Total Plasma Malondialdehyde Levels in 16 Taiwanese College Students Determined by Various Thiobarbituric Acid Tests and an Improved High-Performance Liquid Chromatography-based Method

Abstract

Objectives: In determining the plasma malondialdehyde MDA levels in some Taiwanese college students, we found rather different results by using different thiobarbituric acid TBA tests, even by the high-performance liquid chromatography HPLC-based methods. Here, we re-evaluated four commonly used TBA tests and improved the HPLC-based test. Design and Methods: We used the blood plasma of 16 college volunteers to determine plasma MDA by using four methods: a spectrophotometric measurement of thiobarbituric acid-reactive substances (TBARS) in the TCA-supernatant of plasma (Method A); a fluorescence measurement of plasma lipid peroxides (Method B); and two different HPLC-based measurements of MDA with either 532-nm measurement (Method C, HPLC/532 nm) or fluorescence measurement (Method D, HPLC/fluor.). Results: The levels of MDA or TBA reactive substances obtained from the four methods differed substantially (0.39 ± 0.15; 2.14 ± 0.73; 0.75 ± 0.22; and 0.38 ± 0.15 μM for Methods A, B, C, and D, respectively). The results were positively correlated between Methods A and B ( r = 0.740, p < 0.02) and between Methods C and D ( r = 0.516, p < 0.05). However, results were negatively correlated between Methods B and D ( r = −0.548, p < 0.05). Because most plasma MDA is bound to proteins, we modified the HPLC-based methods (C and D) by adding an alkaline hydrolysis step, and the plasma TBA-MDA adduct detected by HPLC/532 nm was referred to as total MDA. Results show that alkaline hydrolysis was a critical step for measurement of total MDA in plasma because this treatment led to release of MDA from plasma proteins. We also adapted the potassium iodide (KI) treatment of plasma from Method D to reduce lipid hydroperoxides. Our modified method gave a total MDA level in the 16 volunteers of ∼1.5 μM, which was equal to protein-bound MDA plus free MDA. This total MDA level was positively ( p < 0.05) correlated with the level of TBA reactive substances obtained from Methods C ( r = 0.63, p < 0.05) and D ( r = 0.48, p < 0.07), but was not correlated with those from Methods A and B. The recovery (84∼105%), precision (within-assay coefficient of variation: 2.4%, between-assay coefficient of variation: 4∼8%) and sensitivity of the modified procedure were comparable to other HPLC-based methods. Conclusion: By using a validated modification of HPLC-based TBA method, the total plasma MDA in 16 Taiwanese college students was found to be 1.54 μM, which was relatively high compared to those obtained by other HPLC-based method, primarily due to the release of protein-bound MDA by alkaline hydrolysis. This level equaled the sum of protein-bound MDA and free MDA in plasma, confirming that this level represents total plasma MDA.