9

1998by Humana Press Inc.

All rights of any nature, whatsoever, reserved.
0163-4984/98/6201-2-0065 $10.25

Bioavailability, Absorption
Mechanism, and Toxicity
of Microencapsulated Iron (I) Sulfate
Studies in Mice
Jos~ R. BOCClO, *,1 MARCELA B. ZUB1LLAGA,1
RICARDOA. CARO, ~ ALEXISLYSIONEK,~ CARLOS A. GOTELU, 2
MARIANO J. GOTELLI,2 AND RICARDO WEILL3

'Radioisotope Laboratory, Physics Department, School

of Pharmacy and Biochemistry, University of Buenos Aires, Junin
956, 1113-Buenos Aires, Argentina; 2Center of Toxicological
Research, Buenos Aires, Argentina; and 3Center of Research
and Development, La Serenisima, Buenos Aires, Argentina
Received February 10, 1997; Accepted May 1, 1997

ABSTRACT
The iron compounds used for food fortification have to meet certain requisites related to their bioavailability, absorption mechanism,
and toxicity, since they will be consumed by a massive population
group. With these purposes, we evaluated a new product used for the
iron fortification of milk and lacteous derivatives, called SFE-171TM,
which is a ferrous sulfate, miclx~encapsulated with phospholipids. The
bioavailability studies were carried out using four groups of 30 female
mice each. In two groups, we studied the absorption of ferrous ascorbate and ferrous sulfate, both in water as reference standards, which
show absorptions of 13.1 + 4.9% and 13.2 _+ 4 . 3 ~ respectively. W~lth
the third group, we studied the absorption of ferrous sulfate in milk;
its value, 7.9 + 3 . 2 ~ is significantly lower than that of the remaining
groups, with a p < 0.01. The studies with SFE-171 TM in milk, were performed on the fourth group, with a result of 11.6 + 4 . 5 ~ demonstrating that its absorption does not differ significantly from that of the
reference standards. The absorption mechanism was determined b y
means of in vivo self-displacement studies of the ferrous ion and the
*Author to whom all correspondence and reprint requests should be addressed.
Biological Trace Element Research

65

Vol. 62, 1998

Boccio et al.

66

SFE-171TM, taking ferrous sulfate as the reference compound. For this

study, 210 female mice were used, and no significant difference
between the absorption mechanism of both products could be
observed. Toxicity studies of the new product with regard to ferrous
sulfate were carried out with two groups of 70 female mice each and
two groups of 70 male mice each. The lethal dose 50% LDs0 for SFE17U Mand for ferrous sulfate was 1200 and 680 mg/kg for female mice
and 1230 and 670 mg/kg for male mice, respectively, demonstrating
that the toxicity of the first product is substantially lower than that of
the reference standard. We conclude that the iron product under study
has a high bioavailability, an absorption mechanism equal to that of
nonhemic iron, and lower toxicity than ferrous sulfate.
Index Entries: Milk; fortification; iron; mice; toxicity; absorption;
bioavailability.

INTRODUCTION
Iron deficiency is the most frequent nutritional problem in the
world. It affects 24% of the population; its incidence in developed countries is approximately between 4 and 10%, whereas in developing countries, this figure rises to 40%. Studies carried out by the W H O
demonstrated that in developing countries, 47% of the w o m e n in reproductive age are at least slightly anemic (1-5).
This deficiency should be prevented by an adequate diet. If this is
not possible, food fortification has been demonstrated to be the most
appropriate option, since it can be applied to certain groups or to the
whole population. This procedure does not require that the intake has to
be remembered every day, as it is the case with pharmacological administrations, which, precisely owing to this fact, have been demonstrated to
be inefficient in some cases (6-8).
A study of the World Bank clearly demonstrates the economic,
health, and social benefits of food fortification, showing that in those
countries with a high incidence of this and other nutritional deficiencies,
such as those of iodine and vitamin A, losses attributable to incapacities
and deaths represent 5% of the Gross National Product (GNP). The solution of the problem by means of food fortification has a cost of 0.3% of
the GNP (6).
The c o m p o u n d s used for food fortification with iron generally supply nonhemic iron. It is important to take into account the properties of
the c o m p o u n d to be used as well as those of the nutritional vehicle, since
the nature of the food m a y interfere with the iron absorption, consequently decreasing its bioavailability. It should also be taken into account
that as a consequence of iron fortification, the sensorial properties of the
food as well as its cost should not change substantially, to be accessible
to the population, principally those groups of lower incomes (3,9-11).

