Caballero B. (ed.) Encyclopaedia of Food Science, Food Technology and Nutrition. Ten-Volume Set

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7a-hydroxylase, acyl CoA cholesterol acyl trans-

ferase (ACAT) and lecithin cholesterol acyl transfer-

ase (LCAT). The 7a-hydroxylase catalyzes the first

step in the conversion of cholesterol to bile acids,

while ACAT and LCAT are responsible for esterifying

cholesterol in cells and plasma, respectively.

Cholesterol and Lipoprotein Transport

0006 Cholesterol is transported along with other lipids

by the plasma lipoproteins. These are classified

according to their size and density, which are func-

tions of their compositions (Table 2). Each class is

characterized by specific proteins (apoproteins), some

of which have known important functions (Table 3).

Mutations in apoproteins are involved in several

types of hyperlipidemia. Although the liver contains

a large amount of cholesterol because of immense

amounts of membraneous organelles, it does not

store extra cholesterol. Normal nascent (newly se-

creted by the liver) very-low-density lipoprotein

(VLDL) has a cholesterol ester (CE)/triacylglycerol

(TG) ratio of < 1:4, but CE-rich VLDL may have a

ratio of 1:1. CE-rich VLDL is synthesized by the liver

in some experimental animal models fed very high

levels of dietary cholesterol. Such VLDL has extra

apoenzyme E (apoE). This may be important because

macrophages have not only the apoenzyme (apoB)/

LDL receptor, but also a B/E receptor, which will bind

apoB and/or apoE lipoproteins, as discussed below.

0007Circulating VLDL loses much of its triglyceride to

tissues and some of its other components to high-

density lipoprotein (HDL); in the process, VLDL

becomes transformed to the cholesterol-rich LDL

particle. LDL binds to its specific receptor on cells

and is carried into the cell by receptor-mediated endo-

cytosis. The major function of the LDL receptor is

delivery of lipids to cells, but in the liver, it helps

remove LDL and small VLDL from the bloodstream.

The receptor is subject to transcriptional control in

which high [cholesterol] leads to decreased receptor

synthesis, thus downregulating the entry of apoB-

containing lipoproteins. The LDL receptor is ex-

pressed in most cells and is basically the same in all

tissues, but the number of receptors varies greatly

between tissues. The greatest density of receptors

occurs in the adrenal cortex, which requires enor-

mous amounts of cholesterol for steroid hormone

synthesis. It is expressed at low levels in nondividing

cells, presumably because there is less need for chol-

esterol for membrane synthesis. Cholesterol that is

not immediately used in the cell is esterified by

tbl0002 Table 2 Classification and composition of major human plasma lipoproteins

Class Density (g ml

1

) Percentage

protein

Percentage

cholesterol

Percentage

cholesterolester

Percentage

triacylglycerol

Chylomicrons <1.00 2 1 3 85

Very-low-density lipoprotein 0.95–1.006 10 7 12 50

Low-density lipoprotein 1.006–1.063 23 8 37 10

High-density lipoprotein 1.063–1.210 55 2 15 4

The other major constituents of these lipoproteins are phospholipids; minor components are sphingolipids, carotenoids, and other hydrophobic

compounds.

tbl0003 Table 3 Major human apolipoproteins

Apoprotein Associatedlipoprotein Function

ApoA-1 HDL LCAT activation; possibly involved with reverse cholesterol transport

ApoA-II HDL Transport of HDL lipids; unknown

ApoA-IV HDL, chylo Unknown; possibly antiatherogenic

ApoB-48 Chylo Lipid transport

ApoB-100 VLDL, LDL Lipid transport; LDL receptor interaction

ApoC-1 VLDL, HDL Unknown; may function with A-I to activate LCAT

ApoC-II VLDL, HDL, chylo Activatation of lipoprotein lipase

ApoC-1II VLDL, HDL, chylo Inhibition of lipoprotein lipase

ApoD HDL Unknown

ApoE VLDL, HDL, Chylo Clearance of VLDL and chylomicrons from plasma;

abundant in brain and cerebrospinal fluid, modifies risk

of Alzheimer dementia

Apo, apoenzyme; chylo, chylomicron; HDL, high-density lipoprotein; LDL, low-density lipoprotein; VLDL, very-low-density lipoprotein.

1246 CHOLESTEROL/Role of Cholesterol in Heart Disease

ACAT, but normally, there are only small amounts of

the ester within cells. Among the other factors

thought to regulate LDL receptor expression are thy-

roid hormone, insulin, and platelet-derived and other

types of growth factors.

