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Elevated level of plasma homocysteine in patients with slow coronary flow
International Journal of Cardiology 102 (2005) 419 – 423 www.elsevier.com/locate/ijcard
Elevated level of plasma homocysteine in patients with slow coronary flow Ali Riza Erbaya, Hasan Turhana,*, Ayse Saatci Yasara, Selime Ayazb, Onur Sahina, Kubilay Senena, Hatice Sasmaza, Ertan Yetkinc a Department of Cardiology, Turkiye Yuksek Ihtisas Hospital, Ankara, Turkey Department of Biochemistry, Turkiye Yuksek Ihtisas Hospital, Ankara, Turkey c Department of Cardiology, Inonu University Medical Faculty, Malatya, Turkey b
Received 12 January 2004; received in revised form 7 April 2004; accepted 5 May 2004 Available online 22 September 2004
Abstract Background: Elevated plasma levels of homocysteine are currently considered a major, independent risk factor for cardiovascular diseases. Recently, several investigators have suggested that even mild elevation in plasma homocysteine level can severely disturb vascular endothelial function and subsequently impair coronary blood flow. Accordingly, we investigated plasma homocysteine level in patients with slow coronary flow. Method: Study population included 53 patients with angiographically proven normal coronary arteries and slow coronary flow in all three coronary vessels (group I, 21 females, 32 males, mean age=48F9 years), and 50 subjects with angiographically proven normal coronary arteries without associated slow coronary flow (group II, 22 females, 28 males, mean age=50F8 years). Coronary flow rates of all patients and control subjects were documented by Thrombolysis In Myocardial Infarction frame count (TIMI frame count). All patients in group I had TIMI frame counts greater than two standard deviations above those of control subjects (group II) and, therefore, were accepted as exhibiting slow coronary flow. The mean TIMI frame count for each patient and control subject was calculated by adding the TIMI frame counts for each major epicardial coronary artery and then dividing the obtained value into 3. Plasma homocysteine level was measured in all patients and control subjects using commercially available homocysteine kits. Results: There was no statistically significant difference between two groups in respect to age, gender, hypertension, diabetes mellitus, hyperlipidemia and cigarette smoking ( pN0.05). Plasma homocysteine level of patients with slow coronary flow were found to be significantly higher than those of control subjects (15.5F5.7 vs. 8.7F4.2 AM/l, respectively, pb0.001). Moreover, we found a significant positive correlation between plasma homocysteine level and mean TIMI frame count (r=0.660, pb0.001). Conclusion: We have shown that patients with slow coronary flow have raised level of plasma homocysteine compared to control subjects with normal coronary flow. This data suggests that elevated level of plasma homocysteine may play a role in the pathogenesis of slow coronary flow. D 2004 Elsevier Ireland Ltd. All rights reserved. Keywords: Homocysteine; Slow coronary flow; Endothelial dysfunction
1. Introduction The coronary slow flow phenomenon is an angiographic observation characterized by angiographically normal or * Corresponding author. Tel.: +90 312 2867658. E-mail address: [email protected] (A. Turhan). 0167-5273/$ - see front matter D 2004 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.ijcard.2004.05.064
near-normal coronary arteries with delayed opacification of the distal vasculature. It has been reported that coronary microvascular endothelial dysfunction plays an important pathogenetic role in patients with slow coronary flow [1]. Moreover, myocardial biopsy studies have also revealed the presence of coronary microvascular disease in patients exhibiting coronary slow flow [2,3]. However, the mech-
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anisms responsible for this microvascular endothelial dysfunction in patients with slow coronary flow are not known. Homocysteine is a thiol (sulfhydryl-) containing, potentially cytotoxic, 4-carbon alfa-amino acid that is formed during methionine metabolism [4]. Serum homocysteine concentrations are frequently elevated in the elderly; in individuals deficient in folic acid, cyanocobalamin (vitamin B12) or pyridoxal phosphate (vitamin B6); and in the presence of various enzyme abnormalities including deficiencies in cystathionine h-synthase and thermolability of 5,10-methylenetetrahydrofolate reductase [5,6]. Recent prospective studies show that even mild hyperhomocysteinemia is associated with an increased risk of cardiovascular, cerebrovascular and peripheral vascular disease, independently of classical risk factors [7,8]. Besides, elevated plasma level of homocysteine has been reported to be associated with endothelial dysfunction in animals and humans [9,10]. The coronary microvascular endothelium performs essential control on the coronary blood flow. Injury of the coronary microvascular endothelium leads to vasomotor dysfunction with an impairment of endothelial vasoregulatory mechanisms. Cellular and animal studies indicate that homocysteine reduces the bioavailability of endothelium-derived nitric oxide [11]. Furthermore, several important investigations have shown that elevated plasma homocysteine level in healthy subjects strongly predicts the impaired endothelium-dependent vasodilatation [9,10]. Accordingly, in the present study, we aimed to investigate the plasma homocysteine level in patients with slow coronary flow.
