- Email: [email protected]
The influence of simvastatin on lipase and cholesterol esterase activity in the serum of men with coronary heart disease
Pharmacological Research, Vol. 43, No. 4, 2001 doi:10.1006/phrs.2000.0787, available online at http://www.idealibrary.com on
THE INFLUENCE OF SIMVASTATIN ON LIPASE AND CHOLESTEROL ESTERASE ACTIVITY IN THE SERUM OF MEN WITH CORONARY HEART DISEASE ∗ and ANNA PIORUNSKA-MIKOŁAJCZAK ´ ´ MARIA PIORUNSKA-STOLZMANN
Chair and Department of Chemistry and Clinical Biochemistry, Karol Marcinkowski University of Medical Sciences, Grunwaldzka 6, 60-780 Poznan, Poland Accepted 28 November 2000
Several studies have demonstrated that any beneficial effects of 3-hydroxy-3-methylglutarylcoenzyme A (HMG-CoA) reductase inhibitors (statins), of which simvastatin (Merck Sharp & Dohme) is an example, on coronary events are linked to their hypocholesterolemic properties. The in vivo effects of simvastatin treatment on lipase (GEH = glycerol ester hydrolase) and cholesterol esterase (CEase) activity in the serum of men with coronary heart disease (CHD) were examined. GEH and CEase activity in the serum of men with CHD, before simvastatin treatment, was lower than in the control subjects. In our study we have provided evidence that simvastatin increases GEH activity in a time-dependent manner, but has no effect on CEase activity. This suggests that simvastatin can directly affect acylglycerol metabolism by an increase in GEH activity and may therefore be suitable for the treatment of combined lipoprotein disorders characterized by elevation c 2001 Academic Press of triacylglycerols.
K EY WORDS : lipase, cholesterol esterase, simvastatin, serum, CHD men.
INTRODUCTION Coronary heart disease (CHD) remains the leading cause of death in most developed countries. Clinical and experimental investigations [1, 2] have clearly shown that a reduction in plasma cholesterol, particularly in low-density lipoprotein cholesterol (LDL-C), reduces the risk of cardiovascular events in both primary and secondary prevention studies. A number of cholesterol-lowering drugs are currently available for use in humans and of these, statins are the most commonly prescribed agents for the treatment of hypercholesterolemia, because of their efficacy in reducing LDL and their excellent tolerability and safety [3–5]. Different LDL particles are related to differences in the development of coronary artery disease [6, 7]. The LDL phenotype may be controlled by lipolytic enzymes that can alter the lipid composition of lipoproteins [8, 9]. Variations in hepatic lipase (HL) and lipoprotein lipase (LPL) activity have been shown to be related to changes in LDL size and pattern [10]. There is general agreement that the presence of small, dense LDL (pattern B) is associated with an increased risk of CHD due to their physiological properties, such as their high susceptibility to oxidation and low affinity to the LDL receptor, in comparison to pattern A particles [7, 8]. It ∗ Corresponding author. E-mail: [email protected]
1043–6618/01/040359–04/$35.00/0
is known that triacylglycerols (TG) constitute the major determinant of the plasma LDL, and the replacement of cholesteryl esters by triacylglycerols, followed by TG hydrolysis, is a two-step procedure leading to the size reduction of LDL particles [11, 12]. Lipase [glycerol ester hydrolase (GEH), EC 3.1.1.3] hydrolyses triacylglycerides in triglyceride-rich lipoproteins leading to the formation of modified particles that are depleted of triglycerides [13]. Most of the cholesterol in plasma lipoproteins is in the form of cholesteryl esters (CE), which undergo a continual cycle of hydrolysis and re-esterification by the action of cholesterol esterase (CEase, EC 3.1.1.13) and acylcoenzyme A to cholesterol acyltransferase (ACAT, EC 2.3.1.1.26), respectively [14]. Therefore, the ability of CEase to convert large LDL to smaller LDL subspecies, and the relationship between plasma CEase and LDL levels, suggest that high plasma CEase levels may constitute a potential risk factor for atherosclerosis [15]. Although the safety and efficacy of long-term treatment with simvastatin has been demonstrated, the impact of 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase inhibitors on enzymes affecting lipoprotein metabolism in vivo still needs to be fully elucidated. The present study was undertaken to investigate the effect of simvastatin treatment on the GEH and CEase activity in the serum of men with CHD. c 2001 Academic Press
360
Pharmacological Research, Vol. 43, No. 4, 2001 Table I Serum lipid profile of control subjects and of men with coronary heart disease before and after 3 months simvastatin treatment T-C
TG
LDL-C
HDL-C
LDL-C/ HDL-C
mmol l−1 Control n = 10 CHD n = 15
before after
5.0 ± 0.8
1.6 ± 0.5
2.9 ± 0.2
1.6 ± 0.2
2.2 ± 0.7
7.1 ± 0.3a 5.7 ± 0.2b
2.2 ± 0.5a 1.7 ± 0.2b
4.2 ± 0.2a 3.1 ± 0.2b
1.2 ± 0.3a 1.3 ± 0.1
3.7 ± 1.0a 2.8 ± 0.8b
n = number of subjects. The data are mean ± SD. Significant differences: a between the CHD and control groups; and b between the CHD group before and after simvastatin treatment; P ≤ 0.05.
