CholineAn Essential Nutrient for Metabolism

Meeting the body’s need for choline

When free choline is needed in a tissue or organ, it is either biosynthesized, extracted from circulation (1;2), or scavenged from local endogenous supply in the body (3). While the body can produce choline, biosynthesis is insufficient, even under ideal conditions. The efficiency of endogenous choline pro-duction varies with age, gender, and hormonal status, and is itself influenced by diet. All cells synthesize choline via the cytidine diphosphocholine (CDP-choline) mechanism (1;4), known as the Kennedy pathway. Another mode of choline biosynthesis is via the sequential methylation of phosphati-dylethanolamine, catalyzed by the enzyme phosphatidyletha-nolamine N-methyltransferase (PEMT) (5;6). PEMT exists in abundance primarily in the liver, presumably to satisfy the needs incurred there in phospholipid synthesis and to supple-ment the CDP-choline pathway in times of metabolic stress (6). As the efficiency of this enzyme is estrogen-regulated (7;8), it is not surprising that estrogen-deficient segments of the population, particularly men and post-menopausal women, are most likely to become choline deficient with in-adequate dietary intake (9). Despite the abundant estrogen in circulation during gestation, pregnant women are also at increased risk for choline deficiency (10) because of the drain on choline imposed by the fetus (11;12). Choline availability to the fetus (13-15), and later, to the nursing infant, is directly affected by efficiency of PEMT activity (16) and maternal di-etary habits (2;17;18). Dietary intake is especially critical to individuals with loss-of-function variations in their genes for choline metabolism, particularly PEMT (5;7).

Choline is a low molecular weight, positively charged quaternary (2-hydroxyethyl-trimethyl) amine with unique chemical and biochemical properties. It is pres-ent in nature as a component of a number of biological molecules, and can be prepared in water-soluble salt form. Choline’s chemical attributes are the root of its diverse modes of biochemical functionality, and affect its absorption, distribution, metabolism and excretion from the human body.

Choline has many important functions in the healthy structure and function of the human body, being at the center of many convergent biological processes. It is integral in mediating processes of cell growth, func-tion and repair and enabling the metabolism and mo-bilization of other micro-and macronutrients, including vitamin cofactors, amino acids and lipids. Intake of choline profoundly affects the availability of the nutri-ent to the numerous tissues and organs whose viability depend on it. Water-soluble choline salts are an ef-fective delivery form for the nutrient in dietary supple-ments and fortified foods.

acetylcholine

betaine dimethylglycine

homocysteine

S-adenosylmethionine

sphingomyelin

platelet-activating factor

phosphatidylethanolamine

phosphatidylcholine

phosphocholine

acetyl coenzyme A

diacylglycerol

lysophosphatidylcholine

ceramide

methionine

PEM

T pa

thw

ay

Kennedy pathway

Figure 2. Choline: An important metabolic intermediate and precursor

Figure 1. Choline structure

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Choline: Essential for every body

Choline is an irreplaceable material requirement of cells for multiple biological purposes. It is considered an essential nu-trient for humans (19) because biosynthesis does not ordinar-ily supply enough to sustain normal organ function, even in a healthy human body. Choline deficiency results from the convergence of less-than-optimal dietary habits and enzyme impairment at the individual genetic level (20-22). It is com-pounded by an individual’s hormonal status (7;9;23) and avail-ability of partially compensatory B-vitamins.

Deprivation of dietary choline first manifests itself in a decrease in plasma choline. Sustained deprivation will lead to an in-crease in plasma homocysteine, an accumulation of fat in vac-

uoles of liver cells (steatosis), and damage to liver cell mem-branes (9). As deprivation devolves into dietary deficiency, cell suicide is activated in lymphocytes (24), damage is sustained by muscle cells, and organ dysfunction begins (25).

Dietary administration of choline easily prevents and revers-es these symptoms of deficiency (5). In fact, current United States dietary recommendations for choline are based on the daily dosage necessary to prevent these abnormalities in most individuals, i.e. 7 mg/kg body weight/day (19). Based on data from studies of choline-deprived humans and animals, Dietary Reference Intakes (DRIs) were developed for choline nutrition in healthy individuals (19) and published by the Food and Nutrition Board of the Institute of Medicine (US) in 1998.

Substance Function Structure

Phosphatidylcholine

Membrane phospholipid

Sphingomyelin

Platelet-activating factor Cell signaling

Betaine Osmolyte, methyl donor

Acetylcholine Neurotransmitter

Table 1. Biologically important choline derivatives (from references (1-6))

Digestion and metabolism of choline

The metabolism of a meal or ingested supplement begins in the stomach with breakdown of the food matrix by acids and enzymes. The partially digested chyme is moved to the small intestine, where enterocytes bundle up fats, including phospholipids, into chylomicron lipoproteins and release them into the lymph, from where they subsequently enter the blood. Carbohydrates and proteins are broken down into their small-est substituents, namely monosaccharides and amino acids, and are directly released into the bloodstream. Likewise, oral administration of choline readily affects serum choline levels (26-28). When free choline reaches the upper small intestine, a concentration-regulated carrier mechanism transports it into circulation (29). Certain organs and tissues will absorb it in ad-vance of any further metabolism by gut microbiota (26;30-32). It is subsequently stored as the free base or, more commonly, in a phosphorylated form such as phosphocholine (28). In this way, it can be most easily used for the variety of biological purposes for which it is necessary.

