BACKGROUND: Dietary nucleotides are nonprotein nitrogenous compounds that are found in high concentrations in breast milk and are thought to be conditionally essential nutrients in infancy. A high nucleotide intake has been suggested to explain some of the benefits of breastfeeding compared with formula feeding and to promote infant growth. However, relatively few large-scale randomized trials have tested this hypothesis in healthy infants.
OBJECTIVE: We tested the hypothesis that nucleotide supplementation of formula benefits early infant growth.
PATIENTS AND METHODS: Occipitofrontal head circumference, weight, and length were assessed in infants who were randomly assigned to groups fed nucleotide-supplemented (31 mg/L; n = 100) or control formula without nucleotide supplementation (n = 100) from birth to the age of 20 weeks, and in infants who were breastfed (reference group; n = 101).
RESULTS: Infants fed with nucleotide-supplemented formula had greater occipitofrontal head circumference at ages 8, 16, and 20 weeks than infants fed control formula (mean difference in z scores at 8 weeks: 0.4 [95% confidence interval: 0.1–0.7]; P = .006) even after adjustment for potential confounding factors (P = .002). Weight at 8 weeks and the increase in both occipitofrontal head circumference and weight from birth to 8 weeks were also greater in infants fed nucleotide-supplemented formula than in those fed control formula.
CONCLUSIONS: Our data support the hypothesis that nucleotide supplementation leads to increased weight gain and head growth in formula-fed infants. Therefore, nucleotides could be conditionally essential for optimal infant growth in some formula-fed populations. Additional research is needed to test the hypothesis that the benefits of nucleotide supplementation for early head growth, a critical period for brain growth, have advantages for long-term cognitive development.
WHAT'S KNOWN ON THIS SUBJECT:
Dietary nucleotides are nonprotein nitrogenous compounds found in high concentrations in breast milk. A high nucleotide intake has been suggested to explain some of the benefits of breastfeeding, compared with formula feeding, and to promote infant growth.
WHAT THIS STUDY ADDS:
Results of the study revealed that nucleotide supplementation increases weight gain and head growth in formula-fed infants. Therefore, nucleotides may be conditionally essential for optimal infant growth in some formula-fed populations.
Breastfeeding has benefits for growth1 and development,2,3 but whether these effects are attributable to sociobiological differences between breastfed and formula-fed infants or to the nutritional composition of breast milk remains controversial.4 Several nutrients in breast milk have been suggested to contribute to its biological advantages over cow's milk–based formulas, including a higher concentration of nucleotides.5 Nucleotides are nonprotein, nitrogenous compounds that play key roles in many biological processes and are suggested to be conditionally essential nutrients in infancy.5,–,12 However, although nucleotides have been added to some infant formulas for many years, their benefits in humans remain uncertain.
The supply of nucleotides provided through de novo synthesis and less metabolically costly salvage pathways is thought to be insufficient for optimal function of rapidly growing tissues such as those in the gastrointestinal, lymphoid, and hemapoeitic systems.5,7,11 These tissues have limited capacity for de novo nucleotide synthesis, and although a lower nucleotide intake may not result in a clinical deficiency syndrome, an exogenous supply of nucleotides to rapidly growing tissues is important for their optimal function.5,–,12 Consistent with this hypothesis, nucleotide supplementation in rats has been shown to enhance growth and maturation of the gut13; increase villous height, mucosal protein, and disaccharidase activity in the proximal intestine13,14; and enhance gut mucosal recovery after experimentally induced diarrhea.14,15 Dietary nucleotides also increase the gene expression of certain gastrointestinal enzymes,16 and increase postprandial blood flow in mesenteric arteries of infants.17,18 Theoretically, therefore, trophic effects of nucleotides on the gut, along with benefits for immune function and incidence of diarrhea,11,12 could have advantages for infant growth, a hypothesis supported by results of animal studies.19
In humans, however, results of randomized studies have not revealed a beneficial effect of nucleotide supplementation on growth of formula-fed infants.20,–,23 Nevertheless, nucleotide intake was shown to affect biomarkers that could influence catch-up growth in malnourished children24 and to increase growth in weight, length, and head circumference in infants born small for gestational age (SGA).25 Nucleotide supplementation may therefore enhance infant growth at least in some vulnerable populations. Here we report the findings from a study in which we investigated the effects of nucleotide supplementation on colonic microbiota and the incidence of diarrhea,26 and on infant growth. We were particularly interested in the effect of nucleotides on head growth, suggested to be greater in breastfed infants27,28 and related to long-term cognitive function.29,–,32
SUBJECTS AND METHODS
Research nurses recruited infants from 4 hospitals (2 each in Leicester and Nottingham, United Kingdom) between 1999 and 2002. Healthy singletons who were born at ≥37 weeks' gestation without congenital abnormalities, and who had already commenced formula feeding were eligible (n = 200). A reference group of breastfed infants, identified at birth, were eligible if they were still breastfeeding at 8 weeks (n = 101). Informed and written consent was obtained from the mothers of the infants, and the study was approved by a national ethics committee.
