DSM in Animal Nutrition & Health
Properties and Metabolism
Choline is a beta-hydroxyethyltrimethylammonium hydroxide. Pure choline is a colorless, viscous, strongly alkaline liquid that is notably hygroscopic. Choline is soluble in water, formaldehyde and alcohol, and has no definite melting or boiling point. The chloride salt of this compound, choline chloride, is produced by chemical synthesis for use in the feed industry, although there are other forms. Choline chloride consists of deliquescent white crystals, which are very soluble in water and alcohols. Aqueous solutions are almost pH neutral. Choline is ubiquitously distributed in all plant and animal cells, mostly in the form of the phospholipids phosphatidylcholine (lecithin), lysophosphatidylcholine, choline plasmalogens and sphingomyelin–essential components of all membranes (Zeisel, 1990). Lecithin is the predominant phospholipid (>50%) in most mammalian membranes. In the lung, desaturated lecithin is the major active component of surfactant (Brown, 1964), lack of which results in a respiratory distress syndrome in premature infants. Choline is a precursor for the biosynthesis of the neurotransmitter acetylcholine. Glycerophosphocholine and phosphocholine are storage forms for choline within the cytosol and the principal forms found in milk (Rohlfs et al., 1993). Choline is present in the unsupplemented diet mainly in the form of lecithin, with less than 10% present either as the free base or as sphingomyelin. Choline is released from lecithin and sphingomyelin by digestive enzymes of the gastrointestinal tract, although 50% of ingested lecithin enters the thoracic duct intact (Chan, 1991). Choline is released from lecithin by hydrolysis in the intestinal lumen. Both pancreatic secretions and intestinal mucosal cells contain enzymes capable of hydrolyzing lecithin in the diet. Within the gut mucosal cell, phospholipase A1 cleaves the alpha-fatty acid, and phospholipase B cleaves both fatty acids. Quantitatively, digestion by pancreatic lipase is the most important process (Zeisel, 1990). The net result is that most ingested lecithin is absorbed as lysophosphatidylcholine.
Choline is absorbed in the jejunum and ileum mainly by an energy and sodium dependent carrier mechanism. Only one-third of ingested choline in monogastric diets appears to be absorbed intact. Absorbed choline is transported into the lymphatic circulation primarily in the form of lecithin bound to chylomicra; it is transported to the tissues predominantly as phospholipids associated with the plasma lipoproteins (De La Huerga and Popper, 1952). In ruminants, dietary choline is rapidly and extensively degraded in the rumen from studies with both sheep (Neill et al., 1979) and cattle (Atkins et al., 1988; Sharma and Erdman, 1988). Estimates of rumen degradation have ranged from 85% to 99%. In in vivo studies with dairy cows, in which choline intake was increased up to 303 grams per day over controls, there was only a 1.3 gram per day increase in choline flow to the duodenum (Sharma and Erdman, 1988).
Work with sheep (Neill et al., 1979) and goats (Emmanual and Kennelly, 1984) suggests that ruminants must metabolize and utilize choline in a different manner than monogastric animals. Choline absorption must be very limited in all ruminants because of (1) almost complete degradation of dietary choline in the rumen, (2) only limited supplies from any rumen protozoa that might escape rumen degradation and (3) the complete absence of choline in rumen bacteria.
Choline functions in four broad categories in the animal body (Zeisel, 2006; Garrow, 2007):
(a) It is a metabolic essential for building and maintaining cell structure. As a phospholipid component, choline is a structural part of lecithin (phosphatidylcholine), of certain plasmologens and the sphingomyelins. Lecithin is a part of animal cell membranes and lipid transport moieties in cell plasma membranes. Choline is required as a constituent of the phospholipids needed for normal maturation of the cartilage matrix of the bone. Various metabolic functions and synthesis of choline are depicted in Figure 16-1.
