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Quantification of malondialdehyde and 4-hydroxynonenal adducts to lysine residues in native and oxidized human low-density lipoprotein
Open accessAbstract
Malondialdehyde (MDA) and 4-hydroxynonenal (HNE) are major end-products of oxidation of polyunsaturated fatty acids, and are frequently measured as indicators of lipid peroxidation and oxidative stress in vivo. MDA forms Schiff-base adducts with lysine residues and cross-links proteins in vitro; HNE also reacts with lysines, primarily via a Michael addition reaction. We have developed methods using NaBH4 reduction to stabilize these adducts to conditions used for acid hydrolysis of protein, and have prepared reduced forms of lysine-MDA [3-(Nε-lysino)propan-1-ol (LM)], the lysine-MDA-lysine iminopropene cross-link [1,3-di(Nε-lysino)propane (LML)] and lysine-HNE [3-(Nε-lysino)-4-hydroxynonan-1-ol (LHNE)]. Gas chromatography/MS assays have been developed for quantification of the reduced compounds in protein. RNase incubated with MDA or HNE was used as a model for quantification of the adducts by gas chromatography/MS. There was excellent agreement between measurement of MDA bound to RNase as LM and LML, and as thiobarbituric acid-MDA adducts measured by HPLC; these adducts accounted for 70Ő80% of total lysine loss during the reaction with MDA. LM and LML (0.002Ő0.12mmol/mol of lysine) were also found in freshly isolated low-density lipoprotein (LDL) from healthy subjects. LHNE was measured in RNase treated with HNE, but was not detectable in native LDL. LM, LML and LHNE increased in concert with the formation of conjugated dienes during the copper-catalysed oxidation of LDL, but accounted for modification of < 1% of lysine residues in oxidized LDL. These results are the first report of direct chemical measurement of MDA and HNE adducts to lysine residues in LDL. LM, LML and LHNE should be useful as biomarkers of lipid peroxidative modification of protein and of oxidative stress in vitro and in vivo.
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“To our knowledge, MDA modified proteins have not been used as biomarkers in recent clinical investigations although antibodies are commercially available and highly selective MS methods have been established, previously [57].”
From Protein oxidation - Formation mechanisms, detection and relevance as biomarkers in human diseases (2021) · cites this paper as [57]
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“This positive partial charge is attacked by nucleophiles such as cysteine and histidine and stable Michael adducts are formed (Fig. 4B) [50]. Nucleophilic attack of lysine forms unstable Michael adducts, which require reduction, e.g., by sodium borohydride prior to analysis [57].”
From Protein oxidation - Formation mechanisms, detection and relevance as biomarkers in human diseases (2021) · cites this paper as [57]
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Metadata
- Authors
- REQUENA, J. R., FU, M. X., AHMED, M. U., JENKINS, A. J., LYONS, T. J., BAYNES, J. W., & THORPE, S. R.
- Affiliations
- University of South Carolina (US)
- Medical University of South Carolina (US)
- Venue
- Biochemical Journal
- Publisher
- Portland Press Ltd.
- Published
- 1997-02-15
- Volume
- 322
- Issue
- 1
- Pages
- 317-325
- ISSN
- 0264-6021 (ISSN-L)
- 1470-8728
- PMC
- PMC1218194
- OpenAlex
- W1843398852
- Cited by
- 293
- References
- 35
- Citations by year
- 2026: 4 · 2025: 3 · 2024: 3 · 2023: 13 · 2022: 7 · 2021: 6 · 2020: 7 · 2019: 2 · 2018: 5 · 2017: 9 · 2016: 6 · 2015: 5 · 2014: 7 · 2013: 11 · 2012: 14
- FWCI
- 10.07
- Type
- journal article
- Language
- en
- Open access
- bronze
- doi.org
- Locations
- Biochemical Journal (publishedVersion)
- PubMed Central (publishedVersion)
- PubMed (publishedVersion)
- Scholar Commons (University of South Carolina) (submittedVersion)
- Research Portal (Queen's University Belfast) (submittedVersion)
- Funders
- National Institutes of Health (5r01ag011472-04)
- National Institute on Aging (AG-11472)
- National Eye Institute (5R29 EY10697, R29EY010697)
- Topics
- Health Sciences › Medicine › Biochemistry (medical) › Antioxidant Activity and Oxidative Stress
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Redox biology and oxidative stress
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Clinical Biochemistry › Paraoxonase enzyme and polymorphisms
- Keywords
- malondialdehyde, 4-hydroxynonenal, lipid peroxidation, lysine adducts, low-density lipoprotein, protein oxidation, Schiff base adducts, Michael addition, sodium borohydride reduction, oxidative stress biomarkers, conjugated dienes, protein cross-linking
- MeSH terms
- Aldehydes, Amino Acids, Collagen, Copper, Crystallins, Drug Stability, Humans, Lipoproteins, LDL, Lysine, Malondialdehyde, Gas Chromatography-Mass Spectrometry, Oxidation-Reduction, Ribonucleases, Schiff Bases, Skin, Lipid Peroxidation
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