![[Feedback]](/icons/banner/feedbackACT.gif){kind=icon}
![[For Subscribers]](/icons/banner/subscriberACT.gif){kind=icon}
![[Archive]](/icons/banner/archiveACT.gif){kind=icon}
![[Search]](/icons/banner/searchACT.gif){kind=icon}
![[Contents]](/icons/banner/tocACT.gif){kind=icon}
|
|
|
|
|
||
Vol. 294, Issue 2, 735-745, August 2000
Drug Metabolism and Pharmacokinetics Section, DuPont Pharmaceuticals Company, Stine-Haskell Research Center, Newark, Delaware (A.E.M., J.S., R.E., A.D., L.-S.G.); and Chemical and Physical Sciences Division, DuPont Pharmaceuticals Company, Experimental Station, Wilmington, Delaware (N.G.)
 |
Abstract |
|---|
 Top
 Abstract
 Introduction
Materials and Methods
Results
Discussion
References
|
|---|
With the advent of liquid chromatography/mass spectrometry and liquid
chromatography/NMR, it has become easier to characterize metabolites
that were once difficult to isolate and identify. These techniques have
enabled us to uncover the existence of an alternate pathway for the
disposition of glutathione adducts of several structurally diverse
compounds. Studies were carried out using acetaminophen as a model
compound to investigate the role of the glutamic acid pathway in
disposition of the glutathione adducts. Although the mercapturic acid
pathway was the major route of degradation of the glutathione adducts,
it was found that the conjugation of the glutathione, cysteinylglycine,
and cysteine adducts of acetaminophen with the 
-carboxylic acid of
the glutamic acid was both interesting and novel. The coupling of the
glutathione adduct and the products from the mercapturic acid pathway
with the glutamic acid led to unusual peptide conjugates. The natures of these adducts were confirmed unequivocally by comparisons with synthetic standards. This pathway (addition of glutamic acids) led to
larger peptides, in contrast to the mercapturic acid pathway, in which
the glutathione adducts are broken down to smaller molecules. The
enzyme responsible for the addition of glutamic acid to the different
elements of the mercapturic acid pathway is currently unknown. It is
postulated that the -carboxylic acid is activated (perhaps by ATP)
before enzymatic addition to the 
-amino group of cysteine or
glutamate takes place. The discovery of these peptide conjugates of
acetaminophen represents a novel disposition of glutathione adducts of
compounds. The formation of such conjugates may represent yet another
pathway by which drugs could produce covalent binding via their
reactive intermediates.
| |
Introduction |
|---|
|
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
|
|---|
Glutathione
(GSH) is one of the most important molecules in the cellular defense
against chemically reactive toxic compounds or oxidative stress. This
protective function is due in part to its involvement in conjugation
reactions. GSH is a tripeptide composed of L-glutamic acid,
L-cysteine, and L-glycine (-Glu-Cys-Gly). The presence of cysteine in the tripeptide provides a sulfhydryl group
that is nucleophilic, and hence GSH reacts with electrophiles as the
thiolate ion, GS
. These electrophiles may be
chemically reactive and are produced as metabolic products of a phase 1 reaction, or they may be more stable foreign compounds. Hence, GSH
protects cells by removing reactive metabolites. The GSH adducts may
then either be excreted, usually into the bile, or the conjugate may
undergo further metabolism via the mercapturic acid pathway. This
involves sequential removal of the glutamyl and glycine groups and
acetylation of the cysteine amino group to produce the
N-acetylcysteine conjugate or mercapturic acid. Alternate
metabolic pathways or catabolism of these GSH conjugates has not been
described before. In this report, we describe a unique and novel
metabolic pathway for GSH adducts of acetaminophen. Acetaminophen is a
widely used analgesic known to cause hepatotoxicity in animals and in
humans exposed to high doses of this compound. The formation of the GSH
conjugate from acetaminophen has been well documented (Hinson et al.,
1982
). The degradation of the GSH adduct via the mercapturic acid
pathway has been the subject of several publications and reviews
(Commandeur et al., 1995, and the references cited therein).
A previously unreported metabolic pathway for these GSH adducts,
leading to the formation of peptide conjugates, is described in this
report. These polar metabolites were isolated from bile by solid phase
extraction and by reversed phase HPLC. Often, the structures of such
polar metabolites are not elucidated due to the difficulty in isolating
and separating them from endogenous components in the biological
matrices. With the advent of liquid chromatography/mass spectrometry
(LC/MS) and LC/NMR, it has become possible to elucidate the structures
of minor metabolites. Characterization of these polar conjugates can
provide very useful information regarding the metabolic pathways and
evidence for any potentially reactive metabolites of a compound. This
report describes the unequivocal identification of novel peptide
conjugates resulting from conjugation of GSH adducts and its breakdown
products with glutamic acid using LC/MS/MS, LC/NMR, and NMR approaches.
The -carboxylic acid coupling of glutamic acid to other amino acids of GSH-derived adducts was demonstrated by synthesizing authentic standards and comparing spectral data with isolated metabolites. These
metabolites are biologically novel, and the formation of these types of
metabolites may represent a previously unappreciated combination of
drug metabolism reactions.
| |
Materials and Methods |
|---|
|
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
|
|---|
Chemicals and Supplies.
Trifluoroacetic acid (TFA), phenol,
ethanedithiol, thioanisole, acetaminophen,
p-acetamidophenol-
-D-glucuronide,
N-acetylbenzoquinone imine (NAPQI), cysteinylglycine, and
-glutamylcysteine were purchased from Sigma-Aldrich Chemical Co.