0008 Once LDL is delivered to cells, no degradation of

cholesterol can occur in peripheral cells, and removal

of cholesterol appears to be a function of another

lipoprotein. HDL seems to function in ‘reverse chol-

esterol transport,’ in which it helps remove choles-

terol from cells and return it to the liver for excretion

in the bile. The exact mechanism and sequence of

events are not yet understood. High levels of HDL

cholesterol appear to indicate that more reverse chol-

esterol transport is occurring. This topic is discussed

elsewhere in this volume.

Oxidation of LDL

0009 The term oxidized LDL (oxLDL) encompasses a var-

iety of particles, ranging from slight oxidation of LDL

fatty acids to particles with oxidation of fatty acids,

cholesterol and even apoB. LDL that has undergone

certain modifications such as peroxidation or chem-

ical acetylation or maleylation of apoB is taken up

avidly by the modified-LDL receptor, also called the

scavenger receptor. The expression of this receptor is

independent of cell [cholesterol], and there is no

downregulation of receptor number. Cholesterol ac-

cumulation can occur in macrophages and other cells

that have this receptor. In addition, fatty acid oxida-

tion fragments such as malondialdehyde may react

with lysine residues of apoB, which may increase its

ability to accumulate in macrophages. OxLDL is not

a good ligand for the native LDL receptor, but it is

more atherogenic than native LDL. After the discov-

ery of the LDL receptor by Mark Brown and Joseph

Goldstein in the mid-1970s, it gradually became evi-

dent that cholesterol accumulation in arterial lesions

involves some processes other than uptake of native

LDL by its receptors, since subjects with homozygous

familial hypercholesterolemia, whose cells lack the

receptors, accumulate cholesterol in ‘foam’ cells. Fur-

thermore, ‘foam’ cells develop from macrophages/

monocytes and smooth muscle cells, but in vitro

experiments show that neither of these cell types

accumulates much cholesterol from native LDL.

Also, when native LDL delivers cholesterol and

other lipids to cells via receptor-mediated endocyto-

sis, feedback mechanisms decrease the number of

receptors on the cell surface. This is called downre-

gulation of the receptor, a process that normally helps

maintain cholesterol homeostasis. Further experi-

mentation has shown that the macrophages take up

chemically modified LDL by a separate, nonregulated

mechanism that allows accumulation and cellular

damage (Table 4).

0010It was soon learned that in vitro incubation of LDL

with cultured endothelial cells creates oxidatively

modified LDL that is readily bound and accumulated

by the macrophages. Addition of antioxidants in vitro

prevents this modification of LDL. We now know

that the uptake of oxidized LDL occurs via a distinct

receptor, called scavenger receptor A (SRA), on

macrophages/monocytes and smooth muscle cells.

Because this receptor is not downregulated, and

LDL lipids accumulate in these cells, eventually they

become ‘foam cells.’ Even slightly oxidized LDL,

often called ‘minimally modified,’ can be recognized

and taken up by the SRA. More than one type of

scavenger receptor is present on macrophages, and

other types of modification of LDL may also contrib-

ute to increased uptake by the macrophage. The

sequence of events thought to be involved in athero-

genesis is discussed below. Studies with experimental

animals and in vitro cell-culture studies suggest that

the use of antioxidants may help reduce or inhibit

production of oxLDL and therefore may help inhibit

atherogenesis. However, although epidemiologic data

offer evidence of an inverse relationship between diet-

ary antioxidant intake and risk of coronary heart

disease (CHD), effectiveness in preventing athero-

sclerosis has not yet been conclusively demonstrated

in humans.

Atherogenesis

0011Atherosclerosis (Greek athe

‘gruel’ þskle

sis ‘hard-

ness’) occurs within major arteries when cholesterol-

laden lesions build up in the lining and wall, eventu-

ally resulting in narrowing and sometimes occlusion

of the lumen. In addition to decreasing the flow

through the narrowed vessel, the lesions, called

fibrous plaques, can cause thrombosis (blood clots),

which may lead to CHD (also called myocardial in-

farction (MI) or heart attack), decreased oxygen de-

livery to the brain or other organs, or thrombotic

tbl0004Table 4 Detrimental effects attributed to oxidized LDL

Avidly binds scavenger receptors, leading to accumulation of

LDL lipids in arterial macrophages

Cytotoxic to arterial endothelial cells in vitro and possibly in vivo

Stimulates release of mitogenic factor(s) for macrophages and

smooth muscle cells

Stimulates recruitment of additional monocytes/macrophages

from bloodstream into intima

Is immunogenic and leads to autoantibody production

Antagonizes normal nitric oxide-induced vasodilation of

arteries

CHOLESTEROL/Role of Cholesterol in Heart Disease 1247

stroke. Atherosclerosis is usually described as a multi-

factorial disease with numerous risk factors (Table 5).

Although genetics seems to be a predominant risk

factor, attention has focused on those factors that

can be changed, namely hypertension and blood

cholesterol levels. Risk factors appear to be multi-

plicative rather than merely additive. Risk varies be-

tween races, and in America, blacks have a higher

incidence of CHD than whites of the same age.

