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Brain-natriuretic peptide and cyclic guanosine monophosphate as biomarkers of myxomatous mitral valve disease in dogs
The Veterinary Journal 189 (2011) 349–352
Contents lists available at ScienceDirect
The Veterinary Journal journal homepage: www.elsevier.com/locate/tvjl
Short Communication
Brain-natriuretic peptide and cyclic guanosine monophosphate as biomarkers of myxomatous mitral valve disease in dogs Sophia G. Moesgaard a,⇑, Torkel Falk a, Tom Teerlink b, Halldóra H. Guðmundsdóttir a, Sif Sigurðardóttir a, Caroline E. Rasmussen a, Lisbeth H. Olsen a a b
Department of Basic Animal and Veterinary Sciences, The Faculty of Life Sciences, University of Copenhagen, Denmark Department of Clinical Chemistry, VU University Medical Centre, Amsterdam, The Netherlands
a r t i c l e
i n f o
Article history: Accepted 27 July 2010
Keywords: Dog Myxomatous mitral valve disease Biomarkers Heart failure Pro-brain-natriuretic peptide (NT-proBNP) Cyclic guanosine monophosphate (cGMP) L-arginine Asymmetric dimethylarginine (ADMA)
a b s t r a c t Elevations in the plasma concentrations of natriuretic peptides correlate with increased severity of myxomatous mitral valve disease (MMVD) in dogs. This study correlates the severity of MMVD with the plasma concentrations of the biomarkers N-terminal fragment of the pro-brain-natriuretic peptide (NT-proBNP) and its second messenger, cyclic guanosine monophosphate (cGMP). Furthermore, the L-arginine:asymmetric dimethylarginine (ADMA) ratio was measured as an index of nitric oxide availability. The study included 75 dogs sub-divided into five groups based on severity of MMVD as assessed by clinical examination and echocardiography. Plasma NT-proBNP and cGMP concentrations increased with increasing valve dysfunction and were significantly elevated in dogs with heart failure. The cGMP:NT-proBNP ratio decreased significantly in dogs with heart failure, suggesting the development of natriuretic peptide resistance. Although the L-arginine:ADMA ratio decreased with increasingly severe MMVD, this was largely due to the older age of the dogs with heart failure. Ó 2010 Elsevier Ltd. All rights reserved.
Natriuretic peptides (NP) are plasma biomarkers that correlate with the severity of the commonly diagnosed myxomatous mitral valve disease (MMVD) in dogs (Prosek et al., 2007; Chetboul et al., 2009; Tarnow et al., 2009). These peptides act through increasing the synthesis of cyclic 30 ,50 -guanosine monophosphate (cGMP) synthesis. Some studies have indicated that dogs with MMVD have decreased nitric oxide (NO) availability (Pedersen et al., 2003), which may also influence cGMP synthesis. Asymmetric dimethylarginine (ADMA) is an endogenous inhibitor of NO synthase, and the L-arginine:ADMA ratio is used as an index of NO availability (Böger et al., 1998). In this study we tested the hypothesis that the plasma concentrations of the N-terminal fragment of pro-brain NP (NT-proBNP) and cGMP, and of the L-arginine:ADMA ratio, reflect the severity of MMVD. The study included 75 dogs without evidence of clinically significant systemic disease except for a group of dogs with congestive heart failure (CHF). Where dogs in the CHF group were not already being treated, therapy was initiated as soon as a diagnosis of heart disease was confirmed. All dogs were P4 years of age, and had been fasted for 12–18 h prior to sampling. The dogs were categorised into five groups according to their degree of mitral regurgitation (MR): (1) Cairn terriers (CT) with no or minimal (615%)
⇑ Corresponding author. Tel.: +45 35333884; fax: +45 35332525. E-mail address: [email protected] (S.G. Moesgaard). 1090-0233/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.tvjl.2010.07.019
MR; (2) Cavalier King Charles spaniels (CKCS) with no or minimal (615%) MR; (3) CKCS with mild (>15% and 650%) MR; (4) CKCS with moderate to severe (>50%) MR, and (5) dogs of nine different breeds with clinical signs of CHF, diagnosed as previously described except that, in the current study, thoracic radiographs were not used (Tarnow et al., 2009). Patient assessment included blood sampling, clinical examination, electrocardiography and echocardiography (Tarnow et al., 2009). Plasma NT-proBNP and cGMP concentrations were determined using the commercial kits, Canine Cardioscreen (Vetsign, Biomedica) and Acetylation assay (GE Healthcare, Amersham), respectively (Forfia et al., 2007; Tarnow et al., 2009). Intra-assay coefficients of variation were <7% for the NT-proBNP, and <15% for the cGMP assays, respectively. The plasma concentrations of L-arginine and ADMA were determined simultaneously by high performance liquid chromatography (Moesgaard et al., 2007). Continuous response variables were assessed using multivariate analysis (general linear model, SAS statistical software) including relevant co-variates. Least squares means were compared by multiple Student t tests with a Tukey adjustment for multiple comparisons. Relevant correlations were tested by multiple linear regression analyses. For each model, the residuals were tested for normality and homogeneity of variation and both the NT-proBNP concentration and cGMP:NT-proBNP ratio were log-transformed to obtain normality of the residuals. Differences were considered statistically significant when P < 0.05.
