Measurement of Protein Carbonyls in Plasma of Smokers and in Oxidized LDL by an ELISA

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 …