Age and copper intake do not affect copper absorption, measured with the use of 65Cu as a tracer, in young infants

¹ From the Institute of Nutrition and Food Technology, University of Chile, Santiago (MO, FP, and RU); the Department of Nutrition, University of California, Davis (BL); and the US Department of Agriculture/Agricultural Research Service Children’s Nutrition Research Center, Baylor College of Medicine, Houston (SAA).

² Supported by the Copper Risk Assessment Research Program in Chile, which is managed by the Chilean Center for Mining & Metallurgy Research, and the International Copper Association in the form of an unrestricted research grant.

³ Address reprint requests to M Olivares, Institute of Nutrition and Food Technology (INTA), Macul 5540, Santiago 11, Chile. E-mail: molivare{at}uec.inta.uchile.cl.

ABSTRACT  
Background: Copper homeostasis involves a high degree of regulation in which changes in absorption and biliary excretion are the main mechanisms. Whether neonates and small infants can make these changes efficiently is unknown.

Objective: We evaluated the effect of age and copper intake on copper absorption in infants during the first 3 mo of life.

Design: Thirty-nine healthy infants (19 infants aged 1 mo and 20 infants aged 3 mo) were selected. One-half of the subjects were randomly assigned to receive oral supplementation of 80 mg Cu (as copper sulfate) · kg body wt^-1 · d^-1 for 15 d. At the end of the trial, copper absorption was measured by using orally administered ⁶⁵Cu as a tracer and fecal monitoring of recovered ⁶⁵Cu.

Results: Mean (± SD) copper absorption at 1 mo of age was 83.6 ± 5.8% and 74.8 ± 9.1% for the unsupplemented and supplemented infants, respectively. The corresponding figures at 3 mo of age were 77.6 ± 15.2% and 77.7 ± 11.3%. A two-way analysis of variance showed that age, copper supplementation, and the interaction between age and copper supplementation did not have a significant effect on copper absorption. There was an inverse correlation between total fecal copper and the percentage of ⁶⁵Cu absorption (r = -0.50, P < 0.003).

Conclusion: Copper absorption in young infants is high but does not respond to copper intake within the range tested.

Key Words: Copper absorption • human milk • infants • copper supplementation • 65Cu

INTRODUCTION  
A considerable amount of copper is transferred from the mother to the fetus. Copper is accumulated in the fetus mainly at the end of the gestation period, and a substantial portion of the accumulated copper is retained in the liver of the fetus (1). The substantial stores of copper in the liver of the term fetus may aid in preventing copper deficiency during the early months of life. After birth, liver copper concentration decreases and serum copper and ceruloplasmin concentrations increase and become more similar to those in adult serum (2, 3).

Copper homeostasis is believed to involve changes in both intestinal absorption and biliary excretion (4). Studies in human adults in which stable isotopes were used showed an inverse relation between copper intake and copper absorption (5). However, there is insufficient information to ascertain whether the efficiency of copper absorption in infants is up- and down- regulated at low and high copper intakes, respectively. The answer to the question of how well controlled copper absorption is in the first months of life is crucial to determine whether normal infants are at risk of copper excess within the range of acceptable intakes (6, 7). The aim of this study was to evaluate copper absorption in infants during the first 3 mo of life and the effects of age and copper intake on copper absorption.

