Chromatographic separation of autofluorescent and quenching substances from the hydrocarbon in tissue

In spite of the recent progress in fluorometry, the measurement of carcinogenic hydrocarbons in tissue has been carried out with insufficient accuracy because of the presence of substances in the tissue interfering with fluorometry. Such substances, autofluorescent and quenching substances, were removed from ethanol extracts of the tissue by Sephadex LH-20 column chromatography. By elution of the column with ethanol, the interfering substances appeared in fractions of the void volume for 3-methylcholanthrene chromatography. In addition, the excitation wavelength for autofluorescent substances was found to be far from that for 3-methylcholanthrene, for which the excitation was at 300 nm and the luminescence was measured at 400 nm. Chromatographic separation of 3-methylcholanthrene and its metabolites in the bile and veces obtained from rats was done and only one peak consisting of conjugated metabolites was found on the chromatogram of the bile, though three peaks consisting respectively of the parent hydrocarbon with some metabolites, nonconjugated metabolites, and conjugated metabolites were observed in the feces. Total amounts of 3-methylcholanthrene and its metabolites, though the majority of the fluorescent substances were the parent hydrocarbon, in the lung and other organs were measured at various intervals after an intrabronchial application of 1 or 5 mg of 3-methylcholanthrene in Freund's incomplete adjuvant to rats. Preservation of the hydrocarbons in the treated lung was confirmed and this was considered to take part in the enhanced occurrence of epidermoid tumors in the lung.