Characterizing Triacylglycerol in Cell Culture: Can LCMS replace HPTLC when assessing the effect of metformin in salmon in-vitro fatty liver model?

Abstract

Fatty liver, a health complication for aquacultured fish is developed by the deposition of triacylglycerol (TAG) within liver cells. Metformin has been proven to be an effective drug for ameliorating fatty liver in human and rodent model; however, its action in the fish model is yet unknown. The preliminary aim of the present thesis was producing an oleic acidinduced in-vitro salmon fatty liver with excessive TAG accumulation and assessing whether metformin might reduce the TAG level. The cells were analyzed by high performance thin layer chromatography (HPTLC) that indicated the capacity of metformin to attenuate fatty liver. HPTLC has been generally used as a conventional and reliable technique for determining the level of TAG in biological samples. But the determination of individual TAG species and positional distribution of fatty acids on the backbone of TAG demand employing additional method(s) because of the inability of HPTLC to do so. However, due to cost-cutting approach, modern laboratories are in need of finding means to simultaneously measure TAG level and elucidate TAG structure in the same sample by a single technique. Liquid chromatography mass spectrometry (LCMS) has been widely used for structural elucidation of TAG; however, the ability of LCMS to measure the TAG level in salmon liver was not vastly studied. The present thesis, then principally aimed at evaluating the potential of LCMS as an alternative to HPTLC for quantitative analysis of TAG in cell cultures. MATLAB based Chrombox D and QueryTAG algorithmic software tools with several strategies were employed to examine the LCMS chromatogram and spectrum. An MS Excelbased macro-enabled tool has been developed for rapid and easy processing of Chrombox D data for multiple samples. The multivariate chemometric analysis was applied in further data exploration to understand significant effects of the variables. Most of the data analysis strategies indicated that LCMS can reproduce the outcome patterns of HPTLC for some treatments with the experimental condition for one hour with metformin exposure followed by incubation with oleic acid up to 48 hours. Therefore, it can be postulated that LCMS is a potential alternative to the traditional HPTLC for TAG analysis in modern analytical and research laboratories.