Adequacy of Infant Formula With Protein Content of 1.6 g/100 kcal for Infants Between 3 and 12 Months

Abstract

Objectives: 

Infant formulas provide more protein than breast milk. High protein intakes may place infants at risk of later obesity. The present study tested whether a formula with protein content below the regulatory level supports normal growth from age 3 months.

Methods: 

Randomized double-blind trial enrolled healthy infants less than age 3 months. At 3 months, formula-fed infants were assigned to experimental (EXPL, 1.61 g protein/100 kcal; modified bovine whey proteins with caseinoglycomacropeptide removed) or control (CTRL 2.15 g protein/100 kcal; unmodified bovine milk protein with a whey/casein ratio of 60/40) formula; breast-fed (BF) infants were enrolled in a reference group. Complementary foods were allowed in small amounts from 4 to 6 months and unrestricted after 6 months.

Results: 

Weight gain (g/day) from 3 to 6 months was similar in the EXPL and CTRL groups (EXPL-CTRL −0.84 g/day; 95% confidence interval −2.25 to 0.57) and faster in the EXPL and CTRL groups than in the BF group. Weight analyzed longitudinally from 4 to 12 months was lower in the EXPL group than in the CTRL group (P = 0.031) but higher than in the BF group (P < 0.0001). Longitudinal analysis of odds ratios from 4 to 12 months indicated fewer infants with weight >85th percentile in the EXPL group than in the CTRL group (P = 0.015). Length z scores were lower than, and body mass index z scores were similar to, World Health Organization Standards in all of the groups. Serum biochemical parameters in the EXPL group reflected lower protein intake and were closer to parameters in the BF infants than in the CTRL group.

Conclusions: 

A formula with 1.61 g of protein/100 kcal supports normal growth of infants after age 3 months. This protein content is adequate if provided from a high-quality source.

What Is Known

• Infants’ protein needs per kilogram of body weight decrease with age.
• Formulas with the regulatory minimum protein content of 1.8 g/100 kcal provide more protein than infants need after age 3 months.
• High protein intakes in infancy may lead to obesity later.
• What Is New
• Formula with 1.61 g of protein/100 kcal supported normal growth after age 3 months; weight gain (g/day) from 3 to 6 months was similar to higher protein formula.
• Weight analyzed longitudinally from 4 to 12 months was lower in low- versus higher-protein formula.
• Weight >85th percentile was less common with low- versus higher-protein formula.

The protein needs of the infant are high in the first months after birth but decrease during the first year of life. Dewey et al ⁽¹⁾ estimated average safe levels of protein intake at 2.69 g · kg⁻¹ · day⁻¹ in the first month of life, decreasing to 1.37 g · kg⁻¹ · day⁻¹ by 3 to 4 months and to 1.02 g · kg⁻¹ · day⁻¹ by 9 to 12 months. These intake levels formed the basis for the recommendation for minimum protein content of infant formulas (1.8 g/100 kcal) by the Scientific Committee on Food ⁽²⁾. An infant's changing needs for protein are reflected in the protein content of human milk, which decreases from an average of 2.09 g/100 kcal during month 1 to 1.28 g/100 kcal at 3 to 4 months and about 1.24 g/100 kcal by 9 to 12 months ⁽³⁾. Infant formulas are designed to meet the protein needs during the first months of life but inevitably provide unnecessarily high amounts of protein when fed to older infants. In analogy to human milk, an adequate protein content of formulas fed after 3 months could be assumed to be 1.30 g/100 kcal, that is, slightly above the level in human milk, as long as the protein is of high quality. The regulatory lower limit of formula protein concentration is 1.80 g/100 kcal in the European Union and the United States.

