Long-chain PUFA supplementation in rural African infants: A randomized controlled trial of effects on gut integrity, growth, and cognitive development

Background: Intestinal damage and malabsorption caused by chronic environmental enteropathy are associated with growth faltering seen in infants in less-developed countries. Evidence has suggested that supplementary omega-3 (n-3) long-chain PUFAs (LC-PUFAs) might ameliorate this damage by reducing gastrointestinal inflammation. LC-PUFA supplementation may also benefit cognitive development. Objective: We tested whether early n-3 LC-PUFA supplementation improves infant intestinal integrity, growth, and cognitive function. Design: A randomized, double-blind, controlled trial [200 mg DHA and 300 mg EPA or 2 mL olive oil/d for 6 mo] was conducted in a population of 172 rural Gambian infants aged 3-9 mo. The primary endpoints were anthropometric measures and gut integrity [assessed by using urinary lactulose:mannitol ratios (LMRs)]. Plasma fatty acid status, intestinal mucosal inflammation (fecal calprotectin), daily morbidity, and cognitive development (2-step means-end test and an attention assessment) were secondary endpoints. Results: PUFA supplementation resulted in a significant increase in plasma n-3 LC-PUFA concentrations (P < 0.001 for both DHA and EPA) and midupper arm circumference (MUAC) (effect size: 0.31 z scores; 95% CI: 0.06, 0.56; P = 0.017) at 9 mo of age. At 12 mo, MUAC remained greater in the intervention group, and we observed significant increases in skinfold thicknesses (P ≤ 0.022 for all). No other significant differences between treatment groups were detected for growth or LMRs at 9 mo or for secondary outcomes. Conclusions: Fish-oil supplementation successfully increased plasma n-3 fatty acid status. However, in young, breastfed Gambian infants, the intervention failed to improve linear growth, intestinal integrity, morbidity, or selected measures of cognitive development. The trial was registered at www.isrctn.org as ISRCTN66645725. © 2013 American Society for Nutrition. The study was carried out in the West Kiang region of The Gambia from May 2007 to October 2008. The Gambian climate is tropical with 2 main seasons that consist of a hot, rainy season (June to October) and a cooler, dry season (November to May). West Kiang is 145 km inland from the capital and spans an area of 80 km2. Keneba is the largest village in this rural area, where subsistence farming (primarily rice and groundnuts) predominates. A combination of in utero growth retardation, poor-quality and frequently contaminated weaning foods, and high levels of infection cause moderately severe growth faltering in almost all children with an onset at ∼3 mo postpartum. All infants aged 3 mo who were living in the 16 largest villages of West Kiang and not enrolled in any other study were eligible to take part in the current study. In total, 220 infants were assessed for eligibility, of whom 183 infants were randomly assigned (Figure 1). Infants with severe congenital abnormalities that could affect growth and development, infants from multiple births, and infants with known HIV infection were ineligible for participation. Subject flowchart summarized according to Consolidated Standards of Reporting Trials that shows the number of subjects randomly assigned, lost to follow-up, and analyzed by treatment arm. Potential subjects were identified from the West Kiang Demographic Surveillance System (http://www.ing.mrc.ac.uk/research_areas/west_kiang_dss.aspx). Subjects were automatically allocated a study number by the database system on recruitment. Each study number had previously been randomly assigned to one of 4 treatment codes (4 rather than 2 codes were used to promote blinding) represented by 4 simple pictures on the supplements so that infants were allocated to either the n−3 LC-PUFA or the control group in a 1:1 ratio. The trial statistician implemented a permuted block random assignment (block size = 16), which ensured a uniform distribution of treatments across the seasons of birth. After mothers had given their consent, and their infants had been recruited, a fieldworker issued them with a card printed with the appropriate picture. Mothers were asked to bring their picture-coded cards when their infants were brought to be administered the supplement. Fieldworkers were also given a treatment booklet from which to crosscheck for any card swapping. All infants in the study stayed on the same treatment allocations throughout the duration of treatment. Participants, staff, and investigators remained blinded to treatment assignment throughout the duration of the trial. Supplementation started at 3 mo of age and ended at 9 mo of age when all outcome measurements