The Importance of Lipid-Derived Malondialdehyde in Diabetes Mellitus

Diabetologia (2000) 43: 550±557

Ó Springer-Verlag 2000

The importance of lipid-derived malondialdehyde in diabetes mellitus

D.A. Slatter1, C.H.Bolton2, A.J.Bailey1 1 Collagen Research Group, Division of Molecular and Cellular Biology, University of Bristol, Bristol, UK 2 Diabetes and Metabolism Unit, Division of Medicine, Southmead Hospital, University of Bristol, Bristol, UK

Abstract induced by malondialdehyde which result in the stiffening of the collagenous tissues. Our recent studies Malondialdehyde (MDA) is a highly toxic by-product indicate the formation of pyridyl cross-links. formed in part by lipid oxidation derived free radi- Malondialdehyde has been shown to react several or- cals. Many studies have shown that its concentration ders of magnitude faster with the pre-existing col- is increased considerably in diabetes mellitus. lagen enzymic cross-links than the amino acid side- Malondialdehyde reacts both irreversibly and revers- chains. Malondialdehyde modification of basic ami- ibly with proteins and phospholipids with profound no-acid side-chains also results in a change in proper- effects. In particular, the collagen of the cardiovascu- ties, for example, in the charge profile of the mole- lar system is not only stiffened by cross-links mediat- cule resulting in modified cell-matrix interactions. ed by malondialdehyde but then becomes increasing- Although aspects of the biochemistry of malondial- ly resistant to remodelling. It is important in diabetes dehyde are still not fully understood its complex mellitus because the initial modification of collagen chemistry is being unravelled and this should lead to by sugar adducts forms a series of glycation products ways of preventing its damaging reactions, for exam- which then stimulate breakdown of the lipids to mal- ple, through antioxidant therapy. [Diabetologia ondialdehyde and hence further cross-linking by mal- (2000) 43: 550±557] ondialdehyde of the already modified collagen. Some progress is being made into the mechanisms of forma- Keywords Malondialdehyde, cross-linking, diabetes, tion and the nature of the intermolecular cross-links atherosclerosis.

Introduction [1]. It is also found in Alzheimer's disease [2], with effects that extend far beyond the lipid field. Malondialdehyde (MDA) is generated by both lipid Oxidation of complex lipids in vivo is largely caused oxidation and as a by-product of prostaglandin and by oxygen-derived free radicals (OFR) such as OH´. thromboxane synthesis. Its plasma concentration is These radicals are formed by lipoxygenases as a re- increased in diabetes mellitus and it is found in the sponse to cell injury, typically from H2O2, or a metal- atherosclerotic plaque deposits promoted by diabetes ion radical complex. The major targets of these damaging species are the long-chain polyunsaturated fatty acids of cellular phospholipids, which are particularly Corresponding author: Professor A. J. Bailey, Collagen Re- prone to attack because of the arrangement of double search Group, Division of Molecular and Cellular Biology, and single bonds. The resultant lipid peroxide fre- University of Bristol, Bristol BS40 5DS, UK quently decomposes to a radical [3], which reacts with Abbreviations: MDA, Malondialdehyde; OFR, oxygen-de- most biological molecules, including proteins and lip- rived free radicals; ox-LDL, oxidised LDL; NPO, Nd-(2-pyrimidyl)-l-ornithine; b-LAA, Ne-b-lysyl-aminoacrolein; LML, 1,3- ids. Further decomposition of these lipid peroxides di(Ne-lysino)propane NLMDD N-lysyl-4-methyl-2,6 dihydro- produces toxic aldehydes, in particular 4-hydroxynon- pyridine-3,5-dicarbaldehyde. enal (mainly from linoleic acid) malondialdehyde D.A. Slatter et al.: Lipid-derived malondialdehyde cross-linking 551

