The Effect of Excess Dietary Protein on the Need for Folic Acid by the Chick

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1 6 R. D. CREEK AND V. VASAITIS dence. Succinic acid did not influence perosis incidence caused by manganese deficiency. ACKNOWLEDGMENTS This project was supported in part by grant No. A-08 from the National Institute of Arthritis and Metabolic Diseases. The authors are also indebted to Merck & Co., Rahway, New Jersey, for the crystalline vitamins; to Abbot Laboratories of Chicago, Illinois, for the menadione sodium bisulfite; to Monsanto Chemical Co., St. Louis, Missouri, for the Santoquin; and to the Dawes Laboratories, Chicago, Illinois, for the vitamin D. REFERENCES Jukes, T. H., and A. D. Welch, 9. The effect of certain analogs of choline on perosis. J. Biol. Chem. 6: 9-. Leach, R. M., Jr., and A.-M. Muenster, 96. Studies on the role of manganese in bone formation. I. Effect upon the mucopolysaccharide content of chick bone. J. Nutrition, 78: THE phrase, "protein poisoning," exists as a term of the layman, and actually many scientific reports verify that excess protein is detrimental. Patterson (97) concluded that excessive protein and a lack of green feed caused bumble foot in laying hens. In subsequent work (98), he stated that excessive protein caused gout because the birds were Scientific Article No. A0, Contribution No. 0 of the Maryland Agr. Expt. Sta. (Dept. of Poultry Science). Leach, R. M., Jr., L. C. Norris and M. L. Scott, 96. The effect of diethylstilbestrol on choline deficiency in the chick. Poultry Sci. : Pollard, W. O., M. Shorb and R. D. Creek, 96. Alkaline phosphatase, feathering and bone ash in chicks as affected by hemin. Proc. Soc. Exp. Biol. Med. : Snedecor, G. W., 96. Statistical Methods. The Iowa State College Press, Ames, Iowa. White, A., P. Handler, E. Smith and DeWitt Stetten, Jr., 959. Principles of Biochemistry. McGraw-Hill Book Co., Inc. New York. Wiese, A. C, C. A. Elvehjem, E. B. Hart and J. G. Halpin, 99. A study of blood and bone phosphatase in chick perosis. J. Biol. Chem. 7: -0. Wolbach, S. B., and D. M. Hegsted, 95. Perosis. A.M.A. Arch. Path. 56: 7-. Young, R. J., H. M. Edwards and M. B. Gillis, 958. Studies on zinc in poultry nutrition.. Zinc requirement and deficiency symptoms of chicks. Poultry Sci. 7: -07. Young, R. J., L. C. Norris and G. F. Heuser, 955. The chick's requirement for folic acid in the utilization of choline and its precursors, betaine and methyl-amino-ethanol. J. Nutrition, 55: 5-6. The Effect of Excess Dietary Protein on the Need for Folic Acid by the Chick R. D. CREEK AND VALERIA VASAITIS Poultry Science Department, University of Maryland, College Park, Maryland (Received for publication March, 96) unable to eliminate waste nitrogen, which then accumulated as urate deposits throughout the body. Increased hock fluid was also observed. March and Biely (956) noted that high fat-high protein diets had increased need for folic acid. Stoewsand and Scott (96) have recently concluded that excess protein increases the need for dietary A, and also raises the uric acid level of the blood; however, the blood uric acid level was not affected by the dietary vitamin A.