Blood cholesterol levels do not differ between the

two groups, and the higher incidence of CHD is at-

tributed to the greater incidence of hypertension

among blacks.

0012Theories on the etiology of atherosclerosis have

gradually coalesced into the prevailing ‘endothelial

injury’ hypothesis, which encompasses most in vivo

and in vitro observations (Table 6). The endothelium

lining of arteries plays a major role in the develop-

ment of atherosclerosis. The response to endothelial

injury is similar to the inflammatory response to in-

juries elsewhere. This may occur at the bifurcation of

blood vessels where flow is turbulent; it may be in

response to the constant insult of hypertension or

to chemicals such as benzo(a)pyrene in cigarette

smoke. White blood cells invade the area. If high

levels of LDL are present, oxidation of LDL, possibly

by endothelial cells, results in the formation of a

modified (oxidized) LDL. OxLDL triggers a series

of events: it causes more endothelial injury, and

endothelial cells synthesize endothelial-leukocyte ad-

hesion molecules (ELAMs). The latter attract mono-

cytes and T lymphocytes that adhere to the

endothelium. These move below the endothelium

and not only engulf more oxLDL but also secrete

cytokines and growth factors that stimulate move-

ment of smooth muscle cells and macrophages to

the area where all cells begin to accumulate increasing

amounts of lipid, particularly cholesterol esters. Over

a number of years, the process results in pronounced

lesions (Table 7).

tbl0005 Table 5 Primary and secondary risk factors for coronary

heart disease

Primary risk factors for CHD

Serum [cholesterol] >5.2 mmol l

1

[HDL cholesterol] <0.9 mmol l

1

Hypertriglyceridemia

Heredity

Hypertension

Smoking

Age (risk increases at 45 þ years age for men, 55 þ for women)

Diabetes mellitus

Obesity

Physical inactivity

Stress

Secondary risk factors for hyperlipidemia

Nephrotic syndrome

Alcoholism

Various drugs

Certain hormonal disorders (e.g., Cushing’s disease)

Systemic lupus erythematosis

Pregnancy

A normal physiologic response to pregnancy.

tbl0006 Table 6 Likely sequence of events and relationship of LDL and atherogenesis

Dietary, genetic, and environmental factors

Increased blood levels of LDL lipids (cholesterol, triglycerides)

Greater adherence of monocytes to endothelial lining of arteries and more LDL entering lining of arteries

Oxidative modification of LDL to MM-LDL by cells in the intima (lining)

Stimulation of release of MCP-1 from endothelial cells by oxLDL; and simultaneous stimulation of release of MCSF

Movement of more monocytes into the area from the bloodstream; differentiation of these cells into macrophages with increased SRAs

Increased uptake of modified LDL by SRA and accumulation of cholesterol esters

Recruitment of smooth muscle cells from arterial media to intima; induction of SRA expression in these cells

Formation of lipid-laden foam cells from macrophages and smooth muscle cells

Formation of fatty streaks

Continuation of above sequence, leading eventually to localized cell necrosis, atheroma, and finally plaque formation

MCP-1, monocyte chemoattractant protein-1; MCSF, macrophage colony-stimulating factor; MM-LDL, minimally modified low-density lipoprotein; oxLDL,

oxidized LDL; SRA, scavenger receptor-1.

1248 CHOLESTEROL/Role of Cholesterol in Heart Disease

0013 Studies in molecular genetics are proving helpful in

establishing the relationships between lipoproteins,

lipids, apolipoproteins, membrane proteins, and nu-

merous other factors involved in cholesterol metabol-

ism and atherogenesis. Also, the use of transgenic

technology has provided animal models for studying

atherogenesis. There now exist transgenic animals

(mice or rabbits) for overexpression of most of the

apolipoproteins, as well as LCAT, cholesterol ester

transfer protein, and scavenger receptor B-1 protein.

In addition, ‘knockout’ animal models, in which cer-

tain apolipoprotein genes are missing, are being stud-

ied to help determine the functions of the proteins.

Intervention Trials and Current Status

0014 While epidemiologic studies long ago suggested the

relationship between high blood cholesterol levels

and increased incidence of atherosclerosis and CHD,

much of the progress in understanding the etiology,

pathology, and relationships has been made in

the last two to three decades. The positive

correlation between blood cholesterol levels and risk

of CHD-related deaths was established in the 1970–

80s by the Framingham Heart Study and the Lipid

Research Clinics Cornary Primary Prevention Trial.

