Clinical relevance of guanine-derived urinary biomarkers of oxidative stress, determined by LC-MS/MS

Ying-Ming Shih,Marcus S Cooke,Chih-Hong Pan,Mu-Rong Chao,Chiung-Wen Hu

doi:10.1016/j.redox.2018.11.016

Abstract

A reliable and fast liquid chromatography-tandem mass spectrometry method has been developed for the simultaneous determination of three oxidized nucleic acid damage products in urine, 8-oxoguanine (8-oxoGua), 8-oxo-7,8-dihydro-2′-deoxyguanosine (8-oxodGuo) and 8-oxo-7,8-dihydroguanosine (8-oxoGuo). We applied this method to assess the effect of various urine workup procedures on the urinary concentrations of the oxidized nucleic acid products. Our results showed that frozen urine samples must be warmed (i.e., to 37 °C) to re-dissolve any precipitates prior to analysis. We showed that common workup procedures, such as thawing at room temperature or dilution with deionized water, are not capable of releasing fully the oxidized nucleic acid products from the precipitates, and result in significant underestimation (up to ~ 100% for 8-oxoGua, ~ 86% for both 8-oxodGuo and 8-oxoGuo).With this method, we further assessed and compared the ability of the three oxidized nucleic acid products, as well as malondialdehyde (MDA, a product of lipid peroxidation), to biomonitor oxidative stress in vivo. We measured a total of 315 urine samples from subjects with burdens of oxidative stress from low to high, including healthy subjects, patients with chronic obstructive pulmonary disease (COPD), and patients on mechanical ventilation (MV). The results showed that both the MV and COPD patients had significantly higher urinary levels of 8-oxoGua, 8-oxodGuo, and 8-oxoGuo (P < 0.001), but lower MDA levels, compared to healthy controls. Receiver operating characteristic curve analysis revealed that urinary 8-oxoGuo is the most sensitive biomarker for oxidative stress with area under the curve (AUC) of 0.91, followed by 8-oxodGuo (AUC: 0.80) and 8-oxoGua (AUC: 0.76). Interestingly, MDA with AUC of 0.34 failed to discriminate the patients from healthy controls. Emerging evidence suggests a potential clinical utility for the measurement of urinary 8-oxoGuo, and to a lesser extent 8-oxodGuo, which is strongly supported by our findings.

Highlights

Reactive oxygen species (ROS) have been strongly associated with cellular aging, cancer and other degenerative diseases because of their potential to damage cellular constituents, such as nucleic acids, proteins and lipids [1]
For 8oxodGuo (Fig. 1C), its [M +H]+ precursor ion was at m/z 284 and product ions appeared at m/z 168 and m/z 140, resulting from the loss of the neutral 2′-deoxyribose moiety or its combination with CO; a precursor ion at m/z 289 and product ion at m/z 173 characterized the [15N5]-8-oxodGuo. With this online SPE LC-MS/MS method, it was interesting to note that the transitions of m/z 168 → 140 and 168 → 112 were detected at the retention times at 12.4 min and 13.4 min, corresponding to 8-oxoGuo and 8-oxodGuo, respectively (Supplementary Fig. S2), suggesting that artefactual 8-oxoGua could be generated, from 8-oxoGuo and 8-oxodGuo, during the ionization process [21]
This finding highlights the importance of an efficient separation of 8-oxoGua from 8oxodGuo and 8-oxoGuo during chromatography to avoid the overestimation of 8-oxoGua

Summary

Introduction

Reactive oxygen species (ROS) have been strongly associated with cellular aging, cancer and other degenerative diseases because of their potential to damage cellular constituents, such as nucleic acids, proteins and lipids [1]. For oxidatively damaged nucleic acids, guanine (Gua) is the main oxidation target, as it has the lowest redox potential of the nucleobases [2]. Damaged DNA can be Abbreviations: 8-oxodGuo, 8-oxo-7,8-dihydro-2′-deoxyguanosine; 8-oxoGua, 8-oxo-7,8-dihydroguanine; 8-oxoGuo, 8-oxo-7,8-dihydroguanosine; AUC, area under the curve; BER, base excision repair; COPD, chronic obstructive pulmonary disease; crt, creatinine; dGuo, 2′-deoxyguanosine; DNPH, 2,4-dinitrophenylhydrazine; Gua, guanine; Guo, guanosine; ICUs, intensive care units; LODs, limits of detection; LOQs, limits of quantification; MV, mechanical ventilation; MDA, malondialdehyde; NER, nucleotide excision repair; RCC, respiratory care center; ROC, receiver operating characteristic; ROS, reactive oxygen species; RT, room temperature.

Methods

Results

Conclusion