2 FOLIC ACID AND PROTEIN LEVELS 7 Several supplemental nitrogen sources aggravate the incidence of perosis in the chick or poult. Among these are gelatin (Jukes, 9a, b; McGinnis et al, 9; McGinnis, 96; and Lucas et al., 96); casein in excess or creatine also provoke perosis (Jukes, 9b). Evans et al. (9) concluded that higher choline levels counteracted the detrimental effects of excessive protein in the diet of the poult. Jukes (9b) also reported similar results with choline. More recently, Fisher et al. (95) noted that supplementation of a hydrolyzed casein diet with free amino acids brought on perosis which could be overcome by increasing the dietary niacin. The work cited indicates that the harmful effect of excess nitrogen compounds is actually vitamin deficiency brought on by their increased need for protein catabolism. Most of this work was carried out before the discovery of Daniel et al. (96) that folic acid is necessary for prevention of perosis, and that it is intimately associated with the formation of uric acid, (see Cantarow and Schepartz, 97). In addition, Young et al. (955) have shown an interrelationship between folic acid and cholinenamely, that choline had a sparing effect on the folic acid requirement; thus, it would seem reasonable Protein as % of adequacy Cerelose Casein, vit. free Gelatin Corn oil Mineral mix Vit. mix DL-methionine Arginine hydrochloride Glycine to assume that some of the diets utilized in the gelatin studies were borderline in folic acid, particularly those low in choline. In view of these findings, it was decided to test the effect of increased protein levels on the chicks' need for folic acid. EXPERIMENTAL Arbor Acre male chicks were used in these experiments. They were randomized into battery brooders at one day of age, and feed and tap water supplied ad libitum. The basal diets are given in Table. Choline and vitamin B influence the chick's need for folic acid (Schaefer et al., 950; Young et al., 955). The levels of choline and B used in these experiments were believed adequate so that they would not confound the deficiency symptoms of folic acid. When the protein level was raised, all sources of protein were increased in the same proportion so that any given amino acid would remain constant when expressed as a percent of protein. This was done so that amino acid balance would be identical in all diets. The methods for collection and handling of feces and nitrogen, chromic oxide, and uric acid determination have been TABLE.-Basal diets used to measure the effect of protein level on the folic acid requirement of the chick % 5,70,000,000 Diet Type 5%,7,, %,950,000, % 6,068,00 Diety Typ >e %,85,0 8 The mineral mix is identical to that found in footnote, Table, Poultry Sci. 0: 8. The vitamin mix is identical to that found in footnote, Table, Poultry Sci. 0: 8 except for folic acid which was used as a variable in these experiments.

3 8 R. D. CREEK AND V. VASAITIS TABLE.The effect of varying dietary protein and folic acid levels on body weight, perosis index and mortality -*' Body wt. {gm.) Trials Folic acid (mcg./loo gm.) IS IS IS 5 5 ISO Perosis Index Mortality / / / /6 0/6 0/6 0/6 0/6 / 0/ Protein as % of re :qmrement / 0/ / / / / 6/6 5/6 /6 6/6 /6 0/ 0/ Trial was concluded at 8 days. Trials,, and were concluded when the chicks were 0 days of age. Two replicas of birds for each treatment were used in trials and, and two of birds for each in trials and. The perosis index is a weighted mean where the following values are used: completely normal in both legs, slight evidence of enlargement, bowing or slipped tendon in one or both legs, definite evidence of enlargement, bowing or slipped tendon in one or both legs, severe enlargement, bowing, or slipped tendon in one or both legs. previously described (Creek and Vasaitis, 96). At the end of each experiment, the chicks were individually weighed and rated as to hock condition. Analyses of variance for experiments were computed as per Snedecor (96). Folic acid was dissolved in 70% acetic acid and added to the diets in solution. RESULTS Trial. This trial was intended to determine if a relationship existed between the folic acid requirement and the protein level of the diet. The results, (Tables and ), indicate that a high protein level increases the need for folic acid, and amplifies the severity of perosis. The folicprotein interaction was highly significant statistically. Trial. In the previous trial, chicks fed the minimal level of folic acid (no addition to the basal) showed very poor growth and high mortality (Table ). For this experiment it was decided to use as a minimal level, 5 added mcg./loo gm. of diet and to increase the maximum to. These results (Tables and ) again show clearly the folic-protein interaction. Trial. This experiment was similar to trial, but the intermediate levels of folic acid and protein were omitted. The statistical analyses show highly significant differences becauses of protein and folic acid levels, but no significant interaction of the two components. Trial. The growth inhibition caused Trial TABLE.Statistical analyses of weight data given in Table Folic level Protein level Folic X Protein Significant at the % probability level. Non-significant.

