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

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their consumption by the child population does not

imply any toxicological risk for them. Furthermore,

one must remember that some kinds of milk have

been enriched with fluoride.

Tea

0016 Tea leaves also have a high fluoride content. These

values are above 400 mg kg

1

(dry weight), but tea

infusions (tea-bags) have concentrations of around

0.5–1.5 mg l

1

. The amount of F



present in a cup of

tea will depend not only on its volume but also on the

brand of tea, the amount of tea used, how long it is

brewedfor,whether or notitis a dilutionofa previously

prepared cup of tea, and whether or not it has been

made with fluoride-enriched water. The F



intake of

tea drinkers can vary between 0.04 and 2.7 mg day

1

Table 4 shows the fluoride contents of tea.

Fluorides in Solid Foods

0017 Fluorides can be detected in all food. There does not

seem to be a clear relation between the F



content of

most plants and the content found in the soil or

the water in the area where the plants are grown.

Plants grown in acid soil, however, have a higher

concentration of F



. The F



content found in

the leaves of most plants is between 2 and 120 mg of



per kilogram. The fruit and vegetables consumed

by man have a fluoride content of around

0.1–0.4 mg kg

1

and, therefore, are of little interest,

unless there is a prevalence of unusual dietary pat-

terns, although F



concentrations of up to 2.0 and

2.1 mg kg

1

have been found in barley and rice

irrigated with nonfluoride-treated water, and concen-

trations of up to 4.3 and 6.4 mg of F



per kilogram,

respectively, when the water used was fluoride-

enriched. Yams and cassava also make up the main

diet of many tropical regions, and they contain high

levels of F



. However, fluoride contents in meat

tend to be low. Fish products (tinned fish like salmon,

sardines) have fairly high contents, up to 40 mg kg

1

However, the consumption of fluorides from fish

alone, in a combined diet, does not provide enough

fluorides to exceed 0.2 mg day

1

0018The amount of the total fluoride intake accounted

for by solid foods has been considered as low, al-

though this can vary, depending on the composition

of the daily diet and the fluoride content of the water

where the food has been prepared.

0019These quantities of fluorides in food can be

increased by the use of pesticides with added fluoride

and phosphated chemical fertilizers, and because

of the fluoride to be found in the irrigation and wash-

ing waters. Furthermore, one must consider the fluor-

ide contribution made by industrially processed

foods.

0020Table 5 Lists the most significant data collected on

the fluoride contents of solid foods. It can be seen

that the contents are very low, with the exception of

fish products.

tbl0004 Table 4 Fluoride ion content in beers, soft drinks, juices, milk,

and tea

Drink Analytical method Mean

concentration

(mgl

1

)

Beers (1990) Ion-selective electrode 0.51

Beers (1992) Ion-selective electrode 0.16

Bitter soft drinks Ion-selective electrode 0.21

Orange soft drinks Ion-selective electrode 0.30

Lemon soft drinks Ion-selective electrode 0.41

Cola soft drinks Ion-selective electrode 0.34

Carbonated soft drinks Ion-selective electrode 1.06

Natural juices Ion-selective electrode 0.18

Mother’s milk Ion-selective electrode 0.18

Pasteurised milk Ion-selective electrode 0.10

Evaporated milk Ion-selective electrode 0.04

Cows’ milk (raw) Ion-selective electrode 0.26

Powdered milk Ion-selective electrode 0.11

Cream cheese Ion-selective electrode 0.04

Smoked cheese Ion-selective electrode 0.27

Tea-leaves Ion-selective electrode 148.03 mg kg

1

Tea-bags Ion-selective electrode 0.84

tbl0005Table 5 Fluoride content of solid foods

Solid food F



content (mg kg

1

)

Cereals and grain

Wheat germ 0.12

Oats 0.12

Rice 0.05

Precooked rice 0.05

Chick peas 0.08

Fresh vegetables

Beetroot 2.19

Cucumber 0.34

Silver beet 0.25

Carrot 0.06

Garlic 0.15

Cabbage 0.07

Aubergine 0.23

Tinned products

Guayaba pure

e 0.02

Mango pure

e 0.02

Guayaba jam 0.23

Pepper pure

e 0.17

Pastas

Macaronis 0.14

Noodles 0.12

Spaghetti 0.09

Coditos 0.13

Fish

Tuna 3.73 mg l

1

Anchovy, fillets 3.02 mg l

1

Sardines in oil 4.27 mg l

1

2558 FLUORIDE

See also: Beers: Chemistry of Brewing; Dental Disease:

Etiology of Dental Caries; Fluoride in the Prevention of

Dental Decay; Milk: Analysis; Tea: Chemistry; Water

Supplies: Chemical Analysis; Wines: Types of Table

Wine

Further Reading

Hardisson A, Rodrı

guez MI, Burgos A, Dı

az Flores L,

Gutie

rrez R and Va

rela H (2001) Fluoride levels in

public supply and bottled drinking waters in the island

of Tenerife (Spain). Bulletin of Environmental Contam-

ination and Toxicology 67(2): 163–170.

Kjellevold Malde M, Maage A, Macha E, Julshamn K and

Bjorvatn K (1997) Fluoride Content in selected food

items from five areas in East Africa. Journal of Food

Composition and Analysis 10: 233–245.

