Long-chain polyunsaturated fatty acid supplementation in infant formulas
does not influence calcium and magnesium bioavailability in rats
Running tittle:
bioavailability
LCPUFA-supplemented
infant
formula,
Ca
and
Mg
Dr. M Pilar Vaquero, Dr. Beatriz Sarriá
Departamento de Metabolismo y Nutrición, Instituto del Frío, CSIC,
Madrid, Spain
Acknowledgment for research support: this work was supported by the
Spanish Commission of Science and Technology (project ALI 96-0465). The
authors thank Dr. Ana Maria Pérez-Granados for scientific advice and Marcel
Veldhuizen for technical assistance.
Author for correspondence:
Dr. M. Pilar Vaquero
Instituto del Frío, CSIC
C/José Antonio Nováis 10, 28040 Madrid, Spain.
Phone number: 00 34 91 549 00 38 ext. 295
Fax number: 00 34 91 549 36 27
Email: mpvaquero@if.csic.es
Key words: long-chain polyunsaturated fatty acids; infant formula; calcium;
magnesium; mineral bioavailability
ABSTRACT
The influence of infant formula supplementation with long-chainpolyunsaturated fatty acids (LCPUFA) on calcium and magnesium
bioavailability was assessed in rats. Two test diets containing the
unsupplemented (F) or supplemented (SF) infant formula as the fat source and
a control diet (C) were administered to weaning rats for 28 days. Food intake
and body weight were monitored, during the last week faeces and urine were
collected to calculate apparent calcium and magnesium absorption and
retention. Food intake and body weight evolution showed no significant
differences between F and SF but were lower in both groups compared to C.
Calcium and magnesium intake did not differ between F and SF, but both
parameters were significantly lower in SF compared to C. Calcium absorption
efficiency in F and SF was significantly higher than in C. Formula-fed groups
showed higher urinary calcium excretion and thus, no differences were
observed in calcium retention. Magnesium absorption was significantly higher in
F compared to SF and C but there were no significant differences between test
groups for the absorption efficiency. Magnesium apparent retention was similar
in the three groups. The consumption of a diet containing an infant formula
supplemented with LCPUFA compared to the unsupplemented formula does
not affect calcium and magnesium bioavailability in rats.
1
INTRODUCTION
Human milk is the optimal source of nutrients for healthy young infants. As an
alternative to breastfeeding, cow’s milk based infant formulas are consumed.
Modifications to infant formulas are continually being made as the components
of human milk are characterised and as the nutrient needs of diverse groups of
infants are identified. Regarding the fat content, although there is evidence that
infants are able to synthesize arachidonic acid (AA, 20:4, n-6) and
docosahexanoic acid (DHA, 22:6, n-3) from linoleic (18:2, n-6) acid and linolenic
acid (18:3, n-3), respectively, the increased requirements for the longer chain
metabolites during development has raised concern that infants may also
require preformed long-chain polyunsaturated fatty acids (LCPUFA). Therefore,
formulas with LCPUFA in amounts similar to those in human milk have recently
become available1. Beneficial effects of supplementing infant formulas with
LCPUFA, of n-3 and n-6 series, on visual and neural development in rodent
models2 and preterm infants have been reported3,4 whereas in term infants
neutral or positive outcomes have been described 5,6. Controversy exists
concerning the critical period during which the dietary supply of LCPUFA may
influence the maturation of cortical function in term infants. The potential longterm benefits of LCPUFA supplementation are still being explored, biochemical
data indicate that breast-fed infants accumulate DHA in the brain until >12
months of age and at a greater rate than do infants fed formula without DHA7.
Das8 recently suggested supplementation of LCPUFA from the second trimester
of pregnancy to the age of 5 years in order to prevent coronary heart disease in
adult life.