Figure 16-1: Metabolic Pathway for the Synthesis of Choline and Related Compounds
(b) Choline plays an essential role in fat metabolism in the liver. It prevents abnormal accumulation of fat (fatty livers) by promoting its transport as lecithin or by increasing the utilization of fatty acids in the liver itself (Xu et al., 2010). Choline is thus referred to as a “lipotropic” factor due to its function of acting on fat metabolism by hastening removal or decreasing deposition of fat in liver. In broiler liver, fat content was reduced by adding choline at 760 mg per kg (345 mg per lb) of diet for birds fed different energy sources (Rao et al., 2001).
(c) Choline is essential for the formation of acetylcholine, a substance that makes possible the transmission of nerve impulses. Acetylcholine is the agent released at the termination of the parasympathetic nerves. With acetylcholine, there is transmission of nerve impulses from presynaptic to postsynaptic fibers of sympathetic and parasympathetic nervous systems.
(d) Choline is a source of labile methyl groups. Choline furnishes labile methyl groups for formation of methionine from homocystine and of creatine from guanidoacetic acid. Methyl groups function in the synthesis of purine and pyrimidine, which are used in the production of DNA. Methionine is converted to S-adenosylmethionine in a reaction catalyzed by methionine adenosyl transferase. S-adenosylmethionine is the active methylating agent for many enzymatic methylations. A disturbance in folic acid or methionine metabolism results in changes in choline metabolism and vice versa (Zeisel, 1990). The involvement of folic acid, vitamin B12, and methionine in methyl group metabolism, and of methionine in de novo choline synthesis, may allow these substances to substitute in part for choline. A severe folic acid deficiency has been shown to cause secondary liver choline deficiency in rats (Kim et al., 1994).
The demand for choline as a methyl donor is probably the major factor that determines how rapidly a diet deficient in choline will induce pathology. The pathways of choline and 1-carbon metabolism intersect at the formation of methionine from homocysteine. Methionine is regenerated from homocysteine in a reaction catalyzed by betaine: homocysteine methyltransferase, in which betaine, a metabolite of choline, serves as the methyl donor (Finkelstein et al., 1982). Large increases in chick hepatic betaine-homocysteine methyltransferase can be produced under methionine-deficient conditions, especially in the presence of excess choline or betaine (Emmert et al., 1996). To be a source of methyl groups, choline must be converted to betaine, which has been shown to perform methylation functions in some cases. However, betaine fails to prevent fatty livers and hemorrhagic kidneys.
Since choline contains biologically active methyl groups, methionine can partly be spared by choline and homocysteine. Research with lactating dairy cattle suggests that a high proportion of dietary methionine is used for choline synthesis (Erdman and Sharma, 1991). Choline has been shown to influence brain structure and function. For rodents, choline was critical during fetal development, where it influences stem cell proliferation and apoptosis, thereby altering brain structure and function. Memory is permanently enhanced in rodents exposed to choline during the latter part of gestation (Zeisel and Niculescu, 2006).
Choline, unlike most vitamins, can be synthesized by most species, although in many cases not in sufficient amounts or rapidly enough to satisfy all the animal’s needs. The choline requirement for growing poultry of various species ranges from 750 to 2,000 mg per kg (341 to 909 mg per lb) of diet. Generally, adult species of poultry can probably synthesize the vitamin in adequate quantities, with one exception being breeding quail, which require 1,500 mg per kg (682 mg per lb) of diet (NRC, 1994). Dietary factors such as methionine, betaine, myo-inositol, folic acid and vitamin B12 or the combination of different levels and composition of fat, carbohydrate and protein in the diet as well as the age, sex, caloric intake and growth rate of animals, all influence the lipotropic action of choline and the requirement for this nutrient (Mookerjea, 1971). Dietary betaine can spare choline, since choline functions as a methyl donor by forming betaine. In growing chicks, 50% of the dietary choline requirement must be supplied as choline per se, but the remaining 50% could be replaced by betaine (Dilger et al., 2007). It is suggested that the effects of betaine may be to spare methionine by providing labile methyl groups for the synthesis of methylation products, and to reduce abdominal fat by increasing carnitine synthesis and beta-oxidation of fatty acids as well as providing energy (creatine phosphate) for cell metabolism (Kidd et al., 1997; Xu et al., 1998; Dilger et al., 2001).
