Responses in randomised groups of healthy, adult Labrador retrievers fed grain-free diets with high legume inclusion for 30 days display commonalities with dogs with suspected dilated cardiomyopathy

Bakke, Anne Marie; Wood, Joshua; Salt, Carina; Allaway, David; Gilham, Matt; Kuhlman, Gail; Bierer, Tiffany; Butterwick, Richard; O’Flynn, Ciaran

doi:10.1186/s12917-022-03264-x

Research
Open access
Published: 28 April 2022

Responses in randomised groups of healthy, adult Labrador retrievers fed grain-free diets with high legume inclusion for 30 days display commonalities with dogs with suspected dilated cardiomyopathy

Anne Marie Bakke¹,
Joshua Wood¹,
Carina Salt¹,
David Allaway¹,
Matt Gilham¹,
Gail Kuhlman²,
Tiffany Bierer²,
Richard Butterwick¹ &
…
Ciaran O’Flynn¹

BMC Veterinary Research volume 18, Article number: 157 (2022) Cite this article

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A Correction to this article was published on 17 May 2022

This article has been updated

Abstract

Early responses in healthy adult dogs fed grain-free diets with high inclusion of split peas (20%) and lentils (40%) that may lead to canine diet-induced dilated cardiomyopathy (DCM) were investigated. To help understand the clinical relevance of the findings, a survey of electronic health records (EHR) was conducted of dogs with and without suspected DCM for comparison. Control and Test diets were fed to Labrador retriever dogs for 30 days (n = 5 and 6, respectively). Blood and urine samples collected at baseline and days 3, 14 and 28/30 were analyzed for hematology, clinical biochemistry and taurine concentrations. The EHRs of dogs at Banfield® Pet Hospitals in the 2-year period 2018-2019 were surveyed, revealing 420 dogs diagnosed with DCM, which were compared with 420 breed, gender and age-matched healthy control dogs. Compared to baseline values, feeding the Test diet for 28 days caused progressive, significant (p < 0.001) decreases in red blood cell counts (RBC), hematocrit and total hemoglobin by 7.7, 8.3 and 6.3%, respectively, and a 41.8% increase in plasma inorganic phosphate. Commonalities in these parameters were observed in clinical DCM cases. Regarding taurine status, Test dogs transiently increased whole-blood (23.4%) and plasma (47.7%) concentrations on day 14, while taurine:creatinine ratio in fresh urine and taurine in pooled urine were reduced by 77 and 78%, respectively, on day 28/30. Thus grain-free, legume-rich Test diets caused reduced RBC and hyperphosphatemia, findings also indicated in dogs with suspected DCM. Changes in taurine metabolism were indicated. The data will aid in generating hypotheses for future studies.

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Background

Recent years have shown an increasing trend in the use of less traditional (alternative) feed ingredients in pet foods. However, these may change the bioavailability or metabolism of nutrients and other dietary components, potentially leading to health issues. A possible current example is dilated cardiomyopathy (DCM) in dogs in the US following prolonged intake over months or years of grain-free, legume-rich diets [1, 2]. Historically, DCM was most prevalent in genetically predisposed large and giant breed dogs [3,4,5] or as a result of taurine deficiency [6, 7], which in dogs has been limited to certain breeds [7,8,9,10,11,12,13,14,15] suggesting genetic factors influencing taurine biosynthesis/metabolism. Although taurine deficiency was considered as a cause of the recent DCM cases, factors indicate that this may not always be the case: 1) reported diet, whole blood and plasma concentrations of taurine have in most cases been within recommended ranges [1, 2, 16, 17]; and 2) affected dogs are represented across a wider range of breeds/sizes and ages [1, 2, 18]. Further support for a dietary aetiology is provided by improvement of DCM when dogs are transitioned onto conventional, grain-inclusive diets, either with or without additional taurine supplementation [1, 2, 16, 19, 20]. However, the specific pathophysiological events leading to diet-associated DCM in dogs fed grain-free, legume-rich diets is currently not understood.

