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. We present here for the first time evidence that chondrocytes are able to metabolize 2-fluoro-2-deoxy-D-glucose to the UDP-activated sugars UDP-2-fluoro-2-deoxy-D-glucose, UDP-2 fluoro-2-deoxy-D-galactose, and UDP-2-fluoro-2-deoxy-D-glucuronic acid. © 1994 Academic Press, Inc. All rights reserved.