Reactive oxygen species are oxygen free radicals and highly reactive oxygen compounds that are produced during cellular metabolism and can react with any other molecule, leading to changes in molecule configuration and function. Under normal conditions, the antioxidant system of aerobic organisms successfully limits the deleterious effects of ROS in a state called redox homeostasis, which can be disrupted by an excess of ROS, a decrease in antioxidant defense, or a combination of both, causing a state of oxidative stress.1–3 There is growing evidence that oxidative stress substantially impairs the function of organs and has a major role in the etiopathogenesis of several diseases, including a broad range of cardiovascular diseases, in humans and other animals.1,4–8
With cardiovascular diseases, increased formation of ROS is generally associated with oxidative stress and subsequent cardiovascular tissue injury.5,6,9 In human medicine, attention has been paid to the role of oxidative stress in various cardiovascular diseases and to strategies to reduce oxidative stress.5,10–13 Diverse results regarding the amounts of oxidative stress factors in blood samples obtained from human cardiac patients have been demonstrated.14–16 However, a decreased concentration of CoQ10 in plasma and the myocardium seems to be a consistent finding in cardiovascular diseases in humans.17–21
Coenzyme Q10 is an endogenous fat-soluble compound produced in all living cells in humans and most other mammals. In fact, the term CoQ refers to a class of homologous benzoquinones that have been identified in all plants and animals, as well as in most microorganisms. Benzoquinone homologs are composed of a redox active quinoid moiety and a hydrophobic side chain of 6 to 10 isoprenoid units, depending on the species. In humans and most mammals, including dogs, the predominant form of CoQ is CoQ10. Coenzyme Q10, which is also referred to as ubiquinone, has 10 isoprenoid units in the side chain. In rats and mice, the primary form of CoQ is coenzyme Q9, which has 9 isoprenoid units.22–24 Coenzyme Q10 exerts various biological functions. It is an essential cofactor in mitochondrial oxidative phosphorylation and is indispensable for ATP formation. In the inner mitochondrial membrane, it acts as a carrier of electrons from respiratory complexes I and II to complex III.22,23,25,26 It is also present in other subcellular fractions and in plasma. Moreover, at the inner mitochondrial membrane level, CoQ10 is an obligatory co-factor for the function of uncoupling proteins and a modulator of the transition pore. Furthermore, data have revealed that CoQ10 affects the expression of genes involved in human cell signaling, metabolism, and transport.26 The reduced form of CoQ10, ubiquinol, is a powerful antioxidant that inhibits the peroxidation of cell membrane lipids and lipoprotein lipids present in the circulation, inhibits protein and DNA oxidation, and may have a role in recycling tocopherols because it effectively reduces alpha-tocopheryl radicals to alpha-tocopherol, thereby regenerating the active form of vitamin E.26–28 Considering the key role of CoQ10 in cellular energy production and the high energy requirements of cardiac cells, CoQ10 has a potential role in the prevention and treatment of heart ailments by improving cardiac bioenergetics.13 The results of meta-analyses of clinical trials indicate that CoQ10 supplement administration in humans with CHF results in significant improvements in stroke volume, cardiac output, ejection fraction, cardiac index, and diastolic volume index.29,30
Few studies4,7,31 regarding oxidative stress have been conducted in dogs with cardiovascular diseases, and there is only 1 study32 of CoQ10 in dogs with experimentally induced CHF to our knowledge. In dogs with idiopathic DCM, Freeman et al4 found that the oxidant-antioxidant system may have a role in the development of this disease, given that erythrocyte GSH-Px activity was significantly increased in affected dogs, compared with findings in healthy control dogs, and that there was a negative correlation between disease severity and plasma vitamin E concentration in affected dogs. Furthermore, in dogs with CHF in another study,7 some antioxidant defenses were decreased (plasma vitamin E concentration and the ratio between plasma concentrations of reduced and oxidized glutathione) and some were increased (plasma vitamin C concentration and TAC); a biomarker of oxidative stress (plasma 8F2-isoprostane concentration) was also increased. Similarly, Hetyey et al31 found increased TAC in dogs with naturally occurring heart diseases. In dogs with experimentally induced CHF, administration of supplemental CoQ10 has no effects on hemodynamics, but the supplementation does appear to attenuate the hypertrophic response associated with CHF.32 Other indices of CHF were similar in dogs receiving supplemental CoQ10 and control dogs.32
Total antioxidant capacity is used to evaluate the overall antioxidant status of plasma and body fluids resulting from antioxidant intake or production and their consumption by normal or increased amounts of ROS. The capacity of known and unknown antioxidants and their synergistic interaction is assessed, thereby providing insight into the delicate balance between oxidants and antioxidants in vivo.33,34 Measurement of TAC may help in evaluating the physiologic, environmental, and nutritional factors of the redox status.34 The aim of the study reported here was to determine the plasma TAC, erythrocyte SOD activity, whole blood GSH-Px activity, and plasma CoQ10 concentration in dogs with various stages of CHF and in healthy dogs; assess the influence of cardiac treatment on antioxidant variables, plasma CoQ10 concentration, and serum NT-proBNP concentration; and determine any correlation between the disease severity (as indicated by NT-proBNP concentration) and antioxidant variables or CoQ10 concentration. The serum concentration of the cardiac biomarker NT-proBNP was analyzed to evaluate disease severity as a comparative biomarker of myocardial wall stress in dogs.35,36 Our hypotheses were that plasma CoQ10 concentration and the levels of antioxidant variables (plasma TAC, erythrocyte SOD activity, and whole blood GSH-Px activity) are lower in dogs with cardiac diseases than in healthy dogs, that these variables decrease with the progression of cardiac disease, and that plasma CoQ10 concentration and the levels of antioxidant variables are higher in canine cardiac patients receiving cardiac treatment than in untreated patients.
