The present study clarified that in spite of the increase in ARA levels in plasma phospholipids, plasma clinical parameters of cardiovascular, inflammatory and allergic diseases and levels of ARA metabolites in urine and blood were not altered among the healthy elderly participants whose diet was supplemented with an ARA-enriched oil (240 or 720 mg/day ARA) for 4 weeks.
In previous studies, ARA content in plasma phospholipids increased by 0.7% in young females supplemented with 80 mg/day of ARA for 3 weeks , 2.0% in elderly males given 240 mg/day of ARA for 4 weeks , and by 4.3% in adult males given 838 mg/day of ARA for 4 weeks . In the present study, supplementation with 240 and 720 mg/day of ARA increased ARA content by 2.54% and 5.56%, respectively (Figure 2), which was consistent with these findings. These results suggest that age or gender of participants has little effect of ARA supplementation on the increase in plasma ARA content. The results also suggest that the plasma ARA content increases dose-dependently with ARA up to at least around 800 mg/day. The ARA content in plasma phospholipids increased at 2 weeks and was almost the same between at 2 weeks and 4 weeks. The elevated ARA content decreased to almost the initial level during the 4-week washout period regardless of an intake of 240 mg/day or 720 mg/day of ARA. These results were similar to findings seen during intake of 838 mg/day of ARA . The intake of ARA caused a rapid increase in plasma ARA levels but more than 2 weeks of supplementation did not result in any further increases. This is different from DHA, because the velocity of both increases and decreases in plasma DHA content seems slower when fish oil is administered and discontinued during a washout period . The changes in the other fatty acids were also characteristic since plasma DHA and EPA contents remained unchanged throughout the period. The relatively high intake of DHA+EPA (approximately 1 g/day) in the present study (Table 3) might also have contributed to maintaining plasma DHA and EPA levels. The plasma LA content changed in parallel with the plasma ARA content (Figure 2). A previous study also noted this phenomenon . Although ARA and DHA are considered to compete against each other, ARA intake does not reduce plasma DHA content, whereas intake of DHA and EPA reduces both plasma ARA and LA contents [32, 33]. The specificity of incorporation into plasma phospholipids from dietary fatty acids seems to be in the order of DHA, EPA > ARA > LA. The reason for the difference between ARA and DHA incorporation is unclear, but the specificity of some enzymes associated with acylation and/or deacylation of each fatty acid might be involved.
Platelet aggregation remains unaffected by an ARA intake of 1.5 g/day or 838 mg/day in randomized controlled studies [21, 23]. The present study found that parameters of the coagulation system (PT, APTT and ATIII) remained unaltered and within the normal range. Parameters of chronic inflammation such as plasma hs-CRP, TNFα and IL-6, which are risk factors for cardiovascular disease, remained unchanged, as did plasma adiponectin, which is thought to reduce the risk for cardiovascular diseases. These results suggest that ARA intake does not affect the risk for cardiovascular disease. The parameters for inflammatory diseases (CRP, TNFα and IL-6) and allergic diseases (IgE and eosinophil) were similarly unchanged, suggesting that ARA intake does not evoke inflammatory or allergic diseases. Furthermore, general blood biochemical and hematological parameters remained within normal ranges (Additional file 1, Table S1). Thus, ARA intake appears to be safe under the conditions described here.
