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

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Chemical Properties

0002 Cholecalciferol is described in terms of steroid

nomenclature and numbering (Figure 1). The ‘9,10-

seco’ prefix is added to denote the bond cleavage and

the opening of the typical steroidal ring structure, an

essential feature necessary to impart antirachitic ac-

tivity. This cleavage is generally induced by ultravio-

let (UV) irradiation of the precursor compound,

7-dehydrocholesterol (provitamin D

), and occurs in

the skin during exposure to sunlight. The previtamin

so formed subsequently undergoes temperature-

dependent equilibration to vitamin D

. This pro-

cess, along with the requirement for further hydro-

xylation challenges its original classification as

vitamin; it is currently more accurately regarded as a

prohormone.

0003 Any homolog which possesses antirachitic activity

is referred to as a D vitamin and each of the several

compounds sharing this property elicits a unique

and selective biological response (Figure 1). Common

structural features of these substances are the b

stereochemistry of the 3-hydroxy substituent and the

cis conformation of the double bond at C5. While the

3-hydroxy substituent does not have an overwhelm-

ing influence on biological activity, other structural

features, such as the A ring configuration and side-

chain length, appear to be more critical. Thus, while

alterations to the side chain result in diverse activity,

only vitamins D

and D

are of prominence therapeut-

ically and commercially. They are usually obtained via

chemical synthesis, although vitamin D

has trad-

itionally been extracted from fish liver oils.

0004 It is widespread practice to express vitamin D con-

centration in food in international units (IU), rather

than on a weight basis (1 IU is equivalent to 0.025 mg

of either calciferol, although this equivalence in

humans is occasionally challenged). This can be a

useful concept because it reflects the nutritional status

where several components of different biopotency

coexist within a product. Nevertheless, there is some

move back to mass units, particularly in supple-

mented foods where the contribution of chole-

calciferol is dominant.

0005The stability characteristics of cholecalciferol have

received considerable attention because they play a

major part in the shelf-life of marketable products.

Under conditions of low thermal, photochemical, and

oxidative stresses, cholecalciferol is stable for several

years. When added to foods and exposed to industrial

and domestic processes, degradation is minimal.

However, excessive light (UV) causes significant

losses through the production of inactive substances

such as toxisterol, suprasterol, lumisterol, and tachy-

sterol, in a process which is accelerated by heating

(Figure 2). Cooking temperatures above 100



C, even

in the absence of light and air, will cause isomeriza-

tion through ring closure to the pyrocholecalciferols.

Cholecalciferol is also sensitive to low pH and, if

subjected to an acidic environment, will irreversibly

tbl0001 Table 1 Physical properties of the calciferols

Cholecalciferol Ergocalciferol

Molecular weight 384.62 396.63

Empirical formula C

Melting point (



C) 84–85 115–118

max

(nm) 264.5 264.5

Extinction coefficient, E

1cm

in hexane

485 459

Optical rotation in chloroform þ52



þ52



fig0001Figure 1 The chemical structure of cholecalciferol (vitamin D

indicating the carbon-numbering system of the molecule. Related

calciferols with different side-chain configurations are also given,

including ergocalciferol (vitamin D

1206 CHOLECALCIFEROL/Properties and Determination

rearrange to the inactive isotachysterol via the 5,6-

trans isomer. These reactions (illustrated in Figure 3)

are complex and will occur to an extent determined

by the overall environment to which cholecalciferol is

exposed. The reactions largely involve alterations

to ring structure and so affect ergocalciferol and

other D vitamins in a similar manner. It is generally

acknowledged that ergocalciferol is less stable than

cholecalciferol. This may imply that the double bond

in the side chain of ergocalciferol imparts additional

lability to the molecule.

0006Losses during storage are also known to occur in

foods, and vary considerably between food types and

conditions of storage. Thus, low temperatures and

7-Dehydrocholesterol

(provitamin D

)

Previtamin D

Light

Heat

Cholecalciferol

(vitamin D

)

Lumisterol

Tachysterol

fig0002 Figure 2 The major photochemical reactions of cholecalciferol. Ergocalciferol undergoes similar reactions. Overirradiation

products are not shown.

