- Email: [email protected]
Vascular endothelial function and plasma homocysteine levels in Behcet's disease
Vascular Endothelial Function and Plasma Homocysteine Levels in Behcet’s Disease Ramazan Ozdemir, MD, Irfan Barutcu, MD, Alpay T. Sezgin, MD, Nusret Acikgoz, MD, Necip Ermis, MD, Ali Metin Esen, MD, Ergun Topal, MD, Emrah Barıskaner, MD, and Ibrahim Ozerol, MD The purpose of the present study was to test endothelial function and to determine if plasma homocysteine levels are associated with endothelial injury in patients with Behcet’s disease (BD). Flow-mediated dilation in patients with BD was smaller than that of control subjects (p ⴝ 0.001), and mean plasma homocysteine levels in patients with BD were significantly higher (p ⴝ 0.0001). On regression analysis, only mean plasma homocysteine concentration was independently related to flow-mediated dilation (F ⴝ 5.7, p ⴝ 0.001). 䊚2004 by Excerpta Medica, Inc. (Am J Cardiol 2004;94:522–525)
e hypothesized that homocysteine-promoting oxidative stress may be 1 of the mechanisms W responsible for vascular injury in Behcet’s disease (BD). The objective of this study was to test peripheral vascular endothelial function, using a high-resolution ultrasound technique, and to determine if plasma homocysteine levels were associated with the vascular endothelial injury in BD. •••
A total of 36 patients with BD (20 men and 16 women; mean age 36 ⫾ 8 years) who were diagnosed according to International Study Group criteria for BD were included in the study.1 Mean duration of BD was 142 ⫾ 47 months. The control group comprised 30 healthy volunteers (20 men and 10 women; mean age 35 ⫾ 8 years) from the hospital staff who had no disease and were not on any medication. To minimize the confounding effects of endothelial function, the study group did not have coronary risk factors and other systemic diseases. All selected patients were in the chronic state of the disease and were taking colchicine treatment. Patients in the acute phase of the disease or who were being treated with steroids and were on medications known to alter plasma lipid profiles and/or homocysteine levels were also excluded from the study. At the time of study, all medications had been discontinued for ⱖ3 weeks. Patients who had an increase in the degree of symptoms during this discontinuation period were also excluded from the study (4 women and 1 man). Thus, 31 patients with From the Department of Cardiology, Faculty of Medicine, Inonu University, Malaya; Department of Cardiology, Kosuyolu Heart Education and Research Hospital, Istanbul; and Department of Cardiology, Baskent University, Practice and Research Hospital, Adana, Turkey. Dr. Ozdemir’s address is: Department of Cardiology, Inonu University, Turgut Ozal Medical Center, Malatya, Turkı˙ye. E-mail: rozdemir@ inonu.edu.tr. Manuscript received January 16, 2004; revised manuscript received and accepted April 27, 2004.
522
©2004 by Excerpta Medica, Inc. All rights reserved. The American Journal of Cardiology Vol. 94 August 15, 2004
BD (19 men and 12 women) were included in the study. All participants gave their informed consent before the study, and the local ethic committee approved the study protocol. Blood samples were drawn in the morning (8:00 A.M. to 10:00 A.M.) after an overnight fast using a 25-gauge needle from a peripheral vein, avoiding hemolysis into plain tubes. Because synthesis of homocysteine takes place in red cells after sampling, it is very important to centrifuge and separate plasma and serum from the blood cells as soon as possible. Therefore, samples were centrifuged within 30 minutes at 4,000 rpm for 5 minutes and the separated plasma was stored at ⫺70°C until assay. Total homocysteine levels were measured with an enzyme-linked immunosorbent assay using commercially available kits (The Axis, Homocysteine EIA, Hamburg, Germany), which correlate well with the reference high performance liquid chromatography method at a level of ⱖ0.98. The Axis Homocysteine is an enzyme immunoassay for the determination of total homocysteine in blood.2 Briefly, protein-bound homocysteine is reduced to free homocysteine and enzymatically converted to S-adenosyl-L-homocysteine (SAH) in a separate procedure before the immunoassay. The enzyme is specific for the L-form of homocysteine, which is the only form present in the blood. The following solid-phase enzyme immunoassay is based on competition between SAH in the sample and immobilized SAH bound to the walls of the microtitre plate for binding sites on a monoclonal anti-SAH antibody. After removal of the unbound anti-SAH antibody, a secondary rabbit antimouse antibody labeled with the enzyme horseradish peroxidase is added. The peroxidase activity is measured spectrophotometrically after addition of substrate, and the absorbance is inversely related to the