Homocysteine-Induced Oxidative Damage: Mechanisms and Possible Roles in Neurodegenerative and Atherogenic Processes Elke Schlüssel3, Gudrun Preibischa, S. Piitterb and E. F. Elstnera a Lehrstuhl für Phytopathologie, Abt. für Angewandte Biochemie, Technische Universität München, D-85350 Weihenstephan, Bundesrepublik Deutschland b Fa. Medice, D-58638 Iserlohn, Bundesrepublik Deutschland Z. Naturforsch. 50c, 699-707 (1995); received March 27/June 14, 1995 Sulfur Amino Acids, Copper, Iron, Oxidation, Pyridoxalphosphate Increased blood plasma concentrations of the sulphur amino acid homocysteine (“homo- cysteinemia”) have been brought into context with neurodegenerative and arteriosclerotic symptoms and diseases. We recently reported on biochemical model reactions on the prooxidative activity of homo cysteine including the desactivation of Na+/K+-ATPases and hemolysis of erythrocytes (Prei- bisch et al., 1993). In this communication we extend our model reactions including the oxida tion of methionine, metabolization of pyridoxalphosphate and dihydroxyphenylalanine, desactivations of transaminases and peroxidation of low density lipoprotein.
Introduction 1980; Youngman et al, 1982; Youngman and The biosynthesis of methyl group donors such Elstner, 1985; Elstner et al, 1980) we reported that as S-adenosyl-methionine (SAM) and of cysteine methionine in the presence of pyridoxalphosphate (Cys) are essential for the synthesis of methylated is fragmented by certain strong oxidants yielding elements of cell membrane structures such as ethene. This reaction proved as a sensitive indica phosphatidylcholine and of antioxidants such as tor for the production of OH-type radicals. The S-methyl-a-ketobutyric acid (KMB), taurin and transamination product of Met, S-methyl-a-keto- glutathione. These central functions are warranted butyric acid (KMB) is a much less specific but very by the homeostasis of methionine metabolism sensitive indicator for the generation of a wide keeping the overall cellular antioxidative system variety of oxygen radicals including Cys or HC in at high fidelity. If this system is out of balance, the presence of copper or iron ions (Preibisch a vast amount of pathological processes may be et al, 1993). In the absence of PyP no ethene is induced mainly concerning nervous disorders and released from Met by certain biological systems atheromatous developments. Homocysteine (HC) such as membraneous oxidoreductases in the as one member in this amphibolic process, how presence of autoxidizable redox cyclers or by ever, posseses prooxidative functions which are NAD(P)H oxidases. Methionine (Met) in combi biochemically distinct to the reactivities of cys nation with pyridoxalphosphate (PyP) or KMB act teine (Preibisch et al, 1993; Preibisch and Elstner, as scavengers of strong oxidants such as the OH- 1994). As recently reported, the pyridoxalphos- radical. Thus, the conversion of homocysteine into phate-glutamate Schiff-base has strongly antioxi methionine catalyzed by cobalamine (vitamin B12) dative properties preventing damage by organic in cooperation with folic acid clearly shifts a pro hydroperoxides (Meyer et al, 1992). In earlier oxidative towards an antioxidative situation. Vita papers (Konze and Elstner, 1976; Saran et al, min B 6 (PyP) both catalyzes the synthesis of KMB as well as the reactivity of methionine as radical scavenger. Thus, the vitamins folic acid and B 12 Abbreviations: Cys, cysteine; HC, homocysteine; HCTL, indirectly act as antioxidants by removing homo homocysteinethiolactone; Cu, copper; KMB. 4-methyl- cysteine and producing methionine, whereas PyP thio-2-oxobutanoic acid; Met, methionine; PyP, pyrid oxal phosphate; LDL, low density lipoprotein; TA, both in the presence and absence of methionine is transaminase; DOPA, dihydroxyphenylalanine. an effective antioxidant. In this communication we Reprint requests to Prof. Dr. E. F. Elstner. present experimental evidence that HC in the Telefax: 08161-714538. presence of copper or iron ions (see Discussion)
0939-5075/95/0900-0699 $ 06.00 © 1995 Verlag der Zeitschrift für Naturforschung. All rights reserved. 700 E. Schlüssel et al. ■ Oxidative Damage by Homocysteine destroys or alters biomolecules such as transami graphically after a 45-min incubation at 37 °C. The nases (TA), dihydroxyphenylalanine (DOPA) or final concentrations, unless otherwise indicated in low density lipoprotein (LDL) which are actively the legends, were 1.0 mM for Met and PyP, 1.0 mM involved in the above mentioned diseases. Homo for the metal ions and 0.5 mM for the hydrogen cysteine and methionine metabolism is tightly con peroxide. nected and plays a crucial role in a large variety To test the influence of Cys, HC and HCTL on of diseases. An outline of this connection including methionine fragmentation the substances (0.01 up the mentioned damaging reactions as well as their to 2 mM final concentration) were added before counterbalance is presented in Fig. 1. reaction start with metal ions and H 20 2 solu tion .
