Reference intervals for whole blood (WB) and plasma taurine concentrations in dogs have been derived; however, breed-specific differences may exist.1,2 Platelets are rich in taurine,3 and Cavalier King Charles Spaniels (CKCSs) are frequently affected by thrombocytopenia and macrothrombocytosis.4 In addition, increased plasma taurine concentrations have been reported in some dogs with myxomatous mitral valve disease (MMVD),5 with CKCSs strongly predisposed to developing this disease.6–10 Given these factors, further investigation into normal taurine reference intervals in CKCSs is warranted, as breed-specific differences may exist. In addition, determination of whether taurine concentrations change in relation to MMVD status in CKCSs may also have clinical relevance.
Taurine deficiency has been recognized as a potential cause of dilated cardiomyopathy (DCM) in dogs for many years, particularly in a small number of specific dog breeds, including Golden Retrievers, Cocker Spaniels, and Newfoundlands.11–16 More recently, suspected cases of diet-associated DCM have increased in many breeds of dogs,17,18 with the role of taurine deficiency in these cases being of questionable significance.19–21 Decreased taurine concentrations and diet-associated DCM have been reported in Golden Retrievers eating “nontraditional” diets with dietary features associated previously with reports of diet-associated DCM; thus, breed-specific differences may exist.2 When it comes to nutritional assessment of various diets, any number of potential comparisons can be made; however, the World Small Animal Veterinary Association (WSAVA) global nutritional guidelines are considered the gold standard, with established guidelines for the selection of pet food available.22 The impact of diet on taurine concentrations in CKCSs has not been investigated previously.
The objective of our study was to determine breed-specific reference intervals for WB and plasma taurine concentrations in CKCSs and to determine whether taurine concentrations differ across preclinical MMVD stages or between CKCSs eating diets that meet WSAVA nutritional guidelines versus other diets.
Materials and Methods
Clinically healthy adult CKCSs that were not receiving any cardiac medications were recruited prospectively as part of a larger, prospective cross-sectional study.23 This study was approved by the Institutional Animal Care and Use Committee at Texas A&M University, and owners gave informed consent prior to participation.
General health history, demographic data, and diet and supplement history were collected. General health status was assessed by reviewing health history forms and an abbreviated physical examination. Diets from brands that met the following criteria were considered to meet WSAVA nutritional guidelines: full-time, qualified nutritionists are employed and participate in the formulation of foods; feeding trials that meet or exceed the Association of American Feed Control Officials standards are carried out for most diets; foods are produced in facilities owned and operated by the manufacturer; quality control protocols are described; complete nutritional analysis including caloric value per unit of food is readily available; and product research is published in peer-reviewed journals.22 All CKCSs underwent an echocardiogram by a board-certified veterinary cardiologist or third-year veterinary cardiology resident with standard 2-D, M-mode, and Doppler imaging to allow for MMVD staging based on the most recent MMVD consensus guidelines.24 Left ventricular internal diameter at end diastole (LVIDdN) and left ventricular internal diameter at end systole (LVIDsN) were measured on a short-axis M-mode image at the level of the papillary muscles and were normalized to body weight as LVIDdN and LVIDsN, respectively,25 and the left atrial-to-aortic root ratio was measured from a 2-D short-axis view at the heart base.26 Fractional shortening (FS) was also recorded. All echocardiographic measurements were repeated 3 times, with the average of these 3 measurements used for data analysis. Because CKCSs were staged based on echocardiographic measurements, some CKCSs that did not have an audible heart murmur were considered stage B1 based on mitral valve remodeling, with or without mitral regurgitation. Exclusion criteria were age < 1 year, history of receiving taurine supplements, lack of diet or supplementation history provided by owner, insufficient WB and plasma samples for taurine analysis, diagnosis of congenital or other non-MMVD cardiac disease, or history of active or chronically managed noncardiac illness that could affect the cardiovascular system. Cases of DCM were not included in the statistical analysis, but data were reported for descriptive purposes.
