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Article

Chloride Requirement of Kittens for Growth Is Less than Current Recommendations^{1 ,2}

Department of Molecular Biosciences, School of Veterinary Medicine, University of California, Davis, CA 95616

⁴To whom correspondence should be addressed.

	ABSTRACT

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The minimal chlorine (chloride) requirement of kittens for growth was determined in a study in which kittens were given purified diets containing 0.1, 0.4, 0.7, 1.0 or 1.3 g of chloride (Cl) as KCl/kg diet. Each dietary group contained six (three males and three females) specific-pathogen–free kittens; the diets were fed for 30 d. Kittens receiving diets with <0.7 g Cl/kg became alkalotic as evidenced by an increase in blood pH, blood bicarbonate, standard bicarbonate, actual base excess, standard base excess, partial pressure of carbon dioxide (pCO₂₎ and total CO₂. In addition, kittens were hypochloremic and hypokalemic; they had decreased serum ionized calcium and a negative chloride balance. On the basis of the chloride balance measurements, a broken-line nonlinear least-square analysis predicted a Cl requirement as 0.89 g Cl/kg diet (22 kJ/g diet). Because the dietary Cl concentration closest to 0.89 g/kg that we tested was 1.0 g Cl/kg, we recommend a minimum chloride requirement of 1.0 g Cl/kg diet for growing kittens. This value is considerably less than the recommended chloride requirement of the National Research Council of 1.9 g Cl/kg diet, or the allowance of the Association of American Feed Control Officials of 3.0 g Cl/kg diet. Because the bioavailability of chloride is high, the previous estimates appear excessive.

KEY WORDS: • chlorine • chloride • alkalosis • potassium • kittens • requirements

	INTRODUCTION

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Sodium and chloride are the major cations and anions of extra cellular fluid and are the principal osmolites in the maintenance of the volume of extracellular fluid. Although chloride (chlorine) deficiency has been reported in human infants (Grossman et al. 1980 , Hellerstein et al. 1985 , Linshaw et al. 1980 , Rodriguez-Soriano et al. 1983 ), cattle (Fettman et al. 1984 , Neathery et al. 1981 ), poultry (Harms 1982 , Harms and Wilson 1984 ), rats (DeLange et al. 1970 ) and dogs (Felder et al. 1987 ), there have been no reports of chloride deficiency of dietary origin in cats. The clinical signs of chloride deficiency common to all species include anorexia, body weight loss or retarded growth in young animals, hypochloremia, hypokalemia and metabolic alkalosis (Grossman et al. 1980 , Neathery et al. 1981 ).

In estimating a requirement for chloride (Cl) for cats, the NRC (1986) stated that diets containing 1900 mg chloride/kg had been successfully fed without apparent harm to cats, but the minimum chloride requirement of cats had not been defined experimentally. The objective of this study was to determine the minimal chloride requirement of growing kittens.

	MATERIALS AND METHODS

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The experimental protocol adhered to the NIH guidelines (NRC 1985 ) and was approved by the Animal Use and Care Administration Advisory Committee of the University of California, Davis.

Animals and their management.

Specific-pathogen–free domestic short-haired kittens (n = 30) from the Feline Nutrition and Pet Care Center of the University of California at Davis were given a pretreatment purified diet (Table 1 ) containing 3.1 g Cl/kg diet for 5 wk to accustom them to eating a purified diet. At the end of this period, when the kittens ranged from 12 to 14 wk of age, they were divided into two groups on the basis of sex, then within each sex were randomly allocated to the five treatments so that each treatment group contained three male and three female kittens. The experimental dietary treatments had chloride concentrations that ranged from 0.1 to 1.3 g/kg diet. Kittens received the experimental diets for 30 d.

Diets.

