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Biochem J. 1997 Feb 15; 322(Pt 1): 317–325.
PMCID: PMC1218194

Quantification of malondialdehyde and 4-hydroxynonenal adducts to lysine residues in native and oxidized human low-density lipoprotein.


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-(N epsilon-lysino)propan-1-ol (LM)], the lysine-MDA-lysine iminopropene cross-link [1,3-di(N epsilon-lysino)propane (LML)] and lysine-HNE [3-(N epsilon-lysino)-4-hydroxynonan-l-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 70-80% of total lysine loss during the reaction with MDA. LM and LML (0.002-0.12 mmol/ 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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Selected References

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  • Witztum JL, Steinberg D. Role of oxidized low density lipoprotein in atherogenesis. J Clin Invest. 1991 Dec;88(6):1785–1792. [PMC free article] [PubMed]
  • Grundy SM. Role of low-density lipoproteins in atherogenesis and development of coronary heart disease. Clin Chem. 1995 Jan;41(1):139–146. [PubMed]
  • Palinski W, Ylä-Herttuala S, Rosenfeld ME, Butler SW, Socher SA, Parthasarathy S, Curtiss LK, Witztum JL. Antisera and monoclonal antibodies specific for epitopes generated during oxidative modification of low density lipoprotein. Arteriosclerosis. 1990 May-Jun;10(3):325–335. [PubMed]
  • Holvoet P, Perez G, Zhao Z, Brouwers E, Bernar H, Collen D. Malondialdehyde-modified low density lipoproteins in patients with atherosclerotic disease. J Clin Invest. 1995 Jun;95(6):2611–2619. [PMC free article] [PubMed]
  • Ylä-Herttuala S, Palinski W, Rosenfeld ME, Parthasarathy S, Carew TE, Butler S, Witztum JL, Steinberg D. Evidence for the presence of oxidatively modified low density lipoprotein in atherosclerotic lesions of rabbit and man. J Clin Invest. 1989 Oct;84(4):1086–1095. [PMC free article] [PubMed]
  • Salonen JT, Ylä-Herttuala S, Yamamoto R, Butler S, Korpela H, Salonen R, Nyyssönen K, Palinski W, Witztum JL. Autoantibody against oxidised LDL and progression of carotid atherosclerosis. Lancet. 1992 Apr 11;339(8798):883–887. [PubMed]
  • Esterbauer H, Gebicki J, Puhl H, Jürgens G. The role of lipid peroxidation and antioxidants in oxidative modification of LDL. Free Radic Biol Med. 1992 Oct;13(4):341–390. [PubMed]
  • Maggi E, Chiesa R, Melissano G, Castellano R, Astore D, Grossi A, Finardi G, Bellomo G. LDL oxidation in patients with severe carotid atherosclerosis. A study of in vitro and in vivo oxidation markers. Arterioscler Thromb. 1994 Dec;14(12):1892–1899. [PubMed]
  • Steinbrecher UP, Lougheed M, Kwan WC, Dirks M. Recognition of oxidized low density lipoprotein by the scavenger receptor of macrophages results from derivatization of apolipoprotein B by products of fatty acid peroxidation. J Biol Chem. 1989 Sep 15;264(26):15216–15223. [PubMed]
  • Fruebis J, Parthasarathy S, Steinberg D. Evidence for a concerted reaction between lipid hydroperoxides and polypeptides. Proc Natl Acad Sci U S A. 1992 Nov 15;89(22):10588–10592. [PMC free article] [PubMed]
  • Steinbrecher UP. Oxidation of human low density lipoprotein results in derivatization of lysine residues of apolipoprotein B by lipid peroxide decomposition products. J Biol Chem. 1987 Mar 15;262(8):3603–3608. [PubMed]
  • el-Saadani M, Esterbauer H, el-Sayed M, Goher M, Nassar AY, Jürgens G. A spectrophotometric assay for lipid peroxides in serum lipoproteins using a commercially available reagent. J Lipid Res. 1989 Apr;30(4):627–630. [PubMed]
  • Janero DR. Malondialdehyde and thiobarbituric acid-reactivity as diagnostic indices of lipid peroxidation and peroxidative tissue injury. Free Radic Biol Med. 1990;9(6):515–540. [PubMed]
  • Morrow JD, Roberts LJ., 2nd Quantification of noncyclooxygenase derived prostanoids as a marker of oxidative stress. Free Radic Biol Med. 1991;10(3-4):195–200. [PubMed]