The repercussions of LCPUFA supplementation on mineral bioavailability
have been studied to a limited extent. Our research group evaluated the effects
of consuming an infant formula supplemented with LCPUFA on iron absorption
using a rat model, this parameter was found to be unaffected, haemoglobin did
not change but the erythrocytic concentrations of iron9, as well as copper and
zinc10 increased, probably due to increased AA and DHA in erythrocytic
membranes and changes in membrane permeability. Few studies have
investigated the potential for polyunsaturated fatty acids to influence calcium
and bone metabolism during early growth stages. Calcium absorption is
enhanced by linoleic acid compared to oleic acid11, whereas magnesium
absorption is not affected, although thermally treated fats did alter magnesium
bioavailability12. Research in adult rodents fed diets containing varied amounts
of fish oils indicates that a total n-6/n-3 ratio of 3:1 or 1:1 results in higher
amounts of calcium in bone compared with the control, but the ratio 1:3 had no
effect. Feeding the diet with an n-6/n-3 ratio of 3:1 also resulted in significantly
greater absorption of dietary calcium compared with the other diets 13. However,
a higher excess in n-3 fatty acids intake (n-6/n-3 ratio of 1:5) induced growth
retardation and reduced calcium absorption and retention efficiencies in young
rats14. Martinez et al15 assessed in very low weight preterm infants the influence
of LCPUFA supplementation in infant formulas, versus a non-supplemented
formula, on mineral bioavailability and obtained comparable intake and net
retention of calcium and magnesium through a three-day balance. However,
data on term infants are lacking although first age formulas containing LCPUFA
are available in the market.
2
Due to the facts that LCPUFA supplemented infant formulas are widely
consumed and to the difficulties in performing studies in term infants, we
investigated the effects of LCPUFA supplementation of an infant formula on
calcium and magnesium bioavailability in weanling rats. Suckling16-18 and
weanling rat models19 have been extensively used and are well validated to
carry out mineral bioavailability experiments.
METHODS AND MATERIALS
Diets
Two term commercially available infant formulas were obtained from the
same manufacturer (Milupa, Spain). Both were provided in powder and the only
difference was the fat composition. The standard infant formula was devoid of
LCPUFA while the infant formula supplemented with LCPUFA contained egglipid-derived LCPUFA. Details about the nutrient composition of these infant
formulas have been published20.
Three isoenergetic diets were prepared, two contained the
unsupplemented (F) or supplemented (SF) infant formula exclusively as the fat
source (150 g/kg) and the third was a control diet (C) based on the American
Institute of Nutrition recommendations (AIN-93) except for the fat level, which
was also 150g/kg of corn oil. In order to elaborate F and SF diets, the
appropriate amounts of all dietary components were combined to make up with
the control diet. The resulting mixture was divided into two parts and the test
infant formulas were added to make F and SF diets. The control diet (modified
AIN diet, Dyets Inc., Bethlehem, PA, USA) was purchased. The composition of
the experimental diets has been previously detailed 9 and the fatty acid content
is shown in table 1. The three diets were kept at 4ºC until use. The calcium
content of the diets (g/kg) was (mean  standard error of 5 determinations per
diet) 4.790.04, 4.730.05 and 4.590.02 for F, SF and the control,
respectively. Magnesium contents were 0.470.02, 0.440.03 and 0.460.03 for
F, SF, and the control, respectively.
The diets were given to weanling rats for 28 d. During this period, food
intake and body weight were monitored and in the last week faeces and urine
were collected to calculate apparent calcium and magnesium absorption and
retention.
Biological assay
Thirty-four weanling Wistar rats, initial body weight 40.30.1g (mean 
standard error), were housed individually in metabolic cages in an
environmentally controlled room maintained at 20-22ºC, with a 12h light-dark
cycle and 55-70% humidity. F, SF and the control group contained 10, 12 and
12 animals, respectively. An equal number of males and females in each group
were randomly assigned to the dietary treatments. Animals had free access to
food and demineralised water (Milli-Q plus, Ultrapure Water System, Millipore
Corporation, Bedford, U.S.A.) for 28 days. Body weight and food intake were
monitored and faeces and urine were collected and pooled separately from day
21 until day 27. Faeces were dried, weighed and homogenised. Urine was
collected in 0.5% v/v HCl solution, filtered (nº41, ashless Whatman Filter
Papers, Whatman Ltd., England) and diluted.
