Determination of the ultraviolet absorbance and radioactivity of purine compounds separated by high-performance liquid chromatography. Application to metabolic flux rate analysis.

A double detection system for the determination of adenine metabolism in biological tissues using isocratic ion-pair reversed-phase chromatography is presented. Two isocratic ion-pair separations were used: (i) 10 mM NH4H2PO4, 2 mM tetrabutylammonium phosphate (PIC reagent A) and 18% acetonitrile for the determination of nucleotides and (ii) 50 mM KH2PO4, 1 mM PIC reagent A and 1% acetonitrile for the determination of monophosphorylated nucleotides, nucleosides and nucleobases. The parallel detection of ultraviolet absorbance at 254 nm and the radioactivity of separated purine compounds allows the detection of pool sizes and of the specific radioactivities in tracer kinetic experiments. The high-performance liquid chromatography methods were applied to the determination of flux rates during adenine nucleotide metabolism in suspensions of Ehrlich mouse ascites tumour cells. The pathways of adenine metabolism in cells during the proliferation and plateau phases of tumour growth were compared.