Measurement and Meaning of Oxidatively Modified DNA Lesions in Urine

Abstract

Background: Oxidatively generated damage to DNA has been implicated in the pathogenesis of a wide variety of diseases. The noninvasive assessment of such damage, i.e., in urine, and application to large-scale human studies are vital to understanding this role and devising intervention strategies. Methods: We have reviewed the literature to establish the status quo with regard to the methods and meaning of measuring DNA oxidation products in urine. Results: Most of the literature focus upon 8-oxo-7,8-dihydro-2 ¶-deoxyguanosine (8-oxodG), and whereas a large number of these reports concern clinical conditions, there remains (a) lack of consensus between methods, (b) possible contribution from diet and/or cell death, (c) no definitive DNA repair source of urinary 2 ¶-deoxyribonucleoside lesions, and (d) no reference ranges for healthy or diseased individuals. Conclusions: The origin of 8-oxodG is not identified; however, recent cell culture studies suggest that the

Analysis of 8-hydroxy-2′-deoxyguanosine in human urine using hydrophilic interaction chromatography with tandem mass spectrometry

“…fmol [17], 7 fmol (S/N=3) [18], 5 fmol (S/N=4) [19] and 7.5 fmol/injection (S/N=3) [27,31]. The low detection limit of method enables the measurement of basal levels of urinary 8-OHdG in non-exposed healthy subjects that were not quantified in the previous report [27,31].…”

“…The low detection limit of method enables the measurement of basal levels of urinary 8-OHdG in non-exposed healthy subjects that were not quantified in the previous report [27,31]. Furthermore, the analysis can be completed in 10 min and doesn't require washing and re-equilibrating the column, which makes it well suited for continuous analyses.…”

“…Urinary 8-OHdG has been analyzed by several methods, such as enzyme-linked immunosorbent assay (ELISA) [12,13], high-performance liquid 4 chromatography with electrochemical detection (HPLC-ECD) [14], gas chromatography with mass spectrometry (GC/MS) [15,16], and liquid chromatography with tandem mass spectrometry (LC/MS/MS) [17][18][19][20][21][22][23][24][25][26]. The ELISA method suffers from the problem of non-selectivity because the antibody may cross-react with other substances present in urine [12,27,28]. HPLC-ECD has often been used [29], but it suffers from possible interference from the biological matrix, incompatibility of a stable isotope labeled internal standard [16,21].…”

“…In addition, aqueous mobile phases that are used to retain polar compounds on reversed-phase columns are not suited for electrospray ionization (ESI) conditions [30]. Indeed, urinary 8-OHdG levels have even 5 been below the detection limit of LC-MS/MS method (7.5 fmol/injection, S/N= 3) [27,31]. Therefore, a more sensitive LC-MS/MS method is required to measure basal levels of urinary 8-OHdG in non-exposed healthy subjects.…”

Combination of azathioprine and UVA irradiation is a major source of cellular 8-oxo-7,8-dihydro-2′-deoxyguanosine

“…The current understanding is that the most likely and logical source of urinary 8-oxodG is from sanitisation of the 2'-deoxyribonucleotide pool [48], this and the discussion in the paragraph above raise the question, is the dNTP pool a target for 6-TG induced oxidation? It seems highly likely that 6-TG is present in the dNTP pool as, for incorporation into DNA to occur, 6-TG must be converted into 6-TdGTP which is then a substrate for DNA polymerases [49].…”