Wijesinghe et. al. 2014 wanted to investigate the possibility that the CERK-null and wild-type MEFs demonstrated a difference in the amount of eicosanoids produced in the skin following a punch biopsy. Punch biopsies of 10 mm thickness were taken from the shaved dorsums of both wild-type and ablated mice immediately following euthanization. Punch biopsies are when a sharp cutting tool is used to extract a piece of skin and the skin is then further examined. In this case, the skin from the mice was transferred to a culture medium that was then incubated in a medium for 2 and 4 hours. The conditions in which they were incubated include a medium with 2% serum, similar to the first experiment in this section. The eicosanoids that traveled from the skin to the medium were quantified at both 2 hours and 4 hours after the punch biopsy. These eicosanoids were quantified by HPLC ESI-MS/MS, or High-performance liquid chromatography and electrospray ionization mass spectroscopy and the measurement was in nanograms lipid per centimeter of excised skin. See support page 3 for more information on HPLC and ESI-MS/MS.

As a result, the CERK-null mice showed a significant decrease in the amount of arachidonic acid, PGE₂, 5-HETE, 11-HETE, and 15-HETE. PGE₂ and the HETE previously mentioned are specific eicosanoid types. Although there was a significant difference between the production of those eicosanoids in both ablated and wild type mice, the other types of eicosanoids (PGF₂a, 6-keto PGF₁a, and 12-HETE) did not show significant differences. This experiment supported the idea that ablation of ceramide kinase in these mice affected the production of specific eicosanoids when compared to the wild-type mice. This is significant because it shows the importance of ceramide-1-phosphate derived from ceramide kinase in the eicosanoid synthesis response following a wound in the skin.

Fig. 4: Eicosanoid secretion measured in nanograms lipid per square centimeter of skin.