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Plasma concentration of asymmetric dimethylarginine and the risk of coronary heart disease: rationale and design of the multicenter CARDIAC study
Atherosclerosis Supplements 4 (2003) 29–32
Plasma concentration of asymmetric dimethylarginine and the risk of coronary heart disease: rationale and design of the multicenter CARDIAC study Andreas Mügge a , Christoph Hanefeld a , Rainer H. Böger b,∗ CARDIAC study investigators1 a
b
Department of Cardiology, St. Josef Hospital, Ruhr University Bochum, Germany Clinical Pharmacology Unit, Institute of Experimental and Clinical Pharmacology, Center of Experimental Medicine, University Hospital Hamburg-Eppendorf, Martinistr. 52, D-20246 Hamburg, Germany
Abstract There is abundant evidence now that the endothelium plays a crucial role in the maintenance of vascular tone and structure. One of the major endothelium-derived vasoactive mediators is nitric oxide (NO). Asymmetric dimethylarginine (ADMA) is an endogenous inhibitor of NO synthase. ADMA inhibits vascular NO production at concentrations found in pathophysiological conditions; ADMA also causes local vasoconstriction when it is infused intra-arterially. ADMA is increased in plasma of humans with hypercholesterolemia, atherosclerosis, hypertension, chronic renal failure, and chronic heart failure. Increased ADMA levels are associated with reduced NO synthesis as assessed by impaired endothelium-dependent vasodilation. In several prospective and cross-sectional studies, ADMA evolved as a marker of cardiovascular risk in certain populations like hemodialysis patients, non-smoking men, or intensive-care patients. The CARDIAC study is a multicenter case–control study designed to address the hypothesis that ADMA may be a suitable and sensitive marker of cardiovascular risk in a large, unselected population of both sexes, and with a broad range of established cardiovascular risk factors. The population included in the CARDIAC study will be prospectively followed for a scheduled follow-up period of 4 years, and the data of the CARDIAC-PRO study will then provide definite evidence whether ADMA may be prospectively determined as a cardiovascular risk factor. © 2003 Elsevier Ireland Ltd. All rights reserved. Keywords: Endothelium; Nitric oxide; Coronary heart disease; Prognosis; Epidemiology
1. Background: pathophysiological role of ADMA as an endogenous inhibitor of NO synthase
∗ Corresponding author. Tel.: +49-40-42803-9759; fax: +49-40-42803-9757. E-mail address: [email protected] (R.H. Böger). 1 The CARDIAC investigators and study centers: C. Hanefeld, A. Mügge, St. Joseph-Hospital, Ruhr-University Bochum; R.H. Böger, Institute of Experimental and Clinical Pharmacology, University Hospital Hamburg-Eppendorf; K.J. Osterziel, Department of Molecular and Clinical Cardiology, Franz-Volhard Hospital, Charit´e Berlin; M. Kusus, W.G. Daniel, Department of Cardiology, University of Erlangen; C. Schmidt-Lucke, H. Klein, Department of Cardiology and Angiology, Otto-von-Guericke University Magdeburg; H. Lenzen, A. Bartling, Institute of Clinical Pharmacology, Medical School of Hannover; L. Goudeva, Institute of Transfusion Medicine, Medical School of Hannover; D. Strödter, H. Tillmanns, Department of Cardiology, Justus-Liebig-University Giessen; J. Berger, Institute of Mathematics in Medicine, University Hospital Hamburg-Eppendorf.