Epidemiologic evidence links high protein intakes in infancy to obesity in childhood ^(4–7). Also, high protein intakes have been shown in prospective studies to lead to increased weight gain and higher adiposity in infancy ⁽⁸⁾ and childhood ^(9,10). The possible mechanism(s) linking protein intakes to adiposity range from protein inducing higher energy consumption all the way to higher protein intakes providing greater quantities of insulinogenic amino acids. Also, it is evident that only a minority of infants respond to higher protein intakes with increased adiposity. If these high-risk infants could be identified, preventive efforts could be specifically directed at them. It is not known whether infants respond to protein concentrations in feedings or to protein quantities received. Protein intakes in the later parts of infancy have been found in several localities to be very high and to exceed the required intakes ^(4,11–13). Lower protein intake from breast milk compared with formula is thought to be among the reasons why breast-fed infants are at a lower risk of obesity later in life, as the preponderance of evidence indicates ^(14–17). Based on the above evidence, Michaelsen and Greer ⁽¹⁸⁾ concluded that high protein intake during the first 2 years of life is a risk factor for later development of overweight and obesity. Recently, it was proposed that different formulas should be used for different ages during the first year of life to better fit the metabolic needs of infants and to avoid nutrient limitations or excesses ⁽¹⁹⁾. For these reasons, lowering the protein content of a formula fed after age 3 months seems desirable.

Few studies have examined the effects on growth of formulas with protein content <1.80 g/100 kcal. Fomon et al ⁽²⁰⁾ showed that a protein content of 1.25 g/100 kcal led to significantly slowed weight gain between 3 and 4 months. Two studies involving formulas with protein content of 1.65 ⁽²¹⁾ and 1.7 g/100 kcal ⁽²²⁾ provided evidence that, paradoxically, lower-protein formulas may be associated with somewhat greater weight gain than breast-feeding ^(21,22) or formula with higher protein content ⁽²²⁾, although it should be noted that the formulas used in these studies were not comparable to feeding human milk.

The present study was conducted in healthy full-term infants. An earlier study with a similar design with formulas of identical protein composition involved infants whose mothers were overweight (body mass index [BMI] 25–30 kg/m²) or obese (BMI >30 kg/m²). It was found in the earlier study that the low-protein formula slowed weight gain between 3 and 6 months but was adequate in supporting normal growth ⁽²³⁾. The purpose of the present study was to establish the adequacy (safety) of a formula with a protein content of 1.61 g/100 kcal (primary outcome). We also sought to determine whether the low-protein formula may decrease the proportion of infants who were growing rapidly.

METHODS

Study Design

This study tested the hypothesis that a formula with the unconventionally low protein content of 1.61 g/100 kcal supports the growth of infants after age 3 months just as well as a formula with conventional higher protein content. The formulas were compared in double-blind, randomized fashion. A breast-fed reference group was studied concurrently. The specific study hypothesis was that the formula with the low protein content would lead to growth similar to that of infants fed the control formula. Also, it was hypothesized that in infants fed the lower-protein formula, serum, biochemical parameters would be more similar to those of breast-fed infants than those of infants fed the control formula. The experimental low-protein formula (EXPL) had a protein content of 1.61 g/100 kcal, whereas the control formula (CTRL) had a protein content of 2.15 g/100 kcal. The study formulas were fed from 3 to 12 months, with the period from 3 to 6 months considered the primary outcome period because during that period study formulas were the near-exclusive source of nutrients.

Infants were enrolled at or before 3 months (84 ± 4 days) of age. Infants who had been fed formula for ≥2 weeks were randomly assigned to one of the study formulas. Study formulas were provided to the families free of charge. Complementary foods were allowed in small amounts from 4 to 6 months and in unrestricted amounts after 6 months. Infants who were breast-fed at 3 months and whose mothers indicated their intent to breast-feed to ≥6 months were enrolled in the breast-fed (BF) reference group. The study protocols were reviewed and approved by the institutional review boards of each study center, and parents gave written consent at the time of enrollment. The study was conducted between July 2008 and December 2010 at 2 sites: the University of Iowa in Iowa City, which used satellite clinics in Cedar Rapids and Davenport, Iowa, and the University of Oklahoma Health Sciences Center in Oklahoma City. Random allocation sequences were generated with the Nestlé Trial Balance application (Nestlé Research Center, Lausanne, Switzerland) using secure access via the Internet. Randomization was performed with stratification for study center, sex, and BMI of the mother before pregnancy (<25, between 25 and 30, and ≥30 kg/m²).

Sample Size

Weight gain (g/day) between 3 to 6 months of age was considered the primary study outcome. Calculation of sample size was based on weight gain data between 3 to 6 months published by Butte et al ⁽²⁴⁾. A sample size of 88 infants per formula group was calculated using a 2-g/day difference in weight gain, a standard deviation of 4 g/day, a type I error (α) of 5%, and a power of 90%. Recruitment of up to 22 additional infants per group was planned to compensate for an expected 20% dropout rate.

