Background: In several countries cereals are now enriched with
folic acid to reduce the risk of neural tube defects. Human stud-
ies suggest that folic acid interferes with zinc absorption. This
raises concerns about the zinc status of high-risk groups such as
infants, pregnant women, and older persons.
Objective: We sought to determine the effect of added folic
acid on zinc absorption from white bread with high and low
Design: Zinc absorption was measured in 15 healthy women
(22–33 y), each of whom consumed 4 single meals spaced
2 wk apart in a randomized crossover design. The servings of
bread (100 g) differed in zinc and folic acid contents as fol-
lows:A, 1.2 mg Zn and 17 ?g folic acid; B, 1.2 mg Zn and 144 ?g
folic acid; C, 3.0 mg Zn and 17 ?g folic acid; and D, 2.9 mg
Zn and 144 ?g folic acid. Meals were extrinsically labeled
with 65Zn and absorption was estimated from whole-body
retention measurements. Folate status was assessed by meas-
uring plasma and erythrocyte folate and plasma homocysteine
Results: Mean (±SD) zinc absorption did not differ significantly
in relation to the folate content of the breads at either the low
zinc content (38.8 ± 13.5% and 40.6 ± 16.5% for A and B,
respectively; P = 0.74) or the high zinc content (26.7 ± 9.3% and
22.7 ± 6.6% for C and D, respectively; P = 0.16). There was no
significant correlation between folate status and zinc absorption
(r < 0.3, P > 0.1).
Conclusion: Fortification of white bread with a commonly used
amount of folic acid did not appear to influence zinc absorption
at either a high or a low zinc content.
Am J Clin Nutr 2001;
bread, flour, zinc absorption, zinc status, bioavailability, interaction,
radioisotope, whole-body counting,
birth defects, congenital defects
Folate, folic acid, fortification, enrichment,
65Zn, neural tube defects,
The fortification of cereals with folic acid has been imple-
mented in several countries, such as the United Kingdom,
Canada, Australia, and recently the United States. The purpose
of folic acid fortification is to reduce the risk of neural tube
defects in newborns. There is convincing evidence that folic acid
supplementation, when taken from the onset of pregnancy, reduces
the prevalence of neural tube defects (1). An additional effect of
folic acid fortification may be a lowered risk of cardiovascular
diseases. Supplementation with folic acid has been found to
decrease the serum homocysteine concentration, an independent
risk factor for cardiovascular disease (2).
Increasing the intake of folic acid through supplementation or
fortification is generally considered safe. However, concern was
raised in some (3–10) but not all (11–16) studies about a potential
negative effect of folic acid supplementation on zinc absorption.
Decreased zinc absorption could have adverse effects, particularly
in individuals with increased requirements, such as children, ado-
lescents, and pregnant or lactating women. Furthermore, many
elderly persons may already be at risk of marginal zinc status
because of poor appetites and inadequate dietary intakes.
Supplementation with 400 ?g folic acid every other day was
found to influence zinc homeostasis in men during a long-term, par-
allel balance study. At this dose of folic acid, fecal zinc increased
when the diet had a low or moderate zinc content, but not when the
diet was high in zinc. Urinary zinc decreased during the low- and
moderate-zinc diets, which partially compensated for the fecal
losses (5). In another study, 800 ?g folic acid/d was suggested to
have a negative effect on zinc absorption (measured by 65Zn whole-
body counting) only in subjects with a fractional zinc absorption
>30%. These subjects also tended to have lower zinc intakes and
lower plasma zinc concentrations (6). These studies suggest that
folic acid inhibits zinc absorption and that this inhibition is more
pronounced with low-zinc meals than with high-zinc meals.
