Mass spectrometric characterization of circulating covalent protein adducts derived from a drug Acyl glucuronide metabolite: Multiple albumin adductions in diclofenac patients

Article abstract of DOI:10.1124/jpet.114.215079

Covalent protein modifications by electrophilic acyl glucuronide (AG) metabolites are hypothetical causes of hypersensitivity reactions associated with certain carboxylate drugs. The complex rearrangements and reactivities of drug AG have been defined in great detail, and protein adducts of carboxylate drugs, such as diclofenac, have been found in liver and plasma of experimental animals and humans. However, in the absence of definitive molecular characterization, and specifically, identification of signature glycation conjugates retaining the glucuronyl and carboxyl residues, it cannot be assumed any of these adducts is derived uniquely or even fractionally from AG metabolites. We have therefore undertaken targeted mass spectrometric analyses of human serum albumin (HSA) isolated from diclofenac patients to characterize drug-derived structures and, thereby, for the first time, have deconstructed conclusively the pathways of adduct formation from a drug AG and its isomeric rearrangement products in vivo. These analyses were informed by a thorough understanding of the reactions of HSA with diclofenac AG in vitro. HSA from six patients without drug-related hypersensitivities had either a single drug-derived adduct or one of five combinations of 2-8 adducts from among seven diclofenac N-acylations and three AG glycations on seven of the protein's 59 lysines. Only acylations were found in every patient. We present evidence that HSA modifications by diclofenac in vivo are complicated and variable, that at least a fraction of these modifications are derived from the drug's AG metabolite, and that albumin adduction is not inevitably a causation of hypersensitivity to carboxylate drugs or a coincidental association. Copyright

Full text of DOI:10.1124/jpet.114.215079

Supplemental material to this article can be found at:
http://jpet.aspetjournals.org/content/suppl/2014/06/05/jpet.114.215079.DC1.html
1521-0103/350/2/387–402$25.00
THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS
http://dx.doi.org/10.1124/jpet.114.215079
J Pharmacol Exp Ther 350:387–402, August 2014
Copyright ª 2014 by The American Society for Pharmacology and Experimental Therapeutics
Mass Spectrometric Characterization of Circulating Covalent
Protein Adducts Derived from a Drug Acyl Glucuronide
Metabolite: Multiple Albumin Adductions in Diclofenac Patientss
Thomas G. Hammond,1 Xiaoli Meng, Rosalind E. Jenkins, James L. Maggs,
Anahi Santoyo Castelazo, Sophie L. Regan, Stuart N. L. Bennett, Caroline J. Earnshaw,
Guruprasad P. Aithal, Ira Pande, J. Gerry Kenna,2 Andrew V. Stachulski, B. Kevin Park,
and Dominic P. Williams
Medical Research Council Centre for Drug Safety Science, Institute of Translational Medicine, (T.G.H., X.M., R.E.J., J.L.M., A.S.C., S.L.R.,
C.J.E., B.K.P., D.P.W.) and Department of Chemistry (A.V.S.), University of Liverpool, Liverpool, United Kingdom; Nottingham Digestive
Diseases Centre, NIHR Nottingham Digestive Diseases-Biomedical Research Unit, Nottingham University Hospitals NHS Trust and University
of Nottingham (G.P.A.) and Department of Rheumatology, Nottingham University Hospitals NHS Trust (I.P.), Nottingham, United Kingdom; and
AstraZeneca U.K. Ltd (S.N.L.B.) and Safety Assessment, AstraZeneca U.K. Ltd (J.G.K.), Alderley Park, Macclesfield, Cheshire, United Kingdom
Received March 31, 2014; accepted May 29, 2014
ABSTRACT
Covalent protein modifications by electrophilic acyl glucuronide (AG) thereby, for the first time, have deconstructed conclusively the
metabolites are hypothetical causes of hypersensitivity reactions pathways of adduct formation from a drug AG and its isomeric
associated with certain carboxylate drugs. The complex rearrange- rearrangement products in vivo. These analyses were informed by
ments and reactivities of drug AG have been defined in great detail, a thorough understanding of the reactions of HSA with diclofenac AG
and protein adducts of carboxylate drugs, such as diclofenac, have in vitro. HSA from six patients without drug-related hypersensitivities
been found in liver and plasma of experimental animals and humans. had either a single drug-derived adduct or one of five combinations of
However, in the absence of definitive molecular characterization, and 2–8 adducts from among seven diclofenac N-acylations and three AG
specifically, identification of signature glycation conjugates retaining glycations on seven of the protein’s 59 lysines. Only acylations were
the glucuronyl and carboxyl residues, it cannot be assumed any of found in every patient. We present evidence that HSA modifications
these adducts is derived uniquely or even fractionally from AG by diclofenac in vivo are complicated and variable, that at least
metabolites. We have therefore undertaken targeted mass spectro- a fraction of these modifications are derived from the drug’s AG
metric analyses of human serum albumin (HSA) isolated from metabolite, and that albumin adduction is not inevitably a causation of
diclofenac patients to characterize drug-derived structures and, hypersensitivity to carboxylate drugs or a coincidental association.
This work was undertaken principally through a CASE studentship
awarded to T.G.H. that was funded by the BBSRC (Integrative Mammalian
Introduction
Biology award) and Safety Assessment U.K., AstraZeneca U.K. Ltd, as part of
the Centre for Drug Safety Science supported by the Medical Research Council
Acyl glucuronides (AG) are exceptional even among the
numerous electrophilic metabolites of drugs (Stepan et al.,
2011), including the multiple reactive intermediates of car-
boxylic acids (Kenny et al., 2004; Skonberg et al., 2008),
because they can circulate in plasma, principally in non-
covalent association with protein (Williams and Dickinson,
1994) and sometimes at concentrations matching or exceeding
those of the parent compound (Dockens et al., 2000; Zhang
et al., 2011). The circulation of AG, combined with extrahe-
patic expression of uptake transporters of O-glucuronides
(Schiffer et al., 2003), allows for much wider systemic dis-
tribution and potentially much more widespread biologic
effects than are usually associated with chemically reactive
drug metabolites. From the earliest identifications of AG as
unstable and protein-reactive conjugates these commonplace
metabolites have been linked persistently, and at times
almost generically, but always somewhat uncertainly, with
the varied adverse reactions of carboxylate drugs (Regan
et al., 2010; Sawamura et al., 2010). This hypothetical linkage
This article has supplemental material available at jpet.aspetjournals.org. of protein adduction and toxicity has been particularly
[Gant G0700654]. X.M. was funded by the NIHR Biomedical Research Centre
in Microbial Diseases; S.L.R. by Safety Assessment U.K., AstraZeneca U.K.
Ltd; and C.J.E. by the MRC Centre for Drug Safety Science.
Some of the data in this article were published previously in the form of conference
abstracts: Hammond T, Regan S, Meng X, Jenkins R, Kenna G, Sathish J, and
Williams D (2009) An investigation into the in vitro and in vivo fate of acyl
glucuronides. British Pharmacology Society Winter Meeting; 2009 Dec 15–17;
London, UK. British Pharmacology Society; http://www.pA2online.org/abstracts/
Vol7Issue4abst074P.pdf; Hammond TG, Regan SL, Meng X, Maggs JL, Jenkins
RE, Kenna GL, Sathish JG, Williams DP, and Park BK (2010) Protein binding and
pharmacokinetics of reactive acyl glucuronide drug metabolites, in Toxicology;
2010 28–31 Mar; Edinburgh, Scotland. Vol 278, pp 357–358, British Toxicology
Society Annual Congress; Hammond T, Regan S, Meng X, Berry N, Maggs J,
Jenkins R, Kenna G, Sathish J, Williams D, and Park K (2011) In vitro assessment
of the interactions between acyl glucuronide drug metabolites and human serum
albumin. British Pharmacology Society Winter Meeting; 2010 14–16 Dec; London,
UK. British Pharmacology Society; http://www.pA2online.org/abstracts/
Vol8Issue1abst019P.pdf; Hammond TG, Regan SL, Meng X, Maggs JL,
Jenkins RE, Kenna JG, Sathish JG, Williams DP, and Park BK (2011) In vitro
assessment of the interactions between diclofenac and tolmetin acyl glucuronide
drug metabolites and human serum albumin, in Toxicology; 2011 27–30 Mar;
Durham, UK. Vol 290, pp 40–41. British Toxicology Society Annual Congress.
1Current affiliation: Division of Molecular and System Toxicology, Department of
Pharmaceutical Sciences, University of Basel, Pharmacenter, Basel, Switzerland.
2Current affiliation: Safety Science Consultant, Macclesfield, United Kingdom.
dx.doi.org/10.1124/jpet.114.215079.
s
387