Subjects

Infants born ≥37 weeks of gestation with birth weight ≥2500 g and ≤4500 g who were considered otherwise normal and healthy by their parents and by the investigators were enrolled at or before 3 months of age. Infants were born between April 2008 and January 2010. Multiple births were excluded as were infants with illnesses or malformations that could affect growth, infants with suspected or confirmed allergy to cow's-milk protein, and infants who participated in another clinical trial.

Study Feedings

The composition of study formulas is presented in supplementary Table S1 (http://links.lww.com/MPG/A497). The EXPL formula had a protein content of 1.61 g/100 kcal and a caloric density of 67.2 kcal/100 mL, whereas the CTRL formula had a protein content of 2.15 g/100 kcal and a caloric density of 64.6 kcal/100 mL. The CTRL formula was a commercially available formula (NAN 1; Nestlé Nutrition, Nestec Ltd, Vevey, Switzerland), which was manufactured and marketed in Mexico. It contained unmodified bovine milk proteins with a whey/casein ratio of 60/40. The EXPL formula was prepared specifically for the present study. Its protein consisted of bovine whey proteins that were modified by the removal of caseinoglycomacropeptide, ⁽²⁵⁾ which results in higher tryptophan and lower threonine content. It contained essential and branched-chain (insulinogenic) amino acids in amounts close to those of mature breast milk ⁽²⁶⁾. Both formulas contained levels of minerals, vitamins, and trace elements corresponding to specifications of the Codex Alimentarius⁽²⁷⁾. A color-coded formula (Standard Formula) that was identical in composition to the CTRL formula was provided to formula-fed infants between the time of enrollment and randomization and was also provided to the parents of breast-fed infants who wished to give supplemental formula. The carbohydrate was lactose in the EXPL formula and lactose/corn syrup in the CTRL formula. The fat in the formulas was similar except that the CTRL formula also contained sunflower oil. The formulas were provided in powder form in color-coded cans. The identity of the formulas was not known to the investigators or the parents.

The assigned formulas were fed from enrollment until 12 months of age. Parents were requested to feed no complementary foods until age 4 months. Thereafter, up to 3 teaspoons of dry cereal could be fed each day and, beginning at 5 months, up to 2 tablespoons of single-ingredient first or second foods. After age 6 months, there were no restrictions on the amount or kind of complementary foods. The same feeding rules applied to breast-fed infants. If parents wished to initiate supplemental formula feeding in breast-fed infants, which was permitted after age 6 months, standard formula was provided.

Procedures

After enrollment, study visits occurred every 28 days (±4 days) until age 168 days, after which visits occurred within 14 days of ages 240, 300, and 360 days. During visits, weight was determined to the nearest 10 g and length to the nearest 1 mm using established methods ⁽²⁸⁾. Head circumference was determined to the nearest 0.5 cm using a nonstretchable tape applied above the eyebrows and over the largest circumference of the skull. At each visit, parents completed a feeding questionnaire. The information provided was used to determine the amount of formula and complementary foods, if any, that the infant consumed during the preceding 2 days.

During visits at 83, 168, and 360 days, capillary blood was drawn by heel stick using a disposable spring-loaded device (Tenderfoot; International Technidyne Corporation, Edison, NJ). Approximately 1 mL of blood was collected into heparin-coated tubes. All of the laboratory determinations were performed at the Iowa site. Albumin and blood urea nitrogen (BUN) were measured by standard laboratory methods. Insulin-like growth factor 1 was determined with a 2-site immunoradiometric assay (Diagnostic Systems Laboratories, Webster, TX), and C-peptide was analyzed using a sandwich-type immunoassay (ALPCO Diagnostics, Salem, NH). Body composition was determined in a subset of study infants. These results will be reported separately.

Data Analysis

The intention-to-treat (ITT) analysis was conducted using the full analysis set, which included all of the randomized subjects with available data. Per protocol (PP) analysis included subjects who complied with the feeding rules between 3 and 6 months. To be included in the PP analysis, infants could not be hospitalized for >3 days, could not be off the assigned feeding for >3 consecutive days, and could not receive complementary foods in greater than specified amounts between 4 and 6 months of age. Infants were also excluded from PP analysis if they received >240 mL of formula per week (breast-fed infants) or received >1 feeding per week of a nonstudy formula (formula-fed infants). Weight gain between 3 and 6 months (g/day) was assessed by analysis of covariance (ANCOVA) with correction for weight gain between 0 and 3 months, sex, and prepregnancy maternal BMI. Noninferiority was evaluated using a margin of −3 g/day ⁽²⁹⁾.

