Purification of arachidonic acid metabolites from cell cultures by octadecyl reversed phase cartridges.
The use of rat basophilic leukemia (RBL-1) cell-line as a biosynthetic source of arachidonic acid metabolites has been previously described. We have developed a rapid and effective procedure for the extraction of lipoxygenase and cyclooxygenase products. The cells grown in spinner cultures were harvested and washed in phosphate buffer and stimulated by calcium ionophore A23187. The ethanolic extract of the cells was dried, dissolved in 20% methanol acidified with acetic acid and applied to octadecyl reversed phase cartridge (Backer C18). The cartridge was washed with 50% methanol (4 ml). Prostaglandins were eluted with 75% methanol (4 ml) and leukotrienes and related compounds with 100% methanol (4 ml). The identification of the fractions was carried out with pure standards. The recoveries of radiolabelled compounds ranged from 70% to 90%. This system can therefore accomplish an initial separation of major arachidonate metabolites and be applied in investigating metabolic pathways of arachidonic acid in different experimental conditions.
D000818:Animals; D016718:Arachidonic Acid; D001095:Arachidonic Acids / Q000302:isolation & purification* / Q000378:metabolism; D001491:Basophils / Q000378:metabolism; D002458:Cell Fractionation; D002460:Cell Line; D002853:Chromatography, Liquid / Q000379:methods*; D007942:Leukemia, Experimental / Q000378:metabolism; D011453:Prostaglandins / Q000302:isolation & purification; D051381:Rats; D013189:SRS-A / Q000302:isolation & purification; D012821:Silanes
D001095:Arachidonic Acids; D011453:Prostaglandins; D013189:SRS-A; D012821:Silanes; C024779:octadecylsilane; D016718:Arachidonic Acid