Chemical and Apoptotic Properties of Hydroxy-Ceramides Containing Long-Chain Bases with Unusual Alkyl Chain Lengths

Abstract

We analysed four types of free ceramides (Cer 1, Cer 2, Cer 3 and Cer 4) from equine kidneys by electrospray ionization mass spectrometry. Cer 1 was composed of dihydroxy long-chain bases (dLCBs) of (4E)-sphingenine (d18:1), sphinganine and non-hydroxy fatty acids (NFAs); Cer 2 was composed of trihydroxy LCBs (tLCBs) of 4-hydroxysphinganine, t16:0, t18:0, t19:0 and t20:0, and NFAs; Cer 3 was composed of dLCBs, d16:1, d17:1, d18:1, d19:1 and d20:1, and hydroxy FAs (HFAs); and Cer 4 was composed of tLCBs, t16:0, t17:0, t18:0, t19:0 and t20:0, and HFAs. The results indicate all ceramide species containing LCBs with non-octadeca lengths (NOD-LCBs) can be classified into hydroxy-ceramides since these species always consist of tLCBs, and/or HFAs. Furthermore, such species tend to contain FAs with longer acyl chains but contain neither palmitate (C16:0) nor its hydroxylated form (C16:0h). The apoptosis-inducing activities of these hydroxyl-ceramides towards tumour cell lines were compared with that of non-hydroxy-ceramides, dLCB-NFA (Cer 1). Monohydroxy-ceramides, tLCB-NFA (Cer 2) and dLCB-HFA (Cer 3), exhibited stronger activities, whereas dihydroxy-ceramides, tLCB-HFA (Cer 4), exhibited similar or weaker activity than dLCB-NFA (Cer 1), depending on cell lines.

Systematic analyses of free ceramide species and ceramide species comprising neutral glycosphingolipids by MALDI-TOF MS with high-energy CID

“…Preparations of ceramides, sphingomyelins and neutral GSLs from equine kidneys Sphingomyelins and neutral GSLs were purified from equine kidneys as previously described [6,24]. Briefly, lipids were extracted with a chloroform-methanol mixture.…”

“…We previously demonstrated diversities of free ceramides including those with NOD-LCBs from equine kidneys using ESI-MS in negative mode with low-energy CID [6]. However, this system cannot provide enough information to characterize ceramide species in GSLs; therefore, we developed the present method to identify them.…”

“…GSLs, which are also involved in cell signaling processes, mediate cell-cell adhesion and pathogen entry [3,4]. Recent studies have revealed that both fatty acids (FAs) and long-chain bases (LCBs) of each class of lipids are more diverse than expected not only in skin [5], but also in other mammalian organs [6], in terms of i) alkyl chain length, ii) hydroxylation and iii) the presence or absence of double bonds. These factors are considered to affect the geometry of sphingolipids in membrane microdomains of cells [7,8].…”

“…Liquid chromatography-electrospray ionization mass spectrometry (LC/ESI-MS) and matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) have been well utilized to characterize sphingolipids with high throughput [5,6,[12][13][14][15][16][17][18][19][20][21], but these reports did not fully characterize ceramide species including hydroxyl FAs (HFAs) and/or trihydroxy-LCBs (tLCBs) of both free ceramides and the constituent ceramides in GSLs. Namely, in the analyses of GSLs these investigations focused on sugar sequences by releasing sugars from the lipid portions [22,23]; therefore, detailed information regarding the constituent ceramide species in GSLs is limited [20].…”

“…Namely, in the analyses of GSLs these investigations focused on sugar sequences by releasing sugars from the lipid portions [22,23]; therefore, detailed information regarding the constituent ceramide species in GSLs is limited [20]. We recently demonstrated the diversity of free ceramides, including those with non-octadeca LCBs (NODLCBs) using ESI-MS in the negative mode [6]. However, for neutral GSLs, positive mode analysis is more sensitive and informative than negative mode analysis.…”