To the casein-lactalbumin protein-based purified diet (Table 1) , chloride (as KCl) was added at various concentrations at the expense of cornstarch. The experimental diets contained all nutrients except chloride in amounts sufficient to meet the requirements of growing kittens. Dietary chloride concentrations were verified by analysis using a Chloridometer (Model 4–2008, Buchler Instruments, Fort Lee, NJ).

Sample collection.

Food intake was measured daily and body weight weekly. Plastic syringes with potassium heparin as an anticoagulant were used to take blood samples from the jugular veins of nonanesthetized kittens. Hematologic variables were measured within 3 h of blood drawing. Blood samples without anticoagulant were allowed to clot at room temperature for 2 h, and the serum was separated by centrifugation at 1100 x g for 10 min. Samples were stored at -20°C until analysis. For blood gas analysis, ~1.5 mL of jugular blood was taken in plastic syringes that had the dead space filled with heparin. The syringe and needle were sealed with a rubber stopper and placed in ice water until analysis. Blood gas analyses were done on two separate days to keep the time between collection of blood and analysis to within 1.5 h when changes are negligible. Immediately before samples were drawn for blood gas analysis, rectal temperatures were taken (kittens had been trained to the procedure to reduce possible stress). Urine and fecal output was measured on d 21–28 and chloride balance was computed.

Sample analysis.

Serum Na, K and ionized Ca and Mg were measured using NOVA 8 analyzer (NOVA Biomedical, Waltham, MA). Chloride in serum and urine samples was measured directly using a Chloridometer (Model 4–2008, Buchler Instruments). For diet and fecal samples, Cl was extracted with an aqueous mixture of nitric acid (0.1 mol/L) and glacial acetic acid (1.74 mol/L); the extract was used for Cl measurement. Serum aldosterone was assayed as described by Yu and Morris (1997) . Hematologic variables were analyzed using a blood cell counter (Mascot, CDC Technologies, Oxford, CT). Blood gas variables were measured with an Acid-Base Analyzer (ABL30, Radiometer, Copenhagen, Denmark) and corrected for each kitten's rectal temperature and hemoglobin concentration. Chloride balance was computed from ingested Cl from food minus (fecal Cl + urinary Cl). Apparent chloride absorption was computed as (Cl intake - fecal Cl)/Cl intake.

Statistical analysis.

Statistical analyses were performed using SPSS version 8.0 (SPSS 1997 and 1998 ). Unless otherwise indicated, one-way ANOVA was used for each variable at each time of measurement to test the diet effect, followed by Tukey's test for post-hoc comparisons when the variance was equal between dietary groups (Levene's test). The Kruskal-Wallis test was used, followed by Dunnett's T3 test for post-hoc comparisons if variances were not equal. Break points of selected variables were calculated using a nonlinear least-square method (Robbins 1986 ) and SAS statistical software (version 6.03, SAS Institute, Cary, NC) if diet effect was significant. Probability of type I error < 0.05 was considered significant for all tests except those adjusted according to the Bonferroni test because of multiple comparisons. Chloride requirements were determined by breakpoints calculated by nonlinear least-square analysis (Robbins 1986 ).

	RESULTS

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Food intake and final body weight were not affected by the dietary treatment (Table 2 ); however, kittens in the 1.0 g Cl/kg diet group had a significantly higher rate of body weight gain than those in the other groups (Fig. 1 A). Kittens receiving dietary Cl concentrations <0.7g Cl/kg were in negative Cl balance (Fig. 3B) and had a significant reduction in serum chloride concentration (Fig. 1 B) compared with kittens consuming a diet of 1.3 g Cl/kg. Serum potassium and ionized calcium concentrations (Table 3) were also significantly depleted by diets containing <0.7 g Cl/kg, but these diets had no significant effect on serum sodium or ionized magnesium concentrations. There was no difference in serum Cl concentrations between d 21 and 28, indicating that the kittens had reached an equilibrium state before d 21 (Fig. 1 B).

