Abstract
n-3 fatty acids have some benefits in humans and dogs with cardiac disease, and plasma n-3 fatty acid concentrations have been shown to be decreased in dogs with congestive heart failure (CHF). However, there are no published studies reporting fatty acid concentrations in cats with cardiac disease. Therefore, the goal of this study was to compare serum fatty acid concentrations in normal cats and cats with hypertrophic cardiomyopathy (HCM), and determine if fatty acid concentrations correlate with left atrial size or the presence of CHF. Serum fatty acid concentrations were measured in normal cats and cats with HCM by gas chromatography. Twenty-three cats with HCM and 20 healthy controls were enrolled. Compared with healthy controls, cats with HCM had higher concentrations of palmitic acid (P = 0.01), docosahexanoic acid (DHA; P = 0.001) and total n-3 fatty acids (P = 0.03), and lower concentrations of linoleic acid (P = 0.03). Among cats with HCM, there were no differences in plasma fatty acid concentrations, and no association between left atrial dimension and fatty acid concentrations. Cats with HCM have some alterations in plasma fatty acids compared with healthy controls. Given the higher plasma concentrations of DHA, DHA supplementation is unlikely to have benefits in terms of correcting a deficiency; however, other effects of DHA or of supplementation of EPA warrant further investigation.
Introduction
There are numerous effects of n-3 fatty acids that may have cardiovascular benefits, including lipid reduction, anti-inflammatory and anti-arrhythmogenic effects, as well as improvements in endothelial function and reduced platelet aggregability.1–3 As a result of these and other effects, n-3 fatty acid supplementation has been shown to result in decreased incidence of sudden death and improved survival in some, but not all, studies of humans with cardiovascular disease.4–8 In addition, one study of dogs with congestive heart failure (CHF) secondary to dilated cardiomyopathy (DCM) showed that n-3 fatty acid supplementation improved cachexia. 9 Reduced circulating concentrations of the n-3 fatty acids, eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), have been found in dogs with CHF secondary to DCM, and supplementation of n-3 fatty acids can increase plasma concentrations of these fatty acids.9,10 In one of these studies, 9 the increase in n-3 fatty acids was associated with significantly improved muscle mass, but there was no significant change in cardiac size or function. In the second study, 10 dogs that were supplemented with n-3 fatty acids, taurine, carnitine and antioxidants had a decrease in cardiac size, but it is not clear whether this was associated with the n-3 fatty acids, the other nutrients or the combination of the two. Circulating concentrations of fatty acids have not been reported in cats with cardiac disease.
In addition to the aforementioned beneficial effects of n-3 fatty acids is their antithrombotic effects as a result of production of the less potent thromboxane B5 (as opposed to thromboxane B4, which would result from n-6 fatty acid intake).1,2 Cats appear predisposed to thromboembolic disease, with up to 48% of cats with cardiomyopathy developing arterial thromboembolism.11–13 Therefore, n-3 fatty acid supplementation may be of particular benefit in cats with cardiac disease.
Despite all the effects of n-3 fatty acids and numerous studies showing cardiovascular benefits, not all studies have been positive. For example, a recent meta-analysis of human studies found no association between n-3 fatty acid supplementation and lower risk of all-cause or cardiac mortality. 7 The inconsistent results may be the result of differences in study design (eg, species, underlying disease, dose and type of n-3 fatty acids, duration of treatment) and in the outcomes (eg, primary vs secondary prevention of cardiovascular events, mortality, anti-arrhythmic effects, body composition). For dogs, differences may be found between different breeds or underlying diseases. For example, in one study, 14 Doberman Pinschers and Boxers had significantly different plasma concentrations of certain fatty acids. There was a significant correlation between n-3 fatty acids and numbers of ventricular premature complexes in Boxers, but not in Doberman Pinschers. In addition, even if n-3 fatty acids have beneficial effects in dogs and people, supplementation of n-3 fatty acids may not have beneficial effects on cardiac size or function, arrhythmias or mortality in cats. Therefore, studies of n-3 fatty acids in cats are important to determine their relative benefits or lack thereof.
To our knowledge, no studies have been published on fatty acid concentrations in cats with hypertrophic cardiomyopathy (HCM). Therefore, the goals of this study were (1) to measure serum fatty acid concentrations in cats with HCM compared to healthy controls, and (2) to determine if fatty acid concentrations correlate with left atrial size or the presence of CHF in cats with HCM.