3
On day 28, after an overnight fast, animals were anesthetised using
sodium pentobarbital (Abbott Laboratories, S.A., Madrid, Spain) and blood was
drawn from the carotid artery into acid-washed (HNO3 10 mol/L) plastic vials
and allowed to clot. Throughout the study, rats were handled following the
European Science Foundation Statement on the Use of Animals in Research
(http://www.hsus.org/ace/15049).
Analytical Techniques
Diets and faeces were dry-ashed in a muffle furnace at 450ºC. Ashes
were dissolved in a HCl/HNO3/H2O solution (1:1:2) (Suprapur, E.Merck,
Darmstadt, Germany).
Calcium and magnesium analysis in the diets, faeces, urine and serum
was performed in duplicate by flame atomic absorption spectrometry (PerkinElmer 1100B, Norwalk, CT., U.S.A.). A stock standard solution of calcium (1 g l1) and magnesium (1 g l-1) was prepared from Titrisol (E. Merck). Lanthanum
chloride (Merck) was added to the calcium samples and standards (final
concentration: (5 g l-1 lanthanum).
A pool of faeces was used as an internal control for both mineral
determinations. The interassay relative standard deviation was 2.26% and
1.13% for calcium and magnesium, respectively. Certified reference material
(milk powder, CRM 63, Community Bureau of Reference, Brussels) was used to
assess accuracy, values of 12.8±0.4 and 1.11±0.03mg g-1 (mean ± standard
deviation of five determinations) were obtained for calcium and magnesium,
respectively (certified value: 12.6±0.3 mg g-1 and 1,12±0.03 mg g-1 for calcium
and magnesium respectively). Distilled-deionised water (Milli Q plus, Millipore)
was used for the preparation of dilutions and calcium and magnesium
standards.
Indices
The following indices were calculated from data on calcium and
magnesium, intake and faecal and urinary excretion:
Apparent absorption (A)= Intake (I) - Faecal excretion
%A/I = Apparent absorption/ Intake x 100
Apparent retention (R)= Apparent absorption – Urinary excretion
%R/A = Apparent retention/Apparent absorption x 100
%R/I= Apparent retention/Intake x 100
Statistical Analysis
Food intake and body weight data were analysed by analysis of variance
(ANOVA) of repeated measures. The rest of data were studied by means of
one-way ANOVA. Two sample comparisons were made using the Bonferroni
test. Data were processed with the SPSS Statistical Package. Significance of
the results was established at p<0.05.
RESULTS
Food intake and body weight evolution
Throughout the assay, rats fed the C diet showed a higher food intake
compared to those administered the diets containing F and SF (table 3; ANOVA
4
repeated measures p=0.002). The differences between C and both F and SF
were very significant in the periods 3-7 (p=0.002) and 22-27 days (p=0.001) but
not in the period 7-14 days. Animals fed SF showed significantly lower food
intake compared to C in the stage 14-22 days (p=0.014). The differences
between F and SF were not significant, although SF exhibited the lowest values
of food intake.
Accordingly, there were significant differences in the body weight evolution
(table 3; ANOVA repeated measures p=0.002). This parameter was significantly
lower in SF than C (p=0.001). On days 3 and 7, body weight was significantly
lower in both F and SF than C (p=0.007 and p<0.001, respectively), the
differences were not significant on day 14. However, on days 22 and 27 SF rats
presented significantly lower body weights than C animals (p=0.015 and
p=0.004, respectively).
Calcium
Calcium concentration in serum was similar in the three groups (mg/dl)
11.33±0.11, 10.24±0.97 and 10.83±0.66 (mean ± standard error mean) for F,
SF and C, respectively.
