Abstract
Purpose :
Lipidomic studies on the ocular surface are limited, and it is necessary to characterize molecular metabolomics and its implication in pathobiology. Lipid extraction is a critical step in lipidomic analysis and needs to be adjusted to the study model to maximize the analytical power of the method used. Our aim was to optimize the lipid extraction procedure for lipidomic measurements of human corneal epithelial (HCE) and 3T3-J2 mouse fibroblast cell lines, which are widely used in ocular surface research and cell therapy for the limbal stem cell deficiency, using recently described simpler but efficient and trustable methodologies.
Methods :
Two alternative lipid extraction procedures were evaluated in HCE and 3T3-J2 adherent mammal cell lines. Classical Bligh and Dyer (B&D) lipid extraction method based on methanol and chloroform was compared to Isopropanol (IPA) mediated protein precipitation and lipid extraction method. Lipid extracts were analysed by Ultrahigh Performance Liquid Cromatography-Mass Spectrometry (UHPLC-MS/MS) and data were processed to rate critical aspects of sample preparation: simplicity, lipid coverage, reproducibility and recovery efficiencies.
Results :
In terms of recovery, the single-step IPA procedure yielded better values for most of the lipid classes and it was less toxic and simpler than the liquid-liquid extraction method. Regarding lipid coverage, variation in selectivity was observed between methods, providing evidence that IPA was more selective for polar lipids. The reproducibility measured from the coefficient of variation (CV), defined as the standard deviation divided by the mean intensities of each lipid detected in the 10 replicates, showed that IPA has a higher ability to present similar lipid profiles between samples. Overall, IPA precipitation provides excellent results in terms of simplicity of execution, lipid coverage, reproducibility and recovery.
Conclusions :
This study shows that IPA precipitation can be recommended as a sample preparation method for large-scale untargeted UHPLC-MS lipidomic analysis in cell lines. Therefore, this sample preparation method is suitable for exploring biomedical insights into ocular surface research.
This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.