concentration of total homocysteine in the sample. This system has a highly specific enzymatic sample before treatment, a convenient microplate format, a standardized immunoassay procedure, parallel sample processing, and a wide dynamic range (2.0 to 50.0 mol/L); the intra-assay precision is 5.2%. Endothelium-dependent flow-mediated vasodilation in response to reactive hyperemia and endothelium-independent nitroglycerin-induced vasodilation were evaluated in the brachial artery. Ultrasound measurements were taken using a standard technique.3 All studies were performed in a quiet clinical laboratory maintained at 21° to 23°C. Each subject was studied in the morning hours (8:00 A.M. to 10:00 A.M.) after abstaining from alcohol, caffeine, tobacco, and food 0002-9149/04/$–see front matter doi:10.1016/j.amjcard.2004.04.073
for ⱖ8 hours before the study. High-resolution echocardiographic Doppler ultrasound (HDI-5000; ATL, Bothell, Washington) with a 12-MHz linear-array transducer was used to measure the flow velocity and diameter of the right brachial arteries. In all studies, scans were taken at rest during reactive hyperemia (flow-mediated dilation [FMD]: endothelial-dependent stimulus to vasodilation), again at rest, and after sublingual nitroglycerin: endothelium-independent vasodilation). Briefly, patients rested in a supine position for 15 minutes before the first scan and were kept supine throughout the study. A nontortoise segment of the brachial artery was scanned longitudinally 4 to 5 cm above the elbow, where the clearest image was determined; the skin was marked and the arm was kept in a constant position throughout the study. After the baseline scan at rest, a pneumatic tourniquet placed around the forearm distal to the target artery was inflated to a pressure of 250 mm Hg for 5 minutes. The second scan was performed 55 to 60 seconds after cuff deflation. Increased blood flow after sudden cuff deflation, termed reactive hyperemia, resulted in FMD. Fifteen minutes were allowed for vessel recovery; after which another scan at rest was recorded. Sublingual nitroglycerin (400 g) was then administered and 3 to 4 minutes later the last scan was performed. The diameter of the brachial artery was measured from the anterior to posterior interface between the media and adventitia (“m” line) at the end of diastole, incident with the R wave on a continuously recorded electrocardiogram. Diameters for 4 cardiac cycles were determined from images, and the measurements were averaged. All images were recorded on s-VHS videotape using an MD830 videocassette recorder (Sony, Tokyo, Japan). The vessel diameter was measured by 2 observers who were unaware of the study. Endothelium-dependent vasodilation, which is largely mediated by nitric oxide, was determined by the maximal brachial artery diameter after 60 seconds of reactive hyperemia (after cuff deflation) compared with the baseline diameter and expressed as a FMD percentage. Endothelium-independent vasodilation was defined as the maximum brachial artery diameter 4 minutes after administration of nitroglycerin compared with the baseline vessel diameter and expressed as a nitroglycerin-mediated dilation percentage. The inter- and intraobserver variabilities for repeated measurements are 0.1 ⫾ 0.06 and 0.1 ⫾ 0.09 mm, respectively, in our laboratory. Statistical analysis was performed with SPSS for Windows, version 10.0 (SPSS Inc., Chicago, Illinois). Data are presented as mean ⫾ SD. Distribution of the groups was analyzed with the 1-sample KolmogorovSmirnov test. Because both groups showed normal distribution, parametric statistical methods were used to analyze the data. Student’s t test was used for pair-wise comparisons and the chi-square test for categorical changes. Pearson’s correlation analysis was conducted to investigate the association of FMD with contributive risk factors. Then, multivariate regression analysis was undertaken between FMD and various factors, including total homocysteine, total choles-
TABLE 1 General Characteristics and Vascular/Laboratory Study Results of Patients With BD and Control Subjects Variable
BD (n ⫽ 31)
Controls (n ⫽ 30)
Age (yrs) Men Body mass index (kg/m2) Smokers Systolic blood pressure (mm Hg) Diastolic blood pressure (mm Hg) Total cholesterol (mg/dl) LDL cholesterol (mg/dl) HDL cholesterol (mg/dl) Triglycerides (mg/dl) FMD (%) Nitroglycerin-induced dilation (%) Brachial artery diameter (mm) Plasma homocysteine (mol/L)
37 ⫾ 10 19 (61%) 26 ⫾ 4 5 (16%) 127 ⫾ 11 81 ⫾ 11 176 ⫾ 21 121 ⫾ 23 39 ⫾ 3 213 ⫾ 29 1.4 ⫾ 3.0 16.4 ⫾ 3.7 4.0 ⫾ 0.9 14.5 ⫾ 2.6
36 ⫾ 8 20 (64%) 27 ⫾ 4 4 (12%) 126 ⫾ 9 78 ⫾ 9 175 ⫾ 29 115 ⫾ 24 41 ⫾ 4 198 ⫾ 24 4.4 ⫾ 3.4* 16.1 ⫾ 3.9 4.1 ⫾ 0.8 9.2 ⫾ 2.8†
*p ⬍0.001; †p ⬍0.0001. Values are expressed as mean ⫾ SD or percentage. HDL ⫽ high-density lipoprotein; LDL ⫽ low-density lipoprotein.
