CVJA Volume 26 Issue 6 by Clinics Cardive Publishing

NOVEMBER/DECEMBER 2015 VOL 26 NO 6 A Lupin Group Company

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CardioVascular Journal of Africa (official journal for PASCAR)

• Heart-type fatty acid-binding protein as diagnostic marker of non-STEMI • Topical beta-blocker-induced atrioventricular block • Interatrial conduction delay with polycystic ovary syndrome • Protective effects of ginseng extracts on ischaemia–reperfusion injury

• Peri-operative myocardial injury and apoptosis during CABG surgery

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Cardiovascular Journal of Africa . Vol 26, No 6, November/December 2015

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ISSN 1995-1892 (print) ISSN 1680-0745 (online)

Vol 26, No 6, NOVEMBER/DECEMBER 2015

CONTENTS

Cardiovascular Journal of Africa 203

www.cvja.co.za

From the Editor’s Desk P Commerford

Cardiovascular Topics 204

Comparison of a qualitative measurement of heart-type fatty acid-binding protein with other cardiac markers as an early diagnostic marker in the diagnosis of non-ST-segment elevation myocardial infarction DM Gerede • S Güleç • M Kılıçkap • CT Kaya • VK Vurgun • ÖU Özcan • H Göksülük • Ç Erol

210 Management and outcome of topical beta-blocker-induced atrioventricular block KS Özcan • B Güngör • D Osmonov • Aİ Tekkeşin • S Altay • A Ekmekçi • E Toprak • E Yıldırım • N Çalık • AT Alper • K Gürkan • İ Erdinler 214 Autonomic imbalance assessed by time-domain heart rate variability indices in primary Raynaud’s phenomenon K Karabacak • M Celik • E Kaya • M Kadan • G Arslan • U Demirkilic 217 Atrial conduction time, and left atrial mechanical and electromechanical functions in patients with polycystic ovary syndrome: interatrial conduction delay E Gazi • M Gencer • V Hanci • A Temiz • B Altun • A Barutcu • AN Gungor • S Hacıvelioglu • A Uysal • Y Colkesen 222 Protective effects of ginseng extracts and common anti-aggregant drugs on ischaemia– reperfusion injury A Caliskan • O Karahan • S Yazici • S Demirtas • O Guclu • O Tezcan • C Yavuz 227 An open-access, mobile, compatible, electronic patient register for rheumatic heart disease (‘eRegister’) based on the World Heart Federation’s framework for patient registers J van Dam • J Musuku • LJ Zühlke • ME Engel • N Nestle • B Tadmor • J Spector • BM Mayosi

INDEXED AT SCISEARCH (SCI), PUBMED, PUBMED CENTRAL AND SABINET

Editors

SUBJECT Editors

Editorial Board

Editor-in-Chief (South Africa) Prof Pat Commerford

Nuclear Medicine and Imaging DR MM SATHEKGE

prof PA Brink Experimental & Laboratory Cardiology

PROF A LOCHNER Biochemistry/Laboratory Science

PROF R DELPORT Chemical Pathology

PROF BM MAYOSI Chronic Rheumatic Heart Disease

Assistant Editor Prof JAMES KER (JUN) Regional Editor DR A Dzudie Regional Editor (Kenya) Dr F Bukachi Regional Editor (South Africa) PROF R DELPORT

Heart Failure Dr g visagie Paediatric dr s brown Paediatric Surgery Dr Darshan Reddy Renal Hypertension dr brian rayner Surgical dr f aziz Adult Surgery dr j rossouw Epidemiology and Preventionist dr ap kengne Pregnancy-associated Heart Disease Prof K Sliwa-hahnle

PROF MR ESSOP Haemodynamics, Heart Failure DR MT MPE Cardiomyopathy & Valvular Heart Disease DR OB FAMILONI Clinical Cardiology DR V GRIGOROV Invasive Cardiology & Heart Failure

International Advisory Board PROF DAVID CELEMAJER Australia (Clinical Cardiology) PROF KEITH COPELIN FERDINAND USA (General Cardiology) DR SAMUEL KINGUE Cameroon (General Cardiology)

PROF DP NAIDOO Echocardiography

DR GEORGE A MENSAH USA (General Cardiology)

PROF B RAYNER Hypertension/Society

PROF WILLIAM NELSON USA (Electrocardiology)

PROF MM SATHEKGE Nuclear Medicine/Society PROF J KER (SEN) Hypertension, Cardiomyopathy, PROF YK SEEDAT Cardiovascular Physiology Diabetes & Hypertension

DR ULRICH VON OPPEL Wales (Cardiovascular Surgery)

DR J LAWRENSON Paediatric Heart Disease

PROF ERNST VON SCHWARZ USA (Interventional Cardiology)

PROF H DU T THERON Invasive Cardiology

PROF PETER SCHWARTZ Italy (Dysrhythmias)

Vol 26, No 6, NOVEMBER/DECEMBER 2015

CONTENTS

234 High-sensitivity cardiac troponin T is more helpful in detecting peri-operative myocardial injury and apoptosis during coronary artery bypass graft surgery EF Kocak • C Kocak • A Aksoy • OO Isiklar • R Akcilar • IF Ozdomanic • C Unsal • M Celenk • I Altuntas 242 Lack of cardioprotection by single-dose magnesium prophylaxis on isoprenalineinduced myocardial infarction in adult Wistar rats C Garson • R Kelly-Laubscher • D Blackhurst • A Gwanyanya 250

Drug trends in cardiology

251

CARDIO NEWS

PUBLISHED ONLINE (Available on www.cvja.co.za and in Pubmed) Case Reports

e1 Dyspnoea and chest pain as the presenting symptoms of pneumomediastinum: two cases and a review of the literature H Kara • HG Uyar • S Degirmenci • A Bayir • M Oncel • A Ak e5 Iatrogenic left main-stem dissection extending to the circumflex artery and retrogradely involving the left and non-coronary sinuses of Valsalva: iatrogenic aortocoronary dissection R Zwoliński • A Marcinkiewicz • K Szymczyk • R Pietruszyński • R Jaszewski e8 A rare case of aortic dissection presenting as pure transient global amnesia H Kaveeshvar • R Kashouty • V Loomba • N Yono e10 Application of thoracic endovascular dissecting aneurysm repair for secondary type B aortic dissection O Karahan • O Tezcan • S Demirtas • A Caliskan • C Yavuz

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PETER WAGENAAR Cell 082 413 9954 e-mail: skylark65@myconnection.co.za The Cardiovascular Journal of Africa, incorporating the Cardiovascular Journal of South Africa, is published 10 times a year, the publication date being the third week of the designated month. Copyright: Clinics Cardive Publishing (Pty) Ltd.

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CARDIOVASCULAR JOURNAL OF AFRICA • Volume 26, No 6, November/December 2015

203

From the Editor’s Desk This issue provides an array of articles on a variety of cardiovascular topics ranging from clinical registries to basic laboratory science. Most healthcare providers involved in clinical medicine and cardiology in Africa agree that rheumatic heart disease (RHD) remains common, and the consequences for affected individuals are devastating. Despite the perception of its importance, there are a limited number of detailed studies documenting with any degree of accuracy the number of patients affected. Registries serve many useful purposes, one of which describes the scale of the problem and allows planners and providers of healthcare to appropriately allocate resources and to measure whether those allocated are, over time, effective in alleviating the situation. Individual patients identified and recorded in registries may benefit from longitudinal monitoring of compliance with secondary prophylaxis, which is central to control of RHD. The data generated from good registries will greatly benefit research into RHD. Paper-based registries are available but often not used as well as they should be. On page 227, van Dam and co-authors describe the development of an open-access mobile, compatible, electronic patient-register system based on the World Heart Federation paper-based registry forms. The system functions on mobile phones and other mobile devices, as well as on computer systems in clinics and hospitals. This is a description of an exciting development, which if implemented, could potentially change the way we measure and treat RHD. Raynaud’s phenomenon remains one of those unusual, quirky ‘illnesses’, the pathogenesis of which we do not really understand. It causes considerable discomfort to some sufferers without serious long-term sequelae and may be effectively treated by vasodilating calcium-channel blocking agents. Karabacak and others (page 214) report that in a Turkish male cohort there

was evidence of autonomic dysfunction, as measured by heart rate variability, when compared to an age- and gender-matched control cohort. The authors acknowledge that there have been other reports of autonomic dysfunction in this condition. It is not clear why the researchers chose to investigate this condition, which as they report is more common in women, in an exclusively male cohort. Glaucoma and atrioventricular (AV) block both occur more frequently with increasing age and so it is not surprising that conduction disturbances are found in patients treated for glaucoma with β-blocker-containing eye-drops, as reported by Ozcan and co-workers (page 210). Whether the AV block is caused by the drops or not remains, as the authors state, controversial. What is important is that permanent pacing allows continuation of effective therapy for the glaucoma. The goal of cardioprotection in the setting of acute myocardial infarction is important and has received intense attention and interest in the last decades. Despite much basic science effort and the pursuit of theoretically promising avenues, the only interventions that have proven to be clinically effective in humans have been the restoration of blood flow and maintenance of that blood flow by mechanical therapies or pharmacological treatments interfering with blood coagulation. The report by Gwanyanya and others (page 242) of the failure of magnesium to provide protection against experimentally induced myocardial infarction is another example of a theoretically impressive intervention that fails to deliver. Clinicians will continue (hopefully) to implement strategies of proven benefit and avoid untested interventions. PJ Commerford Editor-in-Chief

The management and staff of Clinics Cardive Publishing (publishers of the Cardiovascular Journal of Africa and the South African Journal of Diabetes & Vascular Disease) take this opportunity to thank you for your loyal support during 2015 and we look forward to being of service during 2016. We wish you and your family a merry festive season and a prosperous new year. Please note our offices will close on 17 December and we will be open from 11 January 2016.

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CARDIOVASCULAR JOURNAL OF AFRICA • Volume 26, No 6, November/December 2015

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Cardiovascular Topics Comparison of a qualitative measurement of heart-type fatty acid-binding protein with other cardiac markers as an early diagnostic marker in the diagnosis of non-STsegment elevation myocardial infarction Demet Menekşe Gerede, Sadi Güleç, Mustafa Kılıçkap, Cansın Tulunay Kaya, Veysel Kutay Vurgun, Özgür Ulaş Özcan, Hüseyin Göksülük, Çetin Erol

Abstract Objective: Heart-type fatty acid-binding protein (H-FABP) is a novel cardiac marker used in the early diagnosis of acute myocardial infarction (AMI), which shows myocyte injury. Our study aimed to compare bedside H-FABP measurements with routine creatine kinase-MB (CK-MB) and troponin I (TnI) tests for the early diagnosis of non-ST-elevation MI (NSTEMI), as well as for determining its exclusion capacity. Methods: A total of 48 patients admitted to the emergency room within the first 12 hours of onset of ischaemic-type chest pain lasting more than 30 minutes and who did not have ST-segment elevation on electrocardiography (ECG) were included in the study. Definite diagnoses of NSTEMI were made in 24 patients as a result of 24-hour follow up, and the remaining 24 patients did not develop MI. Results: When various subgroups were analysed according to admission times, H-FABP was found to be a better diagnostic marker compared to CK-MB and TnI (accuracy index 85%), with a high sensitivity (79%) and specificity (93%) for early diagnosis (≤ six hours). The respective sensitivities of bedside H-FABP and TnI tests were 89 vs 33% (p < 0.05) for patients presenting within three hours of onset of symptoms. Conclusion: Bedside H-FABP measurements may contribute to correct early diagnoses, as its levels are elevated soon following MI, and measurement is easy, with a rapid result.

Keywords: acute coronary syndrome, non-ST-elevation myocardial infarction, H-FABP, CK-MB, troponin

Department of Cardiology, Ankara University School of Medicine, Ankara, Turkey Demet Menekşe Gerede, MD, drmeneksegerede@yahoo.com Sadi Güleç, MD Mustafa Kılıçkap, MD Cansın Tulunay Kaya, MD Veysel Kutay Vurgun, MD Özgür Ulaş Özcan, MD Hüseyin Göksülük, MD Çetin Erol, MD

Submitted 26/11/14, accepted 16/3/15 Published online 14/7/15 Cardiovasc J Afr 2015; 26: 204–209

www.cvja.co.za

DOI: 10.5830/CVJA-2015-028

Acute coronary syndrome (ACS) defines the clinical conditions that develop as a result of an abrupt reduction in coronary blood flow. Unstable angina pectoris (UAP), ST-elevation acute myocardial infarction (STEMI), and non-ST-elevation acute myocardial infarction (NSTEMI) are points on this clinical spectrum. All these clinical syndromes should be rapidly diagnosed and treated.1,2 Chest pain contributes to 50% of emergency room admissions and approximately 25% of these patients are hospitalised.3 Patients with ACS are usually admitted with chest pain. Studies have shown that the final diagnoses of patients admitted with chest pain are acute myocardial infarction (AMI) in one-third of patients, UAP in one-third, and non-cardiac chest pain in one-third.4 Early diagnosis of acute chest pain is especially important and difficult in patients without persistent ST-segment elevation. Electrocardiography (ECG) is a valuable and commonly used test for the detection of ACS. The initial ECG is normal or non-diagnostic in 50% of patients with ACS.5 STEMI is readily diagnosed with culprit ECG findings but NSTEMI/ UAP diagnoses are more challenging. Inadequate and delayed diagnoses may lead to inappropriate treatment and delays in the initiation of life-saving therapy. Diagnostic criteria of AMI were reformed after the introduction of more sensitive and specific markers for cardiac injury (cardiac troponins, CK-MB mass) and after a better understanding of the diagnostic and prognostic importance of these markers. In the 2012 report of the European Society of Cardiology (ESC)/ American College of Cardiology (ACC), the essential criterion for MI was defined as elevated cardiac markers.6 Heart-type fatty acid-binding protein (H-FABP) is a recently discovered cardiac biomarker. It is specific to cardiomyocytes and low-molecular weight (15 kDa) cytosolic proteins, which represent five to 15% of the cytosolic proteins of cardiac myocytes.7 H-FABP plays an important role in intracellular transport for β-oxidation of fatty acids in the mitochondria.8,9

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CARDIOVASCULAR JOURNAL OF AFRICA • Volume 26, No 6, November/December 2015

H-FABP is released from the myocardium into the circulation within one to three hours of myocardial injury. Its concentration in the blood peaks within six to eight hours and decreases within 24 to 30 hours.10 Its properties of being abundantly available in myocardial tissue, intra-cytosolic dominancy, relative tissue specificity, and early elevation in blood and urine after AMI suggest that H-FABP may be used in the early diagnosis of ACS.11,12 Its plasma kinetics and secretion are similar to that of myoglobin, therefore, it is used as a marker for the early diagnosis of ACS.13 There are few studies on this topic and the results of previous studies are controversial.14-19 In our study, we aimed to evaluate the diagnostic effectiveness of H-FABP in the early diagnosis of NSTEMI and to compare it with other cardiac markers, including CK-MB and troponin I (TnI) levels.

Methods Forty-eight patients who were admitted to the emergency department within the first 12 hours of onset of ischaemic-type chest pain lasting for longer than 30 minutes, and who did not have ST-segment elevation on ECG, were included in the study. The patients who had newly developed left bundle branch block, who were admitted more than 12 hours after the onset of chest pain, who had chronic renal failure, chronic muscular diseases or heart failure, or who had recently experienced trauma, musculoskeletal injury or shock, were excluded from the study. A detailed medical history was obtained from each patient and a physical examination was performed. Twelve-lead ECGs were obtained and the changes were recorded. A complete blood count, biochemical tests and urgent cardiac profiles (CK-MB mass, myoglobin and TnI levels) were obtained from venous blood. Bedside H-FABP level was also determined from the same blood sample. The patients were monitored for 24 hours, and urgent cardiac profiles and ECG monitoring were performed every six hours. NSTEMI was diagnosed in 24 patients as the result of 24-hour follow up, and the remaining 24 patients did not develop MI. The blood samples were immediately sent to the biochemistry laboratory of our hospital to measure TnI and CK-MB levels. Blood was taken in a 5-cm3 plain tube and centrifuged at 3 000 rpm for 10 minutes. The serum was separated and loaded into a Beckman Coulter Access II device and analysed with chemiluminescence. Measurement of the cardiac markers in each sample was completed within 30 to 45 minutes. The reference values of the cardiac markers were < 0.04 ng/ml for TnI (< 0.04 μg/l) and 0.6–6.3 ng/ml for CK-MB (0.6–6.3 μg/l). All patients were also tested with the CardioDetect® (Med-Rennessens, Niemcy, Poland) H-FABP immunotest. It is a rapid chromatographic immunoassay method designed for qualitative determination of H-FABP levels in blood samples. Three to four drops of capillary blood were dropped onto a CardioDetect kit and left on a flat surface for 15 minutes. Double lines were interpreted as positive, a single line was negative, and no lines was interpreted as inadequate material. H-FABP > 7 μg/l was seen as positive in this test.20 H-FABP was tested only once in each patient, as the number of kits was limited. TnI and/or CK-MB elevation (verified with at least two different measurements) associated with ischaemic-type chest pain for over 30 minutes and without persistent ST-segment

205

elevation was accepted as NSTEMI, regardless of ECG change, as recommended by the ESC/ACC committee.6

Statistical analysis All data were transferred to the SPSS 10.0 statistics program. The Student’s t-test was used for a comparison of the groups when parametric assumptions were realised, and the chi-square and Fisher’s exact tests were used as a comparison and an association of the categorical data, respectively. Screening test results are also given. A p-value of < 0.05 was considered statistically significant. For calculation of sample size, as a guideline we used the results of a study conducted by Ruzgar et al.,21 in which a sensitivity of tnI and H-FABP was 0.38 and 0.95, respectively. However, in order to be more conservative, sample size was calculated based on a sensitivity of tnI of 0.38, sensitivity of H-FABP of 0.8, pre-test probability of 0.6, power of 0.8, and type 1 error rate of 0.05 (with 95% confidence). We found the required sample size to be 43, and our study group consisted of 48 people. For an assessment of the diagnostic performance of cardiac markers in the diagnosis of NSTEMI, sensitivity, specificity, negative predictive value (NPV), positive predictive value (PPV) and the accuracy index (AI) of each marker were calculated according to admission times. Diagnostic sensitivity was calculated by dividing the number of patients who were diagnosed with NSTEMI using H-FABP, CK-MB or TnI levels by the number of patients who were diagnosed with NSTEMI, as recommended by the ESC/ACC committee.6 Diagnostic specificity was calculated by dividing the number of patients who were diagnosed without NSTEMI using H-FABP, CKMB or TnI levels by the number of the patients who were diagnosed without NSTEMI, as recommended by ESC/ACC committee.6 PPV was calculated as the ratio of the number of patients with NSTEMI with positive test results to the number of all patients with positive test results. NPV was calculated as the ratio of the number of patients without NSTEMI with negative test results to the number of all patients with negative test results. Accuracy index was the ratio of the sum of the true-positive (positive marker and NSTEMI) and true-negative (negative marker and no NSTEMI) patients to the number of all patients. The accuracy shows that a cardiac marker can be used as the criterion for an acceptable diagnostic marker for diagnosis of MI.

Test + Test –

Sensitivity = ______   (a + c)    d

Specificity =  ______ (b + d) a

PPV = ______   (a + b)  d

NPV = ______   (c + d)  (a + d)

Accuracy =   ____________ (a + b + c + d) .

NSTEMI +

NSTEMI –

a c

b d

206

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CARDIOVASCULAR JOURNAL OF AFRICA • Volume 26, No 6, November/December 2015

Results In total, 48 patients were included in the study. The demographic and clinical characteristics of the patients are given in Table 1. The mean time of admission was 5.2 (2–10) hours. While ST-segment depression was seen on the ECG of 14 patients (29%), T-wave negativity was seen in 20 (42%) and ST–T segment changes were not detected in 14 patients (29%). A diagnosis of NSTEMI was made in 24 out of 48 patients. Coronary angiography was performed in 40 patients. While H-FABP assessment on admission (two to 10 hours after onset of chest pain) was positive in 20 out of 24 patients whose NSTEMI diagnoses were definite, negative results were obtained in four patients. These four patients constituted the false-negative patient group. H-FABP was found to be negative in 22 out of 24 patients in whom NSTEMI was eliminated, and it was found to be positive in two. These two patients constituted the false-positive patient group. Table 2 summarises the positivity and negativity of the cardiac markers, which were tested on admission. The results of the analyses based on these data showed that diagnostic sensitivity was 83.3%, specificity was 91.7%, NPV was 84.6%, PPV was 90.6%, and AI was 87% for H-FABP in the diagnosis of NSTEMI. Comparisons of these values with other cardiac markers are summarised in Table 3. A comparative analysis of the data obtained when the patients were divided into three groups, according to admission times (≤ three hours, three to six hours, and > six hours after onset of symptoms) is given in Table 4, and two groups (≤ six hours and > six hours after onset of symptoms) is given in Table 5. The sensitivity and specificity of H-FABP for Table 1. Patients’ characteristics Number (%) or mean ± SD Characteristic (minimum–maximum values) Age 60 ± 9 (38–79) Male gender 28 (58) Hypertension 35 (73) Diabetes mellitus 14 (29) Smoking 23 (48) Total cholesterol (mg/dl) 204 ± 57 (97–311) (mmol/l) 5.28 ± 1.48 (2.51–8.05) LDL-C (mg/dl) 124 ± 55 (34–243) (mmol/l) 3.21 ± 1.42 (0.88–6.29) HDL-C (mg/dl) 45 ± 11 (14–69) (mmol/l) 1.17 ± 0.28 (0.36–1.79) Triglycerides (mg/dl) 148 ± 88 (19–533) (mmol/l) 1.67 ± 0.99 (0.21–6.02) Family history 12 (25) History of CAD 15 (31) Admission time (hours) 5.2 ± 2.4 (2–10) ECG on admission ST depression 14 (29) T negativity 20 (42) No ECG changes 14 (29) Coronary angiography findings Normal coronary arteries 7 (17.5) 9 (22.5) Insignificant stenosis (< 50%) Single-vessel disease 13 (32.5) Multiple-vessel disease 11 (27.5) LDL-C, low-density lipoprotein cholesterol; HDL-C, high-density lipoprotein cholesterol; CAD, coronary artery disease.

Table 2. Cardiac markers of the patients who were diagnosed with and without NSTEMI H-FABP positive, n (%) TnI positive, n (%) CK-MB positive, n (%)

NSTEMI + (n = 24) NSTEMI – (n = 24) 20 (83.3) 2 (8.3) 15 (62.5) 4 (16.6) 12 (50) 1 (4.1)

Table 3. Sensitivity, specificity, NPV, PPV and AI of H-FABP, TnI and CK-MB in the diagnosis of NSTEMI

H-FABP TnI CK-MB

Sensitivity Specificity (%) (%) 83.3 91.7 62.5 83.3 50 95.8

NPV 84.6 68.9 65.7

PPV 90.9 78.9 92.3

AI 87.5 72.9 72.9

≤ three hours were calculated as 89 and 100%, respectively, the sensitivity and specificity for three to six hours were 70 and 89%, respectively, and the sensitivity and specificity for > six hours were 100 and 89%, respectively. The respective sensitivities of bedside H-FABP and tnI tests were 89 vs 33% (p < 0.05) for patients presenting within three hours of onset. When H-FABP, CK-MB and TnI were compared according to AI at ≤ three and three to six hours, H-FABP was shown to be a better diagnostic marker (p < 0.01 and p < 0.05, respectively). From the assessment of the two groups of admission times (≤ six hours and > six hours after onset of symptoms), the diagnostic sensitivity and specificity of H-FABP levels were found to be 79 and 93% for ≤ six hours, respectively, while sensitivity and specificity were found to be 100 and 89% for > six hours, respectively. These values indicate that H-FABP is a sensitive and specific marker for the diagnosis of NSTEMI at ≤ six hours (accuracy 85%). When AI values were compared, H-FABP was found to be a better diagnostic marker than TnI (85 vs 65%, p < 0.05) and CK-MB (85 vs 62%, p < 0.05) for the early period (≤ six hours). Table 4. Diagnostic value of H-FABP, TnI and CK-MB in NSTEMI diagnosis, according to admission time after onset of symptoms (≤ 3, 3–6 and > 6 hours)

H-FABP Sensitivity (%) Specificity (%) NPV (%) PPV (%) AI (%) TnI Sensitivity (%) Specificity (%) NPV (%) PPV (%) AI (%) CK-MB mass Sensitivity (%) Specificity (%) NPV (%) PPV (%) AI (%)

≤ 3 hours (n = 15)

3–6 hours (n = 19)

> 6 hours (n = 14)

89 100 86 100 93

70 89 73 88 78

100 89 100 83 92

33 100 50 100 60

70 67 67 70 68

100 89 100 83 92

22 100 46 100 53

50 89 62 83 68

100 100 100 100 100

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Table 5. Diagnostic value of H-FABP, TnI and CK-MB in NSTEMI diagnosis, according to admission time after onset of symptoms (≤ 6 and > 6 hours) ≤ 6 hours (n = 34)

> 6 hours (n = 14)

79 93 78 94 85

100 89 100 83 93

53 80 57 77 65

100 89 100 83 93

37 93 54 88 62

100 100 100 100 100

H-FABP Sensitivity (%) Specificity (%) NPV (%) PPV (%) AI (%) TnI Sensitivity (%) Specificity (%) NPV (%) PPV (%) AI (%) CK-MB mass Sensitivity (%) Specificity (%) NPV (%) PPV (%) AI (%)

On the other hand, a statistically significant difference was not detected between H-FABP, TnI and CK-MB levels in the patient group that was admitted after more than six hours (n = 14). Figs 1 and 2 compare the AI values of the cardiac markers according to admission times after onset of symptoms. False-negative results were obtained in four patients who were tested for H-FABP, and false-positive results were obtained in two. Although all characteristics of these six patients were reviewed in detail with the hope of finding any predictors for false negativity and false positivity, no characteristics could be detected to explain this condition.

Discussion

100 90 80 70 60 50 40 30 20 10 0

p < 0.01

p < 0.05

≤3h

3–6 h

marker than CK-MB and Tn I, with high sensitivity (79%) and specificity (93%) (AI = 85%) for early diagnosis of NSTEMI (≤ six hours). In addition, the sensitivity and specificity of H-FABP for the group admitted ≤ three hours of onset of symptoms were calculated as 89 and 100%, respectively. H-FABP is seen as a novel cardiac marker in the diagnosis of ACS. Nakata et al.22 and O’Donoghue et al.23 have shown it to be an early diagnostic and prognostic marker. It begins to elevate in the plasma within one to three hours following the initial symptoms of ACS and decreases to normal levels within 24 to 36 hours.10 A few immunohistochemical methods are used for the detection of H-FABP levels and these take from 45 minutes to 16 hours. This time decreases to 15 minutes with CardioDetect, a single-step, qualitative bedside test. Values > 7 mg/l are seen as positive.20 In a previous study conducted on 38 patients, this bedside method was compared with the ELISA method, which is used for the quantitative measurement of H-FABP levels, and it was completed in 45 minutes. These two methods were therefore similarly successful in making a diagnosis.20,24 Recent studies in the literature on the diagnostic value of H-FABP in ACS have given controversial results.25,26 Some studies showed H-FABP to be a reliable diagnostic tool for the early diagnosis of ACS/MI,14-16 and others displayed negative results.17-19 In the study by Glatz et al.9 conducted on 83 patients, the diagnostic sensitivity of H-FABP was shown to be better than that of myoglobin in patients who were admitted within six hours of onset of symptoms (78 vs 53%, p < 0.05). Similarly, in the study by Haastrup et al.,27 which was conducted on 130 patients who did not have ST-segment elevation and were admitted in under six hours, the sensitivity of H-FABP was found to be 90–95% and specificity was 81–94% for different reference values. Myoglobin and H-FABP were reported to be useful markers in the early triage of patients with chest pain. In the study by Yoshihiko et al., which was conducted on 129 patients suspected of AMI, the sensitivity of H-FABP was found to be 100% and specificity was 63% in the first three hours.28 Patients with STEMI were included this study. In the same study, the sensitivity of troponin T (TnT) was found to be 50% and specificity was 96% in the first three hours. They concluded that H-FABP was a more valid marker than TnT in

p > 0.05

Accuracy index (%)

In our study, the role of bedside H-FABP measurement was investigated in 48 patients who were admitted to the emergency room within 12 hours of onset of chest pain lasting for more than 30 minutes and who did not have ST-segment elevation on ECG. It was concluded that H-FABP was a better diagnostic

H-FABP

Tnl

>6h

CK-MB

Fig. 1. A I values of H-FABP, TnI, and CK-MB for the diagnosis of NSTEMI according to admission times (≤ 3, 3–6, and > 6 hours).

207

100 90 80 70 60 50 40 30 20 10 0

p < 0.05

p > 0.05

≤6h

H-FABP

>6h

Tnl

CK-MB

Fig. 2. AI values of H-FABP, TnI and CK-MB for the diagnosis of NSTEMI according to admission times (≤ 6 and > 6 hours).

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the diagnosis of AMI.28 We used TnI as a marker in our study. TnI is more commonly used today and it has been found to have greater specificity for myocardial injury in chronic renal failure patients than TnT.29 Similarly, in the study by Ruzgar et al.21 using a qualitative H-FABP measurement, they determined the sensitivity of H-FABP as 95% in the first six hours; however, patients with ST-segment elevation were included in the study. We did not include patients with ST-segment elevation in our study because early diagnosis can be made without waiting for the results of cardiac markers in patients with chest pain accompanied by ST-segment elevation. In a recent large study by McMahon et al.,30 H-FABP was shown to have the highest NPV of all the individual markers in the zero-to-three-hours admission time (93%) for early diagnosis of MI/ACS. According this study, H-FABP was also a valuable rule-out test for patients presenting three to six hours after the onset of chest pain. Unlike our study, H-FABP was measured quantitatively in this study. The study by Figiel et al.31 showed similar results to ours. The difference was that we focused on the time of admission from the onset of symptoms, and determined the sensitivity, specificity, NPV and PPV, since the primary benefit of a new biomarker would be early, rapid and accurate diagnosis. H-FABP seemed to be more sensitive than TnI, with a higher NPV in patients with admission within three hours of onset of symptoms. It would be possible to rule out NSTEMI diagnosis early in the course. In our study, diagnostic sensitivity and specificity, and the NPV and PPV of H-FABP were calculated as 83.3, 91.7, 84.6 and 90.6%, respectively, when all patients who were admitted after less than 12 hours of symptoms were evaluated. When compared with tnI and CK-MB, although the AI of H-FABP was found to be greater, the main time interval of H-FABP was superior to conventional markers at ≤ six hours. While the AI of H-FABP was 85% in this period, the AI of TnI and CK-MB were below this (65 and 62%, respectively, p < 0.05). The importance of our study was that it included only patients who had long-standing ischaemic-type chest pain, it excluded patients with ST-segment elevation, and we used a qualitative bedside method of H-FABP determination (CardioDetect). The limitations of our study include the small number of patients, it was a single-centre study, the study groups consisted of only patients who had ischaemic-type chest pain, and H-FABP was tested once only in every patient.

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Medicine. Philadelphia: WB Saunders, 2001: 1131–1135. 2.

Newby LK, Gibler B, Chriztenson RH. In: Cannon CP, ed. Serum Markers for Diagnosis and Risk Stratification in Acute Coronary Syndromes. NJ: Humana Press, 1999: 147–171.

Hodgson L. Cost containment in the emergency department. CAL/ ACEP Source Guide 1998; 710: 23.

Hargarten K, Chapman PD, Stueven HA, Waite EM, Mateer JR, Haecker P, et al. Prehospital prophylactic lidocaine does not favorably affect outcome in patients with chest pain. Ann Emerg Med 1990; 19: 1274–1279.

Rusnack RA, Stair TO, Hansen K, Fastow JS. Litigation against the emergency physician: Common features in cases of missed myocardial infarction. Ann Emerg Med 1989; 18: 1029–1034.

Thygesen K, Alpert JS, Jaffe AS, Simoons ML, Chaitman BR, White HD. ESC/ACCF/AHA/WHF expert consensus document: Third universal definition of myocardial infarction. Circulation 2012; 126: 2020–2035.

Furuhashi M, Hotamisligil GS. Fatty acid-binding proteins: role in metabolic diseases and potential as drug targets. Nat Rev Drug Discov 2008; 7: 489–503.

Schaap FG, Binas B, Danneberg H, van der Vusse GJ, Glatz JF. Impaired long-chain fatty acid utilization by cardiac myocytes isolated from mice lacking the heart-type fatty acid binding protein gene. Circ Res 1999; 85: 329–337.

Glatz JF, van der Vusse GJ, Simoons ML, Kragten JA, van DieijenVisser MP, Hermens WT. Fatty acid binding protein and the early detection of acute myocardial infarction. Clin Chim Acta 1998; 272: 87–92.

10. Pelsers MM, Hermens WT, Glatz JF. Fatty acid-binding proteins as plasma markers of tissue injury. Clin Chim Acta 2005; 352: 15–35. 11. Yoshimoto K, Tanaka T, Somiya K, Tsuji R, Okamoto F, Kawamura K, et al. Human heart-type cytoplasmic fatty acid-binding protein as an indicator of acute myocardial infarction. Heart Vessels 1995; 10: 304–309. 12. Tanaka T, Hirota Y, Sohmiya K, Nishimura S, Kawamura K. Serum and urinary human heart fatty acid-binding protein in acute myocardial infarction. Clin Biochem 1991; 24: 195–201. 13. Van Nieuwenhoven FA, Kleine AH, Wodzig WH, Hermens WT, Kragten HA, Maessen JG, et al. Discrimination between myocardial and skeletal muscle injury by assessment of the plasma ratio of myoglobin over fatty acid binding protein. Circulation 1995; 92: 2848–2854. 14. Orak M, Üstündağ M, Güloğlu C, Özhasenekler A, Alyan Ö, Kale E. The role of the heart-type fatty acid binding protein in the early diagnosis of acute coronary syndrome and its comparison with troponin I and creatine kinase-MB isoform. Am J Emerg Med 2010; 28: 891–896. 15. Gururajan P, Gurumurthy P, Nayar P, Srinivasa Nageswara Rao G,

Conclusion H-FABP appears to be a good diagnostic tool in the early period of NSTEMI in patients admitted with ischaemic-type chest pain. H-FABP could contribute to early bedside diagnosis in emergency rooms, as it is more sensitive and specific than other routinely used cardiac markers, such as TnI and CK-MB. This is because it becomes elevated soon after MI. Our results need to be confirmed with larger studies before routine use of this procedure.

