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

Подождите немного. Документ загружается.

1a,25(OH)

and 24R,25(OH)

; (4) systemic

transport of the dihydroxylated metabolites

24R,25(OH)

and 1a,25(OH)

to distal target

organs; (5) binding of the dihydroxylated metabol-

ites, particularly 1,25(OH)

, to either a nuclear

receptor or membrane receptor at the target organs

followed by the subsequent generation of appropriate

biological responses (see also Figure 3). An additional

key component in the operation of the vitamin D

endocrine system is the plasma vitamin D binding

protein, which carries vitamin D

and all its metabol-

ites to their various target organs.

0011 The three enzymes responsible for the conversion of

vitamin D

into its two key daughter metabolites

include the hepatic vitamin D

-25-hydroxylase and

the two kidney enzymes, the 25(OH)D

-1a-hydroxyl-

ase and the 25(OH)D

-24R-hydroxylase. All three

enzymes have been demonstrated to be cytochrome

P450 mixed-function oxidases. Both renal enzymes

are localized in mitochondria of the proximal

tubules of the kidney. The 25(OH)D

-1a-hydroxylase

has been cloned and the specific site of mutations

(which result in the appearance of vitamin D-resistant

rickets, type I) identified. Also, the 25(OH)D

-24R-

hydrodroxylase and vitamin D

-25-hydroxylase have

been cloned.

0012The most important point of regulation of the

vitamin D endocrine system occurs through the

stringent control of the activity of the renal

25(OH)D

-1a-hydroxylase. In this way, the produc-

tion of the hormone 1a,25(OH)

can be modulated

according to the calcium and other endocrine needs of

the organism. The chief regulatory factors are

1a,25(OH)

itself, parathyroid hormone (PTH),

and the serum concentrations of calcium and phos-

phate. Probably the most important determinant of

the 1a-hydroxylase is the vitamin D status of the

animal. When the circulating concentration of

1a,25(OH)

is low, production of 1a,25(OH)

by the kidney is high; when the circulating concen-

tration of 1a,25(OH)

is high, the output of

1a,25(OH)

by the kidney is sharply reduced.

0013The process of nuclear receptor-mediated regula-

tion of gene transcription is exquisitely dependent

upon the complimentary relationship between the

unoccupied receptor and its cognate ligand. Thus,

the unoccupied receptor is largely incompetent

to engage in a productive fashion with the

Cell

nucleus

TATA

TBP

RXR Coactivator

DNA

MRNAs

hnRNAs

Transcription

initiation

Vitamin D

induced genes

Translation

Proteins

VDRE

RXRE

Corepressor

9-cis Retinoic acid 1,25(OH)

Corepressor

D R I P S

General

transcription

apparatus

VDR Coactivator

fig0003 Figure 3 Model of 1a,25(OH)

and VDR

nuc

activation of transcription. The VDR, after binding its cognate ligand 1a,25(OH)

, forms

a heterodimer with the retinoid X receptor. This heterodimer complex then interacts with the appropriate vitamin D response element

on the promoter of genes (in specific target cells) which are destined to be up- or downregulated. The heterodimer – DNA complex then

recruits the necessary coactivator proteins, such as TATA, TBP, TFIIB and other proteins to generate a competent transcriptional

complex capable of modulating mRNA production.

1216 CHOLECALCIFEROL/Physiology

transcriptional machinery to effect meaningful regu-

lation of gene transcription. It is only after the ligand–

receptor complex has formed (which results in con-

formational changes in the receptor protein) that a

functional receptor protein is generated. Thus acqui-

sition of a detailed understanding of the complemen-

tarity of the ligand shape with that of the interior

surface of the nuclear VDR receptor ligand binding

domain is believed to be the key not only to under-

standing the structural basis of receptor action and its

formation of heterodimers and interactions with

coactivators (see Figure 3), but also to designing

new drug forms of the various hormones, including

1a,25(OH)

0014 The receptors for all steroid hormones (estrogen,

progesterone, testosterone, cortisol, and aldosterone)

and the nuclear receptors for 1a,25(OH)

, retinoic

acid, and thyroid hormone are all members of the

same super gene family (See Hormones: Steroid Hor-

mones, Figure 5); accordingly, there is a high level of

conservation in their amino acid sequences, particu-

larly in their DNA and ligand binding domains. The

X-ray crystallographic structures of the ligand-bind-

ing domains (LBD) of the thyroid hormone receptor,

the retinoic acid receptor, the estrogen receptor, the

ligand binding and coactivator assembly of the per-

oxisome proliferator-activated receptor-g, and the

progesterone receptor have all been determined with

their respective ligands bound. Also, the crystal struc-

ture of the nuclear receptor for vitamin D bound to its

natural ligand 1a,25(OH)

has been determined at

a 1.8-A

resolution. The VDR

nuc

LBD structure, like

the other nuclear receptors, consists of 12 a-helices

that are arranged to create a three-layer sandwich

that completely encompasses the ligand

1a,25(OH)

in a hydrophobic core. The secondary

and tertiary structural features of this group of

proteins have been found to be remarkably similar.

Nutritional Aspects

Recommended Dietary Allowance

0015 The World Health Organization (WHO) has respon-

sibility for defining the International Unit (IU) of

vitamin D

. Their most recent definition, provided

in 1950, stated that ‘the International Unit of vitamin

D recommended for adoption is the vitamin D activ-

ity of 0.025 mg of the international standard prepar-

ation of crystalline vitamin D

.’ Thus, 1.0 IU of

vitamin D

is 0.025 mg, which is equivalent to

65.0 pmol. With the discovery of the metabolism of

vitamin D

to other active seco-steroids, particularly

1a,25(OH)

, it was recommended that 1.0 Unit

of 1a,25(OH)

be set equivalent in molar terms

to that of the parent vitamin D

. Thus, 1.0 Unit of

1a,25(OH)

has been operationally defined to be

equivalent to 65 pmol.