(mainly from arachidonic acid) [4] and acrolein, RNase, crystallin, and haemoglobin [13±15]. Such ad- whose sources are not yet fully characterised [5]. The ducts diminish these proteins' susceptibility to prote- liver eliminates MDA from the circulation by the ac- ases and they are formed much faster when the pro- tion of aldehyde dehydrogenase and thiokinase such tein is already glycated by sugars [16]. These modi- that injected MDA has a half-life of approximately fied proteins are targeted by antibodies and removed 2 h in rats, but some (10±30%) must bind semi-perma- from the bloodstream. Malondialdehyde can also re- nently to proteins as it is not eliminated within 12 h [6]. act with DNA, having been shown to be both mu- The toxicity of MDA arises from its high reactivity, tagenic and carcinogenic. particularly towards proteins and DNA. Our own particular interest is the high reactivity of Under normal circumstances the extent of lipid MDA with the long-lived proteins of the vascular sys- oxidation is largely controlled by antioxidant concen- tem, collagen and elastin. Due to the slow turnover of tration in the surrounding medium which is usually these proteins in the extracellular matrix and the sufficiently high to prevent propagation of oxidative crystallins of the eye, this eventually becomes a prob- free radical reactions by OFR in blood. In tissue, lem as they become cross-linked and resistant to pro- there is, however, a greater likelihood that localised teolysis. We have shown that the glycation cross-link- deficiencies of antioxidants would allow lipid oxida- ing of the aortic collagen is related to the mechanical tion to occur. This has led to a huge interest in dietary stiffening of the aorta of diabetic subjects [17]. Simi- antioxidants and their protective role in cardiovascu- larly, the physical properties of aortic elastin have in- lar disease [7]. In addition to OFR, glycated collagen creased stiffness and a loss of basic groups with con- has also been shown to increase the oxidative break- sequent conformational rearrangements [18]. The down of lipids compared with normal collagen [8] precise mechanisms are still not clear, encouraging which is one explanation for the increased concentra- new biochemical studies characterising the nature of tions of MDA in serum and tissues in diabetic sub- the additional MDA adducts and cross-links respon- jects. sible for the changes in properties of the aorta. The major carrier of lipid in blood is low density li- In this review we summarise the effects of MDA poprotein (LDL), a potent risk factor for coronary on collagen in relation to the cardiovascular system heart disease (CHD). When sufficient MDA modi- and the finding of a potential biomarker of lipid-de- fies the protein (apolipoprotein B100) of circulating rived oxidative stress which could lead to the identifi- LDL, it no longer reacts with the normal LDL recep- cation of possible treatments. Malondialdehyde is a tor in hepatic and peripheral cells, but only with scav- potential major candidate for the deleterious effects enger receptors of macrophages [9]. Thus, if less than of cross-linking and side-chain modification of the 12% of lysine side-chains are modified, the LDL is collagen of the vascular system. Its reactions with col- still recognisable to its receptor. If more than 15% lagen are only now beginning to be unravelled. are modified, however, only the scavenger receptor can recognise the LDL [10]. The clinical relevance of the reaction between MDA and proteins is high- Structure and chemistry of MDA lighted in atherosclerosis, a disease prevalent in de- veloped cultures, in which the arterial intima be- Malondialdehyde is a white hygroscopic crystalline comes infiltrated with ªfoam cellsº, lipid-laden mac- compound and is typically obtained by acid hydroly- rophages, resulting in thickened, non-resilient arter- sis of 1,1,3,3-tetraethoxypropane [19]. Radioactively ies with reduced blood flow [11]. This is a major cause labelled 14C-MDA can be prepared from 1,3-pro- of CHD and strokes. Malondialdehyde-LDL, in addi- panediol using alcohol dehydrogenase [20]. It is tion to oxidised LDL (oxLDL) mediates several pro- more stable in plasma than might be expected be- inflammatory and pro-atherogenic processes, all of cause it enolises readily, losing a proton at neutral which ultimately lead to foam cell generation [12]. It pH to form a salt (Fig.1). Its reactivity is therefore is important to realise that the term ªoxidised LDLº very dependent on pH. has habitually come to be recognised as any LDL with some degree of modification caused by lipid peroxidation and can range from the so-called minimally Determination of MDA modified product which can be recognised by the LDL receptor to the fully modified protein which is Malondialdehyde is used as a putative marker of lipid recognisable only to the scavenger receptor on mac- oxidation both in plasma and in arterial lesions. rophages. Malondialdehyde is a major, but not the There is now substantial evidence that both ox-LDL only, modifying agent and has been used frequently and MDA occur in the atherosclerotic plaque. as a model compound for making in vitro ªox LDLº. Malondialdehyde also reacts rapidly with other Chemical measurement. Malondialdehyde reacts proteins. It has been shown to cross-link bovine se- strongly with thiobarbituric acid producing fluores- rum albumin to form dimers and also modifies cent thiobarbituric acid reacting substances [21]. 552 D.A. Slatter et al.: Lipid-derived malondialdehyde cross-linking