CHOLECALCIFEROL/Properties and Determination 1207

>150 ⬚C

Pyrocholecalciferol

Isopyrocholecalciferol

Isocholecalciferol

Cholecalciferol

Isomerization

Acid

Metabolites

5,6-trans-cholecalciferol

Isotachysterol

1,25-Hydroxycholecalciferol

25-Hydroxycholecalciferol

fig0003 Figure 3 The major degradation reactions of cholecalciferol as relevant to foods.

1208 CHOLECALCIFEROL/Properties and Determination

absence of light will minimize losses in manufactured

foods, as will vacuum packaging or nitrogen flushing.

Foods containing natural, or added fat-soluble anti-

oxidants such as vitamin E and carotenoids will, in

general, exhibit superior cholecalciferol conservation.

(See Antioxidants: Natural Antioxidants; Antioxi-

dants: Synthetic Antioxidants.)

Occurrence and Forms in Foods

0007 Most natural foods are limited in their content of

active vitamin D components. 7-Dehydrocholesterol

(provitamin D

) and ergosterol (provitamin D

) are

widely distributed within the animal and plant

kingdoms and supply a potentially good dietary

source of vitamin D for humans. In particular, provi-

tamins are abundant in fish, eggs, yeast, liver, milk,

and some vegetables such as cabbage. It has been

suggested that ergosterol only enters the food

chain following fungal contamination. Consequently,

mushrooms are good sources of ergosterol, particu-

larly in the wild.

0008 Vitamin D itself is generally present at low levels in

unfortified foods derived from animal sources. Eggs,

fish, and fish liver oils possess significant amounts,

while meat and dairy products contain lesser quan-

tities. Plants and vegetable oils contain negligible levels

(< 2% recommended dietary allowance). Table 2 pro-

vides a list of the richest food sources of vitamin D

and their approximate concentrations.

0009 In addition, previtamin D isomers invariably coex-

ist with cholecalciferol and ergocalciferol. Previtamin

concentrations are proportional to the calciferols and

are influenced by thermal conditions during food

processing and storage. Higher temperatures increase

the previtamin-to-vitamin ratio. Although the pre-

vitamins are biologically active precursors, they are

not always accounted for in nutritional tables due to

analytical difficulties in their determination.

0010Some foods also contain small but significant quan-

tities of hydroxylated metabolites. These compounds

are highly bioactive and are found in edible tissues

(meat, liver) and fluids (milk) as a result of their

biosynthesis within the live animal. The most pre-

dominant and biologically important compounds

are 25-hydroxyvitamin D and 1,25-dihydroxyvita-

min D, while other trihydroxy metabolites may also

be present.

Use in Food Fortification

0011The supplementation of food products with vitamin

D is a contentious issue because of the toxicological

consequences of overdose. While adequate exposure

to sunlight will generally negate the need for this

practice, modern western lifestyles (including the

recent trend to reduce the intake of dietary fat) and

climatic variables justify its continuation. (See Food

Fortification.)

0012As many diets will not supply the 10 mg (400 IU) of

vitamin D required per day, some additional source is

generally recommended by nutritionists. The balance

of vitamin can be obtained from specially prepared

natural sources (concentrated fish oils) or from con-

sumption of artifically fortified food products. In this

way, the onset of diet-induced bone disease is minim-

ized, particularly in infants, the sick, and the elderly.

Legislation in many countries governs fortification

strategies.

0013Cholecalciferol is the most common D vitamin

additive, ergocalciferol being less frequently used for

human nutrition. Difficulties potentially arise in

delivery of the vitamin to the consumer as a conse-

quence of its intrinsic hydrophobic and labile nature.

Common vehicles are margarine, milk, and milk

powders, while a number of cereal products and vari-

ous dietetic formulations contain vitamin D as part of

their primary purpose.

0014Cholecalciferol can be easily incorporated into fat

and oil-based foods by simple dissolution. It is

common to add both vitamin D and vitamin A (ret-

inol) to these foods. A protective phenolic antioxidant

(e.g., 3-tert-butyl-4-hydroxyanisole, tert-butylhydro-

quinone, tocopherol) is usually incorporated at the

same time. For powdered foods, cholecalciferol is

added in an encapsulated ‘beadlet’ form, protected

by a gelatine or acacia barrier and incorporating

tbl0002 Table 2 Typical vitamin D content of various foods

Vitamin D content

of edible portion

(mgper100g)

Natural food products

Fish liver oils 150–3800

Fish 2–30

Egg yolk 5–8

Mammalian liver 0.5–4

Mushrooms (cultivated) 1–3

Mushrooms (wild) 10–30

Butter 1.5–2

Meat 0.2–2

Cheese 0.1–1

Liquid whole milk (raw) 0.05–0.15

Green vegetables (typical) 0.005 (approx.)

