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REVIEW ARTICLE Scandinavian Journal of Nutrition
Naringsforskning Vol 43:138-146 1999
Folates ood sources analysesretention and bioavailability
By Cornelia M. Witthoft K arin ForssknLena Joha nnesson and
Margaretha Jagerstad
ABSTRACTHealth benefits of folates regarding their prevention of
neural tube defects in babies and occlusivevascular diseases caused
by elevated plasma homocysteine, their link to mental fitness
andpossibly certain forms of cancer have already been recognised.
However, analyses of food folatesis still tedious because of a lack
of validated methods for characterisation and quantitation of
thegreat number of native folate forms, but also due to a lack of
adequate methods for samplepretreatment. Therefore, the assessment
of folate losses through industrial and household foodprocessing is
still incomplete, as well as knowledge on folate bioavailability in
humans. Thispaper reviews the state of art for the occurrence and
analysis of folates in foods. Furthermore,results are summarised
from studies of folate retention during food processing and the
assessmentof folate bioavailability.
Key words: Analysis bioavailabili ty folates food table data
rete ntio do sses
IntroductionFolates represent an important groupamong the
B-vitamins, participating inone-carbon transfer reactions
requiredwithin the cell, especially for purine andpyrimidine
biosynthesis (DNA and RNA)and amino acid interconversions.
Optimalfolate nutrition and status are linked todiminished risk for
neural tube defects,occlusive vascular diseases (1-4) and pos-sibly
some forms of cancer 5). Recently,a relationship to cognitive and
mentalfunction has also been discussed (6).
In the latest edition of the NordicNutritional Recommendations
(1996), thedaily intake for adults was increased from200 pg to 300
pg folate and for pregnantwomen a daily intake of 400 pg was
re-commended 7). When publishing thedietary reference intakes (DRI)
in 1998,the US Food and Nutrition Board includedthe concept of
possible health-protectiveeffects of folate by increasing
recommen-dations for adults to 400 pg/d from the
previous 200 pglday (8). Moreover, theUS Food and Nutrition
Board recom-mends women who plan a pregnancy toconsume an
additional 400 pg syntheticfolic acid from fortified foods or
supp-lements. Such dramatic increases of re-commended intakes for
folate combinedwith the fact that the average daily intakeof folate
among Western populations isgenerally lower than recently set
recom-
Cornelia M. W itthoft Dr, Karin ForssCn MSc,
Lena Johannesson MSc, Margaretha JagerstadProf, Swedish
University of Agricultural Sciences.Correspondence Cornelia M.
Witthoft, SwedishUniversity of A gricultural Sciences, Department
ofFood Science, P.O. Box 7051, SE -750 07 Uppsala,E-mail: Cornelia
Witthoft@lmv slu se
mendations, emphasise the need for acritical evaluation of the
dietary sources offolates. Most of the food folate data derivefrom
microbiological analysis with ofteninsufficient methodological
control. Thereis still today little reliable informationabout which
folate forms and concent-rations are present in food and what
im-pact food processing techniques have onfolate retention.
Moreover, knowledgeabout human folate bioavailability fromnative
food sources or after fortification isstill incomplete (9).
Possible risks andbenefits from food fortification with folicacid
are currently a subject of controversy.
This review aims to give brief infor-mation on folate contents
and analysis infood, folate losses during food processingand the
assessment of folate bioavai-lability (for more detailed
information see10-13).
olate chem istry and stabilityAccording to recommendations of
the
IUNS Committee on Nomenclature(1986), folate should be used as
thegeneric term for the class of compoundshaving similar chemical
and nutritionalproperties to pteroyl-L-glutamic acid(folic acid)
(14). While the pteridine ringof folic acid exists in oxidised
form, nativefolates have either two or four additionalhydrogens in
their pteridine ring formingdihydro- or tetrahydrofolates. Thus,
die-tary folates exist primarily as reduced,one-carbon-substituted
forms of pteroyl-glutamates, with up to seven glutamylresidues
attached to the p-aminobenzoicgroup by y-peptide linkage. Five
differentone-carbon units are known to be linked atN5- and/or
NIo-position of the pteroylgroup: methyl (5-CH,), formyl (5- or
10-
HCO), formimino (5-CHNH), methylene(5,lO-CH,) and methenyl
(5,lO-CH)(Figure 1). Altogether the theoreticalnumber of all native
folate vitamersreaches several hundred (1 I).
