Incorporation of dietary DHA into plasma and other tissues

Wild type C57BL/6 mice, maintained on normal rodent chow, were gavaged daily with 80 µl of corn oil alone (control), or the corn oil containing 1 mg DHA in the form of free fatty acid, sn-1 DHA LPC, or sn-2 DHA LPC for 30 days. The food intake, and the body weights were not significantly different between the 4 groups of mice. Following the cognitive tests (see below), the mice were sacrificed and the fatty acid composition of various tissues was analyzed by GC/MS. The effect of dietary supplementation with different DHA preparations on the DHA content (% of total fatty acids) of plasma, liver, heart, and adipose tissue is shown in Figs 2 and 3. In addition to DHA, the percentages of arachidonic acid (ARA, 20:4) are shown, since DHA has been reported to replace predominantly ARA in most tissues, and since the 22:6/20:4 ratio provides one measure of the anti-inflammatory potential in the tissues18. In the plasma, there was a significant increase in the percentage of DHA by all three preparations of DHA, although the increase was greater in the groups treated with LPC DHA than the mice treated with free DHA (Fig. 2). However, there was also an increase in the percentage of 20:4 with all three DHA preparations, resulting in no significant change in the ratio of 22:6/20:4. The reason for the increase in plasma ARA is not clear, although it is decreased in most tissues where DHA is increased (see below). One possible explanation is that the tissue ARA replaced by DHA is released as free ARA into the plasma. The concentration of LPC DHA in plasma was also determined by LC/MS/MS. As shown in Fig. 2, the LPC DHA concentration of plasma increased significantly in mice treated with either isomer of LPC DHA, but not in mice treated with free DHA. Although we did not analyze the isomer composition of plasma LPC DHA because of insufficient material, the increase was 28% higher after the feeding of sn-1 DHA LPC compared to sn-2 DHA LPC.

Figure 2 Effect of molecular carrier of dietary DHA on plasma levels (% of total fatty acids) of 22:6 (A), and 20:4 (B), on 22:6/20:4 ratio (C), and on LPC-DHA (µg/ml) (D). Results shown are mean ± SD (n = 8). Bars in each panel not sharing a common superscript are significantly different from each other (p < 0.05, one way ANOVA, and post-hoc Tukey test). Full size image

Figure 3 Effect of molecular carrier of dietary DHA on the concentrations of 22:6 and 20:4, and on 22:6/20:4 ratio in liver, heart and adipose tissue. The values shown are mean ± SD (n = 8). Bars in each panel without common superscripts are significantly different from each other (p < 0.05, one way ANOVA, followed by post-hoc Tukey test). Full size image

The molecular species of DHA-containing lipids (PC, PE. TAG) in plasma were analyzed by LC/MS/MS, to determine the metabolic fates of the absorbed DHA. Most of the DHA-PC species were increased by treatment with both the LPC isomers, but not with free DHA (Fig. S1).There was no significant difference between the two LPC isomers, except for a greater increase in 18:1–22:6 PC in the sn-1 DHA LPC group. The molecular species of DHA-PE were also increased by the two LPC DHAs, but not by free DHA (Fig. S2). In contrast to the plasma phospholipids, the plasma TAG species containing DHA were increased more by free DHA than by the LPC DHA (Fig. S3).

The DHA concentration in liver was significantly increased only in the mice fed sn-2 DHA LPC, with a parallel decrease in the concentration of 20:4 and an increase in the 22:6/20:4 ratio (Fig. 3). Although the increase in DHA after feeding sn-1 DHA LPC did not reach statistical significance, the ratio of 22:6/20:4 was significantly increased. All three preparations of DHA increased the percentage of DHA in the heart, at the expense of 20:4, resulting in a significant increase in the ratio of 22:6/20:4 in all three groups of animals, compared to the controls. In contrast to the liver and heart, the adipose tissue showed an increase in DHA concentration, as well as in the 22:6/20:4 ratio after the feeding of free DHA, but not after feeding of either isomer of LPC-DHA. These results show that the dietary free DHA is predominantly directed to adipose tissue and heart in the form of TAG, whereas dietary LPC-DHA was directed to the brain in the form of phospholipid (see below).

Incorporation of DHA into various regions of the brain

The DHA and ARA concentrations of five brain regions are shown in Fig. 4. In contrast to the other tissues, all the brain regions were significantly enriched with DHA after the feeding either sn-1 DHA LPC or sn-2 DHA LPC, but not free DHA. The concentration of ARA was correspondingly decreased after feeding either isomer of LPC, and the 22:6/20:4 ratio more than doubled in all regions of brain. On the other hand, there was no significant increase in this ratio in any brain region after the feeding free DHA. These results show that the brain DHA responds specifically to treatment with dietary LPC-DHA. However, we found no difference between the two isomers of LPC-DHA in their ability to enrich brain DHA, although previous studies suggested that the sn-2 DHA LPC may be preferentially taken up by the brain14, 15.