Babu S, Cherian KM. Heart fatty acid binding protein (H-FABP) as a diagnostic biomarker in patients with acute coronary syndrome. Heart, Lung Circ 2010; 19: 660–664. 16. Xu Q, Chan CP, Cao XY, Peng P, Mahemuti M, Sun Q, et al. Cardiac multi-marker strategy for effective diagnosis of acute myocardial infarction. Clin Chim Acta 2010; 411: 1781–1787. 17. Charpentier S, Ducassé JL, Cournot M, Maupas-Schwalm F, Elbaz M, Baixas C, et al. Clinical assessment of ischemia-modified albumin and heart fatty acid-binding protein in the early diagnosis of non-STelevation acute coronary syndrome in the emergency department. Acad Emerg Med 2010; 17: 27–35.

References 1.

18. Charpentier S, Maupas-Schwalm F, Cournot M, Elbaz M, Ducassé JL, Bottela JM, et al. Diagnostic accuracy of quantitative heart-fatty

Antman EM, Braunwald E. Acute myocardial infarction. In: Braunwald

acid binding protein assays compared with Cardiodetect® in the early

E, Zipes D, Libby P, eds. Heart Disease. A Textbook of Cardiovascular

detection of acute coronary syndrome. Arch Cardiovasc Dis 2011; 104:

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of heart-type fatty acid-binding protein with the degree and extent

524–529.

of atherosclerosis in patients with non-ST elevation acute coronary

19. Banu KY, Niyazi OD, Erdem C, Dpekçi Afşin DH, Ozlem U, Yasemin

syndrome. Arch Turk Soc Cardiol 2013; 41: 610–616.

C, et al. Value of heart-type fatty acid-binding protein (H-FABP) for emergency department patients with suspected acute coronary

26. Freund Y, Chenevier-Gobeaux C, Leumani F, et al. Heart-type fatty acid

syndrome. Afr Health Sci 2014; 14: 757–762.

binding protein and the diagnosis of acute coronary syndrome in the ED. Am J Emerg Med 2012; 30: 1378–1384.

20. Chan C, Sum KW, Cheeung KY, Glatz JF, Sanderson JE, Hempel A, et al. Development of a quantitative lateral-flow assay for rapid detection

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27. Haastrup B, Gill S, Kristensen SR, Jørgensen PJ, Glatz JF, Haghfelt T,

of fatty acid-binding protein. J Immunol Methods 2003; 279: 91–100.

et al. Biochemical markers of ischaemia for the early identification of acute myocardial infarction without ST segment elevation. Cardiology

21. Ruzgar O, Bilge AK, Bugra Z, Umman S, Yilmaz E, Ozben B, et al. The

2000; 94: 254–261.

use of human heart-type fatty acid-binding protein as an early diagnostic biochemical marker of myocardial necrosis in patients with acute

28. Yoshihiko S, Tomita Y, Takano T, Ohbayashi K; Tokyo Rapid-Test

coronary syndrome, and its comparison with troponin-T and creatine

Office Cardiologists (Tokyo-ROC) study. Office cardiologists coopera-

kinase–myocardial band. Heart Vessels 2006; 21: 309–314.

tive study on whole blood rapid panel tests in patients with suspicious acute myocardial infarction. Circ J 2004; 68: 144–148.

22. Nakata T, Hashimoto A, Hase M, Tsuchihashi K, Shimamoto K. Human heart-type fatty acid-binding protein as an early diagnostic

29. Bhayana V, Gougoulias T, Cohoe S, Henderson AR. Discordance

and prognostic marker in acute coronary syndrome. Cardiology 2003;

between results for serum troponin T and troponin I in renal disease. Clin Chem 1995; 41: 312–317.

99: 96–104. 23. O’Donoghue M, de Lemos JA, Morrow DA, Murphy SA, Buros JL,

30. McMahon CG, Lamont JV, Curtin E, McConnell RI, Crockard M,

Cannon CP, et al. Prognostic utility of heart-type fatty acid binding

Kurth MJ, et al. Diagnostic accuracy of heart-type fatty acid-binding

protein in patients with acute coronary syndromes. Circulation 2006;

protein for the early diagnosis of acute myocardial infarction. Am J Emerg Med 2012; 30: 267–274.

114: 550–557. 24. Chan C, Wan T, Watkins K, Pelsers MM, van der Voort D, Tang FP,

31. Figiel Ł, Kasprzak JD, Peruga J, Lipiec P, Drozdz J, Krzemińska-Pakuła

et al. Rapid analysis of fatty acid-binding proteins with immunosen-

M, et al. Heart-type fatty acid binding protein – a reliable marker of

sors and immunotests for early monitoring of tissue injury. Biosens

myocardial necrosis in a heterogeneous group of patients with acute

Bioelectron 2005; 20: 2566–2580.

coronary syndrome without persistent ST elevation. Kardiol Pol 2008; 66: 253–259.

25. Zeren G, Erer HB, Kırıs T, Sahin O, Aksu H, Köprülü D, et al. Relation

Peripheral neuropathy associated with cardiovascular disease and stroke in type 2 diabetes patients Testing for peripheral neuropathy may provide a way to identify individuals at higher risk of cardiovascular events. Jack Brownrigg, a PhD student at St George’s, University of London, UK, who conducted the research at St George’s Vascular Institute, is quoted in a press release from St George’s as saying, ‘While the risk of cardiovascular disease is known to be higher in patients with diabetes, predicting which patients may be at greatest risk is often difficult. We looked at data on individuals with no history of cardiovascular disease and found that those with peripheral neuropathy were more likely to develop cardiovascular disease.’ Robert Hinchliffe, senior lecturer and consultant in vascular surgery at St George’s, who co-led the study with Prof Kausik Ray, said: ‘While loss of sensation in the feet is known to be a key risk factor for foot ulcers, it may also provide additional useful information to guide patient management. This is the first study to show that it can also indicate an increased risk of cardiovascular problems like heart attacks or strokes.’ ‘The good news is that peripheral neuropathy can be easily identified by simple tests carried out in GP surgeries. The results of the study warrant further investigation as to whether even greater control of risk factors, including blood

pressure and blood sugar can prevent or delay the onset of cardiovascular disease. There is likely an unmet potential to reduce cardiovascular disease in this group of patients through greater monitoring and simple treatments.’ The researchers analysed data from 13 000 patients diagnosed with type 2 diabetes with no history of cardiovascular disease. They found that individuals with peripheral neuropathy were more likely to develop cardiovascular disease, noticing that patients who experienced loss of sensation in their feet also tended to have heart and circulatory problems, and so they suggested that the presence of peripheral neuropathy could be used as a simple way to indicate which high-risk patients with diabetes are in need of intensive care and monitoring.

References 1.

Peripheral neuropathy and the risk of cardiovascular events in type 2 diabetes mellitus. Heart doi:10.1136/heartjnl-2014-305657.

http://heart.bmj.com/content/early/2014/08/05/heartjnl-2014-305657. abstract?sid=966c34dc-ea1f-4bc4-8547-d0dd61d23850.

http://www.diabetesincontrol.com/index.php?option=com_content &view=article&id=16752&catid=1&Itemid=17.

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Management and outcome of topical beta-blockerinduced atrioventricular block Kazım Serhan Özcan, Barış Güngör, Damirbek Osmonov, Ahmet İlker Tekkeşin, Servet Altay, Ahmet Ekmekçi, Ercan Toprak, Ersin Yıldırım, Nazmi Çalık, Ahmet Taha Alper, Kadir Gürkan, İzzet Erdinler

Abstract Background: Topical beta-blockers have a well-established role in the treatment of glaucoma. We aimed to investigate the outcome of patients who developed symptomatic atrioventricular (AV) block induced by topical beta-blockers. Methods: All patients admitted or discharged from our institution, the Siyami Ersek Training and Research Hospital, between January 2009 and January 2013 with a diagnosis of AV block were included in the study. Subjects using ophthalmic beta-blockers were recruited and followed for permanent pacemaker requirement during hospitalisation and for three months after discontinuation of the drug. A permanent pacemaker was implanted in patients in whom AV block persisted beyond 72 hours or recurred during the follow-up period. Results: A total of 1 122 patients were hospitalised with a diagnosis of AV block and a permanent pacemaker was implanted in 946 cases (84.3%) during the study period. Thirteen patients using ophthalmic beta-blockers for the treatment of glaucoma and no other rate-limiting drugs were included in the study. On electrocardiography, eight patients had complete AV block and five had high-degree AV block. The ophthalmic beta-blockers used were timolol in seven patients (55%), betaxolol in four (30%), and cartelol in two cases (15%). The mean duration of ophthalmic beta-blocker treatment was 30.1 ± 15.9 months. After drug discontinuation, in 10 patients the block persisted and a permanent pacemaker was implanted. During follow up, one more patient required pacemaker implantation. Therefore in total, pacemakers were implanted in 11 out of 13 patients (84.6%). The pacemaker implantation rate did not differ according to the type of topical beta-blocker used (p = 0.37). The presence of infra-nodal

Department of Cardiology, Derince Training and Research Hospital, Kocaeli, Turkey Kazım Serhan Özcan, MD, serhandr@gmail.com; serhan_oz@ yahoo.com

Department of Cardiology, Siyami Ersek Cardiovascular and Thoracic Surgery Centre, Istanbul, Turkey Barış Güngör, MD Ahmet İlker Tekkeşin, MD Servet Altay, MD Ahmet Ekmekçi, MD Ercan Toprak, MD Ersin Yıldırım, MD Nazmi Çalık, MD Ahmet Taha Alper, MD Kadir Gürkan, MD İzzet Erdinler, MD

Department of Cardiology, Almaty Sema Hospital, Almaty, Kazakhstan Damirbek Osmonov, MD

block on electrocardiography was associated with higher rates of pacemaker implantation. Conclusion: Our results indicate that topical beta-blockers for the treatment of glaucoma may cause severe conduction abnormalities and when AV block occurs, pacemaker implantation is required in a high percentage of the patients. Keywords: beta-blockers, glaucoma, drug-induced block, pacemaker implantation Submitted 17/1/15, accepted 16/3/15 Cardiovasc J Afr 2015; 26: 210–213

www.cvja.co.za

DOI: 10.5830/CVJA-2015-030

Topical beta-blockers have a well-established role in the treatment of glaucoma and are frequently used as first-line therapy for the reduction of associated ocular hypertension.1,2 While systemic concentration after administration of topical beta-blockers is low in comparison to that achieved with oral beta-blockers, cardiovascular, respiratory, central nervous system and metabolic side effects may still occur.3 Topical beta-blockers have been shown to decrease heart rate and blood pressure in comparison to placebo.4 Cardiovascular effects may be augmented with systemic combination therapy with other heart rate-blocking agents, such as beta-blockers and calcium channel blockers.5 In the literature, there are several case reports indicating the possible relationship between topical beta-blockers and the development of severe bradyarrhythmias, such as third-degree atrioventricular (AV) block and sick sinus syndrome.6-9 However, little is known about the incidence and prognosis of severe bradyarrhythmias induced by topical beta-blockers. In this trial, we aimed to investigate the outcome of patients who were hospitalised with a diagnosis of symptomatic AV block while receiving topical beta-blockers.

Methods All patients who were hospitalised in our institution, the Siyami Ersek Training and Research Hospital, between January 2009 and January 2013 with a diagnosis of AV block or symptomatic bradyarrhythmia were reviewed. The site of AV block was diagnosed by surface electrocardiography, as previously described10-13 (Table 1). Patients who had symptomatic (fatigue, faintness, dyspnoea and syncope) type II second- or third-degree AV block, 2:1 AV block, atrial fibrillation with bradycardia (average heart rate ≤ 40 beats/min on 24-hour Holter monitoring) were included in this study. Patients with vasovagal syncope, concomitant myocardial infarction, electrolyte abnormalities or digitalis toxicity were excluded.

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Table 1. Classification of second- and third-degree AV block, and atrial fibrillation with bradyarrhythmia, based on electrocardiographic characteristics Second-degree AV block 2:1 AV block Third-degree AV block Atrial fibrillation and bradyarrhythmia

AV nodal block PR increment preceding a blocked P (Wenckebach) and narrow QRS Conducted impulse has long PR and narrow QRS; PR varies inversely with RP Escape rhythm has narrow QRS and rate ≥ 40 beats/min f waves with irregular narrow QRS

Infra-nodal AV block Constant PR interval preceding blocked P

Undetermined level of AV block PR increment (Wenckebach) preceding a blocked P and wide QRS Conducted impulse has normal PR and wide Conducted impulse has long PR and QRS; PR is constant despite varying RP wide QRS or short PR and narrow QRS Escape rhythm has wide QRS and rate Escape rhythm has wide QRS and rate < 40 beats/min ≥ 40 beats/min f waves with regular wide QRS f waves with irregular wide QRS

AV = atrioventricular.

Subjects using ophthalmic beta-blockers were selected and followed for permanent pacemaker requirement during the hospitalisation period and for three months after discontinuation of the drug. Topical beta-blockers were discontinued after the initial refererral to the hospital. According to the response of the AV conduction after drug withdrawal, the type of adverse drug reaction was identified from the definition of ‘adverse drug reactions’ reported by Edwards and Aronson.14 A permanent pacemaker was implanted in patients in whom AV block persisted beyond 72 hours or recurred during the follow-up period. All of the patients were referred to their primary physicians for treatment of glaucoma after discharge.

Statistical analysis All data were presented as mean ± SD for parametric variables and as percentages for categorical variables, unless stated otherwise. Categorical variables were analysed with the Pearson’s χ2 test and Fisher’s exact test. All statistical studies were carried out using Statistical Package for Social Sciences software (SPSS 16.0 for Windows, SPSS Inc, Chicago, Illinois) and a p-value < 0.05 was considered statistically significant.

Results A total of 1 122 patients were hospitalised with a diagnosis of AV block and a permanent pacemaker was implanted in 946 Table 2. Characteristics of patients with ophtalmic beta-blocker-induced conduction defects Temporary/ Therapy permanent duration Patient’s pacemeker Conduction defect age/gender Drug type (months) 81/M Betaxolol 24 3rd-degree AV block Yes/Yes 58/F Timolol 40 Sinus pause No/Yes 78/F Timolol 52 3rd-degree AV block Yes/No* 82/M Timolol 14 High-degree AV block No/Yes 85/M Timolol 15 3rd-degree AV block No/Yes 62/F Timolol 61 Sinus pause No/No* 56/F Timolol 44 3rd-degree AV block No/Yes 62/M Cartelol 32 High-degree AV block No/Yes** 73/M Betaxolol 16 3rd-degree AV block No/Yes 83/F Betaxolol 18 High-degree AV block No/Yes 72/M Cartelol 11 3rd-degree AV block Yes/Yes 65/F Betaxolol 37 High-degree AV block No/Yes 76/M Timolol 27 3rd-degree AV block No/Yes *Rhythm was improved and conduction disturbance never recurred after drug withdrawal; **Rhythm was improved but recurred one month after drug discontinuation. AV: atrioventricular.

cases (84.3%). Thirteen of the 1 122 patients (1.1%) were using ophthalmic beta-blockers for the treatment of glaucoma. The demographic and clinical characteristics of these patients are summarised in Table 2. None of these 13 patients were using rate-limiting agents (oral beta-blockers, non-dihydropyridine calcium channel blockers, digoxin and anti-arrhythmic drugs). The mean age was 71.7 ± 10.1 years and six patients in this group were male (46%). Nine patients had hypertension (69%) and four (30%) had coronary artery disease. The mean left ventricular ejection fraction was 58.4 ± 11.1% in the study population. The major symptoms were syncope in five subjects, dizziness in four, and bradyarrhythmia-related dyspnoea in four. The ophthalmic beta-blocker used was timolol in seven cases (55%), betaxolol in four (30%), and cartelol in two (15%). The mean duration of ophthalmic beta-blocker treatment was 30.1 ± 15.9 months. On ECG, eight patients had complete AV block and five had high-degree AV block. The level of conduction block, according to ECG criteria, was as follows: four patients on betaxolol (100%) had infra-nodal block; two on cartelol (100%) had undetermined rhythm, four on timolol had infra-nodal block (57%), and three (43%) on timolol had AV node block. After drug discontinuation, in 10 patients the block persisted and a permanent pacemaker was implanted. Three patients (two on timolol and one on cartelol therapy), in whom the AV block had resolved, were discharged without pacemaker implantation. In one patient on previous cartelol treatment, the AV block reccurred one month after hospital discharge and a permanent pacemaker was implanted. Therefore, in total, 11 of 13 patients required permanent pacemaker implantation (84.6%). The implantation rate did not differ according to the type of topical beta-blocker used (p = 0.37). The distribution of ophthalmic beta-blockers in those who required permanent pacemaker implantation were timolol in five cases, betaxolol in four, and cartelol in two cases. The level of conduction block in patients who required permanent pacemaker implantation were infra-nodal block in eight cases, AV node block in one case and undetermined level of block in two cases. Only two patients on timolol therapy, whose ECGs were compatible with AV node block, did not require pacemaker implantation. On the other hand, all of the subjects with infranodal block on ECG required pacemaker implantation.

Discussion Drug-induced AV block is not a well-known clinical entity and there are controversial reports in the literature.15,16 Moreover, little is known about the natural history and prognosis of patients with drug-induced AV block on treatment with topical beta-blockers. The main finding of our study was that most of

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the patients with topical beta-blocker-induced AV block needed pacemaker implantation during follow up. Topical beta-blockers decrease intra-ocular pressure by reducing the inflow of aqueous humour, which is controlled by the adrenergic system in the ciliary epithelium.17 However, only two to 10% of the drugs may penetrate to the inner parts of the eye and the periocular tissues.18 The remainder of the drugs (about 80% for timolol) enter the systemic circulation through the conjunctival vessels and via the nasolacrimal duct through the nasal mucosa, and reach peak plasma concentrations within five to 30 minutes.19,20 Quaranta et al. showed that timolol significantly reduced systolic and diastolic blood pressures.21,22 Beta-blockers decrease sinus node automaticity, prolong sino-atrial, intra-atrial and atrioventricular conduction times, and increase atrioventricular node refractoriness.23 Orzalesi et al. reported that the level of cardiovascular risk was significantly higher in glaucoma patients,24 although in potentially predisposed patients, topical beta-blockers may cause adverse cardiovascular events secondary to systemic effects. In a randomised trial conducted in glaucoma patients using placebo, topical beta-blockers or dual therapy of topical and oral beta-blockers, the pulse rate was significantly lower in the topical beta-blocker group compared to the controls (70.3 vs 76 beats/ min). Heart rate was lowest in the dual-therapy group, which was reported as 58 beats/min.5 In our study, even though none of the patients received dual beta-blocker therapy, severe conduction disturbances were observed in patients using topical betablockers. In most of the patients, even after discontinuation of the drug, the conduction disturbances persisted and permanent pacemaker implantation was required. In a previous study by our group, permanent pacemaker implantation rate was 48% in patients taking rate-limiting drugs except topical beta-blockers.16 However, in this cohort, the rate of permanent pacemaker implantation was significantly higher (84.6%, p = 0.01). The mean age of patients in earlier and more recent articles was similar (72.01 vs 71.7 years, respectively).15 However, the level of AV block was significantly different in two articles: 28 of 108 patients (25.9%) taking rate-limiting drugs except topical betablockers had infra-nodal AV block, while eight of 13 patients (61.5%) on topical beta-blockers had infra-nodal AV block.15 In previous trials conducted on patients with drug-induced AV block, it was shown that infra-nodal block was associated with a higher rate of pacemaker implantation.15,16 Topical betablockers may not disturb electrical conduction as strongly as oral rate-limiting drugs due to their lower dose and the route of administration. However, topical beta-blockers may induce AV block in susceptible patients, and there are case reports of permanent pacemaker implantation in patients receiving them.6-8 Edwards and Aronson classified adverse drug reactions into six types: dose related (augmented), non-dose related (bizarre), dose related and time related (chronic), time related (delayed), withdrawal (end of use), and failure of therapy (failure).14 In our cohort, AV conduction fully recovered after drug withdrawal in two patients who were on fixed-dose timolol for more than four years. In 11 of 13 patients, AV conduction did not recover after drug withdrawal, which means the dose and length of time it was administered had no effect. According to the above drug-reaction classification, we concluded that 11 of 13 of our patients had non-dose-related (bizarre) drug reactions, and

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two of 13 patients had time-related (delayed) drug reactions. We suggest, however, that if AV block occurs during treatment with ophthalmic beta-blockers, it may be an indicator of an underlying severely damaged electrical pathway. Lopez et al. evaluated the prognosis of 12 patients with ophthalmic beta-blocker-induced AV block.8 In their series, seven of the 12 patients recovered during follow up and five needed pacemaker implantation. They concluded that every patient with AV block must be questioned about concomitant use of eye drops. The pacemaker implantation rate in our series was higher than in their series, but the percentage of patients using topical beta-blockers was higher in their series (12 of 243 patients with AV block vs 13 of 1 122 patients with AV block in our series). In their study the age, gender, presence of a bundle branch block, escape rhythm on ECG, or dosage of the drugs did not predict the risk for permanent pacemaker implantation.8

Limitations We evaluated elderly symptomatic patients who required hospitalisation in a tertiary centre. We did not include out-patient clinic patients, asymptomatic cases, those with mild symptoms or patients with transient forms of conduction abnormalities in our study. This may explain the high rate of pacemaker implantation in our cohort, which included patients with more severe forms of conduction abnormalities secondary to topical beta-blocker therapy. Irrespective of the accuracy of the electrocardiographic characteristics in defining the level of the AV block, His-bundle recording was not performed during the course of this study and we could also not determine a causal relationship.

Conclusion This study and previous reports show that patients using topical beta-blockers may suffer severe bradyarrhythmias secondary to AV block, and a significant percentage of these cases required pacemaker implantation. Patients with underlying pathology of the sinus or AV node, or the conduction pathways may develop AV block while on treatment with ophthalmic beta-blockers and these drugs may reveal concealed AV block. It may therefore be beneficial to search for pre-existing conduction abnormalities in patients with glaucoma before initiation of this type of medication.

References 1.

European Glaucoma Society. Treatment principles and options, antiglaucoma drugs. In: Terminology and Guidelines for Glaucoma, 3rd edn. Savona: Dogma, 2008: 121–134.

Zimmerman T. Topical ophthalmic beta-blockers: a comparative review. J Ocul Pharmacol 1993; 9(4): 373–384.

Gerber SL, Cantor LB, Brater DC. Systemic drug interactions with topical glaucoma medications. Surv Ophthalmol 1990; 35(3): 205–218.

Dorigo MT, Cerin O, Fracasso G, Altafini R. Cardiovascular effects of befunolol, betaxolol and timolol eye drops. Int J Clin Pharmacol Res 1990; 10(3): 163–166.

Tattersall C, Vernon S, Singh R. Resting pulse rates in a glaucoma clinic: the effect of topical and systemic beta-blocker usage. Eye (Lond) 2006; 20(2): 221–225.

Sharifi M, Koch JM, Steele RJ, Adler D, Pompili VJ, Sopko J. Third degree AC block due to ophthalmic timilol solution. Int J Cardiol 2001;

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80(2–3): 257–259. 7.

17. Neufeld AH, Bartels SP. Receptor mechanisms for epinephrine and

topical timolol maleate instillation. J Pharmacol Pharmacother 2011;

timolol. In: Lutjen-Drecoll E, ed. Basic Aspects of Glaucoma Research.

Rubín López JM, Hevía Nava S, Veganzones Bayón A, Barriales Alvarez V. Atrioventricular block secondary to topical ophthalmic beta blockers. Rev Esp Cardiol 1999; 52(7): 532.

Pacing Clin Electrophysiol 2012; 35(7): 804–810.

Walia HS, Walia SS, Emanuel ME. Sick sinus syndrome associated with 2(4): 300–302. doi: 10.4103/0976-500X.85946.

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Anguita M, Torres F, Giménez D, Segura J, Aumente D, Suárez de Lezo J, et al. Bradyarrhythmias secondary to the use of ophthalmic timolol. A report of 3 cases. Rev Esp Cardiol 1992; 45(1): 71–73.

10. Rosen KM, Gunnar RM, Rahimtoola SH. Site and type of second degree AV block. Chest 1972; 61: 99–100. 11. Rardon DP, Miles WM, Zipes DP, eds. Atrioventricular Block and Dissociation. Philadelphia, PA: WB Saunders Co, 2000. 12. Narula OS, Samet P. Wenckebach and Mobitz type II AV block due to block within the His bundle and bundle branches. Circulation 1970; 41: 947–965. 13. Langerdorf R, Cohen H, Gozo EG. Observations on second degree atrioventricular block, including new criteria fort the differential diagnosis between type I and type II block. Am J Cardiol 1972; 29: 111–119. 14. Edwards IR, Aronson JK. Adverse drug reactions: definitions, diagnosis, and management. Lancet 2000; 356(9237): 1255–1259.

Stuttgart, Germany: Schattauer, 1982: 113–122. 18. Hopkins GA, Lyle WM. Potential systemic effects of six common ophthalmic drugs. J Am Optometric Assoc 1977; 48: 1241–1245. 19. Korte JM, Kaila T, Saari KM. Systemic bioavailability and cardiopulmonary effects of 0.5% timolol eye drops. Graefes Arch Clin Exp Ophthalmol 2002; 240: 430–435. 20. Salminen L. Review: systemic absorption of topically applied ocular drugs in humans. J Ocul Pharmacol 1990; 6: 243–249. 21. Quaranta L, Miglior S, Floriani I, Pizzolante T, Konstas AG. Effects of the timolol–dorzolamide fixed combination and latanoprost on circadian diastolic ocular perfusion pressure in glaucoma. Invest Ophthalmol Vis Sci 2008; 49(10): 4226–4231. 22. Quaranta L, Gandolfo F, Turano R, Rovida F, Pizzolante T, Musig A, et al. Effects of topical hypotensive drugs on circadian IOP, blood pressure, and calculated diastolic ocular perfusion pressure in patients with glaucoma. Invest Ophthalmol Vis Sci 2006; 47(7): 2917–2923. 23. Fraunfelder FT, Meyer SM. Systemic adverse reactions to glaucoma medications. Int Ophthalmol Clin 1989; 29(3): 143–146. 24. Orzalesi N, Rossetti L, Omboni S; OPTIME Study Group (Osservatorio

15. Zeltser D, Justo D, Halkin A, Rosso R, Ish-ShRalom M, Hochenberg

sulla Patologia glaucomatosa, Indagine Medico Epidemiologica);

M, et al. Drug-induced atrioventricular block: Prognosis after discon-

CONPROSO (Collegio Nazionale dei Professori Ordinari di Scienze

tinuation of the culprit drug. J Am Coll Cardiol 2004; 44:105–108.

Oftalmologiche). Vascular risk factors in glaucoma: the results of

16. Osmonov D, Erdinler I, Ozcan KS, Altay S, Turkkan C, Yildirim E, et

a national survey. Graefes Arch Clin Exp Ophthalmol 2007; 245(6):

al. Management of patients with drug-induced atrioventricular block.

795–802.

Increased platelet activation leads to cardiovascular risk in adolescents with type 2 diabetes Adolescents with type 2 diabetes are at risk of atherosclerosis and cardiovascular disease early on in life. There are wellestablished data that diabetes, platelet hyperactivity and cardiovascular disease (CVD) are causes of mortality in adults with type 1 and type 2 diabetes. The purpose of a pilot study by Israels et al., published in Diabetes Care on 4 June 2014, was to establish whether the same connection was present in adolescents as in adults relative to non-diabetic control subjects. The study examined the expression of the surface and soluble platelet activation markers. In vivo platelet activation was compared in four different groups of adolescents aged 12 to 18 years. These groups comprised type 1 diabetics (n = 15), type 2 diabetics (n = 15), control subjects with normal body mass index (n = 14) and control subjects who were obese/overweight (n = 13). Type 1 and 2 diabetes were classified according to Canadian Diabetes Association criteria. Subjects with Prader–Willi syndrome or hypothyroidism, those who abused alcohol or drugs, had congenital CVD, were pregnant, and/or who used glucocorticoids, lipidlowering agents or platelet-inhibitory agents were all excluded from this study. Measurements of platelet surface and soluble activation markers were performed using the FACSCalibur flow

cytometer. Results were shown as percentage of platelets expressing CD62P and CD63 platelet surface antigen as well as PAC-1 monoclonal antibodies. Results showed that there were significantly higher platelet activation markers in adolescent type 2 diabetics when compared with either the obese or normal control group (p < 0.05). There was a small difference in platelet activation between adolescent type 1 diabetics and the two control groups, although the pattern leaned towards an increase in activation markers for type 1 diabetics. There were no differences in platelet activation markers between the non-diabetic groups. The study showed that in vivo platelet activation was increased in adolescent type 2 diabetics, which can be a potential cause of atherosclerosis, thrombosis and other cardiovascular diseases in early adulthood. Although it was a small study, it raises awareness of the fact that a more aggressive approach should be undertaken when modifying therapeutic interventions for type 2 diabetes in adolescents.

Reference http://www.diabetesincontrol.com/articles/diabetes-news/16447-increasedplatelet-activation-leads-to-cv-risk-in-adolescents-with-type-2-diabetes

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Autonomic imbalance assessed by time-domain heart rate variability indices in primary Raynaud’s phenomenon Kubilay Karabacak, Murat Celik, Erkan Kaya, Murat Kadan, Gokhan Arslan, Ufuk Demirkilic

Abstract Objectives: The pathogenesis of primary Raynaud’s phenomenon (RP) seems to be multifactorial and autonomic nervous dysfunction is one factor. Heart rate variability (HRV) is one of the most reliable parameters to demonstrate autonomic dysfunction. Our aim was to evaluate the time-domain HRV in patients with primary RP. Methods: A time analysis of HRV was performed in patients with primary RP and age- and gender-matched healthy controls. The results of the study and control group were compared. Results: Thirty patients with primary RP [all men, median (IQR) age: 21 (2) years) and 31 age- and gender-matched healthy controls (median (IQR): 21(3) years) were enrolled in the study. We found a statistically significant difference between the primary RP patients and control subjects in terms of time-domain HRV parameters (p < 0.05 for all). Conclusion: Our study showed the presence of autonomic nervous dysfunction of heart function in patients with primary RP. Keywords: heart rate variability, autonomic nervous system, Raynaud’s phenomenon Submitted 7/9/14, accepted 24/3/15 Published online 15/4/15 Cardiovasc J Afr 2015; 26: 214–216

www.cvja.co.za

DOI: 10.5830/CVJA-2015-032

Raynaud’s phenomenon (RP) is a vascular disorder depicted by a repeated course of fading of the fingers and/or toes, which is caused by reversible vasospasm.1 The prevalence of RP varies between three and 4%.2 Primary RP (Raynaud’s disease) occurs as an isolated symptom, whereas secondary RP (Raynaud’s syndrome) is associated with another disease or condition. About 8–9% of all RP are primary cases.2,3 Primary RP has a greater female predominance and is seen at an earlier age than secondary RP.2

Department of Cardiovascular Surgery, Gulhane Military Academy of Medicine, Ankara, Turkey Kubilay Karabacak, MD, kubilaykarabacak@yahoo.com Erkan Kaya, MD Murat Kadan, MD Gokhan Arslan, MD Ufuk Demirkilic, MD

Department of Cardiology, Gulhane Military Academy of Medicine, Ankara, Turkey Murat Celik, MD

The pathogenesis of RP is not fully elucidated. Intravascular and neural mechanisms play a pivotal role in this process.4 Increased sympathetic activity is often thought to be one of the causes in the aetiology of primary RP. Increased sympathetic activation in the chronic phase may also lead to desensitisation of the sinus node to neural input and parasympathetic tone, thereby causing autonomic dysfunction. Heart rate variability (HRV) analysis shows sympathovagal balance and has been used as an easy, non-invasive and reliable test for the identification of dysfunction of the autonomic nervous system in certain diseases. Since sympathovagal balance is affected in favour of sympathetic activation in patients with primary RP, in this study, we evaluated the baseline function of the autonomic nervous system assessed by 24-hour HRV analysis in patients with primary RP.

Methods Patients referred to our Cardiovascular Department for suspicion of primary RP during the period October 2012 to May 2013 were evaluated in this study. The evaluation of past medical history, physical examination, an initial 12-lead ECG and then 24-hour Holter monitoring for HRV analysis were performed in all patients. Diagnosis of Raynaud’s phenomenon was confirmed with the three-phase cold test. All patients were also evaluated for secondary RP. On physical examination, all patients were examined for skin ulceration, telangiectasia, muscle weakness and connective tissue diseases such as scleroderma. In our study, we tried to exclude metabolic factors that may affect the heart rate. Patients with hypothermic or hyperthermic status, injury, anaemia, infection or history of any chronic disease, such as connective tissue disorder or diabetes mellitus, were excluded. Also, structural heart disease, supraventricular or ventricular arrhythmias, atrial fibrillation, sick sinus syndrome, atrioventricular block, haematological or neurological disease, the use of any drug that has an impact on heart rate (such as beta-adrenergic blockers and anti-arrhythmic drugs) were accepted as other exclusion criteria. All HRV measurements were performed at rest. The patients were advised not to take part in vigorous exercise during the HRV measurement. The control group consisted of age- and gender-matched healthy subjects. An informed consent was obtained from all subject enrolled in this study, which was conducted in accordance with the regulations of Declaration of Helsinki. The regional ethics committee of our Institute approved the study protocol.

HRV analysis A 24-hour Holter ambulatory ECG monitoring (Rozinn RZ 152 digital Holter recorder, Rozinn Electronics, Inc, Glendale,

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NY, USA) with a sampling rate of 1 024 Hz was performed on all subjects enrolled in the study. The HRV was automatically analysed with software of the same device. Standard deviation of all R–R intervals (SDNN), standard deviation of the successive N–N differences (SDSD), standard deviation of the averages of the R–R intervals in all five-minute segments of R–R intervals (SDANN), the mean of all the fiveminute standard deviations of N–N (normal R–R) intervals during the 24-hour period (SDNN index), the mean square root of the difference of successive R–R intervals (RMSSD), successive N–N intervals differing more than 50 ms (NN50 count), and the proportion of adjacent normal R–R intervals < 50 ms (pNN50) were defined as the time-domain variables of HRV analysis. HRV analyses of the patients with primary RP were compared with those of healthy controls.

