抄録
Simplified method for extracting leukotrienes (LT's) from cell culture medium was developed, and conditions for their analysis by HPLC were studied. LT, s released into the medium from A23187-stimulated human WBC's and rat alveolar macrophages were extracted and concentrated by passage through a Sep-Pak C18 cartridge after deproteinization with methanol. Human WBC's as well as rat alveolar macrophages secreted only LTC4 and LTB4 into the incubation medium. Productions of LTB4 and LTC4 were dose-dependently stimulated by calcium ionophre (A23187) at up to 10-5M (final conc.) Production of LTB4 reached the maximum value after 15 min incubation and then decreased. On the other hand, the amount of LTC4 in the medium increased throughout the incubation period of 30min. The appropriate conditions for HPLC analysis on three different ODS columns were determined by changing the ratio of solution A in the eluent (A, acetonitrile; B, Methanol: H2O: acetic acid/100: 300: 1, pH 5.6, containing 0.08% EDTA) from 29 to 34% with the use of 2 pumps. Retention times were prolonged and peaks of LT's flattened and widened by decreasing the percentage of solution A in the eluent. And by increasing the ratio of solution A, the peaks became sharper and taller and retention times of LT's shortened, however, separation of peaks became incomplete. The best resolution was obtained with a Novapak C18 column and eluent containing 32-33% solution A. Recovery ratios of standard LT's (LTB4, LTC4, LTD4) were more than 95% except for LTE4 whose recovery was 67.1% through the whole procedures of extraction and HPLC analysis. Relationship between area of peak and amount of LT was linear from 0.5 up to 4ng of each standard sample. We conclude that measurement of LT's by HPLC depends in large part on the quality of the HPLC column, and we obtained the best result by the procedures described here in using a Novapak C18 column.
