Evaluation of the inhibitory effect of docosahexaenoic acid and arachidonic acid on the initial stage of amyloid β1-42 polymerization by fluorescence correlation spectroscopy

Amyloid β(Aβ) _1-42 fibrillation is a crucial step in the development of pathological hallmarks, such as neuritic plaques and neurofibrillary tangles, of Alzheimer’s disease (AD). In this study, we evaluated the effects of free docosahexaenoic acid (DHA), an essential brain polyunsaturated fatty acid (PUFA), on the inhibition of Aβ _1-42 fibrillation by fluorescence correlation spectroscopy (FCS), a technique capable of detecting molecular movements and interactions in solution. We also examined whether free arachidonic acid (AA), eicosapentaenoic acid (EPA), and metabolites of DHA, including neuroprotectin D1 (NPD1, 10S, 17S-dihydroxy-DHA), resolvin D1 (RvD1, 7S, 8R, 17S-trihydroxy-DHA), and didocosahexaenoyl glycerol (diDHA), affect Aβ _1-42 polymerization. The results of the FCS study reveal that DHA and AA significantly reduced the diffusion time of TAMRA (5-carboxytetramethylrhoda-mine)-Aβ _1-42 by 28% and 31%, respectively, while EPA, NPD1, RvD1, and diDHA had no effects on diffusion time. These results indicate that DHA and AA inhibited Aβ _1-42 polymerization and that their inhibitory effects occurred at the initial stage of Aβ _1-42 polymerization. This study will advance the research on PUFAs in preventing AD progression.

• Docosahexaenoic Acid; Arachidonic Acid; Fluorescence Correlation Spectroscopy; Amyloid β Peptide; Fibrillation

1. INTRODUCTION

Alzheimer’s disease (AD) is a progressive neurodegenerative disease characterized by the deposition of amyloid β (Aβ) peptides in neuritic plaques and neurofibrillar tangles in the affected brain regions [1]. Aβ_1-42, which is proteolytically released from membrane-bound amyloid precursor proteins [2], constitutes the foremost component of neuritic plaques and tangles of the affected brains [3] and plays an important role in neurobehavioral impairments in AD [4]. Formation of fibers is thus central to AD pathogenesis, and a great deal of work using various techniques, including transmission electron microscopy [5], atomic force microscopy [6-8], circular dichcoism [9], polyacrylamide gel electrophoresis (PAGE) [10-12], size-exclusion chromatography [13,14], and quantitative fluorimetry [5,15], has been performed to delineate the mechanism. Consistent with the findings of other studies [16-19], we have previously reported that Aβ_1-42 fibrillation involves conformational changes from α helix to β sheet and passes through various phases of fibrillation, including the formation of dimers, trimers, tetramers, oligomers, and finally matured fibrils, using thioflavin T fluorospectroscopy, PAGE, western blot, fluorescence microscopy, and transmission electron microscopy [20-23]. The natural plant compounds includeing curcumin, epigallocatechin-3-gallate and/or Ginkgo biloba extract and also fish oil components such as docosahexaenoic acid (DHA) were reported to inhibit amyloid formation [24-26]. Among these compounds, DHA is the most abundant n-3 polyunsaturated fatty acid (PUFA) in the mammalian brain [27-29], and deficiency of DHA is associated with memory impairment in AD model rats [30] and AD patients [31]. Oral administration of DHA decreases the amyloid burden in the brains of AD model rats [30] and mice [32], with a concomitant in vitro inhibition of the amyloid fibril formation, by acting at various stages of polymerization [20-23]. As one of the mechanism(s) of DHA action, we have previously shown that DHA inhibits in vitro Aβ_1-42 fibrillation at the trimer/tetramer level, and thereby inhibits further progression of lateral stacking of these intermediates and finally prevents mature amyloid fibril formation [20,21]. Thus, DHA is suggested to be a potent therapeutic and preventive agent against Aβ-induced AD. However, the exact mechanisms of action of DHA remain unclear. Thus, in the present investigation, we have used fluorescence correlation spectroscopy (FCS) to delineate the temporal resolution of DHA-induced mechanisms of inhibition of amyloid fibrillation.