It has been 10 years since asymmetric dimethylarginine (ADMA) was first described as an endogenous inhibitor of NO synthase (NOS) in human plasma and urine by Vallance et al. [1]. ADMA has been recognized as an endogenous regulator of NO synthesis and endothelium-dependent vasodilation in vivo [2,3]. Endothelial dysfunction is a common feature of arterial function in patients who are at high risk of developing cardiovascular disease. Recent evidence suggests that the presence of endothelial dysfunction is a prognostic marker for the risk of future major cardiovascular events and death [4,5]. These findings from prospective clinical studies add to experimental evidence suggesting that defective NO production and/or action is involved in the pathogenesis of atherosclerotic vascular disease, as evidenced by animal
1567-5688/$ – see front matter © 2003 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/S1567-5688(03)00031-X
30
A. Mügge et al. / Atherosclerosis Supplements 4 (2003) 29–32
studies showing that inhibition of NO synthesis by synthetic NOS inhibitors like NG -nitro-l-arginine methyl ester (l-NAME) accelerate atherosclerotic plaque formation, whereas supplementation with l-arginine inhibits atherogenesis [6–9]. This latter finding also suggests a competitive mechanism behind the reduced activity of the NO pathway in atherogenesis. ADMA inhibits NO production by displacing the substrate, l-arginine, from the catalytic site of the enzyme. Data from experimental studies suggest that ADMA inhibits vascular NO production at (patho-) physiological concentrations [1,10]. The half-maximal inhibitory concentration of ADMA that is necessary for inihibition of NO synthesis (IC50 ) has been calculated as 1.8 ± 0.1 M in rat cerebellar homogenates [10] and 3.9 mol/l in cultured human endothelial cells.
vascular events and total mortality during a mean 33.4 months of follow-up. Patients with an ADMA concentration in the highest quartile had a 3-fold higher risk of death of any cause than patients with ADMA levels below the median [14]. Valkonen et al. [15] showed in a prospective, nested case–control of middle-aged, non-smoking men from Finland that subjects who had ADMA plasma levels in the highest quartile had a 3.9-fold increase in risk for acute coronary events compared with the other quartiles. Most recently, Nijveldt and co-workers found that ADMA was the strongest predictor of death of patients on an intensive care unit, with a 17-fold excess in mortality for patients in the highest ADMA quartile as compared to those in the lowest quartile [16]. Taken together, these studies provide strong evidence that ADMA may act as novel cardiovascular risk factor.
2. The CARDIAC study: rationale 3. The CARDIAC study: patients and study design The risk of coronary heart disease is of utmost epidemiological importance in the Western world, as mortality from cardiovascular causes still accounts for about 50% of total mortality. Established coronary risk factors like hypercholesterolemia, hypertension, smoking, and diabetes do not account for the total risk in large epidemiological studies. A varying proportion of premature coronary artery disease remains unexplained by these factors. Therefore, the need to identify novel risk factors has been stressed by international scientific organizations in the cardiovascular arena. Among these, factors that may influence the biological actions of NO are strongly supported by pathophysiological evidence. As outlined above, ADMA is one such factor that acts as an endogenous modulator of the NO pathway. Plasma concentration of ADMA is elevated in plasma of humans with hypercholesterolemia, atherosclerosis, hypertension, chronic heart failure, diabetes mellitus, and in other patient groups with evidence of or at high risk of developing cardiovascular disease [11]. From a number of recent clinical studies, evidence has accumulated that ADMA may be a marker for the diagnosis of patients with atherosclerotic vascular disease. ADMA levels were significantly related to carotid artery intima-media thickness in 116 human subjects who had no overt signs of coronary or peripheral arterial disease [12]. ADMA levels were positively correlated with age, mean arterial pressure, and glucose tolerance in this study. In a cohort of 90 patients with end-stage renal disease undergoing chronic hemodialysis treatment, similar observations were recently made [13]. ADMA was highly significantly correlated with carotid intimal thickness in this population; moreover, ADMA and C-reactive protein levels emerged as the sole independent predictors of the progression of intimal lesions in patients with intially normal intima-media thickness during a follow-up period of 1 year. In a prospective trial including 225 hemodialysis patients, ADMA and age were the strongest predictors of cardio-