Materials and Methods
Animals
Seventy-two client-owned dogs of 17 breeds were included in the study. Twenty-nine dogs were healthy controls (11 males [8 sexually intact and 3 neutered] and 18 females (11 sexually intact and 7 spayed) and were aged from 4 months to 9 years (mean age, 35.9 months). The dogs were considered healthy on the basis of history, clinical examination findings, and the results of hematologic and biochemical analyses. The other 43 dogs were patients with confirmed cardiovascular diseases. Among these dogs, 27 were sexually intact males, 3 were sexually intact females, and 13 were spayed females. Cardiovascular disease was confirmed by an experienced cardiologist on the basis of history and results of a clinical examination, radiographic examination of the thorax, standard ECG examination, and echocardiography with 2-D, M-mode, color, or spectral Doppler modes.a In addition, hematologic and biochemical analyses were performed to exclude dogs with concomitant noncardiac diseases. If a candidate dog for the study had a neoplastic, inflammatory, or infectious disease or metabolic disorder, it was not included in the study; therefore, there are no data reported for those dogs. For each dog with cardiac disease, disease severity was classified by use of the ISACHC classification scheme for cardiac disease37; for dogs classified as ISACHC II or III, current cardiac treatment, if any, was noted.
The written consent of the owners was obtained for the dogs’ study participation. All procedures complied with applicable Slovenian governmental regulations (Animal Protection Act University of Ljubljana Republic of Slovenia, 43/2007) and were approved by the Ethical Committee of the Ministry of Agriculture, Forestry and Food of the Veterinary Administration of the Republic of Slovenia.
Blood sample collection and processing
Six blood samples (total volume, 12.5 mL) were collected from a jugular vein from each healthy dog and each dog with ISACHC I, II, or III classification. Blood samples for hematologic analyses (0.5 mL) were collected into tubes containing the anticoagulant EDTA.b Blood samples for assessment of serum biochemical variables and NT-proBNP concentration (4 mL), plasma TAC (2 mL), plasma concentration of CoQ10 (2 mL), whole blood GSH-Px activity (2 mL), and erythrocyte (washed RBC) SOD activity (2 mL) were collected into tubes containing the anticoagulant lithium heparin.c Blood samples for determination of plasma TAC and plasma CoQ10 concentration were centrifuged immediately after collection at 1,500 × g for 15 minutes at 4°C. Plasma was separated from each sample and immediately frozen at −80°C until analysis. Aliquots of whole blood with heparin were prepared and immediately frozen at −80°C until analysis. Immediately after blood sample collection, hemolyzed RBCs were prepared following the manufacturer's instructions of a kitd used for erythrocyte SOD activity determination, and stored at −80°C until analysis. Hemoglobin concentration in the RBC hemolysates was determined spectrophotometrically with the cyanomethemoglobin method and an automated biochemistry analyzer.e Variables of interest were measured at 1 time point only, regardless of whether this was the dog's first examination or recheck examination.
Assessment of antioxidant variables
For all dogs, plasma TAC,f whole blood GSH-Px activity,g and erythrocyte (washed RBC) SOD activityd were determined spectrophotometrically with an automated biochemistry analyzere and commercial kits.d,f,g Results for plasma TAC were expressed as millimoles per liter of Trolox (6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid) equivalents (mmol/L of Trolox). Trolox is a part of the reagent kitf used for TAC measurements. Activities of GSH-Px and SOD were expressed as units/grams of hemoglobin (U/g Hb).
Determination of plasma CoQ10 concentration and serum NT-proBNP concentration
For all dogs, plasma CoQ10 concentrations (total CoQ10) were determined by a high-performance liquid chromatography–atmospheric pressure chemical ionization–tandem mass spectrometryh method with a liquid chromatography quadrupole ion-trap mass spectrometer.i Data processing was done with software.j Lipid standardized CoQ10 was calculated as the ratio between concentrations of plasma CoQ10 (mg/L) and serum total cholesterol (mmol/L) and was expressed as milligrams of CoQ10 per millimoles of total cholesterol (mg/mmol). Serum NT-proBNP concentrations (pmol/L) in samples obtained from dogs with cardiac diseases and from healthy dogs were measured with an ELISAk method.