FCS is a correlation analysis of fluctuations in the fluorescence intensity of fluorescent compounds excited by a sharply focused laser beam in a very tiny space, i.e., the so-called confocal volume. The fluorescence intensity fluctuates because of Brownian motion of the fluorescent particles. In other words, the number of particles in the confocal volume is randomly changing around the average number. This analysis gives the average number of fluorescent particles and average diffusion time when particles are passing through the tiny confocal volume. In practice, the fluorescence of dye-labeled amyloid Aβ_1-42 changes because of diffusion in the confocal volume, thus the diffusion time in the presence or absence of DHA might provide greater insight into the effects of DHA on the molecular interactions of amyloid species undergoing fibrillogenesis. In addition, the effects of other PUFAs such as eicosapentaenoic acid (EPA), a precursor for DHA, and arachidonic acid (AA), the abundant n-6 PUFA in the brain, on amyloid polymerization are also unknown and thus might be studied using this technique. The DHA/AA ratio has been shown to have a significantly negative correlation with long-term memory in Aβ peptide-infused AD model rats [30] and normal rats [33]. Recently, inflammation was also shown to contribute to the amyloid pathogenesis of AD, and metabolites of DHA including neuroprotectin D1 (NPD1) and resolvin D1 (RvD1) were reported to promote anti-inflammation and provide beneficial effects [34]. didocosahexaenoyl phosphopilid species [35-37] are abundant in the brain, and thus, whether the bulky diDHA inhibits Aβ_1-42 polymerization was also tested in the present experiment. Finally, the appearance of Aβ aggregates in solution [38] and the cerebrospinal fluid of AD patients [39] on FCS has been reported. Therefore, the present investigation could be considered of significant interest because it involves use of FCS, an ultrasensitive and non-invasive detection method capable of single-molecule and real-time resolution, for determining whether DHA, AA, EPA, DHA metabolites NPD1 and RvD1, and diDHA inhibit Aβ polymerization in a single experimental setting.

2. MATERIALS AND METHODS

2.1. Materials

The chemical structures of the compounds used in this experiments are indicated in Figure 1. Aβ_1-42 was purchased from the Peptide Institute Inc. (Osaka, Japan). DHA (4Z, 7Z, 10Z, 13Z. 16Z, 19Z-Docosahexaenoic acid), EPA (Icosapentaenoic acid), AA (5, 8, 11, 14-icosatetraenoic acid), NPD1 [Neuroprotectin D1; 10, 17 (S)- dihydro(pero)xydocosahexa-4Z, 7Z, 11E, 13Z, 15E, 19Zenoic acid], and RvD1 [17 (S)-Resolvin D1; 7S, 8R, 17S-trihydroxy-4Z, 9E, 11E, 13Z, 15E, 19Z-docosahexaenoic acid] were purchased from Cayman Chemical Company (MI, USA). diDHA [didocosahexaenoyl glycerol; Didocosahexaenoin (4, 7, 10, 13, 16, 19, -all cis)] was purchased from Larodan Fine Chemicals AB (Malmö, Sweden). Fluorescently labeled Aβ_1-42 [TAM RA (5-carboxytetramethylrhodamine)-Aβ_1-42; TAMRA-la beled β-amyloid_1-42] was purchased from AnaSpec Inc. (CA, USA). All other chemicals were of analytical grade.

2.2. Aβ 1-42 Peptide Preparation for Analysis by FCS

Aβ_1-42 peptide was dissolved in 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP) at a concentration of 100 μM to produce uniform, non-aggregated Aβ and stored at −30˚C until use. On the day of use, the HFIP-dissolved amyloid was blown with N₂ gas at ice cold temperature and redissolved in the assembly buffer [phosphate buffered saline (pH 7.4) containing 0.05% Tween 20].

