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1 From the Department of Nutrition and California National Primate Research Center, University of California, Davis (SLK and BL), and Arla Foods AMBA (DC) and Arla Foods Ingredients, Viby, Denmark (KN).
2 Supported by a grant from Arla Foods, Viby, Denmark, and by NIH base grant RR00169. 3 Reprints not available. Address correspondence to B Lönnerdal, Department of Nutrition, University of California, One Shields Avenue, Davis, CA 95616. E-mail: bllonnerdal{at}ucdavis.edu.
ABSTRACT
Background: Advances in dairy technology make it possible to enrich infant formula with specific bovine milk components that may enhance nutrient status. Glycomacropeptide, a carbohydrate-rich casein peptide, may increase absorption of calcium, iron, or zinc. -Lactalbumin, a major breast-milk protein, may contribute to a balanced amino acid pattern and increase calcium and zinc absorption.
Objective: We determined the effects of glycomacropeptide- and -lactalbumin–supplemented infant formula on growth; trace mineral status; iron, zinc, and calcium absorption; and plasma amino acid, blood urea nitrogen, and plasma insulin concentrations.
Design: Infant rhesus monkeys (n = 5 infants per group) were breastfed or fed control or -lactalbumin– or glycomacropeptide-supplemented formula from birth to 4 mo of age. Hematologic measures and growth were assessed monthly. Mineral absorption was measured with radioisotopes and whole body counting.
Results: Infants fed glycomacropeptide had higher food intake than did other formula-fed infants. Infants fed glycomacropeptide or control formula had higher hematocrit values than did infants that were breastfed or fed -lactalbumin. Infants fed glycomacropeptide or control formula had higher plasma zinc and zinc absorption than did breastfed infants. Where differences were observed, breastfed infants and infants fed -lactalbumin had similar plasma essential amino acid and insulin profiles, which were different from those of infants fed glycomacropeptide or control formula.
Conclusions: Glycomacropeptide- or -lactalbumin–supplemented formula has no adverse effects on nutritional status in infant monkeys. Glycomacropeptide supplementation increases zinc absorption, which may permit the reduction of formula zinc concentrations, and -lactalbumin supplementation promotes a plasma amino acid pattern similar to that of breastfed infant monkeys.
Key Words: Glycomacropeptide • -lactalbumin • formula • infants • monkeys • amino acids • trace minerals
INTRODUCTION
It has long been recognized that breastfeeding provides young infants with a balanced supply of nutrients for optimal health, growth, and development. Breast milk has 2 major advantages over current infant formulas: 1) enhancing nutritional status (various nutrients are better absorbed and utilized from breast milk than from infant formula; 1–3) and 2) providing better defense against infection (4). Because of this disparity, there is interest in improving the quality of infant formula so that formula-fed infants may obtain some of the health benefits currently attained by breastfed infants. Advances in dairy technology have now made it possible to supplement infant formula with specific bovine milk protein fractions that may have biological activity or improve the absorption of nutrients from formula. Two proteins that may have such biological activity are glycomacropeptide and -lactalbumin.
Glycomacropeptide is a carbohydrate-containing peptide, formed from rennin digestion of -casein, that has a large negative charge (5). This negative charge may have a positive effect on mineral and trace element absorption by assisting the chelation and transport of minerals into the intestinal epithelium, thereby improving trace element status. -Lactalbumin is a major protein in the whey fraction of cow and human milk, has a tertiary structure similar to that of c-type lysozymes, and contains a calcium binding site that stabilizes its native configuration (6). The ability of -lactalbumin to bind minerals such as calcium and zinc (7, 8) may positively affect their absorption.
In addition, as a major human whey protein, -lactalbumin may affect the plasma amino acid pattern of breastfed infants because plasma amino acid patterns are affected by protein intake and the amino acid composition of the diet (9, 10). This contribution becomes particularly important because there is evidence that the current protein concentration in infant formula may be unnecessarily high (10). However, reducing the total protein concentration of infant formula may reduce the plasma concentration of the formula’s limiting amino acid (tryptophan), unless the formula is enriched with a tryptophan-rich protein such as -lactalbumin (11). In addition, alterations in the protein composition of infant formula may affect concentrations of blood urea nitrogen, an indicator of amino acid catabolism, or serum insulin concentrations, because of the insulinogenic effect of branched-chain amino acids. In the present study, we used an infant rhesus monkey model and hypothesized that supplementation of infant formulas with glycomacropeptide or -lactalbumin would be safe and would affect the nutritional status of the monkeys by enhancing trace mineral status and promoting a plasma amino acid pattern similar to that of breastfed infants. Secondary outcomes that we were interested in assessing included trace mineral absorption, blood urea nitrogen and insulin concentrations, growth (weight and length), and food intake.
