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Dietary methionine imbalance, endothelial cell dysfunction and atherosclerosis
NutritionResearch, Vol.16,No.7, pp. 1251-1266.1996 Copyright 8 1996Elsevier Science Inc. Printed in the USA. All rights reserved 0271-5317/96$15.00+ .OO
PII SO271-5317(%)00128-5
ELSEVIER
DIETARY METHIONINE
IMBALANCE, ENDOTHELIAL ATHEROSCLEROSIS
CELL DYSFUNCTION
AND
Michal Toborek, M.D. 1 and Bernhard Hemrig, Ph.D.2* Departments
of 1Surgery and 2Nutrition and Food Science, University of Kentucky, Lexington, KY, 40506-0054, USA.
ABSTRACT High fat, high Dietary factors can play a crucial role in the development of atherosclerosis. calorie diets are well known risk factors for this disease. In addition, there is strong evidence that dietary animal proteins also can contribute to the development of atherosclerosis. Atherogenic effects of animal proteins are related, at least in part, to high levels of methionine in these proteins. An excess of dietary methionine may induce atherosclerosis by increasing plasma lipid levels and/or In addition, methionine imbalance by contributing to endothelial cell injury or dysfunction. elevates plasma/tissue homocysteine which may induce oxidative stress and injury to endothelial cells. Methionine and homocysteine metabolism is regulated by the cellular content of vitamins B6, Bl2, riboflavin and folic acid. Therefore, deficiencies of these vitamins may significantly influence methionine and homocysteine levels and their effects on the development of atherosclerosis. KEY WORDS:
Methionine, Homocysteine,
Atherosclerosis,
Endothelial Cells.
INTRODUCTION Amino acids play a crucial role in metabolism of the vascular endothelium (1). Despite their role in protein synthesis, several amino acids exert specific metabolic effects in endothelial cells. For example, glutamate and glutamine are the primary energy sources for endothelial cells (2,3) and arginine, due to its function in the synthesis of nitric oxide, is involved in the regulation of the vascular tone (4,5). Amino acids also control protection of the endothelium against oxidative insult. One of the most important endothelial antioxidant systems is formed by glutathione, a tripeptide synthesized from glycine, glutamate and cysteine. Reduced glutathione is a major nonprotein sulfhydryl compound within cells. Modifications of the glutathione oxdiation/reduction ratio are critical for the overall cellular redox status. In addition, glutathione serves as a non-enzymatic free radical scavenger and a cofactor for glutathione-dependent antioxidant enzymes (6).
* Corresponding
author.
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M. TOBOREK and B. HENNIG
The endothelium which lines blood vessel “communicates” actively with the blood-borne cells and the underlying abluminal tissues. The integrity of the endothelial cells preserves normal structure and function of blood vessels. The endothelium is involved in the regulation of vascular Endothelial cells are constantly tone, coagulation, fibrinolysis and inflammatory responses. exposed to nutrients and other factors which can exert either injurious or protective properties (for review see [7]). It is generally accepted that injury to the vascular endothelium is one of the first events in the development of atherosclerosis (8). Thus, each factor which can cause endothelial damage or dysfunction may be atherogenic. Evidence indicates that dietary sulfur-containing amino acids can exert both protective and injurious effects on endothelial cell metabolism. Dietary methionine is involved in a variety of potentially protective pathways, such as, protein synthesis, polyamine metabolism, methylation processes or glutathione metabolism. On the other hand, methionine is the only dietary source of homocysteine (Figure l), a potent disrupting agent of endothelial integrity (for review see [7,9]). Thus, an imbalance in dietary methionine may contribute to the development of atherosclerosis (10). Common dietary sources of methionine are animal proteins. Moreover, the metabolism of methionine and homocysteine is controlled by vitamins B6, Bl2, riboflavin and folic acid (7). The present review will focus on methionine and its metabolites, e.g., homocysteine, in relation to vascular biology. In addition, because lipids are critical factors in the development of atherosclerosis, the influence of methionine on lipid metabolism also will be addressed.
ENDOTHELIAL CELLS: GENERAL PROPERTIES AND FUNCTIONS The vascular endothelium, which is situated at the vital interface between the circulating blood and the body’s tissues, plays an important role in cardiovascular functions in health and disease (11,12). Endothelial cells participate in almost all aspects of animal and human biology, and most major cardiovascular diseases are associated with pathophysiologic alterations in endothelial cell structural and functional properties. Endothelial cells perform biological functions primarily by sensing and recognizing changes in their environment and by responding to these stimuli via the production of numerous biologically active substances or endothelial cell-derived factors. These substances or factors include small molecules, e.g., histamine, free radicals, endothelium-derived relaxing factor (EDRF) or nitric oxide (NO), lipid derivatives, e.g., prostaglandins and leukotrienes, and proteins, e.g., growth factors, adhesion molecules, matrix proteins, coagulation factors, enzymes and receptors (11). Considering the regulatory properties of these factors, normal endothelial cells maintain a delicate balance in the vasculature between growth promotion and inhibition (13) vasoconstriction and vasodilation (14), blood cell adherence and nonadherence (15), and anticoagulation and procoagulation (16). Thus, the vascular endothelium is able to control vasomotor tone, to regulate vascular structure, to maintain blood fluidity, and to mediate both inflammatory and immunologic responses (12).
ENDOTHELIAL
CELL DYSFUNCTION AND ATHEROSCLEROSIS
There is evidence that events of endothelial cell activation, dysfunction or injury are causative in the development of atherosclerosis (17). Dysfunction of the endothelium can be defined as an imbalance between relaxing and contracting factors, between anti- and pro-coagulant mediators or growth-inhibitory and growth-promoting factors (11).
DIETARY METHIONINE
1253
AND ATHEROSCLEROSIS
Numerous agents have been identified which can trigger endothelial cell activation or dysfunction. These include chemical toxicity (e.g., lipid oxidation derivatives), hemodynamic factors (e.g., shear stress), immunological injury (e.g., immune complexes), viral infection, as well as indirect mediators, such as inflammatory cytokines (e.g., tumor necrosis factor and selected interleukins), which are generated in the vessel wall or blood-borne cells in response to one of the above stimuli (18-20). Some of the consequences of endothelial cell dysfunction include an imbalance in oxidative stress/antioxidant status (2 1,22), expression of binding sites for monocytes (23) and loss of endothelial barrier function (7). These events can exacerbate another critical component in the etiology of atherosclerosis, i.e., the continuous adhesion of circulating monocytes to the endothelial surface (23). Moreover, with the development of endothelial cell dysfunction, there is an associated reduction in the thromboregulation mediated by endothelial cells (16). In addition to increased prothrombin activation and tissue factor synthesis, there is decreased fibrinolysis with increased levels of PAI- (an endothelium-derived inhibitor of plasminogen activators) and loss of the thromboregulation mediated by both prostacyclin and EDRF/NO (12). The long-term clinical outcome of endothelial cell dysfunction and related events is accelerated atherosclerosis, manifested by fatty streak formation via monocyte uptake and conversion to macrophages (foam cells), and ultimate development of advanced fibrous plaques (23,24).
