- Email: [email protected]
Effect of homocysteine and cholesterol in raising plasma homocysteine, cholesterol and triglyceride levels
ELSEVIER
PI1 SOO243205(98)00196-9
Life Sciences, Vol. 62, No. 24, pp. 2191-2194, 19913 f+yTi&ht 0 1998 lasevicr science Inc. Ptintcd in the USA. All ri&htsmewed m24-32osp $19.00 t 03
EFFECT OF HOMOCYSTEINE AND CHOLESTEROL IN RAISING PLASMA HOMOCYSTEINE, CHOLESTEROL AND TRIGLYCERIDE LEVELS Anthony Zulli, Brian Buxton, Laurie Doolan, James J Liu* Vascular Biology Unit, Departments of Cardiac Surgery and Medicine, University of Melbourne Austin Hospital, Heidelberg, VIC 3084, Australia (Received
in find
form March 20,19!48)
Summary A high plasma homocysteine level is a newly regarded risk factor for coronary artery disease. We report a synergistic effect of homocysteine plus cholesterol feeding on further raising total plasma homocysteine, cholesterol and triglycerides levels than each agent alone, which further enhances the risk of coronary artery disease. Key Wovcir: homocysteine,cholesterol, triglyceride,synergisticeffect, plasma levels
A high plasma homocysteine level, hyperhomocysteinemia, is a newly regarded risk factor for coronary artery disease (1). Homocysteine is formed by the demethylation of the essential amino acid methionine. Hyperhomocysteinemia in adults may be acquired by an excess dietary intake of methionine or a decreased intake of folate (2). Individuals with elevated plasma levels of either homocysteine or cholesterol are at increased risk of cardiovascular disease (3). Plasma cholesterol levels are generally normal in hyperhomocysteinemic individuals in clinical observations (4,5,6), however the immediate effects of cholesterol or methionine overload on their relative plasma levels remain uncertain. Therefore, considering methionine and cholesterol are usually consumed together, we hypothesize that combining these two risk factors may affect their relative plasma levels. In the present study, we investigated the effect of methionine feeding alone, cholesterol feeding alone, and both, on plasma cholesterol, homocysteine and triglyceride levels. The effects of methionine induced hyperhomocysteinemia has been previously well documented (7). In the present studies, normotensive rats were fed a control diet, a 2% cholesterol supplemented diet, a 2% methionine supplemented diet and a 2% cholesterol + 2% methionine supplemented diet. The results showed a significant increase in plasma homocysteine level in the cholesterol fed group, a marked increase in plasma homocysteine level in the methionine fed group, and striking increase in the cholesterol + methionine group, indicating a synergistic effect of cholesterol and methionine on raising plasma homocysteine levels. This synergism also raised plasma cholesterol levels and plasma triglyceride levels. These results indicate that homocysteine can act synergistically with cholesterol to further increase plasma homocysteine, cholesterol and triglyceride levels, factors known to promote atherosclerosis and coronary artery disease. *: Address for correspondence: Dr J.J.Liu, Vascular Biology Unit, Departments of Cardiac surgery and Medicine, University of Melbourne Austin Hospital, Heidelberg, VIC 3084, Australia. Telephone 61-3-9496-3652; Fax: 61-3-9459-0971. Email : [email protected]
2192
Homocysteine
and Cholesterol
Vol. 62, No. 24, 1998
Methods Twenty eight week old WKY rats were divided Into four groups of five rats. One group was fed a normal rat chow diet, the other group received a normal rat chow diet supplemented with 2% methionine (Sigma Chemical Co, St Louis). This concentration of methionine has been previously shown to elicit medial hypertrophy and medial fibrosis in the rat aortic wall (8). Another group received a normal rat chow diet supplemented with 2?;0 cholesterol (Sigma Chemical Co, St Louis) and the other group received a normal rat chow diet supplemented with 2% methionine + 2% cholesterol (Sigma Chemical Co, St Louis). After dieting for fifteen weeks, rats were decapitated and blood collected into heparinized tubes Cholesterol and triglyceride was measured using standard biochemical methods and homocysteine via HPLC Sfatisfrcul a~~a~xs Statistical significance was determined by an unpaired Student’s t-test for two group comparison and a one-way analysis of variance for multiple group comparison with Significance was taken at PO 05. Synergy was acknowledged if meansfSEM ((methioninei-cholesterol group ~ control group) > [(methionine alone group - control group) + (cholesterol alone group - control group)] ) Results At the time of sacrifice, control rats had a plasma homocysteine level of 3.8 + 0.7 umol/L. After cholesterol feeding, plasma homocysteine level was raised to 5.6 & 0.4umoVL. Methionine feeding increased plasma homocysteine level to 84 5 -ir 2 I 8 pmol/I+ in addition methionine + cholesterol feeding further increased plasma homocysteine level to 141.4 f 27.6 ).rmol/L (Table I). Synergy was acknowledged since 137 6 > (80 8 + I 7) (See methods section for calculation).
