* CIIT Centers for Health Research, P.O. Box 12137, Research Triangle Park, North Carolina 27709
Received August 16, 2002; accepted October 29, 2002
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ABSTRACT |
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Key Words: acrylamide; glycidamide; N-methylolacrylamide; hemoglobin; adduct.
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INTRODUCTION |
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The accidental release of AM and NMA from a tunnel construction project in Sweden resulted in the exposure of workers, ground water contamination, the death of fish, and poisoning of cattle (Hagmar et al., 2001). In the exposed workers, N-(2-carbamoylethyl)valine, formed by reaction of AM with the N-terminal valine residue of hemoglobin, was measured as an indicator of internal dose. A clear association was described between levels of the hemoglobin adduct and symptoms of peripheral nervous-system toxicity (Hagmar et al., 2001
). The extent of formation of globin adducts from GA was not reported in this study. Whether the N-(2-carbamoylethyl)valine arose from reaction of hemoglobin with AM or from NMA could not be distinguished.
In this study, we investigated the hypothesis that AM and NMA can give rise to the same adducts in globin, but are metabolized quantitatively in a different manner. This hypothesis was tested by measuring the formation of hemoglobin adducts from AM and GA in rats administered AM or NMA by gavage. The specific adducts measured were N-(2-carbamoylethyl)valine, formed by direct reaction of AM with the N-terminal valine residue, and N-(2-carbamoyl-2-hydroxyethyl)valine, formed by reaction of GA with the N-terminal valine residue. To enable a parallel measurement of the extent of metabolism by 13C nuclear magnetic resonance (13C-NMR) spectroscopy reported elsewhere, the AM used was 13C-labeled (Sumner et al., 1992). AAVal and GAVal formed in hemoglobin were measured to compare the internal dose of AM vs. GA on administration of NMA or AM. To accomplish the adduct analyses, a new method was employed for analysis of valine phenylthiohydantoin derivatives using liquid chromatography with tandem mass spectrometry (LC-MS/MS). This new method could distinguish and quantitate adducts derived from both the unlabeled NMA and the labeled AM administered.
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MATERIALS AND METHODS |
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Methylolacrylamide (NMA, N-hydroxymethylacrylamide, CAS No 924425) was obtained from TCI America (Portland, OR) as a powder. The vendor specified a minimum purity of 99%. 1H- and 13C-NMR analysis appeared consistent with the specified purity and did not indicate the presence of any free formaldehyde or acrylamide.
N-(2-carbamoylethyl)valine-leu-anilide (AAVal-leu-anilide) was obtained from Bachem (King of Prussia, PA). Phenylisothiocyanate was obtained from Aldrich (Milwaukee, WI). Valine and valine-13C5 were obtained from Sigma (St. Louis, MO) and Isotec, Inc. (Miamisburg, OH), respectively.
Synthesis of glycidamide.
Glycidamide was synthesized by H2O2 oxidation of acrylonitrile (Payne and Williams, 1961), and stored at 20°C. The 1H nuclear magnetic resonance (1H-NMR) spectrum for GA in CDCl3 (7.24 ppm) contained three 1-proton multiplets centered at 3.45 ppm (CH), 2.95 ppm (CH2a), and 2.79 ppm (CH2b), and a broad 2-proton singlet at 6.2 ppm (NH2). The 13C-NMR spectrum (in CDCl3, 77 ppm) contained signals at 48 ppm (CH2), 50 ppm (CH), and 173 (CONH2) that are consistent with the structure of GA.
Synthesis of N-(2-carbamoylethyl)valine (AAVal).
AAVal was synthesized and purified, as described previously, by Bergmark et al.(1993). AM (10.01 g) and valine (1.76 g) were reacted in 30 ml water and 2.6 ml triethylamine for 6 days at room temperature. The 1H-NMR spectrum of AAVal in D2O (4.9 ppm) had signals at 3.6 ppm (doublet, CH
), 3.4 ppm (multiplet, CH2-N), 2.8 ppm (triplet, CH2-CO), 2.3 (multiplet, CHß), and 1.1 ppm (two doublets, valine CH3). Integration of the signals provided a ratio appropriate for the number of assigned hydrogens. The 13C-NMR spectrum showed 2 carbonyl signals at 176 and 179 ppm, a signal at 72 ppm (CH
), a signal at 47 ppm (CH2-N), 2 signals near 33 ppm (CHß and CH2-CO), and 2 signals near 21 ppm (2 CH3).