Figure 4 Effect of dietary treatment with free DHA, sn-1 DHA LPC, and sn-2 DHA LPC on the levels of 22:6 and 20:4 and on the 22:6/20:4 ratios in various regions of the brain. The values shown are mean ± SD (n = 8, for all regions, except for Amygdala, where n = 6). The bars in each figure not sharing common letter are significantly different from each other (p < 0.05, one way ANOVA, and post-hoc Tukey test). Full size image

The increase in brain DHA content by dietary LPC-DHA, but not free DHA, was also evident when the results are expressed as nmol of DHA per mg wet weight of the tissue. By this measure, the DHA content increased by 2–3 fold in most regions of the brain after LPC-DHA treatment, except in the case of striatum and amygdala where the increase in the sn-1 DHA group did not reach statistical significance (Fig. 5). We also determined the molecular species of DHA-containing PC and PE in the hippocampus by LC/MS/MS, to investigate the incorporation profiles of DHA derived from dietary free DHA and LPC-DHA (Fig. S4). The amount of LPC-DHA, as well as most species of DHA-PC were increased by both isomers of dietary LPC-DHA, but not by free DHA. One exception was 20:4–22:6 PC, which was increased by free DHA, but not by the two LPC-DHAs. The major species of DHA-PE were also increased by the dietary LPC-DHAs, but not by free DHA (Fig. S5). Interestingly, the net increase in the amount of hippocampal PE-DHA was 4-fold higher than the net increase in PC-DHA by both isomers of dietary LPC-DHA, showing that the majority of DHA derived from dietary LPC-DHA was ultimately incorporated into brain PE.

Figure 5 Effect of dietary free DHA, sn-1 DHA LPC, and sn-2 DHA LPC on the DHA concentration in various regions of brain, expressed as nmol/mg tissue. The values shown are n = 8, for all regions, except for Amygdala, where n = 6. Bars with non-identical letters on top are significantly different from each other in each panel (p < 0.05, one way ANOVA, and post-hoc Tukey test). Full size image

Effect of dietary DHA on brain function

Although DHA has been reported to improve markers of cognition in DHA-deficient animals19 or in the disease states20, very few reports show a positive effects of dietary DHA on the cognitive functions of normal adult mice and rats. Even when the effects were demonstrated, the doses used were very high, in the range of 0.6 g to 2.4 g/kg body weight20,21,22,23, relative to what is practical in humans. Since we could more than double the brain DHA levels at very low amounts of LPC DHA (about 40 mg/kg body weight), we assessed whether LPC-DHA also modulated spatial learning and memory, as determined by the Morris water maze test. In the acquisition phase, all groups learned the location of the platform with no significant group differences (Fig. 6A). Notably, however in the probe trial (Fig. 6B), mice treated with either sn-1 DHA LPC or sn-2 DHA LPC located the previous platform area with a shorter latency time and spent longer time in the target quadrant compared to free DHA-treated or control mice (two-way ANOVA followed by Tukey’s post hoc comparisons). For example, both sn-1 and sn-2 DHA LPC treated mice found the previous platform area 7 times faster than the control and free DHA groups, and spent twice as long in the target quadrant.

Figure 6 Improved memory in mice treated with LPC DHA compared to control or free DHA treated mice. In study 1 (A and B), Morris water maze tests of mice treated with vehicle (control), free DHA, sn-1 DHA LPC, and sn-2 DHA LPC for 1 month are shown. LPC DHA-treated mice did not differ from control or free DHA mice in the acquisition phase (A). In the probe trial (B) both sn-1 DHA LPC and sn-2 DHA treated mice reached the previous platform area with shorter latency time, and spent longer in the target quadrant. Data expressed as mean ± S.D (n = 8). *p < 0.05, by two-way ANOVA followed by Tukey’s post hoc comparisons. In a second study (C and D) to validate the above results, only 2 groups of mice were used and were treated either with free DHA or sn-1 DHA LPC for 1 month. As in the first study, sn-1 DHA LPC treated mice reached the previous platform area with a lower latency time, and spent longer in the target quadrant, compared to free DHA group (n = 5 each). Data are expressed as mean ± S.D. *p < 0.05 by two-way ANOVA followed by Tukey’s post hoc comparisons. Full size image

To account for cohort effects and for validation, we conducted a second study in a smaller cohort of mice comparing only the free-DHA treated group with sn-1 DHA LPC treated group (Fig. 6C and D). Since we found no difference between vehicle control and free DHA groups in the above study, we did not include the vehicle control in the second study. Similarly, since there was no significant difference between the two isomers of LPC-DHA, we used only sn-1 DHA LPC. Although the data were more varied in the free-DHA group in this study, they were still consistent with the first study. In the acquisition phase, sn-1 DHA LPC mice learned the location of the platform faster by Day 5 (Day 1 vs days 2,3,4,5 and day 2 vs 5, two-way ANOVA followed by Tukey’s post hoc comparisons). However, the only significant difference for Free DHA mice was for day 1 vs 4 by two-way ANOVA followed by Fisher’s LSD test i.e. no multi-group comparisons. As for the first treatment study, in the probe trial sn-1 DHA LPC mice located the previous platform area with a lower latency time (free DHA- 47.1 seconds, sn-1 DHA LPC- 12.7 seconds) and spent longer time in the target quadrant compared to free DHA (free DHA- 12 seconds, sn-1 DHA LPC- 29.9 seconds).

In both MWM tests there were no group differences in average swim speed or total distance swam in the probe trial, and open field test showed that there was no significant change from the control mice in any of the treatment groups (not shown). Therefore, the beneficial effects of LPC DHA on memory were not related to general changes in locomotion.

Effect of DHA on Brain derived neurotrophic factor (BDNF) levels

BDNF plays an important role in learning and memory, and is a potential downstream target of DHA24,25,26. Therefore, we determined BDNF concentration in the brain regions by ELISA to complement our MWM data. As shown in Fig. 7, while there was no change in BDNF content in any region of the brain after free DHA treatment, its levels were significantly increased by either sn-1 DHA or sn-2 DHA LPC. These results suggest that one of the mechanisms for the augmented memory in the mice treated with LPC-DHA is the increase in the BDNF levels.