T, Kato M, Ichiyama T, Takahashi Y, Tanuma N, Miyata R, Hayasaka K:
Acute encephalopathy with refractory status epilepticus: Bilateral mesial temporal and claustral lesions, associated with a peripheral marker of oxidative DNA damage
J Neurol Sci. 250(1-2):159-61(2006)
33) Roberto Cangemi, Francesco Angelico, Lorenzo Loffredo, Maria Del Ben, Pasquale Pignatelli, Alessandra Martini and Francesco Violi:
Oxidative stress-mediated arterial dysfunction in patients with metabolic syndrome: Effect of ascorbic acid.
Free Radical Biology and Medicine 43(5), p853-859 (2007)
34) Tomasz Dziaman, Daniel Gackowski, Rafal Rozalski, Agnieszka Siomek, Jaroslaw Szulczynski, Romuald Zabielski, Ryszard Olinski:
Urinary excretion rates of 8-oxoGua and 8-oxodG and antioxidant vitamins level as a measure of oxidative status in healthy, full-term newborns.
Free Radical Research, 41(9), p997-1004(2007)
35) Olga Espinosa, Jorge Jimenez-Almazan, Felipe J. Chaves, M. Carmen Tormos, Sonia Clapes, Antonio Iradi, Amparo Salvador, Marta Fandos, Josep Redon, Guillermo T. Saez:
Urinary 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxo-dG), a reliable oxidative stress marker in hypertension.
Free Radical Research 41(5), p546-554 (2007)
36) Parul R Patel, Ruth J Bevan, Nalini MIstry, Joseph Lunec:
Evidence of oligonucleotides containing 8-hydroxy-2'-deoxyguanosine in human urine.
Free Radical Biol Med 42, p552-558(2007)
37) Thompson HJ, Heimendinger J, Haegele A, Sedlacek SM, Gillette C, O'Neill C, Wolfe P, Conry C:
Effect of increased vegetable and fruit consumption on markers of oxidative cellular damage.
Carcinogenesis.20(12),p2261-2266(1999)
Intervention study of vegetable and fruit intake. Urinary 8-OHdG is reported to decrease from 49.6 +/- 12.4 to 21.4 +/- 2.2 ng/mg creatinine. It is also reported that 8-OHdG in white blood cells decreased from 7.9 +/- 1.2 to 6.2 +/- 0.8/106dG.
38) Reiko Nagasaka, Nobuaki Okamoto, Hideki Ushio:
Effects of caloric restriction on post-spawning death of ayu.
Exp Gerontol 40, p556-561(2005)
Detection of 8-OHdG in fish tissues (brain and liver).
39) Nakano M, Onodera A, Saito E, Tanabe M, Yajima K, Takahashi J, Chuyen NV: Effect of Astaxanthin in Combination with alpha-Tocopherol or Ascorbic Acid against Oxidative Damage in Diabetic ODS Rats. J Nutr Sci Vitaminol (Tokyo). 54(4)p329-334 (2008)
Astaxanthin is administrated to diabetes rat. Urinary 8-OHdG decreased in the group administrated mixture of astaxanthin and alpha-tocopherol.
40) Ogawa S, Mori T, Nako K, Ito S: Combination therapy with renin-angiotensin system inhibitors and the calcium channel blocker azelnidipine decreases plasma inflammatory markers and urinary oxidative stress markers in patients with diabetic nephropathy. Hypertens Res 31(6)p1147-1155(2008)
Urinary 8-OHdG is reported to decrease from 8.0 to 7.1 ng/mg creatinine when Ca-channel blocker is administrated to patients with diabetic nephropathy.
41) Yasuda M, Ide H, Furuya K, Yoshii T, Nishio K, Saito K, Isotani S, Kamiyama Y, Muto S, Horie S: Salivary 8-OHdG: a useful biomarker for predicting severe ED and hypogonadism. J Sex Med. 5(6),p1482-1491(2008)
8-OHdG in saliva is detected by 8-OHdG ELISA.
42) 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)
43) 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)
The level of NO, 8-OHdG, hexanoyl-lysine (HEL), SOD activity in seminal plasma were measured.
44) Tanuma N, Miyata R, Hayashi M, Uchiyama A, Kurata K: Oxidative stress as a biomarker of respiratory disturbance in patients with severe motor and intellectual disabilities. Brain Dev. 30(6)p402-409(2008)
8-OHdG concentration in urine samples from patients with severe motor and intellectual disabilities are higher (18.8 ± 9.0 ng/mg creatinine) in comparison with normal controls (10.5 ± 2.9 ng/mg creatinine).
45) 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 8-OHdG is reported to decrease by Etanercept treatent.
