Quantification of malondialdehyde by HPLC‐FL – application to various biological samples

Abstract

Malondialdehyde (MDA) is stabile product of lipid peroxidation (LPO), and therefore MDA is frequently used as a biomarker of LPO. To determine MDA level in various biological samples (human plasma, fish liver tissue and cells in culture), we used an HPLC method with fluorescent detection based on 2-thiobarbituric acid (TBA) assay. The method was validated by the use of spiked pooled plasma samples. In tested concentration range (0.15-3.0 µmol/L) the method was linear (R(2) = 0.9963), the between-day variability (coefficient of variations, CVs) was between 4.7 and 7.6%, the within-day variability CVs was between 2.6 and 6.4% and recovery was between 91.2 and 107.6%. The level of MDA in human plasma (healthy male, non-smokers, 46.3 ± 4.7 years; N = 38) was 2.2 ± 1.4 µmol/L; that in liver tissue of common carp (Cyprinus carpio; N = 12) was 0.02 ± 0.004 µmol/g tissue, and in cultured cells (human laryngeal carcinoma cells; N = 10) it was 0.18 ± 0.02 nmol/mg proteins. The HPLC-FL method is rapid, accurate and reliable to follow the extent of LPO in various biological samples, particularly in samples in which a low level of MDA is expected, such as cells in culture. Owing to the rapid analytical process and run time, it can be used for routine analysis of MDA in clinical laboratory.

Reduced salivary flow rate and high levels of oxidative stress in whole saliva of children with Down syndrome

“…Salivary MDA levels are usually assessed by spectrophotometric quantification of the resulting products of the reaction with thiobarbituric acid (TBA). However, this method has been criticized for low sensitivity, because some noninterest species present in the sample can also react with TBA . In a previous study, using TBA assay, Sousa et al.…”

Protective effects of hydrogen rich water on the intestinal ischemia/reperfusion injury due to intestinal intussusception in a rat model

“…Excess ROS may cause oxidation of protein, lipid and DNA. MDA results from lipid peroxidation of polyunsaturated fatty acids and the degree of lipid peroxidation can be estimated by the amount of malondialdehyde in tissues 19. 8-OHdG is an oxidized derivative of deoxyguanosine and one of the major products of DNA oxidation 20.…”

Measurement and Clinical Significance of Biomarkers of Oxidative Stress in Humans

“…An additional pitfall is the interference of hemolysis that falsely increases the measured MDA levels (Table 2). Thus, many protocols and modifications of the TBA test are available in the literature, and while direct MDA-TBA adduct measurement has a low significance, the determination by HPLC combined with UV or fluorescence detection is a more reliable and reproducible method [149–151]. Despite the methodological bias, MDA measurement could have clinical relevance due to the potential pathogenic role of MDA on to the induction of IL-17 producing cells [152] and a possible link between lipid-peroxidation and T-helper 17 (Th17) cell-mediated diseases, such as inflammatory bowel diseases [153].…”