Eicosapentaenoic acid, unlike arachidonic acid, maintains normal membrane structure and cholesterol distribution under conditions of hyperglycemia as determined by X-ray diffraction

Samuel C.R. Sherratt, B.S.2, R. Preston Mason, Ph.D.1,2

1Brigham and Women’s Hospital, Harvard Medical School, Boston, MA; 2Elucida Research LLC, Beverly, MA

AbstractIntroductionMethods 1

Results 1Results 2

Background/Synopsis: Highly purified, prescription eicosapentaenoic acid (EPA) reduced cardiovascular events in the REDUCE-ITstudy population, including in patients with diabetes. During hyperglycemia, there is abnormal membrane cholesterol aggregationwhich has been linked to oxidative stress and plaque instability. Due to its chemical structure, EPA at a pharmacologic dose maypreserve normal cholesterol distribution as compared to arachidonic acid (AA), an omega-6 fatty acid (FA).

Purpose: To compare the effects of EPA and AA on membrane structure and cholesterol crystalline domain formation underconditions of hyperglycemia and oxidative stress.

Methods: Membrane vesicles were prepared from dilinoleoylphosphatidylcholine (DLPC) at a cholesterol-to-phospholipid mole ratioof 0.6:1 and treated with pharmacologic levels of EPA and AA at a 1:30 FA to phospholipid mole ratio under conditions ofhyperglycemia (200 mg/dL). Changes in membrane lipid organization and width were measured using small angle X-ray diffractionapproaches and correlated with lipid hydroperoxide formation. Cholesterol domains were identified in experimental samples by thepresence of diffraction peaks corresponding to a unit cell periodicity or width of 34 Å.

Results: Membranes containing EPA had a membrane structure characterized by normal width of 55 Å and cholesterol distributionunder conditions of hyperglycemia. By contrast, AA containing samples yielded diffraction patterns consistent with a biphasicmembrane structure containing prominent cholesterol crystalline domains and phospholipid-enriched membrane bilayer domains withunit cell periodicities of 34 Å and 54 Å, respectively. The width of 34 Å corresponds to free cholesterol in an immiscible bilayerstructure within the membrane. Unlike AA, EPA also inhibited lipid peroxide formation compared to vehicle.

Conclusions: EPA preserved normal membrane structure and cholesterol distribution while reducing lipid oxidation under conditionsof hyperglycemia in a manner that was not reproduced with AA. These data indicate physico-chemical differences between these longchain FAs and support a potential benefit for EPA in reducing atherosclerotic cholesterol domain formation and associated pathologyat pharmacologic levels.

Conclusion

Methods 2

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AbstractIntroductionMethods 1

Results 1Results 2Conclusion

Samuel C.R. Sherratt, B.S.2, R. Preston Mason, Ph.D.1,2

1Brigham and Women’s Hospital, Harvard Medical School, Boston, MA; 2Elucida Research LLC, Beverly, MA

• Eicosapentaenoic acid (EPA) is a long-chain omega-3 fatty acid (20:5; n-3) that has been shown to have various biological effects, includingimproved endothelial function, reduced inflammation, decreased platelet aggregation, and lipid-lowering actions.1 An imbalance in the ratio ofEPA to the omega-6 fatty acid arachidonic acid (20:4; n-6) is associated with increased cardiovascular risk.2,3 Prescription EPA has beenshown to significantly increase the EPA/AA ratio.4

• Prescription EPA has been shown to reduce cardiovascular events inpatients with diabetes. The Reduction of Cardiovascular Events withEPA-Intervention Trial (REDUCE-IT) was a randomized, double-blinded, placebo-controlled trial designed to examine the benefits oficosapent ethyl, a prescription-grade, highly-purified ethyl ester ofEPA.5 The objective of this study was to determine if robust EPAtreatment reduces ischemic events in statin-treated patients withelevated and high baseline fasting TG levels (150-499 mg/dL) andelevated risk for clinical cardiovascular events such as diabetes. Theprimary composite endpoint of cardiovascular death, nonfatalmyocardial infarction, nonfatal stroke, coronary revascularization, orunstable angina reported a highly statistically significant (p<0.001) 25%relative risk reduction (RRR) and 4.8% absolute risk reduction (ARR)with a number needed to treat (NNT) of 21 over 4.9 years.

• Atherosclerotic membranes are characterized by elevated cholesteroland lipid oxidation levels which lead to changes in fluidity, width, andcholesterol crystalline domain formation.6,7,8 Cholesterol crystalscontribute to cell death and are a primary activator of nucleotide-binding domain, leucine-rich-containing family, pyrin domain-containing-3 (NLRP3) inflammasomes, which regulate caspase-1 andits associated processing of pro-interleukin 1 beta (IL-1β) into an activecytokine that initiates inflammation in atherosclerosis (Figure 1).9,10

• Previous work in our laboratory has shown EPA to have potent lipid antioxidant effects compared to other TG-lowering agents and a long-chain fatty acid.11 This finding of a distinct antioxidant mechanism for EPA could support a novel benefit for EPA in reducing cardiovascularrisk. In particular, the lipophilic structure and molecular space dimensions of EPA may allow it to insert into and function effectively within lipidbilayers. The impact of EPA versus AA on membrane structure and cholesterol domains formation is not fully understood and serves as thebasis for this study.

