Skip to main content
PMC home page
Biochemical Journal logoLink to Biochemical Journal
. 2004 Jun 1;380(Pt 2):541–548. doi: 10.1042/BJ20040004

A high-throughput and sensitive methodology for the quantification of urinary 8-hydroxy-2'-deoxyguanosine: measurement with gas chromatography-mass spectrometry after single solid-phase extraction.

Hai-Shu Lin 1, Andrew M Jenner 1, Choon Nam Ong 1, Shan Hong Huang 1, Matthew Whiteman 1, Barry Halliwell 1
PMCID: PMC1224185  PMID: 14992687

Abstract

8-hydroxy-2'-deoxyguanosine (8OHdG) is a widely used biomarker for the measurement of endogenous oxidative DNA damage. A sensitive method for the quantification of 8OHdG in urine by single solid-phase extraction and GC-MS (gas chromatography with MS detection) using selective ion monitoring is described in the present study. After solid-phase extraction, samples are freeze-dried, derivatized by trimethylsilylation and analysed by GC-MS. The urinary 8OHdG was quantified using heavy isotope dilution with [18O]8OHdG. The recovery of 8OHdG after the solid-phase extraction ranged from 70 to 80% for a wide range of urinary 8OHdG levels. Using 1 ml of urine, the limit of quantification was >2.5 nM (2.5 pmol/ml) and the calibration curve was linear in the range 2.5-200 nM. This method was applied to measure 8OHdG in urine samples from 12 healthy subjects. The intra- and inter-day variations were <9%. Urinary 8OHdG levels in spot urine samples from four healthy subjects were also measured for 1 week and, again, the variation was small. The presence of H2O2 in urine did not cause artifactual formation of 8OHdG. Since this assay is simple, rapid, sensitive and reproducible, it seems suitable to be used as a routine methodology for the measurement of urinary excretion of 8OHdG in large population studies.