<-. . Methanethiol ”
Formaldehyde
. . -> Hydrogen sulfide
3-Methylthiopropionate
2-Keto-4-Methylthiobutyrate ” TetrahydroGB6 1 c
f”‘ate
METHIONINE 1 demethylation
Vit B,, x Bj_Methyl_ Tetrahydrofolate
f-s.%
1
HOMOC “~T’CThTE -Vit B,
.-) N,N-Dimethylglycine
%.’ ” :a’ fi ,$, .I‘ *\.-_
Serine -
D-r..:..__
cY.,.I:-^
Glycine
l-
Cystathionine
Taurine, + Taurocholic Acid
-
Cysteine
-+
-+
Glutathione
1
1 sod2FIG. 1: Involvment of vitamins in methionine metabolism. Solid arrows reflect pathways active in endothelial cells. Dotted arrows reflect pathways not active in endothelial cells.
M. TOBOREK and B. HENNIG
1254
METHIONINE
METABOLISM
Methionine is an essential amino acid found in high concentrations in animal proteins. It is primarily metabolized in the liver. The major pathway of methionine catabolism is through the transmethylation and transsulfuration pathways (Figure 1). The transmethylation pathway starts from conversion of methionine to S-adenosyl-methionine (SAM). SAM participates in transmethylation processes and in biosynthesis of polyamines. Acceptors for methyl groups from SAM include DNA, RNA, proteins, lipids and numerous micromolecules. In the subsequent reaction, S-adenosyl-homocysteine formed as a results of demethylation of SAM, is converted into homocysteine. Approximately 77% of synthesized homocysteine in liver is remethylated into methionine. The ethyl group which is required for methylation of homocysteine may originate from betaine or 5-methyltetrahydrofalte in reactions catabolized by betaine:homocysteine methyltransferase or 5-methyltetrahydrofalte:homocyteine methyltransferase, respectively. A significant amount of homocysteine is also converted, through the transsulfuration pathway, into cystathionine and then into cysteine, hipotaurine and taurocholic acid (25). In cell cultures, accumulation of cellular homocysteine is prevented by export of this amino acids into culture medium (26). To illustrate this phenomenon, it was reported that after 30 minutes of supplementation with elevated methionine, the cellular homocysteine level was about 60% of that present in medium. However, in cells exposed to methionine for 24 hours, the cellular homocysteine level was only approximately 4% of that found in the surrounding medium. In addition to export mechanisms, cells also may take up homocysteine from the medium (26). The cellular uptake of homocysteine in endothelial cells is regulated by at least two transport systems, ASC and L (27,28). In addition, homocysteine uptake is inhibited by cysteine which competes for ASC transport mechanism (27). Several reactions of the transmethylation and transsulfuration pathways are controlled by vitamins. Folic acid, vitamins B12 and B2 participate in the remethylation of homocysteine into methionine. Moreover, vitamin B6 is a cofactor of cystathionine P-synthase, an enzyme which converts homocysteine to cystathionine. Vitamin B6 also participates in the conversion of cystathionine to cysteine (25). It also was postulated that methionine may be metabolized in the liver via the transamination pathway. Metabolites of methionine degradation through this pathway include, among others, 3-methylthioproprionate and methanethiol(29). However, the significance of the transamination pathway in methionine metabolism is not well characterized (30). Methionine metabolism in endothelial cells is different from that found in the liver. The transamination pathway of methionine degradation is described only in the liver and apparently is not active in endothelial cells. Endothelial cells also lack betaine:homocysteine methyltransferase activity (3 1). One may suggest that this enzyme deficiency may diminish the ability of endothelial cells, as compared to other cell types, to remethylate homocysteine to methionine. In addition, methionine is not converted to cysteine in endothelial cells due to lack of cystathioninase pathway (32). These metabolic deficiencies in endothelial cells may contribute to the atherogenic potential of methionine in vascular tissues.
METHIONINE
IMBALANCE
AND ALTERATIONS
IN LIPID METABOLISM
Plant proteins, e.g., soy protein isolate, may exert hypocholesterolemic effects as compared to animal proteins, e.g., casein (33). This effect is most markedly expressed in experimental animals with cholesterol-induced hypercholesterolemia (34). Mechanisms of hypocholesterolemic
DIETARY METHIONINE
AND ATHEROSCLEROSIS
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effects of plant proteins and the hypercholesterolemic influence of animal proteins are not fully understood. However, they may be related to amino acid profiles of these proteins. For example, feeding animals soy protein elevates plasma tyroxine levels (35) which may increase hydroxymethylglutaryl coenzyme A (HMG CoA) reductase activity (36), elevate excretion of bile salts and decrease plasma triglyceride levels. On the other hand, the effect of animal proteins on lipid metabolism appears to be related to their methionine content and the methionine/protein ratio. Depending on the dietary methioninelprotein ratio, methionine-enriched diets may be hypercholesterolemic, hypocholesterolemic or without any influence on plasma cholesterol (37,38). Evidence suggests that methionine alone is a hypercholesterolemic amino acid, but that its demethylated derivatives (Figure 1) may decrease plasma cholesterol. Therefore, it was concluded that the methyl group was responsible for the hypercholesterolemic influence of methionine (38). This hypothesis was strengthened by the observation that rats fed a diet supplemented with methionine or cysteine preferentially stored energy as lipids or glycogen, respectively. Consequently, it appears that factors which promote demethylation and transsulfuration of methionine may protect against the hypercholesterolemic effect of this amino acid. In fact, glycine, which facilitates utilization of methionine via the transsulfuration pathway (Figure l), lowered plasma cholesterol levels in methionine-fed rats (38). It is also possible that metbionine exerts its hypercholesterolemic effect by down-regulation of hepatic low density lipoprotein (LDL) receptors (39). It has been proposed that the hypocholesterolemic effect of demethylated metabolites of methionine may be mediated by stimulation of bile salt production. Although most of the bile salt pool is conserved normally by intestinal reabsorption, a certain percentage of bile salt is excreted and thus may decrease plasma cholesterol levels. Methionine is metabolized to taurine which reacts with cholic acid to form taurocholic acid, one of the major bile acids. Moreover, methionine serves as a methyl group source for the synthesis of phosphatidilcholine, another component of bile salt micelles. Thus, when methionine is sufftciently metabolized to taurine, it may increase bile acid production and decrease cholesterol levels (9). Apart from the influence on cholesterol levels, a methionine-enriched diet also was shown to cause elevation of triglycerides in experimental animals (37). Both hypercholesterolemia and hypertriglyceridemia are well known risk factors for atherosclerosis. In support of this hypothesis, we have shown recently that lipolytic remnants of triglyceride-rich lipoproteins can be cytotoxic to cultured endothelial cells (40). It was postulated that triglyceride-rich lipoproteins are hydrolyzed in the proximity of the vascular endothelium by lipoprotein lipase. Thus endothelial cells may be directly exposed to elevated levels of free fatty acids (41). Selected free fatty acids cause disruption of endothelial barrier function, increase intracellular free calcium and induce oxidative stress, oxidative stress responsive transcription factors and genes (for review see [ 191). Although this hypothesis needs to be confirmed, it is possible that fatty acid-mediated alterations in endothelial cell metabolism may be a part of the pathological events induced by an imbalance of dietary methionine.
