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

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colored, turbid, or precipitated debris does not inter-

fere with the

output or detection; furthermore,

the scrupulous cleaning of glassware required for

turbidimetric assays is unnecessary. An assay for

pantothenic acid in human milk and blood is based

on the measurement of

produced from

the metabolism of l-[1-

C]methionine or l-[1-

C]-

valine by the yeast Kloeckera brevis (ATCC 9774).

Metabolic CO

can also be measured nonradio-

metrically with the aid of an infrared CO

analyzer,

which measures automatically the infrared radiation

absorbed by the CO

band at 4.2 mm.

Radioimmunoassay

0015 The radioimmunoassay is based on the competition

for a fixed, but limited, number of antibody binding

sites by antigen (a substance capable of binding to a

specific antibody) and a trace amount of radiolabeled

antigen added to the sample extract. In this case, the

antigen is the analyte, pantothenic acid. The presence

of larger amounts of unlabeled analyte results in less

radioactivity being bound to the antibody.

0016 Wyse and colleagues raised antibodies to panto-

thenic acid by coupling a bromoacetyl derivative

of pantothenic acid with reduced and denatured

bovine serum albumin and injecting this immunogen

into rabbits. For the assay, each tube contained

diluted antiserum, pantothenic acid standard solu-

tion or sample extract, and radiolabeled sodium

d-pantothenate. After incubation, neutral saturated

ammonium sulfate was added to facilitate suspension

of the antibody-bound pantothenic acid, which was

then centrifuged. The washed precipitate, containing

antibody-bound pantothenic acid, was dissolved in

tissue solubilizer, and the radioactivity was counted

in a scintillation counter. The amount of pantothenic

acid in each unknown was determined by reference to

a standard curve constructed by plotting on logit-

semilog paper log concentration of nonradioactive

pantothenic acid in the standard vials (ng per

0.50 ml) versus the percentage of the counts bound.

Results from the radioimmunoassay and AOAC

microbiological assay for pantothenic acid in 75 pro-

cessed and cooked foods were highly correlated

(r ¼0.94). However, there was a statistically signi-

ficant difference (p < 0.05) between the two assay

results for all foods and for the subgroups meats,

breads and cereals, and fruits and vegetables. At

p < 0.01, only meats were significantly different. For

breads and cereals, the microbiological assay results

averaged 6.6% higher than those of the radioimmu-

noassay. For fruits and vegetables, the microbiological

assay results were 11.6% higher; for meats, the results

were 23.2% higher. It was postulated that bacterial

enzymes in the assay organism promote further break-

down of bound pantothenic acid, or nonenzymatic

breakdown occurs during the long microbiological

incubation period.

Enzyme-linked Immunosorbent Assay (ELISA)

0017An ELISA is an enzyme-linked immunoassay in which

one of the reactants is immobilized by physical ad-

sorption on to the surface of a solid phase. In its

simplest form, as used in food analysis applications,

the solid phase is provided by the plastic surface of a

96-well microtitration plate. The generally preferred

format for vitamin assays in food analysis is a two-

site noncompetitive assay used in the indirect mode.

This format employs two antibodies: a primary anti-

vitamin antibody raised against an immunogen (in

this case, a pantothenic acid–protein conjugate), and

an enzyme-labeled, species-specific second antibody,

which binds specifically to the primary antibody. To

perform such an assay, a protein conjugate of panto-

thenic acid is immobilized to the well surface of the

microtitration plate, the attached protein being differ-

ent to that used for the immunogen. The protein

adsorbs passively and strongly to the plastic, and,

once coated, plates can usually be stored for several

months. To perform the assay, the standard solution

or sample extract is added to the well, followed by a

limited amount of primary antibody. After incuba-

tion, the antibody becomes distributed between im-

mobilized vitamin and free vitamin according to

the amount of vitamin initially present. After phase

separation, achieved by well emptying and washing,

the second antibody is added in excess, and the plate

is incubated for a second time. Excess unbound

material is removed, and the amount of bound

enzyme is determined by addition of substrate and

spectrophotometric measurement of the colored

product. Unknown samples are quantified by refer-

ence to the behavior of vitamin standards.

0018Finglas and colleagues developed a noncompetitive

ELISA, which is highly specific for pantothenic acid

and does not recognize coenzyme A, panthenol or

pantheneine. The primary antivitamin antibody was

raised in rabbits according to the method of Wyse and

colleagues (see section Radioimmunoassay), and

the enzyme-labeled, species-specific second antibody

(alkaline phosphatase-labeled antirabbit IgG) was

obtained commercially. Microtitration plates were

coated with pantothenic acid–keyhole limpet hemo-

cyanin as the immobilized phase of the assay. A high

correlation coefficient (r ¼0.999) was reported when

ELISA values obtained for six foods were compared

with corresponding values obtained by a microbio-

logical method using L. plantarum. Gonthier and

PANTOTHENIC ACID/Properties and Determination 4335

colleagues improved the sensitivity of the ELISA by

using an immunogen composed of pantothenic acid

coupled to thyroglobulin by a 6-carbon atom linker

(adipoyl dichloride). By contrast, the bromoacetyl lin-

ker used in Finglas’ pantothenic acid–bovine serum

albumin immunogen contains two carbon atoms.

