All solvents and reagents used were commercially available, analytical grade quality unless otherwise stated. Ascorbic acid (AscH), reduced L-glutathione (GSH), and acrolein (ACR) were purchased from Sigma-Aldrich (St. Louis, MO). Pentafluorobenzyl hydroxylamine hydrochloride and acrylic acid were purchased from TCI America (Portland, OR). L-[1-13
C]-AscH and L-[13
]-AscH were purchased from Omicron Biochemicals (South Bend, IN). H2
O was obtained from Cambridge Isotope Laboratories (Andover, MA). HPLC-grade acetonitrile and water were purchased from Honeywell, Burdick and Jackson (Muskegon, MI). Formic acid was obtained from Fluka (Buchs, Switzerland) and K2
was purchased from Mallinckrodt Baker (Phillipsburg, NJ). Acrolein diacetate (allylidene diacetate) was purchased from Pfaltz & Bauer (Waterbury, CT). 1
H NMR spectra (400 MHz) and 13
C NMR spectra (100 MHz) were recorded on a Bruker DPX 400 MHz instrument. The solvent peak was used as an internal standard for reporting chemical shifts. Two-dimensional 1
H COSY, 1
C HMBC, and 1
C HSQC experiments were also performed on the Bruker DPX 400 MHz instrument (see Supporting Information
Liquid Chromatography-Tandem Mass Spectrometry
The HPLC system consisted of two Shimadzu Prominence LC-20AD pumps, a DQU-20A5 degasser, and a Shimadzu SIL-HTc autosampler equipped with two switching valves (Shimadzu, Kyoto, Japan). Three chromatographic systems were employed. System 1 used a Thermo Betasil diol column (150 × 2.1 mm i.d.; particle size, 5 µm; pore size, 100Å; Thermo Fisher Scientific, Waltham, MA) and a linear solvent gradient from 100% solvent B (MeCN containing 0.1% HCOOH) to 5% B in solvent A (0.1% aqueous HCOOH) over 10 min at 0.2 mL/min. The first 2 min of each LC run was diverted to waste. In System 2, the HPLC column was a Synergi HydroRP C18 column (250 mm × 1 mm i.d.; particle size, 4 µm; pore size, 80Å; Phenomenex, Torrance, CA). The HPLC solvents were the same as in System 1. The column was eluted with 5% solvent B in A during the first minute, followed by a linear solvent gradient from 5% B to 95% B over 9 min and then with 95% B for 5 min. After returning to 5% B in 1 min, the column was equilibrated for 10 min before the next injection. The flow rate was 0.1 mL/min. The column effluent was directed to the mass spectrometer between 5 and 20 min of the LC run and to a waste container during the remainder of the LC run. In System 3, chromatographic separations were achieved on a Synergi MaxRP C12 column (250 mm × 2 mm i.d.; particle size, 4 µm) (Phenomenex) at a flow rate of 0.2 mL/min. The HPLC solvents were the same as in HPLC System 1. A linear solvent gradient was used, running from 10% B to 40% B in 10 min, 40 to 90 % B over the next 2 min, held constant at 90% B for 7 min, returned to 10 % B over 1 min, and equilibrated at 10% B for 5 min before the next injection.
The LC-MS/MS instrument consisted of a hybrid triple quadrupole/linear ion trap (4000 QTrap) mass spectrometer equipped with a pneumatically assisted electrospray (Turbo V) source operated at 450 °C (Applied biosystems /MDS Sciex, Concord, Ontario, Canada). Liquid nitrogen was used as the source of heating/nebulizing, curtain, and collision gas. The spray needle was kept at −4.5 kV in the negative ion mode. Q1 mass spectra were recorded by scanning in the range m/z 100–300 at a cycle time of 1 s with a step size of 0.2 u. MS/MS experiments (product ion scan and selective reaction monitoring, SRM) were conducted at unit resolution for both Q1 and Q3 with collision gas set at “medium”, a collision energy of 17 eV, and a declustering potential of 70 V. Peak areas were measured using Analyst 1.4.2 software (Applied Biosystems). The following LC-MS/MS characteristics were used for analysis of cell media and lysates (the first SRM transition was used for quantitative purposes, and subsequent SRMs were used for additional identity confirmation): THO-PFB oxime, t
R 10.4 min (System 2), m/z 400→310, m/z 400→167, m/z 400→112; 13C5-labeled THO-PFB oxime, t
R 10.4 min (System 2), m/z 405→312, m/z 405→167, m/z 405→114; AscACR-PFB oxime, t
R 11.8 min (System 2), m/z 426→115, m/z 426→366, m/z 426→157); GSH-ACR, t
R 5.0 min (System 3), m/z 362→143, m/z 362→306, m/z 400→272, m/z 362→128, m/z 362→179, m/z 400→254; GSH-HP (System 3), t
R 5.0 min, m/z 364→143, m/z 364→128, m/z 364→143; GSH-AA, t
R 5.1 min (System 3), m/z 378→306, m/z 378→143, m/z 378→128.