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S.G. Moesgaard et al. / The Veterinary Journal 189 (2011) 349–352
Table 1 Details of signalment, clinical findings and concentrations of plasma biomarkers pro-brain-natriuretic peptide (NT-proBNP), cyclic guanosine monophosphate (cGMP), L-arginine, asymmetric dimethylarginine (ADMA) and of serum creatinine in dogs sub-divided into five groups based on severity of mitral regurgitation (MR).
n Gender (M/F) Age (years) Bodyweight (kg) Heart rate (beats/min) Serum creatinine (lmol/L) cGMP (pmol/mL) NT-proBNP (pmol/L) cGMP:NT-proBNP ratio ADMA (lmol/L) L-arginine
(lmol/L)
L-arginine:ADMA
ratio
Group 1 CT,a no or minimal MR
Group 2 CKCS,b no or minimal MR
Group 3 CKCS, mild MR
Group 4 CKCS, moderate to severe MR
Group 5 Dogs in heart failure
17 8/9 5.13 (4.70–6.93) 9.00 (7.60–10.30) 104 (96–116)* 76 (72–80) 40.28 ± 14.39 355 (111–600) 119 (44–382) 1.50 ± 0.26 122.35 ± 25.87
17 6/11 4.61 (4.37–6.42) 9.00 (8.00–10.00) 120 (100–132) 61 (59–68) 49.16 ± 13.94 495 (195–696) 109 (79–129) 1.51 ± 0.21 104.84 ± 13.76
13 7/6 6.21 (4.79–7.06) 9.20 (8.40–9.75) 120 (108–140) 61 (56–70) 50.93 ± 11.11 540 (247–920) 83 (63–168) 1.45 ± 0.19 96.90 ± 19.34
12 6/6 8.82 (6.68–9.81)* 8.38 (7.75–9.75) 122 (112–130) 65 (53–68) 56.16 ± 16.88 825 (342–1344) 59 (37–194) 1.52 ± 0.19 96.31 ± 22.61
16 9/7 11.0 (9.75–13.50)* 9.35 (8.05–12.00) 140 (128–156)* 98 (81–110)* 75.53 ± 16.87* 2935 (1224–4008)* 30 (22–155)# 2.13 ± 0.48 108.73 ± 27.64
83.02 ± 17.09*
70.80 ± 14.43
67.87 ± 14.95
63.92 ± 10.85
52.58 ± 15.29#
Data shown as means ± SD or medians with 25–75th percentiles. a CT, Cairn terriers. b CKCS, Cavalier King Charles spaniels. * P < 0.05 compared to all other groups. # P < 0.05 compared to groups 1–3.