Univariate correlation analysis showed that the presence of primary RP was moderately correlated with SDNN (r = 0.287, p = 0.025), RMSDD (r = 0.297, p = 0.020), NN50 count (r = 0.340, p = 0.007), PNN50 count (r = 0.281, p = 0.028) and SDNN index (r = 0.409, p = 0.001). We did not find any significant correlation between the duration of Raynaud’s phenomenon and the timedomain HRV indices (p > 0.05 for all). To demonstrate the independent effect of time-domain HRV indices on the presence of primary RP, we performed a multivariate logistic regression analysis using the LR method, based on independent variables likely to affect the level of mean platelet volume. In multivariate analysis, SDNN index (β = 1.138, 95.0% CI = 1.049–1.235, p = 0.002) and PNN50 (β = 0.881, 95.0% CI = 0.785–0.989, p = 0.032) were the only independent variables (Table 3).

Statistical analysis

Discussion

The data were tested for normal distributions using the Kolmogorov–Smirnov test. The continuous variables are presented as means ± standard deviation (SD) and the categorical variables as percentages. The chi-square test was used to compare categorical data and the independent-samples t-test was used to compare quantitative data. Spearman’s and Pearson’s correlation coefficients were used to perform univariate correlation. A p-value < 0.05 was considered statistically significant. All statistical analyses and calculations were performed using the Statistical Package for Social Sciences version 20.0 (SPSS, Chicago, Illinois, USA).

The pathogenesis of primary RP appears to be multifactorial. The endothelium, smooth muscle, circulating mediators, and autonomic and sensory nerves play a pivotal role in maintaining vasomotor homeostasis. Disturbance in these factors may lead to vasospasm of the small arteries and arterioles and to the manifestation of RP. Since Maurice Raynaud, a French physician, first described Raynaud’s phenomenon in 1862, sympathetic nervous system over-reactivity has been suggested as the most common cause of the disease. Besides sympathetic up-regulation, impaired parasympathetic activation has also been blamed.5 Therefore, it is suggested that the autonomic nervous system seems to have a pivotal role in the pathogenesis of Raynaud’s phenomenon.6-8 However, it should be noted that the autonomic nervous system may not be affected to the same degree in all patients with RP. The cyclic changes in the sinus node rate over time are defined as heart rate variability. HRV analysis provides information on the balance between sympathetic and parasympathetic innervation of the heart and has been extensively used as an indirect method for the determination of cardiac autonomic function.9 Physical and mental stress, exercise, and respiratory and metabolic changes are associated with autonomic tone of heart rate.10,11

Results Thirty patients (median age 21 years, IQR: 2) in whom the three-phase cold test was positive, were diagnosed as primary RP and enrolled in the study. The average duration of primary RP patients’ symptoms was 3 (2.13) years [median (IQR)]. The control group consisted of 31 healthy subjects (median age 21 years, IQR: 3). There was no statistically significant difference between primary RP patients and healthy control subjects regarding basal demographic characteristics (Table 1). All participants were in sinus rhythm without any arrhythmias. Statistical analysis of the HRV analyses showed a significant decrease in time-domain variables of SDNN, RMSDD, NN50 count, PNN50 and SDNN index between the two groups (p < 0.05 for all) (Table 2). Table 1. Basal demographic and clinical characteristics of the two groups Primary RP Control (n = 30) (n = 31) p-value 21 (2) 21 (3) 0.381 30 (100) 31 (100) NA 116.76 ± 10.45 119.41 ± 10.13 0.318 0.615 74.43 ± 7.81 75.54 ± 9.30 10 (33.3) 7 (22.6) 0.258 3 (2.13) – NA

Age (years), median (IQR)* Male, n (%) SBP (mmHg) DBP (mmHg) Smoking, n (%) Duration of primary RP (years), median (IQR)* *Data without normal distribution were expressed as median (interquartile range). RP: Reynaud’s phenomenon, SBP: systolic blood pressure, DBP: diastolic blood pressure IQR: interquartile range.

Table 2. Comparison of time-domain HRV indices between the two groups Primary RP Control (n = 30) (n = 31) p-value Mean heart rate (bpm) 0.857 76.80 ± 12.99 76.22 ± 11.74 SDNN (ms) 0.025 145.09 ± 40.65 177.55 ± 66.18 SDSD (ms) 0.059 46.43 ± 24.03 63.40 ± 42.04 NN50 count (%) 11085.53 ± 10246.89 19302.74 ± 12717.30 0.007 RMSDD (ms) 0.020 42.58 ± 22.76 63.39 ± 42.04 SDANN (ms) 0.437 93.33 ± 65.78 79.06 ± 75.89 SDNN index 0.001 55.71 ± 24.50 82.03 ± 34.25 pNN50 0.028 13.30 ± 12.74 21.92 ± 16.86 SDNN: standard deviation of all R–R intervals, SDSD: standard deviation of the successive N–N differences, NN50 count: successive N–N intervals differing more than 50 ms, RMSSD: the mean square root of the difference of successive R–R intervals, SDANN: standard deviation of the averages of the R–R intervals in all five-minute segments of R–R intervals, SDNN index: the mean of all the five-minute standard deviations of N–N (normal R–R) intervals during the 24-hour period, pNN50: the proportion of adjacent normal R–R intervals < 50 ms.

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Table 3. Univariate analysis and multivariate logistic regression analysis based on independent variables likely to affect the presence of primary RP Univariate analysis Multivariate analysis* Variable r p OR 95% CI p SDNN (ms) 0.287 0.025 0.979 0.955–1.004 0.107 RMSDD (ms) 0.297 0.020 1.012 0.957–1.069 0.684 SDNN index 0.409 0.001 1.138 1.049–1.235 0.002 NN50 count (%) 0.340 0.007 1.000 1.000–1.000 0.711 pNN50 0.281 0.028 0.881 0.785–0.989 0.032 SDNN: standard deviation of all R–R intervals, RMSSD: the mean square root of the difference of successive R–R intervals, SDNN index: the mean of all five-minute standard deviations of N–N (normal R–R) intervals during the 24-hour period, NN50 count: successive N–N intervals differing more than 50 ms, pNN50: the proportion of adjacent normal R–R intervals < 50 ms. *p-value at the last step, where the independent variables remained in the backward LR multivariate regression model.

To define the role of the autonomic nervous system in the pathogenesis of certain diseases, a number of studies have used HRV analysis. However, the results may not agree, presumably due to methodological differences. HRV analysis can be carried out by two different methods, time-domain and frequencydomain methods. The frequency-domain method separates heart rate signals by frequency and density. Although its basic principle is simple, it is technically difficult and complex. The time-domain method is based on analysis of the interval between normal pulses in 24-hour ECG recording. Low-frequency (LF) HRV is an index of sympathetic activity, whereas high-frequency (HF) HRV reflects parasympathetic activity. Increased LF:HF ratio is characterised by an autonomic nervous dysfunction.12 Among time-domain HRV indices, SDNN, SDANN and SDNN index reflect the heart rate, and their decrease is related to diminished vagal and increased sympathetic modulation of the sinus node.13 In this study, we used the time-domain method and found that time-domain HRV variables differed significantly, showing increased sympathetic activity in patients with primary RP. Among these variables, intervals during the 24-hour period (SDNN index) and the proportion of adjacent normal R–R intervals < 50 ms (pNN50) were found to be independent variables in multivariate logistic regression analysis. However, it is obvious that the results of the study will be elucidated more after including frequency-domain HRV indices. Despite this limitation, our study gives inspiration for further larger sample sized prospective studies. Assessment of the autonomic nervous system may play an important role in understanding the underlying mechanism of primary RP. Primary RP patients may have higher resting sympathetic tone or decreased parasympathetic tone. Autonomic parameters of the cardiovascular system can be non-invasively assessed with HRV. The clinical course and prognosis of various cardiac and systemic disorders could be obtained with this assessment. Many articles in the literature have speculated that HRV could be used in various cardiac and non-cardiac diseases for autonomic regulation, but a limited number of studies have used HRV on patients with primary RF. Koszewicz et al. found that patients with primary RP did not have the autonomic stability found in healthy individuals.3 In another study, Ferri et al.14 studied HRV changes in patients with systemic sclerosis.

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They found significantly higher HRV and lower circadian and spectral indices of HRV in systemic sclerosis patients, compared to control subjects. Similarly in our study, we demonstrated an autonomic imbalance suggesting increased sympathetic or reduced parasympathetic activity demonstrated by time-domain HRV indices in primary RP patients compared to controls. Among time-domain variables of HRV indices, the mean of all fiveminute standard deviations of N–N (normal R–R) intervals during the 24-hour period (SDNN index) and the proportion of adjacent normal R–R intervals < 50 ms (pNN50) were found to be most associated with primary RP.

Conclusion The current study demonstrated significant differences in timedomain parameters of HRV analysis during asymptomatic 24-hour intervals and indicated the presence of an autonomic imbalance (increased sympathetic and decreased parasympathetic activity) in patients with primary RP compared to healthy controls. The exact mechanism of the relationship between primary RP and autonomic imbalance remains unclear and needs further studies. Future prospective studies may be helpful to demonstrate the role of HRV analysis in evaluating the progression and treatment effectiveness of patients with primary RP.

References 1. Prete M, Fatone MC, Favoino E, Perosa F. Raynaud’s phenomenon:from molecular pathogenesis to therapy. Autoimmun Rev 2014; 13(6): 665–667. 2. Mikulska D. Raynaud’s phenomenon: pathogenesis and prevalance. Ann Acad Med Stetin 2010; 56(1): 11–14. 3. Koszewicz M, Gosk-Bierska I, Bilińska M, Podemski R, Budrewicz S, Adamiec R, et al. Autonomic dysfunction in primary Raynaud’s phenomenon. Int Angiol 2009; 28(2): 127–131. 4. Gayraud M. Raynaud’s phenomenon. Joint Bone Spine 2007; 74(1): e1–8. 5. Bakst R, Merola JF, Franks AG, Sanchez M. Raynaud’s phenomenon: pathogenesis and management. J Am Acad Dermatol 2008; 59(4): 633–653. 6. Cooke JP, Marshall JM. Mechanisms of Raynaud’s disease. Vasc Med 2005; 10(4): 293–307. 7. Turton EP, Kent PJ, Kester RC. The aetiology of Raynaud’s phenomenon. Cardiovasc Surg 1998; 6(5): 431–440. 8. Herrick AL. Pathogenesis of Raynaud’s phenomenon. Rheumatology (Oxford) 2005; 44(5): 587–596. 9. Stauss HM. Heart rate variability. Am J Physiol Regul Integr Comp Physiol 2003; 285(5): R927–931. 10. Malik M. Heart rate variability. Curr Opin Cardiol 1998; 13(1): 36–44. 11. Pieper SJ, Hammill SC. Heart rate variability: technique and investigational applications in cardiovascular medicine. Mayo Clin Proc 1995; 70(10): 955–964. 12. Ozdemir O, Soylu M, Alyan O, Geyik B, Demir AD, Aras D, et al. Association between mean platelet volume and autonomic nervous system functions: Increased mean platelet volume reflects sympathetic overactivity. Exp Clin Cardiol 2004; 9(4): 243–247. 13. Zaza A, Lombardi F. Autonomic indexes based on the analysis of heart rate variability: a view from the sinus node. Cardiovasc Res 2001; 50(3): 434–442. 14. Ferri C, Emdin M, Giuggioli D, Carpeggiani C, Maielli M, Varga A, et al. Autonomic dysfunction in systemic sclerosis time and frequency domain 24 hour heart rate variability analysis. Br J Rheumatol 1997; 36: 669–676.

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Atrial conduction time, and left atrial mechanical and electromechanical functions in patients with polycystic ovary syndrome: interatrial conduction delay Emine Gazi, Meryem Gencer, Volkan Hanci, Ahmet Temiz, Burak Altun, Ahmet Barutcu, Ayse Nur Gungor, Servet Hacivelioglu, Ahmet Uysal, Yucel Colkesen

Abstract Background: Polycystic ovary syndrome (PCOS) is one of the most common endocrine disorders of women during the reproductive period. Cardiovascular risk factors are more frequent in patients with PCOS. We aimed to investigate the P-wave dispersion (Pd), inter- and intra-atrial conduction time and mechanical functions of the left atrium (LA) in patients with PCOS. Methods: Forty-eight patients with PCOS and 38 normal healthy women were enrolled in this study. A 12-lead surface electrocardiogram was used to evaluate Pd. Left ventricular (LV) functions were measured using conventional and tissue Doppler imaging (TDI) methods. Inter- and intra-atrial conduction times were measured by TDI. LA volumes were measured echocardiographically with the biplane area–length method from the apical four-chamber view. Results: Heart rate (82.02 ± 13.15 vs 74.24 ± 11.02 bpm, p = 0.014) and Pd were significantly increased in the PCOS patients [27 ± 5 vs 24 ± 6 ms, p = 0.035]. Transmitral E/A ratio was significantly lower in the PCOS patients than in the controls (1.5 ± 0.3 vs 1.7 ± 0.4 m/s, p = 0.023). Passive emptying volume (12.54 ± 4.39 vs 15.28 ± 3.85 ml/m2, p = 0.004) and passive emptying fraction [54.4 (21–69) vs 59.1% (28–74), p = 0.008] were significantly decreased in PCOS patients. Total emptying volume was significantly decreased (17.9 ± 5.49 vs 20.67 ± 4.29 ml/m2, p = 0.018) in PCOS patients. Interatrial (19 ± 7.4 vs 15 ± 6.4 ms, p = 0.035) and intra-atrial [8.5 (1–19) vs 5 ms (1–20), p = 0.026] electromechanical delays were found to be significantly higher in PCOS patients. Conclusion: This study showed that patients with PCOS had increased inter- and intra-atrial conduction delays, and decreased LA passive emptying volumes and fractions. Department of Cardiology, Canakkale Onsekiz Mart University, Canakkale, Turkey Emine Gazi, MD, eordulu@hotmail.com Ahmet Temiz, MD Burak Altun, MD Ahmet Barutcu, MD Yucel Colkesen, MD

Department of Obstetrics and Gynecology, Canakkale Onsekiz Mart University, Canakkale, Turkey Meryem Gencer, MD Ayse Nur Gungor, MD Servet Hacivelioglu, MD Ahmet Uysal MD

Department of Anesthesiology and Reanimation, Canakkale Onsekiz Mart University, Canakkale, Turkey Volkan Hanci, MD

Keywords: electromechanical delay, polycystic ovary syndrome, P-wave dispersion, interatrial conduction Submitted 6/11/13, accepted 12/4/15 Cardiovasc J Afr 2015; 26: 217–221

www.cvja.co.za

DOI: 10.5830/CVJA-2015-046

Polycystic ovary syndrome (PCOS) is one of the most common endocrine/hormonal disorders that affect an estimated 5–10% of women of reproductive age. It is characterised by hyperandrogenism, chronic anovulation and polycystic ovaries.1 A high proportion of women with PCOS are associated with a higher than the normal incidence of insulin resistance, hyperlipidaemia and obesity, as well as cardiovascular disease.2 Gonadal steroids have an effect on cardiac ion currents and autonomic function and therefore may cause cardiac arrhythmias.3 About 65–75% of drug-induced ventricular arrhythmia occurs in women, whereas the incidence of atrial fibrillation or sudden death is lower than in men.4,5 It has recently been identified that left atrial (LA) volume and mechanical functional index are potential indicators of atrial arrhythmia and cardiac disease. LA function is an important factor for left ventricular (LV) filling, and atrial emptying pressure and volume may provide important information about LV resistance. As a result, the atrial emptying pattern is strongly affected by LV diastolic function. Intra- and interatrial conduction delay, which are evaluated by tissue Doppler imaging (TDI), as well as power Doppler (PD), are electrophysiological characteristics of the atria that are prone to atrial fibrillation (AF).6,7 Although there are some studies investigating QT dispersion and ventricular diastolic function in women with PCOS, there is no literature, to our knowledge, on the PD and electromechanical properties of patients with this disease. Therefore we aimed to investigate PD, inter- and intra-atrial conduction times and mechanical function of the LA in patients with PCOS.

Methods A total of 86 women were consecutively enrolled in this study. Forty-eight patients with PCOS (mean age 24 ± 4 years) and 38 normal healthy women as controls (mean age 27 ± 5 years), who attended the gynaecology clinic of Canakkale Onsekiz Mart University between March and May 2012, participated in this study. PCOS patients were selected from subjects who were admitted due to oligo-amenorrhea, infertility or hirsutism. The control

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group was selected from healthy women who presented with infertility and the male factor was detected to be the cause of the infertility. PCOS was diagnosed by ultrasound if there were polycystic ovaries [enlarged ovaries (2–8 mm in diameter) with cysts ≥ 8], oligo-amenorrhea (intermenstrual interval > 35 days), hirsutism (Ferriman–Gallwey score ≥ 7) and elevated serum testosterone levels (≥ 2.7 nmol/l, convention factor 0.03467; 80 ng/dl).8 Patients with hypertension, diabetes mellitus, electrolyte imbalance, a history of chronic renal failure or a glomerular filtration rate < 60 ml/min according to the MDRD formula, chronic inflammatory disease, chronic lung disease, heart failure or valve disease, thyroid function disorders, history of arrhythmia, sleep-apnoea syndrome, smoking and drug use in the last three months were excluded. The study protocol was approved by the local ethics committee and written informed consent was obtained from all patients. Laboratory, electrocardiographic and echocardiographic assessments were done on the second or third days of the menstrual cycle, which is the follicular phase. Fasting levels of blood glucose and insulin, lipid profiles and hormone levels were determined by standard laboratory methods. Insulin resistance was assessed using the homeostasis model assessment (HOMA– IR) calculation: fasting serum insulin (μIU/ml) × fasting plasma glucose (mg/dl)/405.9

Analysis of electrocardiography A 12-lead surface electrocardiogram was used to evaluate P-wave parameters. The paper speed was 50 mm/s and amplitude was 20 mm/mV. All electrocardiograms were recorded on the second or third day of the menstrual cycle. P waves were measured manually on all derivations and at least three cardiac cycles were recorded. Pd was defined as the difference between the maximum (Pmax) and minimum (Pmin) P-wave duration. The onset of the P wave was defined as the point of first visible upward slope from baseline for positive waveforms, and as the point of first downward slope from baseline for negative waveforms. The return to baseline was considered as the end of the P wave.

Echocardiography Two-dimensional, M-mode, pulsed and colour-flow Doppler echocardiographic examinations were performed on all patients by one cardiologist on the second or third day of the menstrual cycle (Vivid 7 Pro, GE, Horten, Norway, 2–4 MHz phasedarray transducer). During echocardiography, a single-lead electrocardiogram was recorded simultaneously. Data were recorded from the average of three cardiac cycles. M-mode and Doppler measurements were performed adhering to the American Society of Echocardiography guidelines.10 TDI was performed with transducer frequencies of 3.5–4 MHz. The monitor sweep was set at 100 mm/s. A pulsed Doppler sample volume was placed at the level of the LV septal mitral annulus, lateral mitral annulus and tricuspid annulus in the apical fourchamber view. Peak systolic, early diastolic (E) and late diastolic (A) velocities were obtained at these levels. Atrial electromechanical coupling, the time interval from the onset of the P wave to the beginning of the late diastolic wave,

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was calculated from the lateral mitral annulus (PAlat), septal mitral annulus (PAsep) and tricuspid annulus (PAtri). Interatrial electromechanical delay was defined as the difference between PAlat and PAtri, and intra-atrial electromechanical delay was defined as the difference between PAsep and PAtri.11 LA volumes were measured echocardiographically by the biplane area–length method from the apical four-chamber view. LA maximal volume (Vmax) was calculated at the onset of mitral valve opening, LA minimum volume (Vmin) at the onset of mitral valve closure, and LA presystolic volume (Vp) at the beginning of the P wave on a surface ECG. LA passive emptying volume [(PEV) = Vmax – Vp], LA passive emptying fraction [(PEF) = (Vmax – Vp)/Vmax], LA active emptying volume [(AEV) = Vp – Vmin], LA active emptying fraction [(AEF) = (Vp – Vmin)/Vp], and LA total emptying volume [(TEV) = Vmax – Vmin] were defined as LA emptying function parameters.12,13

Statistical analysis All continuous variables were expressed as mean ± standard deviation and median (interquartile range). All measurements were evaluated with the Kolmogorov–Smirnov and Shapiro–Wilk tests, and comparisons of parametric and non-parametric values between two groups were performed by means of the Mann– Whitney U-test or Student’s t-test. Univariate linear regression and stepwise multiple regression analyses were used to identify the clinical characteristics of interatrial electromechanical delay. Age, body mass index (BMI) and testosterone levels were entered into the model. All statistical studies were carried out with the SPPS program (version 15.0, SPSS, Chicago, Illinois, USA); p < 0.05 was accepted as statistically significant.

Results Clinical and laboratory findings of the subjects are shown in Table 1. Age and serum FSH levels, respectively, were significantly lower in patients with PCOS [24 ± 4 vs 30 ± 7 years, p < 0.01; and 5.07 (2.92–10.1) vs 7.68 (2.02–19.10) mIU/ml, p < 0.001]. BMI (22.5 ± 3.4 vs 25.4 ± 5.4 kg/m2, p = 0.029) and serum estradiol levels (28.8 ± 11.3 vs 43.2 ± 17.8 pg/ml, p < 0.001) were significantly higher in PCOS patients than in the control subjects. Serum testosterone levels were higher in patients with PCOS than in the control group [75.5 (14.7–314) vs 17.2 (2.5–44) ng/dl, p < 0.001]. Heart rate (82.02 ± 13.15 vs 74.24 ± 11.02 bpm, p = 0.014) and Pd were significantly increased in PCOS patients (27 ± 5 vs 24 ± 6 ms). Echocardiographic findings of the study population are given in Table 2. LV diastolic and systolic diameters, ejection fraction, fractional shortening, LA diameters, and E and A waves were similar in both groups. The transmitral E/A ratio was significantly lower in PCOS patients than in the controls (1.5 ± 0.3 vs 1.7 ± 0.4, p = 0.023). The peak systolic myocardial velocity was higher in patients with PCOS (0.09 ± 0.01 vs 0.08 ± 0.01 m/s, p = 0.02). The myocardial early diastolic wave (E’) and E/E’ ratio were similar in both groups. There were no differences in LA Vmax, LA Vmin, and Vp between the groups. The LA active emptying volume and active emptying fraction were similar. The passive emptying volume (12.54 ± 4.39 vs 15.28 ± 3.85 ml/m2, p = 0.004) and passive emptying fraction [54.4 (21–69) vs 59.1 (28–74)%, p = 0.008] were

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Table 1. Clinical and laboratory findings of the study population PCOS (n = 48) Control (n = 38) p-value Age (years) 0.001* 24 ± 4 30 ± 7 0.029* BMI (kg/m2) 25.4 ± 5.4 22.5 ± 3.4 0.071 BSA (m2) 1.71 ± 0.18 1.64 ± 0.13 Waist/hip ratio 0.061 0.80 ± 0.05 0.77 ± 0.01 FSH (mIU/ml) 5.07 (2.92–10.1) 7.68 (2.02–19.10) 0.001* LH (mIU/ml) 6.62 (2.35–39.25) 6.74 (2.03–19.47) 0.442 TSH (μIU /ml) 0.110 2.48 ± 2.36 1.78 ± 0.91 Estradiol (pg/ml) 0.001* 43.2 ± 17.8 28.8 ± 11.3 Testosterone (ng/dl) 75.5 (14.7–314) 17.2 (2.5–44) 0.001* Fasting glucose (mg/dl) 0.945 86 ± 12 87 ± 8 Total cholesterol (mg/dl) 0.557 176 ± 34 181 ± 38 HDL (mg/dl) 0.399 53 ± 15 56 ± 14 LDL (mg/dl) 0.407 96 ± 28 101 ± 33 TG (mg/dl) 0.880 97 ± 74 87 ± 50 Fasting insulin (μIU /ml) 15.28 ± 23.45 12.74 ± 17.57 0.627 HOMA-IR 1.40 (0.37–36.15) 1.44 (0.38–18.99) 0.659 Heart rate (bpm) 82.02 ± 13.15 75.24 ± 11.02 0.014** Systolic blood pressure 0.094 109 ± 7 105 ± 8 Diastolic blood pressure 0.227 70 ± 6 68 ± 8 0.013* Pmax (msn) 97 ± 7 102 ± 8 0.001* Pmin (msn) 70 ± 6 77 ± 7 Pd (msn) 0.035* 27 ± 5 24 ± 6 PCOS = polycystic ovary syndrome; BMI = body mass index; BSA = body surface area; FSH = follicular stimulating hormone; LH = luteinising hormone; TSH = thyroid stimulating hormone; HDL = high-density lipoprotein; LDL= low-density lipoprotein; TG = triglycerides; HOMAIR = homeostasis model assessment of insulin resistance; Pmax = maximum P-wave duration; Pmin = minimum P-wave duration; Pd = P-wave dispersion; *Mann–Whitney U-test; **Independent t-test.

significantly decreased in PCOS patients. The total emptying volume was significantly decreased (17.9 ± 5.49 vs 20.67 ± 4.29 ml/m2, p = 0.018) in PCOS patients (Table 3). Tissue Doppler echocardiography measurements are shown in Table 3. PAlat [57 (34–87) vs 43 (34–77) ms, p < 0.01], PAsep [48 (42–68) vs 34 (25–67) ms, p < 0.01] and PAtri [39 (21–60) vs Table 2. Echocardiographic properties of the study population PCOS (n = 48) Controls (n = 38) p-value LVDD (mm) 0.222 44.3 ± 3.7 43.3 ± 3.6 LVSD (mm) 0.330 27.7 ± 3.3 26.9 ± 4.2 LVEF (%) 0.433 68 ± 4 69 ± 3 LA (mm) 0.171 33 ± 4 32 ± 3 Mitral E wave (cm/s) 0.483 91.7 ± 15.9 94.4 ± 19 Mitral A wave (cm/s) 0.085 59.9 ± 12.8 55 ± 12.4 E/A 0.023* 1.5 ± 0.3 1.7 ± 0.4 DT (ms) 0.114 173 ± 16 180 ± 19 Peak S (m/s) 0.02** 0.09 ± 0.01 0.08 ± 0.01 0.190 LV E′ (cm/s) 14.5 ± 3.5 13.5 ± 2.9 0.116 E/E′ 6.5 ± 1.6 7.1 ± 1.8 LV IVRT (ms) 0.282 112 ± 15 109 ± 15 LV IVCT (ms) 0.790 371 ± 35 373 ± 36 PCOS = polycystic ovary syndrome; LV = left ventricle; DD = diastolic diameter; SD = systolic diameter; EF = ejection fraction; FS = fractional shortening, LA = left atrium, DT = deceleration time, LV E′ = left ventricle early diastolic velocity, IVRT = ısovolumetric relaxation time; IVCT = isovolumetric contraction time; *Independent t-test; ** Mann–Whitney U-test.

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Table 3. Atrial conduction times and left atrial measurements of the study population PCOS (n = 48) Controls (n = 38) p-value PA lateral (ms) 57 (34–87) 43 (34–77) 0.001* PA septal (ms) 48 (24–68) 34 (25–67) 0.001* PA tricuspid (ms) 39 (21–60) 28 (22–65) 0.001* PA lateral tricuspid (ms) 0.035** 19 ± 7.4 15.6 ± 6.4 PA septal tricuspid (ms) 8.50 (1–19) 5 (1–20) 0.026* 2 LA Vmax (ml/m ) 0.201 24.59 ± 6.7 26.31 ± 4.95 6.11 (2.41–19.23) 5.60 (2.33–11.49) 0.164 LA Vmin (ml/m2) 11.06 (6.01–29.23) 10.65 (5.23–20.53) 0.398 LA Vp (ml/m2) 0.004** LA PEV (ml/m2) 12.54 ± 4.39 15.28 ± 3.85 LA PEF (%) 54.4 (21–69) 59.1 (28–74) 0.008** LA AEV (ml/m2) 0.903 5.45 ± 2.18 5.39 ± 2.12 LA AEF (%) 0.252 45.9 ± 10.7 48.7 ± 10.5 0.018** LA TEV (ml/m2) 17.9 ± 5.49 20.67 ± 4.29 PA = time interval from the onset of P wave to the beginning of the late myocardial diastolic velocity, LA = left atrium; Vmax = maximum volume; Vmin = minimum volume; Vp = volume of the beginning atrial systole; PEV = passive emptying volume; PEF = passive emptying fraction; AEV = active emptying volume; AEF = active emptying fraction; TEV = total emptying volume; *Mann–Whitney U-test; **Independent t-test.

28 (22–65) ms, p = 0.001] were significantly longer in patients with PCOS. Interatrial and intra-atrial electromechanical delays were found to be significantly higher in PCOS patients. Values of PAlat – PAtri were 19 ± 7.4 and 15 ± 6.4 ms in PCOS patients and control subjects, respectively (p = 0.035). Median values of PAsep – PAtri were 8.5 (1–19) and 5 (1–20) ms (p = 0.026), respectively. Age and serum testosterone levels were associated with interatrial electromechanical delay in the linear regression analysis (p = 0.071, β = 0.201 and p = 0.052, β = 0.242, respectively). In a multivariate stepwise regression analysis, age was demonstrated to be an independent predictor of interatrial electromechanical delay (p = 0.013, β = –0.321) (Table 4).

Discussion In the present study, we showed that Pd, and inter- and intraatrial conduction times were increased, left atrial mechanical function was impaired, and transmitral E/A ratio was decreased in patients with PCOS. To our knowledge, this is the first

Table 4. Relation between interatrial conduction delay and clinical findings. Multivariate analysis model included age, BMI and testosterone level

Age BMI Estradiol Insulin HOMA-IR Testosterone E/A ratio LA passive fraction Heart rate Total emptying volume LA diameter

Univariate p β 0.201 0.071 0.031 0.802 0.160 0.160 0.047 0.716 0.037 0.779 0.242 0.052 –0.010 0.930 –0.063 0.584 0.034 0.761 0.020 0.861 0.130 0.247

Multivariate p β –0.321 0.013

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study that has assessed the mechanical and electromechanical functions of the LA in patients with PCOS. Previous investigations suggested that endothelial and diastolic dysfunction occurs in patients with PCOS due to insulin resistance and high androgen levels.14,15 Diastolic dysfunction is considered to be an early sign of coronary artery disease. Accordingly, Yarali et al. reported that PCOS patients had a lower peak E velocity and E/A ratio.16 A study showed a negative correlation between insulin levels and E/A ratio, which suggests that PCOS patients may be more prone to develop diastolic dysfunction.17 Another study showed that LV ejection fraction, transmitral E and A waves, E/A ratio, deceleration time, isovolumetric relaxation time and tissue Doppler parameters were not significantly different between patients with PCOS and control subjects.18 In our study, LV systolic function and transmitral E and A waves were similar in the two groups, but E/A ratio was decreased in the study patients, suggesting impaired diastolic function. Atrial fibrillation is the most commonly observed arrhythmia in clinical practice and is associated with cardiovascular morbidity and mortality.19-21 Identifying the risk factors that generate AF is important. It was thought that enlargement and increased pressure of the LA have an effect on P-wave disturbances.22-24 The presence of P-wave dispersion, and interand intra-atrial conduction delays mean that sinus impulses have inhomogeneous propagation. This is a well-known electrocardiographic characteristic of atria that are prone to AF.6,25 The autonomic nervous system plays an important role in heart rate and the conduction system. Imbalance between the sympathetic nervous system and vagal tone is an important predictor of AF.26,27 Gonadal sex hormones affect heart rate and atrioventricular conduction time. Fulop et al. reported that heart rate is moderately reduced after surgical castration in both male and female canines. Heart rate is increased and PQ interval is lengthened with oestrogen replacement in male canines, and with testosterone replacement in female canines. Therefore, oestrogen and testosterone have identical effects on the heart rate and atrioventricular conduction time.3 In our study, Pd, inter- and intra-atrial conduction times were increased in patients with PCOS. Therefore resting heart rate was higher in these patients. Elevated serum levels of oestrogen and testosterone may explain these findings. Ventricular filling pressure is an indicator of LV diastolic function, and LA function is an important determinant of LV diastolic filling.28 LA passive emptying volume is related to increased LV end-diastolic pressure.12 We found that LA mechanical function was impaired in PCOS patients. Insulin resistance, obesity and hyperlipidaemia, risk factors for cardiovascular disease, are more frequent in patients with PCOS.2 Studies showed that arterial stiffness, endothelial dysfunction and LV diastolic dysfunction were increased in obese patients with PCOS.29,30 Additionally, diastolic function may worsen with age. In our study E/A ratio was lower in women with PCOS, even though the PCOS patients were significantly younger. BMI differences were insignificant between the groups. Impairment of LA mechanical and electromechanical function is known to be a risk factor for AF. LA mechanical function, conduction times and Pd have not been evaluated

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previously in patients with PCOS. Although there were no known cardiac risk factors, obesity or insulin resistance, we found not only impaired LV diastolic parameters but also increased LA conduction times and Pd in these patients. Hence, we suggest that decreased LA mechanical function, lengthening inter- and intra-atrial conduction times, and increased Pd may occur before the appearance of cardiovascular risk factors such as hypertension, diabetes mellitus and hyperlipidaemia. Age is an important risk factor for the development of AF.19 However Turhan et al. reported a positive correlation between age and Pd, and LV diastolic parameters.31 In our study, although the patients with PCOS were younger, they still had significant impairment in Pd, LA mechanical dysfunction, and increased electromechanical delay. Our study has some limitations that need to be addressed. It was based on a relatively small group of patients and the patients with PCOS were younger than the controls. There is a need for longitudinal studies to follow up on subjects and controls.

Conclusion This study showed that patients with PCOS had increased inter- and intra-atrial conduction delays, decreased LA passive emptying volume and fraction, and lower E/A ratios. Increased Pd is a risk factor for developing AF, therefore PCOS patients may have a high risk for developing atrial arrhythmias, unless they have other traditional cardiovascular risk factors.

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Fulop L, Banyasz G, Szabo G, et al. Effects of sex hormones on ECG parameters and expression of cardiac ion channels in dogs. Acta Physiol 2006; 188: 163–171.