FIGURE 1. FMD (percent) in patients with BD and control subjects.
terol, high-density and low-density cholesterol, triglyceride and glucose levels, duration of the disorder, body mass index, systolic/diastolic blood pressures, and age. A p value ⬍0.05 was considered statistically significant. General characteristics, and vascular and laboratory study results of the patients with BD and control subjects are shown in Table 1. There were no significant differences between the 2 groups with respect to gender, age, body mass index, and blood pressure. All patients with BD had oral aphtous lesions; genital ulcers were found in 27 patients (87%), eye lesions were found in 20 patients (64%), and skin lesions were present in 21 patients (67%). FMD in patients was found to be significantly smaller than those of controls (Figure 1). Nitroglycerin-induced dilation in patients with BD did not differ significantly from that in healthy control subjects (Figure 2). Mean plasma homocysteine levels in BD were found to be significantly higher than the control group BRIEF REPORTS
523
FIGURE 3. The association between FMD (percent) and plasma homocysteine levels in BD. FIGURE 2. NTG-induced dilation in patients with BD and control subjects. NTG ⴝ nitroglycerin.
TABLE 2 Vascular and Laboratory Study Results of BD With Ocular and Nonocular Involvement
FMD (%) Nitroglycerin-induced dilation (%) Brakial artery diameter (mm) Plasma homocysteine (mol/L)
BD With Ocular Involvement (n ⫽ 20)
BD With Nonocular Involvement (n ⫽ 11)
p Value
0.5 ⫾ 3.0 17.3 ⫾ 3.7
2.7 ⫾ 2.5 15.2 ⫾ 3.4
0.03 0.17
4.1 ⫾ 0.7 15.3 ⫾ 2.8
4.0 ⫾ 0.9 13.3 ⫾ 1.9
0.79 0.02
Values are expressed as mean ⫾ SD.
(Table 1). Table 2 displays the vascular and laboratory results of patients with ocular and nonocular involvement. In patients with BD, FMD of the brachial artery was found to be strongly and inversely correlated with mean plasma homocysteine levels (r ⫽ ⫺0.67, p ⫽ 0.0001) (Figure 3). Similarly, FMD of the brachial artery was found to be inversely correlated with mean duration of the disease (r ⫽ ⫺0.58, p ⫽ 0.001) (Figure 4). When multivariate regression analysis was performed, it showed that only total homocysteine concentration was independently related to FMD (F ⫽ 5.7, p ⫽ 0.001) among total cholesterol, high-density and low-density cholesterol, triglyceride and glucose levels, duration of the disorder, body mass index, systolic/diastolic blood pressures, and age. •••
The principal findings of the present study are that (1) brachial artery FMD is impaired in patients with BD compared with control subjects, (2) plasma homocysteine levels are markedly higher in patients with BD than those of control subjects and significantly associated with the degree of FMD, (3) similar relations were also seen between the duration of disease and FMD, and (4) in ocular BD, FMD is reduced compared with nonocular BD and plasma homocysteine levels are higher in ocular disease than in non524 THE AMERICAN JOURNAL OF CARDIOLOGY姞
VOL. 94
FIGURE 4. The association between FMD (percent) and duration of disease.
ocular disease. This study is the first to show an association between endothelial dysfunction and homocysteine levels in BD. As is known, the pathogenesis of BD is characterized by hypercoagulability and vascular injury4 –7; however, the factors that may contribute to vascular injury have not been well defined. We therefore attempted to measure the plasma homocysteine levels and to determine its contribution to vascular injury. Our results showed that mean plasma homocysteine levels are markedly elevated in patients with BD compared with control subjects and it is well correlated to the degree of endothelial damage. In accordance with our study, mean plasma homocysteine concentrations in patients with BD have been reported to be elevated compared with those in healthy controls, and it has been concluded that hyperhomocysteinemia may be an independent risk factor for venous thrombosis.8 –11 However, these investigators investigated the association between homocysteine levels and development of thrombosis, but not the degree of endothelial function. AUGUST 15, 2004
From the clinical point of view, endothelial dysfunction and elevated homocysteine levels may increase susceptibility to future cardiovascular complications in patients with BD.12–14 This supports the hypothesis that hyperhomocysteinemia may be a risk factor for the development of ocular disease in BD. 1. International Study Group for Behcet’s Disease. Criteria for diagnosis of
Behcet’s disease. Lancet 1990;335:1078 –1080. 2. Frantzen F, Faaren AL, Alfheim I, Nordhei AK. An enzyme conversion
immunoassay for determining total homocysteine in plasma or serum. Clin Chem 1998;44:311–316. 3. Celermajer DS, Sorensen KE, Gooch VM, Spiegelhalter DJ, Miller OI, Sullivan ID, Lloyd JK, Deanfield JE. Noninvasive detection of endothelial dysfunction in children and adults at risk of atherosclerosis. Lancet 1992;340: 1111–1115. 4. Yazici H. Behcet’s syndrome: an update. Curr Rheumatol Rep 2003;5: 195–199. 5. Aydintug AO, Tokgoz G, Ozoran K, Duzgun N, Gurler A, Tutkak H. Elevated levels of soluble intercellular adhesion molecule-1 correlate with disease activity in Behcet’s disease. Rheumatol Int 1995;15:75–78.