Table 2 Details of final multivariate models for logarithmically (log)-transformed N-terminal fragment of the pro-brain-natriuretic peptide (NT-proBNP) and cyclic guanosine monophosphate (cGMP) plasma concentrations and of cGMP:NT-proBNP and Larginine:asymmetric dimethylarginine (ADMA) ratios as response variables, along with details of echocardiographic measurements, age, bodyweight, heart rate and creatinine concentrations as explanatory variables in dogs with clinical evidence of cardiac disease. Variablea
Estimate
P value
r2 b
Log NT-proBNP LA:Ao ratio LVEDDc Jet size
1.397 – 0.017
0.0005 NS <0.0001
0.15 – 0.31
cGMP LA:Ao ratio Age LVEDD Heart rate Jet size
20.94 1.840 1.249 0.192 0.285
0.001 0.02 <0.0001 0.04 <0.0001
0.34 0.38
Log cGMP: NT-proBNP ratio LA:Ao ratio LVEDD Jet size c
– 0.197 0.010
NS 0.0155 0.0018
– 0.08 0.13
L-arginine:ADMA ratio LA:Ao ratio Age LVEDD Age Jet size
4.845 2.943 0.528 2.401 0.247
0.45 0.0004 0.09 0.004 <0.0001
0.27
0.36
0.29 0.29
NS, not significant; LVEDD, left ventricular end-diastolic diameter; LA:Ao, ratio of left atrial to aortic root diameter. a Echocardiographic variables were entered in the model separately along with age, bodyweight, heart rate and creatinine concentration as possible predictors. Bodyweight and creatinine concentration did not significantly influence the response variables. b Reported r2 is for a model with either LA:Ao ratio, LVEDD or jet size together with other significant explanatory variables. c None of the explanatory variables were found to be significant.
Tables 1 and 2 detail the signalment, clinical findings, concentrations of plasma biomarkers and results of regression analyses, relating to the dogs under study. Plasma NT-proBNP and cGMP concentrations were significantly higher in dogs with CHF compared to all other groups (Fig. 1). The cGMP:NT-proBNP ratio was significantly lower in dogs with CHF relative to all groups except dogs with moderate to severe MR (Fig. 1). Echocardiographic mea-
surements were significantly associated with plasma NT-proBNP and cGMP concentrations and with cGMP:NT-proBNP (Table 2). There were significant correlations between the plasma cGMP, NT-proBNP and ADMA concentrations (Fig. 2). Although age was a responsible predictor of decreased L-arginine:ADMA, jet size was also significantly associated with the L-arginine:ADMA, independent of increasing age (Table 2). The findings confirmed that the plasma concentration of NTproBNP increases significantly in dogs with CHF due to MMVD (Chetboul et al., 2009; Tarnow et al., 2009). The fact that increased NT-proBNP did not significantly relate to severe, pre-clinical MR, may reflect the relatively small size of the study group. The NTproBNP concentrations found in the current study were approximately 60% higher than those found previously in similar studies performed at the same laboratory (Tarnow et al., 2009). Such differences in the measurement range of the test kit hinder the comparison of these studies, and we propose that cut-off values should be refined for this assay. Plasma cGMP concentration increased with increasing severity of MMVD and the cGMP:NT-proBNP decreased in dogs with CHF. This finding of reduced cGMP generation despite high NP stimulation is consistent with NP resistance as has been shown in humans and in dogs with experimentally-induced CHF (Forfia et al., 2007; Lourenco et al., 2009). In the present study dogs with CHF had decreased L-arginine:ADMA, whereas this parameter was unchanged in animals without clinical signs. The dogs with CHF were significantly older and this age-effect was greater than the effect of MMVD on the L-arginine:ADMA ratio. This suggests that, in general, dogs might have decreased NO availability with increasing age as occurs in humans (Miyazaki et al., 1999). The biomarker ADMA appeared to be more related to kidney function than to MMVD status, and this might explain the link between ADMA and cGMP, since renal insufficiency can also influence the plasma concentration of cGMP (Sharma et al., 2009). In future studies it will be important to further standardise the age of the dogs assessed given we found that increasing age, in itself, had a significant effect on several biomarkers. Furthermore, it must also be considered that the plasma concentrations of these compounds could have been influenced by the medication given to the animals in CHF on the day of sampling. Future work must consider the assessment of larger groups of dogs, particularly animals in the late, pre-clinical stages of disease, in order to identify if the concentration of any of these biomarkers is significantly altered prior to the onset of clinical signs.
