Involvement of lipid oxidation products in the formation of fluorescent and cross-linked proteins

Chemistry and Physicsof Liptds, 44 (1987) 277-296

277

Elsevier Scientific Publishers Ireland Ltd.

INVOLVEMENT OF LIPID OXIDATION PRODUCTS IN THE FORMATION OF FLUORESCENT AND CROSS-LINKED PROTEINS

KIYOMI KIKUGAWA and MASATOSHI BEPPU

Tokyo Collegeof Pharmacy, 1432-1 Horinouehi, Hachiofi, Tokyo 192-03 (Japan) Received March 20th, 1987 Age-related fluorescent and cross-linked proteins increase with lipid oxidation of tissues. The fluorophores and cross-links have been considered to be conjugated Schiff bases between amino groups of proteins and maionaidehyde. Our recent studies showed that the fluorophores produced in the in vitro reaction of proteins with malonaldehyde are 1,4-dihydropyridine-3,5dicarbaldehydes, whose fluorescence characteristics are similar to but not always the same as those of the age-related fluorescent substances, and that the cross-linking is due to less fluorescent conjugated Sehiff bases. The in vitro reaction of proteins with oxidized lipids produces fluorescent and cross-linked proteins similar to those in the aging cells or tissues. Monofunctional aldehydes such as alkanais, alk-2-enais and alk~-2,4-dienais can also participate in the formation of the fluorophores and cross-links. The fluorescent substances produced from the reaction of primary amines or proteins with these aldehydes showed spectra close to those of the age-related fluorescent substances.

Keywords: protein; fluorescence; c~oss-link; lipid hydroperoxides; malonaldehyde; monofunctional aldehydes.

Introduction Recent studies suggest that lipofuscin pigment is the result o f lipid oxidation o f subcellular components induced by free radicals. Membranes o f mitochondria, lysosomes and endoplasmic reticulum containing a variety of unsaturated fatty acids undergo deterioration in the presence o f free radical initiators or oxygen. According to a widely accepted view obtained so far, lipofuscin pigment is the fluorescent end product o f a free radical-induced cross-linking o f proteins with oxidized lipids including those present in the membranes o f organelles. The mechanisms o f the formation o f fluorescent lipofuscin pigment with aging are now obscure, but a number o f experimental results with respect to this subject have been accumulated. In this paper the present authors intend to sum up the results o f the study o f the mechanisms o f the formation of fluorescent and cross-linked substances in the reaction o f proteins and oxidized lipids.

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© 1987 Elsevier Scientific Publishers Ireland Ltd. Published and Printed in Ireland

278 Lipid Oxidation and its Estimation

Unsaturated fatty acids or lipids undergo oxidation in the presence of oxygen via free radical initiation, propagation and termination [1,2]. Initial products are monohydroperoxides (LOOH). For instance, 8-OOH, 9-OOH, 1043OH and l l-OOH oleate; 9-OOH and 13-OOH linoleate; and 9-OOH, 12-OOH, 13-OOH and 16-OOH linolenate are produced from the corresponding unsaturated fatty acids in the free radical autoxidation [3]. Decomposition of the monohydroperoxides proceeds by hemolytic cleavage of LO-OH to form alkoxyl radicals LO'. These radicals undergo carbon-carbon cleavage to form volatile breakdown products including aldehydes, ketones, alcohols, hydrocarbons, esters, furans and lactones. The monohydroperoxides react again with oxygen to form such secondary products as epoxyhydroperoxides, ketohydroperoxides, dihydroperoxides, cyclic peroxides and bicyclic endoperoxides. These secondary products can in turn decompose to form volatile breakdown products. The monohydroperoxides also can condense into dimers that can break down and produce volatile materials. Recent studies have confirmed the formation of monocyclic peroxides from methyl linoleate [4], methyl linolenate [5,6] and bicycloendoperoxide from methyl linolenate [6]. In autoxidized methyl linolenate, monocyclic peroxides are formed in the same order of magnitude as the monohydroperoxides. Malonaldehyde is suggested to be the breakdown product from the 5-membered cyclic peroxides of linolenate and other fatty acids with more than 2 double bonds [7]. Higher dialdehydes than malonaldehyde also may be derived from dihydroperoxides, 6-membered cyclic peroxides and other polyfunctional secondary products of linoleate and linolenate [8]. The volatile decomposition products have been studied extensively. A generally accepted scheme for the fragmentation of the monohydroperoxides involves carboncarbon cleavage on either side of the alkoxyl radical to produce two types of aldehydes, an olefm radical and an alkyl radical. The radicals can in turn react with either OH" or H" radical to form aldehydes and hydrocarbons. These cleavage reactions explain most of the volatile products identified from the thermal decomposition of the hydroperoxides of oleate, linoleate and linolenate [3]. The progress of the reaction of lipid oxidation can be monitored by determination of (i) lipid hydroperoxides, (ii) secondary products, (iii) reaction products between the oxidized lipids and other substances and (iv) radical species produced during the course of the reactions. However, since each of these methods represents only one aspect of the complex lipid oxidation processes, it is important for reliable evaluation of lipid oxidation to measure several oxidation products by plural methods. Determination of the hydroperoxides is usually performed by the iodometric method developed by Lea and Wheeler [9-13]. The original method suffers from limited sensitivity, but spectrometric determination of iodine [ 14] or potentiometric titration [15,16] increased the sensitivity. Conjugated diene structure formed in the hydroperoxides can be monitored by the absorbance at 234 nm [11,12,17].

