Formation of a vitamin C conjugate of acrolein and its paraoxonase-mediated conversion into 5,6,7,8-tetrahydroxy-4-oxooctanal

Article abstract of DOI:10.1021/tx900452j

Vitamin C (ascorbic acid) has been reported to participate in Michael addition reactions in vitro to form vitamin C conjugates with α,β-unsaturated aldehydes, such as acrolein. This study shows evidence for the formation and metabolism of the vitamin C conjugate of acrolein (AscACR) in cultured human monocytic THP-1 cells exposed to acrolein diacetate. By using ¹⁸O and ¹³C labeling in combination with liquid chromatography-tandem mass spectrometry, AscACR was shown to undergo hydrolytic conversion of the ascorbyl lactone into an intermediate carboxylic acid. Subsequent decarboxylation of the carboxylic acid yielded 5,6,7,8-tetrahydroxy- 4-oxooctanal (THO). When THP-1 cells were pretreated with ascorbic acid (1 mM, 18 h) and then exposed to acrolein diacetate, THO was detected as its pentafluorobenzyl oxime derivative in the cell lysates and medium. Treatment of THP-1 cells with both ascorbic acid and acrolein diacetate was required for THO formation. The formation of THO from AscACR was facilitated by the lactonase enzymes, human recombinant paraoxonases 1 and 2. THP-1 cells exhibited PON activity, which explains the catalytic conversion of AscACR into THO in these cells. THO was formed in addition to metabolites of the glutathione conjugate of acrolein, indicating that THO formation contributes to the elimination of acrolein in a cellular environment.

Full text of DOI:10.1021/tx900452j

836
Chem. Res. Toxicol. 2010, 23, 836–844
Formation of a Vitamin C Conjugate of Acrolein and Its
Paraoxonase-Mediated Conversion into
5,6,7,8-Tetrahydroxy-4-oxooctanal
Nicholas G. Kesinger,† Brandi L. Langsdorf,† Alexandre F. Yokochi,‡
Cristobal L. Miranda,† and Jan F. Stevens*,†
Linus Pauling Institute, and the Departments of Pharmaceutical Sciences and Chemical Engineering,
Oregon State UniVersity, CorVallis, Oregon 97331
ReceiVed December 22, 2009
Vitamin C (ascorbic acid) has been reported to participate in Michael addition reactions in vitro to
form vitamin C conjugates with R,ꢀ-unsaturated aldehydes, such as acrolein. This study shows evidence
for the formation and metabolism of the vitamin C conjugate of acrolein (AscACR) in cultured human
monocytic THP-1 cells exposed to acrolein diacetate. By using 18O and 13C labeling in combination with
liquid chromatography-tandem mass spectrometry, AscACR was shown to undergo hydrolytic conversion
of the ascorbyl lactone into an intermediate carboxylic acid. Subsequent decarboxylation of the carboxylic
acid yielded 5,6,7,8-tetrahydroxy-4-oxooctanal (THO). When THP-1 cells were pretreated with ascorbic
acid (1 mM, 18 h) and then exposed to acrolein diacetate, THO was detected as its pentafluorobenzyl
oxime derivative in the cell lysates and medium. Treatment of THP-1 cells with both ascorbic acid and
acrolein diacetate was required for THO formation. The formation of THO from AscACR was facilitated
by the lactonase enzymes, human recombinant paraoxonases 1 and 2. THP-1 cells exhibited PON activity,
which explains the catalytic conversion of AscACR into THO in these cells. THO was formed in addition
to metabolites of the glutathione conjugate of acrolein, indicating that THO formation contributes to the
elimination of acrolein in a cellular environment.
