Abstract
Non-invasive monitoring of oxidative stress is highly desirable. Urinary 7,8-8-oxo-7,8-dihydro-2’-deoxyguanosine (8-oxodG) is a biologically relevant and convenient analytical target. However, immunoassays can over-estimate levels of urinary 8-oxodG. Measurement of more than one DNA oxidation product in urine would be advantageous in terms of mechanistic information. Urines samples were analysed for 8-oxodG by solid-phase extraction/LC-MS/MS and ELISA. The solid-phase extraction/LC-MS/MS assay was also applied to the analysis of urinary 7,8-dihydro-8-oxo-2’-deoxyadenosine (8-oxodA). Concurring with previous reports, urinary 8-oxodG measured by ELISA was significantly higher than levels measured by LC-MS/MS. However, apparent improvement in the specificity of the commercially available Japanese Institute for the Control of Ageing (JaICA) ELISA brought mean LC-MS/MS and ELISA measurements of urinary 8-oxodG into agreement. Urinary 8-oxodA was undetectable in all urines, despite efficient recovery by solid phase extraction. Exploitation of the advantages of ELISA may be enhanced by a simple modification to the assay procedure, although chromatographic techniques still remain the ‘gold standard’ techniques for analysis of urinary 8-oxodG. Urinary 8-oxodA is either not present or below the limit of detection of the instrumentation.
Highlights
There is growing literature on the role of oxidative stress in a wide variety of malignant and non-malignant conditions [1, 2]
ELISA and LC-MS/MS of urinary 8-oxodG We have attempted to enhance the specificity of N45.1 binding in the Japanese Institute for the Control of Ageing (JaICA) competitive ELISA, by performing the primary antibody incubation step at 4 °C, overnight, rather than for one hour at 37 °C (Figure 2)
Linear regression analysis revealed a significant correlation between 4 °C ELISA 8oxodG values, and those obtained by LC-MS/MS
Summary
There is growing literature on the role of oxidative stress in a wide variety of malignant and non-malignant conditions [1, 2]. Long term storage of samples, such as those in biobanks, are likely to lead to the formation of adventitious damage, further limiting their use These drawbacks may be avoided by using urinary markers of oxidative stress, and the analysis of 8-oxo-7,8-dihydro-2’-deoxyguanosine (8-oxodG), in particular has received widespread use, appearing eminently stable during long term storage [4]. The relatively large number of reports measuring urinary 8-oxodG as a biomarker of oxidative stress or even DNA damage, have been made in many cases without due consideration of the source of this lesion in urine. While the contributions of diet and cell death to the presence of urinary 8-oxoGua and 8-oxodG, for example, continue to be assessed, the prevailing view is that neither of these processes contribute significantly to the presence of these compounds in urine, but a full understanding of their origins is critically important to evaluating their utility as biomarkers. The examination of the origins of other lesions remains largely un-investigated,
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