Although a number of clinical studies report implications of such diets on canine cardiac health [17,18,19,20], fewer report routine health parameters along with taurine status in dogs fed such diets [16, 21, 22].

To this end, a 30-day, longitudinal, small-scale study was initiated with the aim of understanding whether less conventional diets with high inclusion of legumes (60% total) lead to changes in routine hematology, clinical biochemistry, and whole blood, plasma and urinary taurine, the latter considered a more accurate indication of taurine status than blood concentrations [23]. The clinical relevance of the results of the responses were then examined by comparing them to those of dogs with DCM in their diagnosis, along with a breed, gender and age-matched set of healthy controls collected from a survey of electronic health records (EHRs) from Banfield® Pet Hospitals. The hypothesis tested was that routine health parameters and taurine status affected in healthy dogs fed diets with less conventional ingredients, including high levels of split peas and lentils, have clinical relevance for DCM development.

Results

Feeding study

Diets

The results of the nutrient analysis are provided in Table 1. Both Control and Test diets were nutritionally compliant as defined by guidance and regulatory agencies [24, 25], but they differed somewhat in specific nutrient levels. The Control diet was somewhat higher in carbohydrates and ash, while the Test diet was higher in protein, fat and contained twice as much crude fibre. Other nutrients of note were higher leucine, taurine, calcium (Ca), phosphorus (P) and vitamin A in the Control diet, whereas the Test diet contained higher concentrations of lysine, linoleic acid, alpha-linolenic acid, potassium, microminerals and most vitamins.

Table 1 Nutrient analysis results of the experimental diets as fed basis

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Besides the differences in the animal-derived ingredients and legume content, a relatively high level of flaxseed meal was also included in the Test diet. Analysis for cyanogenic glycosides present in flaxseed revealed differences between diets: the Control diet contained 13 mg/kg linamarin, while linustatin and neolinustatin concentrations were below detection limit (< 10 mg/kg). The Test diet contained < 10 mg/kg linamarin, 103 mg/kg linustatin and 128 mg/kg neolinustatin.

Both diets were palatable and well-accepted by the dogs during the trial, with no reports of meal refusals or apparent adverse effects on the dogs’ general health and well-being.

Blood parameters and taurine status

Table 2 summarizes the hematology data. Red blood cell counts (RBC) were significantly (p < 0.05) reduced in Test dogs compared to baseline and dogs fed the Control diet on days 3, 14 and 28, with five and six of six Test dogs demonstrating counts below the lower limit of the reference range (6∙10¹²/L) on days 14 and 28, respectively. Concomitant reductions in hematocrit (HCT) (p < 0.05) were observed, while the mean cell volume (MCV) and reticulocyte distribution width (RDW) (Table 2) were not or only transiently affected. Blood total hemoglobin (HGB) concentration also decreased significantly from baseline on day 28 in the dogs fed the Test diet but remained within the reference range. Mean cell hemoglobin (MCH) and mean cell hemoglobin concentration (MCHC) were generally above the upper limit of the respective reference ranges in both Control and Test-fed dogs. And MCHC increased significantly from baseline on all sampling days reaching an apparent plateau from day 15 for Test dogs. Total white blood cell counts (WBC) were below or within respective physiological ranges, despite some transient, statistically significant diet differences on day 3 (Table 2). Mean platelet volume (MPV) increased compared to baseline in the Test group over the course the feeding trial, significantly so on days 14 and 28, but also remained within the reference range.

Table 2 Mean fasted hematology results with 95% confidence intervals for each diet group (Control, n = 5; Test, n = 6) and sampling time (baseline and 3, 14 and 28 days), as well as results of the statistical analysis (p-values). In the left column, units for each parameter are provided in parentheses and internal lab reference ranges for each parameter are provided in italics