Introduction
Vitamin C (ascorbic acid, AscH1) acts as a cofactor for a
number of 2-ketoglutarate dependent dioxygenases, including
proline hydroxylase, and also as a biological antioxidant (1–3)
and prooxidant (4, 5). Less known is the ability of AscH to
participate in nucleophilic substitution and in Michael addition
reactions. In aqueous solutions at neutral pH, AscH (pKa ) 4.2)
is essentially an enolate and therefore capable of forming C-C
bonds with electrophiles, for which we here use the term
ascorbylation. The best known and most studied ascorbylated
natural product is ascorbigen (6). The natural occurrence,
biosynthesis, and biological activities of 33 ascorbylated natural
products was recently reviewed (7). Ascorbylated products of
synthetic origin have also been reported (8–12). One of these
compounds is ascorbylated acrolein (AscACR), the Michael
addition product of AscH and acrolein (ACR) (Figure 1). This
compound was first synthesized by Fodor and co-workers in
1983 (12). The aldehyde group of AscACR may form a
hemiacetal with either of the two oxygen atoms at positions 2
or 3, depending on the solvent used. For instance, a 5,5,5-
Figure 1. Formation of THO from AscH and ACR via AscACR and
AscACR-acid. The configuration of carbon atoms 6 and 7 of THO is
determined by the configuration of the corresponding carbon atoms in
L-AscH. THO is likely to exist as a mixture of 5- and 6-membered
cyclic hemiketals and hemiacetals in solution but are not shown here
for simplicity.
tricyclic spiro compound is formed when AscACR is crystallized
from water (13, 14) (Figure 1).
* To whom correspondence should be addressed. Tel: 541-737-9534.
Fax: 541-737-3999. E-mail: fred.stevens@oregonstate.edu.
† Linus Pauling Institute and the Department of Pharmaceutical Sciences.
‡ Department of Chemical Engineering.
Humans are primarily exposed to ACR through cigarette
smoking and cooking with vegetable oils in poorly ventilated
kitchens (15). ACR is also produced in vivo as a byproduct of
protein, polyamine, and glucose metabolism, and lipid peroxi-
dation (16). ACR itself is highly electrophilic and is known to
adduct to proteins (17) and DNA (18). Exposure to ACR has
been associated with the development of lung cancer (15, 19).
Several research groups have reported protective effects of AscH
against ACR-induced toxicity. For example, supplementation
1 Abbreviations: ACR, acrolein; ACR(Ac)2, acrolein diacetate; AscH,
ascorbic acid; AscACR, ascorbyl-acrolein conjugate; AUC, area under the
curve; CID, collision-induced dissociation; DHC; dihydrocoumarin; FBS,
fetal bovine serum; GST, glutathione-S-transferase; GSH-ACR, glutathione-
acrolein conjugate; GSH-AA, glutathione-acrylic acid conjugate; GSH-
HP, glutathione-hydroxypropyl conjugate; PON, paraoxonase; PFBHA
HCl, pentafluorobenzyl hydroxylamine hydrochloride PFB, pentafluoroben-
zyl; Q, quadrupole; SRM, selected reaction monitoring; THO, 5,6,7,8-
tetrahydroxy-4-oxooctanal.
10.1021/tx900452j  2010 American Chemical Society
Published on Web 03/30/2010

Biotransformation of Ascorbylated Acrolein
Chem. Res. Toxicol., Vol. 23, No. 4, 2010 837
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) (Phenom-
enex) at a flow rate of 0.2 mL/min. The HPLC solvents were the
same as those 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 quad-
rupole/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 the 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, tR 10.4
min (System 2), m/z 400f310, m/z 400f167, m/z 400f112; 13C5-
labeled THO-PFB oxime, tR 10.4 min (System 2), m/z 405f312,
m/z 405f167, m/z 405f114; AscACR-PFB oxime, tR 11.8 min
(System 2), m/z 426f115, m/z 426f366, m/z 426f157); GSH-
ACR, tR 5.0 min (System 3), m/z 362f143, m/z 362f306, m/z
400f272, m/z 362f128, m/z 362f179, m/z 400f254; GSH-HP
(System 3), tR 5.0 min, m/z 364f143, m/z 364f128, m/z 364f143;
GSH-AA, tR 5.1 min (System 3), m/z 378f306, m/z 378f143,
m/z 378f128.