Full Text

The Full Text of this article is available as a PDF (136.2 KB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Beckman K. B., Ames B. N. Oxidative decay of DNA. J Biol Chem. 1997 Aug 8;272(32):19633–19636. doi: 10.1074/jbc.272.32.19633. [DOI] [PubMed] [Google Scholar]
  2. Bogdanov M. B., Beal M. F., McCabe D. R., Griffin R. M., Matson W. R. A carbon column-based liquid chromatography electrochemical approach to routine 8-hydroxy-2'-deoxyguanosine measurements in urine and other biologic matrices: a one-year evaluation of methods. Free Radic Biol Med. 1999 Sep;27(5-6):647–666. doi: 10.1016/s0891-5849(99)00113-6. [DOI] [PubMed] [Google Scholar]
  3. Chiou Chiuan Chian, Chang Pi Yueh, Chan Err Cheng, Wu Tsu Lan, Tsao Kuo Chien, Wu James T. Urinary 8-hydroxydeoxyguanosine and its analogs as DNA marker of oxidative stress: development of an ELISA and measurement in both bladder and prostate cancers. Clin Chim Acta. 2003 Aug;334(1-2):87–94. doi: 10.1016/s0009-8981(03)00191-8. [DOI] [PubMed] [Google Scholar]
  4. Cooke Marcus S., Evans Mark D., Dizdaroglu Miral, Lunec Joseph. Oxidative DNA damage: mechanisms, mutation, and disease. FASEB J. 2003 Jul;17(10):1195–1214. doi: 10.1096/fj.02-0752rev. [DOI] [PubMed] [Google Scholar]
  5. Cooke Marcus S., Evans Mark D., Lunec Joseph. DNA repair: insights from urinary lesion analysis. Free Radic Res. 2002 Sep;36(9):929–932. doi: 10.1080/1071576021000006635. [DOI] [PubMed] [Google Scholar]
  6. Cooke Marcus S., Lunec Joseph, Evans Mark D. Progress in the analysis of urinary oxidative DNA damage. Free Radic Biol Med. 2002 Dec 15;33(12):1601–1614. doi: 10.1016/s0891-5849(02)01146-2. [DOI] [PubMed] [Google Scholar]
  7. Dizdaroglu M. Application of capillary gas chromatography-mass spectrometry to chemical characterization of radiation-induced base damage of DNA: implications for assessing DNA repair processes. Anal Biochem. 1985 Feb 1;144(2):593–603. doi: 10.1016/0003-2697(85)90158-7. [DOI] [PubMed] [Google Scholar]
  8. Dizdaroglu M. Characterization of free radical-induced damage to DNA by the combined use of enzymatic hydrolysis and gas chromatography-mass spectrometry. J Chromatogr. 1986 Oct 3;367(2):357–366. doi: 10.1016/s0021-9673(00)94856-8. [DOI] [PubMed] [Google Scholar]
  9. Dizdaroglu Miral, Jaruga Pawel, Birincioglu Mustafa, Rodriguez Henry. Free radical-induced damage to DNA: mechanisms and measurement. Free Radic Biol Med. 2002 Jun 1;32(11):1102–1115. doi: 10.1016/s0891-5849(02)00826-2. [DOI] [PubMed] [Google Scholar]
  10. Gackowski D., Rozalski R., Roszkowski K., Jawien A., Foksiński M., Olinski R. 8-Oxo-7,8-dihydroguanine and 8-oxo-7,8-dihydro-2'-deoxyguanosine levels in human urine do not depend on diet. Free Radic Res. 2001 Dec;35(6):825–832. doi: 10.1080/10715760100301321. [DOI] [PubMed] [Google Scholar]
  11. Gackowski Daniel, Speina Elzbieta, Zielinska Maja, Kowalewski Janusz, Rozalski Rafal, Siomek Agnieszka, Paciorek Tomasz, Tudek Barbara, Olinski Ryszard. Products of oxidative DNA damage and repair as possible biomarkers of susceptibility to lung cancer. Cancer Res. 2003 Aug 15;63(16):4899–4902. [PubMed] [Google Scholar]
  12. Halliwell Barry. Effect of diet on cancer development: is oxidative DNA damage a biomarker? Free Radic Biol Med. 2002 May 15;32(10):968–974. doi: 10.1016/s0891-5849(02)00808-0. [DOI] [PubMed] [Google Scholar]
  13. Harman S. Mitchell, Liang Lynn, Tsitouras Panayiotis D., Gucciardo Frank, Heward Christopher B., Reaven Peter D., Ping Wei, Ahmed Alaa, Cutler Richard G. Urinary excretion of three nucleic acid oxidation adducts and isoprostane F(2)alpha measured by liquid chromatography-mass spectrometry in smokers, ex-smokers, and nonsmokers. Free Radic Biol Med. 2003 Nov 15;35(10):1301–1309. doi: 10.1016/j.freeradbiomed.2003.07.003. [DOI] [PubMed] [Google Scholar]