(St. Louis, MO). Resin Fmoc-Gly and Fmoc-Cys (Trt) were obtained from
Advanced ChemTech (Louisville, KY).
Fmoc-L-glutamic acid--t-butyl ester
was obtained from NovaBiochem (San Diego, CA). All other amino acids
and synthesizer reagents were obtained from PE Biosystems (Foster City,
CA). Bond-Elut C18 cartridges (10 g/60 ml) were obtained from Varian
Sample Preparation Products (Harbor City, CA). All general solvents and
reagents were of the highest grade commercially available.
Synthesis of -Glu--Glu-Cys-Gly (Tetrapeptide) and
-Glu--Glu-Cys (Tripeptide).
The tetrapeptide
-Glu--Glu-Cys-Gly and the tripeptide -Glu--Glu-Cys were
made using L-isomers of each amino acid. The -carboxylic
acid was protected on the glutamic acid, allowing the -carboxylic
acid to be coupled with the amino functional group of the coupling
amino acid. The peptides were synthesized on solid phase using Fmoc
protection and 2-(1H-benzotriazol-1-yl)-1, 1, 3, 3, -tetramethyluronium hexafluorophosphate (HBTU) activation on a peptide
synthesizer (model ABI 433A; PE Biosystems). The TFA cleavage of the
peptide from the resin using reagent K (84% TFA, 4%
H2O, 4% thioanisole, 2% EDTA, 6% phenol; King
et al., 1990) was carried out, and the products were precipitated from ethyl ether. After lyophilization of the crude products, HPLC purification was performed on a Rainin Dynamax HPLC system using a
semipreparative C18 column (21.4 × 250 mm). The mobile phase consisted of two components: A (0.1% TFA in water) and B (10:90 v/v
acetonitrile/0.1% TFA). The proportion of component B was increased
from 0 to 15% over 15 min at a flow rate of 18 ml/min. A total of 7.6 mg of -Glu--Glu-Cys-Gly (tetrapeptide) and 1.7 mg of
-Glu--Glu-Cys (tripeptide) were obtained after lyophilizing the
collected fractions.
Synthesis of Tetrapeptide Conjugate (M6) of Acetaminophen.
The synthesis of
3-(-glutamyl-glutathion-S-yl)acetaminophen (M6, see
Scheme 1) was done by reacting freshly
prepared solution of NAPQI with -Glu--Glu-Cys-Gly. NAPQI (1.3 mg)
was dissolved in 167 µl of acetonitrile in a test tube, and 135 µl
was transferred to a vessel containing 2.2 ml of ice-cold water and 270 µl of 0.2 M ammonium acetate solution (pH 6.0).
-Glutamyl--glutamylcysteinylglycine (3.0 mg) was dissolved in 135 µl of water and added to the NAPQI solution. The reaction container
was covered with aluminum foil to protect NAPQI from light, and the
solution was stirred for 1 h at room temperature. After removal of
the solvent under nitrogen at room temperature, the residue was
reconstituted in water (500 µl) and chromatographed on a
semipreparative HPLC column (Waters Symmetry C18, 7.8 × 300 mm, 7 µm). The solvent system consisted of a mixture of acetonitrile and
0.05% TFA. The solvent composition was maintained at 5% acetonitrile
for the first 5 min, followed by a linear ramp to 15% acetonitrile in
the next 8 min. The percentage organic was increased to 60% after 13 min and maintained at this composition for the next 3 min. The column
was re-equilibrated with 5% acetonitrile in 0.1% TFA for 10 min
before the next injection. The solvent was delivered at a rate of 3.5 ml/min. Approximately 4.5 mg of the tetrapeptide conjugate was
recovered after HPLC purification. Mass spectral and NMR analyses were
performed to confirm the identity of the isolated product.
|
Synthesis of Cysteinylglycine Conjugate (M4) of Acetaminophen. The synthesis of 3-(glycinylcystein-S-yl)acetaminophen (M4, see Scheme 1) was done by reacting freshly prepared solution of NAPQI with cysteinylglycine in the same manner as described for metabolite M6. NAPQI (15 mg) was dissolved in 2 ml of acetonitrile in a scintillation vial, and the entire sample was transferred to a vessel containing 5 ml of ice-cold water and 1 ml of 0.2 M ammonium acetate solution (pH 6.0). Cysteinylglycine (18 mg) was dissolved in 2 ml of water and added to the NAPQI solution. The reaction was carried out as described, after which the solvent was removed under nitrogen at room temperature. The residue was reconstituted in water and chromatographed on a semipreparative HPLC column. The HPLC system used to separate the dipeptide conjugate was same as that described for isolation of the tetrapeptide conjugate, M6 (see earlier). A second HPLC purification was done using an isocratic mobile phase (10:90 v/v acetonitrile/0.05% TFA) delivered at a rate of 3.5 ml/min. Approximately 18 mg of the cysteinylglycine conjugate was recovered after purification.
Synthesis of Dipeptide Conjugate (M8) of Acetaminophen.
The
synthesis of 3-(-glutamylcystein-S-yl)acetaminophen (M8,
see Scheme 1) was done by reacting freshly prepared solution of NAPQI
with -glutamylcysteine. NAPQI (2.5 mg) was dissolved in 330 µl of
acetonitrile in a test tube, and 300 µl was transferred to a vessel
containing 4.8 ml of ice-cold water and 600 µl of 0.2 M ammonium
acetate solution (pH 6.0). -Glutamylcysteine (9.1 mg) was dissolved
in 0.5 ml of water, and 300 µl was added to the NAPQI solution. The
reaction container was covered with aluminum foil to protect NAPQI from
light, and the solution was stirred for 1 h at room temperature.
