Quantification of malondialdehyde by HPLC‐FL – application to various biological samples |
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Authors: | Ana‐Marija Domijan Jovica Rali? Sandra Radi? Brkanac Lada Rumora Tihana ?ani?‐Grubi?i? |
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Institution: | 1. University of Zagreb, Faculty of Pharmacy and Biochemistry, Department of Pharmaceutical Botany, Zagreb, Croatia;2. Galapagos Research Center, Zagreb, Croatia;3. University of Zagreb, Faculty of Science, Department of Biology, Zagreb, Croatia;4. University of Zagreb, Faculty of Pharmacy and Biochemistry, Department of Medical Biochemistry and Hematology, Zagreb, Croatia |
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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 (R2 = 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. Copyright © 2014 John Wiley & Sons, Ltd. |
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Keywords: | lipid peroxidation thiobarbituric acid assay plasma cells in culture liver homogenate |
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