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Hammond et al.
enduring but no less contentious in the case of hypersensitivity metabolites frequently circulate in plasma (Benet et al.,
reactions to nonsteroidal anti-inflammatory drugs (NSAIDs), 1993). It was used successfully in early mass spectrometric
such as diclofenac.
studies on protein adduction by NSAID AG in vitro (Ding
Hapten characterization is key to understanding hypersen- et al., 1995; Qiu et al., 1998). Modification sites and structures
sitivity reactions. Protein adducts of carboxylate drugs have were analyzed on tryptic peptides, using mass spectrometry
been located in liver and plasma by radiochemical tracing to search lysines specifically for AG/thioester acylations and
(Masubuchi et al., 2007), immunovisualization (Aithal et al., the glycation adducts derived exclusively from AG. These
2004), and hydrolytic deconjugation (Zia-Amirhosseini et al., systematic searches were informed by detailed understanding
1994). Partial assignments of modified hepatic (Wade of reactions between synthetic diclofenac AG and HSA.
et al., 1997) and plasma (Bailey and Dickinson, 1996) proteins
Liquid chromatography–tandem mass spectrometry (LC-
were achieved by immunoblotting. Antibodies to diclofenac- MS/MS) can identify many peptide modifications from a single
haptenated proteins circulate in some patients (Aithal et al., milligram-scale HSA sample. We have used this technology to
2004). However, neither the metabolic origins nor structures characterize benzylpenicillin haptens in patients (Meng et al.,
2011). Now, for the first time, protein adducts derived from an
of the covalent modifications could be defined completely.
Correlations of plasma protein adduction with AG exposure in AG metabolite have been identified in vivo.
humans implicate direct combination (Castillo et al., 1995),
but definitive identification of adducts derived uniquely from
AG metabolites has proved elusive.
Materials and Methods
Any attempt to deconstruct protein haptenation by carbox-
ylate drugs in vivo must differentiate between adduction
products, some with identical substructures, of multiple bio-
activation pathways: oxidation (Kenny et al., 2004), thio-
esterification (Grillo et al., 2003), and acyl glucuronylation
(Stierlin and Faigle, 1979). Thioester and AG metabolites will
produce indistinguishable N-acyl adducts (Skonberg et al.,
2008). This dilemma is resolvable by exploiting the complex
rearrangements of biosynthetic 1-b AG (Stachulski et al.,
2006). Sequential nucleophilic attacks on the ester carbonyl
by the glucuronyl hydroxyls cause progressive acyl migration
(Fig. 1). The three AG regioisomers undergo deannulation and
anomerization, yielding aldehydes that form hydroxyimine
glycation adducts, and possibly tautomeric ketoamine struc-
tures, via condensation reactions with lysyl «-amine groups
(Ding et al., 1993). Glycation of human plasma proteins by AG
in vivo is suggested by a higher correlation of adduction with
exposure to positional isomers than exposure to 1-b AG
(Hyneck et al., 1988). Therefore, carboxylate deconjugated
from proteins hydrolytically might derive indiscriminately
from side-chain acylations and the ester linkages within
glycation adducts. The fundamental challenge to confirming
protein adduction by drug AG in patients is identification of
complete glycation structures.
Reagents. Diclofenac sodium, zomepirac sodium, dithiothreitol,
iodoacetamide, and HSA (approximately 99% pure, essentially fatty
acid free and globin free; product A3782) were purchased from Sigma-
Aldrich (Poole, Dorset, UK). Protein concentrations were determined
throughout using Bradford assay dye reagent purchased from Bio-
Rad (Hemel Hempstead, Hertfordshire, UK). Sequencing-grade mod-
ified trypsin was obtained from Promega (Southampton, Hampshire,
UK). LC-MS–grade (acetonitrile, ethanol, isopropanol, and methanol)
and high-performance liquid chromatography–grade (diethyl ether
and ethyl acetate) organic solvents were purchased from Fisher
Scientific (Loughborough, Leicestershire, UK). Standard inorganic
chemicals and organic acids were products of either Sigma-Aldrich or
Fisher Scientific.
Synthesis of Diclofenac 1-b AG. The chemical synthesis of
diclofenac 1-b AG via a modified form of the method of Bowkett et al.
(2007) is described in Supplemental Scheme 1.
Human Blood Collection. Blood taken for preparing the single
pool of blank plasma used in the LC-MS/MS assays of diclofenac and
its AG and for the incubations with diclofenac 1-b AG was obtained
from three healthy unmedicated male volunteers, aged 21–25 years,
who gave informed consent according to a procedure approved by the
University of Liverpool Committee of Research Ethics. The blood was
collected into 9-ml lithium heparin–coated Vacuette tubes (Greiner
Bio-One GmbH, Kremsmünster, Austria). Plasma was separated by
centrifugation at 2000g for 10 minutes and stored at 280°C.
Isomerization and Hydrolysis of Diclofenac 1-b Acyl Glucu-
ronide in Phosphate Buffer, HSA Solution, and Human
Plasma In Vitro. The incubation conditions and analytical tech-
niques used for investigating these reactions, including the methods for
identifying the regioisomeric degradation products of diclofenac 1-b AG,
are detailed in Supplemental Methods.
Diclofenac is ideally suited to a search for AG-derived
protein adducts in vivo: its conjugate, having a half-life of
0.51–0.7 hour in pH 7.4 buffer, is among the most reactive AG
of currently prescribed pharmaceuticals (Stachulski et al.,
2006; Sawamura et al., 2010). Diclofenac undergoes covalent
binding to plasma proteins in rats (Masubuchi et al., 2007).
Covalent Binding of Diclofenac Residues to HSA Incubated
with 1-b AG In Vitro. The incubation conditions and analytical
Diclofenac AG circulates in mice (Sparidans et al., 2008) and techniques used for measuring the covalent binding of diclofenac
residues to HSA are detailed in Supplemental Methods and Supple-
mental Table 1.
reacts with human serum albumin (HSA) (Kenny et al., 2004)
and hepatic microsomal protein (Kretz-Rommel and Boelsterli,
1994) in vitro.
Protein adduction by the reactive metabolites of diclofenac
was analyzed using HSA from patients without drug-related
hypersensitivities. HSA has numerous nucleophilic groups,
including 59 «-amines, an elimination half-time of about 19
days (Nicholson et al., 2000) favoring accumulation of mod-
ified protein (Zia-Amirhosseini et al., 1994), and is a physio-
Recruitment of Diclofenac Patients, Blood Sampling, and
Sample Stabilization. The clinical study was designed for analysis
of circulating drug-derived HSA adducts. Ethical approval was
obtained from the National Research Ethics Service Committee East
Midlands-Derby. All the patients were recruited from a rheumatology
clinic in the Nottingham University Hospitals and gave informed
written consent. They were of Northern European ethnic origin and
had been on a stable daily dose of diclofenac (100–150 mg in two or
logically relevant target for adduction by AG because these three divided doses) for at least 1 year (Table 1; additional details in
ABBREVIATIONS: AG, acyl glucuronide; HSA, human serum albumin; LC, liquid chromatography; MRM, multiple reaction monitoring; MS, mass
spectrometry; NSAIDs, nonsteroidal anti-inflammatory drugs.

Circulating Protein Adducts in Diclofenac Patients
389
Fig. 1. (A) Diclofenac. (B) Proposed mechanisms of protein adduction at lysine residues by reactive AG and thioester metabolites of diclofenac in vivo.
Synthetic diclofenac 1-b AG acylated and/or glycated up to ten of HSA’s 59 lysines in vitro depending on the molar ratio (Table 2). Between one and six
modified HSA lysines were identified in each diclofenac patient (Table 4). The acylation adducts and complete glycation adducts were found in all and
three of the six patients, respectively. In vivo, only complete glycation adducts, retaining the glucuronyl and drug carboxyl residues, are unambiguously
formed from AG metabolites. The acylation adducts, as discussed in the text, might also be formed in vivo from thioester metabolites of coenzyme A (CoA)
and glutathione (GSH). Only the 1-a AG, produced from 2-a AG by reverse acyl migration, was seen in vitro, but in principle all of the anomers can be
formed. The 1-b AG, as shown here, is the predominant acylating isomer in vitro, but involvement of the three regioisomers and their anomers cannot be
excluded. Lysine N-«-glycation by the 3-b AG is purely representative; the regioisomers involved were not identified. The extent of any Amadori
rearrangements of hydroxyimine adducts (Schiff bases) of the C-3 and C-4 esters to ketoamines is unknown.
Supplemental Table 3). This extended period of diclofenac therapy
was selected to attain the highest level of plasma protein adduction by
and clinical observation, demonstrated evidence of either liver injury
or drug-related hypersensitivity reactions at the time of the study.
reactive drug metabolites through multiple dosing (Zia-Amirhosseini Some had in the past experienced mild, transient, alanine amino-
et al., 1994). The patients were undergoing regular review, including transferase rises, but those episodes were not clinically relevant and
blood monitoring for their disease every 3 months. None of the pa-
did not lead to drug or dose modifications. No restrictions were placed
tients, based upon routine liver enzyme assays (serum alanine amino- on the patients’ food intake. For patients N01–N03, who had taken
transferase, aspartate aminotransferase, and g-glutamyl transferase)
two tablets daily, single blood samples (18 ml) removed by venipuncture
TABLE 1
Patients dosed with diclofenac
The complete lists of comedications are recorded in Supplemental Table 3. A blank entry signifies that none of the comedications is known to form an AG metabolite in
humans.
Patient
Diclofenac Doses and Formulationsa
Blood Sampling after Last Diclofenac Dose
Comedications Metabolized to AG in Humansb
hrs
N01 (male, 42 yr)
N02 (female, 65 yr)
N03 (female, 52 yr)
N08 (male, 63 yr)
N09 (male, 48 yr)
N10 (female, 77 yr)
50 mg, b.i.d. (EC)
50 mg, b.i.d. (EC)
50/75 mg AM/PM (EC/MR)
50 mg, t.i.d. (EC)
1
1
1
3
2.5
2.5
aspirin, ramipril rosuvastatin
simvastatin
50 mg, t.i.d. (EC)
50 mg, t.i.d. (Voltarol dispersible
tablets)
EC, enteric-coated; MR, modified release.
aDiclofenac sodium formulations except where indicated. All the patients had taken diclofenac for at least 1 year.
bAG metabolites of comedications are formed in vivo and/or in vitro.