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Fig. 1. Plasma concentrations of the biomarkers N-terminal fragment pro-brain-natriuretic peptide (NT-proBNP) (A) and cyclic guanosine monophosphate (cGMP) (B) were elevated in dogs with congestive heart failure (CHF) compared to all other groups. The cGMP:NT-proBNP (C) and L-arginine:asymmetric dimethylarginine (ADMA) (D) ratios were significantly decreased in dogs with CHF compared to dogs with no, minimal and mild mitral regurgitation (MR). The L-arginine:ADMA (D) was significantly higher in Cairn terriers (CT). The decrease in the L-arginine:ADMA in dogs with CHF was largely because dogs with CHF were older. Solid bars represent medians. P < 0.05 compared to all dog groups; #P < 0.05 compared to dogs with no, minimal and mild MR; CKCS, Cavalier King Charles spaniels.
B
150
Plasma cGMP concentration (pmol/mL)
Plasma cGMP concentration (pmol/mL)
A
100
50
0 0
2000
4000
6000
8000
Plasma NT-proBNP concentration (pmol/L)
150
100
50
0 0
1
2
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Plasma ADMA concentration (µM)
Fig. 2. Plasma concentrations of N-terminal fragment pro-brain-natriuretic peptide (NT-proBNP) (A) (r2 = 0.30; P < 0.0001) and asymmetric dimethylarginine (ADMA) (B) (r2 = 0.28; P < 0.0001) correlated significantly with plasma cyclic guanosine monophosphate (cGMP) levels. All biomarkers increased with an increasing degree of myxomatous mitral valve disease, although the increase in ADMA mainly correlated to serum creatinine levels and to advancing age.
In conclusion, we found that the plasma concentration of the biomarkers NT-proBNP and cGMP increased with increasingly severe MR in dogs and both were significantly elevated in dogs with CHF. The cGMP:NT-proBNP ratio was also significantly
decreased in dogs with CHF consistent with the development of NP resistance. The L-arginine:ADMA ratio decreased in dogs with CHF, a finding related largely to the advanced age of these animals.
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S.G. Moesgaard et al. / The Veterinary Journal 189 (2011) 349–352
Conflict of interest statement None of the authors of this paper has a financial or personal relationship with other people or organisations that could inappropriately influence or bias the content of the paper. Acknowledgements This work was supported by: The Danish Medical Research Council (Project Nos. 271-07-0784 and 271-05-0335); The Foundation for Scientific Studies Concerning Diseases of Small Companion Animals and their Treatments; and The Henry and Astrid Moellers’ Foundation. The authors thank Birgitte Holle and Christina Tirsdahl Kjempf of The Department of Basic Animal and Veterinary Sciences, Faculty of Life Sciences, University of Copenhagen, and Sigrid de Jong, Department of Clinical Chemistry, V.U. University Medical Center, The Netherlands for their excellent technical assistance. References Böger, R.H., Bode-Boger, S.M., Szuba, A., Tsao, P.S., Chan, J.R., Tangphao, O., Blaschke, T.F., Cooke, J.P., 1998. Asymmetric dimethylarginine (ADMA): a novel risk factor for endothelial dysfunction – its role in hypercholesterolemia. Circulation 98, 1842–1847. Chetboul, V., Serres, F., Tissier, R., Lefebvre, H.P., Carlos Sampedrano, C., Gouni, V., Poujol, L., Hawa, G., Pouchelon, J.-L., 2009. Association of plasma N-terminal