MATERIALS AND METHODS
Diets
Purified glycomacropeptide and -lactalbumin were provided by Arla Foods (Viby, Denmark) and were produced as described previously (12, 13). The study formulas, which were dry blended, consisted of the following: 1) whey-predominant infant formula (control), 2) control formula with supplemental glycomacropeptide, and 3) control formula with supplemental -lactalbumin. An analysis of the approximate compositions of the formulas showed that they were similar, ie, 5.3 g fat/100 kcal, 2 g protein/100 kcal, and 11 g carbohydrate/100 kcal. There were no differences in vitamin or mineral content; however, minor differences in amino acid composition were found because of the replacement of 20% of the total protein with the supplemented protein (glycomacropeptide or -lactalbumin) during the dry-blending process (Table 1).
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TABLE 1 . Macronutrient, mineral, and amino acid compositions of the experimental formulas, human milk, and rhesus milk1
Animals
Twenty infant rhesus monkeys (Macaca mulatta) were obtained at birth from the breeding colony maintained at the California Regional Primate Research Center at the University of California, Davis. They were maintained indoors under the constant care of nursery and veterinary staff. The infants were either exclusively breastfed or bottle-fed one of the experimental diets ad libitum from birth until they were 4 mo old (n = 5 per group). From birth to 1 mo of age, the formula-fed infants were individually housed in polycarbonate isolettes with a surrogate mother (a terrycloth dummy); from 1 to 4 mo of age, they were paired and housed in stainless steel cages. No solid food was given throughout the course of the study. The study was approved by the Animal Care and Use Committee and the Radiation Use Authorization Committee at the University of California, Davis.
Methods
Weight and crown-rump length measurements were taken biweekly, food intake was recorded daily, and blood was drawn at birth and monthly for analysis. Calcium absorption (at 2 mo of age) and iron and zinc absorption (at 3.5 mo of age) were assessed after radioisotope administration. The monkeys were fasted for 4 h before orogastric gavage of radiolabeled formula: 1 µCi 47Ca (as CaCl2; Risø National Laboratory, Roskilde, Denmark), 59Fe (as FeCl2; Amersham Pharmacia Biotech, Piscataway, NJ), or 65Zn (as ZnCl2; Amersham Pharmacia Biotech) per 3 mL diet. Immediately after the dose, each animal was placed inside a small plastic cage and then inside a whole body counter (Institute of Toxicology and Environmental Health, University of California, Davis), and radioactivity was counted for 10 min to determine the amount of radioactivity administered. To determine background radioactivity, the radioactivity of the empty small plastic cage was counted similarly. No food was given for 2 h after the dose. The monkeys’ radioactivity was recounted 4 (47Ca) or 7 d (59Fe and 65Zn) later to determine retained isotope. The whole body counter was equipped with two 10 x 20-cm sodium iodide crystals and a multichannel analyzer (ND-66; Nuclear Data, Schaumburg, IL) for radioisotope quantification and analysis. Whole body calcium, iron, and zinc absorption was calculated as the amount of radioactivity retained, taking into account isotopic decay.
Hematology
Hemoglobin and hematocrit were quantified with an automated electronic cell counter (Baker 9010 Analyzer; Serono-Baker, Allentown, PA). Heparinized plasma was separated by centrifugation of whole blood at 3000 x g for 15 min at 4 °C. Plasma samples were digested with 0.1% (vol:vol) ultrapure HNO3 as described previously (16). Plasma copper and zinc were analyzed by flame atomic absorption spectrophotometry (Thermo Jarrell Ash, Franklin, MD). Bovine liver preparations were used as reference materials (Standard Reference Material 1577; US Department of Commerce, National Institute of Standards and Technology, Gaithersburg, MD).
Plasma amino acids and blood urea nitrogen
Plasma samples were acidified with 10% (wt:vol) sulfosalicylic acid, mixed by vortex, centrifuged at 21 000 x g and 4 °C for 30 min, and stored at -20 °C until analyzed. Amino acids were analyzed at the Molecular Structure Facility (University of California, Davis) with the use of ion exchange chromatography on a 6300 System Gold (Beckman, Palo Alto, CA) with lithium citrate buffer (17). Blood urea nitrogen was measured by using a commercially available spectrophotometric assay (Sigma, St Louis) based on the reaction of urea with diacetylmonoxime.
Insulin
Plasma insulin was analyzed by using a commercially available kit (Linco, St Louis) that was validated for cross-reactivity with nonhuman primate insulin.
