Arachidonate-derived Dihomoprostaglandin Production Observed in Endotoxin-stimulated Macrophage-like Cells

Eicosanoids, including the prostaglandins, leukotrienes, hydroxyeicosatetraenoic acids, epoxyeicosatetraenoic acids, and related compounds, are biosynthetic, bioactive mediators derived from arachidonic acid (AA), a 20:4(n-6) fatty acid. We have developed a comprehensive and sensitive mass spectral analysis to survey eicosanoid release from endotoxin-stimulated RAW 264.7 macrophage-like cells that is capable of detecting over 70 diverse eicosanoids and eicosanoid metabolites, should they be present. We now address the question: Are biologically significant eicosanoids being overlooked? Herein, we illustrate a general approach to diverse isotope metabolic profiling of labeled exogenous substrates using mass spectrometry (DIMPLES/MS), demonstrated for one substrate (AA) and its resultant products (eicosanoids). RAW cells were incubated in medium supplemented with deuterium-labeled AA. When the cells are stimulated, two sets of eicosanoids are produced, one from endogenous AA and the other from the supplemented (exogenous) deuterium-labeled form. This produces a signature mass spectral “doublet” pattern, allowing for a comprehensive and diverse eicosanoid search requiring no previous knowledge or assumptions as to what these species may be, in contrast to traditional methods. We report herein observing unexpected AA metabolites generated by the cells, some of which may constitute novel bioactive eicosanoids or eicosanoid inactivation metabolites, as well as demonstrating differing metabolic pathways for the generation of isomeric prostaglandins and potential peroxisome proliferator-activated receptor activators. Unexpectedly, we report observing a series of 1a, 1b-dihomologue prostaglandins, products of adrenic acid (22:4(n-6)), resulting from the two-carbon elongation of AA by the RAW cells. Eicosanoids, including the prostaglandins, leukotrienes, hydroxyeicosatetraenoic acids, epoxyeicosatetraenoic acids, and related compounds, are biosynthetic, bioactive mediators derived from arachidonic acid (AA), a 20:4(n-6) fatty acid. We have developed a comprehensive and sensitive mass spectral analysis to survey eicosanoid release from endotoxin-stimulated RAW 264.7 macrophage-like cells that is capable of detecting over 70 diverse eicosanoids and eicosanoid metabolites, should they be present. We now address the question: Are biologically significant eicosanoids being overlooked? Herein, we illustrate a general approach to diverse isotope metabolic profiling of labeled exogenous substrates using mass spectrometry (DIMPLES/MS), demonstrated for one substrate (AA) and its resultant products (eicosanoids). RAW cells were incubated in medium supplemented with deuterium-labeled AA. When the cells are stimulated, two sets of eicosanoids are produced, one from endogenous AA and the other from the supplemented (exogenous) deuterium-labeled form. This produces a signature mass spectral “doublet” pattern, allowing for a comprehensive and diverse eicosanoid search requiring no previous knowledge or assumptions as to what these species may be, in contrast to traditional methods. We report herein observing unexpected AA metabolites generated by the cells, some of which may constitute novel bioactive eicosanoids or eicosanoid inactivation metabolites, as well as demonstrating differing metabolic pathways for the generation of isomeric prostaglandins and potential peroxisome proliferator-activated receptor activators. Unexpectedly, we report observing a series of 1a, 1b-dihomologue prostaglandins, products of adrenic acid (22:4(n-6)), resulting from the two-carbon elongation of AA by the RAW cells. Starting in the early 1960s with the first structural characterization of the prostaglandins (1Bergstrom B. Samuelsson B. J. Biol. Chem. 1962; 237: 3005-3006Abstract Full Text PDF PubMed Google Scholar), mass spectrometry (MS) 2The abbreviations used are: MS, mass spectrometry; AA, arachidonic acid (5Z,8Z,11Z,14Z-eicosatetraenoic acid); AA-d8, deuterated-AA (octadeuterated AA, 5Z,8Z,11Z,14Z-eicosatetraenoic acid, 5,6,8,9,11,12,14,15-d8); adrenic acid, 7Z,10Z,13Z,16Z-docosatetraenoic acid; COX, cyclooxygenase; DiHETE, dihydroxy-eicosatetraenoic acid; DIMPLES/MS, diverse isotope metabolic profiling of labeled exogenous substrates using mass spectrometry; HETE, hydroxyeicosatetraenoic acid; HpETE, hydroperoxyeicosatetraenoic