TR-05536; No of Pages 6 Thrombosis Research xxx (2014) xxx–xxx
Contents lists available at ScienceDirect
Thrombosis Research journal homepage: www.elsevier.com/locate/thromres
Regular Article
Relationship between homocysteine and coronary artery disease. Results from a large prospective cohort study Alon Schaffer a, Monica Verdoia a, Ettore Cassetti a, Paolo Marino a, Harry Suryapranata b, Giuseppe De Luca a,⁎, on behalf of the Novara Atherosclerosis Study Group (NAS) a b
Division of Cardiology, Azienda Ospedaliera-Universitaria “Maggiore della Carità”, Eastern Piedmont University, Novara, Italy Department of Cardiology, UMC St Radboud, Nijmegen, The Netherlands (HS)
a r t i c l e
i n f o
Article history: Received 5 March 2014 Received in revised form 7 May 2014 Accepted 18 May 2014 Available online xxxx Keywords: Homocysteine Coronary atherosclerosis Coronary angiography
a b s t r a c t Background: Coronary artery disease (CAD) still represents the major cause of mortality in developed countries. Large research programs have been focused on the identification of new risk factors to prevent CAD, with special attention to homocysteine (Hcy), due to the known associated increased thrombogenicity, oxidative stress status and endothelial dysfunction. However, controversy still exists on the association between Hcy and CAD. Therefore, aim of the current study was to investigate the association of Hcy with the prevalence and extent of CAD in a large consecutive cohort of patients undergoing coronary angiography. Methods: Our population is represented by a total of 3056 consecutive patients undergoing coronary angiography between at the Azienda Ospedaliera “Maggiore della Carità”, Novara, Italy. Fasting samples were collected for homocysteine levels assessment. Coronary disease was defined for at least 1 vessel stenosis N 50% as evaluated by QCA. Results: Study population was divided according to Hcy tertiles (b13,3, 13,3–18.2, N18.2 nmol/ml). High plasmatic level of homocysteine was related with age (p b 0.001), male gender (p b 0.001), hypertension (p b 0.001) renal failure (p b 0.001), family history of CAD (p b 0.001), previous cerebrovascular accident (p b 0.001), previous MI (p = 0.002), previous CABG (p = 0.003), ejection fraction (p b 0.001), higher baseline creatinine (p b 0.001), in treatment with nitrates (p b 0.001), calcium antagonists (p b 0.001), diuretics (p b 0.001), Ace inhibitors (ACE-I) (p = 0.006), Clopidogrel (p = 0.05), haemoglobin (p = 0.001), white blood cells (WBC) count (p = 0.008), total cholesterol (p = 0.04), Low-Density Lipoproteins (LDL) (p = 0.01). A significant relationship was found between Hcy levels and the extent of coronary artery disease (71.8% vs 77.8% vs 77.4%, OR[95%CI] = 1.18[1.11-1.252.], p b 0.001 and severe CAD (23.6% vs 29.5% vs 32.1%, OR [95%CI] = 1.275 [1.209-1.344], p b 0.001). Elevated Hcy was significantly associated with increased risk of CAD (adjusted OR [95%CI] = 1.087[1.009-1.171], p = 0.02 and severe CAD (adjusted OR [95%CI] = 1.07 [1.01-1.16, P = 0.04]). The results were confirmed in the majority of high risk subsets of patients. Conclusions: This study showed that high levels of plasmatic Hcy are independently associated with CAD. Further large studies are certainly needed to explore the adjunctive benefits from vitamin administration in patients with elevated Hcy to prevent the occurrence and progression of CAD. © 2014 Elsevier Ltd. All rights reserved.
WEAKNESS No data on clinical outcome were provided. The use of intravascular ultrasound might have improved the evaluation and quantification of coronary atherosclerosis.
STRENGHTS Large cohort study Evaluation of CAD by the use of coronary angiography
Summary ⁎ Corresponding author at: Ospedale “Maggiore della Carità”, Eastern Piedmont University, C.so Mazzini, 18, 28100 Novara, Italy. Tel.: + 39 0321 3733141; fax: + 39 0321 3733407. E-mail address: [email protected] (G. De Luca).
● Whether plasmatic total homocysteine (Hcy) is an independent predictor of cardiovascular disease or a bystander phenomenon, is still a matter of debated.
http://dx.doi.org/10.1016/j.thromres.2014.05.025 0049-3848/© 2014 Elsevier Ltd. All rights reserved.