Martı

n M, Hardisson A and A

lvarez R (1992) Fluoride

concentration in beers and soft drinks. Journal of Food

Composition and Analysis 5: 172–180.

Martı

n MM, Hardisson A and A

lvarez R (1993) Direct

potentiometric determination of fluoride in beverage.

Comparative study of different buffering solutions.

Food Chemistry 46: 85–88.

rez-Olmos R, Hardisson A, Elı

as I, Rı

os R and Martı

(1990) Determination of fluoride in Tenerife wines.

Comparative study of conditioning solutions. Belgian

Journal of Food Chemistry and Biotechnology 45(6):

210–214.

Sanz Pe

rez E and Sanz J (1999) Fluoride concentration in

drinking water in the province of Soria (central Spain)

and caries in children. Environmental Geochemistry and

Health 21: 133–140.

Foaming See Aerated Foods

FOLIC ACID

Contents

Properties and Determination

Physiology

Properties and Determination

C J Bates, MRC Nutrition Research, Cambridge, UK

Introduction

0001 In the older literature, it was customary to use the

terms ‘folic acid,’ ‘folate,’ or ‘folacin’ interchangeably

for all of the pteroylglutamic acid derivatives and

their g-polyglutamic acid conjugates. More recently,

the custom has changed so that ‘folic acid’ is now

reserved for the parent and commercially available

pteroylmonoglutamic acid which is in the oxidized

state, whereas ‘folate’ is used to describe the deriva-

tives which typically occur in foods and tissues. The

latter occur in many forms, which may be reduced;

they often carry one-carbon substituents and are

often poly-g-glutamates, especially inside cells. The

advantage of this new practice is that it permits a

shorthand distinction between the commercial folic

acid that is present in supplements and is used in food

fortification, and the folate that is native in living

tissues and in natural foods, and which has a different

bioavailability.

Physical Properties

0002The fully oxidized parent substance of the folate or

folacin group, pteroylglutamic acid or pteroylmono-

glutamic acid (formula weight 441.4) crystallizes

from hot water as yellow-orange platelets. It does

not melt, but darkens and chars at about 250



C. It

is only very slightly soluble in cold water (1–10 mg

1

), increasing to 1% in boiling water, and is much

more soluble in aqueous alkali, acetic acid, phenol,

pyridine, and other basic solvents. A solution for

injection is obtained by dissolving it in aqueous

sodium bicarbonate, or by using the sodium salt,

which is soluble in water. The extinction coefficient

1cm

) in 0.1 mol l

1

alkali is 565 at 255 nm, 350

at 282 nm, and 195 at 365 nm (absorption maxima).

00035-Formyl-5,6,7,8-tetrahydropteroylglutamic acid

(folinic acid: formula weight 473.4) also decomposes

without melting at 250



C; it is sparingly soluble in

water, but is more soluble in aqueous alkali, and the

FOLIC ACID/Properties and Determination 2559

absorption maximum is at 282 nm in alkali. It is more

stable at neutral or slightly alkaline pH than at acid

pH, in solution. The 10-formyl derivative is more

labile, and more sensitive to oxygen.

0004 Tetrahydrofolic acid (formula weight 443.4) is

extremely readily oxidized, and the freeze-dried

solid form must be stored in vacuo. Solutions in

0.5% ascorbate or in 1.0 mol l

1

mercaptoethanol

are moderately stable, and it has an absorption max-

imum at 298 nm in neutral solution.

0005 5-Methyltetrahydrofolic acid (formula weight

459.4) is intermediate in stability to oxidization

between pteroylglutamic acid and tetrahydrofolic

acid. It can be obtained as a white powder, but in

solution it requires ascorbate or another reducing

substance to achieve medium-term stability. The ab-

sorption maximum at 290 nm has a molar extinction

coefficient (e) of 31.7  10

mol

1

,orE

1cm

of 690, at neutral pH.

Chemical Properties

0006 The structural formulae of three of the comm-

only encountered forms of folic acid are shown in

Figure 1.

0007Pteroylglutamic acid and related pterins are readily

hydrogenated in two stages to the dihydro and then the

tetrahydro forms, involving firstly positions 7 and 8 of

the pteridine ring, then positions 5 and 6. These hydro-

genation reactions can be performed either in vitro,

with hydrogen and a platinum oxide catalyst or by

hydrosulfite, or else in vivo, by enzyme-catalyzed re-

actions involving dihydrofolate reductase. Once con-

verted to the fully reduced form, single carbon units

at various levels of oxidation can be transferred to the

nitrogen atoms at positions 5 or 10, yielding methyl,

methylene, methenyl, hydroxymethyl, or formyl

units: the second and third of these form a bridge

between the two acceptor nitrogen atoms. A combin-

ation of a carbon and nitrogen atom (formimino

group, –CHNH–) can also be carried. These transfer

reactions constitute the fundamental basis of the

single-carbon-unit transfer operations, for which the

folic acid cofactors are essential in vivo.(See Cobala-

mins: Physiology; Coenzymes.)