After removal of the solvent under nitrogen at room temperature, the
residue was reconstituted in water and chromatographed on a
semipreparative HPLC column. The HPLC system used to separate the
dipeptide conjugate was same as that described for isolation of
tetrapeptide conjugate M6 (see earlier). Approximately 3 mg of the
dipeptide conjugate was recovered after HPLC purification. Mass
spectral and NMR analyses were performed to confirm the identity of the
isolated product.
Synthesis of Tripeptide Conjugate (M9) of Acetaminophen.
The
synthesis of
3-(-glutamyl--glutamylcystein-S-yl)acetaminophen (M9,
see Scheme 1) was done by reacting freshly prepared solution of NAPQI
with -glutamyl--glutamylcysteine in the same manner as described
for metabolites M6 and M8. Due to the low yield of the product, the
tripeptide conjugate was not isolated for NMR analyses. Instead, the
product was analyzed directly by LC/MS, and comparisons were made with
the in vivo metabolite.
LCMS. LC/MS was carried out by coupling a Hewlett-Packard HPLC system (HP 1100) to a Finnigan LCQ ion trap mass spectrometer. The HPLC eluent was introduced to the mass spectrometer using a pneumatically assisted electrospray source. The mass spectrometer was operated in both the negative and positive ion modes. MSn (MS/MS experiments with n = 0-4) were done with relative collision energy set to 20 to 25%. Metabolites that could not be isolated in pure form for NMR analyses were subjected to several LC/MSn experiments for the purpose of structure elucidation.
For analyses of the acetaminophen metabolites, HPLC was carried out using a Hewlett-Packard HPLC system (HP 1100) coupled in sequence to a Waters symmetry C18 column (150 × 3.9 mm, 5 µm). The metabolites were separated by a gradient solvent system consisting of a mixture of acetonitrile and 0.05% TFA with the flow rate set at 0.7 ml/min. The initial conditions consisted of a mixture of acetonitrile and 0.05% TFA (5:95 v/v) and was maintained for 5 min after the sample was injected. The percentage of acetonitrile was increased from 5 to 30% in the next 15 min. After 20 min, the column was washed with 90% acetonitrile for 5 min before re-equilibrating with the initial mobile phase. Aliquots of bile and urine samples were injected directly onto the HPLC, and the eluent was introduced into the source of the mass spectrometer. To detect the metabolites in the fractions from C18 cartridges and from semipreparative HPLC column, aliquots (20-50 µl) were introduced into the mass spectrometer using the flow injection analysis method. The mobile phase consisted of a mixture of acetonitrile and 10 mM ammonium formate (1:1 v/v) delivered at a rate of 0.25 ml/min.LC/NMR.
The tetrapeptide conjugate of acetaminophen was
characterized by LC/NMR. All of the spectra were obtained using a
Bruker Avance 600 MHz NMR spectrometer equipped with an
1H/13C LC/NMR flow probe
with a cell volume of 120 µl. Suppression of the residual water and
acetonitrile signals was carried out using the WET solvent suppression
method in all of the LC/NMR experiments. Chemical shifts were
referenced to DMSO at 
of 2.49 ppm and to acetonitrile at of 2.0 ppm. An HP 1100 LC system was used with a Bruker Diode-Array detector
set at 254 nm. A 3.9 × 150 mm HPLC Waters Symmetry C18 5-mm
column was used. The initial HPLC conditions consisted of isocratic
elution using 95% D2O and 5%
acetonitrile-d3 for first 5 min, followed by a
gradient from 95% D2O and 5%
acetonitrile-d3 to 70% D2O
and 30% acetonitrile-d3 over 15 min at a flow
rate of 0.7 ml/min. Both solvents contained 0.05% TFA.
Animal Studies.
Six male Sprague-Dawley rats with cannulated
bile ducts (weighing between 250-400 g) were dosed orally with
acetaminophen (100 mg/ml, made in propylene glycol/water 8:2 v/v) once
daily at 500 mg/kg for 2 days. Serial bile and urine samples were
collected over ice and pooled from all of the animals on a daily basis
and stored frozen at 
20°C until analyzed. The dosing volume was 5 ml/kg. In another study, a single male Sprague-Dawley rat was dosed
i.v. (60 mg/kg) with the cysteinylglycine conjugate of acetaminophen (M4), and bile and urine were collected over 0- to 6- and 6- to 24-h
time intervals.
Isolation of GSH-Derived Conjugates from Rat Bile. Bile obtained from the rats was pooled, diluted 1:1 with distilled water, and loaded onto a C18 cartridge (10 g/60 ml). The sample was allowed to elute under gravity at a rate of less than 1 ml/min. After the sample had been loaded, the column bed was washed with 30 ml of deionized water followed by elution with 5, 10, and 15% methanol in water (20 ml volume of each). Aliquots from all the fractions were analyzed by LC/MS. The fractions containing the GSH related adducts were subsequently loaded onto C18 cartridges (10 g/60 ml) for further repurification. The fractions containing the metabolites were pooled and dried under vacuum. The dried residues were reconstituted in water and rechromatographed on a semipreparative HPLC column as described later.