Gas Chromatography

0019 Salts of pantothenic acid present in pharmaceutical

preparations have been analyzed by gas chromatog-

raphy after conversion to volatile acetate, trifluoroa-

cetate, or trimethylsilyl derivatives. An alternative

approach to derivatization is to chromatograph the

pantoyl lactone formed from pantothenic acid by acid

hydrolysis (Figure 2). This approach is applicable to

foodstuffs, because the hydrolysis reaction liberates

the lactone from the free and bound pantothenic acid

in the food matrix with a recovery of at least 95%.

Davı

dek and colleagues applied this technique to the

determination of pantothenic acid in fresh beef liver,

spray-dried egg yolk, soybean flour, whole-grain

wheat flour, and dried bakers’ yeast. Samples were

hydrolyzed by treatment with dilute hydrochloric

acid, and the neutralized hydrolysate, after filtration,

was extracted with dichloromethane. The combined

extracts, to which ethyl laurate was added as an

internal standard, were concentrated by rotary evap-

oration, and then analyzed by gas chromatography

using a polar stationary phase of 10% Carbowax

20M and a flame ionization detector. Gas chromato-

graphic results correlated with results obtained by

the currently accepted microbiological method

(r ¼0.975), and no significant difference was found

between the two sets of results (p > 0.05). Davı

dek

and colleagues used a packed column of dimensions

2.4 m 2 mm i.d. in which the stationary phase was

coated on to a porous support material of diatom-

aceous earth treated with dimethyldichlorosilane.

Woollard and colleagues upgraded the column to a

more efficient 30 m 0.25 mm open-tubular capil-

lary column coated with stationary phase (BPX70).

High-performance Liquid Chromatography (HPLC)

and Capillary Electrophoresis

0020 The pantothenic acid molecule does not contain a

characteristic chromophore, and hence it exhibits

only very weak absorbance at 204 nm, owing to the

presence of carbonyl groups. Detection at wave-

lengths below 220 nm is subject to interference from

the many organic compounds present in a typical

food sample extract prepared for HPLC. The problem

of weak and nonspecific absorbance, coupled with

the low concentrations of pantothenic acid in foods,

has thwarted attempts to apply HPLC to the

determination of pantothenic acid in foodstuffs,

although the technique has been successfully applied

to pharmaceutical products. Chemical treatment of

the pantothenic acid molecule to form a derivative

that fluoresces or absorbs at a higher wavelength is a

possibility, but, so far, reproducible results have not

been obtained using such an approach. Refractom-

etry as a means of detection lacks the required sensi-

tivity and specificity.

0021Recognizing these problems, Woollard and Indyk

in New Zealand developed an HPLC method for

determining free endogenous d-pantothenic acid

in milk and supplemental calcium pantothenate

in infant formulas. Sample preparation simply

entailed addition of acetic acid to the milk or reconsti-

tuted infant formula, followed by centrifugation and

membrane filtration. This treatment resulted in a

protein- and fat-free extract that could be directly

injected (10-ml aliquot). The problems of poor detec-

tion specificity in the low-ultraviolet region of the

spectrum were overcome by the use of a photodiode

array detector that provided multiwavelength detec-

tion (selected wavelengths were 200, 205 and

240 nm) and on-line spectral analysis. The HPLC

system incorporated an on-line mobile phase degasser

– an important feature, as dissolved oxygen consti-

tutes a source of interference at low ultraviolet wave-

lengths. Among several reversed-phase columns

investigated, the column selected for routine use was

of dimensions 250 4.6 mm i.d. and packed with

Luna (Phenomenex) 5 mmC

(octyl) of 100 A

pore

size, 400 m

1

surface area, and 13.5% carbon

load. The Luna material is based on low-acidity silica,

exhaustive end-capping, and shielded bonded phase

ligand. The mobile phase was phosphate buffer

(0.1 M, pH 2.25): acetonitrile (97:3, v/v) delivered

initially at 1.4 ml min

1

and increased to 1.8 ml min

1

at 18 min. Following completion of the sample sched-

ule, the column was purged sequentially with aceto-

nitrile:water (50:50), water (100%),

acetonitrile:water (50:50), and finally acetonitrile

(100%) for column storage between runs. The reten-

tion time of the pantothenic acid was around 15 min,

but the next sample was not injected until a major

unknown peak with a retention time of about 35 min

was removed (Figure 3).

fig0002Figure 2 Pantoyl lactone.