The CARDIAC study (coronary artery risk determination investigating the influence of ADMA concentration) was designed to assess the potential relationship between the plasma concentration of ADMA and the risk for coronary heart disease in a large sample of patients and subjects of both sexes, and with a broad range of established cardiovascular risk factors. Patients and subjects are recruited in eight academic medical centers throughout Germany. Patients are included based on the ascertained evidence of coronary heart disease (prior myocardial infarction or presence of stable angina pectoris, plus objective investigational evidence of the presence of coronary artery stenosis, such as positive exercise ECG, evidence for myocardial ischemia in thallium scintigraphy, or documentation of coronary stenosis by coronary angiography). To reduce biases caused by time-related changes in risk factor treatment, centers are asked to recruit new or recently diagnosed cases wherever possible. Healthy controls are included based on the absence of any acute or chronic cardiovascular or metabolic disease, with the exception of diabetes mellitus (however, diabetic subjects with evidence of diabetic complications will be excluded). Both patients and controls are accepted for inclusion in the study if they are between 25 and 85 years old, and if they do not suffer from any acute or chronic severe disease, such as severe renal failure (creatinine clearance below 30 ml/min; as terminal renal function impairment will lead to elevation of ADMA levels [1,14,17], severe chronic heart failure (NYHA III or above [18]), or hepatic insufficiency [16]. Moreover, patients with acute occlusive cardiovascular events (i.e., within the last 30 days before inclusion into the study), or above the age of 85 years are also excluded from the study. Subjects are recruited from the general population (blood donors and healthy subjects called in through the local
A. Mügge et al. / Atherosclerosis Supplements 4 (2003) 29–32
newspaper) in order to represent as closely as possible the demographic characteristics of the population. Both patients and controls are studied in one of the study centers, and a fasting blood sample will be drawn into vacutainers containing sodium EDTA, centrifuged, and immediately frozen. ADMA, l-arginine, and SDMA analysis is performed by a validated HPLC method in a central core lab. Other laboratory values (lipoprotein profile, uric acid, serum creatinine) are determined locally in the clinical chemical laboratories of the participating institutions, using certified standard laboratory methods.
4. Sample size calculation and definition of end-points Plasma ADMA concentration will be studied as a continuous variable into correlation analyses after checking for normality distribution. If the distribution of ADMA values is skewed, data will be log transformed. For risk analysis, subjects are stratified according to quintiles of ADMA concentration. Relative risks are estimated for subjects in the four upper quintiles, with subjects in the lowest quintile serving as the reference population. Total cholesterol, LDL cholesterol, HDL cholesterol, triglycerides, systolic and diastolic blood pressure will be analyzed as continuous variables. Because of the high number of traditional risk factors and the high portion of patients with treatments directed against these risk factors that can be expected to be present in cases, hypercholesterolemia and hypertension will also be analyzed as categorical variables. For this, hypercholesterolemia is assumed to be present when total cholesterol is 200 mg/dl or more, LDL cholesterol is 160 mg/dl or more, or lipid-lowering medication is prescribed. Hypertension is deemed to be present when repeated (two or more readings) blood pressure recordings have produced blood pressure above 160 mmHg (systolic) or 90 mmHg (diastolic), or blood-pressure-lowering medication is prescribed. Smoking and diabetes mellitus are analyzed as categorical variables. Based on previous publications showing that the difference in ADMA concentration between cases and controls can be expected to be in the order of 0.5 mol/l [2], and considering the necessity to include multiple testing for interacting factors, the sample size needed to significantly discriminate between cases and controls with α = 0.05 and β = 0.9 would be at least 350 subjects per group in the case–control study.
5. Prospective follow-up of the patients: the CARDIAC-PRO study After the initial investigation, patients and controls will be followed for a scheduled follow-up period of 4 years. By telephonic survey, participants will be asked for any severe deteriorations of their health that may have occurred in the
31
Table 1 End-points in the CARDIAC-PRO study Primary end-point Composite end-point of vascular death, myocardial infarction, coronary artery bypass grafting (CABG), percutaneous coronary angioplasty (PTCA), stroke (ischemic or hemorrhagic), or peripheral arterial occlusive disease (PAOD) Secondary end-points Vascular death Myocardial infarction (fatal and non-fatal) Coronary artery bypass grafting (CABG) Percutaneous coronary angioplasty (PTCA) including coronary stenting Stroke (ischemic or hemorrhagic) Peripheral arterial occlusive disease (PAOD; newly diagnosed) Total mortality
meantime. Specifically, major cardiovascular events will be asked, including vascular death, myocardial infarction, coronary artery bypass grafting (CABG), percutaneous coronary angioplasty (PTCA), stroke (ischemic or hemorrhagic), or peripheral arterial occlusive disease (PAOD) (Table 1). The main end-point is defined as the combination of cardiovascular death, non-fatal myocardial infarction, revascularisation (CABG or PTCA), stroke, and PAOD. Each of these end-points will also be determined separately as a secondary end-point, and non-cardiovascular causes of death as well as total mortality will be additional secondary end-points. Anticipating a mean overall event rate of 2% for the main end-point in the general population in Germany, as evidenced by previous trials and surveys [19], and furthermore anticipating a 17% increase in cardiovascular risk with each increase in ADMA by 1 mol/l [14], a sample size of 390 subjects in each group will be sufficient to detect a 50% difference in cardiovascular event rate after 4 years with α = 0.05 and a power of 80%.