0016The vitamin D requirement for healthy adults has

never been precisely defined. Since vitamin D

produced in the skin after exposure to sunlight, the

human does not have a requirement for vitamin D

when sufficient sunlight is available. Man’s tendency

to wear clothes, to live in cities where tall buildings

block adequate sunlight from reaching the ground, to

live indoors, to use synthetic sunscreens that block

ultraviolet rays, and to live in geographical regions of

the world that do not receive adequate sunlight all

contribute to the inability of the skin to biosynthesize

sufficient amounts of vitamin D

. Thus, vitamin D

does become an important nutritional factor in the

absence of sunlight. It is known that a substantial

proportion of the US population is exposed to sub-

optimal levels of sunlight. This is particularly true

during the winter months. Under these conditions,

vitamin D becomes a true vitamin, and therefore

must be supplied in the diet on a regular basis.

0017Since vitamin D

can be endogenously produced by

the body and since it is retained for long periods of

time by vertebrate tissue, it is difficult to determine

with precision the minimum daily requirements for

this seco-steroid. The requirement for vitamin D is

also known to be dependent on age, sex, degree of

exposure to the sun, season, and the amount of

pigmentation in the skin.

0018In the USA, adequate amounts of vitamin D can be

readily obtained from the diet and/or from casual

exposure to sunlight. The UV exposure can be as little

as three 20-min periods of exposure of the face and

hands to ambient sunlight per week. However, in

some parts of the world where food is not routinely

fortified and sunlight is often limited during some

periods of the year, obtaining adequate amounts of

vitamin D becomes more of a problem. As a result,

the incidence of rickets in these countries is higher

than in the United States.

0019Many countries as well as the WHO have prepared

recommendations on the daily intake of essential nu-

trients, including vitamin D. The current ‘adequate

intake’ allowance of vitamin D recommended in 1998

by the Food and Nutrition Board of the US Institute

of Medicine is 200 IU per day (5 mg per day) for

infants, children, and adult males and females up to

age 51. For males and females aged 51–70 or >70, the

adequate indicated level is set at 400 IU per day (10 mg

per day) or 600 IU (15 mg per day), respectively. The

adequate allowance for pregnancy and lactation is set

at 200 IU per day (5 mg per day). The recommenda-

tions of the UK and Canada for adults are about 50%

lower than of the USA. Australia, which is generally

CHOLECALCIFEROL/Physiology 1217

sunny, does not recommend daily supplements of

vitamin D.

Food Sources

0020 Animal products constitute the bulk source of

vitamin D that occurs naturally in unfortified foods.

Marine fish, such as herring, salmon, and sardines,

and fish liver oils are good sources of vitamin D

Small quantities of vitamin D

are also derived from

eggs, veal, beef, butter, and vegetable oils, while

plants, fruits, and nuts are extremely poor sources

of vitamin D. In the USA, artificial vitamin D

fortification of foods such as milk (both fresh and

evaporated), margarine and butter, cereals, and choc-

olate mixes help in meeting the recommended dietary

allowance. Vitamin D

was used in the period of

1940–60 as a food supplement for vitamin D activity.

Functions of Vitamin D

0021 Classical target tissues In the classical target tissues

such as intestine, bone, and kidney, 1,25(OH)

largely in conjunction with PTH, serves to regulate

mineral homeostasis such that serum calcium and

phosphorus levels are maintained within a physio-

logical range that can support normal mineralization

of bone.

0022 In the intestine, 1,25(OH)

primarily stimulates

the active transport of Ca

2þ

and inorganic phosphate,

, via mechanisms that involve a calcium-binding

protein, termed calbindin D. Available evidence sug-

gests that calbindin D may be involved in protecting

the cell (as a buffer) against large fluxes of Ca

2þ

which result from active transport induced by

1,25(OH)

0023 In addition to the genomic effects of 1,25(OH)

recent studies have shown that 1,25(OH)

can

stimulate the rapid (2–4 min) transport of calcium,

termed transcaltachia, via a receptor-mediated pro-

cess, albeit one independent of gene activation.

0024 In the kidney, 1,25(OH)

functions in concert

with PTH to enhance renal Ca

2þ

reabsorption,

besides regulating its own biosynthesis by feedback

inhibition of renal 1-hydroxylase.

0025 1a,25(OH)

plays an important role in bone

growth, development, and differentiation, and sup-

ports bone mineralization indirectly by supplying the

minerals calcium and phosphorus via their enhanced

intestinal absorption. In addition, 24,25(OH)

has

been shown to promote the bone-mineralization pro-

cess and to participate in the process of fracture-

healing.

0026 Nonclassical target tissues The 1,25(OH)

hor-

mone promotes differentiation of cells in the

hemopoietic system. Such effects of 1,25(OH)

offer a therapeutic prospect for leukemia.

0027In the immune system, 1,25(OH)

acts as an

immunomodulator, regulating the functional per-

formance of cells involved in the immune response.

0028Insulin production by the endocrine pancreas is

influenced by vitamin D status in that the blunted

secretion of insulin and impaired glucose tolerance

seen in vitamin D-deficient conditions are corrected

by treatment with vitamin D

and/or 1,25(OH)

0029Myopathy and abnormalities in muscle contraction

seen in patients afflicted with metabolic bone disease

are amenable to correction with vitamin D therapy.

00301a,25(OH)

induces differentiation of keratino-

cytes in skin and exerts antiproliferative effects on

these epithelial cells. Such an effect of 1,25(OH)

in the skin has prompted the use of 1,25(OH)

analogs for treatment of psoriasis, which is a hyper-

proliferative disease of the skin.