acid; Kdo2-Lipid A, (3-deoxy-d-manno-octulosonic acid)2-Lipid A; LC-RT, liquid chromatography-retention time; MS/MS, tandem mass spectrometry; MRM, multiple reaction monitoring; PGD2, prostaglandin D2 (9S,15S-dihydroxy-11-oxo-5Z,13E-prostadienoic acid); PGE2, prostaglandin E2 (9-oxo-11R,15S-dihydroxy-5Z,13E-prostadienoic acid); PGF2α, prostaglandin F2α (9S,11R,15S-trihydroxy-5Z,13E-prostadienoic acid); PGG2, prostaglandin G2 (9S,11R-epidioxy-15S-hydroperoxy-5Z,13E-prostadienoic acid); PGH2, prostaglandin H2 (9S,11R-epidioxy-15S-hydroxy-5Z,13E-prostadienoic acid); PGJ2, prostaglandin J2 (11-oxo-15S-hydroxy-5Z,8Z,13E-prostatrienoic acid); 11β-PGF2α (9S,11S,15S-trihydroxy-5Z,13E-prostadienoic acid); 15d-Δ12,14-PGD2, 15-deoxy-prostaglandin D2 (11-oxo-9S-hydroxy-5Z,12E,14E-prostatrienoic acid); 15d-Δ12,14-PGJ2, 15-deoxyprostaglandin J2 (11-oxo-5Z,9,12E,14Z-prostatetraenoic acid); dihomoprostaglandin, 1a,1b-dihomologue prostaglandin; dihomo-PGD2, dihomoprostaglandin D2 (1a,1b-dihomo-9S,15S-dihydroxy-11-oxo-5Z,13E-prostadienoic acid); dihomo-PGE2, dihomoprostaglandin E2 (1a,1b-dihomo-9-oxo-11R,15S-dihydroxy-5Z,13E-prostadienoic acid); dihomo-PGF2α, dihomoprostaglandin F2α (1a,1b-dihomo-9S,11R,15S-trihydroxy-5Z,13E-prostadienoic acid); dihomo-PGJ2, dihomoprostaglandin J2 (1a,1b-dihomo-11-oxo-15S-hydroxy-5Z,8Z,13E-prostatrienoic acid); dihomo-15d-Δ12,14-PGD2, dihomo-15-deoxyprostaglandin D2 (1a,1b-dihomo-9S-hydroxy-11-oxo-5Z,12E,14E-prostatrienoic acid). has played a critical role in the biochemical study of eicosanoids. More recent advances in electrospray ionization-MS coupled to high-performance liquid chromatography have offered extremely sensitive and quantitative assays for most of the eicosanoids (2Murphy R.C. Barkley R.M. Zemski Berry K. Hankin J. Harrison K. Johnson C. Krank J. McAnoy A. Uhlson C. Zarini S. Anal. Biochem. 2005; 246: 1-42Crossref Scopus (189) Google Scholar), without the need for chemical derivatization prior to analysis as was required by earlier gas chromatography electron ionization-MS methods (3Fischer C. Frohlich J.C. Adv. Lipid Res. 1982; 19: 185-202Crossref PubMed Google Scholar). It is important to recognize, however, that while advances in high-performance LC-MS methods have offered extensive capabilities for surveying a number of different eicosanoid species in a single analysis, to date most efforts have focused on a specific eicosanoid or eicosanoid class, and additionally, with advanced knowledge and assumptions as to the identity of these species (4Margalit A. Duffin K.L. Isakson P.C. Anal. Biochem. 1996; 235: 73-81Crossref PubMed Scopus (65) Google Scholar, 5Kempen E.C. Yang P. Felix E. Madden T. Newman R.A. Anal. Biochem. 2001; 297: 183-190Crossref PubMed Scopus (88) Google Scholar, 6Takabatake M. Hishinuma T. Suzaki N. Chiba S. Tsukamoto H. Nakamura H. Saga T. Tomioka Y. Kurose A. Sawai T. Mizugaki M. Prostaglandins Leukot. Essent. Fatty Acids. 2002; 67: 51-56Abstract Full Text PDF PubMed Scopus (51) Google Scholar, 7Yang P. Felix E. Madden T. Fischer S.M. Newman R.A. Anal. Biochem. 2002; 308: 168-177Crossref PubMed Scopus (62) Google Scholar, 8Nithipatikom K. Laabs N.D. Isbell M.A. Campbell W.B. J. Chromatogr. B. 2003; 785: 135-145Crossref PubMed Scopus (43) Google Scholar, 9Kingsley P.J. Rouzer C.A. Saleh S. Marnett L.J. Anal. Biochem. 2005; 343: 203-211Crossref PubMed Scopus (38) Google Scholar, 10Kita Y. Takahashi T. Uozumi N. Nallan L. Gelb M.H. Shimizu T. Biophys. Res. Commun. 2005; 330: 898-906Crossref PubMed Scopus (33) Google Scholar, 11Kita Y. Takahashi T. Uozumi Shimizu T. Anal. Biochem. 2005; 342: 134-143Crossref PubMed Scopus (107) Google Scholar). Recently, however, investigators have begun to explore the development of theoretical databases and algorithms based on virtual liquid chromatography-UV spectroscopy-tandem mass spectrometry (MS/MS) spectra and chromatograms for identifying potential lipid mediators without synthetic or authentic products as standards (12Lu Y. Hong S. Tjonanhen E. Serhan C.N. J. Lipid Res. 2005; 46: 790-802Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar). Systems biology approaches include a comprehensive quantitative analysis of the manner in which all the components of a biological system interact functionally over time (13Aderem A. Cell. 2005; 121: 511-513Abstract Full Text Full Text PDF PubMed Scopus (312) Google Scholar) and offer the promise of revolutionizing our understanding