4 FOLIC ACID AND PROTEIN LEVELS 9 by high protein levels in previous trials had not been fully reversed by folic acid. For this reason the maximum level of folacin in this experiment was increased to mcg./loo gm., or 5 times the requirement determined by Young et al. (955). In addition nitrogen loss and uric acid output were measured using chromic oxide balance technique. Statistical analysis of the weight data shows a significant interaction of folic acid and protein levels. The growth depression from the high protein was counteracted by the increased level of folic acid. Folic acid deficiency accentuated the excretion of nitrogen and uric acid on both protein levels. Undoubtedly, this is an indirect effect because of the poorer growth. It is interesting to note that uric acid output relative to folic acid intake can be roughly doubled on a molecular basis, even in extreme folic acid deficiency (Table ). Trial 5. In the previous trials all the diets had been similar and of relatively high glycine content. It has been shown that glycine toxicity may be counteracted by folic acid (Naber et al., 95). In order to eliminate the possibility that the effects noted in previous trials were specific for glycine, and not protein, a diet relatively low in glycine was formulated and is designated as type in Table. The results are found in Table 5 and Dietary components Protein as % Folic acid, of requirement mcg./loo gm. 5 5 ISO TABLE 5.The effect of varying folic acid and protein levels on growth of chicks fed a diet relatively low in glycine '* Folic acid (mcg./loo gm.) 5 5 Protein as % of requirement 7 Perosis Index?..08 ISO Individual weights were made when the birds were 0 days of age. Statistical analyses reveal the folic and protein levels and folic X protein to be significant at the % level. See Table for a description of the perosis index. again show a folic X protein interaction, with consistent effects on perosis. DISCUSSION The results of the first four trials indicate that excessive protein depresses growth and elevates the need for folic acid. Trial 5 demonstrates that this is a general protein effect rather than glycine toxicity. Trial indicates that the chick can tolerate large quantities of protein providing adequate vitamins are present. The elevated levels of uric acid output on the high protein diets demonstrate that the protein is absorbed and metabolized, probably for energy. The relative output of uric acid to ingestion of folic acid when expressed on a TABLE.The effect of varying dietary protein and folic acid levels in trial on feed efficiency and excretion of nitrogen and uric acid ' Feed/ Weight % dietary N excreted %of dietary N excreted as uric acid Mg. of uric acid excreted/ meg. of folic acid ingested Moles of uric acid excreted/ mole of folic acid ingested 90,98 75,,8,596 6,786 Molecular weights for uric and folic acid are 68. and., respectively. Chromic oxide was substituted at the rate of 0.% for cerelose in the diets in this experiment. Derived from balance studies using chromic oxide as an indicator. Feces were collected over a hour period on the 7th day.

5 0 R. D. CREEK AND V. VASAITIS molar basis, (Table ), is indeed remarkable in view of the fact folacin is a cofactor contributing the carbon atoms in the and 8 position of the uric acid molecule, and also that it has functions other than the direct synthesis of uric acid. The chicks' ability to increase uric acid output on the folacin-deficient, high protein diet indicates a preferential use of the vitamin for this function. From an evolutionary viewpoint, this would be expected for life is preserved even at the expense of crippling and growth. The accentuation of the perosis problem by high protein diets is obvious, but the pattern is not as well denned as with growth. This is not surprising. The rating of perosis is an arbitrary method at best. In addition, mortality may reduce the average severity of a group as many of the most crippled birds diet. Body weight may also influence the incidence and severity of perosis (Creek et al., 960). In the experiments discussed here, severely stunted birds on many occasions showed relatively normal hocks, primarily because there was not enough weight to produce twisting and bending. Young et al. (955) indicated that the folic acid requirement of the chick, was approximately mcg./loo gm. of diet. The results with the normal protein diets in trials and confirm these findings. Also the relationship of folic acid to protein as noted by March and Biely (956) is confirmed. SUMMARY High levels of dietary protein tend to increase the need for folic acid and aggravate the incidence of perosis in the chick. The chick can metabolize high levels of protein provided adequate vitamins are present. Uric acid output is maintained on folic acid deficient dietsthus, indicating