The results of plasma response studies are conflicting. Plasma
zinc response to a 25-mg dose of zinc was reduced to 49% in
Am J Clin Nutr 2001;74:125–9. Printed in USA. © 2001 American Society for Clinical Nutrition
Folic acid enrichment of bread does not appear to affect zinc
absorption in young women1–3
Marianne Hansen, Samir Samman, Lene T Madsen, Mikael Jensen, Sven S Sørensen, and Brittmarie Sandström
1From the Research Department of Human Nutrition, LMC Centre for
Advanced Food Studies, The Royal Veterinary and Agricultural University,
Frederiksberg, Denmark; the Human Nutrition Unit, the Department of Bio-
chemistry, The University of Sydney, Australia; and the Department of Clin-
ical Physiology and Nuclear Medicine, The National University Hospital,
2Supported by the Danish Food Technology Research Programme,
3Reprints not available. Address correspondence to M Hansen, Research
Department of Human Nutrition, The Royal Veterinary and Agricultural Univer-
sity, Rolighedsvej 30, DK-1958 Frederiksberg, Denmark. E-mail: firstname.lastname@example.org.
Received August 22, 2000.
Accepted for publication November 17, 2000.
10 pregnant women after 2 wk of supplementation with 350 ?g
folic acid and 100 mg Fe/d compared with the baseline response
before supplementation (7). In the same study, the plasma zinc
response to a 50-mg dose of zinc was reduced to 79% of the
baseline value after 2 wk of supplementation with 350 ?g folic
acid/d in 10 young men and women. Other authors who used the
plasma zinc response method found that megadoses of folic acid
[10 mg (11) and 200 mg (17)] had no effect on zinc absorption.
In general, the results of research in this area are inconsistent,
which could be the result of inappropriate methodology and
incomparability of study designs. There is still no scientific basis
for a conclusion regarding the potential effect of folate on zinc
absorption and metabolism.
The aim of the present study was to determine the effect of
folic acid fortification of bread on zinc absorption by using a
radioisotope technique with measurements of whole-body reten-
tion. We chose women of childbearing age as subjects because
they represent a specific target group that is thought to benefit
from fortification with folic acid and that may also be vulnerable
if zinc absorption is compromised.
SUBJECTS AND METHODS
Each subject consumed 4 different bread meals that were
served in random order and spaced 2 wk apart. Two meals had a
low zinc content and 2 had a high zinc content, with or without
added folic acid (2 ? 2 factorial design) in amounts commonly
used in fortification. The meals were extrinsically labeled with
the radioisotope 65Zn. Zinc absorption was calculated by meas-
uring whole-body retention of the isotope. A blood sample was
obtained from each subject before each of the 4 test meals to
measure plasma folate, erythrocyte folate, and serum zinc con-
centrations. Homocysteine concentrations were measured in the
first and last blood samples. The habitual dietary intake of
selected nutrients was assessed by using four 2-d weighed-food
records spread over 2 mo. Nutrient intakes were calculated by
using a national food-composition database (DANKOST 2000,
version 1.4c; Dansk Catering Service, Herlev, Denmark).
Fifteen women aged 22–33 y participated in this study; the
subjects’ mean (±SD) body weight was 66.3 ± 7.9 kg and their
mean body mass index (in kg/m2) was 22.7 ± 3.1. All subjects
were apparently healthy, and none were pregnant or lactating.
None of the subjects took any vitamin or mineral supplements or
donated blood during the study or in the previous 2 mo. None
used medicine regularly or took oral contraceptives. Five smoked
occasionally, whereas one was a regular smoker (<10 cigarettes/d
The study was approved by the Municipal Ethical Committee
of Copenhagen and Frederiksberg (KF 01–238/98) and the
National Institute of Radiation Hygiene, Denmark. The subjects
were given written and oral information about the study and
written consent was obtained from all subjects.