Lithium heparin blood samples were obtained from peripheral veins, a WB aliquot was made, and plasma was separated within 30 minutes by centrifugation at 1,900 relevant centrifugal force for 15 minutes. Plasma and WB were then deproteinized for taurine analysis according to laboratory protocols27 at the Gastrointestinal Laboratory, Texas A&M University. Briefly, samples were deproteinized with the 1:1 (volume/volume) addition of 5% sulfosalicylic acid and 500 µM D-glucosaminic acid as an internal standard, and were filtered subsequently using a 0.2-µm polyvinylidene fluoride centrifugal filter. The filtrate was then placed on a chilled autosampler or stored at –80 °C until further analysis on a Biochrom 30+ lithium high-performance amino acid analyzer (Harvard Biosciences). Injection volume was set to 30 µL with a partial loop fill injection mode, and the acquisition method followed that developed by the manufacturer for taurine analysis. Total run time, including re-equilibration, was 47 minutes per sample. Chromatographic separation was achieved in 14.5 minutes. Acquired data were analyzed using Agilent OpenLab CDS EZ Chrom Edition version A.04.08 (Agilent Technologies). The r2 value for the taurine standard curve in the linear range of 2.5 to 750 µM met or exceeded 0.997 at the time of analysis. The recovery of internal standard from each sample was within 2 SDs of the mean recovery. Six plasma samples and 5 WB samples with taurine concentrations spanning the reference ranges were run in duplicate 2 years apart, and recovery of taurine was between 98% and 107%.
Data were analyzed using statistical software packages (Graph Pad Prism version 9.0.1; JMP Pro version 16.0.0, SAS Institute, Inc.). Shapiro-Wilk normality testing was performed, and nonparametric testing was carried out as a result of non-normal distribution in one or more groups, or because the assumption of equal variances was not met. Kruskal-Wallis tests with Dunn’s post hoc multiple comparisons testing was used to determine whether WB or plasma taurine concentrations varied among groups when dogs were grouped on the basis of MMVD stage (3 groups). Mann–Whitney testing was used to determine whether WB or plasma taurine concentrations varied between groups when dogs were grouped on the basis of diet (2 groups). Spearman’s correlation testing was performed between plasma taurine concentrations (measured in micromoli), WB taurine concentrations (measured in micromoli), LVIDsN, and FS (measured in percentage) to determine correlations among these variables. Breed-specific reference intervals were calculated using the central 95% of the data, without removing outliers, according to American Society for Veterinary Clinical Pathology guidelines.28
Results
A total of 235 CKCSs were enrolled in the study, with 35 excluded for various reasons (lacked both WB and plasma sample for taurine analysis [n = 9], history of receiving taurine supplementation [n = 20], no diet/supplement history provided [n = 1], and other [non-MMVD] cardiovascular diagnoses [n = 5]). Of the non-MMVD cardiovascular diagnoses, there were 2 CKCSs diagnosed with DCM. The final analyzed study population was comprised of 200 CKCSs. Median age was 7.3 years (range, 1.2 to 14.9 years) and median body weight was 8.2 kg (range, 4.4 to 15.6 kg). There were 81 males (14 intact) and 119 females (21 intact).
With regard to MMVD staging, there were 12 CKCSs with stage A (6%) that did not yet have evidence of MMVD, 150 with stage B1 (75%), and 38 with stage B2 (19%) MMVD. Taurine concentrations in WB (stage A: median, 306 µM taurine [range, 191 to 366 µM taurine]; stage B1: median, 262 µM taurine [range, 104 to 482 µM taurine]; stage B2: median, 280 µM taurine [range, 57 to 421]; P = .073) and plasma (stage A: median, 112 µM taurine [range, 101 to 185 µM taurine]; stage B1: median, 110 µM taurine [range, 36 to 362 µM taurine]; stage B2: median, 116 µM taurine [range, 40 to 217 µM taurine]; P = .444) were not significantly different among stages of MMVD in this study population. These data are presented in Figure 1.
Scatterplot depicting plasma (A) and whole blood (B) taurine concentrations for Cavalier King Charles Spaniels grouped based on myxomatous mitral valve disease stage. Red lines indicate median.
