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DSM in Animal Nutrition & Health

Choline

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 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. The remaining two-thirds of choline is metabolized by intestinal microorganisms to trimethylamine, which is excreted in the urine between 6 and 12 hours after consumption (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 invivo 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.

 

Functions

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 plasmalogens 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 15-1. 

 

Figure 15-1: Metabolic Pathway for the Synthesis of Choline and Related Compounds

16Figure_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. 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.

(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. As 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). To be a source of methyl groups, choline must be converted to betaine, which has been shown to perform methylation functions as well as choline 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; Benoit et al., 2010). The amino acid methionine is the source of the methyl donor S-adenosyl methionine, the metabolite that provides methyl groups in a variety of reactions including the de novo synthesis of choline from phosphatidylethanolamine. When choline is oxidized irreversibly to betaine, betaine can provide methyl groups that recycle homocysteine to methionine. Because of these metabolic relationships, dietary supply of either choline or betaine affects requirements, and methionine supply can affect betaine and choline metabolism.

Choline has been shown to influence brain structure and function. For rodents, choline was critical during fetal development where it influenced 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).

 

Requirements

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. 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 have influence on the lipotropic action of choline and thereby requirement of this nutrient (Mookerjea, 1971). Dietary betaine can spare choline, since choline functions as a methyl donor by forming betaine. 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., 2007). Dietary sources and in vivosynthesis from labile methyl groups donated by methionine, creatine or homocysteine meet choline requirements under normal conditions. Folic acid and vitamin B12are 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 the choline requirement have been observed under conditions of folic acid and (or) vitamin B12 deficiency.

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 affects the requirement for 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. 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. For the ruminant, dietary choline in an unprotected form is useless because of extensive ruminal degradation.

Calculations from lactating dairy goats on rates of methyl group transfer revealed that only 6% of methionine methyl groups were derived from choline, while 28% of choline methyl groups were derived from methionine (Emmanual and Kennelly, 1984). This suggests that considerable dietary methionine is used for choline synthesis, but that choline is not a major direct precursor of methionine, although it may spare methionine. Benoit et al. (2010) suggested that ~ 40% of methionine in the mammary gland underwent transmethylation with choline serving as the methy donor.

Typical choline requirements for monogastric animals range from 1,000 to 2,000 mg per kg (454 to 907 mg/lb) of diet. In contrast to monogastric animals, no requirements for choline have been established in ruminants except for milk-fed calves, where 260 mg of choline per liter of synthetic milk prevented choline deficiency signs. The NRC (1989) suggests that milk replacers for calves should contain 0.26% choline.

 

Sources

All naturally occurring fats contain some choline. Egg yolks, glandular meats and brain are the richest animal sources; germ of cereals, legumes and oilseed meals are the best plant sources. Corn is low in choline, with wheat, barley and oats containing approximately twice as much choline as corn. Betaine can spare the requirements of choline. Most feedstuffs contain over twice as much betaine as choline.

Little is known of the biological availability of choline in feedstuffs. When a chick assay method was used, soybean meal was found to contain a high proportion of biologically unavailable choline. Bioavailability of choline is 100% in corn while in dehulled soybean meal bioavailability varied from 60% to 75% (Molitoris and Baker, 1976; McDowell and Ward, 2008). Enogenous choline synthesis makes bioavailability difficult to quantify. Bioavailability of choline for ruminants is of minor importance because both naturally occurring choline in feeds, predominantly found in phospholipids (lecithin) and dietary choline from supplements, such as choline chloride, have been shown to be extensively degraded in the rumen (Sharma and Erdman, 1988; 1989). Choline supplements are only of value if they are resistant to rumen degradation. A rumen-protected choline product has become available (Erdman and Sharma, 1991).

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 as either 70% liquid or 50% to 60% dry dilutions. The 70% liquid choline is very corrosive and requires special storage and handling equipment. It is not suitable for inclusion in concentrated vitamin premixes but can be added directly to mixed feeds. Choline from any source is highly destructive of vitamins in concentrated premixes.