6. Conclusions and outlook There is increasing evidence that ADMA, by inhibiting vascular endothelial nitric oxide synthase, may contribute to the pathogenesis of atherosclerosis. Data from clinical studies that suggest that ADMA is a marker of cardiovascular disease in different populations with varying cardiovascular risk may therefore be extended in the future to show that ADMA may be a causal factor in the initiation and progression of ischemic vascular disease. The CARDIAC study is an attempt to investigate, in a large sample of cases and controls from the general population, of both sexes and with a broad range of established cardiovascular risk factors, the statistical relationship between ADMA and the risk of having cardiovascular disease. This study may help to further define the role of ADMA as a marker of cardiovascular disease. The CARDIAC-PRO study was designed to prove, in a prospective manner, the hypothesis that ADMA is a
32
A. Mügge et al. / Atherosclerosis Supplements 4 (2003) 29–32
prognostic factor in ischemic vascular disease, and may thus have a role as a novel cardiovascular risk factor. Definite evidence favoring or negating this hypothesis will, however, only arise from interventional trials aimed at selectively modifying the risk associated with NO synthase inhibition by ADMA. Acknowledgements The CARDIAC study is supported by the Else Kröner Fresenius foundation.
References [1] Vallance P, Leone A, Calver A, Collier J, Moncada S. Accumulation of an endogenous inhibitor of NO synthesis in chronic renal failure. Lancet 1992;339:572–5. [2] Böger RH, Bode-Böger SM, Thiele W, Junker W, Alexander K, Frölich JC. Biochemical evidence for impaired nitric oxide synthesis in patients with peripheral arterial occlusive disease. Circulation 1997;95:2068–74. [3] Böger RH, Bode-Böger SM, Szuba A, Tangphao O, Tsao PS, Chan JR, et al. Asymmetric dimethylarginine: a novel risk factor for endothelial dysfunction. Its role in hypercholesterolemia. Circulation 1998;98:1842–7. [4] Schächinger V, Britten MB, Zeiher AM. Prognostic impact of coronary vasodilator dysfunction on adverse long-term outcome of coronary heart disease. Circulation 2000;101:1899–906. [5] Heitzer T, Schlinzig T, Krohn K, Meinertz T, Münzel T. Endothelial dysfunction, oxidative stress, and risk of cardiovascular events in patients with coronary artery disease. Circulation 2001;104:2673–8. [6] Cayatte AJ, Palacino JJ, Horten K, Cohen RA. Chronic inhibition of nitric oxide production accelerates neointima formation and impairs endothelial function in hypercholesterolemic rabbits. Arterioscler Thromb 1994;14:753–9. [7] Candipan RC, Wang B, Buitrago R, Tsao PS, Cooke JP. Regression or progression: dependency upon vascular nitric oxide. Arterioscler Thromb Vasc Biol 1996;16:44–50.
