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Nutrient Physiology, Metabolism, and Nutrient-Nutrient Interactions

Differences in Taurine Synthesis Rate among Dogs Relate to Differences in Their Maintenance Energy Requirement^1–3,

Department of ⁴ Molecular Biosciences and ⁵ Department of Chemistry, University of California, Davis, CA 95616 and ⁶ Department of Veterinary Medicine and Surgery, University of Missouri, Columbia, MO 65211

^* To whom correspondence should be addressed. E-mail: backusr{at}missouri.edu.

	ABSTRACT

TOP ABSTRACT Introduction Materials and Methods Results Discussion LITERATURE CITED

 
Diet-induced (taurine deficiency) dilated cardiomyopathy is reported more in large than small dogs possibly because taurine biosynthesis rate (TBR) is lower in large than small dogs. The TBR in 6 mongrels (37.9 ± 2.1 kg) and 6 beagles (12.8 ± 0.4 kg) was determined from the fractional dilution rate of urinary [1,2-²H₂]-taurine, (d4-tau). All dogs were given a 15.6% protein, 0.60% sulfur amino acid (SAA) diet in amounts to maintain an ideal body condition score. After 3 mo, 14.6 mg/kg body weight of d4-tau was given orally and TBR determined from d4-tau to taurine ratio in urine collected each d for 6 d. Enrichments of d4-tau were determined by GC-MS. Thereafter, mongrels and beagles were paired by ranking of SAA intake per metabolic body weight per kg^0.75. Each pair received the same amount of diet/kg^0.75 for 2 wk, then TBR was again determined. Concentrations of taurine in plasma, blood, and urine and concentrations of plasma thiols were measured during each TBR determination. In Expt. 1, TBR and taurine concentrations in plasma and urine of mongrels were lower (P < 0.05) than those of beagles. In Expt. 2, TBR and taurine concentrations in blood and plasma of mongrels were lower (P < 0.05) than beagles. Together, the results support the hypothesis that large compared with small dogs have lower TBR when fed diets near-limiting in dietary SAA, but adequate to maintain ideal body condition.

	Introduction

TOP ABSTRACT Introduction Materials and Methods Results Discussion LITERATURE CITED

Taurine (2-aminoethanesulfonic acid) is a beta, sulfur-containing, amino acid ubiquitously found in animals and reported in especially high concentrations in heart, brain, central nervous system, retina, olfactory bulb, and white blood cells (5). The physiological function of taurine in heart is not fully understood. Proposed mechanisms include osmoregulation, calcium regulation, and inactivation of free-radicals (6).

Taurine is synthesized from the sulfur amino acids, methionine and cyst(e)ine (7), by the activities of the enzymes, cysteine dioxygenase (EC 1.13.11.20) and cysteine sulfinic acid decarboxylase (EC 4.1.1.29) in animals, excluding most carnivores (8). Because of this, taurine is not considered as an essential nutrient in many species. However, it is known that generally, carnivores have a dietary requirement for taurine, and there is evidence that under certain dietary conditions dogs require dietary taurine. Sanderson et al. (9) found a significant decrease in plasma taurine concentration in healthy beagles fed a high-fat, protein-restricted (10% dry matter basis) diet that exceeded the NRC minimum protein requirement of maintenance in adult dogs (10). After feeding the diet for 48 mo, these investigators found 1 dog developed DCM. This indicated that prolonged provision of a protein-restricted diet, although above the minimum protein requirement, could result in taurine deficiency in dogs. More recently, Fascetti et al. (11) reported 12 cases of low blood taurine concentration and DCM in large-breed dogs given apparently nutritionally complete and balanced commercial diets. They suggested that body size may be a factor contributing to development of taurine deficiency in dogs.