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Clinical biochemistry data are summarized in Table 3. Plasma inorganic P concentrations were significantly increased in dogs fed the Test diet compared to baseline, and significantly more than Control-fed dogs, at all three sampling times after baseline. Half the dogs exhibited P concentrations at or above the upper limit of the reference range at days 14 and 28. Plasma total Ca and whole blood ionized Ca (iCa) were transiently and significantly decreased in dogs fed the Test diet on days 3 and 14, with half the dogs exhibiting Ca concentrations below the lower limit of the reference range at those time points. On day 28, however, Ca and iCa concentrations had recovered and did not differ from baseline values (p > 0.05). Total protein, albumin and globulin were not affected by the Test diet. Cholesterol decreased from baseline in the dogs fed the Test diet. However, concentrations were within the reference range and significant differences between diets were not detected (Table 3). Liver enzymes alanine aminotransferase (ALT) and aspartate aminotransferase (AST) activities were increased in all dogs, significantly for AST on all sampling days for the Control group dogs and at day 14 in the Test dogs. AST activities were also often above the upper limit of the reference range in dogs in both diet groups, with all Test group dogs exhibiting values above 41 U/L at day 14. However, no differences between diets were observed and the activities were apparently decreasing in most dogs in both diet groups on day 28.

Table 3 Mean fasted clinical biochemistry results with 95% confidence intervals for each diet group (Control, n = 5; Test, n = 6) and sampling time (baseline and 3, 14 and 28 days), as well as results of the statistical analysis (p-values). In the left column, units for each parameter are provided in parentheses and IDEXX lab reference ranges (unless otherwise specified) for each parameter are provided in italics

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Alkaline phosphatase (ALP) activities were also increased in the Test group dogs from baseline at all sampling times, although within the reference range for all dogs. Bone-specific ALP activities did not reveal differences from baseline or between groups. Creatinine concentrations were transiently decreased in Test dogs on day 3, while no significant changes were observed in urea, and all values for all dogs were within the respective reference ranges (Table 3). Signs of gross hemolysis in the plasma samples were not observed during analysis.

The heart specific biomarker N-terminal pro b-type natriuretic peptide (NTproBNP) concentration in serum, which indicates myocardial stretch when elevated, was not significantly affected by diet and remained within the reference range suggested for Labrador retrievers of 275-2100 pmol/L [26] for all dogs. Nor did any of the dogs fed the control (n = 2) or Test diet (n = 4) show changes of concern in cardiac parameters as evaluated by the radiographer conducting the ultrasound scans (data not shown).

Table 4 summarizes taurine status. Compared to baseline values, whole blood taurine concentrations increased significantly in both Control and Test group dogs, significantly on days 14 and 28 for Control group, while in Test group dogs, a transient increase was observed on day 14 only. No significant differences between diet groups were observed for whole blood, while plasma concentrations increased more in Test compared to Control on day 14. Taurine in fresh urine varied widely, and a significant decrease from baseline was only detected in Test dogs on day 3. When related to creatinine concentrations (taurine:creatinine ratios; Table 4), however, a significant, decrease was detected in Test

Table 4 Taurine status (in μmol/L) as assessed in whole blood, plasma and urine (fresh and pooled). Values are means with 95% confidence intervals for each diet group (Control, n = 5; Test, n = 6) and sampling time (baseline and 3, 14 and 28/30 days), as well as results of the statistical analysis (es). Parameters measured in fresh urine samples are from fasted dogs

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group dogs on both day 3 and 28 compared to baseline, and a diet group difference was observed on day 3. This was substantiated by similar reductions in taurine concentrations measured in pooled urine at baseline and day 30. In the dogs fed the Test diet, pooled urine taurine concentration decreased to 21% of baseline, while the mean taurine-to-creatinine ratio value measured on day 28 decreased to 23%.

EHR interrogation

Figure 1 shows the estimated relative differences and Fig. 2 shows the raw scatter plots for dogs with DCM compared to age and breed-matched controls without DCM for hematology and clinical biochemistry parameters by analyte and age. The HCT, lymphocyte, and albumin data did not converge when the model was fitted. Therefore, the model and subsequent interpretation is unreliable for these analytes.