Studies have shown that vitamin B12 and folic acid reduce the requirement for choline in chicks and rats (Welch and Couch, 1955). Folic acid and vitamin B12 are required for the synthesis of methyl groups and metabolism of the one-carbon unit. Biosynthesis of a labile methyl group from a formate carbon requires folic acid, while B12 plays a role in regulated transfer of the methyl group to tetrahydrofolic acid. Therefore, marked increases in choline requirement have been observed under conditions of folic acid and (or) vitamin B12 deficiency. It is concluded that chicks fed practical ingredient-based diets require 1.3 mg per kg (0.59 mg per lb) of folic acid with low levels of choline, but 1.2 mg per kg (0.54 mg per lb) of folic acid when choline is offered near the NRC recommended level of 1,300 mg per kg (590.9 mg per lb) (Ryu et al., 1995).
The two principal methyl donors functioning in animal metabolism are choline and methionine, which contain “biologically labile methyl groups” that can be transferred within the body. This phenomenon is called transmethylation. Therefore, dietary adequacy of both methionine and choline directly affect requirements of each other. Other than exogenous sources of methyl groups from choline and methionine, methyl group formation from synthesis of formate carbons is reduced with folic acid and (or) vitamin B12 deficiencies.
Most animals can synthesize sufficient choline for their needs provided enough methyl groups are supplied. As an example, methionine in the pig can completely replace that portion of the choline needed for transmethylation. Young poultry, on the contrary, are unable to benefit from methionine or betaine as a dietary replacement for choline unless methylaminoethanol or dimethylaminoethanol is in the diet, as young poultry appear unable to methylate aminoethanol when fed a purified diet (Jukes, 1947; Dilger et al., 2007). Later studies showed that the chick can synthesize microsomal methylaminoethanol and choline from S-adenosylmethionine, but, unlike the pig, at an insufficient rate in relation to needs (Norvell and Nesheim, 1969).
The metabolic needs for choline can be supplied in two ways: either by dietary choline or by choline synthesis in the body that makes use of labile methyl groups. For selected species, body synthesis sometimes cannot take place fast enough to meet choline needs for rapid growth and thus clinical signs of deficiency result. Since choline functions in prevention of fatty livers, hemorrhagic kidneys and perosis, it does not act as a true vitamin since choline is incorporated into phospholipids (via cytidine disphosphocholine). Therefore, unlike a typical B vitamin, the choline molecule becomes an integral part of the structural component of liver, kidney or cartilage cells (Scott et al., 1982).
Excess dietary protein increases the young chick’s choline requirement. Ketola and Nesheim (1974) observed that over three times as much choline was needed for maximum growth of chicks when fed a diet containing 64% protein than when fed 13%. Diets high in fat aggravate choline deficiency and thus increase requirement. Fatty liver is generally enhanced by fats containing a high proportion of long-chain saturated fatty acids (Hartroft et al., 1952). Choline deficiency develops to a greater degree in rapidly growing animals, with deficiency lesions more severe in these animals. Need for supplemental choline is greatest with the starting bird because all facets of use are likely to be maximal. As growth diminishes, the necessity for choline supplementation disappears (NRC, 1994).
Both methionine and choline have been shown to have an immune response in broilers (Swain and Johri, 2000). Chicks on a diet containing 6.5 g per kg (3.0 g per lb) methionine and 1,300 mg per kg (591 mg per lb) choline significantly improved cellular immune response.