were made apart from cognitive function (assessed at 12 mo of age). Field assistants administered the supplement at a central meeting point in each of the villages each day. Sterile, graded pastettes were used to squeeze the oil into the side of the mouth of each infant. Mothers were asked to breastfeed their infants immediately after the oil had been given. Field assistants who administered the dose recorded daily compliance. The intervention group received 2 mL of highly purified fish oil, which supplied 200 mg DHA and 300 mg EPA/d. The control group was given the same volume of olive oil. Both oils were supplied by Nordic Naturals Inc and contained 1.25% lemon oil for blinding and 0.5% rosemary extract and d-α-tocopherol as an antioxidant. On the advice of the trial monitor, the usual tocopherol concentration of 30 IU/5 mL was reduced to 5 IU/5 mL as appropriate to this age group. The dosage of 500 mg combined DHA plus EPA/d was designed to achieve a substantial increase in plasma n−3 PUFA to both eliminate any existing deficiencies and elicit a therapeutic response. A morbidity questionnaire was administered to mothers or caretakers on a daily basis to assess diarrhea, vomiting, cough, fever, and (for safety monitoring) possible abnormal bleeding that might have been related to clotting interference. If a mother felt that her infant was in need of medical attention, she was free to take her infant to the Medical Research Council (MRC) clinic in Keneba to be seen by a doctor or request a visit by a nurse. If the infant was seen by a nurse or doctor, the diagnosis and treatment details were recorded. To reduce intraindividual variance and estimate the reliability of outcome variables, primary endpoints were taken in triplicate (on separate days) at baseline and endpoint visits, and the median value was used for analyses. Mothers were asked to collect a sample of their infant's stool into a pot provided the evening before they were due to come to Keneba for the clinic visit and to continue the collection during the clinic visit if no stool was passed the previous evening. Stools were thoroughly homogenized before freeze drying to a constant weight. Mothers expressed ∼5 mL breast milk from each breast before their infants received their first feed at the clinic and again immediately after they had fed their infants. The 4 breast-milk samples were pooled by mixing together 1 mL of each sample, and an aliquot of this pooled sample was stored at −70°C until analysis. An experienced nurse drew 2 mL venous blood from infants at baseline and endpoint into anticoagulant-coated tubes. At this age, it is not possible to obtain fasted samples. Each month, a basic weaning-food questionnaire was administered to the mother that asked about which foods the infant had been weaned onto the previous month. Anthropometric measures at 3 and 9 mo were performed (by LFvdM). Infant lengths and weights were measured by using a Harpenden Infantometer length board (Holtain Ltd) and electronic baby scale (model 336; Seca), to a precision of 0.1 cm and 0.01 kg, respectively. Left-side triceps, biceps, and subscapular skinfold thicknesses were measured with a skinfold-thickness caliper (Holtain Tanner/Whitehouse) to 2-mm precision, and midupper arm circumference (MUAC) was measured by using a paper measuring tape to a precision of 0.1cm. Head circumference, as a proxy for brain size, was measured to the nearest 0.1 cm with a stretch-proof measuring tape (Model CTM08l Chasmors Ltd) around the maximum circumference of the head (forehead to occiput). Measures for which reference data were available were expressed as z scores on the basis of WHO Growth Reference Data (38, 39). Anthropometric measures were repeated at 12 mo by a fieldworker. Intestinal absorptive capacity and permeability were assessed by using the dual-sugar permeability test (4). Infants were given a 2 mL/kg body weight dose of sugar solution that contained 400 mg lactulose (Lactulose Solution BP; Sandoz Ltd) and 100 mg mannitol (Sigma-Aldrich Co) per 2 mL H2O. The recovery of the monosaccharide mannitol provides a measure of passive intestinal absorption and is reduced by villous atrophy. Paracellular uptake of the nondigestible disaccharide lactulose is a marker of intestinal leakiness. Therefore, intestinal permeability is measured as the urinary lactulose concentration divided by the urinary mannitol concentration. In addition, the ratio of urinary lactulose:lactose is measured as an indication of lactase activity. By measuring the total urine volume passed over 5 h postdose (Hollister U-bag; Abbot Laboratories), the percentage of recoveries of the 2 probes was calculated as a reflection of the amounts taken up by the passive and paracellular intestinal