Naylor Dana Institute, American Health Foundation, 1 Dana Road,Valhalla, NY 10595, USA
![]() |
Abstract |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Abbreviations: ES, electrospray; LC, liquid chromatography; MS/MS, tandem mass spectrometry; Q, quadrupole; SAX, strong anion exchanger; S-PMA, S-phenylmercapturic acid; SRM, selected reaction monitoring; t,t-MA, trans,trans-muconic acid.
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The molecular mechanisms responsible for the toxicity and carcinogenicity of benzene have not yet been fully elucidated. Several studies have indicated that metabolism of benzene to active intermediates is a prerequisite for its toxic and carcinogenic effects (9). The metabolism of benzene is complex. The major metabolic routes consist of cytochrome P4502E1-mediated oxidation of benzene to an epoxide-oxepin intermediate and/or interaction of benzene with a hydroxyl (OH) radical to yield a hydroxy-cyclohexadienyl radical intermediate. These intermediates can lead to the formation of ring-hydroxylated metabolites such as phenol, hydroquinone, catechol and 1,2,4-benzenetriol; open-ring products, such as trans,trans-muconaldehyde and trans,trans-muconic acid (t,t-MA); and also dimeric products, such as biphenyl and hydroxylated biphenyls (Figure 1
) (1020). The phenolic metabolites can undergo further oxidation to the corresponding reactive semiquinones and quinones and then react with cellular macromolecules, or form glucuronide and sulfate conjugates which are excreted in urine (2123). Benzene oxide can also react with glutathione, which is excreted in urine as S-phenylmercapturic acid (S-PMA) (Figure 1
).
|
During the past decade, several HPLC and gas chromatographymass spectrometry (GCMS) methods have been employed for the measurement of urinary t,t-MA and S-PMA (2545). Generally, HPLCUV chromatograms of urinary extracts, especially from smokers, contain several compounds that elute in the vicinity of analytes that can interfere with the exact measurement of these analytes. The GCMS method eliminates such problems and allows the quantification of low levels of S-PMA and t,t-MA. However, the need for derivatization of each analyte to obtain more volatile compounds makes the GCMS method less attractive for large transitional epidemiology studies. LCESMS/MS has, over the past decade, gained widespread use for quantitation of drugs and their metabolites in biological matrices (46). In the present study we have developed an LCESMS/MS assay for simultaneous quantification of two minor urinary benzene metabolites, namely urinary S-PMA and t,t-MA. The feasibility and efficacy of this assay have been examined in a pilot study for cigarette smokers as subjects with low benzene exposure and with non-smokers as a control group.
![]() |
Materials and methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Animals
The Charles River Breeding Laboratories (North Wilmington, MA) supplied male F344/N rats. The rats were 12 weeks old at the onset of experiments.
Synthesis of [13C6]S-PMA internal standard and unlabeled S-PMA
[13C6]S-PMA was prepared from [13C6]aniline by the Gattermann reaction as shown in Figure 2 (47). In brief, [13C6]aniline (0.5 g; 5.3 mmol) was added to concentrated HCl (0.7 ml), diluted with H2O (1.5 ml) and HCl (1 ml) and a solution of NaNO2 (1.4 ml; 6 mmol) was added to the resulting suspension while the temperature was kept below 5°C. While stirring, the formed phenyldiazonium salt mixture was added to an N-acetyl-L-cysteine solution (7.5 ml; 4.8 mmol) and the resulting orange precipitate was centrifuged. The wet solid of N-acetyl-S-phenyldiazol-L-cysteine was dissolved in EtOH (6 ml) and after adding freshly prepared copper (0.64 g) and H2O (14 ml), the suspension was refluxed at 80°C for 1.5 h. The residue was filtered, washed with hot H2O, acidified and extracted with CHCl3. After removing the solvent, the crude [13C6]S-PMA residue was dissolved in 60% EtOH (2.5 ml); upon adding charcoal (0.25 g), it was boiled and filtered. The [13C6]S-PMA was crystallized from aqueous EtOH and characterized by NMR and MS data. Both the NMR and MS were similar to those reported for S-PMA (23). The 360 MHz NMR was: [(CDCl3):
1.87 (3H, s, CH3), 3.34 and 3.54 (2H, dd, cysß and cysß' Jßß'=13.6 Hz), 4.774.82 (1H, m, cys
), 6.2 (1H, d, NH), 7.217.43 (5H, m, aromatic), 12.8 (1H, broad, COOH)]. The electron impact (EI)MS spectrum shows ions at m/z = 245 (M+x) and m/z = 186 (M-59). The purity of synthesized [13C6]S-PMA was >98% by HPLC. Unlabeled S-PMA was prepared as previously described (23).