46) Fujimoto M, Tsuneyama K, Kainuma M, Sekiya N, Goto H, Takano Y, Terasawa K, Selmi C, Gershwin ME, Shimada Y: Evidence-based efficacy of Kampo formulas in a model of non alcoholic fatty liver. Exp Biol Med (Maywood). 233(3),p328-337(2008)
8-OHdG in Rabbit urine is detected. Urinary 8-OHdG is reported to be decreased in Kampo (Chinese traditional medicine) treated rabbits.
47) Chen CM, Liu JL, Wu YR, Chen YC, Cheng HS, Cheng ML, Chiu DT: Increased oxidative damage in peripheral blood correlates with severity of Parkinson's disease. Neurobiol Dis 2008 Dec 9.(in press)
8-OHdG level in peripheral white blood cells from Parkinson's disease is significantly higher than that of control.
48) Yano T, Shoji F, Baba H, Koga T, Shiraishi T, Orita H, Kohno H: Significance of the urinary 8-OHdG level as an oxidative stress marker in lung cancer patients. Lung Cancer 63(1) p111-114 (2009)
Urinary 8-OHdG level in smokers are higher than non-smokers.
49) Michael J Forlenza and Gregory E Miller: Increased serum levels of 8-hydroxy-2'-deoxyguanosine in clinical depression. Psychosomat Med 68, p1-7 (2006)
Serum 8-OHdG concentration in healthy person ranges from 0.20 to 1.26 ng/mL (mean 0.64 ng/mL).
50) Yee Ting Wong, Jan Gruber, Andrew M. Jenner, Mary Pei-Ern Ng, Runsheng Ruan, and Francis Eng Hock Tay: Elevation of oxidative-damage biomarkers during aging in F2 hybrid mice: Protection by chronic oral intake of resveratrol. Free Rad Biol Med 46(6), p799-809(2009)
Oral administration of Resveratrol to mouse is reported to decrease 8-OHdG in liver and heart.
51) Haixiang Su, Mervyn Gornitsky, Ana M. Velly, Hanling Yu, Michael Benarroch and Hyman M. Schipper: Salivary DNA, lipid, and protein oxidation in nonsmokers with periodontal disease. Free Radical Biology and Medicine 46(7), p914-921 (2009)
8-OHdG concentration in saliva from patients of periodontal disease is higher than that from contol. Total antioxidant level in saliva is also reported.
52) 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, 8-OHdG is reported to increase from 1.7 pg/ug DNA to 4.2 pg/ug DNA. In this paper essential role of selenoprotein P in the defense of corneas from oxidative stress.
53) Anna Szaflarska-Popawska, Agnieszka Siomek, Mieczysawa Czerwionka-Szaflarska, Daniel Gackowski, Rafa Rozaliski, Jolanta Guz, Anna Szpila, Ewelina Zarakowska and Ryszard Olinski:
Oxidatively Damaged DNA/Oxidative Stress in Children with Celiac Disease. Cancer Epidemiol Biomarkers Prev 19(8), p1960-1965(2010)
54) Hironori Nagasakaa, Yoshiyuki Okano, Madoka Aizawa, Takashi Miida, Tohru Yorifuji, Go Tajima, Nobuo Sakura, Tomozumi Takatani, Yoshitami Sanayama, Kenji Sugamoto, Mitsufumi Mayumi, Kunihiko Kobayashi, Kenichi Hirano, Masaki Takayanagi, Hirokazu Tsukahara:
Altered metabolisms of mediators controlling vascular function and enhanced oxidative stress in asymptomatic children with congenital portosystemic venous shunt. Metabolism Clinical and Experimental 59, p107-113(2010)
55) Masashi Kato, Machiko Iida, Yuji Goto, Takaaki Kondo, and Ichiro Yajima:
Sunlight Exposure Mediated DNA Damage in Young Adults. Cancer Epidemiol Biomarkers Prev 20(8), p1622-1628 (2011)

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Anti 8-OHdG / 8-oxo-dG antibody
No. Title: Content:
1 Applicable species: Applicable to test tissue samples from many kinds of animals like human, rabbit, rat, and so on. A step is necessary for mouse IgG from mouse tissue sample. For example, Vector M.O.M. Immunodetection kit can be used for mouse tissue sample. It has been reported that the excellent immunohistochemical image of mouse blood vessel with lower background could be observed. The alkaline phosphatase is recommended as a label when endogenous peroxidase activity causes background problems.
2 Protocols: Protocols for immunohistochemical detection are available HERE.
3 Immunohistochemical Conditions: Q: What is the point of 8-OHdG immunohistochemistry?
A: The staining conditions should be decided depending on the kinds of samples or its condition. Generally, 10-6 /dG of 8-OHdG exists in normal cells. Many researches have reported that the rise of oxidative stress increases the amount of 8-OHdG by several percent or several times. If the clear difference between the sample and the control tissues could not be observed, the staining conditions like the concentration of primary antibody or other factors should be changed. In addition, 8-OHdG may be generated by oxidation of normal DNA under the existences of generator of hydroxy radical such as H2O2 or UV. Please avoid using H2O2 for blocking of endogenous peroxidase activity. We recommend alkaline phosphatase as a label. The part stained is mainly nucleus.