Anthropometric data were converted to z scores using the World Health Organization (WHO) Growth Standards ⁽³⁰⁾. Anthropometric parameters (as z scores) at 3 months were compared by ANCOVA correcting for baseline, sex, and maternal BMI as a continuous variable. Between 4 and 12 months, longitudinal analysis of anthropometric parameters and comparison of z scores of anthropometric parameters was performed employing a mixed model to test for different time trends ⁽³¹⁾. Fixed effects were the value at 3 months, sex, maternal BMI as a continuous variable, visit, treatment, and visit × treatment; random effect was the subject. A likelihood ratio test was performed versus a reduced model without the treatment and visit × treatment fixed effects. The percentages of infants in the EXPL and CTRL groups with weight above the 85th percentile of the WHO standards at 4 to 12 months were compared by calculating odds ratios (ORs) and 95% confidence intervals (CIs) by logistic regression correcting for baseline, sex, and maternal BMI. Longitudinal analysis of ORs between 4 and 12 months was performed by a generalized linear mixed-effect model ⁽³²⁾.

Adequacy of growth was considered to be present if the lower bound of the 95% CI around the mean weight-for-age z score of the EXPL group was above −0.5 SD of the WHO standard ⁽³⁰⁾ in the ITT population. Serum biomarkers that showed log-normal distribution were analyzed after logarithmic transformation, but data are presented as arithmetic means and standard deviations. Only PP data are presented because biochemical parameters are strongly influenced by actual nutritional intake. Biomarkers were analyzed by ANCOVA correcting for the measurement value at baseline.

RESULTS

A total of 194 formula-fed infants were randomized at age 3 months (Fig. 1); 183 formula-fed infants completed the study to age 6 months, and 174 formula-fed infants completed the study to age 12 months. Of 112 BF infants enrolled, 109 completed the study to 6 months and 105 completed the study to 12 months. A common reason for discontinuation was parent's desire to feed complementary foods. In the case of breast-fed infants who discontinued, mothers often felt the need to start supplemental formula or discontinued breast-feeding entirely.

FIGURE 1: Flow of study participants (ITT population). BF = breast-fed; CTRL = control formula; EXPL = experimental formula; ITT = intention-to-treat.

Characteristics of study infants at birth and of their mothers are summarized in supplementary Table S2 (http://links.lww.com/MPG/A498). There were no differences between infants in the 3 groups. Mothers who breast-fed, however, had significantly lower weight and BMI than mothers of formula-fed infants. They also tended to smoke less during pregnancy.

At randomization at age 3 months, anthropometric parameters of infants in the EXPL and CTRL groups were similar (ANCOVA). Weight gain between 3 and 6 months (Table 1) was slightly, but not significantly, lower in the EXPL group than in the CTRL group. Weight gain in both formula groups was higher than in the BF group. The lower bound of the 95% CI for difference in weight gain between EXPL and CTRL was above the noninferiority margin of −3 g/day at all of the time intervals, demonstrating noninferiority of weight gain for EXPL.

TABLE 1: Weight gain (g/day) of infants and differences between groups, ITT data set

Weight predicted by the longitudinal analysis (mixed model) was significantly (P < 0.0001) higher in the infants in the EXPL and CTRL groups than in the infants in the BF group between 4 and 12 months (Fig. 2). A likelihood ratio test indicated that the difference in weight between EXPL and CTRL was small but statistically significant (P = 0.031), with EXPL lower than CTRL during the intervention period from 4 to 12 months. ORs indicating the percentage of infants whose weight was above the 85th percentile (+1 SD of the mean) of the WHO Standard ⁽³⁰⁾ are presented in Table 2. ORs tended to be lower in EXPL between 5 and 12 months, but the difference became significant only at 12 months. Longitudinal analysis indicated lower ORs in the EXPL group during the time of the intervention from 4 to 12 months (P = 0.015).