METHIONINE IMBALANCE AND EXPERIMENTAL ATHEROSCLEROSIS Up to date, only few reports have indicated that an excess of dietary methionine may contribute to the development of atherosclerosis. Mrhova et al. (42) reported that oral methionine administered to rats resulted in an increased number of circulating endothelial cells. Fau et al. (43) observed disturbances in arterial wall morphology in rats chronically fed a diet enriched with 2% methionine. Atherosclerotic changes in methionine-fed rabbits also were reported by McCully and Wilson (44). Conversely, feeding monkeys a methionine-enriched diet did not induce atherosclerosis (45).
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M. TOBOREK and B. HENNIG
Recently, we reported the development of a variety of atherosclerotic changes, such as intimal thickening, lipid deposition, and arterial wall calcification in rabbits fed a methionineenriched diet for 6 or 9 months (10). The mechanisms of these effects are not clear. However, they may be related to the induction of oxidative stress. In fact, in rabbits fed the methionineenriched diet, a significant increase in aortic lipid peroxidation was observed (10). The increase in aortic lipid peroxidation was accompanied by elevations in aortic antioxidant enzymes and a decrease in plasma antioxidant activity. Antioxidant activity reflects the potency of extracellular free radical scavengers, such as antioxidant vitamins. A decrease in these vitamins may promote oxidative stress and the development of atherosclerosis. To support this observation, a decrease in antioxidant activity was shown in patients with chronic renal failure, a disease which promotes accelerated atherosclerosis (46). Another mechanism which may promote the development of atherosclerosis in methionine-fed rabbits may be related to alterations in elastin metabolism. Fragmentation of elastic fibers and alterations in elastin synthesis were observed in rabbits fed a methionine-enriched diet (47). Although feeding animals methionine-enriched diets can promote the development of atherosclerosis, this effect may not be fully specific. Methionine toxicity is not limited to the vessel wall. For example, we reported the development of hepatitis in rabbits fed a methionineenriched diet. Methionine-related hepatotoxicity was reflected by inflammatory infiltration of portal triads. In addition, these morphological changes were associated with increased lipid peroxidation and alterations in antioxidant response in liver tissues (48).
METHIONINE IMBALANCE AND ENDOTHELIAL CELL DYSFUNCTION The mechanisms by which methionine may stimulate the development of atherosclerosis are not fully understood. One of the possibilities is the induction of injury to the endothelium. This hypothesis was supported by the observation that administration of methionine to rats resulted in elevated levels of endothelial cells in blood (42). In addition, we observed the disruption of endothelial barrier function in endothelial cells exposed to methionine (Figure 2). Disruption of endothelial cell integrity was further enhanced when endothelial cells were exposed to methionine plus a “physiological” free fatty acid mixture (FFA-M). FFA-M is a mixture of free fatty acids in concentrations similar to those found in serum under physiological conditions. It is possible that homocysteine is at least in part responsible for the atherogenic effects mediated by methionine. In fact, in methionine-fed rabbits we observed changes in the activities of enzymes which regulate the transsulfuration pathway (49). These enzyme alterations might promote accumulation of homocysteine. An increased serum homocysteine level recently has been recognized as a risk factor in atherosclerosis. The relationship between elevated plasma homocysteine levels and peripheral, cerebrovascular and coronary artery disease has been shown clearly in a number of clinical trials and experimental studies (for review see [50]). Moreover, homocysteine toxicity towards endothelial cells has been proposed as the main mechanism responsible for the atherogenic effect of this amino acid (51). A specific enzyme spectrum in endothelial cells may increase the susceptibility of these cells to toxic effects exerted by homocysteine. Endothelial cells express only two of the three crucial enzymes which participate in homocysteine metabolism in other tissues (Figure 1). Active enzymes in endothelial cells are cystathionine P-synthase and 5methyltetrahydrofolate:homocysteine methyltransferase. As it was mentioned previously, betaine:homocysteine methyltransferase is inactive in endothelial cells (3 1). Moreover, under pathologic conditions, such as atherosclerosis or vitamin B6 deficiency, activity of cystathionine P-synthase may be diminished which further contributes to the accumulation of homocysteine
DIETARY METHIONINE
AND ATHEROSCLEROSIS
1257
(52). Recently, mice deficient in cystathionine P-synthase has been generated by specific gene targeting in embryonic stem cells. Homozygous mutants completely lacking cystathionine psynthase activity had plasma homocysteine levels elevated approximately 40 times. However, heterozygous mutants which have approximately 50% of normal cystathionine P-synthase activity showed approximately 2 time increase in plasma homocysteine levels. These mutants may provide excellent experimental models to study metabolic effects of mild or severe homocysteinemia (53).
51
3
2
1
0 Lb
0 fl
without added FFA-M with added FFA-M
“r 1x1 I’,‘,’ ,‘,‘/’ ,‘/‘,’ ,‘/‘/’ ,‘#‘/’ ,‘I’#’ ,‘,‘I’ ,‘/‘I’ /‘/‘/’ I’,‘,’ I’,‘,’ ,‘,‘#’ I’,‘,’ I’,‘,’ ,‘XX\ #‘I’ Control
Methionine Concentration (PM) in cells treated wif*h increasing FIG. 2: Albumin transfer across endothelial monolayers Significantly concentrations of methionine and/or mixture of free fatty acids (FFA-M). higher than corresponding control group. t Significantly higher than values for samples without added FFA-M within each treatment group.
It is conceivable that endothelial integrity is greatly influenced by plasma concentrations of methionine and homocysteine. Methionine and homocysteine metabolism is controlled by the availability of vitamins such as riboflavin, vitamin B6, B12 and folic acid (Figure 1). Dietary deficiencies of any of these vitamins may impair activities of enzymes involved in methionine and homocysteine metabolism and promote accumulation of homocysteine. In fact, elevated homocysteine levels were found to be correlated with decreased levels of dietary and plasma Moreover, a negative correlation between plasma vitamin B6, B12 and folic acid levels. homocysteine and the ratio of dietary vitamin B6 to protein was observed in subjects at high risk for coronary heart disease (54). Plasma pyridoxal5’-phosphate, the active form of vitamin B6 was documented to be lower in atherosclerotic patients (55). Feeding pigs a diet deficient in vitamin B6 elevated homocysteine concentrations and induced atherosclerosis (56). Furthermore, simultaneous administration of riboflavin, vitamins B6, Bl2, folate, choline and troxerutin to myocardial infarct
1258
M. TOBOREK and B. HENNIG
patients caused significant decreases in plasma homocysteine cholesterol, triglycerides and apolipoprotein B concentrations (57).
levels
as well as in plasma
Several lines of evidence support the hypothesis that the atherogenic effect of homocysteine is mediated by injury to the endothelium. In baboons, homocysteine, when continuously infused as homocysteine thiolactone, caused desquamation of the vascular endothelium (58). Although this effect was not observed in pigs (59), and homocysteine thiolactone might have effects unrelated to homocysteine (60), the cytotoxicity of homocysteine was confirmed in a number of experiments (61,62). For example, exposure to homocysteine caused a concentration-dependent detachment of human umbilical artery or vein endothelial cells from polystyrene tissue culture dishes (62). Similar, in our experiments, homocysteine disrupted endothelial barrier function (Figure 3).