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Aydin M, Ozeren A, Bilge M, Dursun A, Cam F, Elbey MA. Effects of dipper and non-dipper status of essential hypertension on left atrial mechanical functions. Int J Cardiol 2004; 96: 419–424.

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paroxysmal atrial fibrillation. Am J Cardiol 1998; 82: 869–874. 28. Rossi A, Zardini P, Marino P. Modulation of left atrial function by ventricular filling impairment. Heart Fail Rev 2000; 5: 325–331. 29. Kosmala W, O’Moore-Sullivan TM, Plaksej R, Kuliczkowska-Plaksej J, Przewlocka-Kosmala M, Marwick TH. Subclinical impairment of left

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3748–3754. 30. Meyer C, McGrath BP, Teede HJ. Overweight women with polycystic ovary syndrome have evidence of subclinical cardiovascular disease. J Clin Endocrinol Metab 2005; 90: 5711–5716. 31. Turhan H, Yetkin E, Sahin O, et al. Comparison of P-wave duration and dispersion in patients aged ≥ 65 years with those aged ≤ 45 years. J Electrocardiol 2003; 36: 321–326.

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Protective effects of ginseng extracts and common anti-aggregant drugs on ischaemia–reperfusion injury Ahmet Caliskan, Oguz Karahan, Suleyman Yazici, Sinan Demirtas, Orkut Guclu, Orhan Tezcan, Celal Yavuz

Abstract Objective: Ginseng is a traditional herbal medicinal product widely used for various types of diseases because of its cellular protective effects. Possible protective effects of ginseng were investigated in blood, cardiac and renal tissue samples and compared with common anti-aggregant agents in an animal ischaemia–reperfusion (I/R) model. Methods: Twenty rats were equally divided into four different groups as follows: control group (I/R-induced group without drug use), group I (acetylsalicylic acid-administered group), group II (clopidogrel bisulfate-administered group), group III (ginsenoside Rb1-administered group). For the groups assigned to a medication, peripheral I/R was induced by clamping the femoral artery one week after initiation of the specified medication. After reperfusion was initiated, cardiac and renal tissues and blood samples were obtained from each rat with subsequent analysis of nitrogen oxide (NOx), malondialdehyde (MDA), paraoxonase 1 (PON1) and prolidase. Results: NOx levels were similar in each group. Significant decrements were observed in serum PON1 levels in each group when compared with the control (p < 0.05). Serum MDA levels were significantly lower in groups II and III (p < 0.05). Ameliorated renal prolidase levels were detected in study groups (p < 0.05) and recovered cardiac prolidase levels were obtained in groups II and III (p < 0.05). Conclusion: These findings indicate that ginseng extracts may have a potential beneficial effect in I/R injury. However, more comprehensive studies are required to clarify the hypothetical cardiac, renal and systemic protective effects in reperfusioninduced oxidative damage. Keywords: ginseng, herbal medicine, anti-aggregant drugs, ischaemia–reperfusion injury Submitted 27/3/14, accepted 12/4/15 Cardiovasc J Afr 2015; 26: 222–226

www.cvja.co.za

DOI: 10.5830/CVJA-2015-047

The meaning of the Chinese word ‘ginseng’ is ‘human seed’. Ginseng has a root-like appearance, and its extracts contain

Department of Cardiovascular Surgery, Medical School of Dicle University, Diyarbakir, Turkey Ahmet Caliskan, MD, drahmetcaliskan@hotmail.com Oguz Karahan, MD Suleyman Yazici Sinan Demirtas Orkut Guclu Orhan Tezcan Celal Yavuz

sponin. Ginseng has been used in traditional medicine for many years, especially in East Asian countries.1 So far, more than 30 ginsenosides have been defined.2,3 Ginsenoid-Rd [dammar24(25)-ene-3β, 12β, 20(S)-triol-(20-O-β-D-glucopyranosyl)3-O-β-D-glucopyranosyl-(1→2)-β-D-glucopyranoside] is one of the basic active substances of ginsenoids. Due to its antioxidant properties, it has been used in ischaemia–reperfusion experiments.4 Yokozawa et al. reported that ginseng had protective effects on rat models in ischaemia–reperfusion experiments.5 The protective effects of anti-aggregant drugs have also been reported in many ischaemia–reperfusion experiments performed on rats.6-8 The preparation of medicines and products containing ginseng varies from region to region and culture to culture. In traditional Chinese medicine, ginseng plants are harvested in their natural state, usually without being subjected to any further processing. In addition, they are only prepared by pulverisation so that they can be eaten with foods that are consumed daily. In modern medicine, ginsenoids obtained from ginseng plants are decomposed in such a manner that they can be used either in vitamin extracts or in hard gelatin capsules that contain a specified dose.1-3 This study was undertaken to evaluate the effects of ginseng extracts on ischaemia–reperfusion injury. Additionally, the protective effects of these extracts were compared with standard anti-aggregant drugs.

Methods Approval for this study was obtained from the local ethics committee and from the Animal Research Committee of Dicle University (2013/6). All procedures were performed according to the Animal Welfare Act and the Guide for the Care and Use of Laboratory Animals. All animal subjects were maintained at the laboratory of the Animal Production Unit at Dicle University in standard humidity- (50 ± 5%) and temperature- (22 ± 2°C) controlled cages with a 12-hour light/dark cycle until the study began. Twenty rats were divided equally into four groups, including one control group. The rats in the control group underwent femoral ischaemia–reperfusion (I/R) without medication (the vehicle control-treated saline). These rats were sacrificed, and blood samples and cardiac and renal tissues were taken to determine the baseline I/R values of oxidative markers. All surgical procedures (without additional intervention) in the control group were designed similarly to the study groups. The ethics committee decided that there was no additional requirement for a sham group for determining the effect of surgical incision. Three study groups were created in order to compare the protective roles of different agents. All rats were anesthetised with ketamine (Ketalar, Pfizer) at a dose of 130 mg/kg and

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xylasine (Rompun, Bayer) at a dose of 20 mg/kg via an intraperitoneal line. Maintenance of anaesthesia was provided with ketamine hydrochloride (50 mg/kg). Three different agents were used for the three separate study groups before I/R, as follows. Group I (n = 5): acetylsalicylic acid (Coraspin®, Bayer, Leverkusen, Germany) was administered orally via gavage at a dose of 30 mg/kg/day, beginning one week prior to the start of the study. I/R was induced after one week of medication administration. Group II (n = 5): clopidogrel bisulfate (Planor®, Koçak Farma, Tekirdağ, Turkey) was administered orally via gavage at a dose of 1 mg/kg/day, beginning one week prior to the start of the study. I/R was induced after one week of drug administration. Group III (n = 5): ginsenoside Rb1 (Panax®, Bayer, Leverkusen, Germany) was administered orally via gavage at a dose of 100 mg/kg/day, beginning one week prior to the start of the study. I/R was induced after one week of drug administration.

Experimental I/R injury modelling The right femoral arteries of all of the rats were explored with simple femoral incision and the femoral artery was rounded with a non-needle suture USP-3/0 metric silk (Dogsan Surgical Sutures, Medical Material Industry Co. Inc, Trabzon, Turkey); thereafter the femoral the artery was clamped for six hours (Fig. 1). The femoral clamp was removed to create reperfusion after six hours. After reperfusion, all rats were sacrificed in the first hour, and blood samples and cardiac and renal tissues were obtained from each rat in each group. All study protocols were designed according to previously published protocols.9 The drug utilisation and surgical protocols are outlined in Fig. 1.

Laboratory analyses NOx measurement: the Griess reagent method, which is based on a modified cadmium reaction, was used to determine nitrogen oxide (NOx) levels. This method measures platelet-derived nitric oxide as described by Yavuz et al.10 NOx levels were calculated as μM/g protein for tissue extracts and as μmol/l for blood samples. MDA measurement: malondialdehyde (MDA) levels were evaluated according to the method described by Ohkawa et al., which is based on the determination of the levels of thiobarbituric acid reactive products.11 MDA values were expressed as μM/g protein for tissue extracts and as μmol/l for blood samples. PON1 measurement: the spectrophotometric modified Eckerson method was used for the detection of paraoxonase 1 A

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(PON1) activity.12 The activity of PON1 was expressed as U/g protein for tissue extracts and as U/l for blood samples. Prolidase measurement: prolin (expressed as U/l protein for tissue extracts and as U/g for blood samples), which is produced by prolidase, was measured spectrophotometrically according to the method described by Myara et al.13

Statistical analysis Oxidative markers in each group were analysed with SPSS software version 15.0 (SPSS Inc., Chicago, IL), and p < 0.05 was considered to be statistically significant. Obtained values were presented as mean ± standard deviation (SD). The Kolmogorov– Smirnov test was used to assess the normality of the distributions. Differences in the mean values between groups were assessed with a one-way analysis of variance (ANOVA) test and a Tukey HSD was used as a post hoc test; p < 0.05 was considered to be statistically significant.

Results In the control group, NOx levels were 8.99 ± 5.01 μmol/l, 25.36 ± 3.69 μM/g protein, and 14.06 ± 4.12 μM/g protein for blood, cardiac and renal samples, respectively. The control group’s MDA values were 24.63 ± 3.23 μmol/l, 28.38 ± 4.87 μM/g protein, and 13.11 ± 3.90 μM/g protein for blood, cardiac and renal samples, respectively. The activities of PON1 in the control group’s blood, cardiac and renal samples were 256.55 ± 19.06 U/l, 18.89 ± 7.41 U/g protein, and 20.75 ± 5.01 U/g protein, respectively. The control group’s prolidase levels were 1283.52 ± 545.44 U/l for blood samples, 63.47 ± 11.51 U/g protein for cardiac tissue extract, and 96.26 ± 4.12 U/g protein for renal tissue extract. There were no significant differences between the groups in terms of NOx levels. The comparison of NOx values between the groups is presented in Fig. 2. The PON1 activity in the blood from each drug group was significantly different from that of the control group [control vs group I (acetylsalicylic acid), (p < 0.05); control vs group II (clopidogrel), (p < 0.05); control vs group III (ginsenoside), (p < 0.05)]. There was a significant difference (p < 0.05) between the control group and group III (ginsenoside) in terms of blood MDA levels. However, blood MDA levels of group II (clopidogrel) were significantly lower than those of the controls (p = 0.045). There were no significant differences between the control group and any of the study groups in terms of cardiac oxidative markers (p > 0.05). C

Fig. 1. A . Gavage set (injector, mouth brace, gavage catheter); B. gavage application; C. femoral incision; D. exploration of femoral artery and rounding of vascular structure with non-needled silk sutures.

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p = 0.091 26.87 ± 17.65

p = 0.437 25.24 ± 2.16

25.36 ± 3.69

256.55 ± 19.06

25 p = 0.838 11.75 ± 13.36

p = 0.937 25.36 ± 3.69 p = 0.297 22.58 ± 3.99

14.06 ± 4.12

p = 0.459 12.03 ± 1.12

p = 0.654 16.91 ± 1.57

*p = 0.001 166.72 ± 34.43 *p = 0.004 176.92 ± 28.58

p = 0.640 12.92 ± 2.82

p = 0.441 18.65 ± 0.73

p = 0.513 17.25 ± 3.10

p = 0.069 11.19 ± 1.11

*p = 0.000 140.05 ± 38.56

20.75 ± 5.01

18.89 ± 7.41

10 p = 0.402 12.53 ± 1.90

9.41 ± 4.89

8.99 ± 5.01

p = 0.725 21.91 ± 8.35

p = 0.917

*p = 0.026 11.45 ± 2.34

0 C

III

Blood μmol/l

III

Blood U/l

Cardiac μM/g protein Renal μM/g protein

III

Cardiac U/g protein

III

Renal U/g protein

PON1 levels

NOx levels

Fig. 2. C omparison of blood, renal and cardiac nitrogen oxide (NOx) levels in each group. C: control group; I: group I; II: group II; III: group III.

The MDA levels in each group are compared in Fig. 3. Both renal prolidase and PON1 levels were significantly lower in group II (clopidogrel) than in the control group (p < 0.05). Similarly, renal prolidase levels were also markedly lower in group III (ginsenoside, p < 0.05). The PON1 activities of each group are presented in Fig. 4. Renal prolidase levels were significantly lower in group I (acetylsalicylic acid) than in the control group (p < 0.05). In addition, cardiac prolidase levels were significantly lower in groups II (clopidogrel) and III (ginsenoside) (p < 0.05). Prolidase levels are compared between groups in Fig. 5.

Discussion Our results suggest that experimental I/R induced oxidative markers in blood, cardiac and renal samples. NOx values were similar in both the study and control groups. Blood MDA values

Fig. 4. Comparison of blood, renal and cardiac paraoxonase 1 (PON1) activity in each group. C: control group; I: group I; II: group II; III: group III.

were markedly lower in the clopidogrel and ginsenoside groups when compared with the control group. Decreased PON-1 levels were found in the clopidogrel group when compared with other groups. Significantly decreased cardiac prolidase levels were detected in the clopidogrel and ginsenoside groups when compared with the control group. Renal prolidase levels were markedly decreased in all study groups that were treated with acetylsalicylic acid, clopidogrel or ginsenoside. For centuries, herbal products have been widely used to treat or alleviate the symptoms of many diseases. Moreover, some of these herbs are currently used for traditional disease management.14,15 According to the World Health Organisation (WHO) report, it is estimated that more than 80% of the world’s population is dependent on herbal medicine.14 Most believe that it is safe to use these natural products, although side effects, toxicity and adverse drug interactions have been reported. p = 0.670 1557.32 ± 1230.56

40 p = 0.078 33.73 ± 2.52

35 30

p = 0.403 26.26 ± 3.76

p = 0.940 24.12 ± 2.39

25 20

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p = 0.043 23.38 ± 4.87 16.36 ± 5.89 24.63 ± 3.23

p = 0.286 17.48 ± 2.53

p = 0.084 22.81 ± 2.89

p = 0.459 11.33 ± 2.61

*p = 0.045 17.03 ± 5.37

p = 0.224 16.33 ± 1.88 13.11 ± 3.90

5 0 C

Blood μmol/l

III

Cardiac μM/g protein Renal μM/g protein

MDA levels

Fig. 3. C omparison of blood, renal and cardiac malondialdehyde (MDA) levels in each group. C: control group; I: group I; II: group II; III: group III.

1500 1400 1300 1200 1100 1000 800 600 400 200 0

1283.52 ± 545.44 p = 0.720 1167.32 ± 371.32

p = 0.177 848.75 ± 255.46

Blood U/l

III

100 90 80 70 60 50 40 30 20 10 0

96.26 ± 4.12 *p = 0.000 75.85 ± 3.47

*p = 0.000 76.48 ± 4.77 63.47 ± 11.51

*p = 0.000 75.16 ± 5.53

*p = 0.016 45.72 ± 2.93 p = 0.179 55.77 ± 5.02 *p = 0.012 42.44 ± 3.65

III

Cardiac U/g protein

III

Renal U/g protein

Prolidase levels

Fig. 5. Comparison of blood, renal and cardiac prolidase activity in each group. C: control group; I: group I; II: group II; III: group III.

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Most drugs originated from or are derived from herbs. However, dosages and usable forms of the drugs must be investigated with expensive clinical trials before they become commercialised.14,16 Although there are conflicting reports, herbal products, while they still have risks, may be safer for prophylaxis and the treatment of diseases. In addition, they are inexpensive and readily available.17 The quality and quantity of herbal products may vary depending on seasonal and regional growing conditions, and may therefore have different effects.18 Supervision and quality control should therefore be required during their production. Ginseng is a commonly studied therapeutic herbal product.18 The protective role of ginseng extracts with several metabolic mechanisms has been reported in cardiovascular events.19 It has been hypothesised that ginseng extracts may protect the cardiovascular system by acting as an antioxidant, antihypertensive, antidiabetic and antinociceptive agent.15,19 The protective effects of acetylsalicylic acid and clopidogrel bisulfate on ischaemia–reperfusion injury have been previously described.6,7,20,21 In addition, similar findings have been reported for ginsenosides.22 However, to our knowledge, there has been no definitive comparison of these three agents in the literature. In this study, the systemic, cardiac and renal protective effects of the well-known anti-aggregant agents acetylsalicylic acid and clopidogrel bisulfate were compared with ginsenoside Rb1 (Panax) against oxidant stress in a peripheral ischaemia– reperfusion model. MDA serves as a biomarker for detection of peroxidative damage in reperfused organs; it is a product of enzymatic and oxygen radical-induced lipid peroxidation.9 Reduced MDA levels have previously been reported with acetylsalicylic acid- or clopidogrel-treated patients in ischaemia–reperfusion studies.23,24 Although, PON-1 is mainly produced by the liver, it has been identified in other tissues such as the kidney, heart and brain.25 An inverse relationship was reported between PON-1 activity and antiplatelet agents.26 Prolidase is a marker for collagen metabolism that is related to increased levels of nuclear hypoxia-inducible factor-1 alpha (HIF-1).9,27 Increased prolidase levels were reported in acute ischaemic events.27 In the current study, the serum MDA levels were partially improved in the clopidogrel bisulfate and ginsenoside Rb1 groups, while the serum PON1 levels were markedly decreased in all three groups. Renal PON1 levels were only significantly expressed in the clopidogrel bisulfate group. Renal prolidase levels were significantly decreased in all groups compared to the control I/R group. Cardiac prolidase levels were significantly decreased in the clopidogrel bisulfate and ginsenoside Rb1 groups. According to our results, it appears that ginsenoside Rb1 had a beneficial effect on the oxidative stress induced by I/R by antioxidant mechanisms. Mannaa et al. reported that Panax had a neuroprotective effect in acrylamide-induced neurotoxicity.15 In another study, Basha et al. reported the renoprotective effects of ginsenosides against oxidative stress in streptozotocin-induced diabetic nephrotoxicity in mice.22 In addition, it has been reported that ginsenosides can play a protective role in decreasing lipid peroxidation and ameliorating oxidative damage.28 Kim reported that ginsenosides have possible protective mechanisms in cardiovascular events.19 He described these

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mechanisms as follows: • Ginsenosides inhibit Ca2+ entry, and therefore may ameliorate cardiac function.19 However, acetylsalicylic acid can stimulate the Ca2+ entry pathways.29 • Ginseng normalises blood pressure and improves blood circulation.19 Previous reports noted that acetylsalicylic acid and clopidogrel can alter blood flow in tissues.30 • Ginsenoids can protect against myocardial damage via nitric oxide-mediated cardiac protection, antioxidant and intracellular calcium homeostasis, and attenuation of calcineurin activation.19 Similarly, some literature has suggested that clopidogrel and acetylsalicylic acid improve endothelial nitric oxide.31,32 • Ginseng saponin has a protective role on endothelial cells via a cellular signalling pathway.19 Similar cellular mechanisms were reported for clopidogrel and acetylsalicylic acid.33,34 • Ginseng has a cardiovascular protective role in inhibiting oxidative damage due to the prevention of reactive oxygen species generation.19 Also, the anti-oxidant effects of clopidogrel and acetylsalicylic acid have been described in previous reports.7, 34 There are some limitations that need to be addressed in this study. An experimental I/R model was created for this study in healthy rats. Therefore, our results are pertinent only to the rat model and these results should be confirmed in human subjects. The other limitation is that only oxidative markers were studied and PCR and Western blot analysis were not applied. Because of this, our findings are lacking in cellular reflections.

Conclusion Herbal medicine is still important for the majority of the world’s population. Traditional ginseng extracts may have beneficial effects on ischaemia–reperfusion injury. However, we caution that herbs should not replace traditional drugs. Ginseng can be beneficial as a drug supplement when controlled by healthcare organisations. In addition, future cardiovascular studies are needed to clarify the drug interactions and the proper dose of ginseng extracts. We are grateful to Dicle University DUBAP for their sponsorship of the English editing of this manuscript.

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Fu YQ, Hua C, Zhou J, Cheng BR, Zhang J. Protective effects of ginseng total saponins against hepatic ischemia/reperfusion injury in experimental obstructive jaundice rats. Pharm Biol 2013; 51(12): 1545–1551. doi: 10.3109/13880209.2013.802352.

Shibata S, Tanaka O, Shoji J, Saito H. Chemistry and Pharmacology of Panax. In: Wagner H, Hikino H, Farnsworth NR, eds. Economic and Medicinal Plant Research, Vol. 1. London, Orlando, San-Diego, New York, Toronto, Montreal, Sydney, Tokyo: Academic Press, 1985; 217–287.

Bruneton J. Ginseng, Panax ginseng C.A. Meyer, Araliaceae. ln: Pharmacognosy, Phytochemistry, Medicinal Plants. Paris: Lavoisier, 1995: 563–565.

Wang Y, Li X, Wang X, Lau W, Wang Y, Xing Y, et al. Ginsenoside Rd attenuates myocardial ischemia/reperfusion injury via Akt/GSK-3β signaling and inhibition of the mitochondria-dependent apoptotic pathway. PLoS One 2013; 8(8): e70956. doi: 10.1371/journal.pone.0070956.

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Yokozawa T, Wu Liu ZW, Dong E. A study of ginsenoside-Rd in a renal ischemia-reperfusion model. Nephron 1998; 78(2): 201–206.

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ginseng) & banaba (Lagerstroemia speciosa) on mitochondrial oxidative 23. Ulubaş B, Cimen MY, Apa DD, Saritaş E, Muşlu N, Cimen OB. The

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24. Taher MA, Nassir ES. Beneficial effects of clopidogrel on glycemic indi-

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Factor-Xa inhibitors protect against systemic oxidant damage induced by peripheral-ischemia reperfusion. J Thromb Thrombolysis 10.1007/ s11239-013-1019-4.

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paraoxonase polymorphism: identification of phenotypes by their response to salts. Am J Hum Genet 1983; 35: 214–227. 13. Myara I, Charpentier C, Lemonnier A. Optimal conditions for prolidase assay by proline colorimetric determination: application to iminodipeptiduria. Clin Chim Acta 1982; 125: 193–205. 14. Wong A, Townley SA. Herbal medicines and anaesthesia. Contin Educ Anaesth Crit Care Pain 2011; 11 (1): 14–17. doi: 10.1093/bjaceaccp/mkq046

21: 1000–1004. 28. Ali MB, Hahn EJ, Paek KY. Protective role of Panax ginseng extract on lipid peroxidation and antioxidant status in polyethylene glycol induced Spathiphyllum leaves. Biochem. Eng J 2006; 32(3): 143–148. 29. Suzuki Y, Inoue T, Ra C. NSAIDs, mitochondria and calcium signaling: special focus on aspirin/salicylates. Pharmaceuticals 2010; 3: 1594–1613; doi:10.3390/ph3051594.

15. Mannaa F, Abdel-Wahhab MA, Ahmed HH, Park MH. Protective role

30. Bruning RS, Dahmus JD, Kenney WL, Alexander LM. Aspirin and

of Panax ginseng extract standardized with ginsenoside Rg3 against

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18. Kizil S, Toncer O, Ipek A, Arslan N. Saglam S, Khawar KM. Blooming stages of Turkish hyssop (Hyssopus officinalis L.) affect essential oil composition. Acta Agr Scand B-P S 2008; 58: 273–279. 19. Kim JH. Cardiovascular diseases and Panax ginseng: A review on molecular mechanisms and medical applications. J Ginseng Res 2012; 36(1): 16–26. doi: 10.5142/jgr.2012.36.1.16.

1648–1652. 32. Taubert D, Berkels R, Grosser N, Schröder H, Gründemann D, Schömig E. Aspirin induces nitric oxide release from vascular endothelium: a novel mechanism of action. Br J Pharmacol 2004; 143(1): 159–165. 33. Bonello L, Harhouri K, Sabatier F, Camoin-Jau L, Arnaud L, Baumstarck-Barrau K, et al. Level of adenosine diphosphate receptor

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the extent of endothelial injury, assessed by circulating endothe-

cylic acid on long-term histologic damage in a rat model of testicular

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An open-access mobile compatible electronic patient register for rheumatic heart disease (‘eRegister’) based on the World Heart Federation’s framework for patient registers Joris van Dam, John Musuku, Liesl J Zühlke, Mark E Engel, Nick Nestle, Brigitta Tadmor, Jonathan Spector, Bongani M Mayosi

Abstract Background: Rheumatic heart disease (RHD) remains a major disease burden in low-resource settings globally. Patient registers have long been recognised to be an essential instrument in RHD control and elimination programmes, yet to date rely heavily on paper-based data collection and non-networked data-management systems, which limit their functionality. Objectives: To assess the feasibility and potential benefits of producing an electronic RHD patient register. Methods: We developed an eRegister based on the World Heart Federation’s framework for RHD patient registers using CommCare, an open-source, cloud-based software for health programmes that supports the development of customised data capture using mobile devices. Results: The resulting eRegistry application allows for simultaneous data collection and entry by field workers using mobile devices, and by providers using computer terminals in clinics and hospitals. Data are extracted from CommCare and are securely uploaded into a cloud-based database that matches the criteria established by the WHF framework. The application can easily be tailored to local needs by modifying existing variables or adding new ones. Compared with traditional paper-based data-collection systems, the eRegister

Novartis Institutes for BioMedical Research, Cambridge, Massachusetts, USA Joris van Dam, PhD, joris.vandam@novartis.com Brigitta Tadmor, PhD Jonathan Spector, MD, MPH

Department of Paediatrics & Child Health, University Teaching Hospital, Lusaka, Zambia John Musuku, BSc HB, MBcHB, MMed

Western Cape Paediatric Cardiac Services, Red Cross War Memorial Children’s Hospital, University of Cape Town, Cape Town, South Africa Liesl J Zühlke, MB ChB, DCH, FCPaeds (SA), Cert Cardiology (Paeds), MPH, FESC

Department of Medicine, Groote Schuur Hospital and University of Cape Town, Cape Town, South Africa Liesl J Zühlke, MB ChB, DCH, FCPaeds (SA), Cert Cardiology (Paeds), MPH, FESC Mark E Engel, BSc (Med) Hons, MPH, PhD Bongani M Mayosi, BMedSci, MB ChB, DPhil, FCP (SA), FESC

Dimagi Inc, South Africa Nick Nestle, BSEE

reduces the risk of data error, synchronises in real-time, improves clinical operations and supports management of field team operations. Conclusions: The user-friendly eRegister is a low-cost, mobile, compatible platform for RHD treatment and prevention programmes based on materials sanctioned by the World Heart Federation. Readily adaptable to local needs, this paperless RHD patient register program presents many practical benefits.

Keywords: rheumatic heart disease, registries, mobile health, open-source model Submitted 21/11/14, accepted 10/6/15 Published online 6/10/15 Cardiovasc J Afr 2015; 26: 227–233

www.cvja.co.za

DOI: 10.5830/CVJA-2015-058

Rheumatic heart disease (RHD) was largely eliminated from most high-income countries decades ago but it remains a major cause of cardiovascular disease in sub-Saharan Africa, indigenous Australia, south-central Asia, the Pacific region, and other low-resource settings globally.1,2 At least 15 to 20 million people are affected and more than 280 000 new cases are diagnosed each year,3 although recent data from populationbased screening using echocardiography suggest that the true prevalence could be up to tenfold higher.4–6 The World Heart Federation (WHF), an association of worldwide heart foundations and medical societies, is the leading international non-governmental organisation concerned with cardiovascular disease prevention.7 A key strategic target put forth by WHF is the use of comprehensive register-based control programmes in regions where RHD is endemic.7 Patient registers are instrumental in helping to organise the medical care of patients with RHD, minimising the loss to follow up, and maximising the likelihood of compliance with therapeutic regimens.7-11 This is particularly important in patients with RHD, many of whom require regular antibiotic therapy for decades in order to mitigate the progression of heart disease. Registers also facilitate monitoring of longitudinal patient outcomes, and can be used to compile epidemiological data for use in programme planning and advocacy activities.7,11,12 Furthermore, patient registers can be used to collect, organise and report data required by national health authorities should RHD be considered a reportable disease. Examples such as the

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multi-national REMEDY study13 demonstrate the important roles that register data can play in research and in the design of effective RHD control programmes. A register variant can also be used to organise and store data in large-scale screening programmes to identify individuals with RHD who were previously undiagnosed. Until now, however, virtually all patient registers used in RHD programmes have relied on paperbased data collection and non-networked data-management systems, which limit their utility. The emergence of mobile and cloud technologies, together with the increasing availability of low-cost mobile phones, computer tablets and data storage, offer the opportunity to explore the use of electronic patient registers in RHD control programmes in high-priority countries. We have developed such an electronic register tailored for specific use in a largescale comprehensive public–private effort to combat RHD in Zambia. This tool was demonstrated in February 2014, at the 2nd All Africa Workshop on Rheumatic Fever and Rheumatic Heart Disease in Livingstone, Zambia,14,15 and the delegates (representing 13 African countries) appealed for a version of the tool that could be incorporated into their own RHD programmes.15 To address this need, we sought to adopt the WHF’s framework for RHD patient register databases for an open-

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access, mobile, compatible electronic platform (‘eRegister’) that would be user-friendly, modifiable to local contexts, inexpensive to operate, and straightforward to distribute. We then sought to assess the practical benefits of deploying such a system, and to make the eRegister freely available to potential users.

Methods World Heart Federation’s RHD patient database tools The WHF has developed patient register database tools in support of RHD control programmes.16 Core components are downloadable from the WHF website and include (1) a datacollection form (Figs 1, 2) meant to be printed and used by health workers to record a patient’s medical history, management plan and clinical outcomes; and (2) a complementary electronic Microsoft Access® database template that contains the same fields as the data-collection form. Using these tools, data would normally be entered by hand onto a printed data-collection form and then copied into the electronic database, which has inbuilt functionality to provide a rich variety of data reports, but in most cases would be non-networked and therefore accessible only from a single computer terminal.

Fig. 1. D ata-collection form for the WHF patient register database, page 1.

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Fig. 2. Data-collection form for the WHF patient register database, page 2.

Electronic register system: development and testing We aimed to replace the paper-based data-collection forms with electronic forms that could be deployed on inexpensive and widely available mobile devices (e.g. phones and tablets), and to associate these forms with a version of the WHF’s register database that would automatically and continuously be updated and readily accessible to multiple users simultaneously for patient care, data analysis and reporting purposes, as illustrated in Fig. 3. We developed the electronic data-collection forms on the CommCare platform,17 an open-source, cloud-based software platform for health programmes that supports the development of customised data-capture tools using mobile devices. Our lead programmer (JVD) had general software programming experience only, with no prior expertise specific to CommCare. To become acquainted with the platform, the CommCare online training course was completed.18 We then developed a number of electronic data-collection forms by dividing the various sections of the WHF data-collection form into smaller sub-forms,16 as illustrated in Table 1. This approach provides greater flexibility for subsequently updating individual elements of the form and tailoring them to local needs. Once the forms were developed, we created data-extraction jobs that export data from CommCare and map them to a spreadsheet in Microsoft Excel, with a table structure and data labels that are identical to the WHF database. We then created a copy of the WHF database in Microsoft Access® 2010 and used the standard data import features of the program to ‘link’ the

database to the spreadsheet. Once established, subsequent dataextraction jobs can overwrite the spreadsheet with new data, which will automatically be visible in the WHF database. We identified a number of minor technical issues for which we implemented adjustments in our copy of the WHF database, mainly to account for the different ways that data are represented in Microsoft Access® versus CommCare, such as the representation of missing values (‘ ’ or ‘–’), the representation of binary values (‘yes/no’ or ‘true/false’) and the use of date formats. Table 1. Overview of what forms are used to collect the data elements in the WHF patient register database and the eRegister. The data elements in the table refer to Figs 1 and 2. WHF patient register database eRegister Data collected on the Data collected on the Data elements following form following form Personal details Data-collection form Data-collection form Secondary prophylaxis Data-collection form Data-collection form Follow-up Data-collection form Schedule follow-up appointments appointments Diagnosis Data-collection form Diagnosis Cardiac surgery Data-collection form Surgery Review results Data-collection form Review results Benzathine penicillin Data-collection form Benzathine penicillin injection delivery delivery Death Data-collection form Death Notes Data-collection form Data-collection form

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Fig. 3. O verview of the targeted eRegister system. Data are collected through electronic forms on a variety of devices, securely managed in the cloud, and made accessible to multiple users for varying clinical care and clinical research purposes.

To pretest the resulting application, we used the sample data available in the WHF sample database.16 We entered the values from the sample database into our newly developed electronic data-collection forms, running on two different mobile devices: a general purpose seven-inch tablet running on the Android operating system (purchased commercially for US$59.99) and a general-purpose Android smartphone (purchased commercially for US$29.99). We extracted the data from CommCare into our copy of the WHF database, reviewed the data and the reports against the original WHF sample database, and confirmed their being identical.

Field testing A version of the eRegister was adapted to the specific needs of a school-based RHD screening programme in Lusaka, Zambia, in which health workers conducted clinical and echocardiographic assessment of schoolchildren in order to detect those with previously unrecognised RHD. Eight health professionals (including local nurses and radiographers, and programme management staff) were orientated on the use of the eRegister over two half-day training sessions and then received ongoing support as needed to utilise the tool. The eRegister was deployed to support screening of 261 children in pilot screening sessions conducted from June to November 2014, and 1 022 children in full-scale screening that was conducted during February and March 2015. The mobile devices

used were Samsung Galaxy Tab 2 tablets, which were sourced locally. Data were entered into the eRegister using a combination of tablets and laptops on site at schools and at the referral hospital.

Results The resulting eRegister application enables simultaneous data collection and entry. For example, field workers can directly enter patient data into the systemâ&#x20AC;&#x2122;s electronic data-collection forms, which then automatically populate the cloud-based database, using either handheld mobile devices or computer terminals in clinics and hospitals. When data are entered on mobile devices, synchronisation with the central database takes place securely the next time a cellular or internet connection is established (i.e. wireless connection is not necessary at the time of data entry). Thus the database is continually updated, which streamlines its various clinical and research functions (Fig. 3). The eRegister variables match those in the WHF register. Variables relating to existing data fields (e.g. names of villages or clinics) can easily be tailored to local needs and new data fields can be added. The system application itself can be installed to mobile devices by downloading from https://sites.google.com/ site/rhderegister/home. All access to the CommCare platform including mobile submissions is achieved through Hypertext Transfer Protocol Secure (HTTPS) and is cryptographically secure. Data stored in the eRegister is confidential and password-protected at all times.