390
Hammond et al.
were collected into lithium heparin–coated Vacuette tubes 1 hour after before. The pellet was redissolved in ammonium bicarbonate solution
the first tablet of the day. Samples were collected similarly from (50 mM, 30 ml) and assayed for protein content. The remainder of the
patients N08–N10 2.5–3 hours after the first tablet of the day. Blood solution (protein concentration, 3.2 mg/ml) was digested with trypsin
was collected over melting ice to minimize AG degradation and protein (5 mg) at 37°C overnight. The digests were desalted using 0.6-ml bed
adduction ex vivo and centrifuged immediately at 2000g for 10 minutes C18 Zip-Tip pipette tips (Millipore, Billerica, MA), eluted with
at 4°C. A drug AG in human plasma can be stabilized effectively
acetonitrile-0.1% trifluoroacetic acid (1:1, v/v; 10 ml), and dried by
through cooling alone (Matthews and Woolf, 2008). Plasma aliquots centrifugation under vacuum (SpeedVac; Eppendorf UK Ltd, Cam-
(60 ml) intended for mass spectrometric analyses of drug-derived HSA bridge, UK) before LC-MS/MS analysis.
adducts were frozen immediately at 280°C. Plasma containing drug
Isolation of HSA from Diclofenac Patients. HSA was isolated
AG is conventionally acidified to stabilize the conjugates (Hyneck by affinity chromatography from stored unacidified plasma samples
et al., 1988; Matthews and Woolf, 2008), but these aliquots remained (60 ml; equivalent to approximately 2.4 mg HSA) of six diclofenac pa-
unacidified to avoid selective hydrolytic loss of AG-derived glycation tients (Table 1) and control plasma, stored under the same conditions.
adducts from the serum albumin (Smith et al., 1990) and interference The samples were processed immediately after they were thawed. HSA
with the isolation of HSA by affinity chromatography. Aliquots for
from patients N01, N02, N03, and N08, and also HSA from the control
quantitative LC-MS/MS analyses of diclofenac and its AG metabolite plasma samples, was captured at room temperature on a 2-ml (4.6 Â
(100 ml) were acidified immediately through addition of 2 M acetic 50 mm) POROS anti-HSA affinity cartridge installed in a PerSeptive
acid (final concentration, 4% v/v), a procedure known to stabilize di-
clofenac AG (Kenny et al., 2004; Sparidans et al., 2008). They were
frozen at 280°C. All of the plasma aliquots were frozen within 1 hour
of blood collection, transported from the clinic to the laboratory buried
in dry ice, and stored at 280°C. Control plasma for the quantitative
analyses was obtained from the healthy unmedicated volunteers. The
BioSystems Vision Workstation (Applied Biosystems, Foster City, CA)
and was eluted with 12 mM HCl as described previously (Greenough
et al., 2004; Jenkins et al., 2009). This cartridge expired before any more
plasma samples were processed and could not be replaced because the
manufacturer had discontinued production. Therefore, HSA from
patients N09 and N10 was captured using an Affinity Removal System
plasma samples used as controls for the analyses of drug-derived HSA column (HSA only, 4.6 Â 50 mm; Agilent Technologies, Santa Clara,
adducts were obtained independently from male and female adult CA) according to the same general procedure and eluted with the
subjects who had not taken diclofenac but, in common with the
proprietary acidic elution buffer. In all cases, the eluted protein
diclofenac patients, were taking a variety of prescribed comedications. fractions were immediately neutralized with 0.1 M Tris-HCl buffer,
None of them exhibited hypersensitivity reactions to those medications.
LC-MS/MS Analysis of Diclofenac and Diclofenac AG in
Clinical Plasma Samples. The sample processing and analytical
pH 9. Protein was precipitated with ice-cold methanol, processed, and
digested with trypsin overnight as described for HSA modified in vitro.
The digest was subjected to ion exchange chromatography on a Poly-
techniques used for assaying diclofenac and diclofenac AG in human SULFOETHYL A strong cation-exchange column (200 Â 4.6 mm, 5 mm,
plasma are detailed in Supplemental Methods and Supplemental
Table 2.
Mass Spectrometric Assessment of Covalent Modification of
300 Å; PolyLC, Columbia, MD), a procedure that enhances sub-
stantially the sensitivity of the peptide analyses by LC-MS/MS (Jenkins
et al., 2009). Peptides were eluted with a linear gradient (0–50% over
HSA by Diclofenac AG In Vitro. The concentration- and time- 75 minutes) of 10 mM KH2PO4 containing 1 M KCl and acetonitrile
dependent covalent modifications of HSA in vitro were investigated (3:1, v/v), pH , 3, against 10 mM KH2PO4 and acetonitrile (3:1,v/v),
by mass spectrometric analysis of modified tryptic peptides. Diclofenac pH , 3, at a flow rate of 1 ml/min. The eluate was monitored at 214 nm.
1-b AG (400 nM, 4, 40, and 400 mM, and 2 mM) in 0.1 M potassium Approximately 15 peptide-containing fractions (2 ml) were collected per
phosphate buffer, pH 7.4, was incubated with HSA (40 mM) at 37°C for
elution. They were dried by centrifugation under vacuum, reconstituted
16 hours. The molar ratios of AG:HSA (0.01–50) were not chosen to in 0.1% (v/v) trifluoroacetic acid, desalted using a Macroporous Reversed-
replicate ratios achieved in plasma in vivo, which were unknown at
that time, but expected to be substantially lower than any ratio that
yielded detectable adducts in vitro. Rather, the higher AG concen-
trations were intended to ensure production and characterization of
all the HSA adducts that might be formed in vivo and consequently to
facilitate a thorough, systematic, targeted search for HSA adducts in
Phase C18 High-Recovery column (4.6 Â 50 mm; Agilent Technologies)
installed on a Vision Workstation, and finally dried under vacuum for
LC-MS/MS analysis.
Mass Spectrometric Characterization of Adducted Tryptic
Peptides of HSA. Adducted tryptic peptides in the Zip-Tip eluates
(HSA modified in vitro) and the desalted ion-exchange fractions (HSA
patients. In a separate experiment, the 1-b AG (2 mM) was incubated modified in vivo) were analyzed on a 5500 QTRAP hybrid triple-
with HSA (40 mM) under the same conditions, and aliquots were
removed at intervals between 30 minutes and 16 hours. Additionally,
quadrupole/linear ion trap instrument fitted with a Nanospray II source
(AB Sciex, Foster City, CA). From earlier mass spectrometric and
the relative contributions of the 1-b AG and its positional (acyl spectrophotometric studies on adductions of HSA by NSAID AG in vitro
migration) isomers collectively to the N-acylation of HSA were (Ding et al., 1993, 1995; Qiu et al., 1998), «-amino groups of lysine
assessed by first allowing 1-b AG (2 mM) to isomerize in phosphate residues were known to be the principal identified sites of modification by
buffer, pH 7.4, at 37°C for 3 hours. Separately, it was shown that only
these AG even in the absence of imine-adduct stabilizing reagents.
5.5 6 1.4% (mean 6 S.D., n 5 3) of the 1-b AG remained at the end of
Although Cys34 of HSA is also recognized as a nucleophilic residue,
an incubation under these conditions, yielding a mixture of 2-, 3-, and standard thiol-masking agents have failed to block covalent modification
4-isomers and small amounts of diclofenac (Fig. 2B). HSA (40 mM; of the protein by NSAID AGs (Smith et al., 1990). None of the previous
final AG:HSA molar ratio, 0.01–50:1) was incubated in this pre- studies that characterized adducts of NSAID AGs on HSA reported
degraded solution at 37°C for 16 hours and simultaneously for
a thioester adduct (Ding et al., 1993, 1995; Qiu et al., 1998). The
comparison in freshly prepared phosphate-buffered solutions of 1-b established chemistry of thioesters certainly suggested stable S-acylation
AG. Aliquots of the various incubations (100 ml) were added to nine of Cys34 by diclofenac AG was an improbable expectation under the
volumes of ice-cold methanol, mixed by vortexing, and centrifuged at experimental conditions employed. Nonetheless, attempts were made
24,000g and 4°C for 15 minutes. The supernatant was removed, and using LC-MS/MS to identify an adduct at Cys34 in the tryptic digest but
the protein pellet was washed with ice-cold methanol (60 ml  3) to they revealed no evidence for covalent modification by the AG in vitro
extract noncovalently bound AG and diclofenac. The protein was
(data not shown). Therefore, multiple reaction monitoring (MRM) tran-
dissolved in 50 ml of 0.1 M phosphate buffer, pH 7.4, reduced with
sitions specific for peptides containing N-acylated or glycated lysines
dithiothreitol (10 mM) for 15 minutes at room temperature, and were selected as follows: the mass values of all HSA peptides with
alkylated (carboxyamidomethylated) with iodoacetamide (55 mM) for a missed trypsin cleavage at a modified lysine residue (Jenkins et al.,
a further 15 minutes at room temperature. It was precipitated and 2009; Meng et al., 2011; Whitaker et al., 2011) and mass additions of
washed with ice-cold methanol and recovered by centrifugation, as either 277 amu (acylated peptide) or 453 amu (acyl-glucuronide glycated

Circulating Protein Adducts in Diclofenac Patients
391
Fig. 2. Acyl migration, anomerization, and hydrolysis of synthetic diclofenac 1-b AG in vitro at 37°C. (A) LC-UV chromatogram (l = 254 nm) of diclofenac
1-b AG; its anomer; and C-2, C-3, and C-4 positional isomers and liberated diclofenac after a 90-minute incubation of the 1-b AG (2 mM) in 0.1 M potassium
phosphate, pH 7.4. (B) 1-b AG (2 mM) in 0.1 M potassium phosphate buffer, pH 7.4. (C) 1-b AG (2 mM) in HSA solution (40 mM; 2.66 mg/ml) buffered with
0.1 M potassium phosphate buffer, pH 7.4. (D) 1-b AG (400 mM) in HSA solution (40 mM) buffered with 0.1 M potassium phosphate, pH 7.4. (E) 1-b AG
(2 mM) in pooled human plasma. (F) 1-b AG (400 mM) in pooled human plasma. The relative proportions (peak areas) of the AG isomers and diclofenac
were determined by LC-UV. Analytes were identified fundamentally by LC-MS. The AG positional isomers were assigned from their chronological order
of appearance. Only the anomers of the 1-isomer were seen to be resolved. The 1-a anomer was assigned by chromatographic comparisons with fully
characterized 1-O-acyl anomers of other AG. Data shown represent means 6 S.D. as error bars (n = 3 separate experiments).