Synthesis of N-(2-carbamoylethyl)valine-13C5 (AAVal-13C5).
Labeled material for use as an internal standard was prepared on a smaller scale, essentially as described above for the unlabeled standard. 13C5-Valine (62.5 mg) was reacted with AM (88.19 mg) in 1 ml of water and 87.5 µl of triethylamine for 6 days at room temperature. The NMR spectra of AAVal prepared from 13C5-Val contained 13C-1H and 13C-13C coupling patterns for signals derived from the valine portion of the molecule, while signals derived from the AM portion of the molecule did not contain these patterns. The 1H-NMR spectrum of 13C5-AAVal had signals at 3.4 ppm (multiplet, CH2-N) and 2.8 ppm (triplet, CH2-CO), the same as those located in spectra for unlabeled AAVal. A doublet of multiplets (at 3.4 and 3.8 ppm) were centered on 3.6 ppm, the shift for the CH proton. A doublet of multiplets (at 2.1 and 2.5 ppm) were centered on 2.3 ppm, the shift for the CHß proton. A doublet of multiplets (at 0.9 and 1.35 ppm) were centered at 1.1 ppm, consistent with the shift for the 2 CH3 protons. Integration of the signals provided a ratio appropriate for the number of assigned hydrogens. Signals derived from the valine-labeled carbons were located in the 13C-NMR spectrum near 176 ppm (CO, doublet), 72 ppm (CH
, 2 doublets), 33 ppm (CHß, 2 doublets), and 21 ppm (CH3, 2 doublets).
Synthesis of N-(2-carbamoyl-2-hydroxyethyl)valine.
N-(2-carbamoyl-2-hydroxyethyl)valine (GAVal) was synthesized and purified as described previously by Bergmark et al.(1993). GA (1.25 g) and valine (0.583 g) were reacted in 5 ml of water and 87 µl of triethylamine for 24 h at 45°C. In addition to the expected signals for Val CH
(3.65 ppm, 2 doublets), CHß (2.35 ppm, multiplet), and CH3 (1.1 ppm, 2 doublets), multiplets for the GA-derived protons were observed at 4.6 ppm (CHOH) and at 3.33.6 ppm (CH2) in the 1H-NMR spectrum of GAVal (in D2O). Integration of the spectrum provided the appropriate integration ratios for the assigned signals. In the 13C-NMR spectrum, Val-derived 13C signals were present at shifts similar to those observed for AAVal, as described above (72, 33, and 21 ppm), while the GA-derived carbon signals were observed at 52 ppm (N-CH2) and 74 ppm (CHOH).
Synthesis of N-(2-carbamoyl-2-hydroxyethyl)valine-13C5(GAVal-13C5).
Synthesis of N-(2-carbamoyl-2-hydroxyethyl)valine-13C5 (GAVal-13C5) for use as an internal standard material was conducted as described above, but on a smaller scale. 13C5-Valine (57.3 mg) was reacted with GA (0.121 g) in 0.976 ml water and 85.4 µl of triethylamine for 6 days at room temperature. Signals in the 13C-NMR spectrum of GAVal-13C5 were at shifts consistent with those obtained for unlabeled GAVal, and contained splitting patterns and relative signal intensity consistent with incorporation of the labeled carbons of 13C5-valine.
Synthesis of AAVal phenylthiohydantoin.
To prepare the AAVal phenylthiohydantoin derivative (AAVal PTH), AAVal (12.2 mg) was incubated with phenylisothiocyanate (30 µl) in 1.5 ml of 0.5 M potassium bicarbonate:1-propanol (2:1), pH 8.6, for 2 h at 45°C. The reaction product was isolated by extracting with n-heptane (2 x 2 ml), drying under N2, redissolving in toluene, drying under N2, redissolving in methanol, and purifying by HPLC using a Beckman Ultrasphere ODS column (0.45 x 25 cm) eluted with 35% methanol/water. The 1H-NMR spectrum of AAVal PTH (CDCl3) contained signals for the phenyl ring protons at 7.27.6 ppm. Additional signals were observed at 4.3 ppm (doublet, CH), 3.85 and 4.35 ppm (multiplets, CH2-N), 2.6 and 3.0 ppm (multiplets, CH2-CO), 2.5 ppm (multiplet, CHß), and 0.95 and 1.25 ppm (2 doublets, valine CH3). Nonequivalent CH2 signals suggest hindered rotation of the AM side chain in AAVal PTH compared with AAVal. The 13C-NMR spectra of AAVal PTH contained signals for the Val portion of the molecule at 68 ppm (CH
) 30 ppm (CHß), and 16 and 18 ppm (2 CH3). Signals for AM-derived carbons were at 34 ppm (CH2-N), 42 ppm (CH2-CO), and 129130 ppm for the phenyl ring carbons.