Citation: Journal of the American Veterinary Medical Association 260, S3; 10.2460/javma.22.07.0280
Scatterplot depicting plasma (A) and whole blood (B) taurine concentrations for Cavalier King Charles Spaniels grouped based on myxomatous mitral valve disease stage. Red lines indicate median.
Citation: Journal of the American Veterinary Medical Association 260, S3; 10.2460/javma.22.07.0280
Scatterplot depicting plasma (A) and whole blood (B) taurine concentrations for Cavalier King Charles Spaniels grouped based on myxomatous mitral valve disease stage. Red lines indicate median.
Citation: Journal of the American Veterinary Medical Association 260, S3; 10.2460/javma.22.07.0280
Dogs represented in the study population ate a variety of different diets. There were 78 CKCSs (39%) eating diets that met WSAVA nutritional guidelines and 116 CKCSs (58%) that were not. There were 6 additional dogs that ate a combination of diets that did and did not meet WSAVA nutritional guidelines. Because of their mixed-diet status, these 6 dogs were not included in statistical comparisons related to diet. In the remaining 194 CKCSs, taurine concentrations were not significantly different between CKCSs eating diets that met WSAVA nutritional guidelines versus those eating diets that did not meet WSAVA nutritional guidelines in either WB (WSAVA diet: median, 263 µM taurine [range, 104 to 411 µM taurine]; non-WSAVA diet: median, 266 µM taurine [range, 57 to 482 µM taurine]; P = .527) or plasma (WSAVA diet: median, 110 µM taurine [range, 47 to 224 µM taurine]; non-WSAVA diet: median, 114 µM taurine [range, 36 to 362 µM taurine]; P = .345). These data are presented in Figure 2.
Scatterplot depicting plasma (A) and whole blood (B) taurine concentrations for Cavalier King Charles Spaniels eating diets that met World Small Animal Veterinary Association (WSAVA) nutritional guidelines versus other diets. Red lines indicate median.
Citation: Journal of the American Veterinary Medical Association 260, S3; 10.2460/javma.22.07.0280
Scatterplot depicting plasma (A) and whole blood (B) taurine concentrations for Cavalier King Charles Spaniels eating diets that met World Small Animal Veterinary Association (WSAVA) nutritional guidelines versus other diets. Red lines indicate median.
Citation: Journal of the American Veterinary Medical Association 260, S3; 10.2460/javma.22.07.0280
Scatterplot depicting plasma (A) and whole blood (B) taurine concentrations for Cavalier King Charles Spaniels eating diets that met World Small Animal Veterinary Association (WSAVA) nutritional guidelines versus other diets. Red lines indicate median.
Citation: Journal of the American Veterinary Medical Association 260, S3; 10.2460/javma.22.07.0280
Given the lack of significant differences of WB or plasma taurine concentrations across preclinical MMVD stages or between CKCSs eating diets meeting WSAVA nutritional guidelines versus those that ate other diets, the entire study population with available taurine concentrations was used for the development of breed-specific reference intervals. In 2 CKCSs, sample quantity was insufficient for measuring WB taurine (only plasma concentrations were available), and in another 2 dogs only a WB taurine concentration was available. Therefore, 198 dogs were used to calculate a breed-specific reference interval for WB and plasma taurine concentrations in CKCSs. Any outliers were not removed from the data set. Distribution is shown in Figure 3. Plasma taurine concentrations had non-normal distribution (Shapiro-Wilk W = 0.906, P < .001) and WB taurine concentrations had normal distribution (Shapiro-Wilk W = 0.989, P = .146). Reference intervals were calculated from the central 95% of the data according to American Society for Veterinary Clinical Pathology guidelines28 using the nonparametric percentile method, resulting in a reference interval of 152 to 373 µM (90% CI lower limit, 104 to 161 µM; 90% CI upper limit, 361 to 421 µM) for WB, and 51 to 217 µM (90% CI lower limit, 40 to 60 µM; 90% CI upper limit, 172 to 230 µM) for plasma.