2. Method 2.1. Study population Study population included 53 patients with angiographically proven normal coronary arteries and slow coronary flow in all three coronary vessels (group I, 21 females, 32 males, mean age=48F9 years), and 50 subjects with angiographically proven normal coronary arteries without associated slow coronary flow (group II, 22 females, 28 males, mean age=50F8 years). All patients with slow coronary flow were selected from individuals who underwent coronary angiography in our hospital with a suspicion of coronary artery disease and diagnosed as having angiographically normal coronary arteries. Control group consisted of 50 consecutive subjects with atypical chest pain admitted to the hospital for elective coronary angiography and subsequently found to have angiographically normal coronary arteries. Patients with coronary artery disease, previous history of myocardial infarction, left ventricular dysfunction, echocardiographically proven left ventricular hypertrophy, uncontrolled hypertension and renal dysfunction were not included in the study.
2.2. Documentation of slow coronary flow All patients underwent selective coronary angiography and left ventriculography by means of judkins technique. We used Iohexol (Omnipaque, Nycomed Ireland, Cork, Ireland) as contrast agent during coronary angiography in all patients and control subjects. Coronary flow rates of all subjects were documented by Thrombolysis In Myocardial Infarction frame count (TIMI frame count). TIMI frame count method is a simple, reproducible, objective and quantitative index of coronary flow velocity [12]. It has been suggested that a higher TIMI frame count may reflect disordered resistance vessel function [12]. TIMI frame count was determined for each major coronary artery in each patient and control subject according to the method first described by Gibson et al. [12]. Briefly, the number of cineangiographic frames, recorded at 30 frames/s, required for the leading edge of the column of radiographic contrast to reach a predetermined landmark, is determined. The first frame is defined as the frame in which concentrated dye occupies the full width of the proximal coronary artery lumen, touching both borders of the lumen, and forward motion down the artery. The final frame is designated when the leading edge of the contrast column initially arrives at the distal landmark. In the left anterior descending (LAD) coronary artery, the landmark used is the most distal branch nearest the apex of the left ventricle, commonly referred as the bpitchforkQ or whale’s tail. LAD coronary artery is usually longer than the other major coronary arteries [13], the TIMI frame count for this vessel is often higher. To obtain corrected TIMI frame count for LAD coronary artery, TIMI frame count was divided by 1.7 [12]. The right coronary artery (RCA) distal landmark is the first branch of the posterolateral RCA after the origin of the posterior descending artery, regardless of the size of this branch. The branch of the left circumflex (LCx) artery that encompassed the greatest total distance traveled by contrast was used to define the distal landmark of the LCx artery. TIMI frame count in the LAD and LCx arteries were assessed in a right anterior oblique projection with caudal angulation and RCA in left anterior oblique projection with cranial angulation. The mean TIMI frame count for each patient and control subject was calculated by adding the TIMI frame counts for LAD, LCx and RCA and then dividing the obtained value into 3. Diameters of the epicardial coronary arteries were measured by use of a computerized quantitative coronary angiography analysis system (Vepro, Medimage, The image managing system, Pfungstadt, Germany). 2.3. Diagnostic criteria for slow coronary flow Patients with a corrected TIMI frame count greater than two standard deviations from normal published range for the particular vessel were considered as having slow coronary flow while those whose corrected TIMI frame count fell
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within two standard deviations of the published normal range were labelled to have normal coronary flow. 2.4. Blood sampling and measurement of plasma homocysteine level Blood samples were obtained after an overnight fasting period and were collected in EDTA-treated tubes and immediately centrifuged. Plasma was stored at 70 8C in aliquots until analyses. Fasting plasma homocysteine concentrations were measured with IMX-Abbott machine (LR 30179; 04667-102) by Fluorescence Polarization Immunoassay (FPIA) method using homocysteine kids (Abbott Laboratories, diagnostic division, Abbott Park, IL 60064, USA).
Fig. 1. Correlation of plasma homocysteine and mean TIMI frame count.
3. Results 2.5. Statistical analysis Continuous variables were expressed as meanFS.D. and categorical variables were expressed as percentage. Comparison of categorical and continuous variables between two groups was performed using chi-square test and unpaired ttest, respectively. The correlation between the plasma level of homocysteine and mean TIMI frame count was assessed by the Pearson correlation test. A P-value of b0.05 was considered statistically significant.
