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© 2006 American Society for Nutrition J. Nutr. 136:2502-2505, October 2006

Nutrient Physiology, Metabolism, and Nutrient-Nutrient Interactions

Copper Deficiency Does Not Lead to Taurine Deficiency in Rats1

Kwang Suk Ko2, Cristina L. Tôrres2, Andrea J. Fascetti2, Martha H. Stipanuk3, Lawrence Hirschberger3 and Quinton R. Rogers2,*

2 Department of Molecular Biosciences, School of Veterinary Medicine, University of California Davis, CA 95616 and 3 Division of Nutritional Sciences, Cornell University, Ithaca, NY 14853

* To whom correspondence should be addressed. E-mail: qrrogers{at}ucdavis.edu.


   ABSTRACT
TOP
 ABSTRACT
Introduction
 Materials and Method
 Results
 Discussion
 LITERATURE CITED
 
Copper deficiency has been reported to cause a decrease in urinary taurine excretion in rats. We determined whether Cu deficiency would decrease taurine status and the hepatic activities of cysteine dioxygenase (CDO) and/or cysteine sulfinic acid decarboxylase (CSAD) in rats. Ten weanling male rats were assigned to either a Cu-adequate (+Cu) or Cu-deficient (–Cu) group. All rats consumed a Cu-deficient purified diet and water ad-libitum for 16 wk. The water for the +Cu group contained 20 mg Cu/L as CuSO4. At wk 16, the groups differed (P < 0.05) in the following variables (means ± SEM, –Cu vs. +Cu): body weight (BW), 375 ± 19 vs. 418 ± 2.9 g; food intake, 16.2 ± 0.7 vs. 18.5 ± 0.4 g/d; hematocrit, 0.294 ± 0.027 vs. 0.436 ± 0.027; hemoglobin, 95.2 ± 9 vs 134 ± 10 g/L; liver Cu, 8.7 ± 2.0 vs. 65.9 ± 2.5 nmol/g; plasma Cu, 0.38 ± 0.09 vs. 13.4 ± 0.61 µmol/L; plasma ceruloplasmin activity, 1.75 ± 1.0 vs. 67.9 ± 8.4 IU; relative heart weight, 0.56 ± 0.04 vs. 0.35 ± 0.02% BW; relative liver weight, 4.06 ± 0.23 vs. 3.37 ± 0.06% BW; and liver CSAD activity, 18.8 ± 1.37 vs. 13.5 ± 1.11 nmol · min–1 · mg protein–1. The groups did not differ at wk 16 in: plasma taurine, 249 ± 14 vs. 298 ± 63 µmol/L; whole blood taurine, 386 ± 32 vs. 390 ± 25 µmol/L; urinary taurine excretion, 82.5 ± 15 vs. 52.0 ± 8.3 µmol/d; liver taurine, 2.6 ± 0.7 vs. 2.8 ± 0.4 µmol/g; liver total glutathione, 6.9 ± 0.48 vs. 6.3 ± 0.40 µmol/g; liver cyst(e)ine, 96 ± 7.1 vs. 99 ± 5.3 nmol/g and liver CDO activity, 2.19 ± 0.33 vs. 2.74 ± 0.21 nmol · min–1 · mg protein–1. These findings support the conclusion that Cu deficiency does not affect body taurine status.


   Introduction
TOP
 ABSTRACT
 Introduction
Materials and Method
 Results
 Discussion
 LITERATURE CITED
 
Taurine (2-aminoethanesulfonic acid) is a beta-amino sulfur amino acid, but it is neither an essential amino acid in most animals nor a building block of proteins. Taurine is known to be synthesized from the sulfur amino acids, methionine/cyst(e)ine (1) at a sufficient rate to meet biological needs in most animals. However, since taurine deficiency was found to be a cause of dilated cardiomyopathy (DCM)4 in cats (2), taurine deficiency has been considered by many nutritionists and veterinarians as a possible causative factor for DCM in dogs.

Moise et al. (3) reported that taurine deficiency was linked to DCM in foxes, a canid, which suggests that taurine deficiency may occur in dogs under certain metabolic conditions, even though it has been shown that with many diets no dietary taurine is required for normal taurine status. Clinical signs of DCM associated with taurine deficiency in dogs have been reported by various cardiologists. Although the metabolic basis for the taurine deficiency has not been elucidated, it is thought to involve abnormal energetics via calcium channel disregulation in mitochondria (4). The majority of clinical signs of DCM in dogs were in large-breed dogs that had been fed commercial dog foods for long periods of time that were composed primarily of lamb meal and rice (5). This suggests a dietary link between certain dog foods and the development of DCM in dogs.