Our research group recently found that plasma taurine concentration and taurine biosynthesis rate (TBR) in Newfoundland dogs, a giant dog breed, are substantially lower than those in beagles when both breeds are fed the same diet (12). We hypothesize that the greater incidence of taurine-deficiency DCM reported in large relative to small dogs is the result of lower TBR in large dogs. In the present study, we compare the abilities of large and small dogs to synthesize taurine when intake of diet is controlled to maintain ideal body condition and when intake is controlled to provide similar dietary sulfur amino acid (SAA) intake on a metabolic body weight (MBW, kg^0.75) basis.

	Materials and Methods

TOP ABSTRACT Introduction Materials and Methods Results Discussion LITERATURE CITED

 
    Animals and diet. Husbandry and treatment of the dogs were in compliance with the NRC Guide for Laboratory Animals (13), and were approved by the Animal Use and Care Administrative Advisory Committee at University of California, Davis. Six sexually intact male beagles (12.8 ± 0.4 kg, 5–7 y) and 6 male mongrels (37.9 ± 2.1 kg, 5 intact and 1 neutered, 6–8 y), owned by the University, were designated small dogs and large dogs, respectively. The dogs were individually housed simultaneously in semi-open runs in the same building, and they received an allotment of diet each day that was completely consumed by the following day. Body weights (BW) and body condition scores (BCS) were determined each week.

All dogs were given the same, nutritionally complete and balanced, extruded dry-type diet produced for the study (Royal Canin). Dietary protein was limited to 15.6% to provide adequate but not excessive SAA to maintain nitrogen balance and provide for taurine biosynthesis (Table 1). The dietary protein content exceeded recommended allowance for maintenance of adult dogs (10% for 16.7 kJ/g metabolizable energy in the diet, dry matter basis) (10,14). Sulfur amino acid bioavailability of the diet was estimated by cecectomized rooster assay (15).

    Expt. 2. Control of diet presentation to maintain ideal BCS was continued after Expt. 1 so that SAA intake per MBW could be calculated for each dog. The dogs were then ranked from least to highest SAA intake per MBW, and large and small dogs were paired by rank of SAA intake per MBW to make 6 experimental pairs. The mean SAA intake per MBW for each pair was determined, and the quantity of diet it represented was given to the pairs each d for 2 wk. After 1 wk, blood, plasma, and urine were sampled, body composition determined, d4-tau administration and urine collection repeated, and biochemical analyses conducted, as described in Expt. 1.

    Sample collection, processing, and analysis. Blood (5 mL) was collected from the cephalic vein by venipuncture into heparinized syringes (20 µL of sodium heparin solution, 1000 USP kU/L, Baxter Health Care). Urine (5 mL) was collected by free-catch before feeding.

Taurine concentrations in blood, plasma, and urine were determined by the method of Kim et al. (17) using an amino acid analyzer (12). To normalize urinary taurine concentration, urinary creatinine concentrations were determined with a commercial kit (Cold Stable, Pointe Scientific).

The GC-MS analysis and calculation of TBR from enrichment of TTR in urine were conducted using a modification (12) of the method of Fay et al. (18). MS of the deuterated taurine derivative revealed a unique fragment of 241 m/z, which was assumed to be an M+3 rather than the expected M+4 fragment. Deuterium on carbon adjacent to the sulfonate group of the taurine label was assumed to exchange with available protium during the derivatization step. Use of the M+3 fragment was justified because its fractional abundance increased linearly with increasing enrichment of the M+4 tracer in standards.

TBR was calculated using equations,

where D is the dose in mg given, TTR(t₀) is the TTR at time 0 as interpolated from TTR in d 1–5 urine samples, K is the rate constant, P_{M + 3} and P_M are areas of peaks corresponding to ions of tracer and tracee, respectively, and A is natural abundance of the isotope used (²H = 0.00015).

    Body composition. Lean body mass (LBM) and body fat mass (BFM) was estimated by isotopic water dilution (19,20). For this, sterile filtered (0.2 µm/25 mm Anotop, Whatman), salinated (90 g/L NaCl), deuterated-water (99.9%, Sigma-Aldrich) was subcutaneously injected (0.4 g/kg), and after 4.5 h, cephalic venous blood (2 mL) was collected by venipuncture. Deuterium enrichment in serum water was measured as previously described (20).

