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Choline Although choline is not a true vitamin in the classical sense, it is an important nutrient as a source of labile methyl groups. It is synthesized by many animal spe- cies. Certain physiological states or clinical disorders may result in relative deficiencies and subsequent needs for choline dietary supplements, however. Choline was originally isolated from hog bile. NUTRITIONAL ROLE Dietary Requirements of Various Species Dietary requirements for choline have been estab- lished for the young of several species including the chicken, pig, rat, and dog (see the appendix table). Most animals can produce all of the choline they need by he- patic synthesis. This synthesis can be insufficient for the needs of rapidly growing poultry or for the young of other species when fed diets deficient in methyl groups, however. In these cases, dietary supplements of choline are required to alleviate growth depression and/or he- patic steatitis. Choline-deficient chicks and poults also show a condition called chondrodystrophy (perosis). Chondrodystrophy is characterized by hemorrhaging and flattening of the tibiometatarsal joint, rotation of the metatarsus, and, ultimately, displacement of the Achil- les tendon from its chondyles, resulting in crippling (Na- tional Research Council, 1984~. The chick's choline requirement is substantial (aproximately 1,300 mg/kg of diet) until about 13 weeks of age, after which time endogenous synthesis can apparently satisfy physiolog- ical demands. Berry et al. (1943), Marvel et al. (1943), and Mishler et al. (1946) found that the apparent choline requirements of chicks are increased by including soybean meal in the diet. Soybeans contain significant amounts (in excess of 2,500 mg/kg) of choline. Therefore, it has been sug- gested that the biological availability of choline in this feedstuff may be relatively poor. Molitoris and Baker (1976) reported that choline in soybean meal is only 60 to 75 percent available for supporting chick growth. The chick growth data of Fritz et al. (1967) presents a some- what higher estimate of 85 to 89 percent. Biochemical Functions Choline has three important functions in metabolism (Chan, 1984~. As the acetyl ester, acetylcholine, it serves as a neurotransmitter. It is also metabolized to phos- phatidyl choline (lecithin). In this form, choline has structural functions in biological membranes and in tis- sue lipid utilization. Choline is also oxidized to betaine, serving as a source of labile methyl groups for the for- mation of methionine from homocysteine and of cre- atine from guanidoacetic acid. FORMS OF THE VITAMIN Choline occurs in biological tissues in the free form (trimethylethanolamine) and as a component of lecithin, acetylcholine, and other phospholipids (see Figure 18~. The form most commonly used for supplementation of diets is choline hydrochloride, which may be in liquid, deliquescent, or solid form. Various investigators have also used choline dihydrogen citrate and cytidine di- phosphate choline (CDP-choline). ABSORPTION AND METABOLISM Little is known of the absorption of choline. Because intestinal microflora break down choline in the large 77

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78 Vitamin Tolerance of Animals CH3 CH3 N+-CH2-CH2 OH CH3 Choline (,B-hydroxyethyltrimethyl- ammonium hydroxide) -OH FIGURE 18 Chemical structure of choline. intestine to trimethylamine, absorption can be assumed to take place in the small intestine. All species are capable of synthesizing choline in the liver by the methylation of ethanolamine, which uses methyl groups from S-adenosyl methionine. The process occurs in two steps, each involving a different methyl transferase. Limitations in the availability of methyl groups, therefore, can reduce endogenous cho- line production. Methionine has a choline-sparing ef- fect. For swine, it has been estimated that when methionine is fed at levels in excess of that required for normal rates of protein synthesis, 4.3 mg of methionine was equal to 1 mg of choline in providing methylating capacity (National Research Council, 1979~. According to Mookerjea (1971), the hepatic accumu- lation of lipids in both choline- and methyl group- deficient rats is due to decreased formation of low-density lipoproteins, which results from inadequate amounts of lecithin. Choline is important in the transfor- mation of the immunoreactive cells. Nauss and New- berne (1980) reported that the thymus gland was hypocellular in rats born from choline-deficient parents. This