2.3. Preparation of DHA, EPA, AA, NPD1, RvD1 and diDHA

DHA, EPA, AA, NPD1, and RvD1 dissolved in ethanol were stored at −80˚C, and diDHA dissolved in chloroform was stored at −30˚C until use. On the day of use, DHA, EPA, AA, and diDHA were mixed with assembly buffer at a final concentration of 20 μM, and NPD1 and RvD1 were mixed at a final concentration of 50 nM. Only freshly prepared DHA, EPA, AA, NPD1, RvD1, and diDHA were used.

2.4. FCS Measurement

In the present experiment, the FCS measurements were performed on a Fluoro Point Light (Olympus, Tokyo, Japan) at room temperature using the on-board 543- nm helium/neon laser at a laser power of 100 μW for excitation. TAMRA-Aβ_1-42 dissolved in 1% NH₄OH was stored at −30˚C. On the day of use, it was re-dissolved in assembly buffer at 1 nM, with or without DHA, EPA, AA, NPD1, RvD1, and diDHA, and quickly mixed with non-labeled Aβ_1-42. Free rhodamine was used as a reference dye. The measurements were performed in a sample volume of 50 μL in a 384-well glass-bottomed microplate. The samples were sequentially and automatically loaded into the device, the optical system of which was also automatically adjusted for each measurement. Initially, the samples were subjected to FCS measurement at zero time. Afterward, the samples were incubated at 37˚C for 1 h, followed by a second reading using the Fluoro Point Light device. All experiments were performed under identical conditions, with a data acquisition time of 10 s per measurement, and measurements were repeated five times per sample. Only freshly prepared TAMRA-Aβ_1-42 was used.

2.5. Statistical Analysis

Results are expressed as means ± S.E. The data were analyzed by unpaired Student’s t-test and one-way ANOVA. ANOVA followed by Dunnett’s test was used for post hoc comparisons. The statistical program used was PASW Statistics 18.0 (IBM-SPSS, Inc., USA). Statistical significance was set at P < 0.05.

3. RESULTS

3.1. Effect of Various DHA Concentrations on Diffusion Time of Aβ 1-42

Figure 2 shows the results of FCS studies of the dosedependent effect of DHA on Aβ polymerization using rhodamine-labeled Aβ_1-42 (TAMRA-Aβ_1-42). One-way analysis of diffusion time of Aβ_1-42 showed that DHA inhibited Aβ_1-42 polymerization in a concentration-dependent manner. DHA (10 and 20 μM) significantly inhibited Aβ_1-42 polymerization (Figure 2), as indicated by the decreased diffusion times, compared with the control (Aβ_1-42 alone) without DHA.

References

1. Selkoe, D.J. (1991) The molecular pathology of Alzheimer’s disease. Neuron, 6, 487-498. doi:10.1016/0896-6273(91)90052-2

2. Selkoe, D.J. (1993) Physiological production of the β amyloid protein and the mechanism of Alzheimer’s disease. Trends in Neurosciences, 16, 403-409. doi:10.1016/0166-2236(93)90008-A

3. Iwatsubo, T., Odaka, A., Suzuki, N., Mizusawa, H., Nukina, N. and Ihara, Y. (1994) Visualization of A β 42(43) and A β 40 in senile plaques with end-specific A β monoclonals: Evidence that an initially deposited species is A β 42(43). Neuron, 13, 45-53. doi:10.1016/0896-6273(94)90458-8

4. Berman, D.E., Dall’Armi, C., Voronov, S.V., McIntire, L.B., Zhang, H., Moore, A.Z., Staniszewski, A., Arancio, O., Kim, T.W. and Di Paolo, G. (2008) Oligomeric amyloid-β peptide disrupts phosphatidyl-inositol-4, 5-bisphophate metabolism. Nature Neuroscience, 11, 547-554. doi:10.1038/nn.2100

5. Naiki H. and Nakakuki, K. ,et al. (1996)First-order kinetic model of Alzheimer’s β-amyloid fibril extension in vitro Laboratory Investigation 74, 374-83.