392
Hammond et al.
peptide) for the [35Cl2]diclofenac-derived residues were calculated and
these were paired with the m/z values of the dominant fragment ions of
diclofenac, namely m/z 250 and m/z 215, to complete the MRM
transitions. The amino acid sequence of UniProtKB/Swiss-Prot entry
P02768 for HSA (monoisotopic mass, 66,429; 585 residues) was used.
This sequence omits the 24 N-terminal residues of preproalbumin. The
choice of mass spectrometric method was also influenced pragmatically
by the reasonable assumption that only highly selective peptide survey
scans would be sufficiently sensitive to detect any protein modifications
in diclofenac patients. This choice was reinforced somewhat by failures of
tryptic peptide analyses to detect covalently modified serum albumin in
rats administered single large intravenous doses (60 mg/kg) of either
diclofenac or diclofenac AG (unpublished data), plausible indications that
only very low levels of modified serum albumin might be expected in
patients. Sample aliquots (2.4–5.0 pmol) were delivered into the mass
spectrometer by an Ultimate 3000 high-performance liquid chromatog-
raphy system through a 5-mm C18 nano-precolumn, a C18 PepMap
column (75 mm  15 cm; Dionex, Sunnyvale, CA), and a 10-mm i.d.
PicoTip ionspray emitter (New Objective, Woburn, MA). The ionspray
potential was set to 2200–3500 V, the nebulizer gas to level 19, and the
interface heater to 150°C. A gradient from 2% acetonitrile/0.1% formic
acid (v/v) to 50% acetonitrile/0.1% formic acid (v/v) over 60 minutes was
applied at a flow rate of 300 nl/min. MRM transitions were acquired at
unit resolution in both Q1 and Q3 to maximize specificity. The collision
energy was optimized for each MRM transition, and the dwell time was
20 milliseconds. MRM survey scans were set to trigger up to three
enhanced product-ion scans of modified peptides according to the MIDAS
technique (Unwin et al., 2005), with Q1 set to unit resolution and with
dynamic fill of the ion trap. These product-ion spectra were used
variously to confirm the peptide sequence, the site of adduction, and the
identity of the lysyl adduct (acylation or glycation). The relative MRM
peak heights of the modified peptides were computed using MultiQuant
software version 2.0 (AB Sciex) to produce an "epitope profile" for each
type of adducting species, i.e., acylating and glycating. However, the
peak-height ratios are regarded as approximations of molar ratios in the
absence of knowledge of the peptides’ relative ionization and trans-
mission efficiencies. The total ion count for each digest sample was
normalized to that of the HSA adduct produced in vitro at a molar ratio of
diclofenac 1-b AG to protein of 50:1 over 16 hours, thereby allowing the
magnitudes of the MRM signals to be adjusted for differences between on-
column sample loading (Meng et al., 2011). Modified tryptic peptides of
HSA isolated from diclofenac patients were also analyzed on an AB Sciex
Triple TOF 5600. The higher resolution and broader mass range of this
instrument allowed for more confident assignments of some peptide
sequences and adduct structures. Peptide aliquots (2.4–5.0 pmol) were
delivered into the mass spectrometer via a 10-mm i.d. PicoTip (New
Objective) by a direct-flow nano-LC system (Eksigent, Dublin, CA). The
system was comprised of a NanoLC-Ultra chromatograph linked to
a cHiPLC-Nanoflex docking station, ChromXP C18 trap column (200 mm Â
0.5 mm), and ChromXP C18 column (75 mm  15 cm). The ionspray
potential was set to 2200–3500 V, the nebulizer gas to level 5, and the
interface heater to 150°C. A gradient from 2% acetonitrile/0.1% formic
acid (v/v) to 50% acetonitrile/0.1% formic acid (v/v) in 90 minutes was
applied at a flow rate of 300 nl/min. Data were acquired at 25 MS/MS
spectra per cycle with an accumulation time of 100 ms each, sorted in
PeakView (AB Sciex) to highlight spectra containing fragment ions at
m/z 215 and m/z 250, and then interpreted manually.
AG after approximately 1, 2, and 3 hours, respectively.
Similar isomer successions occurred in the phosphate buffer.
The relative proportions of the isomers (C-3 . C-2 . C-4)
stabilized from about 6 hours in the presence and absence of
HSA and remained essentially unchanged for the subsequent
10 hours. Reducing the molar ratio AG:HSA from 50:1 to 10:1
produced a dramatic acceleration of conjugate hydrolysis
without greatly affecting the relative proportions of the
positional isomers (#10% variance of isomer exposure over
16 hours): the quantity of deconjugated diclofenac equaled or
exceeded the individual quantities of the AG isomers after
approximately 4 hours (Fig. 2D). However, as in the buffer
and other HSA incubations, the mixture of degradation prod-
ucts stabilized from about 6 hours. In the human plasma in-
cubations (Fig. 2, E and F), although some acceleration of acyl
migration was also apparent, hydrolysis of 1-b AG and all the
regioisomers was the dominant pathway, outstandingly at the
lower concentration of 1-b AG (400 mM) when the quantity of
diclofenac exceeded the quantities of all the positional isomers
after 1.5 hours and only minor residues of the isomers (# 5%)
remained by 6 hours (Fig. 2F). These observations of faster
migration and hydrolysis reactions in plasma conform with
physicochemical measurements made on other NSAID AG
(Karlsson et al., 2010). In all cases, the 1-a anomer was only
formed in trace amounts (Fig. 1). The half-lives of diclofenac
1-b AG in phosphate buffer, HSA solution (2 mM and 400 mM),
and human plasma (2 mM and 400 mM), estimated by
nonlinear regression analysis of the first-order rates of decay
of the conjugate (Supplemental Table 4), were 46.5, 56.8, 31.9,
7.0, and 5.4 minutes, respectively.
Covalent Binding of Diclofenac Residues to HSA
Incubated with 1-b AG In Vitro. Incubation of diclofenac
1-b AG with HSA at pH 7.4 under the conditions favoring acyl-
group migration over hydrolysis, i.e., a 50:1 molar ratio of AG
to HSA (Fig. 2C), resulted in rapid protein modification, which
was assessed from the quantity of diclofenac liberated by
strong alkaline hydrolysis (Fig. 3A). This method does not
differentiate between glycation structures and acyl residues
on protein side chains (Fig. 1). Covalent binding was mea-
surable from 5 minutes onward and reached an essentially
stable maximum at about 6 hours. The covalent binding at the
lower molar ratio of AG to HSA (10:1), when conjugate hy-
drolysis was substantial (Fig. 2D), followed a similar time
course (Fig. 3B). The maximum measured protein adduction
was 0.62 6 0.10 and 1.78 6 0.28% molar equivalents of
diclofenac (mean 6 S.D., n 5 3) at the lower and higher ratio,
respectively. The corresponding area under the liberated
diclofenac time curve, which represents a measure of protein
modification over the 16-hour incubations (AUC0–16), was
238.37 and 124.13 ng×h/ml, respectively.
Structural Characterization of Diclofenac AG–Derived
Adducts Formed on HSA In Vitro. Incubation of diclofe-
nac 1-b AG with HSA (50:1 molar ratio) for 16 hours under the
conditions conducive to rapid and selective acyl migration
(Fig. 2C) produced two predicted adduct types (Fig. 1) that
Results
Isomerization and Hydrolysis of Diclofenac 1-b AG were identified by LC-MS/MS analysis of tryptic digests:
In Vitro. Synthetic diclofenac 1-b AG in phosphate buffer, lysine residues that were either N-acylated or glycated by the
pH 7.4, at 37°C (Fig. 2B) and also in buffered HSA solution at complete AG (diclofenac carboxyl and glucuronyl residues).
a high molar ratio (50:1; Fig. 2C) underwent rapid rearrange- The use of a high reactant:HSA ratio was known from earlier
ment through acyl migration (Fig. 1) but very little hydrolysis studies on HSA adduction by b-lactams (Meng et al., 2011)
to parent carboxylate. In the HSA solution, the quantities of to provide a secure starting point for the considerably more
C-2, C-3, and C-4 positional isomers exceeded that of the 1-b challenging in vivo analyses. It enables identification under

