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Fluorescent n-3 and n-6 Very Long Chain Polyunsaturated Fatty Acids

2010; Elsevier BV; Volume: 285; Issue: 24 Linguagem: Inglês

10.1074/jbc.m109.079897

ISSN

1083-351X

Autores

Avery L. McIntosh, Huan Huang, Barbara P. Atshaves, Elizabeth A. Wellberg, Dmitry Kuklev, William L. Smith, Ann B. Kier, Friedhelm Schroeder,

Tópico(s)

Lipid metabolism and biosynthesis

Resumo

Despite the considerable beneficial effects of n-3 and n-6 very long chain polyunsaturated fatty acids (VLC-PUFAs), very little is known about the factors that regulate their uptake and intracellular distribution in living cells. This issue was addressed in cells expressing liver-type fatty acid-binding protein (L-FABP) by real time multiphoton laser scanning microscopy of novel fluorescent VLC-PUFAs containing a conjugated tetraene fluorophore near the carboxyl group and natural methylene-interrupted n-3 or n-6 grouping. The fluorescent VLC-PUFAs mimicked many properties of their native nonfluorescent counterparts, including uptake, distribution, and metabolism in living cells. The unesterified fluorescent VLC-PUFAs distributed either equally in nuclei versus cytoplasm (22-carbon n-3 VLC-PUFA) or preferentially to cytoplasm (20-carbon n-3 and n-6 VLC-PUFAs). L-FABP bound fluorescent VLC-PUFA with affinity and specificity similar to their nonfluorescent natural counterparts. Regarding n-3 and n-6 VLC-PUFA, L-FABP expression enhanced uptake into the cell and cytoplasm, selectively altered the pattern of fluorescent n-6 and n-3 VLC-PUFA distribution in cytoplasm versus nuclei, and preferentially distributed fluorescent VLC-PUFA into nucleoplasm versus nuclear envelope, especially for the 22-carbon n-3 VLC-PUFA, correlating with its high binding by L-FABP. Multiphoton laser scanning microscopy data showed for the first time VLC-PUFA in nuclei of living cells and suggested a model, whereby L-FABP facilitated VLC-PUFA targeting to nuclei by enhancing VLC-PUFA uptake and distribution into the cytoplasm and nucleoplasm. Despite the considerable beneficial effects of n-3 and n-6 very long chain polyunsaturated fatty acids (VLC-PUFAs), very little is known about the factors that regulate their uptake and intracellular distribution in living cells. This issue was addressed in cells expressing liver-type fatty acid-binding protein (L-FABP) by real time multiphoton laser scanning microscopy of novel fluorescent VLC-PUFAs containing a conjugated tetraene fluorophore near the carboxyl group and natural methylene-interrupted n-3 or n-6 grouping. The fluorescent VLC-PUFAs mimicked many properties of their native nonfluorescent counterparts, including uptake, distribution, and metabolism in living cells. The unesterified fluorescent VLC-PUFAs distributed either equally in nuclei versus cytoplasm (22-carbon n-3 VLC-PUFA) or preferentially to cytoplasm (20-carbon n-3 and n-6 VLC-PUFAs). L-FABP bound fluorescent VLC-PUFA with affinity and specificity similar to their nonfluorescent natural counterparts. Regarding n-3 and n-6 VLC-PUFA, L-FABP expression enhanced uptake into the cell and cytoplasm, selectively altered the pattern of fluorescent n-6 and n-3 VLC-PUFA distribution in cytoplasm versus nuclei, and preferentially distributed fluorescent VLC-PUFA into nucleoplasm versus nuclear