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We compared attributes of the eRegister and traditional paper-based systems. Benefits of the use of the eRegister system are likely to include: • Electronic data entry. Data are entered in electronic format directly at the point of capture, which obviates the need to manage paper forms, reduces the risk of data error associated with manual transcription of data from paper to electronic sources, and improves the ease of updating data. • Flexible, real-time system. Data collected through mobile devices populate the eRegister as soon as the device synchronises with the CommCare platform. Conversely, changes or local adaptions to data-collection forms to suit local needs can be made in real-time in the CommCare application and then distributed automatically to all mobile devices in the field. The next time the device is synchronised, the newly updated form will automatically be loaded onto the device, offering a level of flexibility in data collection that would be largely unworkable using paper forms. • Improving clinical operations. The eRegister system can be used to track and manage treatment of individual patients. Follow-up workflow plans can be created, penicillin allergies can be tracked and alternative prophylactic treatment can be prescribed, and custom reports and worklists can be created to help health workers manage a cohort of patients (for example, reports can be automatically generated that list patients who missed their last appointment). The system can also be utilised to distribute multimedia-format training modules to field workers. • Improving clinical outcomes. It is known that delivery of secondary prophylaxis within a registry-based programme increases the success of control programmes.19 The eRegister system can provide an integrated method to organise ongoing medical care of patients with RHD, minimising the loss to follow up, and maximising the likelihood of compliance with therapeutic regimens. This simple method also enables the monitoring of patient outcomes, and planning of advocacy and awareness activities in low-resource settings. • Improving treatment adherence. A particularly important example of improving clinical operations is the ease of implementing SMS reminders to the phones of patients, parents and health workers in advance of patient appointments, or in follow up to missed patient appointments. Adherence to prophylactic treatment for RHD has been shown to be low in many populations where RHD is endemic,20,21 and the use of register-based reminder systems can be an important tool to help support and improve adherence.22-24 • Field team management. In addition to patient data, data relating to data-collection processes is automatically captured through the CommCare platform including, for example, the time taken to fill in an individual data-collection form or the number of forms submitted by each user. Data that describe patterns of data collection can be used to identify training needs and opportunities for productivity gains. Data collected by field teams can also be anonymised and reviewed by remote teams to analyse the quality of decision making and identify training needs. Multimedia training materials can be delivered to the field teams through the platform as well. • Research. A key feature of the system is the ability to rapidly generate de-identified data reports that can be used for research purposes. Furthermore, even though such data are

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currently not captured in the WHF register, the platform’s real-time data-collection features and workflow support can also be used to effectively support other processes critical to the conduct of clinical research studies, such as adverse event reporting. Preliminary lessons learned from early field testing of the eRegister in Zambia include demonstration that the tool was overall easy to use, and that local programme staff were able to be trained to use the eRegister in a relatively short time and without specific prior technical knowledge or experience. The field team iteratively modified its work practices after the programme was underway in order to increase efficiency in its task of screening large numbers of children, for example, some data elements were collected in a different order and at a different location than was originally planned. It was straightforward to adapt the content and flow of forms in the eRegister to reflect changes in local work practices, and this was achieved in real-time without interruptions to the screening programme. The study team reported several immediate benefits of the eRegister to programme operations. In particular, the eRegister’s actual and up-to-date status reports that could be generated at any time (including total number of children screened, where the screenings had taken place, how many children had screened positive for RHD, etc.), played important roles for programme monitoring and planning purposes. Another significant benefit was remote access to the eRegister by team members based in different locations, and functionality, which was applied to support data quality-assurance mechanisms. There were also a number of challenges associated with the eRegister. Insufficient use was made of the available features to adapt the eRegister to evolving local work practices. Changes in work practices were not always reflected in corresponding changes in forms and workflows in the eRegister, leading to sub-optimal use of the tool. Poor internet connectivity at the sites in Zambia where the eRegister was used led to another intermittent problem; while the eRegister was always functional, it did on occasion take a long time to update software and upload large files to individual patient records (e.g. ultrasound images).

Discussion Rapid advances in technology over the past decade have made electronic patient resources theoretically within the reach of users in virtually every part of the world, including in low-resource settings where RHD is endemic and where efficient diseasecontrol programmes are most needed. We have adopted the WHF framework for patient register to develop an open-access, mobile, compatible, electronic patient register system. Our aim was not to attempt to develop a ‘one-size-fits-all’ RHD patient register, but rather to develop a platform that could be readily accessed by a wide range of stakeholders and adapted to their individual needs. The main benefit of using an RHD register is to support longitudinal treatment programmes for patients diagnosed with RHD. In our field test of the eRegister in Zambia, we found that the tool could also be adapted to effectively support an RHD field screening programme. In addition to providing an efficient platform for managing data associated with screening

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programmes, the eRegister can foster compliance with the WHF guidelines for diagnosis of RHD25 by reproducing the criteria in the electronic forms, which are therefore readily accessible to health workers at the point of screening. The platform is available at no cost, and provides countries and regions with an opportunity to adopt efficient, standardised patient register tools for the implementation of local RHD control programmes, to conduct RHD research studies, or to satisfy national reporting requirements should RHD be identified as a reportable disease. Moreover, further improvements can be made to the eRegister that perhaps would not be possible with the use of paper-based data-collection forms. There are limitations of the eRegister system relative to traditional paper-based tools. Despite the fact that the ‘technology footprint’ with this system is relatively low, it will still be a barrier in some settings with regard to financial costs, familiarity by users, and need for ongoing troubleshooting and technical support. However, we believe these barriers can be effectively addressed by end-user training, and that the barriers will be lowered as the use of mobile technologies becomes more common among healthcare workers.

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Remenyi B, Carapetis J, Wyber R, Taubert K, Mayosi BM. Position statement of the World Heart Federation on the prevention and control of rheumatic heart disease. Nat Rev Cardiol 2013; 10(5): 284–292. PMID: 23546444.

WHO programme for the prevention of rheumatic fever/rheumatic heart disease in 16 developing countries: report from Phase I (1986–90). WHO Cardiovascular Diseases Unit and principal investigators. Bull World Health Organ 1992; 70(2): 213–218. PMID: 1600581.

Strasser T, Dondog N, El Kholy A, et al. The community control of rheumatic fever and rheumatic heart disease: report of a WHO international cooperative project. Bull World Health Organ 1981; 59(2): 285–294. PMID: 6972819.

10. World Health Organization. The WHO global programme for the prevention of rheumatic fever and rheumatic heart disease: report of a consultation to review progress and develop future activities. 2000. URL:http://whqlibdoc.who.int/hq/2000/WHO_CVD_00.1.pdf. Accessed: 2014-08-20. (Archived by WebCite® at http://www.webcitation.org/6Ry8rkzi0). 11. McDonald M, Brown A, Noonan S, Carapetis JR. Preventing recurrent rheumatic fever: the role of register based programmes. Heart 2005; 91(9): 1131–1133. PMID: 16103536. 12. Rheumatic fever and rheumatic heart disease. World Health Organ Tech

Conclusions The WHF register has been successfully converted to an openaccess eRegister platform. Preliminary results from a local RHD study in Zambia, for which a version of the eRegister was adapted, support the expected benefits of using an eRegister. Future improvements to the system (such as SMS reminders for families and integration with portable echocardiographic devices) can be added to the platform, as dictated by programmatic needs.

Rep Ser 2004 923: 1–122. back cover. PMID: 24217043. 13. Zühlke L, Engel ME, Karthikeyan G, Rangarajan S, Mackie P, Cupido B, et al. Characteristics, complications, and gaps in evidence-based interventions in rheumatic heart disease: the Global Rheumatic Heart Disease Registry (the REMEDY study). Eur Heart J 2015 May 7; 36(18): 1115–1122. PMID: 25425448. 14. 2nd All Africa Workshop on Rheumatic Fever and Rheumatic Heart Disease. 2014-01-31, Livingston, Zambia. URL:http://www.rhdafrica. org/. Accessed: 2014-07-22. (Archived by WebCite® at http://www. webcitation.org/6RG8GlZNs).

Novartis Institutes for BioMedical Research funded the project. Joris van

15. Mayosi B M, Gamra H, Dangou JM, Kasonde J. 2nd All Africa

Dam led the development of the eRegister and wrote the first draft of the

Workshop on Rheumatic Fever & Rheumatic Heart Disease, 2014.

manuscript. John Musuku, Liesl Zühlke, Mark Engel, Nick Nestle, Brigitta

Rheumatic heart disease in Africa: the Mosi-o-Tunya call to action.

Tadmor, Jonathan Spector and Bongani Mayosi contributed to the develop-

Lancet Glob Health 2, e438–439. PMID: 25103507.

ment of the eRegister and participated in the writing of the manuscript. Joris

16. World Heart Federation. RHD Register Databases. 2014-08-20.

van Dam, Brigitta Tadmor and Jonathan Spector are employees of Novartis.

URL:http://www.world-heart-federation.org/what-we-do/rheumatic-

Nick Nestle is an employee of Dimagi Inc, the company that developed the

heart-disease-network/for-health-professionals/world-heart-federation-

CommCare platform.

rheumatic-heart-disease-resources/rhd-register-databases/. Accessed: 2014-08-20. (Archived by WebCite® at http://www.webcitation.

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19. Lawrence JG, Carapetis JR, Griffiths K, Edwards K, Condon JR. Acute

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Paar JA, Berrios NM, Rose JD, et al. Prevalence of rheumatic heart

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High-sensitivity cardiac troponin T is more helpful in detecting peri-operative myocardial injury and apoptosis during coronary artery bypass graft surgery Emel Fatma Kocak, Cengiz Kocak, Ahmet Aksoy, Ozden Ozben Isiklar, Raziye Akcilar, Ibrahim Fevzi Ozdomanic, Cevher Unsal, Merve Celenk, Irfan Altuntas

Abstract Aim: To determine whether there is a correlation between cardiac markers and peri-operative myocardial injury (PMI) and apoptosis in coronary artery bypass graft (CABG) surgery and to compare the efficacy of cardiac markers to detect PMI. Methods: The study population consisted of 37 patients (24 male, 13 female, mean age 63.4 ± 8.9 years) undergoing elective CABG. Arterial and coronary sinus blood samples were collected just before aortic cross-clamping (pre-ACC) and after aortic declamping (post-ACC). Creatine kinase-MB isoenzyme (CK-MB) activity, and high-sensitivity cardiac troponin T (hs-cTnT), creatine kinase-MB isoenzyme mass (CK-MB mass) and cardiac troponin I (cTnI) concentrations were measured in blood samples. Myocardial injury and apoptosis were examined in atrial biopsies. Results: CABG caused PMI and apoptosis in all cases. Concentrations and net releases of cardiac markers signifi-

cantly increased after aortic declamping (p < 0.001 for CK-MB and CK-MB mass, p < 0.01 for cTnI, p < 0.05 for hs-cTnT). A positive correlation was found between apoptotic index (r = 0.611, p < 0.001 for cTnI; r = 0.806, p < 0.001 for hs-cTnT), myocardial injury score (r = 0.544, p < 0.001 for cTnI; r = 0.719, p < 0.001 for hs-cTnT) and cTnI and hs-cTnT values in the post-ACC period. A positive correlation was found between apoptotic index (r = 0.507, p < 0.001), myocardial injury score (r = 0.416, p = 0.010) and net release of hs-cTnT. Furthermore, a positive correlation was found between aortic cross-clamp (ACC) time (r = 0.448, p = 0.007), cardiopulmonary bypass (CPB) time (r = 0.342, p = 0.047) and net release of hs-cTnT. Conclusion: Although both cTnI and hs-cTnT may be specific and efficacious markers of myocardial apoptosis and injury occurring during CABG with CPB, hs-cTnT may be a more useful marker than cTnI to detect peri-operative myocardial apoptosis and injury. Keywords: apoptosis, creatine kinase, MB form, coronary artery bypass, myocardial reperfusion injury, troponin

Department of Medical Biochemistry, Faculty of Medicine, Dumlupinar University, Kutahya, Turkey Emel Fatma Kocak, MD, dremelk@hotmail.com Irfan Altuntas, MD

Department of Pathology, Faculty of Medicine, Dumlupinar University, Kutahya, Turkey

Submitted 19/4/15, accepted 14/6/15 Published online 14/7/15 Cardiovasc J Afr 2015; 26: 234–241

www.cvja.co.za

DOI: 10.5830/CVJA-2015-052

Cengiz Kocak, MD

Department of Cardiovascular Surgery, Evliya Celebi Education and Research Hospital, Dumlupinar University, Kutahya, Turkey Ahmet Aksoy, MD Ibrahim Fevzi Ozdomanic, MD

Department of Medical Biochemistry, Evliya Celebi Education and Research Hospital, Dumlupinar University, Kutahya, Turkey Ozden Ozben Isiklar, MD

Department of Physiology, Faculty of Medicine, Dumlupinar University, Kutahya, Turkey Raziye Akcilar, PhD

Department of Anesthesiology and Reanimation, Evliya Celebi Education and Research Hospital, Dumlupinar University, Kutahya, Turkey Cevher Unsal, MD

Department of Medical Biochemistry, Evliya Celebi Education and Research Hospital, Dumlupinar University, Kutahya, Turkey Merve Celenk, MD

Coronary artery bypass grafting (CABG) accompanied by cardiopulmonary bypass (CPB) is a safe, routine procedure for the surgical treatment of various heart diseases, including coronary artery disease. Cardiopulmonary bypass and cardioplegic arrest enable the performance of coronary artery anastomosis in a bloodless and motionless field during CABG surgery.1,2 However, peri-operative myocardial injury (PMI) is a major problem and the most common cause of morbidity and mortality during CABG surgery.3 Despite optimal myocardial protective techniques, a certain amount of myocardial injury may occur in the majority of patients undergoing CABG surgery. Various factors can cause myocardial injury during CABG surgery, most importantly CPB, surgical technique, suture placement or manipulation of the heart, coronary dissection, aortic cross-clamping (ACC), and inadequate cardiac protection.4-6 During CPB, the heart is arrested and protected by cardioplegia. This period is associated with oxygen deprivation and the heart is ischaemic during this time. At the end of CPB, the heart is reperfused and cardiac action resumes. These ischaemic and subsequent reperfusion periods cause myocardial injury and

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even necrosis.7 Myocardial ischaemia causes intracellular calcium accumulation and degradation of the membrane lipids, and oedema during ACC. After removal of the aortic cross-clamp, reperfusion causes oxidative stress depending on the production of reactive oxygen (ROS) and reactive nitrogen species (RNS).8 In addition, it has been reported that myocardial ischaemia– reperfusion (I/R) induces cardiomyocytic apoptosis.9,10 Early and accurate detection of PMI may prompt immediate improvement in the perfusion and oxygen demand of the myocardium,which may limit PMI. Therefore, it is important to have a highly specific diagnostic marker to detect PMI. Cardiac surgery may lead to the release of markers of myocardial injury. Interpretation of these elevated cardiac markers in the blood during the peri-operative period is confusing because increases in cardiac markers may be related to direct skeletal muscle injury due to the surgical procedure, or to myocardial I/R injury. It is diffucult to differantiate between increases in cardiac markers related to surgical procedure and pathological myocardial I/R injury.11 In this study, we considered that histopathological examination of myocardial tissue would clearly reveal myocyte damage occurring in the peri-operative period, and increases in cardiac markers could be properly interpreted, comparing them with the results of the histopathological examination. To the best of our knowledge, the relationship between severity of PMI and apoptosis, and the cardiac markers assayed in this study has not been previously studied in CABG surgery with CPB. This study therefore had the following objectives: (1) to examine whether PMI, as occurs during CABG surgery, is associated with myocardial apoptosis and the release of cardiac markers, using biochemical and histopathological analysis; (2) to determine whether there is a direct relationship between the release of cardiac markers and the severity of myocardial injury and apoptosis, as graded histopathologically; and (3) to compare efficacies of cardiac markers to detect PMI rapidly and accurately.

Methods This prospective study was carried out in Dumlupinar University Evliya Celebi Research and Education Hospital, Turkey, between April and September 2014. The study was in accordance with the principles outlined in the Declaration of Helsinki. Ethical approval was received from the local Human Research Ethics Committee (no: 2013/14-122). Written informed consent was obtained from the all patients. The study population consisted of 37 patients (24 male, 13 female, mean age 63.4 ± 8.9 years) undergoing elective CABG who fulfilled the inclusion criteria. Inclusion criteria were age over 18 and less than 80 years, and need for elective myocardial revascularisation for angina pectoris. The exclusion criteria included ejection fraction < 30%; recent anterior myocardial infarction (< one month), the requirement of a concomitant cardiac operation, emergency surgery or re-operation. Demographic, pre-operative and intra-operative data of patients are shown in Table 1.

Anesthesia, CPB and surgical procedure The same surgical and anesthetic team managed all patients. Cardiopulmonary bypass and surgical techniques were

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standardised and did not change during the study period. Pre-medication, general anaesthesia with endotracheal intubation, and transfusions were the same in all cases. Induction of anaesthesia was performed using 5–10 mcg/kg fentanyl, 3–5 mcg/kg thiopental, 0.05 mg/kg midazolam and 0.1 mg/kg vecuronium. Anaesthesia was maintained using 2% sevoflurane and 1–3 mcg/kg/dk remifentanil. A median sternotomy was performed with a midsternal incision, followed by routine aortic and right atrial cannulation. After harvesting the bypass graft conduits (left internal mammary artery and saphenous vein) the patients were prepared for CPB. Anticoagulation was achieved with 400 U/kg heparin. CPB was carried out using membrane oxygenators and moderate systemic hypothermia. Myocardial protection was achieved with combined antegrade and retrograde continuous mild hypothermic (32°C) blood cardioplegia. The contents of the cardioplegia solution were as follows: 80 mEq potassium, 12 mEq magnesium and 44 mEq sodium bicarbonate in 0.9% saline, and this solution was diluted with blood in a ratio of 1:4. Aortic cross-clamping was performed and diastolic arrest was achieved by cardioplegia. After the distal anastomoses were completed, the aortic cross-clamp was removed and the proximal anastomoses were performed on the aorta during myocardial Table 1. Demographic, pre-operative and intra-operative data of the patients Parameters n = 37 Age (years) 63.4 ± 8.9 Male (n) 24 Female (n) 13 Weight (kg) 75.8 ± 13.7 Height (cm) 162.7 ± 8.6 BMI (kg/m2) 28.67 ± 4.8 NYHA classification (n) Class I 21 Class II 14 Class III 2 LVEF (%) Normal (> 50%) 23 Moderate (31–49%) 14 MI history (n) 15 Medication (n) 33 β-Blockers ACE inhibitors 8 Calcium antagonists 11 Statins 30 Acetylsalicylic acid 35 Other anticoagulants 4 ACC time (min) 56.7 ± 15.3 CPB time (min) 101.9 ± 23.4 Grafted vessels (n) 3.17 ± 0.62 Apoptotic index (TUNEL, %) 25.7 ± 8.4 Myocardial injury score 1.5 ± 0.5 BMI: body mass index; NYHA: New York Heart Association; LVEF: left ventricular ejection fraction; MI: myocardial infarction; ACE: angiotensin converting enzyme; ACC: aortic cross-clamping; CPB: cardiopulmonary bypass; TUNEL: terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end-labelling; SD: standard deviation. Data are presented as median ± SD or number.

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perfusion. Before releasing the aortic cross-clamp, warm reperfusion was given (37°C) until the patient’s body temperature was 35–37°C. Heparin was neutralised with protamine in a ratio of 1:1.5 within 10 minutes of the end of CPB.

Blood sample collection and measurement of cardiac markers Blood samples were drawn after atrial cannulation, just before aortic cross-clamping (pre-ischaemic sample), and within 15 minutes of aortic declamping (reperfusion sample). Blood samples were collected from the arterial line of the bypass circuit (arterial sample) and from the pressure-monitoring line of the coronary sinus perfusion catheter (coronary sinus sample). Blood samples were collected into an evacuated serumseparator clot-activator tube (Vacuette®, Greiner Bio-One, Kremsmunster, Austria) and a 2.0-ml dipotassium (K2) ethylene diamine tetra-acetic acid (EDTA) vacuum tube (BD Vacuteiner® BD Plymouth, UK) for creatine kinase-MB isoenzyme (CK-MB), high-sensitivity cardiac troponin T (hs-cTnT), creatine kinaseMB isoenzyme mass (CK-MB mass) and cardiac troponin I (cTnI) measurements. The tubes were centrifuged at 1 500 × g for 15 minutes within one hour to obtain serum samples for the measurement of CK-MB and hs-cTnT concentrations. Whole blood samples, which were collected in the K2 EDTA tubes, were not centrifuged and CK-MB mass and cTnI concentrations were measured in the whole blood samples on the same day as the surgery. Serum CK-MB activities were measured with the immunoinhibition method on a Roche Cobas c501 analyser (Roche Diagnostics GmbH, Mannheim, Germany). The reference range of CK-MB activity measured by this method was < 25 U/l. Serum hs-cTnT concentrations were measured by electrochemiluminescence immunoassays (ECLIA) on a Roche Cobas e 601 analyser (Roche Diagnostics GmbH, Mannheim, Germany). In healthy subjects, the upper reference limit for hs-cTnT concentrations was 14 ng/l (99th percentile) and the measurement range was 3–10 000 ng/l. CK-MB mass and cTnI were measured with the timeresolved fluorescence method on a radiometer AQT90 FLEX (Radiometer Medical ApS, Brønshøj, Denmark). In healthy subjects the upper reference limit for cTnI concentrations was 0.023 µg/l (99th percentile) and the measurement range was 0.010–25 µg/l. In healthy subjects the upper reference limit for CK-MB mass concentrations was < 7.2 µg/l (99th percentile) and the measurement range was 2–500 µg/l.

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The slides were graded histopathologically, according to the severity of myocardial injury, using a previously described scoring system.12 Histological changes (oedema, leukostasis, cell necrosis and focal bleeding) were scored from 0 to 3 as follows: 0 = no changes; 1 = slight changes: focal myocyte damage or small multifocal degeneration with slight degree of inflammation; 2 = moderate changes: extensive myofibrillar degeneration and/or diffuse inflammatory process; 3 = severe changess: necrosis with diffuse inflammatory process.

In situ detection of myocardial apoptosis We used an in situ TUNEL (terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end-labelling) assay to assess the degree of myocardial apoptosis. Formalinfixed sections were deparaffinised in xylene and rehydrated through graded concentrations of ethanol to water. DNA fragmentation during apoptosis was detected using a commercially available kit (ApopTag® peroxidase in situ apoptosis detection kit, Millipore, Billerica, MA, USA) according to the manufacturer’s instructions. Processed samples were examined under a light microscope (Olympus BX51, Tokyo, Japan). For quantitative analysis, TUNEL-positive cells were counted in six random fields per section (80–120 cells per field). The apoptotic index was calculated as the mean of apoptotic (positive-stained) cells.

Statistical analyses Statistical analyses were performed using GraphPad Prism version 6.05 (GraphPad Software, Inc, CA, USA). All data sets were tested for normality using the Shapiro–Wilk test. Data were presented as median and interquartile ranges (IQR) and non-parametric statistical tests were used, as the values were not normally distributed. The net release of cardiac markers was quantified as the arteriovenous difference (coronary sinus concentration minus arterial concentration). The comparison of cardiac marker values between pre-ACC (just before aortic cross-clamping) and post-ACC (within 15 minutes of aortic declamping) periods was analysed using the Mann–Whitney U-test. The correlation between apoptotic index (TUNEL), histopathological myocardial injury score, intra-operative data and cardiac marker values in the post-ACC period was analysed using Spearman’s correlation analysis. An r-value > 0.5 indicated a strong correlation, 0.35–0.5 a moderate correlation, and 0.2–0.34 a weak correlation.13 A p-value < 0.05 was considered statistically significant.

Atrial tissue sample collection and histopathological examinations

Results

Using a sharp scalpel, myocardial biopsy samples from the same site of the right atrial appendage were taken from each patient within 15 minutes of aortic declamping. The area of the biopsy sample in contact with the forceps was removed. Particular care was taken to avoid possible ischaemic areas caused by surgical manipulation. Tissue samples were fixed in 10% formalin, embedded in paraffin, sectioned (4 µm), placed on slides, stained with haematoxylin and eosin (H&E), and examined under a light microscope (Olympus BX51, Tokyo, Japan) by a pathologist who was blinded to the study design.

Demographic, pre-operative and intra-operative data of the patients are shown in Table 1. In the histopathological examinations, our results showed that CABG surgery with CPB and ACC caused slightto-moderate myocardial injury and moderate-to-severe apoptosis in all cases (Table 1). Acute ischaemic changes with interstitial oedema, myofibrillar thinning and wavy pattern consistent with reperfusion injury were observed in histopathological sections of atrial tissue. In addition, neutrophilic-to-mixed inflammatory cell infiltration and transmigration indicating reperfusion injury were observed (Figs 1–4).

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When the cardiac marker values in arterial and coronary sinus blood samples were compared, significant differences were found between the pre-ACC and post-ACC periods (Table 2). On the other hand, when net release of cardiac markers was compared, significant differences were found between the pre-ACC and post-ACC periods for CK-MB, CK-MB mass, cTnI and hs-cTnT (Table 3). A significant positive correlation was observed between apoptotic index and arterial blood CK-MB mass, cTnI and hs-cTnT values in the post-ACC period. A positive correlation was observed between the apoptotic index and coronary sinus blood CK-MB mass, cTnI and hs-cTnT values in the post-ACC period. No correlation was found between apoptotic index and arterial and coronary sinus blood CK-MB values (Table 4).

A significant positive correlation was observed between myocardial injury score and arterial blood cTnI and hs-cTnT values in the post-ACC period. In addition, a positive correlation was found between myocardial injury score and coronary sinus blood cTnI and hs-cTnT values in the post-ACC period. No correlation was observed between myocardial injury score and arterial and coronary sinus blood CK-MB and CK-MB mass values (Table 4). When the relationship between apoptotic index and net release of cardiac markers in the post-ACC period was analysed, a positive correlation was found between apoptotic index and net release of hs-cTnT. No correlation was found between apoptotic index and net release of CK-MB, CK-MB mass and and cTnI (Table 5). A positive correlation was found between myocardial

Fig. 1. H istopathological section of atrial tissue showing acute ischaemic changes with interstitial oedema (thin arrow). In addition, myofibrils show thinning and wavy patterns consistent with reperfusion injury (thick arrow) (Grade 1, H&E × 100).

Fig. 3. Histopathological section of atrial tissue showing neutrophilic-to-mixed inflammatory cell infiltration and transmigration (thin arrow) and necrotic myocytes (thick arrow). Neutrophil activation plays a prominent role in reperfusion injury (Grade 2, H&E × 200).

Fig. 2. H igh-power representation of the histopathological section of atrial tissue showing neutrophilic-tomixed inflammatory cell infiltration and transmigration (arrow). Neutrophil activation plays a prominent role in reperfusion injury (Grade 2, H&E × 200).

Fig. 4. TUNEL-positive cardiomyocytes in an atrial tissue sample obtained during reperfusion after aortic declamping. The positive TUNEL reaction is visible as dark staining in the nucleus (arrow) (TUNEL × 40).

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Table 2. Comparison of the cardiac marker values in arterial and coronary sinus blood samples between the pre-ACC and post-ACC period Cardiac markers CK-MB (U/l) CK-MB mass (µg/l) cTnI (µg/l) hs-cTnT (ng/l)

Pre-ACC arterial blood

Post-ACC arterial blood

16.4

42.0

(12.0–21.0)

(32.0–73.0)

6.5

21.0

(5.5–10.4)

(15.5–28.0)

0.12

0.25

(0.07–0.3)

(0.13–0.42)

125.0

193.0

(59.5–211.6) (91.0–309.0)

Pre-ACC coronary p-value sinus blood < 0.001 < 0.001 < 0.01 < 0.05

Post-ACC coronary sinus blood p-value

19.7

51.5

(14.8–24.0)

(35.7–85.5)

7.3

25.0

(6.0–12.5)

(17.5–34.0)

0.14

0.31

(0.08–0.29)

(0.17–0.49)

159.0

239.0

< 0.001 < 0.001 < 0.01 < 0.05

(66.0–230.7) (95.5–425.0)

ACC: aortic cross-clamping; CK-MB: creatine kinase isoenzyme MB; cTnI: cardiac troponin I; hs-cTnT: high-sensitivity cardiac troponin T. Data are presented as median and interquartile ranges for each group. Data were tested using the Mann– Whitney U-test. A p-value < 0.05 was considered statistically significant.

injury score and net release of cTnI and hs-cTnT in the post-ACC period. No correlation was found between myocardial injury score and net release of CK-MB and CK-MB mass (Table 5). We analysed the relationship between apoptotic index, myocardial injury score and intra-operative data. We found a significant positive correlation between apoptotic index and ACC time, CPB time and number of grafted vessels (Table 6). A significant positive correlation was found between myocardial injury score and ACC time, CPB time and number of grafted vessels (Table 6). Additionally, when the relationship between net release of cardiac markers in the post-ACC period and the intra-operative data were analysed, a positive correlation was observed between net release of hs-cTnT and ACC time. Furthermore, a positive correlation was found between net release of hs-cTnT and CPB time (Table 7). No correlation was found between peri-operative data and net release of other cardiac markers in the post-ACC period (Table 7).

Discussion CABG is a highly complex and risky surgical procedure, and despite well-established myocardial protective procedures, CABG surgery may still cause myocardial damage.14 The incidence of Table 3. Comparison of the net release of cardiac markers between the pre-ACC and post-ACC period Cardiac markers Pre-ACC net release Post-ACC net release p-value CK-MB (U/l) 3.0 7.0 < 0.001 (1.8–5.0) (2.9–15.0) CK-MB mass 1.0 2.0 < 0.001 (0.5–1.4) (2.0–4.0) (µg/l) 0.02 0.03 cTnI (µg/l) < 0.01 (0.01–0.04) (0.02–0.06) hs-cTnT (ng/l) 15.0 26.0 < 0.05 (5.4–42.0) (9.5–79.6) ACC: aortic cross-clamping; CK-MB: creatine kinase isoenzyme MB; cTnI: cardiac troponin I; hs-cTnT: high-sensitivity cardiac troponin T; pre-ACC: just before aortic cross-clamping; post-ACC: within 15 minutes of aortic declamping. The net releases of cardiac markers were quantified as the arteriovenous difference (coronary sinus concentration minus arterial concentration). Data are presented as median and interquartile ranges for each group. Data were tested using the Mann–Whitney U-test. A p-value < 0.05 was considered statistically significant.

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Table 4. The relationship between apoptotic index (TUNEL), histopathological myocardial injury score and cardiac marker values in arterial and coronary sinus blood samples in the post-ACC period CK-MB CK-MB cTnI hs-cTnT (U/l) mass (µg/l) (µg/l) (ng/l) Arterial blood samples Apoptotic index r = 0.019 r = 0.422 r = 0.611 r = 0.809 (TUNEL) p = 0.910 p = 0.009* p < 0.001* p < 0.001* Myocardial r = 0.021 r = 0.316 r = 0.544 r = 0.719 injury score p = 0.900 p = 0.057 p < 0.001* p < 0.001* Coronary sinus blood samples Apoptotic index r = 0.085 r = 0.358 r = 0.623 r = 0.790 (TUNEL) p = 0.616 p = 0.030* p < 0.001* p < 0.001* Myocardial r = 0.087 r = 0.223 r = 0.554 r = 0.695 injury score p = 0.606 p = 0.184 p < 0.001* p < 0.001* ACC: aortic cross-clamping; CK-MB: creatine kinase isoenzyme MB; cTnI: cardiac troponin I; hs-cTnT: high-sensitivity cardiac troponin T; TUNEL: terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end-labelling; post-ACC: within 15 minutes of aortic declamping. Relationships between data were tested using Spearman’s correlation analysis. *A p-value < 0.05 was considered statistically significant.

PMI varies considerably, from three to 30%, because of different diagnostic criteria and variable patient populations.15 Although changes in blood concentrations of cardiac markers, such as CK-MB, myoglobin (Mb) and cardiac troponins are used in the diagnosis of PMI, there are no widely accepted standardised diagnostic criteria.16 Myocardial damage causes disruption of the normal cardiac myocyte membrane integrity and loss of intracellular content into the extracellular space. Therefore, elevated levels of cytosolic and structural proteins, such as CK-MB and cardiac troponins, can be detected in the blood.17 Interpretation of cardiac biomarkers is difficult after CABG surgery because the specificity of some cardiac markers during CABG surgery is limited, depending on skeletal muscle injury occurring in the surgical procedure. Skeletal muscle injury may increase intra-operative concentrations or activities of some cardiac markers, such as CK-MB and CK-MB mass. As a result, increases in these markers due to skeletal muscle damage may confound the diagnosis of PMI. Consequently, it is important to detect PMI using a highly specific marker.18 In this study, the relationship between myocardial apoptosis and injury and the Table 5. The relationship between apoptotic index (TUNEL), histopathological myocardial injury score and net release of cardiac marker values in the post-ACC period CK-MB CK-MB cTnI hs-cTnT (U/l) mass (µg/l) (µg/l) (ng/l) Apoptotic index r = 0.222 r = 0.013 r = 0.283 r = 0.507 (TUNEL) p = 0.185 p = 0.937 p = 0.090 p = 0.001* Myocardial r = 0.260 r = –0.107 r = 0.333 r = 0.416 injury score p = 0.120 p = 0.530 p = 0.044* p = 0.010* ACC: aortic cross-clamping; CK-MB: creatine kinase isoenzyme MB; cTnI: cardiac troponin I; hs-cTnT: high-sensitivity cardiac troponin T; TUNEL: terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end-labelling; post-ACC: within 15 minutes of aortic declamping. Net release of cardiac markers was quantified as arteriovenous difference (coronary sinus concentration minus arterial concentration). Relationships between data were tested using Spearman’s correlation analysis. *A p-value < 0.05 was considered statistically significant.

