Dogs
Forty-seven privately owned dogs were included in the study: 37 of them were cardiovascular patients, and ten of them were control dogs. Cardiovascular patients were further divided into two groups; patients with (26) and without (11) CHF. The progression algorithm of dogs during the inclusion into the study is shown in Figure 1. The baseline demographic characteristics of cardiovascular patients with and without CHF and control dogs are presented in Table 1. Control dogs and cardiac patients with and without CHF did not differ significantly in weight; however, patients with and without CHF were significantly (P < 0.001) older compared to control dogs.
Table 1. Baseline demographic characteristics of canine cardiac patients and control dogs
|
Control
|
All patients
|
No CHF
|
CHF
|
Number
|
10
|
37
|
11
|
26
|
Sex
Male/Female
|
3/7
|
31/6
|
9/2
|
22/4
|
Age (years)
Mean ± SD
Min–Max
|
4.4 ± 2.5*
1.0–12.5
|
8.8 ± 2.8
2.4–14.3
|
9.3 ± 3.6
2.4–14.3
|
8.6 ± 2.5
4.2–12.5
|
Weight
Median (IQR)
|
22.4 (19.2–34.3)
|
27.8 (13.2–41.9)
|
31.4 (13.6–61.5)
|
27.2 (12.8–39.3)
|
Disease
DCM/MMVD
|
/
|
16/21
|
4/7
|
12/14
|
*significant difference when compared to the groups of canine patients with CHF (p < 0.001) and without CHF (p < 0.001), and the group of all cardiac patients (p < 0.001)
Inflammatory and markers of oxidative stress and NT-proBNP concentrations
Concentrations of IL-6 were excluded from the statistical comparison because they were below the lower level of detection.
The results of inflammatory markers and NT–proBNP are presented in Table 2. NT–proBNP concentration was significantly higher in patients with CHF than in patients without CHF (P = 0.009) and control dogs (P < 0.001); however, NT-proBNP concentration between patients without CHF and control dogs did not differ significantly.
Table 2 Inflammatory markers (Median, IQR) and NT-proBNP concentrations (median, IQR) in patients and control dogs
|
Control
(n = 10)
|
No CHF
(n = 11)
|
CHF
(n = 26)
|
TNF–α (pg/mL)
|
3.90; 3.90–10.50
|
3.90; 3.90–3.90
|
3.90; 6.85–11.90a
|
IL–6 (pg/mL)
|
31.3; 31.3–31.3
|
31.3; 31.3–31.3
|
31.3; 31.3–31.3
|
CRP (mg/L)
|
0.90; 0.78–1.33
|
1.45; 0.57–7.74
|
3.53; 1.10–13.18b
|
WBC (x 109/L)
|
6.2; 5.2–8.2
|
7.7; 6.3–8.9
|
10.5; 8.1–13.6c
|
NEUT (x 109/L)
|
3.4; 2.8–5.2
|
5.2; 3.2–6.8
|
7.2; 5.7–10.3b
|
Neutrophils (%)
|
57.3; 51.2–60.7d
|
66.3; 63.2–75.9
|
69.5; 61.3–73.5
|
LYMPH (x 109/L)
|
1.83; 1.47–2.35
|
1.28; 1.18–2.26
|
1.96; 1.57–2.89a
|
Lymphocytes (%)
|
30.7; 24.8–35.0d
|
17.4; 14.2–26.0
|
20.0; 14.7–24.6
|
MONO (x 109/L)
|
0.28; 0.21–0.39
|
0.42; 0.28–0.55
|
0.60; 0.44–0.84c
|
Monocytes (%)
|
4.2; 3.6–6.4
|
5.5; 4.5–6.2
|
5.7; 4.3–7.7
|
NT–proBNP (pmol/L)
|
822; 507–1201
|
1207; 927–3086
|
4773; 2828–8529c
|
aSignificant difference (P < 0.05) when compared to patients without CHF; bSignificant difference (P < 0.05) when compared to control dogs; cSignificant difference (P < 0.05) when compared to control dogs and patients without CHF; dSignificant difference (P < 0.05) when compared to patients without and with CHF
Serum CRP concentration was significantly higher in patients with CHF than in control dogs (P = 0.012). The concentration of TNF-α was significantly (P = 0.030) higher in patients with CHF compared to patients without CHF, while no significant difference was found in comparison to control dogs.
White blood cell and monocyte counts were significantly higher in patients with CHF compared to control dogs (P = 0.001; and P = 0.001, respectively) and patients without CHF (P = 0.024 and P = 0.049, respectively). Neutrophil counts were significantly (P = 0.001) higher in patients with CHF than in control dogs. We found significantly (P = 0.020) higher lymphocyte counts in patients with CHF than in patients without CHF. Furthermore, we found significantly higher neutrophil percentages and significantly lower lymphocyte percentages in patients without (P = 0.002 and P = 0.010, respectively) and with CHF (P = 0.003 and P = 0.006, respectively) in comparison to control dogs.
Regarding oxidative stress parameters (Table 3) , we found no significant difference in malondialdehyde (MDA) concentration between groups of patients and control dogs. On the other side, glutathione peroxidase (GPX) activity was significantly (P = 0.042) higher in patients without CHF than in control dogs.
Table 3 Oxidative stress markers (mean ± SD) in patients and control dogs
|
Control
n = 10
|
No CHF
n = 11
|
CHF
n = 26
|
MDA (µmol/L)
|
1.34 ± 0.31
|
1.77 ± 0.75
|
1.32 ± 0.72
|
GPX (U/g Hgb)
|
393.7 ± 43.6
|
457.6 ± 43.7*
|
429.2 ± 65.6
|
* Significant difference (P < 0.05) when compared to control dogs
Correlations
In CHF patients, tumor necrosis alpha (TNF–α) concentrations correlated significantly positively with MDA concentrations (P = 0.014, r = 0.474) and negatively with GPX activities (P = 0.026, r = - 0.453). Furthermore, we found a significant negative correlation (P = 0.046, r = - 0.412) between IL–6 concentrations and GPX activities. Percentages of neutrophils and monocyte counts correlated significantly negatively with GPX activities (P = 0.024, r = - 0.460 and P = 0.031, r = - 0.441, respectively). On the other hand, percentage of lymphocytes correlated significantly positively with GPX activities (P = 0.006, r = 0.542), and lymphocyte counts correlated significantly negatively with MDA concentrations (P = 0.050, r = - 0.388). In this group of patients, we found a significant positive correlation between NT-proBNP and MDA concentrations (P = 0.011, r = 0.4893).
In patients without CHF, none of the inflammatory and oxidative stress markers correlated significantly.
Additionally, we correlated inflammatory and oxidative stress markers and NT-proBNP in the group of all 37 patients and found several significant correlations. Interestingly, GPX activities correlated significantly negatively with several inflammatory markers, including TNF–α concentrations (P = 0.010, r = - 0.436), IL–6 concentrations (P= 0.026, r = - 0.382), WBC (P = 0.032, r = - 0.369), and neutrophil (P = 0.027, r = - 0.379) and monocyte counts (P = 0.024, r = - 0.386). On the other hand, GPX activities correlated significantly positively with percentages of lymphocytes (P = 0.043, r = 0.348). Furthermore, we found a significant negative correlation (P = 0.009, r = - 0.423) between MDA concentrations and lymphocyte counts. In the group of all pateints, NT–proBNP concentrations correlated significantly negatively with GPX activities (P = 0.050, r = - 0.339) and positively with TNF–α concentrations (P = 0.022; r = 0.378) and monocyte count (P = 0.019, r = 0.385).