Heat processing in infant formulas induces changes in copper tissue levels
in suckling and weanling rats
Running head: Processing infant formulas and copper bioavailability
Sarria Beatriz* and Vaquero M. Pilar
Departamento de Metabolismo y Nutrición
Instituto del Frío (CSIC)
C/José Antonio Nováis, 10
28040 Madrid
Spain
*Author for correspondence:
Dr. Beatriz Sarriá
Phone number: 00 34 91 549 00 38 ext. 295/292
Fax number: 00 34 91 549 36 27
Emails: beasarria@if.csic.es
Key words: Copper, Maillard reaction, bioavailability, infant formula, food
processing, lactulose, erythrocytes, liver
1
Abstract
Aim: To assess the effects of dehydration, conventional-in-bottle-sterilization and
ultra-high-temperature sterilization, involved in the manufacture of infant formulas
on copper bioavailability in rats at two stages. Methods: Two-week-old suckling
rats were fed a reconstituted powder (P1) and an in-bottle-sterilized liquid infant
formula (SC1) in a drinking bottle for 7 days. Weanling rats were fed P1, SC1,
another powder (P2), and a liquid UHT formula (UHT2) complemented with a
standard rat diet. Intake, body weight and % copper absorption were calculated
and whole body, serum, liver, skin and erythrocyte copper concentration were
determined. Results: Food intake, body weight, and copper intake were reduced
in suckling rats consuming SC1, but % copper absorption increased and whole
body and tissue copper concentrations were unaffected, except for the erythrocyte
copper concentration which was significantly higher compared to pups fed P1. In
weanling rats, the only difference obtained was the significantly higher liver copper
concentration in animals fed the diet containing P1 compared to the diets
containing SC1 and UHT2. Conclusion: Consuming the in-bottle-sterilized infant
formula induces high erythrocyte copper levels in suckling rats whereas the
equivalent dehydrated formula induces elevated liver copper concentration in
weanling rats. This is associated to the different Maillard Reaction Products (MRP)
originated in the processing of each infant formula and to the development rat
stage.
2
Introduction
Infant formulas can provide either less copper than breast milk or
substantially more depending on the manufacturer and type of formula but copper
absorption from infant formulas is not as effective as that from breast milk [1,2] .
The degree of heat treatment by which infant formulas are processed varies
considerably both within and among brands [3,4] and this may affect copper
bioavailability. Heat processing applied to dairy products induces the formation of
Maillard reaction products (MRP) as well as alterations in proteins. Infant formulas
obtained using high temperatures for extended periods of time present a relatively
high content of MRP and denatured proteins which have low digestibility [4,5].
Because proteins are involved in mineral absorption, heat damaged proteins may
affect copper bioavailability. In addition MRP might affect copper absorption or
transport. In infant monkeys fed ready-to-feed infant formulas excessive heat
treatment can have a pronounced negative effect on copper status [5]. The effects
of processing infant formulas on calcium [6], zinc and iron [7,8] bioavailability have
been studied in young rats and reported previously by our team.
Developmental changes in copper absorption take place. Studies in rats
indicate that the absorption and retention of copper can be particularly high in the
neonatal period, and that it decreases by the time of weaning [2]. In suckling and
weanling rats copper is absorbed in a concentration-dependant fashion, the
saturable copper transport system becomes evident in early maturity [9,10,11].
Copper is stored in the liver during the intrauterine life and used to satisfy the
metabolic needs in the suckling stage in addition to the copper provided by breastmilk or infant formula feeding. In the weaning process, when human milk or infant
3
formula is gradually replaced by semisolid foods, infants are nutritionally
vulnerable.
In this study, infant formulas produced using different heat processes were
fed to suckling rats as the only source of food and to weanling rats mixed with a
standard rat diet, in order to evaluate copper bioavailability and study
development-related changes in several tissues.
Subjects and methods
Infant formulas and diets
Commercially available infant formulas were used. A powder form (P1) and
a conventionally in-bottle-sterilized liquid (SC1) infant formula (both first age
formulas) provided by Nutricia, Zoetermeer (The Netherlands) and another powder
(P2) and UHT-sterilized infant formula (UHT2) provided by Mead-Johnson,
Nijmegen (The Netherlands) were used. P1 and SC1 contained (g/L):
carbohydrate, 71; protein, 14; and fat, 36. P2 and UHT2 contained (g/L):
carbohydrate, 70; protein, 15; and fat, 37. Two heat markers were analyzed [6] in
P1, SC1, P2 and UHT2 respectively (g/L): lactulose 0.143, 4.56, 0.097 and 1.587;
and furosine (index of early Maillard reaction products), 0.205, 0.105, 0.102 and
0.111.
