Enhancing children’s cognitive function and achievement through carotenoid consumption: The Integrated Childhood Ocular Nutrition Study (iCONS) protocol

Abstract

Lutein and zeaxanthin (L + Z) are carotenoids that accumulate in neural tissue and potentially confer benefits to cognition. Whereas cross-sectional studies have revealed positive associations between macular carotenoids (MC) and cognition, no studies have investigated whether L + Z supplementation impacts MC and cognition in childhood. Accordingly, the Integrated Childhood Ocular Nutrition Study aims to investigate the impact of L + Z supplementation over 9-months on academic abilities, attentional control, memory, and MC among preadolescent children. Children 8–10 years (N = 288) will enroll in a 9-month double-blind, placebo-controlled, randomized trial. The study is registered and approved as a clinical trial on the U.S. National Library of Medicine http://ClinicalTrials.gov registry (NCT05177679). Participants will be randomized into an active (10 mg lutein+2 mg zeaxanthin) or waitlist placebo-controlled group. Primary outcomes include hippocampal-dependent memory, attentional inhibition, and academic achievement using a spatial reconstruction task, an Eriksen flanker task, and the Kaufman Test of Academic and Educational Achievement 3rd edition, respectively. Secondary outcomes include event-related brain potentials of attentional resource allocation and information processing speed (i.e., P3/P300 amplitude and latency) recorded during the flanker task. Macular pigment optical density (MPOD) will be assessed using heterochromatic flicker photometry. Cognitive assessments will be completed prior to and after completion of the supplementation period. MPOD will be quantified prior to, at the mid-point of (4–5 months), and after (9 months) the supplementation period. It is hypothesized that L + Z supplementation will improve cognition and academic achievement. Further, benefits for cognition and achievement are anticipated to be mediated by increases in MC among treatment group participants.

1. Introduction

Within the last few decades, the interplay between nutrition and brain health has gained increasing interest due to findings that implicate nutritional status early in life as a potential mediating factor for risk of cognitive decline [1-3]. While this phenomenon has been studied primarily in adulthood and early childhood, far less work has aimed to understand this relationship in preadolescence [4,5]. Preadolescence is of particular interest, as this period marks substantial development of brain regions (i.e., prefrontal cortex, hippocampus) that are vital for higher order cognitive processes and scholastic success. Further, optimal development of these brain structures can impact intellectual abilities and academic achievement, which are predictors for quality of life and vocational success in adulthood [6]. The current literature suggests that diet patterns high in omega-3 fatty acids, whole grains, fruits, and green leafy vegetables can mitigate cognitive decline [1]; however, comparatively less is known about why these diet patterns can benefit cognitive function. Thus, identifying nutritional components that could provide support for optimal cognitive function is key for supporting brain health across the lifespan.

Carotenoids, or plant pigments, are a dietary component with the propensity to accumulate in various tissues, including the central nervous system, adipose tissue, skin, liver, and reproductive organs. Specifically, the xanthophyll carotenoid lutein is the predominant carotenoid found in neural tissue, despite accounting for less than half of the dietary intake of carotenoids in typical United States adults [7]. Lutein and its stereoisomers, zeaxanthin and meso-zeaxanthin, also comprise macular pigmentation, a yellow spot in the macula of the eye that is protective against damaging short-wavelength light and age-related macular degeneration [8]. Using a technique known as heterochromatic flicker photometry, researchers and clinicians can non-invasively estimate macular pigment optical density (MPOD), a measure that is strongly associated with brain concentrations of carotenoids [9-11]. MPOD and brain and serum concentrations of lutein have been previously associated with cognitive functioning in children and adults [12-18]. Additionally, neuroelectric indices of attention, measured using event-related brain potentials (ERPs), have shown that children with higher MPOD employ more efficient attentional control mechanisms during completion of a modified flanker task [16], which modulates inhibitory control demands [18]. Further, supplementation of lutein in randomized, controlled trials has resulted in the improvement of cognitive abilities and MPOD in adults and elderly individuals [19-21]. However, no studies have investigated the impact of lutein supplementation on MPOD and cognitive function among children.