|
Optimal condition for clean up of urine samples by solid-phase extraction before LCESMS/MS analysis
The optimal condition for purification of urine samples by solid phase extraction was determined by spiking pooled smokers' urine with either [14C]S-PMA (120 µg/l) or [14C]t,t-MA (0.1 mg/l), or both, followed by elution of the sorbed material from the SAX cartridge with different combinations and volumes of eluates. One milliliter fractions of each eluate were collected and radioactivity measured. Fractions containing radioactive materials were pooled and analyzed by HPLC as described below. The optimal condition for maximal recovery of both analytes from the SAX cartridge, shown in Figure 3, was used for clean up of human urine samples as described below.
|
LCESMS/MS analysis of S-PMA and t,t-MA
The HPLC features included a Waters Model 600 pump, a Rheodyne model 7120 injector and a Phenomenex Ultramex 5-µ C-18 narrow-bore column (250x2.0 mm). A pre-injector splitter was utilized to reduce the flow-rate from 0.9 ml/min (HPLC pump) to 160 µl/min. A linear gradient from 80% solvent A (0.5% aqueous acetic acid), 20% solvent B (MeOH) to 100% solvent B over 5 min was employed in the elution program for analysis of S-PMA and t,t-MA.
The HPLC was interfaced with a Finnigan TSQ 700 triple-stage quadrupole mass spectrometer (San Jose, CA) via an electrospray source. The mass spectrometer was operated in the negative ion mode. The spray voltage was 4.1 kV, the capillary temperature 220°C. The liquid flow was introduced into the mass spectrometer at a rate of 160 µl/min without post-column splitting. For SRM, the first quadrupole (Q1) was used to select the precursor ion for the reaction and pass it on to Q2 where fragment ions were produced via collision with argon. The product ion of the reaction was monitored by Q3. The argon gas pressure in the collision cell (Q2) was adjusted so that the precursor beam suppression was ~75%. A DEC Station no. 5000120 computer was used to control the instrumentation, data acquisition and data processing.
Standard calibration curves
Stock standard solutions of S-PMA were prepared by dissolving 1 mg of the compound in 10 ml MeOH. t,t-MA solutions were prepared by dissolving 1 mg of t,t-MA in 10 ml of a mixture of 10:90 MeOH:0.5% aqueous acetic acid. The solutions were diluted with MeOH and spiked with internal standards 15 µg/l [13C6]S-PMA and 30 µg/l [13C6]t,t-MA to give a series of working standard solutions in a range generally present in biological samples. All standards were prepared on the day of analysis and the purity of each standard sample was checked by HPLC. Standard calibration graphs were constructed by plotting peak area ratios of analytes to those of the internal standards, measured at each nominal concentration.
Reproducibility of the assay
Inter-assay precision was determined by replicate analysis of pooled smokers' urine spiked with internal standards on different days. In addition, along with each run, a blank sample containing 1 ml H2O and the internal standard was also subjected to solid-phase extraction and LCESMS/MS analysis.