Please cite this article as: Schaffer A, et al, Relationship between homocysteine and coronary artery disease. Results from a large prospective cohort study, Thromb Res (2014), http://dx.doi.org/10.1016/j.thromres.2014.05.025
2
A. Schaffer et al. / Thrombosis Research xxx (2014) xxx–xxx
● Present study is one of the largest reports aiming at defining the role of Hcy in coronary atherosclerosis in a large cohort study. ● Our main finding is that Hcy independently associated with CAD Introduction Cardiovascular disease still represents the leading cause of mortality in developed country. In fact, despite the great reduction in mortality achieved by the improvement in myocardial revascularization techniques [1–4], the results are still unsatisfactory in high-risk subgroups of patients [5,6]. Therefore, large interests have been recently focused on the identification of new risk factors for CAD and its prevention. Hcy is an amino acid which is metabolized either by the remethylation pathway to methionine or the trans-sulfuration pathway to cysteine, each pathway depends upon other series of biochemical enzymes as methionine synthetase (MS) and methylene tetrahydrofolate reductase (MTHFR) as well as on vitamin B12 and folic acid. A later pathway is dependent on the enzyme cystathionine beta synthetase (CBS) and pyridoxine (vitamin B6).Any loss of one of those pathways and / or a dietary deficiency can result in hyperhomocysteinemia. In fact, epidemiological studies have demonstrated that atherosclerosis plaques progression is correlated to elevated circulating Hcy due to increased thrombogenicity, oxidative stress status and endothelial dysfunction [7,8] However, the progressive contribution of hyperhomocysteinemia on coronary disease is still under debate. Therefore, aim of our study was to investigate the relationship between Hcy and the prevalence and extent of coronary artery disease in a large consecutive cohort of patients undergoing coronary angiography. Methods Our population is represented by a consecutive cohort of patients undergoing coronary angiography at Azienda Ospedaliera-Universitaria, “Maggiore della Carità”, Novara, Italy from March 2007 and October 2013. All demographic and clinical data were collected after obtaining written informed consent from the patient and included in a dedicated database. No exclusion criteria were applied. Hypertension was defined as systolic pressure N 140 mm Hg and/or diastolic pressure was N 90 mm Hg or if the individual was taking antihypertensive medications. The diagnosis of diabetes was based on previous history of diabetes treated with or without drug therapies, fasting glycaemia N 126 mg/dL, random glycaemia N 200 mg/dL or HbA1c N 6.5%. ACS was defined as an elevation of cardiac biomarkers beyond the upper limit of normal (ULN) (respectively 0,04 μg/l for Troponin I and 5,00 μg/l for CK-MB) due to angiographically documented critical coronary stenosis (N70%). Biochemical measurements Blood samples were drawn at admission in patients undergoing elective (following a fasting period of 12 h) or urgent coronary angiography. Glucose, Creatinine, blood count and lipid profile were determined by standard methods [9]. Plasma homocysteine was measured using a competitive immunoassay with direct chemiluminescence detection. All the assays were performed on Siemens ADVIA Centaur using ADVIA Centaur HCY Ready Pack. The lower detection limit was 0,5 μmol/L. The upper limit for method linearity without dilution was 65 μmol/L. The analytical variability was 7%. Coronary angiography Coronary angiography was routinely performed, preferring radial approach, using 6-French right and left heart catheters. Quantitative coronary angiography was performed by experienced interventional
cardiologists by automatic edge-detection systems (Siemens Acom Quantcor QCA, Erlangen, Germany), as previously described [10]. Significant coronary artery disease was defined as at least 1 coronary stenosis more than 50%, while severe coronary disease was defined as threevessel disease and/or left main disease, according to literature definition [11].
Statistical analysis Statistical analysis was performed by the use of the SPSS 17.0 statistical software. Continuous data were expressed as mean + SD and categorical data as percentage. Analysis of variance and chi-square test were used for continuous and categorical variables, respectively. Patients were grouped according to tertiles of Hcy. Multiple logistic regression analysis was performed to evaluate the relationship between Hcy and coronary artery disease, after correction for baseline confounding factors that were entered in the model in block. A p value b 0.05 was considered statistically significant.
Results Our population is represented by 3056 consecutive patients undergoing coronary angiography. Patients were divided in three groups according to Hcy tertiles (b13,3, 13,3–18.2, ≥ 18.2 nmol/ml). Baseline characteristics are shown in Table 1. Higher Hcy plasmatic levels were associated with age (p b 0.001), male gender (p b 0.001), hypertension (p b 0.001), renal failure (p b 0.001), family history of CAD (p b 0.001), previous CVA (p b 0.001), previous MI (p b 0.001), previous CABG (p = 0.003), ejection fraction (p b 0.001), higher baseline creatinine (p b 0.001), haemoglobin (p = 0.001), WBC count (p = 0.008), total cholesterol (p = 0.04), LDL (p = 0.01). Patients with higher Hcy levels were more often on nitrates (p b 0.001), calcium antagonists (p b 0.001), diuretics (p b 0.001), ace inhibitors (ACE-I) (p = 0.006), Clopidogrel (p = 0.05). Hcy levels were significantly associated with prevalence of coronary artery disease (71.8% vs 77.8% vs 77.4%, OR [95%CI] = 1.18 [1.11-1.252.], p b 0.001, Fig. 1). Similar results were also observed for severe CAD (23.6% vs 29.5% vs 32.1%, OR [95%CI] =1.275 [1.209-1.344], p b 0.001), Fig. 2) (See Table 2). This association was confirmed after correction for confounding factors (age, gender, hypertension, renal failure, family history of CAD, previous CVA, previous MI, previous CABG, ejection fraction, higher baseline creatinine, haemoglobin, WBC count, total cholesterol, LDL cholesterol, nitrates, calcium antagonists, diuretics, ace inhibitors, Clopidogrel) for both CAD (adjusted OR[95%CI] = 1.087[1.01-1.171], p = 0.02) and severe CAD (adjusted OR [95%CI] = 1.07 [1.01-1.16, P = 0.04]. Our results were confirmed at subgroup analysis of higher risk subsets of patients. As shown in Fig. 3, we found an impact of Hcy elevation (above the median, 15.7 nmol/ml) on the risk of CAD in patients presenting with acute coronary syndrome and in electives (elective patients: OR [95% CI] = 1.09 [0.98-1.22], p = 0.13; ACS: OR [95% CI] = 1.83 [1.47 -2.27], p b 0.001, p interaction =0.09), according to patients’ age (N75 years: OR [95% CI] = 1.03[0.87-1.2], p = 0.67; b75 years: OR [95% CI] = 1.18 [1.05-1.3], p = 0.003, p int = 0.22), renal function (renal failure: OR [95% CI] = 1.26 [1.05-1.53], p = 0.01; normal renal function (OR [95% CI] = 1.08 [0.9-1.12], p = 0.13, p int = 0.17), arterial hypertension (hypertensive status (OR [95% CI] = 1.04 [0.93-1.16], p = 0.45; normal arterial pressure OR [95% CI] = 1.26 [1.06-1.49], p = 0.006, p int = 0.07) and left ventricular dysfunction (EF b 35%: OR [95% CI] = 1.25 [1.01-1.54], p = 0.04; EF N 35%: OR [95% CI] = 1.37 [1.21-1.52], p b 0.001, p int = 0.23), and according to diabetic status (diabetes (OR [95% CI] = 1.33 [0.89-1.96], p = 0.39; non diabetics (OR [95% CI] = 1.44 [1.28-1.6], p b 0.001, p interaction = 0.19).