Thus, the Integrated Childhood Optical Nutrition Study (iCONS) aims to investigate the impact of lutein and zeaxanthin supplementation over nine months on academic abilities, attentional control, memory, and macular carotenoids in preadolescent children. This will be achieved through the completion of a randomized, placebo-controlled, double-blind study over the course of five years. Participants will be randomized into the treatment and placebo groups with stratification according to age, sex, socioeconomic status (SES), and race/ethnicity, as these factors are known to impact nutritional status and cognitive outcomes of interest [6,22,23]. Our central hypothesis is that children receiving the carotenoid supplement will exhibit greater gains in cognitive function and achievement relative to the waitlist placebo group. Our secondary hypothesis is that the effects of supplementation will be mediated by an improvement in macular carotenoid status, thus directly linking carotenoids to childhood cognitive health. The study will address three specific aims. Aim 1 is to investigate the effect of lutein and zeaxanthin supplementation on cognitive function. Outcome measures of this aim include behavioral performance on a spatial reconstruction paradigm to assess relational memory abilities, as well as behavioral and neuroelectric indices of attention measured by a modified flanker task and ERPs. We hypothesize that lutein and zeaxanthin supplementation over nine months will improve behavioral and neuroelectric measures of attentional control and memory. Aim 2 will determine whether lutein and zeaxanthin supplementation improves composite academic achievement, with secondary outcomes examining the subcomponents of academic achievement (i.e., math, reading, written language, and reading fluency achievement). The hypothesis is that individuals who receive lutein and zeaxanthin supplementation will exhibit greater gains in composite academic achievement, math, reading, written language, and language fluency abilities. Aim 3 will assess the mediating role of changes in macular xanthophyll status, as indexed by MPOD, on effects of carotenoid supplementation on improvements in cognitive and scholastic abilities. We hypothesize that benefits to cognition and academic achievement will be mediated by improvement in MPOD in the carotenoid supplementation group, thus providing insight into the causal implications of carotenoids impacting cognitive outcomes.

2. Material and methods

2.1. Study design

2.1.1. Overview

iCONS is a 9-month lutein and zeaxanthin supplementation randomized-controlled, double-blind trial. All study personnel involved in day-to-day subject interactions and data analyses will be blinded until the conclusion of the study and analyses. Following baseline testing, participants will be randomized into an active supplement or a waitlist, placebo-controlled group. Consumption of active treatment and placebo will occur daily for nine months over the course of the school year. During the study, participants will complete three baseline laboratory visit, one mid-intervention visit at 4–5 months, and three follow-up testing identical to the baseline visits upon completion of the 9-month intervention. An outline of the study design can be found in Table 1 and Fig. 1. The study is registered as a clinical trial on the U.S. National Library of Medicine http://ClinicalTrials.gov registry under the number NCT05177679. All laboratory visits will be completed at the Department of Kinesiology and Community Health at the University of Illinois Urbana-Champaign. The Department of Kinesiology and Community Health has a complete human research laboratory (2296ft²) specifically designed to conduct a variety of cognitive testing techniques among both child and adult research participants. The laboratory consists of rooms designated for informed consent/assent and interviews. In addition, we have a participant waiting room equipped with computer monitors that allow parents to observe their children during the cognitive task administration. Within the laboratory unit, we have two testing chambers, each equipped with an independent electroencephalogram (EEG) data collection system. The availability of two testing chambers allows for high throughput testing of multiple participants at the same time. In the event that unanticipated problems involving risks to subjects occurs, the lead investigator will notify the institutional review board within 24 h of the event using the adverse event form and maintain a digital record of any possible events. Additionally, a data safety and monitoring board (DSMB) will be established to ensure the safety of all participants involved in the study and to ensure the validity and integrity of the collected data. Two individuals will be identified that vary in their expertise related to the proposed project. These two individuals will have differential expertise related to pediatric nutrition and children’s cognitive development. The committee will be identified and convened prior to the beginning of the study to ensure the safety of all participants involved in the study and to ensure the validity and integrity of the collected data. The investigation team will meet every year with the DSMB throughout the award period with the purpose of evaluating all study protocols and the safety of the study participants. These meetings will occur via telephone conferencing. The items evaluated at these meeting will be to 1) review the protocol and make decisions regarding change (especially related to safety); and 2) review study progress and data quality (i.e., integrity, intervention efficacy). Several protocols for participant safety and data integrity are currently in place in our laboratories. However, should the need arise to report a serious adverse event, the appropriate form for the University of Illinois Institutional Review Board will be completed with copies forwarded to the NIH. Further, the Data Safety and Monitoring Board will be notified in all instances of adverse events and will determine the necessary procedures to execute.