Disease States in Humans Related to

Vitamin D

0031In humans, diseases related to vitamin D can arise

because of (1) altered availability of vitamin D; (2)

altered hepatic conversion of vitamin D

; (3)

impaired renal metabolism of 25(OH)D

, or (4) vari-

ation in end organ responsiveness to 1,25(OH)

(see Figure 4).

Renal Disorders

0032Chronic renal failure, also known as renal osteody-

strophy, is characterized by impaired renal produc-

tion of 1,25(OH)

and intestinal malabsorption

of calcium, which can often lead to derangements

in skeletal metabolism and hyperparathyroidism.

These symptoms are alleviated by 1,25(OH)

ad-

ministration. In 1977, in the USA, the Food and Drug

Administration approved the prescription use of

1,25(OH)

(calcitriol or Rocaltrol

) for renal

osteodystrophy.

Vitamin D-resistant Rickets

0033Also known as familial X-linked hypophosphatemic

rickets, vitamin D-resistant rickets is characterized

by a primary phosphate leak in the kidney, skeletal

deformities, and hypophosphatemia. A combination

of oral phosphate and 1,25(OH)

is effective in

treating these patients.

Vitamin D-dependent Rickets

0034Vitamin D-dependent rickets is also referred to as

hereditary hypocalcemic vitamin D-resistant rickets,

and is classified into Type I and Type II disease states.

1218 CHOLECALCIFEROL/Physiology

0035 Type I rickets is believed to arise as a result of an

inborn error in the renal 1-hydroxylase enzyme. The

clinical features include hypocalcemia, hypophos-

phatemia, and several rachitic lesions. These symp-

toms can be treated with pharmacological doses of

vitamin D

or low doses of 1,25(OH)

0036 Point mutations in the vitamin D receptor gene

have been shown to be responsible for the defective

receptors seen in these children with Type II rickets.

The clinical manifestations are defective bone miner-

alization, decreased intestinal calcium absorption,

hypocalcemia, and increased serum 1,25(OH)

levels. To date, a point mutation in a steroid receptor

gene resulting in the loss of functional activity has

been demonstrated only for the vitamin D receptor

and is therefore unique in this respect.

Diseases of Parathyroid

0037 Hypoparathyroidism Hypoparathoidism exhibits

hypocalcemia as a major clinical consequence and is

corrected with large doses of vitamin D or physio-

logical doses of 1,25(OH)

0038 Hyperparathyroidism Increased 1,25(OH)

levels, enhanced intestinal absorption of calcium

(contributing to hypercalciuria), and nephrolithiasis

are typical of this disorder.

0039 Pseudohypoparathyroidism Pseudohypoparathyroid-

ism results from a state of resistance to PTH. The

biochemical abnormalities are hypocalcemia, hyper-

phosphatemia, elevated serum PTH, and decreased

serum 1,25(OH)

levels.

0040Bone disorders Clinically, a deficiency in vitamin D

manifests as rickets in children and osteomalacia in

adults. Hypocalcemia, hypophosphatemia, increased

serum alkaline phosphatase, and decreased serum

25(OH)D

levels are some of the salient biochemical

abnormalities, all of which can be ameliorated by

vitamin D administration.

Conclusion

0041Current evidence supports the concept that the clas-

sical biological actions of the nutritionally important

fat-soluble vitamin D in mediating calcium homeo-

stasis are supported by a complex vitamin D endo-

crine system which coordinates the metabolism

of vitamin D

into 1a,25(OH)

and

24R,25(OH)

. It is now clear that the vitamin D

endocrine system embraces many more target tissues

than simply the intestine, bone, and kidney. Notable

additions to this list include the pancreas, pituitary,

breast tissue, placenta, hematopoietic cells, skin, and

cancer cells of various origins. Key advances in under-

standing the mode of action of the 1a,25(OH)

have been made by a thorough study of nuclear recep-

tors as well as emerging studies describing a mem-

brane receptor for this steroid hormone. Integral to

Hyperparathyroidism

Diabetes Medullary

carcinoma

Chronic renal

disease

Hypophosphatemic

VDRR

Vitamin D-

dependent rickets

Cirrhosis

Drug induced

metabolism

Obstructive

jaundice

Hypoparathyroidism

Pseudohypo-

parathyroidism

Secondary hyper-

parathyroidism

Tuberculosis

Sarcoidosis

PTH

, Ca

,1a,25(OH)

1a,25(OH)

and

24R,25(OH)

1a,25(OH)

25(OH)D

1a,25(OH)

25(OH)D

PTH Ca

25(OH)D

1a,25(OH)

24R,25(OH)

Psoriasis

Glucocorticoid

antagonism

Idiopathic

hypercalcemia

Malabsorption

syndrome

Steatorrhea

Tropical sprue

Anticonvulsant

treatment

Fibrogenesis

imperfecta ossium

Osteitis fibrosa cystrica

Osteomalacia

Osteoporosis

Osteopenia

Osteosclerosis

Renal osteodystrophy

Rickets

Bone

Parathyroid

Pancreas

Liver Thyroid

Kidney

Lung

Intestine

Diet

Skin

Blood

25(OH)D

PTH CT

1a,25(OH)

/24R,25(OH)

fig0004 Figure 4 Human disease states related to the vitamin D endocrine system. Under the boxed headings (e.g., parathyroid, liver, bone,

etc.) are listed disease states occurring in man which have been shown, or are believed, to have some functional linkage between

some aspect of the vitamin D endocrine system and that particular organ. The information associated with the arrows indicates the

direction of flow of calcium, phosphate or the calcium-regulating hormones (CT (calcitonin), PTH (parathyroid hormone), vitamin D

1a, 25(OH)

, 24R, 25(OH)

). Ca

2þ

, calcium; P

, inorganic phosphate; VDRR, vitamin D-resistant rickets.

