Rapid Fluorimetric Method to Detect Total Plasma Malondialdehyde with Mild Derivatization Conditions

Malondialdehyde (MDA), an oxidation product of polyunsaturated fatty acids, is used as an in vivo marker to assess lipid peroxidation in diseases such as atherosclerosis and diabetes (1)(2)(3)(4). In biological matrixes, MDA is measured after derivatization with thiobarbituric acid (TBA) (5). Because TBA reacts with many other aldehydes (6), results are expressed as TBA-reactive substances (TBARS). Several problems are associated with TBARS analysis, in particular, low reproducibility and a lack of specificity that leads to overestimations. To overcome these difficulties, more specific methods have been proposed that require sample pretreatment to precipitate proteins and extract MDA-reactant adducts (6)(7)(8)(9). This additional step is time-consuming and adversely affects precision. The aim of the present study was to develop a rapid and sensitive method to measure MDA in plasma, avoiding sample pretreatment.Tetraethoxypropane (TEP), TBA, and bilirubin were obtained from Fluka. Fatty acid-free bovine serum albumin (BSA), Total Protein Reagent, and Protein Standard were from Sigma, and 2,2′-azobis(2-amidinopropane) (ABAP) was from Wako.We prepared an aqueous stock solution of 1 mmol/L TEP. A 10 μmol/L MDA solution was obtained by diluting TEP in 0.1 mol/L HCl. A 0.025 mol/L TBA solution was prepared daily by dissolving TBA in water. BSA solutions were prepared in 0.1 mol/L HCl. To remove protein-bound MDA, BSA solutions were heated at 80 °C for 1 h and dialyzed for 3 days against 0.1 mol/L HCl in a 3500-Da cutoff dialysis membrane (Spectrapore; Spectrum Medical Industries). The actual protein concentration was verified using the Total Protein Reagent Kit. A 50 mmol/L ABAP solution was prepared in water.Six MDA solutions ranging from 0.05 to 0.5 μmol/L were prepared by diluting the 10 μmol/L stock solution with 0.1 mol/L HCl. Triplicate solutions were used to obtain a dose–response …