Violin plot showing the distribution of plasma and whole blood taurine concentrations in dogs (n = 198) used to form the reference intervals. Dashed line represents median; dotted lines represent first and third quartiles.
Citation: Journal of the American Veterinary Medical Association 260, S3; 10.2460/javma.22.07.0280
Violin plot showing the distribution of plasma and whole blood taurine concentrations in dogs (n = 198) used to form the reference intervals. Dashed line represents median; dotted lines represent first and third quartiles.
Citation: Journal of the American Veterinary Medical Association 260, S3; 10.2460/javma.22.07.0280
Violin plot showing the distribution of plasma and whole blood taurine concentrations in dogs (n = 198) used to form the reference intervals. Dashed line represents median; dotted lines represent first and third quartiles.
Citation: Journal of the American Veterinary Medical Association 260, S3; 10.2460/javma.22.07.0280
To investigate a possible relationship between taurine concentrations and echocardiographic measurements reflective of systolic function further, Spearman’s correlations were explored between taurine concentrations and both LVIDsN and FS. The median LVIDsN was 0.93, with an interquartile range of 0.83 to 1.02. The median FS was 40.4%, with an interquartile range of 36% to 45.4%. The only significant correlations found were between WB and plasma taurine concentrations themselves (P < .001, ρ = 0.452), and between LVIDsN and fractional shortening (P < .001, ρ = –0.450), which was to be expected (Table 1).
Spearman’s correlations between heart function indicators and whole blood or plasma concentrations of taurine.
| Variable | By variable | Spearman ρ | Probability > |ρ| |
|---|---|---|---|
| Fractional shortening | LVIDsN | –0.450 | < .000 |
| Plasma taurine | LVIDsN | –0.073 | .310 |
| Plasma taurine | Fractional shortening | 0.057 | .424 |
| Whole blood taurine | LVIDsN | –0.005 | .944 |
| Whole blood taurine | Fractional shortening | 0.077 | .283 |
| Whole blood taurine | Plasma taurine | 0.452 | < .000 |
The 2 CKCSs diagnosed with DCM were not receiving nutritional supplements, and both were eating diets that did not meet WSAVA nutritional guidelines. Neither dog had low taurine levels, with the first DCM CKCS having normal WB (213 µM) and plasma (101 µM) taurine concentrations, and the second DCM CKCS having a normal WB taurine concentration (159 µM) and an elevated plasma taurine concentration (238 µM).
Discussion
In this population of CKCSs, taurine concentrations were not significantly different across dogs in various preclinical stages of MMVD, including those in stage A, which are considered predisposed to the disease but not yet affected. Whether differences might exist in CKCSs that have progressed to the point of congestive heart failure remains unknown. In contrast, a previous study5 suggested that dogs with MMVD had greater taurine concentrations than either control dogs or dogs with DCM; however, the sample size of dogs with MMVD was relatively small (n = 28). The same study also found no difference in taurine concentrations in dogs with MMVD (or DCM) that were or were not receiving cardiac medications, or that were or were not in congestive heart failure, suggesting that the stage of acquired cardiac disease may not affect taurine concentrations significantly.5
From a broad perspective, there was also no indication from our data that CKCSs eating diets that met or did not meet WSAVA nutritional guidelines had significantly different taurine concentrations. This does not exclude the possibility that any individual diet could affect taurine concentrations in CKCSs, but it does provide a real-world perspective. Given the multitude of diets consumed by our study population, analysis of individual diet brands, ingredients, or nutritional breakdown was beyond the scope of this work. Of note, in the 2 CKCSs that were diagnosed with DCM, WB or plasma taurine deficiency was not identified. Because CKCSs are not a breed that is predisposed to the development of heritable DCM, and both dogs were eating a diet that did not meet WSAVA nutritional guidelines, diet-associated DCM was considered a plausible etiology in these 2 dogs.
With regard to echocardiographic assessment, there was no significant correlation between 2 echocardiographic measurements of systolic function (LVIDsN and FS) and taurine concentrations in CKCSs. This is contrary to what has been reported previously in the Golden Retriever.2 These results, along with the lack of WB or plasma taurine deficiency in the 2 DCM-affected dogs, suggest that taurine levels are unlikely to be playing an important role in cases of diet-associated DCM in CKCSs.