Significant relative differences (p < 0.002) between dogs with DCM and healthy controls for the hematology parameters RBC, HCT, HGB, RDW, WBC, lymphocytes, monocytes, and platelet counts were observed. Compared to matched controls, RBC count, HCT and HGB were reduced, especially in younger dogs. More complex relationships were observed for RDW, WBC, lymphocytes, monocytes and platelet counts, with variable relative values below, at and above those of matched controls, depending on age. Significant relative differences (p < 0.002) were also observed between dogs with DCM and healthy controls for the clinical biochemistry parameters total protein, globulin, ALT, ALP, blood urea nitrogen (BUN), creatinine and inorganic P. Compared to matched controls, ALT, ALP, BUN and inorganic P were often increased. More complex relationships were observed for total protein, globulin and creatinine, with variable relative values below, at and above those of matched controls, depending on dogs’ age.

Discussion

The data from the feeding study indicate that feeding healthy adult Labrador retriever dogs a diet containing a combination of the main ingredients kangaroo, peas, red and green lentils – with a total legume inclusion of 60% – sunflower oil, and flaxseed meal for a period of 30 days caused a rapid reduction in RBC, hyperphosphatemia, and possible implications on taurine status, as indicated by reduced urinary concentrations of taurine. The clinical relevance of the hematology and clinical biochemistry findings from the feeding study was at least partially substantiated by interrogation of EHRs of dogs at Banfield® Hospitals with DCM in their diagnosis: significantly decreased RBC counts, HCT and total HGB, and increased plasma inorganic phosphate were observed compared to age and breed-matched healthy controls. While compelling, limitations with the EHR data were a lack of diet history, limited information on how DCM was diagnosed, and potential inaccuracies and confounding factors that may influence measured analyte values. When building the master dataset, only the current patient status was considered, while any previous diagnosis or co-morbidities that may influence measured analytes were not assessed. Attempts to apply such additional filters would require considerable resource in interrogating written records, substantially reduce the dataset, and detrimentally impact the model accuracy, and were therefore not undertaken when the survey was conducted. Also, additional exclusion for comorbidities is often not possible when applying the model in a practice or screening application. For some scenarios it is therefore our preference to define cases and controls as described.

Given the assumption that the Banfield® dogs diagnosed with DCM reflect all causes with differing mechanisms leading to DCM, it is compelling that the blood analytes from the EHR of DCM cases largely mirrored those of the dogs fed the Test diet in the feeding study. Breeds genetically predisposed to DCM were relatively under-represented among the those included (Fig. 3A), which may suggest that many of the DCM cases were diet-related during the time that the survey was conducted. On the other hand, the mixed etiologies were a likely source of dilution of the data and may explain inconsistencies within and between the datasets.

A similar rapid decrease in RBC and increase in plasma inorganic P as observed from day 3 of the feeding study was also recently observed in beagles following 7 days of feeding a high protein diet containing split peas (9th listed ingredient) and lentils (11th listed ingredient) by Reis et al. [22]: mean RBC of ~ 6*10¹²/L and serum inorganic P of 1.67 mmol/L were observed. Specific inclusion levels of the legumes were not provided. However, the fava bean-containing diets at 30% inclusion level tested in the study also decreased RBC similarly compared to the same diets containing fermented fava beans, while plasma P was largely unaffected [22]. On the other hand, no changes in RBC and only a transient increase in plasma inorganic P at 13 weeks were observed in Labrador Retrievers in the 6-month Donadelli et al. study [21]. This may be due to different legume levels in the test diet in that study, which were not specified, although whole lentils and peas were the third and sixth listed ingredient, respectively. Also, the first sampling time in the Donadelli study [21] was at 13 weeks following transition to the test diet, and any earlier, transient changes were therefore not detected.