As soon as possible after birth, formula-fed infants were randomly assigned to be fed with a nucleotide-supplemented infant formula (31 mg/L) (n = 100) or a control formula with <5 mg/L nucleotides (n = 100). A random permuted-block design, stratified according to center (Leicester or Nottingham), was allocated by an independent statistician, and allocation was concealed by the use of numbered, sealed, opaque envelopes. Infants not withdrawn from the study continued to receive the assigned formula milk until the age of 5 months. Research staff and mothers were blind to the identity of the formula.
The composition of supplemented formula was based on the concentration of free nucleotides and nucleosides in human milk as reflected in European regulations in force at the time of the study. Supplemented formula contained: cytidine monophosphate (15 mg/L), adenosine monophosphate (6 mg/L), uridine monophosphate (5 mg/L), inosine monophosphate (3 mg/L), and guanosine monophosphate (2 mg/L), whereas the control formula (Farley's First Milk) had <3 mg/L measurable cytidine monophosphate. The formulas (manufactured by H. J. Heinz Company Ltd, Hayes, United Kingdom) were identical except for their nucleotide concentrations (Table 1) and met European guidelines for formula composition.
Clinical, social, demographic, and anthropometric data were collected in formula-fed infants at the time that they were randomly assigned to formula groups (baseline) and in breastfed infants at the age of 8 weeks. Social class identification was coded according to the registrar general's classification and was based on the occupation of the parent who provided the main financial support for the family. Weight was measured by using electronic scales (Seca, Hamburg, Germany) that were accurate to 1 g, and length was measured by using a Rollametre (Raven, Dunmow, United Kingdom) that was accurate to 1 mm. Occipitofrontal head circumference (OFC) was measured by using a nonstretchable tape accurate to 1 mm. Anthropometric measurements were repeated at follow-up home visits at ages 8, 16, and 20 weeks.
Research nurses asked mothers of the infants who participated in the study to record clinical information on their infants, including episodes of illness, any treatment given, medication use, and consultations with health care professionals. Mothers also recorded the age at which foods other than milk were first introduced and the volume of formula-milk intake, bowel function, and food intake for a 3-day period before each follow-up visit. Mothers were provided with 3-day dietary intake diaries and trained in their use. Tolerance to study formulas was monitored by asking mothers to record crying time per 24-hour period, the number of nighttime waking periods, pacifier use, and episodes of colic (according to mother's interpretation of crying symptoms). Research nurses verified all recorded information at each home visit.
The a priori efficacy study outcome was the effect of nucleotide supplementation on stool microbiota and incidence of diarrhea.26 Infant growth was both a safety and efficacy outcome. Anthropometric measurements were expressed as SD scores (z scores) calculated by using published centiles.33 Growth was assessed as attained size at ages 8, 16, and 20 weeks and as the change in size between these ages. To allow comparison with previously published data,25 growth was also expressed as the rate of gain in weight, length, and OFC in the first 8 weeks per anthropometric variable at baseline.25 Sample size was calculated to detect a 0.4 SD difference in anthropometric outcomes between randomized formula-fed groups with 80% power and a P value of <.05.
Student's t test was used to compare randomly assigned formula-fed groups for variables that were normally distributed. The Mann-Whitney U test was used for variables that were not normally distributed, and χ2 test for dichotomous variables. Initial analyses were on an intention-to-treat basis. Multiple linear-regression analysis was then used to adjust for potential confounding factors (gender, socioeconomic status expressed as manual or nonmanual social class, birth weight z score, and length of gestation) together with the anthropometric variable of interest at baseline (randomization). In a secondary analysis, growth in breastfed infants was compared with growth in infants in the 2 formula-fed groups by using 1-way analysis of variance and Bonferroni corrections. Statistical analyses were conducted by using SPSS 12.0 (SPSS Inc, Chicago, IL) for Windows (Microsoft, Redmond, WA) and statistical significance taken as P < .05.