FIG. 3: Albumin transfer across endopelial monolayers in cells treated with increasing concentrations of homocysteine. Significantly higher than control group.
Homocysteine also interferes with coagulation. Homocysteine inhibits the expression thrombomodulin on the surface of endothelial cells. Thrombomodulin is an inhibitor procoagulant activity of thrombin. It is a cofactor for thrombin-modulated activation of protein and activated protein C inactivates coagulation cofactors Va and VIIIa. Consequently, inhibition of thrombomodulin diminishes the antithrombotic potential of endothelial cells (63).
of of C, the
In addition, homocysteine may significantly affect metabolism of vascular smooth muscle cells. For example, it was shown that homocysteine infusion into baboons induces myointimal proliferation (64). In in vitro studies homocysteine increased DNA synthesis and promoted proliferation of rat aortic smooth muscle cells. Homocysteine also elevated cyclin Dl and cyclin A
DIETARY METHIONINE
AND ATHEROSCLEROSIS
1259
which are important factors of the reentry of quiescent smooth muscle cells into the cell cycle (65). Thus, homocysteine-induced stimulation of smooth muscle cell proliferation may be another mechanism of atherogenic effect of this ammo acid. It appears that cellular oxidative stress may be a main mechanism of methionine, and homocysteine imbalance and autoxidation of compounds with a free thiol group may be the primary source of free radicals when methionine is administrated in excessive amounts. The initial and rate limiting reaction in thiol autoxidation is the electron transfer from the thiolate anion to a transition metal ion. As a result of the subsequent steps a wide spectrum of both oxygen and thiyl radical is generated (66). Due to their high reactivity thiyl radicals are capable of degrading and inactivating a variety of enzymes and other biological important compounds. They may also initiate lipid peroxidation (67). Moreover, thiol compounds were shown to oxidize LDL (6869). Although methionine itself does not undergo autoxidation, its metabolites, such as homocysteine or protein-bound thiols, are susceptible to this type of oxidative reaction (66,70). In fact, Starkebaum and Harlan (5 1) documented that the cytotoxicity of homocysteine to cultured endothelial cells was mediated by the formation of hydrogen peroxide during the autoxidation of homocysteine to homocystine. Homocysteine was not toxic to endothelial cells in the absence of copper and ceruloplasmin which catalyze the oxidation of homocysteine to the disultide form. Furthermore, the addition of catalase prevented homocysteine-induced injury to endothelial cells (51). To support the observation that increased oxidative stress may be a main mechanism of homocysteine-induced injury to endothelial cells, we observed an increase in fluorescence of 2’,7’dichlorofluorescein (DCF) (Figure 4), and Jones et al. (71) reported elevated thiobarbituric acid reactive substances (TBARS) levels in cells exposed to this amino acid. DCF fluorescence is a new cell imaging technique visualizing cellular oxidative stress in intact cells. This technique can quantitate primarily oxidative stress mediated by hydrogen peroxide (72). Thus our results further indicate the critical role of hydrogen peroxide in homocysteine-mediated endothelial cell dysfunction. However, hydrogen peroxide is a relatively weak oxidant and its toxicity may be significantly related to the generation of hydroxyl radicals, the most reactive free radical produced in biological systems. These radicals are generated as a result of reactions between hydrogen peroxide and superoxide anion radical in the presence of transition metal ions, such as iron or copper (67). The role of hydroxyl radical in homocysteine-induced toxicity was supported by the observation that desferal, a potent metal chelator, efficiently inhibited oxidative stress in cells exposed to this amino acid (71).
HYF’ERHOMOCYSTEINEMIA
AND ATHEROSCLEROSIS:
CLINICAL STUDIES
Numerous studies indicate that the prevalence of hyperhomocysteinemia in patients with coronary heart disease is between 20-40% (73-76). Hyperhomocysteinemia also is observed in a similar range of patients with premature peripheral and cerebral arterial occlusive disease or thromboembolism (77-80). Several pathologic conditions may induce hyperhomocysteinemia. Classic homocystinuria is caused by homozygosity for cystathionine P-synthase, and it is characterized by severe accumulation of homocysteine. It was estimated that homozygotes for cystathionine P-synthase have a 50% chance to develop vascular disease before the age of 30 years (81). Hyperhomocysteinemia also may be caused by other genetic defects. Plasma homocysteine may be moderately elevated (approximately 30-50% of normal range) due to heterozygosity for cystathionine P-synthase (82) or in the presence of a thermolabile variant of methylenetetrahydrofolate reductase (83). It was reported that this latter defect occurred in 17% of patients with coronary artery disease (84). In addition, nutritional deficiencies may lead to the
M. TOBOREK and B. HENNIG
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In fact, decreased levels of vitamin B 12, vitamin B6 and development of hyperhomocysteinemia. folic acid were demonstrated in apparently healthy men with hyperhomocysteinemia (83). Such a Vitamin therapy also is a common therapeutic approach to treatment of homocysteinemia. therapy usually starts with administration of vitamin B6 for up to 6 weeks. If vitamin B6 is ineffective in the treatment of hyperhomocysteinemia, a combined treatment with vitamin B6 and/or folic acid and/or betaine is introduced. This therapeutic approach was reported to be effective in more than 90% patients with hyperhomocysteinemia (86).
0
Control
Homocysteine
Treatment Groups FIG. 4: Cellular oxidative s$ess (DCF fluorescence) in cultured endothelial mM homocysteine. Significantly higher than control group.
cells treated with 5
CONCLUSION Atherosclerosis and related vascular disorders are the leading cause of death in the United States. Evidence strongly suggests that overconsumption of protein of animal origin can contribute to the development of this disease. The mechanisms of atherogenic effects of dietary animal proteins are not fully understood. However, as discussed above, high concentrations of methionine in animal proteins may be critical to their atherogenicity. Methionine may influence lipid composition and affect integrity of endothelial cells directly or indirectly through homocysteine metabolism. It appears that one of the main mechanisms of the atherogenic effects of dietary methionine is its ability to contribute to an increase in oxidative stress in vascular endothelial cells.