Statistical analysis
Because of the expense of conducting infant monkey studies and the limited amount of experimental fractions available for evaluation, outcomes were divided into primary and secondary variables. Power analysis was performed to determine the number of infant monkeys required to detect significant differences in our primary variables of plasma amino acid concentrations, insulin concentration, hematocrit, plasma zinc and copper concentrations, and zinc and iron absorption. With an effect size of 1.25 SDs and a significance level of 5%, 5 infants per group were required to obtain 80% power. An additional power analysis was performed to determine the number of infant monkeys required to detect significant differences in our secondary variables of weight, length, blood urea nitrogen and hemoglobin concentrations, and calcium absorption. With a similar effect size of 1.25 SDs and a significance level of 5%, 8 infants per group were required to detect significant differences between means of hemoglobin concentration, calcium absorption, and weight to obtain 80% power. Twelve infants per group were required to detect significant differences between means of blood urea nitrogen concentration to obtain 80% power.
Statistical analyses were performed with GRAPHPAD PRISM software, version 3.02 (Graph Pad, San Diego). Statistical analysis was performed by two-factor repeated-measures analysis of variance or one-way analysis of variance (mineral absorption) with a Tukey-Kramer post hoc test. Main effects of diet, age, and the interaction between diet and age were determined. Because of the significant effect of diet on food intake, analysis of covariance was also performed. Significance was determined at P < 0.05.
RESULTS
There was a significant effect of age (P < 0.0001) and diet (P = 0.039) on weight (Figure 1), and a significant interaction between age and diet was observed (P < 0.0001). The formula-fed infants weighed significantly more than did the breastfed infants. There was a significant effect of age (P < 0.0001) and diet (P = 0.044) on food intake (Figure 2), and a significant interaction between age and diet was observed (P = 0.049).
FIGURE 1. . Mean (± SD) weight from birth through 4 mo of age of breastfed infant monkeys (•) and of infant monkeys fed control formula (*) or control formula supplemented with -lactalbumin () or glycomacropeptide () (n = 5 infants per group). Two-factor repeated-measures ANOVA indicated significant effects of age (P < 0.0001) and diet (P = 0.036) and a significant interaction between age and diet (P < 0.0001). The breastfed group was significantly different from the 3 formula groups, P < 0.05.
FIGURE 2. . Mean (± SD) food intake from birth through 4 mo of age of infant monkeys fed control formula (*) or control formula supplemented with -lactalbumin () or glycomacropeptide () (n = 5 infants per group). Two-factor repeated-measures ANOVA indicated significant effects of age (P < 0.0001) and diet (P = 0.044) and a significant interaction between age and diet (P = 0.049). The glycomacropeptide-supplemented group was significantly different from the other 2 groups, P < 0.05.
There were significant effects of age on hemoglobin concentrations, hematocrit values, and plasma copper concentrations (P < 0.0001) (Table 2). There was a significant interaction effect between age and diet on hematocrit values (P < 0.0001) and plasma copper concentrations (P = 0.031). We observed no significant interaction effect between age and diet on hemoglobin concentrations, which may have been a consequence of the limited number of infants used in this study. There was a significant effect of age (P < 0.0001) and diet (P < 0.0001) on plasma zinc concentrations (Figure 3), and a significant interaction between age and diet was observed (P < 0.0001). We observed no differences in iron or calcium absorption between the groups; however, the infants fed glycomacropeptide- or -lactalbumin–supplemented formulas had significantly (P < 0.05) higher zinc absorption than did the breastfed infants (Figure 4).
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TABLE 2 . Hemoglobin concentrations, hematocrit values, and plasma copper concentrations in breastfed infant monkeys and infant monkeys fed control formula or formula supplemented with glycomacropeptide (GMP) or -lactalbumin (-LAC) from birth to 4 mo of age1
FIGURE 3. . Mean (± SD) plasma zinc concentrations from birth through 4 mo of age in breastfed infant monkeys (•) and in infant monkeys fed control formula (*) or control formula supplemented with -lactalbumin () or glycomacropeptide () (n = 5 infants per group). Two-factor repeated-measures ANOVA indicated significant effects of age (P < 0.0001) and diet (P < 0.0001) and a significant interaction between age and diet (P < 0.0001). The breastfed group was significantly different from the glycomacropeptide and control groups, P < 0.0001.
FIGURE 4. . Mean (± SD) retention of 47Ca, 65Zn, and 59Fe given to breastfed infant monkeys () or to infant monkeys fed control formula () or formula supplemented with -lactalbumin () or glycomacropeptide () as an orogastric gavage in 3 mL infant feed at 2 mo of age (47Ca) or 3.5 mo of age (65Zn and 59Fe) (n = 5 infants per group). Values with different letters are significantly different, P < 0.05 (one-way ANOVA with Tukey-Kramer post hoc test).
