The role of human heart-type Fatty Acid Binding Protein in the early detection of myocardial injury in Acute Coronary Syndrome

The Abstract Objectives: To evaluate the use of H-FABP as a novel marker in early detection of cardiac damage (MI) in patients presenting with symptoms of the acute coronary syndrome (ACS). Methods: This cross-sectional study included 250 subjects; 125 patients of ACS presenting within four hours of the onset of chest pain and/dyspnea and 125 age and sex-matched healthy controls. An initial blood sample was taken from patients at presentation. Blood samples of healthy control subjects were also taken. The blood samples of both groups were centrifuged and stored at -200 C for H-FABP analysis. All the patients and control subjects were thoroughly examined and detailed history was taken. The diagnostic test was troponin-T.H-FABP concentrations of all samples were measured by ELISA-kit. The results were analyzed statistically. A p-value≤0.05 was considered statistically significant. Results: In 125 patients of ACS, H-FABP showed a sensitivity of 85% and specificity of 83.3% for acute MI diagnosis at a cut-off level of 16 ng/ml. Positive predictive values and negative predictive values were 96.8% and 48.4% respectively. The accuracy was 84.4%and the area under the ROC curve was 0.842. Conclusion: Evaluation of heart-type fatty acid-binding Protein (H-FABP) within four hours of onset ACS symptoms may be a valuable tool in the diagnosis of AMI.


Introduction
Cardiovascular disease (CVD) has emerged as a serious health issue and according to recent statistics, it almost contributes to one in every three deaths globally and this number will surely rise in both under-developed and developed countries. 1 The inhabitants of Indo-Asian origin have a huge burden of coronary artery disease (CAD) which is the leading cause of death in the Indo-Pakistan subcontinent. 2 The incidence of ischemic heart disease (IHD) is increasing in Pakistan and one in every four individuals with age ≥ 40 years may be having underlying coronary artery disease in urban areas of Pakistan. 3 Inflammation is increased in case of risk factors and their reduction, elimination or treatment may result in slowing inflammation and decreasing the threat of MI. 4 ACS refers to a group of clinical diseases as a result of acute myocardial ischemia and includes unstable angina (UA), non-ST-segment elevation myocardial infarction (NSTEMI), and ST-segment elevation myocardial infarction (STEMI). 5 The annual incidence of NSTEMI has increased compared to STEMI as it is difficult to diagnose and hence to manage effectively. 6 Also, it has been seen that a 12-lead ECG has low sensitivity and specificity for the diagnosis of coronary artery ischemia and has shown a range of accuracy from 58.5% to 62% in identifying ACS. 7 In such clinical situations cardiac biomarkers play a critical role for diagnosis, risk assessment and disease management Currently, cardiac troponins (T or I) are the preferred biomarkers to establish the diagnosis of acute MI. 8 But a major limitation of this current protocol is that troponin levels do not raise to a detectable level until six hours after the start of symptoms. 9 It has also been observed that patients with NSTEMI at high risk will benefit from earlier revascularization if it is done within four hours of the onset of occluding coronary thrombus but unfortunately, troponins do not confirm NSTEMI due to their low sensitivity within initial four hours. 10 There has been a great interest in seeking alternative or additional markers which could detect cardiac damage earlier. 11 Heart-type fatty acid-binding protein (H-FABP) may have the potential to fulfill the requirement. 12 After the damage to cardiomyocytes, it is quickly released in the blood from injured myocardium, detectable within 20 minutes of myocardial injury, reaches to the maximum level in three-four hours and comes to basal level within twenty-four hours due to rapid renal excretion. 13 H-FABP is a low molecular weight membrane-bound protein present in high concentration in heart tissues and carries out transportation of insoluble long-chain fatty acids (LCFA) from the blood into the cardiomyocytes for energy. Due to cardiac cell injury, it appears in circulation even earlier than troponin. 14 Due to its rapid release and early detection in the blood there has been a great interest in its utility as an early indicator of MI. 15 It been found that H-FABP can detect early cardiac damage within three hours of symptoms onset and can rule out non-ischemic acute chest pain. 16 The major issue of H-FABP availability to use it in routine testing is that its assay has not been standardized and the validity of effectiveness of this assay has not been established so far. 17