envelope, especially for the 22-carbon n-3 VLC-PUFA, correlating with its high binding by L-FABP. Multiphoton laser scanning microscopy data showed for the first time VLC-PUFA in nuclei of living cells and suggested a model, whereby L-FABP facilitated VLC-PUFA targeting to nuclei by enhancing VLC-PUFA uptake and distribution into the cytoplasm and nucleoplasm. IntroductionHumans cannot synthesize n-3 and n-6 VLC-PUFAs 2The abbreviations used are: VLC-PUFAvery long chain polyunsaturated fatty acidMPLSMmultiphoton laser scanning microscopyA5c5E,7E,9E,11Z,14Z-eicosapentaenoic acidE6c5E,7E,9E,11Z,14Z,17Z-eicosahexanoic acidD7c4E,6E,8E,10Z,13Z,16Z,19Z-docosaheptaenoic acidL-FABPliver-type fatty-acid binding proteinH-FABPheart-type fatty-acid binding proteinCEcholesterol estersTGtriacylglyceridesFFAfree fatty acidPEphosphatidylethanolaminePCphosphatidylcholineHPLChigh pressure liquid chromatographyi-PrOHisopropyl alcohol. and must therefore obtain these essential fatty acids from the diet (1Norris A.W. Spector A.A. J. Lipid Res. 2002; 43: 646-653Abstract Full Text Full Text PDF PubMed Google Scholar, 2Anderson B.M. Ma D.W. Lipids Health Dis. 2009; 8: 33Crossref PubMed Scopus (256) Google Scholar, 3Simopoulos A.P. Biomed. Pharmacother. 2006; 60: 502-507Crossref PubMed Scopus (772) Google Scholar, 4Morris D.H. Morris D.H. FLAX: A Health and Nutrition Primer. Flax Council of Canada, Winnipeg, Manitoba, Canada2007: 22-33Google Scholar, 5Morris D.H. Morris D.H. FLAX: A Health and Nutrition Primer. Flax Council of Canada, Winnipeg, Manitoba, Canada2007: 34-43Google Scholar). The n-3 and n-6 VLC-PUFAs impact key physiological processes that regulate the levels of blood lipids, cardiovascular and immune function, insulin action, brain development, and neuronal as well as retinal function (2Anderson B.M. Ma D.W. Lipids Health Dis. 2009; 8: 33Crossref PubMed Scopus (256) Google Scholar). Dietary VLC-PUFA supplementation, especially fish oil (rich in 20:5n-3 and 22:6n-3) significantly increases the 20:5n-3 and 22:6n-3 pool size and elicits beneficial effects in chronic diseases such as insulin resistance, cardiovascular disease, and cancer (2Anderson B.M. Ma D.W. Lipids Health Dis. 2009; 8: 33Crossref PubMed Scopus (256) Google Scholar). VLC-PUFAs influence membrane structure and function, serve as substrates for eicosanoids involved in signaling, and regulate nuclear gene expression (2Anderson B.M. Ma D.W. Lipids Health Dis. 2009; 8: 33Crossref PubMed Scopus (256) Google Scholar, 6Schroeder F. Petrescu A.D. Huang H. Atshaves B.P. McIntosh A.L. Martin G.G. Hostetler H.A. Vespa A. Landrock D. Landrock K.K. Payne H.R. Kier A.B. Lipids. 2008; 43: 1-17Crossref PubMed Scopus (177) Google Scholar, 7Jump D.B. Curr. Opin. Lipidol. 2008; 19: 242-247Crossref PubMed Scopus (335) Google Scholar).In contrast to our knowledge of saturated fatty acid uptake, cytoplasmic transport, intracellular distribution, and targeting (6Schroeder F. Petrescu A.D. Huang H. Atshaves B.P. McIntosh A.L. Martin G.G. Hostetler H.A. Vespa A. Landrock D. Landrock K.K. Payne H.R. Kier A.B. Lipids. 2008; 43: 1-17Crossref PubMed Scopus (177) Google Scholar, 8McArthur M.J. Atshaves B.P. Frolov A. Foxworth W.D. Kier A.B. Schroeder F. J. Lipid Res. 1999; 40: 1371-1383Abstract Full Text Full Text PDF PubMed Google Scholar, 9Schaffer J.E. Lodish H.F. Trends Cardiovasc. Med. 1995; 5: 218-224Crossref PubMed Scopus (47) Google Scholar, 10Bradbury M.W. Berk P.D. Adv. Mol. Cell Biol. 2003; 33: 47-80Crossref Scopus (12) Google Scholar, 11Abumrad N.A. J. Clin. Invest. 