279 The utility of glutathione peroxidases in the estimation of lipid hydroperoxides in vitro was first appreciated by Heath and Tappel [12,13,18]. This method is not specific to lipid hydroperoxides and also sensitive to hydrogen peroxide and other organic hydroperoxides. Yamaguchi [19] observed that methyl linoleate hydroperoxide as well as hydrogen peroxide and other organic peroxides can oxidize 4-arninoantipyrine and N,N-dimethylaniline in the presence of horse radish peroxidase and estimated the colored oxidation product at 565 nm. These assays, however, could not be used for the determination of lipid hydroperoxides in human serum because of the presence of interfering substances [12]. Lipid hydroperoxides can oxidize N-methylindole to a fluorescent compound in the presence of peroxygenase from pea seeds [20]. The use of hemoglobin is an alternative method for determination of lipid hydroperoxides. A methylene blue derivative was converted into a 675 rim-absorbing oxidation product by lipid hydroperoxides and hemoglobin [21,22]. Sesamol dimer was converted into a 550 nm-absorbing quinone by lipid hydroperoxides in the presence of a small amount of hemoglobin [23-25]. The limitations for the use of hemoglobin have been shown [26,27]. A use of dichlorofluorescein in the presence of heroin was developed for the fluorescence determination of lipid hydroperoxides [28,29]. Marshall and Lands developed more selective determination of lipid hydroperoxides by use of cyclooxygenase [12,30,31]. Since Chan and Levett [32] developed HPLC for isolation of the isomers of methyl linoleate hydroperoxides, HPLC has become a useful method for determination of lipid hydroperoxides [33,34]. Malonaldehyde has received much attention in the biochemical and food science literatures [ 11.35,36]. When tetramethoxypropane (TMP) that generates malonaldehyde by acid treatment is heated with 2-thiobarbituric acid (TBA) in an acidic medium, a red pigment with an absorption maximum at 532 nm is produced [37]. Oxidized lipids in tissues produced a red pigment with the same absorption spectrum upon treatment with TBA in the acidic medium [38]. Thus, the malonaldehyde precursors or malonaldehyde reaction products that liberate malonaldehyde by acid treatment must have been present in the oxidized lipid samples. The TBA reaction is not specific to malonaldehyde or malonaldehyde derivatives, and many lipid oxidation products and their interaction products give positive reactions [39-42]. Our recent studies [43,44] demonstrate that the reaction of alkanals and alk-2-enals with TBA produced yellow, orange and red pigments depending upon the reaction conditions. Alkanals can be converted into alk-2-enals by dehydration in the presence of TBA. The 1 : 1 reaction of alk-2-enals with TBA initiallyproduced colorless 1 : 1 adducts, which were subsequently converted into the yellow and red pigment by heating under aerobic conditions. The reaction of alk-2-enals with TBA in excess might initially produce other colorless 1:2 adducts, which might be converted into the yellow, orange and red pigments under aerobic conditions. The red color due to these aldehydes may contribute in part to the color formed in the TBA test of lipid oxidation. Frankel and Neff [45 ] provided the acid decompositionacetylation procedure for malonaldehyde precursors, which is more specific than the TBA reaction to evaluate malonaldehyde. There is no correlation between the

280 malonaldehyde contents determined by the TBA test and the acid decompositionacetylation procedure. Hirayama et al. [46] developed an acid-dansyl pyrazoleHPLC method for determination of malonaldehyde precursors, and they can detect only 30% of the malonaldehyde obtained by the TBA test for oxidized methyl linolenate. Presence of free malonaldehyde in the oxidized tissue samples is not claimed, but quantification of free malonaldehyde by HPLC can be accomplished [47,48]. Nevertheless, the TBA reaction may be useful for evaluation of overall lipid oxidation. Aldehydes other than malonaldehyde are considered to be more important lipid oxidation products due to their toxic effects. Detection and determination of alkanals, alk-2-enals and 4-hydroxyalkenals are performed by use of 2,4-dinitrophenylhydrazine [41,49] or 1,3-cyclohexanedione-ammonium sulfate under acidic conditions [50]. Not only the free form but the bound form of the aldehydes may be detected. Estimation of these aldehydes by use of HPLC [51,52] and gas chromatography-mass spectrometry [53] is proposed. Formation of the volatile hydrocarbons such as ethane and pentane originated from the lipid oxidation has been proposed as a sensitive index for lipid oxidation in toxicological studies [54,55]. Secondary products can form fluorescent substances by reaction with other components as described in the following sections. Fluorescence measurement has become an excellent index of lipid oxidation of biological materials [56-58]. Detection and determination of the degree of the protein damage by the lipid oxidation products can be monitored by the incorporation of tritium by reduction with tritiated borohydride [59]. As shown in Fig. 1, erythrocyte ghosts treated with tert-butylhydroperoxide (t-BuOOH) incorporate tritium, on reduction with tritiated borohydride, depending upon the degree of lipid oxidation. Both the protein and lipid fractions are labeled by the reduction. Lipid hydroperoxides generate singlet oxygen or activated carbonyl compounds that emit chemiluminescence. Determination of lipid hydroperoxides by chemiluminescence has been extensively studied [60,61 ].