Calcium intake was significantly lower in SF compared to C (p<0.001). In
the test groups, calcium faecal excretion was 3-4 times lower (p<0.001) and
therefore the apparent absorption was elevated, although the differences were
only significant between F and C. However, urinary calcium was also 3-4 times
higher in SF and F compared to C (p<0.001), therefore the differences between
groups in calcium apparent retention were not significant. The ratios %A/I and
%R/I were significantly higher in both SF and F (p<0.001 and p=0.001,
respectively) respect to C, while %R/A was significantly lower in F and SF
(p<0.001) than in C.
Magnesium
Magnesium concentration in serum was similar in the three groups (mg/dl)
2.15±0.14, 1.9±0.24 and 2.22±0.15 (mean ± standard error mean) for F, SF and
C, respectively.
Magnesium intake was significantly lower in SF compared to C (p<0.001)
but there were not significant differences between F and SF. The experimental
groups eliminated significantly less of this mineral in faeces (p<0.001) and F
showed significantly higher magnesium absorption than SF and C (p=0.016).
However, in F the urinary excretion of magnesium was slightly higher compared
to SF and C, therefore the apparent retention was similar to the other groups
and no significant differences were obtained for this parameter. The digestive
ratio, %A/I, was significantly higher in the test groups compared to the control
(p<0.001). The percentages %R/A and %R/I were within the same range in the
three groups, not showing any statistical differences.
DISCUSSION
The standard infant formula used in the present study (F) contains a mixture
of polyunsaturated and saturated fatty acids, which is also present, although in
slightly different quantities, in the supplemented infant formula, together with
LCPUFA from egg-lipid, which is one of the richest and most commonly used
5
source of these fatty acids21. In contrast, diet C contained approximately 5 times
more linoleic acid than F and SF, very low amounts of saturated fatty acids,
particularly myristic and palmitic acids, and was devoid of LCPUFA.
Considering that diet C is designed for rats, it is not surprising that food intake
and body weight were the highest in the group that consumed it. [Similar values
were obtained for these parameters in F and SF, although lower in SF, through
the experimental period].
Supplementation with LCPUFA did not affect food intake and growth
significantly, although lower values were observed in FS which tended to ingest
less calcium and magnesium. In other animal models, such as mouse pups,
reduced growth was observed after dietary supplementation with up to 9% DHA
(wt/wt), regardless of dietary AA or total n-6:n-3 ratios22. On the contrary,
feeding piglets infant formula supplemented with DHA and AA (0.1% and 0.5%
wt/wt total fat, respectively) did not compromise growth but furthermore, greater
whole body weight, bone mineral content and density were observed compared
to feeding an isoenergetic standard formula23. The suggested mechanism for
the stimulated growth and bone mass is that a greater amount of precursor, AA,
is available for synthesis of prostaglandin E2, a potent stimulator of bone
formation, in response to the calciotropic hormones during growth 24.
Results in human infants are very limited and not conclusive, although no
influence of consuming infant formulas with LCPUFA supplements on standard
growth indexes were reported25,26,27, specific negative associations were
observed in few studies. An infant formula supplemented with DHA and
eicosapentaenoic acid (EPA, C20:5) (both n:3) reduced concentrations of AA in
plasma phospholipids that were significantly associated with reduced weight
and length growth in preterm babies26. In a different study using a supplement
with DHA and AA, infant growth was not affected, although a small association
between DHA status at 16 weeks of age and weight at 1 and 2 years was
observed27. As indicated by Innis28, inconsistencies in the findings of n-6 and n3 requirements of infants may be attributed to the different necessities of
preterm/term infants, differences in the types of oils used to provide DHA and
AA, differences in the AA:DHA ratios, and even to the home environment.
Expert committees and advisory panels recommend that the ratio linoleic acid: α
linolenic acid not be more than 16:1 nor less than 6:1 which points to the
possible adverse effects of excess n-3 fatty acids (EPA, AA and DHA) on
growth29. According to these recommendations, the supplemented infant
formula used in the present study included fatty acids in the adequate amounts
and ratios, and accordingly food intake and body weight were not affected
compared to the unsupplemented formula.
