Choline is a essential nutrient for humans it is consumed
in many foods.
It is a constituent of all cell membranes
and is necessary for growth and development. Also, as
the major precursor of betaine, it is used by the kidney
to maintain water balance and by the liver as a source of
methyl groups for the removal of homocysteine in methionine
formation. Finally, choline is used to produce the
important neurotransmitter (nerve messenger chemical)
acetylcholine, which is involved in memory and other
nervous system functions (Fig. 1). Maternal diets deficient
in choline during the second half of pregnancy in
rodents caused decreased neurogenesis and increased
neuronal apoptosis in fetal hippocampus (the memory
center), resulting in permanent behavioral (memory)
modifications in the offspring. Dietary deficiency of
choline in rodents causes development of liver cancer in
the absence of any known carcinogen. In humans, dietary
deficiency of choline is associated with fatty liver and liver
The dietary requirement for choline is influenced
by gender as well as by genetic polymorphisms.
Using a comprehensive database of the choline content of foods, a
number of epidemiological studies identified associations
between dietary choline intake and plasma homocysteine
levels (risk factor for cardiovascular disease), cancer, and
BIOCHEMISTRY AND RELATIONSHIPS
WITH OTHER NUTRIENTS
Choline is needed for synthesis of several major phospholipids
(phosphatidylcholine and sphingomyelin) in cell
membranes and is also involved in methyl metabolism,
cholinergic neurotransmission, transmembrane signaling,
and lipid–cholesterol transport and metabolism (1)
(Fig. 2). Choline can be acetylated, phosphorylated, oxidized,
or hydrolyzed. There are several comprehensive
reviews of the metabolism and functions of choline (1).
Cells absolutely require choline and die by apoptosis
when deprived of this nutrient (2,3). Humans derive
choline from foods, as well as from the de novo biosynthesis
of the choline moiety via the methylation of phosphatidylethanolamine
using (S)-adenosylmethionine as
the methyl donor (most active in the liver). This ability
to form choline means that some of the demand for
choline can, in part, be met by using methyl groups derived
from one carbon metabolism (via methyl-folate and
methionine). Several vitamins (folate, vitamin B12, vitamin
B6, and riboflavin) and the amino acid methionine interact
with choline in 1-carbon metabolism. There has been
renewed interest in these pathways during the past several
years, engendered by recent insights that indicate that
modest dietary inadequacies of the above-mentioned nutrients,
of a degree insufficient to cause classical deficiency
syndromes, can still contribute to important diseases
such as neural tube defects, cardiovascular disease, and
Perturbing the metabolism of one of these pathways
results in compensatory changes in the others (1).
For example, methionine can be formed from homocysteine
using methyl groups from methyl-tetrahydrofolate
(THF), or using methyl groups from betaine that are
derived from choline. Similarly, methyl-THF can be
formed fromone-carbon units derived fromserine or from
the methyl groups of choline via dimethylglycine, and
choline can be synthesized de novo using methyl groups
derived from methionine [via (S)-adenosylmethionine].
Pathways of choline metabolism
Choline can be a methyl-group donor and interacts with methionine
and folate metabolism. It can be acetylated to form the neurotransmitter
acetylcholine, and it can be phosphorylated to form
membrane phospholipids such as phosphatidylcholine (lecithin)
and sphingomyelin. Choline can be formed via the methylation of
phosphatidylethanolamine (forming phosphatidylcholine, which
can be hydrolyzed to make choline).
use more methyl-THF to remethylate homocysteine in the
liver and increase dietary folate requirements. Conversely,
when they are deprived of folate, they use more methyl
groups from choline, increasing the dietary requirement
for choline (5). The availability of transgenic and knockout
mice has made possible additional studies that demonstrate
the interrelationship of these methyl sources (6).
