Detection of metabolites of polycyclic aromatic hydrocarbons in human urine

A non-invasive assay has been developed for the recovery of r-7,t-8,t-9,c-10-tetrahydroxy-7,8,9,10-tetrahydrobenzo[α]-pyrene (BP-7,10/8,9-tetrol) from human urine. This tetrol is excreted as a metabolite of benzo[α]pyrene (BP) in a process catalyzed by cytochrome P450 enzymes and epoxide hydrolases. Urine was hydrolysed to release activated benzo[α]-pyrene-diol-epoxides covalently bound to macromolecular species or conjugated tetrols. The relatively non-polar organic molecules from urine hydrolysates were collected on octadecasilane chromatography columns (Sep-Paks). Materials eluted in solvent (80% CH₃OH), were further purified on immunoaffinity columns with antibodies raised against anti-N2-[10(7,8,9-trihydroxy-7,8,9,10-tetra-hydrobenzo[α]pyrenyl)]-guanosine. HPLC was then used to isolate BP-7,10/8,9-tetrol, which was quantitated by synchronous fluorescence spectroscopy (SFS). This assay detected 0.24-3.12 pmol BP-7, 10/8,9-tetrol per ml urine (limit of detection 0.01 pmol/ml, given 10 ml urine), in four study subjects. Reproducibility was assessed by adding tritium labeled BP-7, 10/8,9-tetrol (1500 fmol) to a urine sample previously identified to contain the tetrol at levels below the limit of detection of the fluorescence assay; a recovery of >30% of the added radioactivity was achieved (510 ± 64 fmol, mean ± SD, n = 3). Because HPLC alone was not sufficient to isolate materials for quantitation by SFS directly from human urine, immunoaffinity chromatography was found to be a necessary preparatory step in BP-7,10/8,9-tetrol isolation. These data demonstrate the presence of tetrahydrotetrol metabolites of BP in human urine and suggest that measurement of BP-7,10/8,9-tetrol and other polycyclic aromatic hydrocarbon-tetrols may prove to be valuable dosimeters of human internal exposure to polycyclic aromatic hydrocarbons.