© 1992 Oxford University Press
research-article |
ortho-Toluidine Blood Protein Adducts: HPLC Analysis with Fluorescence Detection after a Single Dose in the Adult Male Rat1
Department of Health and Human Services, Public Health Service, Centers for Disease Control, National Institute for Occupational Safety and Health, Division of Biomedicai and Behavioral Science 4676 Columbia Parkway. Cincinnati, Ohio 45226
Received April 15, 1991; accepted November 12, 1991
Hemoglobin (Hb) and albumin (Alb) adducts of the suspect human carcinogen ortho-toluidine (OT) were quantified in blood samples collected from rats after a single i.p. injection. Mild alkaline hydrolysis of Hb adducted with [14C]OT followed by extraction with ethyl acetate resulted in recovery of 63% of the bound radioactivity. HPLC analysis revealed a single radiolabeled peak which was identified as OT by GC-MS. In subsequent experiments Hb and Alb adduct levels were determined by HPLC analysis of this cleavage product using fluorescence detection. 4-Ethylaniline was used as internal standard. The detection limit for OT was approximately 450 pg/injection or 5 pmol/mg Hb. Mean adduct levels for Hb increased rapidly over the first 4 hr with the highest (ng/mg Hb ± SD) 3.7 ± 0.5 detected 24 hr after OT administration at 50 mg/kg body wt. In contrast, adduct levels for pooled Alb samples increased from 0.7 ng/mg Alb at 2 hr to 2.5 ng/mg Alb at 4 hr, but were not detectable 24 hr after dosing. Hb adducts showed a linear relationship for OT doses of 10, 20, 40, 50, and 100 mg/kg body wt. The Hb adduct t
(11 days) was determined after a single 100 mg/kg OT dose. Hb adduct levels were quantifiable (1.3 ± 0.2 ng/mg Hb) by HPLC/fluorescence 28 days after 100 mg/kg OT. Although Hb and Alb adducts differ in stability, a ratio of such OT adducts may be useful in long-term industrial biomonitoring for evaluation of OT exposure.