0008Chemical syntheses of pteroylglutamic acid have

been achieved, first by condensation of guanidine

with ethyl cyanoacetate to give a pyrimidine ring,

then continuing either by reacting p-aminobenzoyl-

glutamic acid with 2,4,5-triamino-6-hydroxypyrimi-

NH C NH CH

COOH

Pterdine ring

Pteroylglutamic acid

(folic acid)

5-Formyltetrahydrofolic acid

(folinic acid)

5-Methyltetrahydrofolic acid

(prefolic A)

Glutamatep-Aminobenzoyl

NH C NH CH

COOH

CHO

NH C NH CH

COOH

910

fig0001 Figure 1 Structural formulae of commonly encountered forms of folic acid.

2560 FOLIC ACID/Properties and Determination

dine and a,b-dibromopropionaldehyde, or by reacting

2,4,5-triamino-6-hydroxypyrimidine with pyridine,

a,b-dibromopropionaldehyde, potassium iodide, and

p-aminobenzoylglutamic acid. For pteroylglutamic

acid there is only one asymmetric center which is

within the glutamic acid moiety (l form), but after

reduction a second asymmetric center appears at

carbon 6, and chemical synthesis produces both

d and l forms, whereas the natural product has the

l configuration. Degradation of folic acid in vivo

involves an initial cleavage at the 9–10 bond.

0009 Folate polyglutamates are the forms of folic acid

(including the reduced, C

-substituted cofactor forms

in vivo) which have g-linked chains of poly-l-glutamic

acid. These chains can extend up to at least 10 l-

glutamyl units, and different tissues differ in their

spectrum of chain lengths. The glutamate residues

are added by the enzyme, pteroylpolyglutamate

synthetase (pteroylpolyglutamate ligase), and can be

removed by pteroylpolyglutamate hydrolase (alias

folate conjugase or g-glutamyl carboxypeptidase).

The latter enzyme is present, inter alia, in serum, in

duodenal and jejunal mucosal cells and in kidney,

especially within the lysosomes. The synthetase is an

intracellular enzyme, responsible for building the

active forms of the folate cofactors. Tetrahydrofolate

and the formyl folates are preferred to methyltetrahy-

drofolate for the synthetase reaction, which helps to

explain the poor retention of folate in vitamin B

deficiency, when such nonmethylated folate species

decline in concentration, leading to a change in the

balance towards shorter-chain polyglutamates.

Occurrence in Food

0010 Awide variety of forms of folate occur in food, and this

complexity has resulted in major problems, both for

devising accurate analytical methods to measure the

folate content of foods as eaten, and in measuring the

bioavailability of the different forms. As noted previ-

ously, the source of variability is threefold: (1) the

oxidation state of the pteridine ring; (2) the presence

and type of the one-carbon substituent carried, and (3)

the presence and length of the polyglutamate side-

chains. (See Bioavailability of Nutrients.)

0011 Because monoglutamates and short-chain polyglu-

tamates are more readily available for absorption

than long-chain polyglutamates, an operational def-

inition of ‘free folate’ was established, which included

only the monoglutamates and short-chain poly-

glutamates that are most readily available to promote

the growth of the test organism Lactobacillus rham-

nosus (formerly L. casei). However, this category has

not, in practice, proved to be very useful, because the

proportion of ‘free folate’ can vary enormously with

the history of the food sample, the assay conditions

(especially the initial folate extraction, if endogenous

conjugases are present), and with the duration of

exposure to the assay microorganism. The original

studies on this subject made use of polyglutamates

derived from the oxidized form, pteroylglutamate,

not those of the natural forms of folate, so that the

results were not entirely applicable to the forms of

folate in foods. (See Lactic Acid Bacteria.)

0012Table 1 shows the folate content of ‘typical’ (aver-

age) samples of food items as eaten. These folate

values are still being revised, usually in an upward

direction, as the assays are optimized. Nearly all of

the existing food table folate values have been

obtained by microbiological assay procedures.

0013It is clear that, although folate contents per 100 g

vary considerably, folate is fairly widely distributed

between different food types. Despite this, there are

very considerable variations in folate content between

diets which, on the one hand, depend heavily on

unfortified cereal staples (traditionally ‘poor’ diets)

and, on the other, include the richer plant and animal

sources. Of the folates in a mixed diet, over 90% are

in polyglutamates of varying chain length; 60% are

methylated, and 30% have a formyl group. Foods

containing substantial amounts of vitamin C also

tend to be rich folate sources, partly because of the

preservative effect of vitamin C on folates during

storage, preparation, and cooking. (See Ascorbic

Acid: Physiology; Cereals: Dietary Importance.)

0014In the UK, vegetables and cereals each provide

about 30% of food folate intake; milk and milk prod-

ucts provide about 10%; meat, fruit, and beverages

each provide about 5%; eggs, fish, and other

foods each provide 1–4%.

Use in Food for Fortification

0015If the vitamin is added to manufactured foods, it is

almost always as pteroylglutamic acid (folic acid),

because this is by far the cheapest commercial form.

Infant formulae usually contain added folic acid, in

amounts between 3 and 15 m g 100 ml

1

, as fed. The

practice of adding folic acid to manufactured foods is

becoming much more common than it was, especially

for breakfast cereals. In the USA, folic acid fortifica-

tion of cereal grains for bread making has now

become mandatory, at 1.4 mgg

1

, from the beginning

of 1998. (See Food Fortification; Infant Foods: Milk

Formulas.)