In the suckling rat assay, rats drank the infant formulas P1 (group IF-P1)
and SC1 (group IF-SC1) from a bottle. The powder form P1 was reconstituted with
deionized water to 127 g/ L (following the manufacturer’s instructions) in order to
be used in liquid form. Copper levels were 0.58±0.02 and 0.63±0.03 mg/L (mean 
4
standard error for five determinations) for the powder and liquid infant formula,
respectively.
In the weanling rat assay, all infant formulas from both manufactures (P1,
SC1, P2, UHT2) were used in powder, for that reason SC1 and UHT2 were
lyophilised, and complemented with AIN 76A diet (American Institute of Nutrition,
1977) (Dyets Inc., Bethlehem, PA, USA). Four diets, D-P1, D-SC1, D-P2 and DUHT2, were prepared by adding each infant formula to the AIN-76A in the
proportion 40/60. One group of rats was fed exclusively AIN 76A as the control
diet. The theoretical content of carbohydrates, protein and fat in the prepared diets
was: 600, 160 and 140 g/kg, respectively. Copper levels were: 9.40.2, 8.90.1,
9.60.2, 10.10.3 and 10.00.2 mg/kg (mean  standard error for five
determinations) for the diets D-P1, D-SC1, D-P2, D-UHT2 and the control
respectively.
Animal assays
Rat assays were approved by the Ethical Committee of the Faculty of
Pharmacy and the Spanish Commission of Science and Technology (CICYT).
Wistar rats were obtained from the animalarium of the Instituto de Nutrición y
Bromatología (CSIC, Madrid) and housed in metabolic cages in an
environmentally controlled room, maintained at 20-22oC, with a 12 h light-dark
cycle and 55-70% humidity. The animals were randomly assigned to the dietary
treatments.
Suckling rat assay. Twenty-four two-week-old litter-mate, Wistar suckling rat pups
(half males and half females; initial body weight 30.1±0.4 g, mean ± standard
5
error) were used. They could freely access the infant formulas in a drinking bottle
during 7 days.
Food intake and body weight were monitored. Faeces were dried, weighed
and homogenized. On the 7th day, 6 rats from each group were anaesthetized
using sodium pentobarbital (Abbott Laboratories, S.A., Madrid, Spain) and blood
was drawn from the carotid artery into acid-washed (HNO3 10N) plastic vials and
allowed to clot. Erythrocytes and serum were obtained after centrifugation for 15
min at 1000 g (J.P. Selecta S.A., Barcelona, Spain). A segment of dorsal skin and
liver were removed, weighed and stored at -20ºC until analysis. The other 6 rats
were sacrificed on the same day and their bodies were digested in 6N HCl, which
was heated for one week, in order to analyze the whole body content of copper.
Weanling rat assay. Sixty three-week-old litter-mate Wistar weanling rats,
half males and half females, weighing initially 40.3±0.3 (mean ± standard error
mean), were used. The rats were kept in metabolic cages for a 4-d adaptation
period followed by a 7-d experimental period. They had free access to their
respective diet and to ultrapure water, (Milli-Q plus, Millipore Iberica, S.A. Madrid,
Spain). Body weight and food intake were monitored and during the last 7-d faeces
were collected, dried, weighed and homogenized. Finally the rats were sacrificed
and tissues were collected and bodies were digested as indicated in the suckling
rat assay.
Analytical techniques
The infant formulas, diets, faeces, livers, skins, and erythrocytes were dry-ashed in
a muffle furnace at 500° C. Ashes were dissolved in an acid solution
(HCl/HNO3/H2O: 1/1/1; Suprapur, Merck, Darmstadt, Germany). Copper was
6
determined in all the indicated samples and serum by atomic absorption
spectrophotometry (Perkin-Elmer 1100B Norwalk, CT, USA). Stock standard
solutions of copper (1 g/L) were prepared from Tritrisol (Merck). Calibration
solutions were prepared from the stock standard solutions by serial dilution with
ultrapure water (Milli-Q plus), and a blank solution was also used.