  • Spadaro AC, Draghetta W, Del Lamma SN, Camargo AC, Greene LJ. A convenient manual trinitrobenzenesulfonic acid method for monitoring amino acids and peptides in chromatographic column effluents. Anal Biochem. 1979 Jul 15;96(2):317–321. [PubMed]
  • Esterbauer H, Schaur RJ, Zollner H. Chemistry and biochemistry of 4-hydroxynonenal, malonaldehyde and related aldehydes. Free Radic Biol Med. 1991;11(1):81–128. [PubMed]
  • Fu MX, Requena JR, Jenkins AJ, Lyons TJ, Baynes JW, Thorpe SR. The advanced glycation end product, Nepsilon-(carboxymethyl)lysine, is a product of both lipid peroxidation and glycoxidation reactions. J Biol Chem. 1996 Apr 26;271(17):9982–9986. [PubMed]
  • Osborne JC., Jr Delipidation of plasma lipoproteins. Methods Enzymol. 1986;128:213–222. [PubMed]
  • Baker JR, Zyzak DV, Thorpe SR, Baynes JW. Mechanism of fructosamine assay: evidence against role of superoxide as intermediate in nitroblue tetrazolium reduction. Clin Chem. 1993 Dec;39(12):2460–2465. [PubMed]
  • Knecht KJ, Dunn JA, McFarland KF, McCance DR, Lyons TJ, Thorpe SR, Baynes JW. Effect of diabetes and aging on carboxymethyllysine levels in human urine. Diabetes. 1991 Feb;40(2):190–196. [PubMed]
  • Young IS, Trimble ER. Measurement of malondialdehyde in plasma by high performance liquid chromatography with fluorimetric detection. Ann Clin Biochem. 1991 Sep;28(Pt 5):504–508. [PubMed]
  • LOWRY OH, ROSEBROUGH NJ, FARR AL, RANDALL RJ. Protein measurement with the Folin phenol reagent. J Biol Chem. 1951 Nov;193(1):265–275. [PubMed]
  • Szweda LI, Uchida K, Tsai L, Stadtman ER. Inactivation of glucose-6-phosphate dehydrogenase by 4-hydroxy-2-nonenal. Selective modification of an active-site lysine. J Biol Chem. 1993 Feb 15;268(5):3342–3347. [PubMed]
  • Chio KS, Tappel AL. Inactivation of ribonuclease and other enzymes by peroxidizing lipids and by malonaldehyde. Biochemistry. 1969 Jul;8(7):2827–2832. [PubMed]
  • Dunn JA, McCance DR, Thorpe SR, Lyons TJ, Baynes JW. Age-dependent accumulation of N epsilon-(carboxymethyl)lysine and N epsilon-(carboxymethyl)hydroxylysine in human skin collagen. Biochemistry. 1991 Feb 5;30(5):1205–1210. [PubMed]
  • Wells-Knecht MC, Huggins TG, Dyer DG, Thorpe SR, Baynes JW. Oxidized amino acids in lens protein with age. Measurement of o-tyrosine and dityrosine in the aging human lens. J Biol Chem. 1993 Jun 15;268(17):12348–12352. [PubMed]
  • Palinski W, Tangirala RK, Miller E, Young SG, Witztum JL. Increased autoantibody titers against epitopes of oxidized LDL in LDL receptor-deficient mice with increased atherosclerosis. Arterioscler Thromb Vasc Biol. 1995 Oct;15(10):1569–1576. [PubMed]
  • Lopes-Virella MF, Virella G. Atherosclerosis and autoimmunity. Clin Immunol Immunopathol. 1994 Nov;73(2):155–167. [PubMed]
  • Uchida K, Toyokuni S, Nishikawa K, Kawakishi S, Oda H, Hiai H, Stadtman ER. Michael addition-type 4-hydroxy-2-nonenal adducts in modified low-density lipoproteins: markers for atherosclerosis. Biochemistry. 1994 Oct 18;33(41):12487–12494. [PubMed]
  • Yeo HC, Helbock HJ, Chyu DW, Ames BN. Assay of malondialdehyde in biological fluids by gas chromatography-mass spectrometry. Anal Biochem. 1994 Aug 1;220(2):391–396. [PubMed]
  • Loidl-Stahlhofen A, Spiteller G. alpha-Hydroxyaldehydes, products of lipid peroxidation. Biochim Biophys Acta. 1994 Mar 3;1211(2):156–160. [PubMed]
  • Mlakar A, Spiteller G. Reinvestigation of lipid peroxidation of linolenic acid. Biochim Biophys Acta. 1994 Sep 15;1214(2):209–220. [PubMed]
  • Wells-Knecht KJ, Zyzak DV, Litchfield JE, Thorpe SR, Baynes JW. Mechanism of autoxidative glycosylation: identification of glyoxal and arabinose as intermediates in the autoxidative modification of proteins by glucose. Biochemistry. 1995 Mar 21;34(11):3702–3709. [PubMed]

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