    Statistical analysis. Effect of body-size (large and small) and means of food intake control (that supporting ideal body condition [Expt. 1] and that supporting similar SAA intakes between large and small dog pairs [Expt. 2]) on food and SAA intakes, TBR, BW, LBM, BFM, and circulating amino acid and thiol concentrations were evaluated using mixed-model ANOVA (PROC MIXED, version 9.1, SAS Institute). Body-size and means of food intake control were assigned as fixed and random effects, respectively, and Tukey multiple comparisons adjustment were used in post-hoc analyses. Significance of correlations between food intake and taurine entry on taurine concentrations in blood, plasma, and urine were determined by regression analysis. Percentage data were transformed [2 x arcsin x (observation)^–1/2] prior to analyses. Differences were considered significant at P 0.05 or a noteworthy trend at P > 0.05 and <0.10. Results are expressed as means ± SEM unless otherwise stated.

	Results

TOP ABSTRACT Introduction Materials and Methods Results Discussion LITERATURE CITED

 
Clearly, BW and food intake in large dogs were greater (P < 0.01) than those in small dogs in Expt. 1 and Expt. 2 (Table 2). However, mean SAA intake per MBW of large dogs was 23.2% less (P < 0.05) than that of small dogs in Expt. 1 and exactly the same for large and small dogs in Expt. 2 because SAA intake was intentionally controlled for each pair of dogs in Expt. 2 to provide the same amount of the precursor for taurine synthesis (Table 2).

The TBR were normalized to BW, MBW, relative liver weight (RLW, kg^0.87), LBM, and metabolic LBM (MLBM, LMB kg^0.75) (Table 4). TBR was normalized to RLW for comparison between large and small dogs because the liver is the major organ of taurine biosynthesis in dogs (21). For Expt. 1, all normalized TBR were lower (P < 0.05) in large compared with small dogs, where the per MBW, LBM, MLBM, and RLW TBR were lower by 49, 37, 48, and 43%, respectively. In Expt. 2, TBR/LBM was lower (P < 0.03) and TBR/BW tended to be lower (P = 0.06) in large than in small dogs.

With decreasing percentage of food intake relative to that expected based on BW (10,22), concentrations of taurine in Expt. 1 decreased (P < 0.05) in plasma, blood, and urine (Supplemental Table 2 and Supplemental Fig. 2). The same relations between food intake and indicators of taurine status were not significant (P > 0.33) in Expt. 2, where the range in food intake was substantially less (32–44 g/MBW) than that in Expt. 1 (17–35 g/MBW).

	Discussion

TOP ABSTRACT Introduction Materials and Methods Results Discussion LITERATURE CITED

 
The major difference between the 2 experiments of this study was the way in which food intake (i.e., SAA intake) was controlled. In Expt. 1, all dogs were given enough diet to maintain an ideal BCS for 3 mo including the period when TBR was determined. This feeding condition resulted in similar body fat percentages among the small and large dogs (Table 2) and body fat percentages consistent with previously reported ideals in dogs (16). Thus, in Expt. 1, TBR associated with maintenance energy intake of small and large dogs was determined. The most salient finding of Expt. 1 was that, although large dogs consumed 67% more diet than small dogs (Table 2), their TBR were similar to those of small dogs (Table 4), whereas there was a trend for lower plasma taurine concentrations (P = 0.06) than those of small dogs (Table 3). It is noteworthy in this context that mean plasma and blood taurine concentrations in the large, but not the small dogs, were indicative of marginal taurine status (11). The plasma taurine concentration observed in 1 large dog (15 µmol/L) was similar to concentrations reported in dogs with DCM that was corrected by taurine supplementation (11,12). These results support the hypothesis that large compared with small dogs are at greater risk for development of taurine deficiency when dietary SAA concentrations are marginal.