Iron deficiency was considered as a potential cause of the rapid decrease in HGB and RBC in the Test group. However, the iron concentration was higher in the Test compared to Control diet. The iron sources were from raw materials only in the Control and a combination of raw materials and a premix containing an iron amino acid chelate in the Test diet. Thus iron bioavailability was considered high for both diets. Hemolysis as a cause cannot be completely ruled out, although it was not registered as an issue during plasma analysis or signs observed (see also below) in the EHR survey’s total bilirubin (TBILI) data. However, this area deserves further investigation as released heme can cause oxidative damage by radical oxygen species, impaired nitric oxide signalling, endothelial dysfunction and systemic vasoconstriction, which may impact heart structure and function [27]. Feeding grain-free diets and diets containing peas, lentils or potatoes to dogs has recently been linked to increased median cardiac troponin I and plasma taurine, both suggested by Adin et al. to originate from damaged cardiomyocytes, while no changes in whole-blood taurine or NTproBNP were observed [18].

Although anemia has not previously been suggested as a cause of DCM in dogs, reduced RBC numbers can reduce oxygen transport, which may put undue strain on the heart muscle when increased blood volumes are required to meet cardiac and peripheral tissue needs for oxygen. Anemia may also exacerbate signs and complicate recovery in DCM patients if not discovered and treated. On the other hand, the data from the feeding study and EHRs do not indicate clinically apparent anemia, as reductions in both RBC and HCT below reference ranges (RBC < 6*10¹²/L and HCT < 40) were observed in only a small proportion of test animals (Table 2, Fig. 2).

High choline supplementation of 8-10 mg choline chloride/kg BW have been reported to cause reductions in RBC in dogs [28, 29]. However, the basal diet without supplementation was not tested in those studies and other dietary factors can therefore not be ruled out as a cause of the reduced RBC. On the other hand, McKibben et al. [30, 31] supplemented puppy diets with 1500 and 2000 mg choline chloride/kg diet, respectively, without observing reductions in RBC. In the current study, the average choline intake was 24 mg/kg BW for the dogs fed the Control diet and 44 mg/kg BW for those fed the Test diet, both higher than the supplementation concentrations provided for the dogs in the studies by Davis [28, 29], but far lower than in the McKibben et al. studies [30, 31].

Anti-nutritional factors (ANFs) present in the legumes of the Test diet cannot be ruled out as causes of the reduced RBC. Although not analysed in the diets, phytic acid can bind minerals such as iron and thereby reduce bioavailability. The amphipathic nature of saponins that are present in some legumes are known to cause hemolysis by disrupting cell membranes and changes in intestinal permeability in various animals tested [32,33,34,35,36]. Likewise, legume lectins can agglutinate cells, including red blood cells, by binding to carbohydrate moieties on the cell surface, and also affect intestinal permeability [37]. However, reduced RBC or other hematological changes due to flaxseed inclusion in the form of linseed cake and its inherent cyanogenic glycosides linostatin and neolinostatin in canine diets have not previously been described [38]. The anti-nutritional factors and/or fibre present in the legume-rich Test diet may also impact taurine and other nutrient bioavailabilities, and thus impact cardiac health. If one or more deficiencies in nutrients specifically affecting heart function and health take longer to manifest, this may explain the development of DCM in dogs when fed such diets over longer periods of time.

The increasing number of individuals affected and increasing severity of hyperphosphatemia observed in the Test diet-fed dogs from day 3 of the feeding trial may be a sign of hemolysis, with the disruption of red blood cells causing intracellular phosphate to leak into the plasma. However, and as mentioned above, no direct signs of hemolysis were observed. Alternatively, the increased plasma inorganic phosphate may also be a sign of increased P availability for absorption. Changes in mineral interactions in the intestine, possibly caused by e.g. high phytic acid and fibre in legume-rich diets, may explain differences in P availability. This because: 1) the high plasma P was most likely not caused by dietary P levels, as the Test diet contained about 1.4 g/1000 kcal (0.49%) compared to 2.5 g/1000 kcal (0.91%) in the Control diet, and 2) transient disruptions in plasma concentrations of Ca and iCa were also evident. A hormonal response, e.g. parathyroid hormone (PTH), fibroblast growth factor 23 (FGF-23) and/or vitamin D metabolites, elicited by the low plasma Ca/iCa and/or high plasma P, would be expected in an attempt to restore homeostasis, as indicated by more or less restored concentrations of blood Ca at day 28. Unfortunately, hormonal regulation was not investigated in the current trial. Despite the hyperphosphatemia, no signs of renal marker changes were observed in the Test group dogs, with serum creatinine and urea, as well as urine specific gravity (data not shown) within reference ranges. A small increase in ALP was observed in the Test diet-fed dogs, however as bone-specific ALP did not differ between the dog groups, it is unlikely that resorption of P from bone was a cause of the increased plasma inorganic phosphate concentrations. Again, the inorganic phosphate and ALP data differed in the Donadelli et al. study [21], in which the transient increase in inorganic phosphate at 13 weeks did not exceed the upper reference range and decreases in ALP were observed in dogs fed the legume-rich Test diet following 13 and 26 weeks of feeding.