Biological Trace Element Research

Vol. 62, 1998

Iron Bioavailability Studies

67

The iron bioavailability is determined by its solubility in the gastric juice, the presence of absorption activators or inhibitors in the food,
as well as the individual nutritional condition for this element. Highsolubility compounds, such as ferrous sulfate or ferrous gluconate, supply a highly bioavailable iron. However, if they are used in highly
humid foods, the liberated iron interacts with the food components,
provoking changes in its sensorial properties. Iron catalyzes oxidative
processes, producing therefore the oxidation of unsaturated fatty acids
and the food lipids rancidity. This catalytic oxidation affects other nutrients, such as vitamins and amino acids, decreasing significantly the
food nutritional value. In contrast, compounds, such as ferric
orthophosphate and ferric pyrophosphate, widely used to fortify solid
foods, have a low solubility, and therefore, even though they do not
produce significant changes in the sensorial properties or the nutritional value of the food, they have the disadvantage of a poor absorption and, consequently, a very low bioavailability, being for this reason
of little use from the nutritional point of view (3,11-13).
Taking into account the foregoing considerations, it may be concluded that the ideal method for the iron fortification of food should be
the utilization of a compound that supplies a highly bioavailable iron,
but at the same time does not alter either the sensorial properties or the
nutritional value of the fortified food and, most importantly, that follows
an absorption mechanism in agreement with the iron physiological
requirements of the organism. The compound should also resist the technological treatments to which the food is submitted.
It has been possible to obtain by a new technological process a compound that meets, in principle, these requisites. It is a ferrous sulfate
microencapsulated with phospholipids, called SFE-171TM**. When it is
added to fluid milk and derivatives, we obtain a food with a high nutritional value, fortified with iron and at a cost that does not differ substantially from that of the nonfortified product. In previous works, we studied
the stability and the metabolism of SFE-171TM fortified milk, as well as its
behavior when the product is taken together with different products, such
as coffee or tea (10,14,15). In the present work, we study the bioavailability and the absorption mechanism of microencapsulated iron, as well as
its toxicity, when fluid milk fortified with this new product is used.

MATERIALS AND METHODS

We used 470 female and 140 male Swiss strain mice, with weights
ranging from 30-36 g. They were maintained in a stainless-steel cages of
315 x 445 x 240 mm, with grated floor and collecting tray of stainless
**SFE-171TM: US patent # 08/264974.

Biological Trace Element Research

Vol. 62, 1998

68

Boccio et al.