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Toborek M, Chmurzynska W, Manteuffel-Cymborowska M, Sikora E, GrzelakowskaSztabert B. Enzymes of homocysteine remethylation and transsulfuration in methionine or cholesterol induced atherosclerosis in rabbits. In: Curtis HC, Ghisla S, Blau N, eds, Chemistry and Biology of Pteridines 1989, Berlin, New York: Walter de Gruyter & Co. 1990; 876-80.
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Accepted
for
publication
March
30,
1996.
PII SO271-5317(%)00128-5
ELSEVIER
DIETARY METHIONINE
IMBALANCE, ENDOTHELIAL ATHEROSCLEROSIS
CELL DYSFUNCTION
AND
Michal Toborek, M.D. 1 and Bernhard Hemrig, Ph.D.2* Departments
of 1Surgery and 2Nutrition and Food Science, University of Kentucky, Lexington, KY, 40506-0054, USA.
ABSTRACT High fat, high Dietary factors can play a crucial role in the development of atherosclerosis. calorie diets are well known risk factors for this disease. In addition, there is strong evidence that dietary animal proteins also can contribute to the development of atherosclerosis. Atherogenic effects of animal proteins are related, at least in part, to high levels of methionine in these proteins. An excess of dietary methionine may induce atherosclerosis by increasing plasma lipid levels and/or In addition, methionine imbalance by contributing to endothelial cell injury or dysfunction. elevates plasma/tissue homocysteine which may induce oxidative stress and injury to endothelial cells. Methionine and homocysteine metabolism is regulated by the cellular content of vitamins B6, Bl2, riboflavin and folic acid. Therefore, deficiencies of these vitamins may significantly influence methionine and homocysteine levels and their effects on the development of atherosclerosis. KEY WORDS:
Methionine, Homocysteine,
Atherosclerosis,
Endothelial Cells.
INTRODUCTION Amino acids play a crucial role in metabolism of the vascular endothelium (1). Despite their role in protein synthesis, several amino acids exert specific metabolic effects in endothelial cells. For example, glutamate and glutamine are the primary energy sources for endothelial cells (2,3) and arginine, due to its function in the synthesis of nitric oxide, is involved in the regulation of the vascular tone (4,5). Amino acids also control protection of the endothelium against oxidative insult. One of the most important endothelial antioxidant systems is formed by glutathione, a tripeptide synthesized from glycine, glutamate and cysteine. Reduced glutathione is a major nonprotein sulfhydryl compound within cells. Modifications of the glutathione oxdiation/reduction ratio are critical for the overall cellular redox status. In addition, glutathione serves as a non-enzymatic free radical scavenger and a cofactor for glutathione-dependent antioxidant enzymes (6).
* Corresponding
author.
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M. TOBOREK and B. HENNIG
The endothelium which lines blood vessel “communicates” actively with the blood-borne cells and the underlying abluminal tissues. The integrity of the endothelial cells preserves normal structure and function of blood vessels. The endothelium is involved in the regulation of vascular Endothelial cells are constantly tone, coagulation, fibrinolysis and inflammatory responses. exposed to nutrients and other factors which can exert either injurious or protective properties (for review see [7]). It is generally accepted that injury to the vascular endothelium is one of the first events in the development of atherosclerosis (8). Thus, each factor which can cause endothelial damage or dysfunction may be atherogenic. Evidence indicates that dietary sulfur-containing amino acids can exert both protective and injurious effects on endothelial cell metabolism. Dietary methionine is involved in a variety of potentially protective pathways, such as, protein synthesis, polyamine metabolism, methylation processes or glutathione metabolism. On the other hand, methionine is the only dietary source of homocysteine (Figure l), a potent disrupting agent of endothelial integrity (for review see [7,9]). Thus, an imbalance in dietary methionine may contribute to the development of atherosclerosis (10). Common dietary sources of methionine are animal proteins. Moreover, the metabolism of methionine and homocysteine is controlled by vitamins B6, Bl2, riboflavin and folic acid (7). The present review will focus on methionine and its metabolites, e.g., homocysteine, in relation to vascular biology. In addition, because lipids are critical factors in the development of atherosclerosis, the influence of methionine on lipid metabolism also will be addressed.
ENDOTHELIAL CELLS: GENERAL PROPERTIES AND FUNCTIONS The vascular endothelium, which is situated at the vital interface between the circulating blood and the body’s tissues, plays an important role in cardiovascular functions in health and disease (11,12). Endothelial cells participate in almost all aspects of animal and human biology, and most major cardiovascular diseases are associated with pathophysiologic alterations in endothelial cell structural and functional properties. Endothelial cells perform biological functions primarily by sensing and recognizing changes in their environment and by responding to these stimuli via the production of numerous biologically active substances or endothelial cell-derived factors. These substances or factors include small molecules, e.g., histamine, free radicals, endothelium-derived relaxing factor (EDRF) or nitric oxide (NO), lipid derivatives, e.g., prostaglandins and leukotrienes, and proteins, e.g., growth factors, adhesion molecules, matrix proteins, coagulation factors, enzymes and receptors (11). Considering the regulatory properties of these factors, normal endothelial cells maintain a delicate balance in the vasculature between growth promotion and inhibition (13) vasoconstriction and vasodilation (14), blood cell adherence and nonadherence (15), and anticoagulation and procoagulation (16). Thus, the vascular endothelium is able to control vasomotor tone, to regulate vascular structure, to maintain blood fluidity, and to mediate both inflammatory and immunologic responses (12).
ENDOTHELIAL
CELL DYSFUNCTION AND ATHEROSCLEROSIS
There is evidence that events of endothelial cell activation, dysfunction or injury are causative in the development of atherosclerosis (17). Dysfunction of the endothelium can be defined as an imbalance between relaxing and contracting factors, between anti- and pro-coagulant mediators or growth-inhibitory and growth-promoting factors (11).
DIETARY METHIONINE
1253
AND ATHEROSCLEROSIS
Numerous agents have been identified which can trigger endothelial cell activation or dysfunction. These include chemical toxicity (e.g., lipid oxidation derivatives), hemodynamic factors (e.g., shear stress), immunological injury (e.g., immune complexes), viral infection, as well as indirect mediators, such as inflammatory cytokines (e.g., tumor necrosis factor and selected interleukins), which are generated in the vessel wall or blood-borne cells in response to one of the above stimuli (18-20). Some of the consequences of endothelial cell dysfunction include an imbalance in oxidative stress/antioxidant status (2 1,22), expression of binding sites for monocytes (23) and loss of endothelial barrier function (7). These events can exacerbate another critical component in the etiology of atherosclerosis, i.e., the continuous adhesion of circulating monocytes to the endothelial surface (23). Moreover, with the development of endothelial cell dysfunction, there is an associated reduction in the thromboregulation mediated by endothelial cells (16). In addition to increased prothrombin activation and tissue factor synthesis, there is decreased fibrinolysis with increased levels of PAI- (an endothelium-derived inhibitor of plasminogen activators) and loss of the thromboregulation mediated by both prostacyclin and EDRF/NO (12). The long-term clinical outcome of endothelial cell dysfunction and related events is accelerated atherosclerosis, manifested by fatty streak formation via monocyte uptake and conversion to macrophages (foam cells), and ultimate development of advanced fibrous plaques (23,24).
