Identification of 1-hydroxypyrene glucuronide as a major pyrene metabolite in human urine by synchronous fluorescence spectroscopy and gas chromatography-mass spectrometry

Humans are exposed to polycyclic aromatic hydrocarbons (PAHs)from various occupational, environmental, medicinal and dietarysources. The measurement of specific PAH metabolites, particularly1-hydroxypyrene, in human urine treated with deconjugating enzymes(e.g. ß-glucuronidase) has been extensively used asa means of assessing recent exposure to PAHs. We have examinedpyrene metabolites in human urine prior to enzymatic deconjugationin order to determine the relative proportions of conjugatedand unconjugated pyrene metabolites. The analytical method utilizedimmuno-affinity chromatography, high performance liquid chromatography(HPLC) and the complementary techniques of synchronous fluorescencespectroscopy (SFS) and gas chromatography-mass spectrometry(GC-MS) to measure pyrene-containing metabolites. SFS analysisof immunoaffinity-purified urine samples showed fluorescencespectra characteristic of the pyrene moiety (using wavelengthdifferences of 34 nm, 54 nm and 102 nm). These spectra are producedby several PAHs containing the pyrene moiety. HPLC analysiswith fluorescence detection indicated that the major fluorescentmetabolite in immunoaffinity-purified urine was much more polarthan simple hydroxylated metabolites of pyrene (1-hydroxypyrene)or benzo[a]pyrene (benzofa] pyrene-diols or -tetrols). Followingdigestion with ß-glucuronidase, this metabolite co-chromatographedwith authentic 1-hydroxypyrene and exhibited fluorescence spectracharacteristic of 1-hydroxypyrene, suggesting that the majormetabolite was a glucuronide conjugate of 1-hydroxypyrene. Thiswas subsequently confirmed by GC-MS analysis of trimethylsilylderivatives of the major metabolite; both 1-hydroxypyrene andglucuronic acid were detected independently as derivatized products.Since 1-hydroxypyrene glucuronide is approximately 5-fold morefluorescent than 1-hydroxypyrene, it may provide a more sensitivebiomarker for assessing exposure to pyrene in mixtures of PAHs.