Extraction and Clean-up

0016Two problems dominate the procedures for extraction

of folic acid from biological tissues: the prevention of

FOLIC ACID/Properties and Determination 2561

oxidation mainly by molecular oxygen, and the

preservation, or alternatively the complete removal,

of the polyglutamate chain. Prevention of oxidation

can be achieved by a variety of reducing agents,

the most common choice being ascorbic acid, usu-

ally at 1% w/v final concentration, either as the

free acid or else partially neutralized, e.g., to a pH

around 4.

0017 Removal of the polyglutamate side chain can be

achieved with partly purified preparations of folate

conjugase (polyglutamyl hydrolase). The ones which

are most commonly used for food folate release are

the hog kidney enzyme and the chicken pancreas

enzyme. The former hydrolyzes the polyglutamate

chain and thus releases monoglutamates from the

food folates more completely than the latter, which

tends to produce diglutamates. Although different

workers have generally taken care to optimize the

assay procedures with their particular selected

enzyme sources, it is important to consider carefully

the implications of using an enzyme which may not

liberate monoglutamates, because this can seriously

affect the relative molar response of the standard

(such as pteroylglutamic acid) against the more com-

plex and variable folate species obtained from the

food matrix. One study indicated that the folate

content of foods as measured after deconjugation

with the hog kidney enzyme can be at least 50%

higher than the values achieved after deconjugation

with chicken pancreas enzyme. A ‘triple enzyme’ mix-

ture, comprising chicken pancreas conjugase plus

a-amylase and a protease, is widely used.

0018 The release of monoglutamyl folates from the poly-

glutamates in red cell extracts has generally been

achieved by incubating the red cell lysates in the

presence of their own plasma, because plasma con-

tains a very active conjugase. This is why red cell

folate assays in whole blood, rather than separated

red cells, are preferred, since the cellular components

contain little or no conjugase. Most of the published

procedures for the red cell folate assay have assumed

that the monoglutamates are completely released

after a brief period of incubation with plasma

enzyme. Another assumption, highlighted recently,

that is made with the assay of folate in red cell hemo-

lysates, is that it is unaffected by the extent of binding

to hemoglobin or the extent of physical trapping,

which varies with the degree of oxygenation of the

sample. Although red cell folate is a preferred index,

because it measures long-term status, there are still

serious practical problems in achieving accurate

measurements and in obtaining acceptable inter-

laboratory agreement.

0019If the polyglutamyl folates are to be preserved, in

order to measure the polyglutamyl profile that is char-

acteristic of the undamaged tissue, then adventitious

conjugase activity must be inactivated at the time of

extraction. One way of achieving this is by using a

boiling ascorbic acid solution during homogenization.

0020If extremely labile folates, such as tetrahydrofolic

acid, are to be extracted and preserved intact, then

special precautions such as the exclusion of oxygen

during the extraction procedure, and the use of low

temperatures, may be necessary.

Analytical Procedures

0021Once the folates have been extracted from the tissues,

there are three main approaches to the problem of

their estimation and characterization. The oldest gen-

eral procedure depends on the extent of growth of a

specific folate-dependent bacterial species. Its growth

tbl0001 Table 1 The folate content of typical food items (mg 100 g

1

wet weight)

Cereals and nuts Dairy products Fruit

Boiled rice 4–10 Milk 6 Oranges 30

Boiled spaghetti 4–7 Cream 7–12 Grapefruit 26

Wheat flour 20–50 Butter trace Apples 1–4

Peanuts 50–110 Cheese 15–100 Pears 2–4

Walnuts 70 Icecream 7–9 Plums 3

Coconut 9 Eggs 50 Strawberries 20

Cereal products Meat and fish products Vegetables

Bread 15–90 Roast beef 15–17 Cabbage (boiled) 25–35

Biscuits 7–40 Roast chicken 7–13 Cauliflower (boiled) 50

Cake 7–100 Roast pork 6–7 Brussel sprouts 110

Breakfast cereals 2–400 Fried liver 200–600 Lettuce 55

Porridge 4–7 Fried kidney 80 Spinach (boiled) 90

Pastry 7–35 Sausage 2–4 Potatoes (boiled) 10–25

Custard 5 White fish 10–14 Carrots (raw) 12–30

Fish fingers 16 Peas (boiled) 27

Data compiled from McCance and Widdowson’s The Composition of Foods, 4th and 5th edns.

2562 FOLIC ACID/Properties and Determination

medium contains optimum amounts of all the neces-

sary growth factors except folate, and a small amount

of the food extract is then added, to provide sufficient

folate for partial suboptimum growth of the test or-

ganism. The second general approach, which has now

been available for nearly three decades, depends on

the specific interaction of the extracted folate with a

fixed amount of a folate-binding protein plus radio-

active- or fluorescence-labeled folate, after which the

protein-bound folate is separated from the unbound

folate and the radioactivity or fluorescence of the

bound folate is measured. The amount of label

bound depends on the total amount of folate present.

This approach can be subdivided into those assays

which depend on naturally occurring folate binders

obtained from body fluids such as milk, and those

which depend on binders which are artifically

induced in an animal, for instance by using folate

bound to a hapten as an antigen. (See Immunoassays:

Radioimmunoassay and Enzyme Immunoassay.)