Because Gray and Daniel (6) reported that urinary taurine excretion was reduced in Cu-deficient rats and suggested that it may be the result of a decreased synthesis of taurine, we examined the Cu content of the dog foods reported to be associated with taurine deficiency. The lamb and rice diet, which most of the affected dogs were consuming, was not supplemented with Cu [3.1mg/1000 kcal (4184 kJ) ME], but was supplemented with Zn at several-fold (84mg/1000 kcal ME) the minimum requirement for the dog. This resulted in a relatively high Zn to Cu ratio of a magnitude known to induce metallothionein formation in some species (7) which, in turn, binds Cu and decreases Cu bioavailability (8). We hypothesized that the high Zn to Cu ratio present in the diet may have decreased the availability of Cu and thereby had an effect on taurine status via the activity of cysteine dioxygenase [CDO, Enzyme Commission(EC) 1.13.11.20] and/or cysteine sulfinic acid decarboxylase (CSAD, EC 4.1.1.29), key enzymes for the synthesis of taurine from cysteine.

To test this hypothesis, Cu deficiency was induced in male weanling rats and taurine status and the activities of the 2 enzymes involved in taurine synthesis were examined as a model to determine whether Cu deficiency in dogs may be involved in causing DCM in dogs.


   Materials and Method
TOP
 ABSTRACT
 Introduction
 Materials and Method
Results
 Discussion
 LITERATURE CITED
 
    Rats and diets. The husbandry and treatment of the rats were approved by the Animal Use and Care Administrative Advisory Committee at University of California, Davis and were in compliance with the NRC guidelines for laboratory animals (9). Ten male weanling rats were purchased (Harlan-Sprague-Dawley) and were divided into 2 groups. Both groups, Cu deficient group (–Cu) and Cu adequate group (+Cu), were fed the same Cu-deficient diet (Table 1), throughout the entire experimental period. The mineral composition of the Cu deficient diet was based on the AIN-76A diet (10) except that the diet contained no added Cu. The diet provided protein at 180 g/kg with no supplementation of methionine to avoid excess substrates for taurine biosynthesis. In addition to the diet, the +Cu group was given Nanopure water (Barnstead Nanopure II System, Barnstead International) containing 20 mg Cu/L as CuSO4. To ensure the consumption of satisfactory amounts of Cu for +Cu group, the amount of Cu-supplemented water consumed for 3 d was recorded once every 4 wk, and the amount of Cu consumed was calculated to be adequate. The mean Cu consumption by the +Cu group was 0.155 mg/d, which exceeds the Cu requirements of growing rats. The –Cu group was given Nanopure water without any supplementation. All rats had free access to food and water throughout the experiment. The rats were housed in hanging stainless-steel cages with a 12-h light-dark cycle. The room temperature ranged between 14 and 29°C.


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  		TABLE 1 Composition of copper-deficient diet

 
    Sampling. To determine the Cu status of the rats, ~500 µL of blood was collected every 2 wk from the saphenous vein (11), using heparinized Microvette CB300 (Sarstedt) blood collection tubes. When the –Cu group showed hematological and biochemical signs of Cu deficiency (hematocrit <40, hemoglobin concentration <120g/L, and/or ceruloplasmin activity <10 IU), the rats were placed in metabolic cages to collect urine for taurine analysis (3 d for adaptation and 4 d for collection). At the end of the urine collection period, the rats were anesthetized with ethyl ether, and the blood, liver, and heart collected. Blood was taken from abdominal aorta using heparinized syringes (~20 µL of sodium heparin solution, 1000 USP kU/L, Baxter HealthCare). A portion of blood was centrifuged, at 15,800 x g for 15 min, immediately after collection to obtain plasma for determination of taurine concentration and ceruloplasmin activity. Liver samples for assays of CDO and CSAD activities and metabolite concentrations were frozen at –80°C until analyses. Other samples were stored at –20°C.

