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Technical Briefs |
a author for correspondence: fax 214-648-8037, e-mail jialal.i{at}pathology.swmed.edu
Protein carbonyls are considered as markers of oxidative stress (1). The conventional colorimetric assay (2) that measures reactive protein carbonyls using 2,4-dinitrophenylhydrazine (DNPH) is labor-intensive, time-consuming, and uses a large quantity of solvents. Recently, a new ELISA method for protein carbonyls was developed (3). However, the validity of this assay in smokers, who clearly have increased lipid peroxidation (4), and as a measure of LDL oxidation has not been studied. Hence, in this study we evaluated a modification of this ELISA method for protein carbonyls and compared it with the conventional colorimetric assay. We also validated the ELISA as an oxidative stress measurement in the plasma of smokers and as a measure of LDL oxidation.
Native bovine serum albumin (BSA; 50 g/L) was oxidized with 0.42 mmol/L Fe2+ and 0.73 mol/L H2O2 in phosphate-buffered saline (PBS) containing EDTA (0.1 g/L) for 2 h at 37 °C as reported previously (5). Oxidation was stopped with 40 µmol/L butylated hydroxytoluene. The carbonyl content of the oxidized BSA was measured spectrophotometrically (2). Aliquots of oxidized BSA were stored at -70 °C. Before use, an aliquot was thawed and diluted to a carbonyl content of 2 nmol/L in PBS. The ELISA method used was a modification of the original assay described by Buss et al. (3). Because in our hands reduced BSA gave high background values, producing negative values, PBS was used as blank, blocking agent, and diluting agent for the calibrator instead of reduced BSA; antibodies of a different nature were used for a more sensitive detection system [Dako anti-DNP antibody (1:1000) and Sigma goat anti-rabbit IgG peroxidase conjugate (1:20 000) vs DNP conjugate of keyhole limpet hemocyanin and streptavidin biotinylated horseradish peroxidase (1:3000)]. Each plate contained a six-point calibration curve with oxidized BSA. Carbonyl concentration was calculated from the calibration curve after subtraction of a reagent blank containing PBS and then expressed as nmol/mg protein (plasma and LDL). There was a very good correlation (n = 73; r = 0.93; P <0.001) between the ELISA and the conventional colorimetric method (2). The intraassay coefficients of variation (CVs), obtained by measuring a native sample and an oxidized sample 18 times in duplicate on the same day in the same plate, were 5.2% and 2.6%, respectively. The interassay CVs for two different pools of native and oxidized plasma assayed in duplicate on 5 different days were 9.3% and 21%, respectively. The method was linear up to 1 nmol/L, and the limit of detection was 0.02 nmol/L. Dilutions of an oxidized pool with an initial carbonyl concentration of 0.72 nmol/L gave the expected concentrations with serial dilutions in PBS (99% ± 15%).
All participants in the study were healthy volunteers who were not taking any antioxidant supplements. Fasting blood (12-h fast) was obtained in Vacutainer Tubes containing EDTA (Becton Dickinson). Total protein and lipid profiles were measured on the Paramax. To assess plasma oxidizability, oxidation was performed for 4 h using 200 mmol/L 2,2'-azobis(2-amidinopropane) hydrochloride (an aqueous free radical generator that thermally decomposes at a constant rate) in PBS at 37 °C and stopped by addition of 40 µmol/L butylated hydroxytoluene and refrigeration before derivatization for carbonyl assessment.
Immediately after the collection of blood, lipoprotein isolation was performed by sequential flotation ultracentrifugation similarly to that described previously (6). The LDL was then desalted using a Bio-Rad column, and the protein content was assessed and adjusted to 0.5 g protein/L. Oxidation of the LDL was induced by addition of a freshly prepared 12.5 µmol/L CuCl2 · 2 H2O solution. At 30-min intervals over a 5-h period, oxidation was arrested and both carbonyls and lipid peroxides were measured, the latter being assessed by the ferrous ammonium sulfate/xylenol orange method (FoX reagent; Pierce).
