Measurement of 1-Nitropyrene Metabolites in Human Urine: A Potential Marker for Exposure to Diesel Exhaust

Abstract

P-156 Introduction: Diesel exhaust represents a major source of ambient fine particulate air pollution (PM), and has been associated with both acute and chronic health effects. The nitro-PAH, 1-nitropyrene (1-NP) has been proposed as a unique marker for diesel exhaust, and could be used to quantify exposure to diesel exhaust. In this paper we describe development and application of an analytical method for measuring 1-NP metabolites in human urine samples. Methods: Personal PM samples were collected from taxi and lorry drivers with elevated occupational exposures to diesel exhaust (n=17), and from subjects exposed to ambient levels of diesel exhaust (n=10). First morning void urine samples were obtained concurrent with the PM samples. PM2.5 exposures in the taxi and lorry drivers were in the range 67–165 μg/m3, and 1–46 μg/m3 in the subjects exposed to ambient levels of diesel exhaust. 1-NP in PM samples was measured by using HPLC with fluorescence detection. To facilitate fluorescence detection the 1-NP is reduced to 1-aminopyrene in an on-line reduction column packed with Pt/Rh catalyst. Using this technique we obtained a detection limit of 0.25 pg for 1-NP. An analytical method based on GC-NICI-MS was developed for the sensitive and specific detection of 1-NP metabolites in human urine. The analytical method involved two solid phase extraction steps using Blue Rayon and Alumina, nitro-reduction using granular zinc, and derivatization with heptafluorobutyric anhydride. The limit of detection for this assay is ∼0.1–0.5 fmol/mL assuming a 50 mL urine sample. Deuterium-labelled nitropyrene metabolites were synthesized and used as recovery standards. Results: NP metabolites were ammended into human urine from subjects in Seattle, WA and Kanazawa, Japan without known exposure to elevated levels of diesel exhaust. Using the above method, recoveries of the 1-NP metabolites ranged from 35% for 1-aminopyrene to >90% for the hydroxynitropyrene isomers. 1-NP concentrations were then measured in the urine specimens obtained from taxi and lorry drivers and from subjects exposed to ambient levels of diesel exhaust. We were able to detect the hydroxylated metabolites of 1-NP in human urine samples in the range 1–50 fmol/mL. Based on rat studies the major urinary metabolites of 1-NP are anticipated to be hydroxylated metabolites, however this is the first report to identify these compounds in human urine. Seidel et al. (Int. J. Hyg. Environ. Health, 2002, 204(5–6):333–8) previously reported concentrations of 1-aminopyrene in the range 8–800 fmol/mL in urine from underground mine workers, but their analytical method was not suitable to detect the hydroxylated metabolites. Conclusions: A sensitive and specific analytical method was developed to measure hydroxylated metabolites of 1-NP in human urine samples. The assay has a limit of detection of ∼0.1–0.5 fmol/mL assuming a 50 mL urine sample, and recoveries ranged from 35% for 1-aminopyrene to >90% for the hydroxynitropyrene isomers. Hydroxylated metabolites of 1-NP were detected in human urine samples in the range 1–50 fmol/mL. These metabolites offer the possibility of a specific biomarker of exposure to diesel exhaust, and their association with diesel exhaust exposures will be investigated.