folic acid is preferentially used for this process at the expense of normal growth. ACKNOWLEDGMENTS This project was supported in part by grant No. A-08 from the National Institute of Arthritis and Metabolic Diseases. The authors are also indebted to Merck & Co., Rahway, New Jersey, for the crystalline Bvitamins; to Abbot Laboratories, Chicago, Illinois, for the menadione sodium bisulfite; to the Dawes Laboratories, Chicago, Illinois, for the vitamin D ; to Chas. Pfizer & Co., Terre Haute, Indiana, for the vitamin A and oleandomycin phosphate; to Distillation Products, Rochester, New York, for the d- alpha tocopherol acetate; to Dow Chemical Co., Midland, Michigan, for the DLmethionine; to Monsanto Chemical Co., St. Louis, Missouri, for the Santoquin; and to Commercial Solvents Corporation, Terre Haute, Indiana, for the choline chloride. REFERENCES Cantarow, A., and B. Schepartz, 957. Biochemistry, W. B. Saunders Company, Philadelphia, Pennsylvania. Creek, R. D., H. E. Parker, S. M. Hauge, F. N. Andrews and C. W. Carrick, 960. The influence of body weight on the experimental production of perosis by manganese deficiency. Poultry Sci. 9: Creek, R. D. and V. Vasaitis, 96. Uric acid excretion in the chick as related to the intake of its precursors and nitrogen. Poultry Sci. 0: Daniel, L. J., F. Farmer and L. C. Norris, 96. Folic acid and perosis. J. Biol. Chem. 6: Evans, R. J., M. Rhian and C. J. Draper, 9. Perosis in turkey poults and the choline content of their diets. Poultry Sci. : Fisher, H., H. M. Scott and B. C. Johnson, 95. Quantitative aspects of the niacin-tryptophan relationship in the chick. Poultry Sci. : 055. Jukes, T. H., 9a. The influence of certain organic compounds on perosis. J. Nutrition, : Supplement p.. Jukes, T. H., 9b. The effect of certain organic

6 FOLIC ACID AND PROTEIN LEVELS compounds and other dietary supplements on perosis, J. Nutrition, : 5-5. Lucas, H. L., L. C. Norris and G. F. Heuser, 96. The effect of gelatin on the antiperotic properties of choline, betaine, and yeast. Poultry Sci. 5: 9-9. March, B., and J. Biely, 956. Folic acid supplementation of high protein-high fat diets. Poultry Sci. 5: McGinnis, J., L. C. Norris and G. F. Heuser, 9. Factors other than manganese required to prevent perosis in chicks. Poultry Sci. : 7. McGinnis, J., 96. The influence of gelatin on prevention of perosis in turkeys fed choline and betaine. Poultry Sci. 5:9-9. Naber, E. C, E. E. Snell and W. W. Cravens, 95. The effect of folic acid on glycine toxicity in the chick. Arch. Biochem. Biophys. 7: THE normal chicken has relatively few microorganisms in its air sacs, according to the work of Smibert et al. (958). Recent work of Tarver and May (96) supports these findings. A majority of commercially slaughtered poultry is generally submerged in heated water to facilitate the removal of feathers, thus certain processing techniques may contribute to an increased bacterial count of remote Journal Paper No. 75, College Experiment Station, College of Agriculture Experiment Stations, University of Georgia, Athens, Georgia. This paper is part of a thesis by the senior author in partial fulfillment for the Ph.D. Current address: Poultry Department, University of Florida, Gainesville, Florida. Patterson, F. D., 97. Nutritional bumblefoot. Vet. Med. : Patterson, F. D., 98. Gout in poultry. Vet. Med. :7-7. Schaefer, A. E., W. D. Salmon, D. R. Strength and D. H. Copeland, 950. Interrelationship of folacin, vitamin B, and choline. J. Nutrition, 0: 95-. Snedecor, G. W., 96. Statistical Methods. The Iowa State College Press, Ames, Iowa. Stoewsand, G. S., and M. L. Scott, 96. Effect of protein on utilization of vitamin A in the chick. Proc. Soc. Exp. Biol. Med. 06: Young, R. J., L. C. Norris and G. F. Heuser, 955. The chick's requirement for folic acid in the utilization of choline and its precursors, betaine and methylaminoethanol. J. Nutrition, 55: 5-6. Effect of Slaughter Technique, Immersion Scald, and Refrigerated Storage on Bacterial Counts of Poultry Air Sacs FRED R. TARVER, JR., AND K. N. MAY Food Technology and Poultry Departments, University of Georgia, Athens, Georgia (Received for publication March, 96) air sac areas of chickens as a result of the immersion scald. Even though the water for scalding may be relatively free of microorganisms prior to its use, the bacterial count will increase with each scalded bird, (Tarver and May, 96). A source of microorganisms is then available to permeate the innermost regions of the respiratory system. According to Thomson and Kotula (959) the air sacs of chicken had lower bacterial counts after scalding when slaughtered by the Kosher method than by the regular or outside cut method. This study was designed to determine the influence of slaughter techniques, immersion scald and refrigerated storage upon the microbial concentration in the