When blood glucose is elevated, the pancreas increases the peptide hormone insulin, which regulates carbohydrate and fat metabolism in the body by directing glucose uptake by the liver, muscle, and adipose tissue. Choline, like glucose, enters the liver via the portal vein (32). It is used in the liver primarily for the construction of phospholipid membranes needed to package lipids and export them to adipose tissue for storage. Primarily in the liver and in the kidney, choline is converted into the osmolyte betaine in an irreversible oxidation process. In this form, it regulates influx and efflux of water in cells, which is particularly important in the kidney.

Against all odds, free choline crosses the blood-brain-barrier (2;33;34) to enter the brain. The concentration of free cho-line available at neuron terminals in the brain helps regulate choline’s conversion to the important neurotransmitter ace-tylcholine. While comparatively little choline meets this fate, as compared to other conversions (4), the route is important

in that it is the only synthetic means to generate this impor-tant agent of cellular messaging (2;35). While the process certainly predominates in the brain, acetylcholine synthesis is significant elsewhere in the body, operating in the non-neural cholinergic mechanism present in other cells, tissues and or-gans, including the epithelium, endothelium, and mesothe-lium, mesenchyme, and immune cells (36). This pathway may be significant in mediating cellular inflammation, as will be presented later in this summary.

Dietary choline is important in that it fills the nutrient pools that can attenuate the need to catabolize and recycle other choline-containing molecules, including membrane phospho-lipids (2;4;37;38), during periods of high metabolic activity. If choline is ingested in bound (rather than free base) form, for example as cytidine diphosphocholine (CDP-choline) (2;39) or glycerylphosphorylcholine (α-GPC choline) (40), it is quickly hydrolyzed and metabolized as free choline is, suggesting that there is no special benefit to its delivery this way. Lecithin, the naturally-occurring lipid-bound source of choline (as phospha-tidylcholine), contains comparatively little of the active choline moiety by weight. Upon ingestion, lecithin is hydrolyzed to a form that is circulated in the lymph in the form of chylomicrons (41). It is transported to multiple organs and tissues, such as the brain, kidney, liver and spleen, and is progressively broken down by multiple enzymes, eventually yielding the free choline molecule (32).

Nutrient metabolism by gut microbes is a current area of ac-tive biomedical research. Gut bacterial populations and metabolism are highly variable within populations, but also per individual. The activity of gut microbes is known to be influenced by diet (42) and other physiological stressors (43). Choline’s biological conversion into trimethylamine (44) and derivative compounds in the gut is regarded as a competitive fate for the molecule, in that it keeps the nutrient from fulfilling its important biochemical purposes. This route is significant in the disposition of choline when it is in considerable excess.

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4

Role of choline in one-carbon metabolic balance Regulation of homocysteine metabolism

Choline supports metabolic balance by its contribution to the so-called cellular “methyl pool.” In this way, it influences the structure, organization, repair, and transcription of genes, as well as the production and function of the proteins encoded by those genes (45). Methyl groups [CH3-] are tags that are transferred between biological macromolecules. They func-tion as “epigenetic markers,” which are functional tags that can activate or suppress the expression of genes and the ac-tivity of catalytic proteins. Studies of the effect of prenatal choline availability on DNA methylation show its profound and lifelong importance; the changes in DNA that the placement of a methyl group brings about are heritable and retained over generations (13;46-48).

Once choline is oxidized in the body, it can donate its methyl groups. Methylation of homocysteine, a cellular metabolite which circulates in the blood of all individuals, converts it to methionine. This essential amino acid is needed to repair and build proteins and to serve as a precursor for other substanc-es, such as creatine and S-adenosylmethionine (SAdoMet), the archetypal biological methyl donor (49;50). (Choline’s remaining two methyl groups are released in subsequent oxi-dation steps and diverted to other metabolic processes (51).) Choline insufficiency in humans, even those with adequate methionine, results in an accumulation of unmetabolized ho-mocysteine (52). A lack of choline creates an imbalance of SAdoMet and its immediate precursor (53). Such conditions thwart the optimal activity of the enzymes that assist in methyl group transfer from one macromolecule to another. Increas-

ing choline intake has been shown to reduce plasma homo-cysteine in healthy and homocysteinemic subjects (54;55).

Homocysteine is currently debated by the biomedical re-search community as a biomarker, by-product, risk-factor or active agent (56) of cellular biochemical change. Its alleged link to numerous health conditions (45) is attributed to cyto-toxic and vascular effects (54;55;57;58). Its mechanism of action appears to be related to its oxidative potential and reac-tivity of its thiol group (51;59). In cell culture studies, unabated homocysteine has been shown to have direct and second-ary oxidative effects (45), generating reactive oxygen species (ROS) that trigger degradative chain reactions in cells and tis-sues (e.g. vascular endothelium), and overwhelming the innate protective mechanisms conferred by antioxidant enzymes and nitric oxide. By its reaction with the thiol groups of amino acid side chains, homocysteine can affect the structure and function of proteins and enzymes. These mechanisms (59) are thought to collectively contribute to fibrosis of the liver (60), cardiovascular occlusion (61;62) and the age-related cogni-tive decline (63). Dysfunctional homocysteine metabolism has also been associated with negative outcomes in pregnancy, such as low birth weight, pre-eclampsia, placental abruption and recurrent pregnancy loss (64-67).

• Choline may help reduce levels of plasma homocysteine.