When considering dietary requirements it is important to
realize that methionine, methyl-THF, and choline can be
fungible sources of methyl groups.
esterified forms such as phosphocholine, glycerophosphocholine,
sphingomyelin, and phosphatidylcholine
(7). Lecithin is a term often used interchangeably
with phosphatidylcholine, whereas the compound is a
phosphatidylcholine-rich mixture added as an emulsifying
agent in the food industry. Pancreatic enzymes can liberate
choline from dietary phosphocholine, glycerophosphocholine,
and phosphatidylcholine. Before choline can
be absorbed in the gut, some is metabolized by bacteria
to form betaine and methylamines (which are not methyl
There is no estimate for percentage absorption of the
various forms of choline in humans. The water-soluble
choline-derived compounds (choline, phosphocholine,
and glycerophosphocholine) are absorbed via the portal
circulation, whereas the lipid-soluble compounds
(phosphatidylcholine and sphingomyelin) are absorbed
as chylomicrons. Lecithin is the most abundant choline containing
compound in the diet. About half of the lecithin
ingested enters the thoracic duct, and the remaining is metabolized
to glycerophosphocholine in the intestinal mucosa
and then to choline in the liver. The liver takes up
the majority of choline and stores it in the form of phosphatidylcholine
and sphingomyelin. The kidney and the
is excreted with urine, most is oxidized in the kidney to
form betaine, which is responsible for maintaining the osmolarity
in the kidney. A specific carrier is needed for the
transport of free choline across the blood–brain barrier;
the capacity is especially high in neonates.
Choline and Epigenetics
Choline and other methyl donors are important dietary
modulators of epigenetic marks on genes. The term “epigenetics”
defines heritable changes in gene expression
that are not coded in the DNA sequence itself. Epigenetic
mechanisms include DNA methylation and histone modification.
DNA methylation occurs predominantly at the
cytosine bases followed by a guanosine (CpGs). When it
occurs in promoter regions that regulate DNA transcription,
the expression of the associated gene is altered (8,9).
Although there are exceptions, increased methylation is
usually associated with gene silencing, whereas decreased
methylation with induced gene expression. Another epigenetic
mechanism is histone modification (10). Histones
are proteins around which DNA is tightly wound, forming
the dynamic structure called chromatin. Chromatin can be
either an inactive state or an active state at which transcription
factors can pass through. Histone acetylation predominantly
promotes active chromatin, whereas histone
methylation can be associated with both transcriptionally
active and inactive chromatin. Furthermore, the degree of
methylation (mono-, di-, or tri-) results in distinct effects
on chromatin state (11). Methylation of DNA and histone
requires (S)-adenosylmethionine to methylate cytosines
in DNA and lysine and arginine residues in histones, respectively.
The availability of (S)-adenosylmethionine is
directly influenced by dietary choline and other methyl
Examples of epigenetic effects of choline and other
methyl donors include experiments in rodents in which
pregnant dams were fed diets that were choline deficient
versus normal, and DNA methylation in fetal brain was
modified, resulting in over expression of genes that inhibit
cell cycling in neural progenitor cells of developing brain
(12,13). Gestational choline availability also affects histone
methylation in the developing embryo, resulting in
changes in expression of genes that regulate methylation
and neuronal cell differentiation (14). Feeding pregnant
Pseudoagouti Avy/a mouse dams a choline and methylsupplemented
diet altered epigenetic regulation of agouti
expression in their offspring, as indicated by increased
agouti/black mottling of their coats and by lean body
phenotype (15,16). In another example, choline and
methyl donor supplementation to dams increased DNA
methylation of the fetal gene Axin fused [Axin(Fu)] and
reduced incidence of tail kinking in Axin(Fu)/+ offspring
by 50% (17). Thus, dietary manipulation of choline and
methyl donors (either deficiency or supplementation) can
have a profound impact upon gene expression.
