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Myxomatous mitral valve disease (MMVD) accounts for approximately 75% of all heart diseases in dogs. The prevalence of the disease is higher in small dogs (< 20 kg) than in large breeds, and disease progression takes years.[2, 3] Various methods to stage the severity of mitral valve disease are used in the current literature. Modified New York Heart Association (NYHA), International Small Animal Cardiac Health Council (ISACHC), and Canine Heart failure International Expert Forum (CHIEF) are classification methods based on clinical and radiographic changes. The CHIEF system is a newer approach based on recent guidelines of the American College of Cardiology and the American Heart Association. In this system, and in contrast to previous staging methods, asymptomatic dogs are divided into 2 groups, one group with previous signs of congestive heart failure (CHF) and the other group that never showed signs of CHF. A similar staging method subdividing asymptomatic dogs into 2 subgroups according to cardiac size is recommended by the consensus panel for therapy of MMVD of the American College of Veterinary Internal Medicine. Traditionally, for the diagnosis of MMVD and assessment of disease severity, echocardiographic and radiographic examinations are relied on. To quantify the amount of mitral valve regurgitation, several echocardiographic approaches based on 2-dimensional (2-D) ultrasonography and Doppler-derived methods are available.[3, 9, 10]
In addition, several studies investigated concentrations of natriuretic peptides (NPs) in dogs at different stages of MMVD, and there is general consent that NPs are useful to detect patients with respiratory symptoms due to CHF.[11, 12] Both B-type natriuretic peptide (BNP) and atrial natriuretic peptide (ANP) are released by myocardial tissue in response to volume or pressure overload and consecutive increased ventricular and atrial wall stretch.[13, 14] In circulation, BNP and ANP increase natriuresis and diuresis, and decrease systemic vascular resistance. BNP is synthesized as proBNP and further cleaved into BNP and the biological inactive N-terminal end NT-proBNP. ANP is encoded as a 126-amino acid precursor molecule, which is cleaved into its 98-amino acid fragment amino-terminal pro-atrial natriuretic peptide (NT-proANP) and ANP, similar to BNP. NT-proANP is further cleaved into the 3 molecules proANP 1-30, proANP 31-67, and proANP 68-98, which appear to have all physiologic functions similar to those of ANP, while some authors do not describe any biologic activity. Importantly, the half-lives of NT-proBNP and NT-proANP fragments are significantly longer than the ones of BNP and ANP, which make them preferable as diagnostic markers.[20, 21] Furthermore, studies in people suggest that proANP is more sensitive compared to ANP in detecting mild increases in atrial filling pressures and that proANP is the most sensitive marker among ANP, BNP, C-type NP, and NT-proBNP in discriminating NYHA Class I individuals (patients with no limitations of physical activity) from healthy individuals. Interestingly, NP concentrations in people seem to be dependent on body mass index (BMI), age, and androgen levels,[24-26] but no consistent veterinary reports about age-, gender- and weight-related variations in healthy dogs exist.[27-30]
So far, there is no consensus regarding the diagnostic power of NP concentrations to discriminate dogs without cardiac disease from dogs with subclinical stages of MMVD, and to differentiate between dogs in different asymptomatic stages.[31-35] Therefore, one aim of the present study was to investigate the diagnostic accuracy of NT-proBNP and proANP determinations in a large study population of dogs in different stages of MMVD, using a modified CHIEF system. In addition, we sought to compare the diagnostic power of NT-proBNP with that of proANP, and to investigate the influence of age, body size, and sex on NT-proBNP and proANP concentrations in a healthy dog population.
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A total of 559 samples of 352 dogs were collected as part of this study between February 2005 and December 2010. The control group consisted of 116 healthy dogs of various breeds (mean weight 22.5 kg, median 17.6 kg; mean age 5.7 years, median 3.9 years). All dogs in the control group were only examined once and by one blood sample. The group included 30 cross-bred dogs, 6 Labrador Retrievers, 6 Great Danes, 6 Cavalier King Charles Spaniels (CKCS), 6 Dachshunds, 6 Australian Shepherds, 5 Boxers, 5 Golden Retrievers, 4 Jack Russel Terriers, 4 Beagles, 3 Irish Wolfhounds, 3 Yorkshire Terriers, 2 Pugs, 2 Newfoundland Dogs, 2 Flat Coated Retrievers, and one each of 26 other breeds.
