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Brief communication |
Address correspondence to Tsan-Zon Liu, PhD, Graduate Institute of Medical Biotechnology, Chang Gung University, Kwei-Shan, Taoyuan, Taiwan; tel 886 3 211 8800 x5205; fax 886 3 211 8047; e-mail: tzliu{at}mail.cgu.edu.tw.
Abstract
Urine p-aminophenol (PAP) concentration serves as a biological marker for occupational exposures to aniline. We report the development of a rapid, simple spectrophotometric method for quantification of urine PAP concentration using a chemical autoanalyzer (Olympus Reply). The method involves oxidative coupling of PAP with an aromatic compound, xylenol, that contains an electron-donating group, based on an electrophilic aromatic substitution reaction catalyzed by sodium periodate. A calibration curve is constructed in the same matrix, urine, as the unknown samples to be analyzed. In this way, potential matrix interferences are largely avoided. The linearity range of the method is 20 to 400 mg/L. Time-course studies show that the color formation by reaction of PAP with xylenol is rapid and essentially complete within 5 min. Within-run and day-to-day reproducibility data at medium (50 mg/L) and high (200 mg/L) concentrations yield CV’s <5.0%. Several prescription drugs and drugs of abuse, as well as related compounds, gave negative tests for interference in the procedure. Clinical applications of the method are illustrated by data for (a) PAP concentrations in 255 urine samples from workers at a rubber plant, and (b) PAP elimination in serial urine samples from 5 volunteers after an oral dose (500 mg) of acetaminophen. In summary, the new method has the advantages of automation, operational simplicity, and suitability for monitoring workers for exposures to aniline.
(received 8 May 2004; accepted 24 May 2004)
Keywords: Aniline exposure, p-aminophenol, acetaminophen, biological monitoring, automated analysis
Introduction
Aniline is a constituent of printing inks, cloth-marking inks, dyes, paints, and paint removers, and it has wide industrial application in the production of compounds used to vulcanize rubber [1] Aniline is also a chemical intermediate for the synthesis of certain pharmaceuticals. Its manifold applications in industrial processes makes occupational exposure to aniline an important health hazard.
About 15 to 60% of an absorbed dose of aniline is oxidized by a cytochrome P-450-dependent reaction to p-aminophenol (PAP), which is excreted in urine mainly as glucuronide and sulfate conjugates [2–5]. In exposed workers, urinary PAP concentration is directly related to blood methemoglobin levels [6]. Urine PAP has been accepted as a specific biological marker to screen for human exposure to aniline. However, certain other industrial chemicals, such as nitrobenzene, acetanilide, and Fenuron, can also result in urinary elimination of PAP.
Numerous methods have been developed for quantitative analysis of PAP in urine. There are gas chromatographic techniques with a variety of detecting systems, including mass spectrometry and atomic emission [7–8], and HPLC procedures involving UV, fluorometric, or electrochemical detection [9–10]. Notwithstanding their merits for the quantification of PAP, such methods are lengthy, because they all required an extraction procedure.
We report the development of a specific spectrophotometric method for urine PAP, based on oxidative coupling of the analyte with xylenol in an electrophilic aromatic substitution reaction catalyzed by sodium periodate (Fig. 1
). The procedure is automated and provides a simple, rapid screening test for use in toxicology and occupational medicine.
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Reagents. Unless otherwise stated, commercial reagents of the highest available quality were used. Doubly distilled water was used throughout the procedure. p-Aminophenol, 2,5-dimethylphenol (p-xylenol), sodium periodate, ß-glucuronidase, and arylsulfatase were purchased from Sigma Chemical Co (St. Louis, MO). Xylenol-sodium periodate solution was prepared by dissolving 6.7 mmol of xylenol and 1.5 mmol of sodium periodate in 100 ml of 0.4 M KOH solution. This solution was stable for one week at 4°C or 24 hr at 25°C. PAP stock standard solution was prepared by dissolving 1.0 g of PAP in 100 ml of PAP-free human urine. Working standard solutions with graded PAP concentrations were prepared from the stock solution by appropriate dilutions with PAP-free human urine. To avoid deterioration, the stock and working standards were prepared daily in brown glassware, immediately before use.