6. Stine, W.B. Jr., Snyder, S.W., Ladror, U.S., Wade, W.S., Miller. M.F., Perun, T.J., Holzman, T.F. and Krafft, G.A. (1996) The nanometer-scale structure of amyloid-β visualized by atomic force microscopy. Journal of Protein Chemistry, 15, 193-203. doi:10.1007/BF01887400

7. Harper, J.D., Wong, S.S., Lieber, C.M. and Lansbury, P.T. (1997) Observation of metastable Aβ amyloid protofibrils by atomic force microscopy. Chemistry & Biology, 4, 119-125. doi:10.1016/S1074-5521(97)90255-6

8. Harper, J.D., Lieber, C.M. and Lansbury Jr., P.T. (1997) Atomic force microscopic imaging of seeded fibril formation and fibril branching by the Alzheimer’s disease amyloid-β protein. Chemistry & Biology, 4, 951-959. doi:10.1016/S1074-5521(97)90303-3

9. Terzi, E., Holzemann, G. and Seelig, J. (1995) Self-association of β-amyloid peptide (1-40) in solution and binding to lipid membranes. Journal of Molecular Biology, 252, 633-642. doi:10.1006/jmbi.1995.0525

10. Hilbich, C., Kisters-Woike, B., Reed, J., Masters, C.L., and Beyreuther, K. (1991) Aggregation and secondary structure of synthetic amyloid β A4 peptides of Alzheimer’s disease. Journal of Molecular Biology, 218, 149-163. doi:10.1016/0022-2836(91)90881-6

11. Burdick D., Soreghan B., Kwon M., Kosmoski J., Knauer M., Henschen A., Yates J., Cotman C. and Glabe, C. ,et al. (1992)Assembly and aggregation properties of synthetic Alzheimer’s A4/β amyloid peptide analogs Journal of Biological Chemistry 267, 546-554.

12. Sweeney, P.J., Darker, J.G., Neville, W.A., Humphries, J. and Camilleri, P. (1993) Electrophoretic techniques for the analysis of synthetic amyloid β-A4-related peptides. Analytical Biochemistry, 212, 179-184. doi:10.1006/abio.1993.1310

13. Garzon-Rodriguez, W., Sepulveda-Becerra, M., Milton, S. and Glabe, C.G. (1997) Soluble amyloid Aβ-(1-40) exists as a stable dimer at low concentrations. Journal of Biological Chemistry, 272, 21037-21044. doi:10.1074/jbc.272.34.21037

14. Shen, C.L., Fitzgerald, M.C. and Murphy, R.M. (1994) Effect of acid predissolution on fibril size and fibril flexibility of synthetic β-amyloid peptide. Biophysical Journal, 67, 1238-1246. doi:10.1016/S0006-3495(94)80593-4

15. LeVine, H. III. (1993) Thioflavine T interaction with synthetic Alzheimer’s disease β-amyloid peptides: Detection of amyloid aggregation in solution. Protein Science, 2, 404-410. doi:10.1002/pro.5560020312

16. Serpell, L.C. (2000) Alzheimer’s amyloid fibrils: Structure and assembly. Biochimica et Biophysica Acta, 1502, 16-30. doi:10.1016/S0925-4439(00)00029-6

17. Barrow, C.J. and Zagorski, M.G. (1991) Solution structures of β peptide and its constituent fragments: Relation to amyloid deposition. Science, 253, 179-182. doi:10.1126/science.1853202

18. Shao, H., Jao, S., Ma, K. and Zagorski, M.G. (1999) Solution structures of micelle-bound amyloid β-(1-40) and β-(1-42) peptides of Alzheimer’s disease. Journal of Molecular Biology, 285, 755-773. doi:10.1006/jmbi.1998.2348