Table 1.

Overview of tasks and timepoints of administration. BIA: Bioelectrical impedance analysis; EEG: Electroencephalography; FFQ: Food Frequency Questionnaire; KTEA-3: Kaufman Test of Academic and Educational Achievement 3rd Edition; MPOD: Macular Pigment Optical Density; WJ-IV: Woodcock Johnson-IV Tests of Cognitive Abilities.

	Phone Screening	Baseline	Randomization	4–5 months	9 months
Parent Consent		✓
Child Assent		✓
Demographics	✓	✓
Child Health History	✓	✓
Tanner Staging		✓			✓
Block FFQ		✓			✓
Anthropometrics		✓			✓
BIA		✓			✓
WJ-IV		✓			✓
MPOD		✓		✓	✓
KTEA-3 Achievement		✓			✓
Hippocampal-Dependent Relational Memory Task		✓			✓
Eriksen flanker Task with EEG		✓			✓

Fig. 1.

Outline of study procedures and collection timeline for a 9-month, placebo-controlled, double-blind study of lutein + zeaxanthin supplementation in preadolescent children. BIA: Bioelectrical Impedance Analysis; WJ-IV: Woodcock Johnson-IV Tests of Cognitive Abilities; IQ: Intelligence quotient; MPOD: Macular pigment optical density; KTEA-3: Kaufman Test of Academic and Educational Achievement 3rd Edition; EEG: Electroencephalography.

2.1.2. Randomization

Following baseline testing, participants will be randomly allocated to either the active or placebo-controlled supplementation group. To blind the participants and research staff, one member of the research team will not be involved in day-to-day operations or data collection procedures and will randomize participants without revealing their condition (active or waitlist control). Participants will be stratified according to age, sex, socioeconomic status (herein, defined by levels of income), and six NIH-defined racial categories. Income and age will be dummy-coded 0 and 1 (i. e., values below the [recruitment wave’s] sample mean of each variable will be recoded as 0 and those above or equal to the mean will be recoded as 1). To balance the number of participants across random group assignment at each level of the background variables, the randomization procedure was repeated 5000 times. At the end of each stage of iteration, the number of participants at the same level of the background variables was compared between groups. Participants will be subsequently algorithmically-assigned to groups among the 5000 randomization outcomes that produces the greatest number of levels (out of 12) that are equivalent across groups.

2.1.3. Treatment and placebo

Supplements will be provided in the form of gummies. Active treatment supplements will contain 10 mg lutein and 2 mg of zeaxanthin (Flora-Glo^®, Kemin Foods, Des Moines, IA, USA). Placebo supplements will be identical in formulation to the active treatment without the inclusion of lutein and zeaxanthin. Gummies will be sugar-free, plant-based, and produced by TopGum Industries Ltd. (Sderot, Israel). The daily dosage of both placebo and active supplements will be given as two flavored gummies to be consumed per day with a meal (breakfast, lunch, or dinner). Active treatment supplements will match in size, color, and flavor to the placebo-control supplements and be provided in identical opaque, sealed bottles. Each batch shipment of gummies will be verified for lutein and zeaxanthin concentrations prior to deployment to subjects. Given the timeline of the study, supplement storage, and frequency of production, it is not expected that these concentrations will degrade before the supplementation period. Following the 9-month supplementation period, the wait-list control group participants will be given the opportunity to receive the active treatment supplements for the subsequent nine months but not be asked to participate in any additional testing.