pro-B-type natriuretic peptide concentration with mitral regurgitation severity and outcome in dogs with asymptomatic degenerative mitral valve disease. Journal of Veterinary Internal Medicine 23, 984–994. Forfia, P.R., Lee, M., Tunin, R.S., Mahmud, M., Champion, H.C., Kass, D.A., 2007. Acute phosphodiesterase 5 inhibition mimics hemodynamic effects of B-type natriuretic peptide and potentiates B-type natriuretic peptide effects in failing but not normal canine heart. Journal of the American College of Cardiology 49, 1079–1088. Lourenco, P., Araujo, J.P., Azevedo, A., Ferreira, A., Bettencourt, P., 2009. The cyclic guanosine monophosphate/B-type natriuretic peptide ratio and mortality in advanced heart failure. European Journal of Heart Failure 11, 185–190. Miyazaki, H., Matsuoka, H., Cooke, J.P., Usui, M., Ueda, S., Okuda, S., Imaizumi, T., 1999. Endogenous nitric oxide synthase inhibitor – a novel marker of atherosclerosis. Circulation 99, 1141–1146. Moesgaard, S.G., Holte, A.V., Mogensen, T., Mølbak, J., Kristensen, A.T., Jensen, A.L., Teerlink, T., Reynolds, A.J., Olsen, L.H., 2007. Effects of breed, gender, exercise and white-coat effect on markers of endothelial function in dogs. Research in Veterinary Science 82, 415. Pedersen, H.D., Schutt, T., Sondergaard, R., Qvortrup, K., Olsen, L.H., Kristensen, A.T., 2003. Decreased plasma concentration of nitric oxide metabolites in dogs with untreated mitral regurgitation. Journal of Veterinary Internal Medicine 17, 178– 184. Prosek, R., Sisson, D.D., Oyama, M.A., Solter, P.F., 2007. Distinguishing cardiac and noncardiac dyspnea in 48 dogs using plasma atrial natriuretic factor, B-type natriuretic factor, endothelin, and cardiac troponin-I. Journal of Veterinary Internal Medicine 21, 238–242. Sharma, M., Zhou, Z.M., Miura, H., Papapetropoulos, A., McCarthy, E.T., Sharma, R., Savin, V.J., Lianos, E.A., 2009. ADMA injures the glomerular filtration barrier: role of nitric oxide and superoxide. American Journal of Physiology – Renal Physiology 296, F1386–F1395. Tarnow, I., Olsen, L.H., Kvart, C., Hoglund, K., Moesgaard, S.G., Kamstrup, T.S., Pedersen, H.D., Haggstrom, J., 2009. Predictive value of natriuretic peptides in dogs with mitral valve disease. The Veterinary Journal 180, 195–201.
Contents lists available at ScienceDirect
The Veterinary Journal journal homepage: www.elsevier.com/locate/tvjl
Short Communication
Brain-natriuretic peptide and cyclic guanosine monophosphate as biomarkers of myxomatous mitral valve disease in dogs Sophia G. Moesgaard a,⇑, Torkel Falk a, Tom Teerlink b, Halldóra H. Guðmundsdóttir a, Sif Sigurðardóttir a, Caroline E. Rasmussen a, Lisbeth H. Olsen a a b
Department of Basic Animal and Veterinary Sciences, The Faculty of Life Sciences, University of Copenhagen, Denmark Department of Clinical Chemistry, VU University Medical Centre, Amsterdam, The Netherlands
a r t i c l e
i n f o
Article history: Accepted 27 July 2010
Keywords: Dog Myxomatous mitral valve disease Biomarkers Heart failure Pro-brain-natriuretic peptide (NT-proBNP) Cyclic guanosine monophosphate (cGMP) L-arginine Asymmetric dimethylarginine (ADMA)
a b s t r a c t Elevations in the plasma concentrations of natriuretic peptides correlate with increased severity of myxomatous mitral valve disease (MMVD) in dogs. This study correlates the severity of MMVD with the plasma concentrations of the biomarkers N-terminal fragment of the pro-brain-natriuretic peptide (NT-proBNP) and its second messenger, cyclic guanosine monophosphate (cGMP). Furthermore, the L-arginine:asymmetric dimethylarginine (ADMA) ratio was measured as an index of nitric oxide availability. The study included 75 dogs sub-divided into five groups based on severity of MMVD as assessed by clinical examination and echocardiography. Plasma NT-proBNP and cGMP concentrations increased with increasing valve dysfunction and were significantly elevated in dogs with heart failure. The cGMP:NT-proBNP ratio decreased significantly in dogs with heart failure, suggesting the development of natriuretic peptide resistance. Although the L-arginine:ADMA ratio decreased with increasingly severe MMVD, this was largely due to the older age of the dogs with heart failure. Ó 2010 Elsevier Ltd. All rights reserved.