routes. Urine samples were stored with 2–3 drops of chlorhexidinegluconate (5% wt:vol) as a bacteriostatic agent. At 12 mo of age (±7 d), infants were brought in to the MRC Keneba field station for their final follow-up visit, during which cognitive development was tested by using a 2-step means-end problem-solving test (Willatts’ Infant Planning Test) (40–42) and a single-object task attention assessment (toddler attention assessment) (43). The 12-mo time point for assessments was determined in a pilot study that evaluated the frequency distribution of test scores in 75 West Kiang infants aged 10–12 mo. On the morning of the test, mothers were asked to ensure that their infant had been fed before the 15-min test. For standardization, the 2-step Infant Planning Test was performed first and was followed by the single-object task toddler assessment. If an infant did not respond during the Infant Planning Test, the attention assessment was completed first, and a second attempt at the planning test was made thereafter. The assessment was completed in a restricted-access room with few distractions. For both tests, the mother was asked to sit on a chair with her infant on her lap facing a table placed in front of her. She was asked not to help, encourage, or prompt her child by either words or actions and not to encourage the infant's eye contact when he or she looked up at the mother. A Gambian fieldworker (SD) carried out all tests. The tests were filmed and saved for later scoring. The aim of the Willatts’ 2-step Infant Planning Test is to present a challenge to an infant and observe whether the infant is capable of planning and executing a solution. The challenge is to retrieve a toy, which is either concealed or out of reach. The infant needs to manipulate an intermediary object (cloth or cover) to retrieve the goal, and the intentionality which the child uses to retrieve this goal (toy) is assessed. This problem-solving test is simple to perform and has proven sensitive to nutritional interventions in infants (40, 41). It has been successfully used in high-income countries and also in middle- and low-income countries (44, 45). The test was performed and scored as described by Willatts et al (46). For the first outcome measure, trial behavior scores were averaged to give a mean total intention score. Each trial in which there was a score of ≥1 for all 3 behaviors was considered to be an intentional solution. The number of trials in which the child showed some sign of intention became the second outcome measure of the total intentional solution score. By conducting this test at 12 mo of age, it was possible to combine it with the toddler attention assessment. This test involves a free-play task, whereby the toddler's attention to a complex toy is assessed. Infant habituation, which is a decrease in attention to a repeatedly presented stimulus, is considered a basic tool for the assessment of cognition or processing speed in infancy. The test was performed by using previously described methods and equipment (43). VirtualDub Mpeg-2, FccHandler software (version 1.6.19, SourceForge.net), which allows frame-by-frame playback viewing, was used for scoring. The 2 outcome measurements were as follows: 1) the mean length of looks at the toy (or mean look duration = the total looking time divided by the number of looks at the toy) and 2) the inattention rate (the number of looks away from the toy per minute). A graduate student (KEH) who trained in the Willatts Laboratory scored all cognitive tests. Because the scoring of the Infant Planning Test relies in part on subjective interpretation, cross-validation was achieved by using a 30% sample of Infant Planning Tests scored by a second observer (LFvdM) who also trained in the Willatts Laboratory. Urinary lactulose, mannitol, and lactose concentrations were measured at MRC Keneba by using a 96-well microplate enzymatic assay that was based on previously published methods (47–50). All samples were measured in duplicate. The average intraassay percentage CV, which was calculated across all replicates run during sample analysis, was 3.01% (urine samples) and 3.97% (standards) for the lactulose assay and 1.89% (samples) and 2.36% (standards) for the mannitol assay. The repeatability of the LMR within individual subjects at each time point gave an intraclass correlation coefficient of 0.26 and an estimated reliability of an individual mean of 0.49. Plasma and breast-milk samples were flushed with nitrogen to minimize the oxidation of volatile PUFAs before storage at −70°C. Samples were later transported to MRC Human Nutrition Research, Cambridge, United Kingdom. To measure plasma and breast-milk fatty acid (FA) concentrations, total lipid extracts were prepared