of cultured human bronchial epithelial cells with AscH has been
shown to strongly inhibit ACR-induced apoptosis, an observa-
tion the authors attributed to a general antioxidant effect of AscH
and to a “more direct and specific effect” of AscH (20). Arai
and co-workers (21) demonstrated that AscH suppresses ACR
modification of apolipoprotein E in human very low density
lipoprotein (VLDL) in vitro. The protective effect of AscH
against ACR-induced neuronal damage in spinal cord white
matter isolated from guinea pigs was also attributed to the
antioxidant effects of AscH (22). In these studies, the ability of
AscH to react directly with ACR was not addressed as a possible
detoxification mechanism.
The metabolism of ACR by enzyme-mediated conjugation
with glutathione (GSH) is well documented (23, 24). Major
metabolites of ACR found in human urine are hydroxypropyl
mercapturic acid and carboxyethyl mercapturic acid (25, 26).
Given the high intracellular concentrations of AscH in humans
(e6 mM) (27) and the ubiquitous presence of ACR in vivo
(16), we hypothesized that AscACR formation may be biologi-
cally significant. However, our previous attempts to detect
AscACR in THP-1 cells exposed to AscH and ACR were
unsuccessful, which led us to suspect that AscACR is subject
to chemical and/or metabolic transformation. Here, we show
evidence for the formation of AscACR and its biotransformation
into 5,6,7,8-tetrahydroxy-4-oxooctanal (THO) in AscH-adequate
human monocytic THP-1 cells exposed to ACR diacetate
[ACR(Ac)2], an ACR precursor that provides an intracellular
source of ACR. This unusual biotransformation represents a
complementary pathway for ACR detoxification that may
contribute to the protective effects of AscH against ACR-
induced toxicity.
Experimental Procedures
General Methods. All solvents and reagents used were com-
mercially available and of 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-[1-13C]-
AscH and L-[13C6]-AscH were purchased from Omicron Biochemi-
cals (South Bend, IN). H218O 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 K2CO3 was purchased from Mallinckrodt Baker (Phillipsburg,
NJ). Acrolein diacetate (allylidene diacetate) was purchased from
Pfaltz & Bauer (Waterbury, CT). 1H NMR spectra (400 MHz) and
13C 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 1H-1H COSY,
1H-13C HMBC, and 1H-13C HSQC experiments were also
performed on the Bruker DPX 400 MHz instrument (see Supporting
Information for spectra).
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 autosam-
pler 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, 100 Å; 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, 80 Å; Phenomenex, Torrance, CA). The HPLC
solvents were the same as those in System 1. The column was eluted
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 of 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
1
data for AscACR (12): H NMR (400 MHz, DMSO-d6) δH 6.60
(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); 13C
NMR (100 MHz, DMSO-d6) δC 175.08, 106.13, 99.68, 87.67, 85.34,
74.87, 73.72, 32.06, 29.39. [13C6]- and [1-13C]-AscACR were
produced by treating 1 mg [13C6]-AscH and [1-13C]-AscH, respec-
tively, 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 (Figure 1)
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 of H218O. The
solution was immediately analyzed by LC-MS using HPLC System
1. The sample was analyzed at 30 min intervals over 4 h using Q1
scanning from m/z 100 to 300.

838 Chem. Res. Toxicol., Vol. 23, No. 4, 2010
Kesinger et al.
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, Figure 5): 310.0503 (found
310.0484).
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 500g for 5 min, and then cotreated 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 supple-
ments. 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 500g for 5 min, and the media were collected. No-cell controls
were terminated by placing the treated media on ice and im-
mediately freezing at -80 °C prior to analysis of ACR metabolites.
The cell pellet was washed by resuspension in D-PBS and
recentrifugation at 500g 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 of 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
3000g 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.