FIGURE 2: Longitudinal analysis of weight from 4 to 12 months of age. Graph shows predicted means and 95% confidence intervals estimated by the mixed model. Significant differences between groups: P = 0.031 for EXPL versus CTRL, P < 0.0001 for BF versus EXPL and BF versus CTRL. BF = breast-fed; CTRL = control formula; EXPL = experimental formula.

TABLE 2: ORs for infants to be above the 85th percentile (+1 SD) of WHO weight-for-age z scores

Weight-for-age z scores (Table 3) of formula-fed infants were significantly greater than those of BF infants between 5 and 12 months. The latter were below the WHO standards between 3 and 12 months. Weight-for-age z scores were slightly lower in EXPL than in CTRL infants (ANCOVA), but the difference became statistically significant only at 12 months. Boys and girls behaved differently in that z scores of boys were significantly lower in the EXPL group than in the CTRL group between 8 and 12 months, whereas in girls feeding-related differences were not significant. Comparison of weight-for-age z scores with WHO standards showed that in the EXPL group, the lower bound of the 95% CI around the mean weight-for-age z scores was above −0.5 SD of the WHO standard. This indicates that infants in the EXPL group had adequate growth compared with the WHO standards.

TABLE 3: Weight-for-age z scores and differences between groups, ITT data set

The length predicted by the longitudinal analysis is shown in supplementary Figure S1 (http://links.lww.com/MPG/A499). The length of the 2 formula groups was nearly identical, but was higher (P < 0.0001) than the length of the BF infants. Length-for-age z scores were similar in the EXPL and CTRL groups until 12 months (supplementary Table S3, http://links.lww.com/MPG/A500) but were significantly higher in formula-fed infants than in BF infants between 6 and 12 months. Length-for-age z scores were below the WHO standards in the formula and BF groups at almost all of the time points (even though at birth values were significantly higher than WHO standards). In all of the 3 groups, the lower bound of the 95% CI was below −0.5 SD at several time points. Thus, the equivalence margins were not met for length. Length-for-age z scores, however, in the EXPL group were not significantly different from CTRL and were significantly higher than in the BF group.

Supplementary Figure S2 (http://links.lww.com/MPG/A501) shows BMI as predicted by the longitudinal analysis. Differences between groups were not statistically significant. BMI-for-age z scores showed small nonsignificant differences between formula groups (supplementary Table S4, http://links.lww.com/MPG/A502). CTRL infants had higher BMI z scores than BF infants between 8 and 12 months. BMI-for-age z scores of the formula groups were at or above the WHO standards but in the BF group were below the WHO standards until 6 months. Mean z scores for head circumference at 6 months and at the end of the intervention (12 months) were not significantly different between the infants fed EXPL or CTRL (data not shown).

Concentrations of serum biomarkers are indicated in supplementary Table S5 (http://links.lww.com/MPG/A503). At 6 months, markers reflected nearly exclusively the effects of the assigned feedings, whereas at 12 months the effects of the assigned feedings were somewhat diluted by the diversified complementary feedings. Differences in albumin concentration at 6 months (the end of near-exclusive formula feeding) were not significant between the 3 groups. At 12 months, albumin concentration was significantly higher in the CTRL group than in the BF group. Concentrations of BUN were higher at 6 months in the CTRL group than in the EXPL group or the BF group; the difference between EXPL and BF was not significant. At 12 months, BUN was still higher in the CTRL group than in any other group, although the difference between CTRL and BF was no longer significant. Concentrations of C-peptide at 6 months were higher in both formula groups compared with the BF group, but at 12 months concentrations were higher in the CTRL group than in any other group. Similarly, concentrations of insulin-like growth factor 1 were higher at 6 and 12 months in the formula groups compared with the BF group.