Protein Damage by Oxidized Lipids A variety of reactive species such as lipid radicals, hydroperoxides and their secondary products react with adjacent molecules in biological systems [62]. In biomembranes many proteins exist in contact with or in close proximity to lipids, and thus they are highly susceptible to chemical modification by the oxidized lipids. This modification, in contrast to enzymatically regulated post-translational protein modifications, is random, non-specific, and damaging to the structure and function of the proteins. The protein damage caused by oxidized lipids so far known [6264] is: formation of fluorescent chromophores, lipid-protein adducts, and proteinprotein cross-links, protein scission, and amino acid damage. Our recent work [59] has shown that borohydride-reducible functions are formed in membrane proteins during lipid oxidation (Fig. 1). Some of these protein modifications may share common chemical reactions.

281

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Fig. 1. Incorporation of tritium into the ghosts oxidized with t-BuOOH. Ghosts were incubated with the indicated concentrations of t-BuOOH in the presence of 2.5 ~M hemoglobin at 370C for lh, followed by reduction with NaB3H4. Thiobarbituric acid-reactive substances (A), fluorescence (B), and 3H incorporation (C) were measured for the oxidized ghosts (o) and their protein (z~) and lipid (A) fractions. Radioactivities are expressed for the ghosts containing 1 Aagprotein.

Two types of reactions, namely radical reactions and the reactions involving the secondary products, are possible in the protein damage by oxidized lipids [62]. The protein damage due to radical reactions is initiated by formation of protein radicals [65]. Protein radicals (P') formed in the reaction of proteins (PH) with lipid hydroperoxides (LOOH) or lipid radicals (LOO',LO') eventually cause protein damage such as protein to protein cross-links, lipid to protein cross.links, protein scission, and amino acid damage. Involvement of radical reactions in the protein damage caused by oxidized lipids has been reviewed in detail by Gardner [62]. Of the secondary products of lipid oxidation, aldehydes are the most probable candidates for the molecules involved in the protein damage because they are highly reactive with proteins, and various types of covalent modifications of the proteins are expected [66]. As shown in the following sections, a variety of aldehydes form fluorescent and cross-linked proteins. We hereafter discuss focusing on the role of aldehydes in the formation of fluorescent and cross-linked proteins by oxidized lipids.

Fluorescent Lipofuscin Pigments and Lipid Oxidation Siakotos and his associates [67,68] isolated two types of fluorescent lipofuscin pigment from human brain. They were different in their fluorescence excitation and emission maxima; one has an emission peak at 435 nm while the other has a

282 peak at 450 nm. Taubold et al. [69] demonstrated the fluorescence maximum of the isolated human brain lipofuscin pigment was 465 nm when excited at 395 nm. Thus, the isolated lipofuscin pigment emitted blue fluorescence. Fletcher et al. [70] found that the fluorescent substances in aging tissues can be extracted with chloroform/methanol (2:1) and the fluorescence intensity relates to the aging of the tissues. Aging tissues of 16-month-old mice, i.e. testes, heart and brain, contain fluorescent substances [71]. Tissues of rat and mouse with vitamin E deficiency produce higher amounts of fluorescent substances [72-74]. Trombly and Tappel [75] and Trombly et al. [76] purified lipid soluble fluorescent substances in human testes. This fluorescence was quenched in alkaline media and by a metal chelator. Csallany and Ayaz [77] isolated lipid-extractable high molecular weight fluorescent substances of 5000-10 ()00 daltons from rat and mouse tissues. Shlmasaki et al. [78,79] obtained age-related fluorescent substance from rat testes by organic solvent extraction and two-dimensional thin-layer chromatography. Fluorescence of this age-related fluorescent substance was quenched in alkaline media and by a metal chelator. Seligman et al. [80] reported that fluorescent substances in the tissues extractable with the organic solvents increased with the injury of the tissue. The studies by Tsuchida et al. [81] demonstrated that water-soluble fluorescent substances were present in the protein fraction of mouse and human sera. The relationship between the formation of these fluorescent substances and lipid oxidation has been demonstrated by Chio et al. [82] and DiUard and Tappel [83]. Thus, incubation of microsomes, mitochondria and lysosomes of rat liver under aerobic conditions produced water-soluble fluorescent substances in the supernatants. Goldstein and McDonagh [84] treated human erythrocytes with ozone and ultraviolet-light and recognized the formation of fluorescent substances extractable with chloroform/2-propanoI. Oxidation of mitochondria of Neurospora crassa [85] produced similar fluorescent substances. All these results support the idea that the formation of fluorescent substances is due to lipid oxidation of tissues or cells. Fluorescent and Cross-linked Proteins Derived from Malonaldehyde