Semipreparative HPLC separation of the polar metabolites of acetaminophen isolated from bile was carried out on a Waters Symmetry C18 (300 × 7.5 mm, 7 µm) using acetonitrile and 0.05% TFA as the eluent. The initial HPLC conditions consisted of isocratic elution using 95% H2O and 5% acetonitrile for first 5 min, followed by a gradient from 95% H2O and 5% acetonitrile to 85% H2O and 15% acetonitrile over 8 min. The percentage of acetonitrile was increased to 60% over the next minute and maintained at this for an additional 5 min before re-equilibrating the column with the initial mobile phase. The flow rate was set at 3.5 ml/min. The eluent was monitored at 254 nm using a variable wavelength detector. Five peaks were collected corresponding to metabolites M1 (m/z 328), M2 (m/z 232), M3 (m/z 271), M4 (m/z 328), and M5 (m/z 457). Samples corresponding to each metabolite were pooled and dried under vacuum. Final purification was done by rechromatographing each isolated metabolite on C18 cartridges. Repurification of M5 on an analytical column (3.9 × 150 mm, Waters Symmetry C18, 5 µm) resulted in separation of the tetrapeptide conjugate, M6 with MH+ at m/z 586 (see later). After pooling the fractions containing the metabolites, the samples were dried under vacuum and submitted for NMR analyses. Approximately 1 to 2 mg of each metabolite M1 to M5 were isolated for NMR analyses.Isolation of Tetrapeptide Conjugate of Acetaminophen from Rat Bile. The tetrapeptide conjugate, M6 (MH+ at m/z 586), coeluted with the GSH adduct of acetaminophen during the initial purification steps. This metabolite was isolated from the GSH adduct on an analytical column (Waters Symmetry C19, 3.9 × 150 mm) using an isocratic mobile phase consisting of a mixture of acetonitrile and 0.05% TFA (5:95 v/v) delivered at 0.7 ml/min. The peak corresponding to this metabolite (11.2 min) was collected, pooled from several injections, dried, and submitted for LC/NMR analyses.
Incubations of
3-(-Glutathion-S-yl)acetaminophen with Rat
Liver/Kidney Subcellular Fractions and Bile.
The purified GSH
conjugate of acetaminophen, M5, was incubated in the presence of rat
bile and rat liver/kidney subcellular fractions. The absence of any
other metabolites in the purified metabolite, M5, was confirmed by
LC/MS experiments before incubations. Approximately 250 µg of M5 was
incubated with 0.5 ml of freshly collected rat bile or with freshly
prepared liver/kidney S9 fractions (5 mg/ml). The samples were
incubated at 37°C for 1 h, after which the samples were analyzed
by LC/MS. Bile samples (25 µl) were analyzed by LC/MS without any
sample treatment. The liver/kidney incubations were precipitated with 3 ml of acetonitrile, and the supernatant was dried, reconstituted in the
HPLC mobile phase, and injected onto LC/MS.
| |
Results |
|---|
|
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
|
|---|
LC/MS and NMR Characterization of Acetaminophen Metabolites in Rat
Bile.
LC/MS analyses of bile samples from rats dosed with
acetaminophen showed that the polar metabolites, including the
glucuronide (M1), sulfate (M2), cysteine (M3), cysteinylglycine (M4),
and GSH (M5) conjugates, were well resolved from each other and from the endogenous materials using a gradient HPLC system. The metabolites were monitored by using the LCQ mass spectrometer and a
variable-wavelength UV detector. A representative total ion current
(TIC) and an HPLC/UV trace obtained simultaneously during the analysis
of a bile sample are shown in Fig. 2. A
number of unusual metabolites (M6-M9) were also found during the
analysis. These metabolites either coeluted with other known
metabolites of acetaminophen and/or were present in very low
quantities. The extracted ion chromatograms showing the presence of
these metabolites in bile are shown in Fig. 2.
|
|
). The
1H NMR data showed proton signals of an intact
aromatic ring [ 7.05 (d) and 7.43 (d)]. The aromatic signals were
characteristic of a para-substituted ring system. The signal
for the amide proton appeared at 9.82, whereas the acetyl group
appeared as a singlet at 2.0 ppm.
Metabolite M3 was found to be the cysteine conjugate with
MH+ at m/z 271. The
1H NMR data and the mass spectral fragmentation
pattern confirmed the structure of this metabolite. The
1H NMR spectrum showed signals at 7.60 (1H,
Ar-H, d), 7.24 (1H, Ar-H, dd), 6.80 (1H, Ar-H, d), 4.00 (1H, cys ,
m), 3.30 (1H, cys , m), 3.20 (1H, cys ', m), 2.00 (3H,
COCH3, s). The aromatic proton signals had
changed as a result of substitution on the ring.
Metabolite M4 was found to be the cysteinylglycine adduct of
acetaminophen. The 1H NMR showed signals for the
cysteine and glycine protons as reported for cysteinylglycine
conjugates (Mutlib et al., 1999). The 1H NMR
spectrum showed signals at 7.63 (1H, Ar-H, d), 7.24 (1H, Ar-H, dd),
6.82 (1H, Ar-H, d), 4.00 (1H, cys , m), 3.78 (2H, gly , m), 3.30 (1H, cys , m), 3.18 (1H, cys ', m), 2.00 (3H, COCH3, s). The aromatic proton signals were found
to be similar to those obtained for the cysteine conjugate. The
MH+ for the cysteinylglycine adduct was found at
m/z 328. The LC/MS retention times and mass
spectral fragmentation patterns for the metabolite and the synthetic
standard were found to be identical.
Metabolite M5 was the GSH adduct of acetaminophen with the mass
spectral data showing the pseudomolecular ion
(MH+) at m/z 457. The
1H NMR of the isolated metabolite clearly showed
the presence of the GSH and the loss of one of the aromatic protons.