HUMAN REQUIREMENT FOR CHOLINE
In one of the first clinical nutrigenomics studies, humans
were phenotyped with respect to their susceptibility
to developing organ dysfunction when fed a low choline
diet (18–21). Adult men and women (pre- and
postmenopausal) aged 18 to 70 years were fed a standard
diet containing a known amount of choline (550 mg/
70 kg/day; baseline) for 10 days. On day 11 subjects were
placed on a diet containing less than 50 mg choline/day
for up to 42 days. Blood and urine were collected to measure
various experimental parameters of dietary choline
status, and markers of organ dysfunction and liver fat
were assessed. If at some point during the depletion period,
functional markers indicated organ dysfunction associated
with choline deficiency, subjects were switched
to a diet containing choline until replete.
Most men and postmenopausal women fed the low choline
diets developed reversible fatty liver (measure by
mass resonance spectroscopy) as well as liver and muscle
damage, whereas 56% of premenopausal women were
resistant to developing choline deficiency (21). The fatty
liver occurred due to lack of phosphatidylcholine synthesis
in liver, which is required for very low density lipoprotein
(VLDL) synthesis needed for export of excess triacylglycerol
from liver (22). Choline deficiency liver damage
was characterized by elevated serum aminotransferase
(23) and muscle damage was characterized by elevated
plasma serum creatine phosphokinase (19): both were due
to increased rates of apoptosis in these tissues (also occurred
in peripheral lymphocytes (24)). Choline-deficient
subjects also had impaired ability to handle a methionine
load, resulting in elevated plasma homocysteine concentrations
Only 44% of premenopausal women develop
signs of choline deficiency when deprived of dietary
choline as compared with most adult men and postmenopausal
women, suggesting their higher resistance
to choline deficiency (19,20). Premenopausal women required
less dietary choline because estrogen induces the
gene to enhance the de novo biosynthesis of choline moiety
(26). Estrogen binds to its receptors, and this complex
interacts with estrogen response elements (EREs) in the
promoter of the PEMT gene, resulting in an upregulation
in PEMT mRNA expression and in hepatic enzyme activity
(26). Estrogen as the mediator of increasing PEMT activity
in women is important, especially during pregnancy
when fetal development uses a great deal of choline. Estradiol
concentration rises from 1 to 60 nMduring pregnancy
(27,28), suggesting that the capacity for endogenous synthesis
most. Pregnancy and lactation are stages of life that demand
high dietary choline intake and leave mothers extremely
vulnerable to choline deficiency (29). In utero,
the fetus is exposed to very high choline concentrations,
with a progressive decline in blood choline concentration
until adult levels of choline concentration are achieved
after the first weeks of life (30). Plasma or serum choline
concentrations are 6–7°ø higher in the fetus and newborn
than those in adults (31,32). High circulating choline in
the fetus and neonate ensures the availability of choline to
Less than 15% of pregnant women consume the recommended
adequate intake for choline (33), and in casecontrol
studies in California, women eating diets in the
lowest quartile for choline were at fourfold increased risk
for having a baby with a neural tube defect and at almost
twofold increased risk for having a baby with a cleft
palate; these risks were calculated after controlling for folate
Genetics of Choline Requirements
Although premenopausal women are more resistant to
choline deficiency, a significant portion of them (44%) still
develops organ dysfunction when deprived of choline,
suggesting individual differences in susceptibility to
choline deficiency. In fact, some men and women require
more than 850 mg/70 kg/day choline in their diet,
whereas others require less than 550 mg/kg/day (21).
Genetic variation likely underlies the differences in these
dietary requirements. A single-nucleotide polymorphism
(SNP) is a genetic variation occurring when a single nucleotide
(A, T, C, or G) in the genome sequence is altered.
These variations may affect metabolism. Only a few
reports investigate whether SNPs in the genes involved
in one carbon metabolism have roles in choline requirements
(36,37). Premenopausal women with a SNP in 5,10-
methylenetetrahydrofolate dehydrogenase (MTHFD1
rs2236225) were 15°ø more susceptible to choline deficiency
than did noncarriers. This variant increased the
use of choline perhaps by limiting the availability of
methyl-folate for Hcy remethylation and increasing the
demand for choline as a methyl-group donor. In addition,
individuals with a SNP in PEMT (rs12325817) were more
susceptible to choline deficiency, and women harboring
this SNP were more affected than did men. SNPs in
the PEMT gene alter endogenous synthesis of choline,
thereby increasing the dietary requirement for choline.
