Multiple UDP-glucuronosyltransferases in human liver microsomes glucuronidate both R- and S-7-hydroxywarfarin into two metabolites

Article abstract of DOI:10.1016/j.abb.2014.10.006

The widely used anticoagulant Coumadin (R/S-warfarin) undergoes oxidation by cytochromes P450 into hydroxywarfarins that subsequently become conjugated for excretion in urine. Hydroxywarfarins may modulate warfarin metabolism transcriptionally or through

Full text of DOI:10.1016/j.abb.2014.10.006

Archives of Biochemistry and Biophysics 564 (2014) 244–253
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Archives of Biochemistry and Biophysics
journal homepage: www.elsevier.com/locate/yabbi
Multiple UDP-glucuronosyltransferases in human liver microsomes
glucuronidate both R- and S-7-hydroxywarfarin into two metabolites
C. Preston Pugh a, Dakota L. Pouncey a,b, Jessica H. Hartman a, Robert Nshimiyimana b,
Linda P. Desrochers b, Thomas E. Goodwin b, Gunnar Boysen c, Grover P. Miller a,
⇑
a Department of Biochemistry and Molecular Biology, University of Arkansas for Medical Sciences, Little Rock, AR, USA
b Department of Chemistry, Hendrix College, Conway, AR, USA
c Department of Environmental and Occupational Health, University of Arkansas for Medical Sciences, Little Rock, AR, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 4 August 2014
and in revised form 8 October 2014
Available online 19 October 2014
The widely used anticoagulant Coumadin (R/S-warfarin) undergoes oxidation by cytochromes P450 into
hydroxywarfarins that subsequently become conjugated for excretion in urine. Hydroxywarfarins may
modulate warfarin metabolism transcriptionally or through direct inhibition of cytochromes P450 and
thus, UGT action toward hydroxywarfarin elimination may impact levels of the parent drugs and patient
responses. Nevertheless, relatively little is known about conjugation by UDP-glucuronosyltransferases in
warfarin metabolism. Herein, we identified probable conjugation sites, kinetic mechanisms and hepatic
UGT isoforms involved in microsomal glucuronidation of R- and S-7-hydroxywarfarin. Both compounds
underwent glucuronidation at C4 and C7 hydroxyl groups based on elution properties and spectral char-
acteristics. Their formation demonstrated regio- and enantioselectivity by UGTs and resulted in either
Michaelis–Menten or substrate inhibition kinetics. Glucuronidation at the C7 hydroxyl group occurred
more readily than at the C4 group, and the reaction was overall more efficient for R-7-hydroxywarfarin
due to higher affinity and rates of turnover. The use of these mechanisms and parameters to model in vivo
clearance demonstrated that contributions of substrate inhibition would lead to underestimation of
metabolic clearance than that predicted by Michaelis–Menten kinetics. Lastly, these processes were
driven by multiple UGTs indicating redundancy in glucuronidation pathways and ultimately metabolic
clearance of R- and S-7-hydroxywarfarin.
Keywords:
Glucuronidation
UGT (UDP-glucuronosyltransferase)
Metabolism
Warfarin
7-Hydroxywarfarin
In vitro
Ó 2014 Elsevier Inc. All rights reserved.
Introduction
involves the addition of a glucuronic acid moiety from the cofactor
UDPGA catalyzed by the superfamily of conjugative enzymes UDP-
Coumadin (R/S-warfarin) is a commonly prescribed anticoagu-
lant for more than 20 million Americans to treat atrial fibrillation,
mechanical heart valves, venous thromboembolism, and other
coagulopathies; however, optimal dosing requires adequate con-
sideration of metabolic pathways that determine levels of the
active drugs R-warfarin and especially S-warfarin [1,2]. Warfarin
is essentially inactivated through oxidation by several cyto-
chromes P450 (P450 or CYP for specific isoforms) into 6-, 7-, 8-,
10- and 40-hydroxywarfarin [3,4]. While hydroxywarfarins are
commonly reported in patient plasma [5–7], there is indirect evi-
dence for extensive conjugation of hydroxywarfarins, most notably
to glucuronides, in patient urine samples [3,8]. Glucuronidation
glucuronosyltransferases (UGTs)1 [9]. UGT metabolic processes rank
second in clinical relevance for the top 200 drugs on the market after
those by cytochromes P450 [10].
Recent studies have implicated two possible mechanisms for
hydroxywarfarins to modulate warfarin metabolism and thus
impact patient responses to the drug. First, these warfarin metab-
olites may interact with receptors to control expression of many
genes involved in drug metabolic clearance including enzymes
and transporters. Hydroxywarfarins can bind to the pregnane X
receptor (PXR) and induce expression of CYP2C9 and CYP3A4 and
to a less extent the transporter P-glycoprotein 1 (also known as
multidrug resistance protein 1 (MDR1) based on gene reporter
assays with transformed HepG2 cells and treatment of primary
⇑
Corresponding author at: Department of Biochemistry and Molecular Biology,
University of Arkansas for Medical Sciences, 4301 W. Markham, Slot 516, Little
Rock, AR 72205, USA. Fax: +1 501 686 8169.
1
Abbreviations used: UGT, UDP glucuronosyltransferase; UDPGA, UDP-glucuronic
acid; OHWAR, hydroxywarfarin; 7-OHWAR-4-GLUC, 7-hydroxywarfarin b-D-4-glucu-
ronide; 7-OHWAR-7-GLUC, 7-hydroxywarfarin b-D-7-glucuronide.
E-mail address: MillerGroverP@uams.edu (G.P. Miller).
http://dx.doi.org/10.1016/j.abb.2014.10.006
0003-9861/Ó 2014 Elsevier Inc. All rights reserved.