  14. Holmberg I., Stål P., Hamberg M. Quantitative determination of 8-hydroxy-2'-deoxyguanosine in human urine by isotope dilution mass spectrometry: normal levels in hemochromatosis. Free Radic Biol Med. 1999 Jan;26(1-2):129–135. doi: 10.1016/s0891-5849(98)00181-6. [DOI] [PubMed] [Google Scholar]
  15. Inoue T., Mu Z., Sumikawa K., Adachi K., Okochi T. Effect of physical exercise on the content of 8-hydroxydeoxyguanosine in nuclear DNA prepared from human lymphocytes. Jpn J Cancer Res. 1993 Jul;84(7):720–725. doi: 10.1111/j.1349-7006.1993.tb02035.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Jenner A., England T. G., Aruoma O. I., Halliwell B. Measurement of oxidative DNA damage by gas chromatography-mass spectrometry: ethanethiol prevents artifactual generation of oxidized DNA bases. Biochem J. 1998 Apr 15;331(Pt 2):365–369. doi: 10.1042/bj3310365. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Kasai H., Iwamoto-Tanaka N., Miyamoto T., Kawanami K., Kawanami S., Kido R., Ikeda M. Life style and urinary 8-hydroxydeoxyguanosine, a marker of oxidative dna damage: effects of exercise, working conditions, meat intake, body mass index, and smoking. Jpn J Cancer Res. 2001 Jan;92(1):9–15. doi: 10.1111/j.1349-7006.2001.tb01041.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Kvasnicová Vladimira, Samcová Eva, Jursová Alice, Jelínek Ivan. Determination of 8-hydroxy-2'-deoxyguanosine in untreated urine by capillary electrophoresis with UV detection. J Chromatogr A. 2003 Jan 24;985(1-2):513–517. doi: 10.1016/s0021-9673(02)01527-3. [DOI] [PubMed] [Google Scholar]
  19. Long L. H., Evans P. J., Halliwell B. Hydrogen peroxide in human urine: implications for antioxidant defense and redox regulation. Biochem Biophys Res Commun. 1999 Sep 7;262(3):605–609. doi: 10.1006/bbrc.1999.1263. [DOI] [PubMed] [Google Scholar]
  20. Long L. H., Halliwell B. Coffee drinking increases levels of urinary hydrogen peroxide detected in healthy human volunteers. Free Radic Res. 2000 May;32(5):463–467. doi: 10.1080/10715760000300461. [DOI] [PubMed] [Google Scholar]
  21. Mei Su-Rong, Yao Qing-Hong, Cai Ling-Suang, Xing Jun, Xu Guo-Wang, Wu Cai-Ying. Capillary electrophoresis with end-column amperometric detection of urinary 8-hydroxy-2'-deoxyguanosine. Electrophoresis. 2003 May;24(9):1411–1415. doi: 10.1002/elps.200390181. [DOI] [PubMed] [Google Scholar]
  22. Nakano Masaru, Kawanishi Yoshikazu, Kamohara Seika, Uchida Yoshiko, Shiota Mami, Inatomi Yuka, Komori Toyonori, Miyazawa Katsumi, Gondo Kazumi, Yamasawa Ikuhiro. Oxidative DNA damage (8-hydroxydeoxyguanosine) and body iron status: a study on 2507 healthy people. Free Radic Biol Med. 2003 Oct 1;35(7):826–832. doi: 10.1016/s0891-5849(03)00432-5. [DOI] [PubMed] [Google Scholar]
  23. Pilger A., Germadnik D., Schaffer A., Theiler A., Pils P., Sluka F., Winker N., Rüdiger H. W. 8-Hydroxydeoxyguanosine in leukocyte DNA and urine of quartz-exposed workers and patients with silicosis. Int Arch Occup Environ Health. 2000 Jul;73(5):305–310. doi: 10.1007/s004200000117. [DOI] [PubMed] [Google Scholar]
  24. Pilger A., Ivancsits S., Germadnik D., Rüdiger H. W. Urinary excretion of 8-hydroxy-2'-deoxyguanosine measured by high-performance liquid chromatography with electrochemical detection. J Chromatogr B Analyt Technol Biomed Life Sci. 2002 Oct 5;778(1-2):393–401. doi: 10.1016/s0378-4347(01)00449-2. [DOI] [PubMed] [Google Scholar]