<-. . Methanethiol ”
Formaldehyde
. . -> Hydrogen sulfide
3-Methylthiopropionate
2-Keto-4-Methylthiobutyrate ” TetrahydroGB6 1 c
f”‘ate
METHIONINE 1 demethylation
Vit B,, x Bj_Methyl_ Tetrahydrofolate
f-s.%
1
HOMOC “~T’CThTE -Vit B,
.-) N,N-Dimethylglycine
%.’ ” :a’ fi ,$, .I‘ *\.-_
Serine -
D-r..:..__
cY.,.I:-^
Glycine
l-
Cystathionine
Taurine, + Taurocholic Acid
-
Cysteine
-+
-+
Glutathione
1
1 sod2FIG. 1: Involvment of vitamins in methionine metabolism. Solid arrows reflect pathways active in endothelial cells. Dotted arrows reflect pathways not active in endothelial cells.
M. TOBOREK and B. HENNIG
1254
METHIONINE
METABOLISM
Methionine is an essential amino acid found in high concentrations in animal proteins. It is primarily metabolized in the liver. The major pathway of methionine catabolism is through the transmethylation and transsulfuration pathways (Figure 1). The transmethylation pathway starts from conversion of methionine to S-adenosyl-methionine (SAM). SAM participates in transmethylation processes and in biosynthesis of polyamines. Acceptors for methyl groups from SAM include DNA, RNA, proteins, lipids and numerous micromolecules. In the subsequent reaction, S-adenosyl-homocysteine formed as a results of demethylation of SAM, is converted into homocysteine. Approximately 77% of synthesized homocysteine in liver is remethylated into methionine. The ethyl group which is required for methylation of homocysteine may originate from betaine or 5-methyltetrahydrofalte in reactions catabolized by betaine:homocysteine methyltransferase or 5-methyltetrahydrofalte:homocyteine methyltransferase, respectively. A significant amount of homocysteine is also converted, through the transsulfuration pathway, into cystathionine and then into cysteine, hipotaurine and taurocholic acid (25). In cell cultures, accumulation of cellular homocysteine is prevented by export of this amino acids into culture medium (26). To illustrate this phenomenon, it was reported that after 30 minutes of supplementation with elevated methionine, the cellular homocysteine level was about 60% of that present in medium. However, in cells exposed to methionine for 24 hours, the cellular homocysteine level was only approximately 4% of that found in the surrounding medium. In addition to export mechanisms, cells also may take up homocysteine from the medium (26). The cellular uptake of homocysteine in endothelial cells is regulated by at least two transport systems, ASC and L (27,28). In addition, homocysteine uptake is inhibited by cysteine which competes for ASC transport mechanism (27). Several reactions of the transmethylation and transsulfuration pathways are controlled by vitamins. Folic acid, vitamins B12 and B2 participate in the remethylation of homocysteine into methionine. Moreover, vitamin B6 is a cofactor of cystathionine P-synthase, an enzyme which converts homocysteine to cystathionine. Vitamin B6 also participates in the conversion of cystathionine to cysteine (25). It also was postulated that methionine may be metabolized in the liver via the transamination pathway. Metabolites of methionine degradation through this pathway include, among others, 3-methylthioproprionate and methanethiol(29). However, the significance of the transamination pathway in methionine metabolism is not well characterized (30). Methionine metabolism in endothelial cells is different from that found in the liver. The transamination pathway of methionine degradation is described only in the liver and apparently is not active in endothelial cells. Endothelial cells also lack betaine:homocysteine methyltransferase activity (3 1). One may suggest that this enzyme deficiency may diminish the ability of endothelial cells, as compared to other cell types, to remethylate homocysteine to methionine. In addition, methionine is not converted to cysteine in endothelial cells due to lack of cystathioninase pathway (32). These metabolic deficiencies in endothelial cells may contribute to the atherogenic potential of methionine in vascular tissues.
METHIONINE
IMBALANCE
AND ALTERATIONS
IN LIPID METABOLISM
Plant proteins, e.g., soy protein isolate, may exert hypocholesterolemic effects as compared to animal proteins, e.g., casein (33). This effect is most markedly expressed in experimental animals with cholesterol-induced hypercholesterolemia (34). Mechanisms of hypocholesterolemic
DIETARY METHIONINE
AND ATHEROSCLEROSIS
1255
effects of plant proteins and the hypercholesterolemic influence of animal proteins are not fully understood. However, they may be related to amino acid profiles of these proteins. For example, feeding animals soy protein elevates plasma tyroxine levels (35) which may increase hydroxymethylglutaryl coenzyme A (HMG CoA) reductase activity (36), elevate excretion of bile salts and decrease plasma triglyceride levels. On the other hand, the effect of animal proteins on lipid metabolism appears to be related to their methionine content and the methionine/protein ratio. Depending on the dietary methioninelprotein ratio, methionine-enriched diets may be hypercholesterolemic, hypocholesterolemic or without any influence on plasma cholesterol (37,38). Evidence suggests that methionine alone is a hypercholesterolemic amino acid, but that its demethylated derivatives (Figure 1) may decrease plasma cholesterol. Therefore, it was concluded that the methyl group was responsible for the hypercholesterolemic influence of methionine (38). This hypothesis was strengthened by the observation that rats fed a diet supplemented with methionine or cysteine preferentially stored energy as lipids or glycogen, respectively. Consequently, it appears that factors which promote demethylation and transsulfuration of methionine may protect against the hypercholesterolemic effect of this amino acid. In fact, glycine, which facilitates utilization of methionine via the transsulfuration pathway (Figure l), lowered plasma cholesterol levels in methionine-fed rats (38). It is also possible that metbionine exerts its hypercholesterolemic effect by down-regulation of hepatic low density lipoprotein (LDL) receptors (39). It has been proposed that the hypocholesterolemic effect of demethylated metabolites of methionine may be mediated by stimulation of bile salt production. Although most of the bile salt pool is conserved normally by intestinal reabsorption, a certain percentage of bile salt is excreted and thus may decrease plasma cholesterol levels. Methionine is metabolized to taurine which reacts with cholic acid to form taurocholic acid, one of the major bile acids. Moreover, methionine serves as a methyl group source for the synthesis of phosphatidilcholine, another component of bile salt micelles. Thus, when methionine is sufftciently metabolized to taurine, it may increase bile acid production and decrease cholesterol levels (9). Apart from the influence on cholesterol levels, a methionine-enriched diet also was shown to cause elevation of triglycerides in experimental animals (37). Both hypercholesterolemia and hypertriglyceridemia are well known risk factors for atherosclerosis. In support of this hypothesis, we have shown recently that lipolytic remnants of triglyceride-rich lipoproteins can be cytotoxic to cultured endothelial cells (40). It was postulated that triglyceride-rich lipoproteins are hydrolyzed in the proximity of the vascular endothelium by lipoprotein lipase. Thus endothelial cells may be directly exposed to elevated levels of free fatty acids (41). Selected free fatty acids cause disruption of endothelial barrier function, increase intracellular free calcium and induce oxidative stress, oxidative stress responsive transcription factors and genes (for review see [ 191). Although this hypothesis needs to be confirmed, it is possible that fatty acid-mediated alterations in endothelial cell metabolism may be a part of the pathological events induced by an imbalance of dietary methionine.