STRUCTURE-FUNCTION CLAIM

DNA RNAProtein orEnzyme

Catalysis orother event

H3C—N

CH3

CH3

OH

Figure 3. One-carbon transfer is an important regulatory mechanism

Synergy with B-vitamins

Choline works in synergy with B-vitamins in homocyste-ine metabolism because of their shared roles in methylation and amino acid synthesis (52;68). Changing availability of B-vitamins shifts the dynamics of reactions requiring free cho-line (69). For instance, adequacy of folate nutrition has been demonstrated to be inextricably linked to choline need (23;70-74). When folate is deficient in the diet, choline is decreased in the liver (72). Likewise, when choline is deficient in the diet, folate is found to be depleted in the liver (75). The activity of the choline-mediated pathway that turns over cellular homo-cysteine spares the complementary nutrient folate for its other very important use in DNA synthesis (74).

Vitamin B2 (riboflavin) is a key component of the cofactor flavin adenine dinucleotide (FAD) for methylene tetrahydrofolate re-ductase (MTHFR), the enzyme which catalyzes the first steps

of the biosynthesis of sulfur-containing amino acids, including that of methionine via homocysteine (76). Vitamin B12 (cobal-amin) is a cofactor for methionine synthase, the enzyme which directly catalyzes the actual conversion (77). Vitamin B6 is involved in an enzymatic reaction that offers a complementary route of disposal for excess homocysteine.

The dietary choline requirement is apparently greater in indi-viduals with certain variations in genes involved in B-vitamin metabolism and one-carbon transfer, particularly the gene en-coding the enzyme MTHFR (9;73). It has been observed that drugs that disrupt the metabolic balance of choline and B-vitamins in the body tend to increase homocysteine levels (78), suggesting another important opportunity for dietary interven-tion. The interrelationship between choline and B-vitamins is highly significant and must be considered in assessing nutrient adequacy.

5

methionine homocysteine

betainedimethylglycine

5-methyl-tetrahydrofolatetetrahydrofolate

amino acid available for

protein synthesis

5,10-methylenetetrahydrofolatedihydrofolate

monomer available for

DNA synthesis and repair

Folatedeoxy-pyrimidine base

cysteineVitamin B6

Vitamin B12

Vitamin B2

SAMe (SAdoMet)for biological

methyla on reac ons

Figure 4. Choline and the methionine cycle

6

Fat metabolism & liver health

The manufacture of very low density lipoproteins occurs in the liver as a mechanism to transport non-polar lipids away from the organ within the aqueous environment of circulating blood (79-81). VLDLs are effectively a cellular packaging system designed to transport fat to adipose tissue for storage or to muscles for immediate use (43). VLDLs are composed of an inner hydrophobic core of lipids, primarily triglycerides and cholesterol esters, and an outer layer of amphiphilic mol-ecules, mostly phospholipids, cholesterol, and the membrane protein apolipoprotein B. As a precursor of the phospholipid phosphatidylcholine, free choline is an important component of the structure of VLDLs. The choline that is used by the liver for this purpose is synthesized by a liver-specific pathway (PEMT, discussed earlier), by a partially compensatory general pathway common to all cells (CDP-choline/Kennedy), and the necessary balance must be obtained from the diet (1;6).

The net effect of dietary choline deficiency is to decrease tri-glyceride export from the liver in VLDLs, resulting in excess fat deposition (steatosis) in that organ (82). Certain individu-als are, by nature, more prone to this outcome by virtue of their inherent biosynthetic ability. As discussed previously, this is hormonally and genetically dictated to a significant extent, and may also be influenced by metabolism by gut bacteria (42). However, the outcome of fatty liver appears not to be related to a simple reduction in phosphatidylcholine synthesis, but rather an apparent effect on the size, shape and viabil-ity (ease of enzymatic breakdown) of the VLDL particles (83). Nor is the derangement of lipid metabolism limited to the liver; choline deprivation affects cellular structural integrity and lipid metabolism in other tissues, as well. Most notably, muscle cell membranes are catabolized and cellular suicide is acti-vated in the absence of choline (25). Triglycerides accumulate in choline deficient muscle cells, not because of accelerated lipogenesis from glucose, but rather from the increased as-sembly of the molecules from the pool of components (fatty acids, diacylglycerol) scavenged from broken down cell mem-branes (84).

The effect of choline deficiency in humans has been most un-mistakably illustrated in individuals on controlled diets, such total parenteral nutrition (TPN) (85). Plasma free choline is observed to decrease significantly with administration of TPN that is inadequate in choline, but sufficient in B-vitamins and methionine (86;87), suggesting a unique importance of cho-line for which these complementary nutrients cannot compen-sate. The degree of bioavailability of the choline source (free versus bound) also appears to be important (60) in maintaining normal choline status as measured in serum and whole blood.

Longer-term deficiency, in TPN patients as well as in other-wise healthy individuals (88-90), is associated with the clini-cal onset of liver dysfunction, as indicated by abnormally high measurable serum transaminase activity. Alanine- and as-partate aminotransferases are enzymes which are released from liver cells when their membrane integrity is compromised (88). These metrics are considered to be a general surrogate indicator of hepatocyte damage and steatosis (accumulation of excess fat), which cannot ordinarily be easily clincally ob-served (19) without invasive biopsy and/or diagnostic imaging.

The etiology of liver dysfunction (91) is thought to be related to the rupture of cell membranes caused by steatosis, as well as the induction of apoptosis (controlled cell death) (92) and compensatory uncontrolled cell division in liver tissue (19;93;94). Hepatotoxicity may arise from infiltration of the liver by fatty acids, some of which are oxidized and recycled, collaterally generating reactive oxygen species (ROS) which may interact with macromolecules (e.g. DNA) or membranes (43;95). Though deficiency in choline and deficiency in lipo-tropes (Vitamin B12, folate, methionine) both overtly result in fatty liver, the two states are clinically distinguishable from one another. Lipotrope deficiency is associated with wasting and with steatosis which escalates to fibrosis of the liver. Sus-tained absence of choline in the diet will, remarkably, induce hepatocellular necrosis, and eventually hepatocarcinoma, even in the absence of exogenous carcinogens (91;96). It appears that the absence of the choline moiety itself, rather than a lack of contributed methyl groups, is significant in this regard (97).

