0.0 0.1 0.2 0.3 0.4
Fig. 4. Calibration curves of GSH conjugates in rat serum and deionized water.
Ion suppression effects were eliminated after we applied isotope-dilution quantification. Isotope-substituted standard was added at a level of 10 ng/mL for each GSH conjugate. (A): AA-GSH ; (B): GA2-GSH; (C): GA3-GSH. Labels:
●Standard/Internal Standard area ratio in serum; ○ Standard/Internal Standard area ratio in de-ionized water.
3.3 Determination of AA-GSH, GA2-GSH and GA3-GSH in rat blood
Our newly-developed LC-MS/MS method was sensitive to measure the AA- and GA-GSH in blood of rats treated with 5 mg/kg AA through ip injection (Fig. 5). After 2 h of AA treatment, AA-GSH, GA2-GSH, and GA3-GSH were 1651.1 ± 374.5, 18.4
± 6.3 and 75.3 ± 31.3 ng/mL, respectively. The results demonstrate that our method successfully analyzes the detoxication products of AA and GA in rat blood. The analysis of AA- and GA- GSH conjugates can clarify the species differences in AA detoxication. 265.850-266.550] MS Genesis Male-HighDose-R3-T3
NL: 3.19E5
TIC F: + c ESI SRM ms2 398.000 [122.750-123.450, 250.750-251.450, 268.750-269.450] MS Genesis Male-HighDose-R3-T3
Fig. 5. Representative LC-MS/MS chromatograms in rat blood. GSH conjugates were detected successfully in rat treated with 5 mg/kg AA. SRM channel: A: AA-GSH, m/z 379 → 250; B: (13C3)-AA-GSH, m/z 382 → 253; C: GA-GSH, m/z 395.0 →
Analysis of AA-GSH was used to investigate the detoxication of AA in vitro.
Pernice at al. studied the effect of sulforaphane (SFN) on the detoxication of AA in Caco-2 cell. SFN was reported to induce GST expression (~1.9 fold) in mouse liver.
Although GST activity decreased after incubation with AA, AA-GSH concentration was about 1,000-fold higher in cells co-incubated with AA and SFN than that in situ chemical synthesis using similar concentration of AA and SFN. This result suggested that formation of GSH was catalyzed by GST. Watzek et al. reported that AA-GSH was generated much earlier than GA (0.5 h to 16 h) for hepatocytes incubated with 2 μM of AA, suggesting that detoxication of AA by GST could be faster than CYP-mediated epoxidation of AA. Aside from the formation of AA-GSH, AAMA also occurred in a concentration and time-dependent manner in rat hepatocytes.
Though rat livers only play a minor role in MAs formation, this evidence demonstrated that primary rat hepatocytes can metabolize AA-GSH to AAMA. Since GSH conjugate appears faster and serves as the direct evidence of primary detoxication, further studies of AA- and GA-GSH toxicokinetics would help to capture AA and GA detoxication in vivo. Therefore, in the present work, an isotope-dilution LC-MS/MS method was developed to accurately quantify AA- and GA-GSH conjugates with excellent sensitivity and specificity.
Mercapturic acids of AA and GA has also been well studied in rodents and
humans. These studies revealed that the majority of AA was metabolized to MAs (29~71 % in rats, 26~50% in humans), supposedly through GST detoxication. Based on AA toxicokinetics in SD rat, AA-GSH measured was 27.4% of measured AA at 2h, which proportion was significantly lower than those estimated by using urinary mercapturic acids The discrepancy between urinary MAs and serum GSH-conjugates might result from the reactions between AA and GA with free N-acetyl-L cysteine (NAC) in blood and urine to form AAMA and GAMA, which might lead to overestimation of detoxification of AA and GA by GST, when measured as MAs. In addition, the half lives of AAMA and GAMAs are much greater than those of AA and GA, which could also result in overestimation if urinary AA metabolites were used to estimate the proportion of detoxification of AA and GA by GST. In order to fully characterize AA metabolism and detoxification, direct analysis of AA- and GA-GSH will generate appropriate estimation. In this study, our objective was to synthesize, characterize, and quantify AA- and GA-GSH. Analysis of AA- and GA-GSH in rats treated with AA was to validate our newly-developed analytical methods. Indeed, our results reveal that this method could quantify AA- and GA-GSH in blood samples with excellent selectivity and sensitivity. AA-, GA2- and GA3-GSH in blood of rats treated with 5 mg/kg AA were detected with LODs of 0.017, 0.075 and 0.15 ng/mL, respectively. AA- and GA-GSH were analyzed at 2 hrs after AA treatment, and
interpretation of these results is very limited. Therefore, our future work is to study the toxicokinetics of AA- and GA-GSH so that the role of GST in AA and GA
detoxification will be fully illustrated.