METHIONINE IMBALANCE AND EXPERIMENTAL ATHEROSCLEROSIS Up to date, only few reports have indicated that an excess of dietary methionine may contribute to the development of atherosclerosis. Mrhova et al. (42) reported that oral methionine administered to rats resulted in an increased number of circulating endothelial cells. Fau et al. (43) observed disturbances in arterial wall morphology in rats chronically fed a diet enriched with 2% methionine. Atherosclerotic changes in methionine-fed rabbits also were reported by McCully and Wilson (44). Conversely, feeding monkeys a methionine-enriched diet did not induce atherosclerosis (45).
1256
M. TOBOREK and B. HENNIG
Recently, we reported the development of a variety of atherosclerotic changes, such as intimal thickening, lipid deposition, and arterial wall calcification in rabbits fed a methionineenriched diet for 6 or 9 months (10). The mechanisms of these effects are not clear. However, they may be related to the induction of oxidative stress. In fact, in rabbits fed the methionineenriched diet, a significant increase in aortic lipid peroxidation was observed (10). The increase in aortic lipid peroxidation was accompanied by elevations in aortic antioxidant enzymes and a decrease in plasma antioxidant activity. Antioxidant activity reflects the potency of extracellular free radical scavengers, such as antioxidant vitamins. A decrease in these vitamins may promote oxidative stress and the development of atherosclerosis. To support this observation, a decrease in antioxidant activity was shown in patients with chronic renal failure, a disease which promotes accelerated atherosclerosis (46). Another mechanism which may promote the development of atherosclerosis in methionine-fed rabbits may be related to alterations in elastin metabolism. Fragmentation of elastic fibers and alterations in elastin synthesis were observed in rabbits fed a methionine-enriched diet (47). Although feeding animals methionine-enriched diets can promote the development of atherosclerosis, this effect may not be fully specific. Methionine toxicity is not limited to the vessel wall. For example, we reported the development of hepatitis in rabbits fed a methionineenriched diet. Methionine-related hepatotoxicity was reflected by inflammatory infiltration of portal triads. In addition, these morphological changes were associated with increased lipid peroxidation and alterations in antioxidant response in liver tissues (48).
METHIONINE IMBALANCE AND ENDOTHELIAL CELL DYSFUNCTION The mechanisms by which methionine may stimulate the development of atherosclerosis are not fully understood. One of the possibilities is the induction of injury to the endothelium. This hypothesis was supported by the observation that administration of methionine to rats resulted in elevated levels of endothelial cells in blood (42). In addition, we observed the disruption of endothelial barrier function in endothelial cells exposed to methionine (Figure 2). Disruption of endothelial cell integrity was further enhanced when endothelial cells were exposed to methionine plus a “physiological” free fatty acid mixture (FFA-M). FFA-M is a mixture of free fatty acids in concentrations similar to those found in serum under physiological conditions. It is possible that homocysteine is at least in part responsible for the atherogenic effects mediated by methionine. In fact, in methionine-fed rabbits we observed changes in the activities of enzymes which regulate the transsulfuration pathway (49). These enzyme alterations might promote accumulation of homocysteine. An increased serum homocysteine level recently has been recognized as a risk factor in atherosclerosis. The relationship between elevated plasma homocysteine levels and peripheral, cerebrovascular and coronary artery disease has been shown clearly in a number of clinical trials and experimental studies (for review see [50]). Moreover, homocysteine toxicity towards endothelial cells has been proposed as the main mechanism responsible for the atherogenic effect of this amino acid (51). A specific enzyme spectrum in endothelial cells may increase the susceptibility of these cells to toxic effects exerted by homocysteine. Endothelial cells express only two of the three crucial enzymes which participate in homocysteine metabolism in other tissues (Figure 1). Active enzymes in endothelial cells are cystathionine P-synthase and 5methyltetrahydrofolate:homocysteine methyltransferase. As it was mentioned previously, betaine:homocysteine methyltransferase is inactive in endothelial cells (3 1). Moreover, under pathologic conditions, such as atherosclerosis or vitamin B6 deficiency, activity of cystathionine P-synthase may be diminished which further contributes to the accumulation of homocysteine
DIETARY METHIONINE
AND ATHEROSCLEROSIS
1257
(52). Recently, mice deficient in cystathionine P-synthase has been generated by specific gene targeting in embryonic stem cells. Homozygous mutants completely lacking cystathionine psynthase activity had plasma homocysteine levels elevated approximately 40 times. However, heterozygous mutants which have approximately 50% of normal cystathionine P-synthase activity showed approximately 2 time increase in plasma homocysteine levels. These mutants may provide excellent experimental models to study metabolic effects of mild or severe homocysteinemia (53).
51
3
2
1
0 Lb
0 fl
without added FFA-M with added FFA-M
“r 1x1 I’,‘,’ ,‘,‘/’ ,‘/‘,’ ,‘/‘/’ ,‘#‘/’ ,‘I’#’ ,‘,‘I’ ,‘/‘I’ /‘/‘/’ I’,‘,’ I’,‘,’ ,‘,‘#’ I’,‘,’ I’,‘,’ ,‘XX\ #‘I’ Control
Methionine Concentration (PM) in cells treated wif*h increasing FIG. 2: Albumin transfer across endothelial monolayers Significantly concentrations of methionine and/or mixture of free fatty acids (FFA-M). higher than corresponding control group. t Significantly higher than values for samples without added FFA-M within each treatment group.
It is conceivable that endothelial integrity is greatly influenced by plasma concentrations of methionine and homocysteine. Methionine and homocysteine metabolism is controlled by the availability of vitamins such as riboflavin, vitamin B6, B12 and folic acid (Figure 1). Dietary deficiencies of any of these vitamins may impair activities of enzymes involved in methionine and homocysteine metabolism and promote accumulation of homocysteine. In fact, elevated homocysteine levels were found to be correlated with decreased levels of dietary and plasma Moreover, a negative correlation between plasma vitamin B6, B12 and folic acid levels. homocysteine and the ratio of dietary vitamin B6 to protein was observed in subjects at high risk for coronary heart disease (54). Plasma pyridoxal5’-phosphate, the active form of vitamin B6 was documented to be lower in atherosclerotic patients (55). Feeding pigs a diet deficient in vitamin B6 elevated homocysteine concentrations and induced atherosclerosis (56). Furthermore, simultaneous administration of riboflavin, vitamins B6, Bl2, folate, choline and troxerutin to myocardial infarct
1258
M. TOBOREK and B. HENNIG
patients caused significant decreases in plasma homocysteine cholesterol, triglycerides and apolipoprotein B concentrations (57).
levels
as well as in plasma
Several lines of evidence support the hypothesis that the atherogenic effect of homocysteine is mediated by injury to the endothelium. In baboons, homocysteine, when continuously infused as homocysteine thiolactone, caused desquamation of the vascular endothelium (58). Although this effect was not observed in pigs (59), and homocysteine thiolactone might have effects unrelated to homocysteine (60), the cytotoxicity of homocysteine was confirmed in a number of experiments (61,62). For example, exposure to homocysteine caused a concentration-dependent detachment of human umbilical artery or vein endothelial cells from polystyrene tissue culture dishes (62). Similar, in our experiments, homocysteine disrupted endothelial barrier function (Figure 3).