MATERIALS AND METHODS The patients included in this study were 15 men, aged 44–62 years, with a diagnosis of coronary heart disease. None had had any cardiovascular episodes. All the patients were given 10 mg simvastatin (Merck, Sharp & Dohme) orally, once daily over a period of 3 months. All the patients had given their written informed consent. Patients with any known abnormalities of carbohydrate metabolism were excluded from this study. The control group consisted of ten healthy male blood donors. Blood samples were taken from all the subjects after an overnight fast. Serum was separated by centrifugation and used in the assays of enzyme activity. The following concentrations in serum were determined: total-, HDL- and LDL-cholesterol (T-C, HDL-C and LDL-C, respectively) and TG, using the appropriate enzyme kits (Boehringer Mannheim Biochimica). The blood sera of both the controls and the men with CHD were processed in order to obtain acetone-butanol powders by the method described in detail previously [16]. The powders were extracted with water and then spun using a Dacie electric cell suspension mixer, at 4 ◦ C for 10 min. The extracts were filtered, kept at 4 ◦ C and used for the enzyme activity assays. Protein content was determined by the method of Lowry et al. [17]. Fresh hydrosols of trioleylglycerol and oleylcholesterol were prepared for each experiment [16]. The levels of CEase and GEH activity were determined under optimal experimental conditions for each enzyme, namely pH 8.6 and pH 8.0, respectively [18, 19]. Enzyme catalytic activity was determined by means of a two-point method based on measuring changes in the concentration of free fatty acids released and extracted from the reaction mixture both before and 20 min after incubation. The results are expressed in nanomoles of fatty acid released × min−1 (mg protein)−1 = mU mg−1 of the enzyme preparations. Statistical analysis of the results (means, standard deviations) was carried out using INSTAT (Sigma) software. The means between groups were compared by Student’s t-test or by the Cochran–Cox test. Statistical significance
Table II Serum GEH and CEase activity of control subjects and of men with CHD before and after 3 months simvastatin treatment Lipase (GEH)
Cholesterol esterase (CEase)
mU mg−1 Control n = 10 CHD n = 15
before after
8.7 ± 1.0
7.9 ± 0.9c
7.1 ± 1.2a 12.1 ± 0.8b
7.0 ± 0.8a 7.5 ± 0.7c
The data are mean ± SD. Significant differences: a between the CHD and control groups; b between CHD group before and after simvastatin treatment; and c between GEH and CEase activity within the groups; P ≤ 0.05.
was set at the level of P < 0.05. This study was approved by the Bioethics Committee of the University of Medical Sciences in Pozna´n, Poland.
RESULTS The serum lipid profiles of the patients are summarized in Table I. Significant differences between groups were observed in the T-C, TG, LDL-C, HDL-C and LDLC/HDL-C ratios. Decreases were observed in the T-C, TG levels, LDL-C and LDL-C/HDL-C ratio in patients with CHD following simvastatin treatment. Serum GEH and CEase activity in the controls and in the men with CHD, before and after 3 months treatment with simvastatin, are presented and compared in Table II. There were differences in the mean activities between GEH and CEase in the control group but not in the CHD group before simvastatin treatment. However, either GEH or CEase activity was reduced in the CHD group compared to the controls. GEH activity in the serum of men with CHD increased significantly throughout the entire period of treatment with simvastatin (Fig. 1). Catalytic CEase activity, as studied in the serum of men with CHD, was not affected by simvastatin and did not significantly differ from that found in their serum before treatment (Table II).
Pharmacological Research, Vol. 43, No. 4, 2001
361
14
### &
GEH (mU mg− 1)
12 10 8 6 4 2 0
Before
1 Month
2 Months Simvastatin treatment
3 Months
Fig. 1. Effect of simvastatin treatment on serum GEH activity in men with coronary heart disease. Mean ± SD. ∗∗∗ —statistically significant difference vs ‘Before’, P < 0.001. ###, &—statistical difference vs ‘1 Month’ (P < 0.001) or ‘2 Months’ (P < 0.05), respectively.