Statistical analysis
Statistical analyses were performed with the use of commercially available software.l No sample size calculation was performed because there were no published data with which to make such a calculation. However, post hoc power analyses with a general linear model and univariate approach were performed.38,39 To test whether the data were normally distributed, histograms were generated and inspected visually and Shapiro-Wilk tests were performed. Kruskal-Wallis analysis followed by post hoc multiple comparisons were used to test for significant differences in all measured variables and age between each of the 3 groups of dogs with cardiac disease (ISACHC I, II, and III groups) and the group of healthy dogs. The same statistical method was used to test for significant differences in all measured variables (with the exception of serum NT-proBNP concentration) and age among the dogs with cardiac disease that were or were not treated and healthy dogs. A Mann-Whitney U test was performed to test for significant differences in serum NT-proBNP concentration between the dogs with cardiac disease that were or were not receiving cardiac treatment. Spearman rank correlation coefficient analysis was performed to determine the correlation between disease severity (ie, serum NT-proBNP concentration) and antioxidant variables (level of plasma TAC, whole blood GSH-Px activity, and erythrocyte SOD activity) or plasma CoQ10 concentration in dogs with different stages of cardiovascular diseases and in dogs with cardiac disease that were or were not receiving cardiac treatment. Results for plasma TAC, erythrocyte SOD activity, and whole blood GSH-Px activity are reported as median and range. Results for plasma CoQ10 concentration, CoQ10(LS), and serum NT-proBNP concentration are reported as median and interquartile range (25th to 75th percentile). A value of P < 0.05 was considered significant.
Results
Dogs with cardiac disease were assigned to 3 groups on the basis of their ISACHC classification. The ISACHC I group included 16 dogs (9 sexually males, 4 spayed females, and 3 sexually intact females); their ages ranged from 3.6 months to 12.3 years (mean age, 76.05 months [6.34 years]). Among these 16 dogs, 6 had myxomatous mitral valve disease, 6 had SAS (mild in 3 dogs, moderate in 2 dogs, and severe in 1 dog), 1 had mild pulmonic stenosis, 2 had pulmonic stenosis in a combination with other cardiac disorders (ie, 1 dog also had severe SAS and 1 dog also had severe SAS and arrhythmogenic right ventricular cardiomyopathy), and 1 had postoperative patent ductus arteriosus. The ISACHC II group included 16 dogs (10 sexually intact males and 6 spayed females); their ages ranged from 4.4 to 14 years (mean age, 124.50 months [10.38 years]). Among these 16 dogs, 12 had myxomatous mitral valve disease, 1 had ventricular tachycardia of unknown cause, 1 had DCM, 1 had primary atrial fibrillation, and 1 had mitral dysplasia. The ISACHC III group included 11 dogs (8 sexually intact males and 3 spayed females); their ages ranged from 5 to 14.3 years (mean age, 97.09 months [8.09 years]). Among these 11 dogs, 2 had primary atrial fibrillation, 4 had myxomatous mitral valve disease, 4 had DCM, and 1 had severe SAS. Dogs that had developed clinical signs as a consequence of their cardiovascular disease (ISACHC II and III classification) were assigned to 1 of 2 groups depending on whether they were or were not currently receiving cardiac treatment. The treated group included 17 dogs (10 sexually intact males and 7 spayed females); the age range was 4.4 to 14.3 years (mean age, 119.15 months [9.93 years]). Twelve treated dogs were classified as ISACHC II and 5 treated dogs were classified as ISACHC III. The untreated group included 10 dogs (8 sexually intact males and 2 spayed females); the age range was 5.9 to 12.5 years (mean age, 103.44 months [8.62 years]). Four untreated dogs were classified as ISACHC II and 6 untreated dogs were classified as ISACHC III. Among the dogs receiving treatment, medication regimens included administration of diuretics (furosemide or spironolactone), angiotensin-converting enzyme inhibitors (ramipril or benazepril), β-adrenergic receptor antagonists (atenolol or sotalol), a calcium channel blocker (diltiazem), an inodilator (pimobendan), and digoxin. Median duration of therapy in our dogs was 113 days (range, 1 to 824 days).
The healthy dogs in the control group were significantly younger than dogs in the ISACHC I (P = 0.048), II (P < 0.001), and III (P = 0.04) groups. Mean ages of the dogs in the 3 ISACHC groups did not differ significantly. There was no significant difference in age between dogs that were or were not receiving cardiac treatment.
Compared with the value for the healthy dogs, the median plasma TAC for the ISACHC I group was significantly (P = 0.046) lower (Table 1). Median plasma TAC for the ISACHC II or III group was not significantly different from that of the healthy control group. There were no significant differences in erythrocyte SOD and whole blood GSH-Px activities between the healthy control group and each of the 3 ISACHC groups. Median plasma TAC did not differ among the 3 groups of dogs with cardiac disease.