[8] Böger RH, Bode-Böger SM, Mügge A, Kienke S, Brandes R, Dwenger A, et al. Supplementation of hypercholesterolaemic rabbits with l-arginine reduces the vascular release of superoxide anions and restores NO production. Atherosclerosis 1995;117:273–84. [9] Böger RH, Bode-Böger SM, Phivthong-ngam L, Böhme M, Brandes R, Mügge A, et al. Dietary l-arginine reduces the progression of atherosclerosis in cholesterol-fed rabbits—comparison with lovastatin. Circulation 1997;96:1282–90. [10] Faraci FM, Brian JE, Heistad DD. Response of cerebral blood vesels to an endogenous inhibitor of nitric oxide synthase. Am J Physiol 1995;269:H1522–7. [11] Böger RH. The emerging role of asymmetric dimethylarginine (ADMA) as a cardiovascular risk factor. Cardiovasc Res (in press). [12] Miyazaki H, Matsuoka H, Cooke JP. Endogenous nitric oxide synthase inhibitor. A novel marker of atherosclerosis. Circulation 1999;99:1141–6. [13] Zoccali C, Benedetto FA, Maas R, Mallamaci F, Tripepi G, Malatino L, et al. Asymmetric dimethylarginine (ADMA), C-reactive protein, and carotid intima media-thickness in end-stage renal disease. J Am Soc Nephrol 2002;13:490–6. [14] Zoccali C, Bode-Böger SM, Mallamaci F, Benedetto FA, Tripepi G, Malatino L, et al. Asymmetric dimethylarginine (ADMA): an endogenous inhibitor of nitric oxide synthase predicts mortality in end-stage renal disease (ESRD). Lancet 2001;358:2113–7. [15] Valkonen VP, Päivä H, Salonen JT, Lakka TA, Lehtimäki T, Laakso J, et al. Risk of acute coronary events and serum concentration of asymmetrical dimethylarginine. Lancet 2001;358:2127–8. [16] Nijveldt RJ, Teerlink T, Van der Hoven B, Siroen MP, Kuik DJ, Rauwerda JA, et al. Asymmetrical dimethylarginine (ADMA) in critically ill patients: high plasma ADMA concentration is an independent risk factor of ICU mortality. Clin Nutr 2003;22:23–30. [17] Kielstein JT, Böger RH, Bode-Böger SM, Schäffer J, Barbey M, Koch KM, et al. Asymmetric dimethylarginine plasma concentrations differ in patients with end-stage renal disease: relationship to treatment method and atherosclerotic disease. J Am Soc Nephrol 1999;10:594– 600. [18] Usui M, Matsuoka H, Miyazaki H, Ueda S, Okuda S, Imaizumi T. Increased endogenous nitric oxide synthase inhibitor in patients with congestive heart failure. Life Sci 1998;62:2425–30. [19] Heidrich J, Wellrich J, Hense HW, Siebert E, Liese AD, Löwel H, et al. Klassische Risikofaktoren für Herzinfarkt und Gesamtsterblichkeit in der Bevölkerung. 13-Jahres-Follow-up der MONICA Augsburg-Kohortenstudie. Z Kardiol 2003;92:445–54.
Plasma concentration of asymmetric dimethylarginine and the risk of coronary heart disease: rationale and design of the multicenter CARDIAC study Andreas Mügge a , Christoph Hanefeld a , Rainer H. Böger b,∗ CARDIAC study investigators1 a
b
Department of Cardiology, St. Josef Hospital, Ruhr University Bochum, Germany Clinical Pharmacology Unit, Institute of Experimental and Clinical Pharmacology, Center of Experimental Medicine, University Hospital Hamburg-Eppendorf, Martinistr. 52, D-20246 Hamburg, Germany
Abstract There is abundant evidence now that the endothelium plays a crucial role in the maintenance of vascular tone and structure. One of the major endothelium-derived vasoactive mediators is nitric oxide (NO). Asymmetric dimethylarginine (ADMA) is an endogenous inhibitor of NO synthase. ADMA inhibits vascular NO production at concentrations found in pathophysiological conditions; ADMA also causes local vasoconstriction when it is infused intra-arterially. ADMA is increased in plasma of humans with hypercholesterolemia, atherosclerosis, hypertension, chronic renal failure, and chronic heart failure. Increased ADMA levels are associated with reduced NO synthesis as assessed by impaired endothelium-dependent vasodilation. In several prospective and cross-sectional studies, ADMA evolved as a marker of cardiovascular risk in certain populations like hemodialysis patients, non-smoking men, or intensive-care patients. The CARDIAC study is a multicenter case–control study designed to address the hypothesis that ADMA may be a suitable and sensitive marker of cardiovascular risk in a large, unselected population of both sexes, and with a broad range of established cardiovascular risk factors. The population included in the CARDIAC study will be prospectively followed for a scheduled follow-up period of 4 years, and the data of the CARDIAC-PRO study will then provide definite evidence whether ADMA may be prospectively determined as a cardiovascular risk factor. © 2003 Elsevier Ireland Ltd. All rights reserved. Keywords: Endothelium; Nitric oxide; Coronary heart disease; Prognosis; Epidemiology