    Measurements. During the experiment, daily food intakes were recorded and body weights (BW) were measured every 3 d. Hematocrits and hemoglobin concentrations were measured every 2 wk. The weights of hearts and livers were measured immediately after collection. A portion of the collected blood was prepared by centrifugation in a model MB micro-capillary centrifuge (IEC) at 10,285 x g for 4 min before hematocrit measurements were taken. Hemoglobin concentration was measured as described by van Kampen and Zijlstra (12). Cu concentrations in the diets, plasma, and liver were measured by atomic absorption spectrometry (AAnalyst 800, Perkin Elmer Instrument) and samples were prepared as described by Clegg et al. (13). Taurine concentrations in whole blood, plasma, and urine were determined using an amino acid analyzer (Beckman 7300 Analyzer C7 Model, Beckman Instruments) (14). Plasma ceruloplasmin activity was measured as its oxidase activity using the modified o-dianisidine dihydrochloride method (15). Liver samples were transported on dry ice from the University of California to Cornell University. Then, CDO and CSAD activities in the livers and concentrations of taurine, total glutathione, and cyst(e)ine in the livers were measured. CDO activity was measured as described by Bagley et al. (16). CSAD activity was measured as described by Bella et al. (17). Total glutathione and cyst(e)ine were quantified by the HPLC method of Fariss and Reed (18) as modified by Stipanuk et al. (19). Protein concentration was determined by the method of Smith et al. (20).

All results are expressed as means ± SEM. Differences between groups at wk 16 were compared using 1-way ANOVA (SYSTAT 10.2, SYSTAT Software). For all analyses, differences were considered significant at P < 0.05. Probability values in the range of 0.05 ≤ P < 0.1 indicated a noteworthy trend.


   Results
TOP
 ABSTRACT
 Introduction
 Materials and Method
 Results
Discussion
 LITERATURE CITED
 
The diets were prepared 3 times during the experiment. The Cu concentrations in the 3 batches of the experimental diets were 1.16, 0.11, and 0.13 mg/kg diet (as-fed basis), respectively. All were lower than the minimum Cu requirement for growing rats (5.0 mg/kg diet) as listed by the NRC (21).

The –Cu group consumed 12% less food and had a 10% lower BW than the +Cu group (P < 0.05; Table 2). However, relative heart (P < 0.01) and liver (P < 0.05) weights were greater in the –Cu group than in the +Cu group (Table 2).


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  		TABLE 2 Anthropometric variables and indicators of copper and taurine status in rats fed –Cu or +Cu diets for 16 wk1

 
Several metabolic indicators of Cu deficiency differed between the groups at wk 16 (P < 0.01, Table 2). The hematocrit and hemoglobin concentrations of the –Cu group were 67 and 71%, respectively, of those of the +Cu group. Liver and plasma Cu concentrations in –Cu group were only 13 and 3%, respectively, of those of the +Cu group. The plasma ceruloplasmin activity in the +Cu group was about 40 times that of the –Cu group.

Taurine concentrations in plasma and whole blood did not differ between the groups but urinary taurine excretion tended to be greater in the –Cu group than in the +Cu group (P = 0.09, Table 2). The groups did not differ (–Cu vs. +Cu) in liver taurine (2.6 ± 0.7 vs. 2.8 ± 0.4 µmol/g ), cyst(e)ine (96 ± 7.1 vs. 99 ± 5.3 nmol/g), and total glutathione (GSH + GSSG) (6.9 ± 0.48 vs 6.3 ± 0.40 µmol/g) concentrations.

Hepatic CDO activity did not differ between the groups whether expressed relative to the total liver, g liver, liver protein, or body weight (Table 3). The CSAD activity was greater in the +Cu group, regardless of the base used for calculation than in the –Cu group (P < 0.005, Table 3).


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  		TABLE 3 Hepatic cysteine dioxygenase and cysteine sulfinic acid decarboxylase activities in rats fed –Cu and +Cu diets for 16 wk1

 

   Discussion
TOP
 ABSTRACT
 Introduction
 Materials and Method
 Results
 Discussion
LITERATURE CITED
 
In this study we focused on the relatively high ratio of Zn to Cu in some lamb and rice diets as a possible factor causing taurine deficiency in dogs. After failing to induce Cu deficiency in 12 medium-to-large mixed breed adult dogs fed a commercial type diet with a Cu chelating agent, Syprine (trientine hydrochloride), for 1 y, we decided to examine a cheaper, more expedient model, the albino rat, to examine the effect of Cu deficiency on taurine status.

The lower BW and food intake in the –Cu group than in the +Cu group and the greater relative heart and liver weights in –Cu group than in the +Cu group (Table 2) are typical and consistent with other reports for Cu-deficient rats (6,22,23). All metabolic indicators of Cu deficiency were significantly lower in the –Cu group than in the +Cu group, confirming that the –Cu group was Cu-deficient after a period of 16 wk (6,23).

Taurine homeostasis is maintained predominantly by the regulation of renal taurine reabsorption so that excess dietary taurine is excreted in the urine (24). Therefore, it is generally assumed that the amount of taurine excreted in urine reflects the extent of excess taurine in the taurine pools of animals. The taurine status of the rats was determined by evaluating plasma and whole blood taurine concentrations and urinary taurine excretion (Table 2). The fact that none of these values were significantly different between the –Cu and +Cu group, and the finding that there was a trend for a higher urinary taurine excretion in the –Cu group, which is the opposite of that found by Gray and Daniel (6), negates our hypothesis that Cu deficiency causes taurine deficiency.