View larger version (17K): [in this window] [in a new window]   Figure 1. Body weight gain (panel A) and serum chloride concentration (panel B) of kittens given a purified diet with various levels of added chloride. Each point represents the mean of six kittens (three males and three females); vertical bars are SEM There was a significant diet effect (P = 0.018, one-way ANOVA) of diet on body weight gain. Points not sharing a letter are significantly different (P < 0.05, Tukey's test). Serum Cl concentrations are significantly different in wk 4, (P = 0.001, Kruskal-Wallis test). No significant difference was found between week 3 and 4 of each dietary group (P > 0.01, paired t test; significant level was adjusted to 0.01 using Bonferroni's methods because of multiple comparisons). The points (wk 4) not sharing a letter are significantly different (P < 0.05, Dunnett's T3 test).

View larger version (17K): [in this window] [in a new window]   Figure 3. Serum aldosterone concentration (panel A) and chloride balance (panel B) of kittens fed a purified diet with various levels of chloride. Each point represents the mean of six kittens (three males and three females); vertical bars are SEM. There was a significant diet effect on serum aldosterone concentrations on d 29-30 of the study (P < 0.01, Kruskal-Wallis test). Points not sharing the same letter are significantly different (P < 0.05, Dunnett's T3 test). There was a significant diet effect on Cl intake (P < 0.05, Kruskal-Wallis test), urinary Cl output and Cl retention (P < 0.05, one-way ANOVA), but there was no such effect on fecal Cl excretion (P > 0.05, Kruskal-Wallis test). Points not sharing the same letter are significantly different (P < 0.05, Tukey's test for urinary Cl output and Cl retention and Dunnett's T3 test for Cl intake). Cl retention was defined as Cl intake - (fecal Cl + urinary Cl).

As dietary Cl concentration decreased from 1.3 to 0.1 g/kg, there was a progressive development of alkalosis in the kittens. This was evidenced by a significant elevation of blood pH (Fig. 2 A), an increase in blood HCO₃ (Fig. 2 B), and increases in standard bicarbonate (SBC),⁵ actual base excess (ABE) and standard base excess (SBE) (Table 4). Both blood pH and HCO₃ were similar at d 22–23 and d 29–30, indicating that the kittens had attained a stable state. Partial pressure of carbon dioxide (pCO₂) and total CO₂ in blood were also elevated in kittens given the lower concentrations of Cl in the diet. There was no significant effect of Cl concentration in the diet on partial pressure of oxygen (pO₂), oxygen concentration or oxygen saturation rate (Table 4) .

View larger version (17K): [in this window] [in a new window]   Figure 2. Blood pH (panel A) and HCO3 (panel B) of kittens given a purified diet with various levels of chloride. Each point represents the mean of six kittens (three males and three females); vertical bars are SEM There was a significant diet but not time effect on blood pH and HCO3 when the data of d 22 through 30 were analyzed by ANOVA (diet P < 0.01, time P > 0.05). Points not sharing the same letter are significantly different (P < 0.05, Tukey's test for both blood pH and HCO3 on d 29 and 30).

Calculated break points using the nonlinear least-square analysis for selected variables ranged from 0.512 to 0.840 g Cl/kg diet; these are given in Table 6 .

	DISCUSSION

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The changes that we observed in kittens are similar to those reported in chloride deficiency in humans and other animals (Grossman et al. 1980 , Linshaw et al. 1980 , Neathery et al. 1981 ). Secondary potassium deficiency became the predominant clinical manifestations of kittens with progression of chloride deficiency. In an earlier report (Yu and Morris 1998a ), kittens given a diet similar to the 0.1 g Cl/kg diet used in this study exhibited ventroflexion of the head and other locomotive disturbances typical of potassium deficiency. These changes were reversed within several hours after either KCl or K₂HPO₄ was given orally, even though the dietary K concentration was 0.8%, which is twice the requirement given by the NRC (1986) for growing kittens. These signs recurred if chloride deficiency was not corrected.