TABLE I
a p
2%
methionine, respectively
Plasma cholesterol levels were also measured. The plasma cholesterol level of the control group was 1.8 k 0.2 mmol/L, the plasma cholesterol level of the cholesterol group significantly increased to 3.3 k 0.1 mmol/L, the plasma cholesterol level of the methionine group was 1.9 + 0.1 mmol/L and the plasma cholesterol level of the cholesterol + methionine group increased to 4.2 + 0.3 mmol/L (Table II). Synergy was acknowledged since 2.4 > (1.5 + 0.12) (See methods section for calculation)
Vd 62, No. 24.19%
Homocysteine and Cholesterol
2193
TABLE II Plasma Cholesterol Levels Accordin
Z%Methionine
+ 2% Cholesterol
b p (0 + 0) (See methods section for calculation). TABLE ZII
Z%Methionine
+ 2% Cholesterol
c pcO.05 compared to control, 2% cholesterol and 2% methionine, respectively Discussion Homocysteine is an independent risk factor for coronary artery disease (3). Increased circulating levels of homocysteine is known to inhibit endothelial dependant vasodilatation (6), stimulate platelet aggregation (9) and smooth muscle cell proliferation (lo), damage endothelial cells (I 1) and inhibit its regeneration (lo), suppress prostacyclin release (12), promote thrombogenesis (13) and oxidize lipoproteins (14). These are known events involved in the initiation of vascular disease. In humans, hyperhomocysteinemia can arise from different causes. The most dramatic but least common is an enzyme abnormality involved in the metabolic pathway, which leads to vascular lesions at a young age (15). The more common, but least severe, is a genetic alteration in enzymes involved in homocysteine metabolism (16), which has a frequency of 30% in the population (17) or from inadequate dietary folate intake (18). Excess methionine and cholesterol feeding act synergistically to increase plasma homocysteine, cholesterol and triglyceride levels, to much higher levels than feeding cholesterol or methionine alone, This underlines the importance of diet in the outcome of hyperhomocysteinemia - excess intake of animal products which are high in methionine coupled with an excess intake of cholesterol could markedly increase plasma homocysteine levels up to the 95th percentile in humans, a status known to increase the risk of myocardial infarction three fold (5).
2194
Homoeysteine and Cholesterol
Vol. 62, No. 24,19!%
Elevated serum cholesterol is an accepted independent risk factor for coronary artery disease, We report a synergistic effect of cholesterol and methionine in raising plasma cholesterol levels compared to cholesterol alone. These results further verify homocysteine as a risk factor for cardiovascular disease. Plasma triglyceride is also a risk factor for coronary artery disease, even after making allowances for high-density lipoprotein (HDL) (19). We have shown a 25% increase in plasma triglyceride levels in the methionine + cholesterol group compared to control, cholesterol alone and methionine alone groups. This increase in triglyceride level may further increase the risk of coronary artery disease. In conclusion these studies suggest a multi factorial effect of excess dietary methionine and cholesterol on their serum concentration, compared to either agent per se. The measured effects include an increase in plasma cholesterol, homocysteine and triglycerides, factors known to increase the risk of coronary artery disease. References
1, S-S. KANG, P.W. WONG and M.R. MALINOW. Annu. Rev. Nutr. 12 279-98 (1992) 2. J.B. UBBINK, W.J. HAYWARD VERMAAK, A.V.D. MERWE and P.J. BECKER. Am. J. Clin. Nutr. 55 1154-l 160 (1992) 3. P.M.UELAND, HREFSUM and L. BRATTSTROM, Plasma Homocysteine and Cardiovascular Disease, DB Francis Jr, (Eds), 183-236, Marcel Dekker, New York (1992) 4. I.J. PERRY, H. REFSUM, R.W. MORRIS, S.B. EBRAHIM, P.M. UELAND and A.G. SHAPER. Lancet. 346 13951398 (1995) 5. M.J. STAMPFER, M.R. MANILOW, W.C. WILLWT, L.M. NEWCOMER, B. UPSON, D. ULLMAN, P.V. TISHLER and C.H. HENNEKENS. J.A.M.A. 268 877-881 (1992) 6. A TAWAKOL, T. OMLAND, M. GERHARD, J.T. WU and M.A. CREAGER. Circulation, 95 1119-1121 (1997) 7. D. MATTHIAS, C.H. BECKER, R. RIEZLER and P.H. KINDLING. Atherosclerosis. 