Measurement of HNE-protein adducts in human plasma and serum by ELISA—Comparison of two primary antibodies

Abstract

There is increasing evidence that non-enzymatic post-translational protein modifications might play key roles in various diseases. These protein modifications can be caused by free radicals generated during oxidative stress or by their products generated during lipid peroxidation. 4-Hydroxynonenal (HNE), a major biomarker of oxidative stress and lipid peroxidation, has been recognized as important molecule in pathology as well as in physiology of living organisms. Therefore, its detection and quantification can be considered as valuable tool for evaluating various pathophysiological conditions.The HNE-protein adduct ELISA is a method to detect HNE bound to proteins, which is considered as the most likely form of HNE occurrence in living systems. Since the earlier described ELISA has been validated for cell lysates and the antibody used for detection of HNE-protein adducts is non-commercial, the aim of this work was to adapt the ELISA to a commercial antibody and to apply it in the analysis of human plasma samples.After modification and validation of the protocol for both antibodies, samples of two groups were analyzed: apparently healthy obese (n=62) and non-obese controls (n=15). Although the detected absolute values of HNE–protein adducts were different, depending on the antibody used, both ELISA methods showed significantly higher values of HNE–protein adducts in the obese group.

Oxidative damage and the pathogenesis of menopause related disturbances and diseases

“…4-HNE-protein adducts can be accurately measured by time-and labor-consuming GC/ MS [40] or by immunoblotting (qualitative) methods. An ELISA test was introduced recently but has not been validated for measurements in blood samples [41]. MDA is also considered an effective marker of lipid peroxidation and is easily measured in serum/plasma samples by a spectrophotometric assay based on complex formation with thiobarbituric acid (TBA) to form TBA reacting substances (or TBARS) [42].…”

Association of 4-hydroxynonenal with classical adipokines and insulin resistance in a Chinese non-diabetic obese population

“…Although the detected absolute values of HNE-protein adducts were different, depending on the antibody used, both ELISA methods showed significantly higher values of HNE-protein adducts in the obese group (21). Intracellular HNE reacts rapidly with the thiol groups of glutathione and cysteine, with the ε-amino groups of lysine, and with the histidine residues of proteins (21). In this study, we used commercial ELISA kits to test the total endogenous 4-HNE bound to protein; our data showed that 4-HNE might be involved in the pathogenesis of non-diabetes obesity, as supported by two novel findings.…”

“…Although several animal experiments have suggested 4-HNE as an important regulator of glucose metabolism and insulin sensitivity, the clinical relevance of these findings in humans remains poorly characterized (18,20). The HNE-protein adduct ELISA is a method to detect HNE bound to proteins, which is considered as the most likely form of HNE in living systems (21). Although the detected absolute values of HNE-protein adducts were different, depending on the antibody used, both ELISA methods showed significantly higher values of HNE-protein adducts in the obese group (21).…”

“…The HNE-protein adduct ELISA is a method to detect HNE bound to proteins, which is considered as the most likely form of HNE in living systems (21). Although the detected absolute values of HNE-protein adducts were different, depending on the antibody used, both ELISA methods showed significantly higher values of HNE-protein adducts in the obese group (21). Intracellular HNE reacts rapidly with the thiol groups of glutathione and cysteine, with the ε-amino groups of lysine, and with the histidine residues of proteins (21).…”

Thermoluminescence as a complementary technique for the toxicological evaluation of chemicals in photosynthetic organisms

“…However, there are other sources of malondialdehyde, so this test is not completely specific for lipid peroxidation. In recent years, the development of immunochemical detection of 4-hydroxynonenal histidine adducts has opened more advanced methodological possibilities for qualitative and quantitative detection of lipid peroxidation in various human and animal tissues (Weber et al, 2013). These chemical methods cannot be however routinely used for the assessment of lipid oxidation in phytoplankton because the analysis requires high amounts of biological material, and they are often time-consuming and, therefore, not adequate for high throughput screening.…”