British Journal of Nutrition (2011), 105, 16521659

q The Authors 2011

doi:10.1017/S0007114510005490

Efficacy of a microencapsulated iron pyrophosphate-fortified fruit juice:

a randomised, double-blind, placebo-controlled study in Spanish
iron-deficient women
Ruth Blanco-Rojo1, Ana M. Perez-Granados1, Laura Toxqui1, Carmen Gonzalez-Vizcayno2,
Marco A. Delgado3 and M. Pilar Vaquero1*
1

Department of Metabolism and Nutrition, Institute of Food Science and Technology and Nutrition (ICTAN),
Spanish National Research Council (CSIC), C/Jose Antonio Novais 10, 28040 Madrid, Spain
2
Clinical Analysis Laboratory, Madrid Salud, Madrid, Spain
3
Grupo Leche Pascual, Aranda de Duero, Burgos, Spain

British Journal of Nutrition

(Received 12 July 2010 Revised 29 November 2010 Accepted 30 November 2010 First published online 8 February 2011)

Abstract
Fe-deficiency anaemia is a worldwide health problem. We studied the influence of consuming an Fe-fortified fruit juice on Fe status in
menstruating women. A randomised, double-blind, placebo-controlled study of 16 weeks of duration was performed. Subjects were randomised into two groups: the P group (n 58) or the F group (n 64), and consumed, as a supplement to their usual diet, 500 ml/d of a
placebo fruit juice or an Fe-fortified fruit juice, respectively. The Fe-fortified fruit juice, containing microencapsulated iron pyrophosphate,
provided 18 mg Fe/d (100 % of the RDA). At baseline and monthly, dietary intake, body weight and Fe parameters were determined: total
erythrocytes, haematocrit, mean corpuscular volume (MCV), red blood cell distribution width (RDW), Hb, serum Fe, serum ferritin, serum
transferrin, transferrin saturation, soluble transferrin receptor (sTfR) and zinc protoporphyrin (ZnPP). The fruit juice consumption involved
increased intake of carbohydrates and vitamin C, and increased BMI within normal limits. Ferritin was higher in the F group after week 4
(P,005) and became 80 % higher than in the P group after week 16 (P, 0001), and transferrin decreased in the F group compared with
the P group after week 4 (P,0001). RDW was higher at weeks 4 and 8 in the F group compared with the P group (P,005). Transferrin
saturation increased after week 8, and haematocrit, MCV and Hb increased after week 12, in the F group compared with the P group. Serum
Fe did not change. sTfR and ZnPP decreased in the F group at week 16 (P,005). Iron pyrophosphate-fortified fruit juice improves Fe
status and may be used to prevent Fe-deficiency anaemia.
Key words: Fortification: Ferric pyrophosphate: Iron-deficiency anaemia: Iron status: Women

Nutritional Fe deficiency has been identified as one of the ten

leading factors for disease, disability and death in the world
today. An estimated two billion people are affected, and the
population at risk includes women of child-bearing age and
children. It is the only highly frequent nutritional deficiency
in developing and developed countries(1).
Dietary strategies for combating Fe deficiency include Fe
supplementation, dietary modification and diversification,
and food fortification(2). Our research group has recently
observed in young women that the current RDA of 18 mg
Fe/d(3,4) was not easily reached, even though the volunteers
consumed five portions of red meat and two portions of poultry per week(5). Supplementation with doses of Fe $ 100 mg/d
is efficacious to increase Fe status, but its major limitation is

low compliance due to gastrointestinal discomfort(2). Finally,

fortification is widely considered to be the most practical
and cost-effective prevention programme(6). However, Fe is
the most challenging micronutrient to add to foods, because
the Fe compounds that have the best bioavailability tend
to be those that interact most strongly with food constituents
producing undesirable organoleptic changes(7). Among Fe
fortificants, ferric pyrophosphate allows appropriate food
processing, and it is easily and effectively absorbed while
producing negligible colour and palatability changes(8 10).
It is well known that the food matrix strongly affects Fe
bioavailability(11). Therefore, in addition to the Fe salt, the
effectiveness of consuming Fe fortificants is highly dependent
on the type of food used.

Abbreviations: AA, ascorbic acid; F, fortified group; P, placebo group; RDW, red blood cell distribution width; STfR, soluble transferrin receptor; ZnPP, zinc
protoporphyrin.
* Corresponding author: M. P. Vaquero, fax 34 915943627, email [email protected]

British Journal of Nutrition

Iron-fortified fruit juice on iron status

Ascorbic acid (AA) is the most important enhancer of Fe

absorption, both for its ability to improve Fe absorption in
the lumen and for overcoming the negative effect of inhibitors(12 14). However, there are doubts concerning the applicability of single-meal results to the practical diet.
Cook & Reddy(15) studied Fe absorption from three diets
varying in AA and concluded that the effect of vitamin C on
Fe absorption from a complete diet was far less pronounced
than that from single meals. A better Fe status is reached
when AA is consumed with meals containing substantial
amounts of added Fe(10,13).
Nevertheless, studies that used foods with a naturally high
content of AA are scarce(16). Fruit juices can contain high
quantities of vitamin C, low pH and no Fe absorption inhibitors, and should be considered as target products to fortify
with Fe.
An orange juice fortified with iron sulphate (2 mg Fe/100 ml)
given to Brazilian preschool children during 4 months (mean
Fe intake 57 mg/d) increased Hb levels and decreased the
percentage of anaemic children from 60 to 20 %(16).
Other studies have been performed using multiple-micronutrient-fortified powdered beverages containing iron bis-glycinate.
These beverages reduced the overall prevalence of anaemia in
children, adolescent girls and pregnant women of developing
countries(17 21).
The present study therefore aims to investigate the influence
of the consumption, as a part of the usual diet, of an Fefortified fruit juice on Fe metabolism in young Spanish
Fe-deficient women.
This trial was registered at clinicaltrials.gov as NCT0
1135576.