Human specimens
The Metropolitan Life Insurance Company's Testing Laboratories (Elmsford, NY) kindly provided urine specimens from men and women smokers and non-smokers. These specimens were collected on the spot from clients and each contained one tablet of a preservative that is generally used in clinical laboratories. Each tablet contained 84 mg potassium phosphate, 42 mg sodium benzoate, 54 mg benzoic acid, 8 mg sodium bicarbonate, 0.66 mg mercuric oxide, 1.5 mg polyvinylpyrrolidone (PVP), 1.7 mg magnesium stearate. All samples were frozen immediately and stored at 20°C until analysis.
Creatinine and cotinine determination
Urinary creatinine was determined with a Kodak Ektachem 500 Computer-Directed Analyzer and urinary cotinine was quantified by radioimmunoassay at the American Health Foundation's Clinical Biochemistry Facility by the previously described methods (25,26).
Statistical analysis
The interrelationship between S-PMA and t,t-MA was assessed using linear regression, version 6.12 of the Statistical Analysis System (SAS), including 95% group and individual confidence limits. Since the two measures were found to be highly skewed and non-normally distributed, the logarithmic (base 10) transformation was used on the data points. For variables found below the detection limit, zero was imputed as a data point. All other statistical analyses (determination of mean, median, standard errors and linear regression for calibration graphs) were performed on a PC using Excel software; all reported P-values are two-tailed.
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The full scan mass spectrum of S-PMA showed an (M-1) ion at m/z = 238 [100, relative intensity] and a major product ion that resulted from loss of CO2 and CH2=CH-NHCOCH3 to yield an ion at m/z = 109 [41]. The internal standard [13C6]S-PMA also gave corresponding ions at (M-1) at m/z = 244 [100] and 115 [40] (data not shown). This reaction was thus chosen for quantitation of S-PMA. Ions at m/z = 238 for S-PMA and m/z = 244 for the internal standard [13C6]S-PMA were selected in the Q1 analyzer; product ions at m/z = 109 and 115, respectively, were monitored by the second analyzer (Figure 4).
|
|
|
The detection limit for S-PMA standard samples was 0.02 ng/injection and that for t,t-MA was 0.2 ng/injection, and the ratio of signal to noise was >5:1. For S-PMA, the lowest value quantified so far was 0.4 µg/l urine (corresponding to 0.08 ng/injection), the highest was 1950 µg/l (390 ng/injection; unpublished data from our current study for validation of the assay). For t,t-MA, the lowest concentration measured in this study was 0.003 mg/l urine (corresponding to 0.6 ng/injection). Blank samples subjected to the entire analytical procedure showed no detectable levels of either analyte. The analytical recoveries of spiked [14C]S-PMA (120 µg/l urine) and [14C]t,t-MA (0.1 mg/l urine), from the anion exchange cartridge, as determined by HPLC, were ~75 and 85% (n = 2), respectively. The total recovery of spiked [13C6]S-PMA (15 µg/l urine) in samples analyzed by LCMS/MS was 43%, n = 28 (ranging from 27 to 55%); the recovery of spiked [13C6]t,t-MA (0.03 mg/l urine) was 65%, n = 28 (ranging from 40 to 107%).
Quantitation of urinary S-PMA and t,t-MA in cigarette smokers and non-smokers
Figure 7 shows the concentrations of urinary S-PMA and t,t-MA in smokers and non-smokers. As shown in Table I
, the mean ± SE concentration of S-PMA in smokers was 9.1 ± 1.7 µg/g creatinine and the median was 5.8 µg/g [ranging from non-detectable (1 out of 28) to 33.4 µg S-PMA/g]; the level of t,t-MA was 0.15 ± 0.03 mg/g creatinine and the median was 0.11 mg/g (ranging from 0.005 to 0.34 mg/g). In non-smokers, the mean was 4.8 ± 1.1 µg S-PMA/g creatinine, the median was 3.6 µg/g (ranging from 1.0 to 19.6 µg/g); the mean of t,t-MA was 0.07 ± 0.02 mg/g creatinine, the median was 0.03 mg/g [ranging from non-detectable (1 out of 18) to 0.48 mg/g]. The levels of urinary S-PMA and t,t-MA in smokers were significantly higher than those in non-smokers, at P = 0.02 and 0.03, respectively. The mean ± SE concentration of urinary cotinine in smokers was 5.4 ± 0.5 mg/g creatinine and the median was 3.4 mg/g (ranging from 0.56 to 15.5 mg/g). In non-smokers, corresponding data were 0.02 ± 0.009 µg cotinine/mg creatinine, the median was 0.009 (ranging from non-detectable to 0.14 µg/g creatinine).