2.1.4. Participants

Participants will be recruited from the East-Central Illinois region by distributing flyers throughout the local community (i.e., local schools and libraries), attending community events, and utilizing email lists from within the University and the laboratory recruitment database. Participants will be male and female children ages 8–10 years old. This age range was chosen given our lab’s previous studies which validated the use of heterochromatic flicker photometry in preadolescent children for this age range [10]. Using parent report during screening procedures, children will be excluded if they have any neurological disorders and/or uncorrected vision. Further, parents will complete the modified Tanner Staging Scale [24] and children will be excluded if they are not prepubertal (score > 2). Lastly, participants will be excluded if they have received lutein supplementation within six months prior to enrollment, including multivitamins containing >1 mg lutein/day. All identifiable data will be handled and databased in a HIPPA protected, locked file by the study coordinator, who is not involved in the day-to-day participant interactions. All participants are assigned an alphanumeric identifier upon enrollment which is shared with the research staff. Inclusion and exclusion criteria are listed in more detail in Table 2.

Table 2.

Inclusion and exclusion criteria for participation.

Inclusion	Exclusion
1.Child assent and parent/guardian consent	1. Non-assent of child or non-consent of guardian
2. 8–10 years of age	2. Above/below 8–10 years of age
3. No lutein supplementation within 6-months prior to enrollment (exception of multivitamins containing <1 mg lutein/day)	3. Lutein supplementation within 6-months prior to enrollment (including multivitamins containing >1 mg lutein/day)
4. Absence of learning disability (parent-reported)	4. Identified learning disability (parent-reported)
5. Modified Tanner Staging score ≤ 2	5. Modified Tanner Staging score > 2
6. 20/20 or corrected vision	6. Not 20/20 or uncorrected vision

All participants and their parents/guardians will be asked to provide written assent and consent, respectively, prior to enrollment. Consent and assent are completed online using REDCap data management software, which is HIPAA protected. Study procedures have been approved by the University of Illinois Urbana-Champaign Institutional Review Board (IRB #21066) and conform to the guidelines of the Declaration of Helsinki. In the case of adverse reactions to the supplements, participants will discontinue consumption and participation in the study, however we do not foresee this occurring as supplementation at this dosage is known to be safe given that there are no upper-level intake recommendations for lutein and zexanthin and that no adverse effects have been observed in previous research supplementing with up to 40 mg/day of lutein [25]. We used a conservative approach to estimate our target sample size by relying on a lower effect size (i.e., Cohen’s d = 0.35) for the direct effect of carotenoids on primary outcomes. Similarly, the effect of supplementation on MPOD was estimated to be 0.59 (Cohen’s d) [26] and the effect of MPOD on the outcomes was estimated to be 0.40 (Cohen’s d) [18]. A Monte Carlo power [27] analysis based on these assumptions and testing the effect of group (x) on MPOD (m), and their direct and indirect effects on a singular (y) outcome revealed that 240 participants (120/group) would be necessary to conduct mediation analyses (Aim 3) while achieving a power of 0.8 (beta) with a 95% Confidence Interval (0.77 to 0.82). Assuming an attrition rate of 20%, we aim to recruit a total sample of 288 children (144/group) over the course of the project.

2.2. Data collection

2.2.1. Academic achievement

To assess academic achievement, the Kaufman Test of Academic and Educational Achievement 3rd edition (KTEA-3) [28] will be administered by a trained research staff member. This is a pencil and paper based academic achievement test, that will provide a composite achievement score and sub-scores for math, reading, written language, and reading fluency abilities. Scores are standardized with a possible range of 40 to 160, where a higher score indicates better performance.

2.2.2. Cognitive function

2.2.2.1. Hippocampal-dependent relational memory.

A spatial reconstruction task will be administered to assess hippocampal-dependent relational memory. Participants will study an array of five ambiguous creatures (objects) created in Spore Creature Creator (Electronic Arts Inc., Redwood City, CA) in randomized locations on the screen for 20 s. Afterwards, the creatures will disappear and reappear at the top of the screen and participants will be instructed to reconstruct the original array to the best of their ability. The task will be scored on two error metrics, object-location binding and misplacement [29]. Object-location binding is a metric of assessing a participant’s ability to correctly place a previously studied creature within a predefined radius around its original location. Misplacement refers to the pixel-distance the participant places the creature relative to its original location. For a visual description of the task and errors, see Fig. 2.

Fig. 2.

Task description for spatial reconstruction task. Participants will study a randomized array of ambiguous creatures (objects) for 20 s. After a 4 s delay, participants will have unlimited time to reconstruct the array. 20 trials of the task will be completed, and performance will be averaged across trials for error metrics of misplacement and object-location binding. Misplacement is distance, in pixels, a creature is placed from its original location. Object-location binding (correct responses designated by red circles) calculates whether the participant correctly placed a creature within a predefined radius surrounding the original location.

