A Double Blinded Randomized Control Trial to Aid in Determining the Dose Dependent Relationship of Docosahexaenoic Acid (DHA) on Eicosapentaenoic Acid (EPA) Feedback Inhibition Using Carbon 13 as a Biomarker

Docosahexaenoic acid (DHA) is vital for the structure of cell membranes, essential for brain and eye development, found at high levels throughout the brain and nervous system and is important for optimal cognitive development in children, the growth of the fetus during pregnancy and healthy breast milk during lactation. DHA has also been shown to aid in the protection against primary and secondary symptoms of cardiovascular disease as well as macular degeneration. However, whether DHA is deemed to be essential in the diet remains controversial. Meanwhile, the Institute of Medicine (now the National Academy of Medicine) set an adequate intake for DHA's precursor, alpha-linolenic acid (ALA), at 1.6 g/d and 1.1 g/d for healthy males and females, respectively. The investigators believe the confusion arises from the approach to studying omega-3 fatty acids, particularly DHA requirements. For these studies, DHA is supplemented, and often complex, multifactorial disease outcomes are used as endpoints. The doses of DHA used in these studies are often subjective and participants, including controls, have varying DHA intakes and levels. Supplementing a population, like vegetarians or vegans whose levels are similarly low would be useful. Classically, it was believed that that when DHA is consumed a portion is converted "backwards" to eicosapentaenoic acid (EPA) which then accumulates in a process called "retroconversion." However, using a novel approach called compound-specific isotope analysis (CSIA), the investigators observed that the rise in EPA is not primarily from retroconversion, but rather from a backlog of EPA primarily originating from ALA. Initially demonstrated in rodent models by CSIA, the team confirmed by similar modeling that DHA consumption in humans results in the same backlog of EPA. Critically, DHA induces a backlog in metabolism of EPA and dietary ALA appears to be essential for this backlog. Thus, the increase in EPA represents a biochemical feedback pathway that slows DHA synthesis in response to sufficient DHA.

Significance: By precisely determining the lowest amount of DHA required to activate this feedback mechanism it may be useful for future estimation of DHA requirements. Moreover, the problem with the amounts suggested for the unofficial recommendations is that if the global population were to consume these amounts it would be environmentally concerning as this nutrient is ecologically sensitive with its major source coming from already stressed ocean fish stocks, encouraging unsustainable fishing practices, something environmentalists and researchers have said are creating legitimate climate and health concerns.

Hypothesis: The dose at which EPA increases with increased DHA is the point at which elongation slows, indicating a significant feedback pathway is present.

Objectives: 1. To determine the dose-response for DHA to increase EPA. 2. Investigate the DHA dose and time that increases EPA using CSIA. 3. To measure EPA, DPAn-3, and DHA turnover and loss rates. 4. Provide data for exploratory analysis related to PUFA metabolism, ALA consumption and its effect on EPA and DHA levels, and DHA's effects on disease related biomarkers, C-Reactive Protein (CRP), and blood clotting.

Trial Design: Co-principle investigators Dr. Richard Bazinet and Dr. John Sievenpiper and co-investigators, Drs. David Jenkins and Adam Metherel along with PhD student Amy Symington, will conduct a double blinded, placebo-controlled, dose-response supplementation trial. Seventy-two healthy vegans or vegetarians who are 18-50 years old will be randomly assigned to 1 of 6 groups. Prior to randomization, participants will undergo a 2-week run-in phase to gather their questionnaire data. Once randomized, each group will take DHA supplements of either 0mg (placebo), 100mg, 200mg, 400mg, 800mg and 1000mg per day over the course of 8 weeks. These doses represent the range of DHA intake levels spanning average population intakes (<100 mg/d), frequently recommended intakes (250 - 500 mg/d), and DHA intake levels known to increase plasma EPA levels (1000 mg/d). A questionnaire will be administered to obtain information in relation to approximate ALA, EPA and DHA intake before and after the supplementation period. A 3-day weighed diet diary will be provided to obtain food intake over the course of the supplementation period to gauge ALA consumption. Participants will be asked to refrain from DHA rich foods during the supplementation period. This will not be an issue as this population generally do not consume DHA rich foods. Baseline intake of the essential fatty acid ALA will be assessed via questionnaire and verified by blood samples.