CHOLECALCIFEROL/Physiology 1219

these observations are efforts to define the signal

transduction systems, which are subservient to the

nuclear and membrane receptors for 1a,25(OH)

and to obtain a thorough study of the tissue distribu-

tion and subcellular localization of the gene products

induced by this steroid hormone. There are clinical

applications for 1a,25(OH)

or related analogs for

treatment of the bone diseases of renal osteodystro-

phy and osteoporosis, psoriasis, and hypopara-

thyroidism; other clinical targets for 1a,25(OH)

currently under investigation include its use in

leukemia, breast, prostate, and colon cancer as well

as use as an immunosuppressive agent.

See also: Cholecalciferol: Properties and Determination;

Dietary Reference Values; Hormones: Steroid

Hormones; Renal Function and Disorders: Nutritional

Management of Renal Disorders

Further Reading

Bikle DD (1995) Vitamin D: New actions, new analogs,

new therapeutic potential: update 1995. Endocrine

Reviews 4: 77–83.

Bouillon R, Okamura WH and Norman AW (1995) Struc-

ture–function relationships in the vitamin D endocrine

system. Endocrine Reviews 16: 200–257.

Haussler MR, Whitfield GK, Haussler CA et al. (1998) The

nuclear vitamin D receptor: Biological and molecular

regulatory properties revealed. Journal of Bone and

Mineral Research 13: 325–349.

Norman AW (2001) Vitamin D. In: Bowman B and Russell

R (eds) Present Knowledge in Nutrition (PKN), 8th edn.,

pp. 134–143. Washington, DC: International Life Sci-

ences Institute.

Norman AW and Litwack G (1997) Calcium-regulating

hormones: Vitamin D, parathyroid hormone, and calci-

tonin. In: Hormones, 2nd edn., pp. 251–280. San Diego,

CA: Academic Press.

Norman AW, Henry HL, Bishop JE et al. (2001) Different

shapes of the steroid hormone 1a,25(OH)

-vitamin D

act as agonists for two different receptors in the vitamin

D endocrine system to mediate genomic and rapid

responses. Steroids 66(3–5): 147–158.

Reichel H, Koeffler HP and Norman AW (1989) The role of

the vitamin D endocrine system in health and disease.

New England Journal of Medicine 320: 980–991.

Rochel N, Wurtz JM, Mitschler A, Klaholz B and Moras D

(2000) The crystal structure of the nuclear receptor for

vitamin D bound to its natural ligand. Molecular Cell

5(1): 173–179.

Weatherman RV, Fletterick RJ and Scanlon TS (1999)

Nuclear receptor ligands and ligand-binding domains.

Annual Review of Biochemistry 68: 559–582.

CHOLESTEROL

Contents

Properties and Determination

Absorption, Function, and Metabolism

Factors Determining Blood Cholesterol Levels

Role of Cholesterol in Heart Disease

Properties and Determination

A J Sheppard and R G O’Dell, United States Food and

Drug Administration, Washington DC, USA

J A T Pennington, National Institute of Health,

Bethesda, MD, USA

This article is reproduced from Encyclopaedia of Food Science,

Background

0001 Cholesterol is a well-known and commonly deter-

mined lipid component and an important intermediate

in the synthesis of steroid hormones. It is a sterol

OH) that occurs notably in animal fats and

oils, bile, gallstones, nerve tissues, blood, brain,

plasma, and egg yolk. Cholesterol is the most

common animal sterol and is also found in trace

amounts in vegetable fats and oils, seaweeds, and

green leaves. Cholesterol was first found in gall-

stones and derives its name from the Greek khole

(bile) and stereos (solid). The determination of chol-

esterol in serum and foods is of significance because

of the implication of cholesterol in the etiology of

arteriosclerosis and coronary heart disease. (See Ath-

erosclerosis; Coronary Heart Disease: Intervention

Studies.)

1220 CHOLESTEROL/Properties and Determination

Structure

0002 A systematic study of the chemistry of cholesterol

began at the end of the nineteenth century. The classic

work of Wieland, Vindaus, Diels, Rosenheim, and

King led to the formulation of the structure of choles-

terol in 1932. The fundamental carbon skeleton of

the cholesterol molecule is the cyclopentanoperhydro-

phenanthrene ring. The structure of cholesterol is

shown in Figure 1. The hydroxyl group on C3 is

connected by a ‘solid’ bond (b orientation) and the

hydrogen by a ‘dashed’ bond (a orientation), depicting

the naturally occurring b-cholesterol. Atoms con-

nected by solid bonds (b orientation) are regarded as

projecting in front of the plane of the steroid ring, and

those connected by dashed bonds (a orientation) as

lying behind the plane. The molecular weight of chol-

esterol is 384.64. Cholesterol consists of 83.87%

carbon, 11.99% hydrogen, and 4.145% oxygen by

weight. The formal chemical name of the molecule is

cholest-5-en-3b-ol. Because the cholesterol nucleus

contains eight centers of asymmetry, approximately

240 isomers of the molecule are possible. However,

only two carbon centers (C3 and C5) appear to be

involved in naturally occurring cholesterol isomers. In

some of the earlier scientific literature, cholesterol is

referred to as ‘cholesterin.’