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Table 6. The relationship between apoptotic index (TUNEL), histopathological myocardial injury score and ACC time, CPB time and graft number ACC time CBP time Number of (min) (min) grafted vessel Apoptotic index r = 0.876 r = 0.694 r = 0.445 (TUNEL) p < 0.001* p < 0.001* p = 0.007* Myocardial r = 0.867 r = 0.725 r = 0.555 injury score p < 0.001* p < 0.001* p < 0.001* ACC: aortic cross-clamping; CPB: cardiopulmonary bypass; TUNEL: terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end-labelling. Relationships between data were tested using Spearman’s correlation analysis. *A p-value < 0.05 was considered statistically significant.

release of biochemical cardiac markers during CABG surgery accompanied by CPB were investigated using histopathological examinations and biochemical measurements. Although optimal current myocardial protective techniques were applied during surgery in all study populations, our histopathological results revealed that CABG surgery accompanied by CPB and ACC led to slight-to-moderate PMI as well as moderate-to-severe myocardial apoptosis in all cases. Apoptosis is considered one of the mechanisms of cardiomyocyte loss during CPB and cardioplegic arrest during CABG surgery.19,20 Myocardial apoptosis is induced immediately after cardioplegic arrest and CPB.21 Apoptosis during CPB and cardioplegic arrest can be induced by several mechanisms, including I/R injury and the release of cytokines and inflammatory factors.20,22 Previous studies have reported that cardioplegic cardiac arrest could stimulate pro-inflammatory cytokines, induce cardiomyocytic apoptosis, and impair postoperative cardiac performance.23,24 In our study, a positive correlation was found between myocardial apoptotic index and ACC or CPB time. Schmitt et al. and Ruifrok et al. found a positive correlation between the apoptotic index and duration of ACC and CPB, which was consistent with our study.25,26 Conversely, Wu et al. reported that myocardial apoptosis showed no correlation with ACC and CPB time.27 We observed significant increases in CK-MB, CK-MB mass, cTnI and hscTnT levels within 15 minutes of the reperfusion period. The concentrations of coronary sinus CK-MB, CK-MB mass, cTnI and hscTnT were higher than the corresponding arterial concentrations after aortic declamping, indicating considerable myocardial release of these markers after reperfusion. In addition, we found significant myocardial net release of CK-MB, CK-MB mass, cTnI and hs-cTnT into the coronary circulation after aortic declamping within 15 minutes of reperfusion. Myocardial net release of CK-MB, CK-MB mass, cTnI and hscTnT indicated that myocardial damage had occurred during ACC and cardioplegic cardiac arrest, as well as a very rapid release from the myocardium with the onset of reperfusion. Although cardiac troponins are structurally bound proteins of striated muscles, the cytosolic pool of cTnT and cTnI may account for the rapid early myocardial release in parallel with CK-MB and CK-MB mass.28 Coronary sinus sampling allows direct sampling of the blood draining the heart, therefore the arteriovenous difference provides the closest correlation between cardiac marker release and ischaemic time. These data obtained during CABG are in accordance with the results of previous studies.29,30 Bleier et al. found significant

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Table 7. The relationship between net release of cardiac marker values and intra-operative data in the post-ACC period Net release of cardiac markers CK-MB (U/l)

ACC time CPB time Number of (min) (min) grafted vessel r = 0.110 r = 0.187 r = 0.128 p = 0.529 p = 0.280 p = 0.448 CK-MB mass r = 0.110 r = 0.155 r = –0.015 (µg/l) p = 0.526 p = 0.374 p = 0.931 cTnI (µg/l) r = 0.157 r = 0.121 r = 0.052 p = 0.366 p = 0.489 p = 0.759 hs-cTnT (ng/l) r = 0.448 r = 0.342 r = 0.200 p = 0.007* p = 0.047* p = 0.249 ACC: aortic cross-clamping; CPB: cardiopulmonary bypass; CK-MB: creatine kinase isoenzyme MB; cTnI: cardiac troponin I; hs-cTnT: high-sensitivity cardiac troponin T; post-ACC: within 15 minutes of aortic declamping. Net release of cardiac markers was quantified as arteriovenous difference (coronary sinus concentration minus arterial concentration). Relationships between data were tested using Spearman’s correlation analysis. *A p-value < 0.05 was considered statistically significant.

myocardial net release of CK-MB mass, cTnT and cTnI into the coronary circulation after aortic declamping within 20 minutes of reperfusion.29 Koh et al. reported that cTnT concentrations increased in every patient after aortic declamping, and were higher in coronary sinus blood than in arterial blood, indicating net myocardial release of troponin T during the period of reperfusion.30 Despite the fact that a positive correlation was found between myocardial apoptotic index and CK-MB mass, cTnI and hs-cTnT concentrations, as well as a positive correlation between degree of myocardial injury and cTnI and hs-cTnT concentrations after aortic declamping, the strongest correlation was observed between hs-cTnT and myocardial apoptosis and injury. In a previous study, myocardial biochemical markers demonstrated no correlation with myocardial apoptosis, unlike in our study.27 When we examined the relationship between myocardial apoptosis and net release of cardiac markers, a significant positive correlation was found between apoptosis and net release of hs-cTnT after reperfusion. A positive correlation was observed between degree of myocardial injury and net release of cTnI and hs-cTnT, but hs-cTnT demonstrated a stronger correlation with myocardial injury than cTnI after aortic declamping. We also examined the relationship between ACC and CPB time and net release of cardiac markers. Duration of ischaemic time, which is expected to be a strong predictor of release of biochemical markers, correlated with net release of hs-cTnT but not with that of CK-MB, CK-MB mass and cTnI. In several previous studies, net cTnT release and peak cTnT concentrations were correlated with ACC time, which was consistent with our study.30,31 Cardiac troponins are highly specific markers of cardiac myocyte damage and seem to better indicate myocardial injury occurring during CABG surgery.32-34 High-sensitivity troponin assays have recently been developed, being more sensitive than contemporary assays at detecting lower troponin levels. The high-sensitivity troponin T assay has lowered the detection threshold for myocardial necrosis and therefore permits more rapid diagnosis of MI.35,36 Recently, several studies have been reported related to the ability of hs-cTnT to diagnose PMI after CABG or non-cardiac surgery.37,38 Wang et al. reported that post-operative hs-cTnT

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as a parameter alone, independently predicted medium-term mortality and morbidity.37 Nagele et al. reported that pre-operative hs-cTnT concentrations were significantly associated with postoperative myocardial infarction and long-term mortality in high-risk patients undergoing major non-cardiac surgery, and they suggested that pre-operatively measured hs-cTnT concentrations may be useful to identify patients at high risk for peri-operative acute MI and increased long-term mortality after non-cardiac surgery.38 In our study, although cTnI and hs-cTnT concentrations were well correlated with severity of myocardial injury and apoptosis, hs-cTnT showed a better correlation than cTnI. In addition, hs-cTnT concentrations were correlated with ACC and CPB time. The results of this study indicate that increases in hs-cTnT concentration seem to best reflect myocardial cell damage and apoptosis. Although both cTnI and hs-cTnT may be specific and efficacious markers of myocardial apoptosis and injury occurring during CABG with CPB, hs-cTnT may be a more useful marker than cTnI to detect peri-operative myocardial apoptosis and injury.

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Analg 2012; 18: 70–74. 9.

Fischer UM, Tossios P, Huebner A, Geissler HJ, Bloch W, Mehlhorn U. Myocardial apoptosis prevention by radical scavenging in patients undergoing cardiac surgery. J Thorac Cardiovasc Surg 2004; 128: 103–108.

10. Ramlawi B, Feng J, Mieno S, Szabo C, Zsengeller Z, Clements R, et al. Indices of apoptosis activation after blood cardioplegia and cardiopulmonary bypass.Circulation 2006; 114 (1 Suppl): I257–1263. 11. Di Stefano S, Casquero E, Bustamante R, Gualis J, Carrascal Y, Bustamante J, et al. Plasma troponins as markers of myocardial damage during cardiac surgery with extracorporeal circulation. Tohoku J Exp Med 2007; 213: 63–69. 12. Qing M, Vazquez-Jimenez JF, Klosterhalfen B, Sigler M, Schumacher K, Duchateau J, et al. Influence of temperature during cardiopulmonary bypass on leukocyte activation, cytokine balance, and post-operative organ damage. Shock 2001; 15: 372–377. 13. Ong SC, Lim SG, Li SC. Reliability and validity of a Chinese version’s health-related quality of life questionnaire for hepatitis B patients. Value Health 2010; 13: 324–327. 14. Preeshagul I, Gharbaran R, Jeong KH, Abdel-Razek A, Lee LY, Elman E, et al. Potential biomarkers for predicting outcomes in CABG cardio-

Conclusion Despite optimal current myocardial protection techniques, PMI may occur during CABG surgery with CPB. Moreover, CABG surgery may cause myocardial apoptosis. Highsensitivity troponin T assay has lowered the detection threshold for myocardial damage and therefore it may provide rapid and specific detection of myocardial injury during CABG surgery with CPB. Measurement of the change in hs-cTnT concentrations may be useful to quantify the severity of perioperative myocardial injury.

thoracic surgeries. J Cardiothorac Surg 2013; 18: 176. 15. Koniari I, Koletti B, Apostolakis E. Perioperative myocardial infarction following coronary artery bypass grafting. Interact Cardiovasc Thorac Surg 2011; 12: 599. 16. Berkan O, Sagban M. Sialic acid or troponin T to detect perioperative myocardial damage in patients undergoing elective coronary artery bypass grafting. Circ J 2002; 66: 1019–1023. 17. Jaffe AS, Babuin L, Apple FS. Biomarkers in acute cardiac disease: the present and the future. J Am Coll Cardiol 2006; 48: 1–11. 18. Peivandi AA, Dahm M, Opfermann UT, Peetz D, Doerr F, Loos A, et al. Comparison of cardiac troponin I versus T and creatine kinase MB after coronary artery bypass grafting in patients with and without peri-

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Andries Brink-Kaye award The winners of the Andries Brink-Kaye award for a most outstanding scientific publication in the Cardiovascular Journal of Africa for 2014 were Dr Roisin Finola Kelly-Laubscher and her team from the Hatter Institute for Cardiovascular Research in Africa, University of Cape Town. The article, titled Cardiac preconditioning with sphingosine-1-phosphate requires activation of signal transducer and activator of transcription-3, was published in Cardiovasc J Afr 2014; 25(3): 118–123. Dr Kelly-Laubscher is seen here receiving the award on behalf of the team.

Award recipient, Roisin F Kelly-Laubscher.

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Lack of cardioprotection by single-dose magnesium prophylaxis on isoprenaline-induced myocardial infarction in adult Wistar rats Christie Garson, Roisin Kelly-Laubscher, Dee Blackhurst, Asfree Gwanyanya

Abstract Aim: Magnesium (Mg2+) is effective in treating cardiovascular disorders such as arrhythmias and pre-eclampsia, but its role during myocardial infarction (MI) remains uncertain. In this study, we investigated the effects of Mg2+ pre-treatment on isoprenaline (ISO)-induced MI in vivo. Methods: Rats divided into four groups were each pre-treated with either MgSO4 (270 mg/kg intraperitoneally) or an equivalent volume of physiological saline, prior to the ISO (67 mg/kg subcutaneously) or saline treatments. One day post-treatment, the electrocardiogram and left ventricular blood pressures were recorded. Infarcts were determined using 2,3,5-triphenyltetrazolium chloride staining, and serum markers of lipid peroxidation were measured with spectrophotometric assays. Results: Mg2+ pre-treatment neither altered the ISO-induced infarct size compared with ISO treatment alone (p > 0.05), nor reversed the low-voltage electrocardiogram or the prominent Q waves induced by ISO, despite a trend to decreased Q waves. Similarly, Mg2+ did not prevent the ISO-induced decrease in peak left ventricular blood pressure or the decrease in minimal rate of pressure change. Mg2+ did not reverse the ISO-induced gain in heart weight or loss of body weight. Neither ISO nor Mg2+ altered the concentrations of lipid peroxidation markers 24 hours post MI induction. Conclusion: Although Mg2+ had no detrimental effects on electrical or haemodynamic activity in ISO-induced MI, the lack of infarct prevention may detract from its utility in MI therapy. Keywords: cardiac, isoprenaline, magnesium, myocardial infarction Submitted 21/10/13, accepted 2/7/15 Published online 15/7/15 Cardiovasc J Afr 2015; 26: 242–249

www.cvja.co.za

DOI: 10.5830/CVJA-2015-055

Department of Human Biology, Faculty of Health Sciences, University of Cape Town, Cape Town, South Africa Christie Garson, MSc Roisin Kelly-Laubscher, PhD Asfree Gwanyanya, MB ChB, PhD, asfree.gwanyanya@uct.ac.za

Department of Clinical Laboratory Sciences, Faculty of Health Sciences, University of Cape Town, Cape Town, South Africa Dee Blackhurst, PhD

Magnesium (Mg2+) is used in the treatment of life-threatening cardiovascular disorders such as arrhythmias and pregnancyinduced hypertension.1 However, there is uncertainty regarding its role in myocardial infarction (MI), a common and lethal complication of many cardiovascular disorders. A meta-analysis of early clinical trials showed that intravenous Mg2+ reduced mortality and arrhythmias in acute MI.2 In large trials, beneficial effects of Mg2+ were also found in the second Leicester Intravenous Magnesium Intervention trial (LIMIT2), in which Mg2+ infusion preceded thrombolytic therapy,3 as well as in studies involving high-risk patients unfit for thrombolysis.4 By contrast, Mg2+ did not improve survival in the fourth International Study of Infarct Survival (ISIS-4) trial, in which Mg2+ was given after thrombolytic therapy,5 and in the more recent Magnesium in Coronaries (MAGIC) trial,6 which included high-risk patients not eligible for reperfusion therapy. In animal studies, Mg2+ reduced infarct size7-11 and inhibited myocardial apoptosis12 under certain conditions, but not others.13,14 There is therefore a need for further experimental and clinical studies on Mg2+ therapy. Mg2+ is proposed to modulate MI through its antithrombotic,15 antioxidant16 and anti-arrhythmic effects.17 Through its ability to block Ca2+ channels,18 Mg2+ prevents cytosolic Ca2+ overload,19 and decreases both systemic and coronary vascular tone.20 At a cellular level, Mg2+ is an essential co-factor for several enzymes, including those involved in ATP synthesis and utilisation. Furthermore, it is a co-factor for ATP activity in the form of MgATP.21 Mg2+ preconditions the myocardium through the activation of ATP-dependent K+ channels22 and also confers resistance to mitochondrial membrane depolarisation,23 thereby minimising mitochondrial Ca2+ overload. Mitochondrial Ca2+ overload attenuates ATP synthesis and augments ATP hydrolysis, particularly that of MgATP.24 In the form of an orotate salt, Mg2+ prevents the opening of the mitochondrial permeability transition pore, which is lethal to cells.25 However, while acute MI is associated with decreased serum Mg2+ levels,26 the conditions under which Mg2+ is cardioprotective remain uncertain. The synthetic catecholamine, isoprenaline (ISO), has been widely used to induce infarcts mimicking human global MI. Overstimulation of β-adrenergic receptors by ISO induces MI through the generation of free radicals,27,28 intracellular Ca2+ overload,29 and apoptosis.30 In addition, the stress due to the MI itself causes further release of catecholamines and worsens the MI. Catecholamine-mediated β-adrenergic receptor stimulation also induces Mg2+ efflux,31,32 thereby potentially depleting intracellular Mg2+. Mg2+ is also known to inhibit catecholamine release.33 The framework of the current study was therefore to investigate the role of Mg2+ prophylaxis in cardiac stress conditions, in which extracellular and intracellular Mg2+ homeostasis may be altered.

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We investigated the effects of Mg2+ pre-treatment on cardiac morphological, electrical and haemodynamic changes, and on the lipid peroxidation profile in a rat model of acute MI induced by ISO.

Methods Adult male Wistar rats, weighing 250–300 g, were obtained from the University of Cape Town animal unit and housed in an air-conditioned animal facility under standard laboratory conditions (12-hour light/dark cycle, illumination of 323 lux and temperature of ~22°C). The rats were fed standard rat chow (Afresh Vention 1, Cape Town, South Africa) and had free access to food and water. Experimental procedures were approved by the animal ethics committee of the Faculty of Health Sciences, University of Cape Town. All protocols were carried out in compliance with the Guide for the Care and Use of Laboratory Animals [NIH Publication No. 85 (23), revised 1996].

Animal procedures and experimental protocol Thirty-five rats were divided into four groups and treated according to the experimental protocol described below, for which the timeline is shown in Fig. 1A. Subcutaneous (sc) injection of ISO at 67 mg/kg in rats is known to produce histologically detectable MI within 24 hours.34 In preliminary tests, we observed that using higher doses of ISO, such as 85 mg/ kg and above, resulted in high mortality rates in our rats. The ISO-induced MI group (n = 9) was pre-treated with intraperitoneal (ip) injection of physiological saline (2.7 ml/kg) two hours prior to injection with ISO (67 mg/kg sc), and the ISO + Mg2+ group (n = 10) was pre-treated with MgSO4 (270 mg/kg ip), which is effective in neuroprotection,35 two hours prior to injection with ISO (67 mg/kg sc). The two-hour wait was meant to avoid possible direct interactions between Mg2+ and ISO when co-administered at Mg2+ peak levels. It was also to allow adequate time for the onset of any downstream cellular effects of Mg2+ treatments that may have occurred prior to the induction of MI, but before the return of serum Mg2+ to the baseline levels expected after 3.5 hours.35 The Mg2+ group (n = 8) was pre-treated with MgSO4 (270 mg/kg ip) two hours prior to saline injection (3.3 ml/kg sc), and the control group (n = 8) was injected with two drug-equivalent volumes of saline (ip and sc) two hours apart. Haemodynamic and other in vivo measurements were performed under anaesthesia 24 hours after the treatments. Rats were anaesthetised with sodium pentobarbitone (60 mg/kg ip), intubated and mechanically ventilated with room air at 70 strokes/min and 2.5 ml/stroke using a rodent ventilator (Model 681, Harvard Apparatus, Holliston, Massachusetts, USA). The depth of anaesthesia was adjusted to achieve loss of pedal withdrawal reflexes, and top-up doses of sodium pentobarbitone (12 mg/kg ip) were administered where necessary. Rats were placed on a heating pad (37°C) and the body temperature was monitored using a rectal probe connected to a T-type pod transducer (ML312, ADInstruments, Bella Vista, Australia).

Electrocardiogram and haemodynamic recordings Lead II of a three-lead surface electrocardiogram (ECG) was used to monitor cardiac electrical changes and compute heart

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rate, and was recorded via an animal bio-amplifier (ML136, ADInstruments, Bella Vista, Australia). Left ventricular blood pressure was measured with a Millar Mikrotip manometer (SPC320, Millar, Houston, Texas, USA) inserted through the right carotid artery in the neck and connected to a bridge amplifier (ML221, ADInstruments, Bella Vista, Australia). To prevent drift of pressure from the baseline during recording, the manometer was cleaned with a physiological detergent (TergA-Zyme, Alconox, New York, USA) and zeroed in water at 37°C. Clot formation around the manometer was prevented by injecting the rats with heparin (100 IU) intravenously. After 20 minutes of recordings, the heart was rapidly excised and retrogradely flushed with cold (4°C) saline through a cannula inserted into the aorta. The heart was then blotted, weighed and stored at –20°C for histochemical staining. To prevent damage of the epicardium due to freeze-drying, the hearts were wrapped in cling film before being frozen. Pulmonary trunk blood was collected during heart excision and centrifuged to obtain plasma, which was snap frozen in liquid nitrogen and stored at –80°C for lipid peroxidation studies. The other organs such as the liver, A Time

24h

Mg or Saline ISO or Saline

Measurements

Control

ISO

ISO + Mg

25 Infarct size (%)

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15 10 5 0 Control

ISO ISO + Mg Treatment

Fig. 1. ISO-induced MI and the effects of Mg2+ pre-treatment. A: Timeline of experimental protocol. The horizontal bar represents a 24-hour timeline with a break in the scale. The times at which rats were treated and at which in vivo and tissue measurements were done are indicated by arrows. B: Pictures of TTC-stained ventricular slices cut from four different hearts of rats treated with saline only (control), ISO and saline (ISO), ISO and Mg2+ (ISO + Mg), or Mg2+ and saline (Mg). Viable myocardium stained red (TTC positive), whereas areas of irreversible infarcts appeared white (TTC negative). C: Summary data of infarct size in whole ventricles. The infarct size is expressed as a percentage of the TTC‑negative area to the total ventricular area. Data are presented as mean ± SEM (n = 8–10 rats per group); *p < 0.05 (treatment vs control).

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lungs, kidneys and adrenal glands were also excised and weighed. Haemodynamic parameters, ECG and temperature measurements were recorded onto the computer using the PowerLab 4/30 data-acquisition system and the LabChart 7.3.5 software (ADInstruments, Bella Vista, Australia). Haemodynamic and ECG data were analysed using LabChart 7 Pro BP and ECG analysis modules (ADInstruments, Bella Vista, Australia). The ECG analysis module was preset to the rat waveform and Bazett’s formula (QTc = QT/√RR) was used to calculate the QT interval, corrected for heart rate (QTc).

Infarct size quantification A series of 2-mm-thick ventricular transverse slices of the frozen heart were cut from apex to base and thawed for 2,3,5-triphenyltetrazolium chloride (TTC) staining. The slices were incubated in a solution of 1% TTC in phosphate buffer (pH 7.4) at 37°C for 20 minutes and agitated periodically while protected from light. The slices were then washed with the buffer and fixed with 10% formalin to enhance contrast and stored in the dark at room temperature for 24 hours. The slices were placed between two glass slides and scanned on both sides using a flatbed scanner. The ventricular infarct size was measured as an average of the TTC-negative areas on the slices from each heart using ImageJ software (Version 1.44p, NIH, USA) and was expressed relative to the total ventricular area.

Lipid peroxidation assays Markers of oxidative stress, measured as by-products of lipid peroxidation, namely conjugated dienes (CD) and thiobarbituric acid-reactive substances (TBARS), were quantified in the plasma using spectrophotometric assays. CD assays were carried out using the methods described by Esterbauer.36 Briefly, 100 µl of plasma was added to 405 µl chloroform: methanol (2:1). After centrifugation at 6 000 g for 15 minutes, the top aqueous layer was removed and the organic layer was isolated and dried under nitrogen. Cyclohexane (250 µl) was added to solubise the dry organic residue and the absorbance was read at 234 nm on a spectrophotometer (Spectramax Plus 384, Molecular Devices and Labotec, Johannesburg, South Africa) using Softmax Pro (Version 4.4) software. A molar extinction coefficient of 2.95 × 104 /M/cm was used. TBARS were measured using the method described by Jentzsch et al.37 Briefly, 6.25 µl of 4 mM butylated hydroxytoluene/ ethanol and 50 µl of 0.2 M ortho-phosphoric acid were added to 50 µl of plasma samples and vortexed. TBA reagent (6.25 µl), dissolved in 0.1 M NaOH, was added and the mixture was centrifuged at 3 000 g for two minutes to collect small volumes at the bottom of the Eppendorf tube. The volumes were heated at 90°C for 45 minutes, placed on ice for two minutes and then left at room temperature for five minutes before n-butanol (500 µl) was added. Phase separation was enhanced by the addition of 50 µl of saturated NaCl. The samples were vortexed and centrifuged at 12 000 g for two minutes and 300 µl of the top butanol phase was transferred into wells and read at 532 nm on the spectrophotometer. A molar extinction coefficient of 1.54 × 105 /M/cm was used. The measurements of CD and TBARS were performed in triplicate and the mean value was taken as the final result.

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Chemicals and reagents ISO and MgSO4 were each dissolved in physiological saline. The TTC buffer was made up of one part 0.1 M monosodium phosphate (NaH2PO4) and four parts 0.1 M disodium phosphate (Na2HPO2). Sodium pentobarbitone was purchased from Kyron Laboratories, Johannesburg, South Africa. All other drugs and chemicals were obtained from Sigma, Johannesburg, South Africa.

Statistical analysis Data are expressed as mean ± standard error of the mean (SEM), with n indicating the number of rats studied under each condition. Statistical analysis was conducted using Prism 5 (GraphPad, USA). A box-plot analysis was conducted to exclude outliers. The distribution of data was checked using the Kolmogorov–Smirnov, D’Agostino and Pearson, and the Shapiro–Wilk normality tests. Differences among multiple groups were evaluated using analysis of variance (ANOVA), followed by a Tukey post hoc test. For data not normally distributed and for normally distributed data that failed the Bartlett’s test, a Kruskal–Wallis test was conducted followed by a Dunns post hoc test; p ≤ 0.05 was taken as the threshold for statistical significance.

Results Effects of Mg2+ on ISO-induced infarct size Fig. 1B shows typical pictures of TTC-stained ventricular slices cut from four different hearts. Whitish-looking, TTC-negative areas were more prominent in the ISO-treated hearts, indicating the presence of irreversible infarction. The infarcted areas were patchy and more diffusely located on the myocardium, consistent with a global type of infarction compared to well-demarcated infarcts due to coronary artery ligation. In contrast to the effects of ISO, the control and Mg2+-only treated hearts appeared mostly red (TTC positive), suggesting tissue viability. The quantification of infarct size in whole ventricles is summarised in Fig. 1C, which confirms that ISO induced significant increases in infarct size (12.79 ± 5.97 vs 6.84 ± 1.54% in the controls; p < 0.05). Pre-treatment with Mg2+ did not prevent or enhance the ISO-induced infarction compared with ISO-treated rats (infarct size: 11.67 ± 6.63%; p > 0.05). Treatment with Mg2+ alone did not cause injury to the myocardium compared with the controls (infarct size: 6.94 ± 2.1%; p > 0.05).

Effects of ISO and Mg2+ on body and organ weights To examine the systemic and organ-specific effects of the various treatments, the body weight, heart weight and the weights of the other organs were quantified (Table 1). ISO caused a significant increase in the heart weight:body weight ratio compared with the controls (p < 0.001). Pre-treatment with Mg2+ did not prevent the ISO-induced increase in heart weight:body weight ratio. Compared with the control, ISO also caused a loss in body weight (p < 0.05), and pre-treatment with Mg2+ did not rectify this weight loss. Compared with ISO‑treated rats, Mg2+ co-treatment also did not affect the weights or the gross appearances of the liver,

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Table 1. Effects of chemical treatments on body weight (BW) and on the weights of various organs Treatment groups Weight parameter

Control (n = 8)

ISO (n = 9)

% BW lost

0.54 ± 0.16 –3.65 ± 0.61*

Heart:BW (mg/g)

3.33 ± 0.06

Liver:BW (mg/g)

ISO + Mg2+ (n = 10) –3.77 ± 1.31*

4.82 ± 0.06***

50.68 ± 1.11 47.34 ± 2.34

4.74 ± 0.13***

Mg2+ (n = 8) –0.74 ± 0.79 3.34 ± 0.05

44.08 ± 1.32

50.54 ± 1.87

Lung:BW (mg/g)

3.62 ± 0.20

3.21 ± 0.10

3.35 ± 0.10

3.46 ± 0.20

Kidney:BW (mg/g)

6.86 ± 0.10

6.58 ± 0.10

6.38 ± 0.10**

6.57 ± 0.10

Adrenal:BW (mg/g)

0.20 ± 0.01

0.24 ± 0.01

0.23 ± 0.01

0.22 ± 0.01

Body weight: BW. The percentage (%) BW lost was calculated from the differences between the BW measured on the day of treatment and 24 hours later. The organ:BW ratios (mg/g) were based on the BW measured 24 hours post-treatment at organ extraction. Values are mean ± SEM; *p < 0.05; **p < 0.01 and ***p < 0.001 (treatment vs control).

lungs, kidneys or adrenal glands. However, co-treatment with ISO and Mg2+ significantly decreased the kidney weight:body weight ratio compared to the controls (p < 0.01). When Mg2+ was administered alone, it did not significantly affect the body weight or the weights of any of the organs compared with the control rats.

Effects of Mg2+ on ISO-induced ECG changes Representative traces of lead II ECG waveforms recorded from individual rats are shown in Fig. 2. Qualitatively, ISO produced a low-voltage ECG recording, with qualitatively large Q waves compared with the controls (Fig. 2A, B). The changes in ECG A

Effects on haemodynamic parameters The effects of chemical treatments on haemodynamic parameters are shown in Fig. 3. ISO significantly decreased

–0.5 –1.0 –1.5

0.0

0.1

0.2 Time (s)

0.3

0.4

0.0

ISO + Mg

–0.5 –1.0 –1.5

0.1

0.2 Time (s)

0.3

0.4

0.1

0.2 Time (s)

0.3

0.4

0.0 Voltage (mV)

ISO

0.0 Voltage (mV)

Voltage (mV)

0.0

characteristics produced by various treatments are summarised in Table 2. ISO did not significantly alter the heart rate, the P-wave amplitude and duration, the S-wave amplitude, or the ST-segment height. The drug also had no effects on the PR interval, QRS duration, QT interval or QTc interval. ISO, however, decreased the R-wave amplitude compared with the controls (p < 0.001), an effect consistent with the low-voltage ECG waveform illustrated in Fig. 2B. ISO also produced prominent Q waves compared with the controls (p < 0.01), suggesting the presence of an evolving infarct.38 The drug also altered ventricular repolarisation by significantly decreasing both the T-wave amplitude and the Tpeak–Tend interval compared with the controls (p < 0.05 in each case). Pre-treatment with Mg2+ did not affect the ISO-induced changes in ECG voltage or the heart rate, P-wave amplitude and duration, PR interval, QRS duration, QT interval, QTc interval, or the ST-segment height. However, Mg2+ decreased the S-wave amplitude (p < 0.05), and further decreased the T-wave amplitude (p < 0.001) in ISO‑treated hearts compared with the controls, but without causing further changes to the Tpeak–Tend interval. Although not statistically significant, as illustrated in Fig. 2C, Mg2+ also tended to reduce the size of the Q waves induced by ISO. Mg2+ alone did not affect the ECG characteristics compared with those in control rats.

Control

245

–0.5 –1.0 –1.5

0.0

0.1

0.2 Time (s)

0.3

0.4

0.0

Fig. 2. E ffects of various treatments on the ECG waveforms. Segments of original lead II ECG traces recorded from individual rats under various treatments, A: saline (control), B: ISO, C: ISO + Mg2+, and D: Mg2+.

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the left ventricular (LV) maximum blood pressure (101.7 ± 2.2 vs123.4 ± 4.5 mmHg in the controls; p < 0.01), but not the LV end-diastolic blood pressure. Mg2+ pre-treatment did not reverse or worsen the ISO-induced decrease in the LV maximum blood pressure (107.4 ± 6.4 mmHg; compared with the controls, p = 0.34; compared with ISO-treated, p = 0.98), or affect the LV end-diastolic blood pressure. ISO significantly decreased the minimal rate of LV pressure change (dP/dt min; –5479 ± 203 vs –7921 ± 435 mmHg/s in controls; p < 0.001), but not the maximal rate of LV pressure change (dP/dt max). Mg2+ pre-treatment did not reverse the ISO effects on dP/dt min or change the dP/dt max. Mg2+ pre-treatment did not affect diastolic duration, but decreased the systolic duration in ISO-treated rats. Mg2+ alone did not alter LV blood pressures, LV maximal/minimal dP/dt, or systolic/diastolic duration.

Effects of ISO and Mg2+ on markers of lipid peroxidation Plasma CD and TBARS were measured 24 hours post-treatment to evaluate the effects of ISO and Mg2+ on oxidative stress. Fig. 4 shows that ISO did not alter CD and TBARS plasma concentrations significantly, suggesting that infarction occurred early, in which case the measured concentrations of CD and TBARS may not have reflected the concentrations of these markers at the time of infarction. In addition, Mg2+ pre-treatment prior to ISO or treatment with Mg2+ alone did not alter the concentrations of these markers.

Discussion Despite the advances in modern medical therapy, the mortality rate due to MI remains high. In this study, we used a catecholamineinduced MI model and found that Mg2+ prophylaxis did not alter the infarct size, as quantified by TTC staining. Mg2+ also had no effect on the ISO-induced ventricular hypotension or disruption of electrophysiological signals. Therefore, while Mg2+ did not worsen MI, the preconditions for its therapeutic indications remain unclear.

Table 2. Summary data on the effects of chemical treatments on ECG parameters Treatment groups ECG parameter Heart rate (bpm)

Control (n = 8)

ISO (n = 9)

406.9 ± 9.5

416.6 ± 14.2

418.4 ± 7.2

0.165 ± 0.009

0.167 ± 0.014

0.162 ± 0.013

–0.107 ± 0.021*

–0.025 ± 0.008

P amplitude (mV) 0.184 ± 0.010

Q amplitude (mV) –0.024 ± 0.008 –0.111 ± 0.020** R amplitude (mV) 0.590 ± 0.056

0.193 ± 0.030***

S amplitude (mV) –0.300 ± 0.073 –0.129 ± 0.060 T amplitude (mV) 0.123 ± 0.010

ISO + Mg2+ (n = 10)

0.216 ± 0.031*** –0.050 ± 0.022*

Mg2+ (n = 8) 405.8 ± 15.4

0.619 ± 0.044 –0.248 ± 0.054

0.087 ± 0.009*

0.023 ± 0.018***

0.134 ± 0.008

ST height (mV)

0.054 ± 0.032

0.081 ± 0.008

0.061 ± 0.007

0.109 ± 0.014

P duration (s)

0.184 ± 0.010

0.164 ± 0.009

0.166 ± 0.015

0.161 ± 0.013

PR interval (s)

0.046 ± 0.003

0.050 ± 0.003

0.046 ± 0.002

0.050 ± 0.002

QRS interval (s)

0.014 ± 0.001

0.013 ± 0.001

0.015 ± 0.001

QT interval (s)

0.061 ± 0.003

0.046 ± 0.005

0.056 ± 0.007

0.054 ± 0.002

QTc (s)

0.157 ± 0.009

0.123 ± 0.016

0.148 ± 0.017

0.140 ± 0.004

Tpeak-Tend (s)

0.040 ± 0.003

0.025 ± 0.003*

0.024 ± 0.003*

0.030 ± 0.001

ECG parameters were sampled from lead II recordings. QTc was calculated using Bazett’s formula. Values are mean ± SEM; *p < 0.05; **p < 0.01 and ***p < 0.001 (treatment vs control).