1. Background: pathophysiological role of ADMA as an endogenous inhibitor of NO synthase
∗ Corresponding author. Tel.: +49-40-42803-9759; fax: +49-40-42803-9757. E-mail address: [email protected] (R.H. Böger). 1 The CARDIAC investigators and study centers: C. Hanefeld, A. Mügge, St. Joseph-Hospital, Ruhr-University Bochum; R.H. Böger, Institute of Experimental and Clinical Pharmacology, University Hospital Hamburg-Eppendorf; K.J. Osterziel, Department of Molecular and Clinical Cardiology, Franz-Volhard Hospital, Charit´e Berlin; M. Kusus, W.G. Daniel, Department of Cardiology, University of Erlangen; C. Schmidt-Lucke, H. Klein, Department of Cardiology and Angiology, Otto-von-Guericke University Magdeburg; H. Lenzen, A. Bartling, Institute of Clinical Pharmacology, Medical School of Hannover; L. Goudeva, Institute of Transfusion Medicine, Medical School of Hannover; D. Strödter, H. Tillmanns, Department of Cardiology, Justus-Liebig-University Giessen; J. Berger, Institute of Mathematics in Medicine, University Hospital Hamburg-Eppendorf.
It has been 10 years since asymmetric dimethylarginine (ADMA) was first described as an endogenous inhibitor of NO synthase (NOS) in human plasma and urine by Vallance et al. [1]. ADMA has been recognized as an endogenous regulator of NO synthesis and endothelium-dependent vasodilation in vivo [2,3]. Endothelial dysfunction is a common feature of arterial function in patients who are at high risk of developing cardiovascular disease. Recent evidence suggests that the presence of endothelial dysfunction is a prognostic marker for the risk of future major cardiovascular events and death [4,5]. These findings from prospective clinical studies add to experimental evidence suggesting that defective NO production and/or action is involved in the pathogenesis of atherosclerotic vascular disease, as evidenced by animal
1567-5688/$ – see front matter © 2003 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/S1567-5688(03)00031-X
30
A. Mügge et al. / Atherosclerosis Supplements 4 (2003) 29–32
studies showing that inhibition of NO synthesis by synthetic NOS inhibitors like NG -nitro-l-arginine methyl ester (l-NAME) accelerate atherosclerotic plaque formation, whereas supplementation with l-arginine inhibits atherogenesis [6–9]. This latter finding also suggests a competitive mechanism behind the reduced activity of the NO pathway in atherogenesis. ADMA inhibits NO production by displacing the substrate, l-arginine, from the catalytic site of the enzyme. Data from experimental studies suggest that ADMA inhibits vascular NO production at (patho-) physiological concentrations [1,10]. The half-maximal inhibitory concentration of ADMA that is necessary for inihibition of NO synthesis (IC50 ) has been calculated as 1.8 ± 0.1 M in rat cerebellar homogenates [10] and 3.9 mol/l in cultured human endothelial cells.
vascular events and total mortality during a mean 33.4 months of follow-up. Patients with an ADMA concentration in the highest quartile had a 3-fold higher risk of death of any cause than patients with ADMA levels below the median [14]. Valkonen et al. [15] showed in a prospective, nested case–control of middle-aged, non-smoking men from Finland that subjects who had ADMA plasma levels in the highest quartile had a 3.9-fold increase in risk for acute coronary events compared with the other quartiles. Most recently, Nijveldt and co-workers found that ADMA was the strongest predictor of death of patients on an intensive care unit, with a 17-fold excess in mortality for patients in the highest ADMA quartile as compared to those in the lowest quartile [16]. Taken together, these studies provide strong evidence that ADMA may act as novel cardiovascular risk factor.