Supplemented food products

Dietary formulae (milk- and soya-based) 3–14

Whole-milk powders 6–12

Margarine 8–10

Infant formulae 5–9

Liquid milk 0.75–1.25

To convert to IU per 100 g, multiply by 40.

CHOLECALCIFEROL/Properties and Determination 1209

stabilizers and carrier. If the blending of the vitamin

and food is performed by dry mixing, then cholecal-

ciferol is maintained in its protected environment,

ensuring enhanced shelf-life. However, attaining uni-

form dispersion remains a problem and particle sizes

must be carefully controlled to avoid redistribution

during packaging, transport and storage. Alterna-

tively, these difficulties can be overcome using ‘wet-

blending’ procedures, but the vitamin is inevitably

released from within its stabilized form into direct

contact with the bulk food. Consequently, the receiv-

ing environment must be designed to minimize the

potentially rapid degradation of the cholecalciferol

supplement. (See Retinol: Physiology.)

Analytical Considerations

0015 While it is clinically important to measure the

hydroxylated metabolites, food scientists have been

largely concerned with vitamin D itself, at both

endogenous and supplemental levels.

0016 The quantification of the parent secosteroid in

foods is complicated by several factors, including

low concentration, overwhelming excesses of other

lipophilic components, limiting spectral properties

(nonselective l

max

, low absorptivity), absence of

native fluorescence, thermal instability, and the

requirement to differentiate cholecalciferol from

ergocalciferol.

0017 Historically, curative and prophylactic bioassay

techniques, based on the antirachitic quality of vita-

min D-containing food, have been used extensively

and have the advantage of estimating the true

(species-specific) physiological response. Although

sensitive, disadvantages of time, cost, and poor preci-

sion moderate against the use of bioassays in routine

food analysis.

0018 Competitive protein-binding radioassay tech-

niques, utilizing a vitamin D receptor, have been

exploited more recently, especially in clinical assays.

Such biochemical recognition methods offer advan-

tages in sensitivity and specificity, but extensive

sample purification is mandatory in order to avoid

endpoint interference from food artifacts. Lack of

binding discrimination between vitamins D

and D

as well as the need for radioactive tracers and lengthy

incubation periods remain problematic within the

food industry. (See Immunoassays: Radioimmuno-

assay and Enzyme Immunoassay.)

0019 Physicochemical determination by UV spectropho-

tometry or colorimetry without prior separation is

clearly impracticable except for highly simplified

food matrices, since the spectral properties of the

parent or derivative calciferol species are insufficiently

characteristic. Even high-potency pharmaceutical

preparations still require scrupulous clean-up proced-

ures in order to minimize spectral interference.

0020The development of instrumental chromatographic

techniques has revolutionized the task of vitamin D

estimation in both food and clinical samples. Al-

though novel detection methods exploiting selective

UV, fluorescence derivatization, or electrochemical

techniques are presently under investigation, contem-

porary UV approaches still require the inclusion of

rigorous purification procedures, since this detection

mode remains inherently nonspecific.

Isolation, Extraction, and Clean-up

0021Vitamin D is vulnerable to oxygen, light, low pH and

is also subject to reversible isomerization when

heated. Therefore, adequate precautions are essential

throughout any analytical procedure to avoid loss of

the target analytes.

0022All chemical extraction techniques exploit the

inherent lipophilic property of this vitamin and a

vitamin-rich fraction is separated from other food

components either by saponification or by total lipid

extraction.

Saponification

0023Alkaline hydrolysis is a convenient way to eliminate

the bulk of neutral lipids and is particularly favored in

high-fat foods and those of an intractable nature. This

popular technique is also advantageous for products

containing encapsulated supplements and when rela-

tively large sample quantities are needed for reasons

of assay sensitivity or analyte heterogeneity.