C. Preston Pugh et al. / Archives of Biochemistry and Biophysics 564 (2014) 244–253
245
human hepatocytes [11]. Second, hydroxywarfarins could inhibit
warfarin metabolism by cytochromes P450. We have shown that
hydroxywarfarins, including 7-hydryoxywarfarin metabolite, one
of the most abundant metabolites in human plasma [5,7], can
potently inhibit CYP2C9, the major pathway for S-warfarin metab-
olism [12]. Therefore, the second phase of metabolism by UGTs
may alter the levels of hydroxywarfarin levels in vivo and ulti-
mately impacts the metabolism of the parent drug warfarin. For
example, studies in mice have shown that R-warfarin clearance
likely involves a coupled process between P450 and UGT metabo-
lism of the drug [13]. Further studies are clearly needed to advance
an understanding of the potential clinical relevance of UGT
metabolic pathways in warfarin metabolic clearance.
Recent efforts have begun to identify possible glucuronidation
pathways for hydroxywarfarins and the UGTs responsible for them.
Recombinant UGT1A1, 1A3, 1A8, 1A9, and 1A10 display regioselec-
tive activity toward racemic warfarin and hydroxywarfarins [14],
although only 6-, 7-, and 8-hydroxywarfarin underwent apprecia-
ble metabolism. The enantioselectivity of these reactions were
investigated in a subsequent study using enantiomers of the parent
drugs and hydroxywarfarins and an emphasis on recombinant
UGT1A1 and 1A10. In addition, high correlations were reported
between with bilirubin glucuronidation (UGT1A1) and UGT1A1
protein content for 6- and 7-hydroxywarfarin, whereas the poorest
correlations occurred with UGT2B7 and 2B15 [15].
Despite these advances, there remain significant gaps in our
understanding of hepatic glucuronidation pathways in humans.
Neither of the studies identified the structure of glucuronide
metabolites generated in those reactions [14,15]. The parent drug,
warfarin, possesses a C4 hydroxyl group, and upon P450 oxidation,
another hydroxyl group becomes available for modification. Both
of these sites are possible targets for glucuronidation as shown
for 7-hydroxywarfarin in Fig. 1. Studies with rat hepatocytes
suggest both sites could be accessible for glucuronidation and/or
sulfonation leading to the formation of mono- and di-conjugates
[16]. Knowledge of the actual products of conjugative reactions is
necessary to guide the synthesis of authentic standards for direct
quantification of glucuronide metabolites and facilitate metabo-
lite-profiling approaches for use as biomarkers [4]. The identifica-
tion of the structures for the respective glucuronides would
enable the mapping of specific metabolic pathways for hydroxy-
warfarins as contributors to overall drug clearance. The reliance
on recombinant UGTs for in vitro metabolic mechanism and
parameters fails to reflect the complexity of a hepatic microsomal
system in which phospholipid and UGT dimerization impact UGT
activity (reviewed in [17]). Lastly, the typical normalization of