Formulation of test meals and serving procedure
Each test meal consisted of 2 rolls made from white (refined)
wheat flour (total wt: 100 g), 30 g raspberry jam, 10 g butter, and
300 g ultrapure water. The rolls were formulated with 2 different
amounts of zinc in the presence or absence of added folic acid as
follows: A, low zinc; B, low zinc + folic acid; C, high zinc; and
D, high zinc + folic acid. Each type of roll was prepared in one
batch and stored at ?20?C. The basic recipe (without zinc or
folic acid added) was 1500 g white wheat flour, 900 g ultra-pure
water, 30 g fresh yeast, 24 g butter, and 21 g NaCl. For the
batches fortified with folic acid, 10.8 mg crystalline folic acid
(Sigma Chemical Co, St Louis) was mixed with 3000 g flour in
a food processor for 90 min at low speed. This equals 262 ?g
folic acid/serving (2 rolls), calculated as the value before baking,
ie, without baking loss. The zinc content was adjusted to 1 and
3 mg/serving in the low-zinc (A) and high-zinc (C) rolls, respec-
tively, by adding a ZnCl2solution (prepared from ZnO and 37%
HCl; Merck, Darmstadt, Germany) to the dough. After mixing,
the dough was allowed to rise for 1 h at room temperature and
rolls consisting of 60 ± 0.5 g dough were prepared. The subjects
received the meal after fasting for 12 h and were instructed to
consume it within 15 min and to alternate between eating and
drinking. The meals were served on disposable material. The sub-
jects were told not to eat or drink for 4 h after the meal.
Isotopes and labeling procedures
For each test meal, the rolls were extrinsically labeled with
0.04 MBq 65Zn by adding drops of the isotope solution (1 mL
total) to the top of the baked rolls. The radioisotopes were pur-
chased at Risø National Laboratory (Roskilde, Denmark) as
65ZnCl2in 0.1 mol HCl/L with a specific activity of 35 MBq/mg
Zn. The total zinc contribution to each meal from the radioisotope
was in the order of 1 ?g, which represents <0.1% of the total
zinc. The labeling was undertaken in the presence of an observer
?18 h before the test meal was served. The calculated total radi-
ation dose from the 4 meals for each subject was 0.56 mSv.
Whole-body counting and calculation of zinc absorption
Whole-body retention of 65Zn was measured in a whole-body
counter on day 12 or 13 after each test meal. This time period was
chosen to allow for the excretion of unabsorbed isotope. The results
were corrected for background radiation in the chamber and the
subject’s background radioisotope level (40K). Zinc absorption was
calculated from the whole-body retention value, which was cor-
rected for endogenous excretion from days 0 to 12 or 0 to 13. This
correction was performed by using a mean retention function
developed from measurements of whole-body retention over time
in a group of healthy subjects after they had received an intravenous
dose of 65Zn (18). When performing calculations for the second,
third, and fourth meals, allowance was made for residual activity in
the subjects from the preceding test meals by using the same equa-
tion. All values were corrected for physical decay.
The whole-body counter, located at the National University
Hospital in Copenhagen, consisted of a lead-lined steel chamber
with 4 plastic scintillator blocks (NE110; Nuclear Enterprises
Limited, Edinburgh), 2 placed above the subject and 2 placed
below. The counting efficiency and the setting of the energy
window were established by measuring 3 water-filled phantoms,
each containing 0.04 MBq 65Zn. The phantoms weighed 55, 66,
and 77 kg and had outlines of typical humans of corresponding
weights. A slight decrease in counting efficiency was observed
with increasing weight. A linear interpolation was used to calcu-
late the actual counting efficiency for each individual subject
according to her weight. The overall counting efficiency for the
66-kg phantom was 0.2 counts·s?1·Bq?1 65Zn. The energy
126HANSEN ET AL
window was set at a lower level of ?80 keV, allowing detec-
tion of the compton events (inelastic scattered ?-photons) in the
plastic scintillators from both 511 and 1115 keV radiation from
65Zn. No upper energy limit was used. To minimize contamina-
tion by atmospheric background activity, the subjects showered
and washed their hair and then dressed in hospital clothing
before each measurement. Each measurement of whole-body
retention lasted for 10 min.