In 1998, the Institute of Medicine (IOM) made recommendations
for choline intake in the diet (4). At the time, there
were insufficient data with which to derive an estimated
average requirement for choline, thus only an adequate intake
(AI) could be estimated. The IOM report cautioned,
“this amount will be influenced by the availability of methionine
and methyl-folate in the diet. Itmaybe influenced
by gender, and it may be influenced by pregnancy, lactation,
and stage of development. Although AIs are set for
by endogenous synthesis at some of these stages.”
In foods, there are multiple choline compounds that contribute
phosphocholine, phosphatidylcholine, and
sphingomyelin) (7). The U.S. Department of Agriculture
(USDA) maintains a database of choline content in
foods (38). Excellent sources of dietary choline are foods
that contain membranes, such as eggs and liver. Average
dietary choline intake on ad libitum diets for males and
females are 8.4 and 6.7 mg/kg choline per day.
commercially available infant formulas. Until recently
some infant formulas had inadequate choline content (especially
soy-derived infant formulas), but in 2007–2008,
many infant formula companies increased the choline content
of their formulas so that they matched mature breast
milk. These formulas still have different mixtures of the
esters of choline than are present in human milk, perhaps
resulting in different bioavailability as compared to human
High doses of choline (>6 g) have been associated with excessive
cholinergic stimulation, such as vomiting, salivation,
sweating, and gastrointestinal effects (4). In addition,
fishy body odor results from the excretion of trimethylamine,
a choline metabolite from bacterial action (24). The
tolerable upper limit for choline has been set at 3 g/day (4).
Assessing Choline Status
in estimating choline status, but the measure is not definitive.
Plasma choline concentration varies in response to
diet and can rise as much as twofold after a two-egg
meal. Fasting plasma choline concentrations vary from7 to
15 M, with most subjects having concentrations of
10 M. Individuals that have starved for up to seven
days have diminished plasma choline, but levels never
drop below 50% of normal, probably because tissue phospholipids
are “cannibalized” to prevent concentrations
of choline from falling further (42). Note that children
during the first year of life have normal plasma choline
concentrations that are higher than 10 to 15 M (43).
Plasma phosphatidylcholine concentration also decreases
in choline deficiency (44), but these values are also influenced
by factors that change plasma lipoprotein levels.
Fasting plasma phosphatidylcholine concentrations
are approximately 1 to 1.5 mM. Thus, measurements of
choline or phosphatidylcholine in blood identify subjects
with low dietary choline intake, but provide little help in
differentiating the degree of deficiency.
CHOLINE AND CARDIOVASCULAR DISEASE
Choline and betaine may benefit heart health by lowering
blood pressure, altering blood lipid profiling, and reducing
plasma Hcy, a risk factor for cardiovascular disease
(CVD) (45). Dietary choline intake was found to have a statistically
significant inverse relationship to circulating Hcy
concentrations in the Framingham Heart Study (46) and in
the Nurse’s Health Study (25), suggesting a protective effect
of choline intake. However, when looking at the association
between dietary choline intake and CVDincidence,
no association was found (14) in the European Prospective
Investigation into Cancer and Nutrition (EPIC) study
(47), and a marginal positive association was found in the
Atherosclerosis Risk in Communities (ARIC) study (48).
It is important to note that in the ARIC study, most individuals
in the cohort had choline intake below AI (49).