Subjects and methods

The present study was designed and carried out following
the statement guidelines of the Consolidated Standards of
Reporting Trials(22).

Subjects
Volunteers were recruited by different announcements in
the press, university campus and web pages of Madrid. The
study was also verbally promoted at public events.
The principal variable for the calculation of sample size was
ferritin, with a mean value for the deficient population of 11
(SD 5) ng/ml. It was calculated that a minimum of sixty-three
subjects with low Fe stores would be required in each
group to demonstrate a difference of 25 units in serum
ferritin between two treatments at 80 % power and confidence
level at 95 %.
Women aged 18 35 years, non-smoker, non-pregnant, nonbreast-feeding, with low Fe stores, defined as serum ferritin
, 40 ng/ml and Hb $110 g/l, were included in the present
study. The cut-off value for serum ferritin was selected
because a normal or elevated ferritin value does not exclude
the presence of Fe deficiency, and cut-off ranges between
25 and 50 ng/ml are usually considered in studies dealing
with predisposition to anaemia(5,23). Subjects were excluded

1653

from the study if they had amenorrhoea, menopause or any

known health problems likely to influence Fe status including
Fe metabolism-related diseases (Fe-deficiency anaemia, thalassaemia and haemochromatosis), chronic gastric diseases
(inflammatory bowel disease, Crohns disease, gastric ulcers,
coeliac disease and haemorrhagic diseases), renal disease or
allergy to any of the components of the assay juices. Other
exclusion criteria were blood donors or to have regularly consumed Fe or AA supplements within the 4 months before participating in the intervention.
A group of 259 women contacted the research group in
order to participate in the study, but only 163 underwent
screening. Out of the 163 women, thirty-three were excluded
(twenty-eight did not meet the inclusion criteria and five
refused to participate). Finally, a total of 130 women agreed
to participate in the nutritional intervention. They were randomised into two groups: fortified (F) and placebo (P). All
volunteers of the F group completed the assay while eight participants of the P group abandoned the intervention (Fig. 1).
Participating women were instructed not to deviate from
their regular habits and to maintain their normal diet and exercise level during the 4 months.
The present study was conducted according to the guidelines laid down in the Declaration of Helsinki, and all procedures involving human subjects/patients were approved
by the Clinical Research Ethics Committee of Hospital Puerta
de Hierro, Madrid. Written informed consent was obtained
from all subjects.

Study design
The study consisted of a randomised double-blind study controlled by placebo of 16 weeks duration.
The volunteers recruited were randomly allocated into two
groups, using the RAND function in Excel (Microsoft Office
2003). One group consumed, as a supplement to their usual
diet, 500 ml/d of the Fe-fortified fruit juice (F group, n 64),
whereas the other consumed 500 ml/d of the placebo fruit
juice (P group, n 66). Fortified and placebo juices were manufactured in 500 ml cartons and in two different tastes (orange
and peach apple) to achieve compliance. The fortified juice
supplied 18 mg Fe/500 ml carton, in the form of microencapsulated iron pyrophosphate coated with lecithin, equivalent
to 100 % of the RDA/d(3,4). All juices were fortified with vitamin C. Nutritional composition of the juices was facilitated
by the manufacturer (Grupo Leche Pascual, Burgos, Spain).
Orange juices provided (per 100 ml) 188 kJ, 06 g of protein,
105 g of carbohydrate and 19 mg of vitamin C; the Fe-fortified
orange juice provided 36 mg of Fe, whereas the placebo juice
had 0084 mg. Peach apple juices provided (per 100 ml) 201 kJ,
06 g of protein, 113 g of carbohydrate and 19 mg of vitamin C;
the Fe-fortified peach apple juice provided 36 mg of Fe,
whereas the placebo juice had 0136 mg.
Participants were instructed to alternate between juice flavours (orange juice one day, peach apple juice the next
day). The 500 ml carton had to be drunk all at once separately
from meals (by at least 2 h) and shaken before consumption.

1654

R. Blanco-Rojo et al.

Contacted the
research group (n 259)

Assessed for
eligibility (n 163)

British Journal of Nutrition

Randomised
(n 130)

Allocated to P group (n 66)

Received allocated
intervention (n 62)
Did not receive allocated
intervention (n 4):
(1) Refused to participate (n 3)
(2) Kidney infection before
the study (n 1)

Lost to follow-up; give reasons

(n 4)
(1) Knee sprain (n 1)
(2) Discomfort caused by juice (n 1)
(3) Change of residence (n 2)

Analysed (n 58)
Excluded from analysis
(n 0)

Excluded (n 33)
Not meeting inclusion
criteria (n 28)
Refused to participate
(n 5)

Allocated to F group (n 64)

Received allocated
intervention (n 64)
Did not receive allocated
intervention (n 0)

Lost to follow-up
(n 0)

Analysed (n 64)
Excluded from analysis
(n 0)

Fig. 1. Diagram of the Consolidated Standards of Reporting Trials.