It is well known that a high polyunsaturated/saturated ratio of dietary fat
favours calcium and magnesium absorption30. Pérez-Granados et al11 observed
that linoleic acid favours calcium absorption more than oleic acid. In contrast,
long chain saturated fatty acids and erucic acid (C22:1) reduced calcium
bioavailability31, the former forming more insoluble soaps32. Moreover, the
addition of oleic acid to stearic acid improves the absorption of this saturated
fatty acid33, the mechanism involves increasing the solubility of stearic acid in
the bile salt-monoglyceride micelle. Concerning palmitic acid, if this fatty acid is
predominantly esterified in sn-2 position (beta-position), higher fat digestibility
and enhanced calcium absorption has been shown 34. Lopez-Lopez et al35
evaluated the influence of the position of the long chain fatty acid in triglycerides
6
on the fatty acid, calcium and magnesium content of term newborn faeces and
found that those infants that consumed human milk and infant formula with
palmitic acid predominantly esterified in sn-2 position (beta-position) showed
reduced contents of calcium and total fatty acids in faeces, without yielding any
differences on magnesium.
In the present experiment, although the differences between the two infant
formulas were small, SF contained more stearic acid, less palmitic acid, oleic
and linoleic acids but more LCPUFA. To these differences the slightly lower
bioavailability of calcium and magnesium may be attributed.
Magnesium is affected by dietary fat to a lower extent than calcium12. It is
known that, long chain saturated fatty acids also form insoluble soaps with this
mineral33, and that magnesium soaps produced with oleic and linoleic fatty
acids are 10 to 20 times more soluble than the corresponding ones formed by
saturated fatty acids36. The fact that magnesium absorption was higher in F
compared to SF may be associated to the higher food intake and to the
differences in fatty acid composition between the two infant formulas: F
contained lower levels of long chain fatty acids, either saturated or unsaturated,
and higher levels of palmitic acid.
The main differences obtained in the present study are between the C group
and the infant formula groups. The reference group showed 3 to 4 times higher
faecal excretion of the two studied minerals inducing lower absorption in C
particularly respect to F. This may be explained because infant formulas contain
16 times more palmitic acid, slightly more oleic acid but only 5 times less linoleic
acid. In S and SF although more calcium and magnesium was absorbed, at
later stages higher proportion of these minerals was excreted by urine, possibly
in response to animal’s physiological needs. Consequently, the calculated
retention of both minerals were similar in the three groups.
CONCLUSION
Consumption of an term infant formula supplemented with LCPUFA compared
to the unsupplemented formula does not interfere with calcium and magnesium
bioavailability in rats. Other beneficial metabolic effects should be overlooked
when recommending this formula for term infants.
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9
Table 1. Fatty acid composition of the experimental diets (g per 100g fatty acids).
Fa
SFa
Controlb
Butyric acid, C4:0
0.04
1.10
-
Caproic acid, C6:0
0.09
0.65
-
Caprylic acid, C8:0
0.90
1.30
-
Capric acid, C10:0
0.78
1.55
-
Lauric acid, C12:0
6.22
5.6
-
Myristic acid, C14:0
3.27
6.05
0.57
Palmitic acid, C16:0
