Abstract
This chapter presents a procedure for the purification and characterization of leukotrienes from mastocytoma cells. Two procedures can be used to purify leukotrienes from CXBGABMCT-1 cells, one for large-scale incubations and another for smaller incubations. In method I, the large-scale procedure involves an initial adsorption to XAD resin followed by silicic acid chromatography and two steps of reversed- phase high-pressure liquid chromatography (reversed-phase HPLC). In method II, small incubation volumes of CXBGABMCT-1 cells are amenable to a much simplified leukotriene isolation procedure. After the XAD-7 adsorption step, the ethanol eluent is evaporated to dryness and dissolved in 2 ml of 30% methanol. It is often necessary to centrifuge (10,000 g) this sample to remove insoluble material. This is injected directly onto an analytical Nucleosil Cls column, and leukotrienes are eluted with methanol-water-acetic acid mobile phase as indicated by the UV monitor set at 280 nm. This procedure permits the isolation and quantitation of both LTC4 and LTB4.
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