2. The CARDIAC study: rationale 3. The CARDIAC study: patients and study design The risk of coronary heart disease is of utmost epidemiological importance in the Western world, as mortality from cardiovascular causes still accounts for about 50% of total mortality. Established coronary risk factors like hypercholesterolemia, hypertension, smoking, and diabetes do not account for the total risk in large epidemiological studies. A varying proportion of premature coronary artery disease remains unexplained by these factors. Therefore, the need to identify novel risk factors has been stressed by international scientific organizations in the cardiovascular arena. Among these, factors that may influence the biological actions of NO are strongly supported by pathophysiological evidence. As outlined above, ADMA is one such factor that acts as an endogenous modulator of the NO pathway. Plasma concentration of ADMA is elevated in plasma of humans with hypercholesterolemia, atherosclerosis, hypertension, chronic heart failure, diabetes mellitus, and in other patient groups with evidence of or at high risk of developing cardiovascular disease [11]. From a number of recent clinical studies, evidence has accumulated that ADMA may be a marker for the diagnosis of patients with atherosclerotic vascular disease. ADMA levels were significantly related to carotid artery intima-media thickness in 116 human subjects who had no overt signs of coronary or peripheral arterial disease [12]. ADMA levels were positively correlated with age, mean arterial pressure, and glucose tolerance in this study. In a cohort of 90 patients with end-stage renal disease undergoing chronic hemodialysis treatment, similar observations were recently made [13]. ADMA was highly significantly correlated with carotid intimal thickness in this population; moreover, ADMA and C-reactive protein levels emerged as the sole independent predictors of the progression of intimal lesions in patients with intially normal intima-media thickness during a follow-up period of 1 year. In a prospective trial including 225 hemodialysis patients, ADMA and age were the strongest predictors of cardio-
The CARDIAC study (coronary artery risk determination investigating the influence of ADMA concentration) was designed to assess the potential relationship between the plasma concentration of ADMA and the risk for coronary heart disease in a large sample of patients and subjects of both sexes, and with a broad range of established cardiovascular risk factors. Patients and subjects are recruited in eight academic medical centers throughout Germany. Patients are included based on the ascertained evidence of coronary heart disease (prior myocardial infarction or presence of stable angina pectoris, plus objective investigational evidence of the presence of coronary artery stenosis, such as positive exercise ECG, evidence for myocardial ischemia in thallium scintigraphy, or documentation of coronary stenosis by coronary angiography). To reduce biases caused by time-related changes in risk factor treatment, centers are asked to recruit new or recently diagnosed cases wherever possible. Healthy controls are included based on the absence of any acute or chronic cardiovascular or metabolic disease, with the exception of diabetes mellitus (however, diabetic subjects with evidence of diabetic complications will be excluded). Both patients and controls are accepted for inclusion in the study if they are between 25 and 85 years old, and if they do not suffer from any acute or chronic severe disease, such as severe renal failure (creatinine clearance below 30 ml/min; as terminal renal function impairment will lead to elevation of ADMA levels [1,14,17], severe chronic heart failure (NYHA III or above [18]), or hepatic insufficiency [16]. Moreover, patients with acute occlusive cardiovascular events (i.e., within the last 30 days before inclusion into the study), or above the age of 85 years are also excluded from the study. Subjects are recruited from the general population (blood donors and healthy subjects called in through the local
A. Mügge et al. / Atherosclerosis Supplements 4 (2003) 29–32
newspaper) in order to represent as closely as possible the demographic characteristics of the population. Both patients and controls are studied in one of the study centers, and a fasting blood sample will be drawn into vacutainers containing sodium EDTA, centrifuged, and immediately frozen. ADMA, l-arginine, and SDMA analysis is performed by a validated HPLC method in a central core lab. Other laboratory values (lipoprotein profile, uric acid, serum creatinine) are determined locally in the clinical chemical laboratories of the participating institutions, using certified standard laboratory methods.