6. Bozkurt E, Erol MK, Keles S, Acikel M, Yilmaz M, Gürlertop Y. Relation of plasma homocysteine levels to intracoronary thrombus in unstable angina pectoris and in non–Q-wave acute myocardial infarction. Am J Cardiol 2002;90: 413– 415. 7. Ozdemir R, Sezgin AT, Topal E, Kutlu R, Barutcu I, Gullu H. Findings of ambulatory blood pressure monitoring and heart rate variability in patients with Behcet’s disease. Am J Cardiol 2003;92:646 – 648. 8. Schmitz-Huebner U, Knop J. Evidence for an endothelial cell dysfunction in association with Behcet’s disease. Thromb Res 1984;34:277–285. 9. Wall RT, Harlan LM, Harker LA, Striker GE. Homocysteine-induced endothelial cell injury in vitro:a model for the study of vascular injury. Thromb Res 1980;18:113–121. 10. Blundell G, Jones BG, Rose FA, Tudball N. Homocysteine mediated endothelial cell toxicity and its amelioration. Atherosclerosis 1996;122:163–172. 11. Blacher J, Benetos A, Kirzin JM, Malmejac A, Guize L, Safar ME. Relation of plasma total homocysteine to cardiovascular mortality in a French population. Am J Cardiol 2002;90:591–595. 12. Sakane T. New perspective on Behcet’s disease. Int Rev Immunol 1997;14: 89 –96. 13. Hankey GJ, Eikelboom JW. Homocysteine and vascular disease. Lancet 1999;354:407– 413. 14. Er H, Evereklioglu C, Cumurcu T, Türköz Y, Özerol E, S¸ ahin K, Doganay S. Serum homocysteine level is increased and correlated with endothelin-1 and nitric oxide in Behçet’s disease. Br J Ophthalmol 2002;86:653– 657.
Characteristics and Prevalence of Intrapulmonary Shunt Detected by Contrast Echocardiography With Harmonic Imaging in Liver Transplant Candidates Byung Jin Kim, MD, Sang-Chol Lee, MD, Seung Woo Park, MD, Moon-Suk Choi, Kwang Cheol Koh, MD, Seung Woon Paik, MD, Sang Hoon Lee, MD, Kyung Pyo Hong, MD, Jeong Euy Park, MD, and Jung Don Seo, MD We investigated the prevalence and characteristics of intrapulmonary shunt using contrast echocardiography with harmonic imaging in 130 liver transplant candidates. We found a high prevalence of intrapulmonary shunts and a significant correlation between the degree of intrapulmonary shunt and the ChildPugh classification score. 䊚2004 by Excerpta Medica, Inc. (Am J Cardiol 2004;94:525–528)
revious studies have suggested that intrapulmonary shunts are not associated with various paP rameters of liver disease. We studied the prevalence 1–3
and clinical characteristics of intrapulmonary shunts by contrast echocardiography with harmonic imaging in liver transplant candidates. •••
From November 1999 to September 2002, 130 patients (89 men and 41 women; mean age 47 ⫾ 10 years) awaiting liver transplantation due to end-stage From the Department of Medicine, Cardiac and Vascular Center, the Division of Gastroenterology, and Cardiac Center, Kangbuk Samsung Hospital, Sungkyunkwan University School of Medicine, Samsung Medical Center, Seoul, Korea. Dr. Lee’s address is: Department of Medicine, Samsung Medical Center, Sungkyunkwan University School of Medicine, Ilwon-Dong 50, Kangnam-Ku, Seoul, Korea 135-710. E-mail: [email protected]. Manuscript received January 19, 2004; revised manuscript received and accepted April 19, 2004. ©2004 by Excerpta Medica, Inc. All rights reserved. The American Journal of Cardiology Vol. 94 August 15, 2004
MD,
liver disease were studied. The most frequent etiology (87%) of liver disease in these patients was chronic hepatitis B, and other etiologies were primary sclerosing cholangitis (5%), chronic hepatitis C (4%), cryptogenic disease (2%), and Wilson’s disease (2%). Patients with active upper gastrointestinal bleeding, combined valvular heart disease, primary pulmonary parenchymal disease, or patent foramen ovale were excluded from the study. Contrast echocardiographic studies were performed 3 times by injecting an agitated solution of 5 ml of normal saline solution and 1 ml of room air using a peripheral intravenous line and two 10-ml syringes connected by a 3-way stopcock. Echocardiography was performed by 3.5-MHz harmonic imaging (Siemens Medical Solutions, Mountain View, California) using 2-dimensional apical 4-chamber views. Positive contrast echocardiography for an intrapulmonary shunt was defined as the delayed appearance of microbubbles in the left-sided heart chambers through the pulmonary veins within 4 to 6 heart beats after their appearance in the right side of the heart. According to the intrapulmonary shunt grading scales of Vedrinne et al,4 the degree of an intrapulmonary shunt was assessed as from grade 0 to IV (Figure 1). Patients with grades II to IV were considered to have a significant intrapulmonary shunt. Liver function tests, blood coagulation tests, arterial blood gas analyses, and pulmonary function tests were performed on the day of the echocardiographic study. The Child-Pugh classi0002-9149/04/$–see front matter doi:10.1016/j.amjcard.2004.04.074