Two rolls from each batch were pooled, freeze-dried, homog-
enized, and analyzed in duplicate for zinc content by using
atomic absorption spectrometry (Spectr-AA 200; Varian, Mul-
grave, Victoria, Australia) after microwave digestion. A refer-
ence material [Standard Reference Material 1548, total diet
(mixed food sources); National Institute of Standards and Tech-
nology, Gaithersburg, MD] was analyzed for zinc content in the
same run as the meal samples. The zinc content of the standard
was measured as 26.45 ?g/g, compared with the certified value
of 24.6 ± 1.79 ?g/g. The folate-fortified (n = 5) and unfortified
(n = 2) rolls were analyzed in duplicate for folic acid content by
turbidimetric measurement on a spectrophotometer (UV160A;
Shimadzu Corp, Kyoto, Japan) by using Lactobacillus casei as a
test organism, according to the method of Finglas et al (19). The
analyses of the unfortified rolls represented the background ana-
lytic amounts of folate equivalents (analyzed as folic acid) in the
rolls. An internal reference was included (0.68 ?g/g with an
average laboratory value of 0.62 ?g/g).
Four blood samples (1 for each of the 4 test meals) were
obtained from each subject after a 12-h fast and 10 min of rest
in the supine position. Subjects were asked to refrain from phys-
ical exercise for 36 h and to refrain from using alcohol or
medication for 24 h before blood sampling. Serum zinc concen-
trations were analyzed by using flame atomic absorption spec-
trometry (Spectra AA-200; Varian). The intraassay CV was
2.0% and the interassay CV was 7.6%. An internal control was
included in all runs (15.3–15.5 ?mol/L with an average labora-
tory value of 15.2 ?mol/L). Hemoglobin concentrations and
hematocrit values were determined on a Cobas Minos ABX
automatic cell counter (Groupe Hoffmann, Montpellier, France).
The intraassay CVs were 1.0% and 1.6% for hemoglobin and
hematocrit, respectively, and the corresponding interassay CVs
were 1.7% and 2.4%, respectively. Blood collected in heparin-
containing tubes was used for plasma and erythrocyte folate
analyses. For erythrocyte folate, 50 ?L blood was incubated
with 1 mL ascorbic acid (L-ascorbic acid; Merck) solution
(0.5% wt:vol in distilled water) for 20 h in the dark at room tem-
perature. Erythrocyte and plasma folate concentrations were
analyzed fluorometrically (Delfia 1232; Wallac OY, Turku, Fin-
land) with a time-resolved fluoroimmunoassay kit (Delfia
Folate Kit A072–101; Wallac OY) (20). The interassay CVs
were 6.5% and 3.4% for plasma and erythrocyte folate, respec-
tively. Plasma homocysteine concentrations were analyzed flu-
orometrically with a fluorescence polarization immunoanalysis
kit (IMx Homocysteine kit 3D3920–20; Abbott Laboratories,
Chicago) (21). An internal control was included (12.3 ?mol/L
with an average laboratory value of 12.5 ?mol/L; intraassay
CV, 1.9%; interassay CV, 4.1%).
Values are expressed as means ± SDs. To compare fractional
zinc absorption, we performed pairwise comparisons (paired t
tests) between the 2 high-zinc meals and also between the
2 low-zinc meals. Pairwise comparisons were performed because
of the inherent inverse relation between zinc dose and fractional
absorption. Absolute zinc absorption values for all 4 meals were
compared by using a repeated-measures two-factor analysis of
variance with an interaction term. When significance was
reached, we performed a modified t test according to the Bon-
ferroni method. MICROSOFT EXCEL 2000 software (Microsoft
Corp, San Francisco) and the STATISTICAL ANALYSIS SYS-
TEM (SAS) statistical package, version 6.12 (SAS Institute Inc,
Cary, NC) were used for the statistical analyses.
The zinc and folate contents of the test meals are shown in
Table 1. We had added 262 ?g folic acid to each serving of rolls,
and 144 ?g folic acid was recovered in the rolls after baking.