The assignment of the proton signals were done by TOCSY experiments;
7.45 (1H, Ar-H, d), 7.22 (1H, Ar-H, dd), 6.75 (1H, Ar-H, d), 4.48 (1H, cys , m), 3.98 (1H, glu , m), 3.76 (2H, gly , d), 3.23 (1H, cys , m), 2.98 (1H, cys ', m), 2.41 (1H, glu , m), 2.35 (1H, glu ', m) 2.05 (2H, glut , ', m), 2.00 (3H,
COCH3, s). The 1H NMR
results obtained for M5 was found to be consistent with data reported
previously (Hinson et al., 1982). The MS/MS fragmentation ions are
shown in Fig. 3. A number of MSn
experiments with the fragment ions derived from
MH+ (m/z 457) and [M H] (m/z 455) were
done, and the results are shown in Tables
1 and 2.
Hence, M5 was confirmed as
3-(-glutathion-S-yl)acetaminophen.
|
|
|
-carboxyl group of the glutamic acid with the -amino group of the
glutamate present in the GSH adduct of acetaminophen. The proposed
structure of this metabolite is shown in Scheme 1. The mass spectral
data showed MH+ at m/z 586, which was consistent with an addition of a glutamic acid onto the GSH
adduct of acetaminophen. LC/MS analysis of bile samples obtained from
rats dosed with acetaminophen showed that this metabolite eluted after
the GSH conjugate on the HPLC column. The structure of this adduct was
supported by MSn experiments that showed that
the glutamic acid was linked to the GSH adduct via the -carboxylic
acid. The analyses were carried out in both positive and negative ion
modes to support the proposed structure. The fragment ions obtained
from these experiments are listed in Tables 1 and 2. The fragmentation
pathways confirming the structure of this conjugate is shown in Fig. 3.
Based on the product ion spectra of a series of model GSH adducts,
Haroldsen et al. (1988) identified routes of fragmentation of these
conjugates. Subsequent studies with other GSH adducts have confirmed
the generality of these pathways, regardless of ionization technique
adopted to generate the pseudomolecular ion. The MS/MS favors the
cleavage of specific peptide bonds in the GSH residue, notably the
-glutamyl linkage to yield ion a (Fig. 3). In the
positive ion mode, MS/MS of the pseudomolecular ion
(m/z 457) of the GSH conjugate produced the major
fragment ion at m/z 328 formed by the loss of the
glutamate moiety. Loss of glycine residue (75 amu) from the parent
ion led to a fragment ion at m/z 382. This
characteristic loss of glycine residue from the GSH adduct has been
described previously (Haroldsen et al., 1988) and was found to be very
diagnostic in confirming the structures of M6 and M7 (see later). The
ion at m/z 182 is derived from the cleavage of
carbon-sulfur bond leading to retention of sulfur on the aromatic ring
of acetaminophen. Such fragmentation pathway has been described for GSH
adducts of aromatic compounds (Haroldsen et al., 1988). The aromatic
thioether conjugates usually display a fragment resulting from the
cysteinyl C---S bond with proton transfer to the aromatic moiety to
yield a stabilized aryl-SH2+
species (ion b, Fig. 3). The MS/MS fragmentation of the
pseudomolecular ion (m/z 586) of the tetrapeptide
produced ions at m/z 457 and 511 formed by losses
of glutamate and glycine residues, respectively. Loss of glutamate from
ion m/z 511 yielded a significant ion at m/z 382. The fragmentation pattern produced from
different ions after several MSn experiments was
consistent with the structure proposed in Fig. 3 and Scheme 1. The
negative ion MS showed a fragmentation pattern dominated by fragments
resulting from the cleavage of amino acid residues from the
Cys-Gly-Glu entity. For comparison, the MS/MS data for the GSH
adduct is shown in Table 2. The MS/MS of the pseudomolecular ion
([M H] at m/z
584) of the tetrapeptide conjugate M6 produced a major fragment ion at
m/z 401 (Table 2). This ion represents the
Gly-Cys-Glu-Glu fragment with the sulfur of cysteine being
retained by the aromatic ring of the acetaminophen (fragment ion at
m/z 182). The MS/MS data obtained from
MSn experiments with different fragment ions are
shown in Table 2. The MS/MS data for intact reduced GSH is included in
Table 2 for comparison purposes. The fragment ions arising from MS/MS of ions at m/z 401 and 272 confirmed that the GSH
moiety linked to acetaminophen was intact (compare data with those of
M5 and GSH). Results from further MS/MS experiments confirming the
structure of the tetrapeptide is shown in Table 2.
The 1H NMR and TOCSY data for the tetrapeptide
conjugate M6 isolated from rat bile are shown in
Figs. 4 and 5, respectively. The
1H NMR spectrum clearly showed the presence of
additional resonances for the extra glutamic acid at 4.28 (1H, glu
, m), 2.3 (2H, glu , m), and 1.90-2.00 (2H, glu , m), in
addition to the usual GSH protons. The correlation between the ,
, and protons of the two glutamic acids in the molecule is shown
by TOCSY data (see Fig. 5). The LC/MS and NMR data obtained for the
synthetic standard were found to be identical with those obtained from
M6. Based on the spectroscopic data and comparison with synthetic standard, M6 was confirmed as
3-(-glutamyl-glutathion-S-yl)acetaminophen.
|
|
H] at
m/z 713. The structure of this conjugate was
determined to be
3-(-glutamyl--glutamyl-glutathion-S-yl)acetaminophen
as shown in Scheme 1. MS/MS of ion at m/z 713 produced sequential losses of two glutamates resulting in ions at
m/z 584 and 455 (see Table 2). Further MS/MS
studies with ions at m/z 584 and 455 supported the proposed structure shown in Scheme 1. M7 was not produced in
sufficient quantities to be isolated for characterization by NMR.
