A radiochemical high-performance liquid chromatographic method for the analysis of 2-fluoro-2-deoxy-D-glucose-derived metabolites in human chondrocytes.
A high-performance liquid chromatography method with on-line radioactivity monitoring was developed for the measurement of 2-fluoro-2-deoxy-D-[U-14C]-glucose-derived metabolites in a cell culture system of human chondrocytes embedded in soft agarose. To optimize the chromatographic procedure, glucose-analogous substrates derived from 2-fluoro-2-deoxy-D-glucose by enzymatic synthesis in vitro were used. The synthesized metabolites could be separated by anion-exchange chromatography on a Partisil 10 SAX cartridge with a LiChrosorb RP 18-5 guard column eluted with a 35-min ion-strength/pH gradient performed from 15 mM NH4H2PO4, pH 3.8, to 0.75 M NH4H2PO4, pH 4.8, at a flow rate of 2 ml/min. Only by using an on-line radioactivity monitor instead of an off-line counting procedure was the resolution obtained sufficient for the determination of these intermediates. This method was applied to studying the metabolic pathway of 2-fluoro-2-deoxy-D-glucose in human chondrocytes. Due to the resistance of the chondrocytes embedded in soft agarose, the usual cell-lysing methods could not be used; therefore, an extraction procedure for acid-stable glucose metabolites, which may also be applied to other resistant cell lines or critical cell culture systems, was developed. With the procedure presented here, the existence of metabolites of 2-fluoro-2-deoxy-D-glucose resulting from enzymatic reactions following the hexokinase reaction could be proven.(ABSTRACT TRUNCATED AT 250 WORDS)
Fedders
G
G
Institute of Clinical Chemistry and Pathobiochemistry, Medical Faculty, University of Technology Aachen, Germany.
Kock
R
R
Van de Leur
E
E
Greiling
H
H
D000328:Adult; D002250:Carbon Radioisotopes; D002356:Cartilage / Q000166:cytology / Q000378:metabolism*; D002478:Cells, Cultured; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D003847:Deoxyglucose / Q000031:analogs & derivatives* / Q000378:metabolism; D019788:Fluorodeoxyglucose F18; D006801:Humans; D008297:Male; D011854:Radiochemistry; D014531:Uridine Diphosphate Galactose / Q000031:analogs & derivatives / Q000378:metabolism; D014535:Uridine Diphosphate Glucuronic Acid / Q000031:analogs & derivatives / Q000378:metabolism
D002250:Carbon Radioisotopes; D019788:Fluorodeoxyglucose F18; C081992:UDP-2-fluoro-2-deoxygalactose; C081993:UDP-2-fluoro-2-deoxyglucuronic acid; D014535:Uridine Diphosphate Glucuronic Acid; D014531:Uridine Diphosphate Galactose; C041530:uridine-2-deoxy-2-fluoro-D-glucose diphosphate ester; D003847:Deoxyglucose