Median (range) plasma TAC, erythrocyte SOD activity, and whole blood GSH-Px activity in 29 healthy dogs and 43 dogs with various types and stages of cardiac diseases (congenital and acquired) that were grouped according to the ISACHC cardiac disease classification scheme as ISACHC I (n = 16), ISACHC II (16), and ISACHC III (11).
| Group | ||||
|---|---|---|---|---|
| Variable | Healthy dogs (n = 29) | ISACHC I (n = 16) | ISACHC II (n = 16) | ISACHC III (n = 11) |
| Plasma TAC (mmol/L of Trolox) | 1.18 (1.00–1.48) | 1.11* (0.88–1.21) | 1.18 (0.85–1.38) | 1.12 (0.90–1.34) |
| Erythrocyte SOD activity (U/g Hb) | 1,860 (1,423–2,234) | 1,815 (1,380–2,341) | 1,939 (1,547–2,637) | 1,777 (1,185–2,810) |
| Whole blood GSH-Px activity (U/g Hb) | 403.2 (306.9–570.7) | 429.7 (326.6–535.2) | 430.8 (311.1–508.1) | 497.1 (363.9–554.4) |
Blood samples were collected once from all dogs at the first examination or a recheck examination. Values for the antioxidant variables were determined spectrophotometrically with an automated biochemistry analyzer and commercial kits; hemoglobin concentration in RBC hemolysates was determined spectrophotometrically with the cyanomethemoglobin method and an automated biochemistry analyzer.
For a given variable, value is significantly different (P < 0.05) from that of the healthy dogs.
Hb = Hemoglobin.
Median plasma concentration of CoQ10 (Figure 1) and CoQ10(LS) (Figure 2) were not significantly different between dogs with cardiovascular disease and healthy dogs or among groups of cardiac patients (the ISACHC I, II, and III groups). However, the value of plasma CoQ10 concentration (Figure 3) was significantly higher in dogs receiving cardiac treatment than in untreated dogs (P < 0.001) or healthy dogs (P = 0.008). Significant differences in plasma CoQ10 (LS) between dogs that were or were not receiving cardiac treatment (P < 0.001) and between treated dogs and healthy dogs (P = 0.029) were also identified (Figure 4).
Box-and-whisker plots of plasma CoQ10 concentration in 29 healthy dogs and 43 dogs with various types and stages of cardiac diseases (congenital and acquired) that were grouped according to the ISACHC cardiac disease classification scheme as ISACHC I (n = 16), ISACHC II (16), and ISACHC III (11). Blood samples were collected once from all dogs at the first examination or a recheck examination. Plasma CoQ10 concentration was determined with a high-performance liquid chromatography–atmospheric pressure chemical ionization–tandem mass spectrometry method. For each box, the horizontal line represents the median and the upper and lower boundaries represent the 75th and 25th percentiles, respectively. Whiskers represent the minimum and maximum values, and circles represent outlier values.
Citation: American Journal of Veterinary Research 78, 4; 10.2460/ajvr.78.4.447
Box-and-whisker plots of plasma CoQ10(LS) in 29 healthy dogs and 43 dogs with cardiac diseases (16 ISACHC I dogs, 16 ISACHC II dogs, and 11 ISACHC III dogs). Lipid standardized plasma CoQ10 was calculated as the ratio between concentrations of plasma CoQ10 (mg/L) and serum total cholesterol (mmol/L). An extreme outlier value is indicated by an asterisk. See Figure 1 for remainder of key.
Citation: American Journal of Veterinary Research 78, 4; 10.2460/ajvr.78.4.447
Box-and-whisker plots of plasma CoQ10 concentration in 29 healthy dogs and 27 dogs with cardiac diseases (ISACHC II and III dogs) that were (n = 17) or were not (10) receiving cardiac treatment. Among the dogs receiving treatment, medication regimens included administration of diuretics, angiotensin-converting enzyme inhibitors, β-adrenergic receptor antagonists, a calcium channel blocker, an inodilator, and digoxin. aMedian value for the dogs receiving treatment was significantly higher than that for the dogs not receiving treatment (P < 0.001) and that for healthy dogs (P = 0.008). See Figures 1 and 2 for remainder of key.
Citation: American Journal of Veterinary Research 78, 4; 10.2460/ajvr.78.4.447
Box-and-whisker plots of plasma CoQ10(LS) in 29 healthy dogs and 27 dogs with cardiac diseases (ISACHC II and III dogs) that were (n = 17) or were not (10) receiving cardiac treatment. bMedian value for the dogs receiving treatment was significantly higher than that for the dogs not receiving treatment (P < 0.001) and that for healthy dogs (P = 0.029). See Figures 1, 2, and 3 for remainder of key.