2005; 115: 2965-2967Crossref PubMed Scopus (67) Google Scholar, 12Hamilton J.A. Curr. Opin. Lipidol. 2003; 14: 263-271Crossref PubMed Scopus (151) Google Scholar, 13Atshaves B.P. McIntosh A.M. Lyuksyutova O.I. Zipfel W. Webb W.W. Schroeder F. J. Biol. Chem. 2004; 279: 30954-30965Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar, 14Weisiger R.A. Mol. Cell. Biochem. 2002; 239: 35-43Crossref PubMed Scopus (72) Google Scholar, 15Lawrence J.W. Kroll D.J. Eacho P.I. J. Lipid Res. 2000; 41: 1390-1401Abstract Full Text Full Text PDF PubMed Google Scholar, 16Huang H. Starodub O. McIntosh A. Atshaves B.P. Woldegiorgis G. Kier A.B. Schroeder F. Biochemistry. 2004; 43: 2484-2500Crossref PubMed Scopus (88) Google Scholar, 17McIntosh A.L. Atshaves B.P. Hostetler H.A. Huang H. Davis J. Lyuksyutova O.I. Landrock D. Kier A.B. Schroeder F. Arch. Biochem. Biophys. 2009; 485: 160-173Crossref PubMed Scopus (43) Google Scholar), there is a significant gap in our understanding of the factors that regulate uptake, transport, and intracellular distribution of very long chain fatty acids, especially n-3 and n-6 VLC-PUFA, in living cells and of the role of liver fatty-acid binding protein (L-FABP). Based on a common role established for the cytoplasmic L-FABP in facilitating the uptake, intracellular transport, and nuclear targeting of saturated fatty acids (6Schroeder F. Petrescu A.D. Huang H. Atshaves B.P. McIntosh A.L. Martin G.G. Hostetler H.A. Vespa A. Landrock D. Landrock K.K. Payne H.R. Kier A.B. Lipids. 2008; 43: 1-17Crossref PubMed Scopus (177) Google Scholar, 8McArthur M.J. Atshaves B.P. Frolov A. Foxworth W.D. Kier A.B. Schroeder F. J. Lipid Res. 1999; 40: 1371-1383Abstract Full Text Full Text PDF PubMed Google Scholar, 13Atshaves B.P. McIntosh A.M. Lyuksyutova O.I. Zipfel W. Webb W.W. Schroeder F. J. Biol. Chem. 2004; 279: 30954-30965Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar, 14Weisiger R.A. Mol. Cell. Biochem. 2002; 239: 35-43Crossref PubMed Scopus (72) Google Scholar, 15Lawrence J.W. Kroll D.J. Eacho P.I. J. Lipid Res. 2000; 41: 1390-1401Abstract Full Text Full Text PDF PubMed Google Scholar, 16Huang H. Starodub O. McIntosh A. Atshaves B.P. Woldegiorgis G. Kier A.B. Schroeder F. Biochemistry. 2004; 43: 2484-2500Crossref PubMed Scopus (88) Google Scholar, 17McIntosh A.L. Atshaves B.P. Hostetler H.A. Huang H. Davis J. Lyuksyutova O.I. Landrock D. Kier A.B. Schroeder F. Arch. Biochem. Biophys. 2009; 485: 160-173Crossref PubMed Scopus (43) Google Scholar), it is postulated that L-FABP may have a similar function for n-3 and n-6 VLC-PUFAs. This possibility is supported by the fact that L-FABP binds n-3 and n-6 VLC-PUFAs with high affinity (Kd ∼10−8 to 10−7 m) and protects VLC-PUFAs (e.g. 20:4n-6) from intracellular peroxidation (1Norris A.W. Spector A.A. J. Lipid Res. 2002; 43: 646-653Abstract Full Text Full Text PDF PubMed Google Scholar, 18Richieri G.V. Ogata R.T. Zimmerman A.W. Veerkamp J.H. Kleinfeld A.M. Biochemistry. 