Please cite this article as: Schaffer A, et al, Relationship between homocysteine and coronary artery disease. Results from a large prospective cohort study, Thromb Res (2014), http://dx.doi.org/10.1016/j.thromres.2014.05.025
A. Schaffer et al. / Thrombosis Research xxx (2014) xxx–xxx
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Table 1 Baseline clinical features. Baseline clinical features
b13.3 nmol/ml n = 997
13,3–18.2 nmol/ml n = 1007
N18.2 nmol/ml n = 1044
P
Age (mean ± SD) Male Sex (%) Arterial hypertension (%) Hypercholesterolemia (%) Diabetes (%) Renal failure (%) Family history of CAD (%) Smokers (%) Active smokers Previous smokers History of MI (%) Previous PCI (%) Previous CABG (%) Previous CVA (%) Ejection fraction (%, mean ± SD) Indication to angiography Stable angina/ silent ischemia (%) ACS (%) DCM – Heart valve disease (%)
64,28 ± 11,2 61,6 65,8 56,2 36,8 7,2 33,4
67,4 ± 11, 71,9 72,6 59,7 38,8 13 29,3
71,2 ± 9,9 73,3 79,8 53,9 35,2 29,5 23,9
b0,001 b0,001 b0,001 0,292 0,453 b0,001 b0,001 0,17
26,5 19,1 21 23,5 10,1 4,1 52.8 ± 12.2
25,6 21 23,8 24,7 11,5 7,4 49.8 ± 13.3
22,6 23,4 28,4 24,5 14,4 9,5 46.7 ± 14.5
22,1 65,3 12,6
25 56,7 18,2
20,7 53,3 26
Therapy at admission ACE inhibitors (%) ARB (%) Statins (%) Beta-blockers (%) Nitrates (%) Calcium antagonists (%) Diuretics (%) ASA (%) Clopidogrel (%) Ticlopidine (%)
35,8 18,1 49,8 50,2 30 16,7 22,8 56 22,7 5,7
38,7 23,4 51,6 51,7 35,8 20 28,9 61,5 24,2 6,1
41,7 21,6 47,7 53,9 41,4 24,5 41,1 58,5 22,3 7,8
0,006 0,06 0,3 0,09 b0,001 b0,001 b0,001 0,26 0,8 0,05
Baseline blood features Glycemia (mg/dl, mean ± SD) HbA1c (mg/dl, mean ± SD) Creatinine (mg/dl, mean ± SD) Total cholesterol (mg/dl, mean ± SD) HDL-C (mg/dl, mean ± SD) LDL-C (mg/dl, mean ± SD) Triglycerides (mg/dl, mean ± SD) Uric acid (mg/dl, mean ± SD) C-reactive protein (mg/dl, mean ± SD) Haemoglobin (g/dl , mean ± SD) Platelets (mg/dl, mean ± SD) White blood cells (×10 6/μL,mean ± SD)
127,9 ± 53,8 6,39 ± 4,1 0,95 ± 0,6 160 ± 42 40,96 ± 12,8 99 ± 53 132 ± 78,5 5,94 ± 6,1 1,49 ± 9,7 13,2 ± 1,6 216 ± 60 7,92 ± 2,8
124,9 ± 49,8 6,33 ± 2,2 1,±0,49 165 ± 41,1 41 ± 11,8 105,3 ± 55 138 ± 97,2 6,3 ± 3,87 1,21 ± 2,4 13,5 ± 1,65 216 ± 68,4 8 ± 3,4
122,2 ± 19 6,19 ± 1,3 1,34 ± 0,95 162 ± 41,5 40,7 ± 13 105,2 ± 49,6 133,5 ± 75,6 7,31 ± 7,8 1,41 ± 2,7 13,29 ± 1,89 216,9 ± 68,8 7,65 ± 2,4
0.44 0.265 b0,001 0.04 0.86 0.01 0.23 b0,001 0.55 0,001 0.98 0.008
0,002 0,6 0,003 b0,001 b0,001 b0,001
CAD = Coronary Artery Disease; MI = Myocardial Infarction; CVA = Cerebrovascular Accident; PCI = Percutaneous Coronary Interventions; CABG = Coronary Artery Bypass Grafting; STEMI = ST-Elevation Myocardial Infarction; ACS = Acute Coronary Syndrome; CMD = Dilated Cardiomiopathy; LV = Left Ventricle; EF = Ejection Fraction; ACE = Angiotensin Converting Enzyme; ARB = Angiotensin Receptor Blockers; ASA = Acetylsalicylic Acid; LDL = Low-Density Lipoproteins, HbA1c = Glycated hemoglobin
Discussion Present study represents an attempt to define a role of plasmatic levels and the extent of coronary atherosclerosis in a large consecutive population of patients undergoing coronary angiography. Our main finding is that elevated Hcy was significantly associated with increased risk of CAD and severe CAD. In the last decades great efforts have been done in order to improve antithrombotic and revascularization therapies, and reduce cardiovascular mortality, especially in the setting of ACS [12]. However, suboptimal results are still observed in some high-risk patients [13–15]. Therefore, large attention has been paid to the identification of new risk factors aiming to prevent CAD. Arteriosclerosis is a continuous inflammatory damage to the arterial intima with increased permeability to plasma, deposition of plasma lipids in plaques and fibrosis and calcification of plaques. Hcy increases vascular oxidative burden and increase of superoxide radicals production leading to a lower NO bioavailability and endothelial dysfunction [16,17], favouring inflammatory and pro-atherogenic mechanisms inside the vascular wall [18] leading eventually to thrombotic events [19].
