Plasma Essential Amino Acids and Gastrointestinal Tolerance

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ORIGINAL ARTICLE a-Lactalbumin-rich infant formula fed to healthy term infants in a multicenter study: plasma essential amino acids and gastrointestinal tolerance

AM Davis1, BJ Harris1, EL Lien2, K Pramuk1 and J Trabulsi1

1Wyeth, Collegeville, PA, USA and 2Department of Food Science and Human Nutrition, University of Illinois, Urbana, IL, USA

Objective: To evaluate the efficacy and safety of an a-lactalbumin-enriched formula with a protein profile and total protein concentration closer to human milk (HM) and lower than conventional formulas. Subjects/methods: Two hundred and sixteen healthy, term infants, p14 days post-natal age were enrolled and 166 (76.9%) completed the study. Timed post-prandial plasma essential amino-acid levels were determined after 8 weeks of ad libitum study feeding. Study events were assessed every 2 weeks. Results: At 8 weeks, all mean plasma essential amino-acid levels in the experimental formula (EF) group were as high as the standard formula (SF) and HM groups. The incidence of feeding-related gastrointestinal (GI) events varied significantly (P ¼ 0.025) across groups: SF (31.3%), EF (17.2%) and HM (13.6%), with SF being significantly higher than HM (P ¼ 0.015). Study withdrawals due to feeding-related GI events were significantly different (P ¼ 0.001) across groups: SF (12.5%), EF (4.7%), and HM (0%). The timing of GI events was also significantly different across groups (P ¼ 0.010). Conclusion: The study demonstrated that feeding a higher quality, lower protein concentration formula (a-lactalbumin- enriched) met all essential amino acid and protein requirements of infants. The GI tolerance profile of infants receiving the EF was similar to HM-fed infants. European Journal of Clinical Nutrition (2008) 62, 1294–1301; doi:10.1038/sj.ejcn.1602848; published online 25 July 2007

