Phytoceramide in Vertebrate Tissues: One Step Chromatography Separation for Molecular Characterization of Ceramide Species

Abstract

Ceramide is a precursor for complex sphingolipids in vertebrates, while plants contain phytoceramide. By using a novel chromatography purification method we show that phytoceramide comprises a significant proportion of animal sphingolipids. Total ceramide including phytoceramide from mouse tissue (brain, heart, liver) lipid extracts and cell culture (mouse primary astrocytes, human oligodendroglioma cells) was eluted as a single homogenous fraction, and then analyzed by thin layer chromatography, and further characterized by gas chromatography-mass spectrometry (GC-MS). We detected a unique band that migrated between non-hydroxy fatty acyl ceramide and hydroxy fatty acyl ceramide, and identified it as phytoceramide. Using RT-PCR, we confirmed that mouse tissues expressed desaturase 2, an enzyme that has been reported to generate phytoceramide from dihydroceramide. Previously, only trace amounts of phytoceramide were reported in vertebrate intestine, kidney, and skin. While its function is still elusive, this is the first report of phytoceramide characterization in glial cells and vertebrate brain, heart, and liver.

Quantitative structural multiclass lipidomics using differential mobility: electron impact excitation of ions from organics (EIEIO) mass spectrometry

“…Cers and SMs had a similar acyl chain profiles (Figure 5(a) and (b)), but the galactosylceramides had a different profile (Figure 5c), which was very rich hydroxylated species with the longer chains (29)(30), otherwise known as cerebrosides. Phytoceramides were not observed in our sample at this time though it should be contained in the brain (31).…”

Regulation ofChlamydomonasflagella and ependymal cell motile cilia by ceramide-mediated translocation of GSK3

“…Using liquid chromatography–tandem mass spectrometry (LC-MS/MS), we found that Chlamydomonas generates very long chain C26:0 (11 ± 3 pmol/mg cells) and C28:0 (8 ± 2 pmol/mg cells) phytoceramides, which are common plant sphingolipids ( Figure 2C ). In comparison, glial cells contain relatively small proportions of phytoceramide (<10% of total ceramides; Dasgupta et al. , 2013 ) but larger amounts of ceramide (800 ± 150 pmol/mg cells), with C18:0 ceramide (35%) and C24:1 ceramide (25%) being the most abundant ( Wang et al.…”

Phytosphingosine Increases Biosynthesis of Phytoceramide by Uniquely Stimulating the Expression of Dihydroceramide C4‐desaturase (DES2) in Cultured Human Keratinocytes

“…It is known that phytoceramide (ceramide NP) was produced through conversion of dihydroceramide by DES2 (Mizutani et al, ). Dasgupta’s group found that DES2 was expressed in various tissues such as skin, kidney, and heart, as well as phytoceramide was detected in these tissues (Dasgupta et al, ). In this research, the expression of DES2 was exceptionally increased by PHS treatment.…”