Natriuretic peptides (NP) are plasma biomarkers that correlate with the severity of the commonly diagnosed myxomatous mitral valve disease (MMVD) in dogs (Prosek et al., 2007; Chetboul et al., 2009; Tarnow et al., 2009). These peptides act through increasing the synthesis of cyclic 30 ,50 -guanosine monophosphate (cGMP) synthesis. Some studies have indicated that dogs with MMVD have decreased nitric oxide (NO) availability (Pedersen et al., 2003), which may also influence cGMP synthesis. Asymmetric dimethylarginine (ADMA) is an endogenous inhibitor of NO synthase, and the L-arginine:ADMA ratio is used as an index of NO availability (Böger et al., 1998). In this study we tested the hypothesis that the plasma concentrations of the N-terminal fragment of pro-brain NP (NT-proBNP) and cGMP, and of the L-arginine:ADMA ratio, reflect the severity of MMVD. The study included 75 dogs without evidence of clinically significant systemic disease except for a group of dogs with congestive heart failure (CHF). Where dogs in the CHF group were not already being treated, therapy was initiated as soon as a diagnosis of heart disease was confirmed. All dogs were P4 years of age, and had been fasted for 12–18 h prior to sampling. The dogs were categorised into five groups according to their degree of mitral regurgitation (MR): (1) Cairn terriers (CT) with no or minimal (615%)
⇑ Corresponding author. Tel.: +45 35333884; fax: +45 35332525. E-mail address: [email protected] (S.G. Moesgaard). 1090-0233/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.tvjl.2010.07.019
MR; (2) Cavalier King Charles spaniels (CKCS) with no or minimal (615%) MR; (3) CKCS with mild (>15% and 650%) MR; (4) CKCS with moderate to severe (>50%) MR, and (5) dogs of nine different breeds with clinical signs of CHF, diagnosed as previously described except that, in the current study, thoracic radiographs were not used (Tarnow et al., 2009). Patient assessment included blood sampling, clinical examination, electrocardiography and echocardiography (Tarnow et al., 2009). Plasma NT-proBNP and cGMP concentrations were determined using the commercial kits, Canine Cardioscreen (Vetsign, Biomedica) and Acetylation assay (GE Healthcare, Amersham), respectively (Forfia et al., 2007; Tarnow et al., 2009). Intra-assay coefficients of variation were <7% for the NT-proBNP, and <15% for the cGMP assays, respectively. The plasma concentrations of L-arginine and ADMA were determined simultaneously by high performance liquid chromatography (Moesgaard et al., 2007). Continuous response variables were assessed using multivariate analysis (general linear model, SAS statistical software) including relevant co-variates. Least squares means were compared by multiple Student t tests with a Tukey adjustment for multiple comparisons. Relevant correlations were tested by multiple linear regression analyses. For each model, the residuals were tested for normality and homogeneity of variation and both the NT-proBNP concentration and cGMP:NT-proBNP ratio were log-transformed to obtain normality of the residuals. Differences were considered statistically significant when P < 0.05.
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S.G. Moesgaard et al. / The Veterinary Journal 189 (2011) 349–352
Table 1 Details of signalment, clinical findings and concentrations of plasma biomarkers pro-brain-natriuretic peptide (NT-proBNP), cyclic guanosine monophosphate (cGMP), L-arginine, asymmetric dimethylarginine (ADMA) and of serum creatinine in dogs sub-divided into five groups based on severity of mitral regurgitation (MR).
n Gender (M/F) Age (years) Bodyweight (kg) Heart rate (beats/min) Serum creatinine (lmol/L) cGMP (pmol/mL) NT-proBNP (pmol/L) cGMP:NT-proBNP ratio ADMA (lmol/L) L-arginine
(lmol/L)
L-arginine:ADMA
ratio
Group 1 CT,a no or minimal MR
Group 2 CKCS,b no or minimal MR
Group 3 CKCS, mild MR
Group 4 CKCS, moderate to severe MR
Group 5 Dogs in heart failure
17 8/9 5.13 (4.70–6.93) 9.00 (7.60–10.30) 104 (96–116)* 76 (72–80) 40.28 ± 14.39 355 (111–600) 119 (44–382) 1.50 ± 0.26 122.35 ± 25.87
17 6/11 4.61 (4.37–6.42) 9.00 (8.00–10.00) 120 (100–132) 61 (59–68) 49.16 ± 13.94 495 (195–696) 109 (79–129) 1.51 ± 0.21 104.84 ± 13.76
13 7/6 6.21 (4.79–7.06) 9.20 (8.40–9.75) 120 (108–140) 61 (56–70) 50.93 ± 11.11 540 (247–920) 83 (63–168) 1.45 ± 0.19 96.90 ± 19.34
12 6/6 8.82 (6.68–9.81)* 8.38 (7.75–9.75) 122 (112–130) 65 (53–68) 56.16 ± 16.88 825 (342–1344) 59 (37–194) 1.52 ± 0.19 96.31 ± 22.61
16 9/7 11.0 (9.75–13.50)* 9.35 (8.05–12.00) 140 (128–156)* 98 (81–110)* 75.53 ± 16.87* 2935 (1224–4008)* 30 (22–155)# 2.13 ± 0.48 108.73 ± 27.64
83.02 ± 17.09*
70.80 ± 14.43
67.87 ± 14.95
63.92 ± 10.85
52.58 ± 15.29#
Data shown as means ± SD or medians with 25–75th percentiles. a CT, Cairn terriers. b CKCS, Cavalier King Charles spaniels. * P < 0.05 compared to all other groups. # P < 0.05 compared to groups 1–3.