Keywords: infant formula; amino acids; a-lactalbumin; protein composition

Introduction tion. Mature (41 month lactation) HM provides 9–11 g/l protein (Dewey et al., 1996), while conventional infant Human milk (HM) is the preferred sole source of nutrition formulas contain 15 g/l. A key objective of infant formula for at least the first 6 months of life (World Health development is to reduce the total protein concentration Organization, 2003). If a mother cannot or chooses not to while meeting the essential amino acid needs of the infant. breast-feed during this period, an infant formula should be a-Lactalbumin is the predominant whey protein found in fed as the alternative. The composition and physiological HM with a concentration of 2–3 g/l (Jackson et al., 2004) effects of infant formula differ from HM and, therefore, the while it is a relatively minor component of bovine milk goal of infant formula development is to mimic both the whey. b-Lactoglobulin, a protein not normally found in HM, composition and function of HM. HM and infant formula is the primary bovine whey protein (Rudolff and Kunz, differ substantially in protein composition and concentra- 1997). a-Lactalbumin has both a biochemical role and a nutritional role for the mother–infant dyad. During lactation, the mammary gland produces a-lactalbumin and Correspondence: Dr BJ Harris, Wyeth Nutrition, 200 Campus Drive, galactosyltransferase. These two proteins form the enzyme Collegeville, PA 19426, USA. E-mail: [email protected] complex lactose synthase, which catalyzes the synthesis of Guarantor: B Harris. lactose from glucose and galactose (Brodbeck et al., 1967). Contributors: AMD, BJH and ELL designed the study. AMD, BJH and the Multi- After the completion of lactose synthesis, a-lactalbumin Center Group collected the data. AMD, BJH, ELL, KP and JT conducted data dissociates from the enzyme complex and becomes an analysis and interpretation. AMD, BJH, ELL, KP and JT wrote the manuscript. Received 15 November 2006; revised 21 May 2007; accepted 18 June 2007; integral part of the protein content of HM (approximately published online 25 July 2007 41% of the whey protein and 28% of the total protein; Heine a-Lactalbumin-rich infant formula AM Davis et al 1295 et al., 1991). The nutritional value of a-lactalbumin lies in its formula to a protein hydrolysate or soy formula, had high proportion of essential amino acids, specifically trypto- conditions that required feedings other than those specified phan, cysteine and lysine (Lonnerdal, 1994). Generally, in the protocol, had any major congenital malformation, had tryptophan is the limiting essential amino acid in infant suspected or documented systemic or congenital infections formula (Heine et al., 1996). In addition to being an essential [such as Human immunodeficiency virus (HIV)]; had cardiac, amino acid required for protein synthesis, tryptophan is also respiratory, hematological, gastrointestinal (GI) or other the precursor to the neurotransmitter serotonin (Lien, 2003). systemic diseases; or had participated in another clinical trial. Recent advances in dairy milk separation technology have All routine immunizations, vitamin supplements and over- resulted in the development of a process that yields whey the-counter medications were permitted. Mineral, protein protein fractions with a substantially higher concentration and herbal supplements (except fluoride) were prohibited. of a-lactalbumin (69%) than standard bovine whey and a 59% reduction of b-lactoglobulin. a-Lactalbumin-enriched formulas produced with this whey fraction can have lower Study feedings protein levels than conventional formulas due to its higher Total protein concentration was 15 g/l (2.2 g protein/ protein quality (amino-acid profiles more similar to HM). 