Fig.1. Enol and salt forms of malondialdehyde AB Fig.2A, B. Reaction products of MDA with (A) Lysine and (B) arginine

This reaction, although simple and reproducible, is cannot react with more autoantibody this could lead unfortunately rather non-specific because thiobarbi- to a falsely low assessment of ox-LDL antibody sta- turic acid reacts with many other carbonyl-containing tus when assayed by the convential ELISA method. compounds. Plasma fatty acids can also oxidise dur- It has also been suggested that autoantibodies which ing the 95C heating step with thiobarbituric acid, bind MDA-LDL trigger autoimmune responses [31]. generating artificially high results [22, 23]. The use of EDTA-containing plasma and high performance liquid chromatography coupled with post-column Reaction of MDA with amino acids thiobarbituric acid derivitisation can, however, iden- tify the specific malondialdehyde-thiobarbituric acid Model reactions with amino acids. At neutral pH, complex, providing a relevant assay for MDA in bio- MDA hydrolyses to acetaldehyde and formic acid logical fluids [24±26]. over a few weeks, a reaction that is catalysed by the With these precautions, normal concentrations of presence of amino acids such as lysine or arginine MDA in plasma have been shown to be in the range [32] but at the same time forms adducts with these 0±1 mM/l. Concentrations of free MDA alone are as amino acid side-chains. These basic amino acids are low as 50 nmol/l [27], but chemical work suggests the only ones whose side-chains react with MDA to that at least 80% of MDA is protein-bound. These form adducts. The e-amino group of lysine reacts concentrations are increased in diabetes mellitus with MDA to form the semi-stable Ne-b-lysyl-amino- where hyperglycaemia is known to accelerate lipid acrolein (b-LAA) (Fig.2A), [19, 33], whereas argin- oxidation [28], partially explaining the increased risk ine reacts roughly an order of magnitude slower to of atherosclerosis. form the stable Nd-(2-pyrimidyl)-l-ornithine (NPO), (Fig.2b) [34, 35]. The side-chain of cysteine reacts Immunological measurement. The presence of both with oxidised fatty acids but not directly with MDA ox-LDL and MDA-LDL in the circulation is diffi- [36]. Schiff bases formed between MDA and tryp- cult to confirm directly although autoantibodies to tophan or histidine are much more stable than b- ox-LDL have been shown in plasma and can give LAA [33]. an indirect measure. Evidence reviewed recently suggests that the relation between circulating ox- Mechanism of formation of potential model cross- LDL autoantibodies and risk of cardiovascular dis- links. Malondialdehyde is best viewed as two entirely ease is inconclusive [29]. Oxidised-LDL will inevita- different molecules depending on the pH. For exam- bly be a mixture of subunits with different levels of ple, at a pH below its pKa MDA reacts with lysine oxidation or MDA complexes or both. Assays to de- to give firstly b-LAA, then 1,3-di(Ne-lysino)propane tect autoantibodies to ox-LDL only react with a spe- (LML) (Fig.3) which can be isolated after sodium cific antigen. For example, autoantibodies to other borohydride reduction of the Schiff-base adduct of forms of ox-LDL will not necessarily bind MDA- two blocked lysine molecules and MDA [37]. When LDL prepared in vitro. An additional complication either the concentration of MDA is lowered to physi- is that autoantibodies to ox-LDL already complexed ological levels [33] or when the pH is neutral [19], with ox-LDL (immune complexes) have also been LML is, not observed unless high concentrations of detected in the circulation of diabetic patients [30]. sodium borohydride are present in the MDA/lysine Because the complexed autoantibodies to ox-LDL mixture [13]. D.A. Slatter et al.: Lipid-derived malondialdehyde cross-linking 553