steel, thus preventing the excrements from coming in contact with the
animals. They had free access to water and were nourished with a
normalized diet with the following composition: proteins: 20.0% w / w
minimum; fat: 2.0% w / w minimum; fibers: 11.0% w / w maximum;
ashes: 10.0% w / w maximum; calcium: 0.6% w / w ; phosphorus: 0.7%
w / w ; iron: 48 ppm; moisture: 13.0% w / w . The animals were maintained with cycles of 12 h with light and 12 h in darkness during all
of the experiment.
Previous to the administration of the products, the animals were
deprived of any solid food for 10 h, which was restored 1 h after the
product intake. The preparations were administered by means of a
syringe coupled to a plastic catheter, which allowed standardization of
the intake volume to 0.1 mL. For the labeling of the products, 59Fe (NEN,
DuPont, catalog no. NEZ 037) was used. In all the cases, pasteurized
whole cow milk was used, which has 3 g/100 mL of fat, 3.1 g/100 mL
of protein, 4.7 g/100 mL of carbohydrates, 0.12 g/100 mL of calcium,
0.09 g/100 mL of phosphorus, and which is fortified with 200 IU of vitamin A/100 mL and 40 IU of vitamin D/100mL.
For the bioavailability studies, four groups of 30 female mice each
were used. Four micrograms of iron and 3 ~tCi of 59Fe were administered per mouse for all the products. Each of the four groups received one
of the following preparations: ferrous sulfate in milk, SFE-171TM in milk,
ferrous sulfate in water, and ferrous ascorbate in water. These last two
groups were taken as reference standards. The standard of aqueous ferrous ascorbate was prepared with ferrous sulfate and ascorbic acid with
a molar ratio of Fe/ascorbic acid equal to 1. The used ferrous sulfate
solution was freshly prepared with bidistilled water under nitrogen
atmosphere. The samples of ferrous sulfate and SFE-171TM in milk were
prepared by adding each compound to the whole milk.
For the absorption mechanism studies carried out in fluid milk, we
used seven groups of 30 female mice each. The first group received 1 lxg
of 59Fea+-labeled ferrous sulfate (59Fe2+) stabilized with ascorbic acid
(molar ratio Fe/ascorbic acid = 1). The iron absorption obtained with this
product was considered as the maximal value under these experimental
conditions. Each of the other three groups received 1 lxg of 59Fe2+, but
together with 1, 10, and 100 txg of nonradioactive Fea+ also stabilized
with equimolar ascorbic acid. Each of the remaining three groups
received 1 ~tg of 59Fe2+ together with 1 ~tg, 10 ~tg, and 100 Ixg of Fe2+ as
SFE-171TM, respectively.
The absolute activity of each sample was determined by means of an
ionization chamber (RADX Model 255 Remote). The activity retained by
each mouse as a function of time was measured by a 7-spectrometer with
a 5 x 5 cm NaI(T1) well crystal in optimal electronic conditions. The measured activity was always compared to a 6~ standard in order to detect
any efficiency fluctuation; this parameter (2.4%) remained constant during all the experiments. The 59Fe radioactivity in the mice was measured
Biological Trace Element Research

VoL 62, 1998

Iron Bioauailability Studies

69

using a whole-body geometry, introducing each animal in a covered

lucite box, the size of which was similar to that of the animal and adequate to the detector geometry. In this way, detection errors attributable
to eventual movements of the animal could be minimized. The radioactivity determinations of a 59Fe standard in the same electronic and geometric conditions demonstrated the reliability of this technique. Taking
into account the measurement efficiency and the 59Fe specific activity, the
mass of absorbed iron was calculated, which allowed determination of
the percentage of absorbed iron with regard to the amount of administered iron.
The toxicity studies were carried out with two groups of 70 female
mice each and two groups of 70 male mice each, which received
increasing doses of iron from SFE-171 or ferrous sulfate, which was
used as the reference compound. For the determination of the lethal
dose 50% LD50, we used the method proposed by Litchfield and
Wilcoxon (16), which, as is known, is considered to be the reference
method for these studies.
The data are presented as mean _+SD. The results were evaluated by
a one-way analysis of variance (ANOVA). To test the differences among
the means, the Student-Newman-Keuls method was used. Only probability levels <0.01 were considered to be statistically significant (17).