100 kcal) in the SF and 14 g/l (2.08 g protein/100 kcal) in Lien et al. (2004) conducted a 12-week growth and safety the EF. The essential amino-acid composition of EF was study comparing a formula enriched in a-lactalbumin closer to that of HM than SF for six amino acids (Table 1). No [experimental formula (EF)] to a standard formula (SF). free amino acids were added to either formula. Final formula Growth and serum albumin levels were comparable between concentration of a-lactalbumin was 2.2 g/l in EF and 1.3 g/l the formulas even though the EF had lower total protein in SF, while the b-lactoglobulin concentration of EF was than the SF. The current study was undertaken as follow-up substantially reduced in comparison to SF and other current research to the aforementioned study (Lien et al., 2004), to whey-dominant infant formulas. Mean non-protein nitrogen evaluate the protein quality and quantity of the a-lactalbu- (NPN) content of EF was 12.61% (87.39% protein nitrogen) min-enriched formula (experimental formula, EF), by mea- compared to the mean NPN content of SF of 16.55% (83.45% suring post-prandial plasma essential amino-acid levels in protein nitrogen). The carbohydrate (lactose), fat and caloric healthy term infants receiving EF, SF or HM during an 8-week content of the EF and SF were the same (73.0 g/l, 36.0 g/l period. In addition, the present study conducted a rigorous and 672 kcal/l, respectively). EF and SF had identical levels of assessment of GI tolerance. vitamins, nucleotides, taurine and the long-chain omega-3 and omega-6 polyunsaturated fatty acids, docosahexaenoic and arachidonic acids (DHA and AA). The composition of Materials and methods the fat blend was the same for both the EF and SF (a blend of palm olein, oleic, coconut and soy oils with 0.21 w% and A prospective, multicenter, masked, randomized clinical trial 0.34 w% of total fatty acids as DHA and AA, respectively). was undertaken to compare the plasma essential amino-acid The nucleotide concentration was the same for both EF and levels of infants fed one of three regimens: a standard whey- SF (cytidine-, uridine-, adenosine-, guanosine- and inosine- dominant infant formula (SF); an EF containing lower total monophospate concentrations were 16.5, 5.0, 4.0, 2.0 and protein and increased a-lactalbumin concentrations (EF) and 2.0 mg/l, respectively). Calcium concentration of EF was HM-fed. The Institutional Review Boards (IRBs) of the participating centers approved the study protocol. Written informed consent was obtained from the parent or legal Table 1 Essential amino-acid composition of HM and study formulas guardian of each infant before enrollment into the study. Amino acid Standard a Experimental a HM b

Arginine 3.7c 3.6 4.0 Study population Cystine 1.5 1.9 1.8 Infants p14 days post-natal age were included if they were Histidine 2.5 2.5 2.6 healthy, born at term and appropriate for gestational age; Isoleucine 5.8 5.7 5.3 Leucine 9.4 9.9 10.0 had weight for length at birth within the 10th and 90th Lysine 7.5 7.5 6.8 percentiles according to growth charts provided by the US Methionine 2.4 2.2 1.6 Center for Disease Control (CDC); had weight for length at Phenylalanine 4.1 4.5 4.2 enrollment within the 10th and 90th percentiles according Threonine 5.4 5.4 4.7 Tryptophan 1.5 1.8 1.8 to CDC growth charts; and were exclusively fed infant Tyrosine 3.6 4.1 4.5 formula or exclusively breast fed. Infants in the formula Valine 5.8 6.0 5.7 groups were excluded if they were partially or exclusively fed HM within 2 days of enrollment were routinely fed either Abbreviation: HM, human milk. aWyeth Nutrition internal data. baby food or solid foods, had siblings with feeding problems bLiterature average. that had necessitated a change from a bovine milk-based cPercentage of amino acids.