Preparation of AscACR
AscACR was synthesized following the method published by Fodor (12
). ACR (1 mmol) was added to a solution of AscH (1 mmol) in water (1 mL). After 2 h stirring at room temperature, the solution was placed at 4 °C for 16 h. The resulting precipitate was filtered, washed with cold water, and dried. NMR spectra of the precipitate were in agreement with published data for AscACR (12
H NMR (400 MHz, DMSO-d
6.6 (1H, s), 6.53 (1H, d, J
=5.6 Hz), 5.61 (1H, d, J
=4.4 Hz), 5.56-5.53 (1H, m), 4.45 (1H, s), 4.28-4.26 (1H, m), 4.21-4.19 (1H, m), 3.86 (1H, dd, J
=4.8, 4.4 Hz), 2.45-2.40 (1H, m), 2.02-2.00 (1H, m), 1.87-1.83 (1H, m), 1.81-1.78 (1H, m); 13
C NMR (100 MHz, DMSO-d
175.08, 106.13, 99.68, 87.67, 85.34, 74.87, 73.72, 32.06, 29.39. [13
]- and [1-13
C]-AscACR were produced by treating 1 mg [13
]-AscH and [1-13
C]-AscH, respectively, with an equimolar amount of ACR in aqueous solution.
Structure Determination of AscACR by X-ray Diffraction Analysis
ACR (5 mmol) was slowly added to a stirred solution of AscH (5 mmol) in water (5 mL). The reaction mixture was stirred for 2 h at room temperature and then placed at 4 °C for 5 days. After this period, a crystalline precipitate was collected and recrystallized from water. The structure of the material was determined to be the 5,5,5-tricyclic form of AscACR () by single crystal X-ray diffractometry (see Supporting Information
), in agreement with the structure of AscACR published by Eger and colleagues (28
Hydrolysis of AscACR
An aliquot of an aqueous solution of AscACR (10 µl, 100 µM) was added to 200 µl H2
18O. The solution was immediately analyzed by LC-MS using HPLC System 1. The sample was analyzed at 30 minute intervals over 4 h using Q1 scanning from m/z 100 to 300.
Decarboxylation of AscACR Acid to obtain THO
AscACR (0.11 mg) was dissolved in 1 mL of an aqueous solution of K2CO3 (0.18 M). After 2 h of incubation at room temperature, the sample containing THO was analyzed by LC-MS using HPLC System 1.
Derivatization of THO and AscACR
A 10 µl aliquot of the above solution containing THO was treated with 1 mL of a 500 mM solution of pentafluorobenzyl hydroxylamine hydrochloride (PFBHA HCl) in NaOAc buffer (pH 5.5, 1 M) for 1 h at room temperature. Similarly, [13C5]-labeled THO was prepared by treating 10 µl of [13C6]-AscACR with aqueous K2CO3 (0.18 M; 200 µl) for 2 h. The reaction mixture containing [13C5]-labeled THO was subsequently treated with 1 mL of a 500 mM solution of PFBHA HCl in NaOAc buffer (pH 5.5, 1 M) for 1 h at room temperature. Treatment of sample solutions containing AscACR with PFBHA resulted in the conversion of AscACR into its PFB oxime. PFB oxime derivatives were analyzed by LC-MS using HPLC System 2. Exact mass calculated for C15H15NO6F5 (THO-PFB oxime, [M-H]−): 400.0820 (Found 400.0819). Exact mass calculated for C12H9NO3F5 (prominent fragment ion, ): 310.0503 (Found 310.0484).