0024While some authors report the use of high-

temperature saponification, strategies such as direct

measurement or use of conversion factors are then

needed to account for the consequent elevated levels

of previtamin D. These concerns can significantly

complicate the assay and may be avoided through

the use of overnight hydrolysis at ambient tempera-

ture, which additionally offers operational simplicity.

It is considered mandatory during extraction to

include a protective antioxidant and to purge with

inert gas. Vitamin D, along with the other fat-soluble

vitamins, sterols, carotenoids and hydrocarbons,

remains in the nonsaponifiable fraction.

0025Enzymatic hydrolysis with lipase has been sug-

gested as an alternative to saponification, thereby

minimizing possible degradation and facilitating

concurrent recovery of the alkali-unstable vitamin

K, if required.

0026The nonsaponifiable fraction containing the calcif-

erol is rapidly and conveniently partitioned into a

solvent mixture of hexane and diethylether with

good recovery, although other solvent systems have

1210 CHOLECALCIFEROL/Properties and Determination

also been cited in the literature. Following washing to

remove hydrophilic remnants and excess alkali, the

organic phase is dried and evaporated to recover an

enriched crude extract. This will generally require

further clean-up prior to quantification, dependent

on the vitamin D concentration and level of coextrac-

tives.

Total Lipid Extraction

0027 The risks of thermal equilibration with previtamin D

and potential degradation of vitamin D may be

avoided through an initial total lipid extraction. A

variety of solvent systems have been recommended

depending upon sample type and fat content. A con-

sequence of employing such a technique for foods is

the need to isolate the vitamin components from

high-molecular-weight fractions (triglycerides, phos-

pholipids) by gel permeation and/or adsorption

chromatography. Various protocols have been advo-

cated which, while successful, contribute complex-

ities to the assays which are difficult for most

quality control laboratories to manage.

0028 This extraction route has been commonly applied

to low-fat clinical specimens (such as plasma), and

fat-reduced fortified foods (e.g., skim milk). It is also

useful when vitamin D and its metabolites are to be

estimated concurrently. The literature consensus

seems to support the view that saponification is

more appropriate when complex and poorly charac-

terized foods are to be assayed.

0029 The concentrations of cholecalciferol are generally

low, even after isolation of the crude extracts. Further

clean-up to remove substantial quantities of sterols

and other unsaponifiable material is usually incorpor-

ated prior to analysis. Such strategies may be as

simple as cholesterol precipitation or as complex

as multistage semipreparative chromatography,

depending on both sample type and the sophistication

of subsequent analytical techniques. Often at this

stage, analysts may expediently utilize the conveni-

ence of prepacked solid-phase extraction cartridges.

Usually, the adsorption mode with activated silica is

selected, producing an extract of higher vitamin D

content and fewer potential interferences. These dis-

posable cartridges are now widely replacing the

earlier techniques of open-column or thin-layer chro-

matography (although the latter is still occasionally

used for clean-up or qualitative ‘spot testing’).

Chromatography

0030 Quantification of enriched vitamin extracts is achiev-

able by applying gas–liquid chromatography (GLC)

techniques, although the thermal instability of vita-

min D at operating temperatures results in formation

of pyro and isopyro peaks of both parent or derivative

forms. This problem has been successfully avoided by

prior conversion to the thermostable isotachysterol

isomers. Modern GLC now benefits from the use of

capillary columns and ultrasensitive mass-selective

spectrometric detectors, but the extensive manipula-

tive procedures still make GLC less attractive than

other technologies for routine food analysis. (See

Chromatography: Gas Chromatography.)

0031High-performance liquid chromatography (HPLC)

has led to continuing improvements in the assay of

vitamin D (and its metabolites) in foods and has now

superseded GLC. While additional clean-up is not

always unavoidable, derivatization procedures are

unnecessary in the majority of schemes. Furthermore,

the ambient and nondestructive features inherent in

HPLC are more compatible with the lability proper-

ties of this, and other, vitamins. Recently, variants

of micellar capillary electrophoresis (CE) have been

applied to fat-soluble vitamin separations. Although

representing an alternative to HPLC techniques, the

application of CE to the hydrophobic vitamins is

currently in its infancy. (See Chromatography: High-

performance Liquid Chromatography.)

0032In the absence of useful native fluorescence or

stable electrochemical viewing modes and the current

infancy of commercial online liquid chromatography–

mass spectroscopy (LCMS) interfaces, the use of UV

spectrophotometric detection is universal.