Triacylglycerol

Apoprotein

Phosphatidylcholine

VLDL

Figure 5. Very low density lipoprotein

• Choline may promote healthy liver function.

STRUCTURE-FUNCTION CLAIM

7

Role in mediating inflammation

Recent research suggests that choline availability may be im-portant in mediation of cellular inflammation, a line of defense against invasion and infection. A recent epidemiological study of adult men and women suggests that levels of typical cel-lular markers of inflammation in blood (e.g. C-reactive protein, interleukin-6, tumor necrosis factor-α) are decreased in indi-viduals with the greatest exposure to choline in their diets, ver-sus those individuals with low choline diets (98). The possible regulatory mechanism of action of choline in cellular inflamma-tory processes is currently the subject of research.

Choline’s role in reduction of circulating homocysteine is like-ly a partial contributor to this effect. By its metabolism of homocysteine, choline contributes to the generation of SA-doMet. This has many potential implications, as SAdoMet influences a variety of cellular processes involved in immune response (45;98). SAdoMet suppresses the production of several inflammatory “first responders,” activates an addition-al, complementary route of disposal for cellular homocysteine (cystathionine β-synthase, with Vitamin B6 as a cofactor), and promotes the production of the antioxidant glutathione and the necessary activity of many significant methyltransferase enzymes (45;98;99).

Choline’s use by cells as a precursor of several key phospho-lipids may be important in mediating inflammatory response, as well. By the mechanism of base exchange, phosphatidyl-choline derived from choline can subsequently be intercon-verted to phosphtidylethanolamine (PE), phosphatidylserine (PS), or phosphatidylinositol (PI) (100), which are involved in cell signaling and membrane structure and transport (101).

The non-neural cholinergic system may also be significant in choline’s putative anti-inflammatory effect (102). Acetylcho-line, synthesized from free choline and acetyl coenzyme A, is the primary neurotransmitter of the vagus nerve, which runs most of the length of the human body and connects the cen-tral nervous system with distal organs and tissues. Signal-ing by the vagus nerve involves release of acetylcholine and uptake of the neurotransmitter by macrophages and other immune cells possessing acetylcholine receptors (103;104). The receipt of the neurotransmitter message inhibits the syn-thesis and release of cytokines and other pro-inflammatory agents (105). The process operates in tissues such as the airway and lungs, intestines, pancreas, and adipose tissue, which all respond to the magnitude of these signals (106;107), with a variety of clinical repercussions.

It has been hypothesized that functional breakdown of the this mechanism contributes to pathogenesis by loss of control over the body’s inflammatory response (36;108). In the case of obese adipose tissue, for instance, rampant inflammation has been shown to be related to pre-diabetic insulin resis-tance (107). Ensuring the ready availability of acetylcholine is likely to be important in efficient operation of the non-neural cholinergic system, via availability of functional transporters and receptors and a well-stocked supply of the precursor molecule choline.

Acetylcholinereceptors

Release ofpro-inflammatory

molecules

Cellular stressBrain

CellCell

Figure 6. Choline’s putative role in attenuating inflammation

8

Conclusion

Choline is currently underappreciated for its central role in the promotion of human health. It is a material requirement of cells for membrane structure and metabolic control, and its presence is integral in maintaining a balance of key biochemi-cals needed for cell growth, function and repair. Choline is significant in the metabolism and mobilization of other key micro- and macronutrients, including vitamin cofactors, amino acids and lipids.

The participation of choline in one-carbon transfer reactions underlies its cellular regulatory epigenetic function. It is also the mechanism by which it reduces the amount of homocys-teine circulating in the blood, ostensibly heading off oxidative processes and other undesirable biochemical interactions that could otherwise compromise cognitive function and prenatal and cardiovascular health.

Dietary intake of choline is known to affect the availability of the nutrient to the tissues and organs where it is utilized. Choline intake is absolutely necessary, as the body does not produce enough, even under ideal conditions. Choline chloride and choline bitartrate are highly soluble, stable, bioavailable forms that can be incorporated into foods, beverages, and supple-ments to easily deliver important lifelong nutritional benefits to consumers.____________________________________________________

This article is for informational purposes only and is not meant to be construed as authoritative legal or medical advice. Balchem makes no representations as to its accuracy and assumes no liability or respon-sibility for the content of this article.

Choline supplements are not intended to diagnose, treat, cure or prevent any disease.

©2011 Balchem Corporation. All rights reserved.

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(1) Michel V, Yuan Z, Ramsubir S, Bakovic M. Choline transport for phospho-lipid synthesis. Exp Biol Med 2006;231:490-504.

(2) Wurtman RJ, Cansev M, Ulus IH. Choline and its products acetylcholine and phosphatidylcholine. In: Tettamani G, Goracci G, editors. Handbook of Neurochemistry and Molecular Neurobiology: Neural Lipids. 3 ed. New York: Springer; 2009. p. 443-500.

(3) Li Z, Agellon LB, Vance DE. Choline redistribution during adaptation to choline deprivation. J Biol Chem 2007;282(14):10283-9.