Materials and Methods Assays for transaminase activity Chemicals and Biochemicals For spectrophotometric determination of trans Chemicals were obtained from SIGMA Chemi aminase activities the procedures of Mavrides cal Co. (Munich) in the greatest available purity. (1987) and SIGMA (quality control test procedure Likewise, the enzymes glutamic-oxalacetic trans 10/'93; available upon request) were performed in aminase EC 2. 6 .1.1 from porcine heart G-7005, a slightly modified manner: The substrates for the glutamic-pyruvic transaminase EC 2. 6 .1.2 from transaminase and the dehydrogenase were pi porcine heart G-8255, malic dehydrogenase EC petted into half micro disposable cuvettes and the 1.1.1.37 from Thermits flavua M-7032, lactic dehy reaction was started by adding transaminase solu drogenase EC 1.1.1.27 from porcine heart L-2625 tion. The final concentrations of the glutamic-oxal and polyphenoloxidase (tyrosinase) EC 1.14.18.1 acetic transaminase assay were 50 mM L-aspartate, from mushroom T-7755 were purchased from 10 mM a-ketoglutarate, 0.2 mM ß-NADH, 2.4 U SIGMA (Munich). malic dehydrogenase and 0.3 U transaminase. For the glutamic-pyruvic transaminase assay 200 mM M et/PyP-system D,L-alanine, 10 mM a-ketoglutarate, 0.2 mM ß- Met and PyP were allowed to react at room tem NADH, 7.5 U lactic dehydrogenase and 0.1 U perature in 0.2 m phosphate-bufferd solution (pH transaminase were added. (Unit definition: One 7.4) for 10 min forming the Schiff-base. Then, Fe2+ International Unit (U) is defined as that amount or Cu2+ ions (FeCl 2 -4H20 or CuS0 4 -5H20 dis of enzyme that will form 1 (.imol of glutamate per solved in aqua bidest.) and H 20 2 were added. minute at pH 7.5 and 25 °C.). After stirring the Ethene production was measured gaschromato- decrease in absorbance at 340 nm was recorded
antioxidant toxicity
JCu, Fe
KMR homocysteine- antioxidant thiolactone »6 B12 m ethionine homocysteine - cysteine glutathione folate »6 I Cu. Fe | Cu, Fe methionine + pyridoxal slow oxidation rapid oxidation phosphate
antioxidant Fig. 1. Methionine-homo- cvsteine metabolism. E. Schlüssel et al. ■ Oxidative Damage by Homocysteine 701
for approximately 15 min to obtain the maximum determined by agarose gel electrophoresis (ac linear reaction rate (A/l/min = activity). cording to Cooper, 1983). To study the influence of the test substances (Cys, HC, HCTL) on enzyme activity a preincuba Results tion of the transaminases with the sulphur com The combination of both Met and PyP is appa pounds at 37 °C for 15 min preceeded the addition rently producing an antioxidant rapidly reacting to the reaction mixture. Activities were compared with the potent oxidant of the OH' type, including as percentages of control activity. transition metal-oxygen complexes of the “Fen- ton-type” i.e. Fe2+ in the presence of a peroxide. Reactions with o-quinone products from We assayed this reaction with respect to the effects PPO-catalyzed dihyroxyphenylalanine (DOPA) of iron or copper ions, which are under increasing oxidation discussion as initiators and/or cofactors of the de A mixture containing 9.6 mM DOPA, 0.8 mM velopments of neurodegenerative (Youdim and Cys/HC/HCTL respectively and 783 