DISCUSSION The present study showed that patients with established CHD who are on statin therapy have significant changes in their serum GEH activity. To our knowledge, this simvastatin effect has not been described previously in humans and such an effect on GEH activity is apparently unique for this class of lipid-lowering medication. This finding supports the idea that some of the beneficial effects of HMG-CoA reductase inhibitors may be mediated via a mechanism other than cholesterol reduction alone [20–22]. Although the liver is the target organ for the action of statins, our study provides evidence that serum enzymes may also be affected. It has been shown in a rat model that after simvastatin administration, there is increased lipoprotein lipase mRNA and an increase in lipase lipoprotein activity in adipose tissue and heart [23]. Statins are thought to be ineffective in directly modifying the size and density of LDL [1], but in our study we found that an increase in serum GEH activity can affect lipoprotein metabolism. Moreover, statin therapy in moderate hypercholesterolemia is associated with changes throughout the apoB-containing lipoprotein, which has been observed by other authors [24]. When lipolytic activity is high, the circulating levels of large TG-rich lipoproteins remain low. On the other hand, if the enzyme activity is low, the TG-rich lipoprotein remains the major species in plasma. The mechanism by which simvastatin may increase GEH activity is not yet known, but we speculate that this phenomenon might also be related to the influence of simvastatin on acylenzyme formation. Simvastatin displays close pharmacokinetic parameters with other statins [25], but the precise mechanism of action of these compounds, however, has not been fully elucidated. It cannot be excluded that, due to the presence of a free electron pair on the oxygen atom in the structure of simvastatin, it may influence an enzyme–substrate interaction by nucleophile interference. Migration and proliferation of arterial myocytes, together with the deposition of lipids, mainly cholesteryl esters, in the arterial wall are the key events in the athero-
genic cascade [12, 26]. Some authors have postulated that plasma CEase activity contributes to the formation and accumulation of atherogenic lipoproteins and a positive correlation has been observed between CEase activity and plasma LDL levels [15]. In our study we did not observe these changes in CEase activity after simvastatin treatment, and suggest that changes may not occur due to inhibition of statin ACAT activity, as proposed by other authors [27]. Statins inhibit cholesteryl ester accumulation in monocyte-derived macrophages either by reducing the availability of free cholesterol to the enzyme acyl-coenzyme A cholesterol acyltransferase by trapping it in phospholipid-containing pools, or by inhibiting LDL endocytosis related to reduced synthesis of the mevalonate or mevalonate byproducts required for cholesterol esterification [20]. It has also been shown that simvastatin inhibits CE accumulation from aggregated LDL [28]. However, while there is a growing body of evidence from case controlled and prospective studies to suggest that plasma triacylglycerols are an independent risk factor for the development of CHD, the risk associated with elevations in plasma TG still remains controversial. Experiments that might help in solving these problems are worth undertaking. Taken together, these studies suggest that simvastatin can directly affect acylglycerol metabolism by an increase in GEH activity and may therefore be suitable for the treatment of combined lipoprotein disorders characterized by elevation of triacylglycerols.
REFERENCES 1. Maron DJ, Fazio S, Linton MF. Current perspectives on statins. Circulation 2000; 101: 207–13. 2. Auer J, Eber B. Current aspects of statins. J Clin Basic Cardiol 1999; 2: 203–8. 3. Pedersen T et al. Safety and tolerability of cholesterol lowering with simvastatin during 5 years in the Scandinavian Simvastatin Survival Study. Arch Intern Med 1996; 156: 2085–92.