All folates are in danger of oxidativedegradation enhanced by
oxygen, lightand heat, resulting in a splitting of themolecule into
biologically inactive forms,of which p-aminobenzoylglutamate is
onemajor form. There are considerable differ-ences in stability
between various reducedfolate forms; the order of stability
is:5-HCO-H4folate > 5-CH,-H4folate> 10-HCO-H4folate >
H,folate.Moreover, the stability is pH-dependent.
Folic acid exhibits substantially greaterstability than the
reduced folate forms.The chemistry of folates makes the vita-min
one of the most vulnerable to lossesduring food processing. If
present inadequate amounts, antioxidants, e.g. as-corbic acid and
thiols, protect folates. Therate of reaction for folate breakdown
in the
presence of oxygen depends on the type offolate derivative and
the nature of the foodmatrix, in particular with respect to
pH,buffer composition, catalytic trace ele-ments and antioxidants
(1 1 13).
ist of abbreviations5-HCO-H4folate
5-formyltetrahydrofolate5-CH3-H4folate
5-methyl-tetrahydrofolate10-HCO-PG A 10-formyl-folic acidFBP
folate-binding proteinHPL C high performance liquid
chromatographyPBA competitive)
protein-binding assayPteGlu pteroylglutamic acidRIA radioimmuno
assayH,folate tetrahydrofolate
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ietary folates a nd bioavailabilily
olate content in foodsA brief look into various food tables
fromdifferent countries (Table 1) shows thatmany vegetables and
pulses are rich sour-ces of folate, with folate concentrations upto
600 pg/100 g in some beans and chickpeas and around 200 pgI100 g in
leafy
vegetables. A sort of general rule is that thelower the water
content in the vegetablethe higher the folate concentration,
andmoreover that leafy vegetables are goodfolate sources (folium
means leaf). Folateconcentrations in fruits and berries areusually
one-tenth those of vegetables,ranging from a few pg to approx. 50
pg/100 g. The highest concentrations are to befound in frozen
concentrated orange andgrapefruit juice, strawberries and
severalnuts with folate concentrations of about50-1 00 pg/ 100 g or
more.
Meat and meat products, except liver
which is the storage organ, contain littlefolate, while chicken
and fish are mode-rate sources (1 5). Folate concentrations inmilk
are only around 5 pg1100 ml, but milkis however of interest due to
its content ofa specific high-affinity folate-bindingprotein. This
may play an important role inthe regulation of absorption and
bioavaila-bility of dietary folates from the gastro-intestinal
tract (16). An increase of thefolate content usually results from
fer-mentation of milk and whey products. Inhard cheeses, up to 40
pg/ 100 g and more
were quantified. Soft cheeses like Ca-membert and Brie contain
60-100 pg/lOOg (17). High in folate is egg yolk, withup to 90
pg/100 g (15).
Cereals are also an important dietarysource for folate. Some
cereal fractionslike bran and germs contain a few hundredpg folate
per 100 g, while bread fromwholemeal flour contains 50-1 00 pg/100
g(Table 1). Baker's yeast with its extremelyhigh folate
concentration (approx . 1000-4000 pg/100 g) contributes to the
folatecontent in soft bread. Commonly, foodsare ordered into groups
being rich ,
good and moderate sources, with folateconcentrations of > 100
pg ,SO- 100 pg and15-50 pg per serving, respectively (1 8).
Folate analysis by HPLC can provideinformation on individual
folate formspresent in food, but currently only fewdata are
available; some examples fromvegetables, fruits and dairy products
aregiven in Table 2. The sum of concent-rations from individual
folate forms ana-lysed by HPLC cannot be directly com-pared with
total folate concentrationsassessed by microbiological methods,
asthe latter response to nearly all tetrahydro-,dihydro- and fully
oxidised folate forms inthe food sample. It is still unclear,
how-ever, which concentration best reflect theamount of folate
bioavailable for humans.
Figure 1. Structure of tetrahydropteroylpoly y glutamate.
Substituent {R Position-CH3 methyl) 5-HCO formyl) 5 or 10-CH=NH
formimino)-CH2- methylene) 5 and 10-CH= methenyl and 10
Both tables show notable discrepanciesbetween folate data for
some foods, whichcannot only be explained by differenceswith folate
content but rather by methodo-logical differences in respect to
folatequantification and sample pretreatment,emphasising the need
for re-assessmentusing better controlled methods.
olate analysisTraditionally, microbiological assay pro-cedures
with Lactobacillus casei (ATCC
7469), which responds to most folatederivatives with up to three
polyglutamateresidues, are used for folate quantification(19). Most
folate values published in foodtables today were established by
micro-biological methods.