After we accounted for the endogenous folate content (17 ?g
folate equivalents) of the unenriched rolls, the recovery of added
folic acid was calculated to be 48%. Consequently, the baking
loss was ?52%.
Addition of folic acid did not influence zinc absorption
from either the low-zinc or the high-zinc meal (Table 1). Frac-
tional zinc absorption was higher from the 2 low-zinc meals
(38.8–40.6%) than from the 2 high-zinc meals (22.7–26.7%),
whereas in absolute amounts, zinc absorption was higher from
the high-zinc meals. There was no significant interaction
between zinc content and addition of folic acid.
The subjects’ habitual daily intakes of energy and selected
nutrients were 9.9 ± 1.2 MJ, 12 ± 2% of energy as protein,
26 ± 3% of energy as fat, 58 ± 5% of energy as carbohydrate,
23 ± 5 g fiber, 9.3 ± 2.0 mg Zn, and 308 ± 60 ?g folate. Dietary
zinc and folate intakes met the Nordic Nutrient Recommenda-
tions of 7 mg and 300 ?g, respectively (4). The subjects’ con-
centrations of serum zinc, plasma and erythrocyte folate, and
plasma homocysteine were within reference ranges for the Dan-
ish population (Table 2). Zinc absorption was not affected by
folate status, because neither erythrocyte folate nor plasma folate
correlated with fractional zinc absorption (r < 0.3, P > 0.1).
ZINC–FOLATE INTERACTION 127
Folate and zinc contents of the test meals (bread rolls) per serving and
?g mg % (mg)
A: Low zinc
B: Low zinc + folic acid
C: High zinc
D: High zinc + folic acid
38.8 ± 13.5 (0.47 ± 0.16)a,4
40.6 ± 16.5 (0.49 ± 0.20)a
26.7 ± 9.3 (0.78 ± 0.27)b
22.7 ± 6.6 (0.68 ± 0.20)a,b
144 ± 13
144 ± 13
1Endogenous folate was analyzed in unfortified bread (A and C), n = 2.
Folate in B and D (n = 5) includes endogenous folate and added folic acid.
2n = 2 for each kind of bread.
3For % absorption, there were no significant differences between A and
B or between C and D (paired t tests). For mg absorption, values with dif-
ferent superscript letters are significantly different, P < 0.05 (ANOVA fol-
lowed by multiple comparisons). The differences between A and D and
between B and D were marginally significant (P = 0.05).
Folic acid is highly bioavailable from bread, according to pre-
vious research (22). In the present study, we added folic acid to
test meals (bread rolls) in amounts typically used in fortification.
The zinc contents of the test meals also corresponded to realistic
dietary intakes. Two different amounts of zinc (low and high)
were used because previous studies suggested that folic acid
inhibits zinc absorption from low-zinc diets (5, 6). Consistent
with previous results (23), the fractional absorption of zinc was
substantially higher from the low-zinc meals than from the high-
zinc meals (Table 1) and the absolute amount of zinc absorbed
was approximately 1.5-fold greater from the high-zinc meals.
The extent of zinc absorption in the present study is consistent
with previous research that included a range of zinc intakes from
high-bioavailability diets (24). Our results showed no significant
differences in zinc absorption from bread with and without folic
acid fortification at either low or high zinc contents. However, a
small inhibitory effect of folic acid on zinc absorption from the
high-zinc meals cannot be excluded. A posthoc power analysis
showed that with a power of 80% (? = 0.05 and ? = 0.8), we
would be able to detect only an 8% or larger difference. How-
ever, it is unlikely that the observed 4% lower zinc absorption
would be of any nutritional significance.
The mechanism for a potential folate-zinc interaction could
operate over the long term, rather than at one moment in time. If
a high folate content of a single meal does inhibit zinc absorp-
tion, then it is possible that a high folate intake over the long
term would lead to low zinc absorption from a single meal. In
accordance with the lack of effect of folic acid content on zinc
absorption in this single-meal study, there was no correlation
between markers of folate status and fractional zinc absorption.
