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1 From the Department of Nutrition and the California Regional Primate Research Center, University of California at Davis, and Wyeth Nutritionals International, Philadelphia.
2 Supported in part by an NIH base grant to the California Regional Primate Research Center. 3 Address reprint requests to B Lönnerdal, Department of Nutrition, University of California, One Shields Avenue, Davis, CA 95616-8669. E-mail: bllonnerdal{at}ucdavis.edu.
ABSTRACT
Background: Infant rhesus monkeys are excellent models in which to study the effect of infant formulas on trace element absorption and status. Infants fed powdered formula from birth exhibit normal growth and have blood variables similar to those of breast-fed infants.
Objectives: The objectives were to evaluate the effects of feeding ready-to-feed (RTF) formulas exposed to different heat treatments to infant monkeys, and, for one of these formulas, to compare the effect of fortification with 2 iron concentrations.
Design: From birth to age 5 mo, infant monkeys (n = 6/group) were fed one of the following formulas exclusively: 1) 12 mg Fe/L processed in cans (RTF-12), 2) formula in glass bottles with 12 mg Fe/L and manufactured by an ultrahigh-temperature (UHT) process (UHT-12), or 3) formula manufactured by a standard thermal process (STP), containing either 8 (STP-8) or 12 (STP-12) mg Fe/L. All formulas had similar copper concentrations (0.6 mg Cu/L). Anthropometric measures and venous blood samples were taken monthly.
Results: Weight and length gain did not differ among groups; however, the STP-12 group weighed less than the UHT-12 group at ages 2, 4, and 5 mo. Hemoglobin values were significantly lower in the RTF-12 group than in all other groups at ages 4 and 5 mo and serum ferritin was lower in the RTF-12 group than in the STP-12 group at age 5 mo. Copper status was lower in STP-12 infants than in STP-8 infants. There was a progressive and significant decline in plasma copper, ceruloplasmin, and Cu/Zn superoxide dismutase activity in infants fed canned formula (RTF-12). Furthermore, coat color changed from normal brown to silver. These outcomes suggest that the canned formula induced copper deficiency in infant monkeys.
Conclusions: Excessive heat treatment of formula can have a pronounced negative effect on copper status. High iron concentrations did not improve iron status but may adversely affect copper status.
Key Words: Thermal processing • heat treatment • infant formula • iron • copper • copper deficiency • infant rhesus monkeys
INTRODUCTION
The primary objective of this study was to evaluate the applicability of using infant rhesus monkeys as models of human infants when assessing the appropriate iron concentration in infant formula. We used infant rhesus monkeys previously to study mineral and trace element absorption from milk and infant formulas (1–5). In several cases we found that the results correlated well with human data. Infant rhesus monkeys were chosen as a model because the composition of monkey milk is similar to that of human milk (6, 7), infant monkeys' gastrointestinal physiology is similar to that of human infants, and infant monkeys are breast-fed for 5–6 mo, which is similar to the duration of breast-feeding in human infants in many industrialized countries. Furthermore, we found that infant rhesus monkeys can be reared from birth exclusively on standard infant formula without any modifications and that their growth pattern is similar to that of breast-fed rhesus infants (5). Thus, we studied genetically homogenous rhesus infants under very controlled circumstances, which is often difficult to do in human infant populations. In this study, we evaluated the effect of feeding formula containing 8 or 12 mg Fe/L, because it was proposed that the current concentration of iron fortification in some formulas (12 mg Fe/L) is unnecessarily high (8).
Much of the published literature shows the effect of various levels of iron fortification on the iron status of formula-fed infants (9–12). In virtually all of these studies the iron concentration of the formula was the variable and the formulas studied were processed by the same method. However, the degree of heat treatment by which infant formulas are processed varies considerably (13), both within and among brands. In the United States and Canada, liquid formula is most commonly used, whereas in most other countries powdered formula is conventionally used. Ready-to-feed (RTF) formulas must be sterile and therefore receive extensive heat treatment. Specific temperatures and the duration of treatment vary among manufacturers and products (eg, from 120 mL glass bottles to 960 mL cans), but usually range from 120 to 130oC for 5–10 min. More recently, ultrahigh-temperature (UHT) processing of infant formula was introduced in Europe. This process is very short in duration (3–10 s) but uses a high temperature (130–150oC). It is known that the extent of processing to which infant formulas are exposed can affect protein digestibility. For example, we have showed in vitro that proteins in powdered formulas are more easily digested than are proteins in liquid formulas and that UHT formulas have high protein digestibility (14). Processing formulas at high temperatures for extended periods of time are correlated to an increased formation of Maillard products, eg, lysinoalanine and lactoselysine (15), which have a negative effect on protein digestibility and protein quality (16). In our previous study, we found that formulas with a high lysinoalanine content also had low protein digestibility (14). In the present study we evaluated the effect of heat processing on formula and iron status because mineral and trace element absorption are affected by the extent of protein digestion.