 

Deficiency

Although animals are able to synthesize choline internally, the young ruminant may require supplementation, at least in milk replacer. An apparent choline deficiency syndrome was produced in calves with a synthetic milk diet containing 15% casein (Johnson et al., 1951). Within six to eight days, calves developed extreme weakness and labored breathing and were unable to stand. Supplementation with 260 mg of choline per liter of milk replacer prevented these deficiency signs. Improved performance of feedlot cattle has been reported in response to supplemental choline in some studies. Several reports from Washington (Swingle and Dyer, 1970) and Maryland (Rumsey, 1975) have shown increased gains by as much as 6% to 7% and improved feed efficiency by 2.5% to 8% in finishing cattle when supplemented with 500 to 700 ppm dietary choline. In other experiments with growing cattle, no response to choline occurred (Wise et al., 1964; Harris et al., 1966). A study with a rumen-stabilized choline reported some influence on blood metabolites in beef finishing heifers (Bindel et al., 1998). Ruminally protected choline has improved growth performance of finishing cattle without negatively affecting carcass characteristics (Drouillard et al., 1998; Bryant et al., 1999; Bindel et al., 2000). Drouillard et al. (1998) observed an interaction between dietary fat and supplemental choline, but results of Bindel et al. (2000) contradicted this finding. Feeding rumen-protected (RP) choline to lambs has increased liveweight gain by 10 percent (Bryant et al., 1999). Nunnery et al. (1999) compared two levels of two sources of rumen-stabilized choline in finishing beef steers and found no effect of either source or level of rumen-stabilized choline on feedlot performance, although choline reduced carcass fatness and yield grade. 

Two studies with Angora goats found no effect on performance of doelings but an increase in growth rate in wethers in response to feeding 3 g per day of rumen-protected (RP) choline (Puchala et al., 1999; Shenkoru et al., 1999). A study of the effects of choline on rumen function in sheep found that 0.5, 1 or 2 g per day of choline reduced rumen volatile-fatty-acid concentrations with either a roughage or high-concentrate diet (Flachowsky et al., 1988b).

Choline may be a limiting nutrient for milk fat synthesis. Lactating cows fed supplemental choline showed an increase in milk fat percentage and fat-corrected milk (Erdman et al., 1984), although a subsequent experiment found no effect of supplemental choline (Atkins et al., 1988). In another experiment, Erdman and Sharma (1991) were unable to show a positive effect on milk fat percentage from choline by postruminal infusion. Grummer et al. (1987) reported no effect of abomasal infusion of choline on milk fat synthesis in lactating cows, but found that milk fat yield was increased significantly by infusion of soy lecithin compared to soy oil.

In dairy cattle, choline supplementation has improved lactational performance (Sharma and Erdman, 1989; Erdman and Sharma, 1991; Pinotti et al., 2003; Emanuele et al., 2007; de Ondarza et al., 2007; Toghdory et al., 2007). Supplementing betaine in the diet of goats has increased milk yield by 12 to 36 percent (Fernández-Figareset al., 2004). However, production responses to supplemental choline or betaine have been inconsistent. Feeding RP choline at 0.078%, 0.156% or 0.234% of ration dry matter increased milk yield from 1 to 2.6 kg per day in dairy cows with no consistent effect on milk fat yield (Erdman and Sharma, 1991). Feeding RP choline for 28 days prior to calving had variable effects on dry matter intake and milk production (Hartwell et al., 1999). Cows were fed 0, 6 or 12 g of rumen-protected choline per day with either a 12% crude protein or 14% crude protein diet. In cows fed the 12% protein diet prepartum, feeding 6 g per day of RP choline decreased milk yield over 120 days postpartum, while 12 g per day fed with the same prepartum diet, increased milk yield. Conversely, when fed the 14% crude protein diet prepartum, cows fed 6 g per day of RP choline had increased 120-day milk yield, while cows fed 12 g per day of RP choline had significantly lower milk production (Hartwell et al., 1999). In a study with the same RP choline product in primiparous dairy heifers, feeding 15 g per day of RP choline during lactation had no effect on dry matter intake or milk yield, although there was a tendency for an increase in milk fat yield (Vasquez et al., 1999). Deuchler et al. (1998) reported that a RP choline used in several other cited studies was effective in elevating milk choline secretion when fed to lactating dairy cows.