The folic acid content of the fortified bread rolls used in the
present study was 144 ?g/100 g. After correction for the endoge-
nous folate content of the flour, the baking loss of added folic
acid was 52%. This loss was substantially higher than the losses
from bread reported by others [8–14% (25) and 30% (22)]. The
baking loss of added folic acid may be related to the size and
shape of the bread. The 144 ?g folic acid in 100 g bread was
higher than the amount that would be obtained by using flour for-
tified with 140 ?g folic acid/100 g, the standard of the Food and
Drug Administration (26). Thus, our data show that even with
folic acid fortification at the high end of the recommended range,
there was no significant interaction with zinc absorption.
Although several studies investigated the effect of folic acid
on zinc absorption, limitations in the study designs and method-
ologies may have contributed to the inconsistent findings. In sev-
eral studies, the plasma zinc response to an oral zinc challenge
was measured to assess the effect of folic acid on zinc absorption
(7, 11, 17). Some disadvantages of this method include the use
of pharmacologic doses of zinc and the confounding effects of
large fluctuations in the rate of gastric emptying and blood clear-
ance and urinary excretion of zinc. These limitations are
reflected by large inter- and intraindividual variations (27, 28)
and poor concordance with isotope methods (27). Pharmacologic
doses of folic acid were also used commonly in studies of folate-
zinc interaction (11, 13, 17). At unphysiologically high doses of
folate, zinc, or both, other factors may be responsible for an
observed interaction. These factors may include saturation or
alternation of uptake mechanisms in the brush border membrane
because of stressing of the absorptive mechanisms. This may
explain why inconsistent results were obtained in studies with
folate-to-zinc molar ratios ranging from 1:1 (17) to 1:975 (7).
Another methodologic problem is the lack of sensitive and valid
indexes of zinc status; this may explain why no consistent rela-
tion between folate intake or status and zinc status has been
found (10, 12–14, 29). In accordance with this, several studies
found no effect of folate supplementation on serum zinc concen-
trations during pregnancy (13, 14, 29).
The strengths of the present study were the use of isotope
labeling and a whole-body counting technique for measurement
of zinc absorption and the use of nonpharmacologic doses of zinc
and folic acid.
Isotope techniques generally provide more accurate and precise
results than do other methods, but few published studies used iso-
tope techniques. Milne (6) used 65Zn labeling and the whole-body
counting technique for determining zinc absorption from single
meals. Our data are in accordance with those of Milne, who found
no significant difference in zinc absorption between a control
meal providing 2.7 mg Zn (equivalent to our high-zinc meal) and
the same meal supplemented with 800 ?g folic acid (a 5.5-fold
greater dose than that used in our test meals). In Milne’s study,
65Zn absorption from the control meal was plotted against the dif-
ference in absorption between the folic-acid-supplemented and
control meals, and a linear correlation was found. On the basis of
this finding, Milne concluded that zinc absorption is inhibited by
folic acid; however, by using the author’s reasoning it can be
argued equally well that folic acid promotes zinc absorption.
The long-term effect of folic acid on zinc status per se cannot
be evaluated effectively in humans because reliable biomarkers
of zinc status are not available. Therefore, we must rely on zinc
absorption studies. On the basis of the present results obtained
with radioisotope labeling and whole-body counting, it is not
likely that fortification with the currently used concentrations of
folic acid is cause for any concern. However, it would be relevant
to study the effects of higher doses of folic acid, as are found in
supplements, on zinc absorption with the same method.
We thank Susanne Svalling and Hedi Sønneland Pedersen for performing
the whole-body counting measurements and Hanne Lysdal Pedersen for con-
ducting the chemical analyses.
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6.4 ± 1.9
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ZINC–FOLATE INTERACTION 129