Synthesis of AAVal PTH-13C5.
AAVal-13C5 (9.1 mg) was incubated with phenylisothiocyanate (20 µl) in 1.5 ml of 0.5 M potassium bicarbonate:1-propanol (2:1), pH 8.6, for 2 h at 45°C, and the product was isolated as described above. The 1H-NMR spectrum of 13C5 AAVal PTH contained signals (multiplets near 4.3, 3.8, 3.0, and 2.6 ppm) for the AM portion of the molecule consistent with those detected for the unlabeled AAVal PTH. Patterns consistent with 13C-1H coupling were observed for signals from the Val-13C5 portion of the molecule. The valine 13CH gave rise to multiplets at 4.0 and 4.5 ppm (centered at 4.3 ppm). The valine 13CHß gave rise to multiplets at 2.3 and 2.7 ppm (centered at 2.5 ppm). The valine methyl groups give rise to multiplets at 0.7 and 1.2 (centered at 0.95), and 1.0 and 1.45 ppm (centered at 1.2 ppm). Signals in the 13C NMR spectrum of 13C5 AAVal PTH were consistent with the AAVal PTH, containing the 13C-13C splitting patterns and relative intensities consistent with the presence of 13C5-Val.
Synthesis of GAVal PTH.
GAVal (12.0 mg) was incubated with phenylisothiocyanate (30 ml) in 1.5 ml of 0.5 M potassium bicarbonate:1-propanol (2:1), pH 8.6, for 2 h at 45 °C. The reaction product was isolated by extraction with n-heptane (2 x 2 ml), drying under N2, redissolving in toluene, drying under N2, redissolving in methanol, and then isolating on HPLC using a Beckman Ultrasphere ODS column (0.45 x 25 cm) eluted with acetonitrile/water at 30% acetonitrile for 10 min, 40% acetonitrile for 3.5 min, and 100% acetonitrile for 1.5 min. The 1H-NMR spectrum of GAVal PTH (in CDCl3) contained a sharp signal at 4.6 ppm (CHOH) and two broader signals at 4.0 and 4.5 ppm (CH2) derived from the GA-protons. Signals for the Val-derived protons were located near 0.9 and 1.3 ppm (CH3, 2 doublets) 2.5 ppm (CHß, multiplet), and 4.3 ppm (CH). Ring protons were present between 7.2 and 7.6 ppm. The occurrence of nonequivalent CH3 protons and GA-CH2 protons suggest structural hindrance of this molecule. The 13C0-NMR spectrum of GAVal PTH had signals at 72 ppm (CHOH) and 49 ppm (CH2-N), derived from the GA portion of the structure, consistent with signals observed for GAVal. The Val portion of the molecule had signals at 68 ppm (CH
), 30 ppm (CHß), and 15 and 18 ppm (2 CH3).
Synthesis of GAVal PTH-13C5.
GAVal-13C5 (10.5 mg) was incubated with phenylisothiocyanate (20 ml) in 1.5 ml of 0.5 M potassium bicarbonate:1-propanol (2:1), pH 8.6, for 2 h at 45°C, and the product was isolated as described above. Signals in the 1H NMR spectrum of GAVal PTH-13C5 (in CDCl3) contained a sharp signal at 4.6 ppm (CHOH) and two broader signals near 4.0 and 4.5 ppm (CH2) derived from the GA-protons. Signals for the Val-derived protons were centered at 0.9 ppm (CH3, doublet of multiplets, 0.75 and 1.15) and 1.3 ppm (CH3, doublet of multiplets, 1.05 and 1.45), 2.5 ppm (CHß, doublet of multiplets, 2.3 and 2.7 ppm ), and 4.3 ppm (CH, doublet of multiplets, 4.5 and 4.0 ppm). Ring protons were present between 7.2 and 7.6 ppm. The 13C NMR spectrum of GAVal PTH13C5 (in CDCl3) has intense multiplets for the Val-13C5 portion of the molecule at 69 ppm (CH
), 30 ppm (CHß), and 15 and 18 ppm (2 CH3).
