Elsevier

Annals of Epidemiology

Volume 14, Issue 10, November 2004, Pages 793-797
Annals of Epidemiology

Original Report
Epidemiological marker for oxidant status: comparison of the ELISA and the gas chromatography/mass spectrometry assay for urine 2,3-dinor-5,6-dihydro-15-F2t-isoprostane

https://doi.org/10.1016/j.annepidem.2004.03.003Get rights and content

Purpose

A biomarker of oxidant status applicable to epidemiological research is essential to studying the relationship between free radicals and chronic disease risk. Gas chromatography with mass-spectrometry detection (GC/MS) is the gold standard for measurement of urinary F2-isoprostanes (F2-isoPs), a non-invasive marker of oxidant status. However, this method is laborious and costly, which prohibits its use in large epidemiological studies.

Methods

We compared GC/MS assay with an inexpensive quick enzyme-linked immunoassay (ELISA) in measurements of 2,3-dinor-5,6-dihydro-15-F2t-isoprostane (F2-isoPM), an abundant β-oxidation metabolite of 8-iso-prostaglandin-F. We measured F2-isoPM in urine of 52 participants of the Insulin Resistance Atherosclerosis Study by both methods.

Results

The ELISA measurements showed approximately 30-fold greater mean and median (22.10, SD 12.92, and 18.49 ng/mg creatinine) than the GC/MS measurements (0.703, SD 0.468, and 0.597 ng/mg creatinine). We found low linear correlation (Pearson correlation coefficient 0.51; 95% CI, 0.28–0.70) and weak agreement in ranking subjects by tertiles (weighted Kappa statistic 0.34) between a GC/MS and ELISA.

Conclusions

We conclude that the current ELISA method is not a valid substitute for the GS/MS assay.

Introduction

A biomarker of oxidant status applicable to epidemiological research is essential to studying the relationship between free radicals and chronic disease risk 1., 2., 3., 4.. Prostaglandin F2-like compounds, F2-isoprostanes (F2-isoPs) are formed by non-enzymatic free radical-induced peroxidation of arachidonic acid in humans in vivo and have been intensively studied as an index of oxidant status 5., 6.. F2-isoPs are present in detectable quantities in all normal biological fluids including blood and urine. Therefore, F2-isoPs can be measured not only at pathologically high, but also at normal and low ranges.

Measurement of F2-isoPs in urine presents a non-invasive method to assess oxidant status and can be used in epidemiological studies. Beside feasibility of specimen collection and long-term stability, this biomarker has several favorable qualities. Excretion levels of urinary F2-isoPs are not sensitive to dietary intake of lipids (7). Measurement in a spot urine sample with correction for diluteness by urine creatinine can be validly used to assess oxidant status, since urinary F2-IsoPs do not show significant diurnal variation 8., 9., 10.. Finally, mean urinary levels of F2-IsoPs are stable (11) and the intra-subject coefficient of variation is low (12).

We selected an abundant β-oxidation metabolite of 8-iso-prostaglandin-F, 2,3-dinor-5,6-dihydro-15-F2t-isoprostane (F2-isoPM), as a urinary biomarker of oxidant status. A new assay based on gas chromatography with mass-spectrometry detection (GC/MS) was developed for measurement of F2-isoPM (13). This assay is very laborious and time consuming, which compromises its feasibility to assess oxidant status in large-scale epidemiological studies. In search for a less laborious substitute we compared the GC/MS assay with inexpensive quick enzyme-linked immunoassay (ELISA) 14., 15..

Section snippets

Study population

Fifty-two subjects were selected from the Insulin Resistance Atherosclerosis Study (IRAS) cohort. A full description of the design and methods of the IRAS have been published previously (16). Briefly, the IRAS study is a multicenter, population-based study. A total of 1625 men and women, 40 to 69 years of age, were recruited from four US communities between 1992 and 1993 and underwent baseline examination. A follow-up examination was conducted 5 years later and 80% of the cohort participated.

Results

This study included a diverse group of participants: men and women of three ethnic groups from 40 to 69 years of age with various obesity and smoking status (Table 1).

The ELISA method showed several-fold greater values for urine concentration of F2-isoPM compared with the GC/MS assay (Table 2). Mean and median of the ELISA results were approximately 30-fold greater than for the GC/MS results. For individual urine samples, difference between the two measurements ranged from 9- to 138-fold. We

Discussion

The etiological role of free radicals in chronic diseases was postulated by a large body of experimental data 19., 20., 21.. An obvious gap in this area of research is lack of epidemiological evidence, which requires a reliable biomarker of oxidant status in vivo 2., 3.. Urine F2-isoPs have several favorable qualities of a biomarker suitable for epidemiological research: these compounds are stable, present in detectable quantities (21), show little diurnal 8., 9., 10. and intra-individual

References (24)

  • B. Halliwell

    Lipid peroxidation, antioxidants and cardiovascular disease: How should we move forward?

    Cardiovasc Res

    (2000)
  • D. Steinberg et al.

    Is the oxidative modification hypothesis relevant to human atherosclerosis? Do the antioxidant trials conducted to date refute the hypothesis?

    Circulation

    (2002)
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    This research was supported by venture grant from the Wake Forest University School of Medicine and by NIH Grants: GM 15431, CA7783, DK48831 to JDM, and CA57707-07 to the Comprehensive Cancer center of Wake Forest University. J.D.M. is the recipient of a Burroughs Wellcome Fund Clinical Scientist Award in Translational Research.

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