Spectrophotometric measurement of plasma 2-thiobarbituric acid-reactive substances in the presence of hemoglobin and bilirubin interference.

Abstract

The 2-thiobarbituric acid reaction with malondialdehyde has been used to assess lipid peroxidation in a variety of biologic systems. However, in an attempt to measure plasma thiobarbituric acid-reactive substances (TBARS) during extracorporeal membrane oxygenation, a form of sustained cardiopulmonary bypass, it became apparent that the absorbance signal at the 532-nm wavelength was composed not only of the peak absorbance of TBARS, but also of interfering substances from heme pigments and bilirubin. A method of subtracting interfering substances was developed and applied to normal human plasma. The method was tested by adding varying amounts of red blood cell hemolysate, bilirubin, and 1,1,3,3-tetramethoxypropane (TMP) standard to plasma and determining TBARS in the resulting mixture. In addition, varying the amount of added desferoxamine was investigated to determine the effects of iron chelation on the assay. This was important because the different samples would have varying amounts of free iron from hemoglobin to catalyze the reaction. It was found that the following equation could be used in this system to determine that amount of 532-nm absorption due to TBARS: MDA532 = 1.22[(A532) - (0.56)(A510) + (0.44)(A560)]. Regression analysis revealed an 86.6% recovery of the TMP spike. Analysis of variance showed that the variability in the model could be explained mainly by the additive increments of TMP spike (94.6%).