RESULTS AND DISCUSSION

BioavaUability
The absorption and consequently the bioavailability of nonhemic
iron is strongly dependent on the nutritional matrix. The results of the
bioavailability studies can be observed in Table 1. Even though the mean
iron absorption for $1~-171TM is slightly smaller than that of the reference
standards, this difference is not statistically significant. However, in the
case of nonencapsulated ferrous sulfate in milk, a statistically si~lificant
decrease (p < 0.01) of the iron absorption with regard to SFE-171TM in
milk as well as to the reference standards is observed.
The principal reason for these results should be attributed to the
presence of proteins, calcium, and phosphates in milk. Casein is the
principal protein in milk, representing 80% of the whole protein,
whereas the remaining 20% are whey proteins (18,19). Casein is a
phosphoprotein that accelerates the oxidation of Fe 2+ to Fe 3+, which is
strongly bound to the protein, but these complexes are poorly
absorbed. The phosphate groups of casein are principally responsible
for this oxidative process, since its removal inhibits the process (20).
The hydrolysis of the milk proteins increases substantially the iron
absorption, but unfortunately this procedure implies important
changes of the sensorial properties of the food, making it unacceptable
for its consumption (18). Calcium and phosphates also inhibit iron
Biological Trace Element Research

Vol. 62, 1998

Boccio et al.

70
Table 1
Iron Absorption From Different Iron Sources
Iron source

Ferrous ascorbate Ferroussulfate Ferroussulfate SFE-171

in waterI

in water2

in milk

in milk

Absorption% + SD

13.1+4.9

13.2+4.3

7.9+3.2"

11.6+4.5

N~of aninals

30

30

30

30

1-Molar ratio Fe/ascorbic acid = 1.

2-Under nitrogen atmosphere.
*-Value statistically different from the other groups.

absorption. Ca 2+ competes for the Fe 2+ carrier protein at the level of the

intestinal membrane. The decreasing degree of iron absorption
depends on the calcium concentration, on the affinity of the intestinal
membrane proteins for either ion, as well as on the chemical form of
calcium. Iron and phosphates form poorly soluble compounds from
which iron is slightly absorbed (21,22). These results demonstrate that
in mice, this negative effect of the nutritional matrix interaction on iron
absorption from fluid milk is significantly lower when this new iron
source is used.

Absorption Mechanism
Iron homeostasis in the organism is not regulated by its elimination, but through its absorption, at the level of the intestinal mucous
cells that determine, by means of a specific transport mechanism, the
quantity of the iron that is to be absorbed, according to the physiologic
and metabolic requirements of the organism (12,23-25). Therefore, the
iron to be used for food fortification must have a high bioavailability,
and its absorption mechanism must obey the specific intestinal regulation mechanisms that protect the organism from an excessive iron
absorption.
The results of the experiments carried out in order to study the
absorption mechanism are shown in Fig. 1. It is evident that as the nonradioactive iron increases, the absorption of 59Fe2+ decreases with similar
patterns for ferrous sulfate or SFE-171TM. Therefore, the iron from either
compound competes for its specific acceptor site. This result demonstrates that the iron supplied by SFE-171TM follows the same absorption
mechanism as the ferrous ion. Consequently, the organism absorbs the
amount of iron that it needs, in agreement with the physiological mechanism of the nonheme iron.
Biological Trace Element Research

Vol. 62, 1998

Iron Bioavailability Studies

71

30-

"
0

25-

Q,

"-

20-

15-

0
m

u.

10-

U3

0
N

50

I
I

20

40

60

80

1 O0

l~g of Fe / m o u s e

Fig. 1. In vivo self-displacement studies. (A) Displacement of 59Fe2+by increasing doses of Fe2 from ferrous sulfate as a reference iron compound. (11)
Displacement of 59Fe2+ by increasing doses of Fe2+ from SFE-171 as the studied
iron source.