A pool of faeces was used as an internal control to assess precision. The
interassay coefficient of variation for copper was 3.8%. Lyophilised Liver (certified
reference material CRM 185; Community Bureau of Reference, Brussels, Belgium)
yielded a value of 194±6 g/g for copper (mean ± SD of five determinations)
(certified value 189±6 g/g).
Statistical Analysis
The data were analysed by the one-way analysis of variance (ANOVA) test
followed by the Bonferroni test. The level of significance was established at
p<0.05. Data were processed with the Statistical Package for Social Sciences
(SPSS).
Results
In the suckling rat assay, food intake was significantly lower (p=0.0201) in IF-SC1
rats (10.8±1.1 mL/d) compared with IF-P1 (13.8±0.6 mL/d). Consequently, final
body weight in the former group was significantly lower (26.7±0.4 and 29.5±0.3 g
in IF-SC1 and IF-P1 respectively, p<0.001). Among weanling rats (table 1), food
intake was analogous in the four groups of rats fed diets containing infant formula,
although in all of them it was significantly lower than in the control. Body weight
7
evolution was similar in all five groups, except for D-SC1 which showed
significantly lower body weight than the control group on day 4.
Copper intake was lower (p=0.013) in IF-SC1 suckling pups compared to
IF-P1 (table 2). However, copper absorption was not affected due to a relevant
increase in the % absorption (p=0.010; table 2). In weanling rats, although the
same trends were observed, the differences between D-P1 and D-SC1 were not
significant. These two groups presented significantly lower copper intake
compared to the control. All groups consuming infant formula showed a
significantly higher % absorption than the control. No significant differences were
observed in either body copper content or concentration in either the suckling and
weanling rats.
Serum copper was unaffected by the dietary treatments either in suckling
or weanling rats (table 3). In the younger rats, the consumption of SC1 compared
to P1 did not affect liver or skin copper levels but resulted in higher copper
erythrocyte concentration (p=0.0034). In weanling rats serum, skin and erythrocyte
copper levels were similar in all groups. However, liver copper concentration was
significantly higher in D-P1 compared to D-SC1 and D-UHT2, as well as to the
control (p<0.001).
Discussion
The infant formulas used in this study were commercially available and presented
copper concentration within the regular infant formula range: 0.4-0.6 mg/L [3]. In
the present study consumption of the in-bottle-sterilized infant formula SC1
induced lower food intake and body weight when it was given exclusively to
8
suckling rats [6,7]. There is limited data in the literature on infant formula
palatability and the influence of consuming infant formulas containing different
quantities of MRP on voluntary food intake. Some studies point to the lower body
weight increase in animals and infants fed different infant formulas, but food intake
was not monitored [5,11]. The SC1 formula contained high levels of lactulose, low
levels of early MRP, and more denaturated whey protein than the P1 formula, and
showed a brown colour what points to intense heat damage and the possibility that
the Maillard reaction progressed yielding advanced products [4]. This composition
may explain the reduced food intake and body weight observed specially in the
suckling rats [8].
Copper intake was reduced in IF-SC1 pups as a consequence of voluntary
food intake being lower in this group but apparent copper absorption was not
affected due to an increase in the % absorption. Therefore, body copper was
similar to that of the IF-P1 rat pups. In the weanling animals, copper absorption did
not vary due to the infant formula consumed. Cordano et al [12] described the
association between protein malnutrition and the incidence of copper deficiency. In
the present work, IF-P1 and IF-SC1 pups showed similar whole body nitrogen
concentrations at the end of the experiment (28.7±0.5 and 28.6±0.6 mg/g,
respectively [13]. Previous results in weanling rats also indicate that protein
utilization was only slightly affected, although nitrogen digestibility decreased in IFSC1 rats [4]. Accordingly, body copper was not affected in either experiment.