Nonenzymatic 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 calcu-
lated as µmol of DHC hydrolyzed/min per mL serum or per mg of
protein (for THP-1 cells, PON1, and PON2) using an extinction
coefficient of 1295 M-1 cm-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 of PON1 or 0.2 µg of PON2, instead
of serum. Control incubations that contained appropriate combina-
tions 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 of 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), and 100 µL aliquots of the GSH solution were diluted with
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. Workup of
the reaction mixtures ultilized Strata-X solid-phase extraction (SPE)
columns (60 mg; Phenomenex, Torrance, CA) that were precon-
ditioned 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 the reduction of the 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 centri-
fuged. 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 Analysis. 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
(Figures 7 and 9). Values of p < 0.05 were considered to be
statistically significant.
Results
The investigations described below were driven by two
observations: (1) AscH readily reacts with ACR in aqueous
solution to form AscACR, but AscACR is not detectable in

Biotransformation of Ascorbylated Acrolein
Chem. Res. Toxicol., Vol. 23, No. 4, 2010 839
Figure 2. Hydrolytic conversion of AscACR into AscACR-acid
monitored by LC-MS using HPLC System 1. (A) Q1 Mass spectrum
of a coeluting mixture of AscACR and AscACR-acid obtained by LC-
MS of a freshly prepared solution of AscACR in water. (B) Q1Mass
spectrum of a coeluting mixture of AscACR and AscACR-acid obtained
by LC-MS analysis of a 3 h incubation of AscACR in H218O. The ions
with m/z 205 in panel A and with m/z 205 and m/z 207 in panel B are
due to in-source fragmentation of AscACR-acid.
Figure 3. Product ion mass spectrum of the m/z 250 [M - H]- ion of
[1-13C]-AscACR-acid obtained by LC-MS/MS analysis of a solution
of [1-13C]-AscACR in H2O.
experiments were repeated with [1-13C]-AscACR in unlabeled
H2O. Upon CID, the H2O addition product of [1-13C]-AscACR
with m/z 250 produced a fragment ion with m/z 205 ()
250-45), consistent with the loss of 13CO2 (Figure 3).
Formation of AscACR-acid (Figure 1) is consistent with the
incorporation of three 18O atoms in the 16O/18O exchange
experiment. Hydration of the aldehyde moiety is not in
agreement with the loss of 18OC16O from the [18O3]-isotopomer
of the H2O addition product of AscACR because the hemiacetal
and hemiketal oxygens of AscACR were both exchangeable and
would also be exchangeable in the H2O addition product of
AscACR, leaving the newly formed COOH group as the only
possible third site of 18O incorporation. Taken together, these
findings indicate that AscACR undergoes hydrolysis of the
lactone to form AscACR-acid.
Decarboxylation of AscACR. When AscACR was dissolved
in a solution of K2CO3 (0.18 M), the LC-MS signal with m/z
231 [M - H]- rapidly declined, and a new chromatographic
peak with m/z 205 [M - H]- appeared. The newly formed
species with m/z 205 () 231 + H2O-CO2) did not arise from
in-source fragmentation of AscACR-acid because its retention
time was different from that of AscACR-acid under the
conditions of HPLC System 1. When the incubation experiment
was repeated with [13C6]-AscACR, the corresponding new
product appeared with a peak at m/z 210 () 237 + H2O-13CO2)
in the Q1 mass spectrum. These findings indicate that AscACR
undergoes hydrolysis and decarboxylation in alkaline solution
to form THO (Figure 1) and that [13C6]-AscACR forms [13C5]-
THO due to hydrolysis and loss of 13CO2. The MS/MS spectrum
of THO (m/z 205 [M - H]-) shows a series of fragment ions
that can be rationalized by multiple loss of H2O neutrals and
R-cleavage of enolate products (Figure 4).
AscH-adequate THP-1 cells exposed to ACR, and (2) synthetic
AscACR is detectable in aqueous solution at neutral pH for
several hours, but it rapidly disappears when added to THP-1
cells or to human serum. These findings led us to examine the
fate of AscACR in aqueous solution, in THP-1 cells, and in
human serum. After the identification of THO as a degradation
product of AscACR in alkaline solution, we were able to detect
THO as a metabolite formed from ACR and AscH in THP-1
cells.
