Omega-3 Supplementation During the First 5 Years of Life and Later

NUTRITION EPIDEMIOLOGY HIGHLIGHTS ORIGINAL ARTICLE Omega-3 supplementation during the ﬁrst 5 years of life and later academic performance: a randomised controlled trial

BK Brew1, BG Toelle1,2, KL Webb3, C Almqvist4,5 and GB Marks1for the CAPS investigators

BACKGROUND/OBJECTIVES: Consumption of oily fish more than once per week has been shown to improve cognitive outcomes in children. However, it is unknown whether similar benefits can be achieved by long-term omega-3 fatty acid supplementation. The objective was to investigate the effect of omega-3 fatty acid supplementation during the first 5 years of life on subsequent academic performance in children by conducting a secondary analysis of the CAPS (Childhood Asthma Prevention Study). SUBJECTS/METHODS: A total of 616 infants with a family history of asthma were randomised to receive tuna fish oil (high in long- chain omega-3 fatty acids, active) or Sunola oil (low in omega-3 fatty acids, control) from the time breastfeeding ceased or at the age of 6 months until the age of 5 years. Academic performance was measured by a nationally standardised assessment of literacy and numeracy (National Assessment Program Literacy and Numeracy (NAPLAN)) in school years 3, 5, 7 and 9. Plasma omega-3 fatty acid levels were measured at regular intervals until 8 years of age. Between-group differences in test scores, adjusted for maternal age, birth weight and maternal education, were estimated using mixed-model regression. RESULTS: Among 239 children, there were no significant differences in NAPLAN scores between active and control groups. However, at 8 years, the proportion of omega-3 fatty acid in plasma was positively associated with the NAPLAN score (0.13 s.d. unit increase in score per 1% absolute increase in plasma omega-3 fatty acid (95% CI 0.03, 0.23)). CONCLUSIONS: Our findings do not support the practice of supplementing omega-3 fatty acids in the diet of young children to improve academic outcomes. Further exploration is needed to understand the association between plasma omega-3 fatty acid levels at 8 years and academic performance. European Journal of Clinical Nutrition (2015) 69, 419–424; doi:10.1038/ejcn.2014.155; published online 13 August 2014

INTRODUCTION supplementation for 412 months in children. Furthermore, Long-chain omega-3 polyunsaturated fatty acids (LCPUFAs), evidence about the long-term effects of omega-3 LCPUFA eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) supplementation on cognitive performance after supplementation 9,18 are essential for neural growth and development, cell signalling has ceased is inconsistent, as is the evidence about the 1,2 correlation between plasma omega-3 LCPUFA concentration and and memory development. They accumulate rapidly in the brain 19–22 in the last trimester of pregnancy and the first 18 months of life.3 cognitive performance. Observational studies have found that consumption of oily fish, The objective of the study presented here was to test the effect fi fi a natural source of omega-3 LCPUFAs, more than once per week is of long-term supplementation with sh oil during the rst 5 years associated with improved cognitive performance in children, even of life on later cognitive performance in school years 3, 5, 7 and 9. after adjusting for the potential confounding effect of socio- In addition, we investigated whether plasma omega-3 LCPUFA fi economic factors.4–7 As a consequence of these observations, levels during the rst 8 years of life are associated with later there has been substantial commercial interest in promoting the cognitive performance. This question was addressed by conducting a secondary analysis of a randomised controlled trial: the cognitive benefits of supplementation with fish oil containing Childhood Asthma Prevention Study. LCPUFA during pregnancy, lactation, formula feeding or childhood. Some individual randomised controlled trials (RCTs) have shown small beneficial effects of omega-3 LCPUFA SUBJECTS AND METHODS supplementation on some cognitive outcomes among infants and children.8–11 However, two Cochrane reviews (identifying 20 Study design and study population relevant RCTs) and a meta-analysis of 12 RCTs have concluded The Childhood Asthma Prevention Study (CAPS) is a factorial, parallel- that there is no evidence to support claims that LCPUFA group randomised controlled trial designed to test the effectiveness of fi supplementation of lactating mothers and in infant formulae house dust mite avoidance and fatty acid supplementation during the rst fi 12–14 5 years of life as strategies for preventing asthma and allergy in high-risk have bene cial effects on cognitive function. Similarly, other children. The study design and outcomes have been previously fi – short-term RCTs of sh oil supplementation for children, either in reported.23 25 In brief, 616 pregnant mothers and their unborn babies capsule form or as fortified foods, have also found no cognitive were recruited between September 1997 and November 1999 from – benefits.15 17 No study has tested the effect of omega-3 LCPUFA hospitals in Sydney, Australia. The main eligibility criteria were that either