Mean NT-proBNP in the control group was 455.3 pmol/L (median 384.3) and proANP was 945.5 fmol/mL (median 308.3). Age and body size had no significant influence on NP levels. However, sex and neutering status had a significant influence on NT-proBNP and proANP concentrations. Intact female dogs had significantly higher NP values compared with intact male dogs (P = .03 and .01, respectively), whereas NP values of male neutered dogs were not significantly different from female spayed dogs. Also, no difference was found between intact and castrated male dogs (Table 2).
Table 2. Effect of sex and neutering on N-terminal pro-brain natriuretic peptide (NT-proBNP, pmol/L) and pro-atrial natriuretic peptide 31-67 (proANP, fmol/L) concentrations in male and female dogs
|Sex|| n ||NT-proBNPa||Percentile 05||Percentile 95||Min||Max|
|Female spayed||32||444 (81)||280||609||33||2630|
|Male neutered||18||528 (74)||371||685||15||1181|
| || n ||proANPa||Percentile 05||Percentile 95||Min||Max|
|Female spayed||32||971 (54)||861||1081||549||1881|
In the MMVD group, a total of 443 NT-proBNP and proANP measurements of 236 dogs (mean weight 13 kg, median 9.7 kg, mean age 10.5 years, median 2.9 years) were performed at different time points. Breeds of the MMVD group included 75 cross-bred dogs, 45 Dachshunds, 16 CKCS, 12 Poodles, 9 Jack Russell Terriers, 6 Pekinese, 5 Yorkshire Terriers, 4 Beagles, 3 Berger des Pyrenees, 3 Airedale Terriers, 3 Border Collies, 3 Chihuahuas, 3 German Spitz, 3 Maltese, 3 Miniature Pinschers, 3 Shi Tzu, 2 each of 8 other breeds, and one each of 24 other breeds. The numbers of NP measurements within the different CHIEF classification groups are listed in Table 3.
Table 3. Ranges of N-terminal pro-brain natriuretic peptide (NT-proBNP, pmol/L) and pro-atrial natriuretic peptide 31-67 (proANP, fmol/L) concentrations in groups of dogs assigned to different stages of myxomatous mitral valve disease according to the Canine Heart failure International Expert Forum (CHIEF)
| ||CHIEF|| n ||Mean (SD)||Percentile 05||Percentile 95||Min||Max|
Concentrations of both NPs increased with advanced disease severity. NT-proBNP values of all disease stages (B1-D) were significantly higher than NT-proBNP of the control group (Figure 1). ProANP concentration was not significantly different between the control group and B1, while proANP concentrations in dogs at stages B2–D1 were significantly higher than the control and B1 group dogs (Figure 2). For both NT-proBNP and proANP, the differences between stage B1 and B2 were significant (P = .006 and < .001, respectively). Both NT-proBNP and proANP discriminated patients with acute heart failure (C2, C3, D) significantly from all asymptomatic stages (B1, B2, P < .001).
Figure 1. Box plots illustrating the concentration of N-terminal pro-brain natriuretic peptide (NT-proBNP) in groups of dogs with myxomatous mitral valve disease categorized according to the Canine Heart failure International Expert Forum (CHIEF) classification. The whiskers indicate the range of values, the boxes represent the inter quartile range (IQR), and the lines within the boxes indicate the median. Outliers are indicated by individual points. *Statistically significant difference from all other stages. + Statistically significant difference from controls, B1, B2, C3. ¤ Statistically significant difference from controls, B1, B2, C1-2. ¥ Statistically significant difference from controls, B1, B2.
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Figure 2. Box plots illustrating the concentration of pro-atrial natriuretic peptide 31-67 (proANP) in groups of dogs with myxomatous mitral valve disease categorized according to the Canine Heart failure International Expert Forum (CHIEF) classification. The whiskers indicate the range of values, the boxes represent the inter quartile range (IQR) and the lines within the boxes indicate the median. Outliers are indicated by individual points. ˄ Statistically significant difference from B2, C1-3, D. ‡ Statistically significant difference from controls, B1, C1-3, D. ¥ Statistically significant difference from controls, B1, B2.
