Isotope effects in enzymatic and non-enzymatic oxygenation of 6,9,12-octadecatrienoic acid

Synthesis of intramolecularly labeled 6,9,12-[11,11-2H2,1-14C]octadecatrienoic acid is described. The doubly labeled acid was added to 6,9,12-[9,10-3H2]octadecatrienoic acid and the mixture was incubated with soybean and human platelet lipoxygenases and was subjected to autoxidation. Methyl 13-hydroxyoctadecanoate obtained in the three sets of experiments had a considerably reduced 14C/3H ratio compared to that of the starting material, i.e., 20-24% (autoxidation) and 12-15% (lipoxygenase oxygenations). This indicates that subtraction of a hydrogen atom from the methylene group of the cis,cis-pentadiene structure is the rate-limiting step in autoxidation as well as in the enzymatic oxygenations.