from 200 μL thawed plasma or homogenized breast milk by using an adaptation of the method of Folch (51), and FA methyl esters were prepared by using acid-catalyzed esterification. A BPX70 column [70% cyanopropylpolysilphenylene-siloxane (SGE)], with polarity designed specifically for FA methyl ester analysis, was used for gas chromatography separation. Elution profiles for each sample were obtained, which consisted of 38 peaks. Each of the FA components was quantified against a calibration range of external standards of known concentration (ng/μL) by using linear regression. Plasma C-reactive protein was measured by using a commercial colorimetric immunoassay (DimensionCardioPhase high-sensitivity C-reactive protein method; Siemens Healthcare Diagnostics Ltd). α-1-Glycoprotein was measured with an immunoturbidimetric specific reaction by using a commercial kit (Sentinel Diagnostics). The Flex reagent cartridge (Siemens Healthcare Diagnostics Ltd) was used for the measurement of plasma albumin. Fecal calprotectin concentrations were measured at MRC Keneba by using a commercial ELISA kit (Phical; CALPRO AS). The kit is used primarily for the determination of disease activity and monitoring the response to treatment in adult patients with inflammatory bowel disease and patients with colorectal cancers. The protocol was adapted for use in this population of infants and on freeze-dried stool. To enable the comparison with reference values, results were expressed per kilogram of wet stool. A central database with specific access entry forms and automatic range checks was created for the trial. All data were double entered, and real-time checks were performed to enable a rapid resolution of queries. Sample-size calculations were based on growth measurements of 1621 Gambian children and, subsequently, the first 25% collected urine samples. The main analysis was an intention-to-treat analysis. Multiple and negative binomial regression analyses, where appropriate, were used to test the effect of treatment on all primary and secondary endpoints. All endpoints for which baseline values were collected were fitted as covariates. In a subsequent analysis, breast-milk DHA and EPA, sex, season of birth, age of treatment commencement, and, only for cognitive development outcomes, the highest level of maternal schooling were added as extra covariates to the regression models. These variables were identified as potential variance inflators and/or effect modifiers. To look at the possibility of dose effects, an additional analysis that fitted the number of doses of treatment, with compliance controlled for, in the regression models were done for all primary and secondary endpoints. Compliance was measured as the percentage of doses taken compared with the doses offered during each infant's supplementation period. With adjustment for the compliance and number of doses, the effect of treatment as it was received in each individual case was measured on the various outcomes. A linear regression model was used to test the relation between each outcome at endpoint with the various FAs at 9 mo. We used the statistical package Stata (version 10; StataCorp LP) throughout. Log transformations were used to both normalize the distribution and stabilize the variance of skewed variables. For the main LC-PUFAs, we analyzed relative percentage and absolute concentration differences between study groups separately. For other FAs and combinations, we used absolute concentrations. Sex differences between cognitive development test scores were assessed by using regression analysis. For the assessment of test validity, correlations between scores of a second observer who scored a sample of the Infant Planning Test and those of the first observer were examined by using Pearson's product-moment correlation coefficients. Bias-corrected 95% CIs for correlation coefficients were estimated by using the bootstrap approach. The bias between markers was tested by using a paired t test. The trial observed MRC UK Good Clinical Practice guidelines, the current version of the Declaration of Helsinki, and applicable local ethical and legal requirements. Ethics approval was obtained from the London School of Hygiene and Tropical Medicine and the joint Gambian Government/MRC ethics boards. The Gambian National Nutrition Agency gave their written approval to carry out the study, and informed assent was given by the village elders. Written informed consent was provided by the parents or caretaker of each child. Independent trial and safety monitors were appointed to monitor and supervise the progress and safety of the trial throughout and ensure the study abided by MRC UK Good Clinical Practice standards. The trial was registered as ISRCTN66645725.