0033The modest spectral properties of vitamin D often

require a semipreparative HPLC fractionation step

before an analytical HPLC stage can be undertaken.

Spectral selectivity is further gained by the judicious

use of either wavelength rationing or full-spectrum

(e.g., diode array) detection. Alternative choices to

attain additional specificity and selectivity include

the use of offline competitive protein-binding assay

or the precolumn conversion of cholecalciferol to its

bathochromically shifted isotachysterol.

0034In view of the differing selectivities of normal-

phase and reversed-phase HPLC, many researchers

have concluded that the assay benefits by combining

the two modes. This multidimensional technique ex-

ploits the fact that cholecalciferol and ergocalciferol

are unresolved on silica columns yet are completely

separated using C

reversed-phase columns. There

are a number of reported methods which employ

reversed-phase chromatography during clean-up,

either low-pressure or HPLC, followed by normal-

phase quantitation. This regimen is generally of use

where both calciferols are known to coexist in the

sample or where the sample is not well defined

in terms of its vitamin D content. Alternatively,

normal-phase silica chromatography can be used

during the preparative step and the two vitamins

CHOLECALCIFEROL/Properties and Determination 1211

collected in a single fraction before application to a

reversed-phase column. This allows one vitamin to be

used as an internal standard for the other, greatly

decreasing the cumulative assay errors introduced

though potential isomerization and manipulative

losses. The method can be used where only one vita-

min is present, as is usually encountered in the food

industry. Chromatograms of a fish oil taken through

this analytical procedure are shown in Figure 4.

The endogenous vitamin is cholecalciferol, allowing

internal standardization with ergocalciferol.

0035The elution positions of previtamin D

and pro-

vitamin D

are shown in Figure 4a. They are well

separated from vitamin D

, thus allowing potential

differentiation of the species. Other calciferols shown

in Figures 2 and 3 can be similarly resolved. On silica

columns, using hydrocarbon/alcohol mobile phases,

the major isomers elute in the sequence previtamin

< trans-vitamin D

< lumisterol

< isotachysterol

< vitamin D

< tachysterol

< provitamin D

hydroxymetabolites. The pyrocalciferols elute close

to lumisterol

. The most likely interfering isomer is

tachysterol

which elutes close to vitamin D

in both

normal and reversed-phase analyses. Interestingly, the

retention sequence of previtamin D

, vitamin D

and provitamin D

is the same, regardless of which

separation mode is used.

See also: Antioxidants: Natural Antioxidants; Synthetic

Antioxidants; Chromatography: High-performance

Liquid Chromatography; Gas Chromatography; Food

Fortification; Immunoassays: Radioimmunoassay and

Enzyme Immunoassay; Retinol: Physiology

Further Reading

Ball GFM (1988) Fat-Soluble Vitamin Assays in Food

Analysis. A Comprehensive Review. London: Elsevier.

Bates CJ (2000) Vitamins: fat and water soluble: analysis.

In: Meyers RA (ed.) Encyclopedia of Analytical Chemis-

try, pp. 1–35. Chichester: John Wiley.

Belitz H-D and Grosch W (1987) Food Chemistry, pp.

305–319. Berlin: Springer-Verlag.

Collins ED and Norman AW (1991) Vitamin D. In: Machlin

LJ (ed.) Handbook of Vitamins, pp. 59–98. New York:

Marcel Dekker.

Eitenmiller RR and Landon WO (1999) Vitamin D. In:

Eitenmiller R and Landon WO (eds) Vitamin Analysis

for the Health and Food Sciences, pp. 77–107. Boca

Raton, FL: CRC Press.

Friedrich W (1988) Vitamin D. In: Vitamins, pp. 143–

216.Berlin: de Gruyter.

Indyk H and Woollard DC (1984) The determination of

vitamin D by high performance liquid chromatography.

New Zealand Journal of Dairy Science and Technology

19: 1–6.

Jones G, Seamark DA, Trafford DJH and Makin HLJ

(1985) Vitamin D: cholecalciferol, ergocalciferol and

hydroxylated metabolises. In: De Leenheer AP, Lambert

WE and De Ruyter MGM (eds) Modern Chromato-

graphic Analysis of the Vitamins, pp. 73–128. New

York: Marcel Dekker.