Volunteers who could not drink the juice one day were
instructed to consume two juice cartons the following day.

Dietary control and compliance

Each subjects dietary intake was evaluated at baseline and
monthly to control any possible changes in energy and nutrient intakes. They completed a 72 h detailed dietary intake
report, previously validated and proved valuable to assess
nutrient intake(24,25), specifying the types of food consumed
and serving weights. Daily food, energy intake, nutrient
intake and energy provided by macronutrients were calculated by a computer application using the Spanish Food Composition Database(26). The compliance of the study was
assessed monthly by questionnaires and personal interview
when volunteers underwent blood sampling.

Anthropometric, blood pressure and physical activity

determinations
Once a month, anthropometric measures were taken using
standardised procedures. Body weight was measured with a
calibrated Seca scale (to a precision of 100 g), and height

was measured at baseline with a stadiometer incorporated

into the scale. Duplicate waist circumference was taken to
the nearest 01 cm using an inelastic, flexible tape measure.
BMI was calculated as weight/height squared (kg/m2). To
avoid inter-examiner variability, one trained member of the
research team did all anthropometric determinations. International manual procedures were used(27).
Physical activity was assessed by a validated questionnaire
after weeks 4 and 12. This questionnaire was completed
during face-to-face interviews conducted by a trained dietitian.
Women were asked about their occupation, sleeping hours
and additional activities at work and during the rest of the
day. The physical activity questionnaire included representative values expressed as multiples of resting energy expenditure. Average daily exercise was calculated taking into
account the intensity level and time spent on each activity.
Activities were divided into five categories and expressed as
an activity factor (resting 1, very light 15, light 25,
moderate 5 and heavy 7)(28).
In addition, every 4 weeks, women completed a questionnaire about physical discomfort or health problems, medication use and changes in their normal routine.

Iron-fortified fruit juice on iron status

Blood sampling and biochemical assays

Volunteers attended the laboratory facilities at baseline, 4, 8,
12 and 16 weeks. Blood samples were collected by venepuncture after a 12 h fasting period. Serum and plasma were
obtained after centrifugation at 1000 g for 15 min and stored
at 2808C.
Total erythrocytes, haematocrit, mean corpuscular volume,
red blood cell distribution width (RDW) and Hb were determined following standard laboratory techniques using the
Symex NE 9100 automated haematology analyser (Symex,
Kobe, Japan). Serum Fe, serum ferritin and serum transferrin
were determined by the Modular Analytics Serum Work Area
analyser (Roche, Basel, Switzerland). Total iron-binding
capacity (TIBC) and transferrin saturation were calculated as
follows:

British Journal of Nutrition

TIBC mmol=l 251 transferrin g=l;

Transferrin saturation serum Fe mmol=l=TIBC 100:
Serum soluble transferrin receptor (sTfR) concentration was
determined using an ELISA technique (sTfR Human ELISA;
Biovendor, Heidelberg, Germany) and erythrocyte zinc
protoporphyrin (ZnPP) by haematofluorophotometry (haematofluorophotometer AVIV 206; Izasa, Barcelona, Spain). Values
of ZnPP mg/mg Hb were converted to mmol/mol haem of
erythrocyte protoporphyrin by using a factor of 2576.
All determinations were subjected to the ISO 9001-2000
requirements, except for the transferrin receptor; the intraassay CV of this determination was 35 % and the inter-assay
CV was 43 %.

Statistical analysis
Data are presented as means and standard deviations.
A normal distribution of variables was determined by
the Kolmogorov Smirnov test. Serum ferritin values were
log-transformed for statistical testing. A two-way repeatedmeasures ANOVA was applied. Because a significant group
time interaction was found for the main variables (ferritin,
transferrin and Hb, P# 0001), the repeated-measures ANOVA
and the Bonferroni post hoc test were used to study the time
effect within groups. Comparisons were also made between
the F group and the P group using ANOVA. A P value of
, 005 was considered significant. Data were analysed using
the SPSS statistical package for Windows (version 17.0; SPSS
Inc., Chicago, IL, USA).

Table 1. Energy and macronutrient intakes of iron-deficient women

consuming placebo and iron-fortified fruit juices during 16 weeks
(Mean values and standard deviations)
Baseline
Groups

A total of 122 volunteers completed the study (Fig. 1). Ages

of the volunteers were 245 (SD 51) and 242 (SD 46) years
for the P and F groups, respectively. Energy and nutrient
intakes at baseline and week 16 are shown in Table 1.
No significant differences between groups were found
in the baseline characteristics of subjects (Tables 2 and 3).
Compliance rate was confirmed to be very high (approximately 100 %).