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The ROC curve analyses were used to calculate cut-off values for reliably predicting different clinical stages. The best cut-off value to predict whether a dog with dyspnea but without a murmur (control vs C2, C3 and D) had CHF or not was > 1181 pmol/L for NT-proBNP, with a sensitivity of 83%, a specificity of 98%, and an AUC of 0.95. For proANP, the cut-off value was > 1366 fmol/mL with a sensitivity of 91%, a specificity of 90%, and an AUC of 0.96.
To distinguish between dogs with acute heart failure due to MMVD (C2, C3, and D) and dogs with MMVD (B1 and B2) but without heart failure, an NT-proBNP cut-off value of 1207 pmol/L and a proANP cut-off of 1578 fmol/mL provided the best sensitivity (83% and 83%, respectively) and specificity (85% and 86%, respectively; Figure 3). AUC was 0.89 and 0.9, respectively. Using the higher NT-proBNP cut-off value of > 1800 pmol/L recommended by the manufacturer increased the specificity to 91%, but resulted in a sensitivity of only 70%.
Figure 3. Interactive dot diagrams displaying sensitivity (Sens) and specificity (Spec, in%) of pro-brain natriuretic peptide (NT-proBNP, top) and pro-atrial natriuretic peptide 31-67 (proANP, bottom) at diagnostic cut-off values (horizontal lines) of 1207 pmol/L and 1578 fmol/mL, respectively, to distinguish groups of dogs with myxomatous mitral valve disease categorized according to the Canine Heart failure International Expert Forum (CHIEF) as asymptomatic dogs (CHIEF B, n = 138) from dogs with acute congestive heart failure (CHIEF C2, C3, D, n = 78). The dots above the line in the CHIEF B group indicate false-positive test results, and dots below the line in the CHIEF C2, C3 and D groups represent false-negative test results.
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Although there was a significant difference between B1 and B2, the calculated cut-off value for NT-proBNP (518 pmol/L) and proANP (1065 fmol/mL) could not reliably detect patients with cardiac enlargement, because sensitivity (83% and 64%, respectively) and specificity (50% and 55%, respectively) were comparatively low, and there was a large overlap between the 2 groups (AUC was 0.7 and 0.7, respectively; Figure 4). An NT-proBNP cut-off of 545 pmol/L and a proANP cut-off of 1246 fmol/mL were not useful to differentiate dogs in stage B from controls due to low sensitivity (60% and 39%, respectively) and specificity (75% and 87%).
Figure 4. Interactive dot diagrams displaying sensitivity (Sens) and specificity (Spec, in%) of pro-brain natriuretic peptide (NT-proBNP, top) and pro-atrial natriuretic peptide 31-67 (proANP, bottom) diagnostic cut-off values (horizontal lines) of 518 pmol/L and 1065 fmol/mL, respectively, to distinguish dogs with myxomatous mitral valve disease categorized according to the Canine Heart failure International Expert Forum (CHIEF) with heart failure class B1 (n = 86) from dogs with heart failure class B2 (n = 52). The dots above the line in the CHIEF B1 group indicate false-positive test results, and dots below the line in the CHIEF B2 group represent false-negative test results.
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No statistically significant differences were found between the NT-proBNP and proANP values in the long term (n = 149, controls: n = 31, CHIEF B1: n = 30, CHIEF B2: n = 29, CHIEF C1: n = 18, CHIEF C2: n = 24, CHIEF C3: n = 13, CHIEF D: n = 4) and short term (n = 99, controls: n = 41, CHIEF B1: n = 16, CHIEF B2: n = 13, CHIEF C1: n = 18, CHIEF C2: n = 6, CHIEF C3: n = 5, CHIEF D: n = 0) samples. CHIEF D groups were not compared.
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Biomarkers might be beneficial in situations where conventional radiographic examinations are not sensitive enough to allow an accurate diagnosis or when echocardiography is not available or too expensive. The aim of this study was to evaluate whether NPs are a useful diagnostic variable to differentiate between the various disease stages in a comparatively large study population of dogs with MMVD and to investigate the influence of age, body size, and sex on NT-proBNP and proANP concentrations in a healthy dog population.
