HNE-ELISA: detecting lipid peroxidation in biological systems
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Free radicals and oxidants are involved in physiological signaling pathways, although an imbalance between pro-oxidant and anti-oxidant systems in favor of the former leads to major biomolecular damage. This is the so-called oxidative stress, a complex process that affects us all and is responsible for the development of many diseases. Lipids are very sensitive to oxidant attack and to-date, malondialdehyde (MDA), 4-hydroxy-2-nonenal (4-HNE) and F2-isoprostane are the main biomarkers for lipid peroxidation assessment. They all derive from polyunsaturated fatty acids (PUFAs) either by enzyme-catalyzed reactions (physiological) or by non-enzyme reactions (pathological). The profile of PUFAs present in the tissue will determine the proportion of each biomarker. In this review we aim to discuss the proper method for MDA determination using HPLC. We also offer reference MDA values in humans in physiological and pathological conditions.
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Lipid peroxidation (LP) is determined by quantifying the malondialdehyde (MDA) content in the homogenate supernatant in the caudal adjacent segment to epicenter by colorimetric reaction with thiobarbituric acid (TBA) at high temperatures. Malondialdehyde is the principal and most studied product of polyunsaturated fatty acid peroxidation.
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Malondialdehyde
Thiobarbituric acid
TBARS
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Unsaturated aldehydes such as acrolein and hydroxyalkenals are produced in vivo through lipid peroxidation chain reactions under conditions of oxidative stress or carcinogenic insult, and are causally involved in the pathogenesis of numerous diseases (,). The ability of cytotoxic and genotoxic compounds to inactivate various biologically important macromolecules has been well documented (, , ). For enzymes and proteins, lysine, cysteine, histidine, and other amino acid residues that play key roles in their functionings are preferentially modified. The complicated chemistry and biochemistry of the reactive molecules occurring in vivo in micro- or ultramicroquantities remain unclear, because direct determination of such unstable compounds is problematic. An alternative approach is the immunochemical quantification of reaction products accumulating in vivo, for which monoclonal antibodies (MAbs) specific for the acrolein-modified lysine and 4-hydroxy-2-nonenal (HNE)-modified histidine epitopes have been prepared (Fig. 1) (,). However, several difficulties are encountered with enzyme-linked immunosorbent assay (ELISA) determination of haptenic molecules. One is the low avidity of MAbs against the small epitopes in general. The other is that the most widely used sandwich method is unapplicable to the quantitation because monovalent antigens are undetectable in principle. Open image in new window Fig. 1. Structures of acrolein and HNE (A) and their amino acid adducts (B).
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Lipid peroxidation is the oxidative deterioration of polyunsaturated fatty acids (PUFAs), which is a free radical related process. Studies showed that reactive oxygen species (ROS) are involved in a diversity of biological phenomena including atherosclerosis, neurodegenerative diseases, carcinogenesis etc. ROS and other pro-oxidant agents have the capacity to exhibit oxidative decomposition of PUFAs of membrane phospholipids leading to the formation of toxic end products, including malonyldialdehyde (MDA), 4-hydroxy-2-nonenal (HNE) etc. Drug-induced lipid peroxidation contributes to significant toxicity. Antioxidants have the ability to reduce generation of free radicals in the body by inhibiting lipid peroxidation process. In view of this concept, the present in vivo study is carried out to evaluate the inhibitory effect of ascorbic acid on lipid peroxidation induced by gentamicin, an aminoglycoside antibiotic. Levels of MDA, HNE, reduced glutathione (GSH) and nitric oxide (NO) are used as markers of lipid peroxidation. Rabbits divided into different experimental groups are treated with drug and co-administered with ascorbic acid. The levels of MDA, HNE, GSH and NO in the blood are estimated and compared with the control. The results revealed that gentamicin significantly increases MDA and HNE contents, but decreases GSH and NO levels; and ascorbic acid on co-administration has capability to inhibit drug-induced lipid peroxidation.
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TBARS
Thiobarbituric acid
4-Hydroxynonenal
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