symptom suggests that defective cellular prolifer- ation is due to impaired DNA synthesis. Beisel (1982) reported impaired chemotaxis in macrophages and de- pressed T-lymphocyte response. Plasma choline may be used as a criterion of ade- quacy. Growth rate, satisfactory reproduction, and lipid levels of livers and kidneys also have been used. HYPERVITAMINOSIS Neumann et al. (1949), Kroenig and Pond (1967), and Dobson (1971) have reported no adverse effects of cho- line supplementation in swine. In fact, the growth rate improved (see Table 17~. Studies with poultry indicated similar tolerance to high levels of choline. Ketola and Nesheim (1974) pro- vided levels of 500 to 2,500 mg of choline (as choline dihydrogen citrate)/kg of purified diet to day-old leghorn-type of chickens. They noted no adverse effects on growth over a 21-day period. In fact, they noted that increasing the level of the vitamin above 500 mg/kg of diet reduced chondrodystrophy to zero. Some leghorn- type of laying hens at 30 weeks of age and 80 percent of egg production were fed purified diets containing either no choline chloride or 1,400 mg/kg. Ketola and Nesheim noted no adverse effects on egg production during a 12- week period. Supplementation of the diet with choline chloride led to significant increases in both BW gain and feed intake. Jukes (1941) fed diets containing either no choline or 2,000 mg/kg (presumed to be the chloride form) to turkey poults from hatching. [ukes reported an improvement in growth and a reduction in chondrodys- trophy. Crawford et al. (1969) and March and MacMil- lan (1979) reported no deleterious effects in laying hens fed diets containing up to 5, 730 mg choline/kg (added as choline HCl). Other studies have revealed adverse effects of high levels of choline. Saville et al. (1967) fed day-old broilers graded levels of choline chloride from 400 to 2,200 ma/ kg of diet. They noted hyperexcitability and muscular incoordination after 7 weeks in those animals fed the 2,200 mg/kg. Similar signs developed later in the other treatment groups. Growth after 6 weeks of age was de- pressed in the groups receiving 1,320, 1,760, and 2,200 mg/kg. The problem was overcome by withdrawal of the choline chloride or by provision of additional pyri- doxine. Deeb and Thornton (1959) fed semipurified di- ets containing supplementary choline chloride at levels up to 8,800 mg/kg to day-old broiler chickens to 4 weeks of age. Growth rate was maximized with 880 to 1,760 mg choline chloride/kg of diet. They reported a depres- sion in BW and feed efficiency with a dietary level ex- ceeding 2,200 mg/kg. This level was slightly above the one Jukes (1941) found to be well tolerated by broilers. Davis (1944a) showed that daily oral administration of 5 g of soybean lecithin (equivalent to 150 mg of choline) to dogs resulted in a maximum number of erythrocyte reduction that took place after 12-25 days. This condi- tion persisted for at least 10 days after the cessation of lecithin feeding. Similarly, daily doses of 8 mg of choline hydrochloride/kg of BW also significantly reduced red blood cell numbers. In this case, more than 10 days were required for maximum red blood cell number depres- sion to occur. Davis (1944a) suggested that choline ad- ministration depressed erythropoeisis by increasing the oxygen supply to the bone marrow. Davis (1944b) found that choline chloride induced a hyperchromic anemia in about 15 dogs. The anemia was produced by giving the dogs single doses of 10 mglkgl day of choline chloride by stomach tube. Once the ane- mia was established, the same dose was continued twice daily. When the erythrocyte numbers were further re- duced, a third daily dose was added. One dog was placed

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t32 Vitamin Tolerance of Animals on an accelerated program. It was dosed twice daily 7 days after the start, and then 3 times daily on the twenty- sixth day. Anemia was established in that animal Bore rapidly. Two other dogs were given doses of 10 mg/kg 3 times daily from the beginning of the experiment. As a result of 3 daily doses of choline hydrochloride, five dogs showed 30 to 43 percent reductions in red blood cell counts. Two other dogs exhibited milder anemias. Hodge (1945) added choline chloride to the diet and drinking water of rats. He found that levels of up to 1 percent of choline chloride in the diet produced no evi- dence of toxicity. Growth was depressed at higher lev- els, however. When choline chloride was given in the drinking water, growth depression was observed at the 1 