A total of 301 infants were recruited, and of these 88 of 100 infants fed nucleotide-supplemented formula, 85 of 100 infants fed control formula, and 96 of 101 breastfed infants were still being followed at the age of 5 months (Fig 1). Infant participation the study was discontinued for reasons that were not given or were social and nonclinical. Mothers of 5 infants (1 infant in the nucleotide-supplemented formula group and 4 in the control-formula group) removed them from the study because of perceived problems with the milk, such as apparent hunger (n = 2) or vomiting (n = 3). Mothers changed the diets of these infants to formulas different from those used in the study. Mothers who discontinued their infant's participation in the study did not allow follow-up visits, so these infants were not included in analyses performed when participating infants were 5 months old.
Both trial formulas were well tolerated, and no serious adverse events were reported. The time spent crying, incidence of colic, night-time wake periods, and use of pacifiers did not significantly differ between randomized groups.26 No infant had a diagnosis of persistent diarrhea or other chronic illness likely to affect growth.
Nucleotide-supplemented formula and control-formula groups were closely matched at the time of randomization (Table 2). There were no significant differences between randomized groups in incidence of eczema or asthma, use of antibiotics during the follow-up period (data not presented), or volume of formula intake (Table 3). The mean age at which solid food was introduced was ∼14 weeks for formula-fed infants and 15 weeks for breastfed infants.
Infant Growth in Randomly Assigned Formula-Fed Groups
Infants randomly assigned to be fed with a nucleotide-supplemented formula had greater OFC at ages 8, 16, and 20 weeks than infants fed with control formula (Table 3) (mean difference in z scores at the age of 8 weeks: 0.4 [95% confidence interval (CI): 0.1–0.7]; P = .006). This difference remained after adjustment for OFC z score at randomization, together with potential confounding factors (gender, socioeconomic status, birth weight z score, and gestation), at 8 weeks of age (adjusted mean difference: 0.2 z scores [95% CI: 0.1–0.4]; P = .005) and at 20 weeks of age (adjusted mean difference: 0.2 z scores [95% CI: 0.001–0.5]; P = .049), but not at the age of 16 weeks (adjusted mean difference: 0.2 z scores [95% CI: −0.04 to 0.4]; P = .1). Additional adjustment for mother's OFC z score did not attenuate this finding (adjusted mean difference at the age of 8 weeks: 0.2 z scores [95% CI: 0.1–0.4]; P = .008).
The mean z score for weight was greater in infants fed with nucleotide-supplemented formula than infants fed with control formula at 8 weeks of age but not at 16 and 20 weeks (mean difference at 8 weeks of age: 0.3 z scores [95% CI: 0.01–0.7]; P = .04) (Table 3). This difference remained after adjustment for potential confounding factors (gender, socioeconomic status, and gestation) together with weight z score at randomization (adjusted mean difference: 0.2 z scores [95% CI: 0.04–0.4]; P = .02). Length did not significantly differ between randomized formula-fed groups at any age (Table 3).
Infants fed nucleotide-supplemented formula showed greater increases in weight and OFC between the time of random assignment to formula group and 8 weeks of age than infants fed control formula (mean difference for weight: 0.3 z scores [95% CI: 0.05–0.4]; P = .02) (mean difference for OFC: 0.2 z scores [95% CI: 0.05–0.4]; P = .01) (Table 3), even after adjustment for potential confounding factors (as above), together with anthropometric variables at randomization (adjusted mean difference for weight: 0.2 z scores [95% CI: 0.04–0.4]; P = .02) (adjusted mean difference for OFC: 0.2 z scores [95% CI: 0.07–0.4]; P = .005). Changes in z scores for weight and OFC between 8 and 16 or 16 and 20 weeks of age did not significantly differ between randomly assigned formula-fed groups (Table 3).