Citation: American Journal of Veterinary Research 78, 4; 10.2460/ajvr.78.4.447
The results of statistical analyses indicated no significant difference in median plasma TAC, erythrocyte SOD activity, or whole blood GSH-Px activity between dogs that were or were not receiving cardiac treatment (Table 2). In addition, plasma TAC, erythrocyte SOD activity, or whole blood GSH-Px activity values did not differ between healthy dogs and dogs that were or were not receiving cardiac treatment.
Median (range) plasma TAC, erythrocyte SOD activity, and whole blood GSH-Px activity in 29 healthy dogs and 27 dogs with cardiac diseases (ISACHC II and III dogs) that were (n = 17) or were not (10) receiving cardiac treatment.
| Group | |||
|---|---|---|---|
| Variable | Healthy dogs (n = 29) | Not treated (n = 10) | Treated (n = 17) |
| Plasma TAC (mmol/L of Trolox) | 1.18 (1.00–1.48) | 1.11 (0.85–1.38) | 1.17 (1.00–1.38) |
| Erythrocyte SOD activity (U/g Hb) | 1,860 (1,423–2,234) | 1,779 (1,185–2,271) | 1,922 (1,348–2,810) |
| Whole blood GSH-Px activity (U/g Hb) | 403.2 (306.9–570.7) | 446.6 (311.1–532.3) | 449.1 (311.5–554.4) |
Among the dogs receiving treatment, medication regimens included administration of diuretics, ACE inhibitors, β-adrenergic receptor antagonists, a calcium channel blocker, an inodilator, and digoxin. See Table 1 for remainder of key.
The median serum NT-proBNP concentration in healthy dogs (511.50 pmol/L; range, 311.77 to 1,044.70 pmol/L) was significantly lower compared with findings for the ISACHC II group (4,212.00 pmol/L [range, 2,171.80 to 9,721.48 pmol/L]; P < 0.001) and the ISACHC III group (6,450.06 [range, 2,656.58 to 36,787.60 pmol/L]; P < 0.001), but not compared with findings for the ISACHC I group (1,842.96 pmol/L [range, 1,184.18 to 6,462.87 pmol/L]; P = 0.219). The median serum NT-proBNP concentration was significantly higher in the ISACHC II (P = 0.007) and ISACHC III (P < 0.001) groups, compared with the value in the ISACHC I group (Figure 5). The NT-proBNP concentration was higher in the ISACHC III group than in the ISACHC II group, but the difference was not significant (P = 0.587). Median serum NT-proBNP concentration did not differ between dogs that were or were not receiving cardiac treatment (4,967.55 pmol/L [range, 2,656.58 to 12,865.10 pmol/L] vs 5,524.54 pmol/L [range, 2,171.80 to 36,787.60 pmol/L], respectively; Figure 6).
Box-and-whisker plots of serum NT-proBNP concentration in 43 dogs with various types and stages of cardiac diseases (congenital and acquired) that were grouped according to the ISACHC cardiac disease classification scheme as ISACHC I (n = 16), ISACHC II (16), and ISACHC III (11). Serum NT-proBNP concentrations were measured with an ELISA method. cMedian value for the ISACHC I dogs was significantly lower than that for the ISACHC II dogs (P = 0.007) and the ISACHC III dogs (P < 0.001). See Figures 1 and 2 for remainder of key.
Citation: American Journal of Veterinary Research 78, 4; 10.2460/ajvr.78.4.447
Box-and-whisker plots of serum NT-proBNP concentration in 27 dogs with cardiac diseases (ISACHC II and III dogs) that were (n = 17) or were not (10) receiving cardiac treatment. See Figures 1, 2, and 5 for key.
Citation: American Journal of Veterinary Research 78, 4; 10.2460/ajvr.78.4.447
With regard to serum NT-proBNP concentration, there was a significant, negative correlation with plasma CoQ10 concentration (r = −0.571; P = 0.017) and with plasma CoQ10 (LS) (r = −0.505; P = 0.039) in the group of dogs receiving cardiac treatment. However, no significant correlations were found between serum NT-proBNP concentration and any of the antioxidant status variables or plasma CoQ10 concentration in dogs with different stages of cardiovascular disease or in cardiac patients that were not receiving treatment.
