Indian Journal of Animal Research

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Indian Journal of Animal Research, volume 56 issue 2 (february 2022) : 234-238

Comparative Assessment of Troponin T, Atrial Natriuretic Peptide, B-type Natriuretic Peptide and Echocardiography in the Diagnosis of Cardiac and Renal Disorders in Canine

K. Mahendran1,*, Neeraj Thakur1,2, G.E. Chethan1,3, Priyanka1,4, Shyam Sundar Choudhary1,5, S. Dey1, A.C. Saxena6, Sumit Mahajan1,7, K. Kavitha1
1Division of Medicine, Indian Veterinary Research Institute, Izatnagar, Bareilly-243 122, Uttar Pradesh, India.
2Department of Veterinary Clinical Complex, Faculty of Veterinary and Animal Sciences, RGSC- Banaras Hindu University, Barkachha, Mirzapur-231 001, Uttar Pradesh, India.
3Department of Veterinary Medicine, CVSc and AH, Central Agricultural University, Selesih, Aizawl-796 015, Mizoram, India.
4Indian Veterinary Research Institute, Sanjaynagar, Bengaluru-560 024, Karnataka, India.
5National Research Centre on Yak, West Kameng-790 101, Arunachal Pradesh, India.
6Division of Surgery and Radiology, Indian Veterinary Research Institute, Izatnagar, Bareilly-243 122, Uttar Pradesh, India.
7Central Institute for Research on Cattle, Meerut-250 001, Uttar Pradesh, India.
Cite article:- Mahendran K., Thakur Neeraj, Chethan G.E., Priyanka, Choudhary Shyam Sundar, Dey S., Saxena A.C., Mahajan Sumit, Kavitha K. (2022). Comparative Assessment of Troponin T, Atrial Natriuretic Peptide, B-type Natriuretic Peptide and Echocardiography in the Diagnosis of Cardiac and Renal Disorders in Canine . Indian Journal of Animal Research. 56(2): 234-238. doi: 10.18805/IJAR.B-4159.
Background: Canine cardiovascular problems are commonly seen in veterinary medicine. Cardiac abnormalities in canine can be diagnosed by various means like electrochardiography, radiography, blood pressure measurement, echocardiography and biomarkers like troponins and peptides specific for heart.

Methods: The present study was conducted to evaluate different biomarkers such as cardiac troponin T (cTnT), atrial natriuretic peptide (ANP) and B-type natriuretic peptide (BNP) for the diagnosis of cardiac diseases in comparison to echocardiography in canine. A total of 40 cases were included in the study, out of which 26 cases found to be positive for cardiac affections as confirmed by echocardiographic examination. 

Result: The serum concentration of cTnT and BNP were found significantly (P<0.05) elevated in dogs having cardiac as well as cardiac along with renal affections. ANP values were significantly (P<0.05) increased in dogs having both cardiac and renal diseases as compared to dogs with cardiac diseases only. Biomarkers like cTnT, ANP and BNP were compared with echocardiography (gold standard). The BNP was having the highest sensitivity (88.46%) and specificity (78.57%) as compared to cTnT and ANP. From the study, it is concluded that BNP can be used as suitable biomarker than cTnT and ANP for the diagnosis of cardiac diseases in canine. It can be used as a biomarker for cardiac diseases under field conditions where facilities for echocardiography are not available.
Cardiomyopathies include the diseases which primarily affect cardiac muscles and may comprise dilated cardiomyopathy (DCM) or arrhythmogenic right ventricular cardiomyopathy (ARVC). DCM is characterized by chamber dilation and myocardial systolic and diastolic dysfunction and is common in some of the dog breeds such as Doberman Pinscher, Great Dane and Irish Wolfhound (Tidholm and Junsson, 2005; Nelson and Couto, 2014). Complex neurohumoral responses are triggered by the abnormally functioning heart through increased circulatory volume (Cheng et al., 2016). Other conditions associated with neoplasia, renal disease, immune mediated hemolytic anemia, myocardial infarction and mitral insufficiency may result in ischemic myocardial disease. Hypertrophic cardiomyopathy (HCM) occurs less commonly in canines (Tilley et al., 2008).
        