European Journal of Clinical Nutrition a-Lactalbumin-rich infant formula AM Davis et al 1296 420 mg/l, slightly lower than that of SF, 460 mg/l. Both those, in the opinion of the study physician, were definitely, formulas were manufactured by Wyeth Nutrition and probably or possibly related to study feeding. Primary safety supplied in 250 ml Ready-to-Feed Tetrabrik packs. To ensure parameters were recorded during scheduled visits at weeks 4 blinding, EF was labeled with one of two different colors and and 8, and by telephone contacts at weeks 2, 6 and 10. SF was labeled with one of two different colors. The sponsors, Growth parameters, infant weight (g), length (cm), weight- investigators and parents were not aware of which formula for-length ratio and head circumference (cm) were measured each infant consumed. at baseline (enrollment) and weeks 4 and 8. Infants were weighed without diapers to the nearest 0.01 kg. Growth velocity parameters, change in weight (g/day), length Randomization (cm/week) and head circumference (cm/week), were calcu- A computerized randomization/enrollment system was used lated using anthropometric data from baseline (enrollment) to assign study formula. Infants were randomly assigned to and week 8. Secondary safety parameters included evalua- receive either EF or SF ad libitum for 8 weeks. The HM group tion of the acceptability and tolerance of study formulas was assigned study numbers, but not randomized. If an by caregivers at each study time point. infant was withdrawn from the study, the randomization number and corresponding formula assignment were not reassigned to another infant. Statistical analysis Data were analyzed according to the intent-to-treat (ITT) principle. The ITT set included data from all infants who Efficacy and safety parameters entered the study and received at least one feeding of study Primary efficacy parameters were post-prandial plasma formula. All data were analyzed using SAS statistical software essential amino-acid levels (arginine, cysteine, histidine, (SAS, Cary, NC, USA). Categorical variables were summarized isoleucine, leucine, lysine, methionine, phenylalanine, with frequencies and percentages. Continuous variables were threonine, tryptophan (free), tyrosine and valine) at week summarized with sample number (n), mean, s.d. and 95% 8. For plasma amino-acid evaluation, a 1.5 ml blood sample confidence interval around the mean. Three-way analysis of was collected by venipuncture in a heparinized tube, variance (ANOVA) and three-way analysis of covariance between 1.5 and 2.5 h after the infant’s most recent study (ANCOVA) were used to assess anthropometric data (weight, feeding (HM or study formula). Amino-acid analysis was length and head circumference) at birth, baseline and weeks conducted by automated ion-exchange chromatography 4 and 8; gestational age and gender at baseline; protein status and compared to reported normative values (Lepage et al., (albumin) and laboratory safety data at baseline and week 8. 1997). Secondary efficacy parameters included markers A Student’s t-test was used to test for differences in plasma of protein status (albumin, blood urea nitrogen (BUN), amino-acid levels between EF and SF. Given the multiple creatinine and complete blood count with differential). t-tests performed, significant results were calculated and For protein status evaluation, a 1.5 ml blood sample presented at the traditional level (a ¼ 0.05) and using the

was collected either by venipuncture or heel stick in Bonferroni adjustment for multiplicity (aADJ ¼ 0.004). A an ethylenediaminetetraacetic acid treated vacutainer three-way ANOVA was used to test for differences in GI or microtainer tube. Blood samples were centrifuged at study event incidence and study withdrawals across groups. 3000 r.p.m. Â 15 min, plasma was transferred to labeled vials Where differences were found, a Fisher’s exact test was used and shipped frozen (À201C) to Mayo Central Laboratory for to test for differences between two groups. A Kalpan–Meier Clinical Trials (Rochester, MN, USA). survival curve was used to estimate the total GI study events Primary safety parameters were the study events associated and their occurrences over the study period for each study with each study feeding and growth. This study was group (including continued estimated GI study events for conducted in accordance with the International Conference study withdrawal infants) as a method to look at cumulative on Harmonization (ICH) and Good Clinical Practice (GCP) incidence. Log-rank test was used to rank order the guidelines for the registration of pharmaceutical products cumulative incidence of GI study events per group over the for human use. Consistent with ICH/GCP guidelines, any study period and to compare the differences in the three unintended change in pathology or in anatomic, metabolic study groups over time. or physiologic functioning temporally associated with con- Weight-for-age measurements at baseline and weeks 4 and sumption of formula or HM during the study was defined 8 were used to assess body weight of study infants relative to as a study event. These changes were reflected by physical CDC reference curves (Dibley et al., 1987; Kuczmarski et al., signs, reported symptoms or laboratory data. Any event 2000). Length-for-age, and weight-for-length z-scores were consistent with the above definition reported by the evaluated at baseline and week 8. Epi-Info 2000 software caregiver or noted by study site personnel were collected (Version 1.1, CDC and Prevention, 2000) was used to as a study event. Study events were categorized as ‘Any determine z-score values. Any infant with a missing value Causality’, meaning all events regardless of causality, or for a particular variable was not included in the analyses ‘feeding-related’. Feeding-related events were defined as involving that variable.