Malonaldehyde, one of the secondary products of lipid autoxidation, produces fluorescence and cross.links in proteins. Two models have been proposed for the fluorescent substances: conjugated Schiff bases and 1,4-dihydropyridine-3,5dicarbaldehydes. The fluorogens that may be formed from malonaldehyde in biological systems seem to be the latter class of compounds, since they can be produced under physiological conditions and their structures were unambiguously established as discussed below. Tappel and his associates [71,86,87] treated amino-containing compounds with malonaldehyde prepared by acid hydrolysis of TMP under relatively strongly acidic conditions far from the physiological, and obtained fluorescent amorphous 2:1 conjugated Schiff bases (N,NLdisubstituted-l-amino-3-iminopropenes) II along with

283 CH.~O OCH 3 o ~CHCH_CH~ CH30 TMP" OCH3

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Fig. 2. Schiff bases I and conjugated Schiff bases II.

the non-fluorescent 1 : 1 Schiff bases I [88] (Fig. 2). Elemental analysis and ultraviolet absorption spectra supported the structure of II. Compounds II were reduced to non-fluorescent N,N'-disubstituted.l,3-diaminopropanes by treatment with borohydride, mass spectra of which supported the structure of II. The conjugated Schiff bases II exhibited fluorescence with excitation maxima at 350-400 nm and emission maxima at 450-470 nm, fluorescence intensity being lower than that of quinine sulfate. The fluorescence was quenched at alkaline pH values and by a metal chelator, europium tris (2,2,6,6-tetramethyl-3,5-heptanedionate) [87,89]. The fluorescence characteristics of II resembled those of the fluorescent substances produced in tissues. Hence, it has been assumed that fluorescent substances in lipofuscin or aging tissues are the conjugated Schiff bases between amino groups of proteins and malonaldehyde [71,87]. Because of its bifunctionality, malonaldehyde cross-links ribonuclease [90-92], coUagen [93], and erythrocyte membrane proteins [94-96]. The cross-links in proteins can be explained by the formation of the conjugated Schiff bases. Both fluorescence and cross-links are considered to be due to the conjugated Schiff bases. Tappe1's theory for the formation of fluorescent substances in aging tissues is summarized in Fig. 3. Thus, malonaldehyde reacts with amino groups of enzymes to form intra- and intermolecular fluorescent cross-links. Buttkus and Bose [97] pointed out that more rigorous assignment of the structure of the conjugated Schiff bases II is required because the compounds are unstable and obtained in an amorphous state. Studies of the chemistry of malonaldehyde are complicated by its tendency to undergo self-condensation reactions [98,

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Fig. 3. Pathwayfor the formationof fluorescencefrom malonaldehyde(Tappers model).

284

99], and /$-methoxyacrolein and 3,3-dimethoxy-l-propanal are formed as side products of acid hydrolysis of TMP [100]. Nair et al. [101,102] demonstrated that the reaction of amino acids with pure malonaldehyde under mildly acidic conditions gave conjugated Schiff bases II and Schiff bases I. The purified and crystalline conjugated Schiff bases II of aromatic amines [97,103,104] showed little or no fluorescence [97,105]. Some of them fluoresced only after derivatization by treatment with dimethylformamide and aqueous alkali [103,105]. We investigated the reaction of primary amines with malonaldehyde under physiological conditions and obtained another type of highly fluorescent compounds III [106-109]. An excess amount of methylamine was reacted with malonaldehyde prepared by acid hydrolysis of TMP under various pH conditions (pH 1-8). Two fluorescent compounds (IIIa and IVa) and two non.fluorescent ultraviolet absorbing compounds (Ia and Va) were isolated (Fig. 4). Compounds Ilia, IVa and Va were isolated in crystalline forms. The structure of these compounds was elucidated by analysis of their ZSCand 1H-NMR spectra, ultraviolet absorption spectra, mass spectra and elemental analysis. They were found to be 1,4-dimethyl-l,4-dihydropyridine-3,5-dicarbaldehyde (IIIa), 1.methyl-4-(dimethoxyethyl)-l,4-dihydropyridine-3,5-dicarbaldehyde (IVa), and 2,6-dimethyl-2,6~iiazabicyclo [3.3.1] 3,7-nonadiene-4,8-dicarbladehyde (Va). Compound Ia obtained as an oil was a mixture of trans.s.trans and cis-s-cis form of methylaminoacrolein. The reaction conducted at neutral pH ranges produced compounds Ia, IIIa and IVa. When the reactions of methylamine and malonaldehyde were conducted in a molar ratio of 1 : 2 and 2 : 1 under neutral conditions, both Ilia and IVa were produced accompanying no other fluorescent compounds. The malonaldehyde preparation may contain malonaldehyde and its intermediCH3NH, ,H CH3NHH~.~HO H;C=~cHO la trans-s-trans cis-s-cis H~,~CH3 OHC~N~]~CHO III TMP

~ (OHCCH2CHO) ÷ RNH2

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a: R=CH3 b (CH2)5 CH3 c CH2COOH d CH2COOC2H5 e CH2CH2OH f CH2 CH2CH2OH

H.~(.CH~H!~H3 OHC']~N~ CHO IVa CH3 H v NCH3 O H C ~ Va CH3 CHO Fig. 4. Reactionof primaryaminesand malonaldehyde.

