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DSM in Animal Nutrition & Health

Choline

Properties and Metabolism

Choline is a beta-hydroxyethyl trimethylammonium 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 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 grams 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 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 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 itself. 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 pre-synaptic to post-synaptic 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 (Zeisel and da Costa, 2009). 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 new 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 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).

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, 2006). The involvement of folic acid, vitamin B12, and methionine in methyl group metabolism, and of methionine in new 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. 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, when 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).

 

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 of the animal’s needs. Dietary sources can meet metabolic requirements for choline, or choline can be synthesized in vivo from labile methyl groups. Methyl groups can originate from methionine (in excess of that required for protein synthesis), and therefore, level of dietary methionine affects the requirement for choline. Kroening and Pond (1967) fed 5 kg (11 lb) pigs a low-protein (12%) diet supplemented with three levels of dl-methionine (0, 0.11% or 0.22%). The addition of 1,646 mg of choline per kg (748 mg per lb) of diet improved the weight gains and feed conversion of pigs fed the control and the 0.11% but not the 0.22% methionine-supplemented diet. Nesheim and Johnson (1950) reported that baby pigs supplied with diets containing 1.6% methionine did not require dietary choline supplementation based on growth and feed efficiency. Likewise, for starting, growing and finishing pigs, the North Central Region-42 Committee on Swine Nutrition (1980) reported that added choline was not beneficial as assessed by performance of pigs fed corn-soy-lysine diets. In their evaluation, performance of starter, grower and finisher pigs fed diets supplemented with up to 86, 172 and 344 mg choline per kg (39, 78 and 156 mg per lb) diet, respectively, was compared with that of pigs that did not receive supplemental choline. Russett et al. (1979b) reported that starter pigs do not require more than 520 kg choline per kg (236 mg per lb) of diet when fed an 18% crude protein, corn-isolated diet (0.23% cystine) that provided 0.22% methionine. The basal diet was analyzed to provide 440 mg choline per kg (200 mg per lb) of diet. Russett et al. (1979b) found no benefit of choline supplementation on performance. These results are in agreement with those of Stockland and Blaylock (1974), who reported that 412 mg of supplemental choline per kg (187 mg per lb) of ration was adequate to provide optimum sow and gilt performance. Seerley et al.(1981) found no beneficial effect on piglet survival or lipid mobilization when 500 ppm supplemental choline was included in the diets of sows throughout lactation. The choline requirement for growing pigs ranges from 300 to 600 mg per kg (136 to 272 mg per lb) of diet, while adult swine require 1,000 to 1,250 mg per kg (455 to 568 mg per lb) (NRC, 1998). Estimates of choline requirements are based on the assumption that the diets contained an adequate level of methionine. Requirements for choline have generally been determined through the use of purified diets, and recommendations often do not take into account bioavailability from feedstuffs, individual animal variation or effects of other dietary factors. In addition to methionine or other sulfur amino acids, additional dietary factors, such as betaine, myoinositol, folic acid and vitamin B12; the combination of different levels and composition of fat, carbohydrate and protein in the diet; and the age, sex, caloric intake and growth rate of animals, all influence the lipotropic action of choline and thereby the requirement for this nutrient (Mookerjea, 1971). Dietary betaine can spare choline, since choline functions as a methyl donor by forming betaine. In relation to protein level, a larger choline effect on litter size and piglet and litter weight was observed for gilts fed a 12% protein diet than for those fed a 16% protein diet (Maxwell et al., 1987). 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 vitamin B12 plays a role in regulated transfer of the methyl group to tetrahydrofolic acid (THF). Therefore, marked increases in choline requirements have been observed under conditions of folic acid and (or) vitamin B12 deficiencies.

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 methionine and choline directly affect requirements of each other (Russett et al., 1979a). Other than exogenous sources of methyl groups from choline and methionine, methyl group formation from de novo synthesis of formate carbons is reduced with folate 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). A later study 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 need for choline can be supplied in two ways: either by dietary choline or by choline synthesis in body, which 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 acts to prevent fatty livers and hemorrhagic kidneys, it does not act as a true vitamin, as it is incorporated into phospholipids (via cytidine diphosphocholine). 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).

In general for some species, males are more sensitive to choline deficiency than females (Wilson, 1978). Growth hormone seemed to increase the choline requirement in rats independent of its ability to promote growth and increase food intake (Hall and Bieri, 1953). Cortisone and hydrocortisone have been reported to decrease severity of renal necrosis, and hydrocortisone reduced the amount of hepatic lipid choline deficiency in rats (Olson, 1959).