Materials and methods
This study was approved by the Tufts Cummings School of Veterinary Medicine Clinical Studies Review Committee. Based on results from previous studies in dogs, sample size calculations determined that 24 cats per group would achieve adequate statistical power to test the null hypothesis of no difference between the two groups. The goal for enrollment, to account for higher than anticipated variance, was 30 cats per group. Cats for the control group were recruited from staff and student-owned cats and were determined to be eligible based on history, physical examination, blood testing (packed cell volume [PCV], total solids [TS] and platelet count) and echocardiography. Owing to financial constraints, none of the control group had a complete blood count, biochemistry profile or T4. Cats with HCM were recruited from clients, staff and students. Physical examination and blood testing (complete blood count, biochemistry profile, T4 if >6 years old) were performed. Cats were excluded from both HCM and control groups if they had significant concurrent disease (eg, cancer, diabetes, chronic kidney disease), were receiving fish oil supplementation or if incomplete diet information was available. A diet history was collected from all cats’ owners to determine the specific cat foods provided to the cat, including the form (dry or canned). Cats were classified in one of four categories for food type: eating all dry food, mostly dry food, all canned food or mostly canned food. Although diets were not analyzed for EPA + DHA content, the amount of EPA + DHA in each cat’s main diet was determined from the manufacturer. For the purposes of this study, and based on our experience, foods were considered to have minimal EPA + DHA content if they contained <25 mg/100 kcal EPA + DHA or had no apparent EPA + DHA sources on the ingredient list. Foods were considered to have moderate EPA + DHA content if they contained 25–75 mg/100 kcal. Foods containing >75 mg/100 kcal EPA + DHA were classified as having a high EPA + DHA content.
Echocardiography (two dimensional [2D], M-mode, and color flow, spectral and tissue Doppler [GE Vivid 7 Dimension; General Electric Healthcare]) was performed on all cats without sedation. For echocardiography, 2D right parasternal long- and short-axis, 2D left parasternal and M-mode right parasternal short-axis views were obtained. Left ventricular, left atrial and aortic M-mode dimensions were measured in right parasternal short-axis views.15,16 The 2D left atrial and aortic dimensions were obtained in the right parasternal short-axis view in diastole, 17 and the 2D interventricular septum and left ventricular free wall measurements were obtained in the right parasternal shor- or long-axis view of the left ventricle in end-diastole. To be classified as normal, cats had to have both an interventricular septal thickness in diastole (IVSd) and left ventricular free wall in diastole (LVWd) <0.6 cm on M-mode, short-axis and long-axis 2D measures; a subjectively normal left atrial size; no systolic anterior motion of the mitral valve; and an aortic velocity ⩾1.5 ms, with no subjective evidence of left ventricular hypertrophy or papillary muscle hypertrophy. Normal cats also had to have normal left ventricular contractile function, normal left ventricular and left atrial chamber dimensions, and no evidence of congenital heart disease. Cats were diagnosed with HCM if they had either an IVSd or LVFWd >0.6 cm, measured by 2D and/or M-mode echocardiographic, and concurrent findings indicative of HCM (ie, some combination of diffuse or focal concentric hypertrophy of the left ventricle, systolic anterior motion of the mitral valve, left atrial enlargement or increased aortic velocity).
Blood was collected by jugular venepuncture into serum and ethylenediamine tetra-acetic acid (EDTA) tubes. Automated platelet count, PCV and TS were determined from EDTA-anticoagulated blood. Manual platelet count was used if an automated count was not given owing to microscopic clumping (n = 4). Serum tubes were centrifuged within 30 mins for biochemical profile and T4 analysis. Additional serum was immediately stored at −80°C until fatty acid analysis. Serum fatty acid analysis was performed at the Vascular Biology Laboratory at the Jean Mayer USDA Human Nutrition Research Center on Aging at Tufts University via chloroform methanol extraction and gas chromatography. 18
Distribution of the data was assessed with the Kolmogorov–Smirnov test. Normally distributed data are presented as mean ± SD, and skewed data are presented as median (range). If possible, skewed data were logarithmically transformed prior to analysis. Fatty acid concentrations were compared between groups (ie, HCM vs normal cats, CHF vs no CHF) using independent t-tests (for normal data) or Mann–Whitney U-tests (skewed data). Categorical data were compared with χ2 tests. Correlations between fatty acid concentrations and left atrial size or age were determined using Spearman correlation. Statistical significance was set at P <0.05. All statistical analyses were performed with commercially available software (Systat 13.0; Systat Software).