Mean

Week 16

SD

Energy (kJ)
Placebo
9452
2179
Fortified
8807
2108
Protein (% energy)
Placebo
152
39
Fortified
146
24
Carbohydrate (% energy)
Placebo
412
73
Fortified
412
62
Lipid (% energy)
Placebo
399
78
Fortified
397
65
Fe (mg)
Placebo
151
47
Fortified
137*
59
Vitamin C (mg)
Placebo
1459
601
Fortified
1183
593

Mean
9826
9285

SD

2493
2292

Time effect (P )
NS
NS

130
136

24
23

, 0001
0004

450
446

73
66

0001
, 0001

373
364

63
68

NS
0001

129
304***

47
75

0001
, 0001

649
660

, 0001
, 0001

1998
1902

Mean values were significantly different from the placebo group at each point
(measured using one-sided tests): * P, 005, *** P, 0001.
Time-point differences were analysed by repeated-measures ANOVA.

Although energy intake did not show significant differences

between baseline and week 16 in both groups, an increase
between week 4 and baseline was observed to be significant
in the F group (9767 v. 8807 kJ/d for week 4 and baseline,
respectively, P,005). A significant decrease in the energy
percentage from proteins and an increase in that from carbohydrates were observed during the study in both groups, and
a decrease in the energy percentage from lipids was observed
only in the F group. The differences between the groups were
not significant (Table 1).
At baseline, Fe intake was slightly lower in the F group
compared with the P group, and due to the Fe-fortified juice
consumption, it was about twice that of the P group for the
duration of the intervention. In contrast, Fe intake of the P
group decreased with time, and it was significantly lower at
week 16 compared with baseline. Vitamin C intake increased
Table 2. Anthropometric values of iron-deficient women consuming
placebo and iron-fortified fruit juices during 16 weeks
(Mean values and standard deviations)
Baseline
Groups

Results

1655

Mean

Weight (kg)
Placebo
575
Fortified
596
2
BMI (kg/m )
Placebo
216
Fortified
218
Waist circumference (cm)
Placebo
687
Fortified
696

Week 16
SD

Mean

SD

Time effect (P )*

64
76

585
605

66
78

, 00001
, 00001

22
23

220
221

22
24

, 00001
, 00001

47
54

688
698

46
54

NS
NS

Mean values were not significant between the placebo and fortified groups at each
point.
* Time-point differences were analysed by repeated-measures ANOVA.

1656

R. Blanco-Rojo et al.

Table 3. Haematological and biochemical markers of iron-deficient women consuming placebo and iron-fortified fruit juices during 16 weeks
(Mean values and standard deviations)
Baseline
Groups

Mean

Week 4
SD

Mean

Week 8
SD

Mean

Week 12

Week 16

SD

Mean

SD

Mean

SD

Time effect (P )

029
032

451
456b

034
031

445
453a,b

030
030

NS
0046

British Journal of Nutrition

212

Total erythrocytes ( 10 /l)

Placebo
450
035
449
Fortified
451a,b
030
449a
Haematocrit (%)
Placebo
393
29
392
Fortified
390a
28
394a
Mean corpuscular volume (fl)
Placebo
874a
48
873a
Fortified
866a
51
879b,c
Red blood cell distribution width (%)
Placebo
127a
08
128a
Fortified
130a
12
137b**
Hb (g/l)
Placebo
133
9
133
Fortified
132a
9
133a
Zinc protoporphyrin (mmol/mol haem)
Placebo

793a,b
Fortified

765a
Serum Fe (mmol/l)
Placebo
140
71
164
Fortified
155
65
147
Serum ferritin (ng/ml)
Placebo
269
179
250
Fortified
254a
165
307b*
Serum transferrin (mg/l)
Placebo 3213a
676
3114b
Fortified 3102a
519
2799b**
Transferrin saturation (%)
Placebo
192
79
196
Fortified
197
107
237
Soluble transferrin receptor (mg/l)
Placebo
141
037

Fortified
148a
060

033
032

448
453a,b

28
25

389
394a

25
27

389
401b**

26
26

387
399a,b**

26
24

NS
0006

43
39

870a,b
871a,b

43
42

865b
881c*

41
36

870a,b
881c

42
40

0011
0001

09
23

129a
135b*

09
19

129a
130a

09
12

128a
127a

08
07

NS
0003

9
8

132
135b

269
286

799a,b
697b,c*

100
72

144
152

174
136

248
341b,c**

628
449
103
153

3208a,b
2875b,c***
178
222*
140
133b

8
9

132
136b*

8
9

294
261

761a
671b

325
241

78
67

150
155

69
58

177
148

231
373c,d***
3104a,b
2806b**

618
486
97
108
035
046

191
234*

162
165
638
438
90
99

132
136b**
861b
746a,c*
142
160
228
407d***
3232a,b
2929c**
178
230*
145
128b*

8
8

NS
, 00001

337
193

0049
, 00001

65
83

NS
NS

149
177

NS
, 00001

718
509
117
113
053
043

0001
, 00001
NS
NS
NS
0001

Mean values were significantly different from the placebo group at each time-point (measured using one-sided tests): * P, 005, ** P#001, *** P#0001.
Mean values within a row with unlike superscript letters were significantly different (repeated-measures ANOVA followed by Bonferroni test).