Table 1 Comparison of baseline clinical characteristics, plasma homocysteine levels and TIMI frame counts of patients with slow coronary flow and control subjects with normal coronary flow
Age (meanFS.D.) Gender (female/male) Hypertension Systolic BP (mm Hg)a Diastolic BP (mm Hg)a Diabetes mellitus Hyperlipidemia Total cholesterol (mg/dl) LDL-cholesterol (mg/dl) HDL-cholesterol (mg/dl) Triglyceride (mg/dl) Cigarette smoking Homocysteine (Amol/l) TIMI frame count LADb LCx RCA Mean TIMI frame count
Control subjects (n=50)
Patients with slow coronary flow (n=53)
p
50F8 22/28 20/50 (40%) 133F18 80F11 5/50 (10%) 27/50 (54%) 198F24 128F16 42F8 166F19 23/50 (46%) 8.6F4.2
48F9 21/32 19/53 (36%) 134F20 83F11 6/53 (11%) 26/53 (49%) 201F24 132F17 41F7 169F22 22/53 (42%) 15.5F5.7
NS NS NS NS NS NS NS NS NS NS NS NS b0.001
27F6 26F7 27F8 27F6
51F12 46F8 47F11 48F9
b0.001 b0.001 b0.001 b0.001
NS: nonsignificant, TIMI: Thrombolysis In Myocardial Infarction, BP: blood pressure. LAD: left anterior descending; LCx: left circumflex; RCA: right coronary artery. a Measured during coronary angiography. b Corrected TIMI frame count was given for left anterior descending coronary artery.
There was no statistically significant difference between two groups in respect to age, gender, hypertension, diabetes mellitus, hyperlipidemia and cigarette smoking ( pN0.05, Table 1). Besides, we found no significant difference between two groups in respect to total cholesterol, LDLcholesterol, HDL-cholesterol and triglyceride levels (Table 1). Systolic and diastolic blood pressures were similar in both groups measured during coronary angiography (Table 1). Patients with slow coronary flow were detected to have significantly higher TIMI frame count for each major epicardial coronary artery compared to control subjects (Table 1). Mean TIMI frame count was found to be significantly higher in patients with slow coronary flow compared to control subjects (Table 1). However, epicardial coronary artery diameters were detected to be similar in patients and control groups (LAD: 3.23F0.43 vs. 3.28F0.44 mm, respectively, p=0.6; LCx: 2.93F0.40 vs. 2.96F0.43 mm, respectively, p=0.7; RCA: 3.08F0.46 vs. 3.16F0.48, respectively, p=0.5) Plasma homocysteine levels of patients with slow coronary flow were found to be significantly higher than those of control subjects (Table 1). TIMI frame count for each major epicardial coronary artery was detected to be significantly correlated with each other (LAD-LCx: r=0.951, pb0.001; LAD-RCA: r=0.861, pb0.001; and LCx-RCA: r=0.864, pb0.001). In addition, we found a significant positive correlation between mean TIMI frame count and plasma homocysteine level (r=0.660, pb0.001, Fig. 1). However, no significant correlation was detected between lipid parameters and TIMI frame count.
4. Discussion The present study shows that patients with slow coronary flow have significantly higher plasma homocysteine level compared to subjects with normal coronary flow. Besides, there was a significant positive correlation between plasma homocysteine level and mean TIMI frame count.
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The normal coronary system consists of large epicardial vessels that normally offers little intrinsic resistance to coronary blood flow and small intramyocardial vessels (microcirculation), which, because of their small diameters and well-developed media, are the major source of coronary vascular resistance [14]. Because of their well-developed media, they have the capacity to alter profoundly the resistance to coronary blood flow. Thus, coronary blood flow is considered to be inversely related to microvascular resistance. Coronary microvascular endothelial dysfunction and subsequent elevation of microvascular resistance have been implicated in slow coronary flow with its first description [1]. In addition, left and right ventricular biopsy studies have also demonstrated small vessel disease in patients with the coronary slow flow [2,3]. Previously, Kurtoglu et al. [15] have reported that oral dipyridamole therapy relieves microvascular tone and accelerates dye runoff in the coronary arteries in patients with slow coronary flow and suggested a functional increase in microvascular resistance in these patients. In addition, Beltrame et al. [16] have also confirmed the presence of an increased resting coronary vasomotor tone in coronary resistance vessels in patients with slow coronary flow. Besides, Camsari et al. [17] have shown that endothelin-1 is higher and nitric oxide concentration is lower in patients with slow coronary flow than in a matched group of control subjects with normal coronary flow. These findings have suggested that endothelial dysfunction may play an active role in the pathophysiology of slow coronary flow. Recently, Cin et al. [18] have demonstrated that the intima+media area was thickened diffusely throughout given coronary artery in patients with slow coronar flow by using intravascular ultrasound. So, they have shown the presence of diffuse early atherosclerosis which does not cause luminal irregularities in the coronary angiography in patients with slow coronary flow. Patients with isolated coronary artery ectasia have been reported to have slow coronary flow phenomenon [19]. However, we found no significant difference between two groups regarding epicardial coronary artery diameters in the present study. Numerous studies have established an association between elevated plasma homocysteine levels and endothelial dysfunction. Woo et al. [9] and Tawakol et al. [10] have reported that in healthy subjects fasting homocysteine level repeatedly emerged as the strongest predictor for impaired flow-mediated dilatation, independent of age, sex, body mass index, blood pressure and cholesterol. Acute hyperhomocysteinemia using an oral methionine loading test induces substantial linear impairment of flow-mediated dilatation in healthy subjects [20] and leads to a significant increase in vWF in patients with vascular disease, reflecting endothelial dysfunction [21]. Only endothelium-dependent flow-mediated dilatation, which is mainly mediated by nitric oxide, was associated with elevated homocysteine level in these studies, therefore suggesting impaired endothelial nitric oxide bioactivity as the underlying mechanism.
