Are long-chain polyunsaturated fatty acids essential nutrients in infancy ?

We investigated whether the disparity in neural maturation between breastfed and formula-fed term infants could be corrected by the addition of fish oil, a source of docosahexaenoic acid (DHA, 22:6omega3), to infant formula. Healthy, term infants were randomised at birth to receive either a supplemented or placebo formula if their mothers had chosen to bottle feed. Breastfed term infants were enrolled as a reference group. Infant erythrocyte fatty acids and anthropometry were assessed on day 5 and at 6, 16, and 30 weeks of age. Visual evoked potential (VEP) acuity was determined at 16 and 30 weeks.

VEP acuities of breastfed and supplemented-formula-fed infants were better than those of placebo-formula-fed infants at both 16 and 30 weeks of age (p<0-001 and p<0.01). Erythrocyte DHA in breastfed and supplemented-formula-fed infants was maintained near birth levels throughout the 30-week study period but fell in placebo-formula-fed infants (p<0.001). Erythrocyte DHA was the only fatty acid that consistently correlated with VEP acuity in all infants at both ages tested. A continuous supply of DHA may be required to achieve optimum VEP acuity since infants breastfed for short periods (<16 weeks) had slower development of VEP than infants receiving a continuous supply of DHA from either breastmilk or supplemented formula. Erythrocyte arachidonic acid (20:4omega6) in supplemented-formula-fed infants was reduced below that of infants fed breastmilk or placebo formula at 16 and 30 weeks (p<0.001), although no adverse effects were noted, with growth of all infants being similar.

DHA seems to be an essential nutrient for the optimum neural maturation of term infants as assessed by VEP acuity. Whether supplementation of formula-fed infants with DHA has long-term benefits remains to be elucidated.

Lancet 1995; 345:1463-68

Preterm breastfed infants are reported to have advanced neural maturation compared with bottle-fed infants as assessed by electroretinograms, visual evoked potentials (VEP), and psychometric tests.[1-4] It has been proposed that the higher tissue levels of long-chain polyunsaturated fatty acids (LCPUFA), particularly docosahexaenoic acid (DHA, 22:6omega3), reported in breastfed infants may be an important causative factor.[1-3] Breastmilk contains a range of LCPUFAs, whereas bottle-milk formulae are fortified only with precursor essential fatty acids such as linoleic acid (18:2omega6) and alpha-linolenic acid (18:3omega3). There is evidence that formula-fed infants are unable to metabolise their full requirement of LCPUFA from precursors, since they have less DHA and less arachidonic acid (20:4omega6) in their erythrocytes than breastfed infants.[5] The absence of LCPUFA from formula may be further exacerbated by inhibition of incorporation of endogenously produced LCPUFA by the high concentrations of linoleic acid currently in most infant formulae.[6]

The main concern about fatty-acid nutrition in infancy is in preterm infants, because the rate of brain growth is known to be greatest in the last trimester of pregnancy. However, brain growth continues throughout the first year of life and there is evidence that cerebral DHA concentrations are higher in term infants who were breastfed than in those fed formula.[7,8] Further, incorporation of DHA into the brain cortex increases with duration of breastfeeding, at least for the first 40 weeks of life, suggesting that a long-term supply may be necessary.[8] These data may be of functional importance; term infants fed breastmilk have better VEP acuities than those fed formula at 4-5 months of age.[9,10]

We studied, in a randomised trial, whether DHA is an essential nutrient for neural development in term infants by determining whether it was possible to match VEP function and erythrocyte fatty-acid profiles of bottle-fed infants to those of breastfed infants by adding a source of DHA (fish oil) to infant formula. A secondary aim was to investigate the effect of breastfeeding duration on VEP acuity and hence gain insight into the minimum duration of breastfeeding required for maximum beneficial effect on neural maturation.

Women giving birth to healthy infants of 37-42 weeks' gestation at Flinders Medical Centre, who had no history of lipid-metabolism disorders, insulin-dependent diabetes, drug or alcohol abuse, and had babies with appropriate weight for gestation, were approached to enter the study. Written informed parental consent was obtained and the study was approved by the Committee on Clinical Investigations. Socioeconomic status of both parents was determined according to a six-point scale.[11] The long-term unemployed were assigned the lowest rank (the highest score). Maternal education was ranked as 0--no formal education, 1--primary school level, 2--mid-secondary school level, 3--completion of secondary school, 4--completion of a certificate or diploma, 5--tertiary degree, and 6--higher degree.