4336 PANTOTHENIC ACID/Properties and Determination

0022 Reversed-phase columns do not retain acidic

solutes in the ionic state, but if the mobile phase is

buffered to pH 3 or lower, the acidic solute will be

nonionized and act as a neutral solute. This technique

is known as ion suppression. Under these conditions,

residual silanol groups on the silica support will also

be nonionized. The net result is retention of undisso-

ciated acidic solutes with no peak tailing, owing to

the elimination of electrostatic interactions between

acidic solutes and silanol groups. A potential problem

with certain reversed-phase column packings is that

the siloxane (

—

Si—O—Si

—

) bond linking the alkyl

ligand to the silica support is subject to hydrolysis at

low pH, resulting in loss of bonded phase. Although

longer-chain ligands such as C

are relatively stable

at a low pH, short-chain bonded phases, including

small endcapping groups, are especially susceptible.

The problem of hydrolysis and loss of bonded phase

can be minimized by the use of ‘shielded’ stationary

phases, which are sterically protected from attack by

hydrolyzing protons. Results obtained by HPLC cor-

related with those obtained by microbiological assay

utilizing L. plantarum (r ¼0.971), and there was no

significant difference (p > 0.05) between the two sets

of results.

0023Reversed-phase HPLC with ion suppression is

unable to separate the d and l enantiomers in syn-

thetic calcium pantothenate. However, separation

200

240 280

240 nm

205 nm

200 nm

Time (min)

fig0003 Figure 3 Multiwavelength UV chromatogram (200, 205, 240 nm) of a typical infant formula extract obtained with a Luna C

(octyl)

column. The insert illustrates a UV spectral scan of pantothenic acid. Chromatographic parameters are given in the text. Reprinted

from Woollard DC, Indyk HE and Christiansen SK (2000) The analysis of pantothenic acid in milk and infant formulas by HPLC. Food

Chemistry 69: 201–208, with permission from Elsevier Science.

PANTOTHENIC ACID/Properties and Determination 4337

of enantiomers in dl pantothenic acid has been

reported using a chiral selector in capillary electro-

phoresis. Optimum separation was obtained using

a pH 7.0 phosphate buffer containing the chiral

selector (60 mM 2-hydroxypropyl-b-cyclodextrin)

and 10% (v/v) methanol. The enantiomers were

unresolved when a buffer of pH 3.0 was tried, imply-

ing that dissociation of pantothenic acid was neces-

sary for its chiral resolution under the conditions

employed.

Overall Appraisal of Analytical Techniques

0024 The ‘free’ (no enzyme treatment) or ‘total’ (after

enzyme treatment) pantothenic acid content of a

food has been traditionally determined by the turbi-

dimetric microbiological assay using L. plantarum.

This assay has been adopted as an official method

by the AOAC on the basis of collaborative study.

Once the facilities are in place, the microbiological

assay can be used routinely to determine other

B-group vitamins with minor changes in protocol.

Inherent problems include stimulation or inhibition

of bacterial growth by other compounds, and non-

linear response (drift) for various volumes of food

extract analyzed. The radioimmunoassay produces

results that, although correlated with microbiological

assay results, are significantly lower. The use of radio-

isotopes would not be permitted in the vicinity of

commercial food production. The ELISA is a better

substitute for the microbiological assay, as results

from the two techniques are in good agreement. The

high technology of the ELISA is built into the re-

agents, so the assays are simpler to perform than

microbiological assays. Routine use of the ELISA for

determining pantothenic acid will depend on the

commercial availability of standardized assay kits.

Little interest seems to have been taken in gas

chromatography, but the technique of chromato-

graphing the lactone hydrolysis product merits

further investigation using modern capillary columns.

High-performance liquid chromatography is becom-

ing increasingly popular for determining vitamins

in foods, although the poor detectability of panto-

thenic acid limits the sensitivity of this technique.

Capillary electrophoresis also suffers from poor sen-

sitivity but, when used with a chiral selector, has the

advantage of separating active d and inactive l

enantiomers of racemic calcium pantothenate added

to foods.

See also: Bioavailability of Nutrients; Chromatography:

Principles; High-performance Liquid Chromatography;

Gas Chromatography; Immunoassays: Principles;

Radioimmunoassay and Enzyme Immunoassay

Further Reading

AOAC (1995) AOAC Official Method 992.07. Pantothenic

acid in milk-based infant formula. Microbiological

turbidimetric method. In: Official Methods of Analysis

of AOAC International, Method No 50.1.22, 16th edn.

Arlington VA: AOAC International.