in both groups from baseline, due to juice composition,

without significant differences between the P and F groups
(Table 1).
The intervention induced increases in body weight and
BMI, within normal limits (Table 2). Waist circumference did
not change during the intervention. Physical activity was
unchanged during the study and did not show differences
between the groups; it was classified between very light and
light (activity factor, 168 v. 168 in the P and F groups).
Table 3 shows the results of haematological and biochemical markers. Increases in the values of total erythrocytes,
haematocrit, mean corpuscular volume, RDW, Hb, serum Fe,
serum ferritin and transferrin saturation show recovery from
Fe deficiency, while increases in serum transferrin, sTfR and
ZnPP denote deterioration, as detailed below.
Total erythrocytes did not show significant differences
between the groups. However, the F group showed significantly higher values at week 12 compared with week 4.
Similarly, haematocrit values were significantly higher at
week 12 compared with week 4 in this group, and higher haematocrit levels were shown at weeks 12 and 16 in the F group
than in the P group (P001). Mean corpuscular volume
decreased slightly at week 12 in the P group, while it

tended to increase in the F group; thus, differences between

the groups were significant at week 12 (P003). RDW was
higher in the F group than in the P group at weeks 4 and 8
(P, 001 and 005, respectively).
Hb concentrations did not vary in the P group during the
assay (Table 3), but they increased in the F group and were
significantly higher at week 8 compared with baseline, and
at weeks 12 and 16 compared with the P group (P , 005).
ZnPP increased in the P group and decreased in the F
group during the assay, and the differences between the
groups were significant at weeks 8 and 16 (P , 005).
No changes in serum Fe concentrations were observed due
to either group or time (Table 3). Ferritin concentrations, the
principal variable of the present study, significantly increased
from week 4 in the F group, and the values became about
80 % higher compared with the P group at the end of the
assay. In contrast, no changes were observed in the P
group. Serum transferrin fluctuated above 3000 mg/l in the P
group, while in the F group, it markedly decreased from
week 4 to the end of the assay, and the differences between
the groups were significant (P, 001). Accordingly, transferrin
saturation was significantly higher from week 8 in the F group
with respect to the P group (P, 005).

Iron-fortified fruit juice on iron status

sTfR concentrations significantly decreased in the F group

compared with baseline and the P group (significantly at
week 16).

British Journal of Nutrition

Discussion
The present study clearly shows that it is feasible to increase
Fe status in an at-risk population by daily consumption of a
microencapsulated iron pyrophosphate-fortified fruit juice
and that the effects are detected in a short period of time
(4 weeks). This consumption was compatible with the usual
diet, and the extra daily 18 mg of Fe provided in each
500 ml juice carton was 100 % of the RDA(3,4). This quantity
of Fe is within the range of supplemental minerals added in
European commercial foods (20 % of the RDA/100 ml).
The study fruit juice was fortified with micronised encapsulated iron pyrophosphate coated with lecithin. This form of Fe
is dispersible in aqueous solution and has been demonstrated
to be highly bioavailable. Its bioavailability is superior to that
of non-micronised iron pyrophosphate, which has higher
particle size, and to that of non-encapsulated iron pyrophosphate(9 11,29). Roe et al.(30) enriched micronised iron pyrophosphate and ferrous sulphate with different Fe stable
isotopes, and included each Fe form in apple juices to conduct
an absorption experiment using the technique of the incorporation of Fe isotopes to erythrocytes. They concluded that the
bioavailability of micronised iron pyrophosphate was higher
relative to ferrous sulphate, indicating that it could be a
useful fortifier for liquid food products.
Several studies using fortified food that supplied amounts of
Fe similar to the present study have been reported, generally
showing lower efficacy. The consumption of a wheat biscuit
enriched with 10 mg of Fe (as encapsulated sulphate) during
22 weeks increased iron ferritin but not Hb levels in young
women(31). A recent study in female soldiers who received
56 mg of Fe/d in the form of food bars compared with placebo
during 9 weeks has shown no changes in serum ferritin, transferrin saturation, % RDW and sTfR(32). The difference between
these two studies and ours could be explained by the presence of phytates and the absence of AA in their Fe-fortified
products, while the fruit juices of the present study contained
no phytate and were fortified with AA. When AA is present, as
in one study giving 16 mg of Fe-fortified breakfast cereal with
kiwifruit, increases in ferritin and Hb were observed(33), in
agreement with the present study.
In the present study, the food matrix used was an acidic
drink that contained AA with a molar AA:Fe ratio of 17:1.
Several reports have discussed the importance of this ratio;
however, there is no agreement on the optimal ratio to facilitate Fe absorption. A linear relationship between molar AA:Fe
ratio and Fe absorption has been suggested(34), but Cook &
Reddy(15) did not observe differences in Fe absorption
between diets with AA:Fe ratios of 12:1, 24:1 and 45:1.
This was attributed to the fact that Fe absorption was
measured from a complete diet and not from individual
meals. However, their study was criticised by Hunt(35) because
subjects were instructed to select or avoid foods according to
their AA content, which resulted in highly variable estimates of