The rate of gain in OFC in the first 8 weeks (as analyzed according to Cosgrove et al25) was greater in infants fed with nucleotide-supplemented formula (mean [SD]: 16.6 [3.6] mm/OFC at baseline per week) than controls (15.6, 3.1 mm/OFC at baseline per week) (mean difference: 1.0 mm/OFC at baseline per week [95% CI: 0.04–2.0]; P = .04). Similar findings were obtained for the rate of weight gain in the first 8 weeks (mean [SD]: 79.4 [23.7] vs 72.5 [22.2] g/kg weight at baseline per week in nucleotide-supplemented and control formula groups, respectively; mean difference: 7.0 g/kg weight at baseline per week [95% CI: 0.2–13.7]; P = .04). The rate of length gain did not significantly differ between randomized formula-fed groups (data not shown).
The effect of nucleotide supplementation on OFC was not a consequence of greater weight gain alone. Thus mean OFC of nucleotide-supplemented infants was greater than controls at 8 weeks after adjustment for potential confounding factors (as above), OFC z score at randomization, together with weight z score at 8 weeks (adjusted mean difference: 0.2 z score [95% CI: 0.01–0.3]; P = .04). Similarly, infants fed nucleotide-supplemented formula had a significantly greater increase in OFC z score from randomization to the age of 8 weeks even after adjustment for potential confounding factors, OFC z score at randomization, together with the change in weight z score between this period (adjusted mean difference: 0.2 z scores [95% CI: 0.01–0.3]; P = .03).
OFC was greater in infants given nucleotide-supplemented formula than controls after adjustment for birth weight z score (see above) or adjustment for the number of infants born SGA (birth weight <10th centile for gestation and gender)33 (adjusted mean difference: 0.6 z scores [95% CI: 0.2–0.9]; P = .003). However, the effect of nucleotide supplementation on head growth was greater in those born with lower birth weight: the interaction randomized formula-fed group × birth weight z score was statistically significant for z-score change in OFC in the first 8 weeks (P = .04) but not for z-score change in weight in the first 8 weeks (P = .9).
Comparison With Breastfed Infants
In secondary analyses, OFC z score at 8 weeks of age was greater in breastfed infants than infants fed control formula (P = .04), but did not significantly differ from that of infants fed nucleotide-supplemented formula (P = .9) (Table 3). Weight z score at 8 weeks of age did not significantly differ between the 3 dietary groups (P = .1).
The presence of nucleotides in breast milk, but not in unsupplemented formula, is suggested to have benefits for health including improved somatic growth.19,24,25 Our findings are consistent with this hypothesis and suggest that nucleotide supplementation of formula increases infant weight gain and head growth. Nucleotides could therefore be conditionally essential in some formula-fed populations. Importantly, the effect of nucleotide supplementation in increasing OFC was independent of changes in body weight, which suggests a preferential effect of nucleotide supplementation on head growth. Because the rapid increase in head size in early infancy reflects an increase in brain volume during a critical period in development and is related to higher cognitive function later in life,29,–,32 these data raise the hypothesis that nucleotide supplementation of infant formula could have advantages for long-term cognitive development. Theoretically, therefore, higher nucleotide concentrations found in human milk compared with most formulas may contribute to the benefits of breastfeeding for early head growth1,27,28 and hence later cognitive development.2,3
Our data were consistent with those of Cosgrove et al,25 who found substantial and clinically important effects of nucleotide supplementation for weight gain and head growth in formula-fed infants born SGA. The effect size (difference in OFC growth rate between nucleotide-supplemented infants and controls of ∼2 mm/m OFC at baseline per week) was greater than in our study (1 mm/m OFC at baseline per week),25 probably because of greater catch-up growth in infants born SGA. Nevertheless, even this smaller effect in our study resulted in greater OFC in nucleotide-supplemented compared with control-formula–fed infants at 8 weeks (by 0.4 z scores), a substantial effect for populations that amounts to ∼8% of the population variation in head size and is similar to the 10% difference in growth rates attributable to nucleotides according to Cosgrove et al.25 This effect, although greatest at 8 weeks of age, remained at 20 weeks, which suggested a longer-term benefit of nucleotide supplementation on head growth. In contrast, differences in weight between infants in randomly assigned formula-fed groups were not apparent at ages 16 and 20 weeks, probably because of the addition of other dietary sources of nucleotides in both groups with the introduction of complementary feeding (at the age of ∼14 weeks). Nucleotide supplementation seemed to ameliorate the disadvantage of formula feeding compared with breastfeeding for head size at the age of 8 weeks. Because breastfed infants may have faster head growth than formula-fed infants this finding suggests that a lower concentration of nucleotides in formula compared with breast milk may limit the optimal rate of head growth in some formula-fed infant populations.1,27,28 This effect of nucleotides may be greatest in infants born SGA, as suggested previously,25 and was supported by our finding of significantly greater head growth in infants born SGA who were fed with nucleotide-supplemented formula.