Results
Twenty-four cats with HCM and 27 healthy controls were enrolled in the study. However, after excluding cats with insufficient dietary information, 23 cats with HCM and 20 healthy control cats were included in the analysis. Further analyses were performed only on those 43 cats. Age was not significantly different between groups (HCM: median = 8 years, range 2–16 years; controls: median = 6 years, range 1–12 years; P = 0.20). The HCM group included domestic short- (n = 12) and longhairs (n = 5), Persians (n = 3), Ragdoll (n = 1), Devon Rex (n = 1) and Himalayan (n = 1), while the control group included domestic short- (n = 13) and longhairs (n = 4) and one each of Ragdoll, Bengal and Cornish Rex. All cats in both groups were neutered, and there was no significant difference in gender between groups (HCM: 17 males and six females; control: 11 males and nine females; P = 0.19). Body weight was not significantly different between the two groups (HCM: median = 4.5 kg, range 2.9–7.6 kg; controls: median = 4.9 kg, range 3.9–6.7 kg; P = 0.18; body weights were available for all cats in the HCM group, but only 15/20 cats in the control group). Cats with HCM were classified according to the International Small Animal Cardiac Health Council (ISACHC) as stage 1b (n = 8), 2 (n = 7), 3a (n = 3) and 3b (n = 5).
No cats had undergone any dietary changes within the 8 weeks before enrolling the in the study. Most cats ate all dry food (HCM: n = 9; controls: n = 19) or mostly dry food (HCM: n = 12; controls: n = 1). Only two cats ate all canned food (both in the HCM group). The percentage of cats eating all/mostly dry or all/mostly canned was not different between the two groups (P = 0.18). Most cats (n = 37) were eating foods with minimal EPA + DHA content (<25 mg/100 kcal). Six cats (HCM: n = 3; controls: n = 3) were eating foods with moderate EPA + DHA content (25–75 mg/100 kcal). No cats were eating a food with a high EPA + DHA content.
Serum concentrations of palmitic acid (P = 0.01), DHA (P = 0.001) and total n-3 fatty acids (P = 0.03) were significantly higher in the HCM group compared with the controls (Table 1), while linoleic acid was significantly lower in the HCM group (P = 0.03). Among cats with HCM, there was no difference in fatty acid concentrations between cats with CHF (n = 15) and without CHF (n = 8), nor was there a significant correlation between any serum fatty acid concentration and left atrial size.
Fatty acid concentrations in cats with hypertrophic cardiomyopathy (HCM; n = 23) and healthy controls (n = 20). Fatty acids are reported as percent normalized concentrations (median [range])
Discussion
In this study, cats with HCM had higher concentrations of palmitic acid, DHA and total n-3 fatty acids, and lower concentrations of linoleic acid than healthy controls. The higher DHA concentrations are in contrast to a study in dogs with CHF secondary to DCM in which lower n-3 fatty acid concentrations were found compared with healthy controls. 9 In another study comparing dogs with chronic valvular disease (CVD) in ISACHC class 1a or 1b to healthy controls, no differences in n-3 fatty acids, palmitic or linoleic acid were found (Freeman and Rush, unpublished data). There are a number of possible reasons for the contradictory results in the current study. These include species differences in fatty acid metabolism, 19 alterations in substrate metabolism in cardiac disease 20 and differences in disease severity among the studies. All dogs in the 1996 study 9 had CHF (vs only 15/23 in the current feline study) and all animals in the study of dogs with CVD were asymptomatic (Freeman and Rush, unpublished data). Disease severity may influence fatty acid metabolism, although, in this study, fatty acid concentrations were not associated with left atrial size.
Differences in fatty acids also have been found in rodent models and in humans with cardiac disease, with some findings similar to those in the current study. One study of humans with DCM showed a negative correlation between disease severity (as assessed by left ventricular ejection fraction) and palmitic and oleic acids and a positive correlation between disease severity and linoleic acid, similar to this study. 21 However, unlike this study, the study by Rupp et al 21 found a positive correlation between disease severity and DHA in humans with DCM. Rupp et al 21 also reported the results of a study using a rat model of aortic banding, in which palmitic acid was increased and DHA was decreased with cardiac hypertrophy. Palmitic acid may play a role in the induction or progression of cardiovascular disease as it can induce cell death through apoptosis, although further study is needed in cats. 22
It is unclear why linoleic acid was lower in cats with HCM in the current study. One possible explanation could be related to cardiolipin, a phospholipid in the inner mitochondrial membrane that plays a role in myocardial energy metabolism. Linoleic acid comprises 80–90% of cardiolipin’s acyl chains, forming tetralinoleoyl cardiolipin.23,24 Reductions in tetralinoleoyl cardiolipin have been associated with mitochondrial dysfunction and cardiac pathology. 25 Rodent models of left ventricular hypertrophy and humans with CHF have both decreased linoleic acid and tetralinoleoyl cardiolipin.25–27 It is important to note that the current study evaluated only circulating fatty acids; more detailed studies assessing myocardial fatty acids and cardiolipin in cats with HCM would be needed to assess whether any changes in myocardial energy metabolism occur in HCM.