1657

reported AA intakes. Shah et al.(36) compared Fe absorption in

children consuming meals that were accompanied by either
apple or orange juice, to which 5 mg of aqueous ferrous sulphate enriched with a stable isotope was added. They found
that Fe absorption was similar to the orange and apple
juices, even though the orange juice had much higher vitamin
C content.
The effect of the AA:Fe ratio on Fe bioavailability depends
on inhibitors present in the food(34), and several authors
have suggested a ratio of 2:1 for low-phytate content
foods(34,37), which is equivalent to the ratio of the juices
assayed in the present study.
It is also important to consider that volunteers in the present
study drank the 500 ml of juice separately at least 2 h after their
meals; thus, the provided Fe could not interact with inhibitors
present in the diet. Moreover, the low Fe status of these
women at the beginning of the assay constituted a factor to
favour Fe absorption(13). Therefore, the Fe supplement given
in the form of fruit juices was highly bioavailable and was efficacious to improve Fe status in Fe-deficient young women.
The short-term response observed in the present study was
unexpected, and the effect was clearly shown in the principal
variable, ferritin, a marker of Fe stores. To our knowledge, this
is the first study to demonstrate a significant increase in this
parameter after the consumption of an Fe-fortified functional
beverage during 1 month. Therefore, it was found that in a
very short period of time, and using a relatively low additional
intake of Fe, the biomarkers of Fe status improved in nonanaemic Fe-deficient women.
Moreover, it was observed that markers such as Hb or haematocrit, which were within normal ranges, increased after 3
months. The observed increase in RDW in the enriched
group could also indicate a recovery from Fe deficiency.
The RDW levels of the F group became higher in the first
months, indicating greater variation in width, and returned
to baseline levels at the end of the assay, which corresponds
to the average lifespan of erythrocytes(38). These results are
consistent with the observed increase in mean corpuscular
volume and haematocrit after the consumption of the Feenriched fruit juice.
Other indices, sTfR and ZnPP, which are not widely available as standardised clinical determinations, also indicate an
improvement of Fe status. Both reflect marrow Fe status for
erythropoiesis and recovery from Fe deficiency(39).
The present study was performed in a European population
at risk of Fe-deficiency anaemia (i.e. young menstruating
women with low Fe stores), and it is important to indicate
that intake of the juice portions was compatible with the
usual diet, and that the amount of Fe ingested daily via the
enriched juice was one-fifth of the usual therapeutic dose
(100 mg) of Fe. Under these experimental conditions, ferritin
levels increased by 80 %. It should be pointed out that once
the assay started, none of the volunteers receiving the Fe-fortified juice dropped out and that they did not complain about
digestive discomfort. Therefore, these results are outstanding
and suggest that Fe-fortified juices could be used as part of
the dietary treatment of anaemic patients in order to correct
anaemia more effectively.

British Journal of Nutrition

1658

R. Blanco-Rojo et al.

The slight increments of energy intake and body weight

indicate high compliance, as all juices contained 10 g/100 ml
of carbohydrates (non-added sugars). However, mean bodyweight gain was only 1 kg, and BMI remained within normal
limits during the whole intervention. This is a controversial
issue because high consumption of fruit juices should be limited in order to prevent obesity, especially in children and
young people. However, the present results show that the percentage of energy from protein and fat is reduced by the
inclusion of the daily juices in the diet, and the energy profile
tended to be more balanced. In this respect, dietary guidelines
recommend 45 60 % of energy from carbohydrates, and
concerning sugar intake, even added sugar, the European
Food Safety Authority has recently indicated that there were
insufficient data to set an upper limit(40). Moreover, Western
populations show a low consumption of fruits and vegetables(41,42), and the beneficial effects of these fruit juices
on other aspects of health should be explored. Nevertheless,
sugar content could be reduced in future products that
might be used as alternatives in subjects predisposed to anaemia and also obesity or diabetes.
A physiological adaptation appears to occur, since the
increase in body weight was observed at the beginning of
the assay (data not shown); thus, it is possible that energy
from foods other than the juice decreased to compensate for
the energy provided by the juices. It should be pointed out
that none of the Fe-deficient women participating in this intervention were obese, and that their physical activity was
unchanged during the study.
Fe-fortified juice consumption should be recommended to
individuals with predisposition to Fe-deficiency anaemia but
not to those at risk of excessive Fe intake who do not need
to increase their Fe supply, such as patients with Fe overload(43). Therefore, consumption of an Fe-fortified fruit juice
may be considered as a supplement to prevent Fe-deficient
anaemia in population risk groups, such as women of childbearing age, pregnant women or children. These groups
have a high acceptance of fruit juices, and the concept of functional foods also has high acceptance in developed countries.
The present study can have repercussions on public health as
prevention of one of the most widespread diseases will have
important economic impact, decreasing the need to use
public health services and pharmaceutical Fe supplements.
The cost benefits of the consumption of this Fe-fortified
food compared with those of the pharmaceutical therapy
and health care services should be studied. In fact, in the present study, consumption of the Fe-fortified juice led to a
recovery from depleted Fe stores (ferritin ,12 ng/ml), with
only one woman remaining depleted.
Further studies should be focused on the effectiveness of
this Fe-fortified beverage in subjects according to their genetic
background, in the line of previous studies(43,44). Consumer
aspects such as the optimal amount of fruit juice to be
drunk, according to concentration of the Fe salt to be
included, cost, palatable aspects and acceptability should
also be investigated. Finally, the effect of such Fe-enriched
juices on subjects with Fe-deficiency anaemia, obesity,

diabetes or CVD and their corresponding nutrigenomic

aspects should also be studied.