Bell JG (1974) Microbiological assay of vitamins of the B

group in foodstuffs. Laboratory Practice 23: 235–242,

252.

Crawley H (1993) Natural occurrence of vitamins in food.

In: Berry Ottaway P (ed.) The Technology of Vitamins in

Food. Glasgow: Blackie Academic & Professional.

Davı

dek J, Velı

ek J, C

erna

J and Davı

dek T (1985) Gas

chromatographic determination of pantothenic acid in

foodstuffs. Journal of Micronutrient Analysis 1: 39–46.

Finglas PM, Faulks RM, Morris HC, Scott KJ and Morgan

MRA (1988) The development of an enzyme-linked

immunosorbent assay (ELISA) for the analysis of panto-

thenic acid and analogues. Part II – Determination of

pantothenic acid in foods. Journal of Micronutrient

Analysis 4: 47–59.

Goli DM and Vanderslice JT (1989) Microbiological assays

of folacin using a CO

analyzer system. Journal of

Micronutrient Analysis 6: 19–33.

Gonthier A, Boullanger P, Fayol V and Hartmann DJ (1998)

Development of an ELISA for pantothenic acid (vitamin

) for application in the nutrition and biological fields.

Journal of Immunoassay 19: 167–194.

Gonthier A, Fayol V, Viollet J and Hartmann DJ (1998)

Determination of pantothenic acid in foods: influence

of the extraction method. Food Chemistry 63: 287–294.

Guilarte TR (1989) A radiometric microbiological assay

for pantothenic acid in biological fluids. Analytical

Biochemistry 178: 63–66.

Kodama S, Yamamoto A and Matsunaga A (1998)

Direct chiral resolution of pantothenic acid using

2-hydroxypropyl-b-cyclodextrin in capillary electro-

phoresis. Journal of Chromatography A 811: 269–273.

Morris HC, Finglas PM, Faulks RM and Morgan MRA

(1988) The development of an enzyme-linked immuno-

sorbent assay (ELISA) for the analysis of pantothenic

acid and analogues. Part I – Production of antibodies

and establishment of ELISA systems. Journal of Micro-

nutrient Analysis 4: 33–45.

Walsh JH, Wyse BW and Hansen RG (1979) A comparison

of microbiological and radioimmunoassay methods for

the determination of pantothenic acid in foods. Journal

of Food Biochemistry 3: 175–189.

Woollard DC, Indyk HE and Christiansen SK (2000) The

analysis of pantothenic acid in milk and infant formulas

by HPLC. Food Chemistry 69: 201–208.

Wyse BW, Wittwer C and Hansen RG (1979) Radioimmu-

noassay for pantothenic acid in blood and other tissues.

Clinical Chemistry 25: 108–111.

4338 PANTOTHENIC ACID/Properties and Determination

Physiology

G F M Ball, Wembley, London, UK

Background

0001 The biological activity of pantothenic acid is attribut-

able to its incorporation into the molecular structures

of coenzyme A (CoA) and acyl carrier protein. CoA

performs multiple roles in cellular metabolism,

whereas acyl carrier protein is involved in fatty acid

biosynthesis. A wide variety of functional proteins are

modified by the addition of acetyl, acyl, and isoprenyl

groups through reactions that directly or indirectly

involve CoA. These modifications allow the regula-

tion of such important processes as gene transcrip-

tion, signal transduction, and vision.

Metabolism

Intestinal Absorption

0002 Ingested CoA, the major dietary form of pantothenic

acid, is hydrolyzed in the intestinal lumen to pan-

tetheine by the nonspecific action of pyrophospha-

tases and phosphatase. Pantetheine is then split into

pantothenic acid and b-mercaptoethylamine by the

action of pantetheinase secreted from the intestinal

mucosa into the lumen. Within the alkaline medium

of the luminal contents, pantothenic acid exists

primarily as the pantothenate anion. Absorption of

the liberated pantothenate takes place mainly in the

jejunum.

0003 At physiological intakes, pantothenate must move

across the brush-border membrane of the intestinal

epithelium from a region of lower concentration in

the lumen to one of higher concentration in the cyto-

plasm of the absorptive cell (enterocyte). Such ‘uphill’

movement requires active transport – a mechanism

that depends ultimately upon the expenditure of

metabolic energy, i.e., the energy released from the

hydrolysis of adenosine triphosphate produced

during cellular metabolism. The precise mechanism

of pantothenate absorption is secondary active trans-

port in which a transmembrane protein (inappropri-

ately called a carrier) mediates the sodium-coupled

transfer of pantothenate across the brush-border

membrane. The carrier spans the membrane in a

weaving fashion and effects solute transfer through

a conformational change in its molecular structure.