U poly- Lavie, 1994) and atherosclerotic diseases (Meyer phenoloxidase in 0.2 m phosphate buffered solu et al., 1992; Kögl et al., 1994; Lupo et al., 1994), tion (pH 6.0) was allowed to react 2.5 - 5 - 10 - where the sulphur containing amino acid, methio 20 - 30 - 45 - 60 min, respectively, then the reac nine, seems to be in the focus of such oxidative tion was stopped by adding an equal volume of destructions (Preibisch and Elstner, 1994; Gilman methanol. Particles were removed by a 10 min et al., 1993). centrifugation at 1000 xg in an Eppendorf-centri- fuge. After dilution 1 : 5 with the reaction buffer, 1) Reactions with methionine and HPLC analysis was performed. A reversed phase pyridoxalphosphate column Hypersil ODS (particle size 5 |.im, pore In the presence of either iron2+ or copper2+ ions, size 100 A) was used. The flow rate was 1 ml the release of ethene from methionine was tested 50 mM phosphate buffer pH 2.4 / min and products as to the effects of cysteine, homocysteine, or were detected at 254 nm. homocysteine thiolactone. As shown in Fig. 2, in the presence of hydrogen Oxidation o f low density lipoprotein peroxide and either iron or copper, increasing LDL from human venous blood was subjected amounts of ethene are released from increasing to oxidation by 2.5 and 5 ^im Cu 2+ respectively. concentrations of Met in the test system. This reac Dieneconjugation was measured spectrophoto- tion is inhibited by PyP in the copper system and metrically by monitoring increase in absorbance at to a much lesser extent in the slower reacting iron 234 nm, changes in electrophoretic mobility were system (Fig. 2).
cu
Increasing concentrations (0.01 to 2 mM) of the formation of a new absorption band at 328 nm and above mentioned sulfhydryl compounds (Cys. HC, 326 nm. respectively (Figs 4a and 4b). HCTL) to various extents inhibit the transi- tionmetal-hydrogenperoxide driven ethene pro 2) Reactions with transaminases duction from Met (Figs 3a and 3b). The reactions with PyP may reflect the sensitiv Cys and HC can interact with PyP forming thi- ity of certain transaminases towards oxidation by azolidine and thiazine complexes (Schonbeck either copper ions or thiol compound - copper et al., 1975; Cooper, 1983), optically visible as the combinations as demonstrated in Figs 5 and 6 : disappearance of the absorption at 386 nm and the Glutamic-oxalacetic transaminase is only inhibited by high concentrations of HCTL (Fig. 5) while glutamic-pyruvic transaminase is more or less sen sitive towards Cu (Fig. 6 a) as well as Cys and HCTL. Cu-inhibition is reversed by HCTL and Cys while high concentrations (> 10 mM) of Cys or HCTL are inhibitory (Fig. 6b).
Sulphur compound [mM] Sulphur compound [mM] during preincubation Fig. 3. Effects from various amounts of cysteine, homo cysteine or homocysteinethiolactone on the Cu2+-hydro- Fig. 5. Activity of glutamic-oxalacetic transaminase genperoxide-driven (a) and on the Fe2+-hydrogenper- (0.3 U) after preincubation (15 min. 37 °C) with various oxide-driven (b) ethene production from methionine. amounts of cysteine, homocysteine or homocysteinethio H 20 2 0.5 m M ; Met, PyP, Cu2+ or Fe2+ 1.0 m M . lactone: 1; 10; 50; 100 m M .
