Application of improved methods to assess pathways for biosynthesis of long- and very-long-chain fatty acids.

摘要

Improved microscale methods for analysing the individual carbon atoms of long- and very-long-chain fatty acids are described, which allow the quantification of radiolabelling in at least 50% of individual carbon atoms from the carboxyl terminal of such fatty acids. The approach is demonstrated using fatty acids from developing Arabidopsis thaliana, soyabeans cv. Elgin and rape seeds to examine the acetate labelling patterns of lcFA (C16-C18) and vlcFA (C20 and C22) fatty acids. The method is started by HPLC separation of free fatty acids from saponified, total lipid extracts, and subsequently, individual free acids are analysed by Schmidt degradation to reveal the extent of carboxyl labelling or are subjected to alpha-oxidation followed by HPLC separation of free acid homologues. An improvement in the procedure for alpha-oxidation resulted in 0.2-2 times higher recovery of longer homologues. Carboxy-labelling of homologues was then determined using Schmidt degradation. For unsaturated acids, microscale hydrogenation and HPLC purification were conducted prior to alpha-oxidation. It is concluded that long- and very-long-chain fatty acids from these seeds were formed solely by fatty-acid-synthase-mediated reactions. In addition, the observations are consistent with the utilization of more than one pool of precursor oleic acid for the synthesis of 20:1 and 22:1 in rape seeds.