362 4. Stein E, Lane M, Laskarzewski P. Comparison of statins in hypertrigliceridemia. Am J Cardiol 1998; 81(4A): 66B–9B. 5. Jones P, Kafonek S, Laurora I, Hunninghake D. Comparative dose efficacy of atorvastatin versus simvastatin, pravastatin, lovastatin, and fluvastatin in patients with hypercholesterolemia. Am J Cardiol 1998; 81: 582–7. 6. Rainwater D. Lipoprotein correlates of LDL particle size. Atherosclerosis 2000; 148: 151–8. 7. Byrne Ch. Triglyceride-rich lipoproteins: are links with atherosclerosis mediated by a procoagulant and proinflammatory phenotype? Atherosclerosis 1999; 145: 1–15. 8. Grundy SM, Vega GL. Two different views of the relationship of hypertriglyceridemia to coronary heart disease. Arch Intern Med 1992; 152: 28–34. 9. Grundy SM. Hypertriglyceridemia, atherogenic dyslipidemia, and the metabolic syndrome. Am J Cardiol 1998; 81(4A): 18B–25B. 10. Tan CE, Forster MJ, Caslake MJ, Bedford D, Watson TD K, McConnell M, Packard CJ, Shepherd J. Relations between plasma lipids and postheparin plasma lipases and VLDL and LDL subfraction patterns in normolipidemic men and women. Arterioscler Thromb Vasc Biol 1995; 15: 1839–48. 11. Nilsson-Ehle P. Lipolytic enzymes and plasma lipoprotein metabolism. Annu Rev Biochem 1980; 49: 667–93. 12. Brown M, Goldstein J. Lipoprotein metabolism in the macrophage. Implications for cholesterol deposition in atherosclerosis. Annu Rev Biochem 1983; 52: 223–61. 13. Aviram M, Bierman E, Chait A. Modification of low density lipoprotein by lipoprotein lipase or hepatic lipase induces enhanced uptake and cholesterol accumulation in cells. J Biol Chem 1988; 263: 15416–22. 14. Miura S, Chiba T, Mochizuki N, Nagura H, Nemoto K, Tomita I, Ikeda M, Tomita T. Cholesterol-mediated changes of neutral cholesterol esterase activity in macrophages. Mechanism for mobilization of cholesteryl esters in lipid droplets by HDL. Arterioscler Thromb Vasc Biol 1997; 17: 3033–40. 15. Brodt-Eppley J, White P, Jenkins S, Hui D. Plasma cholesterol esterase level is a determinant for an atherogenic lipoprotein profile in normolipidemic human subjects. Biochim Biophys Acta 1995; 1272: 69–72. 16. Patelski J, Pniewska B, Pioru´nska M, Obr ebska M. The arterial acyl-CoA cholesterol ester hydrolase activities. In vitro effect of substrates with fatty acids of different chain length and saturation. Atherosclerosis 1975; 22: 287–91.
Pharmacological Research, Vol. 43, No. 4, 2001 17. Lowry OH, Rosebrough NJ, Farr AL, Randall RJ. Protein measurement with the folin phenol reagent. J Biol Chem 1951; 193: 263–75. 18. Patelski J, Pioru´nska-Mikołajczak A, Pioru´nska-Stolzmann M. Effect of substrate composition and concentration on aortic cholesterol ester hydrolase activity. Enzyme 1978; 23: 135–9. 19. Pioru´nska-Stolzmann M. The substrate specificity of glycerol ester hydrolase from pig aorta and serum. Acta Biochim Pol 1990; 37: 457–62. 20. Rosenson R, Tangney C. Antiatherothrombotic properties of statins. Implications for cardiovascular event reduction. JAMA 1998; 279: 1643–50. 21. Lagrost L et al. Plasma lipoprotein distribution and lipid transfer activities in patients with type IIb hyperlipidemia treated with simvastatin. Atherosclerosis 1999; 143: 415–25. 22. Corsini A, Pazzucconi F, Arnaboldi L, Pfister P, Fumagalli R, Paoletti R, Sirtori C. Direct effects of statins on the vascular wall. J Cardiovasc Pharm 1998; 31: 773–8. 23. Schoonjans K, Peinado-Onsurbe J, Fruchart J, Tailleux A, Fievet C, Auwerx J. HMG-CoA reductase inhibitors reduce serum triglyceride levels through modulation of apolipoprotein C-III and lipoprotein lipase. FEBS Lett 1999; 452: 160–4. 24. Gaw A, Packard Ch, Murray E, Lindsay G, Griffin B, Caslake M, Vallance B, Lorimer A, Shepherd J. Effects of simvastatin on apoB metabolism and LDL subfraction distribution. Arterioscler Thromb 1993; 13: 170–89. 25. Lennernas H, Fager G. Pharmacodynamic and pharmacokinetics of the HMG-CoA reductase inhibitors. Similarities and differences. Clin Pharmacokinet 1997; 32: 403–25. 26. Ross R. The pathogenesis of atherosclerosis: a perspective for the 1990s. Nature 1993; 362; 6423: 801–9. 27. Bocan T, Mueller S, Brown E, Lee P, Bocan M, Rea T, Pape M. HMG-CoA reductase and ACAT inhibitors act synergistically to lower plasma cholesterol and limit atherosclerotic lesion development in the cholesterol-fed rabbit. Atherosclerosis 1998; 139: 21–30. 28. Llorente Cortes V, Martinez Gonzalez J, Badimon L. Differential cholesteryl ester accumulation in two human vascular smooth muscle cell subpopulations exposed to aggregated LDL: effect of PDGF-stimulation and HMG-CoA reductase inhibition. Atherosclerosis 1999; 144: 335–42.