Despite that, controversy still exists about the association between elevated Hcy and coronary artery disease. Large interests have been focused on Hcy starting from 1969 when McCully demonstrated the association between high Hcy plasmatic concentration and arteriosclerosis [20]. In the early nineties, Clarke et.al has demonstrated in a small study group of subjects that hyperhomocysteinemia could be considered as an independent risk factor for vascular disease, including coronary disease [21]. The Framingham risk score (FRS), known as an important instrument in predicting CAD in patients with traditional risk factors, such as dyslipidemia, hypertension, diabetes mellitus (DM), and smoking, however, underestimated the CAD risk in individuals with high Hcy plasmatic levels [22]. Similar evidence was observed in a recent meta analysis of 2 large studies with a total of 6,814 individuals conducted by Veeranna et.al, who demonstrated an enhanced risk prediction of CVD when adding plasma Hcy levels to the FRS [23]. Furthermore, other studies, have evaluated the contribution of some genetic polymorphisms of 5,10-methylenetetrahydrofolate reductase (MTHFR) enzyme on Hcy functionality. In 2002, Klerk et.al, In a large meta analysis containing 40 observational studies with a total of 11,162 cases and 12,758 controls, concluded that MTHFR enzyme TT
Please cite this article as: Schaffer A, et al, Relationship between homocysteine and coronary artery disease. Results from a large prospective cohort study, Thromb Res (2014), http://dx.doi.org/10.1016/j.thromres.2014.05.025
4
A. Schaffer et al. / Thrombosis Research xxx (2014) xxx–xxx
Table 2 Angiographic features according to Hcy tertiles. Angiographic Characteristics
b13,3 nmol/ml n = 997
13,3–18,2 nmol/ml n = 1007
N18,2 nmol/ml n = 1044
P
Multivessel disease (%) Severe CAD (%) Left main disease (%) Left anterior descending disease (%) Left Circumflex artery disease (%) Antero-lateral branch (%) Right artery disease (%) Lesion length (mm ± SD) Target Vessel diameter (mm) % Stenosis Type C lesions (%) Calcifications (%) Thrombus (%) Instent restenosis (%) Chronic occlusion (%) Bifurcations (%) Prossimal vessel tortuosity (%) Thrombus (%) TIMI flow TIMI 0-1 (%) TIMI 2 (%) TIMI 3 (%) CMD (%)
39 23,6 8,3 45,5 36,2 5,7 42.8 19,8 ± 12,9 2,9 ± 0,6 86 ± 15,1 32,9 17,7 5,9 5,8 15,4 21,2 4,1 5,9
48,3 29,5 8,6 52,7 43,4 5,5 49.8 20,3 ± 13,3 2,99 ± 1,6 86 ± 14,7 36,5 22,6 4,1 4,5 18,9 21,7 4 4,1
49,8 32,1 12,4 56,8 42,8 5,8 48.2 19,3 ± 12,3 2,95 ± 1 86 ± 15,2 32,9 27,2 2,8 3,3 19,9 21,5 3,7 2,8
b0,001 b0,001 0,002 b0,001 0,003 0,8 0.017 0,12 0,33 0,62 0,82 b0,001 b0,001 b0,001 0,002 0,86 0,48 b0,001 0,7
2,8 3,7 71,6 10,6
3,4 5,9 69,9 18,7
2,7 4,5 70,7 27,1
b0,001
§ per patient definition. CAD = Coronary Artery Disease; PCI = Percutaneous Coronary Intervention. RCA = Right Coronary Artery, LM = Left Main; TIMI = Thrombolysis in Myocardial Infarction.
genotype had significantly higher risk of CAD particularly in the subjects with low folate status [24]. Similar results, also in 2002, were obtained by Wald et.al, in a large meta analysis of 72 studies with a total of 16,849 subjects strengthening the evidence between raised serum homocysteine concentration and cardiovascular disease [25]. On the contrary, a recent large metaanalysis including a total of 48,175 coronary heart disease cases and 67,961 controls, conducted in order to investigate the causality between moderately elevated blood levels of homocysteine with coronary heart disease in MTHFR enzyme TT genotype, has found a non significant effect on coronary heart disease risk [26]. However, in this meta-analysis, the definition of cardiovascular disease differed among the included studies, with not all patients undergoing coronary angiography, and therefore potential misclassification of patients could have occurred, as the absence of symptoms could not have excluded with certainty the absence of coronary artery disease. In addition, this meta-analysis have included many small studies, with heterogeneous population. Present study is the largest cohort study where the association between Hcy and coronary artery disease was assessed, with all patients
undergoing a coronary angiography. Our main finding is that high plasmatic levels of Hcy are associated with the prevalence and severity of CAD. Our results were confirmed even after correction for baseline differences, suggesting an independent role of Hcy in the prediction of CAD risk. Moreover, this positive association was confirmed even in the majority of high risk subsets of patients. However, there is still contrasting results upon the beneficial effects of Hcy-lowering therapy by vitamin B6, B12 and folate administration. In particular, a recent Cochrane review, has concluded that there are no apparent benefits in terms of prevention of long-term cardiovascular events [27]. Other studies, have even shown an harmful effects on in-stent restenosis, especially among patients with baseline Hcy within normality [28,29]. However, few data have evaluated so far the role of Hcy and the effects of Hcy-lowering therapy in patients at higher risk for CAD. Subgroup analyses conducted in our study showed that elevated Hcy was associated with higher risk of CAD in patients with chronic renal dysfunction In fact, a recent meta-analysis has found significant beneficial effects from folate in such a population [30]. In addition, we found a slightly 35
P = 0.003
80
30
Severe CAD (%)
75 70
CAD (%)
P < 0.001
65
25 20 15 10
60 5 55 0 I TERT
50 I TERT
II TERT
III TERT
II TERT
III TERT
Homocysteine (nmol/ml)
Homocysteine (nmol/ml) Fig. 1. Bar graph shows the prevalence of coronary disease according to Hcy tertiles.
Fig. 2. Bar graph shows the prevalence of severe (left main and/or trivessel) coronary disease according to Hcy tertiles.