Discussion
To our knowledge, the present study was the first to assess circulating antioxidant variables and determine plasma CoQ10 concentration in dogs with various stages of spontaneous cardiovascular diseases. In contrast to the human medical literature, there are few reports of oxidative stress studies in dogs with cardiovascular diseases.4,7,23 No studies of plasma CoQ10 concentration or CoQ10 supplementation in cases of spontaneous canine cardiovascular disease have been published. The publications reporting the effect of oxidative stress on the pathogenesis of cardiovascular diseases in humans and other animals reveal a close relationship between the development of various cardiovascular diseases and increased oxidative stress.4,7,16,40–42
Plasma or serum TAC represents a suitable biochemical measure for evaluating the overall antioxidant status of an idividual.34 Low or high values of TAC have been associated with states of increased oxidative stress.33,43 Low serum TAC values have been detected in humans with coronary heart disease.14 The present study revealed significantly lower plasma TAC in dogs classified as ISACHC I, compared with the finding in healthy dogs. However, there were no significant differences in plasma TAC values between healthy dogs and each of the other 2 groups of dogs with cardiac diseases (the ISACHC II and III groups). Furthermore, plasma TAC did not differ among the 3 groups of cardiac patients. Although the TAC data alone are not sufficient to make inferences about oxidative stress,43 the low level of TAC in the ISACHC I group suggested increased oxidative stress and, consequently, a low level of systemic antioxidant defense in this group of dogs. However, the interpretation of the changes in plasma or serum TAC depends on the conditions under which the plasma or serum TAC is determined. Increased plasma or serum TAC may not necessarily be a desirable condition if it reflects a response to increased oxidative stress. Similarly, a decrease in plasma or serum TAC may not necessarily be an undesirable condition if the measurement reflects decreased production of ROS.33 Hetyey et al31 found no significant differences in TAC values between healthy dogs and dogs with cardiovascular disease (DCM or mitral valve disease) by use of the same method as that used in the present study. However, in that study,31 values of TAC measured by the ferric-reducing ability of the plasma method were significantly higher in dogs with naturally occurring DCM or mitral endocardiosis than in healthy control dogs. Significantly higher plasma TAC in dogs with CHF, compared with the value in healthy control dogs, was also found by Freeman et al7 who used an oxygen radical absorbance capacity assay. The difference between these reported TAC data may be a consequence of the different methods used. The methods vary greatly, and the results of different methods are not comparable.33 In the present study, the TAC assessment method detected both hydrophilic and lipophilic antioxidants, including CoQ10; some other methods can detect only hydrophilic antioxidants (eg, the oxygen radical absorbance capacity method). For the dogs with cardiac disease in the present study, plasma TAC values were higher in those receiving treatment than in those not receiving treatment; however, the difference was not significant. Higher TAC values might be attributed to significantly higher CoQ10 concentrations in treated dogs because CoQ10 contributes to plasma TAC values.34 In addition, higher TAC values might be attributable to administration of certain cardiovascular drugs that have antioxidant properties, such as β-adrenergic receptor blockers, angiotensin-converting enzyme inhibitors, and calcium channel blockers.44–46
By removing ROS, the intracellular antioxidant enzymes SOD, GSH-Px, and catalase represent a primary antioxidant defense.47 In human medicine, it has been demonstrated that the lower the activities of antioxidant enzymes, the worse the outcome of coronary heart disease. A meta-analysis revealed a highly negative and significant association between the activities of antioxidant enzymes (SOD, GSH-Px, and catalase) and the outcome of coronary disease in human patients16; moreover, low activity of GSH-Px is a prognostic indicator of poor outcomes of coronary heart disease in humans.16,48,49 Glutathione peroxidase is the most important antioxidant enzyme, protecting cell membranes against lipid peroxidation.47 Significantly higher GSH-Px activity was found in canine patients with DCM in a study by Freeman et al.4 In the present study, dogs in the ISACHC III group had the highest GSH-Px activity, although this value did not differ from findings for the other ISACHC groups and healthy dogs. Higher GSH-Px activity may indicate the induction of an antioxidant defense system. Treatment had no effect on the GSH-Px activity in dogs with cardiac diseases in the present study. To our knowledge, there are no reports of the effect of cardiac treatment on antioxidant variables in canine cardiac patients; however, similar results were obtained in human patients with class IV CHF (New York Heart Association functional IV classification).50 In that study,50 clinical improvement after intensive medical treatment in patients with decompensated CHF was associated with a decrease in patients’ oxidative stress status (ie, significant decrease in malondialdehyde concentration, compared with the pretreatment value) without change in the activities of the antioxidant enzymes SOD, GSH-Px, or catalase.50
The present study revealed no significant difference in erythrocyte SOD activity among dogs with various stages of heart failure or between dogs with cardiac diseases and healthy control dogs. Treatment had no effect on SOD activity in dogs with cardiac diseases. Freeman et al4 reported similar results for dogs with idiopathic DCM. However, a study42 in humans with DCM revealed significantly lower SOD activity, compared with findings in healthy controls. In other studies,50,51 SOD activity in humans with DCM patients (New York Heart Association class IV) was unchanged by treatment. It can be concluded that SOD activity alone is unlikely to represent a useful marker of cardiac disease progression. Therefore, it is better to determine circulating activities of at least 2 antioxidant enzymes, SOD and GSH-Px or catalase.
