Protein reaction with malondialdehyde

Beyond the initial damage to membranes, reaction of these radicals with double bonds of fatty acids in lipids produces peroxides that give rise to a,15-unsaturated aldehydes including malondialdehyde (MDA), 4-hydroxynonenal (HNE) and acrolein. These aldehydes covalently bind to proteins through reaction with thiol groups and alter their function. We have previously shown that CAi hippocampal neurons were HNE positive by immunohistochemistry after transient cerebral ischemia (Adibhatla and Hatcher, 2006). Recently, elevated levels of an acrolein-protein conjugate were demonstrated in plasma of stroke patients (Adibhatla and Hatcher, 2007, and references cited therein). 

The most popular method involves 2-thiobarbituric acid (TBA) two molecules of 2-thiobarbituric acid are condensed with malonaldehyde. The emergent chromogen — the two tautomeric structures of the red TBA-malonaldehyde adduct — is determined at 532 nm, and also often at 450 nm, to determine aUcenals and aUcanals, respectively. The qualitative Kreis test was based on a similar principle it involved detection of the epihydrine aldehyde — a tautomeric malondialdehyde — in a color reaction with resorcine or phloroglucinol. The popularity of the TBA test stems from a correlation between the results and sensory evaluations. Paradoxically, this is related to the most important drawback of the TBA technique — its lack of specificity. In addition to the reaction with malonaldehyde, TBA forms compounds of identical color with other aldehydes and ketones, products of aldehyde interaction with nitrogen compounds, and also with saccharides, ascorbic acid, creatine, creatinine, trimethylamine oxide, trimethylamine, proteins, and amino acids. For this reason, the TBA test may even be treated as a proteolysis indicator (Kolakowska and Deutry, 1983). Recently, TBA-reactive substances (TEARS) were introduced, primarily to stress that the reaction involves hydroperoxides in addition to aldehydes. Due to the nonspecificity of the TEARS test, its results reflect the rancidity of food better than other conventional methods, especially off-flavor, which is caused by volatiles from lipids as well as being affected by products of lipids interaction with nitrogenous compounds. 

Reaction of a malondialdehyde (MDA) carbonyl group with amino groups leads to the formation of imines, but formation of these structures is of no nutritional concern, because they are hydrolysed at the acidic pH of the stomach. AT-Prop-2-enals, which are absorbed from the gut, are also formed in neutral or acidic aqueous media, but most of the absorbed material is not metaboHsed. A third type of reaction products are unavailable 4-substituted l,4-dihydropyridine-3,5-dicarbaldehydes, which arise in reactions of malondialdehyde with amino compounds, such as lysine, in the presence of alkanals. Examples of malondialdehyde reaction products with lysine are lV -(prop-2-enal)lysine, so-called MDA-lysine, JV -(prop-2-enal)lysine, ArAf -di(prop-2-enal)lysine (3-150) and conjugated cross-hnk in proteins termed lysine-MDA-lysine. An example of the reaction product of lysine with malondialdehyde and acetaldehyde is Ar,Ar-di(4-methyl-l,4-dihydropyridine-3,5-dicarbaldehyde)lysine, which, for example, arises in the reaction of bovine serum albumin with malondialdehyde and acetaldehyde (3-151). 

The reactions described so far do not require the involvement of the apo-B protein, neither would they necessarily result in a significant amount of protein modification. However, the peroxyl radical can attack the fatty acid to which it is attached to cause scission of the chain with the concomitant formation of aldehydes such as malondialdehyde and 4-hydroxynonenal (Esterbauer et al., 1991). Indeed, complex mixtures of aldehydes have been detected during the oxidation of LDL and it is clear that they are capable of reacting with lysine residues on the surface of the apo-B molecule to convert the molecule to a ligand for the scavenger receptor (Haberland etal., 1984 Steinbrecher et al., 1989). In addition, the lipid-derived radical may react directly with the protein to cause fragmentation and modification of amino acids. 

NADH, which enters the Krebs cycle. However, during cerebral ischaemia, metabolism becomes anaerobic, which results in a precipitous decrease in tissue pH to below 6.2 (Smith etal., 1986 Vonhanweh etal., 1986). Tissue acidosis can now promote iron-catalysed free-radical reactions via the decompartmentalization of protein-bound iron (Rehncrona etal., 1989). Superoxide anion radical also has the ability to increase the low molecular weight iron pool by releasing iron from ferritin reductively (Thomas etal., 1985). Low molecular weight iron species have been detected in the brain in response to cardiac arrest. The increase in iron coincided with an increase in malondialdehyde (MDA) and conjugated dienes during the recirculation period (Krause et al., 1985 Nayini et al., 1985). 

This test is used for both in vitro and in vivo determinations. It involves reacting thiobarbituric acid (TBA) with malondialdehyde (MDA), produced by lipid hydroperoxide decomposition, to form a red chromophore with peak absorbance at 532 nm (Fig. 10.1). The TBARS reaction is not specific. Many other substances, including other alkanals, proteins, sucrose, or urea, may react with TBA to form colored species that can interfere with this assay. 

It has been suggested that protein modifications by malondialdehyde, a major product of hpid peroxidation, contribute to the fluorescence formation of lipofuscin (Kikugawa and Beppu 1987, Tsuchida et al. 1987, Yin 1996). However, Itakura and UCHiDA (2001) isolated an aminoenimine, N,N -bis[5-(terf-butoxy-carboxamido)-5-carboxypent]-l-amino-3-iminopropene, formed from the reaction of malondialdehyde with a lysine derivative, N -ferf-butoxycarbonyl-L-lysine, at neutral pH, and confirmed that the purified Ar,hr-bis[5-(tert-butoxy-carboxyamido) - 5- carboxypent] -1 -amino- 3-imino-propene, exhibited no fluorescence. 

Antioxidative activities of the hydrolysates from porcine myofibrillar protein were measured in a linolenic acid oxidation system The hydrolysates at the concentration of 0.02, 0.2 and 2% exhibited antioxidative activities. All hydrolysates exhibited stronger antioxidative activity, as the concentration was higher in the production of hydroperoxides. On the other hand, the addition of 0.2% hydrolysate suppressed the production of TEARS most strongly, and the antioxidative activity in the addition of 2% hydrolysate was lower than that of 0.2% in the method of TEARS. It is well known that 2-thiobarbituric acid (TEA) reacts with aldehyde compounds including malondialdehyde (MDA) formed by Upid peroxidation. Two percent hydrolysates contained a lot of amino confounds including free amino acids and peptides. Therefore, the production of aldehyde compounds seems to be accelerated by amino-carbonyl reaction between hydrolysate and lipid in lipid peroxidation system including 2% hydrolysate. This might be the reason why the antioxidative activity of 2% hydrolysate was lower than that of 0.2 % hydrolysate in the method of TEARS. 

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