Similar to previous reports, the present study shows significantly higher concentrations of NT-proBNP and proANP in patients with CHF in comparison with the healthy control group or asymptomatic dogs with MMVD.[28, 32, 34, 45] In addition, our results suggest that a cardiac dysfunction as the cause of dyspnea can be ruled out at NT-proBNP concentrations of < 1181 pmol/L and proANP concentrations of < 1366 fmol/L with high specificity in dyspneic dogs presenting without a cardiac murmur. However, clinically, this question is rarely relevant, as small dogs without a murmur will not have CHF due to MMVD. Therefore, NPs could be useful as a biomarker for rare cases, such as dogs that are difficult to auscultate.
The results of this study indicate that using an NT-proBNP cut-off value of > 1207 pmol/L and a proANP cut-off value of > 1578 fmol/mL allows the differentiation of dogs with MMVD and CHF from dogs with MMVD without CHF with reasonable sensitivity and specificity. These cut-offs are higher than the ones discussed above likely due to cardiac enlargement present in some asymptomatic dogs with MMVD. Although a different assay was used in the present study, our cut-off value is very similar to those of 2 other reports, which reported a cut-off concentration of NT-proBNP > 1158 pmol/L to detect heart failure due to any kind of cardiac disease and a cut-off of proANP > 1400 pmol/L to differentiate dogs with decompensated MMVD and DCM from dogs with primary respiratory disease. However, as shown in Figure 3, there is some overlap in NP concentrations between symptomatic and asymptomatic patients, similar to the findings in other veterinary and human studies.[28, 47] Using a higher cut-off value of > 1800 for NT-proBNP resulted in an increased specificity of 91%, but lower sensitivity of only 71%. Therefore, under clinical circumstances, chances are better that higher NP concentrations correctly predict CHF. These results show that the measurement of serum NP concentrations in dogs with radiographic and echocardiographic examinations non-diagnostic for CHF can represent a useful diagnostic tool. Furthermore, a rapid (bed side) diagnostic test method for NT-proBNP might be helpful in the assessment of cardiac dysfunction in dyspneic dogs in critical care, as shown in human medicine. However, NP levels must be interpreted with caution because increased concentrations can occur in dogs with primary respiratory disease and pulmonary hypertension.
To the authors' knowledge, this is the first study using the CHIEF system for disease staging. NT-proBNP-concentrations of dogs in B1 and B2 and proANP concentrations of dogs in B2 were significantly higher compared with the healthy control group, which confirm results of another study demonstrating a significant difference between dogs in ISACHC Ia and b, and the healthy control group. Class CHIEF B1 and ISACHC Ia are comparable, because they both refer to asymptomatic patients with no chamber enlargement. Using an NT-proBNP cut-off value of > 545 pmol/L dogs in CHIEF B1 and B2 can be differentiated from the healthy population with a low sensitivity and specificity. This is in contrast to 2 other studies showing comparable NT-proBNP and NT-proANP concentrations between in CKCS with mild and moderate MMVD or dogs of various breeds in ISACHC stage Ia, respectively, and a healthy control group.[21, 33] The reason for these conflicting results is not clear. However, in the present study, the study population was comparatively large, which may have increased the probability of detecting a significant difference between groups.
Due to the low sensitivity and specificity of the calculated cut-offs, NP measurements are not useful to differentiate between healthy and asymptomatic patients with MMVD. Rather, dogs with MMVD can easily be identified by a heart murmur based on auscultation, a very reliable, cost-efficient, and non-invasive diagnostic tool. Using NPs to detect asymptomatic heart disease seems to be more useful in the occult stage of DCM. Many of these dogs have no obvious murmurs and may therefore benefit from NT-proBNP testing.
The prediction of cardiac enlargement in asymptomatic patients is relevant as such dogs might benefit from early therapy. Both NT-proBNP and proANP concentrations were significantly different between CHIEF B1 and B2. An NT-proBNP cut-off value of 518 pmol/L and a proANP value of 1065 fmol/mL allowed discrimination of dogs in stage B1 from dogs in stage B2 with a reasonable sensitivity, but low specificity. Another recent study reported a significant difference in NT-proBNP concentration between patients in ISACHC class Ia and those in class Ib in a cohort of 72 asymptomatic dogs with MMVD. In contrast, NT-proBNP could not discriminate between ISACHC stage Ia and Ib in another report with 44 dogs. This discrepancy might again be attributable to the low number of dogs (44 vs 559) examined. As stage ISACHC Ib includes the CHIEF stages B2 and C1, a direct comparison with the results of the present study is not possible.