The adducts generated on incubation of protein with MDA occur essentially at random along the protein chain, preventing any direct analysis by nuclear magnetic resonance or other spectroscopic method. In an attempt to isolate these potential cross-links we have found that acid hydrolysis results in breakdown of any potential MDA cross-links. So far, devel- opment of an efficient enzyme hydrolysis procedure that reliably breaks down glycated collagen to individual amino acids and cross-links has been unsuc- cessful. As a result, the only method used successfully to date is the stabilisation of some of the products by sodium borohydride reduction followed by acid hydrolysis to achieve separation of the individual products. Using this technique, b-LAA has been shown to react with lysine from RNase to form an imido- propene cross-link as LML [13]. Such a protein Fig.3. Reaction of b-LAA with lysine (or arginine) to form po- cross-link in the Schiff base form could easily hydro- tential intermolecular cross-links LML and NLMDD, respec- lyse at neutral pH to release the lysines and MDA. tively There was, however, no hint of its presence in model lysine-MDA reactions analysed by nuclear magnetic resonance and it is unlikely that the b-LAA-Lysine The product of lysine with MDA, b-LAA, can, equilibrium with LML would shift sufficiently to- however, react further with another molecule of wards LML to favour its formation in proteins. It is MDA and a molecule of acetaldehyde (derived from therefore probable that its detection is an artefact MDA breakdown in vitro, but present in serum) to generated by the sodium borohydride reduction. form a stable fluorescent product, N-lysyl-4-methyl- Other similar investigations with bovine serum albu- 2,6-dihydropyridine-3,5-dicarbaldehyde (NLMDD), min and collagen using smaller quantities of sodium (Fig.3) [19, 31, 32]. The aldehyde side-chains on the borohydride (but still in vast excess) have not result- stable dihydropyridine ring are then capable of fur- ed in the isolation of this link [15,19]. This could be ther reactions with lysine to form a reversible cross- because the conjugated P system in LML and b- link [19]. Such an intermolecular cross-link would LAA is hard to reduce, requiring considerable quan- stiffen the collagen fibres of the aorta or vascular le- tities of reducing agent for successful reduction. sions. The most stable lysine product characterised in Whether this cross-link reaction occurs under chemical work as a potential cross-link is the dihy- physiological conditions is still to be established. It is dro-pyridine derivative, NLMDD (Fig.3), which is possible that other plasma components could react also vulnerable to acid hydrolysis even after reduc- in place of the second MDA and acetaldehyde, for in- tion with borohydride [34]. For this reason it has not stance, fumarate, 4-hydroxynonenal, acrolein, sugars, yet been isolated from proteins treated with MDA. and various Maillard intermediates from the reaction of sugars and proteins. Effect of MDA on the pre-existing collagen cross- links. Collagen fibres are stabilised by a series of lysine-aldehyde cross-links [38] and surprisingly our re- Model reactions of MDA with proteins cent work has shown that MDA reacts with these natural collagen cross-links orders of magnitude faster Protein cross-linking. Malondialdehyde has been than lysine [34]. Presumably MDA cleaves the natu- shown to cross-link proteins by molecular weight ral Schiff-base cross-links by competitive reaction changes and visually, for example, after a short incu- with the lysines and then forms a new intermolecular bation with MDA rat tail tendon becomes rigid. The cross-link. Unfortunately they again form `putative collagen becomes insoluble after only 24 h and the cross-links' that are rapidly degraded by acid hydrol- formation of intermolecular cross-links is shown by ysis as shown by the absence of the C-14 label in the thermal isometric tension studies and differential products after incubation with C-14 MDA [34], hence scanning calorimetry (Fig.4). Polyacrylamide gel the nature of these cross-links is still to be explained. electrophoresis and column chromatography have In summary, despite the evidence of increasing conclusively shown, by the increase in molecular molecular weight no cross-link derived from MDA weight, that protein cross-links are formed after reac- has been isolated and characterised. A potential tion with MDA, at least for RNase [13], a-crystallin product has been identified (NLMDD) but not the fi- [14] and soluble collagen (Fig.4). nal peptide bound product to confirm its role as a 554 D.A. Slatter et al.: Lipid-derived malondialdehyde cross-linking