Acknowledgements
The present study was supported by Grupo Leche Pascual and
Projects reference AGL2006 09519/ALI and reference AGL2009
11437. R. B.-R. was supported by a JAE-predoc grant from
European Social Fund and CSIC, Spain, and L. T. was supported by a predoctorate grant from CONACyT, Mexico. The
authors are grateful to I. Wright for revising the English text
and to the volunteers who participated in the study. The
authors responsibilities were as follows: R. B.-R., A. M. P.-G.
and L. T. contributed to the study design, data collection, analysis and manuscript preparation; R. B.-R. and A. M. P.-G.
implemented the recruitment and follow-up of study participants; C. G.-V. contributed to the analytical determinations;
M. A. D. manufactured the beverages; and M. P. V. was the
principal investigator of the study, contributed to the study
design and manuscript preparation. M. A. D. works for
Grupo Leche Pascual. R. B.-R., A. M. P.-G., L. T., C. G.-V.
and M. P. V. had no conflict of interest.

References
1.

2.
3.

4.

5.

6.
7.

8.

9.

10.

World Health Organization (2008) Worldwide prevalence of

anaemia 1993 2005. In WHO Global Database on Anaemia
[EM de Benoist, I Egli and M Cogswell, editors]. Geneva:
World Health Organization.
Lynch SR (2005) The impact of iron fortification on nutritional anaemia. Best Pract Res Clin Haematol 18, 333 346.
Moreiras O, Carbajal A, Cabrera L, et al. (2009) Ingestas
recomendadas de energa y nutrientes para la poblacion
espanola (Recommended intakes of energy and nutrients
for the Spanish population). In Tablas de composicion
de alimentos (Food Composition Tables), 13th ed.,
pp. 227 230. Madrid: Piramide, Grupo Anaya, SA.
Institute of Medicine and Food and Nutrition Board (2001)
Dietary Reference Intakes for Vitamin A, Vitamin K, Arsenic,
Boron, Chromium, Copper, Iodine, Iron, Manganese, Molybdenum, Nickel, Silicon, Vanadium, and Zinc. Washington,
DC: National Academic Press.
Navas-Carretero S, Perez-Granados AM, Schoppen S, et al.
(2009) Iron status biomarkers in iron deficient women consuming oily fish versus red meat diet. J Physiol Biochem
65, 165 174.
Hurrell RF (2002) Fortification: overcoming technical and
practical barriers. J Nutr 132, 806S 812S.
WHO/FAO (2004) Guidelines on Food Fortification with
Micronutrients [L Allen, B de Benoist, O Dary and Richard
Hurrell, editors]. Geneva: World Health Organization.
Navas-Carretero S, Sarria B, Perez-Granados AM, et al. (2007)
A comparative study of iron bioavailability from cocoa supplemented with ferric pyrophosphate or ferrous fumarate in
rats. Ann Nutr Metab 51, 204207.
Navas-Carretero S, Perez-Granados AM, Sarria B, et al. (2007)
Iron bioavailability from pate enriched with encapsulated
ferric pyrophosphate or ferrous gluconate in rats. Food Sci
Tech Int 13, 159 163.
Fidler MC, Walczyk T, Davidsson L, et al. (2004) A micronised, dispersible ferric pyrophosphate with high relative
bioavailability in man. Br J Nutr 91, 107 112.

Iron-fortified fruit juice on iron status

11.

12.

13.
14.

15.

16.

British Journal of Nutrition

17.

18.

19.

20.

21.

22.

23.

24.

25.

26.

27.

28.

Moretti D, Zimmermann MB, Wegmuller R, et al. (2006) Iron

status and food matrix strongly affect the relative bioavailability of ferric pyrophosphate in humans. Am J Clin Nutr
83, 632 638.
Reddy MB, Hurrell RF & Cook JD (2000) Estimation of nonheme-iron bioavailability from meal composition. Am J Clin
Nutr 71, 937 943.
Hurrell R & Egli I (2010) Iron bioavailability and dietary
reference values. Am J Clin Nutr 91, 1461S 1467S.
Conrad ME & Schade SG (1968) Ascorbic acid chelates
in iron absorption: a role for hydrochloric acid and bile.
Gastroenterology 55, 35 45.
Cook JD & Reddy MB (2001) Effect of ascorbic acid intake on
nonheme-iron absorption from a complete diet. Am J Clin
Nutr 73, 93 98.
Nogueira de Almeida C, Crott G, Ricco R, et al. (2002) Control of iron-deficiency anaemia in Brazilian preschool children using iron-fortified orange juice. Nutr Res 23, 27 33.
Ash DM, Tatala SR, Frongillo EA Jr, et al. (2003) Randomized
efficacy trial of a micronutrient-fortified beverage in primary
school children in Tanzania. Am J Clin Nutr 77, 891898.
Abrams SA, Mushi A, Hilmers DC, et al. (2003) A multinutrient-fortified beverage enhances the nutritional status of children in Botswana. J Nutr 133, 1834 1840.
Makola D, Ash DM, Tatala SR, et al. (2003) A micronutrientfortified beverage prevents iron deficiency, reduces anemia
and improves the hemoglobin concentration of pregnant
Tanzanian women. J Nutr 133, 1339 1346.
Solon FS, Sarol JN Jr, Bernardo AB, et al. (2003) Effect of a
multiple-micronutrient-fortified fruit powder beverage on
the nutrition status, physical fitness, and cognitive performance of schoolchildren in the Philippines. Food Nutr Bull
24, S129 S140.
Hyder SM, Haseen F, Khan M, et al. (2007) A multiple-micronutrient-fortified beverage affects hemoglobin, iron, and
vitamin A status and growth in adolescent girls in rural
Bangladesh. J Nutr 137, 2147 2153.
Moher D, Schulz KF & Altman DG (2001) The CONSORT
statement: revised recommendations for improving the quality of reports of parallel-group randomized trials. Ann Intern
Med 134, 657 662.
Koulaouzidis A, Cottier R, Bhat S, et al. (2009) A ferritin level
.50 microg/L is frequently consistent with iron deficiency.
Eur J Intern Med 20, 168 170.
de Groot CP, van Staveren WA, Dirren H, et al. (1996) Summary and conclusions of the report on the second data
collection period and longitudinal analyses of the SENECA
Study. Eur J Clin Nutr 50, Suppl. 2, S123 S124.
Navas-Carretero S, Perez-Granados AM, Schoppen S, et al.
(2009) An oily fish diet increases insulin sensitivity compared
to a red meat diet in young iron-deficient women. Br J Nutr
102, 546 553.
Dietas y calculos de alimentacion (DIAL) (Diets and food
calculations), Alce Ingenieria, Spain. http://www.
alceingenieria.net/nutricion.htm (accessed December 2009).
National Health and Nutrition Examination Survey
(NHANES) (2007) Anthropometry procedures manual.
http//www.cdc.gov/nchs/data/nhanes/ (accesed September
2008).
National Research Council (1989) Recommended Dietary
Allowances, 10th ed. Washington, DC: National Academic
Press.