The immediate energy source for the transport

mechanism is the concentration gradient of sodium

across the brush-border membrane. The gradient is

maintained by the constant extrusion of sodium from

the enterocyte by the action of the sodium pump at

the basolateral membrane. The sodium pump is

driven by metabolic energy and is the primary driving

force for pantothenate absorption. As the transport

process does not respond to an electrical gradient, it

nust be electroneutral, indicating a 1:1 cotransport of

and pantothenate



by the same carrier. The

mechanism by which pantothenic acid exits the ab-

sorptive cell at the basolateral membrane has not

been established.

0004Intestinal microflora have been reported to synthe-

size pantothenic acid in mice, but the contribution of

bacterial synthesis to body pantothenic acid levels or

fecal loss in humans has not been quantified.

0005Unlike other water-soluble vitamins (ascorbic acid,

biotin and thiamin) that are absorbed by specific

carrier-mediated systems, the absorption of panto-

thenic acid is not regulated by its level of dietary

intake. The absence of clear-cut deficiency symptoms

in humans and the lack of toxicity at high doses could

explain why a regulated absorption mechanism has

not evolved for pantothenic acid.

Tissue Uptake and Metabolism

0006After absorption, free pantothenic acid is conveyed to

the body tissues in the plasma from which it is taken

up by most cells. A so-called sodium-dependent mul-

tivitamin transporter that mediates placental and in-

testinal uptake of pantothenate, biotin and the

essential metabolite lipoate has been cloned from rat

placenta and rabbit intestine. Messenger RNA tran-

scripts of this transporter have been found in many

tissues (intestine, liver, kidney, heart, lung, skeletal

muscle, brain and placenta) suggesting that this car-

rier protein may be involved in the uptake of pan-

tothenate, biotin and lipoate by all cell types.

0007In mammalian tissues (but not in red blood cells),

CoA is synthesized from pantothenic acid in five en-

zymatic steps. Three substrates are needed to synthe-

size CoA: pantothenic acid, ATP, and cysteine. The

rate-controlling step in the synthesis is the conversion

of pantothenic acid to 4

-phosphopantothenic acid by

pantothenate kinase. Tissue levels of CoA are kept in

check by feedback inhibition of pantothenate kinase

by CoA, acetyl-CoA, or a related metabolite.

0008In the event of a drastically reduced intake of

pantothenic acid, such as would occur during food

deprivation, the liver, and possibly other tissues, is

able to maintain nearly constant CoA levels for

some considerable time. In fasting rats, pantothenic

acid uptake by the liver is stimulated by the natural

rise in glucagon, and incorporation of pantothenic

acid into CoA is stimulated by glucagon and cortisol.

PANTOTHENIC ACID/Physiology 4339

In contrast to the liver, uptake of pantothenic acid by

heart and skeletal muscle of fasting rats is reduced, and

yet the rate of pantothenic acid conversion to CoA is

increased. Evidently, myocardial and muscle CoA syn-

thesis is not governed by the availability of pantothenic

acid to these tissues, but rather is controlled intracel-

lularly by regulation of enzymes involved in the CoA

synthetic and/or degradative pathways.

0009 Pantothenic acid derived from the degradation of

CoA is excreted intact in urine. The amount excreted

varies proportionally with dietary intake over a wide

range of intake values. Both fasting and diabetes

result in decreased excretion, thus conserving whole-

body pantothenic acid under these conditions.

Biochemical Functions of Coenzyme A

and Acyl Carrier Protein in Cellular

Metabolism

0010 A molecule of pantothenic acid is incorporated into

the structures of CoA and acyl carrier protein.

Though the functional sulfhydryl group of these co-

enzymes is not part of the pantothenate moiety, the

steric configuration of pantothenic acid is important

for enzymatic recognition.

0011 Acetyl-CoA and succinyl-CoA are energy-rich

thioesters that play important roles in the tricar-

boxylic acid cycle. Acetyl-CoA is also required for

the acetylation of choline to form the neurotransmit-

ter, acetylcholine. The amino sugars d-glucosamine

and d-galactosamine react with acetyl-CoA to form

acetylated products, which are structural components

of various mucopolysaccharides. The biosynthesis

of cholesterol begins with the condensation of

two molecules of acetyl-CoA to form acetoacetyl-

CoA. The latter reacts with acetyl-CoA to form 3-

hydroxy-3-methylglutaryl-CoA (HMG-CoA), which

in turn is reduced to the key intermediate, mevalonic

acid. CoA is required at two steps in each cycle of the

b-oxidation of fatty acids. Acyl carrier protein, as an

integral part of fatty acid synthase, is involved in the

biosynthesis of fatty acids.

Physiological Roles of Coenzyme A in the

Modification of Proteins

0012 Many diverse cellular proteins are modified by acetyl-

ation and/or by the covalent attachment of lipids. The

modifications fall into three main categories: acetyla-

tion, acylation, and isoprenylation. The alterations in

protein structure may be relevant to the association of

proteins with the plasma membrane or with subcel-

lular membranes, protein–protein binding, or the

targeting of proteins to specific intracellular locations.

