The Journal of Nutrition Vol. 128 No. 12 December 1998, pp. 2577S-2580S

Optimizing Dietary Amino Acid Patterns at Various Levels of Crude Protein for Cats¹

Quinton R. Rogers², Timothy P. Taylor, and James G. Morris

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

KEY WORDS: cats · kittens · feline · essential amino acids · dispensable amino acids · crude protein · orotic acid

	INTRODUCTION

Introduction References

Experiments with rats (Heger 1990, Stucki and Harper 1962), chicks (Bedford and Summers 1985, Stucki and Harper 1961), turkeys (Bedford and Summers 1988) and pigs (Wang and Fuller 1989) have shown optimal growth rates when the dietary essential amino acid (EAA)³ nitrogen to total nitrogen (E:T) ratios were between 0.4 and 0.65. It has been consistently found that diets containing only EAA (E:T = 1.0) yield poor growth rates. Taylor et al. (1996) showed that in kittens fed diets that contain EAA as the sole source of nitrogen [E:T = 1.0, with the EAA pattern proportionate to the NRC requirements (NRC 1986)], weight loss results. Plasma amino acid concentrations of methionine (and possibly arginine) suggested toxicities of these amino acids. Also, marked depressions in the concentrations of proline and asparagine suggested that these amino acids might be conditionally essential. A near-maximal growth rate for the group receiving all EAA (E:T = 1.0) was achieved when methionine and arginine were limited to not more than 2.25 times the NRC requirements. No response was obtained with proline and/or asparagine supplementation to the all-EAA diet or when these two amino acids were removed from the control diet. Thus asparagine and proline are clearly dispensable under all conditions tested thus far. These results suggest that the poor growth rates recorded in other species given only EAA (E:T = 1.0) diets are the result of an intolerance of one or more EAA, rather than a metabolic inability of animals to synthesize dispensable amino acids (DAA) at a rate fast enough for rapid growth, as previously suggested by Harper (1974). Here we report the results of weight gains of kittens fed a wide range of crude protein (CP) levels at various E:T ratios in which excesses of essential amino acids that depress weight gains were avoided.

Materials and methods.  A control purified diet (CD) was prepared containing an amino acid mixture that provided 1.5X the accepted EAA requirement (EAAreq) including Tyr and Cys [1X EAAreq is as follows (g/kg diet): Arg, 10; His, 3; Ile, 5; Leu, 12; Lys, 8; Met, 4; Cys, 3.5; Phe, 4; Tyr, 4.5; Thr, 7; Trp, 1.5; Val, 6 (NRC 1986)] and 350g/kg CP (crude protein = nitrogen in the diet × 6.25). The DAA mixture contained (g/kg) L-alanine, 175; Gly, 175; L-glutamine, 175, L- glutamic acid, 75; L-asparagine, 150; L-aspartic acid, 100; and L-proline, 150. Six sets of experimental diets were prepared with each set containing 0.75X, 1.0X, 1.5X, 2.0X and 3.0X EAAreq or all EAA, respectively, and CP levels varying from 140 to 560 g/kg, except that Met and Arg were limited to a maximum of 2.5X and 3X each EAAreq, respectively. The difference between the CP and EAA was made up with the DAA mixture. The EAA and DAA mixtures varied as indicated above, whereas all diets contained the following: 100 g/kg starch (except the 460 g/kg CP⁴ all EAA diet from Experiment 6, which contained 53 g/kg starch), 200 g/kg animal tallow, 50 g/kg hydrogenated beef tallow, 10 g/kg vitamin mixture, 50 g/kg mineral mixture and 1.5 g/kg taurine; sodium acetate was added at equimolar concentrations to that of the hydrochlorides of the basic amino acids to replace an equal weight of starch. Specific-pathogen-free kittens (n = 32 female and 32 male), 8-12 wk old, were individually housed in stainless steel metabolism cages and adapted to the CD. These kittens were divided evenly by sex into six separate experiments, each using two (one for males and one for females) 4 × 4, 5 × 5 or 6 × 6 Latin square designs, with kittens as rows, periods (10 d) as columns and one of the six sets of experimental diets and CD as treatments (i.e., CD, 1.5X EAAreq with 350 g/kg diet CP, was a treatment in all six experiments). When ANOVA within an experiment revealed significant effects (P < 0.05), Tukey's method was used to determine which means were significantly different (PC-SAS, version 6.04, SAS Institute, Cary, NC). Food intake and body weight were recorded daily, urine and feces were collected during the last 7 d of each period for nitrogen and/or orotic acid analysis and blood was taken from each kitten on d 8, 9 or 10 of each period for plasma amino acid analysis. The experimental protocol was approved by the University of California, Davis Animal Use and Care Administrative Advisory Committee and was carried out in accordance with NIH standards (NRC 1985) and the Animal Welfare Act.