Animals.
Male Fischer F344 rats were purchased from Charles River Laboratories (Raleigh, NC). At the time of dosing, the rats were 910 weeks old. They were supplied food (NIH 07 diet, Zeigler brothers, Gardner, PA) and reverse osmosis water ad libitum and maintained on a 12-h light-dark cycle (07001900 h for light phase) at a temperature of 6479°F and relative humidity of 3070%.
Dosing and sample collection.
Male Fischer 344 rats (4 per group) were administered [1,2,3-13C]AM by gavage at a nominal dose of 50 mg/kg body weight. NMA was administered by gavage to four male F344 rats at a nominal dose of 71 mg/kg body weight, targeted to provide an equimolar dose administered for AM and NMA. The AM and NMA dose solutions were prepared in distilled water and delivered at 1 ml/kg body weight. A control group of four additional F344 rats were administered distilled water. The dose administered was calculated by weighing the dosing syringe before and after dosing. The actual doses administered were 59.5 ± 8.0 mg AM/kg (0.80 ± 0.11 mmol/kg) and 73.1 ± 3.9 mg NMA/kg (0.72 ± 0.03 mmol/kg). The control rats and rats treated with [1,2,3-13C]AM were placed in glass metabolism cages after administration of the labeled material, and urine was collected over dry ice for 024 h after dosing. The urine samples were stored at 80°C. The rats administered NMA were placed in polycarbonate cages after dosing. At 24 h after administration, rats were euthanized by exposure to CO2, and blood was collected by cardiac puncture in a heparinized syringe. The blood was separated into red blood cells and plasma, and the red blood cell fraction was washed three times with 0.9 % (w/v) saline and stored at 20°C for adduct analysis.
Analysis of AAVal and GAVal in hemoglobin.
AAVal and GAVal, formed by reaction of AM and GA respectively with the N-terminal valine residue in hemoglobin, were measured by an LC-MS/MS method. Globin was isolated from washed red cells (Mowrer et al., 1986). Globin samples (approximately 20 mg) were derivatized with phenylisothiocyanate (5 µl) in formamide (1.5 ml) with 1 N NaOH (5 µl) to form adduct phenylthiohydantoin derivatives in a manner analogous to the modified Edman degradation (Bergmark, 1997
; Perez et al., 1999
; Törnqvist et al., 1986
). AAVal PTH-13C5 (26.6 pmol) and GAVal PTH-13C5 (36.0 pmol) were added as internal standards, and the samples were extracted using a Waters Oasis HLB 3 ml (60 mg) extraction cartridge (Milford, MA). The derivatized adducts were eluted with methanol, dried, and reconstituted in 100 ml of MeOH:H2O (50:50, containing 0.1% formic acid). Analysis was conducted using a PE Series 200 HPLC system interfaced to a PE Sciex API 3000 LC-MS with a Turboionspray interface. Chromatography was conducted on a Phenomenex Luna PhenylHexyl Column (50 x 2 x 3 mm) eluted with 0.1% acetic acid in water and methanol at a flow rate of 350 µl/min, with a gradient of 4555% methanol in 2.1 min. The elution of adducts was monitored by multiple-reaction monitoring (MRM) in the negative-ion mode for the following ions:
AAVal-PTH: m/z 304 233 (M-H-
M-H- CH2-CH2-CONH2)
GAVal-PTH: m/z 320 233 (M-H-
M-H- CH2-CHOH-CONH2)
AAVal-PTH-13C5: m/z 309 238 (M-H-
M-H- CH2-CH2-CONH2)
GAVal-PTH-13C5: m/z 325 238 (M-H-
M-H- CH2-CHOH-CONH2).
Quantitation of AAVal was conducted using the ratio of analyte to internal standard, with a calibration curve generated using AAVal-leu-anilide. Quantitation of GAVal was conducted using the ratio of analyte to internal standard.
For samples from rats administered a single dose of [1,2,3-13C]AM, the 13CAAVal and 13CGAVal adducts formed can be distinguished in the negative ion mode since the 13C-containing adduct side chain is lost from the adduct PTH in the collision cell. An additional set of ions is monitored to quantitate the adducts formed:
13C3-AAVal-PTH: m/z 307 233 (M-H-
M-H- 13CH2-13CH2-13CONH2)
13C3-GAVal-PTH: m/z 323 233 (M-H-
M-H- 13CH2-13CHOH-13CONH2).
Statistical analysis.