1Woolcock Institute of Medical Research, University of Sydney, Sydney, NSW, Australia; 2Sydney Local Health District, Sydney, NSW, Australia; 3Center for Weight and Health, College of Natural Resources and School of Public Health, University of California, Berkeley, CA, USA; 4Department of Medical Epidemiology and Biostatistics, Karolinska Institutet, Stockholm, Sweden and 5Astrid Lindgren Children’s Hospital, Lung and Allergy Unit, Karolinska University Hospital, Stockholm, Sweden. Correspondence: Dr BK Brew, Woolcock Institute of Medical Research, University of Sydney, PO Box M77, Missenden Road PO, Sydney, NSW 2050, Australia. E-mail: [email protected] Received 3 January 2014; revised 10 June 2014; accepted 13 June 2014; published online 13 August 2014 Omega-3 supplementation and academic performance BK Brew et al 420 the parent or an older sibling had a history of asthma or recurrent Scores obtained were 2008, 2009, 2010 and 2011 reading and numeracy wheezing, and that the child was born at 436 weeks of gestation. scores for each participant when they were in years 3, 5, 7 and 9. Data on the age of the mother at the child’s birth, smoking during pregnancy and both maternal and paternal educational levels were recorded prenatally. Gestational age at birth, birth weight and sex of the Statistical analysis child were obtained from hospital records soon after birth. Information on We conducted an intention-to-treat analysis, based on all randomised breastfeeding and the introduction of solid foods was recorded by nurses subjects, and a per-protocol analysis that was limited to subjects with at regular home visits during the first year of life. capsule compliance 450%. NAPLAN scores were transformed to s.d. units by dividing each score by the NSW-wide s.d. for each year and skill. NAPLAN scores for each year Omega-3 PUFA supplement intervention group and skill were compared between diet groups using the t-test. We The active intervention involved a daily fish oil supplement added to the then tested the overall effect of randomised group allocation using a infant’s formula or food and the supply of monounsaturated cooking oils mixed-model analysis, in which all results (that is, all available school years 23,26 and spreads for use in preparing the child’s food. The fish oil capsules and both skills) were included in a single model. In the mixed-model contained 500 mg of tuna fish oil (Nu-Mega Industries Pty Ltd, Brisbane, analysis, the fixed effects were school year, skill and diet group, and the Australia), which comprised 37% LCPUFA (including 135 mg of DHA and random effect was individual participant. Results are presented as the 32 mg of EPA per capsule) and 6% omega-6 PUFA (linoleic acid, difference between groups in s.d. units and 95% confidence intervals (CIs). arachidonic acid and docosapentaenoic acid). The goal was that formula- A similar mixed model was used in a secondary analysis to investigate the fed infants would receive the same amount of EPA as found in breast milk association between total plasma omega-3 PUFA concentration, measured and 1.5 times the amount of DHA. The canola-based cooking oils and at 18 months, 3 years, 5 years and 8 years with NAPLAN performance. margarines (Goodman Fielder foods, Macquarie Park, NSW, Australia) Potential confounder interaction terms were included in all models to contained approximately 16% omega-6 and 6% omega-3 PUFA. check for potential differences in the effect of omega-3 PUFA supple- The control supplementation regimen was designed to maintain the mentation on either skill type or NAPLAN performance in a school year. distribution of dietary PUFA at that observed in the Australian population A directed acyclic graph (DAG) was created using DAGitty version 1.1 at that time. The control regimen included a daily Sunola oil capsule (500 (Johannes Textor, Utrecht University, Utrecht, The Netherlands)29 to mg, Nu-Mega Industries) added to the child’s food. The capsules contained α explicitly describe the assumed causal model and, on this basis, to select 0.3% omega-3 -linoleic acid and 