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 var ious occupational, environmental, medicinal and dietary sources. The m easurement of specific PAH metabolites, particularly 1-hydroxypyrene, in human urine treated with deconjugating enzymes (e.g. beta-glucuroni dase) has been extensively used as a means of assessing recent exposur e to PAHs. We have examined pyrene metabolites in human urine prior to enzymatic deconjugation in order to determine the relative proportion s of conjugated and unconjugated pyrene metabolites. The analytical me thod utilized immunoaffinity chromatography, high performance liquid c hromatography (HPLC) and the complementary techniques of synchronous f luorescence spectroscopy (SFS) and gas chromatography - mass spectrome try (GC-MS) to measure pyrene-containing metabolites. SFS analysis of immunoaffinity-purified urine samples showed fluorescence spectra char acteristic of the pyrene moiety (using wavelength differences of 34 nm , 54 nm and 102 nm). These spectra are produced by several PAHs contai ning the pyrene moiety. HPLC analysis with fluorescence detection indi cated that the major fluorescent metabolite in immunoaffinity-purified urine was much more polar than simple hydroxylated metabolites of pyr ene (1-hydroxypyrene) or benzo[a]pyrene (benzo[a] pyrene-diols or -tet rols), Following digestion with beta-glucuronidase, this metabolite co -chromatographed with authentic 1-hydroxypyrene and exhibited fluoresc ence spectra characteristic of 1-hydroxypyrene, suggesting that the ma jor metabolite was a glucuronide conjugate of 1-hydroxypyrene. This wa s subsequently confirmed by GC-MS analysis of trimethylsilyl derivativ es of the major metabolite; both 1-hydroxypyrene and glucuronic acid w ere detected independently as derivatized products. Since 1-hydroxypyr ene glucuronide is approximately 5-fold more fluorescent than 1-hydrox ypyrene, it may provide a more sensitive biomarker for assessing expos ure to pyrene in mixtures of PAHs.