Formula intake (mL/day) and protein intake (g/day) are shown in supplementary Table S6 (http://links.lww.com/MPG/A504). During near-exclusive formula feeding (between 4 and 6 months), formula intake was slightly higher in the EXPL group than in the CTRL group, though the difference was not significant at any time point (supplementary Table S7, http://links.lww.com/MPG/A505). Median intake of protein in the CTRL group was 2.26 g · kg⁻¹ · day⁻¹ at 4 months and 1.89 g · kg⁻¹ · day⁻¹ at 6 months, whereas in the EXPL group it was 1.67 g · kg⁻¹ · day⁻¹ at 4 months and 1.50 g · kg⁻¹ · day⁻¹ at 6 months (ITT population). The median protein intake at 4 months was thus higher in the EXPL group than the safe intake level of 1.37 g · kg⁻¹ · day⁻¹ at 3 to 4 months ⁽¹⁾. Similarly, the median protein intake at 6 months was greater than the adequate intake level ⁽¹⁾ at 5 to 6 months. Only 2% of the infants in the CTRL group had protein intakes below the safe levels at 4 and 6 months, whereas among infants in the EXPL group 11% and 16% of infants (ITT population) had protein intakes below the safe levels. None of the serum biomarkers related to protein intake (supplementary Table S5, http://links.lww.com/MPG/A503), however, indicated deficiency of protein intake in the EXPL group when compared with BF infants, suggesting that protein intakes were meeting the needs of individual infants.

DISCUSSION

In the past, the main objective of setting safe protein intake levels has been to ensure that all infants receive sufficient protein intakes, with the realization that safe protein intakes tend to be well above the requirement levels. This is supported by comparison of safe protein levels with intakes achieved by breast-fed infants. Thus, what used to be a concern about too little protein has turned into a concern about possibly too much protein. Safe levels of protein intake for normal infants have been estimated to be 2.69 g · kg⁻¹ · day⁻¹ (2.24 g/100 kcal) at 1 month, decreasing to 1.37 g · kg⁻¹ · day⁻¹ (1.44 g/100 kcal) at 3 to 4 months, 1.19 g · kg⁻¹ · day⁻¹ (1.32 g/100 kcal) at 6 months, and 1.02 g · kg⁻¹ · day⁻¹ (1.13 g/100 kcal) by 9 to 12 months ^(1,2). Therefore, formulas with protein content at the lowest regulatory level (1.8 g/100 kcal), when fed to infants older than 3 months, provide protein intakes that exceed protein needs by a substantial margin (31%–76%) that increases with increasing age of the infant. Formulas also provide greater amounts of protein than are received by the breast-fed infant ⁽³⁾. In the past, when the quality of the protein used in formulas was inferior, it was necessary to keep the protein content of formulas at a level that exceeded that of breast milk by a considerable margin. With advances in milk (whey) protein technology, the protein quality gap between formula and breast milk with regard to essential and branched-chain (insulinogenic) amino acids has been narrowed or possibly eliminated altogether ⁽³³⁾. Therefore, there is little rationale for the protein content of formulas to exceed that of breast milk by more than a modest margin.

The present study assessed the performance of infants fed a formula with a protein content of 1.61 g/100 kcal, which is lower than the regulatory level. The formula was fed from 3 months on. The low-protein formula provided a greater protein concentration (g/100 kcal) than breast milk at 3 months lactation, and protein intakes (g/100 kcal) exceeded safe intake levels for protein ⁽¹⁾. The study was undertaken in the expectation that the low-protein formula would meet the protein needs of all of the infants and would lead to normal growth and serum biochemical parameters. The study showed that the growth of infants fed the EXPL formula was actually slightly faster than that of breast-fed infants and greater than the growth indicated by international growth standards ⁽³⁰⁾. It must be pointed out that in the present study, >50% of mothers of formula-fed infants had a BMI of >25 kg/m², whereas among mothers of breast-fed infants the percentage with BMI >25 kg/m² was substantially lower. The WHO growth standards are based on infants whose mothers had BMIs between 18 and 25 kg/m²⁽³⁰⁾. The CTRL formula with conventional (high) protein content produced a tendency toward slightly faster growth and higher serum markers of protein nutrition.

Few studies have explored the adequacy of formulas with protein content below the regulatory lower limit of 1.8 g/100 kcal. Picone et al ⁽²¹⁾ studied normal infants from birth to 12 weeks of age who were breast-fed or were fed 1 of 3 formulas with protein content 1.65, 1.96, and 2.19 g/100 kcal, respectively. Protein of formulas was provided by unmodified bovine whey proteins and casein in a ratio of 50/50. Infant weight, length, and head circumference did not differ between feeding groups at any age, but gains in weight and length of infants fed the formula with protein content of 1.65 g/100 kcal were significantly (P < 0.05) greater than those of breast-fed infants. In a study by Fomon et al ⁽²⁰⁾, infants were fed between 8 and 112 days varying combinations of 2 low-protein formulas to emulate the declining protein intakes of breast-fed infants. Protein was provided from partially demineralized whey and nonfat milk. During the age interval of 84 to 112 days, the only interval with relevance to the present study, the protein/energy ratio was 1.25 g/100 kcal. The mean weight gain was 21.0 (SD 5.9) g/day compared with 24.1 (SD 7.3) g/day in a reference group of formula-fed infants; the difference was not statistically significant. Gain in length for the entire study period from 8 to 122 days was significantly lower in the low-protein group. Serum albumin concentrations at 112 days were similar, but serum urea nitrogen averaged only 3.4 mg/dL in the low-protein group, which was significantly lower than in the reference group. It was concluded that the protein content of the low-protein formula was below the safe level.