View larger version (51K): [in this window] [in a new window]   Fig 1. The effect of dietary crude protein (CP) and E:T ratio on weight gain of kittens.

Results and discussion.  Increasing CP in diets containing 0.75X and the highest CP group with 1.0X but not 1.5X EAAreq or greater resulted in lower weight gains and nitrogen retentions except for kittens fed the 0.75X EAA 560 g/kg CP diet; that group actually lost weight (Table 1). Orotic acid excretion in the urine was not detected from kittens fed 1.5X EAA or greater, but was found in the urine of all of the kittens fed 0.75X EAA diets and in kittens fed the 1.0X EAA diet containing CP levels of 350 and 560 g/kg. The weight gains in these groups were inversely correlated with urinary orotic acid excretion, indicating that a dietary arginine deficit existed with 0.75X EAA at all CP levels and with 1.0X EAA and CP levels at or above 350 g/kg. These results are consistent with earlier work (Costello et al. 1980) in which 1.05% arginine in the diet was necessary to prevent orotic acid from appearing in the urine when a diet containing ~28% CP was fed. The increasing orotic acid in the urine with increasing CP in the diet when arginine is limiting leads us to suggest that the arginine requirement is proportional to the CP in the diet. These results show that plasma arginine must be at or above 100 µmol/L to prevent orotic acid accumulation and excretion at all levels of dietary protein. Previously, when 280 g CP/kg was fed (Costello et al. 1980, Morris and Rogers 1978, Zicker & Rogers 1990) with arginine at 10 g/kg diet, plasma arginine was ~ 75 µmol/L, which is similar to that found (66-81 µmol/L) for all but the lowest CP level in the current experiments when orotic acid was present in the urine. Orotic acid increases in the urine as a result of excess hepatic ammonia, resulting in a build-up of mitochondrial carbamyl phosphate. This diffuses into the cytosol, stimulating the pyrimidine synthetic pathway down to orotate, which diffuses across cell membranes and is ultimately excreted in the urine. The increase in carbamyl phosphate results from a lack of urea cycle intermediates during amino acid catabolism in the liver. Arginine, the normal source of the urea cycle intermediates in the cat, and its precursors, ornithine and citrulline, are not made de novo at a significant rate in cats (Rogers and Phang 1985).