2.2.2.2. Attentional inhibition.

A modified Eriksen flanker task will be administered to assess inhibitory control [30]. Participants will view a stimulus of five fish presented centrally on a computer screen and instructed to focus their attention on the directionality of the central (i. e., target) fish. Participants will be instructed to press one of two buttons on a response pad corresponding to the direction that the target fish is facing (i.e., left or right). There are two distinct trial types within the flanker task, congruent and incongruent, that are presented in a random order. During the congruent trials, all five fish face the same direction, whereas during the incongruent trial, the target fish faces the opposite direction of the four-flanking fish (Fig. 3). Accurate responses on the incongruent trials necessitate greater upregulation of attentional inhibition relative to the congruent trials. A practice block with 48 trials will be completed first in which participants must get at least a 70% to move on to the experimental blocks, which will consist of 200 trials (2 blocks of 100 trials) with equiprobable distribution of congruent and incongruent trials. The trials will include a 250 ms stimulus presentation with jittered inter-trial intervals of 1600, 1800, and 2000 ms. Congruent and incongruent accuracy, as well as mean and standard deviation of reaction time, will be assessed to determine behavioral performance. Further, EEG will be recorded during the task to measure P3 amplitude and latency, which indexes attentional resource allocation and processing speed, respectively.

Fig. 3.

Task description for the modified Eriksen flanker task. Participants are presented with a randomized sequence of congruent and incongruent trial types and are instructed to selectively attend to and press a button corresponding to the direction of the central (i.e., target) fish. Stimuli are presented with a presentation time of 250 ms with a jittered intertrial interval of 1600, 1800, and 2000 ms.

2.2.3. EEG recording and ERP assessment

Recommendations by Keil et al. (2014) will be used for all EEG recording and ERP assessments [31]. During the flanker task, participants will wear a 64-channel Neuroscan Quik-cap Neo Net, which digitizes continuous EEG readings to a Neuroscan Synamps2 amplifier (Compumedics, Charlotte, NC, USA) at a sampling rate of 500 Hz and amplifies the readings 500 times with a direct current to a 70 Hz filter and a 60 Hz notch filter. Electro-oculographic (EOG) activity will be recorded using a set of four electrodes on the outer canthus of each eye and above and below the left orbit. AFz will serve as the ground electrode and reference electrode, placed between Cz and CPz. InterInterelectrode impedances will be maintained at or below 10 KΩ. All EEG processing will be completed using EEGlab [32] and ERPlab toolbox [33].

For ERP reduction, only correct trials will be used for analyses. During offline processing, data will be re-referenced to the average mastoid electrodes (M1 and M2). Eyeblink artifacts will be addressed using independent component analysis (ICA). The ICA components will be removed if they correlate with a vertical EOG by ≥0.35 [34]. The P3 stimulus-locked epochs will be extracted between −200 and 1000 ms around the stimulus using −200 to 0 ms pre-stimulus for baseline correction. All data will be filtered using a 30 Hz zero-pass filter and a moving window (with 100 ms step) and peak-to-peak amplitude exceeding 100 μV will be rejected as an artifact. To determine a region of interest (ROI) for the P3 component, the collapsed localizer method [35] will be utilized, in which time windows and electrodes of interest are determined after all conditions are averaged, resulting in a grand average. The data will be output in ascii format for statistical analysis.

2.2.4. Carotenoid assessment

For an assessment of retinal lutein and zeaxanthin, MPOD will be measured using customized heterochromatic flicker photometry (cHFP) via a macular densitometer (Macular Metrics Corporation, Rehoboth, MA). A modified protocol for children will be administered, as described by Renzi et al. (2014) [36] and determined to be reliable by McCorkle et al. [10]. During this modified procedure, a trained experimenter will manipulate the radiance of the short-wave component for the participant to correctly identify the “null-zone”, whereas in the adult procedure this ability is given to the participant. Participants will complete two assessments at each time point (i.e., baseline, mid-point, and follow-up) and the average of the two measures will be utilized in the analyses.