0022 The third general approach is the separation of

different folate species from each other and from

interfering substances, using high-performance liquid

chromatography. This approach has the advantage

of specificity, but a disadvantage for tissue folate

analyses is that the available detectors (ultraviolet

absorption, fluorescence, or electrochemical) are

barely sensitive enough to detect all of the folates

found in tissues, and they do not always give a con-

stant molar response with different folate species,

which complicates the problem of calibration. Never-

theless, new data and further refinements continue

to emerge. (See Chromatography: High-performance

Liquid Chromatography.)

0023 The microbiological assay procedures have been

established for long enough to dominate the litera-

ture, for instance, on food analysis and food table

compendia. Total folate has, for several decades,

been measured using L. rhamnosus (previously

L. casei), because this organism can respond equally

well to all the common monoglutumate forms of

folate. However, the use of inappropriate assay con-

ditions, e.g., a poor choice and control of pH, has

often resulted in the underestimation of total folate

contents. Until this source of error has been effect-

ively eliminated, the food table values for folate and

the derived estimates of human dietary intakes of

folate may be too low. Revised values are gradually

being obtained to rectify this problem.

0024 Two other microorganisms have been used,

especially in the earlier studies of folate speciation.

One is now called Enterococcus hirae (formerly

Streptococcus faecalis). This organism grows on

most forms of folate except the 5-methyl derivative.

The other is called Pediococcus pentosaceus

(formerly P. cerevisiae,orLeuconostoc citrovorum),

and grows most readily on ‘citrovorum factor,’

i.e., N

-formyltetrahydrofolic acid, or ‘folinic acid’.

Differential growth of these organisms on tissue

extracts has given useful estimates of the different

monoglutamate forms of folates. However, it is im-

portant to note that some tissue or food extracts

contain growth inhibitors, or promoters, which may

result in errors in the estimation of folate levels, and

species ratios.

0025The binding protein methods have recently proved

very popular, especially for blood status assessment,

because of their ease of operation, but they too can

suffer from errors. One source of error, mentioned

earlier, may be the incomplete deconjugation of

polyglutamate side chains; another may be the partial

oxidation of folates to their degradation products.

Like the microbiological assay, specific binding assays

can also suffer from pH-dependent variations in

response to different folate species, so that the choice

of calibrant and of pH may be critical. There are

many variants, using different binding proteins or

antibodies; some depend on the binding of radio-

active- or fluorescence-labeled folate markers; others

depend on enzyme-linked immunosorbent assays.

However, there is a constant need for interlaboratory

methodological comparisons to achieve good com-

parability of results, and a careful study of the sources

of error in these assays, and of procedures to insure

their elimination. External quality assurance schemes

are available for folate (and for vitamin B

) assays in

serum or plasma, and to a lesser extent for folate

in red cells, because these are widely performed,

especially by hospital hematology laboratories.

0026Those studies which have made the most use of

high-performance liquid chromatography have been

metabolic studies in animals, in which the intertissue

distribution, turnover, and speciation of folates have

been followed, using radioactively labeled precursors.

The use of stable isotope-labeled folates for studies of

folate economy in human subjects has recently become

feasible, and a number of new studies have begun to

examine the relative bioavailability of different folate

congeners, with and without a food matrix, by this

approach. Urinary excretion of undegraded folates

and of the breakdown products derived from para-

aminobenzoic acid has provided suitable material for

isotope ratio analysis. Undoubtedly this approach will

expand and be more widely used, especially to study

folate bioavailability, in the future.

See also: Ascorbic Acid: Physiology; Bioavailability of

Nutrients; Cereals: Dietary Importance;

Chromatography: High-performance Liquid

Chromatography; Cobalamins: Physiology; Coenzymes;

FOLIC ACID/Properties and Determination 2563

Food Fortification; Immunoassays:

Radioimmunoassay and Enzyme Immunoassay; Infant

Foods: Milk Formulas; Lactic Acid Bacteria;

Macrobiotic Diets

Further Reading

Ball GFM (1998) Bioavailability and Analysis of Vitamins

in Foods. London: Chapman & Hall.

Friedrich W (1988) Vitamins. Berlin: Walter de Gruyter.

Gregory JF III, Williamson J, Liao J-F, Bailey LB and Toth

JP (1998) Kinetic model of folate metabolism in non-

pregnant women consuming [

] folic acid: isotopic

labeling of urinary folate and the catabolite para-

acetamidobenzoylglutamate indicates slow, intake-

dependent, turnover of folate pools. Journal of Nutrition

128: 1896–1906.

Phillips DR and Wright AJA (1982) Studies on the response

of L. casei to different folate monoglutamates. British

Journal of Nutrition 47: 183–189.

Wright AJA, Finglas PM and Southon S (1998) Erythrocyte

folate analysis: a cause for concern? Clinical Chemistry

44: 1886–1891.