Please cite this article as: Schaffer A, et al, Relationship between homocysteine and coronary artery disease. Results from a large prospective cohort study, Thromb Res (2014), http://dx.doi.org/10.1016/j.thromres.2014.05.025
A. Schaffer et al. / Thrombosis Research xxx (2014) xxx–xxx
Risk of CAD OR [95%CI]
P value
All patients (n=3056)
0.02
Age>75 (n=956)
0.67
Age< 75 (n=2100)
0.03
Hypertension (n=2222)
0.45
No hypertension (n=834)
0.06
CKD (n=847)
0.01
No CKD (n=2209)
0.13 0.13
Elective (n=1950) EF < 35% (n=579)
0.04
EF > 35% (n=2477)
Contents lists available at ScienceDirect
Thrombosis Research journal homepage: www.elsevier.com/locate/thromres
Regular Article
Relationship between homocysteine and coronary artery disease. Results from a large prospective cohort study Alon Schaffer a, Monica Verdoia a, Ettore Cassetti a, Paolo Marino a, Harry Suryapranata b, Giuseppe De Luca a,⁎, on behalf of the Novara Atherosclerosis Study Group (NAS) a b
Division of Cardiology, Azienda Ospedaliera-Universitaria “Maggiore della Carità”, Eastern Piedmont University, Novara, Italy Department of Cardiology, UMC St Radboud, Nijmegen, The Netherlands (HS)
a r t i c l e
i n f o
Article history: Received 5 March 2014 Received in revised form 7 May 2014 Accepted 18 May 2014 Available online xxxx Keywords: Homocysteine Coronary atherosclerosis Coronary angiography
a b s t r a c t Background: Coronary artery disease (CAD) still represents the major cause of mortality in developed countries. Large research programs have been focused on the identification of new risk factors to prevent CAD, with special attention to homocysteine (Hcy), due to the known associated increased thrombogenicity, oxidative stress status and endothelial dysfunction. However, controversy still exists on the association between Hcy and CAD. Therefore, aim of the current study was to investigate the association of Hcy with the prevalence and extent of CAD in a large consecutive cohort of patients undergoing coronary angiography. Methods: Our population is represented by a total of 3056 consecutive patients undergoing coronary angiography between at the Azienda Ospedaliera “Maggiore della Carità”, Novara, Italy. Fasting samples were collected for homocysteine levels assessment. Coronary disease was defined for at least 1 vessel stenosis N 50% as evaluated by QCA. Results: Study population was divided according to Hcy tertiles (b13,3, 13,3–18.2, N18.2 nmol/ml). High plasmatic level of homocysteine was related with age (p b 0.001), male gender (p b 0.001), hypertension (p b 0.001) renal failure (p b 0.001), family history of CAD (p b 0.001), previous cerebrovascular accident (p b 0.001), previous MI (p = 0.002), previous CABG (p = 0.003), ejection fraction (p b 0.001), higher baseline creatinine (p b 0.001), in treatment with nitrates (p b 0.001), calcium antagonists (p b 0.001), diuretics (p b 0.001), Ace inhibitors (ACE-I) (p = 0.006), Clopidogrel (p = 0.05), haemoglobin (p = 0.001), white blood cells (WBC) count (p = 0.008), total cholesterol (p = 0.04), Low-Density Lipoproteins (LDL) (p = 0.01). A significant relationship was found between Hcy levels and the extent of coronary artery disease (71.8% vs 77.8% vs 77.4%, OR[95%CI] = 1.18[1.11-1.252.], p b 0.001 and severe CAD (23.6% vs 29.5% vs 32.1%, OR [95%CI] = 1.275 [1.209-1.344], p b 0.001). Elevated Hcy was significantly associated with increased risk of CAD (adjusted OR [95%CI] = 1.087[1.009-1.171], p = 0.02 and severe CAD (adjusted OR [95%CI] = 1.07 [1.01-1.16, P = 0.04]). The results were confirmed in the majority of high risk subsets of patients. Conclusions: This study showed that high levels of plasmatic Hcy are independently associated with CAD. Further large studies are certainly needed to explore the adjunctive benefits from vitamin administration in patients with elevated Hcy to prevent the occurrence and progression of CAD. © 2014 Elsevier Ltd. All rights reserved.
WEAKNESS No data on clinical outcome were provided. The use of intravascular ultrasound might have improved the evaluation and quantification of coronary atherosclerosis.
STRENGHTS Large cohort study Evaluation of CAD by the use of coronary angiography
Summary ⁎ Corresponding author at: Ospedale “Maggiore della Carità”, Eastern Piedmont University, C.so Mazzini, 18, 28100 Novara, Italy. Tel.: + 39 0321 3733141; fax: + 39 0321 3733407. E-mail address: [email protected] (G. De Luca).
● Whether plasmatic total homocysteine (Hcy) is an independent predictor of cardiovascular disease or a bystander phenomenon, is still a matter of debated.
http://dx.doi.org/10.1016/j.thromres.2014.05.025 0049-3848/© 2014 Elsevier Ltd. All rights reserved.