In the mid- to-late 1980s, health agencies in many

countries published ranges of ‘desirable’ vs. ‘undesir-

able’ cholesterol levels, along with recommendations

on modifying high levels of total and LDL-cholesterol

levels (Table 8). But whether deliberately lowering

cholesterol levels could decrease the risk of CHD-

related events remained questionable until recent

years. Several intervention studies have now provided

good evidence that reducing plasma cholesterol levels

in patients with established CHD with drugs and/or

diet does in fact reduce the incidence of both fatal and

nonfatal heart attacks. The efficacy of early studies

with some of the statin drugs appeared questionable

because, although the incidence of CHD deaths de-

clined, overall mortality did not. Newer studies, how-

ever, have shown no increase in noncardiovascular

disease deaths. Table 9 summarizes some of these

studies.

0015Current thinking is that there should be aggressive

cholesterol management in most patients with CHD,

irrespective of the etiology of the condition. Very-

low-fat diets diets help to reduce total plasma choles-

terol levels in some hypercholesterolemic individuals,

but recent studies have shown that HDL cholesterol is

lowered by such diets, particularly in women. This is

of concern and deserves further work. Cholesterol

levels increase in older people, but the correlation of

high [blood cholesterol] and CHD appears to be less

prominent in the elderly population. Nevertheless, it

has been reported recently that high total cholesterol

levels do increase CHD risk in the elderly population.

In the USA, current guidelines, supported by the

American Heart Association and the American Col-

lege of Cardiology, and based on experimental evi-

dence and clinical trials, suggest that LDL cholesterol

should be reduced to 100 mg dl

1

. Since LDL chol-

esterol is a calculated value and since the measure-

ment of total and HDL cholesterol may be subject to

some error, the consensus opinion is that two or three

fasting lipoprotein measurements on consecutive days

should be made for an accurate baseline. All patients

with CHD and LDL cholesterol > 100 mg dl

1

should

immediately reduce the daily intake of saturated fats

to 7% of total calories and of cholesterol to

< 200 mg per day. In addition, weight loss is advised

for those who are overweight, and appropriate phys-

ical activity is suggested. If baseline LDL cholesterol

in those with CHD is > 130 mg dl

1

, drug therapy in

addition to the diet is normally advised immediately.

tbl0008 Table 8 Classification of cholesterol levels

Type Desirable Borderline high Highrisk

Total serum cholesterol < 5.2 mM (< 200 mg dl

1

) 5.2–6.2 mM (200–239 mg dl

1

) > 6.2 mM (> 240 mg dl

1

)

Low-density lipoprotein cholesterol < 3.35 mM (< 130 mg dl

1

) 3.35–4.1 mM (130–159 mg dl

1

) > 4.15 mM (> 160 mg dl

1

)

High-density lipoprotein cholesterol > 0.9 mM (> 35 mg dl

1

) < 0.9 mM (< 35 mg dl

1

Based on recommendations of the USA National Cholesterol Education Program.

tbl0007 Table 7 Stages in atherogenesis

Stage Age Characteristics

One Childhood Development of ‘fatty streaks’

in the aorta

Two Early and

Mid-adulthood

Fatty deposition under intima;

migration of smooth muscle

cells from arterial media to

intima; macrophages and

smooth muscle cells become

lipid-laden ‘foam cells’

Three Middle age and

seniors

Formation of frank plaques;

calcification of plaques

CHOLESTEROL/Role of Cholesterol in Heart Disease 1249

0016 The choice of drugs depends largely on the lipopro-

tein patterns (e.g., concomitant high [triglyceride],

low [HDL-C], and baseline [LDL-C]). Drugs most

often used individually or in combination are statins,

bile acid sequestrants, and nicotinic acid. Seques-

trants increase the excretion of bile acids in feces

and reduce blood cholesterol levels by conversion of

more cholesterol to bile acids. Statins inhibit HMG-

CoA reductase and thereby decrease the synthesis of

cholesterol and other products of the isoprenoid path-

way. Nicotinic acid appears to decrease the synthesis

of LDL and increase the synthesis of HDL. In patients

with a [LDL-C] of 100–129 mg dl

1

and low [HDL-

C] (< 35 mg dl

1

), nicotinic acid is probably the first-

choice drug. The efficiency, cost, and side-effects of

these drugs are usually important factors in decisions

about therapy. Each of these groups of drugs may

have undesirable side-effects in some patients, and

compliance is often a problem, but recent advances

in drug therapy have resulted in safer, but effective,

pharmocologic reductions in blood cholesterol levels.

Reports from many of the recent intervention trials

have indicated that in addition to the decreases in risk,

number of CHD deaths, and overall mortality, there

was also a reduction in the need for bypass surgery or

angioplasty. Furthermore, the interventions have

proven effective in reducing high blood cholesterol

levels in both genders, in middle-aged and elderly,

and in those with or without established CHD.