A ii iii

Fig.4. A The effect of MDA cross-linking of collagen as as- abetic subjects and NPO could therefore be a poten- sessed by (i) increasing molecular weight by SDS-PAGE; tial biomarker [34]. a and b represent single and dimeric collagen chains and It could be speculated that NPO reacts with lysine HMW high molecular weight components. (ii) increased ten- sile strength as depicted by stress-strain curve after 24 h of in- aldehyde present in collagen to form new cross-links. cubation (iii) increasing thermal denaturation temperature It is self-evident that the faster reaction of glycated with time of incubation by differential scanning calorimetry. B proteins with MDA [16] must involve more than just Schematic representation of the probable location of cross- lysine or arginine and possibly a reaction with pre-ex- links in the fibril (i) natural immature cross-links within the mi- isting MDA adducts such as NPO. crofibre (I), natural mature inter fibril cross-links ()), (ii) addi-

« Similarly, the MDA-lysine adduct, b-LAA, has tional MDA cross-links within and between microfibrils ( ) been observed as a reduction product [40] and could be an epitope recognised by autoantibodies, as well as a target for elimination by macrophages [9]. The terminal a-amino-groups of proteins have cross-link. Studies to date suggest that MDA cross- also been shown to react with MDA in a manner sim- links are labile and are destroyed by acid hydrolysis. ilar to the e-amino group of peptide bound lysine, for It has been shown that MDA will form cross-links example, the valine N-terminus of haemoglobin [15]. such as LML when the pH is approximately five or less, but there is no convincing evidence that it occurs Modification of cell-matrix interactions. The reactions at the higher physiological pH, where NLMDD and with the side-chains of protein-bound lysine and argi- b-LAA are favoured. It has, however, recently been nine possibly not only affect the functions of collagen suggested [39] that acidic conditions prevail in the im- by making it less susceptible to degradative enzymes mediate vicinity of the foam cell lesions seen in early but could result in the formation of adducts that atheroma. Such conditions could support the forma- change the charge profile of the molecule and affect tion of the di-lysyl derivative. collagen-cell interactions. This effect would be particularly important if the particular arginine residues re- Protein side-chain adducts. Non-cross-linking MDA acting with the MDA to form NPO are sites recognis- adducts are also formed in the reaction of MDA ed by cell surface integrins, such as, the arginine-gly- with proteins. Malondialdehyde reacts with arginine cine-aspartic (RGD) sequences. Preliminary investi- side-chains to form NPO and this compound has gations have shown that MDA-collagen affects both been isolated from in vitro collagen-MDA incuba- the morphology and expression of the cells (unpub- tions as it is reasonably resistant to acid [34]. Prelimi- lished observations). The response is similar to that nary studies have identified its presence in skin of di- reported for the reaction of glyoxal, which has been D.A. Slatter et al.: Lipid-derived malondialdehyde cross-linking 555

Fig.6. Proposed schematic pathway of MDA cross-linking and side-chain modification of cardiovascular collagen in diabetes mellitus. The glucose glycated collagen stimulates oxidation of LDL producing MDA which then cross-links the collagen, stabilising the collagen and rendering it susceptible to further glycation and increased oxidation of the LDL and producing more MDA continuing a cycle of events AB Fig.5A, B. Nucleotide adducts with Malondialdehyde. (A) Malondialdehyde-deoxy-guanosine adduct (pyrimido-(1,2)pu- (Fig.5) with analogous products for cytosine. Ad- rine-10(3H)-one-2-deoxyribose) (B) Malondialdehyde-deoxy- ducts of 3:1 have also been reported for MDA:ade- adenosine adduct (N-6-oxypropenyl-2-deoxyribose) nine at pH 4.2 [50] (Fig.5). Biological cross-links have, however, only been isolated using extensive borohydride reduction [46] which as described above shown to react fairly specifically with arginine in col- has been questioned. The conclusion enforced is that lagen to form imidazolones which block arginine con- there is almost certainly DNA-MDA cross-linking to taining integrin sites (e.g. arginine-glycine-aspartic) some degree but no concrete data or consensus on and lead to decreased adhesion and migration of cells its nature. [41].