Physiology

C J Bates, MRC Human Nutrition Research,

Cambridge, UK

Nutritional Significance of Folic Acid and

Folates

0001 Like most micronutrients, the nutritional significance

of folate was recognized and explored as the result of a

deficiency disease, namely megaloblastic anemia,

which occurred especially among pregnant women

living in India. This was characterized by enlarged

(megaloblastic) red cell precursors in the bone marrow,

by macrocytic erythrocytes and by abnormal polymor-

phonuclear leukocytes with increased numbers of nu-

clear lobes, in the peripheral blood. There may also be

lesions of the tongue (glossitis) and of the mucocuta-

neous margins of the mouth (cheilosis), and malab-

sorption. All of these lesions are shared with other

vitamin B deficiency syndromes, so none of them is

unambiguously diagnostic. Normally, the total body

content of folate is about 5–20 mg. (See Anemia (An-

aemia): Megaloblastic Anemias.)

0002 As the biochemical functions of the folate coen-

zymes became clearer, it was recognized that folate

deficiency had implications for many aspects of

homeostasis, metabolism, and health beyond the

limited confines of blood cell production and matur-

ation. Folate is essential for nucleic acid synthesis

at all sites, and for a wide range of biochemical path-

ways involving one-carbon transfers. Deficiency

states are by no means confined to the developing

countries, although they are commonest and most

severe there. As folate deficiency progresses and be-

comes more severe, there is a characteristic sequence

of events, in which low plasma folate levels are

followed by low erythrocyte folate levels, then in-

creased polymorphonuclear leukocyte nuclear lobe

numbers, macroovalocytosis of the erythrocytes and

bone marrow macrocytosis, then macrocytic anemia.

0003Conflicting evidence about human requirements

has led to considerable fluctuations in official recom-

mendations for folate intakes, and problems with the

assay of folates in foods have created parallel difficul-

ties in the estimation of dietary folate intakes by

individuals and populations.

Physiology and Functions of Folic Acid

0004In order to be absorbed efficiently, the polyglutamate

folates in food need to be broken down to mono-

glutamates, either during food preparation, or at the

brush border of the jejunum, which is the main site of

absorption. It is difficult to give precise figures for the

efficiency of deconjugation, and of absorption, be-

cause they vary considerably with the type of folate,

with other dietary constituents, and with unidentified

physiological factors, especially the pH at the site of

absorption, the optimum being c. pH 6. Many foods

contain inhibitors of the intestinal brush border folate

conjugase and/or folate transport system, thereby re-

ducing the efficiency of absorption. Others, such as

milk, contain folate binders which may stimulate ab-

sorption.

0005Absorption of food folate, which occurs mainly in

the jejunum, is largely attributable to active transport

against the concentration gradient at moderate

intakes, and appears to involve two jejunal brush

border folate-binding proteins. This process is pH-,

sodium-, and glucose-dependent. Recent estimates

suggest that about 50% of the complex mixture of

folates that occur, in food can be absorbed and util-

ized. Although most individual folates, even polyglu-

tamates, are better absorbed than this when given on

their own, the effects of partial degradation and food

borne inhibitors etc. reduce the overall efficiency,

which can be very variable. Small amounts of folate

may arise from the intestinal flora in situ, but in

humans (unlike the rat), this is now known to be only

a very minor source of absorbed folate. Although

mono and short-chain polyglutamates are more read-

ily absorbed and utilized than the long-chain polyglu-

tamates, the difference between them is not as

substantial as was once thought, and the old sub-

division into ‘free’ and ‘bound’ (i.e., short-versus

2564 FOLIC ACID/Physiology

long-chain polyglutamates) has largely been super-

seded. The nutritional status of the subject can affect

the efficiency of folate utilization and its economy:

deficiencies of vitamin B

, iron, zinc, or vitamin C in

particular can have an adverse effect. Folate absorp-

tion is also adversely affected by some drugs, including

diphenylhydantoin, phenytoin, phenobarbital, primi-

done (anticonvulsants); cholestyramine, salicylates

and nonsteroidal antiinflammatory drugs; and salicy-

lazosulfapyridine (used to treat bowel inflammation).

0006 Once absorbed, the monoglutamate folates in the

portal plasma are carried to the liver for processing,

the liver being a major repository of folate coenzymes.

From there, folate is carried to the other tissues. Hep-

atic folate is also secreted in bile, and much of this

biliary folate is reabsorbed. Absorbed folate in excess

of requirements is excreted in the urine, and folate

turnover results in several characteristic degradation

products, such as p-aminobenzoylglutamates, also

destined for excretion in the urine. A substantial

amount of folate turnover also occurs by fecal excre-

tion of endogenous folate. (See Coenzymes.)

0007 Folate transport into the cells of other tissues is also

mainly by carrier-mediated processes. There are two

separate mechanisms: one involves a reduced folate

carrier protein which is a transmembrane transporter,

and the other is an anchored protein called membrane

folate receptor, which has a high affinity for both

reduced folates and folic acid. This is especially active

in tissues like kidney, placenta, and breast, where

efficient folate transport is critically important. It

can be inactivated by certain mycotoxins. There are

specific folate-binding proteins at various sites; some

are involved in folate transport, others may have a

protective function.

0008 Within the cells of mammalian tissues, folate is

converted by the enzyme, folyl polyglutamate synthe-

tase, to g-linked polyglutamates of the reduced

folates. 5-Methyl tetrahydrofolate polyglutamate is

the largest fraction of tissue folate, except in tissues

with especially rapid cell division, where the formyl

derivative tends to be dominant.

