ascorbic-acid and 3-hydroxybenzo(a)pyrene

Previous studies examined the bioavailability and first-pass biotransformation of 3-hydroxy[(3)H]benzo[a]pyrene ([(3)H]-3-OHBaP) in an isolated perfused catfish intestinal model. This work showed that 3-OHBaP, or a metabolite formed in intestine, bound covalently to blood protein. In this study, the blood adducts were characterized in vitro by incubating bovine ferric hemoglobin or albumin with [(3)H]-3OHBaP under various conditions. Incubation of 2 microM [(3)H]-3-OHBaP with hemoglobin for 1 h resulted in 7.49 pmol bound/mg protein, while albumin binding was 1.37 pmol/mg protein. Mild acid hydrolysis released only 5% of the radioactivity from 3-OHBaP-hemoglobin adducts. After gel filtration, the 3-OHBaP-hemoglobin adducts were examined by HPLC analysis. A single peak of radioactivity was detected at the same retention time as the heme component of hemoglobin. Unbound 3-OHBaP was oxidized to BaP-3,6-dione during incubation with ferric hemoglobin. Treatment of hemoglobin with ascorbic acid decreased the formation of hemoglobin adducts by 33%, while hydrogen peroxide treatment increased adduct formation by 44%. Incubation of [(3)H]-BaP-3-beta-D-glucuronide (BaP-3G) with hemoglobin and beta-glucuronidase resulted in greater binding to hemoglobin than incubation with [(3)H]-3-OHBaP alone. The hemoglobin adduct obtained from [(3)H]-BaP-3G also co-migrated with heme. These results indicate that an oxidative process is involved in formation of the heme adduct and that 3-OHBaP or BaP-3G might be a precursor of the bound metabolite.

Topics: Animals; Ascorbic Acid; Benzopyrenes; Cattle; Chromatography, High Pressure Liquid; Glucuronidase; Hemoglobins; Hydrogen Peroxide; Protein Binding; Serum Albumin; Time Factors; Tritium

An improved high-performance liquid chromatographic (HPLC) method for the determination of 3-hydroxybenzo(a)pyrene (3-OHBaP) in urine was developed. The sensitivity and reproducibility of the technique was greatly improved by the addition of 1 mg/L ascorbic acid to the methanol eluent of the HPLC system. This procedure also eliminated the peak splitting and band broadening of the 3-OHBaP peak otherwise observed. Furthermore, it corrected the urine matrix effect on the slope of standard curves. In fact, in the absence of ascorbic acid in the HPLC system, slopes of standard curves were steeper when prepared in a methanolic extract of control rat urine (121 L.nmol-1) than in methanol only (86 L.nmol-1). Both these slopes were smaller than that obtained with the modified mobile phase (244 L.nmol-1). The effect of the latter on the shape and intensity of the 1-hydroxypyrene (1-OHP) chromatographic peak was also investigated. Again, slopes were greater when the standards, prepared in a methanolic extract of urine, were chromatographed with ascorbic acid (380 L.nmol-1) than without (157 L.nmol-1). Therefore, it seems that ascorbic acid, like certain substances in urine, may act by masking specific adsorption sites--probably uncapped silanol residues on the LC 18 column that can retain free 3-OHBaP and 1-OHP metabolites.

Topics: Animals; Ascorbic Acid; Benzopyrenes; Chromatography, High Pressure Liquid; Rats; Urine