Fraction

collected

for analysis

(a)

020

(b)

Time (min)

fig0004 Figure 4 High-performance liquid chromatography (HPLC)

chromatograms showing the analysis of cholecalciferol in halibut

liver oil. An internal standard of ergocalciferol (D

) was used

throughout the analysis. (a) The semipreparative clean-up

stage. A Brownlee (25 cm  10 mm) silica column was used with

a mobile phase of 1% 2-propanol in hexane, flowing at 3 ml

min

1

. Detection was by ultraviolet at 280 nm (0.04 aufs). The

unsaponifiable fraction from 2.5 g of fish oil was dissolved in

1.0 ml of mobile phase and injected. The fraction collected and

evaporated for quantitative analysis is shown. The elution pos-

itions of previtamin D

(þ) and provitamin D

(*) are shown. (b) A

reversed-phase C

column (Waters 5 mm Radial-PAK) was used

for analyte quantification. A mobile phase of methanol:water:te-

trahydrofuran (90:8:2 v/v/v) at a flow rate of 2.0 ml min

1

was

used. The injection volume was 100 ml, representing the chole-

calciferol (D

) content of 0.25 g of oil. Detection was at 280 nm

(0.005 aufs).

1212 CHOLECALCIFEROL/Properties and Determination

Lawson DEM (1985) Vitamin D. In: Diplock AT (ed.) Fat

Soluble Vitamins. Their Biochemistry and Applications,

Chapter 2, pp. 76–153. London: Heinemann.

Luque de Castro MD, F-Romero JM, O-Boyer F and

Quesada JM (1999) Determination of vitamin D

me-

tabolites: state-of-the-art and trends. A review. Journal

of Pharmaceutical and Biomedical Analysis 20: 1–17.

Macrae R (1988) HPLC in Food Analysis, 2nd edn.

London: Academic Press.

Mattila PH, Piironen VI, Uusi-Rauva EJ and Koivistoinen

PE (1996) New analytical aspects of vitamin D in foods.

Food Chemistry 57: 95–99.

Parrish DB (1979) Determination of vitamin D in foods.

CRC Critical Reviews in Food Science and Nutrition 12:

29–57.

Strohecker R and Henning HM (eds) (1965) Vitamin D. In:

Vitamin Assay, Tested Methods, pp. 254–282. Verlag

Chemie.

Physiology

A W Norman, University of California-Riverside,

Riverside, CA, USA

Background

0001 Vitamin D is essential for life in higher animals. Clas-

sically, it has been shown to be one of the most

important biological regulators of calcium homeo-

stasis. It has been established that these important

biological effects are only achieved as a consequence

of the metabolism of vitamin D into a family of

daughter metabolites, including the two key kidney-

produced metabolites 1a,25(OH)

-vitamin D

(1a,25(OH)

) and 24R,25(OH)

-vitamin D

(24R,25(OH)

). 1a,25(OH)

is considered to

be a steroid hormone and there is increasing evidence

that 24R,25(OH)

is also a steroid hormone.

0002 It has become increasingly apparent since the

1980s that 1a,25(OH)

also plays an important

multidisciplinary role in tissues not primarily related

to mineral metabolism, e.g., the hematopoietic

system, effects on cell differentiation and prolifer-

ation including important interactions with keratino-

cytes and cancer cells, and participation in the

processes of parathyroid hormone and insulin secre-

tion. The purpose of this chapter is to provide a

succinct overview of our current understanding of

the important nutritional substance vitamin D and

the mechanisms by which its daughter hormone

1a,25(OH)

mediates biological responses.

Historical Review

0003The first scientific description of rickets, which is the

hallmark of a vitamin D-deficiency, was provided in

the seventeenth century by both Dr. Daniel Whistler

(1645) and Professor Francis Glisson (1650). The

major breakthrough in understanding the causative

factors of rickets was the development of nutrition as

an experimental science and the appreciation of the

existence of vitamins. Considering the fact that now

we accept that the biologically active form of vitamin

D is a steroid hormone, it is somewhat ironic that

vitamin D, through a historical accident, became clas-

sified as a vitamin. It was in 1919/20 that Sir Edward

Mellanby, working with dogs raised exclusively in-

doors (in the absence of sunlight or ultraviolet light),

devised a diet which allowed him to establish un-

equivocally that rickets was caused by a deficiency

of a trace component present in the diet. In 1921, he

wrote, ‘The action of fats in rickets is due to a vitamin

or accessory food factor which they contain, probably

identical with the fat-soluble vitamin.’ Furthermore,

he established that cod-liver oil was an excellent anti-

rachitic agent; this ultimately led to the antirachitic

factor being classified as a vitamin.

