Difference Decoupling Nuclear Magnetic Resonance: A method to Study the Exchange of Fatty Acids between Phospholipid Molecules

A nuclear magnetic resonance technique has been developed to study the exchange of oleic acid moieties between phospholipid molecules of Escherichia coli. The method relies on the splitting of the NMR spectrum of the proton on position 2 of a diglyceride glycerol backbone caused by a ¹³C atom in the carboxyl of the fatty acid esterified to position 2. By subtracting decoupled from coupled spectra, the amount of diglycerides having ¹³C in the carboxyl of the position 2 fatty acid moiety can be measured. An E. coli strain designed to optimize incorporation of exogenous glycerol and oleate is grown on [2-²H]glycerol and [1-¹³C]oleate (phase 1). Diglycerides prepared from the phospholipids of the cells have no position 2 proton spectrum due to the deuterium substitution. The cells are then switched to a medium having unenriched glycerol and oleate supplements and allowed to continue growth (phase 2). Diglycerides from phospholipids synthesized de novo during growth in the second medium will have no ¹³C splitting. Therefore if a proton resonance with ¹³C splitting is observed, then this splitting must result from transfer of a [1-¹³C] oleate moiety from a phospholipid synthesized in phase 1 to a phospholipid molecule synthesized in phase 2 (no significant intracellular pools of either glycerol or oleate are found in these cultures). We have used these techniques to measure the exchange of oleic acid moieties between phospholipid molecules in both norrrtally growing cultures of E. coli and cultures starved for oleic acid. In neither case did we observe fatty acid exchange. The implications of this result and the general usefulness of the technique are discussed.