Concluding remarks MDA-nucleotide adducts The inter-molecular cross-linking of collagen through Several products of the reaction of nucleotides with MDA is important in the late complications of diabe- MDA have been isolated and characterised (Fig.5). tes mellitus because it contributes to the stiffening of Adding MDA to human cell cultures so that de- the cardiovascular tissue, although the nature of the oxyguanosine-MDA is three to six times normal has cross-link remains to be determined. In addition it been shown to induce cell cycle arrest [42]. Two sepa- could be a link between glycation and further lipid rate groups report isolation of deoxyguanosine-MDA peroxidation. The stabilisation of long-lived proteins from rodent livers [43,44]. All the non-cross-linking such as collagen through MDA cross-linking not MDA adducts so far described disrupt base-pairing, only reduces its optimal functioning but reduces its and any MDA-DNA cross-link could easily cause already low turnover and consequently allows further the observed mutagenic and carcinogenic effects of glycation by glucose and its oxidation products. This MDA. Whatever the actual reactivity MDA has with in turn increases the potential of the glycated col- DNA, repair systems must be present to recognise lagen to initiate further oxidation of fatty acids re- and replace it, as it has been shown that deoxygua- leasing more MDA and thereby propagating a posi- nosine-MDA (Fig.4) is present in urine [45]. tive feedback cycle of protein and fatty acid damage Again, there is confusion due to investigations of (Fig.6). the chemical reactions being carried out at a different Changes in both physical properties and charge pH. The fluorescence generated from MDA-DNA profile of the protein would be particularly important reactions at neutral pH has been attributed to DNA for the attachment of cells to the collagenous base- cross-linking but these studies were carried out at ment membrane. Recent studies have shown a pro- pH 5 and involved many processing steps before the nounced change in the cell-matrix interactions after final isolation of deoxycytosine-MDA-deoxygua- glycation of the matrix [41, 51]. This induces platelet nosine [46]. aggregation, increasing the risk of thrombotic disease Incubation of the MDA analogue b-benzyloxyac- in the elderly and diabetic subjects. rolein with calf thymus DNA at pH 6.5, has been reported to produce two adducts, deoxyguanosine- Acknowledgements. The authors are indebted to the British MDA and deoxyadenosine-MDA [47]. Chemically, Diabetic Association for a grant supporting D. A. Slatter. MDA: guanosine adducts have been reported at ra- tios of 1:1 at pH 4.5 [48] and 2:1 at pH 7.0 [49], 556 D.A. Slatter et al.: Lipid-derived malondialdehyde cross-linking