THE INFLUENCE OF SIMVASTATIN ON LIPASE AND CHOLESTEROL ESTERASE ACTIVITY IN THE SERUM OF MEN WITH CORONARY HEART DISEASE ∗ and ANNA PIORUNSKA-MIKOŁAJCZAK ´ ´ MARIA PIORUNSKA-STOLZMANN
Chair and Department of Chemistry and Clinical Biochemistry, Karol Marcinkowski University of Medical Sciences, Grunwaldzka 6, 60-780 Poznan, Poland Accepted 28 November 2000
Several studies have demonstrated that any beneficial effects of 3-hydroxy-3-methylglutarylcoenzyme A (HMG-CoA) reductase inhibitors (statins), of which simvastatin (Merck Sharp & Dohme) is an example, on coronary events are linked to their hypocholesterolemic properties. The in vivo effects of simvastatin treatment on lipase (GEH = glycerol ester hydrolase) and cholesterol esterase (CEase) activity in the serum of men with coronary heart disease (CHD) were examined. GEH and CEase activity in the serum of men with CHD, before simvastatin treatment, was lower than in the control subjects. In our study we have provided evidence that simvastatin increases GEH activity in a time-dependent manner, but has no effect on CEase activity. This suggests that simvastatin can directly affect acylglycerol metabolism by an increase in GEH activity and may therefore be suitable for the treatment of combined lipoprotein disorders characterized by elevation c 2001 Academic Press of triacylglycerols.
K EY WORDS : lipase, cholesterol esterase, simvastatin, serum, CHD men.
INTRODUCTION Coronary heart disease (CHD) remains the leading cause of death in most developed countries. Clinical and experimental investigations [1, 2] have clearly shown that a reduction in plasma cholesterol, particularly in low-density lipoprotein cholesterol (LDL-C), reduces the risk of cardiovascular events in both primary and secondary prevention studies. A number of cholesterol-lowering drugs are currently available for use in humans and of these, statins are the most commonly prescribed agents for the treatment of hypercholesterolemia, because of their efficacy in reducing LDL and their excellent tolerability and safety [3–5]. Different LDL particles are related to differences in the development of coronary artery disease [6, 7]. The LDL phenotype may be controlled by lipolytic enzymes that can alter the lipid composition of lipoproteins [8, 9]. Variations in hepatic lipase (HL) and lipoprotein lipase (LPL) activity have been shown to be related to changes in LDL size and pattern [10]. There is general agreement that the presence of small, dense LDL (pattern B) is associated with an increased risk of CHD due to their physiological properties, such as their high susceptibility to oxidation and low affinity to the LDL receptor, in comparison to pattern A particles [7, 8]. It ∗ Corresponding author. E-mail: [email protected]
1043–6618/01/040359–04/$35.00/0
is known that triacylglycerols (TG) constitute the major determinant of the plasma LDL, and the replacement of cholesteryl esters by triacylglycerols, followed by TG hydrolysis, is a two-step procedure leading to the size reduction of LDL particles [11, 12]. Lipase [glycerol ester hydrolase (GEH), EC 3.1.1.3] hydrolyses triacylglycerides in triglyceride-rich lipoproteins leading to the formation of modified particles that are depleted of triglycerides [13]. Most of the cholesterol in plasma lipoproteins is in the form of cholesteryl esters (CE), which undergo a continual cycle of hydrolysis and re-esterification by the action of cholesterol esterase (CEase, EC 3.1.1.13) and acylcoenzyme A to cholesterol acyltransferase (ACAT, EC 2.3.1.1.26), respectively [14]. Therefore, the ability of CEase to convert large LDL to smaller LDL subspecies, and the relationship between plasma CEase and LDL levels, suggest that high plasma CEase levels may constitute a potential risk factor for atherosclerosis [15]. Although the safety and efficacy of long-term treatment with simvastatin has been demonstrated, the impact of 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase inhibitors on enzymes affecting lipoprotein metabolism in vivo still needs to be fully elucidated. The present study was undertaken to investigate the effect of simvastatin treatment on the GEH and CEase activity in the serum of men with CHD. c 2001 Academic Press
360
Pharmacological Research, Vol. 43, No. 4, 2001 Table I Serum lipid profile of control subjects and of men with coronary heart disease before and after 3 months simvastatin treatment T-C
TG
LDL-C
HDL-C
LDL-C/ HDL-C
mmol l−1 Control n = 10 CHD n = 15
before after
5.0 ± 0.8
1.6 ± 0.5
2.9 ± 0.2
1.6 ± 0.2
2.2 ± 0.7
7.1 ± 0.3a 5.7 ± 0.2b
2.2 ± 0.5a 1.7 ± 0.2b
4.2 ± 0.2a 3.1 ± 0.2b
1.2 ± 0.3a 1.3 ± 0.1
3.7 ± 1.0a 2.8 ± 0.8b
n = number of subjects. The data are mean ± SD. Significant differences: a between the CHD and control groups; and b between the CHD group before and after simvastatin treatment; P ≤ 0.05.
