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.
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Research Article|
June 01 2004
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
;
Hai-Shu LIN
*Department of Biochemistry, Faculty of Medicine, National University of Singapore, MD 7, Medical Drive, Singapore 117597, Singapore
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Andrew M. JENNER
;
Andrew M. JENNER
*Department of Biochemistry, Faculty of Medicine, National University of Singapore, MD 7, Medical Drive, Singapore 117597, Singapore
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Choon Nam ONG
;
Choon Nam ONG
†Department of Community, Occupational and Family Medicine, Faculty of Medicine, National University of Singapore, MD 3, Medical Drive, Singapore 117597, Singapore
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Shan Hong HUANG
;
Shan Hong HUANG
*Department of Biochemistry, Faculty of Medicine, National University of Singapore, MD 7, Medical Drive, Singapore 117597, Singapore
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Matthew WHITEMAN
;
Matthew WHITEMAN
*Department of Biochemistry, Faculty of Medicine, National University of Singapore, MD 7, Medical Drive, Singapore 117597, Singapore
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Barry HALLIWELL
Barry HALLIWELL
1
*Department of Biochemistry, Faculty of Medicine, National University of Singapore, MD 7, Medical Drive, Singapore 117597, Singapore
1To whom correspondence should be addressed (e-mail bchbh@nus.edu.sg).
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Biochem J (2004) 380 (2): 541–548.
Article history
Received:
January 05 2004
Revision Received:
February 24 2004
Accepted:
March 02 2004
Accepted Manuscript online:
March 02 2004
Citation
Hai-Shu LIN, Andrew M. JENNER, Choon Nam ONG, Shan Hong HUANG, Matthew WHITEMAN, Barry HALLIWELL; 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. Biochem J 1 June 2004; 380 (2): 541–548. doi: https://doi.org/10.1042/bj20040004
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