SIMULTANEOUS MEASUREMENT OF DESATURASE ACTIVITIES USING STABLE-ISOTOPE TRACERS OR A NONTRACER METHOD

We report here methods for simultaneous determination of in vitro desa turase activities using stable isotope tracers or using a nontracer me thod in place of radiotracers. Rat microsomal Delta(6)- and Delta(9)-d esaturase activities were assayed using standard incubation medium by monitoring the increase in product due to the reactions [U-C-13]18:2n- 6 --> [U-C-13]18:3n-6 or [U-C-13]16:0 --> [U-C-13]16:1n-7, respectivel y, The stable isotope measurements were made by high-precision gas chr omatography-combustion isotope ratio mass spectrometry. Reaction-depen dent changes in product or substrate concentrations were also monitore d quantitatively by conventional capillary gas chromatography with fla me-ionization detection. Desaturase activities calculated from these d ata are consistent with tracer results. Microsomal Delta(5)-desaturase activity was monitored by quantifying the decrease in unlabeled subst rate mass using the reaction 20:3n-6 --> 20:4n-6. All activities showe d the expected dependencies on time, temperature, pH, and microsome an d substrate concentrations and were well within the range of published activities. Activities in brain, liver, kidney, and heart microsome p reparations measured with either labeled or nonlabeled substrate were not significantly different, but were highly significantly different f rom organ to organ. These methods demonstrate a means to measure desat urase activities conveniently without radiotracers and for reactions i nvolving substrates which are not available in labeled form. (C) 1998 Academic Press.