6% omega-6 linoleic acid PUFA. The which potential confounders should be included in the secondary analysis. control group also received polyunsaturated cooking oils and margarines See Supplementary Figure 1 for a diagram of the DAG used. The potential (Goodman Fielder foods) containing approximately 40% omega-6 and confounders included in the DAG were birth weight, sex, gestational age, 1.2% omega-3 PUFA for use in preparing the child’s food. Subjects and mother’s age at the child’s birth, maternal education level, maternal their parents were blinded to which group they were randomised to. smoking in pregnancy and full breastfeeding for more than 3 months. On Supplementation with oil capsules began when the child commenced the basis of the assumptions of the DAG, we included the following bottle-feeding or at the age of 6 months with their solid foods, whichever potential confounders in the adjusted models: birth weight, maternal age was earlier. Replacement of usual cooking oils and fat spreads began when solid foods were introduced to the child’s diet. The supplementation and maternal education. All statistical analyses were performed using SAS regimen continued until the child was 5 years of age. Enterprise Guide version 9.3 (SAS, Inc, Chicago, IL, USA). A negative control study was used to test for residual confounding of the association between total plasma omega-3 PUFA concentration Assessment of compliance measured at 8 years and NAPLAN scores.30 Body mass index (BMI, Consumption of oil capsules (capsule compliance) was estimated at expressed as quintiles) at the age of 8 years was used as a control exposure 6-month intervals by measuring the difference between the weight of variable to replace total plasma omega-3 PUFA at 8 years in the mixed- capsule bottles dispensed and their weight when returned and by dividing model analysis. BMI was chosen for this purpose as it is potentially this difference by the capsule weight. Subjects were expected to consume associated with the covariates that we tested, that is, birth weight, 1 capsule per day. Therefore, the number of capsules consumed was maternal age and maternal education, as well as with unmeasured divided by the number of days to estimate the percentage compliance confounders such as diet and parental income, and is not known to be over this period. Compliance was averaged over the 5-year duration of associated with academic performance. the study. The per-protocol analysis was based on those who had an average compliance of at least 50% (consuming at least 50% of the oil capsule dose provided). Ethics approval The initial study was approved by the human research ethics committees of the University of Sydney, the Children’s Hospital at Westmead and the Measurement of omega-3 levels in plasma Sydney South West Area Heath Services (Western Zone). The substudy was Blood was collected at ages 18 months, 3 years, 5 years and 8 years for the approved by RPAH Zone as Lead HREC (Human Reference Ethics measurement of plasma phospholipids by means of gas chromatography. Committee) for multisite research. The HREC reference is HREC/08/RPAH/ Results for total omega-3 PUFA were expressed as the percentage 472 and the protocol number is X08-0277. It was also approved by the 27 proportion of total fatty acids. The total omega-3 PUFAs measured in NSW Department of Education and Communities for the acquiring of the plasma included α-linoleic acid (18:3n-3), EPA, docosapentaenoic acid NAPLAN results (SERAP # 2011123). Informed consent was obtained from (22:5n-3), DHA and other minor n-3 fatty acids (16:2n-3, 18:4n-3 and the parents during pregnancy and then again when the children were 20:3n-3). Total omega-6 PUFAs were also measured, including linoleic acid 13–15 years of age to allow access to the historic NAPLAN data held by the (18:2n-6), dihomo-γ-linoleic acid (20:3n-6), arachidonic acid (20;4n-6) and NSW Department of Education and Communities. Consent for access to other minor n-6 fatty acids (18:3n-6, 20:2n-6, 22:2n-6 and 22:4n-6). NAPLAN data was also obtained from non-government schools.