MATERIALS AND METHODS The patients included in this study were 15 men, aged 44–62 years, with a diagnosis of coronary heart disease. None had had any cardiovascular episodes. All the patients were given 10 mg simvastatin (Merck, Sharp & Dohme) orally, once daily over a period of 3 months. All the patients had given their written informed consent. Patients with any known abnormalities of carbohydrate metabolism were excluded from this study. The control group consisted of ten healthy male blood donors. Blood samples were taken from all the subjects after an overnight fast. Serum was separated by centrifugation and used in the assays of enzyme activity. The following concentrations in serum were determined: total-, HDL- and LDL-cholesterol (T-C, HDL-C and LDL-C, respectively) and TG, using the appropriate enzyme kits (Boehringer Mannheim Biochimica). The blood sera of both the controls and the men with CHD were processed in order to obtain acetone-butanol powders by the method described in detail previously [16]. The powders were extracted with water and then spun using a Dacie electric cell suspension mixer, at 4 ◦ C for 10 min. The extracts were filtered, kept at 4 ◦ C and used for the enzyme activity assays. Protein content was determined by the method of Lowry et al. [17]. Fresh hydrosols of trioleylglycerol and oleylcholesterol were prepared for each experiment [16]. The levels of CEase and GEH activity were determined under optimal experimental conditions for each enzyme, namely pH 8.6 and pH 8.0, respectively [18, 19]. Enzyme catalytic activity was determined by means of a two-point method based on measuring changes in the concentration of free fatty acids released and extracted from the reaction mixture both before and 20 min after incubation. The results are expressed in nanomoles of fatty acid released × min−1 (mg protein)−1 = mU mg−1 of the enzyme preparations. Statistical analysis of the results (means, standard deviations) was carried out using INSTAT (Sigma) software. The means between groups were compared by Student’s t-test or by the Cochran–Cox test. Statistical significance
Table II Serum GEH and CEase activity of control subjects and of men with CHD before and after 3 months simvastatin treatment Lipase (GEH)
Cholesterol esterase (CEase)
mU mg−1 Control n = 10 CHD n = 15
before after
8.7 ± 1.0
7.9 ± 0.9c
7.1 ± 1.2a 12.1 ± 0.8b
7.0 ± 0.8a 7.5 ± 0.7c
The data are mean ± SD. Significant differences: a between the CHD and control groups; b between CHD group before and after simvastatin treatment; and c between GEH and CEase activity within the groups; P ≤ 0.05.
was set at the level of P < 0.05. This study was approved by the Bioethics Committee of the University of Medical Sciences in Pozna´n, Poland.
RESULTS The serum lipid profiles of the patients are summarized in Table I. Significant differences between groups were observed in the T-C, TG, LDL-C, HDL-C and LDLC/HDL-C ratios. Decreases were observed in the T-C, TG levels, LDL-C and LDL-C/HDL-C ratio in patients with CHD following simvastatin treatment. Serum GEH and CEase activity in the controls and in the men with CHD, before and after 3 months treatment with simvastatin, are presented and compared in Table II. There were differences in the mean activities between GEH and CEase in the control group but not in the CHD group before simvastatin treatment. However, either GEH or CEase activity was reduced in the CHD group compared to the controls. GEH activity in the serum of men with CHD increased significantly throughout the entire period of treatment with simvastatin (Fig. 1). Catalytic CEase activity, as studied in the serum of men with CHD, was not affected by simvastatin and did not significantly differ from that found in their serum before treatment (Table II).
Pharmacological Research, Vol. 43, No. 4, 2001
361
14
### &
GEH (mU mg− 1)
12 10 8 6 4 2 0
Before
1 Month
2 Months Simvastatin treatment
3 Months
Fig. 1. Effect of simvastatin treatment on serum GEH activity in men with coronary heart disease. Mean ± SD. ∗∗∗ —statistically significant difference vs ‘Before’, P < 0.001. ###, &—statistical difference vs ‘1 Month’ (P < 0.001) or ‘2 Months’ (P < 0.05), respectively.
