APPLICATION OF IMPROVED METHODS TO ASSESS PATHWAYS FOR BIOSYNTHESIS OF LONG-CHAIN AND VERY-LONG-CHAIN FATTY-ACIDS

Improved microscale methods for analyzing individual carbon atoms of l ong- and very-long-chain fatty acids are described. These methods allo w quantitation of radiolabel in at least 50% of individual carbon atom s from the carboxyl terminal of such fatty acids. These methods have b een used to study metabolic pathways leading to long-and very-long-cha in fatty acids of seeds. The approach begins with HPLC separation of f ree fatty acids from saponified, total lipid extracts. Subsequently, i ndividual free acids are analyzed by Schmidt degradation to reveal the extent of carboxyl labeling or are subjected to cl-oxidation followed by HPLC separation of free acid homologues. An improvement in the pro cedure for alpha-oxidation results in 0.2-2 times higher recovery of l onger homologues. Carboxy-labeling of homologues is then determined us ing Schmidt degradation. For unsaturated acids, microscale hydrogenati on and HPLC purification are done prior to alpha-oxidation. Results de tail existing evidence that long-and very-long-chain fatty acids of se eds from various oilseed plants are solely formed by fatty-acid-syntha se-mediated reactions. In addition, observations are consistent with t he utilization of more than one pool of precursor oleic acid for synth esis of 20:1 and 22:1 in Brassica seeds.