Table 2 Details of final multivariate models for logarithmically (log)-transformed N-terminal fragment of the pro-brain-natriuretic peptide (NT-proBNP) and cyclic guanosine monophosphate (cGMP) plasma concentrations and of cGMP:NT-proBNP and Larginine:asymmetric dimethylarginine (ADMA) ratios as response variables, along with details of echocardiographic measurements, age, bodyweight, heart rate and creatinine concentrations as explanatory variables in dogs with clinical evidence of cardiac disease. Variablea
Estimate
P value
r2 b
Log NT-proBNP LA:Ao ratio LVEDDc Jet size
1.397 – 0.017
0.0005 NS <0.0001
0.15 – 0.31
cGMP LA:Ao ratio Age LVEDD Heart rate Jet size
20.94 1.840 1.249 0.192 0.285
0.001 0.02 <0.0001 0.04 <0.0001
0.34 0.38
Log cGMP: NT-proBNP ratio LA:Ao ratio LVEDD Jet size c
– 0.197 0.010
NS 0.0155 0.0018
– 0.08 0.13
L-arginine:ADMA ratio LA:Ao ratio Age LVEDD Age Jet size
4.845 2.943 0.528 2.401 0.247
0.45 0.0004 0.09 0.004 <0.0001
0.27
0.36
0.29 0.29
NS, not significant; LVEDD, left ventricular end-diastolic diameter; LA:Ao, ratio of left atrial to aortic root diameter. a Echocardiographic variables were entered in the model separately along with age, bodyweight, heart rate and creatinine concentration as possible predictors. Bodyweight and creatinine concentration did not significantly influence the response variables. b Reported r2 is for a model with either LA:Ao ratio, LVEDD or jet size together with other significant explanatory variables. c None of the explanatory variables were found to be significant.
Tables 1 and 2 detail the signalment, clinical findings, concentrations of plasma biomarkers and results of regression analyses, relating to the dogs under study. Plasma NT-proBNP and cGMP concentrations were significantly higher in dogs with CHF compared to all other groups (Fig. 1). The cGMP:NT-proBNP ratio was significantly lower in dogs with CHF relative to all groups except dogs with moderate to severe MR (Fig. 1). Echocardiographic mea-
surements were significantly associated with plasma NT-proBNP and cGMP concentrations and with cGMP:NT-proBNP (Table 2). There were significant correlations between the plasma cGMP, NT-proBNP and ADMA concentrations (Fig. 2). Although age was a responsible predictor of decreased L-arginine:ADMA, jet size was also significantly associated with the L-arginine:ADMA, independent of increasing age (Table 2). The findings confirmed that the plasma concentration of NTproBNP increases significantly in dogs with CHF due to MMVD (Chetboul et al., 2009; Tarnow et al., 2009). The fact that increased NT-proBNP did not significantly relate to severe, pre-clinical MR, may reflect the relatively small size of the study group. The NTproBNP concentrations found in the current study were approximately 60% higher than those found previously in similar studies performed at the same laboratory (Tarnow et al., 2009). Such differences in the measurement range of the test kit hinder the comparison of these studies, and we propose that cut-off values should be refined for this assay. Plasma cGMP concentration increased with increasing severity of MMVD and the cGMP:NT-proBNP decreased in dogs with CHF. This finding of reduced cGMP generation despite high NP stimulation is consistent with NP resistance as has been shown in humans and in dogs with experimentally-induced CHF (Forfia et al., 2007; Lourenco et al., 2009). In the present study dogs with CHF had decreased L-arginine:ADMA, whereas this parameter was unchanged in animals without clinical signs. The dogs with CHF were significantly older and this age-effect was greater than the effect of MMVD on the L-arginine:ADMA ratio. This suggests that, in general, dogs might have decreased NO availability with increasing age as occurs in humans (Miyazaki et al., 1999). The biomarker ADMA appeared to be more related to kidney function than to MMVD status, and this might explain the link between ADMA and cGMP, since renal insufficiency can also influence the plasma concentration of cGMP (Sharma et al., 2009). In future studies it will be important to further standardise the age of the dogs assessed given we found that increasing age, in itself, had a significant effect on several biomarkers. Furthermore, it must also be considered that the plasma concentrations of these compounds could have been influenced by the medication given to the animals in CHF on the day of sampling. Future work must consider the assessment of larger groups of dogs, particularly animals in the late, pre-clinical stages of disease, in order to identify if the concentration of any of these biomarkers is significantly altered prior to the onset of clinical signs.