An ophthalmic examination was done when infants were aged 12-16 weeks. The pupils were dilated, the fundus examined, and cycloplegic refraction carried out. Infants with refraction outside the range of -3 to +5 dioptres or with severe astigmatism (>/-1.75 dioptres) were excluded from the VEP aspect of the study.

Mothers who intended to bottle feed their infants were randomly assigned to either a standard or a supplemented formula and were unaware of the formula type. When the study started, fish oil was the most accessible DHA supplement for infant formula. Since low plasma arachidonic acid in preterm infants receiving fish-oil-supplemented formula had been associated with poor growth relative to those fed standard formula,[12] we used a blend of fish oil (to provide omega3 LCPUFA such as eicosapentaenoic acid [20:5omega3] and DHA) and evening primrose oil (to increase arachidonic acid by supplying GLA [18:3omega6], a metabolic precursor of arachidonic acid that by-passes the supposed rate-limiting delta-6 desaturase enzyme) in a 1/1 ratio. The base formula was donated by Nestle Australia. The fatty acid compositions of the two study formulae were analysed before and intermittently throughout the study (table 1).

Mothers who chose to breastfeed were encouraged to do so for as long as possible. The definition of full breastfeeding was no formula in the first 16 weeks of life and no more than 120 mL formula per day from 16 to 30 weeks of age. Infants who did not meet these criteria were removed from the reference breastfed group, provided with placebo formula and continued in the study as a partially breastfed group. Infants with feeding or settling problems were referred, when appropriate, to a dietitian, paediatrician, or lactation consultant. All mothers received nutrition advice according to National Health and Medical Research Council guidelines with the recommendation that solid foods be introduced between 4 and 6 months of age.[13] The fatty-acids contribution of solid foods was not quantified because common weaning foods contain negligible amounts of LCPUFA.[14]

Staff involved with the assessments were unaware of the treatment group. VEP was done at 16 and 30 weeks of age. Infants were seated with their mother 1 m away from a 50 cm monitor presenting high-contrast black-and-white checkerboard pattern reversal (2 Hz) stimuli. The active electrode was placed 3 cm above the inion, the reference electrode at 30% of the nasion-to-inion distance, and the inactive electrode on the forehead. Three recordings were made at each checkerboard pattern (7, 14, 28, 42, and 55' of arc). The peak to peak amplitude of the VEP (N1-P1) response was measured and plotted against log of the angle subtended by each check size. The linear portion of the plot was extrapolated to 0 muV to give the theoretical value that would just elicit a response (log of the minimum angle of resolution, logMAR). Points were excluded from the regression if they were not on the linear portion of the stimulus-response function or represented amplitudes of 2 muV or less. VEP acuity extrapolations were accepted as valid only if there were at least three points and r2 was 0.80 or more and p less than 0.05.

Blood samples were taken by heel-prick (200 muL) on day 5 and weeks 6, 16, and 30. Erythrocytes were separated from plasma and total lipids extracted, methylated, and quantified by capillary gas chromatography.[5] Weight, length, and head circumference were measured at birth, 6, 16, and 30 weeks of age. Infants were weighed undressed on a Seca Baby Balance (Model 727, Seca, Germany). Length was determined in the supine position to the nearest 0.5 cm by two individuals with an infant measuring mat. Head circumference was measured at the largest occipitofrontal circumference to the nearest 0.1 cm with a tape. Growth measurements were standardised for sex differences by calculating z-scores for weight-for-age and length-for-age, from the National Centre for Health Statistics (NCHS) data.[15]

This study was designed to test the hypothesis that VEP acuity and erythrocyte DHA of formula-fed infants could be improved to breastfed levels by adding a source of DHA to formula. Based on data from our laboratory,[5,9] sample size was calculated to allow detection of 0.3 log unit (1 octave) differences in VEP acuity and to detect a mean difference of 3 percentage points in the proportion of erythrocyte DHA. Enrolment of breastfed infants was higher, because we anticipated at least half would be weaned to formula before 30 weeks despite the mother's initial intent to breastfeed for longer.