122 201-216 (1996) 8. M.J. OSBORNE-PELLEGRIN and D. FAU. Exp. Mol. Pathol. 56 49-59 (1992) 9. G. DIMINNO, G. DAVI, M. MARGAGLIONE, F. CIRILLO, E. GRANDONE, G. CIABATTONI, I. CATALANO, P. STRISCIUGLIO, G. ANDRIA, C. PATRON0 and M. MANCINI. J. Clin. Invest. 92 1400-1406 (1993) 10. J.C.TSAI, M.A. PERRELLA, M. YOSI-IIZUMI, C-M. HSIEH, E. HABER, R. SCHLEGEL and M-E. LEE. Proc. Natl. Acad. Sci. USA 91 6369-6373 (1994) 11. G. STARKEBAUM and J.M. HARLAN. J. Clin. Invest. 77 1370-1376 (1986) 12. R.V. PANGALAMALA P.W. CARPEN and A.J. MEROLA. Prost. Leuk. Med. 22 349-356 (1986) 13. M.M. REES and G.M. RODGERS. Thromb. Res. 71 337-359 (1993) 14. S. PARTHASARATHY. Biochim. Biophys. Acta. 917 337-340 (1987) 15. S.H. MUD, LEVY H.L, SKOVBY F, Disorders of Transulfuration, C.S. Striver, A.L. Beaudet, W.L. Sly and D. Valle, (Eds), 6th ed, 693-734, McGraw-Hill International Book Co, New York (1989). 16. S-S.KANG, E.L. PASSEN, N. RUGGIE, P.W.K. WONG and H. SORA. Circulation. 88 1463-1469 (1993) 17. M.J. STAMPFER and R.M. MALINOW. N. Engl. J. Med. 332 328-329 (1995) 18. P. DURAND, L.J. FORTIN, S. LUSSIER-CACAN, J. DAVIGNON, and BLACHE. Clin. Chim. Acta. 252 83-93 (1996) 19. W.P. CASTELLI. Atherosclerosis 124 Supp Sl-S9 (1996)
PI1 SOO243205(98)00196-9
Life Sciences, Vol. 62, No. 24, pp. 2191-2194, 19913 f+yTi&ht 0 1998 lasevicr science Inc. Ptintcd in the USA. All ri&htsmewed m24-32osp $19.00 t 03
EFFECT OF HOMOCYSTEINE AND CHOLESTEROL IN RAISING PLASMA HOMOCYSTEINE, CHOLESTEROL AND TRIGLYCERIDE LEVELS Anthony Zulli, Brian Buxton, Laurie Doolan, James J Liu* Vascular Biology Unit, Departments of Cardiac Surgery and Medicine, University of Melbourne Austin Hospital, Heidelberg, VIC 3084, Australia (Received
in find
form March 20,19!48)
Summary A high plasma homocysteine level is a newly regarded risk factor for coronary artery disease. We report a synergistic effect of homocysteine plus cholesterol feeding on further raising total plasma homocysteine, cholesterol and triglycerides levels than each agent alone, which further enhances the risk of coronary artery disease. Key Wovcir: homocysteine,cholesterol, triglyceride,synergisticeffect, plasma levels
A high plasma homocysteine level, hyperhomocysteinemia, is a newly regarded risk factor for coronary artery disease (1). Homocysteine is formed by the demethylation of the essential amino acid methionine. Hyperhomocysteinemia in adults may be acquired by an excess dietary intake of methionine or a decreased intake of folate (2). Individuals with elevated plasma levels of either homocysteine or cholesterol are at increased risk of cardiovascular disease (3). Plasma cholesterol levels are generally normal in hyperhomocysteinemic individuals in clinical observations (4,5,6), however the immediate effects of cholesterol or methionine overload on their relative plasma levels remain uncertain. Therefore, considering methionine and cholesterol are usually consumed together, we hypothesize that combining these two risk factors may affect their relative plasma levels. In the present study, we investigated the effect of methionine feeding alone, cholesterol feeding alone, and both, on plasma cholesterol, homocysteine and triglyceride levels. The effects of methionine induced hyperhomocysteinemia has been previously well documented (7). In the present studies, normotensive rats were fed a control diet, a 2% cholesterol supplemented diet, a 2% methionine supplemented diet and a 2% cholesterol + 2% methionine supplemented diet. The results showed a significant increase in plasma homocysteine level in the cholesterol fed group, a marked increase in plasma homocysteine level in the methionine fed group, and striking increase in the cholesterol + methionine group, indicating a synergistic effect of cholesterol and methionine on raising plasma homocysteine levels. This synergism also raised plasma cholesterol levels and plasma triglyceride levels. These results indicate that homocysteine can act synergistically with cholesterol to further increase plasma homocysteine, cholesterol and triglyceride levels, factors known to promote atherosclerosis and coronary artery disease. *: Address for correspondence: Dr J.J.Liu, Vascular Biology Unit, Departments of Cardiac surgery and Medicine, University of Melbourne Austin Hospital, Heidelberg, VIC 3084, Australia. Telephone 61-3-9496-3652; Fax: 61-3-9459-0971. Email : [email protected]