The characteristics of the 22 smokers (7 males and 15 females) are
presented in Table 1
. The smokers were matched to their controls by sex, body mass
index, total protein, total cholesterol, triglycerides, and LDL- and
HDL-cholesterol. In native plasma there was a trend toward higher
carbonyl concentrations in smokers than in controls
(P = 0.16, not significant). After oxidation, the
carbonyl concentration in the smokers was significantly higher than in
the controls. Some in vitro studies have shown the ability of cigarette
smoke to induce protein damage. Reznick et al. (7) showed
that whole and gas-phase cigarette smoke caused formation of carbonyls
in human plasma as assessed by the colorimetric method. Eiserich et al.
(8) found a 400 µmol/L increase in protein carbonyl
formation in plasma when exposed to nine puffs of cigarette smoke. This
is consistent with previous findings showing increased oxidative stress
in smokers (4)(9). We also measured LDL
carbonyl content, using this ELISA. During LDL oxidation, carbonyl
increased continuously with time, paralleled lipid peroxides, as shown
in Fig. 1
, and was significantly correlated with lipid peroxides
(r = 0.86; P <0.001). Our results
obtained with a copper/LDL protein ratio of 25 µmol/g protein are
consistent with the results assessed spectrophotometrically with 20
µmol copper/g protein shown by Yan et al. (10), who
reported a significant linear relationship between apo B LDL carbonyl
content and its peroxidation as measured by thiobarbituric
acid-reacting substances. Unlike their method, our method does not
require a blank, precipitation, or delipidation. Both native and
oxidized LDL can be assessed directly by immediate derivatization.
Thus, the ELISA method is not only able to measure oxidative stress in
circulating plasma, but can also be used to monitor LDL oxidation.
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In conclusion, because the ELISA requires a small sample volume and allows a large batch of samples to be run simultaneously, it may be useful in clinical studies aimed at comparing plasma as well as LDL oxidizability in different populations and also following antioxidant supplementation.
Acknowledgments
This work was supported by a grant from the American Diabetes Association and NIH grant M01-RR-00633. We thank R. Levine for useful discussions, Alicia Summers for technical assistance, and Beverly Huet Adams for statistical expertise.
Footnotes
Division of Clinical Biochemistry and Human Metabolism, Department of Pathology, University of Texas Southwestern Medical Center at Dallas, 5323 Harry Hines Blvd., CS3.114, Dallas, TX 75235-9073
References
The following articles in journals at HighWire Press have cited this article:
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  S. Vega-Lopez, K.-J. Yeum, J. L Lecker, L. M Ausman, E. J Johnson, S. Devaraj, I. Jialal, and A. H Lichtenstein Plasma antioxidant capacity in response to diets high in soy or animal protein with or without isoflavones Am. J. Clinical Nutrition, January 1, 2005; 81(1): 43 - 49. [Abstract] [Full Text] [PDF]
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D. J O'Byrne, S. Devaraj, S. M Grundy, and I. Jialal Comparison of the antioxidant effects of Concord grape juice flavonoids {alpha}-tocopherol on markers of oxidative stress in healthy adults Am. J. Clinical Nutrition, December 1, 2002; 76(6): 1367 - 1374. [Abstract] [Full Text] [PDF]
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 S. Devaraj, S. V. Hirany, R. F. Burk, and I. Jialal Divergence between LDL Oxidative Susceptibility and Urinary F2-Isoprostanes as Measures of Oxidative Stress in Type 2 Diabetes Clin. Chem., November 1, 2001; 47(11): 1974 - 1979. [Abstract] [Full Text] [PDF]
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 L. B. Meira, S. Devaraj, G. E. Kisby, D. K. Burns, R. L. Daniel, R. E. Hammer, S. Grundy, I. Jialal, and E. C. Friedberg Heterozygosity for the Mouse Apex Gene Results in Phenotypes Associated with Oxidative Stress Cancer Res., July 1, 2001; 61(14): 5552 - 5557. [Abstract] [Full Text] [PDF]
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