285 ates or polymerized products [98-100]. The reaction of methylamine with pure malonaldehyde [100] in a ratio of 1:2 gave only a single fluorescent compound IIIa in a yield of 8%. Only a single fluorescent compound Ilia was produced in the reaction of methylamine and pure malonaldehyde under our reaction conditions regardless of the ratio of the reactants. Compound IIIa may be formed by the reaction of methylamine and malonaldehyde in a molar ratio of 1:3 probably via via the Hantzsch reaction [110]. Compound IVa may be formed by the reaction of methylamine, malonaldehyde and 3,3-dimethoxy-l-propanol (in a ratio of 1:2 : 1) via the Hantzsch reaction [110]. Reaction of 1-hexylamine, glycine, glycine ethyl ester, ethanolamine and 3amino-l-propanol with malonaldehyde at pH 7 gave fluorescent and crystalline 1substituted-4-methyl-l,4-dihydropyridine-3,5-dicarbaldehydes Illb-f. Under the conditions used no other fluorescent compounds were detected by thin-layer chromatography and HPLC. These results have been confirmed by Yoden et al. [111]. The fluorescence spectrum of compound IIIa revealed an excitation maximum at 403 nm and an emission maximum at 462 nm in phosphate buffer (pH 7) with a relative molar intensity higher than that of quinine sulfate. When fluorescence spectra were measured in methanol, ethanol, 2-butanol and chloroform, both excitation and emission maxima shifted to shorter wavelengths (Table I), with the largest shifts in wavelength in chloroform. The fluorescent substances reportedly obtained by the reaction of 1-hexylamine [86] and wN-acetyl-L.lysine [92] with malonaldehyde showed fluorescence spectra very similar to those of Ill, although they had been assigned as the conjugated Schiff bases II. Excitation and emission maxima of IIIa were not altered between pH 1 and 13 but the fluorescence intensity markedly decreased below pH 4 [108]. Treatment of Ilia with borohydride decreased the fluorescence intensity by about 90%, which may be due to the reduction of one of the two aldehyde groups into the alcohol [ 112]. Treatment of Ilia with europium tris(2,2,6,6-tetramethyl-3,5-heptanedionate) TABLE I FLUORESCENCESPECTRAOF Ilia Solvent

0.1 M phosphate (pH 7) Methanol Ethanol 2-Butanol Chloroform

max (nm)

Relative molar intensitya

Excitation

Emission

403 392 397 397 390

462 451 452 452 446

aRelative to the intensity of quinine sulfate.

1.27 1.18 1.23 1.23 0.93

286

gave no decrease in fluorescence intensity. Other compounds I I I b - f exhibited fluorescence spectra and characteristics similar to those of IIIa [108,109]. Fluorescence spectra of III are close to those of the age-related fluorescent substances produced in rive lipid oxidation. However, not every fluorescence characteristic of III is similar to that of the fluorescent substances in tissues [76,79]. The dissimilarity of the fluorescence is as follows: III exhibited rather shorter wavelengths of excitation and emission maxima than the fluorescent substances; the fluorescence of III was quenched in an acidic medium while that of the age-related substances was quenched in an alkaline medium; the fluorescence of III was not quenched by the metal chelator, while that of the age-related substances was quenched. Treatment of polylysine with 1/3 molar excess malonaldehyde at pH 7.5 gave modified polylysine containing 1,4-dihydropyridine-3,5-dicarbaldehyde residues Ill that fluoresced at 398 nm (excitation maximum) and 470 nm (emission maximum) [113]. The amount of the fluorescent residues was estimated to be less than 0.2% of the e-amino groups. Most of the malonaldehyde was incorporated into the e-amino groups as non-fluorescent Schiff bases 1 (22%). This modification resulted in the formation of cross-links between two molecules of polylysine. Hemoglobin A was modified with malonaldehyde forming a number of less cationic components [114]. Some of these modified components were intermolecularly cross-linked, and showed fluorescence with an excitation maximum at 390 nm and an emission maximum at 460 nm. It is likely that malonaldehyde reacts non-specifically with e-amino and N-terminal groups to produce Schiff base I, cross-links probably due to conjugated Schiff bases II, and strongly fluorescent 1,4-dihydropyridine-3,5-dicarbaldehydes Ill (Fig. 5). Treatment of erythrocyte ghosts with malonaldehyde at neutral pH resulted in the formation of fluorescence and cross-links in ghost proteins [ 115]. The fluorescence spectrum of the modified ghost proteins was characteristic to that of III. The ribonuclease modified with malonaldehyde at neutral pH had fluorescence and cross-links [92]. The fluorescence spectrum, with an excitation maximum at 395 nm and an emission maximum at 470 nm, was close to that of IlI, although the