Academic performance: National Assessment Program—Literacy RESULTS and Numeracy (NAPLAN) scores NAPLAN is conducted to assess all Australian students in school years 3, 5, Of the 616 participants recruited at birth, consent was obtained 7 and 9 (ages 8–9, 10–11, 12–13 and 14–15, respectively).28 The tests from the parents of 239 participants (39%) and their schools to measure proﬁciency in reading, writing, language conventions (spelling, obtain NAPLAN results released by the NSW Government grammar and punctuation) and numeracy, using a national test that has Department of Education and Communities (see Figure 1). been conducted annually on the same day in every Australian school since A comparison of those in the NAPLAN cohort (n = 239) with those 2008. The scores are continuous (from 1 to 1000) and are scaled so that who did not consent (n = 377) is shown in Table 1. The mothers of they can be compared across school year levels and over time; for example, a score of 350 has the same meaning in years 3, 5, 7 and 9, and those in the NAPLAN cohort were slightly older, more likely to be therefore individual progress can be monitored. tertiary educated and more likely to have fully breastfed (no solids NAPLAN data for this analysis were obtained from the NSW Government or formula) for longer than 3 months than the mothers of those Department for Education and Communities (the responsible authority). who did not consent to or provide NAPLAN data. Fathers of those

Enrolled and in the NAPLAN cohort were more likely to be tertiary educated Randomized at Birth than fathers of non-consenters. n = 616 Approximately 50% of the NAPLAN cohort had been randomised to the active intervention (118, 49.4%). The active intervention and control groups were not signiﬁcantly different in any of the baseline characteristics or in the proportion who Withdrawn by 18 months Participated at 18 months were full breastfeeding for ⩾ 3 months (38.4% and 32.7%, n = 64 n = 552 respectively, P = 0.41). In the per-protocol analysis, 146 subjects (61.1%) had taken over 50% of the expected number of capsules over the study period. This included 70 (59.3%) and 76 (62.8%)

Withdrawn by 8 years n = 58 among those who were randomised to the active intervention and control groups, respectively. Not available for clinical assessment at Participated at 8 years 8 years n= 45 n= 449 NAPLAN testing in NSW began in 2008. Therefore, only a minority (90, 38%) of participants in this cohort underwent NAPLAN testing in Year 3. Most of them were older than Year 3 in 2008. Further, only a minority (55, 23%) had reached school year 9 Withdrawn by 11.5 years n= 29 Participated at 11.5 years at the time this secondary analysis was conducted. NAPLAN Not available for clinical assessment N= 370 results were available for 230 (96%) participants in year 5 (age at 11.5 years n= 93 10–11 years) and for 219 (92%) in year 7 (age 12–13 years). Fifteen children had NAPLAN results available for one time point only, 89 had results for two time points and 135 had results for three time Parents declined to give consent for points. No participants had results at all four time points. participation n= 26 Participated in NAPLAN analysis Children randomised to the active supplement intervention had NAPLAN results not available or N=239 total plasma omega-3 PUFA proportions (DHA, docosapentaenoic school declined to give consent n= 25 acid, EPA and -linoleic acid) that were signiﬁcantly higher than Not able to be contacted n= 80 those for children in the control arm at ages 18 months, 3 years and 5 years (see Figure 2).25 The ratio of total omega-3 to omega-6 Figure 1. Flowchart displaying the participation in CAPS for the PUFA in the active intervention group averaged 1:5.1 over the ﬁrst NAPLAN analysis. 5 years and that in the control group averaged 1:7.2. Three years

Table 1. Characteristics at birth of members of the CAPS cohort for whom NAPLAN data were available and those for whom these data were not available (non-consenters)

NAPLAN cohort Non-consenters P-valuea

N = 239 N = 377 Mean ± s.d. or n (%) Mean ± s.d. or n (%)

Randomised diet group Active intervention 118 (49.4) 195 (51.7) 0.32 Control 121 (50.6) 182 (48.3)

Sex Male 126 (52.7) 186 (49.3) 0.46

Mother's education University/TAFE 130 (54.4) 146 (38.7) o 0.01 Year 12 41 (17.2) 69 (18.3) Year 10 60 (25.1) 147 (39.0) ⩽ Year 9 8 (3.4) 15 (4.0)