Recently, the association between slow coronary flow and impaired endothelium-dependent flow-mediated dilatation has been demonstrated indirectly. Sezgin et al. [22] have reported that brachial artery flow-mediated dilatation is impaired in patients with slow coronary flow in the absence of any cardiac risk factors such as smoking, diabetes and hyperlipidemia. The mechanism by which homocysteine causes endothelial dysfunction has previously reported. Damage to the vascular endothelium by homocysteine is believed to be secondary to the generation of reactive oxygen species, such as hydrogen peroxide (H2O2), superoxide anion (O2 ) and hydroxyl radical (HO), mediated through the SH groups during the autooxidation of homocysteine to homocysteine or other mixed disulfides [23,24]. Besides, it has been well established that homocysteine impairs the basal production of nitric oxide by generating hydrogen peroxide and by decreasing intracellular glutathione peroxidase, the enzyme responsible for the reduction of hydrogen and lipid peroxides to their corresponding alcohols [25–27]. These mechanisms potentiate the toxic effects of homocysteine on the vascular endothelium. Coronary endothelial dysfunction has been reported to be improved with floic acid supplementation in hyperhomocysteinemic patients with coronary artery disease [28,29]. Accordingly, homocysteine lowering therapies may have a role in improvement of endothelial function and coronary blood flow in patients with slow coronary flow. 4.1. Study limitation The most important limitation of the present study is that the angiographic definition of normal coronary arteries relies on axial contrast angiograms of the vessel lumen which underestimate the presence of atherosclerotic plaque [30]. A contributing factor to insensitivity of angiography for detection of plaques is that atherosclerosis is associated with medial atrophy and vessel wall dilatation resulting in diffusely diseased coronary arteries appearing to have angiographically normal coronary arteries [31]. Intravascular ultrasound overcomes the limitation of angiography with tomographic images which provide accurate characterization of vessel lumen and wall geometry, as well as the presence and distribution of atherosclerosis [32]. However, we did not have the opportunity to perform intravascular ultrasonogarphy. In summary, we have shown that patients with slow coronary flow have raised levels of plasma homocysteine compared to control subjects with normal coronary flow. Although the number of patients included in this study is so limited, this data suggests that elevated level of plasma homocysteine may play a role in the pathogenesis of slow coronary flow. Larger prospective studies are needed to confirm the role of hyperhomocysteinemia in slow coronary flow and to evaluate the usefulness of homocysteine lowering therapies.
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[16] Beltrame JF, Limaye SB, Wuttke RD, Horowitz JD. Coronary hemodynamic and metabolic studies of the coronary slow flow phenomenon. Am Heart J 2003;146:84 – 90. [17] Camsari A, Pekdemir H, Cicek D, et al. Endothelin-1 and nitric oxide concentrations and their response to exercise in patients with slow coronary flow. Circ J 2003;67:1022 – 8. [18] Cin VG, Pekdemir H, Camsari A, et al. Diffuse intimal thickening of coronary arteries in slow coronary flow. Jpn Heart J 2003;44:907 – 19. [19] Senen K, Yetkin E, Turhan H, et al. Increased thrombolysis in myocardial infarction frame counts in patients with isolated coronary artery ectasia. Heart Vessels 2004;19:23 – 6. [20] Chambers JC, McGregor A, Jean-Marie J, Kooner JS. Acute hyperhomocysteinemia and endothelial dysfunction. Lancet 1998;351:36 – 7. [21] Constans J, Blann AD, Resplandy F, et al. Endothelial dysfunction during acute methionine load in hyperhomocysteinemic patients. Atherosclerosis 1999;147:411 – 3. [22] Sezgin AT, Sigirci A, Barutcu I, et al. Vascular endothelial function in patients with slow coronary flow. Coron Artery Dis 2003;14:155 – 61. [23] Starkebaum G, Harlan JM. Endothelial cell injury due to coppercatalyzed hydrogen peroxide generation from homocysteine. J Clin Invest 1986;77:1370 – 6. [24] Welch GN, Upchurch G, Loscalzo J. Hyperhomocysteinemia and atherothrombosis. Ann N Y Acad Sci U S A 1997;811:48 – 58. [25] Upchurch GR, Welch GN, Ramdev N, et al. The effect of homocysteine on endothelial cell nitric oxide production. FASEB J 1995;9:A876. [26] Upchurch GR, Welch GN, Freedman JF, Loscalzo J. Homocysteine attenuates endothelial glutathione peroxidase and thereby potentiates peroxide-mediated cell injury. Circulation 1995;92:I-228. [27] Upchurch GR, Welch GN, Fabian AJ, et al. Homocysteine decreases bioavailable nitric oxide by a mechanism involving glutathione peroxidase. J Biol Chem 1997;272:17012 – 7. [28] Stanger O, Semmelrock HJ, Wonisch W, Bos U, Pabst E, Wascher TC. Effects of folate treatment and homocysteine lowering on resistance vessel reactivity in atherosclerotic subjects. J Pharmacol Exp Ther 2002;303:158 – 62. [29] Willems FF, Aengevaeren WR, Boers GH, Blom HJ, Verheugt FW. Coronary endothelial function in hyperhomocysteinemia: improvement after treatment with folic acid and cobalamin in patients with coronary artery disease. J Am Coll Cardiol 2002;40:766 – 72. [30] Arnett EN, Isner JM, Redwood DR, et al. Coronary artery narrowing in coronary heart disease: comparison of cineangiographic and necropsy findings. Ann Intern Med 1979;91:350 – 6. [31] Chemarin-Alibelli MJ, Pieraggi MT, Elbaz M, et al. Identification of coronary thrombus after myocardial infarction by intracoronary ultrasound compared with histology of tissues sampled by atherectomy. Am J Cardiol 1996;77:344 – 9. [32] Potkin BN, Bartorelli AL, Gessert JM, et al. Coronary artery imaging with intravascular high-frequency ultrasound. Circulation 1990;81: 1575 – 85.