Fig. 4. Formation of a cyclic thiazolidine deriva tive from pyridoxalphosphate and cysteine (a) and of a cyclic thiazine derivative from py ridoxalphosphate and homocysteine (b). PvP Wavelength [nm] 180 u m ; Cys or HC 2.5 m M ; 37 °C. pH 7.4. E. Schlüssel et al. ■ Oxidative Damage by Homocysteine 703
,___ , 140 o 120 oc o 80 100 «4—o 80 £ o & O) > 60 * 40 o T3 (D 40 ■— Cysteine >. * HCthiolactone cN LU 0
0 20 40 60 80 100 0 10 20 30 40 50 60 Sulphur compound [mM] during preincubation Incubation time [min]
Fig. 6a. Activity of glutamic-pyruvic transaminase Fig. 7. Reactivities of the sulfhydryl groups of cysteine (0.1 U) after preincubation (15 min, 37 °C) with various and homocysteine (100 u m ) with Cu2+ (5 p M ) , pH 7.4, amounts of cysteine, homocysteine or homocysteinethio- 37 °C. lactone: 1; 10; 50; 100 m M in the presence of Cu2+ 5 p M . The activity of the enzyme after preincubation with 5 pM Cu2+ alone is about 10% of the control activity. complex as compared to the unstable Cys-Cu complex. This assumption is deduced from semi- empirical computer based ZINDO-calculations (Kieninger and Elstner, manuscript in prepara tion). This hypothesis may explain some of the dif ferences observed in the pro- and antioxidative activities of Cys and HC.
4) Reactions with dihydroxy phenylalanine The mentioned difference in reactivity between Cys and HC is also observed during the poly- phenoloxidase-catalyzed oxidation of the neuro transmitter precurser dihydroxy-phenylalanine Sulphur compound [mM] during preincubation (DOPA). It is well known that PPO-catalyzed o-diphenol oxidation in the presence of Cys yields Fig. 6b. Activity of glutamic-pyruvic transaminase cysteinyl-DOPA as a Michael-type addition product (0.1 U) after preincubation (15 min, 37 °C) with various of Cys onto the catalysis-derived o-quinone. amounts of cysteine, homocysteine or homocysteinethio- HPLC-analysis of two mayor products (peaks 2a lactone; 1; 10; 50; 100 m M (without Cu2+). and 2b) of this condensations show that the Cys- derived products represent rapidly produced inter 3) Oxidation o f the sulfhydryl groups o f cysteine mediates which slowly disappear after 60 min reac and homocysteine tion time (Fig. 8 a) while the HC-derived products As shown in Fig. 2 transitionmetal-dependent seem to accumulate rather slowly (Fig. 8 b). ethene release from Met in the presence of hydro- genperoxide is inhibited by increasing concentra 5) Oxidation o f low density lipoprotein (LDL) tions of PyP. LDL oxidation can be followed by several During the interaction between copper (Fig. 7) methods, such as dieneconjugation, electropho or iron (data not shown) and Cys or HC, the sulf retic mobility or HPLC-analysis of the composi hydryl group of Cys is rapidly oxidized while the tion of the fatty acid contents (Puhl et al., 1994). one of HC is much more stable (Preibisch and Dieneconjugation as an indicator of peroxi Elstner, 1994). This is probably due to the forma dizing unsaturated fatty acids in LDL can be pho tion of a relatively stable octohedral HC -C u tometrically observed starting ca. 30 min after 704 E. Schlüssel et al. ■ Oxidative Damage by Homocysteine
10 20 30 40 50 60 “i------r 400000 □ Peak 2a
<1) < 200000
100000
0 1
600000 □ Peak 2a CO H H Peak 2b 1 1 ) incubation with 2.5 jim or 5 jim C u 2+. Copper con Table I. /?f-values (agarose gel) of LDL preincubated with Cu2+ 5 jim and several test substances. The relative centrations below 1 jim show no effect and 1 0 (.im mobility of LDL incubated with Cu2+ 5 jim alone is set catalyze identical peroxidation as 5 jam. 500 jam Cys as 1.00; the /?f-value for untreated LDL is 0.89. or HC completely inhibit this process (Fig. 9). Changes in the electrophoretic mobility of LDL Test 50 jim 100 JIM 250 jim 500 jim substance on 0 .8 % agarose gel may represent a modification of the Apo B-100 moiety. We incubated LDL in Cys 1.00 0.92 0.91 0.89 the presence