Excessive dietary protein may increase the young animal’s choline requirement. Diets high in fat aggravate choline deficiency and thus increase the growing animal’s requirement. Fatty liver is generally enhanced by fats containing a high proportion of long-chain saturated fatty acids. Boyd et al. (1982) conducted studies to investigate possible choline-tallow interactions when fed to sows. No choline-tallow interaction or response to supplemental choline at 220 and 770 mg was observed for preweaning pig performance. Choline deficiency develops to a greater degree in rapidly growing animals, with deficiency lesions more severe in these animals.

 

Sources

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. 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 contains over twice as much betaine as choline. Thus, the choline needs of swine or poultry fed wheat-based diets are much lower than those of swine or poultry fed diets based on other grains.

Little is known of the biologic availability of choline in natural feedstuffs. Using a chick assay method, soybean meal was found to contrain a high proportion of biologically available choline. 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). Emmert et al. (1996) reported that this diet, which contained soy protein isolate and 2-amino-2-methyl-1-propanol, was signularly deficient in choline and would elicit a weight response only upon addition of choline. Thus, this diet was suitable for determination of bioavailable choline. Emmert and Baker (1997) used a chick bioassay to assess the bioavailability of choline in canola meal, peanut meal and normal and overheated soybean meal (SBM). They determined that although canola meal had three times the total choline of SBM, it had less bioavailable choline than did SBM. Overheating did not appear to decrease the bioavailability. Average bioavailable choline levels were 83%, 24% and 76% of the analytically determined choline concentrations for SBM, canola meal and peanut meal, respectively.

Choline is made synthetically, and most dietary supplementation is from synthesized choline salts. Choline is synthesized from natural gas via methanol and ammonia, which are reacted to produce trimethylamine (Griffith and Nyc. 1971). This trimethylamine is subsequently reacted with ethylene oxide to produce choline. For feed supplementation purposes, a chloride salt is produced by reacting the alkaline base with hydrochloric acid. 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 swine feeds.

 

Deficiency

In the baby pig, a choline deficiency resulted in slower weight gain and fatty infiltration of the liver (Johnson and James, 1948). Choline deficiency in the young pig results in unthriftiness, poor conformation (short-legged and pot-bellied), lack of coordination in movements, a characteristic lack of proper rigidity in joints (particularly the shoulders), fatty infiltration of the liver, characteristic renal glomerular occlusion and some tubular epithelial necrosis (Cunha, 1977). These clinical signs, which resulted from low-methionine diets (0.8%), were prevented with 1.6% dietary methionine inclusion. ”Spraddled hindleg” is a problem occasionally seen in newborn pigs, and some evidence suggests that the incidence has a strong genetic component. This condition, which is often attributed to choline deficiency, is sometimes prevented by supplementation of the vitamin. However, some reports fail to relate a choline deficiency to incidence of spraddle-legged pigs (Luce et al., 1985). Similarly, Stockland and Blaylock (1974) found no consistent relationship between the number of sows farrowing pigs with spraddled legs and the level of dietary choline. Whether folic acid and vitamin B12 are involved in the condition is unknown, but under conditions of deficiencies of these vitamins, choline requirements are increased. Spraddled legs can be described as a congenital disorder in which the newborn pig cannot stand or walk because of the leg condition (Illus. 15-1). The problem seems to be worst on slippery floors. Nursing is also hindered, which affects weaning weights.

 

Illustration 15-1: Choline Deficiency Spraddled Hind Legs

15Illustration_14-3_Pantothenic_Acid_Deficiency

This condition has been produced with a purified ration and is prevented by choline supplementation. Other factors may be involved in this condition.

 