of cellular biology, personalized medicine, and drug design. A vital component of a systems biology approach is the global quantification of the spatial and temporal changes in lipid metabolites that occur with cellular metabolism, and no such strategy has yet to emerge. We have begun to address such a strategy using a single cell type, the RAW 264.7 macrophage-like cell, and a single stimulus, the high purity saccharolipid Kdo2-Lipid A component of the endotoxin lipopolysaccharide (14Raetz C.R.H. Garrett T.A. Reynolds C.M. Shaw W.A. Moore J.D. Smith D.C. Riberio A.A. Murphy R.C. Ulevitch R.J. Fearns C. Reichart D. Glass C.K. Benner C. Subramaniam S. Harkewicz R. Bowers-Gentry R.C. Buczynski M.W. Cooper R.A. J. Lipid Res. 2005; 46: Full Text Full Text PDF PubMed Scopus Google Scholar) for the of eicosanoids and a of and was eicosanoid release from Kdo2-Lipid RAW cells. should be that to the bioactive mediators derived from arachidonic acid and are in the in and in PGD2, PGE2, and bioactive mediators be to products and are to as of bioactive in the the bioactive mediators and metabolites are to as were detecting liquid chromatography coupled with and by the mass in the multiple reaction we the to over 70 different eicosanoid and inactivation species in a single analysis should they be present. offer the for detecting a number of diverse eicosanoid the of the methods earlier (4Margalit A. Duffin K.L. Isakson P.C. Anal. Biochem. 1996; 235: 73-81Crossref PubMed Scopus (65) Google Scholar, 5Kempen E.C. Yang P. Felix E. Madden T. Newman R.A. Anal. Biochem. 2001; 297: 183-190Crossref PubMed Scopus (88) Google Scholar, 6Takabatake M. Hishinuma T. Suzaki N. Chiba S. Tsukamoto H. Nakamura H. Saga T. Tomioka Y. Kurose A. Sawai T. Mizugaki M. Prostaglandins Leukot. Essent. Fatty Acids. 2002; 67: 51-56Abstract Full Text PDF PubMed Scopus (51) Google Scholar, 7Yang P. Felix E. Madden T. Fischer S.M. Newman R.A. Anal. Biochem. 2002; 308: 168-177Crossref PubMed Scopus (62) Google Scholar, 8Nithipatikom K. Laabs N.D. Isbell M.A. Campbell W.B. J. Chromatogr. B. 2003; 785: 135-145Crossref PubMed Scopus (43) Google Scholar, 9Kingsley P.J. Rouzer C.A. Saleh S. Marnett L.J. Anal. Biochem. 2005; 343: 203-211Crossref PubMed Scopus (38) Google Scholar, 10Kita Y. Takahashi T. Uozumi N. Nallan L. Gelb M.H. Shimizu T. Biophys. Res. Commun. 2005; 330: 898-906Crossref PubMed Scopus (33) Google Scholar, 11Kita Y. Takahashi T. Uozumi Shimizu T. Anal. Biochem. 2005; 342: 134-143Crossref PubMed Scopus (107) Google that assumptions have to be in the species that be to for the our being to a global survey of the eicosanoids by the RAW cells, we now address the question: Are biologically significant eicosanoids being overlooked? are species for which we have no prior knowledge of or of no that be required for the general isotope exogenous substrate which we for diverse isotope metabolic profiling of labeled exogenous substrates using mass a global survey is we the approach using one arachidonic acid (AA), and its RAW cell generated eicosanoids and related RAW cells are incubated in medium supplemented with deuterium-labeled arachidonic acid and with Kdo2-Lipid A. sets of eicosanoid generation one from endogenous AA, the other from the supplemented exogenous This in a “doublet” pattern, with mass allowing for a sensitive and comprehensive eicosanoid search without previous knowledge or assumptions as to what these species may be, in contrast to traditional methods. the metabolites mass spectral that may in and being for novel eicosanoids. is demonstrated in for AA and its should for other substrates and the diverse analysis of we report herein observing metabolic products of AA generated by Kdo2-Lipid RAW cells, which have the potential for being novel eicosanoids. these species and biologically the required to specific be approach different pathways for isomeric prostaglandins in these cells. and of we report observing a number of 1a, 1b-dihomologue prostaglandins products of adrenic acid resulting from the two-carbon elongation of AA by the RAW cells. has that adrenic acid as a substrate to cells, resulting in the of H. M. A. P. J. Biol. Chem. 1982; Full Text PDF PubMed Google Scholar, R.M. PubMed Scopus Google Scholar, W.B. Johnson Biophys. PubMed Scopus Google Scholar), has no of its from the and the were from was from Kdo2-Lipid A was from the standards used to our of eicosanoid including AA used for were from