Hydrolysis of AscACR. When dissolved in water at neutral
pH, AscACR produced an LC-MS signal with m/z 231 ([M -
H]-) that decreased over a period of hours. A simultaneous
increase of a new signal with m/z 249 was observed that
corresponds to the addition of a water molecule to AscACR
(Figure 2A). The addition of a water molecule reached equi-
librium after approximately 24 h. We hypothesized that the
addition of a water molecule was due to hydrolysis of the lactone
moiety and not due to hydration of the aldehyde group. To test
this hypothesis, AscACR was dissolved in H218O, and the
incorporation of 18O was monitored. The aldehyde group of
AscACR is in equilibrium with a spiro-hemiacetal moiety
(Figure 1) and is expected to incorporate one 18O atom following
16O/18O exchange. Likewise, the oxygen atom at carbon-3 of
the hemiketal group is exchangeable. After 3 h of incubation
with H218O, AscACR indeed showed incorporation of one or
two 18O atoms, giving rise to isotopomers with m/z 233 and
m/z 235 (Figure 2B). The molecular species with m/z 249
produced three 18O isotopomers upon 16O/18O exchange, con-
sistent with hydrolysis of the lactone moiety in addition to 18
incorporation at the hemiacetal and hemiketal sites.
O
In order to be able to analyze the hydrophilic THO by
LC-MS using reversed-phase LC columns, samples contain-
ing THO were treated with pentafluorobenzyl hydroxyl amine
(PFBHA) to convert THO into its PFB oxime. THO-PFB
oxime was retained on a Synergy HydroRP C18 column
(HPLC System 2) and showed a molecular ion with m/z 400
in its Q1 mass spectrum. Mass fragmentation of THO-PFB
oxime gave rise to fragment ions resulting from cleavage of
When the H2O addition product of AscACR with m/z 249
was subjected to collision-induced dissociation (CID), the MS/
MS spectrum showed a fragment ion with m/z 205 () 249-44),
consistent with loss of CO2. The product’s 18O3-isotopomer with
m/z 255 produced a fragment ion with m/z 209 () 255-46),
consistent with the loss of 18OC16O. To identify the origin of
the carbon atom that is lost as carbon dioxide, the exchange

840 Chem. Res. Toxicol., Vol. 23, No. 4, 2010
Kesinger et al.
currents for two of the most prominent ion transitions of THO-
PFB oxime (panel A) and the corresponding SRM ion currents
of [13C5]-THO-PFB oxime (panel B) in a medium sample. Panels
A and B show the same chromatographic peak with a retention
time of 10.4 min. We attribute the observed peak splitting to
the presence of diastereoisomers (Figure 1). The SRM ion
currents shown in panel A were not observed for a solution of
[13C5]-THO-PFB oxime alone, and neither for the culture
medium nor the cell lysate samples prepared from cells that
were not pretreated with AscH or not exposed to ACR(Ac)2.
These findings demonstrate that THO is formed from AscH and
ACR(Ac)2.
The relative levels of THO were measured by LC-SRM in
culture medium and cell lysate samples prepared from AscH-
pretreated THP-1 cells following ACR(Ac)2 exposure. In
addition, THO formation was measured in FBS-containing and
FBS-lacking culture media that were coincubated with AscH
and ACR(Ac)2 in the absence of cells. Comparison of areas
under the curve (AUCs0-24 h, Figure 7A) revealed that the
relative amounts of intracellular THO were significantly higher
than the amounts of THO in medium surrounding the cells
(p < 0.001) and higher than that in the no-cell controls (medium
with FBS, p < 0.01; medium w/o FBS, p < 0.001).
Figure 4. Product ion mass spectrum of the m/z 205 [M - H]- ion of
THO obtained by LC-MS/MS analysis of a 2 h incubation of AscACR
in an aqueous solution of K2CO3 (0.18 M).