FIG. 3: Albumin transfer across endopelial monolayers in cells treated with increasing concentrations of homocysteine. Significantly higher than control group.
Homocysteine also interferes with coagulation. Homocysteine inhibits the expression thrombomodulin on the surface of endothelial cells. Thrombomodulin is an inhibitor procoagulant activity of thrombin. It is a cofactor for thrombin-modulated activation of protein and activated protein C inactivates coagulation cofactors Va and VIIIa. Consequently, inhibition of thrombomodulin diminishes the antithrombotic potential of endothelial cells (63).
of of C, the
In addition, homocysteine may significantly affect metabolism of vascular smooth muscle cells. For example, it was shown that homocysteine infusion into baboons induces myointimal proliferation (64). In in vitro studies homocysteine increased DNA synthesis and promoted proliferation of rat aortic smooth muscle cells. Homocysteine also elevated cyclin Dl and cyclin A
DIETARY METHIONINE
AND ATHEROSCLEROSIS
1259
which are important factors of the reentry of quiescent smooth muscle cells into the cell cycle (65). Thus, homocysteine-induced stimulation of smooth muscle cell proliferation may be another mechanism of atherogenic effect of this ammo acid. It appears that cellular oxidative stress may be a main mechanism of methionine, and homocysteine imbalance and autoxidation of compounds with a free thiol group may be the primary source of free radicals when methionine is administrated in excessive amounts. The initial and rate limiting reaction in thiol autoxidation is the electron transfer from the thiolate anion to a transition metal ion. As a result of the subsequent steps a wide spectrum of both oxygen and thiyl radical is generated (66). Due to their high reactivity thiyl radicals are capable of degrading and inactivating a variety of enzymes and other biological important compounds. They may also initiate lipid peroxidation (67). Moreover, thiol compounds were shown to oxidize LDL (6869). Although methionine itself does not undergo autoxidation, its metabolites, such as homocysteine or protein-bound thiols, are susceptible to this type of oxidative reaction (66,70). In fact, Starkebaum and Harlan (5 1) documented that the cytotoxicity of homocysteine to cultured endothelial cells was mediated by the formation of hydrogen peroxide during the autoxidation of homocysteine to homocystine. Homocysteine was not toxic to endothelial cells in the absence of copper and ceruloplasmin which catalyze the oxidation of homocysteine to the disultide form. Furthermore, the addition of catalase prevented homocysteine-induced injury to endothelial cells (51). To support the observation that increased oxidative stress may be a main mechanism of homocysteine-induced injury to endothelial cells, we observed an increase in fluorescence of 2’,7’dichlorofluorescein (DCF) (Figure 4), and Jones et al. (71) reported elevated thiobarbituric acid reactive substances (TBARS) levels in cells exposed to this amino acid. DCF fluorescence is a new cell imaging technique visualizing cellular oxidative stress in intact cells. This technique can quantitate primarily oxidative stress mediated by hydrogen peroxide (72). Thus our results further indicate the critical role of hydrogen peroxide in homocysteine-mediated endothelial cell dysfunction. However, hydrogen peroxide is a relatively weak oxidant and its toxicity may be significantly related to the generation of hydroxyl radicals, the most reactive free radical produced in biological systems. These radicals are generated as a result of reactions between hydrogen peroxide and superoxide anion radical in the presence of transition metal ions, such as iron or copper (67). The role of hydroxyl radical in homocysteine-induced toxicity was supported by the observation that desferal, a potent metal chelator, efficiently inhibited oxidative stress in cells exposed to this amino acid (71).
HYF’ERHOMOCYSTEINEMIA
AND ATHEROSCLEROSIS:
CLINICAL STUDIES
Numerous studies indicate that the prevalence of hyperhomocysteinemia in patients with coronary heart disease is between 20-40% (73-76). Hyperhomocysteinemia also is observed in a similar range of patients with premature peripheral and cerebral arterial occlusive disease or thromboembolism (77-80). Several pathologic conditions may induce hyperhomocysteinemia. Classic homocystinuria is caused by homozygosity for cystathionine P-synthase, and it is characterized by severe accumulation of homocysteine. It was estimated that homozygotes for cystathionine P-synthase have a 50% chance to develop vascular disease before the age of 30 years (81). Hyperhomocysteinemia also may be caused by other genetic defects. Plasma homocysteine may be moderately elevated (approximately 30-50% of normal range) due to heterozygosity for cystathionine P-synthase (82) or in the presence of a thermolabile variant of methylenetetrahydrofolate reductase (83). It was reported that this latter defect occurred in 17% of patients with coronary artery disease (84). In addition, nutritional deficiencies may lead to the
M. TOBOREK and B. HENNIG
1260
In fact, decreased levels of vitamin B 12, vitamin B6 and development of hyperhomocysteinemia. folic acid were demonstrated in apparently healthy men with hyperhomocysteinemia (83). Such a Vitamin therapy also is a common therapeutic approach to treatment of homocysteinemia. therapy usually starts with administration of vitamin B6 for up to 6 weeks. If vitamin B6 is ineffective in the treatment of hyperhomocysteinemia, a combined treatment with vitamin B6 and/or folic acid and/or betaine is introduced. This therapeutic approach was reported to be effective in more than 90% patients with hyperhomocysteinemia (86).
0
Control
Homocysteine
Treatment Groups FIG. 4: Cellular oxidative s$ess (DCF fluorescence) in cultured endothelial mM homocysteine. Significantly higher than control group.
cells treated with 5
CONCLUSION Atherosclerosis and related vascular disorders are the leading cause of death in the United States. Evidence strongly suggests that overconsumption of protein of animal origin can contribute to the development of this disease. The mechanisms of atherogenic effects of dietary animal proteins are not fully understood. However, as discussed above, high concentrations of methionine in animal proteins may be critical to their atherogenicity. Methionine may influence lipid composition and affect integrity of endothelial cells directly or indirectly through homocysteine metabolism. It appears that one of the main mechanisms of the atherogenic effects of dietary methionine is its ability to contribute to an increase in oxidative stress in vascular endothelial cells.
DIETARY METHIONINE AND ATHEROSCLEROSIS
1261
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Accepted
for
publication
March
30,
1996.