See also: Antioxidants: Natural Antioxidants;

Atherosclerosis; Coronary Heart Disease: Etiology and

Risk Factor; Antioxidant Status; Intervention Studies;

Prevention; Fats: Digestion, Absorption, and Transport;

Hypertension: Physiology; Hypertension and Diet;

Lipoproteins

Further Reading

American Heart Association Scientific Statement; AHA

Dietary Guidelines, Revision (2000) A statement for

healthcare professionals from the Nutrition Committee

of the American Heart Association. Circulation 102:

2284.

American Heart Association Task Force on Risk Reduction

(1997) When to start cholesterol-lowering therapy in

patients with coronary heart disease. Circulation 95:

1683–1685.

Blake GH and Triplett LC (1995) Management of hyper-

cholesterolemia. American Family Physician 51(5):

1157–1166.

Haber E (ed.) (1995) Molecular Cardiovascular Disease.

New York: Scientific American Medicine.

Jacobson TA (2000) ‘The lower the better’ in hypercholes-

terolemia therapy: a reliable clinical guideline? Annals of

Internal Medicine 133: 549–554.

Rye KA, Clay MA and Barter PJ (1999) Remodelling of

high density lipoproteins by plasma factors. Atheroscler-

osis 145: 227–238.

Silver DL, Jiang XC, Arai T, Bruce C and Tall A (2000)

Receptors and lipid transfer proteins in HDL metabol-

ism. Annals of New York Academy of Science 902:

103–112.

Steinberg D (1997) Low density lipoprotein oxidation and

its pathobiological significance. Journal of Biological

Chemistry 272: 20963–20966.

tbl0009 Table 9 Summary of recent intervention trials

Name of study Subjects, characteristics Treatment Outcome

WOSCOPS (1995) 6500 males, [Ch] ¼249–295 mg dl

1

no CHD

Low fat diet plus statin drug or placebo 20% #[total Ch]

31% #CHD risk

AFCAPS (1998) Healthy men and women with

average [Ch] * 221 mg dl

1

Low-fat diet plus statin drug or placebo 16% #[total Ch]

40% #MI risk

SSSS (1994) Hypercholesterolemic patients

with diagnosed CHD

Diet plus statin drug or placebo 24% #in MI

CLAS (1991) Middle-aged men with

atherosclerosis (4 years)

Colestipol þniacin þdiet vs. placebo þdiet 16.2% of subjects

had lesion regression

compared to 2.4% of

placebo group

AFCAPS, Airforce Coronary Atherosclerosis Prevention Study; Ch, cholesterol; CHD, coronary heart disease; CLAS, Cholesterol-lowering

Atherosclerosis Study; MI, myocardial infarction; SSSS, 4S, Scandinavian Simvastatin Survival Study; WOSCOPS, West of Scotland Coronary Prevention

Study.

1250 CHOLESTEROL/Role of Cholesterol in Heart Disease

CHOLINE

Contents

Properties and Determination

Physiology

Properties and Determination

S H Zeisel, University of North Carolina, Chapel Hill,

NC, USA

Background

0001 Choline is a common constituent of foods and is

an essential component of the human diet. This

quaternary amine is important for the structural

integrity and signaling functions of cell membranes;

it is the major source of methyl groups in the diet;

it directly affects cholinergic neurotransmission;

and it is required for lipid transport/metabolism.

Most choline in human tissues is found in phospho-

lipids such as phosphatidylcholine and sphingomye-

lin. In some components, such as human milk, the

choline esters phosphocholine and glycerophospho-

choline are the predominant forms of choline.

Though representing a smaller proportion of the

total choline pool, important metabolites of choline

include platelet-activating factor, acetylcholine,

choline plasmalogens, lysophosphatidylcholine, and

betaine (Figure 1).

Measurement of Choline

0002 Careful collection of biological samples to be ana-

lyzed for choline is exceedingly important. In blood

samples, choline concentrations double every 15 min

in the sample at room temperature, probably because

membrane phosphatidylcholine is hydrolyzed. There-

fore, it is important that blood be kept on ice until

plasma is separated by centrifugation. For similar

reasons, tissues and foods should be stored at

70



C. At this temperature, samples are stable for

at least one year. Acetylcholine is readily hydrolyzed

by basic conditions, or by cholinesterases present in

tissues. If acetylcholine is to be determined, rapid

deactivation of tissue enzymes is essential, and micro-

wave irradiation is commonly used. When acetylchol-

ine is not an important analyte (e.g., for tissues other

than brain), it is sufficient to freeze-clamp the tissue

using tongs cooled in liquid nitrogen.