Father's education University/TAFEb 114 (48.5) 151 (40.1) o0.05 Year 12 49 (20.9) 110 (29.2) Year 10 66 (28.1) 129 (34.2) ⩽ Year 9 6 (2.6) 22 (5.8) Mother smoked in pregnancy 51 (21.3) 99 (26.3) 0.17 Full breastfeeding ⩾ 3 monthsc 80/225 (35.6) 90/330 (27.3) o0.05 Birth weight (kg) 3.46 ± 0.50 3.52 ± 0.48 0.14 Gestational age (weeks) 39.55 ± 1.30 39.57 ± 1.24 0.84 Mother's age at time of birth (years) 29.8 ± 4.90 28.4 ± 5.45 o0.01

Plasma omega-3 (% of total fatty acid)d Active intervention 6.40 (±1.25) n = 115 5.96 (±1.38), n = 145 Control 4.95 (±1.06), n = 117e 5.03 (±1.06), n = 134e Abbreviations: CAPS, Childhood Asthma Prevention Study; NAPLAN, National Assessment Program—Literacy and Numeracy. aP value determined by either two-sample t-test or χ2-test. bTechnical and further education. cFull breastfeeding = no solids or artiﬁcial feeding before 3 months. Full breastfeeding data were missing for some infants. dAverage of concentrations measured at 18 months, 3 years and 5 years. Not all children consented to having their blood taken, and hence the denominator is given. eThe P value of the comparison between active intervention and control omega-3 concentration is o0.001.

© 2015 Macmillan Publishers Limited European Journal of Clinical Nutrition (2015) 419 – 424 Omega-3 supplementation and academic performance BK Brew et al 422 after the intervention had ceased, when the children were 8 years associated with overall NAPLAN scores (Table 3). However, old, there were no differences in total plasma omega-3 PUFA at 5 years, there was a positive association between plasma proportion (Figure 2) or in the ratio of total omega-3 to omega-6 omega-3 PUFA and NAPLAN scores in the unadjusted analysis. PUFA between groups. The ratio of total omega-3 to omega-6 This was no longer significant after adjusting for the child’s birth PUFAs and the concentrations of each of the omega-3 and weight, maternal age and maternal education level. At 8 years, the omega-6 PUFAs at each of 18 months, 3 years, 5 years and 8 years positive association between total plasma omega-3 PUFA and fi have been published previously.31,32 NAPLAN scores remained signi cant after adjustment for con- The omega-3 LCPUFA intervention did not improve the NAPLAN founders (Table 3). For every 1% absolute increase in the scores in either reading or numeracy in any school year (Table 2). proportion of total plasma omega-3 PUFA, NAPLAN combined scores were observed to be 0.13 (95% CI 0.03, 0.23) standard The mixed-model analysis combining all scores revealed no deviation units higher. Interaction terms for skill tested and for significant overall effect of the active intervention on NAPLAN school year were nonsignificant in all models. scores. The overall difference in NAPLAN scores between the The negative control study found no significant association active intervention and control groups, expressed in s.d. units, was between BMI at 8 years and academic performance in the − − 0.18 (95% CI 0.41 to 0.05) in the intention-to-treat analysis and unadjusted (P = 0.90) or adjusted results (P = 0.84). This increases − 0.21 (95% CI − 0.51 to 0.08) in the per-protocol analysis. our confidence that the association between total plasma levels of Interaction terms for skill tested and for school year were omega-3 fatty acid at age 8 years and subsequent NAPLAN scores nonsignificant in all models. is not attributable to residual confounding. The proportion of total plasma omega 3 PUFA of total fatty acids at ages 18 months and 3 years was not significantly DISCUSSION

9 In this cohort of 239 children followed up from birth to the age of 14 years, daily omega-3 LCPUFA supplementation for the ﬁrst 5 8 * years of life did not improve long-term academic performance, as measured by numeracy and reading scores. However, we did 7 Control * * Intervention observe an association between observed total plasma omega-3 6 PUFA levels at the age of 8 years and academic performance measured from 8 to 14 years of age. 5 The active omega-3 LCPUFA intervention was successful in