The discovery that humans with various cardiac diseases or heart failure have deficiencies of CoQ10 in blood or cardiac tissue sparked a great deal of interest,17–21 as did the fact that treatment with CoQ10 can improve their cardiovascular function.12,13,29,30,52–54 However, studies in humans with DCM55 and dogs with experimentally induced CHF32 revealed no beneficial role of CoQ10 supplementation. The lack of a beneficial effect of CoQ10 supplementation might be related to the use of the oxidized form of CoQ10 (ubiquinone) in those studies. Results of a bioavailability study56 in humans indicated that the reduced form, ubiquinol, is superior to ubiquinone. In humans, better bioavailability of ubiquinol was reflected by significantly higher plasma CoQ10 concentration as well as in significantly higher plasma CoQ10(LS) (the ratio of plasma concentration of CoQ10 and total cholesterol concentration) after ubiquinol supplementation, compared with the effects of ubiquinone supplementation.56 Plasma CoQ10 concentration reflects not only the amount of absorbed CoQ10 but also tissue CoQ10 content. Although plasma CoQ10 concentration may not necessarily reflect tissue CoQ10 status, it still serves as a useful measure of overall CoQ10 status and also as a guide to CoQ10 dosing.57 In addition, the bioavailability of supplemental CoQ10 in humans56,58 and dogs59 varies with the type and amount of oil in a given preparation and the delivery method. In the study by Permanetter et al,55 the lack of a significant effect of CoQ10 supplementation on measurements of myocardial function in humans with CHF was probably also due to the use of a low dosage of CoQ10 (only 100 mg/d) and the short duration of CoQ10 supplementation (4 months), because 100 mg of CoQ10/d is suboptimal for most human patients and the maximal myocardial function improvement with CoQ10 supplementation is typically achieved in 6 to 12 months.60 In humans, the severity of heart failure correlates with the severity of CoQ10 deficiency.19 Moreover, low plasma CoQ10 concentration has been found to be an independent predictor of death in humans with CHF.61 Coenzyme Q10 is transported bound to circulating lipoproteins; consequently, the plasma concentration of CoQ10 depends on the lipoprotein concentration in blood.62 Therefore, a more accurate expression of the true concentration of CoQ10 in plasma is the ratio of CoQ10 concentration and total cholesterol concentration (ie, CoQ10[LS]). In contrast to most studies involving humans,17,18,20,62 there was no significant difference in plasma CoQ10 concentration or CoQ10(LS) between dogs with cardiac diseases and healthy dogs or among dogs in the 3 ISACHC groups in the present study. However, plasma CoQ10 concentration and CoQ10(LS) were significantly higher in affected dogs that were receiving treatment, compared with findings in affected dogs that were not receiving treatment. Thus, cardiac treatment appeared to have a significant effect on plasma CoQ10 concentrations in dogs with cardiac diseases. The effect of cardiac treatment in the present study might be ascribed to the antioxidant properties of some cardiac medications used. The results of this study have supported the idea that cardiac treatment itself may have an antioxidant effect, thereby sparing some amount of CoQ10 and resulting in higher plasma CoQ10 concentrations in treated dogs. Moreover, the plasma CoQ10 concentration in dogs that were not receiving cardiac treatment was lower than that in healthy dogs and significantly lower than that in dogs that were receiving cardiac treatment, which may be evidence of CoQ10 deficiency in dogs with cardiac disease (ISACHC II and III classification) in the absence of treatment.
In the present study, serum concentration of the cardiac biomarker NT-proBNP increased significantly with the stage of heart failure, although there was no significant difference between dogs in the ISACHC II and III groups or between affected dogs that were or were not receiving treatment. Similarly, Oyama et al63 found no differences in serum NT-proBNP concentration between ISACHC class II and III canine cardiac patients. In contrast, in dogs with CHF as a result of mitral valve disease, treatment resulted in lower plasma NT-proBNP concentration. In that study,64 low plasma NT-proBNP concentration was a predictor of 12-month survival in that plasma NT-proBNP concentration < 965 pmol/L at 7 to 30 days after starting treatment for CHF was associated with a significantly better outcome. However, there were some major differences between that investigation and the present study. The earlier study64 included 26 small-breed dogs with mitral valve disease–associated CHF; the present study included variably sized dogs with various heart disorders, although myxomatous mitral valve disease predominated (22 dogs). The lower serum NT-proBNP concentration in the dogs that survived for 12 months in the earlier study64 could be attributable to the fact that this variable was measured also at 7 and 30 days after initiation of treatment for CHF, whereas the variables of interest in the present study were measured at 1 time point only, regardless of whether this was the dog's first examination or a recheck examination.