All naturally occurring fats contain some choline, and thus, it is supplied by all feeds that contain fat. Egg yolk, glandular meats, and brain are the richest animal sources; germ of cereals, legumes and oilseed meals are the best plant sources (DuCoa L.P., 1994). Corn is low in choline, with wheat, barley and oats containing approximately twice as much choline as corn. Since betaine can spare the requirements of choline, it would be useful to know the concentrations of betaine in feeds. Unfortunately, most feedstuffs contain only small amounts of betaine. However, wheat and wheat by-products apparently contain over twice as much betaine as choline. Thus, the choline needs of swine or poultry fed wheat-based diets would be much lower than those fed diets based on other grains. Sugarbeets are also high in betaine.
Little is known of the biological availability of choline in natural feedstuffs. Using a chick assay method, soybean, canola, and peanut meals were found to obtain a substantial proportion of unavailable choline (Emmert and Baker, 1997). Bioavailability of choline is 100% in corn while in dehulled regular soybean meal and whole soybeans availability ranged from 60% to 75% (Molitoris and Baker, 1976; McDowell and Ward, 2008). However, soybean lecithin products are equivalent to choline chloride in bioavailability (Emmert et al., 1997). Canola meal, although three times as rich in total choline as soybean meal, has less bioavailable choline (Emmert and Baker, 1997). In their work with chicks, production of trimethylamine (resulting from bacterial degradation of choline) in the intestine was greater in chicks fed canola meal than in those fed soybean meal.
Commercially, choline is produced by chemical synthesis, and choline salts are used in dietary supplementation. Choline is available as chloride (86.8%) and bitartrate (48%) salts. Choline chloride is available for feed use as the 70% liquid or 50% to 60% dry dilutions. The 70% liquid is very corrosive and requires special storage and handling equipment. It is not suitable for inclusion in concentrated vitamin premixes, but is most economical to add directly to concentrate feed mixtures.
Choline from dietary sources or supplemental sources, such as choline chloride, would not be of value to ruminants since rumen microorganisms almost completely destroy dietary choline. Choline supplements are only of value if they are resistant to rumen degradation. Recently, a rumen-protected choline product has become available (Erdman and Sharma, 1991).
Growth retardation and perosis result from choline deficiency in young poultry. Perosis is the primary clinical sign of a choline deficiency in chicks and turkey poults, whereas Bobwhite quail develop enlarged hocks and bowed legs (NRC, 1994). Ducks given choline-free diets had reduced growth rates and developed severe perosis (Hsu et al., 1988). Perosis is first characterized by pinpoint hemorrhages about the hock joint, followed by an apparent flattening of the tibiometatarsal joint (Scott et al., 1982). Progressively, the Achilles’ tendon slips from its condyles, thus rendering the bird relatively immobile. Some studies indicated that in prevention of perosis, choline is required for the phospholipids needed for normal maturation of the cartilage matrix of bone. Although perosis commonly refers to many hock abnormalities, true perosis is described as the classic choline deficiency sign. Adult chickens probably synthesize sufficient choline to meet requirements for egg production. Minimal dietary choline does not affect hatchability with either chickens or turkeys, but Japanese quail and their developing embryos readily express general signs of deficiency (Latshaw and Jensen, 1972; NRC, 1994). Supplementary choline may be necessary for maintenance of egg size in quail (NRC, 1994). Contrary to some reports, 500 mg per kg (227 mg per lb) of supplemental choline to Leghorn hens increased egg weight while reducing specific gravity (Tapia Romero et al., 1985). Also, the choline growth requirement for quail is apparently higher than for chicks or poults. The choline requirement of growing chicks decreases with age as it is generally not possible to produce a deficiency at an age over eight weeks. It was observed that methylation of aminoethanol to methylaminoethanol seems to be the rate-limiting step in choline biosynthesis for young birds. High levels of dietary methionine or other methyl donors, therefore, cannot completely spare the chick’s requirement of dietary choline, which is in contrast to the situation with growing mammals such as the pig or the rat.