Urine taurine concentration was determined because it reflects acute changes in taurine status as a result of renal homeostatic modulation of taurine excretion (23). Variances in urine taurine concentrations within dog groups were great compared with variances observed in blood and plasma taurine concentrations. Nonetheless, there was a trend (P = 0.07) for urine taurine concentrations to be lower in large compared with small dogs in Expt. 1(Table 3). This finding is consistent with a trend of a lower taurine status in large compared with small dogs consuming the same diet.

The lower than expected TBR in large dogs appears to be at least partially a result of lower than expected SAA intake by large dogs. Although the large and small dogs were housed in the same environment during the experiments, large dogs consumed less diet (and therefore less SAA) on a MBW basis than small dogs to maintain ideal body condition (Table 2, Expt. 1). Energy intakes of small dogs [555 ± 29 kJ · kg^–0.75 · d^–1] were very close to intakes that would be predicted from body weight using a well established allometric relation [552 kJ · kg^–0.75 · d^–1, (10)]. In contrast, energy intakes of large dogs were substantively less than those that would be predicted [427 ± 37 kJ · kg^–0.75 · d^–1]. Variations in breed attributes other than body weight, such as conformation, hair coat, and physical activity, may account for deviations in scaling of maintenance energy intake (22,24). The observed positive correlations between taurine status indicators (blood, plasma, and urine taurine concentrations) and food intake (Supplemental Fig. 1 and Supplemental Table 2) indicates that food intake differences probably accounted for the observed size-effects on TBR and taurine status.

To the authors' knowledge, the scaling of taurine metabolism with body mass has not been reported. In Expt. 2, exactly the same amount of SAA per MBW was given to each pair of small and large dogs so that effect of metabolic body size on TBR could be evaluated when the same quantity of substrates of taurine metabolism is provided. It was presumed that taurine metabolism scales with MBW as is reported with metabolism of other nutrients (25,26). However, although food intake was controlled according to MBW in Expt. 2, correlations between taurine entry and indicators of taurine status were greatest with taurine entry rate normalization by BW and LBM (Supplemental Table 2). This may indicate taurine entry scales linearly rather than exponentially with body weight.

In Expt. 2, SAA intake relative to that in Expt. 1 was increased in both large and small dogs, but more so in large dogs (69 ± 15 vs. 24 ± 4%). The TBR in large dogs tended to be lower than those in small dogs after normalization to MLBM (P = 0.32) and RLW (P = 0.20) (Table 4). These normalizations were used because most taurine synthesis occurs in the lean mass, especially liver (23), and the percentage body fat in small compared with large dogs tended to be greater, in Expt. 2 (P = 0.31) relative to Expt. 1 (P = 0.99) (Table 2). Together, findings of the experiments indicate that the observed body-size effect on TBR was primarily a result of size-related difference in SAA intake relative to expected energy needs.

Plasma thiol concentrations did not differ between large and small dogs but plasma cysteinyl-glycine tended (P = 0.10) to be higher in large dogs. However, a trend (P < 0.10) of higher cysteinyl-glycine concentration was found in large compared with small dogs in Expt. 1. Lower intake of dietary SAA in large dogs relative to small dogs may result in lower -glutamyl transpeptidase (EC 2.3.2.2) and dipeptidase (EC 3.4.3.5) activities to hydrolyze plasma glutathione and cysteinyl-glycine (23). This should spare plasma glutathione and cysteinyl-glycine, maintaining homeostatic concentrations of these thiols.

Most of plasma amino acid concentrations (Supplemental Table 1) were similar to or greater than those in other reports with healthy dogs (27,28). The exceptions were proline, hydroxyproline and a few other dispensable amino acids. This indicates that the experimental diet and amount consumed were adequate for maintenance of protein and amino acid balance, with the exception of taurine (29). The low-normal plasma concentrations of free cyst(e)ine in dogs in this study are consistent with the experimental diet providing SAA sufficient for protein synthesis, but not sufficient for optimal taurine status in large dogs.