Materials and Methods
This analytical cross-sectional study was carried out at the emergency department (ED) of Punjab Institute of Cardiology (PIC) Lahore from November 2014 to January 2015. The study was approved by the ethical committee of the Postgraduate Medical Institute Lahore and was conducted after approval of the head of institute PIC Lahore. The sample size was calculated using the formula n=Z 2 1-α/2 x SN x (1-SN)/ L 2 ×prevalence. The calculated sample size was 492 (246 patients and 246 healthy control subjects) but due to limited resources, the sample size was reduced and we finally included 250 subjects of both sexes. Among 250 subjects, 125 were patients showing symptoms of ACS presenting to the ED with the final diagnosis of ACS established by the expert panel of doctors and 125 were healthy age and sex-matched blood donors as controls. The inclusion criteria for patients were Age ≥ 40 years with typical chest pain and/or dyspnea suggestive of ischemic origin within 4hours of onset and had the final diagnosis of ACS. The exclusion criteria were patients who had undergone an intervention or received fibrinolytic therapy at an early stage and those with serum creatinine level ≥1.3 mg/dl The control group had no history or clinical findings of cardiac or other systemic diseases. Informed written consent was obtained from all the participants. The values of cardiac biomarkers (Troponin-T, CK-MB, and H-FABP), creatinine and other routine investigations were also documented in the Performa.
At a presentation in ED, three ml of the blood was drawn from each patient and stored for H-FABP estimation as per the recommendation of the manufacturer. Blood for routine investigation was also taken. Another blood sample was drawn from each patient at eight hours after the presentation in ED for the estimation of the Troponin T level. General physical and systemic examination and complete history was taken from all participants. Blood samples for H-FABP and routine investigations were also obtained from the control group. Heart-type fatty acid-binding protein was measured by using human heart-type fatty acid-binding protein (H-FABP) ELISA kit manufactured by Glory Biosciences USA for the quantitative determination of H-FABP with a detection range of 0.4-22ng/ml. The serum samples were stored at -20 0 C until the quantitative determination of H-FABP. Samples were analyzed in batches with three levels of controls in each batch and before analysis, samples were thawed, centrifuged and the supernatant was used for analysis. Repeated freeze-thaw cycles were avoided.
The samples were analyzed according to the manufacturer's instructions. Data were analyzed statistically by SPSS software, version 18. Quantitative variables were presented as mean and standard deviation SD. Qualitative variables were presented as frequency and percentages. For the comparison of quantitative variables between cases and controls independent samples t-test was applied while for the comparison of qualitative variables chisquare test was applied. Sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) were calculated for H-FABP using Trop-T as the gold standard. ROC curve was also drawn and AUC was calculated. A p-value ≤ 0.05 was considered significant.

Results
Two hundred and fifty subjects were included in the study. One hundred and twenty-five patients of ACS were based on clinical features (±ECG changes) and cardiac troponin T. One hundred and twenty-five healthy subjects who served as a control group were also enrolled in the study.  (Table-2). The positive predictive value (PPV) and negative predictive value (NPV) were 96.8% and 48.4% respectively. The accuracy of H-FABP in ACS diagnosis was 84.4% (Table-3

Discussion
Acute coronary syndrome (ACS) is a chief cause of morbidity and mortality worldwide. It is strongly believed that identification and management of AMI at early hours greatly influences the AMI related morbidity and mortality and hence it's related expenditure. 18 It has also been found that the serum levels of cardiac troponins and CK-MB are not sufficient to establish the diagnosis of ACS (NSTEMI-ACS) in the patients seeking urgent medical aid in the casualty department within three hours of the onset of chest pain. So, for decision making, biomarkers facilitating identification of ACS at the initial point of time will gain particular significance. 19 H-FABP is one of the novel biomarkers, which has the potential to reenforcing cardiac troponins in the early confirmation of AMI. 20 At presentation, the combined assessment of H-FABP and high-sensitivity troponins (hs-cTn) has been proposed as an early rule-out strategy for MI for early discharge from ED. 21 So far, it has not given consistent results as a diagnostic tool for MI, though many studies have given excellent results. The main reason may be the lack of standardization of its assay as different methods are being used for its detection in the serum and different researchers have used different assays and cut-off values for diagnosis of MI.
Recently, Vupputuri et al reported the sensitivity of 89.7% and specificity 68% for H-FABP in early six hours for AMI detection. 22 Banuet al have found a sensitivity of 25% and AUC of 0.61 in 36 cases diagnosed with AMI(both STEMI and NSTEMI) presenting within 48 hours of the onset of chest pain of which majority presented within four hours. The diagnostic cut-off was 7ng/m. 23 Ramaiah et al reported a sensitivity of 77%, specificity 91% area under ROC curve equal to 0.83± 0.05 in ACS patients presenting within four hours of symptoms onset by a qualitative H-FABP immune-test with a diagnostic cut-off value of 10ng/L. 24 The other issue concerned with the fact that some studies have not given convincing results may be due to the kinetics of H-FABP i.e. early release and rapid clearance from plasma, hence having its prime diagnostic role in early six hours after which it starts declining. It might be the reason for poor results in studies in which the patients were included with a larger duration of symptoms. The variation in performance of H-FABP in ACS diagnosis may also be due to several other factors like age group, the gender ethnic variations or sample size as this biomarker has not been evaluated at a very large scale for generalization.  28 In our country, a large number of people come to ED with chest pain as the main complaint out of which a small fraction of cases are with true ACS diagnosis. In our study H-FABP at cut-off level, 16ng/ml has 85% sensitivity which makes it a better tool to rule out the disease at an early phase. The specificity of H-FABP is also significant and a more specific test gives a minimum number of false-positive results. In our study the AUC was equal to 0.842 which confirms that the methodology used to analyze H-FABP is good and it can discriminate among ACS and non-ACS cases efficiently. The positive predictive value which helps in confirming ACS was significantly raised which shows H-FABP as a useful early diagnostic tool for the ischemic type of chest pain in an emergency clinical setting.

Conclusion
In conclusion, the measurement of H-FABP is a valuable tool in the early diagnosis of patients with chest pain (4hrs). H-FABP is a promising biomarker for the early detection of ischemic myocardial damage (infarction) in patients presenting with the acute coronary syndrome and seems to be a preferred biomarker in the differential diagnosis of NSTE-ACS. More studies are needed to establish the exact role of this biomarker in diagnosing ACS at the early phase of ischemia.