Metabolite M8 was found to be the dipeptide conjugate of acetaminophen
with MH+ at m/z 400. The
structure of this conjugate was confirmed as 3-(-glutamylcystein-S-yl)acetaminophen (see
Fig. 6). MS/MS of the pseudomolecular ion at
m/z 400 produced an ion at
m/z 271 formed by the characteristic loss of
glutamate residue. The other fragments included ions at
m/z 140, 182, 208, 225, and 254. An ion at
m/z 337 was postulated to be formed by subsequent
losses of NH3 and formic acid from the
-position of the attached glutamic acid. Hence, the data suggested
that the glutamic acid was linked to the amino group of the cysteine
via the -carboxylic terminal group. The structure of this conjugate
was confirmed by synthesizing the dipeptide conjugate of acetaminophen.
The 1H NMR (see Fig. 6) and HMBC experiments
confirmed the structure of the synthetic standard. The LC/MS retention
time and mass spectral data of the metabolite matched those of the
standard as shown in Fig. 7.
|
|
-glutamyl--glutamylcystein-S-yl)acetaminophen (see
Scheme 1). MS/MS of the pseudomolecular ion at
m/z 529 produced a characteristic ion at
m/z 466 that probably was formed by the losses of
ammonia and formic acid from the terminal glutamate moiety. Further
MS/MS experiments with the fragment ions at m/z 400 and 271 confirmed the postulated sequence of amino acids as proposed in Scheme 1.
LC/MS Characterization of Metabolites Produced by Cysteinylglycine
Conjugate (M4) of Acetaminophen.
Analyses of bile samples from the
rat dosed with M4 showed that most of the metabolite was converted to
N-acetylcysteine conjugate via the mercapturic acid pathway.
Interestingly, when acetaminophen was given orally to the rats,
cysteine and not N-acetylcysteine conjugate was detected as
one of the major metabolites. The purpose of administering M4 was to
show that GSH could be formed from this metabolite via the glutamic
acid pathway. Indeed, the GSH adduct was detected as a minor metabolite
in the bile. In addition to these metabolites, the
3-(-glutamylcystein-S-yl)acetaminophen conjugate M8 was
found in significant quantities in bile. Overall, the conversion of M4
via the mercapturic acid pathway was the dominant route of metabolism.
Incubation of 3-(-Glutathion-S-yl)acetaminophen
with Rat Liver/Kidney Subcellular Fractions and Bile.
It was found
that the GSH adduct, M5, was converted to the cysteinylglycine and
cysteine conjugates, M4 and M3, respectively, in the presence of rat
bile and liver/kidney subcellular fractions. The formation of M6 or any
of the peptide conjugates M7, M8, and M9 were not detected in any of
these experiments. The LC/MS profiles suggested that mercapturic acid
pathway predominated under the experimental conditions used during the incubations.
| |
Discussion |
|---|
|
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
|
|---|
Numerous reports have appeared in the literature describing the
formation of GSH adducts and their subsequent catabolism to cysteinylglycine, cysteine, and N-acetylcysteine conjugates
via the mercapturic acid pathway (Commandeur et al., 1995, and the literature cited therein). The GSH conjugates are formed by the interaction of an electrophilic compound, such as those produced by
bioactivation of drugs by cytochrome P450, with the endogenous pool of GSH. These reactions are either mediated by specific GSH transferases or are nonenzymatic in nature (Habig et al., 1974; Ketterer, 1986). The resulting GSH adducts are excreted in bile or are
further catabolized via the mercapturic acid pathway to cysteinylglycine, cysteine, or N-acetylcysteine adducts
(Commandeur et al., 1995). The cleaved cysteine conjugates are often
reabsorbed through the gastrointestinal tract and are converted to the
N-acetylcysteine derivatives by
N-acetyltransferases present in kidneys. The formation of
GSH conjugates of electrophilic compounds has generally been regarded
as a detoxification process. However, conjugation with GSH has also
been implicated in the activation of some xenobiotics to mutagenic and
carcinogenic electrophiles (Lash and Anders, 1986; Vamvaka et al.,
1987; Anders et al., 1988; Monks and Lau, 1989; Koob and Dekant, 1991).
Furthermore, evidence has been presented showing that GSH adducts of a
variety of compounds and/or their corresponding cysteine conjugates are
nephrotoxic (Elfarra and Anders, 1984; Nash et al., 1984; Monks et al.,
1985; Anders et al., 1987; Monks and Lau, 1989).
Acetaminophen, a commonly used analgesic, has been shown to produce a
GSH adduct via an interaction between its metabolic intermediate,
NAPQI, and endogenous GSH (Dahlin et al., 1984). The GSH adduct of
acetaminophen, 3-(glutathion-S-yl)acetaminophen (M5) has
been characterized before (Hinson et al., 1982). In this study, the GSH
adduct of acetaminophen and the products resulting from further
processing of this conjugate via the mercapturic acid pathway were
characterized spectroscopically. In addition to these metabolites, a
number of peptide conjugates were isolated from rat bile and
characterized spectroscopically. The MS and NMR analyses of these
conjugates confirmed the presence of additional glutamic acid moieties
linked to the intact GSH, cysteinylglycine, and cysteine adducts of
acetaminophen. The addition of one and two glutamic acids to the GSH
adduct of acetaminophen produced metabolites M6 and M7, respectively.