2000; 39: 7197-7204Crossref PubMed Scopus (143) Google Scholar, 19Ek B.A. Cistola D.P. Hamilton J.A. Kaduce T.L. Spector A.A. Biochim. Biophys. Acta. 1997; 1346: 75-85Crossref PubMed Scopus (54) Google Scholar).To examine these questions in living cells, several families of fluorescent PUFAs have been synthesized (e.g. C18 parinaric acids; C16, C18, and C22 pentaenoic acids). However, the conjugated tetraene or pentaene fluorophores in these probes are localized in the methyl-terminal half of the fatty acid, a position not consistent with the methylene-interrupted n-3 or n-6 grouping present in naturally occurring n-3 and n-6 VLC-PUFAs (20Kuklev D.V. Smith W.L. Chem. Phys. Lipids. 2004; 131: 215-222Crossref PubMed Scopus (41) Google Scholar, 21Mateo C.R. Souto A.A. Amat-Guerri F. Acuña A.U. Biophys. J. 1996; 71: 2177-2191Abstract Full Text PDF PubMed Scopus (46) Google Scholar, 22Kuerschner L. Ejsing C.S. Ekroos K. Shevchenko A. Anderson K.I. Thiele C. Nat. Methods. 2005; 2: 39-45Crossref PubMed Scopus (149) Google Scholar). Consequently, fluorescent PUFA analogues such as the parinaric acids imperfectly monitor uptake of fatty acids, L-FABP-mediated fatty acid uptake, esterification, and intracellular distribution in living cells (16Huang H. Starodub O. McIntosh A. Atshaves B.P. Woldegiorgis G. Kier A.B. Schroeder F. Biochemistry. 2004; 43: 2484-2500Crossref PubMed Scopus (88) Google Scholar, 23Schroeder F. Jefferson J.R. Powell D. Incerpi S. Woodford J.K. Colles S.M. Myers-Payne S. Emge T. Hubbell T. Moncecchi D. Prows D.R. Heyliger C.E. Mol. Cell. Biochem. 1993; 123: 73-83Crossref PubMed Scopus (53) Google Scholar, 24Heyliger C.E. Kheshgi T.J. Murphy E.J. Myers-Payne S. Schroeder F. Mol. Cell. Biochem. 1996; 155: 113-119Crossref PubMed Scopus (8) Google Scholar). Likewise, the pattern of L-FABP affinities for n-3 and n-6 VLC-PUFA (20:4n-6 and 22:6n-3) established by displacement of parinaric acids does not reflect that established by other assays (1Norris A.W. Spector A.A. J. Lipid Res. 2002; 43: 646-653Abstract Full Text Full Text PDF PubMed Google Scholar, 18Richieri G.V. Ogata R.T. Zimmerman A.W. Veerkamp J.H. Kleinfeld A.M. Biochemistry. 2000; 39: 7197-7204Crossref PubMed Scopus (143) Google Scholar, 19Ek B.A. Cistola D.P. Hamilton J.A. Kaduce T.L. Spector A.A. Biochim. Biophys. Acta. 1997; 1346: 75-85Crossref PubMed Scopus (54) Google Scholar, 25Schroeder F. Jolly C.A. Cho T.H. Frolov A.A. Chem. Phys. Lipids. 1998; 92: 1-25Crossref PubMed Scopus (115) Google Scholar).The purpose of this study was to overcome these issues by use a different family of fluorescent n-3 and n-6 VLC-PUFAs recently developed by Smith and co-workers (26Kuklev D.V. Smith W.L. Chem. Phys. Lipids. 2004; 130: 145-158Crossref PubMed Scopus (12) Google Scholar, 27McIntosh A.L. Atshaves B.P. Wellberg E.K. Smith W.L. Schroeder F. FASEB J. 2005; 19: A292Google Scholar) where the conjugated tetraene fluorophore was positioned near the carboxylate and natural methylene-interrupted n-3 or n-6 grouping. As shown here, these fluorescent VLC-PUFAs were taken up and metabolized to esterified lipids similarly as their nonfluorescent counterparts. MPLSM real time imaging and quantitative analysis of n-3 and n-6 VLC-PUFA uptake and intracellular distribution revealed that the VLC-PUFAs were rapidly taken up and quickly targeted to nuclei, nuclear envelope membranes, and nucleoplasm of living cells. These studies are important because they provide for the first time a dynamic