0009 In most tissues, the dominant functions of the

folate coenzymes are the synthesis of DNA, thus

permitting cell division, growth, and tissue renewal,

and the provision of active methyl groups via

the methionine cycle and the methyl donor S-adeno-

sylmethionine (SAM). Folates have several essential

functions in the synthesis of purine and pyrimidine

building blocks of DNA, but the one function that is

exquisitely sensitive towards folate deficiency is the

conversion of deoxyuridylic acid to thymidylic acid,

catalyzed by thymidylate synthase (Figure 1). For this

reaction, folate must first be converted into 5,10-

methylene tetrahydrofolic acid, which undergoes a

regeneration cycle each time it participates. This func-

tion of folate explains most of the pathological effects

of its deficiency in humans and animals, including

megaloblastic anemia, leukocytopenia and other

white cell abnormalities that arise from disturbances

in DNA synthesis and hence of cell division in bone

marrow. Folate deficiency also reduces growth (in

children), regeneration of intestinal mucosa, and cell

division at other sites which have a rapid turnover.

(See Nucleic Acids: Physiology.)

0010Certain anticancer drugs which interfere in the

folate regeneration cycle (e.g., dihydrofolate reduc-

tase inhibitors such as methotrexate), and some anti-

biotics which are antimetabolities of folic acid, act by

reducing pathological rates of cell division, thus

protecting the host organism against unchecked div-

ision of parasitic cells. A major challenge for future

research will be to target such antimetabolites

specifically to the sites of invasion and damage, in

order to reduce their side-effects on healthy tissues.

Paradoxically, healthy tissues may also be protected

against some carcinogenic agents by adequate folate

nutrition, because removal of damaged DNA requires

folate-dependent metabolic reactions. Recently, there

have been several controlled studies of cancer

susceptibility in human subjects, in which folic acid

has been tested for possible protective effects. These

have focused especially on the large bowel, uterine

cervix, breast, pancreas and, to a lesser degree, lung.

Although a consensus has yet to emerge, interest in

this subject is rapidly expanding. (See Carcinogens:

Carcinogenic Substances in Food: Mechanisms.)

0011Other important functions of the folate

coenzymes include the turnover of histidine, the syn-

thesis of methionine from homocysteine, interconver-

sion of serine and glycine, and other single-carbon

transfers between molecules. As noted above, the

methionine-SAM pathway is of major importance

for provision of methyl groups for a plethora of es-

sential structural and functional molecules. There are

complex allosteric (feedback) interactions which

closely control these pathways which involve folate

coenzyme/cosubstrate participation. The strong

metabolic link between the functions of folate and

those of vitamin B

occurs at the methionine

synthase reaction which transfers the carbon unit

from methyltetrahydrofolate to homocysteine to

form methionine (Figure 1). The requirement for B

cofactor in this reaction leads to a lack of suitable

active single-carbon units for DNA synthesis during

deficiency, so that either folate deficiency or B

deficiency will result in megaloblastic anemia and the

other sequelae of impaired cell division at sensitive

tissue sites. (See Amino Acids: Metabolism; Cobala-

mins: Physiology.)

FOLIC ACID/Physiology 2565

0012 Turnover of folate in the body results in part

from its cleavage to liberate p-aminobenzoyl poly-

glutamate, which is then deconjugated and excreted

as p-acetamidobenzoylglutamate and p-acetamido-

benzoate. These breakdown products, plus some un-

changed folate (at higher circulating levels), are

excreted in the urine; however the essential body

stores of folate are efficiently conserved in the kidney

by reabsorption in the proximal renal tubule, and by

an efficient enterohepatic circulation. Folate that

is found in the feces is mainly what is formed by

intestinal bacteria in the large bowel, unless folic

acid intakes are very large.

Biochemical Status Measurements

0013 Folate biochemical status is monitored most com-

monly by the assay of folate concentrations in plasma

or in the red cells of the peripheral blood.

0014Plasma folate essentially reflects recent intakes and

the magnitude of intertissue transfer, whereas red cell

levels represent longer-term tissue stores, since intracel-

lular (red cell) folate does not readily exchange with

extracellular folate. Infants have much higher serum

and red cell folate levels than those of adults, and this

maybe connected with the greater rate of cell division in

infancy. Assays for folate which are based on competi-

tive protein binding (radioassay or fluorescence assay),

or on antibody binding, are now widely used, and

several commercial kits for competitive-binding assays

are now available. It is commonly accepted that serum

(or plasma) levels below 3 mgl

1

or erythrocyte levels

below 100 mgl

1

are indicative of biochemical defi-

ciency, and should be further investigated or treated.

0015‘Functional’ tests, based on the efficiency of catab-

olism of a histidine load, or on deoxyuridine sup-

pression of preformed thymidine utilization for DNA

synthesis in tissue biopsies such as bone marrow cells,

N N

O O

Deoxyribose 5-phosphate

Deoxyuridylic acid

Deoxyribose 5-phosphate

Thymidylic acid

Thymidylate

synthase (EC 2.1.1.45)

5,10-Methylene

tetrahydrofolic

acid

Dihydrofolic acid

Dihydrofolate

reductase (EC 1.5.1.3)

Tetrahydrofolic

acid

5-Methyltetra-

hydrofolic acid

Homocysteine

Methionine

Methionine synthase (5-methyltetrahydrofolate:

L-homocysteine S-methyltransferase, EC 2.1.1.13)

Methylene tetrahydro-

folate reductase

(NADPH: EC 1.5.1.20)

fig0001 Figure 1 Biosynthesis of thymidylate, regeneration of methylene tetrahydrofolic acid, and biosynthesis of methionine.