The addition of glutamic acid was also observed with the
cysteinylglycine and cysteine conjugates of acetaminophen to produce M5
(GSH adduct) and M8, respectively. Metabolites M8 and M9 were produced
as a result of conjugation of one and two glutamic acids to the
cysteine conjugate (M3) of acetaminophen, respectively. The enzyme
responsible for mediating the addition of glutamic acid to cysteine,
cysteinylglycine, and GSH adducts in the glutamic acid pathway is
currently unknown. It is postulated that -glutamyltranspeptidase
could be involved in the formation of these conjugates. The natural
occurrence of -glutamyl-GSH, a polyanionic tetrapeptide, has been
described previously (Abbott et al., 1986). The formation of
-Glu-GSH from endogenous GSH was demonstrated to have been mediated
by -glutamyltranspeptidase (Abbott et al., 1986). Studies are
ongoing to determine the nature of the enzyme or enzymes responsible
for the "glutamic acid pathway".
The coupling of glutamic acid to other amino acids was demonstrated to
occur via its -carboxylic acid group. This was confirmed by
comparing the NMR and MS data of the isolated metabolites with those of
synthetic standards. The identification of metabolites M8 and M9 was
important because it confirmed that the enzymatic addition of glutamate
was taking place on amino acids other than just the glutamate of the
GSH adducts. Furthermore, it was demonstrated from in vivo studies that
the cysteinylglycine conjugate itself could be converted to the GSH
conjugate via the aforementioned glutamic acid pathway. However, it was
shown that mercapturic acid pathway is more dominant than the glutamic
acid pathway.
The discovery of these tetrapeptide and pentapeptide conjugates of
acetaminophen represents a novel disposition of GSH adducts of
compounds. The degradation of GSH adducts via the mercapturic acid
pathway is the only one that has been described in the literature (Commandeur et al., 1995, and the literature cited therein). GSH adducts undergoing catabolism via mercapturic pathway lead to smaller
molecules such as cysteinylglycine and cysteine conjugates, which are
normally excreted in bile. In this study, we have uncovered an
alternate disposition pathway for GSH adducts (and for their breakdown
products formed via the mercapturic acid pathway) in which the
conjugates were taken as substrates by an unknown enzyme that added
glutamic acids in an ordered manner (see Scheme 1). This pathway seems
to act in the opposite manner compared with the mercapturic acid
pathway. Although the mercapturic acid pathway represented essentially
a degradation route for GSH adducts, the newly discovered glutamic acid
pathway appears to build on the GSH adducts (and their breakdown
products from mercapturic acid pathway) by adding elements of glutamic
acid. As shown in Scheme 1, glutamic acid was added sequentially to the
GSH adducts of acetaminophen to give metabolites M6 and M7. The
addition of glutamic acid was not only confined to the glutamic acid
moiety of the GSH adduct; instead, products were found in which the
glutamic acid was added sequentially to the cysteine to give
metabolites M8 and M9 or to the cysteinylglycine adduct to give M5. The
structures of these dipeptides, tripeptides, tetrapeptides, and
pentapeptides suggested that the -carboxylic acid of glutamic acid
was activated and subsequently added to the -amino group of the
conjugates. This type of reaction has gone unnoticed for a number of
reasons. Analytical techniques, such as LC/MS and LC/NMR, were not
available in the past to characterize these products that were once
considered trivial and nonidentifiable. In addition, because of their
low abundance in bile and their close proximity to the GSH adducts during chromatography, these peptides were easily overlooked (see Fig. 2). Numerous metabolism studies in the past with
radiolabeled compounds have shown the existence of radioactive peaks in
radiochromatograms that were not characterized. It is quite possible
that some of the radioactivity could have been due to products
resulting from glutamic acid pathway. With the advent of LC/MS, LC/NMR,
and high field NMR, it has become much easier to characterize compounds that were once considered unidentifiable by previously existing techniques. LC/MS has been found to be useful tool in characterizing polar metabolites such as the GSH adducts (Pallante et al., 1986; Haroldsen et al., 1988, Draper et al., 1989; Muck and Henion, 1990;
Burlingame et al., 1992, and the references cited therein; Baillie and
Davis, 1993). However, there are limitations with MS in obtaining
complete structural information on these metabolites. More frequently,
NMR has been found to be a complementary tool in elucidating structures
of complex metabolites such as those described in this report.
Metabolites formed as very polar diconjugates have recently been
confirmed by using a combination of such techniques (Spraul et al.,
1993; Mutlib et al., 1995, 1999; Shockcor et al., 1996; Tang and
Abbott, 1996; Lindon et al., 1997).
The characterization of these peptides provides very convincing
evidence of the existence of a competing pathway ("glutamic acid
pathway") for GSH conjugates. Although the levels of these peptides
appeared low in bile compared with the GSH adducts and the mercapturic
acid products, the amount of these products formed intracellularly is
not known. The formation of tetrapeptides and pentapeptides suggests
the possibility that larger peptides were made by cells (probably
elongation of the peptide chain by the sequential addition of glutamic
acid via the -carboxylic acid linkage) that were not detected in
this study. These probably were not identified because they were
confined to cells (i.e., could not be transported out) and hence were
not eliminated in bile. In vivo studies with M4 and in vitro studies
with M5 incubated with rat bile or rat kidney/liver S9 fractions
suggested that the mercapturic acid pathway was the predominant route
for the disposition of GSH adducts. The contribution toward disposition of the GSH adducts by the glutamic acid pathway appears to be minor in
comparison with the mercapturic acid pathway. A number of in-house
discovery compounds that were structurally different from acetaminophen
and were capable of producing GSH adducts were found to produce
metabolites via the glutamic acid pathway.
