title: Method to simultaneously determine the sphingosine 1-phosphate breakdown product (2E)-hexadecenal and its fatty acid derivatives using isotope-dilution HPLC-electrospray ionization-quadrupole/time-of-flight mass spectrometry.
abstract: Sphingosine 1-phosphate (S1P), a bioactive lipid involved in various physiological processes, can be irreversibly degraded by the membrane-bound S1P lyase (S1PL) yielding (2E)-hexadecenal and phosphoethanolamine. It is discussed that (2E)-hexadecenal is further oxidized to (2E)-hexadecenoic acid by the long-chain fatty aldehyde dehydrogenase ALDH3A2 (also known as FALDH) prior to activation via coupling to coenzyme A (CoA). Inhibition or defects in these enzymes, S1PL or FALDH, result in severe immunological disorders or the Sjögren-Larsson syndrome, respectively. Hence, it is of enormous importance to simultaneously determine the S1P breakdown product (2E)-hexadecenal and its fatty acid metabolites in biological samples. However, no method is available so far. Here, we present a sensitive and selective isotope-dilution high performance liquid chromatography-electrospray ionization-quadrupole/time-of-flight mass spectrometry method for simultaneous quantification of (2E)-hexadecenal and its fatty acid metabolites following derivatization with 2-diphenylacetyl-1,3-indandione-1-hydrazone and 1-ethyl-3-(3-(dimethylamino)propyl)carbodiimide. Optimized conditions for sample derivatization, chromatographic separation, and MS/MS detection are presented as well as an extensive method validation. Finally, our method was successfully applied to biological samples. We found that (2E)-hexadecenal is almost quantitatively oxidized to (2E)-hexadecenoic acid, that is further activated as verified by cotreatment of HepG2 cell lysates with (2E)-hexadecenal and the acyl-CoA synthetase inhibitor triacsin C. Moreover, incubations of cell lysates with deuterated (2E)-hexadecenal revealed that no hexadecanoic acid is formed from the aldehyde. Thus, our method provides new insights into the sphingolipid metabolism and will be useful to investigate diseases known for abnormalities in long-chain fatty acid metabolism, e.g., the Sjögren-Larsson syndrome, in more detail.
authors.affiliation: Department of Nutritional Toxicology, Institute of Nutritional Science, University of Potsdam , Arthur-Scheunert-Allee 114-116, 14558 Nuthetal, Germany.
authors.forename: Corinna
authors.initials: C
authors.lastname: Neuber
authors.forename: Fabian
authors.initials: F
authors.lastname: Schumacher
authors.forename: Erich
authors.initials: E
authors.lastname: Gulbins
authors.forename: Burkhard
authors.initials: B
authors.lastname: Kleuser
mesh_terms: D000445:Aldehyde Oxidoreductases / Q000378:metabolism; D000446:Aldehyde-Lyases / Q000378:metabolism; D000447:Aldehydes / Q000032:analysis* / Q000302:isolation & purification; D002234:Carbodiimides / Q000737:chemistry; D002851:Chromatography, High Pressure Liquid; D056945:Hep G2 Cells; D006801:Humans; D006835:Hydrazones / Q000737:chemistry; D008246:Lysophospholipids / Q000378:metabolism*; D010169:Palmitic Acids / Q000032:analysis* / Q000302:isolation & purification; D016111:Sjogren-Larsson Syndrome / Q000175:diagnosis / Q000378:metabolism / Q000473:pathology; D021241:Spectrometry, Mass, Electrospray Ionization; D013110:Sphingosine / Q000031:analogs & derivatives* / Q000378:metabolism; D013237:Stereoisomerism; D014226:Triazenes / Q000737:chemistry
chemical_list: D000447:Aldehydes; D002234:Carbodiimides; D006835:Hydrazones; D008246:Lysophospholipids; D010169:Palmitic Acids; D014226:Triazenes; C060506:sphingosine 1-phosphate; C093821:hexadecenoic acid; C034613:triacsin C; D000445:Aldehyde Oxidoreductases; C038828:long-chain-aldehyde dehydrogenase; D000446:Aldehyde-Lyases; C022649:sphingosine 1-phosphate lyase (aldolase); D013110:Sphingosine; C001647:hexadecanal