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Method for the simultaneous determination of free/protein malondialdehyde and lipid/protein hydroperoxides.

Grintzalis K, Zisimopoulos D, Grune T, Weber D, Georgiou CD.

Free Radic Biol Med. 2013 Jun;59:27-35.

Genetics, Cell, and Developmental Biology Section, Department of Biology, University of Patras, Patras 26100, Greece.

Abstract

A simple and sensitive method is presented for the simultaneous quantification (spectrophotometric and spectrofluorimetric) of the main lipid andprotein peroxidation products after their initial fractionation: free malondialdehyde (FrMDA), protein-bound malondialdehyde (PrMDA), totalhydroperoxides (LOOH), and protein hydroperoxides (PrOOH). FrMDA and PrMDA (released from proteins by alkaline hydrolysis) are measured after the reaction of MDA with thiobarbituric acid (TBA) under acidic conditions, by the specific fluorimetric quantification of the resulting MDA-(TBA)2 adduct chromophore. The measurement of LOOH and PrOOH is based on the reaction of Fe(3+) (resulting from the reaction of LOOH and PrOOH with Fe(2+)) with xylenol orange (XO) and the photometric quantification of the resulting XO-Fe complex. The sensitivity of the assays for FrMDA/PrMDA and LOOH/PrOOH is 20 and 100pmol, respectively. The method was applied successfully on human plasma and can be used for the evaluation of oxidative stress in both basic and clinical research.

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Biomedical significance

Lipids and proteins are modified by a large number of reactions involving reactive oxygen species, which generate certain peroxidation products: lipid hydroperoxides (LOOH), free malondialdehyde (FrMDA), protein-MDA adducts

(PrMDA) and protein hydroperoxides (PrOOH). These have attracted a great deal of attention as useful biomarkers of severe oxidative lipid and protein damage because of their association with any biological dysfunction and disease induced by oxidative damage. Of particular biomedical and biotechnological interest are a large number of human diseases and age-related disorders, such as Parkinson’s and Alzheimer’s disease, cancer, chronic lung disease and renal failure, diabetes and sepsis, skeletal muscle dysfunctions etc. Therefore, the simultaneous quantification of the aforementioned lipid and protein peroxidative modifications, by the protocol of this article, could provide new diagnostic (possibly pre-symptomatic) biomarkers for human diseases and also for testing the oxidative toxicity of related drug treatment protocols.

 

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