All data are expressed as mean (SD). The effects of diet and age on VEP acuity were determined by repeated-measures ANOVA. Significant differences between dietary treatments at each assessment age and between time points within each treatment were identified by least significant difference (LSD). X2 was used to determine whether there were any associations between dietary grouping and the infant's ability to respond at the smallest checkerboard pattern at 16 weeks and dietary grouping and the presence of a successful VEP acuity extrapolation at 30 weeks.

The effects of diet and age on erythrocyte fatty acids of breast, placebo, and supplemented formula-fed infants was also determined by repeated-measures ANOVA. Significant differences due to either dietary grouping or age were subsequently identified by LSD. Infants who were breastfed for part of the 30-week study were not included in the repeated-measures ANOVA model but data from these infants were used to determine associations between length of breastfeeding and VEP acuity and were included in correlations between erythrocyte PUFA and VEP acuity at 16 and 30 weeks. Associations between VEP acuity and erythrocyte PUFA were tested at 16 and 30 weeks by Pearson correlation coefficients.

Comparisons of weight and length z-scores between dietary groups were made with a repeated-measures ANOVA model where dietary grouping and age were treated as the main effects. Post-hoc analysis was by LSD. Possible interactions between standardised growth and erythrocyte fatty acids were investigated with Pearson correlation coefficients at all assessment times. Comparison of non-parametric variables (ie, maternal education and social status) between dietary groups was by Kruskal-Wallis one-way ANOVA on ranks.

89 subjects were enrolled, 10 of whom later withdrew their consent (4 family relocating, 2 lactose intolerance, 2 maternal anxiety about blood taking, 1 work commitments, and 1 diagnosis of congenital heart disease) and were excluded. Of the 79 who completed the trial, 23 were fully breastfed, 24 partially breastfed, 19 were fed placebo formula, and 13 supplemented formula (table 2). Mothers who chose to breastfeed their infants had attained a greater level of formal education than mothers who chose to formula feed.

2 of the 79 subjects were excluded from the VEP assessment because of astigmatism or squint. At 16 weeks, a further 11 subjects had no VEP recorded as a result of technical difficulties with equipment and 2 acuities could not be extrapolated. At 30 weeks, 1 subject was absent and 16 of 76 acuities could not be extrapolated. There was no association between dietary grouping and ability to extrapolate an acuity threshold.

At 16 weeks of age, not all infants were able to evoke cortical responses to our smallest checkerboard pattern (check subtending visual angles of 7' arc). 87% of fully breastfed infants, 82% of partially breastfed infants, 39% of placebo-formula-fed infants, and 100% of supplemented-formula-fed infants produced recordable VEP to checkerboard patterns subtending angles of 7' arc (Pearson X2 value 15.88, df=3, p<0.001).

Breastfed infants had significantly better VEP acuities than infants fed placebo formula at both 16 (p<0.001) and 30 (p<0.01) weeks (figure 1A). Subjects fed supplemented formula had acuities matching those of fully breastfed infants at both time points. Analysis of paired data showed that the VEP acuity of all infants, irrespective of dietary grouping, improved with age but differences due to diet were maintained and there was no evidence of "catch-up" with age (figure lB).

Infants who were breastfed for less than 16 weeks had VEP acuity thresholds at 16 weeks that were intermediate between those of fully breastfed infants and those that had received placebo formula since birth (figure 2). By 30 weeks, the VEP acuity of infants who received breastmilk for less than 16 weeks was no different from that of the placebo-formula group. The effect of breastfeeding for between 16 and 30 weeks (mean 23 [4] weeks) was similar to full breastfeeding for 30 weeks.

At enrolment (day 4-6), breastfed infants already had higher levels of DHA and arachidonic acid and lower linoleic acid than those fed formula (table 3). 21 of 23 fully breastfed infants; 18 of 19 placebo-formula-fed infants; and 12 of 13 supplemented-formula-fed infants had complete sets of serial fatty acid measures. DHA in breastfed infants was reduced at 30 weeks but declined rapidly in infants fed placebo formula. Supplemented formula resulted in DHA concentrations being higher than those of breastfed infants at 16 and 30 weeks. A decrease in arachidonic acid between day 5 and week 6 was observed in all infants, but by 16 and 30 weeks arachidonic acid was lower in the supplemented-formula group than in breastfed and placebo-formula-fed infants. Linoleic acid increased in all dietary groups, being highest in the placebo-formula-fed group. Eicosapentaenoic acid remained low in breastfed or placebo-formula-fed infants but increased tenfold in infants fed supplemented formula.