2566 FOLIC ACID/Physiology

may provide useful functional evidence of tissue folate

adequacy, although they are not used for routine meas-

urement of status. Folate deficiency, like vitamin B

or B

deficiencies, results in raised plasma homocys-

teine (see below), and this functional index can pro-

vide useful information about folate status and dietary

adequacy. (See Immunoassays: Radioimmunoassay

and Enzyme Immunoassay.)

0016 A useful cytological indicator of marginal folate

deficiency, which is more sensitive than overt anemia

or megaloblastosis, is an increase in the lobe average

count of the polymorphonuclear neutrophils in the

circulation. Fewer than 3.3 lobes per cell is con-

sidered normal; more than 3.65 per cell indicates a

significant functional deficiency.

Human Requirements for Folic Acid and

Folates

Recommended Dietary Allowances and Dietary

Reference Values

0017 Several lines of evidence have indicated that for adult

men and adult nonpregnant and nonlactating women,

the minimum requirement for folate (given as a sup-

plement of pteroylglutamic acid) which is needed to

prevent overt clinical deficiency is not greater than

50 mg day

1

. The problem which has faced recom-

mended dietary allowance (RDA) committees is how

much more is needed to cover the needs of nearly all

individuals, when folate is obtained from food? In

1980, for instance, the US committee recommended

400 mgday

1

and 800 mgday

1

during pregnancy. By

the end of the 1980s, the available evidence suggested

that the majority of healthy adults in the USA,

Canada, the UK, and elsewhere appeared to maintain

acceptable folate status on intakes around 150–

200 mgday

1

. During the past decade, there has

been a great deal of research activity which has once

more reopened the debate and has resulted in a shift in

the consensus towards a preference for higher

requirement estimates once more. Metabolic ward

studies of trends in status indices at different folate

intakes have provided some of the evidence; studies of

neural tube defect risk in very early pregnancy plus

the identification of a substantial segment of the

population with a genotype that implies possibly in-

creased folate requirements is another, and the obser-

vation that plasma homocysteine concentrations are

responsive to folic acid supplements even when folate

intakes are as high as 300 mg day

1

or higher, is a third.

Dietary Folate Equivalents (DFE)

0018 The bioavailability of food folate differs from that of

synthetic folic acid, mainly because the latter, being

already oxidized, is not easily damaged, it is already a

monoglutamate, and it is not in a tissue matrix. On

average, the folic acid in supplements and fortified

food is calculated to be 1.7 times as available as food

folate; therefore the US National Academy of Sci-

ences has recently introduced the concept of dietary

folate equivalents (DFEs), analogous to retinol or

niacin equivalents, which is equal to (mg food

folate) þ(1.7 mg synthetic folic acid). This has been

used in calculating the new US (1998) estimated aver-

age requirements or EAR (e.g., 320 mgday

1

for non-

pregnant, nonlactating adults), and RDA (e.g., 400 mg

day

1

for nonpregnant, nonlactating adults), rising to

600 mg day

1

during pregnancy and 500 mg day

1

during lactation. Requirements during late pregnancy

clearly must be higher than those of nonpregnant

adults, because unsupplemented pregnant women,

even in well-nourished societies, often develop folate-

responsive bone marrow megaloblastosis during the

later stages of pregnancy. Folate supplements effect-

ively insure that this functional abnormality does not

occur. (See Dietary Requirements of Adults.)

0019The folates that are present in breast milk

(c.50 mgl

1

) appear to be more readily available

than the pteroylglutamic acid that is added to formula

feeds, since breast-fed babies tend to have higher

blood folate levels for a given folate intake. Folate-

deficiency anemia has been encountered in premature

or growth-retarded infants given inappropriate for-

mulas, but these mistakes have now been rectified

by giving folic acid-fortified feeds. Erythroblastotic

infants have a particularly high folate requirement,

and their growth rate can respond dramatically to

folate supplements. Breast-milk folate levels are gen-

erally well protected, even when the mother’s tissues

and circulating pool have been partially depleted by

moderate dietary folate deficiency. (See Infant Foods:

Milk Formulas; Infants: Breast- and Bottle-feeding.)

0020Certain types of long-term medication, notably

with anticonvulsive drugs, can interfere with folate

economy, and therapeutic doses of folate may make it

more difficult to control epileptic seizures in some

patients. Patients with tropical sprue may, in contrast,

benefit from folate supplements, at least transiently.

Older people are particularly at risk, because their

dietary folate intakes often fall below the safe level,

and because homocysteine levels and the risk of overt

vascular disease rise very steeply in old age. Chronic

alcoholics, smokers, and people with chronic intes-

tinal, renal, or hemolytic disease, or with rare inborn

errors of folate economy, are at increased risk of defi-

ciency. (See Drug–Nutrient Interactions; Elderly: Nu-

tritionally Related Problems.)

0021Vitamin B

deficiency results in impairment of

folate utilization, and hence low intracellular and

FOLIC ACID/Physiology 2567