P38 Investigating the biosynthesis of 6-hydroxyceramides in the stratum corneum
Hawra Alsalman, Alexandra Kendall, Catherine O’Neill, Anna NicolaouAbstract
Introduction and aims
The stratum corneum (SC) is a key component of the epidermal permeability barrier. It is composed of terminally differentiated keratinocytes in lipid lamellae, containing cholesterol, ceramides and free fatty acids. Ceramides are esters of sphingoid bases; to date, > 1200 species belonging to 12 classes were reported in SC. The ceramides are derivatives of sphingosine (S), dihydrosphingosine (DS), phytosphingosine (P), 6-hydroxysphingosine(H) and 4,14-sphingadiene (SD). Although ceramide biosynthesis has been studied, enzymes producing 6-hydroxysphingosine ceramides (CER[H]) are unknown. As SC ceramides are vital for skin barrier function, we explored potential biochemical pathways leading to CER[H].
Methods
Rat epidermal keratinocyte cultures were air-lifted to form SC. Three dimensional (3D) cultures were treated with CER[N(8)DS(18)]; its metabolism to CER[N(8)S(18)], CER[N(8)P(18)], CER[N(8)H(18)] and CER[N(8)SD(18)] was monitored by lipidomics using ultrahigh performance liquid chromatography coupled to electrospray ionization tandem mass spectrometry (UPLC/ESI-MS/MS) and multiple reaction monitoring. Cultures were treated with fenretinide [inhibitor of dihydroceramide desaturases 1/2]. Immunohistochemistry (ki67; keratin14) were used to assess the SC.
Results
3D cultures formed SC as assessed by ki67 and keratin14 expression. UPLC/ESI-MS/MS ceramide profiling confirmed that 3D cultures formed all ceramides of interest, including CER[H]. Although CER[N(8)DS(18)] was metabolized to CER[N(8)S(18)] and CER[N(8)P(18)], CER[N(8)H(18)] was not detected. Fenretinide reduced utilization of CER[N(8)DS(18)] and impacted CER[N(8)S(18)] production, but did not induce CER[N(8)H(18)] formation.
Conclusions
Findings indicate that CER[H] species are not formed via metabolism of precursor CER[NDS]. Desaturation of CER[S] and CER[P] did not appear to be involved in CER[H] biosynthesis either. Alternative targets should be considered.