Urine samples. Urine samples were obtained from (a) healthy adults without exposure to aniline, (b) workers at a local rubber company who were potentially exposed to aniline, and (c) volunteers who took a 500 mg oral dose of acetaminophen. Urine samples were kept in well-capped containers, without additives, in the dark, and refrigerated. If a urine sample could not be tested immediately, it was frozen. The test protocol was approved by the Human Experimentation Committee of Yuan’s General Hospital. Subjects in the acetaminophen experiment all gave informed consent.
Pretreatment of urine samples. Urine samples were adjusted to pH 5.0 with 2 M HCL. Then 900 µl of each urine sample was mixed with 100 µl of H2O (or aqueous standard in the case of calibration samples). To 250 µl of this mixture in a sample cup, 50µl of an enzyme solution (sodium acetate buffer, 1 M, pH 4.5; ß-glucuronidase:arylsulfatase:H2O; 50:5:45 v:v) was added, as described by Van Bocxlaer et al [11]. The cups were closed with parafilm and incubated overnight for 17–20 hr at 37°C.
Spectrophotometric assay. Analysis of urinary PAP was carried out using an Olympus Reply Chemistry Autoanalyzer with the following parameter settings: (a) pipettor settings: reagent volume: 50 µl + 200 µl dilution solution (acetonitrile); sample volume: 10 µl +90 µl dilution solution (acetonitrile); (b) analyzer settings: units: mg/L; reaction mode: end-point; wavelength : 600 nm; temperature 37°C; calibrators: 50, 100, 200 mg/L; (c) absorbance reading: first reading: 144 sec; last: 576 sec; interval: 36 sec.
Results and Discussion
Specimen processing. During the initial phase of our work, we noted that highly alkaline urine was capable of degrading free PAP to form a quinon-imine (a visible brown discoloration). Therefore, acidification of urine specimens was adopted to prevent this potential troublesome situation. Most PAP in human urine is in conjugated forms, such as glucuronides or sulfates. Under these conditions, measured PAP concentrations are stable for a long period of time (>30 day) [11]. Long-term storage of urine specimens is possible in the frozen state. Once urine specimens are thawed, the conjugated forms of PAP should be liberated before the actual quantitation can be performed. We adopted an enzyme mixture (ß-glucuronidase/arylsulfatase) recommended by Van Bocxlaer et al [11] to release PAP from its conjugated forms. The mild enzymatic deconjugation reaction effectively prevented the interferences that may occur with chemical hydrolytic processes.
Spectral scan and time-course studies.
A spectral scan of the reaction product formed by oxidative coupling of PAP with xylenol revealed a sharp absorbance maximum at 605 nm (Fig. 2). The Olympus Reply Chemistry Autoanalyzer has a filter at 600 nm that is suitable for this automated procedure for urine PAP. Time-course studies using 3 levels of PAP indicated that the sodium-periodate-catalyzed oxidative coupling reaction is efficient and the formation of the colored reaction product is virtually complete within 5 min (Fig. 3). For this reason, the initial absorbance reading for the automated procedure was set at 2.4 min (144 seconds). A total of 12 serial readings were recorded and averaged by the instrument.
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). The detection limit of the method was <10 mg/L, a urine PAP level usually found in individuals without occupational exposure to aniline [6].
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, the relative variability about the mean of both PAP control samples was within the acceptable limits (CV <5.0%). Aanalytical recovery of PAP added to urine was 101.5 ± 5.8% (mean ± SD, n=5) at a PAP concentration of 25 mg/L and 99.8 ± 6.2% at a PAP concentration of 50 mg/L. These data indicate that the accuracy of the method is excellent.
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Clinical applications.
This automated method was used by our laboratory in an occupational health screening program for workers at a rubber company who came to our hospital for annual physical checkup. The frequency distribution of the measured PAP concentrations in 255 urine specimens from the rubber workers is shown in Fig. 5. None of these analytical results exceeded the reported alarm limit of 50 mg of PAP/L of urine.
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, the peak urine PAP excretion occurred around 4 hr after intake and then quickly diminished thereafter. Thus, we suggest that measurement of urinary PAP might be a useful indicator in patients with acetaminophen overdosage.
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Footnotes
* These authors contributed equally to this work. 
References
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