Elevated level of plasma homocysteine in patients with slow coronary flow Ali Riza Erbaya, Hasan Turhana,*, Ayse Saatci Yasara, Selime Ayazb, Onur Sahina, Kubilay Senena, Hatice Sasmaza, Ertan Yetkinc a Department of Cardiology, Turkiye Yuksek Ihtisas Hospital, Ankara, Turkey Department of Biochemistry, Turkiye Yuksek Ihtisas Hospital, Ankara, Turkey c Department of Cardiology, Inonu University Medical Faculty, Malatya, Turkey b
Received 12 January 2004; received in revised form 7 April 2004; accepted 5 May 2004 Available online 22 September 2004
Abstract Background: Elevated plasma levels of homocysteine are currently considered a major, independent risk factor for cardiovascular diseases. Recently, several investigators have suggested that even mild elevation in plasma homocysteine level can severely disturb vascular endothelial function and subsequently impair coronary blood flow. Accordingly, we investigated plasma homocysteine level in patients with slow coronary flow. Method: Study population included 53 patients with angiographically proven normal coronary arteries and slow coronary flow in all three coronary vessels (group I, 21 females, 32 males, mean age=48F9 years), and 50 subjects with angiographically proven normal coronary arteries without associated slow coronary flow (group II, 22 females, 28 males, mean age=50F8 years). Coronary flow rates of all patients and control subjects were documented by Thrombolysis In Myocardial Infarction frame count (TIMI frame count). All patients in group I had TIMI frame counts greater than two standard deviations above those of control subjects (group II) and, therefore, were accepted as exhibiting slow coronary flow. The mean TIMI frame count for each patient and control subject was calculated by adding the TIMI frame counts for each major epicardial coronary artery and then dividing the obtained value into 3. Plasma homocysteine level was measured in all patients and control subjects using commercially available homocysteine kits. Results: There was no statistically significant difference between two groups in respect to age, gender, hypertension, diabetes mellitus, hyperlipidemia and cigarette smoking ( pN0.05). Plasma homocysteine level of patients with slow coronary flow were found to be significantly higher than those of control subjects (15.5F5.7 vs. 8.7F4.2 AM/l, respectively, pb0.001). Moreover, we found a significant positive correlation between plasma homocysteine level and mean TIMI frame count (r=0.660, pb0.001). Conclusion: We have shown that patients with slow coronary flow have raised level of plasma homocysteine compared to control subjects with normal coronary flow. This data suggests that elevated level of plasma homocysteine may play a role in the pathogenesis of slow coronary flow. D 2004 Elsevier Ireland Ltd. All rights reserved. Keywords: Homocysteine; Slow coronary flow; Endothelial dysfunction
1. Introduction The coronary slow flow phenomenon is an angiographic observation characterized by angiographically normal or * Corresponding author. Tel.: +90 312 2867658. E-mail address: [email protected] (A. Turhan). 0167-5273/$ - see front matter D 2004 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.ijcard.2004.05.064
near-normal coronary arteries with delayed opacification of the distal vasculature. It has been reported that coronary microvascular endothelial dysfunction plays an important pathogenetic role in patients with slow coronary flow [1]. Moreover, myocardial biopsy studies have also revealed the presence of coronary microvascular disease in patients exhibiting coronary slow flow [2,3]. However, the mech-
420
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anisms responsible for this microvascular endothelial dysfunction in patients with slow coronary flow are not known. Homocysteine is a thiol (sulfhydryl-) containing, potentially cytotoxic, 4-carbon alfa-amino acid that is formed during methionine metabolism [4]. Serum homocysteine concentrations are frequently elevated in the elderly; in individuals deficient in folic acid, cyanocobalamin (vitamin B12) or pyridoxal phosphate (vitamin B6); and in the presence of various enzyme abnormalities including deficiencies in cystathionine h-synthase and thermolability of 5,10-methylenetetrahydrofolate reductase [5,6]. Recent prospective studies show that even mild hyperhomocysteinemia is associated with an increased risk of cardiovascular, cerebrovascular and peripheral vascular disease, independently of classical risk factors [7,8]. Besides, elevated plasma level of homocysteine has been reported to be associated with endothelial dysfunction in animals and humans [9,10]. The coronary microvascular endothelium performs essential control on the coronary blood flow. Injury of the coronary microvascular endothelium leads to vasomotor dysfunction with an impairment of endothelial vasoregulatory mechanisms. Cellular and animal studies indicate that homocysteine reduces the bioavailability of endothelium-derived nitric oxide [11]. Furthermore, several important investigations have shown that elevated plasma homocysteine level in healthy subjects strongly predicts the impaired endothelium-dependent vasodilatation [9,10]. Accordingly, in the present study, we aimed to investigate the plasma homocysteine level in patients with slow coronary flow.
