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Departments of Molecular Biosciences and * Surgical and Radiological Sciences, School of Veterinary Medicine, University of California, Davis, CA 95616
3 To whom correspondence should be addressed. E-mail: ajfascetti{at}ucdavis.edu.
KEY WORDS: • lysine • arginine • feline • cats • herpesvirus
Feline herpesvirus type 1 (FHV-1) is one of the most common feline pathogens in the world (1). Primary infection with FHV-1 in susceptible kittens causes severe upper respiratory and ocular disease with 
100% morbidity particularly in multi-cat environments (2). After primary exposure, FHV-1 establishes lifelong neural latency with periodic reactivation in 80% of cats (2). Presently no medications or vaccines have demonstrated reduced establishment of latency or frequency of reactivation. Oral lysine administration to cats infected with FHV-1 is associated with a significant reduction in the severity of clinical signs and basal viral shedding (3,4). The mechanism of action is hypothesized to be reduced viral replication due to antagonism of arginine by excess lysine (5). The most logical means of controlling viral shedding in multi-cat environments is to reduce viral shedding in latently infected cats. Nutritional control of viral shedding would provide a simple and efficient means of implementing such a management technique.
Determination of the safety of lysine supplementation is imperative due to cats' exquisite sensitivity to arginine deficiency (6). Although previous studies demonstrated that exogenous supplementation with lysine increases plasma lysine without antagonizing plasma arginine concentrations (3,4), the effect was short lived (3 h) in the one study where it was evaluated (3). It is hypothesized that if the lysine content of a commercial diet was increased, cats consuming that diet ad libitum would have and maintain elevated plasma lysine concentrations throughout the day and forgo the variable concentrations observed with exogenous supplementation. This consistent elevation in plasma lysine concentrations may be beneficial in controlling FHV-1. However, to the authors' knowledge, the safety of lysine administration >19 g/kg of diet (7) has never been evaluated. The objective of this study was to determine the safety of excess lysine supplementation to a commercial-type, expanded diet fed to healthy adult cats.
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MATERIALS AND METHODS |
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 TOP MATERIALS AND METHODS RESULTSDISCUSSIONLITERATURE CITED |
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Animals and their management
Healthy intact adult female, domestic short-hair cats (n = 36) from the Feline Nutrition and Pet Care Center of the University of California, Davis were used. Cats were individually housed in metabolic cages (60 x 60 x 60 cm) in rooms with controlled temperature (21 ± 2°C) and a 14:10-h light:dark cycle. The animals had free access to water throughout the study.
Diets
The basal diet was a commercially prepared, experimental, dry expanded diet manufactured by Nestlé Purina PetCare Company. The proximate composition of the diet (in g/kg of diet) was 320 protein, 150 fat, and 13 crude fiber.4 The caloric density of the diet was 409 kcal metabolizable energy (ME)/100 g of diet. The calculated lysine content was 13 g/kg of diet. The analyzed lysine content was 10.7 ± 0.2 g/kg of diet (mean ± SEM, n = 9 samples). The calculated arginine content was 13 g/kg of diet. The analyzed arginine content was 12.9 ± 0.2 g/kg of diet (mean ± SEM, n = 9 samples).