525
e hypothesized that homocysteine-promoting oxidative stress may be 1 of the mechanisms W responsible for vascular injury in Behcet’s disease (BD). The objective of this study was to test peripheral vascular endothelial function, using a high-resolution ultrasound technique, and to determine if plasma homocysteine levels were associated with the vascular endothelial injury in BD. •••
A total of 36 patients with BD (20 men and 16 women; mean age 36 ⫾ 8 years) who were diagnosed according to International Study Group criteria for BD were included in the study.1 Mean duration of BD was 142 ⫾ 47 months. The control group comprised 30 healthy volunteers (20 men and 10 women; mean age 35 ⫾ 8 years) from the hospital staff who had no disease and were not on any medication. To minimize the confounding effects of endothelial function, the study group did not have coronary risk factors and other systemic diseases. All selected patients were in the chronic state of the disease and were taking colchicine treatment. Patients in the acute phase of the disease or who were being treated with steroids and were on medications known to alter plasma lipid profiles and/or homocysteine levels were also excluded from the study. At the time of study, all medications had been discontinued for ⱖ3 weeks. Patients who had an increase in the degree of symptoms during this discontinuation period were also excluded from the study (4 women and 1 man). Thus, 31 patients with From the Department of Cardiology, Faculty of Medicine, Inonu University, Malaya; Department of Cardiology, Kosuyolu Heart Education and Research Hospital, Istanbul; and Department of Cardiology, Baskent University, Practice and Research Hospital, Adana, Turkey. Dr. Ozdemir’s address is: Department of Cardiology, Inonu University, Turgut Ozal Medical Center, Malatya, Turkı˙ye. E-mail: rozdemir@ inonu.edu.tr. Manuscript received January 16, 2004; revised manuscript received and accepted April 27, 2004.
522
©2004 by Excerpta Medica, Inc. All rights reserved. The American Journal of Cardiology Vol. 94 August 15, 2004
BD (19 men and 12 women) were included in the study. All participants gave their informed consent before the study, and the local ethic committee approved the study protocol. Blood samples were drawn in the morning (8:00 A.M. to 10:00 A.M.) after an overnight fast using a 25-gauge needle from a peripheral vein, avoiding hemolysis into plain tubes. Because synthesis of homocysteine takes place in red cells after sampling, it is very important to centrifuge and separate plasma and serum from the blood cells as soon as possible. Therefore, samples were centrifuged within 30 minutes at 4,000 rpm for 5 minutes and the separated plasma was stored at ⫺70°C until assay. Total homocysteine levels were measured with an enzyme-linked immunosorbent assay using commercially available kits (The Axis, Homocysteine EIA, Hamburg, Germany), which correlate well with the reference high performance liquid chromatography method at a level of ⱖ0.98. The Axis Homocysteine is an enzyme immunoassay for the determination of total homocysteine in blood.2 Briefly, protein-bound homocysteine is reduced to free homocysteine and enzymatically converted to S-adenosyl-L-homocysteine (SAH) in a separate procedure before the immunoassay. The enzyme is specific for the L-form of homocysteine, which is the only form present in the blood. The following solid-phase enzyme immunoassay is based on competition between SAH in the sample and immobilized SAH bound to the walls of the microtitre plate for binding sites on a monoclonal anti-SAH antibody. After removal of the unbound anti-SAH antibody, a secondary rabbit antimouse antibody labeled with the enzyme horseradish peroxidase is added. The peroxidase activity is measured spectrophotometrically after addition of substrate, and the absorbance is inversely related to the concentration of total homocysteine in the sample. This system has a highly specific enzymatic sample before treatment, a convenient microplate format, a standardized immunoassay procedure, parallel sample processing, and a wide dynamic range (2.0 to 50.0 mol/L); the intra-assay precision is 5.2%. Endothelium-dependent flow-mediated vasodilation in response to reactive hyperemia and endothelium-independent nitroglycerin-induced vasodilation were evaluated in the brachial artery. Ultrasound measurements were taken using a standard technique.3 All studies were performed in a quiet clinical laboratory maintained at 21° to 23°C. Each subject was studied in the morning hours (8:00 A.M. to 10:00 A.M.) after abstaining from alcohol, caffeine, tobacco, and food 0002-9149/04/$–see front matter doi:10.1016/j.amjcard.2004.04.073
