On the In Vitro and In Vivo Protein-binding of N-2-Fluorenylacetamide and Related Compounds

Abstract

Data are presented on the distribution of radioactivity after one intraperitoneal injection of C¹⁴-labeled N-2-fluorenylacetamide (2-FAA) into rats as a function of dose, from 10 to 0.01 mg/100 g, and of time, from 6 hours to 32 days. The urinary metabolites were qualitatively and quantitatively similar at all time intervals and dosages. The activity in blood, liver, and soluble and insoluble liver proteins decreased with decreasing doses, but not directly proportionally. The isotope in blood and liver, highest at 6 hours, dropped more rapidly in liver than in blood. Maximal labeling of the liver proteins occurred at 1 day. Hydrolysis of the labeled proteins by acid, alkali, or enzymes rendered 50 to 60 percent of the activity extractable into organic solvents. Details of the solvent partition and chromatographic experiments performed on the hydrolysate are presented. Amounts of 0.3 and 0.7 percent of the activity in the labeled protein were due to 2-FAA and 2-fluorenamine, respectively. The balance consisted of at least 6 more polar materials. Thus, with 2-FAA more than one metabolite and protein are involved in the labeling of tissues. Homogenization of rat liver with labeled 2-FAA or hydroxylated metabolites under various conditions gave negligible binding. However, the aminofluorenols studied led to considerable “protein-bound” activity by homogenization or by the mixture of soluble proteins with a solution of the compound. This artifact-binding took place to a larger extent with the ortho-aminofluorenols than with 7-amino-2-fluorenol. In contrast to the binding in vivo, no radioactivity was rendered extractable into organic solvents after hydrolysis of the proteins labeled in vitro. Artifact-binding seems to play only a limited role under conditions in vivo.