Chemical Characteristics

0003 Cholesterol is a glistening, white, soapy, crystalline

substance that is practically insoluble in water (about

0.2 mg per 100 ml). It is slightly soluble in alcohol

(1.29% w/w at 20



C) and more soluble in hot alco-

hol (100 g of saturated 96% alcoholic solution con-

tains 28 g of cholesterol at 80



C). One gram of the

compound dissolves in 2.8 ml of ether, 4.5 ml of

chloroform, or 1.5 ml of pyridine. Cholesterol is

also soluble in benzene, hexane, petroleum ether,

oils, fats, and aqueous solutions of bile salts. It crys-

tallizes easily from absolute alcohol, acetic acid,

ether, and similar solvents as colorless rhombic plates

with one or more characteristic notches in the

corners. Because cholesterol has an unsaturated

bond, it will accept up to two halogen atoms. Choles-

terol is not saponifiable.

0004Cholesterol gives a number of color reactions that

are useful to test for the molecule both qualitatively

and quantitatively. The Salkowski reaction produces

a series of colors when a chloroform solution of chol-

esterol is stratified over concentrated sulfuric acid.

The acid assumes a yellowish color with a green

fluorescence, whereas the chloroform layer first be-

comes bluish red and then gradually changes to a

violet–red. If the chloroform layer is decanted into a

porcelain evaporating dish, it changes from violet–red

to violet, to green, and then to yellow. Another test,

the Liebermann–Burchard reaction, which involves

adding acetic anhydride and concentrated sulfuric

acid (under conditions as nearly anhydrous as pos-

sible) to a chloroform solution of cholesterol, results

in an initial blue to violet color that changes to emer-

ald green. Under carefully controlled conditions, the

intensity of the green color produced is proportional

to the amount of cholesterol present.

0005Free cholesterol unites with digitonin, a glycosidic

saponin, to form cholesterol digitonide; cholesterol

esters do not form such compounds. Cholesterol digi-

tonide is insoluble in petroleum ether; cholesterol

esters are freely soluble in petroleum ether. This dif-

ference in solubilities is useful to test both qualita-

tively and quantitatively for free versus esterified

cholesterol.

0006Fieser showed that the melting point of cholesterol,

which had been purified by recrystallization from

acetic acid, was 149.5–150.0



C. Radin and Gramza

indicated that the acceptable melting point of re-

crystallized cholesterol was 149.3–151.3



C. The

Merck Index indicates that the melting point of

anhydrous cholesterol is 148.5



C, and the boiling

point is 233



C at 0.5 mmHg and 360



C at 1 atm

(760 mmHg). At 360



C, some decomposition occurs.

Primary Cholesterol Standard

0007The requirements for primary standards become pro-

gressively more stringent as methods are developed

that are more sensitive and more compound-specific;

such is the case for cholesterol. Primary standards are

chemical substances that, by virtue of their purity, can

be weighed directly for the preparation of solutions

with known concentrations. Primary cholesterol

standards are expected to be at least 99% pure.

10 9 8

fig0001 Figure 1 Structure of b-cholesterol showing carbon atom

numbering. Reproduced from Cholesterol: Properties and Deter-

mination, Encyclopaedia of Food Science, Food Technology and Nu-

trition, Macrae R, Robinson RK and Sadler MJ (eds), 1993,

Academic Press.

CHOLESTEROL/Properties and Determination 1221

Cholesterol stored at room temperature, unprotected

by a nitrogen cover, will undergo autoxidation over

an extended period. In addition, ultraviolet light will

produce structural changes in cholesterol unless

amber glass containers are used. Consequently, vari-

ous amounts of cholesterol oxidation products may

be present in what was initially pure cholesterol.

Among the cholesterol oxides that have been identi-

fied in stored cholesterol are 7-ketocholesterol,

20-hydroxycholesterol, and 24-, 25-, and 26-hydro-

xycholesterol. Therefore, primary standards that

have been stored must be checked and may need to

undergo repurification before use. Crystalline stand-

ards should be stored in small amounts over a desic-

cant such as silica gel at 20



C in the dark.

0008 Cholesterol may be recrystallized from ethanol or

acetic acid, or as the dibromide. A cholesterol prepar-

ation may be added to absolute ethanol, which is

gently heated until the cholesterol dissolves and is

then cooled to room temperature. The precipitated

cholesterol is collected on a filter, washed with a small

volume of diethyl ether, dried overnight in air, and

dried in an oven at 90



C for 2 h. Cholesterol is re-

crystallized from boiling glacial acetic acid solution,

which is then cooled to room temperature in an ice

bath. The crystals are collected on a filter, washed

with acetic acid and methanol, and dried as described

above.

0009 The recrystallization of cholesterol by the dibro-

mide method (Schoenheimer) is a more arduous task

than the recrystallization from either ethanol or acetic

acid, but it produces a superior product. A bromine

solution is added to the cholesterol solution. A white

paste is produced that is then transferred to a filter

and washed with acetic acid until the filtrate is color-

less. Zinc dust is then added to a suspension of the

white material in diethyl ether and glacial acetic acid

(750:10 v/v); the reaction produces evolution of

hydrogen. The resultant white precipitate of zinc

salts is dissolved in water, and the ether solution is

decanted. The water contains any excess solid zinc.

The ether solution is washed in a separatory funnel

with acid solution and neutralized with sodium hy-

droxide solution. Methanol is added to the ether

solution, and most of the ether is evaporated on a

steam bath as the purified cholesterol begins to crys-

tallize. The crystallization proceeds more rapidly as

room temperature is approached. The product is col-

lected on a filter and dried as described above.

0010 The cholesterol purified by the above methods may

be characterized by the color reactions discussed

above (Salkowski and Liebermann–Burchard). Other

color tests, such as the formaldehyde–sulfuric acid

test, may be used. In this test, formaldehyde–sulfuric

acid solution is added to a solution of cholesterol

dissolved in chloroform. The solution, which turns

cherry red, is poured into another tube, and two to

three drops of acetic anhydride are added. A blue

color develops. These color tests have been adapted

to form the basis of spectrophotometric measure-

ments. The results of the purified products are deter-

mined by reference to a standard of known purity.