In conclusion, this study has synthesized, purified, and characterized AA- and GA-GSHs, and successfully developed a sensitive LC-MS/MS method to directly analyze these conjugates in blood. By applying this method, AA-GSH, GA2-GSH and GA3-GSH has been analyzed to demonstrate AA and GA detoxication by GSH in AA-treated rats. According to our results, the estimated levels of AA-, GA2- and GA3-GSH in blood of rats treated with 0.05 μg/kg of AA will be greater than our LODs, and should be detectable with our newly-developed method. Such exposure level of AA is comparative to the human daily intakes, making this method applicable in human studies. Further studies in AA detoxication across different species can further reduce uncertainty in species extrapolation of internal dose and improve quality of health risk assessment of AA exposure.
Conflict of interest
The authors declare that there are no conflicts of interest.
Acknowledgements
This study was supported by a grant from National Science Council (grant number, NSC 101-2314-B-002-115-MY3).
Supplementary data
Product ion scan spectra of isotope-substituted internal standards (Fig. S1S3), the NMR spectra of GSH conjugates (Fig. S4S11) and the results of method validation (Table S1) are available free of charge via the internet.
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Tables
Table 1. NMR Chemical Shifts and Coupling Constants of GSH and GA-GSH
GSH GA2-GSH* GA3-GSH, Diastereomer A GA3-GSH, Diastereomer B
Position δH (ppm) J (Hz) δC δH (ppm) δC δH (ppm) J (Hz) δC δH (ppm) J (Hz) δC
a 173.6 173.8 173.7 173.9
b 3.77† ( t, 1H) 6.4 53.8 3.70-3.78† ( m, 1H) 54.0 3.72† ( t, 1H) 6.3 53.9 3.72† ( t, 1H) 6.3 54.1
c 2.09-2.14 ( m, 2H) 26.0 2.07-2.10 ( m, 2H) 26.1 2.06-2.10 ( m, 2H) 26.0 2.08-2.12 ( m, 2H) 26.1
d 2.45-2.56 ( m, 2H) 31.2 2.44-2.49 ( m, 2H) 31.3 2.40-2.50 ( m, 2H) 31.2 2.43-2.53 ( m, 2H) 31.4
e 174.9 174.8;174.9 174.8 174.9
f 4.52† ( dd, 1H) 5.4, 6.9 55.6 4.52-4.55† ( m, 1H) 53.0;53.2 4.52† ( dd, 1H) 4.9, 8.7 53.1 4.57† ( dd, 1H) 5.1, 8.8 53.3
g 172.4 172.0 172.5 172.0
h 3.92 ( s, 2H) 41.5 3.80 ( s, 2H) 42.4 3.86 ( s, 2H) 41.8 3.76 ( s, 2H) 43.0
i 173.6 174.8 173.9 175.6
j’ 2.88 ( dd, 1H) 14.2,6.9
25.3 2.87-3.11 ( m, 2H)
32.5;32.7 2.85 ( dd, 1H) 14.3, 8.9
33.5 2.89 ( dd, 1H) 14.1, 8.8
j’’ 2.92 ( dd, 1H) 14.2,5.4 3.05 ( dd, 1H) 14.1, 4.9 3.07 ( dd, 1H) 14.1, 4.9 33.8
3 3.70-3.78 ( m, 2H) 61.5 2.80 ( dd, 1H) 14.3, 6.7 35.8 2.87 ( dd, 1H) 14.1,6.2 36.0
3’ 2.96 ( dd, 1H) 14.1, 3.8 2.95 ( dd, 1H) 14.1, 4.1
2 3.49-3.51† ( m, 1H) 50.0;50.2 4.30† ( dd, 1H) 6.5, 3.8 70.3 4.32 ( dd, 1H) 6.2, 4.2 70.5
1 175.6;175.8 177.8 177.8
Assigning numbers are according to Scheme 1 and are confirmed by COSY, HSQC, HMBC and DEPT experiments.
* Racemic compound.† Chiral center. GSH, GA2-GSH and GA3-GSH was characterized in D2O by 500 MHz NMR.
Table 2. LC-MS/MS parameters used to quantify AA-and GA-GSH
†IT ion transition (m/z) RT retention time CE collision energy (V) TL tube lens (AU) SW scan width (m/z) ST scan time (s)