Figure 5 Product ion mass spectrum of the m/z 400 [M-H]− ion of the pentafluorobenzyl (PFB) oxime derivative of THO obtained by LC-MS/MS analysis (HPLC System 2) of an aqueous solution containing THO and treated with PFB hydroxylamine for 2 h at room temperature. (more ...)
Metabolic Transformation of ACR(Ac)2 by THP-1 Cells
THP-1 cells, obtained from the American Type Culture Collection (Manassas, VA), were grown as suspension cultures in RPMI 1640 medium supplemented with penicillin (100 U/mL), streptomycin (100 µg/mL), 0.05 mM 2-mercaptoethanol and 10% fetal bovine serum (FBS) at 37 °C in an atmosphere of 95% air and 5% CO2. The cells (3 × 106 cells/mL; 2 mL/well) were pretreated with 1 mM AscH for 18 h in phenol red-free RPMI medium with supplements, centrifuged at 500 × g for 5 min, and then co-treated with freshly prepared 1 mM AscH and 0.1 mM ACR(Ac)2 in fresh phenol red-free RPMI medium with supplements. ACR(Ac)2 was prepared as a 100 mM stock solution in 100% ethanol before addition to the culture medium (2 µL ACR(Ac)2 /2 mL medium, 0.1 mM final concentration). A stock solution of 50 mM AscH was freshly prepared in Dulbecco’s Phosphate-Buffered Saline (D-PBS, Invitrogen Cat. no. 14190250) and neutralized with sodium hydroxide prior to use. Control cells were incubated with D-PBS and ethanol (0.1%) in phenol red-free RPMI medium with supplements. No-cell controls consisted of complete RPMI medium containing 1 mM AscH and 0.1 mM ACR(Ac)2. After 3, 6, 12, and 24 h of incubation, the cells were harvested by centrifugation at 500 × g for 5 min and the media were collected. No-cell controls were terminated by placing the treated media on ice and immediately frozen at −80 °C prior to analysis of ACR metabolites. The cell pellet was washed by resuspension in D-PBS and re-centrifugation at 500 × g for 5 min. The pellet was then resuspended in D-PBS and lysed by sonication. The experiments were conducted in replicates of five. The samples were derivatized as described below and then analyzed for THO formation and residual AscACR by LC-MS/MS using HPLC System 2.
Derivatization of THP-1 Samples
An aliquot (200 µL) of each cell medium sample was transferred to an HPLC autosampler vial containing 50 µL PFBHA HCl (500 mM) in NaOAc buffer (pH 5.5, 1 M). Cell lysate samples were vortexed and centrifuged. An aliquot of the supernatant (150 µL) was transferred to an HPLC autosampler vial with a glass insert containing 50 µL of a 500 mM solution of PBFHA HCl in NaOAc buffer (pH 5.5, 1 M). After 1 h of incubation at room temperature, the samples were analyzed by LC-MS/MS using HPLC System 2.
Lactonase Activity of THP-1 cell lysate, FBS, Human Serum and Recombinant Paraoxonases
A venous blood sample (10 mL) was taken from a 43 year old volunteer and centrifuged at 3000 × g for 5 min at room temperature to obtain serum (Study #2599, approved by the Institutional Review Board of Oregon State University). Lactonase activities of THP-1 cell lysate, FBS, human serum, recombinant human paraoxonase-1 (PON1, ProSec, Rehovot, Israel) and recombinant human paraoxonase-2 (PON2, Prospec, Rehovot, Israel) were measured using dihydrocoumarin (DHC) as the substrate (29
) with some modifications. The incubation was carried out on a 96-well UV plate and the reaction mixture consisted of 20 µL of serum [diluted 1:10 in 10 mM Tris-HCl, pH 8, with 1 mM CaCl2
or in RPMI 1640 (for FBS)], THP-1 cells (50 µg protein/well), PON1 or PON2 (0.05 µg protein/well), 1 mM DHC, 1 mM CaCl2
, and 50 mM Tris-HCl (pH 8.0) in a total volume of 0.2 mL/well. The reaction was monitored at 30 °C by the increase in absorbance at 270 nm over a 10 min period after substrate addition. Non-enzymatic hydrolysis of DHC was run on microplate wells without THP-1 cells, serum or recombinant enzyme. Vehicle control wells contained methanol instead of DHC. Lactonase activity was calculated as µmoles DHC hydrolyzed/min per mL serum or per mg protein (for THP-1 cells, PON1 and PON2) using an extinction coefficient of 1295 M−1
. Only the initial linear portion of the curve was used for calculations and all assays were run in quadruplicate.