2192
Homocysteine
and Cholesterol
Vol. 62, No. 24, 1998
Methods Twenty eight week old WKY rats were divided Into four groups of five rats. One group was fed a normal rat chow diet, the other group received a normal rat chow diet supplemented with 2% methionine (Sigma Chemical Co, St Louis). This concentration of methionine has been previously shown to elicit medial hypertrophy and medial fibrosis in the rat aortic wall (8). Another group received a normal rat chow diet supplemented with 2?;0 cholesterol (Sigma Chemical Co, St Louis) and the other group received a normal rat chow diet supplemented with 2% methionine + 2% cholesterol (Sigma Chemical Co, St Louis). After dieting for fifteen weeks, rats were decapitated and blood collected into heparinized tubes Cholesterol and triglyceride was measured using standard biochemical methods and homocysteine via HPLC Sfatisfrcul a~~a~xs Statistical significance was determined by an unpaired Student’s t-test for two group comparison and a one-way analysis of variance for multiple group comparison with Significance was taken at PO 05. Synergy was acknowledged if meansfSEM ((methioninei-cholesterol group ~ control group) > [(methionine alone group - control group) + (cholesterol alone group - control group)] ) Results At the time of sacrifice, control rats had a plasma homocysteine level of 3.8 + 0.7 umol/L. After cholesterol feeding, plasma homocysteine level was raised to 5.6 & 0.4umoVL. Methionine feeding increased plasma homocysteine level to 84 5 -ir 2 I 8 pmol/I+ in addition methionine + cholesterol feeding further increased plasma homocysteine level to 141.4 f 27.6 ).rmol/L (Table I). Synergy was acknowledged since 137 6 > (80 8 + I 7) (See methods section for calculation).
TABLE I
a p
2%
methionine, respectively
Plasma cholesterol levels were also measured. The plasma cholesterol level of the control group was 1.8 k 0.2 mmol/L, the plasma cholesterol level of the cholesterol group significantly increased to 3.3 k 0.1 mmol/L, the plasma cholesterol level of the methionine group was 1.9 + 0.1 mmol/L and the plasma cholesterol level of the cholesterol + methionine group increased to 4.2 + 0.3 mmol/L (Table II). Synergy was acknowledged since 2.4 > (1.5 + 0.12) (See methods section for calculation)
Vd 62, No. 24.19%
Homocysteine and Cholesterol
2193
TABLE II Plasma Cholesterol Levels Accordin
Z%Methionine
+ 2% Cholesterol
b p
Z%Methionine
+ 2% Cholesterol
c pcO.05 compared to control, 2% cholesterol and 2% methionine, respectively Discussion Homocysteine is an independent risk factor for coronary artery disease (3). Increased circulating levels of homocysteine is known to inhibit endothelial dependant vasodilatation (6), stimulate platelet aggregation (9) and smooth muscle cell proliferation (lo), damage endothelial cells (I 1) and inhibit its regeneration (lo), suppress prostacyclin release (12), promote thrombogenesis (13) and oxidize lipoproteins (14). These are known events involved in the initiation of vascular disease. In humans, hyperhomocysteinemia can arise from different causes. The most dramatic but least common is an enzyme abnormality involved in the metabolic pathway, which leads to vascular lesions at a young age (15). The more common, but least severe, is a genetic alteration in enzymes involved in homocysteine metabolism (16), which has a frequency of 30% in the population (17) or from inadequate dietary folate intake (18). Excess methionine and cholesterol feeding act synergistically to increase plasma homocysteine, cholesterol and triglyceride levels, to much higher levels than feeding cholesterol or methionine alone, This underlines the importance of diet in the outcome of hyperhomocysteinemia - excess intake of animal products which are high in methionine coupled with an excess intake of cholesterol could markedly increase plasma homocysteine levels up to the 95th percentile in humans, a status known to increase the risk of myocardial infarction three fold (5).