SUBJECTS AND METHODS  
Thirty-nine healthy infants (19 infants aged 1 mo and 20 infants aged 3 mo) with a birth weight > 2500 g were selected. One-half of the subjects were randomly assigned to receive oral supplementation of 80 mg Cu (as copper sulfate solution) · kg body wt^-1 · d^-1 (1.2 µmol · kg body wt^-1 · d^-1) for 15 d. A daily dose of 0.3 mL solution/kg body wt (commonly 1–1.5 mL) was administered to the infants between feedings by their mothers: a 3 mL syringe was placed at the back of the infant’s tongue because the taste of the copper solution was not masked. To assess compliance with the instructions provided by the investigators, a field worker visited each infant’s home twice a week at a random time to record the volume of the copper solution remaining in the bottle. In addition, the field worker conducted one food-frequency questionnaire for each infant to obtain the volume of cow milk or formula received. Because the concentration of copper in breast milk is not affected by maternal diet or ethnic factors (8), we calculated the copper intake of breast-fed infants on the basis of data published by Butte et al (9) for human milk composition and volume of intake by age. Before the infants were included in the study, informed consent was obtained from their parents, and the study protocol was approved by the Ethics on Human Research Committee of the Institute of Nutrition and Food Technology of the University of Chile.

The stable ⁶⁵Cu isotope, which was 99.7% enriched (Oak Ridge National Laboratory, Oak Ridge, TN), was obtained in the form of copper wire, which was weighed, dissolved in a minimum of ultrapure nitric acid and hydrochloric acid (Merck, Darmstadt, Germany), and diluted with distilled, deionized water. The feeding solutions were made in one batch and diluted so that each dose had 30 or 200 mg ⁶⁵Cu. Single doses were weighed in test tubes and stored frozen until the day of use.

A trained field worker administered the isotope to each infant in a small feeding bottle. On day 1, each infant received one oral dose of 200 mg (3.1 µmol) ⁶⁵Cu (supplemented group) or 30 mg (0.5 µmol) ⁶⁵Cu (unsupplemented group) dissolved in 10 mL distilled, deionized water. The bottle was then refilled and rinsed with 5 mL additional distilled, deionized water, which was again administered to the infant. The isotope was given immediately after the second feeding of the day (1000 to 1100).

On day 1 and 72 h later, infants received in a feeding bottle 150 mg Brilliant Blue FCF marker (Foodsafe, Santiago, Chile) in 20 mL distilled, deionized water to mark the beginning and end of the stool collection period. To ensure the quality of sampling and to avoid the loss of all or part of the samples, mothers were carefully trained before the study in how to collect the fecal samples from their infants. When the mothers’ ability to properly collect fecal samples was tested at the end of the training period, no differences were observed between the mothers in the 4 study groups (ie, 1-mo-old supplemented infants, 1-mo-old unsupplemented infants, 3-mo-old supplemented infants, and 3-mo-old unsupplemented infants). Mothers were provided daily with diapers that were labeled with an indelible code to document the sequence of diaper use. The field worker visited each infant’s home daily to pick up the used diapers and check the sequence to assure that all stools were collected. Stool was extracted from each diaper with a wooden spatula in the laboratory, and the stained liner was cut out. The Brilliant Blue and the diaper liner were checked for copper content to verify negligible amounts of copper. Daily pools of stools and liners were prepared. Glassware was acid-washed before use to reduce the risk of copper contamination, and random tests of the glassware confirmed that it was free of trace elements at the time of use. Fecal pools were weighed in porcelain crucibles, heated in a muffle furnace for 7 h at 90 °C, and then ashed by heating for 12 h at 600 °C. Later, 50 mL of a 5-mol HNO₃/L solution (Merck) was added to the crucibles, and each crucible was covered with a watch glass. The liquid was evaporated on a hot plate in a clear plastic box under a hood. Samples were returned to the muffle furnace for an additional 12 h at 600 °C, removed, covered, and cooled. The resulting ash was dissolved in 10 mL of a 2-mol HNO₃/L solution (Merck) for later analysis.