In some cases, the modifications are cotranslational,

i.e., they take place on the growing polypeptide chain

associated with the ribosome during protein synthesis;

in other cases, they are posttranslational.

0013Most soluble proteins are modified at their amino

termini with an acetate group that is donated by CoA.

Acetylation alters the protein’s binding affinity for

receptors or other proteins.

0014A wide variety of proteins are modified with long-

chain fatty acids donated by CoA. The two fatty acids

most commonly attached to proteins are myristic acid

(14:0) and palmitic acid (16:0). The enzyme linking

myristate to amino-terminal glycine residues by an

amide bond is N-myristoyl transferase. For myristoyl-

ation to take place, the protein substrate must have a

glycine residue at position 2, immediately following

methionine, and preferably a hydroxyamino acid

(typically serine) at position 6. Myristoylated proteins

include G protein a subunits (signal transduction),

ADP-ribosylation factors (vesicular transport), myr-

istoylated alanine-rich C kinase substrate protein

(cytoskeletal rearrangements), recoverin (vision),

proteins of the immune system, and several enzymes.

Palmitoyl transferases link palmitate to the side

chains of cysteine residues by a thioester bond. The

cysteine residues can reside at any point in the pri-

mary structure of the protein; there is little evidence

for any specific sequence requirements. Unlike the

highly stable amide linkages to myristate, modifica-

tions of proteins by palmitate occur in thioester or

oxyester linkages that are subject to hydrolysis by

esterases. Cycles of palmitoylation and depalmitoyla-

tion allow the modified protein to have a regulating

function. Palmitoylated proteins include G protein a

subunits, many plasma membrane-anchored recep-

tors, cytoskeletal proteins, gap junction proteins,

neuronal proteins, and the enzymes acetylcholinester-

ase and glutamic acid decarboxylase. Palmitate modi-

fication is also a prerequisite for the budding of

transport vesicles from Golgi cisternae.

0015Two important isoprenoids, the 15-carbon farnesyl

pyrophosphate and the 20-carbon geranylgeranyl

pyrophosphate (Figure 1), are metabolic products of

mevalonic acid. Attachment of either isoprenoid

chain is the first step in the modification of proteins

bearing a C-A1-A2-X motif, where C is a carboxy-

terminal cysteine residue, A1 and A2 are aliphatic

amino acids, and X is an undefined amino acid. The

attachment is a thioester bond with the terminal cyst-

eine. Isoprenylated proteins include Ras proteins

(signal transduction), Rab proteins (vesicular trans-

port), nuclear lamins A and B (assembly and stabiliza-

tion of the nuclear membrane), G protein g subunits,

and the enzymes phosphorylase kinase and rhodopsin

kinase.

4340 PANTOTHENIC ACID/Physiology

0016 The physiological implications of selected acetyla-

tion and acylation modifications of proteins are

discussed below.

Acetylation of b-Endorphin

0017 Amino-terminal acetylation plays an important role

in regulating the biological activity of the brain

neurotransmitter b-endorphin. This peptide has mor-

phine-like analgesic activity and also affects sexual

behavior and learning. Acetylation deactivates b-

endorphin by rendering it unable to bind to specific

receptors. The modification is posttranslational and

occurs before or during the packaging of the peptide

into the secretory granules of multineurotransmitter

neurons in the pituitary gland.

Histone Acetylation

0018 The DNA in cell nuclei does not exist in the ‘naked’

state – rather, it is compacted into chromatin by

winding around specific DNA-binding proteins called

histones. The fundamental repeating unit of chroma-

tin is the nucleosome, which appears in electron

micrographs as beads on a string. Each nucleosome

consists of core histones (H2A, H2B, H3, and H4),

linker histones (H1 or variants thereof) and variable

lengths of linker DNA. Two molecules each of the

core histones form a barrel-shaped nucleosome core

particle, around which 146 base pairs of DNA are

wrapped in nearly two complete turns. A model of the

octamer of core histones is shown in Figure 2. The

linker histone acts as a clamp, preventing the unwind-

ing of DNA from the octameric complex. Each of

the four types of core histone comprises a globular,

hydrophobic carboxy terminus and an extended

hydrophilic amino-terminal tail containing a number

of positively charged amino acid residues. The tails

lie on the outside of the nucleosome, where they can

interact ionically with the negatively charged phos-

phate groups of the DNA backbone. During the

periods between cell division, the ‘beads on a string’

chromatin filaments form higher-order structures by

winding into a solenoid containg six nucleosomes per

turn. In these structures, the tails of the core histones

still extend outside the nucleosome.