were from and the used with these was from were chromatography and from and 264.7 macrophage-like cells were in medium supplemented with with endotoxin and in and of these cells were in with of the medium supplemented with and without of for a sets of cells were with of Kdo2-Lipid A or of medium as a for cell were Kdo2-Lipid A with and without and with and without medium from was for to cells, supplemented with and to eicosanoid of eicosanoids and fatty was using to a were using of by of were with of with of was to using a and the was with of liquid chromatography A. liquid chromatography was using two high with a was using a with a A was was was using and to by to by and to by and to by and to some a was using and to by and to by and to was for and of were the using a liquid chromatography was coupled to a mass for chromatography was using the system for was on a with a A was was This of in A is in the and was to be vital for and was using and to by to by and to by and to chromatography was coupled to a mass for mass spectral were using mass with a and in MS/MS, and multiple reaction specific to of are all the was in electrospray chromatography the in chemical and the was as and used for to species and from to was in chemical chromatography using the and mass spectral generated were of mass and to R. M. B. B. E. R. R. K. H. S. E. R. Smith R. R.A. PubMed Scopus Google Scholar) using the were with a to in mass over a mass and a of for mass spectral A of was used to a of supplemented and and of and a of mass spectral to the of to cells, Kdo2-Lipid A medium and and LC-MS analysis is in of by RAW cells as a of time was by of medium from a of cells in of medium supplemented with and acid and the of was for a to the however, no cells A of these two a of the of in the medium the different time and the be to by the cells of was used to the of in the as by the its and its time was with a were to the in that a of the in the medium that by the time of Kdo2-Lipid A a all the was the cells. from the cell A with and without and no with and without were for acids, and in of A. A LC-MS analysis was for of the and the mass in for the a number of most a by its and that time with that of a prostaglandin D2 of was with our previous Kdo2-Lipid A RAW cell (14Raetz C.R.H. Garrett T.A. Reynolds C.M. Shaw W.A. Moore J.D. Smith D.C. Riberio A.A. Murphy R.C. Ulevitch R.J. Fearns C. Reichart D. Glass C.K. Benner C. Subramaniam S. Harkewicz R. Bowers-Gentry R.C. Buczynski M.W. Cooper R.A. J. Lipid Res. 2005; 46: Full Text Full Text PDF PubMed Scopus Google Scholar), in which has eicosanoid in high cells. A for the as as a mass from the these two spectra produces a “doublet” by the and in the and and the in the and in the have a different from and we that is to the number of on the deuterated-AA used for the of and the of its A was mass spectral analysis of the used for the a of the the and as well in the of and This was using the the and was in It is that the deuterated-AA was in cell medium for a no in its It is important to while the of the supplemented AA the of the generated of a on of the of a from of its however, the in is is that is being from its PGD2, from or of the other It should be however, the in is a be for from a is the was to a one its to be the in the the of to be as is the a that a significant of the time the to of the two cell a as the in the with a single by its in the were in a that has (14Raetz C.R.H. Garrett T.A. Reynolds C.M. Shaw W.A. Moore J.D. Smith D.C. Riberio A.A. Murphy R.C. Ulevitch R.J. Fearns C. Reichart D. Glass C.K. Benner C. Subramaniam S. Harkewicz R. Bowers-Gentry R.C. Buczynski M.W. Cooper R.A. J. Lipid Res. 2005; 46: Full Text Full Text PDF PubMed Scopus Google Scholar). This that the eicosanoid in the the generated mass for some of the other eicosanoids by the Kdo2-Lipid cells. It should be that for some of these metabolites and the most products for PGE2, and are in the most of these is chromatography Prostaglandins Lipid 2005; PubMed Scopus Google Scholar) coupled to mass spectrometry the species to be A and chromatography A, chromatography coupled to mass spectrometry the species by the Kdo2-Lipid RAW cells to be a of and standards a of in has developed for and and mass spectral sets for the liquid chromatography time and chemical for the AA and the most of the and be A of and to a be to the with a that quantitative on these the and to a of as are for the the the of to the the in the of is the most of the and to the of the from the a of a for the of the eicosanoids to earlier the these of sets from supplemented and Kdo2-Lipid RAW cell were using the and a was generated of the was a species with of and of which in our of We have a comprehensive of eicosanoid which is in the on in that LC-RT, and number E. Subramaniam S. Glass C.K. Murphy R.C. C.R.H. Y. 