Our LC-SRM method allows for the detection of AscACR
as its PFB oxime, but it was not detected in culture medium
and cell lysate samples prepared from AscH-adequate THP-1
cells exposed to ACR(Ac)2. We did detect AscACR in FBS-
containing and FBS-lacking culture media that were coincubated
with AscH and ACR(Ac)2 in the absence of cells. The AscACR
concentration was maximal at 3 h and significantly higher in
the presence of FBS (Figure 7B).
Incubation of AscACR with Human Serum and
Paraoxonase (PON) 1 and 2. AscACR and THO were both
measured by LC-MS/MS in samples of AscACR incubated with
human serum, buffer (control experiment), and buffer containing
PON1 or PON2. At 3 min of incubation, the serum and PON
incubations contained 10-fold higher concentrations of THO
compared to that of the buffer control (Figure 8A), indicating
that PON1 and PON2 both accept AscACR as a substrate. At
30 min of incubation, AscACR was completely consumed in
all incubations, including the buffer control (Figure 8B). The
initial 10-fold difference in THO concentration between the
buffer and PON incubations disappeared during the 3-30 min
incubation period (Figure 8A). After complete consumption of
AscACR at 30 min, the concentration of THO continued to
increase between 30 min and 3 h of incubation in all samples,
which is explained by the transient formation of AscACR-acid
(Figure 1, not measured) and its decarboxylation to form THO.
The corresponding control reaction comparing the amount of
THO produced when AscACR is incubated with buffer, and
the buffer containing heat-denatured PON1 or PON2 showed
no statistical difference at any of the three time points (p > 0.05
for each comparison at a specific time point).
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.
the oxime to form nitrile species and R-cleavage of enolate
products. The odd-mass fragment with m/z 167 is readily
identified as the pentafluorobenzyl anion (Figure 5). In
subsequent LC-MS/MS experiments, the prominent fragment
ions with m/z 310 and m/z 112 were chosen for selected
reaction monitoring (SRM) to detect and semiquantify THO-
PFB oxime in biological samples.
Transformation of ACR(Ac)2 into THO. Human monocytic
THP-1 cells were pretreated with 1 mM AscH in the culture
medium for 18 h and subsequently exposed to 100 µM
ACR(Ac)2 and freshly added AscH (1 mM). At various times
following ACR(Ac)2 exposure, culture medium and cell lysates
were analyzed by LC-MS/MS using SRM. In the experiment
of Figure 6, the samples were also spiked with [13C5]-THO,
prepared by alkali treatment of [13C6]-AscACR, and then
immediately treated with PBFHA. Figure 6 shows the SRM ion
Lactonase Activity of the THP-1 Cell Lysate, FBS,
Human Serum, and Recombinant Paraoxonases. Human
serum, FBS, THP-1 cell lysate, PON1, and PON2 exhibited
lactonase activity as shown by their ability to hydrolyze DHC
(Table 1). DHC was chosen as a substrate to demonstrate the
lactonase activity of the various samples because this compound
has been used to detect the lactonase activity of human PON1,
PON2, and PON3 (38).
GSH Conjugates of ACR in THP-1 Cell Lysates and
Media. GSH conjugates were measured in culture medium and
cell lysate samples prepared from THP-1 cells exposed to

Biotransformation of Ascorbylated Acrolein
Chem. Res. Toxicol., Vol. 23, No. 4, 2010 841
Figure 6. LC-MS/MS analysis of culture medium obtained from AscH-adequate THP-1 cells exposed to ACR(Ac)2 for 3 h, spiked with [13C5]-
THO, and treated with PFB hydroxylamine. (A) Detection of THO-PFB oxime by selected reaction monitoring (SRM). (B) Simultaneous detection
of spiked [13C5]-THO-PFB oxime by using SRM. THO in the medium formed from AscH and ACR is chromatographically indistinguishable from
spiked [13C5]-THO.
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 of THP-1 cells. In the absence of cells, THO (panel A)
and AscACR (panel B) were both detected in FBS-containing and FBS-
lacking media that were coincubated with AscH and ACR(Ac)2.
