Discussion
This randomized controlled trial was performed in healthy volunteers who did not consume DHA or consumed very low amounts. Therefore, it was a vulnerable population that could benefit from its increased consumption and associated metabolic functions. The assayed DHA-supplement was manufactured from algae to be accepted by vegetarians who avoid animal products including fish, the principal dietary source of this fatty acid. The results clearly confirm that the tested supplement, at DHA dose of 250 mg/day, was effective in increasing serum % DHA in all three diet groups (OMN, LOV and VEG) which doubled in the case of vegans compared to placebo and baseline.Volunteers were characterized by anthropometric values, body composition, blood pressure, heart rate, and serum glucose and lipids within normal reference ranges. Nutrient intakes showed the expected differences between vegetarians and non-vegetarians: the former ingested lower saturated fat and more carbohydrates than the latter. In addition, it was confirmed that DHA and EPA intakes were negligible in LOV and VEG and that all participants were correctly classified in one of the three diet groups.Dietary intake did not vary during the trial and the DHA supplementation did not substantially change the differences between the three diet groups. Plant-based diets compared to omnivorous diets involve a higher consumption of PUFA at the expense of SFA, the contribution of which was mainly the essential LA (C18:2n6) [In the present study, results of dietary intake reveal that total fat intake was approximately 35–40% of the energy, as observed in several surveys performed in Spain [Intake of LA was on average 4.3, 5.0 and 5.5% of total energy (%E), while ALA was 0.6, 0.7, and 0.6%E, in OMN, LOV and VEG, respectively. Considering that LA intake is relatively high in Europe and that the lowest average intake is not associated with deficiency, the European Food Safety Authority (EFSA) proposed an Adequate Intake for n-6 PUFA of 4 E% and for ALA of 0.5 E% without risk of deficiencies [In omnivorous, it was estimated an average intake of DHA, at baseline or with placebo, of 440–500 mg/day, and a EPA intake nearly 200 mg/day, which is above the recommended intake of 250 mg for DHA alone or 250–500 mg/day for EPA + DHA [Overall, the dietary intake results support that the usual diet of the volunteers did not change during the assay, and an imbalance in fatty acids intake was observed, with a relative excess of the n-6 LA in detriment of the two n-3, EPA and DHA, which were absent in the diet of vegans, who just depend on the metabolic synthesis of these long-chain fatty acids.Results of serum percentages of fatty acids show that the major fatty acid was LA (C18:2n6) and that it remained similar after consumption of the DHA-supplement without differences due to treatment. Vegans presented the highest %LA values either after the placebo or the DHA-supplement (Table In addition, serum fatty acids confirm that there was some endogenous production of EPA and DHA in vegetarians (Table However, the metabolic efficiency of the n-3 pathway could be partly inhibited in vegetarians. It is known that there is a relationship between the n-3 and n-6 fatty acid pathways, because there is a competition for the same enzymes, desaturases and elongases, that participate in the synthesis of the long-chain fatty acids of the two families. The rate limiting enzyme is the Δ-6 desaturase that has higher affinity for ALA than LA. However, under conditions of high LA intake as in the present study, the n-6 pathway predominates over the n-3 pathway. As shown in Fig. Biosynthesis of long chain polyunsaturated fatty acids in humans and changes due to vegetarian diets and DHA supplementation of the present randomized controlled trial. Fatty acids analyzed in this study are in bold. Δ6: desaturase Δ6; Δ5: desaturase Δ5; Δ: desaturase Δ9; Elon: elongase. Differences due to diet type: open arrows indicate significantly increased or decreased in lacto-ovo vegetarians and/or vegans compared to omnivorous. Changes due to the DHA supplementation: dark arrows indicate significant increase or decrease after consumption of the DHA-supplement in the three diet groupsIn this regard, it was obtained that vegetarians presented lower serum EPA and DHA than omnivorous as expected, and at the same time, they had higher %ALA than omnivorous, which was particularly shown in vegans. However, because their serum LA was elevated, which was clearly seen in vegans, our results support that the conversion of ALA into EPA and DHA was very limited, especially in this diet group, due to the imbalance n-6/n-3 in addition to the virtually no DHA intake. This is in accordance with our previous study comparing LOV and VEG in which we obtained even more elevated serum n-6/n-3 ratios, indicative of metabolic imbalance [Concerning the influence of the intervention with the DHA-supplement on serum fatty acids, it was clearly efficacious in increasing serum DHA, independently of the diet of the volunteers, did not modify serum total n-6 fatty acids or saturated fatty acids, and improved the ratio n-6/n-3, specially in vegans. Stark et al. [Figure Nevertheless, our results show that serum DPA (C22:5 n3) and ADA (C22:4 n6) decreased by the DHA supplementation. Our results are consistent with others who also found lower DPAn3 levels after DHA supplementation [In support to this metabolic change, we observed that both DPA (n-3) and ADA (n-6) decline and that the ratio EPA/ALA increases after the DHA supplementation. In agreement, we previously observed that vegetarians who consumed n-3 supplements presented increases in serum DHA and also in EPA, which initially we had associated to the composition of some supplements made from fish oil that they ingested [Since our design includes the gender effect, we observed that women presented higher serum DHA than men. This agrees with reports suggesting that a sexual dimorphism leads to higher serum DHA in women than in men. Women may have a greater capacity for DHA synthesis from ALA than men, which has been attributed to oestrogens [Figure This crossover randomized controlled trial was the first to investigate the bioavailability of 250 mg of a new algal DHA-supplement in lacto-ovo vegetarians, omnivorous and vegan volunteers. Some studies found significant increases in plasma DHA levels with higher doses than our supplement had and during shorter time of intervention [The strengths of the study were: the sample size planned was achieved and was sufficient to detect very significant effects. The three diet groups, lacto-ovo vegetarian, vegan and omnivore, were characterized and the differences between them were obtained with very important differences in nutrient intakes and serum fatty acids. The crossover randomised controlled trial was successfully performed and there were no carry-over effects. Moreover, the gender effect was also observed but women and men responded similarly to the DHA-supplement. It was confirmed that the placebo made of olive oil resembled the main fat of the usual diet, either omnivorous or vegetarian, of the volunteers. In addition, there are very few studies with a moderate amount of supplement, and the dose used on this one is the recommended for this type of supplement.There are some limitations of this study: analyses were performed in serum and there is no information on membrane fatty acids or tissue concentrations. However, since our objective was to know the bioavailability of DHA from the algae source, we were aware of this limitation from the beginning. In addition, we did not study any DHA function. Finally, the genetic factor associated to fatty acid metabolism was not taken into account.Present results clearly demonstrate that the supplement of DHA from the microalgae 