In this study, we measured TXA2, PGI2, PGE2 and LXA4. TXA2 causes platelet aggregation and vasoconstriction, which are considered to lead to cardiovascular disease, PGI2 competes against TXA2 and suppresses cardiovascular disease and PGE2 has various physiological roles, one of which is an inflammatory trigger in addition to possible involvement in cancer growth. LXA4 has effects opposite to PGE2, and reduces inflammation and cancer growth . Considering their association with diseases, we estimated TXA2 and PGI2 production as urinary 11-dehydro TXB2 and 2,3-dinor-6-keto PGF1α, respectively. The reported urinary concentration of 11-dehydro TXB2 is 1.489 ng/mg Cre in patients with heart failure, 0.632 ng/mg Cre in those with ischemic heart disease, 0.44 ng/mg Cre in healthy controls  and around 0.6 ng/mg Cre in patients with essential hypertension and retinopathy . The mean concentration of 11-dehydro TXB2 throughout the present study was < 0.4 ng/mg Cre, which was lower than the levels in these patients and did not significantly differ among the groups. Urinary 2,3-dinor-6-keto PGF1α was similarly unchanged and did not differ significantly among the groups. These results show that an increase in the ARA content of plasma phospholipids from 8% to 14% did not affect TXA2 and PGI2 contents. This is consistent with the finding that parameters for cardiovascular disease did not change. However, these findings seem to differ from those of a previous study in which both urinary 11-dehydro TXB2 and 2,3-dinor-6-keto PGF1α were slightly increased by intake of 1.5 g ARA/day for 50 days . The larger dose and longer study duration might explain the discrepancies between that study and ours, but the actual reason for the difference remains unclear.
Several reports have described that plasma PGE2 increases in inflammatory diseases. For instance, the plasma PGE2 concentration increases to > 40 pg/mL in patients with ulcerative colitis , and to 54.5 pg/mL in patients with advanced periodontitis  compared with about 10 pg/mL in controls. The plasma PGE2 concentration in the present study was about 10 pg/mL, which was below the values associated with inflammatory diseases. This value did not change or significantly differ throughout the study. Urinary concentrations of tetranor-PGEM, another marker of PGE2 production, are higher in patients with cancer. The reported level is 11.6 ng/mg Cre in patients with colorectal cancer and 7.0 ng/mg Cre in matched controls . Another study indicated a urinary tetranor-PGEM concentration of 15.0 ng/mg Cre in patients with colorectal cancer and 7.17 ng/mg Cre in polyp-free controls . The mean concentration of tetranor-PGEM in the present study was 8 ng/mg Cre in the placebo and the low-ARA group, and 14 ng/mg Cre in the high-ARA group which seems relatively higher compared with normal levels reported previously. The high level of tetranor-PGEM in the high-ARA group was due to the three participants with levels > 30 ng/mg Cre. The higher levels in these participants were not changed by ARA supplementation or during the washout period. Thus, tetranor-PGEM concentration was not significantly changed by ARA supplementation, indicating that ARA intake does not affect a candidate marker of colorectal cancer. Plasma LXA4 in the high-ARA groups was significantly increased after the washout period (Figure 4E). It was considered to be unrelated to ARA supplementation because the increase was also observed in the placebo group. Plasma LXA4 level may be more variable compared to the other metabolites, but the details are unclear. The increase was slight and considered not to affect the state of the participants.
Next, we analyzed correlations between plasma ARA content and ARA metabolites concentrations after 4 weeks of ARA administration (Figure 5). Concentrations of each ARA metabolite were distributed across a wide range, although all participants were healthy volunteers. None of the values correlated with plasma ARA content or ARA dose. Several individual values were high, but these values seemed normal for these patients, because the high levels in those participants were not changed at baseline and at 2 and 8 weeks. These results indicate that the increase in plasma ARA content from 8% to 14% does not increase the production of TXA2, PGI2, PGE2 and LXA4. The production of lipid mediators is not determined primarily by ARA content and seems to be controlled by other factors.
Diet was assessed to ensure that dietary intake of ARA and related fatty acids did not differ among the groups or as a result of the intervention. Participants consumed 170-200 mg/day of ARA from daily meals (Table 3), which is within the normal reported range [1–3] and values did not differ among the groups or with time. The intakes of DHA and EPA were 300-500 mg/day and 500-800 mg/day, respectively, and also did not differ among the groups. Although these values are within the common range in Japan, they are much more than those in Western countries (, ). Energy and macronutrient intake did not differ among the groups (Table 3), and did not seem to affect the present data.
The effects of ARA supplementation on healthy elderly were clarified here, but studies of patients with specific diseases are needed. In summary, blood parameters of cardiovascular, inflammatory and allergic diseases, as well as urinary and plasma ARA metabolites did not change in Japanese healthy elderly participants who consumed ARA-enriched oil (240 or 720 mg/day of ARA) for 4 weeks although plasma ARA levels significantly increased.