Circulating Protein Adducts in Diclofenac Patients
393
Fig. 3. Covalent binding of diclofenac residues
to HSA (40 mM) incubated with synthetic
diclofenac 1-b AG (400 mM and 2 mM) in 0.1 M
potassium phosphate, pH 7.4, at 37°C for 16
hours. (A) Binding at 50:1 molar ratio of AG:
HSA. (B) Binding at 10:1 molar ratio of AG:
HSA. Covalent binding was estimated using
a LC-MS/MS assay of diclofenac liberated from
the protein adducts by alkaline hydrolytic de-
conjugation. Data shown represent means 6 S.D.
as error bars (n = 3 separate experiments).
controlled conditions of (almost) all the selected HSA adducts were not observed invariably. Two of the modified residues,
formed detectably in patients and thereby delivers a working i.e., Lys137 and Lys525, were paired lysines—HSA has four
inventory of adducts for the in vivo study. Because trypsin is lysine pairs—but neither of the adjacent residues (Lys136 and
unable to cleave the protein at adducted amino acids (Jenkins Lys524) was adducted detectably. The marked localized se-
et al., 2009; Meng et al., 2011; Whitaker et al., 2011), all of the lectivity of lysine modification was also exemplified by the
peptides with a modified lysine residue will have either an adduction of only one of the five residues (Lys541) between
N-terminal lysine—as when the residue is one of a pair—or Lys534 and Lys545, inclusive. Nevertheless, derivatization
a single subterminal lysine (Table 2). A priori, any tryptic within short amino acid sequences was highly variable: all
peptide detected by the specific MRM survey-scan method three of the lysine residues between Lys190 and Lys199,
used here will be acylated or glycated with diclofenac inclusive, were adducted. Typical product-ion spectra of the
AG–derived residues (diclofenac AG– and/or thioester-derived N-«-acylated and N-«-glycated forms of a modified miscleaved
residues in vivo) at the lysine that is the site of the missed tryptic peptide, namely those of 182LDELRDEGKASSAK195
enzymic cleavage. Eight of the 59 lysine residues, which con- modified at Lys190, are shown in Fig. 4, A and B, respectively.
stitute 1.4% of the entire sequence and 13.6% of the lysines, Fragment ions at m/z 250 (benzyl moiety) and m/z 215 (m/z
were modified consistently. Seven of the residues were ad- 250-Cl·) were diagnostic of modifications that included the
ducted when the AG:HSA molar ratio was 1:1, and all eight diclofenac residue, namely N-acylations and complete glyca-
when the ratio was $10:1 notwithstanding the extensive AG tions, whereas the ion at m/z 278 (the diclofenac acyl residue)
hydrolysis at 10:1 (Fig. 2D). All except one of those eight was observed less frequently, but all the fragmentations (Fig. 4,
modified residues underwent both types of modification con- C and D) could be rationalized in terms of the two predicted
sistently when the ratio was $10:1. Exceptionally, Lys351 adduct structures (Fig. 1) and are known from the product-ion
was not always found to be acylated (Table 2). Similarly, the spectrum of diclofenac (Galmier et al., 2005). The glycated pep-
glycation of Lys162 and the acylation and glycation of Lys436 tide alone yielded an ion at m/z 839, corresponding to [M 1 2H]21
TABLE 2
Modified tryptic peptides of HSA reacted with diclofenac 1b-AG in vitro
HSA was incubated with synthetic diclofenac 1b-AG in phosphate buffer, pH 7.4, at 37°C for 16 hours. The peptides were
characterized by LC-MS/MS (AB Sciex 5500 QTRAP). Glycated Lys162 was not detected consistently and was only ever
seen at low signal strength (acylated Lys162 was never detected). Acylated Lys351 was not detected consistently and was
only found at the 50:1 molar ratio. Acylated and glycated Lys436 were not detected consistently. Inconsistently observed
adducts are shown as bold letters.
Molar Ratio Diclofenac 1b-AG: HSA
Modified Lysinea
Tryptic Peptideb
0.01:1
0.1:1
1:1
10:1
50:1
137
162
190
195
199
351
432
436
525
541
K*YLEIAR
—
—
G
—
—
—
—
—
—
—
—
—
G
—
—
G+T
G+T
G+T
G
G+T
G
G+T
G+T
G+T
G
G+T
G+T
G+T
G+T
G+T
G
YK*AAFTECCQAADK
LDELRDEGK*ASSAK
ASSAK*QR
G+T
G+T
G+T
G+T
G+T
G+T
G+T
G+T
—
LK*CASLQK
LAK*TYETTLEK
NLGK*VGSK
VGSK*CCK
K*QTALVELVK
ATK*EQLK
G
—
G
—
—
—
G
—
G
G+T
G, complete glycation structure (diclofenac carboxyl and glucuronyl residues); T, diclofenac transacylation adduct.
aBenoxaprofen AG modified Lys159 and Lys199 in vitro without reductive stabilization (Qiu et al., 1998). Tolmetin AG
modified Lys195, 199, 525, and 541 in vitro without reductive stabilization (Ding et al., 1995). Lys159 and Lys199,
respectively, were the principal adduction sites.
bAsterisk indicates lysine modification site on the miscleaved peptide. The methodology of adduct identification is
described under “Mass Spectrometric Characterization of Adducted Tryptic Peptides of HSA” in Materials and Methods.
Cysteine residues of the recovered and reduced protein were carboxyamidomethylated before trypsin digestion.

394
Hammond et al.
Fig. 4. Product-ion spectra of modified HSA peptide 182LDELRDEGKASSAK195 acquired during LC-MS/MS analysis of a tryptic digest of protein
reacted in vitro with synthetic diclofenac 1b-AG (50:1 molar ratio of AG:HSA). (A) The diclofenac-acylated peptide (peptide + 277 amu; parent ion, [M + 3H]3+ at
m/z 599.2). (B) The glycated peptide (peptide + 453 amu; incorporating glucuronyl and drug carboxyl residues; parent ion, [M + 3H]3+ at m/z 658.6). The
MRM survey scans were set up to search for acylated and glycated HSA peptides with missed trypsin cleavages, i.e., covalent modifications, at lysine
residues. The m/z values of the modified peptides and fragments correspond to the 35Cl2 isobars. The y6 ion of the acylated peptide (m/z 868) was
adducted (+277 amu, the diclofenac acyl residue) at Lys190. The glycated peptide did not yield any observable y or b ions bearing the complete glycation
structure. The multiply charged peptide + 158 amu ion was assigned to a whole-peptide species that retained the dehydrated residue of the
dehydroglucuronic acid moiety. The acylated peptide’s parent ion also yielded peptide fragments that had undergone collision-induced elimination of the
entire adduct residue. Fragment ions of the adducts are circled. The principal adduct-derived fragment ions of the modified peptide were rationalized as
shown. (C) Fragment ions of the acyl adduct. (D) Fragment ions of the glycation adduct.

Circulating Protein Adducts in Diclofenac Patients
395
for the unmodified miscleaved peptide plus 158 amu. This ion products after about 3 hours, the glycated tryptic peptides, for
was assigned to a fragment of the glycated peptide that re- the analytical reasons mentioned previously, were not neces-
tained only the dehydrated residue of the dehydroglucuronic sarily more abundant in total.
acid moiety (Fig. 4D). A glucuronate species is apparently
Time- and Isomer-Dependent Adduction of HSA by
more susceptible to this dehydration if it is derived from Diclofenac AG In Vitro. A closer examination of the dynamics
non–1-O-b-AG structures (Karlsson et al., 2010), as it must be of the two types of adduction in vitro provided an important
in this case. The peptide 1 78 amu ion seen in spectra of gly- insight into the mechanism of protein modification by diclofenac
cated peptides (Fig. 4B; Supplemental Fig. 2B) was assigned AG. The rapidity of the isomerization of diclofenac 1-b AG in
by analogy to a pyrylium species ([C5H4O] 5 NR) produced by protein solutions, even when the glucuronide underwent some-
triple loss of water and elimination of CO2 from the deacylated what extensive hydrolysis (Fig. 2, D and E), suggested an ex-
ꢀ
glucuronic acid residue (Jeric et al., 2002; Karlsson et al., planation for early completion of the acylation reactions in HSA
2010). It is putatively diagnostic of a C-N glycation linkage. incubations (Fig. 6A), namely that they were affected principally
The AG-derived structures were evidently more labile in the by the 1-b isomer. In fact, from reaction mechanism theory and
MS collision cell than the peptide bonds, leading to multiple all other considerations being equal, the anomeric acyl group
overlapping and weak spectra within the same analytical will always be a better acylating agent than its regioisomeric
space and difficulties with interpreting amino acid sequences. forms. This proposition was tested by reacting HSA with
Fragment ions corresponding to the full-length peptide minus a preformed mixture of positional AG isomers (∼8:7:3 of the C-2,
the modification were also visible in most spectra, as were C-3, and C-4 isomers, respectively, estimated from LC-UV anal-
full-length peptides with partial adduct structures and pep- ysis; Fig. 2B) generated by incubating 1-b AG in phosphate
tide fragments with either no or partial adducts. Nonetheless, buffer, pH 7.4, at 37°C for 3 hours, by which time only approx-
it was possible to design MRM transitions that detected the imately 5% of the parent isomer remained. Parallel incubations
AG-modified peptides and to interpret the MRM-triggered of HSA with degraded and undegraded glucuronide at pH 7.4 for
MS/MS spectra.
16 hours (AG:HSA molar ratio, 0.01–50:1) revealed that the
Epitope Profiles of HSA Adducted by Diclofenac AG total level of protein acylation in solutions containing rear-
In Vitro. Computation of the relative MS/MS ion count for ranged AG, as represented by summated ion counts of modified
each modified HSA peptide in a tryptic digest provides an peptide ions, was approximately 30% of that observed in solu-
"epitope profile" that is characteristic of the adducting drug or tions containing only 1-b AG at the outset (Fig. 6B). In contrast,
drug metabolite (Jenkins et al., 2009; Meng et al., 2011; the total level of glycated peptide (peptide 1 453 amu) ions, as
Whitaker et al., 2011). These profiles can be used to visualize dictated by the established mechanism of protein glycation
the time and concentration dependency of covalent modifica- (Fig. 1), was unaffected by extensive preincubation depletion of
tions. The acylation and glycation structures derived from parent AG (Fig. 6C). Therefore, it was deduced that the 1-b AG
diclofenac 1-b AG were located on the subset of eight lysine was at least the principal source of lysine acylation adducts
residues described previously (Fig. 5; Table 2). All except two of collectively on HSA in vitro. A detailed representation of the
the 16 observed modifications—acylation of Lys199 and gly- differential adductions of individual peptides in these experi-
cation of Lys351—were detected after 0.5 hour of incubation ments is shown in Supplemental Fig. 1. Collective and in-
at the highest AG:HSA molar ratio, namely 50:1. Acylation of dividual contributions of the positional isomers to acylation
Lys199 and glycation of Lys351 was first detected after 2 and reactions appear to be minor. The data do not reveal which of the
1 hour, respectively (data not shown). In general, the glycated three positional isomers is principally responsible for the gly-
adducts were identified at lower AG:HSA molar ratios cations, although the timescales of isomer formation (Fig. 2, C
(Table 2). The proportions of the normalized ion counts for the and D) and protein glycation (Fig. 6A) in HSA solutions suggest
two adduct types varied considerably between adducted all three isomers might contribute to this modification after the
residues and with time at certain lysines. Thus the lowest fourth hour. Glycations at the anomeric carbon of the C-2
AG:HSA molar ratio at which a glycation adduct was found was regioisomer are likely to be less durable than those of the other
0.01:1, whereas the corresponding ratio for the acyl adducts migration products, however, because their structure prevents
was 1:1. The levels of both modifications were dependent on the a stabilizing Amadori tautomeric rearrangement.
concentration of AG (Fig. 6, B and C). However, because the
Diclofenac and Diclofenac AG in Clinical Plasma
individual proportionalities of ion counts and peptide abun- Samples. All of the acid-stabilized single plasma samples
dances will have been influenced by unknown efficiencies of taken from the six patients 1–3 hours after their last tablet
ionization and ion transmission in the mass spectrometer, it contained detectable concentrations of diclofenac, although in
cannot be assumed the measured counts equate with relative three cases the concentration was below the lower limit of LC-
molar abundances of the peptides. The time courses of totalized MS/MS quantification (Table 3). Tmax values for the narrow
ion counts showed that the acyl adducts collectively were peaks of diclofenac can display considerable variability,
formed early, but no greater acylation occurred after about 2 although Crook et al. (1982) calculated 2.0 6 0.50 hours
hours (Fig. 6A). The glycation structures appeared no more (mean 6 S.D.) for a 50-mg oral dose in rheumatoid patients.
rapidly than the acyl adducts, but the collective level of this Only four of the samples contained detectable concentrations
modification increased continuously to the end of the 16-hour of diclofenac AG, which was assayed without chromatographic
incubation. A representative selection of ion-count time courses resolution of the positional isomers, and in only one patient
for individual modified peptides is shown in Supplemental Fig. 1. (N08) was the conjugate quantifiable. The latter sample was
The representative relationship between AG concentration taken 3 hours after the last tablet. Notably, the concentration
and adduction of the Lys195 peptide is shown in Supplemen- of parent drug in that spot sample was by far the highest
tal Fig. 1C. Although the totalized ion counts for glycation concentration of diclofenac found in any of the patients. Nev-
products were progressively greater than those for acylation ertheless, it was substantially lower than the Cmax of 3.361.4 mM