European Journal of Clinical Nutrition a-Lactalbumin-rich infant formula AM Davis et al 1297 Results similar among groups at baseline (SF: 0.570.2 mg/dl, EF: 0.570.1 mg/dl and HM: 0.570.3 mg/dl) and week 8 (SF: Infant demographic and disposition data are summarized in 0.570.1 mg/dl, EF: 0.470.1 mg/dl and HM: 0.570.2 mg/dl). Table 2. Of the 216 infants enrolled in the study, 64 (29.5%) Serum BUN measured at baseline and 8 weeks were within received SF, 64 (29.5%) received EF and 88 received HM normal ranges for all study groups. There was a statistically (41%). Forty-three (67.2%) infants were fed SF, 49 (76.6%) significant (Po0.001) difference in the BUN concentration were fed EF and 74 (84.1%) were fed HM and completed the among study groups at week 0 (SF: 7.773.3 mg/dl, EF: study. The most common reason for study discontinuation 7.273.0 mg/dl and HM: 10.773.8 mg/dl) and week 8 (SF: was due to study events: 21.9% of the SF group discontinued 9.471.9 mg/dl, EF: 9.571.9 mg/dl and HM: 6.872.6 mg/dl). due to a study event, compared to 10.9% of the EF group; no There were no significant differences in hematocrit or infants in the HM group discontinued due to a study event. hemoglobin concentrations among groups at weeks 0 or 8 Thirty-four (53.1%) infants in the SF group and 35 (54.7%) in (data not shown). the EF group were females (NS). The mean age at enrollment, Table 4 represents the mean post-prandial concentrations and birth growth parameters (birth weight, birth length and of plasma essential amino acids at week 8 among infants birth head circumference) were similar among all three receiving SF, EF and HM for the ITT analysis. There were no groups. The mean gestational age of the HM group was significant differences in plasma amino-acid concentrations higher than the two formula groups (P ¼ 0.036). between infants fed EF versus SF for 8 of 12 amino acids. For There were no statistically significant differences among the remaining four amino acids (cystine, lysine, tryptophan the three treatment groups in growth velocity from baseline and tyrosine), the means were higher in the EF group; to week 4 or week 8 (Table 3). Mean changes from baseline to however, they were within the normal range for healthy week 8 z-scores were similar across study groups. There were infants of similar age (Lepage et al., 1997). no significant differences between the two formula groups in mean formula intake (SF versus EF at 8 weeks: 10327258 versus 9907228 ml/day, respectively). Table 3 Growth velocity and formula intake by feeding group There were no statistically significant differences across Growth parameter Standard Experimental HM treatment groups in the serum concentrations of albumin n ¼ 40 n ¼ 47 n ¼ 70 or creatinine at baseline or week 8. The mean albumin concentration was similar among groups at baseline Weight gain (g/day) 35.078.0a 35.979.5 34.578.7 (SF: 3.870.3 g/dl, EF: 3.870.3 g/dl and HM: 3.770.4 g/dl) Length gain (cm/week) 0.9270.19 0.9070.21 0.8870.21 7 7 7 and week 8 (SF: 4.170.3 g/dl, EF: 4.170.2 g/dl and HM: Head circumference gain 0.52 0.12 0.50 0.09 0.50 0.12 7 (cm/week) 4.0 0.2 g/dl). The mean creatinine concentration was Intake (volume) (ml/day, week 8) 10687342 9847222 N/A Protein intake (g/day, week 8) 16.075.1 13.873.1 N/A

Table 2 Demographic characteristics and disposition of enrolled infants Abbreviation: HM, human milk; N/A, not available. by feeding group aMean7s.d.