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Several animal models have been developed to mimic human MI in vivo, but the lack of reliability, reproducibility or survival remains a problem. Surgical methods such as ligation and cauterisation of coronary arteries produce well-demarcated infarcts compared to the more global infarcts due to ISO (Fig. 1). However, surgical techniques are invasive and associated with post-operative mortality rates as high as 40–50% within 24 hours, and the infarct sizes also vary.39 Pharmacological methods such as ISO-induced MI are non-invasive and the drug doses can be adjusted to minimise mortality. However, the methods produce diffuse global infarcts of variable sizes. The ISO-induced MI disease model mimics cardiovascular stress disorders that not only produce infarction, but in which intracellular Mg2+ deficiency may play a role.31,32 Nevertheless, in our study, Mg2+ pre-treatment did not alter infarct size, suggesting a lack of Mg2+ cardioprotection, as also reported in other studies,13,14 and at the same time, contradicting the results of some previous studies.7-11 The moderate dose of ISO used in our study was optimal to induce infarcts and to minimise mortality in our rats. However, the relatively smaller infarcts induced (~15% of the whole ventricular tissue), compared to coronary ligation models (~50% of the localised region at risk),7,8,11 may have made it more difficult to identify any mild effects of Mg2+, especially with the presence of baseline infarcts that are attributable to tissue handling. The protective effects of Mg2+ may depend on the dose, bioavailability, and the timing of administration as well as on the type of experimental protocol used. In the experimental studies showing Mg2+ protection, Mg2+ was given during reperfusion,7-11 which is a different protocol from the one used in our study. In some studies, Mg2+ was protective only when administered early during reperfusion.8,9 By contrast, Mg2+ may have preconditioned the myocardium through the activation of ATP-dependent K+ channels,22 and also protected it in a cellular model of ischaemia alone without reperfusion.25 In our study, serum Mg2+ was not measured, making it uncertain whether adequate prophylaxis may have been achieved at the onset of MI. However, a similar dose of Mg2+ used in other studies in rats achieved neuroprotection,35,40 and lower doses used in guinea pigs provided cardioprotection.41 In studies where repeated doses of Mg2+ were used, it was re-administered only after four hours,40 a longer period than when ISO was given in our study. Furthermore, in guinea pigs, Mg2+ cardioprotection occurred even if the insult was given at a time when Mg2+ levels in the plasma and heart tissue were no longer significantly elevated,41 indicating that the downstream cellular effects from the adequate initial exposure to Mg2+ may outlast the real elevation of Mg2+ in the tissue or plasma. The low-voltage ECG induced by ISO administration was possibly due to the infarct-related loss of tissue, whereas the presence of pathological Q waves are indicative of an evolving MI.38 We however did not observe an elevation of the ST segment, in contrast to what would be expected in acute infarction, and what has been reported by others.42 The ST segment in rats is difficult to assess because the end of the QRS complex merges with the T wave (Fig. 2), thereby overshadowing the isoelectric portion.43 Therefore, the decreases in the S- and T-wave amplitudes by ISO and Mg2+ in our study may in fact reflect ST-segment modulation. Overall, Mg2+ pre-treatment did not reverse the electrical changes, in keeping with the unaltered infarct size.

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B 10

150 LV end-diastolic pressure (mmHg)

LV max pressure (mmHg)

100 50 0

ISO ISO + Mg Treatment

Control

ISO ISO + Mg Treatment

D 9000

dP/dt min (mmHg/s)

dP/dt max (mmHg/s)

–5 Control

6000 3000 0

Control

ISO ISO + Mg Treatment

0 –3000 –6000 –9000

*** Control

ISO ISO + Mg Treatment

F 0.09

Diastolic duration (s)

Systolic duration (s)

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* 0.06 0.03 0.00

Control

ISO ISO + Mg Treatment

0.09 0.06 0.03 0.00

Control

ISO ISO + Mg Treatment

Fig. 3. E ffects of ISO and Mg2+ treatments on haemodynamic parameters in four different groups of rats. A: The maximum left ventricular (LV) blood pressure, B: LV end-diastolic pressure, C: maximal rate of LV pressure change (dP/dt max), D: minimal rate of LV pressure change (dP/dt min), E: systolic duration, and F: diastolic duration. Data are presented as mean ± SEM (n = 8–10 rats per group); *p < 0.05; **p < 0.01 and ***p < 0.001 (treatment vs control).

B 80 6

60 TBARS (μmol/l)

Conjugated dienes (μmol/l)

40 20 0

Control

ISO ISO + Mg Treatment

4 2 0

Control

ISO ISO + Mg Treatment

Fig. 4. E ffects of ISO and Mg2+ on the plasma concentrations of markers of lipid peroxidation in four different groups of rats. Plasma concentrations of conjugated dienes (A), and thiobarbituric acid-reactive substances (B), measured 24 hours after administration of the drugs. Data are presented as mean ± SEM (n = 8–10 rats per group).

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In this study, although Mg2+ did not worsen the haemodynamic parameters, it did not reverse the ISO-induced left ventricular hypotension or the decrease in ventricular dP/dt min. The physiological significance of the decrease in the systolic duration in ISO and Mg2+ co-treated rats is unclear, but given that the heart rate was unchanged, it is unlikely to be of major impact. By contrast, in an ISO-induced cardiac dysfunction model in dogs, Mg2+ preserved ventricular activity and the effect was proposed to be due to Mg2+-mediated reduction in cardiac afterload.19 In human heart MI, unrelated to ISO, the preservation of ventricular function by Mg2+ has been attributed to the direct action of increased extracellular Mg2+ due to MI-induced efflux of Mg2+.44 It has previously been reported that the action of ISO on the myocardium involves the production of reactive oxygen species,27,28 and that Mg2+ acts as an antioxidant and reduces the infarct size by protecting against free radicals.16 In our study, neither ISO nor Mg2+ administration resulted in any significant changes in the concentration of oxidative stress markers (CD and TBARS) in the circulation after 24 hours. After ischaemic damage to myocardial tissue, the blood will reflect the appearance of specific cardiac markers as well as relatively non-specific markers such as those attributable to lipid peroxidation. The latter products, for example CD, lipid hydroperoxides and TBARS depend not only on oxidative stress, but also on the nature of the lipid substrate. Lipid peroxidation products generally change in concert, and therefore using two of the three commonly used markers should reveal the trend. These changes have been reported up to eight days in ISO-induced MI,45 and therefore the 24-hour interval in our study could be expected to be appropriate. Although infarcts were clearly demonstrated in our study, they did not affect the lipid peroxidation markers, suggesting that the infarct size was either too small to produce detectable markers in the plasma, or the lipid substrate was not very susceptible to oxidative stress. By contrast, in coronary artery ligation-induced infarction in dogs, the markers of lipid peroxidation reached peak concentrations a few hours after the ligation.46 In rats injected with ISO at a higher dose (110 mg/kg ip, once daily for two days), Anandan et al.47 found significant increases in the concentration of TBARS in homogenised heart tissue. With ISO being a broad-acting catecholamine, it is not unexpected for it to produce systemic effects such as the loss of body weight seen in this study. The ISO-related weight loss was previously attributed to the stress of myocardial necrosis or to the catabolic state of altered protein metabolism.48 The increase in heart weight:body weight ratio with ISO may indicate the onset of cardiac hypertrophy49 or oedema. The increase in heart weight was unlikely to be an artifact related to loss of body weight because the relative weights of the other organs were unaltered by ISO treatment alone. The physiological relevance of the decreased kidney weight in ISO and Mg2+ co-treated rats is unclear.

CG was supported by the Oppenheimer Memorial Trust grant and UCT

Conclusion

12. Du C-S. Magnesium lithospermate B protects cardiomyocytes from

Masters Research Scholarship, RK-L by the UCT URC/Carnegie Research Development grant, the South African Heart Association grant and the UCT Health Sciences Faculty Research Committee, and AG by the UCT URC/ Carnegie Research Development grant, National Research Foundation (NRF Grant No 91514) and ADInstruments (Australia) grant. We thank Prof Edward Johns for providing the Mikrotip catheter, Dr Kishor Bugarith for insightful discussions, and Mr Henri Carrara for statistical advice.

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Woods KL, Fletcher S, Roffe C, Haider Y. Intravenous magnesium sulphate in suspected acute myocardial infarction: results of the second Leicester Intravenous Magnesium Intervention Trial (LIMIT-2). Lancet 1992; 339: 1553–1558. PMID:1351547.

Shechter M, Hod H, Chouraqui P, Kaplinsky E, Rabinowitz B. Magnesium therapy in acute myocardial infarction when patients are not candidates for thrombolytic therapy. Am J Cardiol 1995; 75: 321–323. PMID:7856520.

Collins R, Peto R, Flather M, Parish S, Sleight P, Conway M, et al. ISIS-4: a randomised factorial trial assessing early oral captopril, oral mononitrate, and intravenous magnesium sulphate in 58,050 patients with suspected acute myocardial infarction. ISIS-4 (Fourth International Study of Infarct Survival) Collaborative. Lancet 1995; 345: 669–685. PMID:7661937.

MAGIC. Early administration of intravenous magnesium to highrisk patients with acute myocardial infarction in the Magnesium in Coronaries (MAGIC) Trial: a randomised controlled trial. Lancet 2002; 360: 1189–1196. PMID:12401244.

Barros LF, Chagas AC, da Luz PL, Pileggi F. Magnesium treatment of acute myocardial infarction: effects on necrosis in an occlusion/reperfusion dog model. Int J Cardiol 1995; 48: 3–9. PMID:7744535.

Christensen CW, Rieder MA, Silverstein EL, Gencheff NE. Magnesium sulfate reduces myocardial infarct size when administered before but not after coronary reperfusion in a canine model. Circulation 1995; 92: 2617–2621. PMID:7586364.

Herzog WR, Schlossberg ML, MacMurdy KS, Edenbaum LR, Gerber MJ, Vogel RA, et al. Timing of magnesium therapy affects experimental infarct size. Circulation 1995; 92: 2622–2626. PMID:7586365.

10. Leor J, Kloner RA. An experimental model examining the role of magnesium in the therapy of acute myocardial infarction. Am J Cardiol 1995; 75: 1292–1293. PMID:7778564. 11. Matsusaka T, Hasebe N, Jin YT, Kawabe J, Kikuchi K. Magnesium reduces myocardial infarct size via enhancement of adenosine mechanism in rabbits. Cardiovasc Res 2002; 54: 568–575. PMID:12031702.

Our results suggest a lack of reduction in infarct size by single-dose Mg2+, despite the presumed optimal pre-treatment. In future studies, it would be important to evaluate serum, heart tissue or urine Mg2+ levels to better understand the temporal effects, and to use repeated doses of Mg2+ for more sustained prophylaxis. Although Mg2+ did not have adverse cardiovascular effects, the role and indications for Mg2+ therapy in MI still require further clarification.

ischemic injury via inhibition of TAB1-p38 apoptosis signaling. Front Pharmacol 2010; 1: 111. PMID:21607062. 13. Kingma JG, Qiu Y, Hearse DJ. Influence of low-flow infusion and magnesium on tissue necrosis during regional ischemia in the canine myocardium. Can J Physiol. Pharmacol 1992; 70: 1004–1010. PMID:1451022. 14. Schlack W, Bier F, Schäfer M, Uebing A, Schäfer S, Borchard U, et al. Intracoronary magnesium is not protective against acute reperfusion

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injury in the regional ischaemic-reperfused dog heart. Eur J Clin Invest 1995; 25: 501–509. PMID:7556368. 15. Hwang DL, Yen CF, Nadler JL. Effect of extracellular magnesium on platelet activation and intracellular calcium mobilization. Am J Hypertens 1992; 5: 700–706. PMID:1418832. 16. Garcia LA, Dejong SC, Martin SM, Smith RS, Buettner GR, Kerber RE. Magnesium reduces free radicals in an in vivo coronary occlusion-

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32. Young L, Bercute-Dammann A, Weis MT. Mg2+ efflux from the isolated perfused rabbit heart is mediated by two states of the beta1-adrenergic receptor. Naunyn-Schmiedeberg’s Arch Pharmacol 2002; 366: 431–439. PMID:12382072. 33. Vormann J, Fischer G, Classen HG, Thoni H. Influence of decreased and increased magnesium supply on the cardiotoxic effects of epinephrine in rats. Arzneimittelforschung 1983; 33: 205–210. PMID:6682658.

reperfusion model. J Am Coll Cardiol 1998; 32: 536–539. PMID:9708488.

34. Arteaga de Murphy C, Ferro-Flores G, Villanueva-Sanchez O, Murphy-

17. Miyoshi K, Taniguchi M, Seki S, Mochizuki S. Effects of magnesium

Stack E, Pedraza-López M, Meléndez-Alafort L, et al. 99mTc-glucarate

and its mechanism on the incidence of reperfusion arrhythmias follow-

for detection of isoproterenol-induced myocardial infarction in rats. Int

ing severe ischemia in isolated rat hearts. Cardiovasc Drugs Ther 2000; 14: 625–633. PMID:11300363.

J Pharm 2002; 233: 29–34. PMID:11897407. 35. Sameshima H, Ota A, Ikenoue T. Pretreatment with magnesium sulfate

18. Mubagwa K, Gwanyanya A, Zakharov S, Macianskiene R. Regulation

protects against hypoxic-ischemic brain injury but postasphyxial treat-

of cation channels in cardiac and smooth muscle cells by intracellular

ment worsens brain damage in seven-day-old rats. Am J Obstet Gynecol

magnesium. Arch Biochem Biophys 2007; 458: 73–89. PMID:17123458. 19. Jin Y-T, Hasebe N, Matsusaka T, Natori S, Ohta T, Tsuji S, et al. Magnesium attenuates isoproterenol-induced acute cardiac dysfunction and beta-adrenergic desensitization. Am J Physiol Heart Circ Physiol 2007; 292: H1593–1599. PMID:17114241. 20. Altura BM, Altura BT, Carella A, Gebrewold A, Murakawa T, Nishio A. Mg2+–Ca2+ interaction in contractility of vascular smooth muscle: Mg2+ versus organic calcium channel blockers on myogenic tone and agonist-induced responsiveness of blood vessels. Can J Physiol Pharmacol 1987; 65: 729–745. PMID:3300911. 21. Alberty RA, Goldberg RN. Standard thermodynamic formation properties for the adenosine 5’-triphosphate series. Biochemistry 1992; 31: 10610–10615. PMID:1420176. 22. Bazargan M, Faghihi M, Chitsaz M. Importance of timing of magnesium administration in the isolated ischemic-reperfused rat heart: role of K(ATP) channels. Physiol Res 2008; 57: 839–846. PMID:18052684. 23. Faghihi M, Sukhodub A, Jovanovic S, Jovanovic A. Mg2+ protects adult beating cardiomyocytes against ischaemia. Int J Mol Med 2008; 21: 69-73. PMID:18097618.

1999; 180: 725–730. PMID:10076154. 36. Esterbauer H. Estimation of peroxidative damage. A critical review. Pathologie-biologie 1996; 44: 25–28. PMID:8734296. 37. Jentzsch AM, Bachmann H, Fürst P, Biesalski HK. Improved analysis of malondialdehyde in human body fluids. Free Radic Biol Med 1996; 20: 251–256. PMID:8746446. 38. Thygesen K, Alpert JS, White HD. Universal definition of myocardial infarction. Eur Heart J 2007; 28: 2525–2538. PMID:17951287. 39. Pfeffer MA, Pfeffer JM, Fishbein MC, Fletcher PJ, Spadaro J, Kloner RA, et al. Myocardial infarct size and ventricular function in rats. Circ Res 1979; 44: 503–512. PMID:428047. 40. Euser AG, Bullinger L, Cipolla MJ. Magnesium sulphate treatment decreases blood-brain barrier permeability during acute hypertension in pregnant rats. Exp Physiol 2008; 93: 254–261. PMID:17933863. 41. Kusniec F, Fischer G, Sela BA, Ashkenazy Y, Feigel D, Moshonov S, et al. Magnesium protects against anaphylactic shock and cardiac myolysis in guinea-pigs. J Basic Clin Physiol Pharmacol 1994; 5: 45–58. PMID:8736070. 42. Li H, Xie Y-H, Yang Q, Wang S-W, Zhang B-L, Wang J-B, et al.

24. Woods KL. Possible pharmacological actions of magnesium in

Cardioprotective effect of paeonol and danshensu combination on

acute myocardial infarction. Br J Clin Pharmacol 1991; 32: 3–10.

isoproterenol-induced myocardial injury in rats. PLoS One 2012; 7:

PMID:1888638.

e48872. PMID:23139821.

25. Mirica SN, Duicu OM, Trancota SL, Fira-Mladinescu O, Angoulvant

43. Farraj AK, Hazari MS, Cascio WE. The utility of the small rodent

D, Muntean DM. Magnesium orotate elicits acute cardioprotection at

electrocardiogram in toxicology. Toxicol Sci 2011; 121: 11–30.

reperfusion in isolated and in vivo rat hearts. Can J Physiol Pharmacol 2013; 91: 108–115. PMID:23458194. 26. Shechter M. Magnesium and cardiovascular system. Magnes Res 2010; 23: 60–72. PMID:20353903. 27. Mukherjee D, Roy SG, Bandyopadhyay A, Chattopadhyay A, Basu

PMID:21278051. 44. Suzuki N, Tanabe K, Osada N, Yamamoto A, Nakayama M, Yokoyama Y, et al. Magnesium dynamics and relation to left ventricular function in acute myocardial infarction. Jpn Circ J 2000; 64: 377–381. PMID:10834454.

A, Mitra E, et al. Melatonin protects against isoproterenol-induced

45. Radhiga T, Rajamanickam C, Sundaresan A, Ezhumalai M, Pugalendi

myocardial injury in the rat: antioxidative mechanisms. J Pineal Res

KV. Effect of ursolic acid treatment on apoptosis and DNA damage

2010; 48: 251–262. PMID:20210856.

in isoproterenol-induced myocardial infarction. Biochimie 2012; 94:

28. Selvaraj P, Pugalendi KV. Hesperidin, a flavanone glycoside, on lipid peroxidation and antioxidant status in experimental myocardial ischemic rats. Redox Rep 2010; 15: 217–223. PMID:21062537. 29. Hori M, Sato H, Kitakaze M, Iwai K, Takeda H, Inoue M, et al.

1135–1142. PMID:22289617. 46. Röth E, Török B, Zsoldos T, Matkovics B. Lipid peroxidation and scavenger mechanism in experimentally induced heart infarcts. Basic Res Cardiol 1985; 80: 530–536. PMID:4074287.

Beta-adrenergic stimulation disassembles microtubules in neonatal rat

47. Anandan R, Ganesan B, Obulesu T, Mathew S, Kumar RS, Lakshmanan

cultured cardiomyocytes through intracellular Ca2+ overload. Circ Res

PT, et al. Dietary chitosan supplementation attenuates isoprenaline-

1994; 75: 324–334. PMID:7518364.

induced oxidative stress in rat myocardium. Int J Biol Macromol 2012;

30. Communal C, Singh K, Pimentel DR, Colucci WS. Norepinephrine stimulates apoptosis in adult rat ventricular myocytes by activation of the beta-adrenergic pathway. Circulation 1998; 98: 1329–1334. PMID:9751683. 31. Romani A, Marfella C, Scarpa A. Regulation of magnesium uptake and release in the heart and in isolated ventricular myocytes. Circ Res 1993; 72: 1139–1148. PMID:8495544.

51: 783–787. PMID:22829055. 48. Wexler BC. Isoprenaline-induced myocardial infarction in spontaneously hypertensive rats. Cardiovasc Res 1979; 13: 450–458. PMID:487383. 49. Teerlink JR, Pfeffer JM, Pfeffer MA. Progressive ventricular remodeling in response to diffuse isoproterenol-induced myocardial necrosis in rats. Circ Res 1994; 75: 105–113. PMID:8013068.

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Drug Trends in Cardiology Blood glucose levels during acute illness can help predict future diabetes risk Blood glucose levels measured in hospitalised patients during acute illness predicted the risk of developing type 2 diabetes in the following three years, according to a study published in PLos Medicine in August 2014. Scottish researchers measured the blood glucose levels of 86 634 patients, aged 40 years or older, admitted for an acute illness between 2004 and 2008. Patients were followed up to December 2011 to determine their type 2 diabetes risk. The researchers reported that type 2

diabetes risk for patients with a glucose level of less than 90 mg/dl (5 mmol/l) was 1% and the risk increased to approximately 15% among those with a glucose level of 270 mg/dl (15 mmol/l) or more. Plus, the risk of developing diabetes increased with increasing blood glucose levels during admission. Based on the findings, the researchers developed a risk calculator that uses the patient’s age, gender and admission blood glucose level to predict risk of developing diabetes over three years following hospital admission. However,

the risk calculator has not yet been tested in non-white populations or populations outside of Scotland. The researchers said in a press release, ‘These findings can be used to inform individual patients of their long-term risk of type 2 diabetes and to offer lifestyle advice as appropriate.’

Reference http://www.diabetesincontrol.com/index. php?option=com_content&view=article&id=167 90&catid=1&Itemid=17.

Encapsulated beta-cell replacement therapy for type 1 diabetes Beta-cell encapsulation therapy is a procedure that involves implantation of cells, contained in a protective barrier, with the ability to secrete insulin into the body in a glucose-responsive manner. On 17 July 2014, the Juvenile Diabetes Research Foundation (JDRF) announced that its partner, ViaCyte, Inc, had filed an Investigational New Drug (IND) application with the US Food and Drug Administration (FDA), seeking to conduct a phase 1 and 2 clinical trial in patients with type 1 diabetes. The purpose of this trial is to evaluate the safety and efficacy of the VC-01 product, a stem cellderived, encapsulated-cell replacement therapy. In addition to the IND, ViaCyte also submitted a medical device master file (MAF) to the FDA regarding the Encaptra® drug-delivery system, a device component of the VC-01 product. Beta-cell encapsulation therapy is a procedure that involves implantation in a protective barrier of cells with the ability to secrete insulin into the body in a glucose-responsive manner. The advantage of these encapsulated betacells is that they can assess the patient’s blood glucose level and secrete the correct

amount of insulin, while their barrier protects them from being destroyed by the autoimmune system. More importantly, encapsulation therapy also helps prevent the requirement of lifetime administration of powerful and toxic immunosuppressive drugs designed to protect the newly introduced islets from the immune system. VC-01 therapy is the combination of PEC-01 cells (a proprietary pancreatic endoderm cell product derived through directed differentiation of an inexhaustible human embryonic stem cell) and an Encaptra drug-delivery system (a proprietary immune-protecting and retrievable encapsulation medical device.) The VC-01 combination product is expected to be implanted under the skin of the patient through a simple out-patient surgical procedure. Once inside the body, the cells are expected to differentiate and become mature pancreatic cells with the ability to produce and secrete insulin based on the patient’s glucose level. Based on pre-clinical studies, VC-01 therapy has been shown to be effective in mice. Normal blood glucose levels for mice range from 160–200 mg/dl (8.88–11.1 mmol/l) , which are considered

hyperglycaemic in humans. However, when the mice received the VC-01 combination product, their blood glucose levels were closer to human levels. In addition, when these mice received STZ, a chemical designed to kill native mouse beta-cells, the mice still maintained their blood glucose levels. Study of the synergy of cell therapy and the Encaptra medical device also showed positive results. In the mouse study, host blood vessels began to grow into the VC-01 combination product at week four, supplying a steady amount of oxygen and nutrients to PEC-01 cells. At week eight, vascularisation developed quickly. Meanwhile, the Encaptra device protected the PEC-01 cells from immune rejection with a protective permeable membrane. The VC-01 cell replacement therapy could be a potential cure for type 1 diabetes.

Reference http://www.diabetesincontrol.com/index. php?option=com_content&view=article&id=167 27&catid=1&Itemid=17.

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Cardio News National Advisory Committee for the Prevention and Control of Rheumatic Fever and Rheumatic Heart Disease in Namibia In Windhoek, Namibia, Thursday 23 April 2015 marked a historic milestone for the Pan-African campaign to arrest the march of rheumatic fever (RF) and rheumatic heart disease (RHD) throughout our continent. Under the authority of the Minister of Health and Social Services, Dr Bernard Haufiku, the first meeting of the National Advisory Committee on Rheumatic Fever and Rheumatic Heart Disease began to elaborate on a plan for the prevention and control of a heart disease, which, it is estimated, claims the lives of 1.4 million people in less wellresourced countries globally every year. The prevalence in Africa is as high as 30/1 000 among school children. Among survivors, RHD is a major cause of morbidity through heart failure, atrial fibrillation and cerebrovascular accidents. RHD results in school absenteeism in about two-thirds of affected learners, and because the disease progresses during early adulthood and causes chronic disability, it has the potential to undermine national productivity. The economic impact of RHD in the African region is profound and was estimated at US$791 million to 2.37 billion in 2010. Significantly, Namibia is the first African country to tackle the prevention and control of RHD in this manner at a national level. The national programme was launched in March 2014 by Dr Richard Kamwi, the health minister at that time. Advocacy for the national programme had been informed by research conducted by the Namibian National Registry of RF and RHD, which is an important partner in the Global Registry of RF and RHD. The campaign to eliminate RHD in our lifetime has its origins in the first all-Africa workshop on rheumatic fever and rheumatic heart disease, which was supported by the Pan-African Society of Cardiology (PASCAR) and the World Health Organisation African region (WHO-AFRO), and held in the

Drakensberg, South Africa in 2005. At that meeting, four actions were recommended as part of any programme: awareness-raising for both the public and health workers, surveillance (of incidence and prevalence), advocacy for funding and implementing treatment and prevention programmes, and prevention (primary and secondary). From this conversation, the ‘Stop Rheumatic Heart Disease ASAP Programme’, described in the Drakensberg Declaration, was to emerge. Clinicians in 12 countries in Africa took up the surveillance challenge and participated in the Global Registry for RHD (REMEDY), which in 2012 collected robust data on 3 066 children and adults (including 266 Namibian patients) with RHD. A strong coalition for RF and RHD prevention developed over this period. Both the knowledge gathered and the collaboration itself established a powerful platform through which the coalition has been able to influence public policy and advocate for the prevention and control of the most common non-communicable disease affecting the heart in our continent. These intentions were consolidated at the second all-Africa workshop on RF and RHD at Livingstone, Zambia in 2014 and expressed through the ‘Mosi-oTunya (the smoke that thunders) Call to Action’ (2014). This call from PASCAR

was endorsed by the WHO-AFRO and called for the elimination of acute RF and control of RHD in Africa in our lifetime. Persistent in-country advocacy over four years, together with the momentum created by the Pan-African coalition, led to the creation of the National Advisory Committee on Rheumatic Fever and Rheumatic Heart Disease in Namibia. RHD is the end result of acute RF, a consequence of untreated pharyngitis caused by group A Streptococcus (GrAS). Overcrowding, poor housing conditions, under-nutrition and lack of access to penicillin for sore throat are determinants of RHD. With adequate medical care, RHD is preventable, and it is therefore a litmus test for the efficacy of primary healthcare systems. Penicillin prevents rheumatic fever and is the cornerstone of both primary and secondary prevention. Penicillin supply is dependent on health system infrastructure. Penicillin delivery depends on awareness among healthcare providers of the importance of this strategy. Recognising these realities, Namibia has adopted the ‘ASAP’ strategies and will incorporate them into the national programme. The advisory committee will work with the Minister to design the details of the programme, namely raising awareness through public and professional education, establishing a well-tested surveillance system, advocacy work to improve the availability of health services for patients, and promoting adherence to effective measures for the prevention of RF. Dr Christopher Hugo-Hamman Centre for Paediatric and Congenital Heart Disease, Namibia

Delegates of the National Advisory Committee on RF and RHD

Dr Norbert Forster Deputy Permanent Secretary Ministry of Health and Social Services, Namibia

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Case Report Dyspnoea and chest pain as the presenting symptoms of pneumomediastinum: two cases and a review of the literature Hasan Kara, Hasan Gazi Uyar, Selim Degirmenci, Aysegul Bayir, Murat Oncel, Ahmet Ak

Abstract Pneumomediastinum is the presence of air in the mediastinum. It may occur as spontaneous, traumatic, or iatrogenic pneumomediastinum. Although spontaneous pneumomediastinum is usually observed in healthy young men, traumatic pneumomediastinum may be caused by blunt or penetrating trauma to the chest and neck. Pneumomediastinum is a clinical condition with potential complications that cause high morbidity and mortality rates. Pneumomediastinum also may develop without tracheal or oesophageal injury after spontaneous or blunt chest, neck and facial injuries, and it may be accompanied by pneumothorax. We treated two patients who had pneumomediastinum. Case 1 was a 20-year-old man who had pain and dyspnoea around the sternum for one hour, as a result of a blow from an elbow during a football match. Case 2 was a 23-year-old man who had a two-day history of dyspnoea and chest pain with no history of trauma. In both patients, diagnosis of pneumomediastinum was confirmed with thoracic computed tomography scans, and the condition resolved within five days of in-patient observation. In conclusion, the diagnosis of pneumomediastinum should be considered for all patients who present to the emergency department with chest pain and dyspnoea. Keywords: trauma, spontaneous, mediastinum, emergency department Submitted 27/1/15, accepted 25/3/15 Published online 8/10/15 Cardiovasc J Afr 2015; 26: e1–e4

www.cvja.co.za

DOI: 10.5830/CVJA-2015-035

Department of Emergency Medicine, Faculty of Medicine, Selçuk University, Konya, Turkey Hasan Kara, MD, hasankara42@gmail.com Hasan Gazi Uyar, MD Selim Degirmenci, MD Aysegul Bayir, MD Ahmet Ak, MD

Department of Thoracic Surgery, Faculty of Medicine, Selcuk University, Konya, Turkey Murat Oncel, MD

Pneumomediastinum, also known as mediastinal emphysema, is the presence of air or other gas in the mediastinum.1 Pneumomediastinum can be categorised as traumatic, spontaneous, or iatrogenic, and it also may be categorised as spontaneous or secondary. Spontaneous pneumomediastinum may occur in situations that increase alveolar pressure, such as coughing, vomiting, straining, or Valsalva manoeuvre, which may cause spontaneous rupture of the alveoli. These conditions may occur with asthma, chronic obstructive pulmonary disease, diabetic keto-acidosis, excessive exercise, cannabis or cocaine intake, and diffuse interstitial fibrosis. In addition, severe coughing that may cause mediastinal emphysema may occur with pertussis, diphtheria, influenza, bronchiolitis, or acute bronchitis in children. Iatrogenic pneumomediastinum may develop after tracheostomy induced by barotrauma during mechanical ventilation or as a result of rupture of the tracheo-bronchial tree or oesophagus during endoscopy. Traumatic pneumomediastinum may occur as a result of blunt or penetrating chest, head or neck, or eye injuries.2,3 Traumatic and spontaneous pneumomediastinum have similar symptoms, most commonly retrosternal chest pain that begins acutely. In addition, common symptoms and signs include neck pain, neck swelling, dyspnoea, cough, nasal voice, dysphagia, anxiety, increased salivation, hoarseness and fever. The clinical presentation is variable and may range from vague symptoms to life-threatening respiratory failure. The patient may have subcutaneous emphysema present in the neck and chest, a Hamman sign with heart auscultation (crackling sounds synchronous with the heartbeat), or cardiovascular collapse.4 The purpose of this study was to report the experience with two patients who had isolated pneumomediastinum that presented with dyspnoea and chest pain.

Case reports Case 1 A 20-year-old man had pain and dyspnoea around the sternum for one hour as a result of a blow from an elbow during a football match, and he was admitted to the emergency department. His past medical history was non-contributory. The blood pressure was 130/75 mmHg, pulse was 87 beats per minute, respiratory rate was 16 breaths per minute, temperature was 36.9°C, and transcutaneous oxygen saturation was 96% on room air. He had

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Fig. 1. C ase 1: axial thoracic computed tomography showing free air density consistent with pneumomediastinum (A) around the trachea in the upper mediastinum, (B) around the aorta in the lower mediastinum, and (C) at the posterior oesophagus (red arrows).

crepitus on palpation around the sternal notch. Auscultation of the heart revealed a loud crunch-like sound during systole consistent with Hamman sign. Neurological and abdominal examinations showed no abnormalities. Laboratory tests, including cardiac enzymes and an electrocardiogram, were normal. A postero-anterior chest radiograph was normal, but a chest computed tomography (CT) scan showed subcutaneous emphysema and pneumomediastinum (Fig. 1). There was no evidence of pneumothorax, pneumopericardium, pulmonary parenchymal injury, rib fractures, or tracheal or bronchial injuries. The patient was transferred to the thoracic surgery department and admitted to hospital for observation and non-surgical treatment. His progress was uneventful and he was discharged after four days. Written informed consent was obtained from the patient for the publication of this case report.

Case 2 A 23-year-old man was admitted to the emergency department because of a two-day history of dyspnoea and chest pain. He had no history of trauma. The blood pressure was 120/85 mmHg, pulse was 91 beats per minute, respiratory rate was 18

breaths per minute, temperature was 37Â°C, and transcutaneous oxygen saturation was 93% on room air. There was tenderness to palpation in the right hemithorax and around the sternum. The breath sounds were normal and equal in both lungs. Laboratory tests, including cardiac enzymes and an electrocardiogram, were normal. The postero-anterior chest radiograph showed a right pneumothorax and transparency that was consistent with left mediastinal air. Thoracic CT scan showed right pneumothorax and pneumomediastinum (Fig. 2). The patient was transferred to the thoracic surgery department and admitted to hospital for observation and non-surgical treatment. His progress was uneventful and he was discharged after five days. Written informed consent was obtained from the patient for the publication of this case report.

Discussion The chief complaint on presentation to the emergency department in both patients included chest pain and dyspnoea. The first patient had traumatic pneumomediastinum as a result of blunt chest trauma, which is a rare clinical condition. The second patient had spontaneous pneumomediastinum. In this study, we investigated the diagnosis and treatment of the two

Fig. 2. C ase 2: axial thoracic computed tomography showing free air density consistent with pneumomediastinum (A) around the aorta and pulmonary artery, (B) inferior to the heart, and (C) anterior to the heart (red arrows). In addition, pneumothorax was detected (B and C) in the anterior right hemithorax (blue arrows).