0011The purity of cholesterol standards may be assessed

by using melting point and boiling point determin-

ations, microscopic comparison of the crystals with a

pure reference material, and infrared and ultraviolet–

visible spectra. The spectrum of the cholesterol

standard is compared with that of a pure crystalline

reference material.

0012Classic colorimetric tests, microscopic examin-

ations, and melting point determinations have been

supplemented with more modern techniques, such

as mass spectrometry, nuclear magnetic resonance

spectrometry, gas chromatography (GC) and high-

performance liquid chromatography (HPLC) for

determining the purity of prepared crystalline choles-

terol. No one test in itself is sufficient to determine

purity. Confirmation of results requires a minimum of

two tests, which should preferably be chemically

or physically unrelated. (See Chromatography:

High-performance Liquid Chromatography; Gas

Chromatography; Mass Spectrometry: Principles

and Instrumentation; Spectroscopy: Nuclear Mag-

netic Resonance.)

0013A serum reference material for determining serum

cholesterol is normally used for both manual and

automated methods. A serum reference material

may be prepared by filtering pooled human serum

through clarifying and ‘sterilizing’ filters. Stable

serum preparations of cholesterol, with concentra-

tions ranging from 100 to 400 mg dl

1

, may be

made by adding an alcohol-precipitated cholesterol-

rich protein from human serum to bovine, horse, or

human serum. Aliquots of the preparation should be

stored in sealed vials or ampoules at a temperature of

20



C or below. The stability of these preparations

is similar to that of human serum. These sterile prep-

arations may be shipped at room temperature for

periods of up to 5 days. The concentrations of some

commercial reference sera may vary considerably if

the Abell–Kendall method is used as the reference

assay. The Abell–Kendall procedure includes an ini-

tial step in which the serum is treated with alcoholic

potassium hydroxide to liberate the cholesterol from

the lipoprotein complexes and to saponify the choles-

terol esters. The total cholesterol is extracted into a

measured volume of petroleum ether. The cholesterol

in an aliquot of the petroleum ether extract is meas-

ured by means of the Liebermann–Burchard color

reaction. All commercial reference sera should be

1222 CHOLESTEROL/Properties and Determination

checked against serum reference materials and stand-

ardized by the reference cholesterol method (Abell–

Kendall method).

Colorimetry-based Analytical Methods

0014 Routine lipid testing in clinical settings generally in-

cludes serum determination of triglycerides and chol-

esterol, and a more recent trend has been to include

lipoprotein–cholesterol determination. The literature

contains more than 200 methods or modifications for

measuring serum cholesterol. This number is perhaps

an indication of the difficulties of developing a reli-

able assay. Free cholesterol and cholesteryl esters may

be determined separately, but it is common practice to

determine the two together as ‘total cholesterol.’ (See

Spectroscopy: Visible Spectroscopy and Colorimetry.)

0015 The Liebermann–Burchard reagent has had a cen-

tral role in much of the methodological development

of cholesterol measurement systems. Since Grigant

introduced a procedure for the quantitative estima-

tion of cholesterol in 1910 by using the Liebermann–

Burchard reagent (developed between 1885 and

1890), numerous modifications of the method have

appeared in the literature. Most of the deviations

from earlier methods consist of changes in extraction

and color development.

0016 The reaction of cholesterol with sulfuric acid in

acetic anhydride (Liebermann–Burchard) to form a

colored product has provided the basis for many sub-

sequent methods. These methods may be categorized

into three groups: (1) direct, in which the serum is

added directly to the color reagent; (2) extraction,

in which cholesterol is extracted into an organic

solvent before it is added to the color reagent; and

(3) hydrolysis, in which the esterified cholesterol is

hydrolyzed before solvent extraction and color devel-

opment. Many methods utilize acetic anhydride,

acetic acid, or ferric chloride to develop color. Speci-

ficity is enhanced by prior extraction techniques.

Values for cholesterol are usually higher by direct

and automated methods than by manual, extraction,

or hydrolysis methods. The color development of

these various procedures obeys the Lambert–Beer

law, which states that the absorbance is directly pro-

portional to the concentration.

0017 The direct methods are simple and convenient, but

are subject to interference from compounds normally

present in serum, such as bilirubin and proteins.

Much of this interference is eliminated by extracting

the cholesterol with a solvent before the color devel-

opment reaction is initiated. Esterified cholesterol in

serum should be hydrolyzed before a total cholesterol

determination is made because more color is

produced by esterified cholesterol than by free

cholesterol. Failure to hydrolyze results in an over-

estimation of total cholesterol. Standard solutions of

cholesterol must be reacted with the color reagent to

determine when the color has developed its maximum

intensity. Color reagents should be added to test solu-

tions and standard solutions at fixed intervals, for

example, every 30 or 60 s. When the color intensity

of the standard solutions has reached its maximum,

the color intensities of the test solutions should be

read according to the preparation schedule, for

example, every 30 or 60 s. The time required for the

color to develop its maximum intensity is influenced

by the ambient temperature and the composition of

the individual batches of prepared reagents.