References 20. Summerfield FW, Tappel AL (1978) Enzymatic synthesis of malondialdehyde. Biochem Biophys Res Commun 82: 1. Kume S, Takeya M, Mori T et al. (1995) Immunohisto- 547±552 chemical and ultrastructural detection of advanced glyca- 21.Yagi K (1994) Lipid peroxides and related radicals in clini- tion end-products in atherosclerotic lesions of human aorta cal medicine. Adv Exp Med Biol 366: 1±15 with a novel specific monoclonal antibody. Am J Pathol 22. Dahle LK, Hill EG, Holmann RT (1962) The thiobarbitu- 147: 654±657 ric acid reaction and the autoxidation of polyunsaturated 2. Markesberry WR (1995) Oxidative stress hypothesis in fatty acid methyl esters. Arch Biochem Biophys 98: Alzheimer's Disease. Free Radic Biol Med 23: 134±147 253±261 3. Spiteller G (1998) Linoleic acid peroxidation, the dominant 23. Esterbauer H, Schaur RJ, Zollner H (1991) Chemistry and lipid peroxidation process in low-density lipoprotein, and biochemistry of 4-hydroxynonenal, malondialdehyde and its relationship to chronic diseases. Chem Phys Lipids 95: related aldehydes. Free Radic Biol Med 11: 81±128 105±162 24. Wong SHY, Knight JA, Hopfer SM, Zaharia O, Leach CN, 4. Esterbauer H, et al. (1990) Biochemical, structural, and Sunderman FW (1987) Lipoperoxides in plasma as mea- functional properties of oxidized low-density lipoprotein. sured by liquid-chromatographic separation of malondial- Chem Res Toxicol 3: 77±92 dehyde thiobarbituric acid adduct. Clin Chem 33: 214±220 5. Uchida K, Kanematsu M, Morimitsu Y, Osawa T, Noguchi 25. Young IS, Trimble ER (1991) Measurement of malondial- N, Niki E (1998) Acrolein is a product of lipid peroxidation dehyde in plasma by high performance liquid chromatogra- reaction. Formation of free acrolein and its conjugate with phy with fluorimetric detection. Ann Clin Biochem 28: lysine residues in oxidised low density lipoproteins. J Biol 504±508 Chem 273: 16058±16066 26. Fukunga K, Yoshida M, Nakazono N (1998) A simple, rap- 6. Sui SM, Draper HH (1982) Metabolism of malondialde- id, highly sensitive and reproducible quantification method hyde in vivo and in vitro. Lipids 17: 349±355 for plasma malondialdehyde by HPLC. Biomed Chroma- 7. Rimm EB, Stampfer MJ, Ascherio A, Giovannucci E, Col- togr 12: 300±303 ditz GA, Willett WC (1993) Vitamin E consumption and 27. Yeo HC, Helboch HJ, Chuyu DW, Ames BN (1994) Assay the risk of coronary heart disease in men. N Eng J Med of malondialdehyde in biological fluids by gas chromatog- 328: 1450±1456 raphy mass-spectroscopy. Anal Biochem 220: 391±396 8. Hicks M, Delbridge L, Yue DK, Reeve TS (1988) Catalysis 28. Niskanen LK, Salonen JT, Nyyssonen K, Uusitupa HI of lipid peroxidation by glucose and glycosylated collagen. (1995) Plasma lipid peroxidation and hyperglycaemia: a Biochem Biophys Res Commun 151: 649±655 connection through hyperinsulinaemia? Diabet Med 12: 9. Brown MS, Goldstein JL (1983) Lipoprotein metabolism in 802±808 the macrophage: implications for cholesterol deposition in 29. Yla-Herttuala S (1998) Is oxidised low-density lipoprotein atherosclerosis. Ann Rev Biochem 52: 223±261 present in vivo. Curr Opin Lipidol 9: 337±344 10. Haberland ME, Fogelman AM, Edwards PA (1982) Speci- 30. Festa A, Kopp HP, Schernthaner G, Menzel EJ (1998) Au- ficity of receptor-mediated recognition of malondialde- toantibodies to oxidised low density lipoproteins in IDDM hyde-modified low density lipoproteins. Proc Natl Acad are inversely related to metabolic control and microvascu- Sci USA 79: 1712±1716 lar complications. Diabetologia 41: 350±356 11. Ross R (1995) Cell biology of atherosclerosis. Ann Rev 31. Xu D, Thiele GM, Beckenhauer JL, Klassen LW, Sorrell Physiol 57: 791±804 MF, Tuma DJ (1998) Detection of circulating antibodies 12. Berliner J, Heinecke JW (1996) The role of oxidized lipo- to malondialdehyde-acetaldehyde adducts in ethanol fed proteins in atherogenesis. Free Radic Biol Med 20: 707± rats. Gastroenterology 115: 686±692 727 32. Nair V, Offermain RJ, Turner GA, Pryor AN, Baenziger 13. Requena JR, Fu MX, Ahmed MU et al. (1997) Quantifica- NC (1998) Fluorescent 1,4-dihydropyridines ± the malondi- tion of malondialdehyde and 4-hydroxynonenal adducts to aldehyde connection. Tetrahedron 44: 2793±2803 lysine residues in native and oxidised human low-density li- 33. Nair V,Vietti DE, Cooper CS (1981) Degenerative chemis- poprotein. Biochem J 322: 317±325 try of malondialdehyde ± structure, stereochemistry, and 14. Libondi T, Ragone R, Vincenti D, Stiuso P, Auricchio G, kinetics of formation of enaminals from reaction with ami- Colonna G (1994) In vitro cross-linking of calf lens a-cry- no acids. J Am Chem Soc 103: 3030±3036 tallin by malondialdehyde. Int J Pept Protein Res 44: 34. Slatter DA, Paul RG, Murray M, Bailey AJ (1999) Reac- 342±347 tions of lipid-derived malondialdehyde with collagen. J 15. Kautiainen A, Tornqvist M, Svensson K, Osterman-Golkar Biol Chem 274: 19661±19669 S (1989) Adducts of malondialdehyde and a few other alde- 35. King TP (1996) Malondialdehyde reacts with arginine hydes to haemoglobin. Carcinogenesis 10: 2123±2130 to form Nd-(2-pyrimidyl)-L-ornithine. Biochemistry 5: 16. Mooradian A.D, Lung C-C, Pinnas JL (1996) Glycosyla- 3454±3459 tion enhances malondialdehyde binding to proteins. Free 36. Shin BC, Huggins JW, Carraway KL (1971) Effects of pH, Radic Biol Med 21: 699±701 concentration, and aging on the malondialdehyde reaction 17. Sims TJ, Rasmusson LM, Oxlund H, Bailey AJ (1996) The with proteins. Lipids 7: 229±233 role of glycation cross-links in diabetic vascular stiffening. 37. Chio KS, Tappel AL (1969) Synthesis and characterization Diabetologia 39: 946±951 of the fluorescent products derived from malondialdehyde 18. Winlove P,Parker KH, Avery NC, Bailey AJ (1996) The in- and amino acids. Biochemistry 8: 2821±2827 teraction of elastin and aorta with sugars in vitro and their 38. Bailey AJ, Paul RG, Knott L (1998) Mechanisms of matu- effects on biochemical and physical properties. Dia- ration and ageing of collagen. Mech Ageing Dev 106: 1±56 betologia 39: 1131±1139 39. Leake DS (1997) Does the acidic pH explain why low den- 19. Slatter DA, Murray M, Bailey AJ (1998) Formation of a di- sity lipoprotein is oxidised in atherosclerotic lesions? Ath- hydropyridine derivative as a potential cross-link from mal- erosclerosis 129: 149±157 ondialdehyde in physiological systems. FEBS Letts 421: 40. Chancerelle Y, Alban C, Viret R, Tosetti F, Kerganou D-F 180±184 (1991) Immunological relevance of malonic dialdehyde: D.A. Slatter et al.: Lipid-derived malondialdehyde cross-linking 557