Standard Experimental HM Table 4 Post-prandial plasma essential amino acids at week 8 by Enrolled 64 64 88 feeding group Completed (%) 43 (67.2) 49 (76.6) 74 (84.1) Age at enrollment (days) 9.8 (3.8)a 9.3 (3.9) 10.1 (3.4) Amino acid Standard Experimental HM Gestational age (week) 38.9 (1.0) 38.9 (1.2) 39.3 (1.0)b n ¼ 40 n ¼ 47 n ¼ 70 Female (%) 34 (53.1) 35 (54.7) 49 (55.7) Birth weight (g) 3342 (442.7) 3430 (444.9) 3521 (447.6) Arginine 78.2 (22.2)a 84.6 (18.3) 90.3 (25.8) Birth length (cm) 50.2 (2.4) 50.4 (2.5) 51.0 (2.4) Cysteine 28.3 (11.9)b,c 36.0 (10.8)b,c 34.0 (13.4) Birth head circumference (cm) 34.3 (1.61) 34.4 (1.5) 34.8 (1.7) Histidine 76.3 (15.2) 79.9 (20.0) 67.8 (21.7) Isoleucine 70.0 (21.5) 75.8 (15.7) 61.9 (19.2) Study disposition (%) Leucine 115.1 (28.0) 126.2 (26.3) 113.3 (37.3) Discontinued studyc 21 (32.8) 15 (23.4) 14 (15.9) Lysine 195.2 (39.1)b 214.1 (41.8)b 166.0 (39.0) Methionine 34.4 (7.7) 34.7 (8.4) 26.2 (9.0) Reason for study discontinuation (%) Phenylalanine 46.8 (10.1) 52.5 (20.7) 40.6 (19.7) Study eventd 14 (21.9) 7 (10.9) 0 (0) Threonine 182.7 (50.1) 193.2 (52.9) 119.7 (37.5) Protocol violation 2 (3.1) 2 (3.1) 8 (9.1) Tryptophan 55.7 (10.9)b 62.9 (19.1)b 56.8 (15.6) MD/family request 3 (4.7) 3 (4.7) 3 (3.4) Tyrosine 78.6 (20.4)b,c 95.8 (20.8)b,c 79.1 (21.9) Lost to follow-up 1 (1.6) 3 (4.7) 1 (1.1) Valine 182.3 (35.4) 197.6 (39.3) 154.0 (43.9) Other 1 (1.6) 0 (0) 2 (2.3) Abbreviation: HM, human milk. Abbreviations: ANOVA, analysis of variance; HM, human milk. aMean (s.d.) (mmol/l). aMean (s.d.). bStatistically significant difference (Student’s t-test) between standard and bSignificant difference across groups (ANOVA, P ¼ 0.036). experimental formula at a traditional significance level (Po0.05). cMore than one reason could be listed for discontinuation. cStatistically significant difference (Student’s t-test) between standard and dMore than one study event could be listed for discontinuation. experimental formula after adjustment for multiplicity (Po0.004).

European Journal of Clinical Nutrition a-Lactalbumin-rich infant formula AM Davis et al 1298 One hundred and seventy-seven of the 216 infants Figure 3 shows the cumulative incidence (%) of GI study enrolled (81.9%) experienced a study event (see Materials events over time. There was a significant difference and methods section). There was no statistically significant (P ¼ 0.010) in the timing of GI study events incidence among difference in the number of infants reporting a study event all groups, based on the log-rank test. For the SF group, GI (overall) among the treatment groups. The most common study event incidence occurs relatively frequently near the body system reported for study events was the GI system. beginning of the study (between days 1 and 20) and remains The most common GI study events were eructation, higher for the duration of the trial. For the EF group, the constipation, gastroesophageal reflux, abdominal pain, vomiting, diarrhea and regurgitation. Figure 1 depicts the incidence of infants that experienced a GI study event of any causality and the incidence of infants that experienced a 70% feeding-related GI study event. The incidence of GI study 60% Standard events of any causality was highest for the SF group (56.3%) Formula 50% Experimental and lower for the EF and HM groups (39.1 and 40.9%, Formula respectively); however, this difference did not reach statis- Human 40% Milk tical significance (P ¼ 0.092). The incidence of constipation 30%

(SF 18.8%, EF 9.4% and HM 6.8%) and regurgitation (SF Incidence 20.3% 9.4%, EF 3.1% and HM 3.4%), for study events of any 20% 10.9% 12.5% causality, were substantially lower in the EF group than the 10% 4.7% SF group (data not shown). A similar trend was observed 0.0% 0.0% 0% in the incidence of constipation for feeding-related study aAny Casuality bFeeding-related events (data not shown). Feeding-related GI study events Figure 2 Incidence of study withdrawals due to GI study events (Figure 1) were significantly different (P ¼ 0.025) across among all feeding groups. aThe incidence of study withdrawals due treatment groups. The incidence of feeding-related GI study to GI study events of any causality was significantly different across events was highest for the SF group (31.3%), followed by the groups (ANOVA, Po0.001). bThe incidence of study withdrawals EF and HM groups at 17.2 and 13.6%, respectively. A Fisher’s due to feeding-related GI study events was significantly different across groups (ANOVA, P ¼ 0.001). The incidence of study with- exact test showed the incidence of feeding-related GI study drawals due to feeding-related study events was significantly higher events was significantly higher in the SF versus HM group in the SF versus HM group (Fisher’s exact test, P ¼ 0.001). ANOVA, (P ¼ 0.015). Figure 2 depicts the incidence of study with- analysis of variance; GI, gastrointestinal; HM, human milk; SF, drawals due to GI study events. A significant difference standard formula. across treatment groups was found for study withdrawals due to GI study events of any causality (Po0.001) and feeding- related GI study events (P ¼ 0.001) (Figure 2). For both 1.0 variables, the incidence of study withdrawals was highest for the SF group and lowest for the HM group. 0.9 standard experimental human milk 0.8 0.7 70% Standard 0.6 60% 56.3% Formula Experimental 0.5 50% Formula 0.4 40.9% Human 40% 39.1% Milk 0.3 31.3% Cumulative Incidence (%) Cumulative 0.2 30% p=0.010