Please cite this article as: Schaffer A, et al, Relationship between homocysteine and coronary artery disease. Results from a large prospective cohort study, Thromb Res (2014), http://dx.doi.org/10.1016/j.thromres.2014.05.025
2
A. Schaffer et al. / Thrombosis Research xxx (2014) xxx–xxx
● Present study is one of the largest reports aiming at defining the role of Hcy in coronary atherosclerosis in a large cohort study. ● Our main finding is that Hcy independently associated with CAD Introduction Cardiovascular disease still represents the leading cause of mortality in developed country. In fact, despite the great reduction in mortality achieved by the improvement in myocardial revascularization techniques [1–4], the results are still unsatisfactory in high-risk subgroups of patients [5,6]. Therefore, large interests have been recently focused on the identification of new risk factors for CAD and its prevention. Hcy is an amino acid which is metabolized either by the remethylation pathway to methionine or the trans-sulfuration pathway to cysteine, each pathway depends upon other series of biochemical enzymes as methionine synthetase (MS) and methylene tetrahydrofolate reductase (MTHFR) as well as on vitamin B12 and folic acid. A later pathway is dependent on the enzyme cystathionine beta synthetase (CBS) and pyridoxine (vitamin B6).Any loss of one of those pathways and / or a dietary deficiency can result in hyperhomocysteinemia. In fact, epidemiological studies have demonstrated that atherosclerosis plaques progression is correlated to elevated circulating Hcy due to increased thrombogenicity, oxidative stress status and endothelial dysfunction [7,8] However, the progressive contribution of hyperhomocysteinemia on coronary disease is still under debate. Therefore, aim of our study was to investigate the relationship between Hcy and the prevalence and extent of coronary artery disease in a large consecutive cohort of patients undergoing coronary angiography. Methods Our population is represented by a consecutive cohort of patients undergoing coronary angiography at Azienda Ospedaliera-Universitaria, “Maggiore della Carità”, Novara, Italy from March 2007 and October 2013. All demographic and clinical data were collected after obtaining written informed consent from the patient and included in a dedicated database. No exclusion criteria were applied. Hypertension was defined as systolic pressure N 140 mm Hg and/or diastolic pressure was N 90 mm Hg or if the individual was taking antihypertensive medications. The diagnosis of diabetes was based on previous history of diabetes treated with or without drug therapies, fasting glycaemia N 126 mg/dL, random glycaemia N 200 mg/dL or HbA1c N 6.5%. ACS was defined as an elevation of cardiac biomarkers beyond the upper limit of normal (ULN) (respectively 0,04 μg/l for Troponin I and 5,00 μg/l for CK-MB) due to angiographically documented critical coronary stenosis (N70%). Biochemical measurements Blood samples were drawn at admission in patients undergoing elective (following a fasting period of 12 h) or urgent coronary angiography. Glucose, Creatinine, blood count and lipid profile were determined by standard methods [9]. Plasma homocysteine was measured using a competitive immunoassay with direct chemiluminescence detection. All the assays were performed on Siemens ADVIA Centaur using ADVIA Centaur HCY Ready Pack. The lower detection limit was 0,5 μmol/L. The upper limit for method linearity without dilution was 65 μmol/L. The analytical variability was 7%. Coronary angiography Coronary angiography was routinely performed, preferring radial approach, using 6-French right and left heart catheters. Quantitative coronary angiography was performed by experienced interventional
cardiologists by automatic edge-detection systems (Siemens Acom Quantcor QCA, Erlangen, Germany), as previously described [10]. Significant coronary artery disease was defined as at least 1 coronary stenosis more than 50%, while severe coronary disease was defined as threevessel disease and/or left main disease, according to literature definition [11].
Statistical analysis Statistical analysis was performed by the use of the SPSS 17.0 statistical software. Continuous data were expressed as mean + SD and categorical data as percentage. Analysis of variance and chi-square test were used for continuous and categorical variables, respectively. Patients were grouped according to tertiles of Hcy. Multiple logistic regression analysis was performed to evaluate the relationship between Hcy and coronary artery disease, after correction for baseline confounding factors that were entered in the model in block. A p value b 0.05 was considered statistically significant.
Results Our population is represented by 3056 consecutive patients undergoing coronary angiography. Patients were divided in three groups according to Hcy tertiles (b13,3, 13,3–18.2, ≥ 18.2 nmol/ml). Baseline characteristics are shown in Table 1. Higher Hcy plasmatic levels were associated with age (p b 0.001), male gender (p b 0.001), hypertension (p b 0.001), renal failure (p b 0.001), family history of CAD (p b 0.001), previous CVA (p b 0.001), previous MI (p b 0.001), previous CABG (p = 0.003), ejection fraction (p b 0.001), higher baseline creatinine (p b 0.001), haemoglobin (p = 0.001), WBC count (p = 0.008), total cholesterol (p = 0.04), LDL (p = 0.01). Patients with higher Hcy levels were more often on nitrates (p b 0.001), calcium antagonists (p b 0.001), diuretics (p b 0.001), ace inhibitors (ACE-I) (p = 0.006), Clopidogrel (p = 0.05). Hcy levels were significantly associated with prevalence of coronary artery disease (71.8% vs 77.8% vs 77.4%, OR [95%CI] = 1.18 [1.11-1.252.], p b 0.001, Fig. 1). Similar results were also observed for severe CAD (23.6% vs 29.5% vs 32.1%, OR [95%CI] =1.275 [1.209-1.344], p b 0.001), Fig. 2) (See Table 2). This association was confirmed after correction for confounding factors (age, gender, hypertension, renal failure, family history of CAD, previous CVA, previous MI, previous CABG, ejection fraction, higher baseline creatinine, haemoglobin, WBC count, total cholesterol, LDL cholesterol, nitrates, calcium antagonists, diuretics, ace inhibitors, Clopidogrel) for both CAD (adjusted OR[95%CI] = 1.087[1.01-1.171], p = 0.02) and severe CAD (adjusted OR [95%CI] = 1.07 [1.01-1.16, P = 0.04]. Our results were confirmed at subgroup analysis of higher risk subsets of patients. As shown in Fig. 3, we found an impact of Hcy elevation (above the median, 15.7 nmol/ml) on the risk of CAD in patients presenting with acute coronary syndrome and in electives (elective patients: OR [95% CI] = 1.09 [0.98-1.22], p = 0.13; ACS: OR [95% CI] = 1.83 [1.47 -2.27], p b 0.001, p interaction =0.09), according to patients’ age (N75 years: OR [95% CI] = 1.03[0.87-1.2], p = 0.67; b75 years: OR [95% CI] = 1.18 [1.05-1.3], p = 0.003, p int = 0.22), renal function (renal failure: OR [95% CI] = 1.26 [1.05-1.53], p = 0.01; normal renal function (OR [95% CI] = 1.08 [0.9-1.12], p = 0.13, p int = 0.17), arterial hypertension (hypertensive status (OR [95% CI] = 1.04 [0.93-1.16], p = 0.45; normal arterial pressure OR [95% CI] = 1.26 [1.06-1.49], p = 0.006, p int = 0.07) and left ventricular dysfunction (EF b 35%: OR [95% CI] = 1.25 [1.01-1.54], p = 0.04; EF N 35%: OR [95% CI] = 1.37 [1.21-1.52], p b 0.001, p int = 0.23), and according to diabetic status (diabetes (OR [95% CI] = 1.33 [0.89-1.96], p = 0.39; non diabetics (OR [95% CI] = 1.44 [1.28-1.6], p b 0.001, p interaction = 0.19).