percent level. Levels greater than 3 percent were not well tolerated. In general, the effects of choline chloride on the histopathology of the major organs were nega- tive. Choline has been administered as a precursor of ace- tylcholine to humans suffering from various nervous and mental diseases. Hollister et al. (1978) reported that peak plasma concentration was obtained with repeated oral doses of 16 to 20 g/d, with a rapid disappearance following treatment cessation. Some clinical improve- ment and no deleterious effects were noted in those patients. PRESUMED UPPER SAFE LEVELS Insufficient data are available to support precise esti- mates of maximum tolerable dietary levels of choline, although published information suggests that the toler- ance for choline is high in most species. For instance, McKibbin et al. (1944) supplemented the diet of grow- ing pups with up to 1,500 mg of choline chloride/kg with no reported problems. McKibbin et al. (1945) used sup- plements of up to 2,000 mg/kg of diet successfully with growing pups. Hodge (1944) found that the acute intraperitoneal LD50 values of choline chloride for rats ranged from 35 to 74 mg/100 g of BW. Lethality varied according to the concentration of the dosing solution. Hodge also noted that all deaths took place within 20 minutes after the injections. Hodge and Goldstein (1942) determined the LD50 values of choline chloride for both mice and rats. In mice injected intraperitoneally with an aqueous solution of 2 percent choline chloride, the LD50 was 320 mg/kg of BW. In rats given a 67 percent solution of the compound in the same manner, the LD50 was 6.7 g/kg of BW. Neuman and Hodge (1945) administered choline chlo- ride in doses of four concentrations (200, 400, 500, and 670 mg/ml) to rats by stomach tube. The total lethality of the two higher concentrations was significantly greater than that of the two lower concentrations. The pooled LD50 was 3.4 g/kg of BW for the higher concen- tration groups and 6.1 g/kg of BW for the lower concen- tration groups. Studies with mice suggest that choline chloride is rela- tively innocuous when administered orally. According to Agut et al. (1983), the acute oral LD50 value of choline chloride is 3,900 mg/kg of BW. The acute intravenous LD50 is 53 mg/kg of BW. They estimated the oral and intravenous maximum tolerable levels to be 2,000 and 21.5 mg/kg of BW, respectively. The CDP form of cho- line was less toxic. The LD50 values were indeterminate for the oral route and 4,600 mg/kg of BW for the intrave- nous route. The oral and intravenous maximum tolera- ble levels were 14,000 mg/kg of BW and 3,500 mg/kg of BW, respectively. Wecker and Schmidt (1979) have shown that the CDP form is almost completely ab- sorbed. Their work supports other data suggesting that the small amount of chloride in choline chloride may contribute to the toxicity of this compound. SUMMARY 1. Although choline is not a vitamin in the strictest sense, it is an important nutrient as a source of labile methyl groups. Dietary requirements have been estab- lished for young chickens, swine, rats, and dogs for which endogenous synthesis is insufficient for physio- logical demands or is inadequate in circumstances of dietary deficiencies of methyl groups. 2. Data with pigs indicate a high tolerance for choline. Studies with chickens suggest that a dietary level of about twice the dietary requirement is safe and pro- duces no deleterious effects. Some of the chicken data indicate a growth reduction and- interference with the utilization of pyridoxine when the dietary level of cho- line exceeds twice the required level. 3. Studies with dogs suggest a low tolerance for cho- line chloride and lecithin in that species. Adverse effects have been reported for levels of choline chloride equiva- lent to 3 times the apparent choline requirement. 4. Mouse data suggest that choline chloride is rela- tively innocuous when given orally (the LD50 is 3,900 mg/kg of BOO), but appreciably more toxic when given intravenously (the LD50 is 53 mg/kg of BOO). The CDP form of choline is less toxic to mice by the same parame- ters; the oral LD50 is indeterminate and the intravenous LD50 is 4,600 mg/kg of BW. The maximum tolerable levels of choline chloride and CDP choline appear to be 2,000 and 14,000 mg/kg of BW, respectively, when given orally, and 21.5 and 3,500 mg/kg of BW, respec- tively, when given intraperitoneally. 5. The LD50 of choline chloride administered to rats by