for ⱖ8 hours before the study. High-resolution echocardiographic Doppler ultrasound (HDI-5000; ATL, Bothell, Washington) with a 12-MHz linear-array transducer was used to measure the flow velocity and diameter of the right brachial arteries. In all studies, scans were taken at rest during reactive hyperemia (flow-mediated dilation [FMD]: endothelial-dependent stimulus to vasodilation), again at rest, and after sublingual nitroglycerin: endothelium-independent vasodilation). Briefly, patients rested in a supine position for 15 minutes before the first scan and were kept supine throughout the study. A nontortoise segment of the brachial artery was scanned longitudinally 4 to 5 cm above the elbow, where the clearest image was determined; the skin was marked and the arm was kept in a constant position throughout the study. After the baseline scan at rest, a pneumatic tourniquet placed around the forearm distal to the target artery was inflated to a pressure of 250 mm Hg for 5 minutes. The second scan was performed 55 to 60 seconds after cuff deflation. Increased blood flow after sudden cuff deflation, termed reactive hyperemia, resulted in FMD. Fifteen minutes were allowed for vessel recovery; after which another scan at rest was recorded. Sublingual nitroglycerin (400 g) was then administered and 3 to 4 minutes later the last scan was performed. The diameter of the brachial artery was measured from the anterior to posterior interface between the media and adventitia (“m” line) at the end of diastole, incident with the R wave on a continuously recorded electrocardiogram. Diameters for 4 cardiac cycles were determined from images, and the measurements were averaged. All images were recorded on s-VHS videotape using an MD830 videocassette recorder (Sony, Tokyo, Japan). The vessel diameter was measured by 2 observers who were unaware of the study. Endothelium-dependent vasodilation, which is largely mediated by nitric oxide, was determined by the maximal brachial artery diameter after 60 seconds of reactive hyperemia (after cuff deflation) compared with the baseline diameter and expressed as a FMD percentage. Endothelium-independent vasodilation was defined as the maximum brachial artery diameter 4 minutes after administration of nitroglycerin compared with the baseline vessel diameter and expressed as a nitroglycerin-mediated dilation percentage. The inter- and intraobserver variabilities for repeated measurements are 0.1 ⫾ 0.06 and 0.1 ⫾ 0.09 mm, respectively, in our laboratory. Statistical analysis was performed with SPSS for Windows, version 10.0 (SPSS Inc., Chicago, Illinois). Data are presented as mean ⫾ SD. Distribution of the groups was analyzed with the 1-sample KolmogorovSmirnov test. Because both groups showed normal distribution, parametric statistical methods were used to analyze the data. Student’s t test was used for pair-wise comparisons and the chi-square test for categorical changes. Pearson’s correlation analysis was conducted to investigate the association of FMD with contributive risk factors. Then, multivariate regression analysis was undertaken between FMD and various factors, including total homocysteine, total choles-
TABLE 1 General Characteristics and Vascular/Laboratory Study Results of Patients With BD and Control Subjects Variable
BD (n ⫽ 31)
Controls (n ⫽ 30)
Age (yrs) Men Body mass index (kg/m2) Smokers Systolic blood pressure (mm Hg) Diastolic blood pressure (mm Hg) Total cholesterol (mg/dl) LDL cholesterol (mg/dl) HDL cholesterol (mg/dl) Triglycerides (mg/dl) FMD (%) Nitroglycerin-induced dilation (%) Brachial artery diameter (mm) Plasma homocysteine (mol/L)
37 ⫾ 10 19 (61%) 26 ⫾ 4 5 (16%) 127 ⫾ 11 81 ⫾ 11 176 ⫾ 21 121 ⫾ 23 39 ⫾ 3 213 ⫾ 29 1.4 ⫾ 3.0 16.4 ⫾ 3.7 4.0 ⫾ 0.9 14.5 ⫾ 2.6
36 ⫾ 8 20 (64%) 27 ⫾ 4 4 (12%) 126 ⫾ 9 78 ⫾ 9 175 ⫾ 29 115 ⫾ 24 41 ⫾ 4 198 ⫾ 24 4.4 ⫾ 3.4* 16.1 ⫾ 3.9 4.1 ⫾ 0.8 9.2 ⫾ 2.8†
*p ⬍0.001; †p ⬍0.0001. Values are expressed as mean ⫾ SD or percentage. HDL ⫽ high-density lipoprotein; LDL ⫽ low-density lipoprotein.