Toxicity
Taking into account that some food has a massive consumption, the toxicological studies of a new fortification product are essential. The LD50 of
SFE-171 TM and ferrous sulfate after an oral intake was determined, taking
the last compound as reference standard. As can be observed in Table 2, the
LD50 of SFE-171 TM in female as well as in male mice was significantly higher
than that of ferrous sulfate, demonstrating that the toxicity of this new fortification product is smaller than that of the reference standard.
Table 2
Acute Oral Toxicity in Mice. Values of LDs0 and its Confidence Limits for
Ferrous Sulfate and SFE-171. Ferrous Sulfate was Used as Reference Standard.
Iron Source

LDs0* Lower Limit*

Upper Limit" N~ of animals

Sex

Fe~ousSu~ate

680

572

808

70

Female

SFE-171

1200

956

1505

70

Female

Fe~ous Sulfate

670

565

816

70

Ma~

SFE-171

1230

967

1521

70

Male

*Expressed as mg of ferrous sulfate per kg of body weight.

Biological Trace Element Research

Vol. 62, 1998

72

Boccio et al.

These experimental results can be explained by taking into account the

fact that the microencapsulated iron is liberated slowly into the intestinal
lumen, avoiding an abrupt increase of its concentration in this place, as it is
in the case of ferrous sulfate. This property of the microencapsulated compounds to decrease the toxicity of the compounds or drugs that they contain is well known, and it is used to diminish the toxicity of certain drugs
as, for example, taxol and anphotericin B, used in the treatment of the neoplasic and fungal diseases, respectively. In this case, the possibility of using
this procedure allows the use of these drugs in therapeutic doses with a significant decrease in their toxicity and with lower side effects (26-28).

CONCLUSIONS
We conclude that this new iron source has adequate properties to be
used for iron fortification of milk and other lacteous products. The results
obtained here as well as in previous papers show that this product has a
good bioavailable iron, which is absorbed according to the mechanism
proposed for the nonhemic iron. Moreover, the product has a lower toxicity than ferrous sulfate, and when the milk fortified in this way is submitted to the usual technological treatments, no significant changes in
the iron absorption or the sensorial properties of the milk can be
observed (10,14,15). The possibility of fortifying milk and derivatives by
means of an iron with these properties allows a food rich in iron to be
obtained, which provides adequate amounts of this element, covering a
significant fraction of the daily requirements in order to prevent the consequences of the deficiency of this vital micronutrient.

REFERENCES
1. WHO Commission on Health and Environment, Report of the panel on food and
agriculture, World Health Organization, Geneva (1992).
2. First report on the world nutrition situation. A report compiled from information
available to the United Nations Agencies of the ACC/SNC. OPS/OMS. Washington,
DC (1987).
3. P. R. Dallman, Iron. Present Knowledge in Nutrition, 6th ed. International Life Sciences
Institute, ILSI, North America (1990).
4. D. Rose, D. SmaUwood, and J. Blaylock, Socioeconomic factors associated with the
iron intake of preschoolers in the United States, Nutr. Res. 15, 1297-1309 (1995).
5. S. H. Zlotkin, M. Ste-Marie, H. Kopelman, A. Jones, and J. Adam, The prevalence of
iron depletion and iron-deficiency anaemia in a randomly selected group of infants
from four Canadian cities, Nutr. Res. 16, 729-733 (1996).
6. Enriching lives, overcoming vitamin and mineral malnutrition in developing countries.
The International Bank for Reconstruction and Development. The World Bank. (1994).
7. T. Walter, E. Hertrampf, F. Pizarro, M. Olivares, S. Llaguno, A. Letelier, V. Vega, and
A. Stekel, Effect of bovine-hemoglobin-fortified cookies on iron status of school children: a nationwide program in Chile, Am. J. Clin. Nutr. 57, 190-194 (1993).
8. W. Schultink, M. Ree, P. Matulessi, and R. Gross, Low compliance with an ironsupplementation program: a study among pregnant women in Jakarta, Indonesia,
Am. J. Clin. Nutr. 57, 135-139 (1993).