Hypokalemia in kittens with hypochloremic metabolic alkalosis suggests that the homeostatic mechanisms of mineral, acid-base and water regulation of cats are similar to those in other mammals. When the load of chloride filtered by the glomerulus decreases, there is a reduction in active reabsorption of chloride and sodium in the loop of Henle. This results in more sodium reaching the distal tubule where it is reabsorbed in exchange for potassium and hydrogen, leading to potassium depletion (Simopoulos and Bartter 1980 ). Serum ionized calcium concentration decreased in the kittens with the reduction in dietary Cl concentration (Table 3) as a direct result of increased blood pH (Fig. 2 A). However, serum sodium and ionized magnesium concentrations were not affected.

Sodium and chloride are the major ions of the extracellular fluid and play an important role in maintenance of extracellular volume. Chloride deficiency in kittens induced a significant reduction in serum chloride concentration (Fig. 1 B), which presumably resulted in hypovolemia that triggered the renin-angiotention-aldosterone system. Serum aldosterone was significantly elevated (Fig. 3 A) in kittens receiving the diet containing 0.1 g Cl/kg diet compared with a reference value (Yu and Morris, 1998b ). Hypovolemia is further supported by the general inverse dose-response relationship between the concentration of formed elements in the blood (WBC, RBC, Hb and PCV) and dietary Cl concentration. MCHC remained unchanged, and MCV and MCH changed slightly, with no dose-response relationship (Table 5) .

Changes in the blood gas tensions and acid-base variables (Table 4 , Fig. 2 B) are direct responses to changes in extracellular chloride concentration, in response to changes of Cl balance. Oxygen tension in blood, pO₂, O₂ concentration and O₂ saturation were not affected. Serum Cl (Fig. 1 B) as well as blood HCO₃ and pH (Fig. 2 B) reached equilibrium within 3 wk after the kittens were given the diets with different levels of chloride. The rapid equilibrium of chloride status of cats with diet indicates that cats do not have substantial amounts of reserve chloride. Therefore, the 4-wk experimental period that we used appears to be of adequate duration to estimate chloride requirements of growing kittens.

Fecal Cl excretion was not affected by dietary Cl concentrations in the range from 0.1 to 1.3 g Cl/kg (Fig. 3 B), which is similar to that found for fecal sodium excretion (Yu and Morris, 1997 ). This suggests that the efficiency of Cl uptake is high and not dependent on the Cl concentration of the diet. The obligatory loss of Cl in feces was ~0.1 mmol/d. Chloride homeostasis, like sodium, is achieved by renal regulation of the efficiency of resorption of filtered Cl. Urinary Cl excretion increased progressively with the Cl intake >0.6 mmol/d in the kittens. Chloride retention in growing kittens reached a plateau when dietary Cl concentration was >0.7 g/kg (Fig. 3B) .

The broken-line technique, together with nonlinear least-square analysis, (Robbins, 1986 ) was used in this study to calculate the break point objectively, i.e., the chloride requirement of kittens for growth. As is the case for most nutrients, the estimated requirement of Cl depends on the variables selected and ranged from 0.51 to 0.84 g Cl/kg diet (Table 6) . When an upper asymptotic 95% cut-off point is used, the estimated chloride requirement based on chloride retention is 0.89 g/kg. From this study, it appears that the chloride requirement of cats suggested by the NRC (1986) (which was not based on experimental data) is about twice the requirement. The Association of Feed Control Officials cat food nutrient profile (AAFCO 1999 ), which is widely used in formulating cat foods by the pet food industry, estimated a chloride allowance of 3.0 g/kg diet for growth, reproduction and maintenance. This value is more than three times the requirement found in our study. Because the chloride present in ingredients used in the pet food industry should have high bioavailability (Henry 1995 ) and have no problems with stability, an allowance of more than three times the requirement appears excessive. The dietary Cl concentration closest to 0.89 g Cl/kg diet that we tested in kittens was 1.0 g Cl/kg diet; therefore we recommend 1.0 g Cl/kg diet (22 kJ metabolizable energy/g) be used as a requirement for growing kittens.