Although hyperphosphatemia has not been previously cited as a cause of DCM in dogs, it has been associated with myocardial hypertrophy, bone restructuring, and possibly reduced hematocrit in parathyroid hormone-treated, parathyroidectomized and 5/6 nephrectomized Wistar rats fed high-P (1.2%, Ca:P 0.58) diets [39]. Because the experimental conditions differ substantially, including species investigated and dietary P and Ca:P levels, it is difficult to directly compare our results with those reported in Neves et al. [39]. In other studies, high FGF-23 concentrations in human patients suffering from chronic kidney disease has been implicated in the pathogenesis of left-ventricular hypertrophy [40]. FGF-23 was subsequently shown to cause cardiac myocyte hypertrophy in vitro [41], which has later been demonstrated in mice in vivo [42]. Again, how this relates to the current study is difficult to assess at this time, but closer inspection of hormones regulating mineral homeostasis should be included in future trials with dogs fed alternative diets or in clinical trials with dogs with clinically apparent DCM.

Taurine concentrations in whole blood and plasma were not significantly reduced in the Test group dogs, confirming recent observations in dogs suffering from apparent diet-associated DCM [2] as well as healthy dogs fed grain-free diets [16, 21]. Concentrations were transiently increased on day 14 in the current study, possibly due to up-regulated biosynthesis from cysteine. However, decreased urinary taurine concentrations, a trend not observed previously [16, 21], indicated that neither biosynthesis nor taurine potentially released from cardiomyocytes [18] could fully meet taurine requirements when fed the Test diet. This may have been compounded by dietary methionine and cysteine levels only marginally meeting recommended allowances in this diet. An increased need for cholesterol and taurine to meet increased bile salt synthesis as a response to increased fecal bile acid/salt losses, as previously reported [21], may therefore have contributed to the decreases in plasma cholesterol and urinary taurine. Interestingly, the whole blood taurine concentrations at baseline and day 3 for both diet groups and day 28 for the Test diet group were below, while plasma concentrations were within, the reference ranges recently suggested for Golden retrievers (213-377 and 63-194 μmol/L, respectively) [43]. Although these reference ranges established for Golden retrievers need to be validated for other dog breeds, the data provided herein suggest they may be of value for Labrador retrievers. As previously suggested [21, 23], whole blood and urinary taurine excretion, as assessed by taurine:creatinine ratio, appear to be more sensitive measures of taurine status in dogs than plasma taurine, and relevant for evaluating taurine status in future feeding studies.

This study had limitations, including the small number of dogs included in the feeding study; the limited data regarding hemolysis; the absence of reticulocyte count and cardiac troponin I data; and the above-mentioned limitations in the EHR data with the lack of diet history, limited information on how DCM was diagnosed, and factors that may have influenced analyte values.

Conclusions

The data from the 30-day, longitudinal feeding study, indicate that the Test diet containing kangaroo, 60% of the legumes peas (20%) and lentils (40%), and flaxseed (7%) may cause reduced RBC and hyperphosphatemia, as well as possible disturbances in taurine status. EHR interrogation of dogs with DCM in their diagnosis largely corroborated the hematology and clinical biochemistry findings from the feeding study, suggesting these data may be clinically relevant. Further studies are needed to increase our understanding of the relevance of the findings and further details regarding the pathophysiology associated with the development of diet-associated DCM in dogs.