A novel technique for the sensitive quantification of acyl CoA esters fromplant tissues

We report a novel, highly sensitive and selective method for the extraction and quantification of acyl CoA esters from plant tissues. The method detec ts acyl CoA esters with acyl chain lengths from C4 to C20 down to concentra tions as low as 6 fmol in extracts. Acyl CoA esters from standard solutions or plant extracts were derived to their fluorescent acyl etheno CoA esters in the presence of chloroacetaldehyde, separated by ion-paired reversed-ph ase high-performance liquid chromatography, and detected fluorometrically. This derivitization procedure circumvents the selectivity problems associat ed with previously published enzymatic methods, and methods that rely on ac yl chain or thiol group modification for acyl CoA ester detection. The form ation of acyl etheno CoA esters was verified by mass spectrometry, which wa s also used to identify unknown peaks from chromatograms of plant extracts. Using this method, we report the composition and concentration of the acyl CoA pool during lipid synthesis in maturing Brassica napus seeds and durin g storage lipid breakdown in 2-day-old Arabidopsis thaliana seedlings. The concentrations measured were in the 3-6 mum range for both tissue types. We also demonstrate the utility of acyl CoA profiling in a transgenic B. napu s line that has high levels of lauric acid. To our knowledge, this is the f irst time that reliable estimates of acyl CoA ester concentrations have bee n made for higher plants, and the ability to profile these metabolites prov ides a valuable new tool for the investigation of gene function.