2.2.5. Covariates

2.2.5.1. General intellectual ability.

The Woodcock Johnson-IV (WJ-IV) Tests of Cognitive Abilities [37] will be used to assess intelligence quotient (IQ) by having participants complete a series of pencil and paper-based tasks. Participants will be asked to respond in writing and/or verbally to various visual and auditory presentations of different subtests (oral vocabulary, number series, verbal attention, letter-pattern matching, phonological processing, story recall, and visualization).

2.2.5.2. Diet.

Habitual diet (i.e., carotenoid intake) will be measured using the Block Food Frequency Questionnaire [38,39] for children ages 8–17 years, which will be completed by the parent/guardian on behalf of child participants. The questionnaire includes 77 food items and relies on food lists developed from the NHANES 1999–2002 dietary recall data and the USDA nutrient database.

2.2.5.3. Anthropometrics and Adiposity.

Height and weight will be assessed using a stadiometer (model 240; Seca) and a digital scale (Tanita BWB-627A), respectively. They will be measured three times each and averaged for analyses. BMI percentile (BMI%) will be calculated using Centers for Disease Control (CDC) guidelines, accounting for age and sex for BMI% [40]. Body composition measures (i.e., body fat percentage) will be assessed without shoes or socks using bioelectrical impedance analysis (BIA; InBody 270: Biospace, California, USA).

2.3. Timeline

Baseline testing will involve three laboratory visits. On day 1, participants and their parents/guardians will complete informed parental consent and child assent. Following consent and assent, the parent/guardian will complete a modified Tanner Staging Scales [24] questionnaire to confirm eligibility. If the participant is eligible to continue, parents will complete demographic, health history, and food frequency questionnaires. Child participants will complete anthropometric assessments, WJ-IV, and MPOD. On day 2, participants will complete MPOD, KTEA-3, and the spatial reconstruction task. Lastly, on day 3, participants will complete the cognitive tasks with EEG recordings. After completion of baseline assessments, participants will be randomized to the intervention or waitlist control group for nine months. Between 4 and 5 months, participants will complete an MPOD assessment during a mid-intervention visit. Lastly, after nine months, participants will return to the laboratory and complete three visits identical to their baseline visits. Recruitment and enrollment for this study will be initiated in April 2022, with the goal of collecting data in 288 total participants over four years, completing data collection by the in summer of 2026. Each cohort will aim to enroll 70–80 participants per year to account for an estimated 20% attrition.

2.4. Statistical approach

Data will be screened for distributions and outliers in SPSS [41], which in turn will be reduced using transformations and Winsorizing, respectively. Any missing data due to drop-outs over the course of the intervention will be replaced using multiple imputation with an intent to treat approach. To test the primary aim, a path analysis with Bayesian estimation will be conducted in Mplus [42](p value = .05, one-tailed). Specifically, changes in attentional inhibition (accuracy and reaction time), hippocampal-dependent relational memory (accuracy), and academic achievement (KTEA III composite) will be regressed on group (1 = Active Treatment, 0 = Wait-list Control) and relevant covariates (e.g., baseline BMI, dietary intake of lutein and zeaxanthin, and intelligence quotient [IQ]). Secondary aims will be tested with repeated measures ANOVA involving changes in MPOD, the latency and amplitude of the P3 ERP component at the P3 ROI during the flanker task, and the four subscales of the KTEA III; statistical significance will be interpreted after adjusting for multiple comparisons (α = 0.05/ 7). The hypothesized mediation model will assess the potential causal role of changes in MPOD in driving the effects of the intervention. Bootstrapped confidence intervals will be used for direct and indirect effects.