(4) Blusztajn JK, Liscovitch M, Richardson UI. Synthesis of acetylcholine from choline derived from phosphatidylcholine in a human neuronal cell line. PNAS 1987;84:5474-7.

(5) Waite KA, Cabilio NR, Vance DE. Choline deficiency-induced liver damage is reversible in Pemt-/- mice. J Nutr 2002;132:68-71.

(6) Walkey CJ, Yu L, Agellon LB, Vance DE. Biochemical and evolutionary sig-nificance of phospholipid methylation. J Biol Chem 1998;273(42):27043-6.

(7) Fischer LM, daCosta KA, Kwock L, Galanko JA, Zeisel SH. Dietary choline requirements of women: effects of estrogen and genetic variation. Am J Clin Nutr 2010;92(5):1113-9.

(8) Resseguie M, Song J, Niculescu M, daCosta KA, Randall TA, Zeisel SH. Phosphatidylethanolamine N-methyltransferase (PEMT) gene expression is induced by estrogen in human and mouse primary hepatocytes. FASEB J 2007;21:2622-32.

(9) Fischer LM, daCosta KA, Kwock L, Stewart PW, Lu TS, Stabler SP et al. Sex and menopausal status influence human dietary requirements for the nutrient choline. Am J Clin Nutr 2007;85:1275-85.

(10) Caudill MA. Pre- and post-natal health: Evidence of increased choline needs. J Am Diet Assoc 2010;110:1198-206.

(11) Zeisel SH, Mar MH, Zhou Z, daCosta KA. Pregnancy and lactation are associated with diminished concentration of choline and its metabolites in rat liver. J Nutr 1995;125:3049-54.

(12) Zeisel SH. Choline: Critical role during fetal development and dietary re-quirements in adults. Annu Rev Nutr 2006;26:229-50.

(13) Cooney CA, Dave AA, Wolff GL. Maternal methyl supplements in mice affect epigenetic variation and DNA methylation of offspring. J Nutr 2002;132:2392S-400S.

(14) Garner SC, Mar MH, Zeisel SH. Choline distribution and metabolism in pregnant rats and fetuses are influenced by the choline content of the maternal diet. J Nutr 1995;125:2851-8.

(15) Ilcol YO, Urncu G, Ulus IH. Free and phospholipid-bound choline concen-trations in serum during pregnancy, after delivery, and in newborns. Arch Phys Biochem 2002;110(5):393-9.

(16) Zeisel SH. Choline: Clinical nutrigenetic/nutrigenomic approaches for identification of functions and dietary requirements. In: Simopoulos AP, Milner JA, editors. Personalized Nutrition: Translating Nutrigenetic/Nutrig-enomic Research into Dietary Guidelines.Basel: Karger; 2010. p. 73-83.

(17) Holmes-McNary MQ, Cheng WL, Mar MH, Fussell S, Zeisel SH. Choline and choline esters in human and rat milk and in infant formulas. Am J Clin Nutr 1996;64:572-6.

(18) Ilcol YO, Ozbek R, Hamurtekin E, Ulus IH. Choline status in newborns, infants, children, breast-feeding women, breast-fed infants and human breast milk. J Nutr Biochem 2005;16:489-99.

(19) Institute of Medicine. Choline. In: Food and Nutrition Board NAoS, editor. Dietary Reference Intakes for Thiamin, Riboflavin, Niacin, Vitamin B6, Fo-late, Vitamin B12, Pantothenic Acid, Biotin and Choline.Washington, DC: National Academy Press; 1998. p. 390-422.

(20) Song J, daCosta KA, Fischer LM, Kohlmeier M, Kwock L, Wang S et al. Polymorphism of the PEMT gene and susceptibility to nonalcoholic fatty liver disease (NAFLD). FASEB J 2005;19:1266-71.

(21) Zeisel SH. Gene response elements, genetic polymorphisms and epi-genetics influence the human dietary requirement for choline. IUBMB Life 2007;59(6):380-7.

(22) Zhu X, Song J, Mar MH, Edwards LJ, Zeisel SH. Phosphatidylethanol-amine N-methyltransferase (PEMT) knockout mice have hepatic steatosis and abnormal hepatic choline metabolite concentrations despite ingesting a recommended dietary intake of choline. Biochem J 2003;370:987-93.

(23) Christensen KE, Wu Q, Wang X, Deng L, Caudill MA, Rozen R. Steatosis in mice is associated with gender, folate intake and expression of genes of one-carbon metabolism. J Nutr 2010;140(10):1736-41.

(24) daCosta KA, Niculescu M, Craciunescu CN, Fischer LM, Zeisel SH. Cho-line deficiency increases lymphocyte apoptosis and DNA damage in hu-mans. Am J Clin Nutr 2006;84:88-94.

(25) daCosta KA, Badea M, Fischer LM, Zeisel SH. Elevated serum creatine phosphokinase in choline-deficient humans: mechanistic studies in C2C12 mouse myoblasts. Am J Clin Nutr 2004;80:163-70.

(26) Hirsch MJ, Growdon JH, Wurtman RJ. Relations between dietary choline or lecithin intake, serum choline levels, and various metabolic indices. Me-tabolism 1978;27(8):953-60.

(27) Hollister LE, Jenden DJ, Amaral JRD, Barchas JD, Davis KL, Berger PA. Plasma concentrations of choline in man following choline chloride. Life Sci 1978;23:17-22.

(28) Millington WR, Wurtman RJ. Choline administration elevates brain phos-phorylcholine concentrations. J Neurochem 1982;38:1748-52.