19. Kirkitadze, M.D., Condron, M.M. and Teplow, D.B. (2001) Identification and characterization of key kinetics intermediates in amyloid β-protein fibrillogenesis. Journal of Molecular Biology, 312, 1103-1119. doi:10.1006/jmbi.2001.4970

20. Hashimoto, M., Shahdat, H.M., Yamashita, S., Katakura, M., Tanabe, Y., Fujiwara, H., Gamoh, S., Miyazawa, T., Arai, H., Shimada, T. and Shido, O. (2008) Docosahexaenoic acid disrupts in vitro amyloid β1-40 fibrillation and concomitantly inhibits amyloid levels in cerebral cortex of Alzheimer’s disease model rats. Journal of Neurochemistry, 107, 1634-1646. doi:10.1111/j.1471-4159.2008.05731.x

21. Hossain, S., Hashimoto, M., Katakura, M., Miwa, K., Shimada, T. and Shido, O. (2009) Mechanism of docosahexaenoic acid-induced inhibition of in vitro Aβ1-42 fibrillation and Aβ1-42 induced toxicity in SH-SY5Y cells. Journal of Neurochemistry, 111, 568-579. doi:10.1111/j.1471-4159.2009.06336.x

22. Hashimoto, M., Shahdat, H.M., Katakura, M., Tanabe, Y., Gamoh, S., Miwa, K., Shimada, T. and Shido, O. (2009) Effects of docosahexaenoic acid on in vitro amyloid beta peptide 25-35 fibrillation. Biochimica et Biophysica Acta, 1791, 289-296. doi:10.1016/j.bbalip.2009.01.012

23. Hashimoto, M., Katakura, M., Hossain, S., Rahman, A., Shimada, T. and Shido, O. (2011) Docosahexaenoic acid withstands the Aβ25-35 induced neurotoxicity in SH-SY5Y cells. Journal of Nutritional Biochemistry, 22, 22-29. doi:10.1016/j.jnutbio.2009.11.005

24. Ono, K., Hasegawa, K., Naiki, H. and Yamada, M. (2004) Curcumin has potent anti-amyloidogenic effects for Alzheimer’s β-amyloid fibrils in vitro. Journal of Neurosci- ence Research, 75, 742-750. doi:10.1002/jnr.20025

25. Rezai-Zadeh, K., Arendash, G.W., Hou, H., Fernandez, F., Jansen, M., Runfeldt, M., Shytle, R.D. and Tan, J. (2008) Green tea epigallocatechin-3-gallate (EGCG) reduces β-amyloid mediated cognitive impairment and modulates tau pathology in Alzheimer’s transgenic mice. Brain Research, 1214, 177-187. doi:10.1016/j.brainres.2008.02.107

26. Luo, Y., Smith, J.V., Paramasivam, V., Burdick, A., Curry, K.J., Buford, J.P., Khan, I., Netzer, W.J., Xu, H. and Butko, P. (2002) Inhibition of amyloid-β aggregation and caspase-3 activation by the Ginkgo biloba extract EGb761. Proceedings of the National Academy of Sciences of the United States of America, 99, 12197-12202. doi:10.1073/pnas.182425199

27. Lauritzen, L., Hansen, H.S., J?rgensen, M.H. and Michaelsen, K.F. (2001) The essentiality of long chain n-3 fatty acids in relation to development and function of the brain and retina. Progress in Lipid Research, 40, 1-94. doi:10.1016/S0163-7827(00)00017-5

28. Innis S.M. ,et al. (2007)Dietary (n-3) fatty acids and brain development Journal of Nutrition 137, 855-859.

29. Tozuka, Y., Wada, E. and Wada, K. (2009) Bio-communication between mother and offspring: Lessons from animals and new perspectives for brain science. Journal of Pharmacological Sciences, 110, 127-132. doi:10.1254/jphs.09R01CP

30. Hashimoto, M., Hossain, S., Shimada, T., Sugioka, K., Yamasaki, H., Fujii, Y., Ishibashi, Y., Oka, J. and Shido, O. (2002) Docosahexaenoic acid provides protection from impairment of learning ability in Alzheimer’s disease model rats. Journal of Neurochemistry, 81, 1084-1091. doi:10.1046/j.1471-4159.2002.00905.x