Please cite this article as: Schaffer A, et al, Relationship between homocysteine and coronary artery disease. Results from a large prospective cohort study, Thromb Res (2014), http://dx.doi.org/10.1016/j.thromres.2014.05.025
A. Schaffer et al. / Thrombosis Research xxx (2014) xxx–xxx
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Table 1 Baseline clinical features. Baseline clinical features
b13.3 nmol/ml n = 997
13,3–18.2 nmol/ml n = 1007
N18.2 nmol/ml n = 1044
P
Age (mean ± SD) Male Sex (%) Arterial hypertension (%) Hypercholesterolemia (%) Diabetes (%) Renal failure (%) Family history of CAD (%) Smokers (%) Active smokers Previous smokers History of MI (%) Previous PCI (%) Previous CABG (%) Previous CVA (%) Ejection fraction (%, mean ± SD) Indication to angiography Stable angina/ silent ischemia (%) ACS (%) DCM – Heart valve disease (%)
64,28 ± 11,2 61,6 65,8 56,2 36,8 7,2 33,4
67,4 ± 11, 71,9 72,6 59,7 38,8 13 29,3
71,2 ± 9,9 73,3 79,8 53,9 35,2 29,5 23,9
b0,001 b0,001 b0,001 0,292 0,453 b0,001 b0,001 0,17
26,5 19,1 21 23,5 10,1 4,1 52.8 ± 12.2
25,6 21 23,8 24,7 11,5 7,4 49.8 ± 13.3
22,6 23,4 28,4 24,5 14,4 9,5 46.7 ± 14.5
22,1 65,3 12,6
25 56,7 18,2
20,7 53,3 26
Therapy at admission ACE inhibitors (%) ARB (%) Statins (%) Beta-blockers (%) Nitrates (%) Calcium antagonists (%) Diuretics (%) ASA (%) Clopidogrel (%) Ticlopidine (%)
35,8 18,1 49,8 50,2 30 16,7 22,8 56 22,7 5,7
38,7 23,4 51,6 51,7 35,8 20 28,9 61,5 24,2 6,1
41,7 21,6 47,7 53,9 41,4 24,5 41,1 58,5 22,3 7,8
0,006 0,06 0,3 0,09 b0,001 b0,001 b0,001 0,26 0,8 0,05
Baseline blood features Glycemia (mg/dl, mean ± SD) HbA1c (mg/dl, mean ± SD) Creatinine (mg/dl, mean ± SD) Total cholesterol (mg/dl, mean ± SD) HDL-C (mg/dl, mean ± SD) LDL-C (mg/dl, mean ± SD) Triglycerides (mg/dl, mean ± SD) Uric acid (mg/dl, mean ± SD) C-reactive protein (mg/dl, mean ± SD) Haemoglobin (g/dl , mean ± SD) Platelets (mg/dl, mean ± SD) White blood cells (×10 6/μL,mean ± SD)
127,9 ± 53,8 6,39 ± 4,1 0,95 ± 0,6 160 ± 42 40,96 ± 12,8 99 ± 53 132 ± 78,5 5,94 ± 6,1 1,49 ± 9,7 13,2 ± 1,6 216 ± 60 7,92 ± 2,8
124,9 ± 49,8 6,33 ± 2,2 1,±0,49 165 ± 41,1 41 ± 11,8 105,3 ± 55 138 ± 97,2 6,3 ± 3,87 1,21 ± 2,4 13,5 ± 1,65 216 ± 68,4 8 ± 3,4
122,2 ± 19 6,19 ± 1,3 1,34 ± 0,95 162 ± 41,5 40,7 ± 13 105,2 ± 49,6 133,5 ± 75,6 7,31 ± 7,8 1,41 ± 2,7 13,29 ± 1,89 216,9 ± 68,8 7,65 ± 2,4
0.44 0.265 b0,001 0.04 0.86 0.01 0.23 b0,001 0.55 0,001 0.98 0.008
0,002 0,6 0,003 b0,001 b0,001 b0,001
CAD = Coronary Artery Disease; MI = Myocardial Infarction; CVA = Cerebrovascular Accident; PCI = Percutaneous Coronary Interventions; CABG = Coronary Artery Bypass Grafting; STEMI = ST-Elevation Myocardial Infarction; ACS = Acute Coronary Syndrome; CMD = Dilated Cardiomiopathy; LV = Left Ventricle; EF = Ejection Fraction; ACE = Angiotensin Converting Enzyme; ARB = Angiotensin Receptor Blockers; ASA = Acetylsalicylic Acid; LDL = Low-Density Lipoproteins, HbA1c = Glycated hemoglobin
Discussion Present study represents an attempt to define a role of plasmatic levels and the extent of coronary atherosclerosis in a large consecutive population of patients undergoing coronary angiography. Our main finding is that elevated Hcy was significantly associated with increased risk of CAD and severe CAD. In the last decades great efforts have been done in order to improve antithrombotic and revascularization therapies, and reduce cardiovascular mortality, especially in the setting of ACS [12]. However, suboptimal results are still observed in some high-risk patients [13–15]. Therefore, large attention has been paid to the identification of new risk factors aiming to prevent CAD. Arteriosclerosis is a continuous inflammatory damage to the arterial intima with increased permeability to plasma, deposition of plasma lipids in plaques and fibrosis and calcification of plaques. Hcy increases vascular oxidative burden and increase of superoxide radicals production leading to a lower NO bioavailability and endothelial dysfunction [16,17], favouring inflammatory and pro-atherogenic mechanisms inside the vascular wall [18] leading eventually to thrombotic events [19].
