Fluorometric assay using high-pressure liquid chromatography for the microsomal metabolism of certain substituted aliphatics to 1,N6-ethenoadenine-forming metabolites.

Monohaloacetaldehydes and monohalooxiranes are early oxidative metabolites of several carcinogenic haloaliphatics. Since monohaloacetaldehydes and supposedly monohalooxiranes react with adenines to form fluorescent 1,N6-ethenoadenines, it was hypothesized that in vitro metabolic systems that produce an ethenoadenine-forming metabolite could be assayed quantitatively by trapping the metabolite in situ with an adenine and identifying it by its characteristic retention and fluorescence during HPLC. Bromoacetaldehyde was chosen as a model haloacetaldehyde to develop an assay based on this concept for measurements in a microsomal system. The optimal trapping reaction requires a postmetabolic step involving acidification and heating. Cyclic AMP was found to be a suitable adenine for the trapping reaction under these conditions. The chromatographic analysis utilizes tetrabutylammonium phosphate and a nonsilica reversed-phase stationary phase (Hamilton PRP-1). The chromatography is isocratic and allows an analysis time of less than 5 min per sample. The titration of bromoacetaldehyde in a microsomal system is affected by typically studied metabolic conditions: incubation time, pH, and protein concentration. Using this assay, the following were found to be metabolized by rat liver microsomes to etheno-adenine-forming products: 1,2-dibromoethane, 1,2-dichloroethane, cyclophosphamide, vinyl chloride, and acrylonitrile. Chloroacetone and 1,3-dichloroacetone also are fluorochromogenic without metabolism but the latter apparently forms a positively charged, nonetheno adduct. The proposed assay should be useful for in vitro metabolic studies of 1,2-dihaloethanes and mustards and has potential application for similar studies of monohalogenated ethanes, ethanols, and ethenes. The positive results with acrylonitrile suggest also that many types of substituted aliphatics may be studied with this proposed assay.