|
|
|
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The optimal HPLC condition for S-PMA and t,t-MA was assessed with an elution gradient from an acidic pH to an organic solvent; thus the complete elution of t,t-MA from the HPLC column is achieved by acidic solvent elution and, when t,t-MA is introduced into the mass spectrometer, it is predominantly in MeOH. The optimal LCESMS/MS conditions for the S-PMA and t,t-MA assay were obtained in the negative ionization mode. Operating in this mode, both S-PMA and t,t-MA and their corresponding internal standards generate prominently deprotonated molecular ions (M-1).
One problem encountered with the ESMS method in quantitative analysis is the suppression of a monitored analyte ion by a co-eluting matrix component (48). The solid-phase extraction applied prior to the LCESMS/MS analysis eliminated the interfering matrix components to some extent. However, human urine samples, especially from smokers contain many agents that cannot be removed completely from analytes. This could be the reason for the relatively low recoveries of [13C]S-PMA. Stable isotope isomers of [13C6]S-PMA and [13C6]t,t-MA used as internal standards have nearly identical physicochemical properties and HPLC retention times similar to those of the urinary analytes. Thus, it is anticipated that any co-eluting unknown compound present in samples would have similar effects on both analytes and internal standards.
The efficacy of the newly developed LCESMS/MS assay was evaluated by analyzing human urine specimens from 28 smokers and 18 non-smokers as benzene-exposed and non-exposed groups. The number of samples analyzed was based on the availability of specimens at this time. Urinary cotinine was used to assess the intensity of smoking. The mean concentration of cotinine in the urine of smokers was 5.4 mg/g creatinine (~2030 cigarettes/day), suggesting that the subjects in this study were heavy tobacco smokers. This pilot study indicated that the mean concentrations of S-PMA and t,t-MA in the urine of smokers were, respectively, 1.9-fold (P = 0.02) and 2.1-fold (P = 0.03) higher than those in urine of non-smokers (Figure 7; Table I
).
The mean concentration of S-PMA in smokers quantified by LCESMS/MS was 9.1 µg/g creatinine and that in non-smokers was 4.8 µg/g creatinine. The concentrations of urinary S-PMA reported in the literature (analyzed by GCMS and HPLC) are 3.61 and 9.4 µg S-PMA/g creatinine for smokers and 1.99 and 1.5 µg S-PMA/g creatinine for non-smokers (35,38). Similarly, the mean concentrations of t,t-MA in the current study were 0.15 mg/g in smokers and 0.07 mg/g creatinine in non-smokers. Levels of t,t-MA reported so far (mostly analyzed by HPLC) in smokers range from 0.14 to 0.61 mg/g creatinine and those in non-smokers range from 0.05 to 0.21 mg/g creatinine (25,26,33,35, 3745). Thus, current values are in agreement with the literature data. However, it appears that in most studies measurement of t,t-MA by HPLC resulted in higher values. This could partially be due to co-elution of unknown compounds with t,t-MA.
An alternative source of urinary t,t-MA is sorbic acid (CH3CH=CHCH=CHCOOH). This preservative is used in certain foods, cosmetics and pharmaceutical products and its metabolism leads to t,t-MA (27,43,44). For persons in the USA, the uptake of sorbic acid from food is estimated to be 630 mg/day and ~0.12% of the sorbic acid dose is excreted in urine as t,t-MA. About 75% of t,t-MA is excreted within 6 h after ingestion of sorbic acid (43,44). Thus, collecting urine samples many hours after a meal can minimize interference of t,t-MA from ingested sorbic acid. It has been reported that dietary intake of sorbic acid accounts for 525% of the background t,t-MA excretion in smokers and for 1050% in non-smokers (43,44).