In a preliminary study we found that some rhesus infants fed RTF formula had very low copper status (B Lönnerdal, L Jayawickrama, EL Lien, unpublished observations, 1996). In addition, no signs of copper deficiency were observed in rhesus infants fed powdered milk formula. We hypothesized that the degree of heat treatment of an infant formula can affect copper absorption and, therefore, we also evaluated copper status of the infants in this study.
MATERIALS AND METHODS
Study design
Twenty-four newborn infant rhesus monkeys (Macaca mulatta) were obtained from the breeding colony at the California Regional Primate Center, Davis. They were bottle-fed exclusively ad libitum from birth to age 5 mo (n = 6/group) and housed in stainless steel cages with an artificial surrogate mother. Infants were kept in cages pairwise within each group to foster normal socialization. Monthly anthropometric measures (weight and crown-rump length) and fasting (>3 h after eating) venous blood samples were taken. All infants were supervised constantly by veterinarians during the entire study. The study was approved by the Animal Care and Resources Committee at the University of California, Davis.
Diets
The following commercially available formulas were studied: 1) standard thermal process formula (Wyeth Nutritionals, Radnor, PA) with 8 mg Fe/L (STP-8), 2) standard thermal process formula (SMA; Wyeth, St-Laurent, Canada) with 12 mg Fe/L (STP-12), 3) ready-to-feed process formula (Enfamil; Mead Johnson, Evansville, IN) with 12 mg Fe/L (RTF-12), and 4) ultra-high-temperature process formula (S-26; Wyeth, Ireland) with 12 mg Fe/L (UHT-12). All formulas were whey-predominant and had similar lipid and nutrient composition. The RTF-12 formula was purchased in cans (960 mL, or 32 oz) in one batch, whereas the other formulas were purchased in glass containers (120 mL, or 4 oz).
Coat color
To determine the development of coat color, a color index was developed by comparing study animals with a group of nonexperimental, breast-fed, infant rhesus monkeys of the same age. Experimental rhesus infants with the same coat color as control rhesus infants were given a score of 2, infants with a lighter color a score of 1, and those with a much lighter color a score of 0. Before scoring, all labels that identified the experimental diets and groups were removed from the cages. At all times, nonexperimental infants were placed beside the experimental groups during observations. The observations and scores were made separately by 3 individuals that had been trained previously and the results were examined for consistency.
Analyses
A complete blood count [hemoglobin, red blood cells (RBCs), and white blood cells] was taken at the California Regional Primate Research Center. Serum ferritin was measured with the use of the Magic Ferritin kit (Ciba Corning Diagnostics, Medfield, MA); the antibody against human serum ferritin used in this kit cross-reacts with rhesus monkey ferritin (SL Kelleher, B Lönnerdal, unpublished observations, 1998). Ceruloplasmin activity was assayed by the method of Schosinsky et al (17), and the activity of Cu/Zn superoxide dismutase (Cu/Zn SOD) in RBCs was determined by inhibition of the autooxidation of pyrogallol (18). Plasma zinc and copper were analyzed by flame atomic spectrophotometry with the use of an IL 551 instrument (Instrument Laboratories, Mountain View, CA) following wet ashing with concentrated nitric acid as described by Clegg et al (19). A National Bureau of Standards bovine liver sample (Standard reference material 1577; US Department of Commerce, National Bureau of Standards, Washington, DC) was included with the blood samples to ensure accuracy of the analysis. Statistical analysis was done by Tukey's test and by repeated-measures analysis of variance. SAS (version 7; SAS Institute Inc, Cary, NC) was used in the statistical analyses.
RESULTS
There were some significant weight differences among the groups; in particular, the STP-12 group weighed significantly less than the UHT-12 group at ages 2, 4, and 5 mo (Table 1). The STP-12 group also had a significantly lower crown-rump length than did the STP-8 group at age 4 mo. However, there were no significant differences among groups in overall weight or length gain during the study period. The coat color changed dramatically in the RTF-12 group (silvery-gray coat; score: 0.2 ± 0.4) and some change was also observed in the STP-12 group (a more pale than normal color; score: 0.5 ± 0.5), whereas the groups fed STP-8 and UHT-12 had normal coat colors (scores: 1.0 ± 0.0 and 1.3 ± 0.5, respectively). In addition, the tails of the monkeys in the RTF-12 group were completely bare, whereas in all other groups they were covered with hair.