There was a significant effect of age on all plasma amino acid concentrations (P < 0.0001) (Tables 3 and 4). In general, plasma amino acid concentrations in the breastfed infants were not significantly different from those in the infants fed -lactalbumin–supplemented formula. Plasma threonine, isoleucine, valine, and methionine concentrations in the breastfed infants and the infants fed -lactalbumin–supplemented formula were significantly different from those in the infants fed control or glycomacropeptide-supplemented formula; however, most of the differences were observed at 1 mo.
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TABLE 3 . Plasma concentrations of essential amino acids in breastfed infant monkeys and infant monkeys fed control formula or formula supplemented with glycomacropeptide (GMP) or -lactalbumin (-LAC) from 1 to 4 mo of age1
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TABLE 4 . Plasma concentrations of nonessential amino acids in breastfed infant monkeys and infant monkeys fed control formula or formula supplemented with glycomacropeptide (GMP) or -lactalbumin (-LAC) from 1 to 4 mo of age1
There was a significant effect of age on plasma insulin concentrations (P < 0.0001), and a significant interaction between age and diet was observed (P = 0.05). At 4 mo of age, the plasma insulin concentrations in the infants fed -lactalbumin–supplemented formula were significantly different from those in the infants fed glycomacropeptide-supplemented or control formula but were not significantly different from those in the breastfed infants (Table 5). There was a significant effect of age (P < 0.0001) and diet (P = 0.005) on blood urea nitrogen concentrations, but no significant interaction between age and diet was observed.
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TABLE 5 . Plasma insulin and blood urea nitrogen (BUN) concentrations in breastfed infant monkeys and infant monkeys fed control formula or formula supplemented with glycomacropeptide (GMP) or -lactalbumin (-LAC) from birth to 4 mo of age1
DISCUSSION
This study produced several significant findings regarding the effect of supplementing infant formula with specific milk protein fractions on growth and nutrition in an infant rhesus monkey model. We found small differences between the groups in conventional indicators of trace mineral status. At 3 mo of age, the infants fed control or glycomacropeptide-supplemented formula had higher hematocrit values than did the breastfed infants or the infants fed -lactalbumin–supplemented formula. However, these differences disappeared by 4 mo of age, and we observed no significant effect of diet on hemoglobin concentrations, perhaps because of the limited number of animals in each group. Plasma copper concentrations also differed significantly between the diet groups: the infants fed -lactalbumin–supplemented formula had the lowest plasma copper concentrations, and the infants fed glycomacropeptide-supplemented formula had the highest concentrations. Although the mechanism behind the effect of -lactalbumin on plasma copper concentrations is unknown, Greger and Mulvaney (18) observed that rats fed a diet high in -lactalbumin (30%) absorbed less copper than did rats fed a control diet, suggesting an effect on copper metabolism of supplementing infant formula with this specific milk protein fraction.
Plasma zinc concentrations in the infant monkeys fed glycomacropeptide-supplemented or control formula were significantly higher than those in the breastfed infant monkeys. However, the consequences of moderately increased plasma zinc concentration need to be investigated further because there is speculation that high concentrations of supplemental zinc can interfere with immune function (19). Although it has been suggested that some breastfed infants may not receive enough zinc, particularly during periods of accelerated growth (20, 21), Krebs et al (22) found that zinc retention in healthy, term, breastfed infants was adequate to meet their requirement until 4–6 mo of age, possibly as a consequence of the high bioavailability of zinc in breast milk. Furthermore, in contrast with plasma zinc concentrations in the breastfed infant monkeys, which remained unchanged from birth to 4 mo of age, plasma zinc concentrations in the formula-fed infant monkeys increased between 2 and 3 mo of age and then gradually decreased through 5 mo of age to values similar to those of the breastfed infant monkeys (SL Kelleher and B Lonnerdal, unpublished observations, 2001). These differences in plasma zinc concentrations suggest that this response was transient and may have been reflective of the higher observed zinc absorption in the formula-fed infant monkeys than in the breastfed infant monkeys, possibly as a consequence of the addition of specific milk protein fractions such as -lactalbumin. The concentrations of trace minerals, such as zinc, in infant formula are significantly higher than those in human milk (Table 1), and health professionals have been concerned that this may have negative ramifications for infant health. Therefore, the addition to infant formula of specific milk protein fractions that may positively affect trace mineral absorption may permit the reduction of the amount of minerals, such as iron and zinc, in infant formula.
