Quantitative isolation of radiolabeled metabolites without chromatography: Measurements of the biosynthesis of purines, pyrimidines, and urea in isolated hepatocytes

Measurements of the incorporation of radiolabeled precursors into urea, orotic acid, uridine nucleotides, and adenine nucleotides were used to demonstrate the reliability of procedures for the isolation of metabolites by cocrystallization with carrier. Incorporation of precursor into product was carried out with suspensions of isolated hepatocytes. Direct isolation of each radiolabeled product was accomplished essentially by saturating aliquots of the acid-soluble fraction of the incubation mixture with carrier at elevated temperatures, and allowing the radiolabeled metabolite and carrier to cocrystallize as the solutions cooled. When [ 14C]NaHCO 3 was employed as the precursor, the procedure permitted isolation of radiolabeled urea, orotic acid, and uridine nucleotides from the same incubation mixture. Evidence that the radiolabeled metabolite isolated with carrier was of the same chemical identity as the carrier was obtained by recrystallization to constant specific activity and also by the use of enzymes to remove the putative metabolite prior to cocrystallization. Conditions were varied to provide a range of rates of 8.4-fold, 180-fold, and 13-fold for the incorporation of [ 14C]NaHCO 3 into urea, orotic acid, and uridine nucleotides, respectively, and the results of duplicate assays at six different rates over these ranges of activity showed individual determinations to differ from their means by 3.2 ± 1.0, 1.1 ± 0.3, and 3.3 ± 1.5% (average ± SE), respectively. The method of isolation by cocrystallization with carrier has proved inexpensive, reliable, and well suited to the simultaneous analysis of multiple samples. The advantages and limitations of these procedures over more conventional methods are discussed.