0018The American Association of Clinical Chemistry

has suggested three methods for small laboratories

that need a manual procedure for estimating total

serum cholesterol. These procedures are an enzymatic

method, the iron–uranyl acetate method (Parekh–

Jung), and the Liebermann–Burchard reagent

method. The modified Liebermann–Burchard reagent

method, in both manual and automated forms, is

widely used by small clinical laboratories. This dis-

cussion focuses on the manual manipulation of the

method for clarity of the chemistry. The Liebermann–

Burchard color reaction was discussed above with

regard to primary standards. In the modified method,

the cholesterol is extracted from serum test samples

into 2-propanol to eliminate any interfering sub-

stances. A measured aliquot of the extract is evapor-

ated before the Liebermann–Burchard color reagent

is added. If the reagents are added directly to the

extract, it is difficult to control the rate of color reac-

tion under manual conditions. The heat produced

from the exothermic reaction of 2-propanol with

sulfuric acid cannot be controlled sufficiently to

allow reproducible measurements. However, under

stringent automated conditions, this problem can be

circumvented. After a timed incubation and color-

development period, the absorbance is measured at

630 nm. In this method, reference serum and serum

test samples are treated in the same manner.

According to the American Association for Clinical

Chemistry, recoveries of cholesterol added to serum

were 98–101% over a linear range of cholesterol

concentrations of 0.8–4.0 g

1

. This method may be

used to determine cholesterol in food and tissue ex-

tracts that have been cleaned in a separating funnel

before being dried and redissolved in 2-propanol.

Crystalline cholesterol is used as the primary standard

for these test samples.

0019An estimation of the relationship of unesterified

cholesterol to cholesterol ester can be made with

another modification of the Liebermann–Burchard

method. Unesterified cholesterol in serum is

CHOLESTEROL/Properties and Determination 1223

precipitated as the digitonide from ethanol–ether so-

lution. The solvent is evaporated, and the esters are

extracted from the residue by adding petroleum ether,

bringing the solution to a boil, cooling and centrifu-

ging, and collecting the supernatant. The supernatant

is processed as in a total cholesterol determination.

The resultant value is a measure of the esterified

cholesterol in the serum test sample.

0020 The Parekh–Jung manual method is used in many

clinical laboratories. This method is based on the

precipitation of proteins and associated substances

with ferric acetate–uranyl acetate reagent. The mix-

ture is centrifuged, and the resultant supernatant is

treated with sulfuric acid–ferrous sulfate color re-

agent. After a 20-min incubation and color-develop-

ment period, the absorbance is measured at 560 nm.

0021 The enzymatic method for the determination of

total serum cholesterol is frequently used both manu-

ally and in an automated setting. The enzyme method

is of limited value when either aqueous or pure

alcohol standards are used. The method may be cali-

brated accurately with a homogeneous and stable

serum pool. The method provides a direct measure

of serum cholesterol. The enzymatic analytical deter-

mination is calibrated by using serum labeled with

a target value assigned by an accepted reference

method (e.g., the Abell–Kendall method). (See

Enzymes: Uses in Analysis.)

0022 Although cholesterol in serum is primarily free,

cholesterol associated with lipoproteins is esterified.

Cholesteryl esters are freed from the lipoproteins and

enzymatically hydrolyzed by cholesterol-ester hydro-

lase to free cholesterol and fatty acids. The free chol-

esterol is then oxidized by cholesterol oxidase to

cholest-4-en-3-one and hydrogen peroxidase. The per-

oxide, in the presence of peroxidase, oxidatively

couples with phenol and 4-aminoantipyrine to pro-

duce a quinoneimine dye. The absorbance values at

560 nm are proportional to the concentration of total

cholesterol in the serum test sample. The absorbance

obeys the Lambert–Beer law over a wide concentra-

tion of up to about 5.5 g of cholesterol per liter. An

estimate of the relationship of unesterified cholesterol

to cholesterol ester is made by subtracting the free

cholesterol value (the enzyme cholesterol esterase

is withheld from the working test reagent) from

the total cholesterol value. The enzymatic method

has also been used successfully to determine the chol-

esterol content of foods. For this purpose, the choles-

terol is extracted with a chloroform–methanol

solvent, and crystalline cholesterol is used for calibra-

tion. A survey of the literature indicates that the coef-

ficient of variation for this method is about 1–3%.

0023 The Abell–Kendall method is generally accepted

by clinical chemists as the total cholesterol reference

method. The results obtained by other methods

are nearly always compared with those obtained by

the Abell–Kendall method. The method, however,

does not lend itself well to analysis of large numbers

of test samples. A saponification step generally pre-

cludes the development of an automated version

of the method. The serum or plasma is saponified

with alcoholic potassium hydroxide. The free

cholesterol (from both unesterified and esterified

cholesterol) is extracted into petroleum ether and

dried; the cholesterol is determined photometrically

by a modified Liebermann–Burchard reagent (acetic

anhydride–sulfuric acid–acetic acid) at 620 nm. The

results of this method are in good agreement

with those obtained by the Schoenheimer–Sperry

method.

0024No discussion of colorimetric cholesterol method-

ology is complete without a brief discussion of the

separate determination of blood cholesterol and chol-

esterol esters that was originally developed by Bloor

and Knudson in 1916. Practically all methods for

separating free and esterified cholesterol emulate

their work. Bloor and Knudson adapted the Windaus

cholesterol digitonin precipitation method to separ-

ate cholesterol from its esters in small amounts of

blood. The method consists of the determination of

total cholesterol in an aliquot of an alcohol–ether

extract of blood and the determination of cholesterol

esters in another aliquot after precipitation of the free

cholesterol by digitonin. The difference between the

two values represents free cholesterol. The color re-

agent and color development were previously used by

Liebermann–Burchard.

