This student paper was written as an assignment in the graduate course Free Radicals in Biology

Malondialdehyde (MDA) is a naturally occurring product of lipid peroxidation; it can also be generated during prostaglandin biosynthesis in cells. MDA reacts with amino groups on proteins and other biomolecules to form a variety of adducts, including adducts with DNA bases that are mutagenic and possibly carcinogenic. Increased levels of lipid peroxidation products, by measurement of MDA, have been associated with various conditions and pathological states of diseases. Therefore, it is important to understand the characteristics and function of MDA as well as the effective techniques of measurement for using MDA as a tool in clinical monitoring of the patient. S. Jetawattana MDA, a lipid oxidation product 3 Introduction Malondialdehyde, MDA, is a highly reactive three carbon dialdehyde produced as a byproduct of polyunsaturated fatty acid peroxidation [1] and also during arachidonic acid metabolism for the synthesis of prostaglandins [2]. MDA can combine with several functional groups on molecules including proteins, lipoproteins, RNA and DNA [3]. The monitoring of MDA levels in biological materials can be used as an important indicator of lipid peroxidation in vitro and in vivo for various diseases. This will mainly focus on MDA chemistry, biochemistry, routes of formation, detection, and biological health aspects. Routes of formation Malondialdehyde is a naturally occurring product of lipid peroxidation. Lipid peroxidation is a well-established mechanism of cellular injury in both plants and animals and is used as an indicator of oxidative stress in cells and tissues. Lipid peroxides, derived from polyunsaturated fatty acids, are unstable and decompose to form a complex series of compounds, which include reactive carbonyl compounds, including MDA. MDA can be generated during cyclooxygenase (COX) catalysis in human platelets, formation from prostaglandin endoperoxide (PGH2), catalyzed by thromboxane synthase [4] and in liver cells [5] by breakdown of PGH2, Figures 1 and 2. Figure 1. Conversion of prostaglandin endoperoxide (PGH2) to 12-hydroxyheptadecatrienoate (HHT) and malondialdehyde (MDA) [5]. S. Jetawattana MDA, a lipid oxidation product 4 Figure 2. Conversion of arachidonic acid to prostaglandin endoperoxides (PGG2) resulting in formation of MDA and potentially M1G adducts. Step 1 incorporates both oxygenase and peroxidase activities of the cyclooxygenase (COX) enzyme. Step 2 represents breakdown of PGH2 to MDA and hydroxyheptadecatrieonic acid (HHT), which can occur spontaneously or via catalysis by thromboxane synthases or other cytochromes P450. Step 3 is one possible mechanism of formation of the M1G adduct [6]. Chemical and biological properties MDA is also called malonaldehyde or bis(dimethyl acetal). It has the molecular formula C7H16O4 and molecular weight 164.2. The boiling point is 183C, and the freezing point is 130C. It was suggested that the bulk of MDA in human plasma is bound to protein; this would explain the very low levels of MDA in plasma as measured under standard assay conditions [7]. In addition to biological properties, DNA-protein cross-links are another result of the reaction between DNA and MDA. It has been reported that MDA reacts with DNA bases to form a series of adducts such as deoxyadenosine (M1A), and deoxycytidine (M1C) and deoxyguanosine (M1G) [8]. The major adduct is a pyrimidopurinone, abbreviated M1G or M1dG. This adduct possesses a blocked Watson-Crick base-pairing region that has been shown to be mutagenic [2, 8]. Another work reported that it is the first endogenous DNA lesion found to be a target of nucleotide excision repair enzymes, which might be a major endogenous DNA adduct that significantly contributes to cancer [9]. Recently, it has been reported that MDA adducts produced in mammalian cells may block enzyme RNA polymeraseII translocation and be subject to removal from DNA by transcription-coupled repair. M1dG formation results in two 1 http://www.4adi.com/data/protox/mda51.html; accessed on January 25, 2005. S. Jetawattana MDA, a lipid oxidation product 5 chemically distinct DNA adducts, Figure 3. Both M1dG and N-OPdG are highly mutagenic, inducing both frameshifts and base substitution, in bacteria and mammalian cells. Therefore, the efficient repair of MDA adducts is essential for genomic stability [10]. Figure 3. Formation of M1dG and the structures of M1dG and PdG (A synthetic, saturated analog of M1dG). MDA and its tautomer β-hydroxyacrolein react with guanine in DNA to form the pyrimidopurinone adduct (M1dG). When M1dG correctly paired with cytosine in duplex DNA, M1dG undergoes a base-catalyzed conversion to an acyclic structure, N-OPdG, which is known to be highly mutagenic in bacteria and mammalian cells, inducing both frameshifts and base substitutions [10]. . Detection Lipid peroxidation is a well-established mechanism of cellular injury in both plants and animals, and is used as an indicator of oxidative stress in cells and tissues. The measurement of MDA, which is the most abundant of lipid peroxidation products, is a convenient and sensitive method for quantitative estimation of lipid peroxide concentration in many types of samples including drugs, food products and biological tissues from human and animal. S. Jetawattana MDA, a lipid oxidation product 6 The most common method of measuring MDA is based on the reaction with thiobarbituric acid (TBA). The thiobarbituric acid reactive substances (TBARS) assay is the colorimetric method widely used for the detection of lipid peroxidation in biological materials. MDA is formed as a result of lipid peroxidation and reacts with thiobarbituric acid under high temperature (90-100C) and acidic condition. The reaction yields a pink MDA-TBA adduct, the product of 2 mol of TBA plus 1 mol of MDA, Figure 4. The colored complex can be extracted into organic solvents such as butanol and measured by fluorometry or spectrophotometry using wavelength 532 nm with an extinction coefficient ε532 = 1.53 x 10 M cm [11]. Figure 4. Thiobarbituric acid reaction [12]. Another method for the determination of MDA is based on the reaction of a chromogenic reagent, N-methyl-2-phenylindole (NMPI), with MDA at 45°C. One molecule of MDA reacts with 2 molecules of NMPI to yield a stable carbocyanine dye, Figure 5. In HCl, the molar extinction coefficient at 586 nm for malondialdehyde is approximately 1.2 x 10 M cm [12]. S. Jetawattana MDA, a lipid oxidation product 7