4 lowering the total plasma omega-3 PUFA to omega-6 PUFA ratio compared with the ratio in the control group. This was sustained 3 throughout the period of supplementation. This study supports the ﬁndings of a Cochrane review and a meta-analysis that 2 showed that LCPUFA supplementation in infant formula is not ﬁ 13,14 1 associated with cognitive bene ts. Although there is no meta-

Omega-3 as a percentage of Total Fatty Acids analysis on omega-3 LCPUFA supplementation of older children, 15–17 0 several RCTs are consistent with our findings. The difference 18 months 3 years 5 years 8 years between our study and other previous studies is that the Age supplementation period in this study was much longer: 5 years Figure 2. Proportion of omega 3 fatty acids (DHA, EPA, DPA and ALA) compared with 3–12 months in other trials. Therefore, our study of total fatty acids and 95% confidence intervals. Comparison of shows that, even when extended over the first 5 years of life when control versus intervention groups at 18 months, 3 years, 5 years the brain is maturing at a rapid rate, omega-3 LCPUFA and 8 years. *Po0.01. supplementation does not improve cognitive performance.

Table 2. Effect of the active intervention on NAPLAN scores (for intention-to-treat and per-protocol analyses)

Academic measure Intention-to-treat analysis (N = 239) Per-protocol analysisa (N = 146)

N Difference in NAPLAN Difference in s.d. P (t-test) N Difference in NAPLAN Difference in s.d. P (t-test) units (95% CI) units (95% CI) units (95% CI) units (95% CI)

Year 3 Reading 90 − 27.03 (−58.6, 4.53) − 0.34 (−0.73, 0.06) 0.09 57 − 21.5 (−57.9, 15.0) − 0.27 (−0.72, 0.19) 0.24 Numeracy 81 − 25.4 (−52.8, 2.04) − 0.36 (−0.75, 0.03) 0.07 58 − 7.8 (−42.3, 26.7) − 0.11 (−0.60, 0.38) 0.65

Year 5 Reading 230 − 3.2 (−25.1, 18.7) 0.04 (−0.33, 0.25) 0.77 140 − 10.6 (−38.1, 16.9) − 0.14 (−0.50, 0.22) 0.45 Numeracy 230 − 13.7 (−32.1, 4.75) − 0.19 (−0.44, 0.07) 0.15 140 − 19.93 (−44.3, 4.4) − 0.27 (−0.61, 0.06) 0.11

Year 7 Reading 219 − 7.0 (−27.4, 13.3) − 0.1 (−0.39, 0.19) 0.50 131 − 6.21 (−31.7, 19.3) − 0.09 (−0.45, 0.27) 0.63 Numeracy 216 − 11.7 (−31.6, 8.2) − 0.15 (−0.4, 0.1) 0.25 131 − 19.9 (−46.3, 6.49) − 0.25 (−0.59, 0.08) 0.14

Year 9 Reading 55 − 19.9 (−62.8, 23.0) − 0.29 (−0.93, 0.34) 0.36 34 − 16.3 (−73.6, 40.9) − 0.24 (−1.09, 0.66) 0.57 Numeracy 54 − 24.1 (−64.6, 16.3) − 0.30 (−0.82, 0.21) 0.24 34 − 19.2 (−73.4, 34.9) − 0.24 (−0.93, 0.44) 0.48 Abbreviation: NAPLAN, National Assessment Program—Literacy and Numeracy. Differences are intervention group – control group. aPer protocol = 450% compliance for taking capsules.

Table 3. Association between total plasma omega-3 PUFA and NAPLAN results by year of plasma measurement

Age at plasma omega-3 Na Unadjusted results Adjusted resultsb PUFA measurement Change in NAPLAN Change in NAPLAN by P-value Change in NAPLAN Change in NAPLAN by P-value score (95% CI) s.d. units (95% CI) score (95% CI) s.d. units (95% CI)