396
Hammond et al.
Fig. 5. Ion-count epitope profiles of HSA modified at lysine residues by reactions with diclofenac 1-b AG in vitro. (A) Acylation adducts at 0.5 hour. (B)
Glycation adducts at 0.5 hour. (C) Acylation adducts at 16 hours. (D) Glycation adducts at 16 hours. Data represent relative ion intensities of lysine-
modified peptides detected during LC-MS/MS analyses of tryptic digests. Each relative ion intensity was derived from the area under the curve for the
relevant extracted ion chromatogram by normalization to the total ion count of the sample. Synthetic AG was incubated with HSA (AG:HSA molar ratio,
50:1) at pH 7.4 and 37°C. Acylated Lys351 was detected in these analyses, but this modification of Lys351 and also modifications of Lys162 and Lys436
were not detected consistently (Table 2). Lys436 was modified detectably in patient N08.
reported for a single 50-mg dose (Crook et al., 1982). The (affinity chromatography) and tryptic peptide fractionation
molar ratio of AG to drug in N08 was 0.21. Two of the other (cation exchange chromatography) before peptide analysis by
plasma samples that contained detectable concentrations of nano–LC-MS/MS. These steps were essential to achieving the
diclofenac AG also contained quantifiable amounts of diclofe- high sensitivity required for characterization of circulating
nac. The relatively low concentrations of diclofenac in the adducts. Although two of the eight lysines modified consis-
patients’ plasma have no clear explanation. Certainly neither tently in vitro were not modified detectably in vivo (Lys137
the clinical conditions of the patients nor the comedications and Lys351), a collective total of seven adducted residues and
(Supplemental Table 3) suggest an explanation. Most of the ten modifications were identified (Table 4). The detection of
patients took a delayed release formulation, but delaying adducts on 75% of the HSA residues modified consistently in
blood sampling from 1 to 2.5/3 hours had no marked or vitro exceeded expectations. The lower limit of adduct detec-
consistent effect on drug concentrations, notwithstanding the tion in vitro after a 16-hour incubation was an AG:HSA molar
sampling times were within the range of published Tmax for ratio of 0.01:1 (Table 2)—and reaction at that ratio yielded
enteric-coated diclofenac (Willis et al., 1981; Crook et al., only one modification. Contrastingly, the molar ratio in the
1982).
one patient (N08) with a measurable plasma AG concentra-
Characterization of Drug-Derived Adducts Formed tion (90.8 nM; Table 3) was estimated to be only 0.0001:1 3
on HSA in Diclofenac Patients. Plasma samples from the hours after the final drug dose, taking an HSA concentration
six diclofenac patients were processed by HSA concentration of 45 mg/ml for the calculation (Veering et al., 1990). This

Circulating Protein Adducts in Diclofenac Patients
397
Fig. 6. Acylation and glycation of HSA by
synthetic diclofenac 1b-AG and preformed
diclofenac AG positional isomers in 0.1 M
potassium phosphate, pH 7.4, at 37°C over
16 hours demonstrating the transacyla-
tion reactions were effected principally by
the 1-b isomer. (A) Time-dependent acyl-
ation (black line) and glycation (red line)
of HSA (40 mM) by 1b-AG (2 mM). (B)
Concentration-dependent acylation of HSA
(40 mM; final AG:HSA molar ratio, 0.01–50:1)
by freshly prepared solutions of 1b-AG
(black line) and a preformed mixture of posi-
tional isomers (red line). (C) Concentration-
dependent glycation of HSA (40 mM; final
AG:HSA molar ratio, 0.01–50:1) by freshly
prepared solutions of 1b-AG (black line)
and a preformed mixture of positional iso-
mers (red line). The extent of each generic
modification of HSA (acylation or glyca-
tion) in an incubation sample was esti-
mated by summing the normalized ion counts
for all the acylated or glycated peptides
detected during a LC-MS/MS analysis of
a tryptic digest. The mixture of positional
isomers was produced by preincubating
the 1b-AG in phosphate buffer, pH 7.4, at
37°C for 3 hours (see Fig. 2B). The data
are representative of at least two separate
experiments.
disparity is attributed to an accumulation of modified protein vitro although not modified detectably by either acylation or
(Zia-Amirhosseini et al., 1994) and cautions against any glycation in any of the subjects, Lys137, was only modified in
tendency to discount adduct detectability in vivo based on vitro when the AG:HSA molar ratio was $10:1. Acylation of
chemical analysis of adduction. Acylation of one, three, four, the other residue, Lys351, was detected inconsistently in
or five lysine residues was observed on HSA isolated from the vitro, i.e., near the limit of mass spectrometric identification,
patients. The acylated residues detected in each person were although its glycation was detected reproducibly at an AG:
variously one to five from a total of six of the seven lysines that HSA ratio of $1:1. Acylated Lys436 and Lys525 were
underwent this adduction reaction reproducibly in vitro identified exclusively in patient N08. Unusually, Lys436
(Table 2). One of the two residues adducted invariably in was not adducted consistently in vitro. Three of the lysines
(Lys195, 199, and 541) were acylated in four patients. Two of
these residues and two of the other four acylated lysines were
TABLE 3
modified in vitro when the AG:HSA molar ratio was 1:1. A
typical product-ion spectrum of an acylated peptide is shown
in Fig. 7A. Glycation adducts were found on one or three
lysines (Lys195 or Lys199 and Lys195, 199, and 432, re-
spectively) but in only three of the patients (N01, N08, and
N09). All of these residues were among the eight lysines that
were glycated in vitro. Modified peptide 198LKCASLQK205
was detected most readily (Fig. 7B), notwithstanding in-
source fragmentation that resulted in this peptide being
found additionally at m/z 691.9 after loss of water. Two of the
residues glycated in vivo were glycated detectably by
synthetic diclofenac 1-b AG when the AG:HSA molar ratio
was 0.1:1 and all of them when the ratio was $1:1 (Table 2).
Product-ion spectra of other glycated peptides are shown in
Supplemental Fig. 2, A and B. Two of the three glycated
Concentrations of diclofenac and diclofenac AG in plasma of patients
See Table 1 for individual patient data.
Patient ID Number Time of Blood Samplinga Diclofenac AGb Diclofenacb
hrs
nM
N01
N02
N03
N08
N09
N10
1
N.D.
N.D.
N.Q.
90.8
N.Q.
N.Q.
N.Q.
N.Q.
1
1
166.7
423.3
N.Q.
3
2.5
2.5
63.91
N.D., not detected (MRM signal-to-noise ratio ,3 at confirmed Rt of the analyte);
N.Q., not quantified (analyte concentration .lower limit of detection, ,lower limit of
quantification).
aSingle blood samples were removed by venipuncture at the indicated time after
the last diclofenac tablet had been taken.
bDiclofenac and diclofenac AG were assayed by LC-MS/MS.