Spraddled leg started to appear as swine producers began to decrease feed allowances given sows during gestation from 2.7 to 3.2 kg (5.9 to 7.0 lbs) daily to 1.4 to 2.0 kg (3.1 to 4.4 lbs) (Cunha, 1977), which resulted in reduced intakes of both choline and methionine. Studies from Colombia, South America (J. H. Maner, personal communication to Cunha, 1977), revealed death losses due to spraddled legs. Some of these pigs recuperated by the tenth day after birth, which may indicate that the condition can be corrected through the sow’s milk. Other reports have indicated that a high proportion of baby pigs affected by spraddled legs were able to recover after a few days, especially if the hindlegs are bound temporarily to allow them to move and suckle. Research reports have shown that sows without choline had a significantly lower conception rate and farrowing rate and farrowed significantly fewer total pigs and fewer live pigs per litter. No difference was found in the average birth weight, but sows with choline supplementation weaned significantly more pigs per litter, and sows without choline farrowed a slightly higher percentage of pigs with spraddled legs. Pigs from choline-deficient sows were unthrifty in appearance and became increasingly so with age (Ensminger et al., 1947). Stockland and Blaylock (1974) reported that sows fed a diet without choline had significantly lower conception rates (57% versus 73%), lower farrowing rate (62% versus 78% bred sows), fewer total pigs per litter farrowed (9.3 versus 10.1) and fewer live pigs per litter at farrowing (8.0 versus 9.1) than sows that received diets containing 412 or 824 mg choline per kg (187 or 375 mg per lb) ration. The North Central Region-42 Committee on Swine Nutrition (1976) evaluated the effects of supplemental choline at 770 mg per kg (350 mg per lb) of diet during gestation and lactation on litter size at birth and at weaning. Nine stations participated in 22 trials on 551 sows. The diet was a 15% protein corn-soybean meal type during gestation, and 7.5% beet pulp was substituted for an equal amount of corn during lactation. Results indicated that sows fed supplemental choline farrowed more total pigs per litter (10.54 versus 9.89), live pigs per litter (9.33 versus 8.64) and weaned more pigs per litter (7.72 versus 7.29). Kornegay and Meacham (1973) evaluated the effect of adding choline to a fortified corn soybean meal gestation-lactation diet for sows. Sows fed 880 mg of supplemental choline per kg (400 mg per lb) of diet during breeding and gestation farrowed more live pigs than the sows without supplemental choline. The largest response of choline supplementation occurred during the fifth and sixth parities. Kornegay and Meacham (1973) saw no benefit of choline supplementation on number of pigs weaned.

 

Fortification Considerations

Response to dietary supplementation of choline depends on 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. On a synthetic milk diet containing 1.6% methionine, young pigs were found not to require supplemental choline (Firth et al., 1953). Neumann et al. (1949) reported that the young pig requires 0.1% dietary choline when methionine is present at 0.8% to 1% of the diet. Hongtrakul et al. (1997) have suggested that supplemental choline is not required in the diets of weanling pigs, since in their study growth performance was not affected by the addition of 150 g of choline per ton. Hongtrakul et al. (1977) emphasized that supplemental choline is still required for gestating and lactating sows. 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 a choline shortage. Methionine is not used for choline synthesis if there is an adequate level of dietary choline. In formulating typical swine 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 by Ruiz et al. (1983). Lovett et al. (1986) found that when inorganic sulfate (Na2SO4) plus methionine or Na2SO4 plus choline was added to weanling swine diets, daily gains and feed efficiency were increased. Sulfur is present in a number of body metabolites (i.e., mucopolysaccharides), and if it is not adequately supplied in the diet, sulfur amino acids are likely to be degraded. Data suggest that sulfate must be present for choline to spare a maximum amount of methionine (Miles et al., 1983). The practical implication is that sulfate and choline must 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.

Most choline supplementation studies emphasize the production benefits of providing the vitamin to young animals. However, research and observations with adult swine demonstrate improved litter size at weaning and indicate that supplementation may keep sows in the producing herd longer (Cunha, 1977). The exact level of choline needed for sow diets is unknown. Until more research data are available, Cunha (1972) suggested use of the following levels when spraddled hindlegs are likely to occur: (1) during the first part of gestation, 3,000 mg per sow daily and (2) for the last month of gestation, 4,200 to 4,500 mg daily.

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).

 

Vitamin Safety

Limited data with pigs indicated a high tolerance for choline (NRC, 1998). However, a more extensive study indicated that excess choline should be avoided in pig diets if maximum rate of gain is to be achieved (Southern et al., 1986). Excess supplemental choline (2,000 mg per kg; 909 mg per lb) given throughout the weanling, growing and finishing phases of growth (121 to 126 days) reduced daily gain but had no effect on feed utilization. Choline at 2,000 mg per kg (909 mg per lb) did not affect pig gain when given only during the growing and finishing stages (68 to 86 days). Bryantet al. (1977) also found no detrimental effects of choline on performance of young or grow-finish pigs fed diets containing 2002 mg choline per kg (910 mg per lb) of complete feed. Very high levels of choline (>5gm per day) in humans have been associated with a fishy body odor, excessive sweating and salivation, vomiting, and gastrointestinal distress (Garrow, 2007).