There were no significant differences when comparing cats with HCM with and without CHF. While a previous study comparing fatty acid concentrations between dogs with CHF and healthy controls found that dogs with CHF had significantly lower arachidonic acid, EPA and DHA, 9 a study comparing dogs with CHF to those with cardiac disease, but without CHF, has not been published. The lack of significant differences in fatty acid concentrations between cats with and without CHF could be due to the small sample size, to differences in myocardial substrate adaptations seen in CHF 20 or may be related to dietary intake. Cats with CHF, for example, are more likely to have complete or partial anorexia so may be more likely to have a different dietary fatty acid intake than cats without CHF. 28 A future prospective study that controlled for dietary intake of fatty acids would help us to better understand these differences.
Fatty acid concentrations in this study in both the cats with HCM and the healthy controls appear to be similar to baseline fatty acid concentrations reported in a study examining the effects of n-3 fatty acid supplementation on platelet function in healthy cats. 29 In both studies, the predominant fatty acid found was linoleic, followed by fatty acids with 14–18 carbon chain length, arachidonic acid and small percentages of the n-3 fatty acids. Although the percentages of n-3 fatty acids can be modified by supplementation, the fact that cats taking n-3 fatty acid supplementation were excluded from the current study and that no cats were eating diets with a high EPA + DHA content makes the very low plasma concentrations of n-3 fatty acids expected.
The underlying type of cardiomyopathy may affect fatty acid metabolism in cats. Although the current study only included cats with HCM, the effects of underlying disease would be interesting to evaluate in future studies of cats. In one study, Boxer dogs had higher plasma concentrations of γ-linolenic acid, but lower concentrations of arachidonic and total n-6 fatty acids, than Doberman Pinschers. 14 Therefore, comparison of fatty acids among larger groups of cats with differing forms of cardiomyopathy is warranted.
One question raised by this study is whether n-3 fatty acid supplementation in cats would have the benefits seen in humans and dogs, given that DHA concentrations were higher in cats with HCM. Two studies have examined the effects of fish oil supplementation on platelet function in cats. The first showed no significant change in platelet aggregation or bleeding times after 8 weeks in eight cats supplemented with up to 1.689 g EPA and 0.936 g DHA per day, despite significant elevations in plasma concentrations of EPA and DHA. 29 The second study did show a significant difference in bleeding time and platelet aggregation after 16 weeks (but not 8 weeks) in 12 cats supplemented with a diet containing 1.03 g/kg diet of n-3 fatty acid. 30 We are not aware of studies examining possible clinical benefits of fatty acid supplementation in cats with cardiac disease, but further studies are warranted. In addition, more research is needed on the differential effects of EPA versus DHA or the ratio between the two fatty acids.
An important limitation of the current study is the lack of information on, and control of, dietary intake of fatty acids, which could account for differences in plasma n-3 fatty acids seen between the current study and previous canine studies. Although cats receiving n-3 fatty acid supplements were excluded from the study and no diets appeared to be highly enriched in n-3 fatty acids, more subtle differences in the fatty acid profiles could have been present that could result in the different plasma fatty acid concentrations. In addition, as diets in the current study were not analyzed, neither the exact concentration of dietary fatty acids nor dietary fatty acid intake in each individual cat were known. Future studies would benefit from having all cats eating a similar diet to see if plasma fatty acid differences persisted despite eating a similar diet. Alternatively, dietary fatty acids could be analyzed to determine if the dietary intake of fatty acids was different between groups and thus could have accounted for the differences found in plasma fatty acid concentrations.
Another limitation that must be noted is that, owing to financial constraints, testing to ensure eligibility was not as thorough for cats in the control group as for those in the HCM group. Therefore, while control cats had to be overtly healthy, with no history evidence of prior medical issues and had normal cardiac structure and function confirmed using echocardiography and a complete physical examination, blood testing in the controls was limited to a PCV, TS and platelet count. Therefore, cats in the control group may have had underlying diseases that escaped detection because of the limited blood testing, which could have affected the plasma fatty acid results.
Conclusions
Despite the multiple limitations of the study, cats with HCM had higher concentrations of palmitic acid, DHA and total n-3 fatty acids, and lower concentrations of linoleic acid than normal cats. These changes did not appear to be related specifically to the presence of CHF. While these data suggest that supplementation of DHA may not have benefits in terms of correcting a deficiency, investigation of other effects of DHA or of supplementation of EPA warrants further investigation.
Footnotes
Acknowledgements
We are grateful for the assistance of Dawn Meola, Dr Mohsen Meydani, Tahira Hasan and Tim Noble.
Conflict of interest
The authors do not have any potential conflicts of interest to declare.
Funding
This study was funded by a Nestlé Purina PetCare Resident Research Grant.
This article was presented, in part, at the 2011 American College of Veterinary Internal Medicine Forum, Denver, CO, USA.