A lower food intake by the –Cu group provided less total substrate and might have been expected to result in less taurine synthesis. Food intakes relative to metabolic body weights of the rats during the last 3 d of the experiment, were 34.3 ± 1.02 g/kg BW0.75 for the –Cu group and 41.4 ± 0.95 g/kg BW0.75 for the +Cu group (P < 0.01). Perhaps the results would have been different if a less severe Cu deficiency had been induced or if the rats were fed on the diets for a longer period of time.

Cu deficiency had no effect on the taurine, cyst(e)ine, or total glutathione concentrations at the major site of taurine synthesis, the liver. These results indicate that Cu deficiency in rats does not affect the major products of cysteine metabolism in the liver. However, some reports indicate that Cu deficiency in rats increases hepatic GSH concentration (25,26). The cause for this inconsistency is unclear. Perhaps a more prolonged Cu deficiency in the earlier studies is responsible.

The only significant effect of Cu deficiency on sulfur amino acid metabolism was a higher CSAD activity in liver (P < 0.01). The activities of CDO and CSAD are critical to taurine synthesis because they are the key enzymes in the synthesis of taurine from its direct precursor, cysteine. The regulation of these key enzymes in the synthesis of taurine has been reported (27,28). Bagley and Stipanuk (28) demonstrated that, as the dietary protein concentration increases, CDO activity increases and CSAD activity decreases. That is, CDO and CSAD are regulated in a reciprocal manner in response to dietary protein or sulfur amino acid concentration in the diet. In the current study, the reciprocal regulations of activities in the 2 enzymes were not found because CDO did not change. However, the difference in CSAD activity in this study was consistent with previous finding that CSAD activity decreases with higher protein intake (27,28). The food intake/kg BW0.75 of the rats during the last 3 d of the the experiment was higher in the +Cu group (P < 0.01) and the CSAD activity was lower in this group. Although the CDO activity did not differ between groups, it was 10% higher in the +Cu group (P = 0.60), possibly showing a trend for metabolic adaptation of the taurine synthesis system to maintain taurine homeostasis.

In conclusion, Cu deficiency did not affect taurine or other sulfur amino acid metabolites in plasma or in the liver of rats in this study. CSAD activity appeared to be controlled in a normal manner by the amount of dietary protein ingested. We conclude that Cu deficiency does not affect cysteine metabolism or taurine homeostasis in rats and that it is highly unlikely that DCM-induced taurine deficiency in large-breed dogs is the result of a dietary-induced Cu deficiency.


   FOOTNOTES
 
1 Supported by the Center for Companion Animal Health (CCAH), the School of Veterinary Medicine, University of California, Davis. Back

4 Abbreviations used: BW, body weight; CDO, cystein dioxygenase; CSAD, cystein sulfinic acid decarboxylase; +Cu, copper-adequate; –Cu, copper-deficient; DCM, dilated cardiomyopathy. Back

Manuscript received 2 June 2006. Initial review completed 17 June 2006. Revision accepted 25 July 2006.


   LITERATURE CITED
TOP
 ABSTRACT
 Introduction
 Materials and Method
 Results
 Discussion
 LITERATURE CITED
 

1. Awapara J, Wingo WJ. On the mechanism of taurine formation from cysteine in the rat. J Biol Chem. 1953;203:189–94.[Free Full Text]

2. Pion PD, Kittleson MD, Rogers QR, Morris JG. Myocardial failure in cats associated with low plasma taurine—a reversible cardiomyopathy. Science. 1987;237:764–8.[Abstract/Free Full Text]

3. Moise NS, Pacioretty LM, Kallfelz FA, Stipanuk MH, King JM, Gilmour RF, Jr. Dietary taurine deficiency and dilated cardiomyopathy in the fox. Am Heart J. 1991;121:541–7.[Medline]

4. Pion PD, Sanderson SL, Kittelson MD. The effectiveness of taurine and levocarnitine in dogs with heart disease. Vet Clin N Am Small Anim Pract. 1998;28:1495–514.[Medline]

5. 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;223:1137–41.[Medline]

6. Gray LF, Daniel LJ. Effect of copper status on the urinary excretion of tryptophan metabolites and amino acids by the rat. J Nutr. 1973;103:1764–71.[Abstract/Free Full Text]

7. Fischer PW, Giroux A, L'Abbe MR. The effect of dietary zinc on intestinal copper absorption. Am J Clin Nutr. 1981;34:1670–5.[Abstract/Free Full Text]

8. Baker DH, Ammerman CB. Copper bioavailability. In: Bioavailability of nutrients for animals: amino acids, minerals, and vitamins. San Diego, CA: Academic Press; 1995.