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287 authors reported the fluorescence was due to the conjugated Schiff bases II. The in vitro addition of malonaldehyde to normal erythrocytes caused a decrease in bands 1 and 2 of spectrin and an increase in high-molecular-weight protein polymers [94,95]. Similar protein cross-links of erythrocyte membrane by malonaldehyde have been reported [96]. It is likely that cross-links are formed by the mechanisms different from those of the formation of fluorescence, although the earlier workers [92,94-96] ascribed both fluorescence and cross-links to the conjugated Schiff bases II. We found that monofunctional aldehydes stimulated and participated in the formation of fluorescent substances in the reaction of primary amines and malonaldehyde [109]. The reaction of a primary amine together with malonaldehyde, and a monofunctional aldehyde at pH 7 produced fluorescent 1,4-disubstituted. 1,4-dihydropyridine-3,5-dicarbaldehydes. The substituents derived from the monofunctional aldehydes were introduced into the 4-position of the products. These compounds were essentially the same type of compounds as III, and exhibited the same fluorescence with excitation maxima at 386-403 nm and emission maxima at 444-465 nm. The highest yield of the compounds was obtained when a primary amine, malonaldehyde and a monofunctional aldehyde were reacted in a ratio of 1:2:1-2. The reaction of primary amines in which malonaldehyde participated produced no other fluorescent compounds than 1,4-dihydropyridine-3,5-dicarbaldehydes III and their 4-substituted derivatives.

Fluorescent and Cross-linked Proteins Derived from Lipid Hydroperoxides Formation of fluorescence and cross-links in reaction of primary amines and proreins with oxidized fatty acids or lipids has been demonstrated. Lipid-soluble and water-soluble fluorescent chromophores are formed [116,117]. The development of fluorescence was linearly related to oxygen absorption. The fluorochromes had a maximum excitation at 360-365 nm and a maximum emission at 430-440 nm, which were similar to those of the aging tissues. Shimasaki et al. [118] demonstrated that the formation of fluorescent products in the reaction of glycine with methyl linoleate hydroperoxide correlated directly with the decrease in diene conjugation and the increase in TBA-reactive substances. While methyl linoleate hydroperoxide produced fluorescent substances with an excitation maximum at 360-365 nm and an emission maximum at 435-440 nm, oxidized fatty acids With increased unsaturation produced fluorescent substances showing spectra with longer peak wavelengths. The fluorescent substances produced have been ascribed to the conjugated Schiff bases II between the amines and malonaldehyde [116-118]. Tabata et al. [119,120] suggested that the fluorescent substances derived from primary amines and oxidized fatty acids are different from the conjugated Schiff bases II. Our studies demonstrated that linoleic, linolenic and arachidonic acids produced fluorescent substances with excitation maxima at 355-370 nm and emission maxima at 420-440 nm by reaction with methylamine under aerobic conditions [121]. The wavelength of excitation and emission maxima increased

288 with increasing unsaturation of the fatty acids. The fluorescence was little influenced in an acidic medium but was affected differently in an alkaline medium with various fatty acids. The effect of borohydride changed with different fatty acids. The fluorescence characteristics of the products indicate that they differ in structure or composition with different fatty acids, and major fluorescent products are not 1,4-dihydropyridine-3,5-dicarbaldehydes HI or their 4-substituted derivatives. Reaction of Coho salmon myosin with autoxidizing linoleate produced fluores. cent proteins [122]. Reaction of bovine serum albumin with oxidizing methyl linoleate afforded fluorescent and cross-linked albumin with an excitation maximum at 350 nm and an emission maximum at 435 nm [63,64]. Reaction of polylysine with linoleic acid hydroperoxide afforded fluorescent and cross-linked polylysine, which showed fluorescence with maxima at 347 nm (excitation) and at 425 nm (emission) [113]. More than 70% of fluorescence was lost by treatment with borohydride. Reaction of erythrocyte ghosts with linoleic acid hydroperoxide produced fluorescent ghost proteins with cross-links [115]. Ferrous ion and ascorhateinduced lipid oxidation of liposomal membranes containing phosphatidyl ethanolamine led to the formation of fluorescent substances in liposomal membranes [123].

