31] Desaturation of long-chain fatty acids by animal liver

Abstract

Publisher Summary This chapter discusses the determination of desaturation of long-chain fatty acids by animal liver. Stearyl-CoA desaturase activity is estimated from the conversion of [1- 14 C]stearyl-CoA into [1- 14 C]oleate. The incubation mixture is saponified and the liberated [1- 14 C]stearate and [1- 14 C]oleate are separated by argentation thin-layer chromatography. An alternative assay based on formation of 3 H 2 O from [9,10 3 H 2 ]stearyl-CoA is described. One unit of desaturase is defined as the amount of protein catalyzing the formation of 1 μmole of oleate per minute under the conditions of the assay. Specific activity is expressed in milliunits per milligram of protein. For the process of resolution of desaturase, livers from old laying hens are collected in ice at the slaughterhouse. The livers may be kept on ice overnight or processed the same day. Three-hundred grams of liver are blended for 30 seconds with 450 ml of 0.1 M potassium phosphate buffer (pH 7.2 at 0°) in a Waring blender at 5°. Hen liver microsomes desaturate saturated fatty acids of chain lengths 12 through 22 to the corresponding cis -Δ 9 -monoenoic acid. Maximum desaturation occurs with C 14 and C 18 fatty acids. The hydrogens abstracted during the conversion of stearic to oleic acid are cis and are both of the D configurations.