FIGURE 1. FMD (percent) in patients with BD and control subjects.
terol, high-density and low-density cholesterol, triglyceride and glucose levels, duration of the disorder, body mass index, systolic/diastolic blood pressures, and age. A p value ⬍0.05 was considered statistically significant. General characteristics, and vascular and laboratory study results of the patients with BD and control subjects are shown in Table 1. There were no significant differences between the 2 groups with respect to gender, age, body mass index, and blood pressure. All patients with BD had oral aphtous lesions; genital ulcers were found in 27 patients (87%), eye lesions were found in 20 patients (64%), and skin lesions were present in 21 patients (67%). FMD in patients was found to be significantly smaller than those of controls (Figure 1). Nitroglycerin-induced dilation in patients with BD did not differ significantly from that in healthy control subjects (Figure 2). Mean plasma homocysteine levels in BD were found to be significantly higher than the control group BRIEF REPORTS
523
FIGURE 3. The association between FMD (percent) and plasma homocysteine levels in BD. FIGURE 2. NTG-induced dilation in patients with BD and control subjects. NTG ⴝ nitroglycerin.
TABLE 2 Vascular and Laboratory Study Results of BD With Ocular and Nonocular Involvement
FMD (%) Nitroglycerin-induced dilation (%) Brakial artery diameter (mm) Plasma homocysteine (mol/L)
BD With Ocular Involvement (n ⫽ 20)
BD With Nonocular Involvement (n ⫽ 11)
p Value
0.5 ⫾ 3.0 17.3 ⫾ 3.7
2.7 ⫾ 2.5 15.2 ⫾ 3.4
0.03 0.17
4.1 ⫾ 0.7 15.3 ⫾ 2.8
4.0 ⫾ 0.9 13.3 ⫾ 1.9
0.79 0.02
Values are expressed as mean ⫾ SD.
(Table 1). Table 2 displays the vascular and laboratory results of patients with ocular and nonocular involvement. In patients with BD, FMD of the brachial artery was found to be strongly and inversely correlated with mean plasma homocysteine levels (r ⫽ ⫺0.67, p ⫽ 0.0001) (Figure 3). Similarly, FMD of the brachial artery was found to be inversely correlated with mean duration of the disease (r ⫽ ⫺0.58, p ⫽ 0.001) (Figure 4). When multivariate regression analysis was performed, it showed that only total homocysteine concentration was independently related to FMD (F ⫽ 5.7, p ⫽ 0.001) among total cholesterol, high-density and low-density cholesterol, triglyceride and glucose levels, duration of the disorder, body mass index, systolic/diastolic blood pressures, and age. •••
The principal findings of the present study are that (1) brachial artery FMD is impaired in patients with BD compared with control subjects, (2) plasma homocysteine levels are markedly higher in patients with BD than those of control subjects and significantly associated with the degree of FMD, (3) similar relations were also seen between the duration of disease and FMD, and (4) in ocular BD, FMD is reduced compared with nonocular BD and plasma homocysteine levels are higher in ocular disease than in non524 THE AMERICAN JOURNAL OF CARDIOLOGY姞
VOL. 94
FIGURE 4. The association between FMD (percent) and duration of disease.
ocular disease. This study is the first to show an association between endothelial dysfunction and homocysteine levels in BD. As is known, the pathogenesis of BD is characterized by hypercoagulability and vascular injury4 –7; however, the factors that may contribute to vascular injury have not been well defined. We therefore attempted to measure the plasma homocysteine levels and to determine its contribution to vascular injury. Our results showed that mean plasma homocysteine levels are markedly elevated in patients with BD compared with control subjects and it is well correlated to the degree of endothelial damage. In accordance with our study, mean plasma homocysteine concentrations in patients with BD have been reported to be elevated compared with those in healthy controls, and it has been concluded that hyperhomocysteinemia may be an independent risk factor for venous thrombosis.8 –11 However, these investigators investigated the association between homocysteine levels and development of thrombosis, but not the degree of endothelial function. AUGUST 15, 2004
From the clinical point of view, endothelial dysfunction and elevated homocysteine levels may increase susceptibility to future cardiovascular complications in patients with BD.12–14 This supports the hypothesis that hyperhomocysteinemia may be a risk factor for the development of ocular disease in BD. 1. International Study Group for Behcet’s Disease. Criteria for diagnosis of
Behcet’s disease. Lancet 1990;335:1078 –1080. 2. Frantzen F, Faaren AL, Alfheim I, Nordhei AK. An enzyme conversion
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6. Bozkurt E, Erol MK, Keles S, Acikel M, Yilmaz M, Gürlertop Y. Relation of plasma homocysteine levels to intracoronary thrombus in unstable angina pectoris and in non–Q-wave acute myocardial infarction. Am J Cardiol 2002;90: 413– 415. 7. Ozdemir R, Sezgin AT, Topal E, Kutlu R, Barutcu I, Gullu H. Findings of ambulatory blood pressure monitoring and heart rate variability in patients with Behcet’s disease. Am J Cardiol 2003;92:646 – 648. 8. Schmitz-Huebner U, Knop J. Evidence for an endothelial cell dysfunction in association with Behcet’s disease. Thromb Res 1984;34:277–285. 9. Wall RT, Harlan LM, Harker LA, Striker GE. Homocysteine-induced endothelial cell injury in vitro:a model for the study of vascular injury. Thromb Res 1980;18:113–121. 10. Blundell G, Jones BG, Rose FA, Tudball N. Homocysteine mediated endothelial cell toxicity and its amelioration. Atherosclerosis 1996;122:163–172. 11. Blacher J, Benetos A, Kirzin JM, Malmejac A, Guize L, Safar ME. Relation of plasma total homocysteine to cardiovascular mortality in a French population. Am J Cardiol 2002;90:591–595. 12. Sakane T. New perspective on Behcet’s disease. Int Rev Immunol 1997;14: 89 –96. 13. Hankey GJ, Eikelboom JW. Homocysteine and vascular disease. Lancet 1999;354:407– 413. 14. Er H, Evereklioglu C, Cumurcu T, Türköz Y, Özerol E, S¸ ahin K, Doganay S. Serum homocysteine level is increased and correlated with endothelin-1 and nitric oxide in Behçet’s disease. Br J Ophthalmol 2002;86:653– 657.