4. Sample size calculation and definition of end-points Plasma ADMA concentration will be studied as a continuous variable into correlation analyses after checking for normality distribution. If the distribution of ADMA values is skewed, data will be log transformed. For risk analysis, subjects are stratified according to quintiles of ADMA concentration. Relative risks are estimated for subjects in the four upper quintiles, with subjects in the lowest quintile serving as the reference population. Total cholesterol, LDL cholesterol, HDL cholesterol, triglycerides, systolic and diastolic blood pressure will be analyzed as continuous variables. Because of the high number of traditional risk factors and the high portion of patients with treatments directed against these risk factors that can be expected to be present in cases, hypercholesterolemia and hypertension will also be analyzed as categorical variables. For this, hypercholesterolemia is assumed to be present when total cholesterol is 200 mg/dl or more, LDL cholesterol is 160 mg/dl or more, or lipid-lowering medication is prescribed. Hypertension is deemed to be present when repeated (two or more readings) blood pressure recordings have produced blood pressure above 160 mmHg (systolic) or 90 mmHg (diastolic), or blood-pressure-lowering medication is prescribed. Smoking and diabetes mellitus are analyzed as categorical variables. Based on previous publications showing that the difference in ADMA concentration between cases and controls can be expected to be in the order of 0.5 mol/l [2], and considering the necessity to include multiple testing for interacting factors, the sample size needed to significantly discriminate between cases and controls with α = 0.05 and β = 0.9 would be at least 350 subjects per group in the case–control study.
5. Prospective follow-up of the patients: the CARDIAC-PRO study After the initial investigation, patients and controls will be followed for a scheduled follow-up period of 4 years. By telephonic survey, participants will be asked for any severe deteriorations of their health that may have occurred in the
31
Table 1 End-points in the CARDIAC-PRO study Primary end-point Composite end-point of vascular death, myocardial infarction, coronary artery bypass grafting (CABG), percutaneous coronary angioplasty (PTCA), stroke (ischemic or hemorrhagic), or peripheral arterial occlusive disease (PAOD) Secondary end-points Vascular death Myocardial infarction (fatal and non-fatal) Coronary artery bypass grafting (CABG) Percutaneous coronary angioplasty (PTCA) including coronary stenting Stroke (ischemic or hemorrhagic) Peripheral arterial occlusive disease (PAOD; newly diagnosed) Total mortality
meantime. Specifically, major cardiovascular events will be asked, including vascular death, myocardial infarction, coronary artery bypass grafting (CABG), percutaneous coronary angioplasty (PTCA), stroke (ischemic or hemorrhagic), or peripheral arterial occlusive disease (PAOD) (Table 1). The main end-point is defined as the combination of cardiovascular death, non-fatal myocardial infarction, revascularisation (CABG or PTCA), stroke, and PAOD. Each of these end-points will also be determined separately as a secondary end-point, and non-cardiovascular causes of death as well as total mortality will be additional secondary end-points. Anticipating a mean overall event rate of 2% for the main end-point in the general population in Germany, as evidenced by previous trials and surveys [19], and furthermore anticipating a 17% increase in cardiovascular risk with each increase in ADMA by 1 mol/l [14], a sample size of 390 subjects in each group will be sufficient to detect a 50% difference in cardiovascular event rate after 4 years with α = 0.05 and a power of 80%.
6. Conclusions and outlook There is increasing evidence that ADMA, by inhibiting vascular endothelial nitric oxide synthase, may contribute to the pathogenesis of atherosclerosis. Data from clinical studies that suggest that ADMA is a marker of cardiovascular disease in different populations with varying cardiovascular risk may therefore be extended in the future to show that ADMA may be a causal factor in the initiation and progression of ischemic vascular disease. The CARDIAC study is an attempt to investigate, in a large sample of cases and controls from the general population, of both sexes and with a broad range of established cardiovascular risk factors, the statistical relationship between ADMA and the risk of having cardiovascular disease. This study may help to further define the role of ADMA as a marker of cardiovascular disease. The CARDIAC-PRO study was designed to prove, in a prospective manner, the hypothesis that ADMA is a
32
A. Mügge et al. / Atherosclerosis Supplements 4 (2003) 29–32
prognostic factor in ischemic vascular disease, and may thus have a role as a novel cardiovascular risk factor. Definite evidence favoring or negating this hypothesis will, however, only arise from interventional trials aimed at selectively modifying the risk associated with NO synthase inhibition by ADMA. Acknowledgements The CARDIAC study is supported by the Else Kröner Fresenius foundation.
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