Symbols represent the means ( SEM of five replicates (n ) 5).
Figure 8. Biotransformation of AscACR into THO. The formation of
THO was monitored after the incubation of AscACR with human serum,
PON-1, PON-2, and buffer only (panel A). Panel B shows the decrease
of the AscACR concentrations in the same incubations. The increase
of THO following complete consumption of AscACR within 30 min
is presumably due to the formation of the intermediate product,
AscACR-acid. Symbols represent the means ( SEM of three replicates
(n ) 3).
ACR(Ac)2, either with or without AscH preincubation. GSH-
ACR, GS-AA, and GSH-HP were all found to be produced
following the exposure of cells to ACR(Ac)2. Intracellular levels
of these GSH metabolites were maximal at 3 h and subsequently
declined over a period 24 h. GSH-HP (Figure 9A) was found
to be the major metabolite in both the cell lysates (Figure 9B)
and the media (Figure 9C), with a much higher concentration
found in the cells relative to the media. GSH conjugates were
not detected in culture medium exposed to ACR(Ac)2 in the
absence of cells.
Comparison of the areas under the curve (AUC0-24 h) revealed
a slightly lower amount of GSH-HP in cells pretreated with
AscH compared to that in AscH-deficient cells (Figure 9B,
Student’s t-test, p ) 0.037 with n ) 5). In Figure 9C, the average
amount of GSH-HP found in cell medium (AUC0-24 h) was
higher for the AscH-adequate cells, but the difference from

842 Chem. Res. Toxicol., Vol. 23, No. 4, 2010
Kesinger et al.
Table 1. Lactonase Activity of Human Serum, Fetal Bovine
Serum (FBS), and Recombinant Paraoxonases Using
Dihydrocoumarin (DHC) as a Substrate
lysates. These findings suggest either that AscACR is not formed
due to competing pathways such as glutathione-S-transferase
(GST)-mediated GSH conjugation and subsequent metabolism
of the GSH conjugates or that AscACR is formed but not
detectable due to degradation or metabolism. The second
hypothesis was tested by investigating the fate of AscACR in
neutral and in alkaline solution. By using 18O- and 13C-labeling
of AscACR, we found that AscACR undergoes hydrolysis of
the lactone moiety at neutral pH to form AscACR-acid (Figure
2). The carboxyl group of AscACR-acid spontaneously dis-
sociates from the ascorbyl moiety in the collision cell of the
mass spectrometer (Figure 3) and in alkaline solution to form
THO (Figure 4). THO was detected in the medium and lysate
prepared from AscH-adequate THP-1 cells exposed to
ACR(Ac)2 (Figure 6). Taken together, these results suggest that
AscACR is indeed formed but converted into THO.
enzyme source
lactonase activitya
human serum
FBS
THP-1
PON1
6.25 ( 0.16
0.172 ( 0.005
0.08 ( 0.01
34.3 ( 2.2
PON2
58.9 ( 3.8
a Data are expressed as mean ( SE (n ) 4). Lactonase activities of
serum (human and FBS) and recombinant PON1 or PON2 are expressed
as µmol DHC hydrolyzed/min/mL serum and µmol DHC hydrolyzed/
min/mg protein, respectively. Lactonase activity of THP-1 cells is
expressed as µmol DHC hydrolyzed/min/mg protein.
A similar transformation has been observed for ascorbigen,
the most well-known and studied ascorbylated product. Ascor-
bigen is formed during the degradation of the indole glucosi-
nolate, glucobrassicin, in the presence of AscH and is found in
many species of Brassicaceae (6, 32). Studies by Preobrazhen-
skaya and co-workers (33) have demonstrated that ascorbigen
undergoes hydrolysis of the lactone moiety and subsequent
decarboxylation to form a mixture of 1-indolyl-1-deoxytagatose
and 1-indolyl-1-deoxysorbose. Both 1-indolyl-1-deoxyketohex-
oses were found in bovine serum exposed ex vivo to ascorbigen
and in urine of mice after i.p. administration of ascorbigen (34).