In contrast to our data, and to other reported data from infants born SGA,25 results of previous randomized trials have not shown advantages of nucleotide-supplemented formula for somatic growth.20,–,23 There are several potential explanations for this difference, although few data to support a particular hypothesis. One theory is that nucleotides are conditionally essential during periods of rapid growth and hence nucleotide-supplemented formula may have greater effects on growth in populations with faster postnatal growth, such as infants born SGA. However, postnatal growth is determined by genetic and environmental factors34,35 other than size at birth, including the degree that maternal characteristics affect fetal growth. Therefore, an inherently faster rate of postnatal growth could explain growth-promoting effects of nucleotides in the current population, a hypothesis supported by the faster weight gain in the first 8 weeks in infants fed control formula relative to the UK reference population (by 0.5 SD)33 (Table 3).
Another hypothesis to explain the variation in reported benefits of nucleotide-supplemented formula for somatic growth is the existence of a threshold intake, above which additional dietary nucleotides have little effect on growth. Control formulas used in previous studies contained 10 mg/L inherent nucleotides21,23 (compared with <3 mg /L in our study), an amount that may have obscured any advantage of nucleotide supplementation at higher concentrations. Differences in study findings may also be attributable to population differences in absorption, use, and metabolism of nucleotides or beneficial effects of nucleotides on gastrointestinal microbiota,26 although little evidence exists to support this hypothesis.
A lack of evidence for the mechanism of the beneficial effects of nucleotide supplementation is a limitation of our study. Nucleotides may act as flavor enhancers6 and promote growth by increasing formula intake, but like previous investigators,20 we found no evidence for this. Nucleotide supplementation could have protein-sparing effects. For instance, Uauy36 calculated that daily nucleotide synthesis requirements could consume up to 10% of protein requirements in infancy. Nucleotides also enhance immune function5,–,12 and so could improve growth by reducing illness episodes, although, again, we found no evidence to support this hypothesis. A more probable mechanism is a beneficial effect of nucleotides on the gastrointestinal tract, which could enhance epithelial function and nutrient absorption by a trophic effect13,–,15 or via an increase in intestinal blood flow.17,18
Among the few human populations to have a minimal nucleotide intake are infants fed formula without nucleotide supplementation. Our findings of a beneficial effect of nucleotides on head growth in formula-fed infants, together with the previously described benefits of nucleotide supplementation for immune function and diarrhea prevention, support the addition of nucleotides to all cow's milk–based infant formulas. However, controversy exists as to the benefits of faster weight gain, and the slower weight gain that occurs in breastfed infants compared with formula-fed infants may be beneficial.37 Nevertheless, the advantage of nucleotide supplementation for increased head growth during a critical period for brain development may have important long-term implications for formula-fed infants.
This study was funded by the Medical Research Council with a charitable contribution from the H. J. Heinz Company Ltd (Hayes, Middlesex, United Kingdom). We thank the H. J. Heinz Company Ltd for supply of the trial formulas.
- Accepted May 28, 2010.
- Address correspondence to Atul Singhal, MD, MRCP, Institute of Child Health, Medical Research Council Childhood Nutrition Research Center, 30 Guilford St, London WC1N 3EH, United Kingdom. E-mail:
Dr Singhal was the principal investigator and main author; and Ms Clough and Ms Jenkins were responsible for data collection, which was supervised by Ms Lanigan and Ms Kennedy. All authors contributed to study design and preparation of the final manuscript for submission.
FINANCIAL DISCLOSURE: The authors have indicated they have no financial relationships relevant to this article to disclose.
- SGA =
- small for gestational age •
- OFC =
- occipitofrontal head circumference •
- CI =
- confidence interval
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- Copyright © 2010 by the American Academy of Pediatrics