4 Application to cultured cells: Q: The cultured cells come off from the slide glass during the process of antigen retrieval. How to prevent it?
A: That depends on types of cell or experimental conditions, but in some cases the cells come off easily from the slide glass. In this case, Bouin solution is recommended for fixation instead of formalin solution. Bouin-fixed samples can be stained without antigen retrieval.
5 Blocking of endogenous biotin: It may be necessary to block endogenous biotin for kidney, liver, prostate, and intestines tissues. In this case, please block endogenous biotin before primary antibody reaction.
1) Incubate with 0.001% Avidin / PBS for 10-15min.
2) Wash with PBS
3) Incubate with 0.001% Biotin / PBS for 10-15min.
4) Wash with PBS
6 Frozen samples: Anti 8-OHdG monoclonal antibody may be applicable to frozen sections, but not tested.
7 Mouse tissue:
(Detection by texas red.)
1) Blocking of internal biotin (Vector).
2) Blocking of endogenous mouse IgG (MOM kit, Vector).
3) Anti 8-OHdG antibody (5 µg/mL,30 min at room temperature).
4) Biotin-anti mouse IgG for 10 minutes at room temperature.
5) Texas red-avidin (10 µg/mL).
Reference: Fujihara et.al., PLoS ONE 3(9),e3119 (2008)
8 Cultured cells from human:
(Detection by Fluor488.)
1) Fixation by 4% paraformaldehyde.
2) RNase 100 µg/mL, 10mM Tris-HCl pH7.5, 1mM EDTA, 0.4M NaCl (37 °C for 1 hour).
3) Proteinase K 10 µg/mL (7 minutes at room temperature).
4) 4N HCl treatment (7 minutes at room temperature).
5) 0.2% Triton X in PBS.
6) Anti 8-OHdG antibody (dilution by 1:50).
7) Fluor488-anti mouse IgG goat antibody (dilution by 1:200).
Reference: Lee YA, at. al.,Biol Pharm Bull 31(6)p1265-1269(2008)

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List of references:
1) 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)
2) 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 (1996)
3) S.Toyokuni, T.Tanaka, Y.Hattori, Y,Nishiyama, A.Yoshida, K.Uchida, H.Hiai, H.Ochi, and T.Osawa:
Quantitative immunohistochemical determination of 8-hydroxy-2'deoxyguanosine by a monoclonal antibody N45.1: Its application to ferric nitrilotriacetate-induced renal carcinogenesis model.
Lab. Invest. 76(3), p365-374 (1997)
4) S.Takahashi, M.Hirose, S.Tamano, M.Ozaki, S.Orita, M.Takeuchi, H.Ochi, S.Fukuda, H.Kasai, and T.Shirai:
Immunohistochemical detection of 8-hydroxy-2¡¦deoxyguanosine in paraffin-embedded sections of rat liver after carbon tetrachloride treatment.
Toxicol. Pathol. 26, p247-252 (1998)
5) N.U.Ahmed, M.Ueda, O.Nikaido, T.Osawa and M.Ichihashi:
High levels of 8-hydroxy-2'deoxyguanosine appear in normal human epidermis after a singer dose of ultraviolet radiation.
Br. J. Dermatol. 140, p226-231 (1999)
6) Y.Ihara, S.Toyokuni, K.Uchida, H.Odaka, T.Tanaka, H.Ikeda, H.Hiai, Y.Seino, and Y.Yamada:
Hyperglycemia causes oxidative stress in pancreatic -cells of GK rats, a model of type 2 diabetes.
Diabetes 48, p927-932 (1999)
7) 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)
8) S.Toyokuni, Y.Iwasa, S.Kondo, T.Tanaka, H.Ochi, and H.Hiai:
Intranuclear distribution of 8-hydroxy-2'-deoxyguanosine: An immunocytochemical study.
J. Histochem. Cytochem. 47(6), p833-835 (1999)
9) S.Toyokuni:
Reactive oxygen species-induced molecular damage and its application in pathology.
Pathol. Internat. 49, p91-102 (1999)
10) S.Toyokuni:
An in vitro Feton reaction model and the application of immunohistochemistry to detect oxidative damage.