Despite that, controversy still exists about the association between elevated Hcy and coronary artery disease. Large interests have been focused on Hcy starting from 1969 when McCully demonstrated the association between high Hcy plasmatic concentration and arteriosclerosis [20]. In the early nineties, Clarke et.al has demonstrated in a small study group of subjects that hyperhomocysteinemia could be considered as an independent risk factor for vascular disease, including coronary disease [21]. The Framingham risk score (FRS), known as an important instrument in predicting CAD in patients with traditional risk factors, such as dyslipidemia, hypertension, diabetes mellitus (DM), and smoking, however, underestimated the CAD risk in individuals with high Hcy plasmatic levels [22]. Similar evidence was observed in a recent meta analysis of 2 large studies with a total of 6,814 individuals conducted by Veeranna et.al, who demonstrated an enhanced risk prediction of CVD when adding plasma Hcy levels to the FRS [23]. Furthermore, other studies, have evaluated the contribution of some genetic polymorphisms of 5,10-methylenetetrahydrofolate reductase (MTHFR) enzyme on Hcy functionality. In 2002, Klerk et.al, In a large meta analysis containing 40 observational studies with a total of 11,162 cases and 12,758 controls, concluded that MTHFR enzyme TT
Please cite this article as: Schaffer A, et al, Relationship between homocysteine and coronary artery disease. Results from a large prospective cohort study, Thromb Res (2014), http://dx.doi.org/10.1016/j.thromres.2014.05.025
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A. Schaffer et al. / Thrombosis Research xxx (2014) xxx–xxx
Table 2 Angiographic features according to Hcy tertiles. Angiographic Characteristics
b13,3 nmol/ml n = 997
13,3–18,2 nmol/ml n = 1007
N18,2 nmol/ml n = 1044
P
Multivessel disease (%) Severe CAD (%) Left main disease (%) Left anterior descending disease (%) Left Circumflex artery disease (%) Antero-lateral branch (%) Right artery disease (%) Lesion length (mm ± SD) Target Vessel diameter (mm) % Stenosis Type C lesions (%) Calcifications (%) Thrombus (%) Instent restenosis (%) Chronic occlusion (%) Bifurcations (%) Prossimal vessel tortuosity (%) Thrombus (%) TIMI flow TIMI 0-1 (%) TIMI 2 (%) TIMI 3 (%) CMD (%)
39 23,6 8,3 45,5 36,2 5,7 42.8 19,8 ± 12,9 2,9 ± 0,6 86 ± 15,1 32,9 17,7 5,9 5,8 15,4 21,2 4,1 5,9
48,3 29,5 8,6 52,7 43,4 5,5 49.8 20,3 ± 13,3 2,99 ± 1,6 86 ± 14,7 36,5 22,6 4,1 4,5 18,9 21,7 4 4,1
49,8 32,1 12,4 56,8 42,8 5,8 48.2 19,3 ± 12,3 2,95 ± 1 86 ± 15,2 32,9 27,2 2,8 3,3 19,9 21,5 3,7 2,8
b0,001 b0,001 0,002 b0,001 0,003 0,8 0.017 0,12 0,33 0,62 0,82 b0,001 b0,001 b0,001 0,002 0,86 0,48 b0,001 0,7
2,8 3,7 71,6 10,6
3,4 5,9 69,9 18,7
2,7 4,5 70,7 27,1
b0,001
§ per patient definition. CAD = Coronary Artery Disease; PCI = Percutaneous Coronary Intervention. RCA = Right Coronary Artery, LM = Left Main; TIMI = Thrombolysis in Myocardial Infarction.
genotype had significantly higher risk of CAD particularly in the subjects with low folate status [24]. Similar results, also in 2002, were obtained by Wald et.al, in a large meta analysis of 72 studies with a total of 16,849 subjects strengthening the evidence between raised serum homocysteine concentration and cardiovascular disease [25]. On the contrary, a recent large metaanalysis including a total of 48,175 coronary heart disease cases and 67,961 controls, conducted in order to investigate the causality between moderately elevated blood levels of homocysteine with coronary heart disease in MTHFR enzyme TT genotype, has found a non significant effect on coronary heart disease risk [26]. However, in this meta-analysis, the definition of cardiovascular disease differed among the included studies, with not all patients undergoing coronary angiography, and therefore potential misclassification of patients could have occurred, as the absence of symptoms could not have excluded with certainty the absence of coronary artery disease. In addition, this meta-analysis have included many small studies, with heterogeneous population. Present study is the largest cohort study where the association between Hcy and coronary artery disease was assessed, with all patients
undergoing a coronary angiography. Our main finding is that high plasmatic levels of Hcy are associated with the prevalence and severity of CAD. Our results were confirmed even after correction for baseline differences, suggesting an independent role of Hcy in the prediction of CAD risk. Moreover, this positive association was confirmed even in the majority of high risk subsets of patients. However, there is still contrasting results upon the beneficial effects of Hcy-lowering therapy by vitamin B6, B12 and folate administration. In particular, a recent Cochrane review, has concluded that there are no apparent benefits in terms of prevention of long-term cardiovascular events [27]. Other studies, have even shown an harmful effects on in-stent restenosis, especially among patients with baseline Hcy within normality [28,29]. However, few data have evaluated so far the role of Hcy and the effects of Hcy-lowering therapy in patients at higher risk for CAD. Subgroup analyses conducted in our study showed that elevated Hcy was associated with higher risk of CAD in patients with chronic renal dysfunction In fact, a recent meta-analysis has found significant beneficial effects from folate in such a population [30]. In addition, we found a slightly 35
P = 0.003
80
30
Severe CAD (%)
75 70
CAD (%)
P < 0.001
65
25 20 15 10
60 5 55 0 I TERT
50 I TERT
II TERT
III TERT
II TERT
III TERT
Homocysteine (nmol/ml)
Homocysteine (nmol/ml) Fig. 1. Bar graph shows the prevalence of coronary disease according to Hcy tertiles.
Fig. 2. Bar graph shows the prevalence of severe (left main and/or trivessel) coronary disease according to Hcy tertiles.
Please cite this article as: Schaffer A, et al, Relationship between homocysteine and coronary artery disease. Results from a large prospective cohort study, Thromb Res (2014), http://dx.doi.org/10.1016/j.thromres.2014.05.025
A. Schaffer et al. / Thrombosis Research xxx (2014) xxx–xxx
Risk of CAD OR [95%CI]
P value
All patients (n=3056)
0.02
Age>75 (n=956)
0.67
Age< 75 (n=2100)
0.03
Hypertension (n=2222)
0.45
No hypertension (n=834)
0.06
CKD (n=847)
0.01
No CKD (n=2209)
0.13 0.13
Elective (n=1950) EF < 35% (n=579)
0.04
EF > 35% (n=2477)