3. Discussion

With increasing evidence that nutrition in early life could impact brain and cognitive health in later life, identification of dietary patterns and components that can optimize brain health is critical for informing future dietary guidelines. Given the current evidence supporting the cognitive benefits of xanthophyll consumption among adults, it is important to address the gap in literature regarding the effects of xanthophylls on childhood cognitive function and achievement [20,21]. This study will be the first clinical intervention trial to supplement lutein and zeaxanthin in childhood to examine macular and cognitive effects. We will directly test the potential cognitive benefits from lutein and zeaxanthin supplementation during childhood and determine the mediating role played by increased concentrations of carotenoids in central nervous tissue. Comparable to the literature present in adults and cross-sectional evidence in childhood, we anticipate that childhood relational memory and attentional control, at the behavioral and neuroelectric level, will benefit from the 9-month supplementation of lutein and zeaxanthin [14,15,20,21,43]. Further, given cross-sectional evidence that MPOD is associated with academic abilities in children, we anticipate that lutein and zeaxanthin supplementation over the school year will result in greater benefits in academic achievement relative to the placebo control group [43,44]. Utilizing a non-invasive measure of macular carotenoid status can provide additional insights into our sample, as MPOD is strongly correlated with brain carotenoid concentrations in humans and non-human primates [11,45]. Thus, we expect that an increase in MPOD will mediate improvement in cognitive performance, which we can infer is due to an increased concentration of carotenoids within the brain. The major strengths of this study lie in the novelty of the population and our ability to test the mediating effects of improvement in MPOD as a causal factor in any cognitive benefits observed. Currently, no studies have investigated the effects of lutein and zeaxanthin supplementation on cognitive abilities in preadolescent children. Further, utilizing multiple cognitive assessments, neuroelectric outcomes, and standardized achievement tests will provide us with a more comprehensive understanding of the benefits that carotenoid supplementation may have on cognitive function in childhood. Indeed, the behavioral assessments will be strengthened by the inclusion of ERPs, which have high temporal resolution and may provide additional insights into the specificity of the potential cognitive benefits of carotenoid supplementation (i.e., attentional resource allocation and/or information processing speed).

There are multiple hypothesized mechanisms regarding how lutein and zeaxanthin may impact cognitive abilities. Firstly, given that higher density of macular pigmentation can improve overall visual function and processing speed [46] it would be reasonable to assume that improvements in cognitive performance could be linked to the improvements present within the visual system. Further, the anti-oxidant activity of carotenoids in the retina is thought to act prophylactically in the brain thus mitigating oxidative stress and inflammation which can lead to cognitive decline [47]. While this hypothesis is well supported by the literature, childhood cognitive function may also benefit from additional mechanisms of neural carotenoids given the brain is still undergoing developmental changes. For example, in the retina it has been observed that xanthophylls present in the retina comprise raft domains of the phospholipid bilayer, which assist in the regulation of G-protein mediated pathways of signal transduction [48]. If macular xanthophylls are able to mediate more efficient signal transduction in retina, it is possible a similar mechanism is occurring in the brain leading to more efficient signal transduction and thus cognitive processing⁴⁹, which is supported by our work which displays individuals with higher macular pigmentation exhibit improved neural efficiency measured utilizing ERPs [18]. Through this study, we hypothesize the results will display a similar effect thus contributing to this proposed mechanism.

This work has some limitations to consider. First, blood biomarkers of nutritional and metabolic status (e.g., inflammation) are missing from the study and could provide additional insights into the mechanisms by which xanthophyll carotenoids may impact cognitive function. Second, given the length of the intervention trial, we may encounter issues with maintaining compliance during the 9-month period; however, this will be mitigated through a variety of methods. Biweekly check-ins with participants’ parent/guardian will serve as a consistent reminder to take the supplements, and participants will track consumption on the outside of the bottle by marking each day of consumption with a sticker. Additionally, and we will have the ability to check in with participants at their mid-point testing visit. Third, as a supplementation trial, we are unable to control the habitual diet of the individuals enrolled in the study, which could have an additional impact on our results. To account for this, each participant’s parent/guardian will complete the Block food frequency questionnaire, and habitual diet can be included as a covariate if diet patterns changes are observed. Lastly, the addition of neuroimaging techniques (e.g., MRI, fMRI) could provide improved spatial resolution to examine the impact of carotenoids on brain health; however, this is outside the scope of the proposed aims of this study.

Considering the evidence regarding the beneficial effects of lutein and zeaxanthin supplementation on adults, it is crucial to understand these impacts earlier in life. This study will be the first clinical intervention study of lutein and zeaxanthin supplementation in preadolescent children. Our approach will utilize high resolution techniques, including the behavioral and neuroelectric indices of attention, memory assessments, and comprehensive measures of academic achievement. Thus, this study will provide novel evidence using high quality techniques to fill the gap in the literature regarding carotenoid supplementation in preadolescent children.