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.