Erythrocyte DHA correlated with VEP acuity at 16 and 30 weeks (r2=0.23, p<0.001; r2-0.12, p<0.005). These correlations persisted when only data from randomised (formula-fed) infants were analysed (r2=0-37, p<0.001; r2=0.17, p<0.05 at 16 and 30 weeks, respectively). No other fatty acid consistently correlated with VEP acuity.

Weight and length z-scores were similar between dietary groups at all assessment times (figure 3 and figure 4), as was head circumference (at 30 weeks 44.5 [1.2] cm breastfed; 45-2 [1.2] cm placebo formula; 45.0 [1.6] cm supplemented formula). There were no correlations between any standardised growth measurements and erythrocyte PUFA.

Our randomised clinical trial involving term infants shows that omega3 LCPUFA supplementation of formula is important for the development of visual acuity. Supplementation with 0.36% DHA resulted in an improvement in VEP acuity to match that of fully breastfed infants. We[9] and others[10] have previously reported differences in VEP acuity between breastfed and formula-fed term infants and shown an association with dietary DHA, which is present in breastmilk but not in formula. Comparable studies with preterm infants have drawn similar conclusions.[1,3,16]

Differences of approximately 0.3 log units (1 octave) in VEP acuity were apparent between infants who received omega3 LCPUFA (breastfed and supplemented-formula groups) and those fed standard placebo formula at both 16 and 30 weeks of age. The results provide no evidence of "catch-up" with age and are consistent with the visual loss observed in the rhesus monkey model of omega3 fatty acid deficiency.[17] Other studies in preterm and term infants where acuity was electrophysiologically determined have not used the VEP beyond 4 months (16 weeks) corrected age.[3,10] Studies that have assessed the role of omega3 fatty acid nutrition on the visual development of infants born at term were not randomised and have only compared breastfeeding and formula feeding.[10,18,19] Birch et al[10] showed better VEP acuity and forced-choice preferential looking in breastfed compared with formula-fed infants at 4 months of age. These results were confirmed by Jorgensen et al[18] who showed improved behavioural acuity at 2 and 4 months in exclusively breastfed infants over those that were predominantly formula-fed, despite the latter group being initially breastfed for less than 4 weeks. By contrast, Innis et al[9] failed to detect any differences in behavioural acuity between breastfed and formula-fed term infants at 3 months of age. Relative to behavioural methods, VEP acuity has better acuity thresholds and tends to mature more rapidly.[20,21] Alternatively, the disparity in the results may be explained by differences in the availability of alpha linolenic acid (the DHA precursor) in the various formula fat blends tested.

In our study, good discrimination of visual acuity was possible at 16 weeks as the smallest checkerboard pattern had individual checks that subtended visual angles of 7' arc, which is near the sensory threshold for most infants at this age. However, there were difficulties with obtaining VEP acuities at 30 weeks with 16 of 76 VEP recordings not meeting our criteria for a valid extrapolated visual acuity threshold. Reasons for this may include the stimuli being presented too far from the acuity threshold, difficulty in maintaining the infant's attention at 30 weeks, and the fact that amplitude as a function of check size may not always decrease linearly in the near threshold region.[22,23] Unsuccessful extrapolations were distributed amongst the various treatment groups and other research workers have reported similar or greater proportions of unsuccessful extrapolations.[23]

Erythrocyte fatty acids differed between infants who were breastfed and formula-fed, even by day 5. Unsupplemented-formula-fed infants showed a 50% decline in erythrocyte DHA by 16 weeks of age similar to that found in earlier studies in both term[5] and preterm[24,25] infants. DHA was higher in the infants fed supplemented formula than in those breastfed despite our aim to match breastfed levels by designing a formula to mimic DHA in breastmilk. Analysis of breastmilk from mothers in this study showed that we had overestimated the proportion of DHA as breastmilk fat and therefore supplemented-formula-fed infants received 0.36% of their fat as DHA, whereas breastmilk contributed 0.21% DHA. There has been a change in DHA levels in breastmilk over the past decade in Australia from 0.32%[26] to 0.21% in our study and our data demonstrate that this lower level is inadequate to maintain erythrocyte DHA at baseline (day 5) levels. It may be that the influence of maternal fat composition on infant development needs to be considered?