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Lipid 2002; Google Scholar). for with being the most of the components and the for that from for is with being the most of the from no a that is required were to from and for the is to that for the with the of for the most of the components with chromatography the by the RAW cells to be A and It has that is resulting from the G2 It should be that be the of the mass for generated from Kdo2-Lipid as with PGF2α, is the most of its AA metabolites, such and we now eicosanoids are from AA. by the prostaglandin as or on AA is the first in all prostaglandin first one from the of AA to to which with two first and and a the prostaglandin as a on prostaglandin its to a Prostaglandins Lipid 2002; PubMed Scopus Google Scholar, Prostaglandins Lipid 2002; PubMed Scopus Google Scholar). as a substrate for a number of or This to that the different we for and with and are the of that occur the of PGH2, to the of the in the mass of the our A and with being the most of its components to be to and to or This a as in and in the and products of the and the of and most mass the is a that is of a of from its E. L. Murphy R.C. J. PubMed Scopus Google Scholar). a mass resulting from the of mass by the of the and of the and (2Murphy R.C. Barkley R.M. Zemski Berry K. Hankin J. Harrison K. Johnson C. Krank J. McAnoy A. Uhlson C. Zarini S. Anal. Biochem. 2005; 246: 1-42Crossref Scopus (189) Google Scholar, E. L. Murphy R.C. J. PubMed Scopus Google Scholar). A mass from in a in and the as well as mass mass to was with a the such a acid in and with a to be the for some of the in and of to dihomo-PGF2α, a number of other were to be by the Kdo2-Lipid cells. of these other in were using standards of the that from the were using and and these were used to for the other and the of the with was was to that occur to in spectra that the elongation of AA the of the from a is products of adrenic acid to from the two-carbon elongation of AA prior to the of the adrenic acid to the of the cell This is by the of adrenic acid and which from the supplemented with adrenic acid prostaglandins from the endogenous AA A and some of the in the AA elongation and the of by cells, are in It should be that prostaglandins were to be by the RAW cells. has that adrenic acid to cells as a substrate resulting in the of H. M. A. P. J. Biol. Chem. 1982; Full Text PDF PubMed Google Scholar, R.M. PubMed Scopus Google Scholar, W.B. Johnson Biophys. PubMed Scopus Google Scholar), has no of its from the as to the elongation of AA of adrenic acid to cell was to the of AA to prostaglandins PubMed Scopus Google Scholar). It is that the elongation as a a of the of AA derived the may have of and of these is It is that the elongation may be on the however, the of adrenic acid some the generation of some with the of in a prostaglandin two-carbon elongation of should be its of the was on the prostaglandins one to all the being in that is the prostaglandin in most should be the most of the prostaglandin the in dihomoprostaglandin and elongation as in is to the of including to be by the Kdo2-Lipid A RAW cells are PGD2, PGF2α, PGJ2, and It is in the of some for and for on is by the most generated by the Kdo2-Lipid to in is in is by the of its products and This that adrenic acid may be a substrate for or adrenic acid may be a substrate for and This is by the adrenic acid is the of AA, and are the of and PGF2α, of the and the were This of is of and its role as endogenous for the peroxisome proliferator-activated receptor have the of and P. J. R.M. Cell. Full Text PDF PubMed Scopus Google Scholar, M. M. Glass C.K. S. A. PubMed Scopus Google Scholar, T. P. M. J. 2003; PubMed Scopus Google Scholar, J. 2003; PubMed Scopus Google Scholar). is a of dihomo-PGD2, as is to be of PGD2, we to its with and in these that its may be are to quantitative of the and the of the elongation and prostaglandin of other supplemented fatty acids, acid and acid should now be using these methods. We our to of and C. Murphy of of and to of our for with