9. National-Research-Council. Guide for the care and use of laboratory animals. Washington D.C.: National Academy Press; 1996.

10. American Institute of Nutrition. Report of the American institute of nutrition ad hoc committee on standards for nutritional studies. J Nutr. 1977;107:1340–8.[Free Full Text]

11. Hem A, Smith AJ, Solberg P. Saphenous vein puncture for blood sampling of the mouse, rat, hamster, gerbil, guinea pig, ferret and mink. Lab Anim. 1998;32:364–8.[Abstract/Free Full Text]

12. van Kampen E, Zijlstra WG. Standardization of hemoglobinometry II. The hemiglobincyanide method. Clin Chim Acta. 1961;6:538–44.[Medline]

13. Clegg MS, Keen CL, Lonnerdal B, Hurley LS. Influence of ashing techniques on the analysis of trace elements in aminal tissues. 1. Wet ashing. Biol Trace Elem Res. 1981;3:107–15.

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

15. Schosinsky KH, Lehmann HP, Beeler MF. Measurement of ceruloplasmin from its oxidase activity in serum by use of o-dianisidine dihydrochloride. Clin Chem. 1974;20:1556–63.[Abstract]

16. Bagley PJ, Hirschberger LL, Stipanuk MH. Evaluation and modification of an assay procedure for cysteine dioxygenase activity: high-performance liquid chromatography method for measurement of cysteine sulfinate and demonstration of physiological relevance of cysteine dioxygenase activity in cysteine catabolism. Anal Biochem. 1995;227:40–8.[Medline]

17. Bella DL, Stipanuk MH. Effects of protein, methionine, or chloride on acid-base balance and on cysteine catabolism. Am J Physiol 1995;269:E910–E7.

18. Fariss MW, Reed DJ. High-performance liquid chromatography of thiols and disulfides: dinitrophenol derivatives. Methods Enzymol. 1987;143:101–9.[Medline]

19. Stipanuk MH, Bagley PJ, Coloso RM, Banks MF. Metabolism of cysteine to taurine by rat hepatocytes. Adv Exp Med Biol. 1992;315:413–21.[Medline]

20. Smith PK, Krohn RI, Hermanson GT, Mallia AK, Gartner FH, Provenzano MD, Fujimoto EK, Goeke NM, Olson BJ, Klenk DC. Measurement of protein using bicinchoninic acid. Anal Biochem. 1985;150:76–85.[Medline]

21. National-Research-Council. Nutrient requirements of laboratory animals. 4th ed. Washington DC: National Academy Press; 1995.

22. Allen CB. Effects of dietary copper deficiency on relative food intake and growth efficiency in rats. Physiol Behav. 1996;59:247–53.[Medline]

23. Prohaska JR, Heller LJ. Calcium reintroduction decreases viability of cardiac myocytes from copper-deficient rats. J Nutr. 1999;129:1842–5.[Abstract/Free Full Text]

24. Park T, Rogers QR, Morris JG, Chesney RW. Effect of dietary taurine on renal taurine transport by proximal tubule brush border membrane vesicles in the kitten. J Nutr. 1989;119:1452–60.[Abstract/Free Full Text]

25. Chao PY, Allen KG. Glutathione production in copper-deficient isolated rat hepatocytes. Free Radic Biol Med. 1992;12:145–50.[Medline]

26. Kim S, Wilson JJ, Allen KG, Clarke SD. Suppression of renal gamma-glutamylcysteine synthetase expression in dietary copper deficiency. Biochim Biophys Acta. 1996;1313:89–94.[Medline]

27. Bella DL, Hirschberger LL, Hosokawa Y, Stipanuk MH. Mechanisms involved in the regulation of key enzymes of cysteine metabolism in rat liver in vivo. Am J Physiol 1999 FEB;276:E326–E35.

28. Bagley PJ, Stipanuk MH. The activities of rat hepatic cysteine dioxygenase and cysteinesulfinate decarboxylase are regulated in a reciprocal manner in response to dietary casein level. J Nutr. 1994;124:2410–21.[Abstract/Free Full Text]

29. Williams JM, Morris JG, Rogers QR. Phenylalanine requirement of kittens and the sparing effect of tyrosine. J Nutr. 1987;117:1102–7.[Abstract/Free Full Text]





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