0004The chemical structures of the vitamins D were

determined in the 1930s in the laboratory of Profes-

sor A. Windaus at the University of Gottingen. Vita-

min D

, which could be produced by ultraviolet

irradiation of ergosterol, was chemically character-

ized in 1932. Vitamin D

was not chemically charac-

terized until 1936, when it was shown to result from

the ultraviolet irradiation of 7-dehydrocholesterol.

Virtually simultaneously, the elusive antirachitic com-

ponent of cod-liver oil was shown to be identical to

the newly characterized vitamin D

. These results

clearly established that the antirachitic substance

vitamin D was chemically a steroid, more specifically

a seco-steroid (see below).

0005The modern era of vitamin D began during the

period of 1965–70 with the discovery and chemical

characterization of 1a,25(OH)

and its nuclear

receptor, the VDR

nuc

Chemistry of Vitamin D

0006Vitamin D

is the naturally occurring form of vitamin

D and is produced from 7-dehydrocholesterol (see

Figure 1). Vitamin D

is a synthetic form of vitamin

D that is produced by irradiation of the plant yeast

steroid, ergosterol.

0007The structures of vitamin D

(cholecalciferol) and

its provitamin 7-dehydrocholesterol are presented in

Figure 1. Vitamin D is a generic term and indicates a

molecule of the general structure shown for rings A, B,

CHOLECALCIFEROL/Physiology 1213

C, and D with differing side-chain structures. The A,

B, C, and D ring structure is derived from the cyclo-

pentanoperhydrophenanthrene ring structure for ster-

oids. Technically, the steroid vitamin D is classified as

a seco-steroid. Seco-steroids are those in which one of

the rings has been broken; in vitamin D, the 9,10

carbon–carbon bond of ring B is broken, and it is

indicated by the inclusion of ‘9,10-seco’ in the official

nomenclature.

0008 Vitamin D (synonym calciferol) is named

according to the revised rules of the International

Union of Pure and Applied Chemistry (IUPAC).

Because vitamin D is derived from a steroid, the

structure retains its numbering from the parent com-

pound cholesterol (see Figure 1). Asymmetric centers

are designated by using the R,S notation; the config-

uration of the double bonds is indicated as E for

‘entgegen’ or trans, and Z for ‘zusammen’ or cis.

Thus, the official name of vitamin D

is 9,10-seco

(5Z, 7E)-5,7,10(19)cholestatriene-3b-ol. Vitamin D

differs from D

by virtue of the presence of a 22-ene

and 24-methyl group in the side-chain. The official

2221

24 27

Cholesterol

18 10

23 25

AAB

Cortisol

(A classic steroid hormone)

Ergosterol

(Pro-vitamin D

)

7-dehydrocholesterol

(Pro-vitamin D

)

Sunlight

(Natural process)

Vitamin D

Cholecalciferol

Vitamin D

Ergocalciferol

UV Light

(Commercial preparation)

fig0001 Figure 1 Structural relationship of vitamin D

(cholecalciferol) and vitamin D

(ergocalciferol) with their respective provitamins

(7-dehydrocholesterol and ergosterol), cholesterol, and a classic steroid hormone, cortisol (see inset box). The two structural

representations presented at the bottom for both vitamin D

and vitamin D

are equivalent; these are simply different ways of drawing

the same molecule. It should be noted that vitamin D

is the naturally occurring form of the vitamin; it is produced from

7-dehydrocholesterol, which is present in the skin, by the action of sunlight. Vitamin D

(which is equivalently potent to D

in humans

and many mammals, but not birds) is produced commercially by the irradiation of the plant sterol ergosterol with ultraviolet light.

1214 CHOLECALCIFEROL/Physiology

name of vitamin D

is 9,10-seco(5Z,7E)-

5,7,10(19),22-ergostatetraene-3b-ol.