Cardiac troponin-I (cTnI) along with troponin-T (cTnT) and troponin-C (cTnC) form a conglomeration of three myocardial proteins that are bound to the actin backbone within myocardiocytes. The cardiac isoforms of cTnI and cTnT are specific to cardiac tissue and are specific markers of myocardial cell injury or necrosis. cTnI is released in several fold higher concentrations than cTnT after any cardiac injury. This may be attributed to small molecular size of cTnI as well as the strong binding of cTnT to the contractile apparatus (Shaw et al., 2004; Apple et al., 2008). Both pro- B-type natriuretic peptide (proBNP) and pro-atrial natriuretic peptide (proANP) are constitutively produced by atrial and to a lesser extent ventricular myocardiocytes. Stress or stretch of the myocardium (for instance, in response to volume overload) increases the production of proBNP and proANP, particularly within ventricular myocardiocytes. C-terminal BNP (C-BNP) and C-terminal ANP (C-ANP) elicit vasodilation and diuresis through the binding of specific natriuretic receptors found in vascular and renal tissue. N-terminal pro-B-type natriuretic peptide (NT-proBNP) has been reported as a biomarker for the detection of cardiac diseases and to evaluate the severity of congestive heart failure in dogs (Wess et al., 2011; Ettinger et al., 2012). A degradation system affects BNP which is mediated by receptors present in the kidney and vascular system and NT-proBNP is also considered to be cleared by the kidneys (Tsutamoto et al., 2007). The present study was undertaken with the objective of comparatively evaluating cTnT, ANP and BNP for the diagnosis of cardiac diseases in comparison to echocardiography in canine.
Selection of animals
 
The present study was conducted in Referral Veterinary Polyclinic, Indian Veterinary Research Institute, Izatnagar, India from January 2015 to December 2016 following the ethical guidelines of Institutional Animal Ethics Committee. In this study, 40 dogs with cardiac diseases (ventricular dilatation, ventricular hypertrophy, arrhythmia, pericardial effusion, systolic and diastolic dysfunction) along with or without the involvement of renal diseases were studied. Out of 40 cases, 8 were found to have cardiac and renal involvement and 32 were found to have only cardiac involvement. Six healthy dogs were included as control for assessing cardiac biomarkers.
        
The initial diagnosis was made based on clinical examination of the animals and electrocardiography (ECG). ECG was done in standard body position (Tilley, 1985) using an ECG recorder with 6 lead system (Cardiart-408-BPL service power supply Class 1, 220- 240 V ± 10%; 50-60 Hz) at the paper speed of 50 mm/sec and sensitivity (10 mm=1 mV) without the filter. A minimum of 10 complexes on bipolar (I, II and III) limb lead systems were recorded for study. Echocardiography was performed using a two- dimensional and motion mode machine (Pie Medicals, Netherlands) with a 5 MHz annular array transducer.
 
Sample collection
 
For cardiac biomarker estimation, the blood samples were collected from dogs by the venipuncture of cephalic or saphenous vein into vials without any anticoagulant. The blood samples were allowed to clot and centrifuged at 2000 rpm for 10 min at 4°C to retrieve serum and the serum was stored at -20°C till analysis. In the present study, biomarkers like serum ANP, BNP and cTnT were measured using commercial sandwich enzyme immune assay (Uscn Life Scinece Inc., Wuhan, China) as per the method described by Oyama et al., (2007). By plotting the standard curve on log graph paper, with biomarker concentration on X-axis and absorbance on Y-axis, concentration in the samples were determined by regression analysis and expressed as pg/mL for ANP as well BNP and ng/mL for cTnT. The blood urea nitrogen (BUN) and the serum creatinine were estimated by the procedure described by Wybenga et al., (1971) and Frankel et al., (1970), respectively.
 
Statistical analysis
 
The data analysis was done by using standard statistical methods (Snedecor and Cochran, 1994). The evaluation of factors like sensitivity, specificity, overall agreement, kappa statistics (κ), positive and negative predictive values and diagnostic odds ratio were analyzed as per the method suggested by Thrusfield (2006).
Cardiac disease in canines is a common finding which may occur either primarily or most commonly as a concurrent disease. There is increased production and release of cTnT, ANP and BNP in cardiac diseases. Increase in the levels of natriuretic peptides is noticed in the early course of cardiac diseases due to stress on the heart wall. The present study was conducted to evaluate the assessment of cTnT, ANP and BNP for the diagnosis of cardiac diseases in comparison to echocardiography in canine.
 
Biomarkers
 
The present study was conducted on 40 cases, out of which 32 cases were showing only cardiac abnormalities and 8 cases were having cardiac abnormalities with renal affections. Renal involvement was diagnosed based on increased serum BUN and creatinine values. Diagnosis of cardiac diseases was made based on history, clinical examination, ECG findings, echocardiographic findings and concentration of biomarkers like cTnT, ANP and BNP. In dogs having cardiac involvement, the serum concentration of cTnT and BNP were found significantly (P<0.05) elevated in comparison to healthy control as depicted in Table 1. However, the serum concentration of ANP showed a non-significant increase in dogs with various cardiac abnormalities in comparison to healthy control. cTnT is a cytosolic protein and is released quickly whenever there is the destruction of cardiomyocyte, so, it is an early indicator of myocardial insult (Langhorn and Willesen, 2016). ANP and BNP are both released from cardiac chambers but ANP has a shorter half-life as compared to BNP and is cleared earlier from the plasma which may be the reason for non-significant elevation in the ANP level in cardiomyopathies (Kimura et al., 2007). Out of 40 cardiac cases, 26 cases showed cardiac abnormalities on echocardiography. cTnT and BNP levels in diseased animals (cTnT- 0.139±0.017 ng/mL and BNP- 623.3±195.4 pg/mL) were again found to be elevated at a significance level of (P<0.05) and (P<0.01), respectively when compared with healthy control (cTnT- 0.03±0.015 ng/mL and BNP- 64.26±29.62 pg/mL). While, ANP level in diseased animals (83.94±24.44 pg/mL) was having no significant difference as compared to healthy control (70.26±15.62 pg/mL). In cases which were diagnosed as having renal involvement along with cardiac abnormality, cTnT, ANP and BNP concentrations were also significantly (P<0.05) increased as compared to healthy control (Table 1). ANP values were significantly (P<0.05) increased in animals having both cardiac and renal disease as compared to animals with cardiac diseases only, whereas there was no significant difference in cTnT and BNP among these two groups as shown in Table 1. As ANP is a known potent natriuretic peptide and the kidney also acts as a site for its synthesis, so in patients having renal dysfunction along with cardiomyopathy, there may be an elevation in ANP level (Shin et al., 1997).
 