2194
Homoeysteine and Cholesterol
Vol. 62, No. 24,19!%
Elevated serum cholesterol is an accepted independent risk factor for coronary artery disease, We report a synergistic effect of cholesterol and methionine in raising plasma cholesterol levels compared to cholesterol alone. These results further verify homocysteine as a risk factor for cardiovascular disease. Plasma triglyceride is also a risk factor for coronary artery disease, even after making allowances for high-density lipoprotein (HDL) (19). We have shown a 25% increase in plasma triglyceride levels in the methionine + cholesterol group compared to control, cholesterol alone and methionine alone groups. This increase in triglyceride level may further increase the risk of coronary artery disease. In conclusion these studies suggest a multi factorial effect of excess dietary methionine and cholesterol on their serum concentration, compared to either agent per se. The measured effects include an increase in plasma cholesterol, homocysteine and triglycerides, factors known to increase the risk of coronary artery disease. References
1, S-S. KANG, P.W. WONG and M.R. MALINOW. Annu. Rev. Nutr. 12 279-98 (1992) 2. J.B. UBBINK, W.J. HAYWARD VERMAAK, A.V.D. MERWE and P.J. BECKER. Am. J. Clin. Nutr. 55 1154-l 160 (1992) 3. P.M.UELAND, HREFSUM and L. BRATTSTROM, Plasma Homocysteine and Cardiovascular Disease, DB Francis Jr, (Eds), 183-236, Marcel Dekker, New York (1992) 4. I.J. PERRY, H. REFSUM, R.W. MORRIS, S.B. EBRAHIM, P.M. UELAND and A.G. SHAPER. Lancet. 346 13951398 (1995) 5. M.J. STAMPFER, M.R. MANILOW, W.C. WILLWT, L.M. NEWCOMER, B. UPSON, D. ULLMAN, P.V. TISHLER and C.H. HENNEKENS. J.A.M.A. 268 877-881 (1992) 6. A TAWAKOL, T. OMLAND, M. GERHARD, J.T. WU and M.A. CREAGER. Circulation, 95 1119-1121 (1997) 7. D. MATTHIAS, C.H. BECKER, R. RIEZLER and P.H. KINDLING. Atherosclerosis. 122 201-216 (1996) 8. M.J. OSBORNE-PELLEGRIN and D. FAU. Exp. Mol. Pathol. 56 49-59 (1992) 9. G. DIMINNO, G. DAVI, M. MARGAGLIONE, F. CIRILLO, E. GRANDONE, G. CIABATTONI, I. CATALANO, P. STRISCIUGLIO, G. ANDRIA, C. PATRON0 and M. MANCINI. J. Clin. Invest. 92 1400-1406 (1993) 10. J.C.TSAI, M.A. PERRELLA, M. YOSI-IIZUMI, C-M. HSIEH, E. HABER, R. SCHLEGEL and M-E. LEE. Proc. Natl. Acad. Sci. USA 91 6369-6373 (1994) 11. G. STARKEBAUM and J.M. HARLAN. J. Clin. Invest. 77 1370-1376 (1986) 12. R.V. PANGALAMALA P.W. CARPEN and A.J. MEROLA. Prost. Leuk. Med. 22 349-356 (1986) 13. M.M. REES and G.M. RODGERS. Thromb. Res. 71 337-359 (1993) 14. S. PARTHASARATHY. Biochim. Biophys. Acta. 917 337-340 (1987) 15. S.H. MUD, LEVY H.L, SKOVBY F, Disorders of Transulfuration, C.S. Striver, A.L. Beaudet, W.L. Sly and D. Valle, (Eds), 6th ed, 693-734, McGraw-Hill International Book Co, New York (1989). 16. S-S.KANG, E.L. PASSEN, N. RUGGIE, P.W.K. WONG and H. SORA. Circulation. 88 1463-1469 (1993) 17. M.J. STAMPFER and R.M. MALINOW. N. Engl. J. Med. 332 328-329 (1995) 18. P. DURAND, L.J. FORTIN, S. LUSSIER-CACAN, J. DAVIGNON, and BLACHE. Clin. Chim. Acta. 252 83-93 (1996) 19. W.P. CASTELLI. Atherosclerosis 124 Supp Sl-S9 (1996)