0019The organization of chromatin into nucleosomes is

an essential feature in the regulation of gene tran-

scription – the step in protein synthesis in which

messenger RNA is synthesized from DNA. During

transcription, the enzyme RNA polymerase II com-

bines with a host of protein transcription factors to

form a multiprotein complex at a precise site on the

DNA called the promoter. The polymerase moves

along the DNA, temporarily unwinding and separat-

ing the two strands. As it moves along, RNA is

formed by the linking of ribonucleotides under the

influence of the enzyme and using one of the DNA

strands as a template.

0020It is necessary to control gene transcription so that

only those proteins needed by a particular cell for a

specific purpose are synthesized. When a protein is

not needed, nucleosomes prevent transcription by

impeding the access of factors required to initiate

and regulate this process. When protein synthesis is

required, changes in cell physiology cause a partial

and localized alteration of chromatin structure

(chromatin remodeling) in a manner that permits

the binding of initiating and regulatory factors.

0021One important chromatin remodeling system

involves the post-translational modification of core

histones by acetylation. Nuclear histone acetyltrans-

ferases (HATs) catalyze the transfer of an acetyl group

from acetyl-CoA on to the e-amino group of specific

lysine residues present exclusively in the amino-

terminal tails of each of the core histones. Neutraliza-

tion of the positively charged lysines reduces the net

positive charge of the histone tails and weakens

their association with DNA. The displacement of

the flexible tails permits subtle changes in nucleoso-

mal structure and a partial unwinding or loosening

of the core DNA. The result is an increase in accessi-

bility of transcription factors to their DNA-binding

sites. Acetylation does not occur randomly; multiple

HATs have specificities for different lysines in the

histone tails. Histone deacetylases (HDACs) counter

the effects of HATs by restoring the nucleo-

somes to their transcriptionally repressive configur-

ations.

0022In the overall scheme (Figure 3), the chromatin

structure is transiently and reversibly altered to

allow or prevent access of the transcription factors

by targeting HATs or HDACs to the core promoter,

thereby activating or repressing transcription, re-

spectively. It is now clear that transcriptional activa-

tors function by recruiting coactivators, and it is the

coactivators that possess HAT activity. Repressors

inhibit transcription indirectly by recruiting HDACs

via a bridging corepressor.

O-PP

Farnesyl pyrophosphate

Geranylgeranyl pyrophosphate

O-PP

fig0001 Figure 1 Structures of farnesyl pyrophosphate (C15) and

geranylgeranyl pyrophosphate (C20).

PANTOTHENIC ACID/Physiology 4341

a-Tubulin Acetylation

0023 Microtubules are long, stiff, hollow cylinders com-

posed of polymerized a- and b-tubulin dimers. As

constituents of the cytoskeleton, microtubules pro-

vide structural support for the cell. They also act as

lines of transport for the organized movement of

mitochondria and other organelles to desired loca-

tions within the cell, facilitate delivery of transport

vesicles from the Golgi complex to the apical mem-

brane in epithelial cells, and become associated with

the centrioles and chromosomes to form the spindle

during mitosis and meiosis (cell division). A subset of

the a-tubulin is modified, like the histones, by post-

translational acetylation of the e-amino group of spe-

cific lysine residues. In contrast to histone acetylation,

the acetylation of a-tubulin stabilizes the polymeric

structure of the microtubule; deacetylation is coupled

to depolymerization.

Acylation of G Proteins

0024 Peptide hormones, being hydrophilic, cannot cross

the lipid bilayer of the cell plasma membrane. To

overcome this problem, the hormones bind to specific

cell surface receptors, and a member of the family

of guanine nucleotide-binding regulatory proteins

(G proteins) acts as a signal transducer in coupling

these receptors to intracellular effector proteins –

enzymes that generate the second messenger (e.g.,

cyclic 3

-adenosine monophosphate, cAMP). The

second messenger mediates the biological action of the

hormone through the activation of protein kinases.

0025The posttranslational attachment of palmitate to

the G protein a subunit provides the means for revers-

ible translocation of the subunit between the plasma

membrane and the cytoplasm. The model shown in

(Figure 4) applies to the a

subunit responsible for

stimulation of cAMP synthesis. In the unactivated

state, the palmitoylated a subunit–GDP complex,

pal

–GDP, is associated with the b/g subunits and

the plasma membrane. Receptor activation stimulates

release of GDP and binding of GTP to form active

pal

–GTP; the a and b/g subunits dissociate from each

other but remain at the plasma membrane by virtue of

their respective palmitate and isoprenyl attachments.

Palmitate is rapidly cleaved from a

pal

–GTP by a pal-

mitoyl esterase, and the depalmitoylated a subunit

is released from the membrane into the cytoplasm.