of 5 jim Cu2+ with various concentra HC 1.09 1.14 1.14 1.17 1.14 tions of Cys, HC or HCTL ranging from 50 to 500 HCTL 1.17 1.17 1.06 jam for 24 hours at 37 °C. Results are presented as /?f-values (relative mobility) in Table I. Treatment of LDL with 5 jim Cu 2+ clearly in pendent manner. The copper-increased mobility is creases its electrophoretic mobility while Cys an further augmented by HC. HCTL also enhances nihilates the Cu2+- effect in a concentration-de- the copper effect, but no clear correlation between concentration and changes in electrophoretic mobility can be observed. Discussion The transitionmetal ions copper and iron to gether with HC are under discussion in oxidatively causing neurodegenerative and arteriosclerotic diseases. Decreases or imbalances of the vitamins B6, B 12 and folic acid seem to be cooperatively or causally involved in several categories of the above disease fields (Schäfer-Elinder and Waldius, 1994; Santhosh-Kumar et al., 1994; Bell et al., Incubation time [min] 1992). The transamination product of Met, a-keto- Fig. 9. Monitoring of dieneconjugation in low-density methylthiobutyrate (KMB). is one product of Met lipoprotein caused by incubation with Cu2+ (various amounts), cysteine and homocysteine (500 jim) by con metabolism especially in persons with inborn Met tinuous recording changes in absorbance at 234 nm. adenosyltransferase deficiency (Blom et al., 1989). E. Schlüssel et al. ■ Oxidative Damage by Homocysteine 705 It reacts with a wide spectrum of oxidants produc (L-glutamate and D-aspartate) by cerebral cortical ing ethene, methanethiol and other products. As synaptosomes and oxidation of Cys- or Met-resi- reported previously (Preibisch and Elstner, 1994), dues in proteins produces a decrease in synaptic homocysteine in the presence of either copper- or potentials in the hippocampus (Gilman et al., 1993 iron-ions stimulates ethene release from KMB in and refs, therein). A very similar type of reaction a concentration-dependent manner. In the pres is known to inactivate protease inhibitors via Met- ence of either 5 jam Fe2+, 5 |im Fe3+ or 25 |im C u 2+ oxidation through the cooperative reaction of ions KMB is fragmented yielding ethene. The ef iron-thiolate with endogenous histidine residues fects of homocysteine or its thiolactone are en (Schöneich et al., 1993) according to the above hanced by Fe1+ and Cu2+. The stimulating effects reaction sequence a) to d). of cysteine and homocysteinethiolactone are com Reaction d) represents a so-called “Fenton- pletely abolished in the presence of catalase while Type” of strong oxidant production from hydro- superoxide dismutase has less effect. The chain genperoxide catalyzed be either copper- or iron- breaking radical scavenger propylgallate strongly ions, reoxidizing the metal catalyst for reaction a). inhibits the thiolactone effect but has only little These oxidants, similar to the OH’ radical react effect on the cysteine system. Since the hydro- extremely rapidly with KMB and slower with Met genperoxide forming capacity is much stronger producing ethene. As shown in Fig. 2, PyP is a with cysteine as compared to homocysteine or its strong inhibitor of the copper system and is de thiolactone the rapid formation of a free radical in graded during its reaction (data not shown). This the thiolactone ring after reacting with Cu2+ seems protective role is also expressed during the copper- to represent the critical step in the destructive ca catalyzed and Cys- or HCTL-enhanced inactiva pacity of the thiolactone. The following sequence tion of transaminases (TA), where characteristic of events might be operating, where HC'+ or differences between glutamic-oxaloacetic TA and HC(TL) + may represent a HC- or HCTL-thiyl- glutamic-pyruvic TA are observed. The latter en kation radical, i.e. a HC- or HCTL-molecule with zyme seems to be especially sensitive to copper an unpaired