DISCUSSION The present study showed that patients with established CHD who are on statin therapy have significant changes in their serum GEH activity. To our knowledge, this simvastatin effect has not been described previously in humans and such an effect on GEH activity is apparently unique for this class of lipid-lowering medication. This finding supports the idea that some of the beneficial effects of HMG-CoA reductase inhibitors may be mediated via a mechanism other than cholesterol reduction alone [20–22]. Although the liver is the target organ for the action of statins, our study provides evidence that serum enzymes may also be affected. It has been shown in a rat model that after simvastatin administration, there is increased lipoprotein lipase mRNA and an increase in lipase lipoprotein activity in adipose tissue and heart [23]. Statins are thought to be ineffective in directly modifying the size and density of LDL [1], but in our study we found that an increase in serum GEH activity can affect lipoprotein metabolism. Moreover, statin therapy in moderate hypercholesterolemia is associated with changes throughout the apoB-containing lipoprotein, which has been observed by other authors [24]. When lipolytic activity is high, the circulating levels of large TG-rich lipoproteins remain low. On the other hand, if the enzyme activity is low, the TG-rich lipoprotein remains the major species in plasma. The mechanism by which simvastatin may increase GEH activity is not yet known, but we speculate that this phenomenon might also be related to the influence of simvastatin on acylenzyme formation. Simvastatin displays close pharmacokinetic parameters with other statins [25], but the precise mechanism of action of these compounds, however, has not been fully elucidated. It cannot be excluded that, due to the presence of a free electron pair on the oxygen atom in the structure of simvastatin, it may influence an enzyme–substrate interaction by nucleophile interference. Migration and proliferation of arterial myocytes, together with the deposition of lipids, mainly cholesteryl esters, in the arterial wall are the key events in the athero-
genic cascade [12, 26]. Some authors have postulated that plasma CEase activity contributes to the formation and accumulation of atherogenic lipoproteins and a positive correlation has been observed between CEase activity and plasma LDL levels [15]. In our study we did not observe these changes in CEase activity after simvastatin treatment, and suggest that changes may not occur due to inhibition of statin ACAT activity, as proposed by other authors [27]. Statins inhibit cholesteryl ester accumulation in monocyte-derived macrophages either by reducing the availability of free cholesterol to the enzyme acyl-coenzyme A cholesterol acyltransferase by trapping it in phospholipid-containing pools, or by inhibiting LDL endocytosis related to reduced synthesis of the mevalonate or mevalonate byproducts required for cholesterol esterification [20]. It has also been shown that simvastatin inhibits CE accumulation from aggregated LDL [28]. However, while there is a growing body of evidence from case controlled and prospective studies to suggest that plasma triacylglycerols are an independent risk factor for the development of CHD, the risk associated with elevations in plasma TG still remains controversial. Experiments that might help in solving these problems are worth undertaking. Taken together, these studies suggest that simvastatin can directly affect acylglycerol metabolism by an increase in GEH activity and may therefore be suitable for the treatment of combined lipoprotein disorders characterized by elevation of triacylglycerols.
REFERENCES 1. Maron DJ, Fazio S, Linton MF. Current perspectives on statins. Circulation 2000; 101: 207–13. 2. Auer J, Eber B. Current aspects of statins. J Clin Basic Cardiol 1999; 2: 203–8. 3. Pedersen T et al. Safety and tolerability of cholesterol lowering with simvastatin during 5 years in the Scandinavian Simvastatin Survival Study. Arch Intern Med 1996; 156: 2085–92.