In the present study, a significant negative correlation between serum NT-proBNP concentration and plasma CoQ10 concentration or plasma CoQ10(LS) was identified in the dogs that were receiving cardiac treatment. This finding suggests that lower CoQ10 concentrations are associated with greater severity of CHF in dogs, which is consistent with published data indicating that the severity of heart failure correlates with the severity of CoQ10 deficiency in humans.19
Values of the antioxidant variables assessed in the present study did not differ among the 3 groups of dogs with cardiac disease, regardless of their stage of cardiac disease or treatment, but this study had some limitations. These limitations included the lack of sample size determination, low numbers of dogs with various cardiac diseases in the ISACHC groups, no disease matching of dogs across ISACHC groups, no age matching of healthy dogs with dogs in the 3 ISACHC groups, and the use of only 1 method for plasma TAC determinations. Because low numbers of dogs were included in ISACHC groups, post hoc power analyses were conducted. Nonsignificant results may be the result of insufficient statistical power.38,39 Post hoc power analyses indicated quite high power coefficients for the comparison of measured variables among the 4 study groups (ISACHC I, ISACHC II, ISACHC III, and healthy control groups). The highest power coefficient obtained was for serum NT-proBNP concentration (0.928); power coefficients for plasma TAC (0.759), plasma CoQ10 concentration (0.780), and whole blood GSH-Px activity (0.616) almost reached the recommended value of 0.80.38 Power coefficients for plasma CoQ10(LS) and erythrocyte SOD activity were much lower (0.267 and 0.221, respectively). In contrast, quite low power coefficients (much less than the recommended 0.80)38 were obtained for almost all the measured variables when assessing the effect of treatment (plasma TAC, 0.288; erythrocyte SOD activity, 0.145; whole blood GSH-Px activity, 0.264; plasma CoQ10 concentration, 0.467; and plasma CoQ10[LS], 0.276). The exception was serum NT-proBNP concentration with a power coefficient of 0.723. It would have been ideal to have had larger numbers of dogs in each ISACHC group such that differences in measured variables might be greater and that overlaps of the values of antioxidant variables and plasma CoQ10 concentration would not have occurred. We cannot completely exclude the effect of age on plasma CoQ10 concentration, plasma TAC, erythrocyte SOD activity, or whole blood GSH-Px activity in the investigated groups of dogs, but there are no studies on the effect of age on plasma CoQ10 concentration and plasma TAC in dogs and there are only 3 studies65–67 on the effect of age or sex on blood SOD and GSH-Px activities in dogs. Several methods have been developed to measure TAC in different biological samples because of the difficulty in measuring each antioxidant component separately and the interactions between different antioxidant components in a given sample. The methods vary greatly, which can result in incomparable TAC results from studies. It must be emphasized that the relative contributions of the various antioxidant components to the TAC vary considerably depending on the measurement procedure. Hence, all assays have their specific limitations and probably the true TAC of a biological system such as serum or plasma could be best determined by use of a battery of assays simultaneously.33,68 The method used in the present study has been used in many research and routine clinical biochemistry laboratories because of its operational simplicity; however, this assay has certain shortcomings,69 which may be a reason for nonsignificant differences in plasma TAC among the investigated groups of dogs.
Results of the present study indicated that the evaluated antioxidant variables were not altered in the dogs with cardiac disease, regardless of the stage of cardiac disease or treatment. Cardiac treatment was associated with increased plasma CoQ10 concentration, although no significant difference in this variable was found among the 3 ISACHC groups. The study data indicated that lower plasma CoQ10 concentrations were likely associated with greater severity of CHF. Further investigation into the possible effects of CoQ10 supplementation in dogs with advanced stages of CHF is warranted.
Acknowledgments
The authors acknowledge the financial support of the Slovenian Research Agency (research program P4-0053).
The authors declare that there were no conflicts of interest.
Preliminary results were presented in abstract form at the 20th Congress of the European College of Veterinary Internal Medicine-Companion Animal (ECVIM-CA), Toulouse, France, September 9–11, 2010.
ABBREVIATIONS
| CHF | Congestive heart failure |
| CoQ | Coenzyme Q |
| CoQ10 | Coenzyme Q10 |
| CoQ10(LS) | Lipid-standardized coenzyme Q10 |
| DCM | Dilated cardiomyopathy |
| GSH-Px | Glutathione peroxidase |
| ISACHC | International Small Animal Cardiac Health Council |
| NT-proBNP | N-terminal pro-brain natriuretic peptide |
| ROS | Reactive oxygen species |
| SAS | Subaortic stenosis |
| SOD | Superoxide dismutase |
| TAC | Total antioxidant capacity |
Footnotes
Vingmed System Five, GE Healthcare, Milwaukee, Wis.
BD Microtainer, Becton Dickinson, Franklin Lakes, NJ.
Vacuette, Greiner Bio-One, Kremsmunster, Austria.
Ransod Kit, Randox, Crumlin, County Antrim, Northern Ireland.
RX Daytona analyzer, Randox, Crumlin, County Antrim, Northern Ireland.
Total Antioxidant Status kit, Randox, Crumlin, County Antrim, Northern Ireland.
Ransel kit, Randox, Crumlin, County Antrim, Northern Ireland.
Surveyor LC system, Thermo Finnigan, Riviera Beach, Calif.
Finnigan MAT, San Jose, Calif.
Xcalibur, version 1.3, Thermo Finnigan, San Jose, Calif.
VETSIGN, Canine CardioSCREEN NT-proBNP, Biomedica, Vienna, Austria.
SPSS, version 22.0, SPSS Inc, Chicago, Ill.
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