Application of Gas Chromatography – Flame Ionization Detection to Study Cellular Incorporation of Dietary Trans Fatty Acids of Medical Importance

Abstract

Putative health effects of dietary trans fatty acids (TFAs) receive a growing attention; while very little is known about the metabolism of these special food components. In vitro studies carried out in cultured cells provide an efficient and standardizable approach to follow the metabolic fate of TFAs, but it requires suitable techniques for the quantitative measurement of FAs in cell samples. Here, the development and validation of a simple and reliable method for the quantification of a group of relevant FAs by gas chromatography and flame ionization detection is presented. Sample preparation used a fast one-step and chloroform-free process for simultaneous extraction and esterification, and chromatographic separation was achieved in 25 min using a Zebron ZB-88 capillary column. A linear calibration (of R2 >0.99) was obtained in the concentration range of 1-200 µg/mL for each FA. Recovery rate was 82 % for samples of non-esterified FAs and >95 % for complex lipids, such as ceramides, diglycerides and triglycerides. The LOD and LOQ were below 0.5 µg/mL, and a robust method precision was achieved (RSD % was below 6 % for each lipid classes). The present method was also tested on a cultured cell line with or without FA treatment at close to physiological concentration, and the observed changes in the metabolite concentration levels revealed characteristic differences between the metabolism of cis and trans unsaturated FAs.

Molecular Mechanisms Underlying the Elevated Expression of a Potentially Type 2 Diabetes Mellitus Associated SCD1 Variant

“…For chromatographic separation of FAMEs, the following oven time-temperature program was used: the flow velocity was 35 mL/min, the initial temperature was 100 °C and reached 210 °C with an increase of 5 °C/min. 1 µL of extracted sample was injected into the GC [ 60 ].…”

Different Metabolism and Toxicity of TRANS Fatty Acids, Elaidate and Vaccenate Compared to Cis-Oleate in HepG2 Cells

“…The cells were suspended in PBS, and the protein concentration of the cell suspension was measured, as mentioned, in Western blot analysis. 50 µL of each suspension was transferred to a clear crimp vial for GC-FID measurement of fatty acids [41], and 50 µL was transferred to a micro-centrifuge tube for the HPLC-MS/MS analysis of TGs, DGs and ceramides [42].…”

“…The injector and detector temperature was 250 • C and the oven temperature was ramped from 100 • C to 210 • C at a rate of 4 • C/min. Quantitative analysis was based on a five-point calibration in the concentration range of 1-200 µg/mL for each FA [41].…”