The use of biologically prepared radioactive indicators in metabolic studies: metabolism of pentobarbital.

Although isotopic labeling has been of great use in studies of drug metabolism, the necessarily small amounts of material used in such experiments often preclude the identification of unknown metabolites by conventional chemical means. Such difficulties are commonly circumvented by adding a known non-labeled compound, suspected to be a metabolite, to the unknown mixture and reisolating the substance in labeled form. The isotope dilution technique, however, involves guesses about the unknown structures which may not be correct, especially when metabolic alteration has obscured the relationship between precursor and metabolite. There are a number of recorded instances in which difficulties in predicting structure have hampered or prevented the identification of unknown derivatives (2, 3). The use of biologically prepared radioactive indicators to guide the isolation of sufficient material for direct chemical identification offers one means of avoiding structural assumptions. The indicators are prepared as radiochemically pure entities by microfractionation (usually paper chromatography) of the labeled metabolites obtained after administering a radioactive precursor of high specific activity. A single indicator may be added to urine or other large pools of degradation products from animals receiving the unlabeled parent compound, and its radioactivity used to guide fractionation. As in the conventional isotope dilution procedure, isolation of radioactive material satisfying the criteria of homogeneity demonstrates the derivation of the product from the parent substance. When the desired products are present in low concentration, very large pools of material may have to be used to provide tangible quantities in the final isolation. By means of counter-current distribution (4), separations carried out on a small aliquot of the pooled metabolites may be exactly duplicated with any desired volume. Parallel distributions of a small indicator-labeled aliquot and the main body of material may t,hus be run