31. Soderberg, M., Edlund, C., Kristensson, K. and Dallner, G. (1991) Fatty acid composition of brain phospholipids in aging and in Alzheimer’s Disease. Lipids, 26, 421-425. doi:10.1007/BF02536067

32. Grimm, M.O., Kuchenbecker, J., Gr?sgen, S., Burg, V.K., Hundsd?rfer, B., Rothhaar, T.L., Friess, P., deWilde, M.C., Broersen, L.M., Penke, B., Péter, M., Vígh, L., Grimm, H.S. and Hartmann, T. (2011) Docosahexaenoic acid reduces amyloid β production via multiple pleiotropic mechanisms. Journal of Biological Chemistry, 286, 14028-14039. doi:10.1074/jbc.M110.182329

33. Gamoh, S., Hashimoto, M., Sugioka, K., Hossain, S., Hata N., Misawa, Y. and Masumura, S. (1999) Chronic administration of docosahexaenoic acid improves reference memory-related learning ability in young rats. Neuroscience, 93, 237-241. doi:10.1016/S0306-4522(99)00107-4

34. Ariel, A. and Serhan, C.N. (2007) Resolvins and protectins in the terminatrion program of acute inflammation. Trends in Immunology, 28, 176-183. doi:10.1016/j.it.2007.02.007

35. Miljanich, G.P., Sklar, L.A., White, D.L. and Dratz, E.A. (1979) Disaturated and dipolyun-saturated phospholipids in the bovine retinal rod outer segment disk membrane. Biochimica et Biophysica Acta, 552, 294-306. doi:10.1016/0005-2736(79)90284-0

36. Bell, M.V., Dick, J.R. and Buda, C. (1997) Molecular speciation of fish sperm phos-pholipids: Large amounts of dipolyunsatusated phosphatidyl-serine. Lipids, 32, 1085-1091. doi:10.1007/s11745-997-0140-y

37. Bell M.V. and Tocher, D.R. ,et al. (1989)Molecular species composition of the major phos-pholipids in brain and retina from rainbow trout (Salmo gairdneri) Biochemical Journal 264, 909-915.

38. Tjernberg, L.O., Paramanik, A., Bj?rling, S., Thyberg, P., Thyberg, J., Nordstedt, C., Berndt, K.D., Terenius, L. and Rigler, R. (1999) Amyloid β-peptide polymerization stud- ied using fluorescence correlation spectroscopy. Chemistry & Biology, 6, 53-62. doi:10.1016/S1074-5521(99)80020-9

39. Pitschke, M., Prior, R., Haupt, M. and Riesner, D. (1998) Detection of single amy-loid β-protein aggregates in the cerebrospinal fluid of Alzheimer’s patients by fluorescence correlation spectroscopy. Nature Medicine, 4, 832-834. doi:10.1038/nm0798-832

40. Hashimoto M., Tanabe Y., Fujii Y., Kikuta T., Shibata H. and Shido, O. ,et al. (2005)Chronic administration of docosahexaenoic acid ameliorates the impairment of spatial cognition learning ability in amyloid β-infused rats Journal of Nutrition 135, 549-555.

41. Kotani, S., Sakaguchi, E., Warashina, S., Matsukawa, N., Ishikura, Y., Kiso, Y., Sakakibara, M., Yoshimoto, T., Guo, J. and Yamashima, T. (2006) Dietary supplementation of arachidonic and docosahexaenoic acids improves cognitive dysfunction. Neuroscience Research, 56, 159-164. doi:10.1016/j.neures.2006.06.010

42. Song, C. and Horrobin, D. (2004) Omega-3 fatty acid ethyl-eicosapentaenoate, but not soybean oil, attenuates memory impairment induced by central IL-1β administration. Journal of Lipid Research, 45, 1112-1121. doi:10.1194/jlr.M300526-JLR200