0003There are multiple forms of choline in biological

tissues, as discussed above. Assay procedures are

available for the most common forms. For all the

assays, it is important to realize that purified choline

salts are very hygroscopic, and this makes assessing

the true weight of internal standards difficult. Unes-

terified choline can be measured in plasma and red

blood cells using a radioisotopic procedure, but this

difficult assay has largely been supplanted by a pro-

cedure that uses high-pressure liquid chromatography

(HPLC), an enzyme reactor, and electrochemical

detection after a simple sample pretreatment. An

alternative method using HPLC and fluorometric de-

tection has also been used. These methods are accur-

ate, reliable, and inexpensive, but are only suitable

for measurement of the choline (unesterified) and

acetylcholine, and they do not permit the use of

internal standards. A gas chromatography/mass

spectrometry method requires much more expensive

equipment, but has the advantage that it has been

adapted to measure other choline esters. The original

method has been enhanced by preseparation of the

various aqueous soluble choline metabolites using

HPLC. This procedure allows analysis of choline,

acetylcholine, phosphocholine, and glycerophospho-

choline in a single sample at minimum detection

levels of 5–10 pmol applied to the GC/MS (e.g., the

amount of choline present in 1 ml of plasma, but

owing to the complex extraction and derivatization

procedures, 25 ml of plasma is the minimum amount

that can be analyzed). It is a demanding procedure

and requires that the separate HPLC peaks be col-

lected, the metabolite in question be converted to

choline, and then this choline be injected on to the

gas chromatogram/mass spectrometer. It does allow

the inclusion of an isotopically labeled internal stand-

ard for each metabolite, thereby insuring a greater

accuracy and reliability when widely divergent matri-

ces are to be analyzed (e.g., tissues and foods).

0004Neither the electrochemical nor the mass spectro-

metric assays detect betaine. Betaine can be isolated

using HPLC and derivatized with 4

-bromo-phenacyl

triflate so that it can be detected and quantified using

UV absorbance. Phosphatidylcholine and sphingo-

myelin can be isolated by thin-layer chromatography

CHOLINE/Properties and Determination 1251

and quantified using choline or phosphorus determin-

ation, or isolated by HPLC and quantified with ultra-

violet or fluorometric detection. The advantage of

these last methods is the ability to differentiate phos-

pholipids by fatty acid composition.

0005 A method has recently been devised, using liquid

chromatography/electrospray ionization/mass spec-

trometry (LC/ESI/MS), which greatly reduces the

number of steps in the analysis (eliminates the trans-

fer to the gas chromatograph), but the equipment

costs still remain very high. This method requires no

derivatization and allows the use of internal stand-

ards. After sample extraction, the aqueous and or-

ganic phases are separated and analyzed separately

Compound Chemical structure Biologic function

Choline

Betaine

Acetylcholine

Phosphatidylcholine

Platelet-activating factor

Sphingomyelin

Glycerophosphocholine

Phosphocholine

Methyl group donor;

renal osmolyte

Neurotransmitter

Necessary building

block of biomembranes;

very-low−density

lipoprotein component

necessary for hepatic

very-low−density

lipoprotein secretion

Hormone

Necessary building

block of biomembranes

Intracellular storage

pool of choline

Intracellular storage

pool of choline

NCH

fig0001 Figure 1 Biologically important metabolites of choline.

1252 CHOLINE/Properties and Determination

using straight-phase liquid chromatography. This

method requires 2 pmol of choline to be applied to

the LC/MS (e.g., the amount of choline in 0.2 mlof

plasma).

Dietary Sources of Choline

0006 Many foods eaten by humans contain significant

amounts of choline and esters of choline, but there

is currently no comprehensive database of the choline

content of foods. Some of this choline is added during

food processing (especially when preparing infant

formula). The average dietary intake of choline and

choline esters in the adult human is estimated to be 7–

10 mmol per day. When humans are switched from a

diet of normal foods to a defined diet containing

5 mmol per day, plasma choline and phosphatidyl-

choline concentrations decrease in most subjects.

Thus, the average dietary intake of choline seems to

exceed this level in adults. Human, commercially

available infant formulas and bovine and rat milk

contain approximately 1–2 mmol per liter of choline

and choline esters. Human milk has a significantly

higher phosphocholine concentration, the same or

lower glycerophosphocholine concentration, and

similar phosphatidylcholine and sphingomyelin

concentrations compared with either bovine milk or

bovine-derived infant formulas. Soy-derived infant

formulas have lower glycerophosphocholine and

sphingomyelin concentrations and higher phosphati-

dylcholine concentrations than do either human milk

or bovine milk-derived formulas. By assuming that a

newborn infant drinks 150 ml of milk per kilogram

per day, their choline intake would be approximately

200–250 mmol per kilogram per day, two to three

times that ingested by the adult human.