Design
All of the cats were adapted to the basal diet for 1 wk. Individual food-intake and lysine-intake amounts were determined daily throughout the study. Cats were fed once daily at 1100 h. Based on food-intake determinations during wk 1, each cat was fed 120 g of food/d, which was an amount greater than any cat consumed daily. All cats had free access to their food. During wk 2, 12.7 g of deionized water was added daily to each cat's portion before feeding. On wk 3, the cats were assigned to six dietary treatment groups. Cats in the control group (n = 6 cats) were fed 120 g of the basal diet (11 g of lysine/kg of diet) with 12.7 g of added water. Cats in the other five treatment groups (n = 6 cats/group) were fed the basal diet but with lysine added as lysine acetate dissolved in deionized water to yield a total dietary concentration (diet plus supplemental lysine) of 36, 61, 86, 111, or 131 g of lysine/kg of diet. The lysine acetate solution was prepared and applied to the diets on a daily basis. Dissolution of the lysine acetate in water permitted a thorough and even application of the amino acid throughout the basal diet. Cats in the control group and the dietary treatment groups that consumed 36, 61, or 86 g of lysine/kg of diet received a total of 12.7 g of deionized water in their diet daily. The treatment groups with 111 and 131 g of lysine/kg of diet were added after no significant changes were observed in cats that received the diets containing lower lysine concentrations. To get sufficient lysine acetate into solution to provide 111 or 131 g of lysine/kg of diet, it was necessary to add 18.7 or 22.7 g, respectively, of deionized water to these diets. In all aspects of the study other than this, cats in these two dietary treatment groups were treated identically to those in the previous groups. Therefore, the results from all six treatment groups are reported simultaneously.
Cats were observed daily and weighed weekly throughout the study. Blood was collected on d 2 (2 d after beginning the lysine-supplemented or control diets), d 7, and d 14 (end of the project) by routine venipuncture at 1500 h. Blood was collected into heparinized syringes and separated into two tubes (whole blood and plasma). Plasma was obtained by centrifugation of the heparinized blood sample at 10,000 x g for 15 min, immediate deproteinization with an equal volume of 0.24 mol/L sulfosalicylic acid, and centrifugation again at 10,000 x g for 15 min at 4°C. All samples were stored at –80°C until analysis. Sample preparation of the basal diet for analysis was previously described (8). Amino acid analysis of plasma and basal diet samples was performed using an amino acid analyzer (model 6300, Beckman Instruments, Palo Alto, CA).
The effect on food intake of the increased amount of water needed to dissolve the lysine acetate into solution for the treatment groups with 111 and 131 g of lysine/kg of diet was determined in a separate study. Individual food-intake amounts were determined for six cats fed the basal diet with the same amounts of deionized water added as in the initial experiment. Each cat was fed 120 g of basal diet for 1 wk and subsequently received the same amount of diet supplemented with 12.7, 18.7, or 22.7 g of deionized water for 1 wk at each level of water supplementation. Dietary dry-matter intake amounts were determined daily for each cat.
Statistical analysis
All results are expressed as means ± SEM. One-way ANOVA was used to test for differences in means among groups. Repeated-measures ANOVA with two grouping variables (time and treatment) and four repeated measures (amino acid concentration, food intake, lysine intake, or body weight) was used to test for variable interactions and variance of plasma amino acid concentration, food intake, lysine intake, and body weight. Specific differences were determined using a Tukey-Kramer post-hoc test. (Systat 10.2, SPSS, Chicago, IL). For all analyses, differences were considered significant at P 
0.05.
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RESULTS |
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TOPMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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0.05) and tended to increase with increasing lysine intake. There was a significant effect of treatment on mean plasma lysine concentrations (P 0.05, Table 1) but not mean plasma arginine concentrations (P 
1.0). There was no significant time-by-treatment interaction (arginine, P 0.7; lysine, P 0.8) nor a significant main effect of time on either mean plasma arginine (P 0.06) or mean plasma lysine (P 0.7) concentrations. Other mean plasma amino acid concentrations did not differ significantly among dietary treatment groups (data not shown).