Breed-specific WB (range, 152 to 373 µM; median, 266 µM) and plasma (range, 51 to 217 µM; median, 113 µM) taurine reference intervals have now been generated for CKCSs. These CKCS breed-specific taurine reference intervals are generally wider than previously published normal taurine values for WB (200 to 350 µM) and plasma (60 to 120 µM) taurine concentrations of healthy dogs.1,29 The median plasma taurine concentration of 113 µM in CKCSs was substantially greater than the median of 78 µM reported by Delaney et al,1 and the median WB taurine concentration of 266 µM in CKCSs was relatively similar to the previously reported1 260 µM median in healthy dogs. However, a recent study2 reported Golden Retriever breed-specific taurine reference intervals for WB (213 to 377 µM; mean, 297 µM) and plasma (63 to 194 µM; mean, 106 µM), and this range of plasma taurine concentrations was more similar to our study.
One possible influence on the differences in reference intervals is the way in which they were calculated. The Golden Retriever plasma reference interval was calculated in a manner identical to both the WB and plasma CKCS reference intervals in our study—namely, by calculating the central 95% of the data. Although the reference interval for WB taurine in Golden Retrievers was calculated using a different method involving bootstrap confidence intervals, it produced a reference interval relatively similar to the central 95% of the data. It was not specified how the non–breed-specific normal taurine ranges or lowest values for no known risk for taurine deficiency were calculated, but the lower limit of the normal range for plasma is identical to the first quartile reported by Delaney et al,1 meaning it excludes the lowest 25% of the plasma taurine measurements in those presumably healthy dogs.1,29 Furthermore, 4% of the healthy dogs were reported to have plasma taurine concentrations below the lowest value listed for no known risk of taurine deficiency. Neither of these lower limits appears to follow a typical lower reference limit that would only exclude 2.5% of the lowest values. Additional factors that could have contributed to differences in taurine reference intervals and medians include differences in sample processing methods, and differences in acquisition and data analysis methods between laboratories where the taurine was measured.
Although the CKCS reference intervals for WB and plasma taurine appear wider when compared to other reference intervals, preliminary (n = 36), non–breed-specific reference intervals for taurine that were measured in the same laboratory as the CKCS samples were similar to the CKCS-specific reference intervals reported (WB taurine, 137 to 357 µM; plasma taurine, 52 to 144 µM). Therefore, taurine reference intervals may need to be laboratory specific.
There were several limitations to our study. The sample size was not the same across various stages of preclinical MMVD, with the stage A group being relatively small (n = 12). This may have limited our ability to detect more subtle differences in taurine concentration in this population compared to stages B1 and B2. In addition, hematology data were not obtained as part of this study; thus, the percentage of CKCSs with platelet abnormalities and any potential impact of either macrothrombocytosis or thrombocytopenia on taurine concentrations was not investigated specifically. Also, although owners did provide detailed information about current diet and supplement history, the length of time that each dog had been eating their current diet was not provided by the owner; thus, a potential confounding factor of recent diet changes cannot be excluded. Last, CKCSs were not required to be fasted when they presented for study enrollment, with plasma taurine concentrations potentially affected by postprandial sampling, although WB taurine samples should not be.1
In conclusion, taurine concentrations do not appear to differ significantly in CKCSs based on preclinical MMVD stage. Furthermore, taurine concentrations do not differ significantly between CKCSs eating diets that do or do not meet WSAVA nutritional guidelines.
Acknowledgments
This project was part of a larger study funded primarily by a grant from the Cavalier King Charles Spaniel USA Health Foundation, with additional support from the Texas A&M Gastrointestinal Laboratory.
The authors declare there were no conflicts of interest.
The authors acknowledge Patricia Ishii and Robert Kyle Phillips for their assistance in onsite blood sample processing, as well as Dr. Ryan Fries, Dr. Ashley Saunders, Dr. Bruno Boutet, Dr. Jordan Vitt, Dr. Saki Kadotani, and Dr. Jonathan Stack for their role in echocardiographic imaging.