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Choline 83 stomach tube was estimated to be 3.4 to 6.1 g/kg of BW. 6. The fact that choline chloride appears to present some hazard to chickens and dogs when included in the diet at relatively low levels indicates a need for addi tional research on choline with these and other species. REFERENCES Agut, J., E. Font, A. Sacristan, and J. A. Ortiz. 1983. Dissimilar ef- fects in acute toxicity studies of CDP-choline and choline. Arzneim- Forsch/Drug Res. 33:1016. Beisel, W. R.1982. Single nutrients and immunity. Am. J. Clin. Nutr. 35:417. Berry, E. P., C. W. Carrick, R. E. Roberts, and S. M. Hauge. 1943. A deficiency of available choline in soybean oil and soybean oil meal. Poult. Sci.22:442. Chan, M. M. 1984. Choline and carnitine. P. 549 in Handbook of Vitamins, L. J. Machlin, ed. New York: Marcel Dekker. Crawford, J. S., M. Griffith, R. A. Tuckell, and A. B. Watts. 1969. Choline requirement and synthesis in laying hens. Poult. Sci. 48:620. Davis, J. E. 1944a. Depression of the normal erythrocyte number by soybean lecithin or choline. Ant. J. Physiol.142:65. Davis, J. E. 1944b. The experimental production of a hyperchromic anemia in dogs which is responsive to anti-pernicious anemia treat- ment. Am. J. Physiol. 142:402. Deeb, S. S., and P. A. Thornton.1959. The choline requirement of the chick. Poult. Sci. 38:1198. (Abstr.) Dobson, K. J.1971. Failure of choline to prevent splay leg in piglets. Anat. Vet. J.47:587. Fritz, J. C., T. Roberts, and J. W. Boehne.1967. The chick's response to choline and its application to an assay for choline in feedstuffs. Poult. Sci. 46:1447. Hodge, H. C. 1944. Acute toxicity of choline hydrochloride adminis- tered intraperitoneally to rats. Proc. Soc. Exp. Biol. Med. 57:26. Hodge, H. C. 1945. Chronic oral toxicity of choline chloride in rats. Proc. Soc. Exp. Biol. Med. 58:212. Hodge, H. C., and M. R. Goldstein.1942. The acute toxicity of choline hydrochloride in mice and rats. Proc. Soc. Exp. Biol. Med.51:281. Hollister, L. E., D. J. Jenden, J. R. D. Amaral, J. D. Barchas, K. L. Davis, and P. A. Berger. 1978. Plasma concentrations of choline in man following choline chloride. Life Sci.23:17. Jukes, T. H. 1941. Studies of perosis in turkeys. 1. Experiments re- lated to choline. Poult. Sci.20:251. Ketola. H. G.. and M. C. Nesheim. 1974. Influence of dietary choline , and methionine levels on the requirement for choline by chickens. J. Nutr.104:1484. Kroenig, G. H., and W. G. Pond. 1967. Methionine, choline and threonine interrelationships for growth and lipotropic action in the baby pig and rat. J. Anim. Sci.26:352. March, B. E.1981. Choline supplementation of layer diets containing soybean meal or rapeseed meal as protein supplement. Poult. Sci. 60:818. March, B. E., and C. MacMillan.1979. Trimethylamine production in the caeca and small intestine as a cause of fishy taints in eggs. Poult. Sci.58:93. Marvel, J. A., C. W. Carrick, R. E. Roberts, and S. M. Hauge. 1943. The supplementary value of choline and methionine in a corn and soybean oil meal chick ration. Poult. Sci.23:294. McKibbin, J. M., S. Thayer, and F. J. Stare.1944. Choline deficiency studies in dogs. J. Lab. Clin. Med.29:1109. McKibbin, J. M., R. M. Ferry, Jr., S. Thayer, E. G. Patterson, and F. J. Stare. 1945. Further studies on choline deficiency in dogs. J. Lab. Clin. Med.30:422. Mishler, D. H., C. W. Carrick, R. E. Roberts, and S. M. Hauge. 1946. Synthetic and natural vitamin supplements for corn and soybean oil meal chick rations. Poult. Sci.25:479. Molitoris, B. A., and D. H. Baker.1976. Assessment of the quantity of biologically available choline in soybean meal. J. Anim. Sci.42:481. Mookerjea, S.1971. Action of choline in lipoprotein metabolism. Fed. Proc.30:143. National Research Council. 1979. Nutrient Requirements of Swine, 8th rev. ed. Washington, D.C.: National Academy Press. National Research Council. 1984. Nutrient Requirements of Poultry. 8th rev. ed. Washington, D.C.: National Academy Press. Nauss, K. M., and P. M. Newberne. 1980. Effects of dietary folate, vitamin B12 and methionine/choline deficiency on immune func- tion. Adv. Exp. Med. Biol. 135:63. Neuman, M. W., and H. C. Hodge. 1945. Acute toxicity of choline chloride administered orally to rats. Proc. Soc. Exp. Biol. Med. 58:87. Neumann, A. L., J. L. Krider, M. F. James, and B. C. Johnson. 1949. The choline requirement of the baby pig. J. Nutr. 38:195. Saville, D. G., A. Solvyns, and C. Humphries. 1967. Choline-induced pyridoxine deficiency in broiler chickens. Aust. Vet. J. 43:346. Wecker, L., and D. E. Schmidt. 1979. Central cholinergic function: Relationships to choline administration. Life Sci. 25:375.