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1.0) in the mean body weights of cats in any group throughout the study. There were significant main effects of both time and treatment on food-intake values in this study (Fig. 1, P 0.05); however, there was no significant time-by-treatment interaction on food intake (P 0.3). Food-intake amounts in the groups that consumed the diets with 111 or 131 g of lysine/kg of diet began to decrease compared with the other dietary treatments on d 2. The mean food-intake values for cats that consumed 11, 36, 61, 86, 111, or 131 g of lysine/kg of diet on d 2 were 86.0 ± 7.8, 79.3 ± 6.3, 70.8 ± 13.5, 60.5 ± 8.4, 50.3 ± 11.1, and 62.0 ± 10.1 g, respectively. By d 14 of lysine supplementation, food-intake levels in the groups that consumed 111 or 131 g of lysine/kg of diet were still lower, and this difference approached significance (Fig. 1, P 0.08). When cats that consumed 111 or 131 g of lysine/kg of diet began to reduce their food-intake amounts, lysine-intake values were reduced. There was a significant time-by-treatment interaction with respect to lysine intake (P 0.05), and the main effect of treatment was also significant (Fig. 1; P 0.001). There was no significant effect of time on lysine-intake amount (P 0.3). The follow-up study to determine the effect of adding water to the basal diet on food-intake values did not yield any significant differences in dietary dry-matter intake with increasing water supplementation.
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DISCUSSION |
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TOPMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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In this study, plasma lysine values increased with dietary lysine intake. Studies in other species show similar responses to increased dietary lysine concentrations (9,10). The slight variations in mean plasma lysine concentrations within a dietary treatment group over the course of the study are most likely the result of differences in each individual cat's food intake before blood collection. Every cat was offered fresh food 4 h before blood collection. Some cats ate the fresh food immediately, whereas others did not. However, food-intake amount was measured only once daily before fresh food was offered.
Plasma arginine concentrations did not decrease nor were clinical signs of arginine deficiency observed with increasing dietary lysine concentrations. This was also observed in kittens, young pigs, and growing dogs that were fed excess lysine in diets that contained arginine concentrations at or above the recommended minimal requirements (9–11). Furthermore, unlike plasma lysine concentrations, plasma arginine concentrations were not affected by food intake. This is most likely because all dietary treatments contained arginine somewhat in excess of the minimal recommended arginine requirement for adult cats.
The mean body weights of the cats did not differ significantly among or change within the dietary treatment groups throughout the study. However, there was a reduction in food-intake amount in the cats that consumed 111 or 131 g of lysine/kg of diet, and by d 14, this difference approached significance. A longer study period may have yielded a statistically significant difference. Two likely causes for this reduction in food-intake amount are the increased quantities of water needed to incorporate the higher amounts of lysine into solution in the 111 and 131 g of lysine/kg of diet concentrations, or toxic effects of high concentrations of lysine in these two diets. There was no change in dietary dry-matter intake when equivalent amounts of water only were added to the basal diet. Therefore, the reduction in food-intake level in the cats that consumed 111 or 131 g of lysine/kg of diet was most likely the result of the excess dietary lysine and not the water used to incorporate it into solution.
It is hypothesized that the reduction in food intake in this study was the result of an amino acid toxicity. A reduction in food intake can be the result of amino acid imbalance, toxicity, or antagonism (12). Excess dietary lysine was shown to antagonize arginine in chicks (13), rats (14), guinea pigs (15), and growing dogs (9). An amino acid imbalance resulting in a reduction in both food-intake and weight gain was reported for young pigs that consumed diets that contained 34.5 or 46.5 g of lysine/kg of diet and 5.3 g of arginine/kg of diet (11). A reduction in food intake coupled with the absence of reduced plasma amino acid concentrations and clinical signs of arginine deficiency support an amino acid toxicity at the two highest dietary lysine concentrations fed in this study.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Supported by grants from the Nestlé Purina PetCare Company and the George and Phyllis Miller Feline Health Fund, Center for Companion Animal Health, School of Veterinary Medicine, University of California, Davis.