  25. Pourcelot S., Faure H., Firoozi F., Ducros V., Tripier M., Hee J., Cadet J., Favier A. Urinary 8-oxo-7,8-dihydro-2'-deoxyguanosine and 5-(hydroxymethyl) uracil in smokers. Free Radic Res. 1999 Mar;30(3):173–180. doi: 10.1080/10715769900300191. [DOI] [PubMed] [Google Scholar]
  26. Proteggente Anna R., England Timothy G., Rehman Almas, Rice-Evans Catherine A., Halliwell Barry. Gender differences in steady-state levels of oxidative damage to DNA in healthy individuals. Free Radic Res. 2002 Feb;36(2):157–162. doi: 10.1080/10715760290006475. [DOI] [PubMed] [Google Scholar]
  27. Ravanat J. L., Guicherd P., Tuce Z., Cadet J. Simultaneous determination of five oxidative DNA lesions in human urine. Chem Res Toxicol. 1999 Sep;12(9):802–808. doi: 10.1021/tx980194k. [DOI] [PubMed] [Google Scholar]
  28. Renner T., Fechner T., Scherer G. Fast quantification of the urinary marker of oxidative stress 8-hydroxy-2'-deoxyguanosine using solid-phase extraction and high-performance liquid chromatography with triple-stage quadrupole mass detection. J Chromatogr B Biomed Sci Appl. 2000 Feb 11;738(2):311–317. doi: 10.1016/s0378-4347(99)00542-3. [DOI] [PubMed] [Google Scholar]
  29. Rozalski Rafal, Gackowski Daniel, Roszkowski Krzysztof, Foksinski Marek, Olinski Ryszard. The level of 8-hydroxyguanine, a possible repair product of oxidative DNA damage, is higher in urine of cancer patients than in control subjects. Cancer Epidemiol Biomarkers Prev. 2002 Oct;11(10 Pt 1):1072–1075. [PubMed] [Google Scholar]
  30. Rozalski Rafał, Winkler Piotr, Gackowski Daniel, Paciorek Tomasz, Kasprzak Heliodor, Olinski Ryszard. High concentrations of excised oxidative DNA lesions in human cerebrospinal fluid. Clin Chem. 2003 Jul;49(7):1218–1221. doi: 10.1373/49.7.1218. [DOI] [PubMed] [Google Scholar]
  31. Saito S., Yamauchi H., Hasui Y., Kurashige J., Ochi H., Yoshida K. Quantitative determination of urinary 8-hydroxydeoxyguanosine (8-OH-dg) by using ELISA. Res Commun Mol Pathol Pharmacol. 2000;107(1-2):39–44. [PubMed] [Google Scholar]
  32. Shimoi Kayoko, Kasai Hiroshi, Yokota Naoko, Toyokuni Shinya, Kinae Naohide. 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 Prev. 2002 Aug;11(8):767–770. [PubMed] [Google Scholar]
  33. Slot C. Plasma creatinine determination. A new and specific Jaffe reaction method. Scand J Clin Lab Invest. 1965;17(4):381–387. doi: 10.3109/00365516509077065. [DOI] [PubMed] [Google Scholar]
  34. Suzuki J., Inoue Y., Suzuki S. Changes in the urinary excretion level of 8-hydroxyguanine by exposure to reactive oxygen-generating substances. Free Radic Biol Med. 1995 Mar;18(3):431–436. doi: 10.1016/0891-5849(94)00152-a. [DOI] [PubMed] [Google Scholar]
  35. Toyokuni S., Tanaka T., Hattori Y., Nishiyama Y., Yoshida A., Uchida K., Hiai H., Ochi H., Osawa T. 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. 1997 Mar;76(3):365–374. [PubMed] [Google Scholar]
  36. Weimann Allan, Belling Dorthe, Poulsen Henrik E. Quantification of 8-oxo-guanine and guanine as the nucleobase, nucleoside and deoxynucleoside forms in human urine by high-performance liquid chromatography-electrospray tandem mass spectrometry. Nucleic Acids Res. 2002 Jan 15;30(2):E7–E7. doi: 10.1093/nar/30.2.e7. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Yoshida Rie, Ogawa Yasutaka, Kasai Hiroshi. Urinary 8-oxo-7,8-dihydro-2'-deoxyguanosine values measured by an ELISA correlated well with measurements by high-performance liquid chromatography with electrochemical detection. Cancer Epidemiol Biomarkers Prev. 2002 Oct;11(10 Pt 1):1076–1081. [PubMed] [Google Scholar]

Articles from Biochemical Journal are provided here courtesy of The Biochemical Society

RESOURCES