IV. Further evidence about the epitope recognised by anti- 47. Chaudhasry AK, Reddy GR, Blair IA, Marnett LJ (1996) bodies obtained from rabbits immunised with MDA-modi- Characterisation of N6-oxopropenyl-2'-deoxyguanosine fied lysozyme. Biochem Int 24: 157±163 adduct in malondialdehyde-modified DNA using liquid 41. Paul RG, Bailey AJ (1999) The effect of advanced glyca- chromatography/electrospray ionization tandem mass tion end-product formation upon cell-matrix interactions. spectrometry. Carcinogenesis 17: 1167±1170 Int J Biochem Cell Biol 31: 653±660 48. Seto H, Takesue T, Ikemura T (1985) Reaction of malondi- 42. Chuan J, Rouzer CA, Marnett LJ, Pietenpol JA (1998) In- aldehyde with nucleic acids II. Formation of fluorescent duction of cell cycle arrest by the endogenous product of pyrimido-[1,2-a]-purin-10-(3H)-one mononucleotide. Bull lipid peroxidation, malondialdehyde. Carcinogenesis 19: Chem Soc Jpn 58: 3431±3435 1275±1283 49. Marnett LJ, Basu AK, O'Hara SM, Weller PE, Rhaman 43. Argawal S, Draper HH (1992) Isolation of a malondialde- AFMM, Oliver JP (1986) Reactions of malondialdehyde hyde-deoxyguanosine adduct from rat liver DNA. Free Ra- with guanine nucleotides:- formation of adducts containing dic Biol Med 13: 695±699 Oxodiazabicyclononene residues in the base pairing re- 44. Chaudhary AK, Nukubo M, Reddy GR, Yeola SN, Mor- gion. J Am Chem Soc 108: 1348±1352 row JD, Blair IA, Marnett LJ (1994) Detection of endoge- 50. Nair V, Turner GA, Offerman RJ (1984) Novel adducts nous malondialdehyde-deoxyguanosine adducts in liver. from the modification of nucleic acid bases by malondial- Science 265: 1580±1582 dehyde. J Amer ChemSo. 106: 3370±3371 45. Hadley M, Draper HH (1990) Isolation of a guanine-mal- 51. Haitoglou CS, Tsilibary EC, Brownlee M, Charonis AS ondialdehyde adduct from rat and human urine. Lipids 25: (1992) Altered cellular interactions between endothelial 82±85 cells and non-enzymatically glucosylated laminin/type IV 46. Summerfield FW, Tappel AL (1984) Detection and mea- collagen. J Biol Chem 267: 12404±12407 surement by high-performance liquid chromatography of malondialdehyde cross-links in DNA. Anal Biochem 143: 265±271