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Rossner P.,Academy of Sciences of the Czech Republic | Orhan H.,Ege University | Koppen G.,Flemish Institute for Technological Research | Sakai K.,Nikken SEIL Co. | And 9 more authors.
Free Radical Biology and Medicine | Year: 2016

ELISA is commonly used for the detection of urinary 8-oxo-7,8-dihydro-2′-deoxyguanosine (8-oxodG), a marker of whole body oxidative stress. However, the method has been criticized for high inter-laboratory variability and poor agreement with chromatographic techniques. We performed an inter-laboratory comparison of 8-oxodG assessed in 30 urine samples and a urine spiked with four different concentrations of 8-oxodG by ELISA using standardized experimental conditions, including: sample pre-treatment with solid-phase extraction (SPE), performing analysis using a commercial kit from a single manufacturer and strict temperature control during the assay. We further compared the ELISA results with high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) and performed tentative identification of compounds that may contribute to the discrepancy between both methods. For all but one participating laboratory (Data 1) we observed consistent ELISA results lying mostly within 1 SD of the mean 8-oxodG concentration. Mean 8-oxodG levels assessed by ELISA correlated with the data obtained by HPLC-MS/MS (R=0.679, p<0.001). The correlation improved when Data 1 were excluded from the analysis (R=0.749, p<0.001). We identified three outlying urine samples; one with an ELISA 8-oxodG concentration lower, and two with 8-oxodG levels higher, than those measured by HPLC-MS/MS. Omitting these samples further improved inter-methodology agreement (R=0.869, p<0.001). In the outliers with high 8-oxodG estimates various aromatic and heterocyclic compounds were tentatively identified using gas chromatography-mass spectrometry (GC-MS). Application of authentic standards revealed the presence of saccharides, including d-glucose and d-galactose as putative interfering substances. In summary, assay standardization improved ELISA inter-laboratory agreement, although some variability is still observed. There are still compounds contributing to overestimation of 8-oxodG by ELISA, but only in some urine samples. Thus, despite significant improvement, ELISA still should not be considered a robust alternative to chromatographic techniques. © 2016 Elsevier Inc. All rights reserved.

Evans M.D.,University of Leicester | Olinski R.,Nicolaus Copernicus University | Loft S.,Copenhagen University | Cooke M.S.,University of Leicester | And 40 more authors.
FASEB Journal | Year: 2010

Of the DNA-derived biomarkers of oxidative stress, urinary 8-oxo-7,8-dihydro-2′-deoxyguanosine (8-oxodG) is the most frequently measured. However, there is significant discrepancy between chromatographic and immunoassay approaches, and intratechnique agreement among all available chromatography-based assays and ELISAs is yet to be established. This is a significant obstacle to their use in large molecular epidemiological studies. To evaluate the accuracy of intra/intertechnique and interlaboratory measurements, samples of phosphate buffered saline and urine, spiked with different concentrations of 8-oxoG, together with a series of urine samples from healthy individuals were distributed to ESCULA members. All laboratories received identical samples, including 2 negative controls that contained no added 8-oxodG. Data were returned from 17 laboratories, representing 20 methods, broadly classified as mass spectrometric (MS), electrochemical detection (EC), or enzyme-linked immunosorbant assay (ELISA). Overall, there was good within-technique agreement, with the majority of laboratories' results lying within 1 SD of their consensus mean. However, ELISA showed more within-technique variation than did the chromatographic techniques and, for the urine samples, reported higher values. Bland-Altman plots revealed good agreement between MS and EC methods but concentration-dependent deviation for ELISA. All methods ranked urine samples according to concentration similarly. Creatinine levels are routinely used as a correction factor for urine concentration, and therefore we also conducted an interlaboratory comparison of methods for urinary creatinine determination, in which the vast majority of values lay within 1 SD of the consensus value, irrespective of the analysis procedure. This study reveals greater consensus than previously expected, although concern remains over ELISA. © FASEB.

Barregard L.,Gothenburg University | Moller P.,Copenhagen University | Henriksen T.,Laboratory of Clinical Pharmacology Q | Mistry V.,University of Leicester | And 42 more authors.
Antioxidants and Redox Signaling | Year: 2013

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 showed 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 (rp 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. © Copyright 2013, Mary Ann Liebert, Inc.

Sakai K.,Nikken SEIL Co. | Kino S.,Nikken SEIL Co. | Masuda A.,Nikken SEIL Co. | Takeuchi M.,Nikken SEIL Co. | And 6 more authors.
Sub-Cellular Biochemistry | Year: 2014

Published evidences indicate that reactive oxygen species (ROS) can induce lipid peroxidation, which plays important role in the pathophysiology of numerous diseases including atherosclerosis, diabetes, cancer and aging process. Monitoring of oxidative modification or oxidative damages of biomolecules may therefore be essential for the understanding of aging, and age-related diseases. N-epsilon-Hexanoyl-lysine (HEL) is a novel lipid peroxidation biomarker which is derived from the oxidation of omega-6 unsaturated fatty acid. In this chapter, development of HEL ELISA and its applications are reported. Assay range of HEL ELISA was 2-700 nmol/L, and showed good linearity and reproducibility. Accuracy of this assay was validated by recovery test and absorption test. HEL concentration in human urine was 22.9 ± 15.4 nmol/L and it was suggested that HEL exists as low molecular substances, in a free or in the peptide-attached form. In contrast with the urine sample, serum HEL was suggested to exist in the protein-attached form, and hydrolysis by protease might be essential for the accurate measurement of HEL in protein containing samples such as serum and cultured cells. By sample pretreatment with proteases, HEL was successfully detected in oxidized LDL, oxidized serum, and rat serum. In conclusion, HEL ELISA can be applied to measure urine, serum, and other biological samples independent of the animal species, and may be useful for the assessment of omega-6 PUFA oxidation in the living bodies. © Springer Science+Business Media Dordrecht 2014.

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