Models and Methods in Cell Signaling and Gene Expression: Applications to Oxidative Stress Research., Chapter four pp40-54 (2000)
11) K.Yamagami, Y.Yamamoto, M.Kume, Y.Ishikawa, Y.Yamaoka, H.Hiai and S.Toyokuni:
Formation of 8-hydroxy-2'deoxyguanosine and 4-hydroxy-2-nonenal-modified proteins in rat liver after ischemia-reperfusion: Distinct localization of the two oxidatively modified products. Antioxidants & Redox Signaling, 2(1), p127-136 (2000)
12) D.Nakae, H.Akai, H.Kishida, O.Kusuoka, M.Tsutsumi, and Y.Konishi:
Age and organ dependent spontaneous generation of nuclear 8-hydroxydeoxyguanosine in male Fischer 344 rats.
Lab. Invest. 80(2), p249-261 (2000)
13) S.Liardet, C.Scaletta, R.Panizzon, P.Hohlfeld and L.L.Applegate:
Protection against pyrimidine dimmers, p53, and 8-hydroxy-2'deoxyguanosine expression in ultraviolet-irradiated human skin by sunscreens: difference between UVB + UVA and UVB alone sunscreens.
J. Investigative Dermatol. 117(6), p1437-1441 (2001)
14) 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)
15) S Akatsuka, TT Aung, KK Dutta, L Jiang, HW Lee, YT Liu, J Onuki, T Shirase, K Yamasaki, H Ochi, Y Naito, T Yoshikawa, H Kasai, Y Tominaga, K Sakumi, Y Nakabeppu, Y Kawai, K Uchida, A Yamasaki, T Tsuruyama, Y Yamada and S Toyokuni:
Contracting genome-wide distribution of 8-hydroxyguanine and acrolein-modified adenine during oxidative stress induced renal carcinogenesis.
Am. J. Pathol. 169(4), p1328-1342 (2006)
16) Subrata K. Biswas, Jose B. Lopes De Faria:
Which comes first: Renal inflammation or oxidative stress in spontaneously hypertensive rats?
Free Radical Research, 41(2), p216 -224(2007)
17) JUN YAMAKOSHI, FUJIO OTSUKA, ATSUSHI SANO, SHOICHI TOKUTAKE, MAKOTO SAITO, MAMORU KIKUCHI and YOSHIRO KUBOTA:
Lightening Effect on Ultraviolet-Induced Pigmentation of Guinea Pig Skin by Oral Administration of a Proanthocyanidin-Rich Extract from Grape Seeds.
Pigemt Cell Res 16, p629-638(2003)
18) T. Ichiseki, A. Kaneuji, S. Katsuda1, Y. Ueda,T. Sugimori and T. Matsumoto:
DNA oxidation injury in bone early after steroid administration is involved in the pathogenesis of steroid-induced osteonecrosis.
Rheumatology 44,p456-460(2005)
19) Nakajima Y, Inokuchi Y, Shimazawa M, Otsubo K, Ishibashi T, Hara H: Astaxanthin, a dietary carotenoid, protects retinal cells against oxidative stress in-vitro and in mice in-vivo. J Pharm Pharmacol. 60(10), p1365-1374(2008)
20) Fujihara M, Nagai N, Sussan TE, Biswal S, Handa JT: Chronic cigarette smoke causes oxidative damage and apoptosis to retinal pigmented epithelial cells in mice. PLoS ONE. 3(9) e3119(2008)
Immunohistochemistry of 8-OHdG in mouse retinal cells using texas-red.
21) Lee YA, Cho EJ, Yokozawa T: Protective effect of persimmon (Diospyros kaki) peel proanthocyanidin against oxidative damage under H2O2-induced cellular senescence. Biol Pharm Bull 31(6)p1265-1269(2008)
Detection of 8-OHdG in cultured cells using Fluor488. Cells are stimulated by 300uM H2O2 for 2 hours.
22) Shinya Toyokuni, Ayaka Hirao, Tamae Wada, Ryoji Nagai, Akira Date, Takashi Yoshii, Shinya Akatsuka, Yoriko Yamashita and Akira Kawada: Age- and sun exposure-dependent differences in 8-hydroxy-2'-deoxyguanosine and Nε-(carboxymethyl)lysine in human epidermis. J Clin Biochem Nutr: doi 10.3164/jcbn.11-05 (2011)
Immunohistochemical detection of 8-OHdG and CML in sun-exposed human skin.
23) Dhillon H, Chikara S, Reindl KM: Piperlongumine induces pancreatic cancer cell death by enhancing reactive oxygen species and DNA damage. Toxicol Rep.1:309-318(2014)
24) Sawada O, Perusek L, Kohno H, Howell SJ, Maeda A, Matsuyama S, Maeda T: All-trans-retinal induces Bax activation via DNA damage to mediate retinal cell apoptosis. Exp Eye Res. 123,p27-36(2014). doi: 10.1016/j.exer.2014.04.003.

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