Gas Chromatographic (GC) Analysis

0025GC is used to measure cholesterol as free cholesterol,

as the trimethylsilyl ether derivative, or as cholesteryl

butyrate. Although colorimetric methods have been

used in the past to measure the cholesterol content of

foods, body fluids, and tissues, they have been, for the

most part, cumbersome and not compound-specific

in many applications. Agricultural chemists, bio-

chemists, nutritionists, and food scientists began to

explore the use of GC for measuring sterols in the

mid-1970s. Although blood cholesterol determin-

ations by GC can be fairly easy to accomplish, GC is

not widely used as a routine clinical technique for

determining cholesterol. The most probable reasons

for this are the difficulties of automating the prepara-

tive stages and the fact that adequate colorimetric

assays currently exist. Most chromatographic deter-

minations of cholesterol are found in the research

environment, in food and nutrition analytical labora-

tories, and in government regulatory laboratories.

1224 CHOLESTEROL/Properties and Determination

0026 All GC methods use a column stationary phase that

is classified as nonpolar, typically methyl silicone (SE-

30). In one method, the test sample is saponified, and

the unsaponifiable materials are analyzed by GC.

This method performs well for most products, but

not for those that contain measurable amounts of

a-tocopherol in addition to cholesterol. The retention

times of a-tocopherol and cholesterol are nearly

identical and, therefore, an analysis for either com-

pound is not feasible in the presence of the other.

The a-tocopherol may be present naturally, or may

be added as a nutritional supplement or antioxidant.

(See Tocopherols: Properties and Determination.)

0027 Another approach is to saponify the test sample,

extract the unsaponifiables, and derivatize the unsa-

ponifiable compounds before GC analysis. The offi-

cial method of the Association of Official Analytical

Chemists (AOAC) uses this approach to form

trimethylsilyl ethers (Punwar method). Another

well-established method uses the classic butyric an-

hydride–pyridine reaction to attach a C

ester at pos-

ition 3 on the A ring of the sterol to form cholesteryl

butyrate. During GC analyses, both derivatives func-

tion equally with respect to response, reproducibility,

and reliability. The trimethylsilyl ethers create prob-

lems over an extended series of analyses. The detector

sensitivity drops drastically, and the detector must be

disassembled, thoroughly cleaned, and reinstalled to

restore sensitivity. The butyrate derivatives burn

cleanly in the hydrogen flame ionization detector

and do not degrade detector sensitivity. The proced-

ure for preparing the trimethylsilyl derivative is some-

what less tedious than that for preparing the buyrates.

0028 The GC official method of the AOAC determines

cholesterol as the trimethylsilyl ether on a 2.4 m 

3 mm internal diameter silanized glass column packed

with 0.5% Apiezon L on 80–100 mesh Gas-Chrom Q

(Alltech Associates/Applied Science, Deerfield, IL).

An alternative column may be used that consists

of a 1.8 m 4 mm internal diameter glass column

packed with 1% SE-30 on 100–120 mesh Gas-

Chrom Q (Alltech Associates/Applied Science, Deer-

field, IL). In this method, one column is maintained at

230



C, and the carrier gas flow is adjusted to elute

the trimethylsilyl ether of cholesterol in 9–11 min. An

internal standard of 5a-cholestane is used, and test

samples containing 0.5–1 g of fat are extracted with

chloroform–methanol–water (50:100:40 v/v/v).

0029 A GC method based on steryl butyrates has been

incorporated into a total-lipid analytical system

(Sheppard–Hubbard system) for determining total

lipids, fatty acid composition, and cholesterol in

foods from extraction of a single test portion. The

system is widely used in university, food quality

control, commercial, and regulatory laboratories to

determine the lipid components required for fatty

acid and cholesterol labeling. The system has also

been used to identify adulterated foods. This analyt-

ical system uses an aliquot of the fatty acid methyl

ester preparation to prepare cholesteryl butyrate and

other sterols. The fatty acid methyl esters are not

affected by the reaction that forms the butyrate de-

rivatives of the sterols. On the SE-30 column, the

fatty acid methyl esters elute near the solvent front

and before the appearance of the butyrates of the

tocopherols and the sterols. In this system, external

calibration is used. However, internal standards can

be used if the internal standard peak is situated very

close to the peak being measured and if there are no

interfering peaks from compounds such as squalane,

squalene, or cholestane in the test sample. Generally,

in this system, there are too many peaks from the

matrix to have a clear retention time available for

internal standards.

0030The relative retention times for some sterols are 1.0

for cholesteryl butyrate, 1.15 for brassicasteryl butyr-

ate, 1.3 for campesteryl butyrate, 1.4 for stigmasteryl

butyrate, and 1.6 for sitosteryl butyrate. Free choles-

terol and other sterols can be easily separated with a

15 m 0.242 mm, SE-54, wall-coated, fused-silica

capillary column operated at 250



C with a helium

flow rate of 0.74 ml min

1

. The sequence of com-

pound appearance is the same as that for SE-30

packed columns.

High-performance Liquid

Chromatography (HPLC)

0031HPLC is increasingly being used to determine choles-

terol and other sterols in foods and tissue extracts.

However, like GC, HPLC is not generally used in

routine clinical analyses performed with automated

clinical multiple analysis systems based on colorimet-

ric or fluorometric assays. Sterols that can be separ-

ated by GC usually cannot be separated by an HPLC

system. One HPLC system in widespread use deter-

mines the benzoate ester of cholesterol on a

300 3.9 mm internal diameter mBondapak C

column with a 100% methanol mobile phase and a

variable wavelength ultraviolet detector set at 230 nm.

GC and HPLC determinations of cholesterol in a var-

iety of foods show that the two techniques yield statis-

tically identical results. Amounts as low as 10 ng of

cholesterol benzoate can be determined using HPLC.

See also: Atherosclerosis; Chromatography: High-

performance Liquid Chromatography; Gas

Chromatography; Coronary Heart Disease: Intervention

Studies; Enzymes: Uses in Analysis; Mass

Spectrometry: Principles and Instrumentation;

CHOLESTEROL/Properties and Determination 1225