A Rapid Method for Estimating the Levels of Urinary Thiobarbituric Acid Reactive Substances for Environmental Epidemiologic Survey

Malondialdehyde (MDA), used as an oxidative stress marker, is commonly assayed by measuring the thiobarbituric acid reactive substances (TBARS) using HPLC, as an indicator of the MDA concentration. Since the HPLC method, though highly specific, is time-consuming and expensive, usually it is not suitable for the rapid test in large-scale environmental epidemiologic surveys. The purpose of this study is to develop a simple and rapid method for estimating TBARS levels by using a multiple regression equation that includes TBARS levels measured with a microplate reader as an independent variable. Twelve hour urine samples were obtained from 715 subjects. The concentration of TBARS was measured at three different wavelengths (fluorescence: λ-(ex) 530 nm and λ-(ex) 550 nm; λ-(ex) 515 nm and λ-(ex) 553 nm; and absorbance: 532 nm) using microplate reader as well as HPLC. 500 samples were used to develop a regression equation, and the remaining 215 samples were used to evaluate the validity of the regression analysis. The induced multiple regression equation is as follows: TBARS level (μM) = -0.282 + 1.830 × (TBARS level measured with a microplate reader at the fluorescence wavelengths λ-(ex) 530 nm and λ-(em) 550 nm, μM) –0.685 × (TBARS level measured with a microplate reader at the fluorescence wavelengths λ-(ex) 515 nm and λ-(em) 553 nm, μM) + 0.035 × (TBARS level measured with a microplate reader at the absorbance wavelength 532 nm, μM). The estimated TBARS levels showed a better correlation with, and are closer to, the corresponding TBARS levels measured by HPLC compared to the values obtained by the microplate method. The TBARS estimation method reported here is simple and rapid, and that is generally in concordance with HPLC measurements. This method might be a useful tool for monitoring of urinary TBARS level in environmental epidemiologic surveys with large sample sizes.