Intrinsic GTP hydrolysis converts the active GTP-

bound subunits in both membrane and cytoplasm

(a) Octamer of core histones

Nucleosome core particle with linker DNA(b)

Linker DNA

H2B

H2A

H2B

fig0002 Figure 2 Models for (a) the octamer of core histones, and (b) the nucleosome core particle with linker DNA. From Csordas A (1990)

Biochemical Journal 265: 23–38 with permission.

4342 PANTOTHENIC ACID/Physiology

into the inactive GDP-bound forms. Reattachment of

palmitate to the cytoplasmic subunit by a palmitoyl

transferase facilitates the return of the a

pal

–GDP to

the plasma membrane.

0026 The G protein a subunit is further modified by the

cotranslational attachment of myristic acid. This in-

creases the affinity of the a subunit with the b/g

subunits, with the plasma membrane, and with the

effector protein.

Palmitoylation of Asialoglycoprotein Receptors

0027 The hepatic asialoglycoprotein receptor mediates the

endocytosis of desialylated glycoproteins containing

terminal galactose or N-acetylgalactosamine. (Endo-

cytosis refers to the cellular uptake of macro-

molecules by entrapment within inward foldings of

the plasma membrane, which then pinch off to form

intracellular vesicles.) There is evidence that a cycle of

palmitoylation and depalmitoylation regulates the

ligand-binding activity of the asialoglycoprotein re-

ceptor. Inactivation of the receptor by depalmitoyla-

tion prevents the rebinding of dissociated ligand

molecules and ensures that ligand is shuttled to lyso-

somes for degradation rather than nonproductively

recycled back to the cell surface.

Deficiency in Animals and Humans

0028 Pantothenic acid deficiency has been induced experi-

mentally in many species of animals and birds by

feeding diets containing low levels of the vitamin.

The wide range of deficiency signs, histopathological

abnormalities, and metabolic changes indicate dis-

orders of the nervous system, reproductive system,

gastrointestinal tract, and immune system. Rodents

are particularly prone to necrosis and hemorrhage of

the adrenal glands with consequent impairment of

adrenal endocrine function. In young animals, the

earliest sign of deficiency is a decline in the rate of

growth. Distinctive visible signs are depigmentation

of fur in rats and mice, and rough plumage and

exudative lesions around the beak and eyelids of

chickens. ‘Goose-stepping’ of the hind legs in pigs

and ataxia (falling down to one side) in chicks

are associated with demyelination of the motor

neurons.

0029Human pantothenic acid deficiency has been care-

fully studied in healthy male volunteers given an

emulsified artificial diet by stomach tube. In one

study, two subjects received the basic diet devoid of

pantothenic acid, a second pair received the same diet

with added antagonist (o-methyl pantothenic acid),

and a third pair (the controls) received the diet

supplemented with pantothenic acid. After about 4

weeks, subjects receiving the antagonist and those in

the deficient group began to show similar symptoms

of illness. Clinical observations were irritability, rest-

lessness, drowsiness, insomnia, impaired motor co-

ordination, and neurological manifestations such as

numbness and ‘burning feet’ syndrome. The most

persistent and troublesome symptoms were fatigue,

headache, and the sensation of weakness. Among the

laboratory tests, the loss of eosinopenic response to

adrenocorticotropic hormone indicated adrenocorti-

cal insufficiency.

Lysine

X Lysine

CoA

Acetic acid

CoA

HAT

HDAC

Acetyl-CoA

fig0003 Figure 3 Opposing activities of histone acetyltransferases (HAT) and histone deacetylases (HDAC) in the control of transcription

through chromatin remodeling.

PANTOTHENIC ACID/Physiology 4343

Dietary Intake

0030 A recommended dietary allowance (RDA) for a nu-

trient is derived from an estimated average require-

ment (EAR), which is an estimate of the intake at

which the risk of inadequacy to an individual is

50%. In the case of pantothenic acid, no data have

been found on which to base an EAR, and an ad-

equate intake (AI) is used instead of an RDA by the

Food and Nutrition Board of the US Institute of Medi-

cine. The AI for infants up to 12 months old (1.7–

1.8 mg day

1

) reflects the observed mean intake of

breastfed infants. The AI for children aged 1–3 years

(2 mg day

1

) is extrapolated from adult values. The

AIs for children aged 4–13 years (3–4 mg day

1

), and

adolescents and adults of both sexes (5 mg day

1

) are

based on pantothenic acid intake sufficient to replace

urinary excretion. AIs for women during pregnancy

and lactation are 6 and 7 mg day

1

, respectively.

0031 There are no known toxic effects of oral panto-

thenic acid in humans or animals.

See also: Fatty Acids: Metabolism; Oxidative

Phosphorylation; Tricarboxylic Acid Cycle