32.2
25.35
1.8
Palmitoleic acid, C16:1
0.16
1.25
0.29
Stearic acid, C18:0
4.29
5.6
2.10
Oleic acid, C18:1
34.5
33.90
26.5
Linoleic acid, C18:2,n-6
13.5
11.35
58.0
γ-linolenic acid, C18:3 n-6
-
0.35
-
α-linolenic acid, C18:3, n-3
0.55
1.30
0.90
Arachidic acid, C20:0
0.31
0.20
0.29
Eicoseic acid, C20:1
0.33
0.45
0.19
Eicosadienoic acid, C20:2 n-6
-
0.07
-
Homo γ- linolenic, C20:3 n-6
-
0.09
-
Arachidonic acid, C20:4 n-6
-
0.35
-
Behenic acid, C22:0
-
0.10
-
Erucic acid, C22:1
-
0.05
-
Docotetranoic acid, C22:4 n-6
-
0.04
-
Docosapentaenoic acid, C22:5 n-3
-
0.04
-
Docosahexaenoic acid, C22:6 n-3
-
0.20
-
Lignoceric acid, C24:0
-
0.10
-
Polyunsaturated:saturated
0.35
0.37
4.25
n-6:n-3
24.5
8.0
64.4
a
from Milupa, S.A., Spain
b
from Dyets Inc., Bethlehem, PA, USA
10
Table 2. Food intake and body weight evolution in rats fed diets containing a non-supplemented infant formula (F), a long-chain
polyunsaturated supplemented infant formula (SF) and the control diet (C)
Food intake (g/day)*
Body weight evolution (g)**
Group
Days 3-7
Days 7-14
Days 14-22
Days 22-27
Day 3
Day 7
Day 14
Day 22
Day 27
F
8.12  0.28a
10.65  1.18
11.88  0.32
13.89  0.57 a
47.51.1 a
66.61.3 a
104.43.0
147.14.6
162.76.4
SF
7.71  0.32a
9.43  0.28
11.05  0.35a
12.93  0.38 a
47.70.5 a
63.91.2a
98.32.4
133.53.9 a
148.24.8a
C
9.50  0.35
10.84  0.34
13.04  0.63
15.86  0.60
53.21.9
71.41.0
106.92.3
152.95.3
180.17.4
ANOVA
p = 0.002
NS
p = 0.014
p=0.001
p=0.007
p<0.001
N.S.
p=0.015
p=0.004
Values represent means  standard error deviation (n=12, except for F n= 10)
a
denotes significant differences compared to the control group at p<0.05
ANOVA repeated measures: *p=0.002; **p=0.002
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Tabla 3. Intake (I), faecal and urinary excretion, absorption(A), retention (R) and the absorption and retention efficiencies of calcium
in weanling rats fed a diet containing a non-supplemented infant formula (F), a long-chain polyunsaturated supplemented infant
formula (SF) and the control diet (C).
Group
Intake
Faecal
excretion
Urinary
excretion
Absorption
Retention
A/I
(mg/day)
R/A
R/I
%
F
66.51±2.74
8.55±1.00a
4.18±0.74a
57.96±2.36 a
53.78±2.11
87.2±1.3a
92.9±1.1a
81.1±1.9a
SF
61.10±1.79a
5.09±0.91a
3.08±0.31a
56.02±2.27
52.94±2.25
91.4±1.7a
94.4±0.6a
86.4±1.8a
C
72.79±2.74
23.34±1.87
1.06±0.19
49.45±1.72
48.38±1.76
68.3±1.7
97.8±0.4
68.7±1.6
ANOVA
p<0.001
p<0.001
p<0.001
p=0.020
N.S.
p<0.001
p<0.001
p=0.001
Values represent means ± SED (n=12, except for F n=10).
a denotes
significant difference from the control group at p<0.05.
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Table 4. Intake (I), faecal and urinary excretion, absorption (A), retention (R) and the absorption and retention efficiencies of
magnesium in weanling rats fed a diet containing a non-supplemented infant formula (F), a long-chain polyunsaturated
supplemented infant formula (SF) and the control diet (C).
Group
Intake
Faecal
excretion
Urinary
excretion
Absorption
Retention
A/I
(mg/day)
R/A
R/I
%
F
6.52±0.27
0.87±0.07 a
2.40±0.34
5.66±0.28 ab
3.26±0.31
86.5±1.1 a
57.9±5.5
50.3±4.9
SF
5.71±0.17 a
0.83±0.06 a
1.68±0.23
4.89±0.17
3.21±0.20
85.4±1.0 a
66.3±4.4
56.5±3.6
C
7.29±0.27
2.45±0.21
1.51±0.22
4.84±0.18
3.33±0.25
66.8±2.07
68.8±4.4
45.7±2.9
ANOVA
p<0.001
p<0.001
N.S.
p=0.016
N.S.
p<0.001
N.S.
N.S.
Values represent means ± SED (n=12, except for F n=10).
a and b denote significant difference from the control group and the SF group, respectively, at p<0.05
13