visual yet quantitative approach not available for native VLC-PUFAs for the purpose of examining not only 20:4n-6, 20:5n-3, and 22:6n-3 whole cell uptake kinetics but also kinetics involving the cytoplasm, nucleoplasm, nuclear envelope, and total nucleus in real time. Finally, it was shown that L-FABP regulated the uptake, intracellular distribution, and metabolism of the fluorescent VLC-PUFAs in living cells.DISCUSSIONThe uptake, intracellular transport, and intracellular targeting of saturated and monounsaturated fatty acids into nuclei are increasingly well understood (8McArthur M.J. Atshaves B.P. Frolov A. Foxworth W.D. Kier A.B. Schroeder F. J. Lipid Res. 1999; 40: 1371-1383Abstract Full Text Full Text PDF PubMed Google Scholar, 9Schaffer J.E. Lodish H.F. Trends Cardiovasc. Med. 1995; 5: 218-224Crossref PubMed Scopus (47) Google Scholar, 10Bradbury M.W. Berk P.D. Adv. Mol. Cell Biol. 2003; 33: 47-80Crossref Scopus (12) Google Scholar, 11Abumrad N.A. J. Clin. Invest. 2005; 115: 2965-2967Crossref PubMed Scopus (67) Google Scholar, 12Hamilton J.A. Curr. Opin. Lipidol. 2003; 14: 263-271Crossref PubMed Scopus (151) Google Scholar, 13Atshaves B.P. McIntosh A.M. Lyuksyutova O.I. Zipfel W. Webb W.W. Schroeder F. J. Biol. Chem. 2004; 279: 30954-30965Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar, 14Weisiger R.A. Mol. Cell. Biochem. 2002; 239: 35-43Crossref PubMed Scopus (72) Google Scholar, 51Weisiger R.A. Comp. Biochem. Physiol. B. 1996; 115: 319-331Crossref Scopus (38) Google Scholar). In contrast, almost nothing is known regarding these aspects of n-3 and n-6 VLC-PUFA dynamics or the factors that regulate VLC-PUFA uptake, metabolism, and intracellular distribution in living cells. The recent development of fluorescent n-3 and n-6 VLC-PUFA (26Kuklev D.V. Smith W.L. Chem. Phys. Lipids. 2004; 130: 145-158Crossref PubMed Scopus (12) Google Scholar, 27McIntosh A.L. Atshaves B.P. Wellberg E.K. Smith W.L. Schroeder F. FASEB J. 2005; 19: A292Google Scholar), together with MPLSM and L-FABP-expressing cells for the first time, allowed real time quantitative examination of these questions in living cells, yielding the following new insights.First, the fluorescent n-3 and n-6 VLC-PUFA more accurately reflected the uptake of their nonfluorescent native counterparts than earlier fluorescent PUFAs whose conjugated fluorophores were located in the methyl-terminal region of the PUFA. Kinetic analysis at early time points indicated that the fluorescent n-3 and n-6 VLC-PUFAs were taken up very rapidly, appearing in the cytoplasm with essentially the same kinetics as in the whole cell, i.e. detectable in nucleoplasm); and (iii) at longer incubation times the fluorescent VLC-PUFAs were primarily in esterified form and distributed into a variety of membranous structures as well as into lipid droplets. In contrast, earlier nonesterified fluorescent PUFAs with the conjugated tetraene fluorophore in the methyl-terminal region were distributed primarily into bright lipid droplets and diffusely in cytoplasm and less so in nuclei (34Huang H. Starodub O. McIntosh A. Kier A.B. Schroeder F. J. Biol. Chem. 2002; 277: 29139-29151Abstract Full Text Full Text PDF PubMed Scopus (129) Google Scholar). Thus, the intracellular distribution of fluorescent n-3 and n-6 VLC-PUFA much better reflected that of the native fatty