Although in our study there was a statistically significant difference of NT-proBNP and proANP concentrations between CHIEF B1 and B2, the cut-offs cannot be used as a basis for clinical decisions based on the extensive overlap between the groups, and radiographic or echocardiographic examinations remain the appropriate diagnostic modalities of choice to differentiate between CHIEF stages B1 and B2.
The present study indicates that NP testing could be useful to monitor disease progression, as NP concentrations increased with severity of disease. Serial testing of NPs might be useful for monitoring disease progress and identifying patients with clinically significant heart disease; however, the reported weekly or daily variability of NPs might be a limiting factor. This, hypothesis has to be confirmed by further studies.
Another interesting and potentially clinically relevant result of our study were the statistically significant differences in NT-proBNP and proANP concentrations between intact male and female dogs. These results are in agreement with several studies in people, where BNP, ANP, and NT-proBNP concentrations were significantly higher in women than in men.[24, 51, 52] The reason for these results is not completely clear. One recent study suggests that androgens are mediating sex-related differences in BNP and NT-proBNP levels.
There was no difference in NP concentrations between different body size and age groups, which is consistent with findings in other canine studies.[27, 28, 50] However, in one study including only healthy CKCS, NT-proANP was lower in larger breeds and higher in older dogs, and in another study, a population of overweight dogs had significantly lower NT-proANP concentrations than controls with normal BMI. Also, a study in Doberman Pinschers showed significantly increased NT-proBNP concentration in dogs > 8 years of age. These results are similar to those of human studies, where NP levels decrease with increasing BMI and rise in older populations.[24, 26] These discrepancies might be explained by the fact that the dogs in the present study were classified according to their absolute weight and age. BMI and different life expectancy among various breeds were not taken into consideration.
Some of the samples were stored for 3 or more years until analysis. This might represent a limitation of the present study as, to the authors' knowledge, there are no reports on long-term stability of NPs in veterinary medicine. In one human study, there was no substantial loss of immunoreactivity after a 2-year storage of NT-proBNP at −20°C. We found no statistically significant difference between long-term and short-term stored samples. Although this suggests that NPs in our samples were stable at−70○C, a validation study addressing storage stability is warranted.
Some of the apparently healthy dogs and some of the dogs with mild MMVD had NT-proBNP and proANP concentrations in a range usually observed in patients with CHF. About half of the patients with increased NT-proBNP concentrations had concurrent high proANP values. All other dogs had either increased NT-proBNP levels or increased proANP alone. Whether these dogs had a subclinical undiagnosed disease, falsely elevated levels due to assay interference, or whether this represents a wide biological range leading to an overlap between healthy and affected dogs is not clear. No further screening for additional diseases was undertaken, except measurement of urea and creatinine concentrations, 2 parameters known to be of limited sensitivity to indicate chronic disease.
Although this study included a large dog population, the number of animals in class CHIEF D was very low, resulting in low statistical power. Furthermore, to define an optimal cut-off value to discriminate between patients with dyspnea and/or coughing due to CHF, and dogs with respiratory disease, a group with dyspnea related to primary respiratory disease and concurrent asymptomatic MMVD is needed. There was no such group in the present study resulting in the limited reliability of the cut-off value. Finally, mean age and body weight ranges of the control group compared with the MMVD group are limiting the power of the current study.
In summary, this study showed that NT-proBNP and proANP could differentiate dogs with CHF from patients with MMVD not yet affected by CHF. However, due to low sensitivity and specificity, NT-proBNP and proANP testing is not useful to detect asymptomatic dogs with MMVD, and to differentiate CHIEF stages B1 and B2 in asymptomatic dogs. Significant differences between healthy intact male and female dogs suggest that sex-specific reference intervals should be determined to help interpret concentrations of NT-proBNP and proANP.