The use of (radio-) protein binding pro-cedures is common for
clinical diag-nostics in plasma, serum or whole bloodmainly
containing 5-CH3-H4folate (20).These tests are based on
non-specificcompetitive binding of folates from thetest sample and
radiolabelled folic ,acid toa folate-binding protein. Accurate
control
of the assay pH is required, and formylatedfolates are only
bound to a small per-centage (2 1,22). Protein-binding proce-dures
have not often been used success-fully for food folate analyses and
theirapplication might be restricted to foodmatrixes which contain
mainly 5-CH3-H4folate (23-30). Most foods, however,contain a
variety of folate forms (3 1-36).
In recent years, HPLC methods for thesimultaneous determination
of severalindividual folate monoglutamates wereestablished (3
1,37-42). Often fluore-scence detection is used, and
consequentlydetermination is restricted to reducedfolate forms
H4folate, 5-HCO-H4folateand 5-CH3-H4folate, which show
nativefluorescence (43).
Folate stabilisation before and duringanalysis is necessary as
folates are sen-sitive to oxygen, heat, light, pro-oxidantsand
extreme shifts of pH-value (10-13).Optimisation of stabilisation
proceduresis hampered, because individual folatesforms possess
different pH-optima formaximum stability. With the use of
antio-xidants throughout sample preparation,folates are
successfully protected frominterconversion (44) and from
oxidativedegradation (45-48). Additional exclusion
of oxygen by overlay with nitrogen (49-5 1 , as well as use of
low temperatures andshelter from light, should already beapplied
when homogenising the samples.
Commonly, folates are extracted inslightly acidic to slightly
alkaline con-ditions by heat, using autoclaving withmicrobiological
folate assay 19,25,52) ora boiling water bath with HPLC pro-cedures
(40,4 1).
Prior to quantification with both micro-biological and HPLC
procedures, decon-jugation of folate polyglutamates
tomonoglutamates is required (9,lO). As de-
conjugase preparations are not com-mercially available, they
have to be pre-pared by the investigator. Partially puri-fied
suspensions of y-glutamyl-hydro-lases (EC 3.4.22.12) from hog
kidney(37), chicken pancreas (53) and plasmafrom humans or other
species (25,54,55)are used; to a lesser extent enzyme pre-parations
from intestines (56-58). ForHPLC determination, the use of
humanplasma or hog kidney deconjugation isadvisable, because these
exopeptidasesproduce folate monoglutamates (37,59).Procedures for
folate deconjugation haveto be optimised in respect to time,
tem-perature, incubation milieu, folate stabili-sation and
substrate-enzyme ratio depend-ing on the characteristics of the
sample
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Table 1. Total folate contents in foods compiled from different
national food tables.
Food item Folate concentration in pgI100g
Vegetablesaubergine 18asparagus 119
beet red 86beans, green 36beans, white, dried 488broccoli
175Brussels sprouts 61butterhead lettuce 73cabbage, white
57cabbage, fermented, sauerkraut 40cabbage, Chinese 150cabbage, red
2 1carrot 14cauliflower 47chick peas, dried 557cucumber 14garlic
3kale 30leek 9 1lentils, dried 433onion 20parsley 183parsnip
67peas, green 65peppers, sweet, green 14potato 19radishes 27spinach
194squash 22tomato 21Fruit berries nutsalmonds 56apricot 9avocado
62banana 19
black currants 23blueberry 6cherries 3grapes 4hazelnuts 72lemon
11mango 36orange juice 44peach 4peanuts 101rosehip, dried
2strawberries 99Yeast cereals cereal productsbaker's yeast,
compressed 1000corn flour 10rye flour, wholemeal 56wheat bran
260wheat flour 21wheat germs 330wheat flour, plain 21barley, rolled
20bread, white 36oats, rolled 56spaghetti, raw 12rice, parboiled,
raw 3 1Milk and dairy productsBrie, 28 % fat 65Camembert, 23 % fat
62Cheddar, 33 % fat nrcheese, blue, 30 % fat 36cottage cheese
12milk, whole 6yoghurt, plain, 3 % fat 15
whipping cream 4
(119); (120); (121); (122); = analysed by HPLC; + = folate
present in food sample, butno reliable value available; g nr = no
value reported
Table 2. Folate contents and derivates in foods assessed
byHPLC.
Food item Folate concentration in pg1100g
folate folate folate
Vegetables
asparagusbutterhead lettuce
broccoli
Brussels sprouts
cabbage, white
cauliflower
carrot
Chinese cabbage
peas, green, frozen
peppers, sweet
potato
potato, cooked
turnipturnip, cookedtomato
tomato, conserve
Fruits and berriesapple
banana
black currants
orange
orange juice
strawberries
Milk and dairy productsmilk, 1.5 % fat