Incidence 0.1 Log-rank test 20% 17.2% 13.6% 0.0 10% 0 10203040 50 60 Time to GI AE (days) 0% aAny Casuality bFeeding-related Figure 3 Time to incidence of GI study events by feeding group. There was a significant difference (P ¼ 0.010) in the timing of GI Figure 1 Incidence of GI study events among all feeding groups. events among the feeding groups (log-rank test). For the SF group, aThe incidence of GI study events of any causality was not GI events occurred relatively frequently near the beginning of the significantly different across feeding groups (ANOVA, P ¼ 0.092). study (days 1–20) and less frequently near the end of the study (days bThe incidence of feeding-related GI study events was significantly 40–60). For the HM group, GI events occurred relatively frequently different across feeding groups (ANOVA, P ¼ 0.025). The incidence near the beginning of the study (days 1–15) and in a more linear of feeding-related GI study events was significantly higher in the SF manner (days 50–60). For the EF group, the incidence pattern of versus HM group (Fisher’s exact test, P ¼ 0.015). ANOVA, analysis GI events was similar to that of the HM group after day 18. EF, of variance; GI, gastrointestinal; HM, human milk; SF, standard experimental formula; GI, gastrointestinal; HM, human milk; SF, formula. standard formula.

European Journal of Clinical Nutrition a-Lactalbumin-rich infant formula AM Davis et al 1299 incidence pattern of GI study events was similar to that sodium-dependent transport system is required for free of the HM fed infant after an initial period of adjustment amino-acids absorption whereas dipeptides and tripeptides (18 days). (the primary products of intestinal protein digestion) are absorbed without a dependent transport system (Wenzel et al., 2001). Ingestion of small peptides has been found to Discussion appear in plasma more rapidly and uniformly as compared to free amino acids using the same amino-acid pattern A key goal as well as a major challenge of infant formula (Svangerb et al., 1977). HM only contains about 5% research is to design formulas that mimic HM both in its free amino acids (Rerat, 1995) mainly as glutamic acid composition and physiologic effects. Traditionally, infant and taurine (Pamblanco et al., 1989; Agostini et al., 2000), formulas contain a higher protein concentration than HM to and therefore departure from the HM model by adding free provide formula-fed infants with sufficient quantities of each amino acids to formula requires careful consideration. essential amino acid. Data from the present study demon- In the present study, sufficient tryptophan was provided in strated that all essential amino acid requirements were met a lower protein formula (EF) via enrichment with the protein with an a-lactalbumin-enriched formula containing a lower a-lactalbumin. The mean post-prandial serum concentration total protein content than conventional formulas. The of tryptophan in the EF group was higher than that of the plasma amino-acid levels of infants receiving the EF were SF and HM groups, but was within 1 s.d. of the means of within the range of infants receiving SF and HM. Addition- both groups. Heine et al. (1996) found serum tryptophan ally, EF-fed infants had a GI tolerance profile similar to HM- concentrations similar to the levels observed in the EF group, fed infants as demonstrated by the incidence and timing of using a formula with a similar amount of a-lactalbumin GI events. Creating an infant formula with a protein profile in their formula (2.2 g/l, 13.4 g protein/l). closer to HM is an ongoing challenge of infant formula In addition to lowering the protein concentration of EF research. Various studies have evaluated infant formula closer to HM, the enrichment of infant formula with the protein systems in an attempt to more closely imitate HM, whey protein a-lactalbumin results in whey protein compo- both in protein content and in the infant’s post-prandial sition closer to that of HM. HM whey proteins play an plasma amino acid patterns. Janas et al. (1987) evaluated important role in many physiologic activities that benefit formulas with lower protein/energy ratios (1.8 g protein/ the infant such as immune function and nutrient absorption 100 kcal, versus SF levels of 2.2 g protein/100 kcal) and with (Lonnerdal and Lien, 2003). a-Lactalbumin in HM has been varying whey/casein ratios. Although some plasma amino- shown to bind minerals such as calcium (Lonnerdal and acid levels were similar to breast-fed controls, the levels of Glazier, 1985) and zinc (Ren et al., 1993) facilitating their tryptophan in all groups receiving formula were significantly absorption (Lonnerdal and Lien, 2003). Increased absorption lower than the breast-fed group. Picone et al. (1989) of zinc and iron has been shown in rhesus monkeys fed an evaluated two lower protein formulas (11 and 13 g/l) infant formula enriched with a-lactalbumin (Kelleher et al., compared to a standard protein formula group (15 g/l) and 2003). The ability of a-lactalbumin to enhance mineral a breast-fed group. They found that both of the reduced- absorption from our formula matrix warrants further protein formula groups had lower plasma tryptophan levels investigation. as compared to the SF and breast-fed groups. In contrast to Improving the GI tolerance profile of formula-fed infants’ the above results, other studies have found similar plasma closer to that of HM-fed infants is another goal of infant tryptophan levels compared to breast-fed infants after formula development. Formula tolerability is frequently feeding term infants lower protein formulas (Lonnerdal assessed by the comparison of the numbers and kinds of GI and Chen, 1990; Karlsland Akeson et al., 1998; Lonnerdal study events (Alarcon et al., 2002); these are present in all and Hernell, 1998). The inconsistent results may be due to infants and may differ between breast-fed and formula-fed differences between formulas with regards to formula infants (Lloyd et al., 1999). The unique finding in the present processing techniques or heat treatment that affect protein study was that the cumulative GI event profile of EF was digestibility (Lonnerdal and Hernell, 1998). similar to that of the HM profile after study day 18. A number of further studies have evaluated lower protein Additionally, the timing of GI study events suggests that formulas with the addition of free tryptophan. Two studies the SF was not as well tolerated when compared to HM. GI (Fazzolari-Nesci et al., 1992 and Hanning et al., 1992) effects usually present soon after the introduction of a new compared a control whey-dominant protein formula feeding (Lloyd et al., 1999), and to our knowledge, a detailed (15 g/l), both to an EF with added tryptophan (13 g/l protein, time course of study events has never before been reported. whey to casein ratio of 40:60), and to HM. While the mean Of potential clinical importance is that the incidence of plasma tryptophan levels in the group receiving the constipation and regurgitation in the EF group was similar to control formula were significantly lower than breast-fed HM-fed infants, and lower than that of the SF group. The infants, the levels in the supplemented groups were similar improved GI profile observed in the present study may be to the breast-fed infants. However, addition of free amino attributed to a formula matrix that is closer to HM. This acids to infant formula should take into consideration that a outcome complements the findings of the a-lactalbumin

