Changes in fatty acid levels after consumption of a novel docosahexaenoic supplement from algae: a crossover randomized controlled trial in omnivorous, lacto-ovo vegetarians and vegans

Vegetarian diets are associated to low levels of the most relevant risk factors for chronic disease compared to omnivorous [1]. However, they may result in inadequate intake of some nutrients such as essential fatty acids. There are only two essential fatty acids: the polyunsaturated fatty acids (PUFA) linoleic acid (LA, C18:2n6) which belongs to the omega-6 family (n-6), and α-linolenic acid (ALA, C18:2n3) which belongs to the omega-3 family (n-3), with pro-inflammatory and anti-inflammatory properties, respectively. Indeed, both families must be balanced to maintain optimal health and metabolic functions. LA is a precursor of arachidonic acid (AA, C20:4n6), while ALA is a precursor of eicosapentaenoic acid (EPA, C20:5n3) and docosahexaenoic acid (DHA, C22:6n3). These long-chain PUFA n-3 play many important roles, as they are highly concentrated in brain and retinal phospholipids as well as being involved in neurological and cognitive developments. In addition, they exert anti-inflammatory and antithrombotic properties [2]. Nevertheless, the rate of conversion of ALA to EPA and DHA is very low in humans [3‐5]. For this reason, it is advisable to ingest DHA directly, being fish its main dietary source.

Because vegetarian diets do not include fish and fish by-products, it is of great interest to have adequate sources of n-3 long-chain PUFA, and to know their bioavailability and metabolic fate. In this regard, there are many vegetable sources of the precursor ALA: flax-seeds, hemp seeds, chia seeds, nuts, rapeseed, soybean and their derived oils [6]. However, low serum levels of EPA and DHA have been reported in lacto-ovo vegetarians (LOV) and vegans (VEG) [6‐8]. Our research group recently observed that only 10% of the vegetarians participating in a nutritional study consumed n-3 supplements, which was associated with higher serum EPA but no improvement on DHA [8].

In the search for a plant-based alternative to fish, seaweed culture has been developed. Microalgae, which in fact are eaten by fish and consequently they acquire the n-3 fatty acids, nowadays, represent the most promising DHA source for the vegetarian population. Specifically, Schizochytrium sp. oil shows high DHA concentrations [9]. Moreover, the use of this oil has been authorized by The European Commission as a novel food and food supplement to provide a maximum of 250 mg DHA/day for general population [10]. It is stated that an intake of 250 mg of DHA helps maintain normal brain function and normal vision. In addition, a combined intake of 250–500 mg/day of EPA and DHA is recommended in adults based on the prevention of cardiovascular risk [11, 12]. Much higher doses of these n-3 fatty acids have been used to obtain therapeutic effects (> 3 g/day) [13], however, when looking for the most suitable supplement for vegetarians, the non-fish source and the physiological/nutritional quantity are the main determining factors.

Several studies in humans have reported a significant increase in serum DHA after intake of algal oil supplements. Early studies on algal DHA supplementation were conducted using DHA at doses of 1.6 g/day in vegetarians and omnivorous [14] or in two groups of vegetarians, one of them consuming corn oil as control [15]. Increases in DHA in serum phospholipids and platelets were noticed after 3 and 6 weeks. Other works in postmenopausal women used DHA supplements at doses of 2.1 and 2.8 g/day and reported elevations in circulating DHA and decreases in plasma lipids indicative of reduced cardiovascular risk [16, 17]. However, these studies present some limitations such as the small sample size and the need to give several capsules per day to achieve the desired dose. Subsequently, Arterburn et al. [18] evaluated the bioavailability of DHA oils in capsules from two different algae (Crypthecodinium cohnii and Schizochytrium sp.) and found a dose-dependent response with doses from 200 to 1000 mg/day after 4 weeks, and Sarter et al. [19] analyzed the omega-3 index (DHA + EPA in erythrocytes) in a dry blood spot sample after supplementation with a vegetal oil containing 254 mg of DHA plus EPA for 4 months and found a clear increase. In these two works, the algal oil contained a mixture of DHA and EPA that should be taken into account in order to compare their results with others.