The formation of such conjugates may represent yet another pathway by
which drugs could produce covalent binding via their reactive metabolic
intermediates. Previous reports have suggested the direct interaction
of reactive metabolic intermediate of compounds such as acetaminophen
with proteins and cellular components leading to covalent binding and
possibly toxicity (Jollow et al., 1973; Streeter et al., 1984). The
results from these studies suggest an alternate route for the
interaction of reactive metabolites with cellular components may exist.
Because the cellular concentration of endogenous GSH is fairly high
(approximately 5 mM in hepatocytes), there is a good possibility that a
reactive metabolic intermediate will interact with the thiolate of GSH
to a greater extent than with other protein components. This in turn
leads to the GSH adducts such as metabolites M5 of acetaminophen.
Subsequent processing of these adducts via this novel metabolic pathway
could lead to longer peptides (e.g., M6 and M7) in which the
incorporated moiety includes the trapped reactive metabolic
intermediate already linked to cysteine or GSH. The consequence of
having peptides such as the ones described in this report is not known.
It is possible that these peptides could disrupt cellular functions
with toxicological consequences. Furthermore, the retention of
exogenous compounds linked to peptides could potentially have
immunological consequences such as the production of allergies or
idiosyncratic reactions. The metabolic and physiological significance
of existence of such drug-peptide conjugates requires additional study.
One must also consider the possibility that these conjugates have no
function and that their formation represents a side reaction catalyzed by the -glutamyltranspeptidase.
The formation of peptide conjugates of acetaminophen was confirmed by
MS and NMR analyses of isolated metabolites. It was shown that these
peptides were formed by the addition of (activated) glutamic acid via
the -carboxylic acid functional group to the -amino group of the
adducts. The glutamic acid pathway appears to function in the opposite
manner to mercapturic acid pathway. Longer peptide conjugates are
produced via this metabolic pathway compared with the mercapturic acid
pathway, which essentially degrades the GSH adducts to smaller molecules.
| |
Footnotes |
|---|
Accepted for publication April 26, 2000.
Received for publication November 16, 1999.
Send reprint requests to: Dr. A. E. Mutlib, Drug Metabolism and Pharmacokinetics Section, DuPont Pharmaceuticals Company, P.O. Box 30, 1094 Elkton Rd., Newark, DE 19714-0030. E-mail: abdul.mutlib{at}dupontpharma.com
| |
Abbreviations |
|---|
GSH, glutathione;
LC, liquid chromatography;
FMOC, N--fluorenylmethoxycarbonyl;
MS, mass
spectrometry;
SPE, solid phase extraction;
COSY, correlated
spectroscopy;
TOCSY, total correlated spectroscopy;
HMBC, heteronuclear
multiple bond coherence;
HSQC, heteronuclear single quantum
coherence.
| |
References |
|---|
|
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
|
|---|
)-cis-5-fluoro-1-[2-(hydroxymethyl)-1,3-oxathiolan-5-yl]cytosine, a nucleoside analogue active against human immunodeficiency virus (HIV).
Xenobiotica
26:
189-199[Medline].This article has been cited by other articles:
|
|
 |
|
  S. E. Kostrubsky, J. F. Sinclair, S. C. Strom, S. Wood, E. Urda, D. B. Stolz, Y. H. Wen, S. Kulkarni, and A. Mutlib Phenobarbital and Phenytoin Increased Acetaminophen Hepatotoxicity Due to Inhibition of UDP-Glucuronosyltransferases in Cultured Human Hepatocytes Toxicol. Sci., September 1, 2005; 87(1): 146 - 155. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 W. Yin, G. A. Doss, R. A. Stearns, and S. Kumar N-ACETYLATION OF THE GLUTAMATE RESIDUE OF INTACT GLUTATHIONE CONJUGATES IN RATS: A NOVEL PATHWAY FOR THE METABOLIC PROCESSING OF THIOL ADDUCTS OF XENOBIOTICS Drug Metab. Dispos., January 1, 2004; 32(1): 43 - 48. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
V. S. Gopaul, W. Tang, K. Farrell, and F. S. Abbott Amino Acid Conjugates: Metabolites of 2-Propylpentanoic Acid (Valproic Acid) in Epileptic Patients Drug Metab. Dispos., January 1, 2003; 31(1): 114 - 121. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 K. J. Rutherfurd, S. M. Rutherfurd, P. J. Moughan, and W. H. Hendriks Isolation and Characterization of a Felinine-containing Peptide from the Blood of the Domestic Cat (Felis catus) J. Biol. Chem., January 4, 2002; 277(1): 114 - 119. [Abstract] [Full Text]
|
|
|
|
|
|
A. Mutlib, J. Shockcor, S.-Y. Chen, R. Espina, J. Lin, N. Graciani, S. Prakash, and L.-S. Gan Formation of Unusual Glutamate Conjugates of 1-[3-(Aminomethyl)phenyl]-N-[3-fluoro-2'-(methylsulfonyl)-[1,1'-biphenyl]- 4-yl]-3-(trifluoromethyl)-1H-pyrazole-5-carboxamide (DPC 423) and Its Analogs: The Role of gamma -Glutamyltranspeptidase in the Biotransformation of Benzylamines Drug Metab. Dispos., October 1, 2001; 29(10): 1296 - 1306. [Abstract] [Full Text] [PDF]
|
|
| ||||||||