0009 Vitamin D

can be produced photochemically by

the action of sunlight or ultraviolet light from the

precursor sterol 7-dehydrocholesterol that is present

in the epidermis or skin of most higher animals. The

chief structural prerequisite of a provitamin D is that

it be a sterol with a D5–7 diene double bond system

in ring B (see Figure 1). The conjugated double

bond system in this specific location of the molecule

allows the absorption of light quanta at certain wave-

lengths in the UV range; this can be readily provided

in most geographical locations by natural sunlight.

This initiates a complex series of transformations

(partially summarized in Figure 1) that ultimately

result in the transformation into vitamin D

. Thus,

it is important to appreciate that vitamin D

can be

endogenously produced and that as long as the

animal (or human) has access on a regular basis

to sunlight, there is no dietary requirement for this

vitamin.

Physiology and Biochemistry

Vitamin D Endocrine System

0010Vitamin D

is not known to have any intrinsic

biological activity itself. It is only after vitamin D

is metabolized first into 25(OH)D

in the liver

and then into 1a,25(OH)

and 24R,25(OH)

by the kidney, that biologically active molecules are

produced. In toto, some 37 vitamin D

metabolites

have been isolated and chemically characterized.

Figure 2 illustrates the concept of the vitamin D endo-

crine system. The elements of the vitamin D endocrine

system include the following: (1) in the skin, photo-

conversion of 7-dehydrocholesterol to vitamin D

dietary intake of vitamin D

; (2) metabolism of vita-

min D

by the liver to 25(OH)D

, which is the major

form of vitamin D circulating in the blood compart-

ment; (3) conversion of 25(OH)

by the kidney

(functioning as an endocrine gland) to produce the

two principal dihydroxylated metabolites,

24R,25(OH)

1α,25(OH)

(−)(+)

(+)

1-Hydroxylase24-Hydroxylase

Endocrine modulators

Parathyroid

hormone

(−)

Short

feedback

loop

(−)

Long

feedback

loop

, P

, H

Estrogen

Calcitonin

Growth hormone

Prolactin

Insulin

Glucocorticoid

Kidney

25(OH)D

25(OH) D

HO HO

24R,25(OH)

1α,25(OH)

7-dehydrocholesterol

Placental

production

37 Chemically

characterized

metabolites

Macrophages (activated)

Keratinocytes

Astrocytes (activated)

Paracrine production of

1α,25(OH)

24R,25(OH)

Fetus

1α,25(OH)

Mediated

cellular growth and

defferentiation

1α,25(OH)

Rapid actions

1α,25(OH)

Nuclear receptors

Adipose

Adrenal

Bone

Bone marrow

Brain

Breast

Cancer cells (many)

Cartilage

Colon

Eggshell gland

Epididymis

Ganglion

Hair follicle

Intestine

Kidney

Liver (fetal)

Lung

Muscle, (cardiac)

Muscle, (smooth)

Osteoblast

Ovary

Pancreas β cell

Parathyroid

Parotid

Pituitary

Placenta

Prostate

Skin

Stomach

Testis

Thymus

Thyroid

Uterus

Yolk sac (birds)

Intestine

Bone

Parathyroid

Liver

Pancreas β cell

PKC (activation)

Development

Generation of

osteoclasts

Selected biological

responses

(present in skin)

(hormonal origin)

Hematopoietic cells

Skin

Brain

(Sunlight)

Vitamin D

Dietary

sources

∆ Heat

Blood Blood

Blood

1α,25(OH)

Blood

24R,25(OH)

Receptors

Chondrocyte

Fracture-healing callus

Reabsorption of Ca

and P

Mobilization / accretion of Ca

and P

Absorption of Ca

Classic target organs

Bone

Intestine

Kidney

Liver

Blood

fig0002 Figure 2 Summary of the vitamin D endocrine system. In this system, the biologically inactive vitamin D

is activated, first in the liver

to generate 25(OH)D

, which is then converted by the endocrine gland, the kidney, to the hormones 1a ,25(OH)

and 24R,25(OH)

, inorganic phosphate. There is currently much research being conducted to understand structure–function relationships with

respect to chemical synthesis of analogs of 1a,25(OH)

and their interaction with the vitamin D endocrine system. The objective is

to develop new drug forms of 1a,25(OH)

CHOLECALCIFEROL/Physiology 1215