Lipidomics Reveals Triacylglycerol Accumulation Due to Impaired Fatty Acid Flux in Opa1-Disrupted Fibroblasts.

OPA1 is a dynamin GTPase implicated in mitochondrial membrane fusion. Despite its involvement in lipid remodeling, the function of OPA1 has never been analyzed by whole-cell lipidomics. We used a nontargeted, reversed-phase lipidomics approach, validated for cell cultures, to investigate OPA1-inactivated mouse embryonic fibroblasts ( Opa1 -/- MEFs). This led to the identification of a wide range of 14 different lipid subclasses comprising 212 accurately detected lipids. Multivariate and univariate statistical analyses were then carried out to assess the differences between the Opa1 -/- and Opa1 +/+ genotypes. Of the 212 lipids identified, 69 were found to discriminate between Opa1 -/- MEFs and Opa1 +/+ MEFs. Among these lipids, 34 were triglycerides, all of which were at higher levels in Opa1 -/- MEFs with fold changes ranging from 3.60 to 17.93. Cell imaging with labeled fatty acids revealed a sharp alteration of the fatty acid flux with a reduced mitochondrial uptake. The other 35 discriminating lipids included phosphatidylcholines, lysophosphatidylcholines, phosphatidylethanolamine, and sphingomyelins, mainly involved in membrane remodeling, and ceramides, gangliosides, and phosphatidylinositols, mainly involved in apoptotic cell signaling. Our results show that the inactivation of OPA1 severely affects the mitochondrial uptake of fatty acids and lipids through membrane remodeling and apoptotic cell signaling.

Comparison of sample preparation methods, validation of an UPLC-MS/MS procedure for the quantification of tetrodotoxin present in marine gastropods and analysis of pufferfish.

Tetrodotoxin (TTX) is one of the most potent marine neurotoxins reported. The global distribution of this toxin is spreading with the European Atlantic coastline now being affected. Climate change and increasing pollution have been suggested as underlying causes for this. In the present study, two different sample preparation techniques were used to extract TTX from Trumpet shells and pufferfish samples. Both extraction procedures (accelerated solvent extraction (ASE) and a simple solvent extraction) were shown to provide good recoveries (80-92%). A UPLC-MS/MS method was developed for the analysis of TTX and validated following the guidelines contained in the Commission Decision 2002/657/EC for chemical contaminant analysis. The performance of this procedure was demonstrated to be fit for purpose. This study is the first report on the use of ASE as a mean for TTX extraction, the use of UPLC-MS/MS for TTX analysis, and the validation of this method for TTX in gastropods.