Apparently, choline requirement of laying hens can be influenced by choline level in the diet of the growing pullet (Scott et al., 1982). Hens that received choline-free diets after eight weeks of age were able to synthesize all of the choline required for good egg production. Those that had received choline supplements in the growing diet required supplemental choline in the laying diet for maximum egg production. The deficiency signs noted in these hens were a reduction in egg production and an increase in fat content of the liver. Even with choline deficiency, however, choline content of the egg was not affected by low dietary choline.
Despite lack of evidence that laying chickens require a dietary source of choline for maximum egg production, addition of choline to practical diets markedly reduces the amount of fat in the liver (NRC, 1994). However, a number of reports with chicks and turkey poults did not find fatty livers with deficiency (Ruiz et al., 1983). A choline response in laying chickens is likely to occur only if inadequate daily sulfur amino acids are provided.
Addition of 0.1% of supplemental methionine resulted in no response in laying hens to supplemental choline (Crawfordet al., 1969). It appears that benefits from supplemental choline in layer diets occur mainly when supplemental methionine is just adequate to meet methionine requirements. Miles et al. (1983) demonstrated that the addition of 0.11% choline plus 0.1% sulfate could essentially spare all supplemental methionine in broiler diets. However, in turkey poult diets (Harms and Miles, 1984), responses to sulfate and choline addition were not equivalent to the addition of supplemental methionine. Pesti et al. (1980), using young chicks, found that supplementation with methyl donors from either 0.23% choline or 0.23% betaine was equivalent to supplementation with 0.23% methionine in 21-day chick experiments, using basal diets containing 0.31% methionine and 0.43% cystine. Spires et al. (1982) found that supplemental choline could replace up to two-thirds of the supplemental methionine required in broiler diets from 0 to 47 days in diets containing 0.30% methionine and 0.43% cystine in the starter phase, and 0.25% and 0.42% methionine and cystine, respectively, in the finisher phase.
Response to dietary supplementation of choline will be most dependent on species, age of animals, protein and sulfur amino acid intake, dietary choline and other choline sparing nutrients. Unlike most vitamins, choline can be synthesized by various animals, although often in insufficient amounts. It appears that choline deficiency in the young of some species (e.g., chicks) is perhaps not due to the lack of ability to synthesize choline, but more likely to the lack of ability to synthesize it at a rate sufficient for the animal’s needs. Age is an important consideration as, for example, it is difficult to produce a choline deficiency in growing chicks over eight weeks of age. The young of many species (e.g., pig and rat) do not require supplementary choline if dietary methionine level is sufficiently high. On a synthetic milk diet containing 1.6% methionine, young pigs were found not to require supplemental choline (Firth et al., 1953). Neumannet al. (1949) reported that the young pig requires 0.1% dietary choline when methionine is present at 0.8% to 1.0% of the diet. Methionine can furnish methyl groups for choline synthesis for most species. Choline, however, is effective only in sparing methionine which otherwise would be used to make up for the choline shortage. Methionine is not used up for choline synthesis if there is an adequate level of dietary choline. In formulating typical poultry diets, methionine is frequently one of the most limiting amino acids. Therefore, it would be impractical for marginal quantities of methionine to be wasted for synthesis of the vitamin when supplemental choline can be provided more economically. In providing supplements of methionine and (or) choline, a third nutrient, sulfur, must be considered. Significance of a three-way interrelationship among methionine, choline and sulfate has been reviewed (Ruiz et al., 1983; Miles et al., 1986; Miles and Butcher, 1997).
Sulfur is present in a number of body metabolites (e.g., mucopolysaccharides) and if not adequately supplied in the diet, sulfur amino acids would likely be degraded. In feeding broilers, supplemental sulfate accompanied by choline or methionine achieved a greater growth response than when either was fed alone (Miles et al., 1983). Data suggest that sulfate must be present for choline to spare a maximum amount of methionine. The practical implication is that sulfate and choline need to be adequately provided in diets so that the more expensive and often marginally deficient nutrient methionine is not used to provide either of these nutrients.