Intestinal Absorption

0007 The extent to which dietary choline is bioavailable

depends upon the efficiency of its absorption from the

intestine. In adults, some ingested choline is metabol-

ized before it can be absorbed from the gut. Gut

bacteria degrade it to form betaine and to make

methylamines. The free choline surviving these fates

is absorbed all along the small intestine. No other

component of the diet has been identified as compet-

ing with choline for transport by intestinal carriers.

Both pancreatic secretions and intestinal mucosal

cells contain enzymes (phospholipases A

, and

B) capable of hydrolyzing phosphatidylcholine in

the diet. The free choline that is formed enters the

portal circulation of the liver.

0008 In infants, there are differences in the bioavailabil-

ity of the water soluble, choline-derived compounds

(choline, phosphocholine, and glycerophosphocho-

line) and the lipid-soluble compounds (phosphatidyl-

choline and sphingomyelin) present in milk.

See also: Phospholipids: Properties and Occurrence

Further Reading

Ceder G and Schuberth J (1977) In vivo formation and

post-mortem changes of choline and acetylcholine in

the brain of mice. Brain Research 128: 580–584.

Cheng W-L, Holmes-McNary MQ, Mar M-H, Lien EL and

Zeisel SH (1996) Bioavailability of choline and choline

esters from milk in rat pups. Journal of Nutritional

Biochemistry 7: 457–464.

Damsma G and Flentge F (1988) Liquid chromatography

with electrochemical detection for the determination of

choline and acetylcholine in plasma and red blood cells.

Failure to detect acetylcholine in blood of humans and

mice. Journal of Chromatography 428: 1–8.

FASEB Life Sciences Research Office (1981) Effects of

Consumption of Choline and Lecithin on Neurological

and Cardiovascular Systems, Report # PB-82-133257.

Washington, DC: Bureau of Foods, Food and Drug

Administration, Department of Health, Education, and

Welfare.

Fox JM, Betzing H and Lekim D (1979) Pharmacokinetics

of orally ingested phosphatidylcholine. In: Barbeau A,

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Physiology

S H Zeisel, University of North Carolina, Chapel Hill,

NC, USA

Metabolism of Choline

0001 There have been several comprehensive reviews of

the metabolism and functions of choline that describe

its role in the synthesis of the phospholipids in cell

membranes, methyl metabolism, cholinergic neuro-

transmission, transmembrane signaling, and lipid-

cholesterol transport and metabolism. Choline can

be acetylated, phosphorylated, or oxidized.

0002 Choline, methionine, and folate metabolism inter-

act at the point that homocysteine is converted to

methionine (Figure 1). Betaine:homocysteine methyl-

transferase catalyzes the methylation of homocys-

teine using the choline metabolite betaine as the

methyl donor. Elevated plasma homocysteine is an

independent risk factor for cardiovascular disease

and stroke in humans. Treatment with betaine or

choline also lowers elevated plasma homocysteine in

humans. In an alternative pathway, 5-methyl-tetra-

hydrofolate:homocysteine methyltransferase regener-

ates methionine. In addition, tetrahydrofolate is

needed to scavenge one-carbon groups when betaine

is metabolized. Perturbing metabolism of one of the

methyl donors results in compensatory changes in the

other methyl donors owing to the intermingling of

these metabolic pathways. Rats ingesting a choline-

deficient diet have diminished tissue concentrations

of methionine and S-adenosylmethionine, doubled

plasma homocysteine concentrations, and diminished

hepatic methyl-folate content. These effects are re-

versible by refeeding choline. Rats fed diets deficient

in both methionine and choline for 5 weeks have

hepatic folate concentrations that are half of those

present in controls. Rats treated with the antifolate,

methotrexate, have diminished pools of choline me-

tabolites in liver. Severe folate deficiency, induced in

rats by feeding an amino acid-defined diet containing

no folate and succinylsulfathiazole for 4 weeks have

resulted in hepatic choline and phosphocholine con-

centrations that were 65 and 80% lower, respectively

than in controls.