References
- 1. ↑
Delaney SJ, Kass PH, Rogers QR, Fascetti AJ. Plasma and whole blood taurine in normal dogs of varying size fed commercially prepared food. J Anim Physiol Anim Nutr (Berl). 2003;87:236–244. doi:10.1046/j.1439-0396.2003.00433.x
- 2. ↑
Ontiveros ES, Whelchel BD, Yu J, et al. Development of plasma and whole blood taurine reference ranges and identification of dietary features associated with taurine deficiency and dilated cardiomyopathy in golden retrievers: a prospective, observational study. PLoS One. 2020;15:e0233206. doi:10.1371/journal.pone.0233206
- 3. ↑
Paasonen MK, Solatunturi E, Nieminen ML. Blood platelets: accumulation and release of taurine. Prog Clin Biol Res. 1985;179:225–233.
- 4. ↑
Brown SJ, Simpson KW, Baker S, Spagnoletti MA, Elwood CM. Macrothrombocytosis in cavalier King Charles spaniels. Vet Rec. 1994;135:281–283. doi:10.1136/vr.135.12.281
- 5. ↑
Kramer GA, Kittleson MD, Fox PR, Lewis J, Pion PD. Plasma taurine concentrations in normal dogs and in dogs with heart disease. J Vet Intern Med. 1995;9:253–258. doi:10.1111/j.1939-1676.1995.tb01076.x
- 6. ↑
Haggstrom J, Hansson K, Kvart C, Swenson L. Chronic valvular disease in the cavalier King Charles spaniel in Sweden. Vet Rec. 1992;131:549–553.
- 7.
Beardow AW, Buchanan JW. Chronic mitral valve disease in cavalier King Charles spaniels: 95 cases (1987–1991). J Am Vet Med Assoc. 1993;203:1023–1029.
- 8.
Malik R, Hunt GB, Allan GS. Prevalence of mitral valve insufficiency in cavalier King Charles spaniels. Vet Rec. 1992;130:302–303. doi:10.1136/vr.130.14.302
- 9.
Pedersen HD, Kristensen BO, Lorentzen KA, Koch J, Jensen AL, Flagstad A. Mitral valve prolapse in 3-year-old healthy Cavalier King Charles Spaniels: an echocardiographic study. Can J Vet Res. 1995;59:294–298.
- 10. ↑
Lewis T, Swift S, Woolliams JA, Blott S. Heritability of premature mitral valve disease in Cavalier King Charles spaniels. Vet J. 2011;188:73–76. doi:10.1016/j.tvjl.2010.02.016
- 11. ↑
Kittleson MD, Keene B, Pion PD, Loyer CG. Results of the multicenter spaniel trial (MUST): taurine- and carnitine-responsive dilated cardiomyopathy in American cocker spaniels with decreased plasma taurine concentration. J Vet Intern Med. 1997;11:204–211. doi:10.1111/j.1939-1676.1997.tb00092.x
- 12.
Belanger MC, Ouellet M, Queney G, Moreau M. Taurine-deficient dilated cardiomyopathy in a family of golden retrievers. J Am Anim Hosp Assoc. 2005;41:284–291. doi:10.5326/0410284
- 13.
Basili M, Pedro B, Hodgkiss-Geere H, Navarro-Cubas X, Graef N, Dukes-McEwan J. Low plasma taurine levels in English cocker spaniels diagnosed with dilated cardiomyopathy. J Small Anim Pract. 2021;62:570–579. doi:10.1111/jsap.13306
- 14.
Fascetti AJ, Reed JR, Rogers QR, Backus RC. Taurine deficiency in dogs with dilated cardiomyopathy: 12 cases (1997–2001). J Am Vet Med Assoc. 2003;223:1137–1141. doi:10.2460/javma.2003.223.1137
- 15.