electron at the sulphur atom: ions. PyP is also rapidly consumed forming a new a) 2 HC(TL) + 2 Cu2+ -» 2 HC(TL) + + 2 Cul + product in the presence of thiols such as Cys or HC. PyP deficiency in certcain cases, especially in b) 2 Cul+ + 2 0 2 ^ 2 Cu2+ + 2 0 2’~ elderly people frequently exhibiting elevated HC c) 2 0 2- + 2 H+ H20 2 + 0 2 levels (Bell et al., 1992) may thus be explainable not only by nutritional shortcomings but also by d) H 20 2 + Cu,+ — Cu2+ + OH- + OH' derivatization or cooxidation with HC. Similarly, e) OH' + KMB —> ethene + other products the concentrations and relative distribution of aromatic amines such as DOPA, dopamine, 6 -hy- Reaction a) results in the production of excita droxyDOPA and noradrenalin are critical in age- tory neurotransmitters (sulfinic acids, cysteic and associated memory impairment, dementia of de homocysteic acids) on the one hand and inhibitory pressed elderly people as well as Parkinson’s and transmitters (taurine, hypotaurine) on the other Alzheimer’s diseases (Bell et al., 1992; Gottfries, hand (Santhosh-Kumar et al., 1994) via the reac 1985; McEntee and Crook, 1990; Kopin, 1993). As tion as ligands for the NMDA-(N-methyl-D-aspar- shown in Figs 8a and 8b DOPA reacts with both tate-) type of glutamate receptors. HC-induced Cys and HC. The products are different both in elevated extracellular (dialyzable) aspartate and quality and in quantity where the products with taurine levels and reduced GABA (y-amino Cys seem to represent rapidly formed transitory butyric acid) levels suggesting the creation of an states while the ones with HC accumulate slowly. imbalance in these neurotransmitters in the hippo Independent of the monoamine oxidase-catalyzed, campus have recently been reported by Butcher the macrophage-initiated and transition metal-cat- et al., 1991. alyzed autoxidation of these neurotransmitters In this context it is interesting to note that oxi (Youdim and Lavie, 1994) the derivatization of the dativ “stress” releases excitatory amino acids o-dihydroxy aromats by HC may represent an 706 E. Schlüssel et al. ■ Oxidative Damage by Homocysteine other pathway for the induction of neurodegener- tive treatment with copper ions in the presence or ative processes. absence of HC. The LDL preparation from one Atherogenic processes seem to be initiated or donor exhibited the “normal” copper effect but no augmented by oxidative modification of LDL enhancement by HC (data not shown). The LDL (Schäfer-Elinder and Waldius, 1994; Ross, 1993) from the second donor showed a reproducible where structural configuration as well as the chem overadditive HC-copper effect in the electropho ical composition of the macromolecule determines retic test and a complete inhibition of the diene- the oxidation resistance. Especially a dense LDL conjugation by Cys or HC. This inhibitory effects fraction (Dejager et al., 1993) and LDL species are in contrast to the findings by Lupo et al. (Lupo with low sialic acid contents (Tertow et al., 1993) et al., 1994). The difference may be explaind by seem to exhibit diminished oxidation resistance the extremely different concentrations of copper (Ross, 1993) and/or accumulate certain athero and HC described by Kopin, 1993 and/or differ genic lipids such as cholesterol (Tertow et al., ences in the two experimental approaches. Our 1993). We tested two different LDL preparations findings are in perfect agreement with the results from different donors as to their optical (diene- recently published by Hirano et al., 1994 who conjugation, Fig. 9) and chromatographic (gel showed an HC-induced, iron-catalyzed LDL oxi electrophoresis, Table I) behaviours after oxida dation which was prevented by a-tocopherol. Bell J. R., Edman J. S., Selhub J., Morrow F. D., Marby Kögl C., Schneider W. and Elstner E. F. (1994), The in D. W., Kayne H. L. and Cole J. O. 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