In a third study testing a low-protein formula, Fomon et al ⁽²²⁾ fed a formula with a protein content of 1.7 g/100 kcal provided by fat-free cow's-milk solids. From 8 to 112 days, gain in length was similar to that of a formula-fed reference group, but weight gain was significantly higher (37.7 g/day) than that of the reference group (32.2 g/day). Energy intake in the experimental group was similar to that in the reference group. Serum albumin and BUN concentrations at 112 days were similar to those in a breast-fed reference group (the formula-fed reference group did not have serum albumin and urea determined at 112 days). Fomon et al ⁽²²⁾ concluded that the protein content was adequate but may not have been safe for infants between birth and 4 months of age. It must be pointed out that in the above studies ^(20–22), formulas contained unmodified whey proteins and therefore must be assumed to have lacked the improved amino acid profile exemplified by the formula used by Inostroza et al ⁽²³⁾.

The earlier study ⁽²³⁾ testing a formula with a protein content below the regulatory level used a formula with the same modified whey as used in the present study. The study involved infants whose mothers were overweight or obese with a BMI >25 kg/m². The low-protein formula (protein content 1.65 g/100 kcal) was fed from 3 months on. Between 3 and 6 months, growth was judged to be normal. A control formula with higher protein content produced significantly faster weight gain between 3 and 6 months. The present study used a similar design but involved infants of mothers not selected for overweight or obesity. It had the purpose of establishing the adequacy (safety) of a formula with a protein content of 1.61 g/100 kcal.

In the Childhood Obesity Project ⁽⁸⁾, where the differences in formula protein content were far greater than in the present study, the low-protein feeding led to lower gain in weight and BMI in the first year of life. At follow-up at age 6 years ⁽⁹⁾, differences in weight and BMI were found to persist. Children who had received the low-protein formula had lower BMI, and a smaller percentage was classified as obese. At either age, differences in BMI were largely explained by differences in weight, whereas length (height) was unaffected. In the present study, differences in formula protein content were much smaller than in the Childhood Obesity Project ⁽⁸⁾, but still there was a lower OR for high (>85th percentile) weight. It appears possible that a low protein intake in infancy slows weight gain particularly in the fastest growing infants who are at an increased risk of later obesity, as was also observed in the study by Inostroza et al ⁽²³⁾.

In our study, differences between boys and girls were observed in that weight z scores of boys were significantly lower in the EXPL group than in the CTRL group between 8 and 12 months, whereas in girls feeding-related differences were not significant. The endocrine response to a high-protein diet early in life is sex-dependent, although differences did not translate to differences in growth ⁽³⁴⁾.

In the present study, the CTRL formula had a somewhat lower caloric density than the EXPL formula. This small difference is unlikely to have had an effect on energy intake because infants have been shown to compensate completely for even larger differences (6 kcal/100 mL) in caloric density ⁽³⁵⁾. One would have expected intake volumes to be somewhat larger with the CTRL formula, but a trend in the opposite direction was seen, perhaps because of a mechanism similar to that explaining the somewhat higher weight gain of infants fed low-protein formulas ^(20,22).

In conclusion, the present study establishes that a formula containing 1.61 g/100 kcal of a high-quality protein, which is below the regulatory minimum level (1.80 g/100 kcal), supports normal growth of infants after 3 months of age. Although weight gain in infants fed the lower-protein formula, did not differ significantly from that in infants fed standard formula, and was higher than that of breast-fed infants, infant weight analyzed longitudinally from 4 to 12 months was lower in the infants fed the low protein formula compared with those fed the standard formula. Additionally, infants fed lower-protein formula were less likely to have weight >85th percentile than those fed the standard formula.