Significant effects of diet on plasma amino acid concentrations were observed among the groups. Unfortunately, although proper care was taken to recover tryptophan in our samples, we were unable to quantify tryptophan in our amino acid analysis for unknown reasons. Nevertheless, the breastfed infants and the infants fed -lactalbumin–supplemented formula had plasma threonine and methionine concentrations that were significantly different from those of the infants fed glycomacropeptide-supplemented or control formula. Glycomacropeptide is a major component of many whey-based formulas and is particularly enriched in threonine (23). The nonglycosylated form of glycomacropeptide, which represents 50% of the peptide, is very susceptible to cleavage by trypsin in vitro (D Chatterton, unpublished observations, 2000). This proteolysis could release threonine in an absorbable form, which could account for the higher plasma threonine concentrations in the glycomacropeptide-supplemented group at 1 mo. Our results are in agreement with those of other studies, which have shown that in general, formula-fed infants have higher plasma threonine concentrations than do breastfed infants (24). Although the higher plasma threonine concentrations in the glycomacropeptide-supplemented infant monkeys may be reflective of the higher threonine concentration in the glycomacropeptide-supplemented formula, Darling et al (9) observed that breastfed infants had lower plasma threonine concentrations than did formula-fed infants, despite finding no differences in threonine concentration between breast milk and formula in their study. Thus, they speculated that formula-fed infants have a lower capacity to oxidize threonine than do breastfed infants. It has been recognized that because breast milk has a lower concentration of branched-chain amino acids than does formula, breastfed human infants have lower plasma concentrations of branched-chain amino acids than do formula-fed infants (10, 24). The insulinogenic role these amino acids play in metabolism may lead to the differences in body composition observed between formula-fed and breastfed infants (25). In the present study, the breastfed infant monkeys and the infant monkeys fed -lactalbumin–supplemented formula had transiently lower isoleucine and valine concentrations than did the infant monkeys fed control formula. Interestingly, we observed only transient differences in fasting insulin concentration: only at 4 mo did the infant monkeys fed -lactalbumin–supplemented formula have significantly higher plasma insulin concentrations than those of the infant monkeys fed glycomacropeptide-supplemented or control formula. These results may suggest temporary effects of -lactalbumin supplementation on glucose metabolism, which in extension may suggest effects on body composition, or the results may have been a consequence of the small number of infant monkeys used in this study. In addition, the infant monkeys fed -lactalbumin–supplemented formula had plasma lysine, tyrosine, and arginine concentrations that were signifcantly different from those of the infant monkeys fed control formula, suggesting that the supplementation of infant formula with -lactalbumin balances the plasma amino acid profile to that of breastfed infants.
All the infant monkeys fed infant formula weighed more than did the breastfed infant monkeys, which parallels the pattern observed for human infants (26, 27); however, the mechanism is not well understood. This difference does not appear to be related to energy density because the energy density of current infant formulas closely matches that of human milk, as well as rhesus milk (1, 15; Table 1). Dewey (28) has suggested that in human infants, these differences may be due in part to the higher food intake of formula-fed infants. Although it was not possible in the present study to determine the milk intake of the breastfed infant monkeys because, unlike human infants, rhesus infants are not meal feeders but continuously suckle, the food intake of the glycomacropeptide-fed infant monkeys was significantly higher than that of the other formula-fed infant monkeys; however, this difference did not result in significant differences in body weight.
In summary, supplementation of infant formula with glycomacropeptide or -lactalbumin does not appear to adversely affect the nutritional status of growing infant rhesus monkeys. In fact, iron and copper status indicators (hematocrit and plasma copper) were positively affected by the addition of these specific milk protein fractions. Furthermore, enhanced zinc absorption resulted from supplementation of infant formula with these components. If similar results are obtained in human infants, the formula industry may be able to reduce the concentration of trace minerals in formula, thus protecting infants from the potential adverse effects of excess dietary intake. In addition, infant monkeys fed formula supplemented with -lactalbumin had a plasma amino acid profile similar to that of breastfed infant monkeys. If similar observations are made in human infants, these changes may have positive consequences, particularly if the trend continues toward a reduction in protein concentration in infant formula.
ACKNOWLEDGMENTS
We are grateful to Dennis Heuring for assistance in the formulation of the experimental diets and to Gitte Graverholt for generous assistance in the preparation of this manuscript.
All authors participated in the study design. SLK was responsible for data collection and analysis. The manuscript was prepared by SLK and was edited by the other authors. DC and KN were employees of Arla Foods. SLK and BL had no conflict of interest.
REFERENCES