An improved rapid procedure to determine the fatty acid composition of glycerolipids is described. The procedure includes KOH-catalyzed transesterification and high-speed gas chromatography. Glycerolipids (20-40 mg) were mixed with 2 mL of hexane and 0.2 mL of 2 M methanolic KOH at room temperature for 1-2 min. The fatty acid methyl esters in the hexane layer were analyzed by gas chromatography on 10% SP-2340 at 240 degrees C. Methyl linolenate and docosahexaenoate eluted within 2 and 5 min, respectively. Analysis was thus completed within 5 min for common vegetable oils and 8 min for fish oils.
When pDHA4, a vector carrying all five pfaA-pfaE genes responsible for docosahexaenoic acid (DHA; 22:6) biosynthesis in Moritella marina MP-1, was coexpressed in Escherichia coli with the individual pfaA-pfaD genes for eicosapentaenoic acid (EPA; 20:5) biosynthesis from Shewanella pneumatophori SCRC-2738, both polyunsaturated fatty acids were synthesized only in the recombinant carrying pfaB for EPA synthesis. Escherichia coli coexpressing a deleted construct comprising pfaA, pfaC, pfaD and pfaE for EPA and pfaB for DHA produced EPA and DHA. Both EPA and DHA were detected in bacteria that inherently contained pfa genes for DHA. These results suggest that PfaB is the key enzyme determining the final product in EPA or DHA biosynthesis.
We applied a gas chromatography-mass spectrometry (GC-MS) method using dimethyl disulfide (DMDS) adducts and were able to determine the double-bond positions in monounsaturated 2-hydroxy fatty acids (2-HFA). 2-HFA methyl esters, prepared from the hydrolysate of Arabidopsis thaliana leaf glucosylceramides, were acetylated and methylthiolated. GC-MS analysis of the resulting DMDS adducts showed simple mass spectra with recognizable molecular ions and a series of key fragment ions indicating the original double-bond positions in the aliphatic chain. Based on this GC-MS elucidation, we confirmed that Arabidopsis leaf glucosylceramides have C22, C23, C24, C25, and C26 chain length 2-HFA with monounsaturation, and all their double bonds are placed at the n-9 position. This procedure is simple, time efficient, and highly sensitive.
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