398
Hammond et al.
TABLE 4
Modified tryptic peptides of HSA isolated from diclofenac patients
HSA was isolated by affinity chromatography from plasma of patients who had taken diclofenac (100–150 mg/day) as two
or three times daily doses for at least 1 year. The peptides were characterized by LC-MS/MS (AB Sciex 5500 QTRAP and
TripleTOF 5600). See Table 1 for individual patient data.
Patient ID Number
N01
Modified Lysine
Tryptic Peptidea
Modificationb
190
195
199
LDELRDEGK*ASSAK
ASSAK*QR
LK*CASLQK
T
T
T+G
432
541
190
195
199
432
541
NLGK*VGSK
ATK*EQLK
LDELRDEGK*ASSAK
ASSAK*QR
LK*CASLQK
NLGK*VGSK
ATK*EQLK
T
T
T
T
T
T
T
N02
N03
N08
195
199
541
195
199
ASSAK*QR
LK*CASLQK
ATK*EQLK
ASSAK*QR
LK*CASLQK
T
T
T
T+G
T+G
432
436
525
541
NLGK*VGSK
VGSK*CCK
K*QTALVELVK
ATK*EQLK
G
T
T+Glucuronylationb
T
N09
N10
195
195
ASSAK*QR
ASSAK*QR
T+G
T
G, complete glycation structure (incorporating the diclofenac carboxyl and glucuronic acid residues); T, diclofenac
transacylation adduct.
aAsterisk indicates lysine modification site on the miscleaved peptide. Cysteine residues of isolated and reduced HSA
were carboxyamidomethylated before trypsin digestion.
bModified by acylation and glucuronylation/hexuronylation.
lysines (Lys195 and Lys199) were also acylated in the same subjects. Finally, neither the detection nor the chemical char-
patients. The three residues were among the seven lysines acteristics of the drug-protein adducts were associated consis-
acylated and glycated in vitro. Supplemental Table 5 itemizes tently with measured concentrations of drug or AG in plasma
the nine modified HSA tryptic peptides obtained from N08, (Tables 3 and 4). N08 had the greatest number of HSA adducts
the greatest number obtained from any of the patients. and the highest concentrations of diclofenac and AG but there
Exceptionally, peptide 525KQTALVELVK534 from HSA of was not a consistent alignment of the two plasma concentra-
patient N08 (Table 1) was detected with an addition of 176 tions and the number of adducts in the six patients. Thus, N01
amu at Lys525 (Supplemental Fig. 3). Although the biochem- and N02 had nonquantifiable drug concentrations and yet they
ical context of the mass increment immediately suggested each yielded multiple adducts. The spot drug and AG concen-
conjugation with glucuronic acid alone (Fig. 1), the LC-MS/MS trations are unlikely to have any relevance to the detection or
analysis was not able to differentiate between a glucuronyl composition of the HSA adducts found in these patients. Those
and other, isomeric, hexuronyl modifications. Lys525 was also phenomena will have been determined by protracted accumu-
acylated in N08 and in vitro and was glycated in vitro lation and chemical evolution of the adducts during the ex-
(Table 2) but was not modified in any of the other patients. tended pharmacotherapy preceding the study.
The 176-amu modified peptide was recovered in a later cation
exchange fraction than the corresponding acylated peptide
(Supplemental Table 5). On the basis of the retention times of
Discussion
other modified peptides, a corresponding AG-glycated peptide
Mass spectrometric analyses of HSA from diclofenac
would have eluted earlier still from the cation exchange patients have proven definitively for the first time that an
column, suggesting that detection of the unique glucurony- AG metabolite can modify a protein covalently in vivo. This
lated species was not a result of in-source fragmentation of an was achieved through the identification of signature lysyl
AG-glycated precursor ion. This seemingly novel adduct is glycations retaining glucuronyl and carboxyl residues (Fig. 1).
attributed tentatively to a somewhat rare, nonenzymic, post- It was also shown that a bioactivated carboxylate drug can
translational modification occurring in vivo by the slow re- modify multiple lysines via N-acylation and glycation. Ten
action of HSA with glucuronic acid in plasma (Mazzuchin et al., adductions to seven lysines in highly variable combinations
1971; Smith et al., 1990). In a separate study using similar were characterized. Each patient had a unique combination,
analytical methods, we found glucosylated Lys525 (Barnaby and the number of modifications in a subject varied almost
et al., 2011) in vivo (data not shown). Neither glucuronylated continuously between one and eight. Only one modification was
Lys525 nor any of the N-acylations and glycations associable common to all the patients. Apart from an N-acylation, found in
with either diclofenac AG or reactive thioester metabolites of one subject, all of these adductions were predicted from re-
diclofenac were found on HSA isolated from the three control actions of diclofenac AG with HSA in vitro.

Circulating Protein Adducts in Diclofenac Patients
399
Fig. 7. Product-ion spectra of modified HSA peptide 198LKCASLQK205 acquired during a LC-MS/MS analysis of a tryptic digest of the protein isolated
from diclofenac patient N08. HSA was isolated from a 60-ml plasma sample by affinity chromatography. (A) The diclofenac-acylated peptide (peptide
+ 277 amu; parent ion, [M + 2H]2+ at m/z 612.8). (B) The glycated peptide (peptide + 453 amu; incorporating glucuronyl and drug carboxyl residues;
parent ion, [M + 2H]2+ at m/z 700.8). The MRM survey scans were set up to search for acylated and glycated HSA peptides with missed trypsin cleavages,
i.e., covalent modifications, at lysine residues. The m/z values of the modified peptides and fragments (b and y ions) correspond to the 35Cl2 isobars. The
b2 (LK) ion (m/z 519.22) of the acylated peptide and the y7 ion (m/z 556.25) were both adducted (+277 amu, the diclofenac acyl residue) at Lys199. The
y7 ion of the glycated peptide was adducted (+453 amu) but the peptide did not yield any observable b ions bearing the complete glycation structure.
The peptide + 158 amu ion was assigned to a whole-peptide species that retained the dehydrated residue of the dehydroglucuronic acid moiety. The
peptide + 176 amu ion retained the complete dehydroglucuronic acid moiety. The parent ions also yielded peptide fragments that had evidently
undergone collision-induced elimination of the entire adduct residue. Fragment ions of the modifications are circled. The principal adduct-derived
fragment ions of the modified peptide were rationalized as shown in Fig. 4. Cysteine residues of isolated and reduced HSA were
carboxyamidomethylated before trypsin digestion. The presence of an alkylated cysteine in a peptide fragment is indicated by the annotation C + iodo.
Hitherto the identification of protein adducts of diclofenac in et al., 1997; Hermening et al., 2000), has not been applied to
vivo rested on radiotracing, which showed relatively low binding diclofenac patients. Correlations of plasma protein adduc-
in rat liver and plasma (Masubuchi et al., 2007), and immuno- tion with NSAID AG exposure in humans (Castillo et al.,
visualization in rodent (Hargus et al., 1995; Wade et al., 1997) 1995), and especially the higher correlation with exposure to
and human (Aithal et al., 2004) liver. Although some targeted regioisomers (Hyneck et al., 1988), implicated covalent
hepatic proteins were assigned, and glucuronyltransferase combination with AG and possibly intravascular reactions.
catalyzed diclofenac binding to rat liver homogenate (Hargus However, protracted alkalinolysis releases carboxylic acid
et al., 1994), the structures and metabolic derivations of unspecifically from amide and ester side-chain linkages and
modifications were not defined precisely. The method used often ester bonds of isomeric glycations (Smith et al., 1990).
to characterize carboxylate-derived adducts of plasma proteins, Without identification of AG-definitive glycations it cannot
namely alkalinolysis (Zia-Amirhosseini et al., 1994; Sallustio be assumed any of these circulating adducts are derived from