In summary when a low, but adequate, protein diet was given to dogs of varying body size to maintain ideal body condition, a trend of lower taurine concentrations in blood, plasma, and urine was found in large dogs, but not in small dogs. Some large dogs had taurine deficiency (plasma taurine 40 µmol/L) such that, if continued for the long-term, would be at risk for development of taurine-deficiency DCM. Our results support the hypothesis that the rate of taurine synthesis in large dogs is lower than that in small dogs when taurine precursor SAA are not in excess. In general, large relative to small dogs appear to be at greater risk for taurine deficiency because they ingest less diet for their MBW than small dogs. We conclude that the SAA allowance should be increased enough for large-breed dogs and dogs with low maintenance energy requirement to enable them to maintain an optimal taurine status.

	FOOTNOTES

 
¹ Supported by Center for Companion Animal Health (CCAH), School of Veterinary Medicine, University of California, Davis and by Royal Canin, Aimargues, France.

² Author disclosures: K. S. Ko, no conflicts of interest; R. C. Backus, no conflicts of interest; J. R. Berg, no conflicts of interest; M. W. Lame, no conflicts of interest; and Q. R. Rogers, no conflicts of interest.

³ Supplemental Tables 1 and 2 and Supplemental Figures 1 and 2 are available with the online posting of this paper at jn.nutrition.org.

⁷ Abbreviations used: BCS, body condition score; BFM, body fat mass; BW, body weight; d4-tau, [1,2-²H₂]-taurine; DCM, dilated cardiomyopathy; LBM, lean body mass; MBW, metabolic body weight; MLBM, metabolic lean body mass; RLW, relative liver weight; SAA, sulfur amino acid; TBR, taurine biosynthesis rate; TTR, tracer to tracee ratio.

Manuscript received 21 November 2006. Initial review completed 12 December 2006. Revision accepted 27 February 2007.

	LITERATURE CITED

TOP ABSTRACT Introduction Materials and Methods Results Discussion LITERATURE CITED

 

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16. Laflamme D. Development and validation of a body condition score system for dogs. Canine Pract. 1997;22:10–5.

17. Kim SW, Morris JG, Rogers QR. Dietary soybean protein decreases plasma taurine in cats. J Nutr. 1995;125:2831–7.[Abstract/Free Full Text]

18. Fay LB, Metairon S, Montigon F, Ballevre O. Evaluation of taurine metabolism in cats by dual stable isotope analysis. Anal Biochem. 1998;260:85–91.[Medline]

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20. Backus RC, Havel PJ, Gingerich RL, Rogers QR. Relationship between serum leptin immunoreactivity and body fat mass as estimated by use of a novel gas-phase Fourier transform infrared spectroscopy deuterium dilution method in cats. Am J Vet Res. 2000;61:796–801.[Medline]

21. Stipanuk MH. Role of the liver in regulation of body cysteine and taurine levels: a brief review. Neurochem Res. 2004;29:105–10.[Medline]

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26. Rucker RB, Steinberg FM. Vitamin requirements—Relationship to basal metabolic need and functions. Biochem Mol Biol Educ. 2002;30:86–9.

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28. Outerbridge CA, Marks SL, Rogers QR. Plasma amino acid concentrations in 36 dogs with histologically confirmed superficial necrolytic dermatitis. Vet Dermatol. 2002;13:177–86.[Medline]

29. Zicker SC, Rogers QR. Use of plasma amino acid concentrations in the diagnosis of nutritional and metabolic diseases in veterinary medicine. In: Kaneko JJ, editor. Proceedings of the IVth congress of the international society for animal clinical biochemistry; 1990; Davis, CA; 1990. p. 107–21.

This Article Abstract Full Text (PDF) Online Supporting Material Purchase Article View Shopping Cart Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Ko, K. S. Articles by Rogers, Q. R. Search for Related Content PubMed PubMed Citation Articles by Ko, K. S. Articles by Rogers, Q. R.