Figure 1. Schematic illustration showing the role of cholesterol crystals in triggering inflammationassociated with atherosclerosis.8

Methods 2

Eicosapentaenoic acid, unlike arachidonic acid, maintains normal membrane structure and cholesterol distribution under conditions of hyperglycemia as determined by X-ray diffraction

AbstractIntroductionMethods 1

Results 1Results 2Conclusion

Samuel C.R. Sherratt, B.S.2, R. Preston Mason, Ph.D.1,2

1Brigham and Women’s Hospital, Harvard Medical School, Boston, MA; 2Elucida Research LLC, Beverly, MA

Materials:• 1,2-Dilinoleoyl-sn-glycero-3-phosphocholine (DLPC) and cholesterol were purchased from Avanti

Polar Lipids, Inc. (Alabaster, AL) and dissolved in HPLC-grade chloroform.• cis-5,8,11,14,17-Eicosapentaenoic acid (EPA) and cis-5,8,11,14-Eicosatetraenoic Acid

(Arachidonic Acid; AA) were purchased from Sigma-Aldrich (Saint Louis, MO) and solubilized inethanol to 1.0 mM under nitrogen atmosphere.

Figure 2. Method for orienting membranes and MLVs forsmall angle x-ray scattering analysis. The membranesuspension is loaded into a Lucite® sedimentation cell andcentrifuged at 35,000 g. The sedimentation cell is thendismantled and the membrane sample (on the aluminum foilsubstrate) is removed and mounted onto a curved support. Aschematic representation of membrane stacking is depicted inthe inset. Membrane bilayers are separated by water spaceshaving a width that is modulated by relative humidityconditions.

Preparation of Membrane Vesicles for X-Ray Diffraction:• Multilamellar vesicles (MLVs) were prepared as binary mixtures of DLPC and cholesterol, at a

cholesterol-to-phospholipid (C/P) mole ratio of 0.6:1, as previously described.8 Component lipids (inchloroform) were transferred to 13 × 100 mm borosilicate culture tubes and combined with vehicle(ethanol) or an equal volume of EPA or AA stock solution, adjusted to achieve a final fatty acid-to-phospholipid mole ratio of 1:30. Samples were then shell-dried under nitrogen gas and placedunder vacuum for a minimum of 1 hr to remove residual solvent.

• After drying, each sample was resuspended in saline buffer (0.5 mM HEPES, 154 mM NaCl, pH7.32) treated with 200 mg/dL glucose, thus simulating hyperglycemic conditions, to yield a finalphospholipid concentration of 2.5 mg/mL. Lipid suspensions were then vortexed for 3 min atambient temperature to form MLVs.12

• Following MLV formation, samples were placed in a shaking water bath set at 37°C for 96 hours.Membrane samples were collected at the start of the experiment (0 hour) and the conclusion of theexperiment (96 hour) and prepared for X-ray diffraction analysis.

• Samples were oriented for x-ray diffraction analysis as previously described.8 Briefly, aliquots ofeach MLV preparation were transferred to sedimentation cells fitted with a removable aluminum foilsubstrate designed to collect MLVs into a single membrane pellet upon centrifugation. Sampleswere then centrifuged at 35,000 g, 5°C, for 1.5 hr (Figure 2).

Methods 2

Eicosapentaenoic acid, unlike arachidonic acid, maintains normal membrane structure and cholesterol distribution under conditions of hyperglycemia as determined by X-ray diffraction

X-ray Diffraction Analysis:• Following membrane orientation, sample supernatants were aspirated and aluminum foil substrates mounted onto curved

glass slides. The membrane samples were then placed in hermetically sealed containers in which temperature and relativehumidity were controlled during x-ray diffraction analysis. Samples were evaluated at 20°C at constant relative humidity(74%).

• Each oriented membrane sample was subjected to small angle x-ray diffraction analysis as shown in Figure 3.

• A schematic illustration of a diffraction pattern and its relationship to the phospholipid bilayer and cholesterol crystallinedomain (peaks 1′ and 2′) as shown in Figure 4.13

AbstractIntroductionMethods 1

Results 1Results 2Conclusion

Samuel C.R. Sherratt, B.S.2, R. Preston Mason, Ph.D.1,2

1Brigham and Women’s Hospital, Harvard Medical School, Boston, MA; 2Elucida Research LLC, Beverly, MA

Methods 2

Figure 3. Membrane samples were aligned at grazing incidence with respect to a collimated,monochromatic x-ray beam produced by a high-brilliance microfocus generator. Diffraction data werecollected on a one-dimensional, position-sensitive electronic detector spaced 150 mm from the sample site.

Figure 4. Schematic illustration of a typical diffraction pattern collected from a biphasic membrane sample,exhibiting sterol-poor, phospholipid bilayer domains (peaks 1, 2, and 4) and cholesterol crystalline domains(peaks 1′ and 2′), and its relationship to the spatial arrangement of these domains in a representativemembrane bilayer..