Erythrocyte arachidonic acid decreased in all groups between day 5 and week 6. Arachidonic acid remained low only in the supplemented-formula-fed infants. Eicosapentaenoic acid and possibly DHA found in the supplement are known to compete with arachidonic acid for incorporation into cell membranes.[6] Erythrocyte arachidonic acid of supplemented infants did, however, not fall below the level observed at 6 weeks despite continued increases in erythrocyte eicosapentaenoic acid and DHA. Placebo-formula-fed infants showed an increase in arachidonic acid between 6 and 30 weeks despite having no direct dietary source, while levels in breastfed infants returned to baseline (day 5) by 16 weeks. Collectively these data suggest a conservation of erythrocyte arachidonic acid? The addition of evening primrose oil as a metabolic precursor of arachidonic acid was unsuccessful in preventing a fall in arachidonic acid in infants on supplemented formula, since concentrations at 16 weeks were similar to those reported in preterm infants at the equivalent age supplemented with fish oil alone.[25]

There were no differences between dietary groups for weight and length z-scores and there were no associations between any erythrocyte PUFA and growth measurements. Furthermore, all mean weight-for-age and length-for-age z-scores were between the 25th and 75th percentiles,[15] showing normal growth. The only other studies that have assessed long-term fish-oil supplementation have been in preterm subjects, with one study reporting a small adverse effect on growth[12,29] and the other reporting no effect.[30] Sick preterm infants may be more vulnerable to changes in arachidonic acid than term infants.

Our study and others provide evidence that DHA may be the factor associated with improved visual/neural performance of supplemented infants compared with those fed standard formula. Whether dietary fat relates to long-term outcome will be resolved with follow-up studies; however, the controversy regarding the feeding advice for infants of today remains. The best advice seems to be to breastfeed for at least 4 months and, possibly, a year.

This project was partly funded by grants-in-aid from Channel 7 Children's Medical Research Foundation, Nestle Australia, Scotia Pharmaceuticals UK, and the Flinders Medical Centre Research Foundation. We thank Ms Tasha Graham, Ms Ela Zielinski, Ms Nancy Hermsen, and Ms Fleurette Martin for administrative and technical support.

Table 1: Mean (SD) % fatty acid composition of Infant formulae and breastmilk during 16th week of lactation

ND=not detected. (a)Each analysis represents a pooled breast milk sample collected over 7 consecutive days in the 16th week of lactation from mothers of fully breastfed infants. (b)The analysis of infant formulae was checked before the commencement of the study and intermittently throughout the study.

Table 2: Characteristics of study subjects

Values are mean (SD). *Fully breastfed and breastfed for <30 weeks groups significantly different from other two groups (p<0.05); (a)fully breastfed group significantly different from breastfed for <30 weeks (p<0.05).

Table 3: The effect of diet and age on infant erythrocyte LCPUFA (mean % total fatty acids [SD])

Linoleic acid ANOVA: diet p<0-001; age p<0.001; diet x age p<0.001.

Arachidonic acid ANOVA: diet p<0.001; age p<0.001; diet x age p<0.001.

Eicosapentaenoic acid ANOVA: diet p<0.001; age p<0.001; diet x age p<0.001.

DHA ANOVA: diet p<0.001; age p<0.001; diet x age p<0.001.

Superscript letters indicate differences between diet groups at each age; superscript numbers indicate differences between ages within each diet group.

GRAPH : Figure 1: Mean (SD) VEP acuity at 16 and 30 weeks of age (A), and VEP acuity as a function of age (B) *p<0.01; **p<0-001. ANOVA: diet p<0.001; age p<0.001; diet x age NS. Letters indicate differences between diet groups at each age. Numbers indicate differences between ages for each diet group.

GRAPH : Figure 2: VEP acuity as a function of age of breastfed, partially breastfed, and placebo-formula-fed infants ANOVA: diet p<0.001; age p<0.001; diet x age NS. Letters indicate differences between diet groups at each age. Numbers indicate differences between ages for each diet group.

GRAPH : Figure 3: Weight-for-age z-scores ANOVA: diet NS; age p<0.001; diet x age p<0.005. Numbers indicate differences between ages for each diet group.

GRAPH : Figure 4: Length-for-age z-scores ANOVA: diet NS; age p<0-001; diet x age NS. Numbers indicate differences between ages for each diet group.

References