acids.Third, the metabolism of the fluorescent n-3 and n-6 VLC-PUFAs much better represented that of their occurring counterparts than previous fluorescent PUFAs. With increasing incubation time, the fluorescent n-3 and n-6 VLC-PUFA was more representative of their radiolabeled nonfluorescent counterparts in becoming increasingly esterified to the phospholipids rather than neutral lipids. At early time points, the fluorescent VLC-PUFAS mimicked their radiolabeled VLC-PUFA counterparts in becoming primarily incorporated into PC and less so into PE. Although this was also the case at longer time points (overnight incubation) for fluorescent A5c and E6c as well as their radiolabeled nonfluorescent counterparts (20:4n-6, 20:5n-3), both the fluorescent D7c analogue and its radiolabeled nonfluorescent counterpart (22:6n-3) was esterified more into PE than PC. Overall, these findings were consistent with studies of n-3 and n-6 VLC-PUFA distribution in esterified lipids of multiple tissues (39Chen C.T. Green J.T. Orr S.K. Bazinet R.P. Prostaglandins Leukot. Essent. Fatty Acids. 2008; 79: 85-91Abstract Full Text Full Text PDF PubMed Scopus (125) Google Scholar, 40Golovko M.Y. Murphy E.J. J. Lipid Res. 2006; 47: 1289-1297Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar, 41Murphy E.J. Rosenberger T.A. Patrick C.B. Rapoport S.I. Lipids. 2000; 35: 891-898Crossref PubMed Scopus (23) Google Scholar, 42Jump D.B. J. Biol. Chem. 2002; 277: 8755-8758Abstract Full Text Full Text PDF PubMed Scopus (507) Google Scholar, 57Jump D.B. Crit. Rev. Clin. Lab. Sci. 2004; 41: 41-78Crossref PubMed Scopus (288) Google Scholar). Finally, both fluorescent n-3 and n-6 VLC-PUFA analogues as well as their radiolabeled nonfluorescent counterparts were only weakly esterified to the neutral lipids. This finding was due to the CoA thioesters of n-3 and n-6 VLC-PUFAs being poor substrates for diacylglycerol acyltransferase (last step in triglyceride synthesis) and cholesterol acyl-CoA acyltransferase (last step in cholesterol ester synthesis (42Jump D.B. J. Biol. Chem. 2002; 277: 8755-8758Abstract Full Text Full Text PDF PubMed Scopus (507) Google Scholar)). In marked contrast, earlier fluorescent PUFA analogues wherein the conjugated polyene fluorophore was localized in the methyl half of the molecule (interrupting/abolishing the normal methylene interruptions present in naturally occurring n-3 and n-6 PUFAs) were either very slowly esterified (3%/day) or esterified nearly equally to phospholipids and neutral lipids, especially at early time points of incubation (22Kuerschner L. Ejsing C.S. Ekroos K. Shevchenko A. Anderson K.I. Thiele C. Nat. Methods. 2005; 2: 39-45Crossref PubMed Scopus (149) Google Scholar, 24Heyliger C.E. Kheshgi T.J. Murphy E.J. Myers-Payne S. Schroeder F. Mol. Cell. Biochem. 1996; 155: 113-119Crossref PubMed Scopus (8) Google Scholar, 34Huang H. Starodub O. McIntosh A. Kier A.B. Schroeder F. J. Biol. Chem. 2002; 277: 29139-29151Abstract Full Text Full Text PDF PubMed Scopus (129) Google Scholar). Finally, it is important to note that very little of the fluorescent n-3 and n-6 VLC-PUFAs were oxidized or photodegraded in the early time frame of the experiments herein because their fluorescence was relatively stable. Similar low degree of oxidation and/or photodestruction was reported for earlier fluorescent PUFA analogues, regardless of where the conjugated fluorescent polyene resided in the molecule (16Huang H. Starodub O. McIntosh A. Atshaves B.P. Woldegiorgis G. Kier A.B. Schroeder F. Biochemistry. 