Eicosapentaenoic acid, unlike arachidonic acid, maintains normal membrane structure and cholesterol distribution under conditions of hyperglycemia as determined by X-ray diffraction

AbstractIntroductionMethods 1

Results 1Results 2Conclusion

Samuel C.R. Sherratt, B.S.2, R. Preston Mason, Ph.D.1,2

1Brigham and Women’s Hospital, Harvard Medical School, Boston, MA; 2Elucida Research LLC, Beverly, MA

Figure 5. Distinct Effects of EPA versus AA on Membrane Cholesterol Domain Formation after Exposure to Hyperglycemic Conditionsthrough 96 Hours. Cholesterol domains highlighted in red.

Methods 2

Eicosapentaenoic acid, unlike arachidonic acid, maintains normal membrane structure and cholesterol distribution under conditions of hyperglycemia as determined by X-ray diffraction

AbstractIntroductionMethods 1

Results 1Results 2Conclusion

Samuel C.R. Sherratt, B.S.2, R. Preston Mason, Ph.D.1,2

1Brigham and Women’s Hospital, Harvard Medical School, Boston, MA; 2Elucida Research LLC, Beverly, MA

Time point (Hr) Treatment d-Space (Å)

Vehicle 52

0 EPA 52

AA 51

Vehicle 44

96 EPA 52

AA 41

Table 1. Comparative Effects of EPA and AA onMembrane Width (d-Space) under HyperglycemicConditions at 0 and 96 hr.

Figure 6. Distinct Effects of EPA versus AA on Membrane Cholesterol Domain Formation afterExposure to Hyperglycemic Conditions (0 and 96 Hours). Cholesterol domains highlighted in red.

Methods 2

Eicosapentaenoic acid, unlike arachidonic acid, maintains normal membrane structure and cholesterol distribution under conditions of hyperglycemia as determined by X-ray diffraction

AbstractIntroductionMethods 1

Results 1Results 2Conclusion

Samuel C.R. Sherratt, B.S.2, R. Preston Mason, Ph.D.1,2

1Brigham and Women’s Hospital, Harvard Medical School, Boston, MA; 2Elucida Research LLC, Beverly, MA

Summary: EPA prevented the formation of cholesteroldomains and maintained normal membrane width followingextended exposure to hyperglycemic conditions. Bycontrast, samples treated with either vehicle or AA showedprogressive development of cholesterol domains and asubstantial reduction in membrane width by 8 and 10 Å,respectively. The most prominent changes in domainformation and membrane structure were observed with AAtreated samples.

Figure 7. Schematic Illustration of the Proposed Antioxidant and Membrane Structural Effectsof EPA as Determined by Biochemical and Biophysical Approaches. Following its intercalationinto the membrane lipid bilayer, EPA interferes with the propagation of free radicals. The conjugateddouble bonds associated with EPA facilitate an electron stabilization mechanism that quenches thefree radical reaction and preserves membrane lipid structure and organization.

Conclusion: We observed pronounced differences in theeffects of EPA and AA on membrane structure andcholesterol domain formation under conditions ofhyperglycemia. EPA maintained normal membranestructure and cholesterol distribution as compared tovehicle. By contrast, AA treatment was associated with theformation of prominent cholesterol domains and loss ofmembrane integrity as evidenced by a large reduction in membrane width. These data indicate physico-chemical differences between theselong chain FAs and support a potential benefit for EPA in reducing atherosclerotic cholesterol domain formation and associated pathology atpharmacologic levels due to its ideal combination of hydrocarbon chain length and number of double bonds.Acknowledgements: This study was supported by Amarin Pharma Inc, Bedminster, NJ.

1. Mason, RP. Curr Atheroscler Rep. 2019; 21:22. Nishizaki Y et al. Am J Cardiol. 2014;113:441-445.3. Shigemasa T et al. Am J Cardiovasc Drug. 2017. DOI: 10.1007/s40256-017-0238-z.4. Braeckman RA et al. Prostaglandins Leukot Essent Fatty Acids. 2013;89:195-201.5. Bhatt et al. NEJM 2019;380:11-22.6. Mason RP and Jacob RF. Circulation. 2003;107:2270. 7. Self-Medlin Y et al. Biochimica et Biophysica Acta. 2009;1788:1398-1403.

8. Mason RP et al. J. Bio Chem. 2006;281:9337-9345.9. Ridker PM. Circ Res 2016;118:145-156.10. Karasawa T and Takahashi M. J Atheroscler Thromb. 2017;24:443.11. Mason RP et al. J Cardiovasc Pharmacol. 2016;68:33-40.12. Bangham AD et al. J Mol Biol. 1965; 13:238-252.13. Mason RP, et al. J Biol Chem. 2006;281:9337-9345.

References:

Methods 2

Eicosapentaenoic acid, unlike arachidonic acid, maintains normal membrane structure and cholesterol distribution under conditions of hyperglycemia as determined by X-ray diffraction