There is an increased demand for new sources of high-quality n-3 PUFAs for human consumption, and PC-rich lipids from herring roe is a promising product in this regard. Supplementation with DHA- and EPA-rich PC may have additional benefits compared to DHA- and EPA-rich TAG both due to PL being more easily incorporated into cellular membranes, as well as being a source of the essential nutrient choline. In this two-week intervention in young healthy adults with high habitual fish intakes, there was a rapid increase in EPA and DHA in RBC PC, plasma FA and plasma PC. Furthermore, there was a corresponding improved lipid status, including a decrease in plasma TAG and NEFA, and increased HDL-cholesterol, choline, and betaine. These findings demonstrate that MOPL30 had significant biological effects in healthy subjects.

The participants were given 2350 mg EPA and DHA daily, comparable to doses utilized in patients with CVD or metabolic syndrome to achieve a TAG-lowering effect. In line with this, plasma TAG was reduced and HDL-cholesterol increased after only two weeks of treatment. This indicates a high bioavailability of MOPL30 and a subsequent rapid effect on lipid metabolism. It has been shown that the preferred lipid form for transport of DHA to RBC is lyso-PC, which is rapidly converted to PC [14, 15]. This could mean that DHA-rich PC may be preferred for uptake in RBC and putatively in brain. Incorporation of EPA from fish oil into RBC membranes has been shown to reach a steady state after 180 days [12]. Notably, we observed an increase in EPA and DHA in RBC PC similar to that of plasma after only 14 days. In mice, we recently showed that similar amounts of EPA and DHA in liver PL were achieved with krill oil and a two-fold higher dose of EPA and DHA from fish oil, indicating higher bioavailability of EPA and DHA from the PL-source krill oil [16]. Importantly, in a recent study in which subjects were supplemented with 600 mg EPA and DHA from either fish- or krill oil, the omega-3 index was significantly higher in subjects receiving krill oil than in those given fish oil [8]. However, no effect on plasma TAG was observed at 600 mg/day EPA + DHA. A recent study in adults with high TAG levels showed that a dose of 0.5-2 g/day krill oil for 12 weeks significantly reduced TAG [17].

Although the intake of DHA exceeded that of EPA by 2.8 fold, the increase in EPA was higher than DHA in the PC-fraction of both plasma and red blood cells after two weeks of MOPL30. Studies have shown that the DHA level in lipid pools has a less steep dose–response curve than EPA, which is easy to influence by supplementation [18], and our results confirm this. This can partly be due to more efficient liberation of DHA from chylomicrons [19], leaving more EPA in chylomicron remnants and hereby making more EPA available for PL synthesis in liver. In addition, retro-conversion of DHA to EPA is dependent on peroxisomal β-oxidation, and is reported to be at approximately 10% in humans [20, 21]. Hansen et al. showed that 4 g supplement of pure EPA for 5 weeks led to a 6.2 fold increase in EPA in plasma phospholipids, and no change in DHA [18]. In contrast, the same intervention using a pure DHA supplement led to a 1.9 fold increase in DHA and a 1.7 fold increase in EPA. This demonstrates the importance of supplementation with DHA and not only EPA. Despite a dose of 132 mg DPA/day, and the possibility of formation of DPA from EPA, DPA levels were not influenced by two weeks of MOPL30. In line with this, a decrease in DPA after uptake of EPA or DHA has been reported in long-term studies [22, 23].

Indications of differential effects of n-3 PUFA in PL and TAG form have also been found at the gene level in several animal studies [6, 7], including genes involved in glucose metabolism. A recent study reported that EPA and DHA supplements may improve insulin sensitivity in young obese individuals [24]. While some meta-studies have failed to show an effect between n-3 PUFA intake and incident type 2 diabetes mellitus (T2DM) [25, 26], others indicate a reduced risk of T2DM with increased intake of PUFAs [27, 28]. Based on findings in humans and from recent animal studies, PL supplements could be expected to have potent effects on glucose metabolism. The small, but significant, improvement in glucose response in healthy individuals after only two weeks of intervention suggests a potential for the use of MOPL30 in insulin resistant individuals, or patients with diabetes.

The conditionally essential nutrient choline is a quaternary amine, and is mainly utilized for the synthesis of PC and sphingomyelin, as well as lysophosphatidylcholine. In addition, choline can be oxidized to betaine, which is involved in the remethylation of homocysteine to methionine in the one-carbon cycle [29]. Finally, in the neurons choline is a precursor for the important neurotransmittor and vasodilator acetylcholine [30–32], and increased intake has been connected with improved cognition, learning and memory [33–35]. Betaine holds an important role in the liver, and has a potential therapeutic use in the treatment of fatty liver disease as well as homocysteinemia, a risk factor for CVD [36, 37]. In addition, some studies have demonstrated that betaine supplements improve muscle performance [38]. PC biosynthesis is required for VLDL production, both through the CDP-choline (Kennedy) pathway and the phosphatidylethanolamine N-methyltransferase (PEMT) pathway. In general, a balanced diet will provide sufficient amounts of choline, but groups which may benefit from choline supplementation are pregnant and lactating women, infants, and cirrhosis patients [39]. Thus, a PC supplement can both reduce the need for methyl-donors for PC synthesis, and supply betaine for homocysteine remethylation, which will be beneficial in situations where methyl donors are limited [40]. Interestingly, while MOPL30 supplementation led to increased EPA and DHA-rich PC in plasma and RBC, the total level of PC decreased, with a concomitant increase in plasma free choline. This may indicate a higher level of PC degradation as a result of increased dietary intake, ensuring maintenance of the strictly regulated choline balance in the human body [41]. We were unable to measure acetylcholine in plasma due to its short half-life, however, both the one-carbon cycle/remethylation process and the production of acetylcholine may potentially have been stimulated by increased choline levels. DHA has been demonstrated to increase synaptic transmission in mammalian brain cells, and this effect was potentiated by phosphatidylcholine [42]. Thus, MOPL30 may have beneficial effects on cognitive function. It would be valuable to measure plasma choline acetyltransferase activity in future clinical trials to verify if acetylcholine production is stimulated by MOPL30 in humans.

As high plasma levels of long- and medium-chain acylcarnitines are linked to increased heart failure in CVD patients, they have been put forward as potential biomarkers of cardiovascular risk [13]. Incomplete β-oxidation, impaired substrate switching, and dysregulation of mitochondria during insulin resistance can cause elevated levels of intermediate oxidation products, and this can be reflected in plasma acylcarnitine levels [43, 44]. Thus, it is of interest to establish whether dietary intervention with n-3 PUFAs affect these plasma parameters in healthy adults, as EPA and DHA are known to stimulate mitochondrial β-oxidation. We observed a reduction in all acylcarnitines after a two-week intervention with MOPL30, including the risk-associated palmitoylcarnitine.

In further studies it will be interesting to determine if the supplement can benefit patients with insulin resistance, both with regard to plasma TAG levels, mitochondrial function, and glucose tolerance. It will be of particular interest to compare the bioactivity of MOPL30 and TAG EPA and DHA supplements at lower doses of EPA and DHA. In a follow-up study, a double blind comparison to fish oil will be performed to identify possible PL-specific effects of MOPL30. Also, a rodent study is planned to examine bioaccretion of EPA and DHA into brain and other tissues.