Statistical analysis was conducted using Instat 2.01 (Graphpad Software, San Diego, CA). One-way analysis of variance (ANOVA) with the TukeyKramer post test was used to compare control, AM-treated, and NMA-treated groups.
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RESULTS |
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For standardization of the quantitative analysis, AAVal-leu-anilide was added to samples containing unmodified globin. This peptide provides a model for the AAVal in globin, and will cyclize and cleave in a similar manner (Lawrence et al., 1996; Osterman-Golkar et al., 1994
; Törnqvist et al., 1986
). The samples were derivatized with PITC, and after addition of the internal standard AAVal-PTH-13C5, the PTH derivatives were extracted and analyzed by LC-MS/MS. A standard curve for AAVal PTH generated with AAVal-leu-anilide indicated a linear response with a background present in untreated hemoglobin, consistent with the observations of others (Bergmark, 1997
; Perez et al., 1999
; Tareke et al., 2000
). This background limits evaluation of the sensitivity that may be obtained for the analysis method. However, for the analysis of samples of AAVal PTH without added globin, the limit of detection is estimated to be approximately 0.5 fmol/injection. Injection of 5-µl aliquots from a 100-µl sample derived from 20 mg of globin gives an estimated limit of detection of 0.5 fmol/mg globin. Greater sensitivity could be achieved by increasing the volume of the aliquot injected and by increasing the amount of globin analyzed. The standard curve generated was used for the quantitative analysis to convert the ratio of analyte to internal standard to amount of adduct present in each sample. Comparison of the amount of AAVal-leu-anilide added with the amount of internal standard added indicated that approximately 70% of the added analyte was recovered. A similar calibration could not be performed for GAVal with a peptide standard, because this is not commercially available. Therefore, quantitation of GAVal was based on the peak area ratio of analyte:internal standard, and the amount of GAVal-PTH standard added.
Adducts in AM- and NMA-Treated Rats
Samples from rats administered [1,2,3-13C]AM or unlabeled NMA were analyzed for the adducts formed by both the 13C-enriched and natural abundance forms of AA and GA. A chromatogram for AAVal in globin from a rat administered [1,2,3-13C]AM by gavage is shown in Figure 4. Three separate chromatograms are shown for two forms of the analyte and the internal standard. A similar set of chromatograms is shown in Figure 5
for GAVal in the same animal. For quantitation, the two peaks for the isomers of GAVal-PTH were integrated together. Typical chromatograms for AAVal in NMA-treated rats are shown in Figure 6
and for GAVal in Figure 7
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DISCUSSION |
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The considerably higher levels of AAVal in NMA-treated rats suggest that the internal dose of NMA is higher than that of AM. To calculate the rate of elimination (kel) of AM and GA, Bergmark et al.(1991) used the relationship:
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An ambiguity remains in using AAVal and GAVal in assessing exposure to mixtures of AM and NMA. If AAVal data alone were used to estimate exposure to AM but the exposure was in reality to NMA, the exposure using the data generated in this study would be overestimated on a molar basis by a factor of two. The amount of GAVal formed on exposure to AM would be considerably higher per mole of AAVal when compared with NMA.
Further investigations should focus on whether NMA undergoes oxidative metabolism to produce hydroxymethylglycidamide and whether the hemoglobin adducts of NMA and hydroxymethylglycidamide can be detected. The approach used in this study with LC-MS/MS is much more amenable to the analysis of polar adducts than is the more widely used GC-MS approach. Analysis for the presence of NMA and hydroxymethylglycidamide adducts may provide a means to distinguish NMA and AM exposure. However, the stability of these adducts in vivo, and during isolation and preparation for analysis will need to be evaluated.
The approach used in this study with LC-MS/MS of valine hemoglobin adducts has considerable advantages over the more widely used GC-MS approach. This new method requires less labor-intensive sample preparation: sample analysis is more rapid as a result of shorter chromatography runs, and the method much more amenable to the analysis of polar adducts. This method has been applied to the quantitation of adducts from other reactive chemicals, the results of which will be communicated elsewhere.
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ACKNOWLEDGMENTS |
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NOTES |
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2 Present address: RTI International, P.O. Box 12194, Research Triangle Park, NC 27709-2914.
3 Present address: GlaxoSmithKline, 5 Moore Drive, Research Triangle Park, NC 27709.
4 Present address: Paradigm Genetics, Inc., P.O. Box 14528, Research Triangle Park, NC 27709-2548.
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