18 months (n = 183) 183 − 1.07 (−5.86, 3.71) − 0.02 (−0.08, 0.05) 0.62 − 3.72 (−8.52, 1.08) − 0.05 (−0.11, 0.02) 0.16 3 years (n = 197) 197 4.81 (−1.90, 11.52) 0.06 (−0.03, 0.15) 0.17 2.56 (−3.96, 9.08) 0.03 (−0.05, 0.12) 0.46 5 years (n = 200) 200 10.04 ( 2.74, 17.34) 0.13 (0.04, 0.23) 0.01 5.53 (−1.56, 12.63) 0.07 (−0.02, 0.17) 0.13 8 years (n = 177) 177 12.12 (4.42, 19.83) 0.16 (0.06, 0.27) 0.002 9.69 (2.23, 17.16) 0.13 (0.03, 0.23) 0.01 Abbreviations: NAPLAN, National Assessment Program—Literacy and Numeracy; PUFA, polyunsaturated fatty acid. Unadjusted and adjusted increase in NAPLAN scores and in s.d. units per 1% absolute increase in total plasma omega-3 PUFA NAPLAN results are based on years 3, 5, 7 and 9 numeracy and reading scores combined. aNot all participants provided blood samples. bAdjusted for birth weight, maternal age and maternal education.

Another difference between our study and that of others was capsule compliance, total plasma PUFA levels and breastfeeding, that the outcomes were measured from 3 years post supplemen- hence reducing recall bias. tation rather than immediately after, or during, supplementation. One limitation of this analysis was the considerable loss of The study that is most similar to the present one entailed the subjects from the original cohort. Attrition of subjects was because follow-up of 421 bottle-fed children who had been randomly of withdrawals from the study over the 14-year study period and a assigned to receive supplemented formula or non-supplemented lack of consent from parents and principals of non-government formula for the first 6 months of life. There were no differences schools for researchers to access the child’s NAPLAN results. between the randomised groups in cognitive and behavioural However, the reliability of the analysis was improved as more than 18 outcomes at 9 years. half of the participants contributed at least 3 years of NAPLAN We observed that the proportion of total omega-3 PUFA in data to the analysis, and 94% contributed at least 2 years of data. plasma fatty acids at the age of 8 years (but at no earlier age) was At each year there were two scores, one for numeracy and one for correlated with academic performance after adjusting for reading. Therefore, 943 scores were included in the intention-to- maternal age, maternal education and birth weight. Several other treat analysis and 576 scores were included in the per-protocol studies have also found an association between blood plasma analysis. Second, the upper limit of the 95% confidence interval of PUFA proportions and academic outcomes,19,20 whereas others the mixed model testing the difference between the active have not.21,22,33 One possible explanation for finding an associa- intervention and control was only 0.05 of a s.d. unit in the tion between plasma omega-3 PUFA and cognitive outcomes is unmeasured or residual confounding. It seems plausible that intention-to-treat analysis and 0.08 in the per-protocol analysis. parents with a higher socioeconomic background (represented in This demonstrates that despite the loss to follow-up of subjects our study by those with a higher level of education) may provide the analysis was adequately powered to exclude clinically fi an optimal academic or learning environment for their children, as important bene cial effects. well as a more varied diet including seafood and oily fish, which in The other potential problem with loss to follow-up is the Australia tends to be more expensive than other forms of protein. introduction of selection bias. Those who remained in the study However, the negative control study showed that residual and for whom NAPLAN results could be obtained were more likely confounding is a less likely explanation for the association that to have educated parents and therefore may have been more we have observed, and hence it improves the validity of our likely to do well on NAPLAN tests. However, we observed a finding that there is an association between plasma omega-3 significant difference in total plasma omega-3 PUFA at 18 months, PUFA and academic performance. 3 years and 5 years between NAPLAN cohort participants in the A South African study found that students who consumed a fish active intervention and control groups; therefore, the lack of flour spread added to bread on school days for 6 months association between active intervention and academic perfor- performed better on learning ability and memory tests compared mance suggests that supplements are not beneficial in this group. with placebo.34 One explanation for this finding is that omega-3 It is possible that underperforming students may benefit from LCPUFA acts more effectively when given in whole food (fish) omega-3 LCPUFA supplementation8 and that our sample under- rather than as a supplement. This has also been reported in represented this group. Finally, it should be recognised that the relation to the protective effect of omega-3 PUFA on cardio- subjects in this population all had a family history of asthma, and 35 36 vascular disease and in relation to other nutrients. This although the omega-3 LCPUFA supplement made no difference to hypothesis might also explain why we saw an association with asthma outcomes (as measured previously) this may affect the the total plasma omega-3 PUFA at 8 years, when the source was generalisability of our results. entirely dietary, but not at 18 months, 3 years and 5 years, when the source of omega-3 LCPUFA was, in part, the supplements. However, more research is needed to understand whether the CONCLUSIONS source of omega-3 PUFA and timing of measurement is important in understanding the association between total plasma omega-3 Long-term supplementation with omega-3 LCPUFA in early PUFA and cognitive performance. childhood does not improve academic performance in later The strengths of this study were as follows: a long supplemen- childhood. Hence, our findings do not support the practice of tation period, as discussed above; a randomised controlled trial supplementing omega-3 LCPUFA in young children for this design, hence avoiding the potential for selection bias and purpose. The observed association between total plasma confounding; blinding of parents and children to which interven- omega-3 PUFA and academic performance at the same age tion group they were in; and contemporaneous measurement of requires further investigation.