Excretion of t,t-MA can also be affected by simultaneous exposure to toluene and benzene. Brondeau et al. (49) have shown that excretion of urinary t,t-MA by rats was, on average, by 28, 44 and 85% lower after simultaneous exposure to 100, 200 or 1000 p.p.m. toluene with 20 p.p.m. benzene. Thus, measurement of t,t-MA and other urinary metabolites of benzene may underreport the true exposure to benzene in cases where toluene was concomitantly present.
S-PMA appears to be a better biomarker of benzene exposure than t,t-MA (unpublished data). To our knowledge, there is no dietary source that would lead to the formation of urinary S-PMA. A linear regression analysis of logarithmic transformation pointed to a correlation between S-PMA and t,t-MA, r = 0.41, P = 0.005 (Figure 8). Urinary S-PMA in workers exposed to 0.5 p.p.m. benzene has been reported in the range from 7.2 to 25 µg/g creatinine. Similarly, the predicted excretion of t,t-MA ranged from 0.39 to 1.1 mg/g creatinine for 0.5 p.p.m. benzene exposure (34,35,38,45). Assuming that the uptake of benzene by smokers is 1.8 mg/day and the respiratory rate is 10 l air/min, the levels of S-PMA andt,t-MA excreted from uptake of benzene in cigarette smoke were estimated to be 1.635.9 µg/g creatinine and 0.090.26 mg/g creatinine, respectively. The enhanced levels of 4.3 µg S-PMA/g creatinine (from 4.8 to 9.1) and 0.08 mg t,t-MA/g creatinine (from 0.07 to 0.15) observed in smokers compared with non-smokers are in good agreement with the estimated ranges derived from occupational exposure to benzene.
There was inter-individual variation in the ratio of urinary excretion of t,t-MA to S-PMA from 1 to 146. This inter-individual variation may be due to differences in the detoxification of benzene epoxide by glutathione, or these metabolites may be derived from different intermediates, or, alternatively, they may stem from varying amounts of ingested sorbic acid. The median ratio of t,t-MA/S-PMA in smokers was 10; the corresponding ratio among non-smokers was 6. These data suggest that smoking may alter the activation of benzene pathways and enhance the ratio of t,t-MA to S-PMA. In laboratory animal experiments and in a physiological model study, it has been shown that as the concentration of benzene exposure increases, its metabolism is shifted toward production of S-PMA (13,17,34). On the other hand, the formation of t,t-MA has been shown to increase when mice were exposed to lower doses of benzene (18). Thus, one would expect that in smokers, who are exposed to higher levels of benzene than non-smokers, the ratio of t,t-MA to S-PMA should be less than that in non-smokers. However, the current results are contradictory to that expectation. The enhancing effect of cigarette smoking on the t,t-MA to S-PMA ratio could be due to altered benzene metabolism in favor of the hydroxycyclohexadienyl intermediate; indeed tobacco smoke increases the formation of OH (50).
In conclusion, the HPLC separation coupled with an electrospray-interfaced MS/MS method to determine S-PMA and t,t-MA simultaneously is a practical, sensitive and specific assay, from a laboratory standpoint, and enables detection of the background levels of benzene exposure in the general population. These biomarkers of benzene are currently being validated in experiments with known benzene exposures.
![]() |
Acknowledgments |
---|
![]() |
Notes |
---|
2 Present address: Curriculum in Toxicology, University of North Carolina, Chapel Hill, NC 27599, USA
3 Present address: Baxter Healthcare Corporation, Round Lake, IL 60073, USA
4 To whom correspondence should be addressed
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|