Fluorescent and Cross4inked Proteins Derived from Monofunctional Aldehydes Formation of fluorescence and Cross-linksin reaction of proteins with secondary products other than malonaldehyde has been demonstrated. The carbonyl compounds derived from oxidizing lipids, i.e. 2,4-hexadienal and 2,3-butanedione, react with amino groups of bovine serum albumin to give products that have fluorescence very similar to that observed for the albumin modified with oxidizing methyl linoleate [63]. Yoden et al. [11 I] reported similar observations. The aldol condensation reaction of alkanals to give alk-2-enals is catalyzed by amino-containing compounds. Condensation of alkanals with primary amines gave pyridinium salts [124,125]. A mechanism involving the intermediacy of a~unsaturated aldimines is proposed. Incubation of methylamine and 1-butanal produced four fluorescent substances and two ultraviolet absorbing substances, 2-ethyl2-hexenal and pyridinium salt [126]. Two of the four fluorescent substances were derived from the reaction of methylamine and 2-ethyl-2-hexenal. Formation of the fluorescent substances required molecular oxygen. One of the fluorescent substances produced from 2-ethyl-2-hexenal showed fluorescence spectrum with an excitation maximum at 357 nm and an emission maximum at 433 nm. The fluorescence intensity decreased only by about 10% by treatment with borohydride. Exposure of lysozyme to vaporized 1-hexanal resulted in the production of fluorescence with an excitation maximum at 355 nm and an emission maximum at 425 n m [ 127]. Incubation of rat liver microsomes or mitochondria with 4-hydroxynonenal resulted.in a slow formation of a fluorophore with an excitation maximum at 360 nm and an emission maximum at 430 nm [128]. When polylysine was incubated with acetaldehyde or 1-hexanal, the modified polylysines showing fluores-

289 cence with excitation maxima at 340-360 nm and emission maxima at 410-430 nm were obtained [113]. The fluorescence was reduced by 55% and 22%, respectively, by reduction with borohydride. The modified polylysines showed two fluorescent peaks in gel filtration. The modification resulted in the formation of cross.links between two or three molecules of polylysine. While the mechanisms for crosslinking were obscure, it is likely that these aldehydes were condensed into alk-2enals which in turn participated in the cross-linking formation. The formation of fluorophores and cross-links by linoleic acid hydroperoxide can be caused by the secondary products such as these monofunctional aldehydes. Human erythrocyte ghosts modified with monofunctional aldehydes were analyzed for fluorescence formation and protein cross-linking [115]. Fluorescence spectra of the ghost proteins modified with 1-heptanal and 2,4-decadienal were indistinguishable from those observed for the ghost proteins modified with linoleic acid hydroperoxide. Like linoleic acid hydroperoxide and malonaldehyde, the monofunctional aldehydes such as 1-hexanal, 1-heptanal and 2,4-decadienal were capable of cross-linking the ghost proteins as determined by SDS-polyacrylamide gel electrophoresis (Fig. 6). Acetaldehyde, a product of ethanol metabolism, is

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Front Fig. 6. SDS-polyacrylamidegel elec~rophoresisof the ghosts treated with (a) control; (b) 0.5 mM linoleic acid hydroperoxide; (c) 2 mM linoleic acid hydroperoxide; (d) 5 mM malonaldehyde; (e) 25 ram malonaldehyde;(f) 50 mM acetaldehyde;(g) 50 mM 1-hexanal;(h) 25 mM 1-heptanal; (i) 0.5 mM 2,4-decadienal;(j) 0.5 mM glutaraldehyde.

290 been known to cross-link proteins [ 129 ]. The mechanism of the cross-link formation was supposed to be a nucleophilic addRion of a thiol group to the Schiff bases between the aldehyde and amino groups of proteins [130]. 12-Keto oleic acid, one of the secondary products of lipid hydroperoxides, forms fluorescent substances with an excitation maximum at 345 nm and an emission maximum at 400 nm by reaction wRh primary amines [131], whose structures were elucidated [132]. Biological Implications of the Formation of Fluorescent and Cross-linked Proteins Covalent modification of proteins by lipid oxidation products impairs biological functions of the proteins such as enzyme activities [92,133]. Accumulation of fluorescent lipopigments or fluorescent proteins in a tissue is a good sign that the tissue has undergone lipid oxidation or is aged [71,81,95]. However, as described above, formation of fluorophores in polylysine on reaction with malonaldehyde accounts for only a minor part of the modification [113]. Thus, occurrence of fluorescent proteins as a result of lipid oxidation would suggest other concurrent modifications of the proteins such as intra- or intermolecular cross-linking, formation of lipid-protein adducts, and so forth. Protein cross-linking may also be taken as an indicator of lipid oxidation in biological membranes. However, it is not always associated with lipid oxidation. For example, induction of lipid oxidation in erythrocyte ghosts with exogenously added linoleic acid hydroperoxide or t-BuOOH resulted in extensive cross-linking of the membrane proteins [59,115,134,135], while little cross.linking was observed when lipid oxidation was induced by a catalyst, ADP-chelated iron (ADP/Fe 3+) [59]. This is probably due to the difference of the extent of lipid oxidation since lipid oxidation induced by ADP]Fe ~+ was milder than that induced by the hydroperoxides as estimated by TBA-reactive substance and fluorescence formation. Tritium labeling of the ghost proteins modified by oxidized lipids revealed that the membrane proteins which did not undergo intermolecular cross-linking were also modified by oxidized lipids (Fig. 7) [59]. Intact normal erythrocytes resist lipid oxidation and the irreducible membrane protein cross-linking is rarely seen when the cells are moderately oxidized by t-BuOOH [ 136] or periodate [137]. Membrane protein cross-linking of erythrocytes is, however, observed for unusual erythrocytes such as those with abnormal hemoglobin K61n [138], those from ion-deficient rats [139], and thalassemic erythrocytes [140]. Membrane protein cross-linking appears to take place when the oxidative stress is relatively high. Protein crosslinking, either intra- or intermolecular, should result in a substantial change of the structure and physicochemical properties of the proteins as exemplified by an enzymatic cross-linking of the connective tissue proteins, collagen and elastin [ 141 ]. Thus, it is conceivable that the formation of protein cross-links would lead to extensive damage of the membranes and the cells. The modification of 15roteins with oxidized lipids such as fluorophore formation