View this table:
TABLE 1.. Characteristics of infant rhesus monkeys fed different formulas1
Hemoglobin and hematocrit values were significantly lower in the RTF-12 group than in all other groups at ages 4 and 5 mo (Table 2). Serum ferritin concentrations were significantly lower in the RTF-12 group than in the STP-12 group at age 5 mo. Two-factor repeated-measures analysis of variance showed that there was a significant age effect for both hemoglobin and serum ferritin; both indexes decreased with increasing postnatal age.
View this table:
TABLE 2.. Hematologic and serum ferritin concentrations of infant rhesus monkeys at different ages1
Plasma copper concentrations were significantly lower in the RTF-12 group than in the STP-8 and UHT-12 groups at age 4 mo and than in all other groups at ages 3 and 5 mo (Figure 1). The STP-12 group also tended to have lower plasma copper concentrations than did the STP-8 and UHT-12 groups at ages 3–5 mo, but only significantly so at age 4 mo. Plasma ceruloplasmin activity was significantly lower in the RTF-12 group than in the STP-8 and STP-12 groups at age 2 mo, than in all other groups at age 3 mo, and than in the RTF-12 and the STP-12 groups than in the other 2 groups at ages 4 and 5 mo (Figure 2). Similarly, RBC Cu/Zn SOD activity was significantly lower in the RTF-12 and STP-12 groups than in the STP-8 and UHT-12 groups at age 2 mo, whereas STP-8, STP-12, and RTF-12 groups had lower RBC Cu/Zn SOD activity than did the UHT-12 group at ages 3 and 4 mo (Figure 3). At age 5 mo, the UHT-12 group had significantly higher Cu/Zn SOD activity than did all other groups.
FIGURE 1. . Mean (±SD) serum copper concentrations of infant rhesus monkeys fed different infant formulas: STP-8, standard thermal processing, glass bottles, 8 mg Fe/L; STP-12, standard thermal processing, glass bottles, 12 mg Fe/L; RTF-12, ready-to-feed thermal processing, cans, 12 mg Fe/L; UHT-12, ultrahigh-temperature treatment, 12 mg Fe/L. n = 6 animals/group. Means with different superscript letters are significantly different, P < 0.05.
FIGURE 2. . Mean (±SD) serum ceruloplasmin concentrations of infant rhesus monkeys fed different infant formulas: STP-8, standard thermal processing, glass bottles, 8 mg Fe/L; STP-12, standard thermal processing, glass bottles, 12 mg Fe/L; RTF-12, ready-to-feed thermal processing, cans, 12 mg Fe/L; UHT-12, ultrahigh-temperature treatment, 12 mg Fe/L. n = 6 animals/group. Means with different superscript letters are significantly different, P < 0.05.
FIGURE 3. . Mean (±SD) erythrocyte activity of Cu/Zn superoxide dismutase (SOD) in infant rhesus monkeys fed different infant formulas: STP-8, standard thermal processing, glass bottles, 8 mg Fe/L; STP-12, standard thermal processing, glass bottles, 12 mg Fe/L; RTF-12, ready-to-feed thermal processing, cans, 12 mg Fe/L; UHT-12, ultrahigh-temperature treatment, glass bottles, 12 mg Fe/L. n = 6 animals/group. Means with different superscript letters are significantly different, P < 0.05. Hb, hemoglobin.
Data analysis indicated that there were no significant differences in iron status between the 2 STP groups at age 5 mo (Figure 4). However, it is evident that the iron concentration in the formula affects copper status. Infants fed the STP-12 formula had significantly lower plasma ceruloplasmin concentrations than did infants fed the STP-8 formula; serum copper concentrations also tended to be lower, although this difference was only marginally significant (P = 0.07). There were no significant differences in plasma zinc concentrations between any of the groups, although there was a significant increase in zinc concentrations with age in all groups (data not shown).
FIGURE 4. . Indicators of iron and copper status at age 5 mo in infant rhesus monkeys fed the same standard thermal process formula with either 8 or 12 mg Fe/L (STP-8 and STP-12). *Significantly different from STP-12, P < 0.05. SOD, superoxide dismutase.