4 The basal diet was composed of the following ingredients (in g/kg of diet): 235.2 corn gluten meal, 232.2 milled brewers rice, 213.8 whole yellow corn, 183.3 poultry by-product meal, 61.1 fish meal, 48.9 wheat flour, 10.4 potassium chloride; 3.7 mineral mix containing zinc sulfate, ferrous sulfate, manganese sulfate, copper sulfate, calcium iodate, and sodium selenite (remaining ingredients and amounts proprietary, Nestlé Purina PetCare Company); 3.1 calcium carbonate, 3.1 sodium chloride, 2.5 choline chloride, 1.2 taurine; 0.9 vitamin mix containing retinyl acetate, cholecalciferol, dl-
-tochopheral acetate, thiamine mononitrate, riboflavin, nicotinic acid, calcium D-pantothenate, pyridoxine hydrochloride, cyanocobalamine, D(+)biotin, and menadione (remaining ingredients and amounts proprietary, Nestlé Purina PetCare Company); 0.6 vitamin E, and 0.1 DL-methionine.
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LITERATURE CITED |
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TOPMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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1. Gaskell, R. M. & Wardley, R. C. (1978) Feline viral respiratory disease: a review with particular reference to its epizootiology and control. J. Small Anim. Pract. 19: 1–16.[Medline]
2. Gaskell, R. M. & Povey, R. C. (1977) Experimental induction of feline viral rhinotracheitis virus re-excretion in FVR-recovered cats. Vet. Rec. 100: 128–133.[Abstract]
3. Maggs, D. J., Nasisse, M. P. & Kass, P. H. (2003) Efficacy of oral supplementation with L-lysine in cats latently infected with feline herpesvirus. Am. J. Vet. Res. 64: 37–42.[Medline]
4. Stiles, J., Townsend, W. M., Rogers, Q. R. & Krohne, S. G. (2002) Effect of oral administration of L-lysine on conjunctivitis caused by feline herpesvirus in cats. Am. J. Vet. Res. 63: 99–103.[Medline]
5. Maggs, D. J., Collins, B. K., Thorne, J. G. & Nasisse, M. P. (2000) Effects of L-lysine and L-arginine on in vitro replication of feline herpesvirus type-1. Am. J. Vet. Res. 61: 1474–1478.[Medline]
6. Morris, J. G. & Rogers, Q. R. (1978) Ammonia intoxication in the near adult cat as a result of dietary deficiency of arginine. Science 199: 431–432.
7. Anderson, P. A., Baker, D. H. & Corbin, J. E. (1979) Lysine and arginine requirements of the domestic cat. J. Nutr. 109: 1368–1372.
8. Spitze, A. R., Wong, D. L., Rogers, Q. R. & Fascetti, A. J. (2003) Taurine concentrations in animal feed ingredients; cooking influences taurine content. J. Anim. Physiol. Anim. Nutr. (Berl.) 87: 251–262.[Medline]
9. Czarenecki, G. L., Hirakawa, D. A. & Baker, D. H. (1985) Antagonism of arginine by excess dietary lysine in the growing dog. J. Nutr. 115: 743–752.
10. Taylor, T. P., Morris, J. G., Willits, N. H. & Rogers, Q. R. (1996) Optimizing the pattern of essential amino acids as a sole source of dietary nitrogen supports near-maximal growth in kittens. J. Nutr. 126: 2243–2252.
11. Edmonds, M. S. & Baker, D. H. (1987) Failure of excess dietary lysine to antagonize arginine in young pigs. J. Nutr. 117: 1396–1401.
12. Harper, A. E., Benevenga, N. J. & Wohlhueter, R. M. (1970) Effects of ingestion of disproportionate amounts of amino acids. Physiol. Rev. 50: 428–558.
13. Allen, N. K., Baker, D. H., Scott, H. M. & Norton, H. W. (1972) Quantitative effect of excess lysine on the ability of arginine to promote chick weight gains. J. Nutr. 102: 171–180.
14. Jones, J. D., Wolters, R. & Burnett, P. C. (1966) Lysine-arginine-electrolyte relationships in the rat. J. Nutr. 89: 171–188.
15. O'Dell, B. L. & Regan, W. O. (1962) Effect of lysine and glycine upon arginine requirement of guinea pigs. Proc. Soc. Exp. Biol. Med. 112: 336–337.
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