(29) Kamath AV, Darling IM, Morris ME. Choline uptake in human intestinal Caco-2 cells is carrier-mediated. J Nutr 2003;133:2607-11.

(30) Cohen EL, Wurtman RJ. Brain acetylcholine: Increase after systemic cho-line administration. Life Sci 1975;16(16):1095-102.

(31) Cohen EL, Wurtman RJ. Brain acetylcholine: Control by dietary choline. Science 1976;191(4227):561-2.

(32) Zeisel SH. Dietary choline:Biochemistry, physiology, and pharmacology. Annu Rev Nutr 1981;1:95-121.

(33) Sweiry JH, Yudilevich DL. Characterization of choline transport at ma-ternal and fetal interfaces of the perfused guinea-pig placenta. J Physiol 1985;366:251-66.

(34) Zeisel SH. Dietary influences on neurotransmission. Adv Pediatr 1986;33:23-48.

(35) Lockman PR, Allen DD. The transport of choline. Drug Dev Indust Pharm 2002;28(7):749-71.

(36) Wessler I, Kirkpatrick CJ. Acetylcholine beyond neurons: the non-neuro-nal cholinergic system in humans. Brit J Pharmacolog 2008;154:1558-71.

(37) Blusztajn JK, Wurtman RJ. Choline and cholinergic neurons. Science 1983;221:614-20.

References

10

(38) Leventer SM, Rowell PP. Investigation of the rate-limiting step in the synthesis of acetylcholine by the human placenta. Placenta 2005 May;5(3):261-70.

(39) Conant R, Schauss AG. Therapeutic applications of citicoline for stroke and cognitive dysfunction in the elderly: A review of the literature. Alt Med Rev 2004;9(1):17-31.

(40) Lopez CM, Govoni S, Battaini F, Bergamaschi S, Longoni A, Giaroni C et al. Effect of a new cognition enhancer, alpha-glycerylphosphorylcholine, on scopolamine-induced amnesia and brain acetylcholine. Pharm Bio-chem Behavior 1991;39(4):835-40.

(41) Parthasarathy S, Subbaiah PV, Ganguly J. The mechanism of intestinal absorption of phosphatidylcholine in rats. Biochem J 1974;140:503-8.

(42) Spencer MD, Hamp TJ, Reid RW, Fischer LM, Zeisel SH, Fodor AA. As-sociation between composition of the human gastrointestinal microbiome and development of fatty liver with choline deficiency. Gastroenterology 2011;140(3):976-86.

(43) Dumas M-E, Barton RH, Toye A, Cloarec O, Blancher C, Rothwell A et al. Metabolic profiling reveals a contribution of gut microbiota to fatty liver phenotype in insulin-resistant mice. PNAS 2006;103(33):12511-6.

(44) de la Huerga J, Popper H. Factors influencing cholineabsorption in the intestinal tract. J Clin Investig 1952;31(6):598-603.

(45) James SJ, Melnyk S, Pogribna M, Pogribny I, Caudill MA. Elevation in S-adenosylmethionine and DNA hypomethylation: Potential epigenetic mechanism for homocysteine-related pathology. J Nutr 2002;132:2361S-6S.

(46) Meck WH, Williams CL. Metabolic imprinting of choline by its availabil-ity during gestation: implications for memory and attentional processing across the lifespan. Neurosci Biobehav Rev 2003;27:385-99.

(47) Mellott TJ, Follettie MT, Diesl V, Hill AA, Lopez-Coviella I, Blusztajn JK. Prenatal choline availability modulates hippocampal and cerebral cortical gene expression. FASEB J 2007;21:1311-23.

(48) Zeisel SH. Importance of methyl donors during reproduction. Am J Clin Nutr 2009;89S:673S-7S.

(49) Lokk J. Association of vitamin B12, folate, homocysteine and cognition in the elderly. Scand J Nutr 2003;47(3):132-8.

(50) Ulrey CL, Liu L, Andrews LG, Tollefsbol TO. The impact of metabolism on DNA methylation. Hum Molec Genetics 2005;14(Review Issue 1):R139-R147.

(51) Shinozuka H, Katyal SL. Pathology of choline deficiency. In: Sidransky H, editor. Nutritional Pathology.New York: Marcel Dekker; 1985. p. 279-320.

(52) daCosta KA, Gaffney CM, Fischer LM, Zeisel SH. Choline deficiency in mice and humans is associated with increased plasma homocysteine concentration after a methionine load. Am J Clin Nutr 2005;81:440-4.

(53) Dudman NP, Wilcken DE, Wang J, Lynch JF, Macey D, Lundberg P. Dis-ordered methionine/homocysteine metabolism in premature vascular disease: Its occurrence, cofactor therapy and enzymology. Arteriocler Thrombosis 1993;13:1253-60.

(54) Cho E, Zeisel SH, Jacques PF, Selhub J, Dougherty L, Colditz GA. Dietary choline and betaine assessed by food-frequency questionnaire in relation to plasma total homocysteine concentration in the Framingham Offspring Study. Am J Clin Nutr 2006;83:905-11.

(55) Olthof MR, Brink EJ, Katan MB, Verhoef P. Choline supplemented as phosphatidylcholine decreases fasting and postmethionine-loading plasma homocysteine concentrations in healthy men. Am J Clin Nutr 2005;82:111-7.

(56) Hustad S, Midttun O, Schneede J, Vollset S, Grotmol T, Ueland PM. The methylenetetrahydrofolate reductase 677 C->T polymorphism as a modulator of a B vitamin network with major effects on homocysteine metabolism. Am J Hum Genet 2007;80:846-55.

