Here we report several novel findings that will be considered in turn. First, our results demonstrate that, in association with EPA + DHA supplementation, concentrations of the EPA and DHA species of LPC increased substantively while the 22:4n-6 LPC species decreased. These data are the first demonstration, to our knowledge, that LPC molecular species are directly influenced by diet. Second, although LPC is currently thought to be the major precursor for plasma LPA, concentrations of each LPC species did not consistently correlate with their respective LPA species at all time points. Third, EPA + DHA supplementation did not alter concentrations of any LPA species, including those containing EPA and DHA. Fourth, we found no consistent relationship between plasma autotaxin activity and LPC or LPA species using a highly sensitive fluorimetric assay. Although a few LPC species positively correlated with autotaxin at different time points, these relationships are not robust due to the issue of multiple statistical testing and should be interpreted with caution. These data challenge the widely held assumption that plasma LPA species derive from LPC as a precursor, and suggest that regulation of lysophospholipid metabolism in human subjects is more complex than suggested from in vitro studies and animal models.
The ingestion of the fish oil-derived long-chain polyunsaturated omega-3 fatty acids EPA and DHA is associated with substantial beneficial health effects[17
] as are other unsaturated fats.[23
] The benefits of EPA and DHA intake include the prevention of sudden cardiac death, a leading cause of death in Western populations.[32
] In contrast, 3 clinical trials have generated conflicting findings regarding the effects of high-dose EPA+DHA supplementation on ICD discharges in individuals with cardiomyopathy and an implantable cardioverter defibrillator.[33
] As no clear and convincing explanation for these divergent facts exists, a possible explanation lies in the metabolism of fatty acids. In fact, unsaturated LPA species (16:1, 18:1, and 18:2) have been implicated in the atherosclerotic process in an in vitro
model by inducing vascular smooth muscle cell dedifferentiation, migration, and proliferation whereas saturated species (12:0, 14:0, 16:0, and 18:0) do not.[15
] In an in vivo
model, the unsaturated 18:1 LPA has been shown to induce neointimal vascular remodeling but the saturated 18:0 species has not.[16
] Data from our study suggest that EPA + DHA supplementation does increase EPA- and DHA-LPC concentrations, but this does not translate directly into differences in LPA composition. The ability to increase DHA LPC with fish oil ingestion may be important given that this source of DHA appears to be its preferred carrier to the central nervous system[36
] and thus an additional relevant biomarker for adequate intake of EPA and DHA.[37
] Whether alterations in specific LPC species underlie any of the cardioprotective effects of omega-3 fatty acid supplementation will require further study. Our findings also suggest that the production of EPA and DHA LPA species is a highly regulated process, and that steady-state LPA levels are not influenced by the ingestion and concentration of these dietary fatty acids. Recent studies suggest that plasma LPA has a very short half-life in vivo[38
]. Since we did not conduct an extensive kinetic analysis of LPC or LPA species after ingestion of the dietary supplement, we can not absolutely exclude the possibility that a very transient spike in plasma EPA or DHA LPA species occurs soon after ingestion (e.g. within minutes). However, we would question the biological relevance of such transient LPA “remodeling”, in contrast to the alterations in LPC species, which appear to be sustained. Interestingly, the proportions of 22:6n3 (DHA) LPC and LPA plasma species is higher than the proportion of DHA in red blood cell membranes (). This suggests that DHA may concentrate in lysophospholipids, providing a rich source for a variety of tissues.
In serum or plasma, LPC is formed from phospholipids via phospholipases which include circulating phospholipase A2. Oxidized LDL (which is highly atherosclerotic) is one source of the phospholipids from which LPC is derived. Current thinking is that hydrolysis of LPC by autotaxin is the major source of extracellular LPA.[1
] However, we did not observe positive associations between plasma autotaxin activity and LPA concentrations at any time point. Since LPA can also be produced from phosphatidic acid via phospholipase D and other pathways, we favor the hypothesis that plasma LPA derives at least in part from an LPC- and autotaxin-independent pathway. Our results contrast with previous studies showing positive correlations between plasma LPA concentrations and serum autotaxin activity in healthy subjects.[29
] and in patients with hematologic malignancies.[39
] Reasons for these apparent discrepancies between studies are not immediately apparent, but could relate to differences in subject groups studied or methods of analysis. Autotaxin antagonists are under active development and entering clinical trials for patients with cancer and other diseases.[40
] Future studies with these compounds should help clarify the role of autotaxin in regulation of plasma LPA levels in human subjects.