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Fig. 1. Plasma concentrations of the biomarkers N-terminal fragment pro-brain-natriuretic peptide (NT-proBNP) (A) and cyclic guanosine monophosphate (cGMP) (B) were elevated in dogs with congestive heart failure (CHF) compared to all other groups. The cGMP:NT-proBNP (C) and L-arginine:asymmetric dimethylarginine (ADMA) (D) ratios were significantly decreased in dogs with CHF compared to dogs with no, minimal and mild mitral regurgitation (MR). The L-arginine:ADMA (D) was significantly higher in Cairn terriers (CT). The decrease in the L-arginine:ADMA in dogs with CHF was largely because dogs with CHF were older. Solid bars represent medians. P < 0.05 compared to all dog groups; #P < 0.05 compared to dogs with no, minimal and mild MR; CKCS, Cavalier King Charles spaniels.
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Fig. 2. Plasma concentrations of N-terminal fragment pro-brain-natriuretic peptide (NT-proBNP) (A) (r2 = 0.30; P < 0.0001) and asymmetric dimethylarginine (ADMA) (B) (r2 = 0.28; P < 0.0001) correlated significantly with plasma cyclic guanosine monophosphate (cGMP) levels. All biomarkers increased with an increasing degree of myxomatous mitral valve disease, although the increase in ADMA mainly correlated to serum creatinine levels and to advancing age.
In conclusion, we found that the plasma concentration of the biomarkers NT-proBNP and cGMP increased with increasingly severe MR in dogs and both were significantly elevated in dogs with CHF. The cGMP:NT-proBNP ratio was also significantly
decreased in dogs with CHF consistent with the development of NP resistance. The L-arginine:ADMA ratio decreased in dogs with CHF, a finding related largely to the advanced age of these animals.
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Conflict of interest statement None of the authors of this paper has a financial or personal relationship with other people or organisations that could inappropriately influence or bias the content of the paper. Acknowledgements This work was supported by: The Danish Medical Research Council (Project Nos. 271-07-0784 and 271-05-0335); The Foundation for Scientific Studies Concerning Diseases of Small Companion Animals and their Treatments; and The Henry and Astrid Moellers’ Foundation. The authors thank Birgitte Holle and Christina Tirsdahl Kjempf of The Department of Basic Animal and Veterinary Sciences, Faculty of Life Sciences, University of Copenhagen, and Sigrid de Jong, Department of Clinical Chemistry, V.U. University Medical Center, The Netherlands for their excellent technical assistance. References Böger, R.H., Bode-Boger, S.M., Szuba, A., Tsao, P.S., Chan, J.R., Tangphao, O., Blaschke, T.F., Cooke, J.P., 1998. Asymmetric dimethylarginine (ADMA): a novel risk factor for endothelial dysfunction – its role in hypercholesterolemia. Circulation 98, 1842–1847. Chetboul, V., Serres, F., Tissier, R., Lefebvre, H.P., Carlos Sampedrano, C., Gouni, V., Poujol, L., Hawa, G., Pouchelon, J.-L., 2009. Association of plasma N-terminal
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