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patients who had pneumomediastinum with similar clinical symptoms but different causes. Pneumomediastinum may have varied causes, including tracheobronchial or oesophageal rupture that may cause an air leak into the mediastinum.5 Pneumomediastinum is a potentially ominous sign because it may have severe complications. The differential diagnosis of chest pain, dyspnoea and subcutaneous emphysema may include acute pulmonary and cardiac conditions, such as pericarditis, pulmonary embolism, pneumonia and pneumothorax, and oesophageal perforation, spasm and reflux disease. These potential causes may be less likely in patients who have traumatic or spontaneous pneumomediastinum.4 Spontaneous pneumomediastinum is a rare clinical condition that typically is observed in young men, and symptoms usually resolve spontaneously after diagnosis.6,7 Traumatic pneumomediastinum may be accompanied by subcutaneous emphysema, pneumothorax, rib fractures and pneumopericardium. Iatrogenic pneumomediastinum may develop as a result of bronchial or oesophageal rupture during endoscopy, barotrauma during mechanical ventilation, or after tracheostomy.8 The clinical course of pneumomediastinum is variable. Patients may have mild complaints or life-threatening respiratory distress. Patients usually present with chest pain localised to the sternum. They also may have dysphagia, hoarseness, a foreign body sensation in the throat, and dyspnoea. Subcutaneous emphysema detected on physical examination may occur as a result of the spread of extra-alveolar air to the neck, face and anterior chest wall. In addition to subcutaneous emphysema, physical examination may show a crackling sound synchronous with the heartbeat (Hamman sign), which is pathognomonic for pneumomediastinum.8 Although the reasons for hospital admission were the same in both patients (chest pain and dyspnoea), the fact that blunt chest trauma was accompanied by subcutaneous emphysema in the first case was an important finding in the diagnosis of pneumomediastinum. Patients who are suspected of having pneumomediastinum should be evaluated with postero-anterior and lateral chest radiography that includes the cervical area. Although CT scan is more sensitive than ordinary chest radiography in detecting pneumomediastinum, the diagnosis is often verified with a careful history and chest radiographs. Radiographs may show a vertical lucent line on the left side of the heart and aortic arch, lucent line through the retrosternal, pericardiac and paratracheal areas, or subcutaneous emphysema of the shoulders and neck.9-11 Suggestive radiographic signs may include the thymic sail sign (appearance of thymus as a triangular sail), ring-around-the-artery sign (lucency along the right pulmonary artery on the lateral radiograph caused by mediastinal air), tubular artery sign (air outlining the major aortic branches), double bronchial wall sign (air outlining the bronchial wall), continuous diaphragm sign (lucency above the diaphragm), and extrapleural sign (pulmonary opacity with oblique margins). The CT scan should be reserved for evaluation of underlying lung disease or other accompanying conditions. In the present study, postero-anterior chest radiography was normal in the first patient, but the second patient had a right apical pneumothorax and left hyperlucency, with the appearance of a linear band that suggested the presence of mediastinal air. Chest radiography may be normal in 30% of patients. Therefore,

the most sensitive method, thoracic CT scan, could be useful in diagnosing pneumomediastinum when there is clinical suspicion but non-contributory radiographs. In addition, bronchoscopy and oesophagoscopy can be considered because they may show possible ruptures in the bronchial tree and oesophagus (Boerhaave syndrome); in such cases, surgical intervention should be considered. In some cases, contrast studies and mediastinoscopy may be helpful. In the treatment of pneumomediastinum, supportive care should be considered when there is no bronchial injury, oesophageal injury, or bullous structure from lung disease that may cause air leakage.11 The treatment of pneumomediastinum in the emergency department includes airway and haemodynamic stabilisation, and treatment to prevent further complications such as tension pneumomediastinum and mediastinitis. Patients who have pneumomediastinum should be observed and provided with supplemental oxygen. Treatment should be non-surgical until symptoms disappear within four to five days.12,13 Both of our patients received supportive treatment in the thoracic surgery department and were followed with daily postero-anterior chest radiography. They were discharged on hospital day four and five, respectively, without complications.

Conclusion Pneumomediastinum is a clinical condition that can vary from a mild to life-threatening clinical situation. This diagnosis should be considered for all patients who present to the emergency department with chest pain and dyspnoea. Pneumomediastinum also may develop spontaneously or after blunt chest, neck, facial, or eye injury, with or without tracheal or oesophageal injury. Despite normal chest radiographs, patients suspected of having traumatic or spontaneous pneumomediastinum should have a CT scan. Patients who have pneumomediastinum should be hospitalised for observation because the condition may be associated with complications, including death.

References 1.

Kikuchi N, Ishii Y, Satoh H, Ohtsuka M, Hizawa N, Ohta Y. Spontaneous pneumomediastinum after air travel. Am J Emerg Med 2008; 26: 116.e1–2. (PMID: 18082810).

Maravelli AJ, Skiendzielewski JJ, Snover W. Pneumomediastinum acquired by glass blowing. J Emerg Med 2000; 19: 145–147. (PMID: 10903462).

Özhasenekler A, Gökhan Ş, Yilmaz F, Tan Ö, Nasir A. Pneumomediastinum and pneumothorax after blunt neck trauma [in Turkish]. J Acad Emerg Med Case Reports 2010; 1: 17–19. doi: 10.5505/jaemcr.2011.39974.

Caceres M, Ali SZ, Braud R, Weiman D, Garrett HE Jr. Spontaneous pneumomediastinum: a comparative study and review of the literature. Ann Thorac Surg 2008; 86: 962–966. doi: 10.1016/j.athoracsur.2008.04.067. (PMID: 18721592).

Wintermark M, Schnyder P. The Macklin effect: a frequent etiology for pneumomediastinum in severe blunt chest trauma. Chest 2001; 120: 543–547. (PMID: 11502656).

De Luca G, Petteruti F, Tanga M, Luciano A, Lerro A. Pneumomediastinum and subcutaneous emphysema unusual complications of blunt facial trauma. Indian J Surg 2011; 73: 380–381. (PMID: 23024550).

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Esayag Y, Furer V, Izbicki G. Spontaneous pneumomediastinum: is a chest X-ray enough? A single-center case series. Isr Med Assoc J 2008; 10: 575–578. (PMID: 18847153).

chest trauma: a case report and review of the literature. Case Rep Emerg Med 2014; 2014: 685381. doi: 10.1155/2014/685381. (PMID: 25114811). 11. Kaneki T, Kubo K, Kawashima A, Koizumi T, Sekiguchi M, Sone S.

Vanzo V, Bugin S, Snijders D, Bottecchia L, Storer V, Barbato A.

Spontaneous pneumomediastinum in 33 patients: yield of chest comput-

Pneumomediastinum and pneumopericardium in an 11-year-old rugby

ed tomography for the diagnosis of the mild type. Respiration 2000; 67:

player: a case report. J Athl Train 2013; 48: 277–281. doi: 10.4085/10626050-48.1.11. (PMID: 23672393). 9.

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Pekcan S, Gokturk B, Uygun Kucukapan H, Arslan U, Fındık D. Spontaneous pneumomediastinum as a complication in human bocavirus infection. Pediatr Int 2014; 56: 793–795. doi: 10.1111/ped.12475. (PMID: 25336003).

10. Mansella G, Bingisser R, Nickel CH. Pneumomediastinum in blunt

408–411. (PMID: 10940795). 12. Abrahamian FM, Pollack CV. Traumatic pneumomediastinum caused by isolated blunt facial trauma: a case report. J Emerg Med 2000; 19: 43–46. (PMID: 10863117). 13. Freixinet J, García F, Rodríguez PM, Santana NB, Quintero CO, Hussein M. Spontaneous pneumomediastinum long-term follow-up. Respir Med 2005; 99: 1160–1163. (PMID: 16085218).

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Case Report Iatrogenic left main-stem dissection extending to the circumflex artery and retrogradely involving the left and non-coronary sinuses of Valsalva: iatrogenic aortocoronary dissection Radosław Zwoliński, Anna Marcinkiewicz, Konrad Szymczyk, Robert Pietruszyński, Ryszard Jaszewski

Abstract We present the case of a 57-year-old female who experienced iatrogenic left main-stem (LMS) dissection during elective coronary angiography. The dissection immediately affected the circumflex artery (Cx), causing its total distal occlusion, and the left anterior descending artery (LAD), in which a metal stent, implanted six months earlier, provided blood flow. The dissection spread retrogradely to the left and non-coronary sinuses of Valsalva (SV). Ventricular fibrillation (VF) occurred but the patient was successfully defibrillated. The subsequent introduction of a catheter resulted in recurrent VF, again successfully defibrillated. Total arterial myocardial revascularisation with double skeletonised internal thoracic arteries was performed without complications and SV repair was avoided. At the one-year follow up, a control multi-slice CT (MSCT) angiography was conducted, revealing complete healing of the SV and LMS dissections. It also showed native blood flow, the left internal thoracic artery (LITA) graft to the Cx occlusion, and a patent right internal thoracic artery (RITA) graft implanted to the LAD.

Keywords: left main-stem dissection, limited aortic dissection, coronary angiography Submitted 25/11/14, accepted 26/7/15 Cardiovasc J Afr 2015; 26: e5–e7

www.cvja.co.za

DOI: 10.5830/CVJA-2015-060

Department of Cardiac Surgery, Clinical Teaching Centre, Medical University of Lodz, Lodz, Poland Radosław Zwoliński, MD, PhD Anna Marcinkiewicz, MD, annamar87@o2.pl Ryszard Jaszewski, MD, PhD

Department of Radiology: Imaging Diagnostics, Norbert Barlicki Memorial Teaching Hospital No 1, Medical University of Lodz, Lodz, Poland Konrad Szymczyk, MD PhD

Department of Vascular Diagnostics and Procedures, Military Teaching Hospital, Veterans Central Hospital, Lodz, Poland Robert Pietruszyński, MD, PhD

According to the simplified classification, iatrogenic left main-stem (LMS) dissection involving the aortic root is the rarest and most life-threatening type of dissection. The incidence of iatrogenic aortic root dissection is estimated to be 0.02%. In the majority of cases it remains confined to the coronary sinus of Valsalva (SV).1 Although therapeutic management of LMS dissection involving SV is demanding, there are few published works in this field.2-4 There are neither guidelines nor consensus among experts on how to manage LMS and retrograde SV dissection during percutaneous procedures. The published literature on iatrogenic dissection describes the following therapeutic solutions: surgical revascularisation with SV repair, including usage of an autologous patch; percutaneous angioplasty with stent(s) implantation to cover the ostial dissection; and conservative treatment, which can provide spontaneous healing of the dissection.1-4

Case report A 57-year-old patient with well-controlled arterial hypertension, hyperlipidaemia and anterolateral ST-elevation myocardial infarction (STEMI) was treated with a primary bare-metal stent (BMS) implantation to the left anterior descending artery (LAD). Six months later, she underwent an elective coronary angiography due to exacerbated angina pectoris. On admission, she was classified as class III according to the Canadian Cardiovascular Society classification (CCS). During coronary angiography, the angiogram showed both the positive long-term effect of the BMS implantation and the absence of any significant progression of previously described atherosclerotic lesions. After completing the left coronary artery examination, the patient started complaining of chest pain. At the same time the electrocardiogram (ECG) showed ST-segment elevation. As the patient became symptomatic, catheterisation of the left coronary artery was repeated. A standard diagnostic coronary catheter, Impuls 6-Fr JL3.5 Boston (guidewire 6-Fr JL3.5 Medtronic Launcher) was used. This time the examination revealed LMS dissection, antegrade dissection of the circumflex artery (Cx) causing distal occlusion of blood flow, and contrasting of the left aortic bulb (Fig. 1). Instant ventricular fibrillation (VF) occurred but the patient was successfully defibrillated. The cardiologist immediately attempted to cover the dissection with a stent, but VF recurred, and was again successfully treated with defibrillation.

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Fig. 1. A . Coronary angiogram showing LMS dissection extending antegradely to the Cx with its distal total occlusion, and retrogradely to the left and non-coronary SV. A bare-metal stent implanted in the LAD provided contrast flow. Persistent staining after the contrast cleared shows the vessel lumen present. B. Progression of the dissection to almost total contrast flow occlusion in the left coronary artery branches. Persistent staining is seen in the false lumen. The dissection was classified as F according to the NHLBI. LMS: left main stem, Cx: circumflex artery, SV: sinus of Valsalva, LAD: lateral anterior descending artery.

On the ECG, signs of anterolateral myocardial ischaemia were observed, and the patient became severely symptomatic. The dissection, leading to total occlusion of the coronary lumen without distal antegrade flow, was classified as class F according to the National Heart, Lung, Blood Institute classification (NHLBI). A continuous infusion of nitroglycerin and heparin, as well as a lignocaine infusion, were administered. Within a few minutes, the patient’s condition stabilised and she was transferred to the cardiac surgery department. On admission there, her systolic blood pressure was 110 mmHg and the heart rate was 80 beats/min. The patient complained of chest pains. Her cardiac necrosis markers were elevated: creatine kinase-MB (CK-MB) 99 U/l and troponin T (Ths) 449.4 ng/l. The continuous nitroglycerin and heparin infusion was maintained. Transthoracic echocardiography (TTE) revealed hypokinesis of the apex and para-apical segments of the anterior, lateral and postero-inferior cardiac walls. The ascending aorta was 3.1 cm, the aortic bulb was 3.2 cm, and signs of dissection on the mitral side were observed. The ejection fraction was 52%. Multi-slice CT (MSCT) angiography showed significant ostial LMS stenosis (intraluminal diameter 2 mm, area 5 mm2 ) without signs of atherosclerosis. Persistent staining of the left sinus of Valsalva, extending to the aortic annulus and non-coronary sinus of Valsalva, was found. The scan indicated limited aortic dissection (Fig. 2A). The 12-lead ECG indicated anterolateral STEMI. Laboratory tests showed significant increase in levels of cardiac markers: CK-MB 538 U/l and Ths 7 034 ng/l. A decision was made to

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Fig. 2. A. Multi-slice CT angiography performed during acute aortic root dissection. Non-enhanced computed tomography (calcium score sequence) presents a hyperdense and thickened aortic root wall, which corresponds to the intramural haematoma (white arrow). B. At the one-year follow up, control MSCT angiography showed self-healing of the dissected aorta. Note the reduced wall thickness.

intervene surgically. The time period between the iatrogenic dissection and surgical intervention was approximately six hours. An intra-operative view confirmed the presence of a dissection of the left and non-coronary sinuses of Valsalva. There was also a haematoma along the proximal portion of the LAD, which extended to the surrounding epicardium. On a beating heart, without extracorporeal circulatory assist, total arterial myocardial revascularisation with double skeletonised internal thoracic arteries was performed. The right internal thoracic artery (RITA) was anastomosed to the LAD. The left internal thoracic artery (LITA) was anastomosed to the Cx. Further hospitalisation was uneventful. At the one-year follow up, the patient was feeling well and remained asymptomatic. Control MSCT angiography (Fig. 2B) revealed complete healing of the limited aortic and LMS dissection. Competitive native blood flow, the LITA graft occlusion and the patent RITA graft were also seen on MSCT angiography.

Discussion Iatrogenic dissection of a coronary artery during a percutaneous procedure can be triggered by many factors, including unusual anatomy of the LMS, atherosclerosis of the LMS, difficulty when introducing a catheter, vigorous contrast infusion, inexperience of the operator, catheter type, inappropriate catheter position or sub-intimal passage of the guidewire.5 The choice of treatment strategy in the case of an iatrogenic coronary artery dissection depends on many factors, including haemodynamic stability, the patient’s clinical state, extension of the dissection, the number of dissected vessels, and SV involvement.5 When dealing with an LMS dissection, urgent surgical myocardial revascularisation is preferred. Many authors emphasise the unpredictable nature of a dissected flap.6

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Although stenting gives the opportunity of a fast and direct approach, more than one stent is usually required to obtain proper blood flow in the LAD and Cx. Furthermore, percutaneous angioplasty may be associated with extension of the dissection area. While forming a haematoma, the coronary artery lumen may become occluded. In a long-term follow up by Unal and co-workers, after salvage PCI of the dissected LMS, rates of in-stent restenosis and repeated revascularisation were high.6 In addition, LMS dissection can easily affect the main branches, causing ischaemia in a large mass of myocardium, and clinical recurrence of angina pectoris.7 In our case, it seems that the previously implanted BMS provided blood flow through the LAD but the dissection resulted in haematoma formation. Although an attempt at covering the dissection with a stent was undertaken, recurrent VF occurred with each introduction of the catheter. There was no opportunity to protect the dissection percutaneously, therefore, a clinical approach focused on the patient’s stabilisation and transfer to the cardiac surgery department. The results of laboratory tests and changes in the ECG suggested progression of ischaemia. In addition to recurrent VF, it showed that there was only temporary haemodynamic stability. In such a scenario, surgical treatment, especially in the case of limited aortic root dissection and persistent myocardial ischaemia, was the only reasonable and possible solution. ECG evolution indicated that the haematoma along the LAD could have been causing occlusion of the medial and distal segments, and anterior myocardial ischaemia. Iatrogenic aortocoronary dissection (IACD) may be treated conservatively, especially in cases of high-risk patients, provided that entry of the dissected coronary artery is covered with a stent and the patient can be carefully monitored.8 On the other hand, IACD is unpredictable by nature. A stent implantation may not prevent type A ascending aortic dissection early after the primary procedure, therefore, sudden clinical deterioration may be observed.9 Spontaneous resolution of the SV dissection, even within 24 hours post procedure, has also been reported.8 Some authors suggest the surgical approach when the dissection extends into the ascending aorta for more than 4 cm.1 A decision on total, no-touch arterial revascularisation was made during the surgery. It allowed the blood supply to the ischaemic myocardial areas to be restored without manipulation of the ascending aorta. An unchanged sino-tubular junction and ascending aorta allowed SV repair to be avoided. The one-year follow up and results of the control MSCT angiography confirmed the appropriateness of the intra-operative decision. Healing of the dissection probably caused competitive native and bypass flow, which contributed to occlusion of the LITA graft.

The percentage of self-healing dissections is unknown, as is the mechanism of this process. Almafragi et al.10 suggested that the healing of a coronary artery dissection is stimulated by retrograde flow and intravascular pressure augmentation caused by a bypass implantation. This corresponds with the high rate of in-stent restenosis. Our decision was confirmed by a favourable outcome in the patient, and the positive impact of the competitive flow.

Conclusions IACD poses therapeutic difficulties and individual risk evaluation may confirm the treatment strategy. Surgical intervention limited to myocardial revascularisation, performed as a no-touch technique, and conservative management of the limited aortic dissection may give satisfactory long-term results. Careful patient follow up is also required.

References 1.

Garg P, Buckley O, Rybicki FJ, Resnic FS. Resolution of iatrogenic aortic dissection illustrated by computed tomography. Circ Cardiovasc Interv 2009; 2(3): 261–263.

Barbero C, Di Rosa E, Devotini R, Attisani M, Rinaldi M. Left main coronary artery ostial repair with autologous ring-shaped aortic patch for iatrogenic aortic dissection. J Card Surg 2014; 29(6): 821–823.

Burstow D, Poon K, Bell B, Bett N. Anterior ECG changes following iatrogenic dissection of the right coronary artery into the aortic root: exclusion of left coronary obstruction with transoesophageal echocardiography. Cardiovasc Revasc Med 2013; 14(2): 102–105.

Kagoshima M, Kobayashi C, Owa M. Aortic dissection complicating failed coronary stenting. J Invasive Cardiol 2002; 14(5): 263–265.

Celik M, Yuksel UC, Yalcinkaya E, Gokoglan Y, Iyisoy A. Conservative treatment of iatrogenic left main coronary artery dissection: report of two cases. Cardiovasc Diagn Ther 2013; 3(4): 244–246.

Unal M, Korkut AK, Kosem M, Ertunc V, Ozcan M, Caglar N. Surgical management of spontaneous coronaryartery dissection. Tex Heart Inst J 2008; 35(4): 402–405.

Auer J, Punzengruber C, Berent R, Weber T, Lamm G, Hartl P, Eber B. Spontaneous coronary artery dissection involving the left main stem: assessment by intravascular ultrasound. Heart 2004; 90(7): e39.

Saito T, Noguchi K, Oikawa T. Iatrogenic dissection of the anomalousorigin right coronary artery and left sinus of Valsalva. J Invasive Cardiol 2011; 23(3): E51–53.

Takahashi Y, Tsutsumi Y, Monta O, Kohshi K, Sakamoto T, Ohashi H. Closure of the left main trunk of the coronary artery and total arch replacement in acute type A dissection during coronary angiography. Ann Thorac Surg 2010; 89(2): 618 –621.

10. Almafragi A, Convens C, van den Heuvel P. Spontaneous healing of spontaneous coronary artery dissection. Cardiol J 2010; 17(1): 92–95.

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Case Report A rare case of aortic dissection presenting as pure transient global amnesia Hirsh Kaveeshvar, Rabih Kashouty, Vivek Loomba, Noor Yono Case report

Abstract Transient global amnesia (TGA) is a well-described neurological phenomenon. Clinically, it manifests with the sudden onset of a paroxysmal, transient loss of anterograde memory and disorientation but with intact consciousness. Typically, symptoms last for only a few hours. We present an unusual case of aortic dissection presenting with pure TGA in a patient, who had a positive outcome. This is the second case report of a patient with aortic dissection presenting with pure TGA syndrome, but it is the first case in which the patient survived. Keywords: transient global amnesia, aortic dissection, TIA, emergency, Valsalva Submitted 28/1/15, accepted 26/7/15 Cardiovasc J Afr 2015; 26: e8â&#x20AC;&#x201C;e9

www.cvja.co.za

DOI: 10.5830/CVJA-2015-061

Transient global amnesia (TGA) is a well-described neurological phenomenon. Clinically, it manifests with a sudden onset of a paroxysmal, transient loss of anterograde memory and disorientation but with intact consciousness.1 Typically symptoms last for only a few hours. The aetiology of TGA remains unclear.1,2 Few case reports have described a link between TGA and aortic dissection (AD). We present an unusual case of AD presenting with pure TGA in a patient who had a positive outcome.

Department of Neurology, Henry Ford Hospital, Detroit, USA Hirsh Kaveeshvar, DO, hkavees2@hfhs.org

Department of Neurology, Icahn School of Medicine, Mount Sinai Beth Israel, New York, USA Rabih Kashouty, MD

Department of Anaethesiology and Pain Medicine, Henry Ford Hospital, Detroit, USA Vivek Loomba, MD

Department of Neurology, North Shore University Hospital, Hofstra University, New York, USA Noor Yono, MD

A 63-year-old man with a history of hypertension and hyperlipidaemia suddenly developed anterograde and retrograde amnesia and was admitted to our hospital. The patient, who was in the passenger seat of a car, with his colleague driving, suddenly became pale and dizzy and was not aware of his surroundings. He repeatedly asked the reason they were in the car. The patient denied any complaints, including chest pain, but asked repetitive questions. On general examination, the patient was afebrile. His blood pressure was 94/53 mmHg. His neurological examination was unremarkable, except for anterograde amnesia. Blood tests revealed mild leukocytosis (15 700 cells/mm3). A chest X-ray showed no abnormalities, a head computed tomography was unremarkable, and an electroencephalography revealed no epileptiform discharges. The anterograde amnesia resolved 10 hours after onset. However, he remained hypotensive and a mild diastolic murmur was noted over the aorta. Urgent cardiac echocardiography revealed Stanford type A AD. The patient was immediately taken to the operating room and successfully rescued. He was discharged without any significant neurological symptoms.

Discussion This case illustrates an example of painless AD presenting with pure TGA with no focal neurological deficits. The persistence of hypotension and the presence of an aortic murmur after the resolution of TGA raised the suspicion of AD. The aetiology of TGA remains unclear.1,2 Traditionally, it is believed to be due to transient cerebral ischaemia, particularly in the hippocampal formation and mesiotemporal structures; however, evidence is now accumulating that fails to show diffusion-weighted imaging hyperintensities within 24 hours of onset of symptoms.1,3 AD is a life-threatening emergency and prompt clinical recognition is essential for treatment.4 Acute neurological syndromes in AD are uncommon and typically present with focal neurological deficits.5 It can mimic a large group of neurological symptoms, including TGA, despite the absence of chest pain.6 In a series of 977 patients, Park et al.7 observed only 63 (6.4%) patients with painless AD, which may mislead the physician and delay the treatment. The existence of a pathogenic link between pure TGA and AD is still unclear.8 Transient episodes of increased intrathoracic pressure can potentially precipitate AD.9 Similarly, transient episodes of increased intrathoracic pressure due to the Valsalva manoeuvre

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have been noted to be a precipitating factor of TGA. Ultrasound studies during the Valsalva manoeuvre demonstrate a reduction in lumen diameter of the superior vena cava due to increased intrathoracic pressure, which causes obstruction of the venous blood flow transmitting venous back pressure towards the brain.10 While the association between AD and TGA remains unclear, the shared common precipitant of raised intrathoracic pressure may provide a link between the two processes.

Gass A, Gaa J, Hirsch J, Schwartz A, Hennerici MG. Lack of evidence of acute ischemic tissue change in transient global amnesia on singleshot echo-planar diffusion-weighted MRI. Stroke 1999; 30: 2070–2072. PMID: 10512909.

Gaul C, Dietrich W, Tomandl B, Neundörfer B, Erbguth FJ. Aortic dissection presenting with transient global amnesia-like symptoms. Neurology 2004; 63: 2442–2443. PMID: 15623727.

Isselbacher EM. Diseases of the aorta. In: Zipes DP, Libby P, Bonow RO, Braunwald E, eds. Braunwald’s Heart Disease: a Textbook of Cardiovascular Medicine, 7th edn. Philadelphia, PA: Elsevier Saunders,

Conclusion This report emphasises the importance of detailed physical examination in patients with pure TGA. Physicians should be aware of such a possibility and suspect AD in patients with pure TGA. To our knowledge, this is the second case report of a patient with AD presenting with pure TGA syndrome.10 In the only other case reported, the patient had a fatal outcome. This is therefore the first case report of a patient surviving after admission with AD presenting as pure TGA syndrome.

2005. 6.

In: Aminoff MJ, ed. Neurology and General Medicine, 4th edn. Philadelphia, PA: Churchill Livingstone, 2008: 23–44. 7.

Park SW, Hutchison S, Mehta RH, Isselbacher EM, Cooper JV, Fang J, et al. Association of painless acute aortic dissection with increased mortality. Mayo Clin Proc 2004; 79: 1252–1257. PMID: 15473405.

Mondon K, Blechet C, Gochard A, Elaroussi D, Fetissof F, De Toffol B, et al. Transient global amnesia caused by painless aortic dissection. Emerg Med J 2007; 24: 63–64. PMID: 17183052.

References

Goodin DS. Neurological complications of aortic disease and surgery.

Baydin A, Nural MS, Güven H, Deniz T, Bildik F, Karaduman A. Acute aortic dissection provoked by sneeze: a case report. Emerg Med J 2005; 22: 756–757. PMID: 16189052.

Huber R, Aschoff AJ, Ludolph AC, Riepe MW. Transient global amne-

10. Attubato MJ, Katz ES, Feit F, Bernstein N, Schwartzman D, Kronzon

sia. Evidence against vascular ischemic etiology from diffusion weighted

I. Venous changes occurring during the Valsalva maneuver: evaluation

imaging. J Neurol 2002; 249: 1520–1524. PMID: 12420091.

by intravascular ultrasound. Am J Cardiol 1994; 74: 408–410. PMID:

Lewis SL. Aetiology of transient global amnesia. Lancet 1998; 352:

8059711.

397–399. PMID: 9717945.

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Case Report Application of thoracic endovascular dissecting aneurysm repair for secondary type B aortic dissection Oguz Karahan, Orhan Tezcan, Sinan Demirtas, Ahmet Caliskan, Celal Yavuz

Abstract Type A aortic dissection is an emergency condition that requires immediate surgery. Graft replacement of the ascending aorta is the main treatment for this disorder. However, after ascending aortic replacement, the dissection flap may progress to the distal side (to the descending aorta) and a new intimal tear may develop. In this study, we report on a 66-year-old woman who had a history of ascending aortic replacement six months earlier. She was admitted to hospital with a new onset of back pain. Computed tomography revealed a new dissection tear originating from the distal side of the subclavian artery orifice. Thoracic endovascular dissecting aneurysm repair (TEVDAR) was carried out on the patient. Additional complications were not observed in the postoperative period. Complete cure was provided and the patient was discharged on the fourth day after the operation. TEVDAR may be safe and effective in preventing progression of the aortic flap and the formation of a new intimal tear in type A aortic dissections. Optional hybrid interventions could ameliorate the outcomes in aortic dissection cases.

Keywords: type A aortic dissection, surgery, endovascular intervention, hybrid procedure

Surgical replacement of the ascending aorta is indicated as the most appropriate curative therapy for Stanford type A AD. However, type B AD may initially be treated medically, with subsequent surgery or endovascular intervention.2,3 During the postoperative period, close monitoring of the progression of the flap, organ perfusion and other systemic events is critical. A rigorous postoperative follow up is required if the dissection flap involves the abdominal aorta or if the dissection has progressed significantly.4 Failure to closely monitor the disease progression in patients with type A AD undergoing surgical replacement of the aorta can result in significant clinical complications, such as secondary type B AD, as presented in the current case. The use of supplementary medication or hybrid interventions may improve the success rate of the initial ascending aortic graft replacement surgery. Here, we report on a secondary type B AD patient who had previously been operated on for a type A AD. Thoracic aneurysm repair with endovascular graft is usually an elective procedure, but a dissecting aneurysm of the thoracic aorta is a more progressive and serious condition. We therefore undertook thoracic endovascular dissecting aneurysm repair (TEVDAR) instead of thoracic endovascular aneurysm repair (TEVAR). The presentation, management and clinical outcomes of the case are presented in the context of the current clinical literature.

Submitted 20/7/15, accepted 25/8/15 Cardiovasc J Afr 2015; 26: e10–e12

www.cvja.co.za

DOI: 10.5830/CVJA-2015-067

Aortic dissection (AD) is a life-threatening emergency situation that progresses rapidly. Early mortality rates are as high as 50%, even under optimal treatment conditions.1-3 Alternate treatment approaches may be used according to the specific AD subtype.2 The standard AD classification system used in clinical practice is Stanford’s classification. This system categorises AD into two classes, type A and B, according to the presence or absence of ascending aortic involvement.

Medical School of Dicle University, Department of Cardiovascular Surgery, Diyarbakir, Turkey Oguz Karahan, MD, oguzk2002@gmail.com Orhan Tezcan, MD Sinan Demirtas, MD Ahmet Caliskan, MD Celal Yavuz, MD

Case report A 66-year-old woman was admitted to hospital with severe backache. This patient had undergone ascending aortic replacement surgery to treat type A AD six months prior to the presentation (Fig. 1). The medical history of the patient included hypertension for the past 25 years, nephrectomy due to nephrolithiasis eight years earlier, polio sequela and a motor deficit of the left leg. Her systolic blood pressure was 130 mmHg on the right arm and 110 mmHg on the left arm. All arterial pulses were determined by manual examination. Contrast-enhanced computed tomography revealed a type B dissection flap involving the left subclavian artery with retrograde progression. The diameter of the true lumen had narrowed significantly to < 10 mm, and the total diameter (with false lumen) was 43.7 mm at the widest section (Fig. 2). The peak aortic diameter was measured at 67.2 mm. We therefore initiated preparation for the TEVDAR surgery. The patient underwent surgery under general anaesthesia. During the operation, an initial exploration of the right common

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Fig. 1. D issection flap without tear during the initial diagnosis of type A AD.

Fig. 3. View of the flap tear on the distal side of the subclavian artery.

femoral artery was conducted. A 5-F pigtail catheter was inserted into the ascending aorta through the right brachial artery. This artery was chosen to facilitate proximal imaging, as delivery of the catheter through the left brachial artery could have been inhibited by the presence of the thoracic aortic stent placed six months previously. Contrast imaging of the aortic arch revealed the brachiocephalic truncus sourced from the left common carotid artery (Fig. 3) (bovine arch). Moreover, the origin of the retrograde dissection flap was identified 1 cm distal to the left subclavian artery in the contrast view (Fig. 3). Following the completion of the measurements, a 40 × 212-mm tube stent–graft was implanted into the descending aorta, including the proximal subclavian section. The placement of the tube stent–graft was challenging because of the narrowing of the true lumen and the high-angled aortic progression. The graft was placed using forced external manoeuvres. An extension tube stent–graft with a diameter of 42 × 112 mm was

placed through the right common femoral artery. The correct placement of the extension tube stent–graft was confirmed with angiography and the application was concluded (Fig. 4). Primary repair of the right common femoral artery was conducted. After surgery, no pulse deficit was observed in the left limb. The patient recovered in the intensive care unit and hydration was administered for deficient blood urine nitrogen and creatinine levels (due to the patient’s nephrectomy history). She was discharged on the fourth day after surgery.

Fig. 2. D issection flap with tear (flow can be observed between the true and false lumen) nine months after replacement of the ascending aorta.

Discussion All current treatment strategies for AD are associated with a high mortality rate. This risk is further increased by the extended patient transfer times. However, recent advances in surgical procedures may improve the overall morbidity and mortality rates in AD.2,3,5 In the present case, a brachiocephalic truncus in the left common carotid artery (bovine arch) was detected by contrast-enhanced computed tomography.

Fig. 4. Closed flap tear after TEVDAR application.

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Hornick et al. retrospectively analysed a series of AD cases and they concluded that bovine arch anatomy is not associated with increased incidence of AD compared with the normal anatomical configuration. Therefore bovine arch anatomy was likely not a significant factor in the development of AD in the present case.6 The primary treatment strategy for type A AD is graft replacement of the ascending aorta.5,7 However, dissection or distension of the distal aorta may be neglected in most cases. In the present case, the patient had undergone replacement of the ascending aorta due to type A dissection nine months earlier. Upon presentation, contrast-enhanced computed tomography (CT) detected a progression of the previous flap to the level of the renal artery. No additional progression of the dissection flap had been observed in the previous CT images (Fig. 3). In the earlier CT examination, the true lumen had retained the contrast. However, our examination nine months later showed the contrast agent passing through the false lumen. Despite the absence of a rupture in the aorta, these recent flap changes could have caused organ ischaemia due to the narrowing of the lumen diameter. Therefore, we proposed that the type B dissection was closely related to the previous type A dissection in this case. Moreover, the same risk factors that resulted in the previous dissection of the ascending aorta (hypertension, etc) could also have resulted in disruption of the distal section of the aortic intima, even though the damage to the proximal aorta had been completely repaired.

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Conclusion TEVDAR application is beneficial to most patients diagnosed with type B AD. Although this procedure is associated with higher costs, the benefits of this intervention include reduced risk of complications, shorter recovery time in the intensive care unit and a more rapid return to normal quality of life. Additionally, the management of type A AD using surgical and medical hybrid therapy may be critical to the prevention of secondary complications, such as the development of a secondary type B AD. In high-risk AD cases, TEVDAR may result in improved outcomes and a better quality of life.

References 1.

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