362 4. Stein E, Lane M, Laskarzewski P. Comparison of statins in hypertrigliceridemia. Am J Cardiol 1998; 81(4A): 66B–9B. 5. Jones P, Kafonek S, Laurora I, Hunninghake D. Comparative dose efficacy of atorvastatin versus simvastatin, pravastatin, lovastatin, and fluvastatin in patients with hypercholesterolemia. Am J Cardiol 1998; 81: 582–7. 6. Rainwater D. Lipoprotein correlates of LDL particle size. Atherosclerosis 2000; 148: 151–8. 7. Byrne Ch. Triglyceride-rich lipoproteins: are links with atherosclerosis mediated by a procoagulant and proinflammatory phenotype? Atherosclerosis 1999; 145: 1–15. 8. Grundy SM, Vega GL. Two different views of the relationship of hypertriglyceridemia to coronary heart disease. Arch Intern Med 1992; 152: 28–34. 9. Grundy SM. Hypertriglyceridemia, atherogenic dyslipidemia, and the metabolic syndrome. Am J Cardiol 1998; 81(4A): 18B–25B. 10. Tan CE, Forster MJ, Caslake MJ, Bedford D, Watson TD K, McConnell M, Packard CJ, Shepherd J. Relations between plasma lipids and postheparin plasma lipases and VLDL and LDL subfraction patterns in normolipidemic men and women. Arterioscler Thromb Vasc Biol 1995; 15: 1839–48. 11. Nilsson-Ehle P. Lipolytic enzymes and plasma lipoprotein metabolism. Annu Rev Biochem 1980; 49: 667–93. 12. Brown M, Goldstein J. Lipoprotein metabolism in the macrophage. Implications for cholesterol deposition in atherosclerosis. Annu Rev Biochem 1983; 52: 223–61. 13. Aviram M, Bierman E, Chait A. Modification of low density lipoprotein by lipoprotein lipase or hepatic lipase induces enhanced uptake and cholesterol accumulation in cells. J Biol Chem 1988; 263: 15416–22. 14. Miura S, Chiba T, Mochizuki N, Nagura H, Nemoto K, Tomita I, Ikeda M, Tomita T. Cholesterol-mediated changes of neutral cholesterol esterase activity in macrophages. Mechanism for mobilization of cholesteryl esters in lipid droplets by HDL. Arterioscler Thromb Vasc Biol 1997; 17: 3033–40. 15. Brodt-Eppley J, White P, Jenkins S, Hui D. Plasma cholesterol esterase level is a determinant for an atherogenic lipoprotein profile in normolipidemic human subjects. Biochim Biophys Acta 1995; 1272: 69–72. 16. Patelski J, Pniewska B, Pioru´nska M, Obr ebska M. The arterial acyl-CoA cholesterol ester hydrolase activities. In vitro effect of substrates with fatty acids of different chain length and saturation. Atherosclerosis 1975; 22: 287–91.
Pharmacological Research, Vol. 43, No. 4, 2001 17. Lowry OH, Rosebrough NJ, Farr AL, Randall RJ. Protein measurement with the folin phenol reagent. J Biol Chem 1951; 193: 263–75. 18. Patelski J, Pioru´nska-Mikołajczak A, Pioru´nska-Stolzmann M. Effect of substrate composition and concentration on aortic cholesterol ester hydrolase activity. Enzyme 1978; 23: 135–9. 19. Pioru´nska-Stolzmann M. The substrate specificity of glycerol ester hydrolase from pig aorta and serum. Acta Biochim Pol 1990; 37: 457–62. 20. Rosenson R, Tangney C. Antiatherothrombotic properties of statins. Implications for cardiovascular event reduction. JAMA 1998; 279: 1643–50. 21. Lagrost L et al. Plasma lipoprotein distribution and lipid transfer activities in patients with type IIb hyperlipidemia treated with simvastatin. Atherosclerosis 1999; 143: 415–25. 22. Corsini A, Pazzucconi F, Arnaboldi L, Pfister P, Fumagalli R, Paoletti R, Sirtori C. Direct effects of statins on the vascular wall. J Cardiovasc Pharm 1998; 31: 773–8. 23. Schoonjans K, Peinado-Onsurbe J, Fruchart J, Tailleux A, Fievet C, Auwerx J. HMG-CoA reductase inhibitors reduce serum triglyceride levels through modulation of apolipoprotein C-III and lipoprotein lipase. FEBS Lett 1999; 452: 160–4. 24. Gaw A, Packard Ch, Murray E, Lindsay G, Griffin B, Caslake M, Vallance B, Lorimer A, Shepherd J. Effects of simvastatin on apoB metabolism and LDL subfraction distribution. Arterioscler Thromb 1993; 13: 170–89. 25. Lennernas H, Fager G. Pharmacodynamic and pharmacokinetics of the HMG-CoA reductase inhibitors. Similarities and differences. Clin Pharmacokinet 1997; 32: 403–25. 26. Ross R. The pathogenesis of atherosclerosis: a perspective for the 1990s. Nature 1993; 362; 6423: 801–9. 27. Bocan T, Mueller S, Brown E, Lee P, Bocan M, Rea T, Pape M. HMG-CoA reductase and ACAT inhibitors act synergistically to lower plasma cholesterol and limit atherosclerotic lesion development in the cholesterol-fed rabbit. Atherosclerosis 1998; 139: 21–30. 28. Llorente Cortes V, Martinez Gonzalez J, Badimon L. Differential cholesteryl ester accumulation in two human vascular smooth muscle cell subpopulations exposed to aggregated LDL: effect of PDGF-stimulation and HMG-CoA reductase inhibition. Atherosclerosis 1999; 144: 335–42.