2. Method 2.1. Study population Study population included 53 patients with angiographically proven normal coronary arteries and slow coronary flow in all three coronary vessels (group I, 21 females, 32 males, mean age=48F9 years), and 50 subjects with angiographically proven normal coronary arteries without associated slow coronary flow (group II, 22 females, 28 males, mean age=50F8 years). All patients with slow coronary flow were selected from individuals who underwent coronary angiography in our hospital with a suspicion of coronary artery disease and diagnosed as having angiographically normal coronary arteries. Control group consisted of 50 consecutive subjects with atypical chest pain admitted to the hospital for elective coronary angiography and subsequently found to have angiographically normal coronary arteries. Patients with coronary artery disease, previous history of myocardial infarction, left ventricular dysfunction, echocardiographically proven left ventricular hypertrophy, uncontrolled hypertension and renal dysfunction were not included in the study.
2.2. Documentation of slow coronary flow All patients underwent selective coronary angiography and left ventriculography by means of judkins technique. We used Iohexol (Omnipaque, Nycomed Ireland, Cork, Ireland) as contrast agent during coronary angiography in all patients and control subjects. Coronary flow rates of all subjects were documented by Thrombolysis In Myocardial Infarction frame count (TIMI frame count). TIMI frame count method is a simple, reproducible, objective and quantitative index of coronary flow velocity [12]. It has been suggested that a higher TIMI frame count may reflect disordered resistance vessel function [12]. TIMI frame count was determined for each major coronary artery in each patient and control subject according to the method first described by Gibson et al. [12]. Briefly, the number of cineangiographic frames, recorded at 30 frames/s, required for the leading edge of the column of radiographic contrast to reach a predetermined landmark, is determined. The first frame is defined as the frame in which concentrated dye occupies the full width of the proximal coronary artery lumen, touching both borders of the lumen, and forward motion down the artery. The final frame is designated when the leading edge of the contrast column initially arrives at the distal landmark. In the left anterior descending (LAD) coronary artery, the landmark used is the most distal branch nearest the apex of the left ventricle, commonly referred as the bpitchforkQ or whale’s tail. LAD coronary artery is usually longer than the other major coronary arteries [13], the TIMI frame count for this vessel is often higher. To obtain corrected TIMI frame count for LAD coronary artery, TIMI frame count was divided by 1.7 [12]. The right coronary artery (RCA) distal landmark is the first branch of the posterolateral RCA after the origin of the posterior descending artery, regardless of the size of this branch. The branch of the left circumflex (LCx) artery that encompassed the greatest total distance traveled by contrast was used to define the distal landmark of the LCx artery. TIMI frame count in the LAD and LCx arteries were assessed in a right anterior oblique projection with caudal angulation and RCA in left anterior oblique projection with cranial angulation. The mean TIMI frame count for each patient and control subject was calculated by adding the TIMI frame counts for LAD, LCx and RCA and then dividing the obtained value into 3. Diameters of the epicardial coronary arteries were measured by use of a computerized quantitative coronary angiography analysis system (Vepro, Medimage, The image managing system, Pfungstadt, Germany). 2.3. Diagnostic criteria for slow coronary flow Patients with a corrected TIMI frame count greater than two standard deviations from normal published range for the particular vessel were considered as having slow coronary flow while those whose corrected TIMI frame count fell
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within two standard deviations of the published normal range were labelled to have normal coronary flow. 2.4. Blood sampling and measurement of plasma homocysteine level Blood samples were obtained after an overnight fasting period and were collected in EDTA-treated tubes and immediately centrifuged. Plasma was stored at 70 8C in aliquots until analyses. Fasting plasma homocysteine concentrations were measured with IMX-Abbott machine (LR 30179; 04667-102) by Fluorescence Polarization Immunoassay (FPIA) method using homocysteine kids (Abbott Laboratories, diagnostic division, Abbott Park, IL 60064, USA).