	ACKNOWLEDGMENTS

 
The vitamin mixture was a gift from Hoffman La Roche, Nutley, NJ and is gratefully acknowledged.

	FOOTNOTES

 
¹ Presented in part in poster form at Experimental Biology 98, April 1998, San Francisco, CA [Yu, S. & Morris, J. G. (1998) Hypokalemia in kittens induced by a chlorine-deficient diet. FASEB J. 12: A219 (abs.)].

² Supported in part by a grant from the George and Phyllis Miller Feline Health Fund, Center for Companion Animal Health, School of Veterinary Medicine, University of California, Davis and Hill's Pet Nutrition, Topeka, KS.

³ Current address: Research and Development, Heinz Pet Products, 212 Terminal Way, San Pedro, CA 90731.

⁵ Abbreviations used: ABE, actual base excess; Hb, hemoglobin concentration; MCH, mean corpuscular hemoglobin; MCHC, mean corpuscular hemoglobin concentration; MCV, mean corpuscular volume; pCO₂, partial pressure of carbon dioxide; PCV, packed cell volume; pO₂ partial pressure of oxygen; SBC, standard bicarbonate; SBE standard base excess; WBC, white blood cell count.

Manuscript received May 13, 1999. Initial review completed June 25, 1999. Revision accepted July 14, 1999.

	REFERENCES

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 

1. Association of American Feed Control Officials (1999) AAFCO Official Publication, pp.143.

2. DeLange W. E., Visser J.W.E., Van Woudenberg F., Doorenbos H. Chloride space and sodium and potassium metabolism in chloride deficiency; an experimental study in rats. Folia Med. Neerl. 1970;13:157-161[Medline]

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4. Fettman M. J., Chase L. E., Bentinck-Smith J., Coppock C. E., Zinn S. A. Nutritional chloride deficiency in early lactation Holstein cows. J. Dairy Sci. 1984;67:2321-2335

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6. Harms R. H. Chloride requirement of young turkeys. Poult. Sci. 1982;61:2447-2449[Medline]

7. Harms R. H., Wilson H. R. The chloride requirement of the broiler breeder hen. Poult. Sci. 1984;63:835-837[Medline]

8. Hellerstein S., Duggan E., Merveille O., Scarth L. Follow-up studies on children with severe dietary chloride deficiency during infancy. Pediatrics 1985;75:1-7[Abstract/Free Full Text]

9. Henry P. H. Sodium and chlorine bioavailability. Ammerman C. B. Baker D. H. Lewis A. J. eds. Bioavailabilty of Nutrients for Animals 1995:337-348 Academic Press San Diego, CA.

10. Linshaw M. A., Harrison H. L., Gruskin A. B., Prebis J., Harris J., Stein R., Jayaram M. R., Preston D., DiLiberti J., Baluarte J., Elzouki A, Carroll N. Hypochloremia alkalosis in infants associated with soy protein formula. Pediatrics 1980;96:635-640

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12. National Research Council Nutrient Requirements of Cats 1986 National Academy Press Washington, DC.

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14. Robbins K. R. A method, SAS program, and example for fitting the broken-line to growth data. The University of Tennessee Agricultural Experiment Station Research Report 1986:86-89 Knoxville, TN.

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20. Yu S., Morris J. G. The minimum sodium requirement of growing kittens defined on the basis of plasma aldosterone concentration. J. Nutr. 1997;127:494-501[Abstract/Free Full Text]

21. Yu S., Morris J. G. Hypokalemia in kittens induced by a chlorine-deficient diet. FASEB J 1998;12:A219(abs.)

22. Yu S., Morris J. G. Plasma aldosterone concentration of cats. Vet. J. 1998;155:63-68[Medline]

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