Hence, the effects of choline supplementation on CVD
risk remain unknown. Some human studies suggested
that high betaine supplementation increases plasma lowdensity
lipoprotein (LDL) cholesterol and triacylglycerol
concentrations (50–52), effects that might counterbalance
its Hcy lowering effects. However, the changes in serum
lipid concentrations were not associated with higher risk
of CVD. Moreover, the rise in LDL concentration may
be an artifact of increasing VLDL and triacylglycerol
excretion from fatty liver to plasma, which is not an adverse
outcome (for critical review see Ref. 53). The relationship
between choline and heart health warrants more
The choline-containing phospholipid phosphatidylcholine
has been used as a treatment to lower the
cholesterol concentrations because lecithin-cholesterol
acyltransferase has an important role in the removal of
cholesterol from tissue. Betaine, the oxidized product
of choline, has been used to normalize the plasma
homocysteine and methionine levels in patients with
homocystinuria, a genetic disease caused by 5,10-
methylenetetrahydrofolate reductase deficiency. Therefore,
dietary choline intake might be correlated with
cardiovascular disease risk. Many epidemiologic studies
have examined the relationship between dietary folic acid
and cancer or heart disease. It may be helpful to also consider
choline intake as a confounding factor because folate
and choline methyl donation can be interchangeable (7).
CHOLINE DEFICIENCY AND CANCER
An interesting effect of dietary choline deficiency in rats
and mice has never been studied in humans. Dietary
deficiency of choline in rodents causes development of
hepatocarcinome in the absence of any known carcinogen
(54). Choline is the only single nutrient for which
this is true. It is interesting that choline-deficient rats not
only have a higher incidence of spontaneous hepatocarcinoma
but also are markedly sensitized to the effects of
administered carcinogens. Several mechanisms are suggested
for the cancer-promoting effect of a choline-free
diet. These include increased cell proliferation related to
regeneration after parenchymal cell death occurs in the
choline-deficient liver; hypomethylation of DNA (alters
expression of genes); reactive oxygen species leakage from
mitochondria with increased lipid peroxidation in liver;
activation of protein kinase C signaling due to accumulation
of diacylglycerol in liver; mutation of the fragile
histidine triad (FHIT) gene, which is a tumor suppressor
gene; and defective cell-suicide (apoptosis) mechanisms
(54). Loss of PEMT function may also contribute to malignant
transformation of hepatocytes (55).
Only a handful of epidemiologic studies explore
how choline and betaine intakes alter cancer risk in populations.
This was perhaps due to the absence of food composition
data, which has not been developed until recently
(7). The Long Island Breast Cancer Study found that high
choline consumption was associated with reduced breast
cancer risk (56), and high choline and betaine consumption
was associated with reduced breast cancer mortality
(57). Moreover, individuals with PEMT rs12325817 and
CHDH rs12676 SNPs had lower risk of developing breast
cancer, whereas BHMT rs3733890 had lower breast cancer
mortality. These data suggest the importance of nutrients
and genetic interactions in the etiology of cancer. Alternatively,
the Nurse’s Health Study II found no association
between choline intake and breast cancer risk (58), but a
positive association between choline intake and colorectal
cancer risk (59), suggesting different etiologies between
breast and colorectal cancer. More research is warranted.
CHOLINE AND BRAIN
Choline and Brain Development
In rodents, maternal dietary choline intake during late
pregnancy modulated mitosis and apoptosis in progenitor
(stem) cells of the fetal hippocampus and septum
and altered the differentiation of neurons in fetal hippocampus
(60). Variations in maternal dietary choline
during late pregnancy were also associated with significant
and irreversible changes in hippocampal function in
the adult animal, including altered long-term potentiation
(LTP) and altered memory (61). More choline (about 4°ø
dietary levels) during days 11–17 of gestation in the rodent
increased hippocampal progenitor cell proliferation,
decreased apoptosis in these cells, enhanced LTP in the
offspring when they were adult animals, and enhanced
visuospatial and auditory memory by as much as 30%
in the adult animals throughout their lifetimes (61). The
enhanced maze performance appears to be due to cholineinduced
improvements in memory capacity. Indeed, adult
rodents decrement in memory as they age, and offspring
exposed to extra choline in utero do not show this
“senility” (62). In contrast, mothers fed choline-deficient
diets during late pregnancy have offspring with diminished
progenitor cell proliferation and increased apoptosis
in fetal hippocampus, insensitivity to LTP when they
were adult animals, and decremented visuospatial and
auditory memory (61).