Fig. 1. Correlation of plasma homocysteine and mean TIMI frame count.
3. Results 2.5. Statistical analysis Continuous variables were expressed as meanFS.D. and categorical variables were expressed as percentage. Comparison of categorical and continuous variables between two groups was performed using chi-square test and unpaired ttest, respectively. The correlation between the plasma level of homocysteine and mean TIMI frame count was assessed by the Pearson correlation test. A P-value of b0.05 was considered statistically significant.
Table 1 Comparison of baseline clinical characteristics, plasma homocysteine levels and TIMI frame counts of patients with slow coronary flow and control subjects with normal coronary flow
Age (meanFS.D.) Gender (female/male) Hypertension Systolic BP (mm Hg)a Diastolic BP (mm Hg)a Diabetes mellitus Hyperlipidemia Total cholesterol (mg/dl) LDL-cholesterol (mg/dl) HDL-cholesterol (mg/dl) Triglyceride (mg/dl) Cigarette smoking Homocysteine (Amol/l) TIMI frame count LADb LCx RCA Mean TIMI frame count
Control subjects (n=50)
Patients with slow coronary flow (n=53)
p
50F8 22/28 20/50 (40%) 133F18 80F11 5/50 (10%) 27/50 (54%) 198F24 128F16 42F8 166F19 23/50 (46%) 8.6F4.2
48F9 21/32 19/53 (36%) 134F20 83F11 6/53 (11%) 26/53 (49%) 201F24 132F17 41F7 169F22 22/53 (42%) 15.5F5.7
NS NS NS NS NS NS NS NS NS NS NS NS b0.001
27F6 26F7 27F8 27F6
51F12 46F8 47F11 48F9
b0.001 b0.001 b0.001 b0.001
NS: nonsignificant, TIMI: Thrombolysis In Myocardial Infarction, BP: blood pressure. LAD: left anterior descending; LCx: left circumflex; RCA: right coronary artery. a Measured during coronary angiography. b Corrected TIMI frame count was given for left anterior descending coronary artery.
There was no statistically significant difference between two groups in respect to age, gender, hypertension, diabetes mellitus, hyperlipidemia and cigarette smoking ( pN0.05, Table 1). Besides, we found no significant difference between two groups in respect to total cholesterol, LDLcholesterol, HDL-cholesterol and triglyceride levels (Table 1). Systolic and diastolic blood pressures were similar in both groups measured during coronary angiography (Table 1). Patients with slow coronary flow were detected to have significantly higher TIMI frame count for each major epicardial coronary artery compared to control subjects (Table 1). Mean TIMI frame count was found to be significantly higher in patients with slow coronary flow compared to control subjects (Table 1). However, epicardial coronary artery diameters were detected to be similar in patients and control groups (LAD: 3.23F0.43 vs. 3.28F0.44 mm, respectively, p=0.6; LCx: 2.93F0.40 vs. 2.96F0.43 mm, respectively, p=0.7; RCA: 3.08F0.46 vs. 3.16F0.48, respectively, p=0.5) Plasma homocysteine levels of patients with slow coronary flow were found to be significantly higher than those of control subjects (Table 1). TIMI frame count for each major epicardial coronary artery was detected to be significantly correlated with each other (LAD-LCx: r=0.951, pb0.001; LAD-RCA: r=0.861, pb0.001; and LCx-RCA: r=0.864, pb0.001). In addition, we found a significant positive correlation between mean TIMI frame count and plasma homocysteine level (r=0.660, pb0.001, Fig. 1). However, no significant correlation was detected between lipid parameters and TIMI frame count.
4. Discussion The present study shows that patients with slow coronary flow have significantly higher plasma homocysteine level compared to subjects with normal coronary flow. Besides, there was a significant positive correlation between plasma homocysteine level and mean TIMI frame count.