We also studied the effects of acute ingestion of a 650 mg dose of aspirin on plasma lysophospholipids and autotaxin activity. We found that aspirin did not significantly affect concentrations of LPC, LPA, or autotaxin activity, whether taken before EPA + DHA supplementation (time point 2) or after four weeks of daily EPA + DHA supplementation (time point 4). We did not analyze ATX expression (e.g. by Western blot analysis of plasma samples), thus although it remains possible that aspirin may affect ATX expression per se we conclude that this did not translate into effects on ATX activity, arguably a more relevant biological readout. In a study that used the same EPA + DHA and aspirin intervention protocol (n=10), aspirin and EPA + DHA ingestion led to reduced platelet function (in response to ADP and collagen agonists) that was more potent than for each agent alone.[23
] The absence of effect of aspirin and EPA + DHA on LPA concentrations in the current study suggests that these agents do not, alone or in combination, influence LPA production. As both aspirin and LPA are known to affect platelet function,[23
] and platelet activation leads to LPA production, it is likely that the lack of effect of these agents on LPA was independent of their effects on platelet function. The ingestion of 100 mg of aspirin for 1 month has been associated with a reduction in plasma LPA in individuals with cerebrovascular disease,[19
] but we did not observe acute effects of a higher dose of aspirin in our study. It is important to note that aspirin doses of 75-150 mg/d have been associated with a lower risk of vascular events than higher or lower doses.[41
] In addition, a dose of 81mg/d of aspirin has been shown to increase the production of 15 epi-lipoxin A4, a metabolite of arachidonic acid with potent anti-inflammatory and tissue-protective effects, whereas doses of 325 and 650 mg do not.[44
] Thus we can not exclude the possibility that lower doses of aspirin or longer durations of therapy would affect LPA metabolism in vivo. Future studies will be needed to address this possibility.
Our study has some limitations. First, the sample size was small (n=15), but the statistical power was substantially increased for sequential variable measurements by the fact that each subject acted as their own control. However, we acknowledge the possibility that reduced statistical power may have led to the lack of association between LPC, LPA species, and autotaxin activity, but note that we did not observe associations even when pooling data across all time points (n=60 data points). Second, although red blood cell content of fatty acids correlates well with dietary intake[45
] and cardiac tissue content,[46
] this lipid pool may not be as relevant to lysophospholipid metabolism as is the content of plasma phosphatidylcholines (PC), a known source for LPC.[8
] Future studies that measure plasma PC fatty acids and lysophospholipid concentration and autotaxin activity in relevant end-organs (e.g. heart muscle or endovascular tissues) should be revealing. Since the participants were healthy adults, further research will be required to determine the effects of EPA +DHA and aspirin on lysophospholipid metabolism in diseased individuals. Current evidence supports the fact that the effects of low-dose aspirin (81-100 mg/d or less) on lipid metabolism and inflammation differ from that of higher doses.[47
] In fact, we are currently analyzing changes in the LPA and LPC species described in this manuscript after a single dose of 81 mg aspirin in a separate study. We cannot completely exclude the possibility that concentrations of lysophospholipids vary significantly depending on the duration between EPA+DHA ingestion and phlebotomy. As dephosphorylation of LPA by lipid phosphate phosphohydrolase type 1 has been shown to be one potential mechanism for the rapid metabolism of LPA,[38
] it is possible that a transient change in LPA due to EPA+DHA or aspirin ingestion was missed in this study. However, the main point of this research is that mean concentrations of EPA- and DHA-LPC species increase with EPA+DHA supplementation while their corresponding LPA species do not, and these conclusions are unlikely to be negated by a more consistent time-frame between dosing and phlebotomy. As the physiologic relevance of short term changes are not defined, the importance of this variation is unknown. The kinetic profile, over hours or days, of LPA concentrations in human blood after EPA+DHA ingestion would be an important focus of future research. Another limitation is the possibility that hydrolysis of the flourogenic LPC substrate used in the autotaxin activity assay in this study may be accomplished by molecules other than autotaxin and that our assay lacks specificity. However, in this study no effects on autotaxin were seen and thus this issue should not have negated the hypotheses tested.
In conclusion, our study is the first to describe the effects of EPA + DHA supplementation and acute aspirin ingestion, alone and combined, on plasma concentrations of lysophospholipid species in individuals without chronic disease. By comprehensively measuring RBC total fatty acids and plasma LPC and LPA species and autotaxin activity at each study point, we documented a complex relationship between circulating lysophospholipid species and fatty acids that would not have been readily predictable from paradigms established using in vitro or animal models. Our findings indicate that plasma LPA species in healthy subjects are not easily influenced by dietary intervention with EPA/DHA, in contrast to LPC concentrations and species. These findings may help in the interpretation of future clinical trials using autotaxin and other pathway antagonists. It should be noted that the health effects of altering blood concentrations of LPA and LPC species are currently unknown. We also conclude that plasma content of EPA and DHA LPC species may be reliable biomarkers for adequate omega-3 fatty acid intake, but LPA species may not.