Limited research indicates that supplemental choline may reduce fatty liver in transitional dairy cows (Jurlin, 1965; Hartwell et al., 2001; Cooke et al., 2007; Zom et al., 2010). Feeding RP choline was very effective in increasing milk production in fat cows (Zahra et al., 2006) and for those receiving a methionine limited diet (Davidson et al., 2008). Overall, cows that received RP choline produced 1.2 kg (2.6 lb) per day more milk in the first 60 days of lactation, but this effect was attributable to an increase in milk production of 4.4 kg per g (9.7 lb) among cows with a body condition score ≥4 at three weeks before calving; fat cows that received RP choline ate 1.1 kg (2.4 lb) of dry matter per day more from week three before calving through week four after calving (Zahra et al., 2006). Multiparous cows fed RP choline on a diet low in methionine had higher milk yields and increased milk protein yield than controls, but not cows that also received additional methionine (Davidson et al., 2008).

Feeding dairy cows RP choline and methionine indicated an improved reproductive performance (Ardalan et al., 2010). The RP choline and methionine fed cows had the lowest open days, days to first estrus and services per conception compared with other groups.

Carnitine is synthesized in part from methyl groups derived from choline and other donors via S-adenosylmethionine (Lehninger, 1975). Carnitine is a required component of carnitine-acyltranferases I and II, two molecules required for transport of long-chain fatty acids into the mitochondria for oxidation. Choline may affect lipid metabolism indirectly by influencing synthesis of carnitine. A series of studies with lactating dairy cows found that plasma and liver carnitine could be increased either by feeding or abomasal infusion of carnitine, but that milk yield and dry matter intake were not affected by carnitine supplementation (LaCount et al., 1995, 1996a, b). However, total tract digestibility of lipids and energy increased in response to carnitine supplementation by either route (LaCount et al., 1995).

 

Fortification Considerations

Any response to dietary supplementation of choline in ruminants would depend on factors such as age, body condition, level of productivity, sulfur amino-acid status and other choline-sparing nutrients. Folic acid, vitamin B12 and manganese status affect choline metabolism and absorption. Supplemental choline has been shown to be beneficial to young ruminants (e.g., young calves, prior to ruminal development). For high producing lactating dairy cows or other ruminants that may be marginal in choline supply, a RP choline source is required to consistently increase plasma choline levels. Both choline and betaine are susceptible to rapid ruminal degradation, the amounts available for absorption are limited. Therefore, dairy cows may benefit from RP supplementation of choline or betaine. Emmanuel and Kennelly (1984) reported that 28% of absorbable methionine was used for choline synthesis in lactating goats and Benoit et al. (2010) suggested ~ 40% of methionine was used to provide choline. Therefore, in dairy cattle diets, if methionine is limited then choline is likely limited as well, and a portion of the dietary methionine requirement is used to provide choline. 

Fatty liver is a metabolic disorder that can affect up to 50% of high-producing dairy cows during the transition period, potentially compromising health, production, and reproduction (Jorritsma et al., 2000). Fatty liver is associated with elevated nonesterified fatty acids (NEFA) resulting from depressed feed intake and endocrine changes that occur in the periparturient period (Grummer, 1995). The result is accumulation of the fat triacylglycerol (TAG) in liver, which has been shown to decrease the rate of hepatic ureagenesis, gluconeogenesis, hormonal clearance, and responsiveness (Strang et al., 1998a, b). Therefore, prevention of fatty liver may be necessary to maintain optimal hepatic function during the periparturient period. Fatty liver incidence could be reduced either by suppressing fatty acid mobilization from adipose tissue, or by enhancing the TAG export as lipoproteins from the liver or by both mechanisms. Feeding RP choline can prevent and possibly alleviate fatty liver induced by feed restriction. Various experiments have reported RP choline to have a positive effect on reducing liver fat (Julin, 1965; Hartwell et al., 2001; Cooke et al., 2007; Zomet al., 2010).

Response to choline by feedlot cattle has been a mixture of positive and no response. The decision whether to fortify rations with choline will depend on assessment of diet, production level or rate of growth, and projected return on investment.

Choline chloride is itself stable in multivitamin premixes but highly destructive of vitamins in the premix (Frye, 1978; Gadient, 1986). This is due to both the high reactivity and the hygroscopicity of choline. Choline is stable during feed processing and storage in pelleted and extruded feeds.

 

Safety

Insufficient data are available to support precise estimates of maximum tolerable dietary levels of choline in ruminant species. Data from swine and poultry indicate a high tolerance for choline (NRC, 1987; Leeson and Summers, 2001). 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. The tolerance of ruminants to native choline would be expected to be high due to extensive rumen degradation.