Accordingly, the aims of this study were to: (1) determine the fatty acid profile of vegetarians and omnivorous (OMN), (2) establish the possible differences due to the type of vegetarian diet, either including eggs and milk products (LOV) or excluding them (VEG), and (3) study the effects of consuming a DHA-supplement of vegetable origin on fatty acid profile of LOV, VEG and OMN. To this end, a randomized double-blind, placebo-controlled cross-over study was carried out in healthy Spanish participants.

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) [24], although the intake of the other essential fatty acid, ALA (C18:3n3), was also high in vegetarians as we observed throughout the assay (Table 3).

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 [25, 26]. This has been related to the Mediterranean diet and the spread use of olive oil. Almost all our volunteers specified that they consumed olive oil as culinary fat and food ingredient. This fact was reflected on the estimation of OA intake that contributed to the highest dietary fatty acid, approximately 25–30 g/day, with moderate differences between diet groups (Table 2).

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 [27]. Therefore, results of LA and ALA intake of the present study indicate that volunteers ingested sufficient amounts of both essential fatty acids. However, the proposed reference n-6/n-3 ratio below 10:1 [28] was at the upper level in LOV and above in vegans who ingested more LA compared to the other groups and whose n-3 intake was almost exclusively ALA (Tables 2 and 3). This is in line with findings indicating that the abundance of LA in the diet reduces the transformation of ALA into their long chain fatty acid derivatives, EPA and DHA [8, 29]. Therefore, although vegans ingested enough ALA its metabolism could be inhibited in a certain extent, due to interaction between the n-3 and n-6 pathways, as explained below.

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 [10‐12]. However, this was probably overestimated, as these volunteers had to be low consumers of fish to be selected for the trial (maximum two times per week). Lower values of EPA and DHA intakes have been reported in an exploratory study in a small group of Australian omnivorous and vegan endurance athletes [30]. In any case, these results also show that the dose of DHA in the tested algae supplement was at the lowest threshold of what was consumed by the average omnivorous participant, whose maximum intake was two servings of fish per week.

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 4). Likewise, as for the second most abundant fatty acid in serum, OA (C18:1, n9), there was no effect of treatment. This should be related to the predominant use of olive oil by the participants, independently of their diet classification, and with the composition of the DHA-supplement that did not include OA. Consumption of the placebo capsules did not further increase serum %OA from baseline as in fact the intake of OA from the placebo represented only a small fraction of the total dietary intake of this fatty acid. It should be noticed that fatty acid profiles of the present work are consistent but slightly differ from the global fatty acid database reported by Brenna [31] (see also supplemental Table S3). In the present Spanish study, the percentage of OA in serum resulted higher than of palmitic acid (C16:0), that was the second more abundant fatty acid in the mentioned report which primarily collected data from Nord American laboratories. Furthermore, our EPA and DHA data were relatively low and should be used as a reference for low-fish consumers and vegetarians living in a Mediterranean country.

In addition, serum fatty acids confirm that there was some endogenous production of EPA and DHA in vegetarians (Table 4). Although the diets LOV and VEG did not provide appreciable amounts, both fatty acids were detected in serum, and this should be attributed to biosynthesis from the common precursor ALA (C18:3n3). Interestingly, serum DHA was much higher in women than in men and this difference was unaffected by dietary classification or DHA-supplement. The literature consistently reports that this is due to hormonal differences, probably related to reproduction and infant protection since DHA is crucial for brain development [32].

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. 3, EPA (C20:5n3) and AA (C20:4n6) result from sequential desaturation and elongation steps using the same enzymes. These fatty acids can be converted to metabolic mediators or continue through the n-3 and n-6 cascades to yield C24:6n3 and C24:5n6, then transported to peroxisomes for beta-oxidation, and finally produce DHA (C22:6n3) and docosapentaenoic acid (C22:5n6) [33, 34]. The conversion rate of ALA to DHA has been estimated to be lower than 1% or as low as 0.01%, depending on the used models [5, 35].

In 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 [8]. This low metabolic production of DHA was estimated by the DHA/ALA ratio that was lower in VEG and LOV compared to OMN after placebo, with marked differences between the three diet groups, suggesting that the biosynthesis of DHA form its precursor was compromised more in vegans than in the other vegetarian group. However, these estimations in terms of ratios do not give information on the relative content in tissues, such as red cell membranes and heart, and much more research is needed in animal models and humans to know the effects of varying n-3 and n-6 fatty acid intakes into tissue levels and biological function [29, 36].