Early postnatal choline supplementation significantly
attenuated the effects of prenatal alcohol on a learning
task, suggesting that early dietary interventions may
also influence brain development (63). The mechanisms
for these developmental effects of choline are not yet clear.
Fetal alcohol syndrome (FAS) is an important concern of
pediatricians, with 1 in every 750 infants born with FAS
each year in the United States. Rats exposed to alcohol during
the perinatal period had poor performance on memory
tasks, which were improved by either prenatal or postnatal
choline supplementation (64,65). Rett syndrome (RTT),
a neurodevelopmental disorder associated with mutations
in the methyl-CpG-binding protein 2 (MeCP2) gene, is the
second leading cause of mental retardation in girls. RTT
girls experience a variety of deficits in cognitive, motor,
and social functions. In mouse models of RTT, enhancing
maternal or postnatal choline supplementation attenuates
motor coordination deficits and improves neuronal
integrity, proliferation, and survival (66,67). Choline supplementation
also ameliorates the symptoms in rodent
models of traumatic brain injury (68), status epilepticus
(69–71), and schizophrenia (72).
Are these findings in animals likely to be true in humans?
We do not know. Human and rat brains share many
elements of brain development but they mature at different
rates. In terms of hippocampal development, the embryonic
days 12–18 in the rat correspond to approximately
the last trimester in humans. Rat brain is comparatively
more mature at birth than is the human brain, but human
hippocampal development may continue for months or
years after birth.
Choline and Adult Brain
Acetylcholine is one of the most important neurotransmitters
used by neurons in the memory centers of brain
(hippocampus and septum). Choline accelerates the synthesis
and release of acetylcholine in nerve cells. Choline
used by brain neurons is largely derived from membrane
lecithin, or fromdietary intake of choline and lecithin. Free
choline is transported across the blood–brain barrier at a
rate that is proportional to serum choline level; lecithin
may be carried into neurons as part of an ApoE lipoprotein.
Choline derived from lecithin may be especially important
when extracellular choline is in short supply, as
might be expected to occur in advanced age because of
decreased brain choline uptake (73).
Results from studies using choline or phosphatidylcholine
to treat adults with brain disorders have been
very variable. Single doses of choline or lecithin in adult
humans may enhance memory performance in healthy
individuals, perhaps with greatest effect in individuals
with the poorest memory performance. Studies in students
showed that lecithin or choline treatment improved
memory transiently for hours after administration (74). In
humans with Alzheimer-type dementia, some studies report
enhanced memory performance after treatment with
lecithin (75), whereas other studies did not observe this.
Buchman et al. recently reported that humans on longterm
total parenteral nutrition may have verbal and visual
memory impairment, which may be improved with
choline supplementation (76). If lecithin is effective, it is in
a special subpopulation in the early stages of the disease.
Choline and lecithin have also been effectively used to
treat tardive dyskinesia, presumably working by increasing
cholinergic neurotransmission (77).
Choline in the diet is important for many reasons. Humans
deprived of it develop liver and muscle dysfunction, and
parenterally nourished patients need a source of choline.
As our understanding of the importance of folate and homocysteine
nutrition increases, there should be increased
interest in how choline interacts with these compounds.
Recent findings about choline in brain development in
animals should stimulate comparable studies in humans.
The availability of food composition data now makes it
possible to examine interactions between choline, folate,
and methionine when considering epidemiological data.
This work was supported by grants from the National
Institutes of Health (AG09525, DK55865). Support for this
work was also provided by grants from the NIH to the
UNC Clinical Nutrition Research Unit (DK56350).
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