400
Hammond et al.
AG rather than reactive thioester metabolites (Skonberg et al., 2008). Consequently acylations by multiple metabolites
et al., 2008). The strongest evidence of protein modification by as well as glycations might occur before and after HSA is
diclofenac AG within biologic systems was the observation transported to the blood. However, unlike AG, highly reactive
that glucuronidation inhibitors reduced [14C]diclofenac bind- S-acyl metabolites are improbable plasma constituents. The
ing to rat hepatocytes (Kretz-Rommel and Boelsterli, 1993).
known susceptibility of a thioester prodrug to biochemical
Diclofenac AG incubated with HSA under conditions fa- hydrolysis implies any diclofenac thioester entering the
voring acyl-group migration produced the dominant adduct circulation will be degraded in the same way (Bentley et al.,
types, N-acyl and glycation, obtained when tolmetin AG (Ding 2012). In fact, in rat liver homogenate, diclofenac does not
et al., 1993; Ding et al., 1995) and benoxaprofen AG (Qiu et al., undergo acyl-CoA–dependent covalent binding to protein
1998) were reacted similarly with HSA. The half-lives of (Hargus et al., 1994).
tolmetin and R/S-benoxaprofen AG in phosphate buffer are
The adduction pathways of AG are now better character-
0.26 and 2.0/4.1 hours, respectively (Stachulski et al., 2006). ized, but relative extents of acylation versus glycation and of
The values of the half-life of diclofenac AG reported here, and glycation by individual regioisomers remain uncertain. Oxaprozin
by Ebner et al. (1999) and Sawamura et al. (2010), of 0.78, 0.51, AG modifies HSA only through transacylation (Ruelius et al.,
and 0.7 hour, respectively, all rate the conjugate as highly 1986). Five NSAID AG modify insulin only through glycation
reactive. Several of the modified residues are common targets (Liu et al., 1998). Whereas HSA transacylation was effected
of electrophilic compounds and, thereby, potentially frequent principally by the diclofenac 1-b AG, the rank order of binding
locations of antigen formation. Lys195, adducted in every of diflunisal AGs to HSA is C-4 . .C-3 . C-2 . C-1
diclofenac patient, also reacts with b-lactams (Jenkins et al., (Dickinson and King, 1991). High reactivity of diclofenac C-4
2009; Meng et al., 2011; Whitaker et al., 2011), a cyclic imide AG would, however, be counterbalanced by the isomer’s lower
(Meng et al., 2007), and tolmetin AG (Ding et al., 1995), al- abundance. In contrast, (S)-naproxen C-2 AG modifies HSA
though not benoxaprofen AG without reductive imine-adduct slower than the 1b-conjugate (Iwaki et al., 1999). Apparently,
stabilization (Qiu et al., 1998). AG of diclofenac, benoxaprofen, the balance of acylation versus glycation differs substantially
and tolmetin modify, respectively, three, two, and four lysines between AG and proteins. Further analyses might expand the
detectably without stabilization (Table 2). Lys195 and Lys199 diclofenac adduct inventory by finding structures derived
(adducted in four patients) lie at the wide and flexible entrance from hydroxydiclofenac AG (Kumar et al., 2002) and cyto-
to HSA drug site 1, a predominantly apolar ligand binding pocket chrome P450–generated metabolites (Boerma et al., 2012).
(Ghuman et al., 2005) where the regioisomers of diflunisal AG
Although HSA was chosen for adduct analysis because of
react selectively (Williams and Dickinson, 1994). They and abundance, experience suggests it will be the principal target
Lys432 were the only residues glycated detectably in patients. in vivo. Several undefined rat plasma proteins are adducted by
Lys414, in HSA site 2, diclofenac’s high-affinity binding site zomepirac and diflunisal, but the major target is albumin
(Chamouard et al., 1985), is modified by the cyclic imide but (Bailey and Dickinson, 1996). Ketoprofen AG reacts selectively
not by diclofenac AG or benzylpenicillin. Lys199 and Lys414, with HSA in vitro (Dubois et al., 1993), without detectable
but neither Lys195 nor Lys432, are glycated spontaneously by adduction of fibrinogen and g-globulins and only low-level
glucose (Barnaby et al., 2011) via the same pathway of revers- binding to a- and b-globulins. HSA adducts were found in every
ible imine formation and slow, cumulative, Amadori rearrange- patient notwithstanding diclofenac AG underwent rapid hy-
ment. Diclofenac AG glycations were identified notwithstanding drolysis in plasma ex vivo and even when the glucuronide was
incubations did not contain cyanoborohydride, which reduces undetectable in plasma. Five of eight lysines modified re-
imines and enhances binding of AG, including diclofenac AG producibly in vitro at high AG:HSA molar ratios were also
(Kenny et al., 2004), to HSA (Smith et al., 1990). HSA adducts modified in two or more patients, one in all of them. Because
of diclofenac AG are unstable at pH 7.4 (Ebner et al., 1999). the patients received diclofenac daily for $12 months, adduct
Stabilizing additives were excluded to avoid compromising levels will probably have stabilized; t1/2 of plasma protein
prediction of HSA’s glycations in patients. Contrarily, even adducts of carboxylate drugs in humans are approximately
without additives, five of the eight consistent glycations were 5–13.5 days (Zia-Amirhosseini et al., 1994).
not detected in patients. Detection of six from seven re-
We have obtained direct evidence of multiple, variable, drug-
producible N-acylations suggests, hypothetically, a relatively derived protein modifications in diclofenac patients without
low abundance of AG isomers or greater instability of gly- adverse reactions. However, studies on b-lactams have
cation adducts in vivo. Although C-3 and C-4 lysyl aldimines established that albumin adduction per se is insufficient to
of AG might stabilize via rearrangements (Fig. 1), and this induce drug hypersensitivity (Jenkins et al., 2009; Meng et al.,
has been proposed (Smith et al., 1990; Ding et al., 1993), no 2011; Whitaker et al., 2011). Induction of an immune response
experimental confirmation is available. Hydroxyimine and is a function of the chemistry of the drug and the biology of the
ketoamine peptide adducts are in equilibrium (Acharya and patient. Moreover, multiple pathways have been proposed for
Sussman, 1984) and therefore probably indistinguishable by initiation of hypersensitivity, including haptenation, phar-
LC-MS/MS methods. Because progressive acyl migrations, macological interaction, and altered self-peptide presentation
cyclization, hydrolysis, and even back migrations (Johnson (Louis-Dit-Sully and Schamel, 2014). Therefore, to determine
et al., 2008), might occur after adduction, the glycation the clinical consequences of AG-protein adduct formation, it
adducts are prospectively a complex, dynamic, set of regioiso- will be necessary to test T cells and antibodies from hyper-
meric structures.
sensitive and nonhypersensitive patients for recognition of
The N-acyl adducts could have derived additionally and the epitopes defined in this study. Such studies are essential
independently from the minor glutathione and CoA thioesters to confirm or dispel the notion that AG are metabolite alerts
of diclofenac formed in human hepatocytes (Grillo et al., for idiosyncratic toxicity and should be considered a liability
2003), other NSAID thioesters acylate HSA in vitro (Skonberg when identified during drug discovery.

Circulating Protein Adducts in Diclofenac Patients
401
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Acknowledgments
The authors thank Marie-Josephe Pradere, Research Nurse, De-
partment of Rheumatology, Nottingham University Hospitals NHS
Trust, for identifying eligible patients, drawing blood samples, and
undertaking numerous administrative duties associated with clinic
aspects of the study; Rawinder Banwait and Melanie Lingaya, Re-
search Technicians, NIHR Nottingham Digestive Diseases-Biomedical
Research Unit, Nottingham University Hospitals NHS Trust and
University of Nottingham, for additional handling of the plasma
samples from diclofenac patients; Emma J. Williams, Steve Swallow,
and the Flexi Chemistry Team, Alderley Park, AstraZeneca U.K., for
contributing to the synthesis of diclofenac acyl glucuronide; Claire
Prince, Research Nurse, The Wolfson Centre for Personalised Medi-
cine, Department of Molecular and Clinical Pharmacology, University
of Liverpool, for blood collections from healthy volunteers; and Laura
Dickinson, Department of Molecular and Clinical Pharmacology, for
advice on WinNonlin analyses. The authors thank Prof. Ann K. Daly,
Institute of Cellular Medicine, Newcastle University, Newcastle upon
Tyne, UK; James E. Sidaway, AstraZeneca U.K., Safety Assessment;
and Ian D. Wilson, Drug Metabolism and Pharmacokinetics IM,
Alderley Park, AstraZeneca U.K., for their advice and assistance. The
authors are indebted to Pfizer Global Research and Development, U.K.,
and AstraZeneca for gifts of mass spectrometric (API 2000) and
chromatographic equipment, respectively.
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Authorship Contributions
Participated in research design: Hammond, Meng, Jenkins, Maggs,
Aithal, Pande, Kenna, Stachulski, Park, Williams.
Conducted experiments: Hammond, Meng, Jenkins, Maggs, Santoyo
Castelazo, Regan, Earnshaw.
Contributed new reagents or analytic tools: Meng, Jenkins, Bennett.
Performed data analysis: Hammond, Meng, Jenkins, Maggs,
Pande.
Wrote or contributed to the writing of the manuscript: Hammond,
Meng, Jenkins, Maggs, Bennett, Aithal, Pande, Kenna, Stachulski,
Park, Williams.
Jenkins RE, Meng X, Elliott VL, Kitteringham NR, Pirmohamed M, and Park BK
(2009) Characterisation of flucloxacillin and 5-hydroxymethyl flucloxacillin
haptenated HSA in vitro and in vivo. Proteomics Clin Appl 3:720–729.
ꢀ
Jeric I, Versluis C, Horvat S, and Heck AJR (2002) Tracing glycoprotein structures:
electrospray ionization tandem mass spectrometric analysis of sugar-peptide
adducts. J Mass Spectrom 37:803–811.
Johnson CH, Athersuch TJ, Wilson ID, Iddon L, Meng X, Stachulski AV, Lindon JC,
and Nicholson JK (2008) Kinetic and J-resolved statistical total correlation NMR
spectroscopy approaches to structural information recovery in complex reacting
mixtures: application to acyl glucuronide intramolecular transacylation reactions.
Anal Chem 80:4886–4895.
Karlsson ES, Johnson CH, Sarda S, Iddon L, Iqbal M, Meng X, Harding JR, Stachulski
AV, Nicholson JK, and Wilson ID, et al. (2010) High-performance liquid chromatography/
mass spectrometric and proton nuclear magnetic resonance spectroscopic studies of
the transacylation and hydrolysis of the acyl glucuronides of a series of phenyl-
acetic acids in buffer and human plasma. Rapid Commun Mass Spectrom 24:
3043–3051.
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Address correspondence to: Dr. Dominic P. Williams, Translational Safety
Department, Drug Safety and Metabolism, Room 23S57, Mereside, Alderley
Park, Macclesfield, Cheshire, SK10 4TG, UK. E-mail: dominic.williams@
astrazeneca.com