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.
There are substantial inter-laboratory variations in the levels of DNA damage measured by the comet assay. The aim of this study was to investigate whether adherence to a standard comet assay protocol would reduce inter-laboratory variation in reported values of DNA damage. Fourteen laboratories determined the baseline level of DNA strand breaks (SBs)/alkaline labile sites and formamidopyrimidine DNA glycosylase (FPG)-sensitive sites in coded samples of mononuclear blood cells (MNBCs) from healthy volunteers. There were technical problems in seven laboratories in adopting the standard protocol, which were not related to the level of experience. Therefore, the inter-laboratory variation in DNA damage was only analysed using the results from laboratories that had obtained complete data with the standard comet assay protocol. This analysis showed that the differences between reported levels of DNA SBs/alkaline labile sites in MNBCs were not reduced by applying the standard assay protocol as compared with the laboratory's own protocol. There was large inter-laboratory variation in FPG-sensitive sites by the laboratory-specific protocol and the variation was reduced when the samples were analysed by the standard protocol. The SBs and FPG-sensitive sites were measured in the same experiment, indicating that the large spread in the latter lesions was the main reason for the reduced inter-laboratory variation. However, it remains worrying that half of the participating laboratories obtained poor results using the standard procedure. This study indicates that future comet assay validation trials should take steps to evaluate the implementation of standard procedures in participating laboratories.
Objective Investigation of increased oxidative stress in early pregnancy and association with an increased risk of small-forgestational-age (SGA) fetus.Design Longitudinal case-control study.Setting University Hospitals of Leicester NHS Trust, Leicester, UK.Population Low-risk pregnant women with no current or preexisting medical illness were recruited at a large teaching hospital from 2004 to 2006.Methods Recruitment performed at the time of the dating ultrasound scan (12 ± 2 weeks of gestation). Spot urine samples collected at 12 ± 2 and 28 ± 2 weeks of gestation were analysed for 8-oxo-7,8-dihydro-2#-deoxyguanosine (8-oxodG) by liquid chromatography with tandem mass spectrometry). SGA was defined as birthweight <10th centile based on customised centile calculator (www.gestation.net). This identified the cases (n = 55), whereas controls (n = 55) were mothers whose babies were appropriate for gestational age (AGA, birthweight 10th-90th centile). Statistical analysis was performed using GraphPad Prism v.5. The relationship between maternal urinary 8-oxodG at different gestations and customised SGA was investigated by nonparametric tests.Main outcome measures Customised SGA and AGA pregnancies.Results Urinary 8-oxodG concentrations were significantly increased in pregnancies with subsequent SGA compared with concentrations in normal pregnancies; 12 weeks: 2.8 (interquartile range [IQR] 1.96-3.67) versus 2.2 (IQR 1.26-3.28) pmol 8-oxodG/ mmol creatinine (P = 0.0007); 28 weeks: 2.21 (IQR 1.67-3.14) versus 1.68 (IQR 1.16-2.82) pmol 8-oxodG/mmol creatinine (P < 0.0002). Concentrations decreased significantly between week 12 and 28 (P = 0.04 and P = 0.02 for controls and cases).Conclusions In this study, urinary 8-oxodG at 12 and 28 weeks were elevated in SGA compared with AGA pregnancies. This may reflect early placental changes predating clinical features of SGA.Keywords 8-oxo-7, 8-dihydro-2#-deoxyguanosine, DNA damage, fetal growth restriction, oxidative stress, placenta, pregnancy.Please cite this paper as: Potdar N, Singh R, Mistry V, Evans M, Farmer P, Konje J, Cooke M. First-trimester increase in oxidative stress and risk of small-forgestational-age fetus. BJOG 2009;116:637-642.
The alkaline comet assay is an established, sensitive method extensively used in biomonitoring studies. This method can be modified to measure a range of different types of DNA damage. However, considerable differences in the protocols used by different research groups affect the inter-laboratory comparisons of results. The aim of this study was to assess the inter-laboratory, intra-laboratory, sample and residual (unexplained) variations in DNA strand breaks and formamidopyrimidine DNA glycosylase (FPG)-sensitive sites measured by the comet assay by using a balanced Latin square design. Fourteen participating laboratories used their own comet assay protocols to measure the level of DNA strand breaks and FPG-sensitive sites in coded samples containing peripheral blood mononuclear cells (PBMC) and the level of DNA strand breaks in coded calibration curve samples (cells exposed to different doses of ionising radiation) on three different days of analysis. Eleven laboratories found dose-response relationships in the coded calibration curve samples on two or three days of analysis, whereas three laboratories had technical problems in their assay. In the coded calibration curve samples, the dose of ionising radiation, inter-laboratory variation, intra-laboratory variation and residual variation contributed to 60.9, 19.4, 0.1 and 19.5%, respectively, of the total variation. In the coded PBMC samples, the inter-laboratory variation explained the largest fraction of the overall variation of DNA strand breaks (79.2%) and the residual variation (19.9%) was much larger than the intra-laboratory (0.3%) and inter-subject (0.5%) variation. The same partitioning of the overall variation of FPG-sensitive sites in the PBMC samples indicated that the inter-laboratory variation was the strongest contributor (56.7%), whereas the residual (42.9%), intra-laboratory (0.2%) and inter-subject (0.3%) variations again contributed less to the overall variation. The results suggest that the variation in DNA damage, measured by comet assay, in PBMC from healthy subjects is assay variation rather than variation between subjects.
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