In suckling rats copper concentration in the different tissues analyzed did
not vary except erythrocytes copper which was higher in the IF-SC1 pups. High
levels of iron, zinc and copper in erythrocytes have been associated with increased
red cell membrane permeability due to membrane oxidation caused by elevated
9
polyunsaturated fatty acid intake [14, 15]. Both infant formulas contained exactly
the same fatty acids (manufacturer information) and iron and zinc levels in red
cells were unaffected [7,9]. However, it is known that certain Maillard Reaction
products induce lipid peroxidation in vivo which can affect membrane phosholipids
in erythrocytes [16], consecuently affecting erythrocytes function including
decreased deformability [17] and altered permeability. Another hypothesis for the
elevated erythrocyte copper concentration, is that Cu, Zn-superoxide dismutase is
increased in erythrocytes as an antioxidant mechanism. Although the mechanism
is not known, results of the present study indicate that only copper distribution
among tissues was affected since total body copper content and concentration
tended to be lower in the IF-SC1 animals, as indicated before. Therefore, the
indicated effect of the in-bottle-sterilized infant formula, when consumed as the
only food by suckling rats, is copper specific and deserves further investigation in
relation to oxidation status and membrane function.
The results here presented concerning absorption efficiency and body
copper concentrations in the suckling and weanling animals confirm the relatively
high absorption capacity and body copper stores in the pups. Mistilis and Mearrick
[18] described 100% absorption of an intragastric dose of 64Cu in 7-10 day old rat
pups with subsequent decreases during the suckling period and even further
during the weaning stage. According to Ehrenkranz et al [19], there is a significant
correlation in copper absorption where the stable isotopic extrinsic tag method and
the chemical balance technique are compared. Lönnerdal et al [20] obtained
copper absorption values between 32% and 40%, depending on the dietary
treatment, using 67Cu in 14-day-old rat pups. Olivares et al [21] described in young
infants a high variability, from 46% to 95%, using 65Cu as a tracer and the faecal
10
monitoring technique, pointing to copper absorption not being down-regulated
within the range of copper intake tested and to the trapping of copper in the
intestine and an incomplete release through desquamation over the study period.
The suckling rat mucosa retains a considerable fraction of absorbed copper but
this fraction decreases considerably with increasing age [1,10] in agreement to the
present study.
In addition, the fact that food and copper intakes were reduced in the IFSC1 pups may have elevated copper absorption efficiency compared to the IF-P1
pups as a compensatory mechanism. However, other factors could be also
responsible for enhanced copper absorption, such as the lactulose and/or MRP
contents in this infant formula. We have previously reported that the IF-SC1
suckling rats increased % calcium absorption but not that of iron and zinc [6,7].
Other authors have also obtained an increase on the apparent absorption in
different minerals associated with the presence of MRP [6, 22, 23, 24] indicating
that the breakdown of the complexes formed between MRP and the mineral by the
microflora in the colon, allows the metal to become available for absorption. The
present results are in agreement with Delgado-Andrade et al [25], who observed
increased copper absorption efficiency in rats fed a diet containing a mixture of
glucose and methionine heated at 150º C during 90 min. These authors stated that
the complexes formed might have other properties such as competing with
inhibition factors for copper absorption, therefore enhancing the digestive process.
Accordingly, the degradation of melanoidins in the large intestine may also play a
role in the binding and/or release of other dietary components [26]. The effects of
MRP on gut microflora may be similar to those of lactose and other poorly
digestible carbohydrates [27]. Rats fed lactulose presented a higher retention of
11
iron, calcium, magnesium, copper and zinc, which has been associated to their
higher caecum bifid bacteria flora [28, 29].
It is known that copper concentration in liver, skin and erythrocytes is
higher in suckling rats compared to weanling. Liver copper was reported to be
highest in pups 9–16 days of age, in older pups both liver and small intestine 64Cu
uptake was lower [1]. Klein et al [30] described similar copper changes during
development in foetal and neonatal human liver. By the time of weanling, most of
the dose of copper given is found in the caecum-colon (unabsorbed copper) and
not in the small intestine or the liver [2].
The high liver copper concentration in the weanling animals that consumed
powder infant formulas, particularly P1, may be attributed to the elevated content
of early MRP. Other authors [31] have shown that there was an uptake of early
Maillard reaction compounds into the cells of the liver, as well as kidneys and
muscles. Accordingly, infant formula P1 contained twice as much furosine that the
formula P2. Absorbable MRP with low molecular weight may be able to bind
copper and increase liver copper storage. Studies on the influence of MRP
obtained by heating glucose-lysine and methionine-lysine showed that early MRP
and melanoidines have a chelating effect on copper [32, 33]. Moon et al [34]
described that copper binds to soluble MRP in vitro. Delgado et al [25] also
reported copper accumulation in various tissues. Our study shows that copper
concentration in liver was higher in the D-P1 rats that consumed the infant
formula with higher early MRP level but that this did not induce important
changes in total body content.