A glass chromatography column (1 x 20 cm), with a 40-mL reservoir in the top, was filled with cation exchange resin (AG 50W X8, 100–200 mesh; Bio-Rad Laboratories, Richmond, CA), which was washed with 30 mL of a 6-mol ultraclean HCl/L solution and with 20 mL distilled, deionized water and was reconditioned with 30 mL 60% (by vol) acetone. Samples were loaded onto the column with 4 mL 60% acetone and passed through the column, which was then washed first with 30 mL 60% acetone and then with 30 mL 75% acetone. Copper was extracted from the column with 30 mL 90% acetone. The solution collected was dried on a hot plate at a subboiling temperature, resuspended, and redissolved in 1–2 mL of a 6-mol HCl/L solution to be used as the sample for the next anion exchange procedure. A polyethylene column (8 cm x 0.4 cm) with a 4-mL reservoir in the top was filled with anion exchange resin (AG-1 X8, 100–200 mesh; Bio-Rad Laboratories). The resin in the column was cleaned with 4 mL of a 6-mol ultraclean HCl/L solution and with 4 mL distilled, deionized water. The column was then reconditioned in 2 mL of a 6-mol HCl/L solution before the sample solution was loaded. The sample was loaded with 1 mL of a 6-mol HCl/L solution. After the sample solution had passed through the column, it was washed with 6 mL of a 6-mol HCl/L solution before copper was extracted from the column with 4 mL of a 3-mol HCl/L solution. The solution collected was dried on a hot plate at a subboiling temperature, resuspended in 0.03 mol HNO₃, and loaded onto the filament for mass spectrometric analysis.

Copper content was measured by atomic absorption spectrometry (model 2280; Perkin Elmer, Norwalk, CT), and the ratio of ⁶⁵Cu to ⁶³Cu was measured as described by Turnlund et al (10) with the use of a magnetic-sector thermal-ionization mass spectrometer (MAT 261; Finnigan, Bremen, Germany). Measurement precision for the ratio of ⁶⁵Cu to ⁶³Cu was 0.1%. Apparent absorption results are based on changes in fecal copper isotope ratios. Indexes of mass balance were not derived because copper intake was not directly measured. In the first 4 infants who were recruited, the ratio of ⁶⁵Cu to ⁶³Cu was measured but the total stool copper was not, because the samples for these latter measurements were lost. Values in the tables and the text are given as means ± SDs. Statistical analysis included two-way analysis of variance, Student’s t test, stepwise multiple regression, linear regression, and Pearson correlation. When analysis of variance was statistically significant, identification of significant differences between groups was based on Scheffe’s post hoc test. Statistical analyses were performed by using STATISTICA for WINDOWS (release 4.5; StatSoft Inc, Tulsa, OK).

RESULTS  
Except for initial age, the characteristics of the subjects in the supplemented and unsupplemented groups were not significantly different (Table 1). Twenty-eight of the 39 infants were exclusively breast-fed, 9 were partially breast-fed, and 2 were totally weaned. Total derived copper intakes in the unsupplemented and supplemented infants at 1 mo of age were 60.3 ± 0.2 µg · kg body wt^-1 · d^-1 (268.2 ± 30.2 µg/d; 0.9 ± 0.0 µmol · kg body wt^-1 · d^-1; 4.2 ± 0.5 µmol/d) and 86.3 ± 1.9 µg · kg body wt^-1 · d^-1 (362.4 ± 33.8 µg/d; 1.4 ± 0.0 µmol · kg body wt^-1 · d^-1; 5.7 ± 0.5 µmol/d), respectively. The corresponding figures at 3 mo of age were 35.6 ± 0.3 µg · kg body wt^-1 · d^-1 (226.8 ± 35.5 µg/d; 0.6 ± 0.0 µmol · kg body wt^-1 · d^-1; 3.6 ± 0.6 µmol/d) and 55.9 ± 1.7 µg · kg body wt^-1 · d^-1 (353.9 ± 32.0 µg/d; 0.9 ± 0.0 µmol · kg body wt^-1 · d^-1; 5.6 ± 0.5 µmol/d), respectively.