Metabolic Conversion of AscACR to THO by Human Serum, PON1 and PON2
The incubation mixture contained 0.1 mM AscACR (from a 50 mM stock, dissolved in ethanol:water, 2:1 v/v), 50 mM Tris–HCl (pH 8.0), 1 mM CaCl2, and 25 µl of serum (diluted 1:10), in a total volume of 0.25 mL. Other incubations contained 0.2 µg PON1 or 0.2 µg PON2, instead of serum. Control incubations that contained appropriate combinations of reagents and either no enzyme or heat denatured enzyme (boiled at 100 °C for 10 min) were also conducted. The reaction was carried out for 3 min, 30 min and 3 h at 37 °C. At each time point, an aliquot (200 µl) of the reaction mixture was transferred to an HPLC autosampler vial containing 50 µL PFBHA HCl (50 mM) in NaOAc buffer (pH 5.5, 1 M). After 1 h of incubation at room temperature, the samples were analyzed for THO formation by LC-MS/MS using HPLC System 2.
Preparation of GSH Adducts
GSH adducts of ACR (GSH-ACR) and acrylic acid (GSH-AA) were prepared and characterized by LC-MS/MS following the method of Miranda et al. (30
). Briefly, a solution of GSH (10 mM) was prepared in 0.1 M phosphate buffer (pH 8). To 100 µL aliquots were added 400 µL of the same phosphate buffer and 400 µL of water. These solutions (900 µL) were mixed with 100 µL of a 1 mM solution of ACR or acrylic acid in EtOH, and the reaction mixtures stirred for 2 h at 37 °C and then acidified to pH 3 with 1 M HCl. Work-up of the reaction mixtures ultilized Strata-X solid-phase extraction (SPE) columns (60 mg; Phenomenex, Torrance, CA) that were preconditioned with 1.2 mL of MeCN containing 0.1% HCOOH and equilibrated with 1.2 mL of 0.1% aqueous HCOOH. After sample loading and washing with 0.1% aqueous HCOOH (1.2 mL), the SPE column was eluted with MeCN-0.1% aqueous HCOOH (1:1, v/v) to obtain the GSH adducts. Hydroxypropyl-S
-GSH (GSH-HP) was prepared by reduction of GSH-ACR adduct with 10 µL of a 5 M sodium borohydride solution in 1 M NaOH. The reaction mixture was stirred for 30 min at room temperature and then acidified to pH 3 with 1 M HCl. The reduction product was isolated by SPE as described above.
LC-MS/MS Analysis of GSH Adducts in Cell Lysates and Media
Cell lysate (50 µl) and medium (400 µl) were mixed with a 2-fold volume of MeCN containing 0.1% HCOOH and centrifuged. The supernatant was analyzed by LC-MS/MS using HPLC System 3.
Calculation of Intracellular Metabolite Levels
Intracellular levels of metabolites were presented as peak areas and calculated using a THP-1 cell volume of 473 µm3
or 4.73 ×10−7
µl per cell (31
). Cells were counted using a hemacytometer.
Statistical differences were determined by ANOVA followed by Tukey-Kramer multiple comparison tests or by the Student’s t-test. Comparisons between the amounts of metabolites in different incubations were based on areas under the curve (AUC) between the start and the end of the incubation periods ( and ). Values of p < 0.05 were considered to be statistically significant.
Figure 7 Relative concentrations of THO and AscACR following exposure to AscH and ACR(Ac)2. (A) AscH-adequate THP-1 cells and the surrounding media were analyzed at various time points for AscACR and THO by LC-MS/MS using SRM. AscACR was not detected in the presence (more ...)
Figure 9 Relative concentrations of GSH-HP in THP-1 cells (panel B) and surrounding media (panel C) following exposure to ACR(Ac)2, in the presence and absence of AscH. GSH-HP was measured by LC-MS/MS using SRM and HPLC System 3. Symbols represent means ± (more ...)