TY - JOUR
T1 - Quantitative analysis of prostaglandins in cell culture medium by high-resolution gas chromatography with electron-capture detection
AU - Berens, Michael E.
AU - Salmon, Sydney E.
AU - Davis, Thomas P.
N1 - Funding Information:
This study was supported by Grants CA 17094, CA 21839, from the National Institute of Health, Bethesda, MD, U.S.A.
PY - 1984
Y1 - 1984
N2 - Prostaglandins have been shown to be important modulators of haemostatis, immune responses, and growth of normal and neoplastic cells. In order to investigate the cell origin and metabolic profile of the endogenous prostaglandins in human tumours, a convenient extraction and gas chromatographic method for measuring the various classes of prostaglandins was developed. Infiltrating macrophages from human tumours were isolated using adherence to plastic. Macrophage-enriched and macrophage-depleted cell populations were then cultured in vitro and the media supernatant was studied for the presence of prostaglandins E1, E2, F2α, and 6-keto-F1α (the spontaneous breakdown product of prostacyclin, PGI2). Routinely, 1 ml of medium containing 106 cells was studied. The eicosanoids were extracted using commercially available octadecylsilyl silica reversed-phase columns prior to derivatization. Standards and samples were prepared as pentafluorobenzyl ester (methoxime) trimethylsilyl ether derivatives for analysis on an OV-101 (25 m × 0.2 mm) fused-silica capillary column. Recovery of standards ranged from 93% to 37%, with linear recovery in all instances (regression coefficients greater than 0.98). Detection limits were 20 pg for each of the prostaglandins. Analysis of cell subpopulations from six human tumours revealed that infiltrating macrophages produce various prostaglandin profiles and are largely responsible for the prostaglandin production in human cancer. The described analytical method is the first application of high-resolution gas chromatography with electron-capture detection to the quantitative profiling of prostaglandins from human cell culture.
AB - Prostaglandins have been shown to be important modulators of haemostatis, immune responses, and growth of normal and neoplastic cells. In order to investigate the cell origin and metabolic profile of the endogenous prostaglandins in human tumours, a convenient extraction and gas chromatographic method for measuring the various classes of prostaglandins was developed. Infiltrating macrophages from human tumours were isolated using adherence to plastic. Macrophage-enriched and macrophage-depleted cell populations were then cultured in vitro and the media supernatant was studied for the presence of prostaglandins E1, E2, F2α, and 6-keto-F1α (the spontaneous breakdown product of prostacyclin, PGI2). Routinely, 1 ml of medium containing 106 cells was studied. The eicosanoids were extracted using commercially available octadecylsilyl silica reversed-phase columns prior to derivatization. Standards and samples were prepared as pentafluorobenzyl ester (methoxime) trimethylsilyl ether derivatives for analysis on an OV-101 (25 m × 0.2 mm) fused-silica capillary column. Recovery of standards ranged from 93% to 37%, with linear recovery in all instances (regression coefficients greater than 0.98). Detection limits were 20 pg for each of the prostaglandins. Analysis of cell subpopulations from six human tumours revealed that infiltrating macrophages produce various prostaglandin profiles and are largely responsible for the prostaglandin production in human cancer. The described analytical method is the first application of high-resolution gas chromatography with electron-capture detection to the quantitative profiling of prostaglandins from human cell culture.
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U2 - 10.1016/S0378-4347(00)84097-9
DO - 10.1016/S0378-4347(00)84097-9
M3 - Article
C2 - 6429180
AN - SCOPUS:0021242951
SN - 0378-4347
VL - 307
SP - 251
EP - 260
JO - Journal of Chromatography B: Biomedical Sciences and Applications
JF - Journal of Chromatography B: Biomedical Sciences and Applications
IS - C
ER -