(57) McCully KS. Homocysteine and vascular disease: The rold of folate, cho-line and lipoproteins in homocysteine metabolism. In: Zeisel SH, Szuhaj BF, editors. Choline, Phospholipids, Health and Disease.Champaign, IL: AOCS Press; 1998. p. 117-30.

(58) Verhoef P, de Groot LCPGM. Dietary determinants of plasma homocyste-ine concentrations. Seminars in Vascular Medicine 2005;5(2):110-23.

(59) Perna AF, Ingrosso D, Lombardi C, Acanfora F, Satta E, Cesare CM et al. Possible mechanisms of homocysteine toxicity. Kidney Intl 2003;63(Sup-pl. 84):S137-S140.

(60) Sentongo TA, Kumar P, Karza K, Keys L, Iyer K, Buchman AL. Whole-blood-free choline and choline metabolites in infants who require chronic parenteral nutrition therapy. JPGN 2010;50(2):194-9.

(61) Malinow MR. Plasma homocysteine and arterial occlusive diseases: a mini-review. Clin Chem 1995;41(1):173-6.

(62) Malinow MR. Plasma homocysteine: a risk factor for arterial occlusive dis-eases. J Nutr 1996;126(4):1238S-43S.

(63) Garcia A, Zanibbi K. Homocysteine and cognitive function in elderly peo-ple. Can Med Assoc J 2004;171(8):897-904.

(64) Molloy AM, Mills JL, Cox C, Daly SF, Conley M, Brody LC et al. Choline and homocysteine interrelations in umbilical cord and maternal plasma at delivery. Am J Clin Nutr 2005;82:836-42.

(65) Velzing-Aarts FV, Holm PI, Fokkema MR, van der Dijs FP, Ueland PM, Muskiet FA. Plasma choline and betaine and their relation to plasma ho-mocysteine in normal pregnancy. Am J Clin Nutr 2005;81:1383-9.

(66) Vollset S, Refsum H, Irgens L, Emblem BM, Tverdal A, Gjessing HK et al. Plasma total homocysteine, pregnancy complications and adverse pregnancy outcomes: the Hordaland Homocysteine study. Am J Clin Nutr 2000;71:962-8.

(67) Zeisel SH. Choline, homocysteine and pregnancy. Am J Clin Nutr 2005;82:719-20.

(68) Tanaka T, Scheet P, Giusti B, Bendinelli S, Piras MG, Usala G et al. Ge-nome-wide association study of vitamin B6, vitamin B12, folate and ho-mocysteine blood concentrations. Am J Hum Genet 2009;84:477-82.

(69) Melse-Boonstra A, Holm PI, Ueland PM, Olthof MR, Clarke R, Verhoef P. Betaine concentration as a determinant of fasting total homocysteine concentrations and the effect of folic acid supplementation on betain con-centrations. J Clin Nutr 2005;81:1378-82.

(70) Craciunescu CN, Johnson AR, Zeisel SH. Dietary choline reverses some, but not all, effects of folate deficiency on neurogenesis and apoptosis in the fetal mouse brain. J Nutr 2010;doi: 10.3945/jn.110.122044.

(71) Jacob RA, Jenden DJ, Allman-Farinelli MA, Swendseid ME. Folate nutri-ture alters choline status of women and men fed low choline diets. J Nutr 1999;129:712-7.

(72) Kim Y-I, Miller JW, daCosta KA, Nadeau MR, Smith D, Selhub J et al. Severe folate deficiency causes secondary depletion of choline and phos-phocholine in rat liver. J Nutr 1994;124:2197-203.

(73) Kohlmeier M, daCosta KA, Fischer LM, Zeisel SH. Genetic variation of folate-mediated one-carbon transfer pathway predicts susceptibility to choline deficiency in humans. PNAS 2005;102(44):16025-30.

(74) Niculescu M, Zeisel SH. Diet, methyl donors and DNA methylation: Interactions between dietary folate, methionine and choline. J Nutr 2002;132:2333S-5S.

(75) Selhub J, Seyoum E, Pomfret EA, Zeisel SH. Effects of choline deficiency and methotrexate treatment upon liver folate content and distribution. Cancer Res 1991;51:16-21.

(76) Institute of Medicine. Riboflavin. In: Food and Nutrition Board NAoS, edi-tor. Dietary Reference Intakes for Thiamin, Riboflavin, Niacin, Vitamin B6, Folate, Vitamin B12, Pantothenic Acid, Biotin and Choline.Washington, DC: National Academy Press; 1998. p. 87-122.

(77) Institute of Medicine. Vitamin B12. In: Food and Nutrition Board NAoS, editor. Dietary Reference Intakes for Thiamin, Riboflavin, Niacin, Vitamin B6, Folate, Vitamin B12, Pantothenic Acid, Biotin and Choline.Washing-ton, DC: National Academy Press; 1998. p. 306-56.

(78) Kang SS. Treatment of hyperhomocyst(e)inemia: physiological basis. J Nutr 1996;26(4 Suppl.):1273S-5S.

(79) Gibbons GF, Wiggins D, Brown AM, Hebbachi AM. Synthesis and function of hepatic very-low-density lipoprotein. Biochem Soc Trans 2004;32(1):59-64.

(80) Yao Z, Vance DE. The active synthesis of phosphatidylcholine is required for very low density lipoprotein secretion from rat hepatocytes. J Biol Chem 1988;263(6):2998-3004.

(81) Yao Z, Vance DE. Head group specificity in the requirement of phospha-tidylcholine biosynthesis for very low density lipoprotein secretion from cultured hepatocytes. J Biol Chem 1989;264(19):11373-80.