© 2015 Macmillan Publishers Limited European Journal of Clinical Nutrition (2015) 419 – 424 Omega-3 supplementation and academic performance BK Brew et al 424 CONFLICT OF INTEREST 16 Nemo Study Group. Effect of a 12-mo micronutrient intervention on learning and GB Marks sits on an advisory board for Novartis, who pay an honorarium on his memory in well-nourished and marginally nourished school-aged children: behalf. The unit GB Marks works in has conducted the research funded by 2 parallel, randomized, placebo-controlled studies in Australia and Indonesia. AstraZeneca. The remaining authors declare no conflict of interest. Am J Clin Nutr 2007; 86:1082–1093. 17 Muthayya S, Eilander A, Transler C, Thomas T, van der Knapp HCM, Srinivasan K ACKNOWLEDGEMENTS et al. Effect of fortification with multiple micronutrients and n-3 fatty acids on growth and cognitive performance in Indian schoolchildren: the CHAMPION We thank the children and parents of the CAPS cohort, without whose participation (Children's Health and Mental Performance Influenced by Optimal this study would have been impossible to perform. Kitty Ng was instrumental in Nutrition) Study. Am J Clin Nutr 2009; 89: 1755–1775. communicating with families, obtaining consents and blood samples over many 18 de Jong C, Kikkert HK, Fidler V, Hadders-Algra M. Effects of long-chain years. This was not an industry-supported study. Bronwyn Brew is supported by an polyunsaturated fatty acid supplementation of infant formula on cognition and NHMRC research scholarship (#1038533), Guy Marks by NHMRC Practitioner behaviour at 9 years of age. Dev Med Child Neurol 2012; 54: 1102–1108. fellowships (#1003500 & 402827) and the CAPS study by NHMRC Project Grants 19 Ryan A, Nelson E. Assessing the effect of docosahexaenoic acid on cognitive #974097, #991101, #211081, #352409 and #570919, and the CRC for Asthma. Catarina functions in healthy, preschool children: a randomized, placebo-controlled, Almqvist was supported by the Swedish Research Council, the Swedish Heart-Lung double-blind study. Clin Pediatr 2008; 47:355–362. foundation and the regional agreement on medical training and clinical research 20 Jacobson JL, Jacobson SW, Muckle G, Kaplan-Estrin M, Ayotte P, Dewailley E. (ALF) between Stockholm County Council and Karolinska Institutet, Stockholm Beneficial effects of a polyunsaturated fatty acid on infant development: evidence Sweden. Contributions of goods and services were made by Allergopharma Joachim from the Inuit of Arctic Quebec. J Pediatr 2008; 152: 356–364. Ganzer KGGermany, John SandsAustralia, Hasbro, TollRefrigerated, AstraZeneca 21 Bakker EC, Ghys AJA, Kester ADM, Vles JS, Dubas JS, Blanco CE et al. 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