Characteristics and Prevalence of Intrapulmonary Shunt Detected by Contrast Echocardiography With Harmonic Imaging in Liver Transplant Candidates Byung Jin Kim, MD, Sang-Chol Lee, MD, Seung Woo Park, MD, Moon-Suk Choi, Kwang Cheol Koh, MD, Seung Woon Paik, MD, Sang Hoon Lee, MD, Kyung Pyo Hong, MD, Jeong Euy Park, MD, and Jung Don Seo, MD We investigated the prevalence and characteristics of intrapulmonary shunt using contrast echocardiography with harmonic imaging in 130 liver transplant candidates. We found a high prevalence of intrapulmonary shunts and a significant correlation between the degree of intrapulmonary shunt and the ChildPugh classification score. 䊚2004 by Excerpta Medica, Inc. (Am J Cardiol 2004;94:525–528)
revious studies have suggested that intrapulmonary shunts are not associated with various paP rameters of liver disease. We studied the prevalence 1–3
and clinical characteristics of intrapulmonary shunts by contrast echocardiography with harmonic imaging in liver transplant candidates. •••
From November 1999 to September 2002, 130 patients (89 men and 41 women; mean age 47 ⫾ 10 years) awaiting liver transplantation due to end-stage From the Department of Medicine, Cardiac and Vascular Center, the Division of Gastroenterology, and Cardiac Center, Kangbuk Samsung Hospital, Sungkyunkwan University School of Medicine, Samsung Medical Center, Seoul, Korea. Dr. Lee’s address is: Department of Medicine, Samsung Medical Center, Sungkyunkwan University School of Medicine, Ilwon-Dong 50, Kangnam-Ku, Seoul, Korea 135-710. E-mail: [email protected]. Manuscript received January 19, 2004; revised manuscript received and accepted April 19, 2004. ©2004 by Excerpta Medica, Inc. All rights reserved. The American Journal of Cardiology Vol. 94 August 15, 2004
MD,
liver disease were studied. The most frequent etiology (87%) of liver disease in these patients was chronic hepatitis B, and other etiologies were primary sclerosing cholangitis (5%), chronic hepatitis C (4%), cryptogenic disease (2%), and Wilson’s disease (2%). Patients with active upper gastrointestinal bleeding, combined valvular heart disease, primary pulmonary parenchymal disease, or patent foramen ovale were excluded from the study. Contrast echocardiographic studies were performed 3 times by injecting an agitated solution of 5 ml of normal saline solution and 1 ml of room air using a peripheral intravenous line and two 10-ml syringes connected by a 3-way stopcock. Echocardiography was performed by 3.5-MHz harmonic imaging (Siemens Medical Solutions, Mountain View, California) using 2-dimensional apical 4-chamber views. Positive contrast echocardiography for an intrapulmonary shunt was defined as the delayed appearance of microbubbles in the left-sided heart chambers through the pulmonary veins within 4 to 6 heart beats after their appearance in the right side of the heart. According to the intrapulmonary shunt grading scales of Vedrinne et al,4 the degree of an intrapulmonary shunt was assessed as from grade 0 to IV (Figure 1). Patients with grades II to IV were considered to have a significant intrapulmonary shunt. Liver function tests, blood coagulation tests, arterial blood gas analyses, and pulmonary function tests were performed on the day of the echocardiographic study. The Child-Pugh classi0002-9149/04/$–see front matter doi:10.1016/j.amjcard.2004.04.074
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