Kaplan JL, Stern JA, Fascetti AJ, et al. Taurine deficiency and dilated cardiomyopathy in golden retrievers fed commercial diets. PLoS One. 2018;13:e0209112. doi:10.1371/journal.pone.0209112
- 16. ↑
Backus RC, Cohen G, Pion PD, Good KL, Rogers QR, Fascetti AJ. Taurine deficiency in Newfoundlands fed commercially available complete and balanced diets. J Am Vet Med Assoc. 2003;223:1130–1136. doi:10.2460/javma.2003.223.1130
- 17. ↑
FDA investigation into potential link between certain diets and canine dilated cardiomyopathy. 2019. Accessed July 1, 2022. https://www.fda.gov/animal-veterinary/outbreaks-and-advisories/fda-investigation-potential-link-between-certain-diets-and-canine-dilated-cardiomyopathy
- 18. ↑
FDA investigation into potential link between certain diets and canine dilated cardiomyopathy - February 2019 update. 2019. Accessed July 1, 2022. https://www.fda.gov/animal-veterinary/news-events/fda-investigation-potential-link-between-certain-diets-and-canine-dilated-cardiomyopathy-february
- 19. ↑
Adin D, DeFrancesco TC, Keene B, et al. Echocardiographic phenotype of canine dilated cardiomyopathy differs based on diet type. J Vet Cardiol. 2019;21:1–9. doi:10.1016/j.jvc.2018.11.002
- 20.
Freid KJ, Freeman LM, Rush JE, et al. Retrospective study of dilated cardiomyopathy in dogs. J Vet Intern Med. 2021;35:58–67. doi:10.1111/jvim.15972
- 21. ↑
Freeman L, Rush J, Adin D, et al. Prospective study of dilated cardiomyopathy in dogs eating nontraditional or traditional diets and in dogs with subclinical cardiac abnormalities. J Vet Intern Med. 2022;36:451–463. doi:10.1111/jvim.16397
- 22. ↑
WSAVA. WSAVA Global Nutrition Committee: guidelines on selecting pet foods. 2021. Accessed August 19, 2022. https://wsava.org/wp-content/uploads/2021/04/Selecting-a-pet-food-for-your-pet-updated-2021_WSAVA-Global-Nutrition-Toolkit.pdf
- 23. ↑
Wesselowski S, Gordon S, Saunders A, et al. Use of physical examination, electrocardiography, radiography and biomarkers to predict stage B2 myxomatous mitral valve disease in preclinical Cavalier King Charles Spaniels. J Vet Intern Med. 2021:3096–3097. doi:10.1111/jvim.16289
- 24. ↑
Keene BW, Atkins CE, Bonagura JD, et al. ACVIM consensus guidelines for the diagnosis and treatment of myxomatous mitral valve disease in dogs. J Vet Intern Med. 2019;33:1127–1140. doi:10.1111/jvim.15488
- 25. ↑
Cornell CC, Kittleson MD, Della Torre P, et al. Allometric scaling of M-mode cardiac measurements in normal adult dogs. J Vet Intern Med. 2004;18:311–321. doi:10.1111/j.1939-1676.2004.tb02551.x
- 26. ↑
Hansson K, Haggstrom J, Kvart C, Lord P. Left atrial to aortic root indices using two-dimensional and M-mode echocardiography in cavalier King Charles spaniels with and without left atrial enlargement. Vet Radiol Ultrasound. 2002;43:568–575. doi:10.1111/j.1740-8261.2002.tb01051.x
- 27. ↑
Blake A. Canine/feline serum or plasma deproteinization for amino acid analysis on Biochrom. Accessed September 9, 2022. https://www.protocols.io/view/canine-feline-serum-or-plasma-deproteinization-for-877hzrn
- 28. ↑
Friedrichs KR, Harr KE, Freeman KP, et al. ASVCP reference interval guidelines: determination of de novo reference intervals in veterinary species and other related topics. Vet Clin Pathol. 2012;41:441–453. doi:10.1111/vcp.12006
- 29. ↑
UC Davis Veterinary Medicine. Amino Acid Laboratory. Accessed September 9, 2022. https://www.vetmed.ucdavis.edu/labs/amino-acid-laboratory