Biological Trace Element Research

Vol. 62, 1998

Iron Bioavailability Studies

73

9. Meat and meat products in human nutrition in developing countries, Food and Agriculture Organization of the United Nations, Rome (1992).
10. J. Boccio, M. Zubillaga, R. Caro, C. Gotelli, M. Gotelli, and R. Weill, New procedure
to fortify fluid milk and derivatives with iron. Comparative study in mice, J. Nutr.
Sci. Vitaminol. 41, 619-626 (1995).
11. H. Brise and L. Hallberg, Iron absorption studies, Acta Medica Scandinavica Suppl.
376, 23--45 (1962).
12. M. W. Gavin, D. M. McCarthy, and J. P. Garry, Evidence that iron stores regulate iron
absorption--A set point theory, Am. J. Clin. Nutr. 59, 1376-1380 (1994).
13. J.E. Oliveira, M. L. S. Freitas, J. E Ferreira, A. L. Gon~alves, and J. S. Marchini, Iron from
complex salts and its bioavailability to rats, Int. J. Vit. Nutr. Res. 65, 272-275 (1995).
14. M. Zubillaga, R. Caro, J. Boccio, C. Gotelli, M. Gotelli, and R. Weill, New procedure
to fortify fluid milk with iron: Metabolic and biochemical study in rats, Nutr. Res. 16,
131-137 (1996).
15. J. Boccio, M. Zubillaga, R. Caro, C. Gotelli, M. Gotelli, and R. Weill, Bioavailability
and stability of microencapsulated ferrous sulfate in fluid milk, J. Nutr. Sci. Vitaminol.
42, 233-239 (1996).
16. J. T. Litchfield and E Wilcoxson, A simplified method of evaluating dose-effect experiments, J. Pharmacol. Exp. Ther. 96, 99-113 (1949).
17. R. R. Sokal and F. J. Rohlf, Biometry, WH Freeman, San Francisco, CA (1969).
18. R. F. Hurrell, S. R. Lynch, P. T. Trinidad, S. A. Dassenko, and J. Cook, Iron absorption
in humans as influenced by bovine milk proteins, Am. J. Clin. Nutr. 49, 546-552 (1989).
19. H. D. Beltiz and W. Grosch, Food Chemistry, Springer Verlag, Berlin (1987).
20. T. Emery, Iron oxidation by casein. Biochem. Biophys. Ress. Commun. 182, 1047-1052 (1992).
21. L. Hallberg, L. Rossander, M. Brune, and A. Gleerup, Calcium and iron absorption:
mechanism of action and nutritional importance, Eur. J. Clin. Nutr. 46, 317-327 (1992).
22. P. L. Minotti, S. M. Buchonski, and D. D. Miller, Effects of calcium supplementation,
calcium source and lactose on iron absorption in the rat, Nutr. Res. 13, 1173-1181 (1993).
23. O. Han, M. L. Failla, A. D. Hill, E. R. Morris, and J. C. Smith, Reduction of Fe (III) is
required for uptake of nonheme iron by caco-2 cells, J. Nutr. 125, 1291-1299 (1995).
24. S. Castro del Pozo, Metabolismo del hierro normal y patol6gico, Segunda edici6n, Masson, Barcelona, Espa~a (1995).
25. Y. Ikeda-Moore, H. Orimo, S. Hisayasu, and Y. Yoshino, Characteristics of iron binding to solubilized brush border membrane of the rat intestine, J. Nutr. Sci. Vitaminol.
41, 419-432 (1995).
26. R. M. Straubinger, A. Sharma, M. Murray, and E Mayhew, Novel taxol for formulations: Taxol-containing liposomes, Monogr. Natl. Cancer Inst. 15, 69-78 (1993).
27. A. Gabizon, Tailoiring liposomes for cancer drug delivery: from the bench to the
clinic, Ann. Biol. Clin. Paris 51, 811--813 (1993).
28. H. J. Schmitt, New methods for delivery of Anphotericin B, Clin. Infect. Dis. 17,
501-506 (1993).

Biological Trace Element Research

Vol. 62, 1998

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.