The chemical transformation reported for ascorbigen in bovine
serum is essentially identical to the transformation of AscACR
into THO in cell culture medium and in human serum. The
observed splitting of the chromatographic peak that represents
THO (Figure 6) may be due to the formation of cyclic hemiketal
isomers, analogous to the degradation of ascorbigen in bovine
serum (34).
AscACR was detected in culture medium that was coincu-
bated with AscH and ACR(Ac)2 (Figure 7B) but not in culture
medium when THP-1 cells were present. Furthermore, the
maximum concentration of AscACR was higher when the
culture medium contained FBS. These findings indicate that
ACR(Ac)2 hydrolyzes spontaneously in culture medium and that
the hydrolysis is facilitated by components of FBS, presumably
by enzymes with esterase activity. In the presence of cells, levels
of THO in the media surrounding the cells were much lower
than the intracellular concentrations of THO (Figure 7A),
suggesting an intracellular source of THO via hydrolysis of
ACR(Ac)2, formation of AscACR, and metabolic conversion
of AscACR into THO.
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 the means
( SEM of five replicates (n ) 5).
AscH-deficient cells did not reach significance (Student’s t-test,
p ) 0.28 with n ) 5).
The conversion of AscACR into AscACR-acid involves
hydrolytic opening of the lactone ring of the ascorbyl moiety
(Figure 1). Therefore, we hypothesized that the conversion
is mediated by an enzyme or enzymes with lactonase activity.
Paraoxonases are a family of enzymes capable of hydrolyzing
the organophosphate paraoxon, the bacterial N-3-oxodode-
canoyl homoserine lactone, the δ-lactone dihydrocoumarin,
and, with the exception of PON2, a series of γ- and
δ-hydroxy alkanoic acid lactones (29, 35–38). PON1 is found
in serum associated with high-density lipoprotein (HDL),
while PON2 is found in human and murine macrophages but
not in serum (39). We tested the hypothesis that AscACR is
a substrate for PON1 and PON2. Both paroxonase isoenzymes
exhibited lactonase activity with DHC as a substrate (Table
1) and facilitated the conversion of AscACR into THO
(Figure 8). Human serum exhibited lactonase activity by
facilitating the hydrolysis of the PON-specific substrate DHC
Discussion
THP-1 cells provide an ideal biological environment in which
to investigate the covalent interaction between AscH and ACR
because these cells accumulate AscH at concentrations of up
to 9 mM and are capable of forming GSH conjugates of
2-alkenals and their metabolites (30) via enzyme-mediated
pathways that will compete with AscACR formation. ACR(Ac)2
was used as a pro-drug form of ACR to minimize adduct
formation of ACR with components of the cell culture medium
and to maximize intracellular concentrations of ACR by
enzyme-mediated release of ACR from ACR(Ac)2. Another
reason for not using free ACR is that it could react with AscH
in the medium despite the presence of cells. Exposure of AscH-
adequate THP-1 cells to ACR(Ac)2, however, did not yield
detectable concentrations of AscACR in the medium or cell

Biotransformation of Ascorbylated Acrolein
Chem. Res. Toxicol., Vol. 23, No. 4, 2010 843
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We have shown that ascorbylation of ACR and subsequent
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elimination of ACR that coexists with GSH conjugation of
ACR in THP-1 cells. The conversion of AscACR into THO
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Acknowledgment. We are grateful for support of this work,
in whole or in part, by National Institutes of Health Grants R01
HL081721 and S10 RR022589, as well as by USANA Health
Sciences, Inc., Salt Lake City, UT. We acknowledge the use of
the Mass Spectrometry Facility (Mr. Jeffrey Morre´) and the
Integrated Health Core (Ms. Mary Garrard) of the Environmental
Health Sciences Center at Oregon State University (NIH Grant
P30 ES000210).
Supporting Information Available: 1H, 13C, COSY, HMBC,
and HSQC NMR spectra and X-ray crystallographic data for
AscACR. This material is available free of charge via the
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