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. [37, 38] calculated EPA + DHA in erythrocytes (omega-3 index, O3I) from the values of EPA + DHA in different blood fractions, including total plasma/serum, to unify data and identify countries potentially at an increased risk of chronic disease. We have estimated the O3I from our data in serum and we have obtained that serum EPA + DHA is approximately 2% in omnivorous after placebo and ≥ 3% after the DHA supplementation. These values are equivalent to the lowest erythrocyte O3I category of ≤ 4% given by these authors indicative of low cardiovascular protection. The LOV and VEG diet groups had serum EPA + DHA of 1–1.5% with placebo and approximately 2% after the DHA-supplement. This means that all our participants, including the diet group OMN and even after consuming the DHA-supplement, would be in the lowest category of O3I which would imply the highest disease risk. However, we are aware of the limitations of this estimation and our results are far from supporting a recommendation in terms of cardiovascular risk.

Figure 2 shows that the DHA increment in serum was much higher in VEG, followed by LOV and OMN, and it is important to note that DHA significantly increased in the three diet groups. Moreover, it was observed that the values increased similarly in men and women who presented generally higher DHA concentrations than men. Therefore, these results lead us to the conclusion that all participants with low or null intake of DHA would benefit from the consumption of the tested DHA-supplement. Other attempts to improve the efficiency of the n-3 pathway, such as reducing LA intake and increasing that of ALA, for example by consumption an ALA supplement, were not able to increase DHA levels [5, 29, 35, 39].

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 [14, 17, 40]. To explain this result, on one hand, it could be argued that the n-3 pathway is partly inhibited with the DHA supplementation, as abundance of the product inhibits its own synthesis (Fig. 3). Our results suggest that the n-3 pathway slows down at the EPA level as the immediate metabolite DPA is markedly decreased, probably because the elongase-2 activity is reduced. On the other hand, it has been suggested that this is likely due to increased competition for incorporation into complex lipids by acyltransferases due to higher amounts of free DHA [14, 17]. However, the exact mechanism is unknown and this deserves further investigation.

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 [8]. Other authors have reported an increase in circulating EPA after DHA supplementation, but in their studies, the DHA dose was higher than in the present study. This opened the possibility of a retroconversion of DHA to EPA [14, 15, 17], although this has not been demonstrated and it is more likely that EPA is spared from further metabolism to DHA [40, 41].

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 [32]. However, both genders responded with similar elevations of serum DHA after supplementation (Fig. 2).

Figure 3 summarises the main findings of the current study. Serum long chain fatty acids vary due to the usual diet and the DHA supplementation. Vegetarian diets compared to omnivore diets involve higher serum values of the two precursors of the n-3 and n-6 pathways (ALA and LA) but lower n-3 EPA and DHA. This was particularly shown with the vegan diet that in addition leads to higher n-9 fatty acids, OA and EA, than the other type of diets. The tested DHA-supplement compared to placebo produces a marked increase in serum DHA, independently of usual diet and sex, a decrease in the two intermediate metabolites DPAn3 and ADAn6, and appears to spare EPA for metabolic functions apart from that of DHA synthesis.

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 [22, 42, 43]. We observed that ingestion of one capsule containing of 250 mg of DHA during 5 weeks was acceptable by the volunteers without discomfort, was compatible with the usual diet, and delivered highly bioavailable DHA. Moreover, reports on intakes of fatty acids and circulating levels of vegetarian compared with omnivorous populations are very scarce and lacking in Spain.

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 Schizochytrium sp. was bioavailable and useful for raising circulating levels of this important n-3 long-chain fatty acid. This novel DHA-supplement has a great potential, as a variety of consumers of plant-based diets, could benefit from it. Innovation in this field has been reviewed in detail [44]. Furthermore, among omnivorous, those who avoid fish or should not ingest fish products, due to pathologies such as allergy to Anysakis sp. for example could also benefit from its consumption, as well as the general population who is open to new formulations, supplements and functional foods.