Schutte et al. (1997) reported an experiment investigating the interrelationship of betaine and methionine for broiler chicks. The addition of methionine significantly increased body weight; the addition of betaine resulted in only a small numerical response when compared with the appropriate level of methionine. It is concluded that in diets deficient in choline, betaine may spare methionine from methyl donor functions and result in an improvement in growth rate. While betaine is an efficient methyl donor, choline is significantly cheaper and would be the methyl donor of choice for use in practical broiler feeds. Neither betaine or choline can produce a significant net increase in available methionine for use in protein synthesis or other metabolic activities, unless quantities of homocystine are present, which is not usually the case.
Betaine may have beneficial effects in alleviating coccidiosis infections in chicks (Augustine et al., 1997; Matthews et al., 1997). Coccidiosis is associated with osmotic and ionic disorders, which are probably caused by dehydration and diarrhea that are characteristic of coccidial infection. Remus and Virtanen (1996) reported that betaine and methionine were equally efficacious in diets for coccidiosis-infected chicks. Zimmerman et al. (1996) reported similar results for chicks fed 0.05 mg per kg (0.023 mg per lb) of betaine, but not in chicks fed 0.1 mg per kg (0.045 mg per lb) of betaine. In both of these studies, all chicks were infected with coccidiosis, and in the report by Zimmerman et al.(1996), all chicks received an anticoccidial medication. Virtanen et al. (1996) reported that betaine in combination with salinomycin was more effective in preventing the signs of coccidiosis (lesion scores and decreased feed efficiency) than salinomycin alone. As in the aforementioned research, all chicks were infected with coccidiosis.
The most widely used supplemental form of choline is choline chloride. Choline is available for feed use as the 70% or 75% liquid or 25% to 60% dry powder. Choline chloride can be available on a cereal carrier; the product is obtained by spraying and thoroughly mixing aqueous choline chloride on a suitable cereal carrier and then drying to a low moisture content. The 70% to 75% liquid is very corrosive and requires special storage and handling equipment. It also is not suitable for inclusion in concentrated vitamin premixes, but rather is most generally added singly to concentrate mixtures.
Currently, a variety of lecithin products that are derived from soybeans are available for use in feeds. The products range from crude fluid lecithin to 50% lecithin co-dried with corn syrup solids to a newly developed dry de-oiled soy lecithin (Meyers, 1990). A number of studies suggest that choline from soybean meal is close to 100% available (Menten et al., 1997). The advantage of a de-oiled soybean lecithin is that it can be handled as a dry feed ingredient. The dry de-oiled lecithin product also possesses those advantageous properties of soybean lecithin in general that impart important dietary formulation features, including vitamin stabilization (thus protecting vitamins from oxidation); improvement of fat and vitamin utilization; and providing a source of choline, inositol, and growth-stimulating compounds.
Choline chloride is stable in multivitamin premixes but is highly destructive to various other vitamins in the premix (Frye, 1978). Choline is stable during processing and storage in pressure-pelleted and extruded feeds. Since the material is hygroscopic, containers of choline should be kept closed when not in use. Some research has shown that supplemental betaine, a product of choline oxidation, is beneficial to swine and poultry production (Lowry et al., 1987; Odle, 1996). Supplemental betaine is available as betaine hydrochloride (98.0% betaine on an anhydrous basis).
Derilo and Balnave (1980) reported a reduced gain and efficiency in young broiler chicks fed a level of choline only slightly in excess of the requirement. Studies with chickens suggest that dietary choline at twice the requirement is safe (NRC, 1987). Some of the chicken data indicates a growth reduction and interference with the utilization of vitamin B6when the dietary level of choline exceeds twice the required level. Other research indicates that birds tolerate high levels of choline because 20,000 to 30,000 mg per kg (9,091 to 13,637 mg per lb) of diet was needed to induce toxicity (Leeson and Summers, 2001). Clinical signs included a reduced red blood cell number.