Nevertheless, a similar influence on copper liver was not observed in the
suckling assay, in which the animals consumed exclusively infant formula. We
12
hypothesised that these products may have chelated to copper interfering with its
mobilization from the liver which takes place during later stages of development.
Faist and Erbersdobler [35] explained different metabolic transit mechanisms of
premelanoidins and melanoidins by intestinal degradation by digestive and
microbial enzymes, absorption of these compounds or their degradates, and tissue
retention. Further experiments aimed at studying the metabolic pathway of Maillard
reaction products need to be performed.
Acknowledgements
This work was supported by the Spanish Commission of Science and Technology
and The National Plan (projects ALI 96-0465 and AGL 2002-04411-C02-01 ALI).
The authors thank Laura Barrios MD, for her statistical assistance and Santiago
Navas for his help in editing this manuscript.
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Table 1.
Food Intake
Body weight (g)
Groups
g/day
Day 1
Day 4
Day 11
D-P1
9.2±0.4a
40.1±0.2
48.8±2.1
84.4±3.2
D-SC1
8.9±0.3a
40.3±0.3
46.6±1.5a
80.3±2.1
D-P2
9.1±0.3a
40.5±0.2
51.2±2.1
85.3±2.3
D-UHT2
9.1±0.4a
40.4±0.3
52.3±0.6
86.9±1.9
Control
12.6±0.3
40.2±0.3
54.1±0.4
85.5±2.3
One-way ANOVA
p<0.001
N.S.
p=0.005
N.S.
Values are means ± SEM (n=12).
Superscript a denotes significant difference with respect to the control group
19
Table 2.
Group
Intake (µg/day)
Absorption (µg/day)
%Absorption/Intake
Body copper
Cu (µg)
Cu (µg/g)
Suckling rats
IF-P1
7.9±0.3
4.2±0.2
53.6±3.2
117.2±6.5
3.9±0.2
IF-SC1
7.0±0.2
4.4±0.1
63.4±1.3
99.9±8.0
3.7±0.2
One-way ANOVA
p=0.013
N.S.
p=0.010
N.S.
N.S.
D-P1
82.0±4.4a
32.2±2.7
38.9±2.3a
222.5±18.2
2.6±0.2
D-SC1
78.4±3.0a
32.9±1.9
42.0±2.1 a
214.2±9.6
2.6±0.1
D-P2
87.3±3.1
32.4±3.0
36.8±2.9 a
213.0±8.7
2.5±0.1
D-UHT2
91.7±3.5
40.3±3.1a
43.7±2.5 a
196.0±8.3
2.4±0.1
Control
100.6±3.4
23.5±2.9
22.8±2.3
199.0±5.6
2.5±0.1
One-way ANOVA
p<0.001
p=0.003
p<0.001
N.S.
N.S.
Weanling rats
Values are means ± SEM (n=12 and n=6 for body copper parameters)
Superscript a denotes significant difference with respect to the control group
20
Table 3.
Cu (µg/ml)
Cu (µg/g)
Group
Serum
Liver
Skin
Erythrocytes
IF-P1
0.6±0.1
18.0±2.9
6.9±0.5
7.8±2.1
IF-SC1
0.5±0.1
17.1±1.4
6.5±0.3
13.0±3.3
One-way ANOVA
N.S.
N.S.
N.S.
p=0.0034
D-P1
0.5±0.1
11.6±1.03a
2.3±0.1
5.0±1.3
D-SC1
0.4±0.1
6.2±0.4*
2.1±0.2
5.2±0.9
D-P2
0.4±0.1
9.3±0.4
1.9±0.2
7.9±2.1
D-UHT2
0.4±0.1
6.3±0.6*
2.1±0.2
6.4±1.1
Control
0.3±0.0
6.5±0.7
2.2±0.1
7.8±1.3
One-way ANOVA
N.S.
p<0.001
N.S.
N.S.
Suckling rats
Weanling rats
Values are means ± SEM (n=6)
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Superscripts a and * denote significant difference with respect to the control and the P-1 group, respectively
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