Strickland P T, Kang D, Bowman E D, Fitzwilliam A, Downing T E, Rothman N, Groopman J D, Weston A
Department of Environmental Health Sciences, Johns Hopkins School of Hygiene and Public Health, Baltimore, MD 21205.
Carcinogenesis. 1994 Mar;15(3):483-7. doi: 10.1093/carcin/15.3.483.
Humans are exposed to polycyclic aromatic hydrocarbons (PAHs) from various occupational, environmental, medicinal and dietary sources. The measurement of specific PAH metabolites, particularly 1-hydroxypyrene, in human urine treated with deconjugating enzymes (e.g. beta-glucuronidase) has been extensively used as a means of assessing recent exposure to PAHs. We have examined pyrene metabolites in human urine prior to enzymatic deconjugation in order to determine the relative proportions of conjugated and unconjugated pyrene metabolites. The analytical method utilized immunoaffinity chromatography, high performance liquid chromatography (HPLC) and the complementary techniques of synchronous fluorescence spectroscopy (SFS) and gas chromatography-mass spectrometry (GC-MS) to measure pyrene-containing metabolites. SFS analysis of immunoaffinity-purified urine samples showed fluorescence spectra characteristic of the pyrene moiety (using wavelength differences of 34 nm, 54 nm and 102 nm). These spectra are produced by several PAHs containing the pyrene moiety. HPLC analysis with fluorescence detection indicated that the major fluorescent metabolite in immunoaffinity-purified urine was much more polar than simple hydroxylated metabolites of pyrene (1-hydroxypyrene) or benzo[a]pyrene (benzo[a]pyrene-diols or -tetrols). Following digestion with beta-glucuronidase, this metabolite co-chromatographed with authentic 1-hydroxypyrene and exhibited fluorescence spectra characteristic of 1-hydroxypyrene, suggesting that the major metabolite was a glucuronide conjugate of 1-hydroxypyrene. This was subsequently confirmed by GC-MS analysis of trimethylsilyl derivatives of the major metabolite; both 1-hydroxypyrene and glucuronic acid were detected independently as derivatized products. Since 1-hydroxypyrene glucuronide is approximately 5-fold more fluorescent than 1-hydroxypyrene, it may provide a more sensitive biomarker for assessing exposure to pyrene in mixtures of PAHs.
人类会通过各种职业、环境、医疗和饮食来源接触多环芳烃（PAHs）。在用去结合酶（如β-葡萄糖醛酸酶）处理的人类尿液中，特定PAH代谢物的测量，尤其是1-羟基芘，已被广泛用作评估近期PAH暴露的一种方法。我们在酶促去结合之前检测了人类尿液中的芘代谢物，以确定结合型和非结合型芘代谢物的相对比例。分析方法利用免疫亲和色谱、高效液相色谱（HPLC）以及同步荧光光谱（SFS）和气相色谱-质谱联用（GC-MS）等互补技术来测量含芘代谢物。对免疫亲和纯化的尿液样本进行SFS分析，显示出芘部分的荧光光谱特征（使用34 nm、54 nm和102 nm的波长差）。这些光谱由几种含芘部分的PAHs产生。荧光检测的HPLC分析表明，免疫亲和纯化尿液中的主要荧光代谢物比芘（1-羟基芘）或苯并[a]芘（苯并[a]芘二醇或四醇）的简单羟基化代谢物极性大得多。用β-葡萄糖醛酸酶消化后，这种代谢物与纯1-羟基芘共色谱，并表现出1-羟基芘的荧光光谱特征，表明主要代谢物是1-羟基芘的葡萄糖醛酸共轭物。随后通过对主要代谢物的三甲基硅烷基衍生物进行GC-MS分析证实了这一点；1-羟基芘和葡萄糖醛酸均作为衍生化产物被独立检测到。由于1-羟基芘葡萄糖醛酸酯的荧光比1-羟基芘强约5倍，它可能为评估PAHs混合物中芘暴露提供更敏感的生物标志物。
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