European Journal of Clinical Nutrition a-Lactalbumin-rich infant formula AM Davis et al 1300 growth and safety study where Lien et al. (2004) found Brodbeck U, Denton WL, Tanahashi N, Ebner KE (1967). The improved tolerance in infants fed with a-lactalbumin- isolation and identification of the B protein of lactose synthetase enriched formula, as demonstrated by superior acceptability as alpha-lactalbumin. J Biol Chem 242, 1391–1397. Dewey KG, Beaton G, Fjeld C, Lonnerdal B, Reeds P (1996). and tolerance (% satisfactory) ratings in EF versus SF Protein requirements of infants and children. Eur J Clin Nutr 50, after week 2, and a significantly higher rating at week 8 5119–5150. (EF: 97% and SF: 89%). Dibley MJ, Goldsby JB, Staehling NW, Trowbridge FL (1987). In conclusion, this study of a lower protein infant formula Development of normalized curves for the international growth reference: historical and technical considerations. Am J Clin Nutr with increased a-lactalbumin concentration represents a 46, 736–748. major improvement in the infant formula protein matrix Fazzolari-Nesci A, Domianello D, Sotera V, Raiha NCR (1992). to more closely match the protein composition and GI Tryptophan fortification of adapted formula increases plasma tolerance profile of HM. The formula enriched with tryptophan concentrations to levels not different from those found in breast-fed infants. J Pediatr Gastroenterol Nutr 14, a -lactalbumin delivered lower total protein with higher 456–459. protein quality as demonstrated by the comparable plasma Hanning RM, Paes B, Atkinson SA (1992). Protein metabolism and essential amino-acids profiles compared to a group who growth of term infants in response to a reduced-protein, 40:60 received a conventional formula. Moreover, given that the whey:casein formula with added tryptophan. Am J Clin Nutr 56, 1004–1011. mean post-prandial plasma concentrations of nearly all Heine W, Radke M, Wutzke KD, Peters E, Kundt G (1996). Alpha- amino acids in EF-fed infants were higher than HM-fed lactalbumin-enriched low-protein infant formulas: a comparison infants, a further reduction of the protein concentration of to breast milk feeding. Acta Paediatr 85, 1024–1028. this formula may be feasible. Lastly, the a-lactalbumin- Heine WE, Klein PD, Reeds PJ (1991). The importance of alpha- J Nutr enriched (EF) formula demonstrated improved GI tolerance lactalbumin in infant nutrition. 121, 277–283. Jackson JG, Janszen DB, Lonnerdal B, Lien EL, Pramuk KP, Kuhlman over SF with significantly fewer GI study events and a CF (2004). A multinational study of alpha-lactalbumin concentra- similarity in the cumulative incidence and timing of GI tions in human milk. J Nutr Biochem 15, 517–521. study events to that of HM-fed infants. Janas LM, Picciano MF, Hathch TF (1987). Indices of protein metabolism in term infants fed either human milk or formulas with reduced protein concentration and various whey/casein ratios. J Pediatr 110, 838–848. Acknowledgements Karlsland Akeson PM, Axelsson IEM, Raiha NCR (1998). Protein and amino acid metabolism in three-to twelve-month old infants fed human milk or formulas with varying protein concentrations. This study was funded by a grant from Wyeth Nutrition J Pediatr Gastroenterol Nutr 26, 297–304. (Collegeville, PA, USA). This study would not have been Kelleher SL, Chatterton D, Nielsen K, Lonnerdal B (2003). Glycoma- possible without the diligent work from each of the cropeptide and alpha-lactalbumin supplementation of infant following 12 investigators and their study staff at each site: formula affects growth and nutritional status in infant rhesus monkeys. Am J Clin Nutr 77, 1261–1268. Jeffrey Adelglass, MD (Dallas, Texas); William Brown, MD Kuczmarski RJ, Ogden CL, Grummer-Strawn LM et al. (2000). CDC (Murfreesboro, Tennessee); Gloria Durelli, MD (Stratford, growth charts: United States. Advance data from vital and health New Jersey); Martin Greenberg, MD (Savannah, Georgia); statistics; no. 314. Hyattsville, Maryland: National Center for Frank Hughes, MD (Bossier City, Louisiana); John A Fling, Health Statistics. Lepage N, McDonald N, Dallaire L, Lambert M (1997). Age-specific MD (Fort Worth, Texas); Cyndy Lively, MD (Winston-Salem, distribution of plasma amino acid concentrations in a healthy North Carolina); Douglas K. Mitchell, MD (Norfolk, pediatric population. Clin Chem 43, 2397–2402. Virginia); Kathleen Salter, MD (Chapel Hill, North Carolina); Lien EL (2003). Infant formulas with increased concentrations of Richard Schwartz, MD (Vienna, Virginia); John Spitzer, MD alpha-lactalbumin. Am J Clin Nutr 77 (Suppl), 1555S–1558S. Lien EL, Davis AM, multi-center group (2004). Growth and safety of a (Kalamazoo, Michigan); Tracy Stewart, MD (Little Rock, reduced protein formula enriched with bovine alpha-lactalbumin Arkansas). in term infants. J Pediatr Gastroenterol Nutr 38, 170–176. A special thanks to Nicole Stouffer, MS, Biostatistician Lloyd B, Halter RJ, Kuchan MJ, Bags GE, Ryan AS, Masor ML (1999). consultant, and Robert Paul Felton, Biostatistician from Formula tolerance in post-breastfed and exclusively formula-fed infants. Pediatrics 103, E7. Biotechnical Services Inc., N Little Rock, AR and Susan Lonnerdal B (1994). Digestibility and absorption of protein in Troemel, BS, Wyeth, Collegeville, PA, USA, for their expert infants. In: Raiha NCR (ed.), Protein Metabolism During Infancy. contributions. Raven Press: Vevey 33, pp 53–65. Lonnerdal B, Chen CL (1990). Effects of formula protein level and ratio on infant growth, plasma amino acids and serum trace elements. Acta Paediatr Scand 79, 257–265. References Lonnerdal B, Glazier C (1985). Calcium binding by alpha-lactalbumin in human milk and bovine. J Nutr 115, 1209–1216. Agostini C, Carratu B, Boniglia C, Riva E, Sanzini E (2000). Free Lonnerdal B, Hernell O (1998). Effects of feeding ultrahigh- amino acid content in standard infant formulas: comparison with temperature (UHT)-treated infant formula with different protein human milk. J Am Coll Nutr 19, 434–438. concentrations or powdered formula, as compared with breast- Alarcon PA, Tressler RL, Mulvaney A, Lam W, Comer GM (2002). feeding, on plasma amino acids, hematology, and trace element Gastrointestinal tolerance of a new infant milk formula in healthy status. Am J Clin Nutr 68, 350–356. babies: an international study conducted in 17 countries. Nutrition Lonnerdal B, Lien E (2003). Nutritional and physiologic significance 18, 484–489. of alpha-lactalbumin in infants. Nutr Rev 61, 295–305.

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