View this table:
TABLE 1 . Characteristics of the study groups  
The effect of age and of copper supplementation on total fecal copper and apparent ⁶⁵Cu absorption was tested by two-way analysis of variance (Table 2), which showed a significant effect of copper supplementation on total fecal copper. However, apparent ⁶⁵Cu absorption was not significantly different by age group or copper supplementation group. In the entire group of infants, fecal copper and apparent ⁶⁵Cu absorption for the 3 d ranged from 178 to 1347 mg Cu (from 2.8 to 21.2 µmol Cu) and from 46% to 95%, respectively. Copper absorption in exclusively breast-fed, partially breast-fed, and totally weaned infants was 79.7 ± 10.2%, 78.5 ± 13.3%, and 58.6% (no SD because there were only 2 infants in this category), respectively.

View this table:
TABLE 2 . Fecal copper and apparent ⁶⁵Cu absorption in copper-supplemented and unsupplemented young infants¹  
Copper intake was not significantly correlated with the percentage of copper absorption. As shown in Figure 1, there was an inverse, simple correlation between fecal copper and the percentage of ⁶⁵Cu absorption (r = -0.50, P < 0.003). When fecal copper, type of feeding, copper intake, and age were included as independent variables, stepwise multiple regression for the percentage of copper absorption indicated R² = 0.29 (P < 0.02). Of the variables tested, only fecal copper met the significance level for entry into the model (P < 0.02).

View larger version (16K):
FIGURE 1. . Correlation between fecal copper and the percentage of ⁶⁵Cu absorption in young breast-fed (•), partially breast-fed (), and weaned () infants. r = -0.50.

 

DISCUSSION  
Copper homeostasis is believed to involve a high degree of regulation in which modifications in absorption and endogenous excretion are the main mechanisms. Whether neonates and small infants can make these modifications efficiently is unclear.

Composition of the diet, copper intake, and copper nutritional status influence copper absorption (5, 10–12). The type of feeding and the amount of copper supplied affect copper balance in early life. We observed a high apparent copper absorption (80%) in infants aged 1–3 mo. This effect may be the consequence of breast-feeding or a developmental phenomenon. Breast-fed infants absorb more copper, perhaps because of the lower casein content of human milk or because of factors associated with human milk that enhance copper absorption (13). In the present study, copper absorption in breast-fed and partially breast-fed infants was higher (79.7 ± 10.2% and 78.5 ± 13.3%) than in 2 totally weaned infants who received only cow milk (58.6%). There is a basis for attributing an absorption-enhancing effect to breast milk that could account for the absorption of exogenous ⁶⁵Cu as well. Although the isotope in the present study was administered after the meal, the high absorption could also have been due, in part, to the isotope being administered in water rather than in milk. Using the suckling rat pup model, Lönnerdal et al (13) found a higher copper absorption from human milk than from cow milk. In chemical balance studies and absorption studies in which stable isotopes were used, infants had higher retention and absorption of copper from human milk than from cow milk formula (14, 15). ⁶⁵Cu absorption from human milk in premature infants aged 1 mo was significantly greater (69.8 ± 14.0%) than ⁶⁵Cu absorption from formula in premature infants (39.6 ± 21.6%) and full-term infants (26.5 ± 6.9%) (14). Studies in rats showed that copper absorption is very high during the neonatal period but that it decreases by the time of weaning (13). It is possible that this also occurs in humans, but studies by Turnlund et al (5, 10) showed that copper absorption is high in adults as well, even when copper intakes are very low. We found that the percentage of apparent copper absorption at 1 mo of age was modestly, but nonsignificantly lower in the infants who were supplemented with copper than in the unsupplemented infants, but this pattern was not observed at 3 mo of age. The dose of ⁶⁵Cu given to the supplemented infants was 7-fold that given to the unsupplemented infants; hence, the apparent copper absorption in the supplemented group may have been even lower.