2004; 43: 2484-2500Crossref PubMed Scopus (88) Google Scholar, 22Kuerschner L. Ejsing C.S. Ekroos K. Shevchenko A. Anderson K.I. Thiele C. Nat. Methods. 2005; 2: 39-45Crossref PubMed Scopus (149) Google Scholar). Taken together, these data suggested that the fluorescent n-3 and n-6 VLC-PUFAs were taken up and remained primarily in unesterified form at early time points, preferentially esterified to phospholipids (not neutral lipids) at longer time points, and relatively stable to oxidation/photodestruction under the conditions of the real time MPLSM imaging studies in living cells used herein.Fourth, L-FABP binding of fluorescent VLC-PUFAs mirrored the natural nonfluorescent counterparts better than earlier fluorescent PUFAs (1Norris A.W. Spector A.A. J. Lipid Res. 2002; 43: 646-653Abstract Full Text Full Text PDF PubMed Google Scholar, 28Frolov A. Cho T.H. Murphy E.J. Schroeder F. Biochemistry. 1997; 36: 6545-6555Crossref PubMed Scopus (93) Google Scholar, 45Wolfrum C. Börchers T. Sacchettini J.C. Spener F. Biochemistry. 2000; 39: 1469-1474Crossref PubMed Scopus (73) Google Scholar). L-FABP bound all three fluorescent VLC-PUFAs with high affinity and reflected the specificities for their nonfluorescent counterparts (1Norris A.W. Spector A.A. J. Lipid Res. 2002; 43: 646-653Abstract Full Text Full Text PDF PubMed Google Scholar, 28Frolov A. Cho T.H. Murphy E.J. Schroeder F. Biochemistry. 1997; 36: 6545-6555Crossref PubMed Scopus (93) Google Scholar, 45Wolfrum C. Börchers T. Sacchettini J.C. Spener F. Biochemistry. 2000; 39: 1469-1474Crossref PubMed Scopus (73) Google Scholar). The high affinities exhibited by L-FABP for both the fluorescent and naturally occurring n-3 and n-6 VLC-PUFAs reflected the natural distribution of the endogenous bound native nonfluorescent fatty acids (32Murphy E.J. Edmondson R.D. Russell D.H. Colles S. Schroeder F. Biochim. Biophys. Acta. 1999; 1436: 413-425Crossref PubMed Scopus (45) Google Scholar) as follows. (i) the n-3 and n-6 VLC-PUFAs included over 40% of the endogenous L-FABP-bound fatty acid, consistent with the higher affinity of L-FABP for such fatty acids than saturated or monounsaturated fatty acids. (ii) 20:4n-6 and 22:6n-3 included 25 and 2% of the total endogenous bound fatty acid, whereas 22:5n-3 was not detectable, consistent with the relative composition of hepatic unesterified fatty acid pool (32Murphy E.J. Edmondson R.D. Russell D.H. Colles S. Schroeder F. Biochim. Biophys. Acta. 1999; 1436: 413-425Crossref PubMed Scopus (45) Google Scholar, 42Jump D.B. J. Biol. Chem. 2002; 277: 8755-8758Abstract Full Text Full Text PDF PubMed Scopus (507) Google Scholar, 57Jump D.B. Crit. Rev. Clin. Lab. Sci. 2004; 41: 41-78Crossref PubMed Scopus (288) Google Scholar). In contrast, earlier fluorescent PUFAs with conjugated polyene fluorophore in the methyl-terminal region (e.g. parinaric acids) do not interact with L-FABP in the same manner as naturally occurring n-3 and n-6 VLC-PUFA (20:4n-6, 22:6n-3). For example, although several studies previously established that L-FABP has higher or equal affinity for 22:6n-3 than 20:4n-6, parinaric acid displacement assays showed the opposite (1Norris A.W. Spector A.A. J. Lipid Res. 2002; 43: 646-653Abstract Full Text Full Text PDF PubMed Google Scholar, 18Richieri G.V. Ogata R.T. Zimmerman A.W. Veerkamp J.H. Kleinfeld A.M. Biochemistry. 