Table 1: Mean±SE values of cardiac biomarkers in healthy control in comparison with diseased animals.


 
Comparison of biomarkers with echocardiography
 
Echocardiographic examination was performed in animals and 26 out of 40 cases were found to be having cardiac abnormality, which were further selected for comparative study. Out of 26 positive cases in echocardiography, cTnT, BNP and ANP values were corresponding with 21, 23 and 10 cases, respectively. Both cTnT and echocardiography were in agreement for 21 and 8 cases (out of 40) as being positive and negative, respectively. Both ANP and echocardiography agreed for 10 and 9 cases as being positive and negative, respectively. Both BNP and echocardiography were in agreement for 23 and 11 cases as being positive and negative, respectively.
        
The sensitivity of cTnT, ANP and BNP was 80.76%, 38.46% and 88.46%, respectively and specificity was 57.14%, 64.28% and 78.57%, respectively when compared with echocardiography which is considered as a gold standard test. The overall agreement between echocardiography and BNP was 85% which was higher as compared to that of cTnT (72.5%) and ANP (47.5%) values as shown in Table 2. Among the three studied biomarkers, BNP was having higher sensitivity and specificity as compared to cTnT and ANP. Similarly, Kumar et al., (2014) also reported higher sensitivity (72.2%) and specificity (93.9%) of NT-proBNP for diagnosis of cardiac diseases in canine and having an overall agreement of 89.3% with echocardiography. Oyama et al., (2007) also reported that BNP is a better cardiac biomarker over ANP and cTnI. Low concentration of BNP or its parent protein proBNP is found in cases of non-cardiac origin whereas elevated values of these signify disease of cardiac origin (Oyama, 2015). In this particular study, significant (P<0.05) increase in the concentrations of cTnT, ANP and BNP were present in cases suffering with renal impairment along with cardiac involvement and similar findings are reported by Miyagawa et al., (2013) in the case of acute or chronic renal diseases.
 

Table 2: Comparative evaluation of cTnT, ANP and BNP with echocardiography for the detection of cardiac abnormalities in dogs.


        
In the cTnT study, 0.03±0.01 ng/mL was considered as a cut-off for healthy animals, which gave a positive predictive value of 0.77 and a negative predictive value of 0.61. Herman et al., (1999) considered 0.02±0.01 ng/mL as the cut-off value for cTnT in healthy animals. For BNP levels 64.26±29.62 pg/mL was considered as a cut-off in this study for healthy dogs, which meant all dogs having higher than this value of BNP were considered as suffering with cardiac disease which leads to a positive predictive value and negative predictive value of 0.88 and 0.78, respectively. While Kumar et al., (2014) and Boswood et al., (2008) considered the cut-off values for NT-proBNP as 783.26±102.62 pg/mL and 210 pmol/L, respectively. For ANP, cut-off level was considered as 70.26 15.62 pg/mL in healthy animals, as a result a positive predictive value of 0.66 and a negative predictive value of 0.36 were obtained. Whereas Smith et al., (2015) considered the cut-off value for ANP 18.5 pg/mL in healthy dogs. Kappa value, positive likelihood ratio and diagnostic odds ratio were higher for BNP than cTnT and ANP. Kappa value for BNP was 0.67, which falls in the category of substantial agreement of the test with that of the gold standard test (echocardiography). So, based on the various markers examined in this study, the BNP was having substantial agreement with echocardiography.
From the present research study, it is concluded that BNP is having higher sensitivity and specificity than cTnT and ANP and can be used as a suitable biomarker for the diagnosis of cardiac diseases in canine. BNP can be used as a biomarker in cardiac diseases under field conditions where facilities for echocardiography are not available. In cases having renal involvement along with cardiac disease, cTnT, ANP and BNP values were significantly increased. Further investigation is required to find any correlation of these elevated values with renal involvement or it is merely due to cardiac involvement.
The authors are thankful to Director, Indian Veterinary Research Institute, Izatnagar for providing the necessary research facilities.
The authors declare that they have no conflict of interest.

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