The second factor that contributed to poor performance when cats were fed low quantities of EAA at high levels of crude protein (thus high dietary levels of DAA) was high plasma glutamate (Table 1). Low dietary concentrations of EAA appeared to enhance the effect of high levels of dietary DAA on plasma glutamate. Deady et al. (1981) showed that dietary levels of glutamic acid >60 g/kg diet caused an increase in plasma glutamate in excess of 200 µmol/L. When plasma glutamate levels exceeded 250 µmol/L (dietary glutamate 90 g/kg), adverse effects such as growth depression and emesis occurred. Because there were adequate EAA, growth was not severely depressed. Growth rates that have been reduced by limiting the dietary EAA result in a lower rate of removal of DAA for tissue protein synthesis; thus more nitrogen is available from the catabolism of the DAA, resulting in an increase of various intermediates, especially glutamate, an obligatory intermediate in the disposal of nitrogen. It is known that dietary glutamate is transaminated in the small intestine to -keto glutarate and alanine; therefore the nitrogen is absorbed as alanine, preventing the absorption and toxicity of large amounts of free glutamate. If crude protein is increased by adding only DAA rather than both DAA and EAA, then large amounts of alanine are added. It is probable that the slower removal of circulating DAA, as the result of slower growth, results in high levels of alanine in the intestinal mucosa and interferes with the complete transamination of glutamate to alanine. These conditions result in an increase in the absorption of glutamate and an elevation of plasma glutamate. At the same level of dietary glutamate (or the same level of total DAA), higher plasma glutamate occurred when the level of essential amino acids was low (Table 1).

Kittens fed diets containing 2.0X EAAreq and 560 g/kg CP diet had somewhat lower growth rates than kittens fed the 2.0X EAAreq diet and 280 g/kg CP. It is not clear why the growth rates of these kittens were depressed. Plasma arginine and glutamate were well within the tolerable range; even proline concentration in the plasma of kittens fed the 2.0X EAAreq diet and 560 g CP/kg was less than threefold higher than that in kittens fed the 2.0X EAAreq diet and 280 g/kg CP, which, from other work (Taylor et al. 1996 and 1998), would not appear high enough to depress weight gains. However, the level of proline required in the diet (or the level in plasma) to cause a growth depression is not known. It should be pointed out that when dietary EAA were limiting, with a low plasma arginine and high plasma glutamate, plasma proline was also very high, perhaps contributing to the adverse effect of a very low E:T ratio. More work is required to establish whether high plasma proline alone will inhibit growth.

Kittens fed the diet containing all EAA and 140 g CP/kg diet did not grow. In contrast, when the E:T ratio was 0.47, the same CP content resulted in weight gains (14.7 g/d) that were ~60% of those of kittens fed CD. Weight gains were also somewhat lower in kittens fed only EAA and 210 or 280 g CP/kg compared with kittens fed 1.5X-3.0X EAAreq plus DAA. These results indicate that the nitrogen requirement is higher when only EAA are the source of nitrogen. Apparently the conversion of nitrogen from EAA to DAA is inefficient in the liver as a result of the lack of down-regulation of the urea cycle enzymes, resulting in a high rate of amino acid catabolism and high obligatory nitrogen loss as urea. When the diet contains DAA, some of the amino acids bypass the liver, thus initially escaping catabolism and increasing efficiency of utilization for nonhepatic protein synthesis and therefore growth.

When the results of all six experiments are plotted in a three-dimensional plot, weight gain vs. E:T ratio vs. crude protein (Fig. 1), it is apparent that there is a broad plateau in which weight gains are maximal at wide dietary E:T ratios and wide dietary crude protein levels. The broad plateau is in contrast to narrow peaks observed when similar plots are made from results with chicks (Stucki and Harper 1961) or rats (Stucki and Harper 1962). The difference could be the result of a species difference; however, we suggest that both rats and chicks would be more similar to cats if adverse effects of excesses of certain amino acids were avoided. From Figure 1, it can also be seen that at an E:T ratio of 1.0 (limiting methionine and arginine to not more than 2.5X and 3.0X each requirement, respectively), a higher CP level in the diet is required for kittens to reach maximal weight gains compared with that at lower E:T ratios (0.3-0.7). As expected, weight gains were submaximal at low CP levels and low E:T ratios because at low ratios, the EAA requirements and crude protein requirements were not met. However, at high levels of crude protein, when the EAA requirements and crude protein requirements were both met or exceeded, the weight gains were not maximal, apparently because of one of the following three factors: 1) arginine deficiency resulting in hyperammonemia (documented by high urinary orotic acid); 2) glutamate intolerance (documented by high plasma glutamate); and 3) the possibility of proline intolerance (documented by high plasma proline). Further work is required to establish whether maximal growth can be achieved at very low E:T ratios in kittens fed high CP levels when glutamate and proline concentrations in plasma do not exceed the levels found to depress growth in kittens fed an E:T ratio of 0.5 at a CP level of 300 g/kg.