291

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bc

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Fig. 7. SDS-polyacrylamidegel electrophoresis of the ghosts oxidized with t-BuOOH (A and B) and the oxidized ghosts reduced with tritiated borohydride (C). Ghosts were incubated with t-BuOOH, and then reduced with NaBSH+.(A) Coomassie blue staining of the proteins of the t-BuOOH-treated ghosts. (B) Periodic acid-Schiff reagent (PAS) staining of the sialoglycoproteins of the t-BuOOH-treatedghosts. (C) Fluorography of the t-BuOOH-treatedghosts reduced with NaBSH+.(a-e) the ghosts incubated with 0, 0.01,0.1, 1 and 10 mM t-BuOOH,respectively. Positions corresponding to the major protein and sialoglycoproteinbands axe indicated. The band running faster than the gel front correspondsto lipids.

and intra- or intermolecular cross-linking appears to restrict the life-span of the proteins in vivo. It is known that serum albumin modified with formaldehyde [142, 143] or maleic anhydride [144] is bound and taken up by macrophages. Horiuchi et al. [145] extended this observation to albumin modified with various aliphatic aldehydes. The recognition and endocytosis by macrophages are mediated by the specific receptors called scavenger receptors [144-147]. Low density lipoprotein (LDL) modified with acylating agents [144] or malonaldehyde [148,149] is also recognized and endocytosed through the scavenger receptors on macrophuges. This phenomenon is of special interest from the clinical point of view since the accumulation of cholesteryl esters in macrophages by the intake of the modified LDL may be relevant to the development of the lipid-laden foam cells found in atherosclerotic lesions. The possibility of in vivo occurrence of aldehyde-modified LDL and its clearance from the circulation has been suggested by the fact that LDL incubated uvder the conditions in which lipid oxidation takes place was effectively endocytosed by the cells [150]. These findings suggest that the self-defence system in the body including macrophages has an ability to recognize and remove the proteins damaged by aldehydes originating from in vivo lipid oxidation. Some investigators [145,151,152] emphasize the importance of the modification of the peptide

292 regions of particular primary sequences, but it is possible that certain chemical structures produced on the proteins by the reaction with aldehydes provide the recognition determinants. In vivo formation of fluorescence and cross-links in proteins has been known to take place by a mechanism irrelevant to lipid oxidation, non-enzymatic glycosylation (Maillard reaction ) [66]. In patients of diabetes mellitus, glycosylated proteins such as albumin, hemoglobin, lens protein and collagen are often observed [153]. These proteins are regarded as analogous to the aldehyde-modified proteins. The reaction proceeds to form brown fluorescent pigments which cross-link the protein (advanced glycosylation end products) [ 154]. The glycosylation tends to occur in long-lived proteins, and the relationship between the development of the modified proteins and aging of proteins has been suggested [155]. It is noteworthy that proteins with the advanced glycosylation end products are recognized and taken up by macrophages [156,157]. This phenomenon may also be thought to be involved in the recognition and clearance of aldehyde-modified proteins that are produced as a consequence of aging or physiological disorders. Concluding Remarks The present authors describe here the characteristics of the fluorescent substances formed in the in vitro reactions of lipid oxidation products, and the mechanisms of their formation. One mechanism for the production of these pigments is the reaction of malonaldehyde and proteins, producing conjugated Schiff bases II that fluorescence and form cross-links in proteins. An alternative but more reasonable mechanism for malonaldehyde reaction can be proposed. Thus, malonaldehyde in the presence or absence of monofunctional aldehydes produces highly fluorescent 1,4-dihydropyridine-3,5-dicarbaldchydes III and their 4-substituted derivatives, non-fluorescent Schiff bases I, and cross-links probably due to formation of 1I. While fluorescence spectra of III and their 4-substituted derivatives were similar to those of the fluorescent components in lipofuscin, they were not always identical. Monofunctional aldehydes alone can produce fluorescence and crosslinks in protein molecules, whose fluorescence spectra were close to those of the hydroperoxide-modified proteins or lipofuscin pigments. Secondary products other than malonaldehyde may play an important role in the formation of fluorescence and cross-links in protein molecules.

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