The lack of a major difference in the percentage of copper absorption observed between the supplemented and unsupplemented infants may be explained in different ways. One explanation is that copper intake was not high enough to trigger homeostatic adaptation of intestinal absorption. In fact, Turnlund et al (5) observed that when the copper intake of adults was 10-fold that of the previous intake, the percentage of copper absorption decreased, whereas Milne et al (11) showed no such effect when the copper intake of adults was 1.5-fold that of the previous intake. The copper intake of our supplemented subjects was 1.4- to 1.5-fold that of the unsupplemented infants. Another explanation is that the latency in copper absorption adaptation may be > 15 d. A third explanation is the inability of young infants to regulate copper absorption. Studies in animals and humans support the existence of developmental changes in copper absorption. In kinetic studies, Varada et al (16) found that copper absorption was saturable only in adolescent rats, whereas copper absorption was linear and nonsaturable in suckling and weanling animals. A similar efficiency of copper absorption was observed in rats when the copper content of human milk was increased to 10 times the normal content by the addition of a copper salt (13). Furthermore, Dörner et al (15) found a linear relation between copper intake and copper retention in balance studies in infants, supporting the suggestion from studies in rats that copper absorption is nonsaturable during early infancy. We observed an inverse linear relation between fecal copper (an indirect measure of copper intake) and the percentage of copper absorption. It could be speculated that infants, given the smaller total absorptive surface of their intestines, may have a substantial residual absorptive capacity and thus may be at a greater risk of copper overload than are older individuals.

Copper homeostasis is mediated not only by absorption but also by modifications in the endogenous secretion of copper. However, the methodology used in our study did not allow estimations of the endogenous secretion of copper or its adaptation. The high apparent absorption may also be explained by a systematic error in the balance technique, because more complete stool collections will necessarily be associated with higher ⁶⁵Cu recovery from the feces and higher apparent absorption. We took all possible measures to assure complete stool collection. However, although Brilliant Blue is a good marker for stool collection, it is not truly quantitative. Continuous markers are better but are harder to detect.

Our study was conducted in the subject’s homes and thus may have been affected by confounders that are better controlled in studies conducted in metabolic wards under rigidly controlled conditions. The ethics standards at our institutions restrict balance studies in the metabolic ward to those that are absolutely essential for the medical care of infants and do not allow balance studies in healthy infants. Thus, the possibility of incomplete stool collection is a limitation of this study. However, because this study was essentially a paired study, there is no reason to think that incomplete stool collections affected one group more than the other.

The results of our study show that copper absorption was highly variable (ranging from 46% to 95%) under the conditions of this study and that absorption is not down-regulated within the range of copper intakes tested. These intakes were 2- to 3-fold the most recent adequate intake defined for 1-mo-old breast-fed infants by the Food and Nutrition Board of the National Academy of Sciences and 1.2- to 2-fold the adequate intake defined for 3-mo-old breast-fed infants (7). It may be possible that down-regulation of absorption does occur at higher intakes, which would protect against excess. On the contrary, if absorption remains high, toxicity could be possible even at moderately high exposures. Our in vitro results suggest that copper absorption at the apical surface is not down-regulated by chronic exposure to high copper concentrations. We have shown that Caco-2 cells respond to copper by increasing basolateral copper efflux. Thus, homeostatic control is not dependent on regulation of copper uptake but rather on the release from the enterocyte to the portal vein (17). This leads us to speculate that intestinal cell desquamation over time may contribute to whole-body copper homeostasis as copper stored in enterocytes is lost. The high percentage of apparent absorption observed in our study may be explained by trapping of copper in the intestine and an incomplete release through desquamation over the study period. Our stool collection period was 72 h, whereas enterocyte turnover in humans takes 3–5 d (18). The experimental design of the present study may be considered inadequate because intakes were not sufficiently high; yet, because of the potential for increased susceptibility to excess copper in very young infants, the ethical imperative of "do no harm" precluded us from testing higher intakes. Further research is required to elucidate the full range of homeostatic regulation and the contribution of copper absorption to this process during the first months of life. Such elucidation is crucial to determine the safe range of copper exposure in young infants.

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