DISCUSSION
There were no significant differences in any of the hematologic indexes (hemoglobin, hematocrit, and mean corpuscular volume) or iron status (serum ferritin) between groups receiving formula manufactured with identical thermal processing but that contained 2 different iron concentrations (STP-12 compared with STP-8). This shows that a concentration of 8 mg Fe/L is adequate to meet the iron requirements of infant rhesus monkeys exclusively formula-fed for the first 5 mo of life. In addition, it shows that there is no further advantage to iron status of fortifying formula with 12 mg Fe/L. We previously showed that, similar to human infants, exclusive breast-feeding or feeding infant formula containing 12 mg Fe/L to infant rhesus monkeys results in satisfactory iron status at age 5 mo, whereas feeding a low-iron formula (1.5 mg Fe/L) results in significantly lower iron status (20). We therefore believe that the infant rhesus monkey is a sensitive model to assess the capacity of infant diets to provide iron and to obtain valuable information before clinical trials are conducted in human infants. A more recent study in human infants showed that infant formula containing 8 mg Fe/L resulted in satisfactory iron status and that formula with 12 mg Fe/L provided no advantages with regard to hematology or iron status (8). These investigators, using a stable isotope of iron, showed that the percentage of erythrocyte incorporation of iron was higher from the formula containing 8 mg Fe/L than from the same formula containing 12 mg Fe/L. It is well known that the amount of iron in the diet will affect iron absorption (21) and this study showed that the net amount of accumulated iron was the same for both formulas.
We previously showed in human infants that feeding a higher iron concentration (7 mg Fe/L) in powdered formula results in lower plasma copper and ceruloplasmin concentrations at age 6 mo than in infants fed the same formula with a lower iron concentration (4 mg Fe/L) (10). Similarly, Haschke et al (22) showed that infants fed formula with a high iron concentration (10.2 mg Fe/L) had a significantly lower copper balance than did infants fed formula with a low concentration of iron (1.5 mg Fe/L). Other studies in low-birth-weight infants and children also showed an interaction between iron and copper status (23, 24). In the present study, we found significantly lower copper status, as indicated by plasma copper, ceruloplasmin activity, and RBC Cu/Zn SOD activity in rhesus infants fed the STP-12 than in those fed the STP-8 formula. Thus, it appears evident that even modestly higher iron concentrations in infant formula have a negative effect on copper status. To illustrate this point, we compiled the results for iron, copper, and zinc status in Figure 4. The mechanism behind this interaction between iron and copper is not yet known but it is likely to occur at the level of absorption. Several studies in rats documented such an interaction (25, 26).
There appears to be no negative effect of the higher iron concentration in the STP-12 formula on zinc status as assessed by plasma zinc. This is also similar to our observations in human infants fed formula with 2 different iron concentrations (10). Although it is known that iron can affect zinc absorption, this appears to occur only at a relatively high iron-to-zinc ratio and only when the 2 elements are given in a water solution (27). In a previous study, an infant formula with a very low zinc concentration (1 mg Zn/L) was fed to infant rhesus monkeys for 4 mo with either a low (1 mg Fe/L) or a somewhat higher (4 mg Fe/L) concentration of iron (5). No significant differences in plasma zinc concentrations were found between the groups at any time point. In addition, zinc absorption, as assessed with the use of 65Zn and whole-body counting (5), was similar in both groups at all time points. Thus, it seems plausible that iron fortification at concentrations conventionally used in infant formula does not interfere with zinc absorption or zinc status (plasma zinc).
A surprising and striking observation in the present study was the very low copper status of the infants fed the RTF-12 formula. These infants had very low plasma copper, ceruloplasmin, and RBC Cu/Zn SOD activity, clearly showing a state of copper deficiency. Consistent with these biochemical markers, coat color was dramatically altered from a normally reddish-brown color to a pale silvery-gray color. Copper deficiency is wellknown to affect the pigmentation of skin and hair through a decrease in melanin formation (28). The cause of the remarkable and complete hair loss on the monkey's tails is not known, but it is known that copper deficiency affects connective tissue (29), possibly causing hair loss in areas of the body where skin turnover is rapid, eg, the tail. Additionally, the infants fed this formula had significantly low hemoglobin and hematocrit values at ages 3, 4, and 5 mo, which is consistent with the known anemia of copper deficiency (28). This anemia is caused by a reduction in ceruloplasmin activity; ceruloplasmin should be more accurately called ferroxidase I because it is required for the proper oxidation of iron in the pathway of releasing iron from the liver (+II) into circulating transferrin (+III). Thus, iron accumulates in body stores, primarily in the liver, and hemoglobin synthesis is impaired during copper deficiency.
It is not yet known what caused the low copper status in the infants fed the RTF-12 formula. One contributing factor may be the type of thermal processing used. The temperature used during formula manufacture was possibly higher or the duration of the heat exposure longer during processing of the RTF formula in the large cans than that used for the smaller cans; however, detailed information is considered proprietary and not available from the manufacturers. It is possible that complexes are formed between copper and some other constituent of the formula; that the oxidation of copper is altered, thereby affecting absorption; or that some type of interaction is induced that interferes with copper absorption, transport, or excretion. Further studies at a biochemical level are needed to explore these potential mechanisms.
REFERENCES