(82) Gruffat D, Durand D, Graulet B, Bauchart D. Regulation of VLDL synthesis and secretion in the liver. Reprod Nutr Dev 1996;36:375-89.

(83) Verkade HJ, Fast DG, Rusino AE, Scraba DG, Vance DE. Impaired bio-synthesis of phosphatidylcholine causes a decrease in the number of very low density lipoprotein particles in the Golgi but not in the endoplasmic reticulum of rat liver. J Biol Chem 1993;268(33):24990-6.

(84) Michel V, Singh RK, Bakovic M. The impact of choline availability on mus-cle lipid metabolism. Food Funct 2011;2:53-62.

(85) Buchman AL, Moukarzel A, Jende DJ, Roch M, Rice K, Ament ME. Low plasma free choline is prevalent in patients receiving long term parenteral nutrition and is associated with hepatic aminotransferase abnormalities. Clin Nutr 1993;12:33-7.

(86) Burt ME, Hanin I, Brennan MF. Choline deficiency associated with total parenteral nutrition. Lancet 1980 September 20;316(8195):638-9.

(87) Sheard NF, Tayek JA, Bistrian BR, Blackburn GL, Zeisel SH. Plasma cho-line concentration in humans fed parenterally. Am J Clin Nutr 1986;43:219-24.

(88) Buchman AL, Dubin MD, Moukarzel A, Jenden DJ, Roch M, Rice K et al. Choline deficiency: A cause of hepatic steatosis during parenteral nutrition that can be reversed with intravenous choline supplementation. Hepatol-ogy 1995;22:1399-403.

(89) Buchman AL, Ament ME, Sohel M, Dubin MD, Jenden DJ, Roch M et al. Choline deficiency causes reversible hepatic abnormalities in patients receiving parenteral nutrition: Proof of a human choline requirement: A placebo-controlled trial. JPEN 2001;25(5):260-8.

(90) Shronts EP. Essential nature of choline with implications for total paren-teral nutrition. J Am Diet Assoc 1997;97(6):639-45, 649.

(91) Ghoshal AK, Farber E. Biology of disease: Choline deficiency, lipotrope deficiency and the development of liver disease including liver cancer: A new perspective. Lab Investig 1993;68(3):255-60.

(92) Albright CD, Liu R, Bethea TW, daCosta KA, Salganik RI, Zeisel SH. Cho-line deficiency induces apoptosis in SV40-immortalized CWSV-1 rat he-patocytes in culture. FASEB J 1996;10:510-6.

(93) Yen CL, Zeisel SH. Choline phospholipids and cell suicide. In: Zeisel SH, Szuhaj BF, editors. Choline, Phospholipids, Health and Disease.Cham-paign, IL: AOCS Press; 1998. p. 11-32.

(94) Yen CL, Mar MH, Craciunescu CN, Edwards LJ, Zeisel SH. Deficiency in methionine, tryptophan, isoleucine, or choline induces apoptosis in cul-tured cells. J Nutr 2002;132:1840-7.

(95) Trauner M, Arrese M, Wagner M. Fatty liver and lipotoxicity. Biochim Bio-phys Acta 2010;1801:299-310.

(96) Canty DJ, Zeisel SH. Lecithin and choline in human health and disease. Nutr Rev 1994;52(10):327-39.

(97) Shin O-H, Mar MH, Albright CD, Citarella MT, daCosta KA, Zeisel SH. Methyl-group donors cannot prevent apoptotic death of rat hepatocytes induced by choline-deficiency. J Cell Biochem 1997;64:196-208.

(98) Detopolou P, Panagiotakis DB, Antonopolou S, Pitsavos C, Stefanidis C. Dietary choline and betaine intakes in relation to concentrations of in-flammatory markers in healthy adults: the ATTICA study. Am J Clin Nutr 2008;87:424-30.

(99) Selhub J. Homocysteine metabolism. Annu Rev Nutr 1999;19:217-46.

(100) Zeisel SH. Choline phospholipids: signal transduction and carcinogenesis. FASEB J 1993;7(551):557.

(101) Vicinanza M, D’Angelo G, DiCampli A, DeMatteis MA. Function and dys-function of the PI system in membrane trafficking. EMBO J 2008;27:2457-70.

(102) Tracey KJ. Physiology and immunology of the cholinergic antiinflamma-tory pathway. J Clin Investig 2007;117(2):289-96.

(103) Pavlov VA, Tracey KJ. Controlling inflammation: the cholinergic anti-in-flammatory pathway. Biochem Soc Trans 2006;34(6):1037-40.

(104) Wang H, Yu M, Ochani M, Amella CA, Tanovic M, Susarla S et al. Nicotinic acetylcholine receptor α 7 subunit is an essential regulator of inflamma-tion. Nature 2003;421:384-8.

(105) Tracey KJ. The inflammatory reflex. Nature 2002;420:853-9.

(106) Racke K, Juergens UR, Matthiesen S. Control by cholinergic mecha-nisms. Eur J Pharmacol 2006;533:57-68.

(107) Wang XF, Yang ZG, Xue B, Shi H. Activation of the cholinergic antiinflam-matory pathway ameliorates obesity-induced inflammation and insulin resistance. Endocrinology 2011;152(3):836-46.

(108) Li D-J, Evans RG, Yang Z-W, Song S-W, Wang P, Ma X-J et al. Dysfunc-tion of the cholinergic anti-inflammatory pathway mediates organ damage in hypertension. Hypertension 2011;57:298-307.

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