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The normal coronary system consists of large epicardial vessels that normally offers little intrinsic resistance to coronary blood flow and small intramyocardial vessels (microcirculation), which, because of their small diameters and well-developed media, are the major source of coronary vascular resistance [14]. Because of their well-developed media, they have the capacity to alter profoundly the resistance to coronary blood flow. Thus, coronary blood flow is considered to be inversely related to microvascular resistance. Coronary microvascular endothelial dysfunction and subsequent elevation of microvascular resistance have been implicated in slow coronary flow with its first description [1]. In addition, left and right ventricular biopsy studies have also demonstrated small vessel disease in patients with the coronary slow flow [2,3]. Previously, Kurtoglu et al. [15] have reported that oral dipyridamole therapy relieves microvascular tone and accelerates dye runoff in the coronary arteries in patients with slow coronary flow and suggested a functional increase in microvascular resistance in these patients. In addition, Beltrame et al. [16] have also confirmed the presence of an increased resting coronary vasomotor tone in coronary resistance vessels in patients with slow coronary flow. Besides, Camsari et al. [17] have shown that endothelin-1 is higher and nitric oxide concentration is lower in patients with slow coronary flow than in a matched group of control subjects with normal coronary flow. These findings have suggested that endothelial dysfunction may play an active role in the pathophysiology of slow coronary flow. Recently, Cin et al. [18] have demonstrated that the intima+media area was thickened diffusely throughout given coronary artery in patients with slow coronar flow by using intravascular ultrasound. So, they have shown the presence of diffuse early atherosclerosis which does not cause luminal irregularities in the coronary angiography in patients with slow coronary flow. Patients with isolated coronary artery ectasia have been reported to have slow coronary flow phenomenon [19]. However, we found no significant difference between two groups regarding epicardial coronary artery diameters in the present study. Numerous studies have established an association between elevated plasma homocysteine levels and endothelial dysfunction. Woo et al. [9] and Tawakol et al. [10] have reported that in healthy subjects fasting homocysteine level repeatedly emerged as the strongest predictor for impaired flow-mediated dilatation, independent of age, sex, body mass index, blood pressure and cholesterol. Acute hyperhomocysteinemia using an oral methionine loading test induces substantial linear impairment of flow-mediated dilatation in healthy subjects [20] and leads to a significant increase in vWF in patients with vascular disease, reflecting endothelial dysfunction [21]. Only endothelium-dependent flow-mediated dilatation, which is mainly mediated by nitric oxide, was associated with elevated homocysteine level in these studies, therefore suggesting impaired endothelial nitric oxide bioactivity as the underlying mechanism.
Recently, the association between slow coronary flow and impaired endothelium-dependent flow-mediated dilatation has been demonstrated indirectly. Sezgin et al. [22] have reported that brachial artery flow-mediated dilatation is impaired in patients with slow coronary flow in the absence of any cardiac risk factors such as smoking, diabetes and hyperlipidemia. The mechanism by which homocysteine causes endothelial dysfunction has previously reported. Damage to the vascular endothelium by homocysteine is believed to be secondary to the generation of reactive oxygen species, such as hydrogen peroxide (H2O2), superoxide anion (O2 ) and hydroxyl radical (HO), mediated through the SH groups during the autooxidation of homocysteine to homocysteine or other mixed disulfides [23,24]. Besides, it has been well established that homocysteine impairs the basal production of nitric oxide by generating hydrogen peroxide and by decreasing intracellular glutathione peroxidase, the enzyme responsible for the reduction of hydrogen and lipid peroxides to their corresponding alcohols [25–27]. These mechanisms potentiate the toxic effects of homocysteine on the vascular endothelium. Coronary endothelial dysfunction has been reported to be improved with floic acid supplementation in hyperhomocysteinemic patients with coronary artery disease [28,29]. Accordingly, homocysteine lowering therapies may have a role in improvement of endothelial function and coronary blood flow in patients with slow coronary flow. 4.1. Study limitation The most important limitation of the present study is that the angiographic definition of normal coronary arteries relies on axial contrast angiograms of the vessel lumen which underestimate the presence of atherosclerotic plaque [30]. A contributing factor to insensitivity of angiography for detection of plaques is that atherosclerosis is associated with medial atrophy and vessel wall dilatation resulting in diffusely diseased coronary arteries appearing to have angiographically normal coronary arteries [31]. Intravascular ultrasound overcomes the limitation of angiography with tomographic images which provide accurate characterization of vessel lumen and wall geometry, as well as the presence and distribution of atherosclerosis [32]. However, we did not have the opportunity to perform intravascular ultrasonogarphy. In summary, we have shown that patients with slow coronary flow have raised levels of plasma homocysteine compared to control subjects with normal coronary flow. Although the number of patients included in this study is so limited, this data suggests that elevated level of plasma homocysteine may play a role in the pathogenesis of slow coronary flow. Larger prospective studies are needed to confirm the role of hyperhomocysteinemia in slow coronary flow and to evaluate the usefulness of homocysteine lowering therapies.
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