Dietary Requirements

0003Though there is a pathway (in all tissues, but most

active in the liver) for the de novo biosynthesis of the

choline moiety via the sequential methylation of phos-

phatidylethanolamine using S-adenosylmethionine

AdoHcy

Sphingomyelin

ceramide

AdoMet

Phosphatidylcholine

Phosphorylcholine

CDP-Choline

Choline

Acetylcholine

Betaine

methyl-groups

for methyl-THF

Sarcosine

methyl-THF

Homocysteine

Vit. B12

DNA methylation

other methylations

PtdEtn

Methionine

Tetrahydrofolate

methylenetetra-

hydrofolate

reductase defect

fig0001Figure 1 Choline, folate, and homocysteine metabolism

are closely inter-related. The pathways for the metabolism of

these three nutrients intersect at the formation of methionine

from homocysteine. PtdEtn, phosphatidylethanolamine; AdoHcy,

S-adenosylhomocysteine; AdoMet, S-adenosylmethionine

1254 CHOLINE/Physiology

as the methyl donor (Figure 1), only some of the

demand for choline can be met by using methyl groups

derived from one carbon metabolism. Animals and

humans fed a choline deficient diet deplete choline

stores and develop liver dysfunction. Healthy male

humans with normal folate and vitamin B

status

fed a choline-deficient diet have diminished plasma

choline and phosphatidylcholine concentrations, and

develop liver damage (elevated plasma alanine ami-

notransferase). Liver cell death occurs in choline defi-

ciency, because hepatocytes initiate programmed cell

death (apoptosis) when deprived of choline. Because

methyl supplementation with betaine, methionine,

folate, or vitamin B

does not prevent apoptotic

death induced by choline deficiency in hepatocytes,

it must be that depletion of intracellular choline moi-

eties rather than depletion of methyl groups is the

critical parameter involved in induction of apoptosis.

Some humans (male and female) fed with total paren-

teral nutrition solutions devoid of choline, but ad-

equate for methionine and folate, develop fatty liver

and liver damage that resolves when a source of diet-

ary choline is provided. Fatty liver occurs because

choline is required to make the phosphatidylcholine

portion of the very-low-density lipoprotein particle.

Animals fed a choline-deficient diet may also develop

growth retardation, renal dysfunction and hemor-

rhage, or bone abnormalities. A metabolite of choline,

betaine, is used in the kidney glomerulus as an osmo-

lyte, and for this reason, choline-deficient animals

have trouble excreting concentrated urine.

0004 Human studies of choline requirements in women,

children, or infants have not been conducted. Thus,

we do not know whether choline is needed in the diet

of these groups. Female rats are less sensitive to

choline deficiency than are male rats perhaps because

estrogen enhances females’ capacity to form the

choline moiety de novo from S-adenosylmethionine.

Pregnancy may be a time when dietary supplies of

choline are especially limiting. Though female rats are

resistant to choline deficiency, pregnant rats are as

vulnerable to deficiency, as are males. During preg-

nancy, large amounts of choline are delivered to the

fetus across the placenta, and this depletes maternal

stores of the various forms of this nutrient. At birth,

humans and other mammals have plasma choline

concentrations that are much higher than those in

adults. Also, the need for choline is increased during

lactation, because so much must be secreted into

milk; lactating rats are more sensitive to choline

deficiency than are nonlactating rats.

0005 The Institute of Medicine (IOM) recently made

recommendations for choline intake in the diet.

There were insufficient data with which to derive an

estimated average requirement for choline, and so

only an adequate intake can be estimated. The IOM

report cautioned, ‘this amount will be influenced by

the availability of methionine and methyl-folate in the

diet. It may be influenced by gender, and it may

be influenced by pregnancy, lactation, and stage of

development. Although AIs are set for choline, it may

be that the choline requirement can be met by en-

dogenous synthesis at some of these stages.’ The

IOM recommendations are shown in Table 1.

0006The recent report by Jacob that folate deficiency in

humans exacerbates choline deficiency highlights

why studies of choline–folate–homocysteine inter-

relationships in humans are important for the future

refinement of these recommendations.

0007Plasma choline concentration varies in response to

diet and can rise as much as twofold after a two-egg

meal. Fasting plasma choline concentrations vary

from 7 to 20 mM, with most subjects having concen-

trations of 10 mM. Individuals that have starved for

up to 7 days have diminished plasma choline, but

levels never drop below 50% of normal. Plasma

phosphatidylcholine concentration also decreases in

choline deficiency, but these values are also influ-

enced by factors that change plasma lipoprotein

levels. Fasting plasma phosphatidylcholine concen-

trations are approximately 1–1.5 mM.

Choline and Brain Development

0008Choline availability during embryogenesis and peri-

natal development may be especially important. There

is a sensitive period in rodent brain development

during which treatment with choline (about three

times the dietary intake) produces long-lasting en-

hancement of spatial memory that is lifelong. This

period occurs during embryonic days 12–17 in the

rat (rats give birth on day 21; mice give birth one

day earlier and probably have a slightly different

period of susceptibility). Choline supplementation

tbl0001Table 1 Institute of Medicine recommendations for choline

intake in the diet

AI for infants 0–6 months 125 mg per day,

18 mg per kilogram

6–12 months 150 mg per day

AI for children 1–3 years 200 mg per day

4–8 years 250 mg per day

9–13 years 375 mg per day

AI for males 14–18 years 550 mg per day

19 and older 550 mg per day

AI for females 14–18 years 400 mg per day

19 years and older 425 mg per day

AI for pregnancy All ages 450 mg per day

AI for lactation All ages 550 mg per day

AI = adequate intake level.

CHOLINE/Physiology 1255