29.

30.

31.

32.

33.

34.

35.
36.

37.

38.

39.

40.

41.

42.

43.

44.

1659

Navas-Carretero S, Perez-Granados AM, Sarria B, et al. (2009)

Iron absorption from meat pate fortified with ferric pyrophosphate in iron-deficient women. Nutrition 25, 20 24.
Roe MA, Collings R, Hoogewerff J, et al. (2009) Relative bioavailability of micronized, dispersible ferric pyrophosphate
added to an apple juice drink. Eur J Nutr 48, 115 119.
Zimmermann MB, Winichagoon P, Gowachirapant S, et al.
(2005) Comparison of the efficacy of wheat-based snacks
fortified with ferrous sulfate, electrolytic iron, or hydrogenreduced elemental iron: randomized, double-blind, controlled trial in Thai women. Am J Clin Nutr 82, 1276 1282.
Karl JP, Lieberman HR, Cable SJ, et al. (2010) Randomized,
double-blind, placebo-controlled trial of an iron-fortified
food product in female soldiers during military training:
relations between iron status, serum hepcidin, and inflammation. Am J Clin Nutr 92, 93 100.
Beck K, Conlon CA, Kruger R, et al. (2011) Gold kiwifruit
consumed with an iron-fortified breakfast cereal meal
improves iron status in women with low iron stores: a
16-week randomised controlled trial. Br J Nutr 105,
101 109.
Lynch SR & Stoltzfus RJ (2003) Iron and ascorbic acid:
proposed fortification levels and recommended iron
compounds. J Nutr 133, 2978S 2984S.
Hunt JR (2001) How important is dietary iron bioavailability?
Am J Clin Nutr 73, 3 4.
Shah M, Griffin IJ, Lifschitz CH, et al. (2003) Effect of orange
and apple juices on iron absorption in children. Arch Pediatr
Adolesc Med 157, 1232 1236.
Hurrell RF, Lynch S, Bothwell T, et al. (2004) Enhancing the
absorption of fortification iron. A SUSTAIN Task Force
report. Int J Vitam Nutr Res 74, 387401.
Dugdale AE (2006) Predicting iron and folate deficiency
anaemias from standard blood testing: the mechanism and
implications for clinical medicine and public health in developing countries. Theor Biol Med Model 3, 34.
Labbe RF & Dewanji A (2004) Iron assessment tests: transferrin receptor vis-a`-vis zinc protoporphyrin. Clin Biochem 37,
165 174.
European Food Safety Authority (EFSA) Panel on Dietetic
Products, Nutrition and Allergies (2010) Scientific opinion
on dietary reference values for carbohydrates and dietary
fibre. EFSA J 8, 1462 (epublication 25 March 2010).
Casagrande SS, Wang Y, Anderson C, et al. (2007) Have
Americans increased their fruit and vegetable intake? The
trends between 1988 and 2002. Am J Prev Med 32, 257 263.
Lloyd-Jones DM, Hong Y, Labarthe D, et al. (2010) American
Heart Association Strategic Planning Task Force and Statistics
Committee. Defining and setting national goals for cardiovascular health promotion and disease reduction: the American Heart Associations strategic Impact Goal through 2020
and beyond. Circulation 121, 586 613.
Toxqui L, De Piero A, Courtois V, et al. (2010) Iron
deficiency and overload. Implications in oxidative stress
and cardiovascular health. Nutr Hosp 25, 350365.
Sarria B, Lopez-Parra AM, Navas-Carretero S, et al. (2007)
Hepcidin, transferrin (exon 7) and hemochromatosis genotyping suggests that haplotype block analysis is the best
strategy for predicting iron deficiency phenotype in
women. Nutr Res 27, 672678.