Human and Methodological Sources of Variability in the Measurement of Urinary 8-Oxo-7,8-dihydro-2′-deoxyguanosine

Abstract

Aims: Urinary 8-oxo-7,8-dihydro-2¢-deoxyguanosine (8-oxodG) is a widely used biomarker of oxidative stress. However, variability between chromatographic and ELISA methods hampers interpretation of data, and this variability may increase should urine composition differ between individuals, leading to assay interference. Furthermore, optimal urine sampling conditions are not well defined. We performed inter-laboratory comparisons of 8-oxodG measurement between mass spectrometric-, electrochemical-and ELISA-based methods, using common within-technique calibrants to analyze 8-oxodG-spiked phosphate-buffered saline and urine samples. We also investigated human subject-and sample collection-related variables, as potential sources of variability. Results: Chromatographic assays showed high agreement across urines from different subjects, whereas ELISAs Kronos Science, Phoenix, Arizona. ANTIOXIDANTS & REDOX SIGNALINGVolume 18, Number 18, 2013 ª Mary Ann Liebert, Inc. DOI: 10.1089/ars.2012.4714 2377showed far more inter-laboratory variation and generally overestimated levels, compared to the chromatographic assays. Excretion rates in timed 'spot' samples showed strong correlations with 24 h excretion (the 'gold' standard) of urinary 8-oxodG (r p 0.67-0.90), although the associations were weaker for 8-oxodG adjusted for creatinine or specific gravity (SG). The within-individual excretion of 8-oxodG varied only moderately between days (CV 17% for 24 h excretion and 20% for first void, creatinine-corrected samples). Innovation: This is the first comprehensive study of both human and methodological factors influencing 8-oxodG measurement, providing key information for future studies with this important biomarker. Conclusion: ELISA variability is greater than chromatographic assay variability, and cannot determine absolute levels of 8-oxodG. Use of standardized calibrants greatly improves intra-technique agreement and, for the chromatographic assays, importantly allows integration of results for pooled analyses. If 24 h samples are not feasible, creatinine-or SG-adjusted first morning samples are recommended.

Association between 8-oxo-7,8-dihydro-2′-deoxyguanosine Excretion and Risk of Postmenopausal Breast Cancer: Nested Case–Control Study

“…A larger case-control study with 1,066 cases and a prospective study with 350 cases split in pre-and postmenopausal cancer found no association between 8-oxodG excretion and breast cancer risk in logistic regression analysis (19)(20)(21). Nevertheless, these studies used ELISA-based measurement of 8-oxodG, which show substantially less precision, accuracy, and ability to discriminate patients and healthy subjects compared with the chromatographic assays (17,18). This could increase measurement error in exposure assessment biasing risk toward null.…”

“…However, only one spot sample of urine was collected at enrolment and this may not be representative for the long period at risk. (18,49,50). The concentrations of 8-oxodG had to be adjusted by creatinine concentration (18).…”

“…(18,49,50). The concentrations of 8-oxodG had to be adjusted by creatinine concentration (18). Creatinine excretion is affected by muscle mass possibly explaining higher levels of 8-oxodG per creatinine with increasing age, whereas 24-hour urinary collection show decreased excretion with age (32,50).…”

“…Several epidemiological studies have addressed risk of breast cancer associated with these biomarkers in urine samples assayed by chromatographic methods with electrochemical or mass spectrometrybased detection or by ELISA. Large-scale interlaboratory and other validation studies have documented that ELISA-based measurement of 8-oxodG is unspecific and imprecise, not discriminating between healthy subjects and patients (17,18). Indeed, one case-control study with approximately 1,100 cases and one nested-in-cohort prospective case-control study with 326 cases using ELISAbased assay of 8-oxodG found no association between the level and risk of breast cancer (19)(20)(21).…”

Urinary phthalate metabolites among elementary school children of Korea: Sources, risks, and their association with oxidative stress marker

“…Oxidative stress markers such as MDA can be influenced by numerous factors including physical, chemical, and biological parameters, hence certain extent of within-individual variation is inevitable. For example, urinary 8-oxo-7,8-dihydro-2′-deoxyguanosine (8-oxodG), another widely used biomarker of oxidative stress, showed a within-individual variation of 20% CV in the first morning void samples collected from 26 healthy nonsmoking adults (Barregard et al, 2013). While quantitative explanation is not possible, day-of-the-week variation of stress biomarkers may also contribute to the observation of the present study, e.g., the 1st set of samples were collected on Tuesday (Day 2) morning following weekend vs. the 2nd set of samples were collected on Friday (Day 5).…”