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Quantification of Fatty Acids in Mammalian Tissues by Gas Chromatography-Hydrogen Flame Ionization Detection.

Bio-protocol (2021-03-05)
Fumie Hamano, Suzumi M Tokuoka, Tomomi Hashidate-Yoshida, Hideo Shindou, Takao Shimizu, Yoshihiro Kita
ABSTRACT

In mammalian organisms, fatty acids (FAs) exist mostly in esterified forms, as building blocks of phospholipids, triglycerides, and cholesteryl esters, while some exist as non-esterified free FAs. The absolute quantification of FA species in total lipids or in a specific lipid class is critical in lipid-metabolism studies. To quantify FAs in biological samples, gas chromatography-hydrogen flame ionization detection (GC-FID)-based methods have been used as highly robust and reliable techniques. Prior to GC-FID analysis, FAs need to be derivatized to volatile FA methyl esters (FAMEs). The derivatization of unsaturated FAs using classical derivatization methods that rely on high reaction temperature requires skill; consequently, the quantification results are often unreliable. The recently available FA-methylation procedure rapidly and reliably derivatizes a variety of FA species, including poly-unsaturated FAs (PUFAs). To analyze FAs in mammalian tissue samples, lipid extraction and fractionation are also critical for robust analysis. In this report, we describe a whole protocol for the GC-FID-based FA quantification of mammalian tissue samples, including lipid extraction, fractionation, derivatization, and quantification. The protocol is useful when various FAs, especially unsaturated FAs, need to be reliably quantified.

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
Tricosanoic acid, ≥99% (capillary GC)
Supelco
Supelco 37 Component FAME Mix, certified reference material, TraceCERT®, in dichloromethane (varied conc.), ampule of 1 mL
Sigma-Aldrich
Dulbecco′s Phosphate Buffered Saline, 10×, Modified, without calcium chloride and magnesium chloride, liquid, sterile-filtered, suitable for cell culture