We conclude the following: 1) the arginine requirement of kittens increases with increasing dietary crude protein; 2) glutamate intolerance increases as dietary dispensable amino acids increase; 3) proline intolerance must be examined in kittens; and 4) when amino acids (some essential and some dispensable) that cause growth depression when fed in excess are limited in the diet of kittens, optimal weight gains can be achieved at broad E:T ratios and a wide range of dietary crude protein levels.

	FOOTNOTES

	LITERATURE CITED

Introduction References

• Bedford M. R., Summers J. D. Influence of the ratio of essential to non-essential amino acids on performance and carcass composition of the broiler chick. Br. Poultry Sci. 1985; 26:483-491[Medline]
• Bedford M. R., Summers J. D. The effect of the essential to nonessential amino acid ratio on turkey performance and carcass composition. Can. J. Anim. Sci. 1988; 68:899-906
• Costello M. J., Morris J. G., Rogers Q. R. Effect of dietary arginine level on urinary orotate and citrate excretion in growing kittens. J. Nutr. 1980; 110:1204-1208
• Deady J. E., Anderson B., O'Donnell J. A. III, Morris J. G., Rogers Q. R. Effects of level of dietary glutamic acid and thiamin on food intake, weight gain, plasma amino acids, and thiamin status of growing kittens. J. Nutr. 1981; 111:1568-1579
• Harper A. E. "Nonessential" amino acids. J. Nutr. 1974; 104:965-967
• Heger, J. (1990) Non-essential nitrogen and protein utilization in the growing rat. Br. J. Nutr. 64:653-661.
• Morris J. G., Rogers Q. R. Arginine: an essential amino acid for the cat. J. Nutr. 1978; 108:1944-1953
• National Research Council (1985) Guide for the Care and Use of Laboratory Animals. Publication no. 85-23 (rev.), National Institutes of Health, Bethesda, MD.
• National Research Council (1986) Nutrient Requirement of Domestic Animals. Nutrient Requirements of Cats, rev. ed. National Academy Press, Washington, DC.
• Rogers Q. R., Phang J. M. Deficiency of pyrroline-5-carboxylate synthase in the intestinal mucosa of the cat. J. Nutr. 1985; 115:146-150
• Stucki W. P., Harper, A..E. Importance of dispensable amino acids for normal growth of chicks. J. Nutr. 1961; 74:377-383
• Stucki W. P., Harper A. E. Effects of altering the ratio of indispensable to dispensable amino acids in diets for rats. J. Nutr. 1962; 78:115-119
• Taylor T. P., Morris J. G., Kass P. H., Rogers Q. R. Increasing dispensable amino acid in diets of kittens fed essential amino acids at or below their requirement increases the requirement for arginine. Amino Acids 1997; 13:257-272
• Taylor T. P., Morris J. G., Willits N. H., Rogers Q. R. Optimizing the pattern of essential amino acids as the sole source of dietary nitrogen supports near-maximal growth in kittens J. Nutr. 1996; 126:2243-2252
• Wang T. C., Fuller M. F. The optimum dietary amino acid pattern for growing pigs. 1. Experiments by amino acid deletion. Br. J. Nutr. 1989; 62:77-89[Medline]
• Zicker, S. C. & Rogers Q. R. (1990) Use of plasma amino acid concentrations in the diagnosis of nutritional and metabolic diseases in veterinary medicine. In: Proceedings of IVth Congress of International Society for Animal Clinical Biochemistry (Kaneko, J. J., ed.), pp. 107-121.

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