43. Hashimoto, M., Hoss-ain, S., Tanabe, Y., Kawashima, A., Harada, T., Yano, T., Mizuguchi, K. and Shido, O. (2009) The protective effect of dietary eicosapentaenoic acid against impairment of spatial cognition learning ability in rats infused with amyloid β1-40. Journal of Nutritional Biochemistry, 20, 965-973. doi:10.1016/j.jnutbio.2008.08.009

44. Ishiguro, J., Tada, T., Ogihara, T., Ohzawa, N., Murakami, K. and Kosuzume, H. (1988) Metabolic disposition of ethyl eicosapentaenoate and its metabolites in rats and dogs. Journal of Pharmacobio-Dynamics, 11, 251-261. doi:10.1248/bpb1978.11.251

45. Martins J.G., Bentsen H. and Puri, B.K. ,et al. (2012)EPA in major depressive disorder: Eicosapentaenoic acid appears to be the key omega 3 fatty acid component associated with efficacy in major depressive disorder: A critique of Bloch and Hannestad and updated metaanalysis Molecular Psychiatry 17, 1-6.

46. Stillwell, W. and Wassall, S.R. (2003) Docosahexaenoic acid: Membrane properties of a unique fatty acid. Chemistry and Physics of Lipids, 126, 1-27. doi:10.1016/S0009-3084(03)00101-4

47. Yonezawa Y., Hada T., Uryu K., Iijima H., Yoshida H. and Mizushina, Y. ,et al. (2006)Inhibitory action of C22-fatty acids on DNA polymerases and DNA topoisomerases International Journal of molecular Medicine 18, 583-588.

48. Mizushina, Y., Dairaku, I., Yanaka, N., Takeuchi, T., Ishimaru, C., Sugawara, F., Yoshida, H. and Kato, N. (2007) Inhibitory action of polyunasaturated fatty acids on IMP dehydrogenase. Biochimie, 89, 581-590. doi:10.1016/j.biochi.2007.01.009

49. Lukiw, W.J. and Bazan, N.G. (2008) Docosahexaenoic acid and the aging brain. Journal of Nutritioin, 138, 2510-2514. doi:10.3945/jn.108.096016

50. Serhan C.N., Gotlinger K., Hong S., Lu Y., Siegelman J., Baer T., Yang R., Colgan S.P. and Petasis, N.A. ,et al. (2006)Anti-inflammatory actions of neuroprotectin D1/ Protectin D1 and its natural stereoisomers: Assignment of dihydroxy-containing docosatrienes Journal of Immunology 176, 1848-1859.

51. Lukiw, W.J., Cui, J.G., Marcheselli, V.L., Bodker, M., Botkjaer, A., Gotlinger, K., Serhan, C.N. and Bazan, N.G. (2005) A role for docosahexaenoic acid-derived neuroprotectin D1 in neural cell survival and Alzhemier disease. Journal of Clinical Investigation, 115, 2774-2783. doi:10.1172/JCI25420

52. Serhan, C.N. and Chiang, N. (2008) Endgenous pro-resolving and anti-inflammatory lipid mediators: A new pharmacologic genus. British Journal of Pharmacology, 153, S200-S215. doi:10.1038/sj.bjp.0707489

53. Wiegand, R.D. and Anderson, R.E. (1983) Phospholipid molecular species of frog outer segment membranes. Experimental Eye Research, 37, 159-173. doi:10.1016/0014-4835(83)90075-1

54. Neil, A.R. and Masters, C.J. (1973) Metabolism of fatty acids by ovine spermatozoa. Journal of the Society for Reproduction and Fertility, 34, 279-287. doi:10.1530/jrf.0.0340279

55. Breckernridge, W.C., Gombos, G. and Morgan, I.G. (1972) The lipid composition of adult rat brain synaptosomal plasma membranes. Biochimica et Biophysica Acta, 266, 695-707. doi:10.1016/0005-2736(72)90365-3