2000; 39: 7197-7204Crossref PubMed Scopus (143) Google Scholar, 19Ek B.A. Cistola D.P. Hamilton J.A. Kaduce T.L. Spector A.A. Biochim. Biophys. Acta. 1997; 1346: 75-85Crossref PubMed Scopus (54) Google Scholar, 25Schroeder F. Jolly C.A. Cho T.H. Frolov A.A. Chem. Phys. Lipids. 1998; 92: 1-25Crossref PubMed Scopus (115) Google Scholar). L-FABP exhibits higher affinity for the straight chain trans-parinaric acid (saturated analogue) as compared with the kinked chain cis-parinaric acid (unsaturated analogue), opposite to the known preference of L-FABP for kinked chain unsaturated fatty acids versus straight chain saturated fatty acids (8McArthur M.J. Atshaves B.P. Frolov A. Foxworth W.D. Kier A.B. Schroeder F. J. Lipid Res. 1999; 40: 1371-1383Abstract Full Text Full Text PDF PubMed Google Scholar, 28Frolov A. Cho T.H. Murphy E.J. Schroeder F. Biochemistry. 1997; 36: 6545-6555Crossref PubMed Scopus (93) Google Scholar, 58Paulussen R.J.A. Veerkamp J.H. Hilderson H.J. Subcellular Biochemistry. Plenum Publishing Corp., New York1990: 175-226Google Scholar). Thus, the pattern of L-FABP affinities for the fluorescent n-3 and n-6 VLC-PUFAs, but not that established with parinaric acid displacement assays, better reflects that of naturally occurring n-3 and n-6 VLC-PUFA.With regard to specificity of L-FABP for VLC-PUFA binding, the cytosolic fatty acid-binding protein family is composed of more than a dozen members with overlapping specificity for binding a broad variety of fatty acids. The rodent FABPs bind n-3 and n-6 VLC-PUFAs in the following order of affinities: L-FABP, H-FABPs ≫ intestinal type FABP, adipocyte-type FABP ≫ cellular retinoic acid-binding proteins I and II (18Richieri G.V. Ogata R.T. Zimmerman A.W. Veerkamp J.H. Kleinfeld A.M. Biochemistry. 2000; 39: 7197-7204Crossref PubMed Scopus (143) Google Scholar). The liver L-FABP has high affinity for VLC-PUFAs (as compared with most other FABP family members), and it is highly expressed in tissues active in fatty acid uptake and metabolism (i.e. liver, intestine, kidney). L-FABP also enhances the uptake/metabolism of saturated and monounsaturated fatty acids (43Atshaves B.P. Martin G.G. Hostetler H.A. McIntosh A.L. Kier A.B. Schroeder F. J. Nutr. Biochem. 2010; (in press)PubMed Google Scholar). Although it is tempting to speculate that differences in binding affinities to L-FABP may account for the differences in predominant products formed by the n-3 and n-6 PUFA synthetic pathways, there is as yet no evidence that this is the case (1Norris A.W. Spector A.A. J. Lipid Res. 2002; 43: 646-653Abstract Full Text Full Text PDF PubMed Google Scholar). Conversely, however, by binding long chain fatty acid peroxidation products of PUFAs, the L-FABP (more so than other FABPs tested) modulates availability of these PUFAs to intracellular oxidative pathways and controls the amount of reactive oxygen species released within the cell (19Ek B.A. Cistola D.P. Hamilton J.A. Kaduce T.L. Spector A.A. Biochim. Biophys. Acta. 1997; 1346: 75-85Crossref PubMed Scopus (54) Google Scholar, 59

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