Analysis of urinary 8-oxo-7,8-dihydro-purine-2’-deoxyribonucleosides by LC-MS/MS and improved ELISA

Abstract

Non-invasive monitoring of oxidative stress is highly desirable. Urinary 7,8-dihydro-8-oxo-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 our instrumentation.

Analytics of oxidative stress markers in the early diagnosis of oxygen DNA damage

“…Therefore the preparation of a urine sample for analysis by HPLC-ECD for the 8-OHdG content relies heavily on solid-phase extraction (SPE) technology, which allows for significant enrichment of the analyte. [49][50][51][52][53][54] If the analyte is 8-hydroxy-2'-deoxyguanosine -considered as a product of hydrolysis of cellular DNA -it is necessary to carry out hydrolysis of the DNA, and digestion with the appropriate enzymes: nuclease P1 and alkaline phosphatase. Nuclease P1 cuts the DNA, leading to the formation of the corresponding nucleosides.…”

“…These tests are performed using commercially available kits. 51,[62][63][64][65][66] The alkaline elution method is another indirect method. Before using this method, it is necessary to use formamidopyrimidine-DNA glycosylase (FPG), which creates single DNA strand breaks in the modified purine bases -the alkaline elution test is used for the detection of such breaks.…”

A commentary on “Urea, the most abundant component in urine, cross-reacts with a commercial 8-OH-dG ELISA kit and contributes to overestimation of urinary 8-OH-dG”. What is ELISA detecting?

“…The authors conclude that other, as yet unidentified, cross-reacting substances may be present in urine, which account for the remaining disagreement with chromatographic techniques. They also note that, in their study, only a 7-fold greater concentration of 8-oxoGuo is required to compete for 8-oxodG, unlike the previous reports of one or two orders of magnitude [25,26]. …”

“…Indeed, this does appear to be the case. The authors also exploited a recent development in which performing the JaICA ELISA kit's primary antibody step at 4 ºC, rather than 37 ºC, improved the agreement between ELISA and LC-MS/MS [25]. This further decreased the recognition of urea by N45.1, and demonstrated the most significant correlation with HPLC-EC, to date (r = 0.98; p < 0.0001).…”

“…This, in part, derives from recognition of the hydroxylated C8 position of Gua, it would appear to discriminate the C6 carbonyl group of 8-oxoGua (and C2 NH 2 ), from the C6 amino group of 8-oxoAde. Furthermore, the closest competitors, other than 8-oxodG, for the antibody is 8-oxo-7,8-dihydroguanosine (8-oxoGuo) and 8-oxodGMP [25], the former of which needs to be present in concentrations two orders of magnitude higher than 8-oxodG, in order to compete to the same extent [26], a situation that does not appear to occur in vivo [27]. It is unknown whether 8-oxodGMP is present in urine.…”