recombinant activity to total protein concentration does not allow
a correction for differences among preparations and corresponding
metabolic rates. This strategy makes it impossible to directly com-
pare metabolic clearance rates among different recombinant UGT
preparations.
In this study, we identified probable conjugation sites, kinetic
mechanisms and hepatic UGT isoforms involved in microsomal
glucuronidation of R- and S-7-hydroxywarfarin. For these studies,
we synthesized R- and S-7-hydroxywarfarin substrates enantiose-
lectively using novel green chemistry techniques. Experiments
revealed for the first time the formation of two glucuronide metab-
olites and their corresponding spectral properties were used to
identify whether glucuronidation occurred at the C4 or C7
hydroxyl group (Fig. 1). We then measured steady-state kinetics
for R- and S-7-hydroxywarfarin using human liver microsomes
pooled from 150 donors (HLM150) as a model for the metabolic
capacity of the average human liver [18]. The corresponding
kinetic profiles mostly deviated from the relationship reported by
Michaelis–Menten [19]. Consequently, we compiled the data and
globally fit it to six possible mechanisms involving either a single
binding site (Michaelis–Menten) or two binding sites and identi-
fied the most statistically probable one using DynaFit software
[20] as described recently [21]. Inhibitor phenotyping experiments
were carried out to determine relative roles for UGT1A1, 1A4, 1A6,
and 1A9 in microsomal 7-hydroxywarfairn metabolism. Lastly, we
assessed the potential impact of these mechanisms and parameters
on the prediction of in vivo hepatic metabolic clearance of R- and
S-7-hydroxywarfarin.
Methods
Materials
All chemicals used in this study were ACS grade or higher. The
following chemicals were purchased from Sigma–Aldrich (St. Louis,
MO): 4,7-dihydroxycoumarin, E-4-phenylbut-3-en-2-one, (R,R)-
and (S,S)-1,2-diphenylethylenediamine, tetrahydrofuran, glacial
acetic acid, ethyl acetate, CD3OD, ethanol, UDP-glucuronic acid
(UDPGA), perchloric acid, serotonin (inhibitor), hecogenin
(inhibitor), bilirubin (inhibitor), and mefenamic acid (inhibitor).
R/S-7-Hydroxywarfarin and R/S-warfarin (internal standard) were
purchased from Toronto Research Chemicals (Toronto, Canada).
HPLC Grade methanol, alamethicin, magnesium chloride, and sac-
charolactone were purchased from Fisher Scientific (Wilmington,
MA). Pooled human liver microsomes were purchased from BD
Biosciences (San Jose, CA).
Synthesis of R- and S-7-hydroxywarfarin through green chemistry
Fig. 1. Structures of coumarin derivatives possessing a 7-hydroxyl group. Panel A,
The 7-hydroxywarfarin metabolite of warfarin is pictured with arrows indicating
the hydroxyl groups on the coumarin that serve as potential sites of glucuronida-
tion. The asterisk shows the chiral center for the compound resulting in R and S
enantiomers of the drug metabolite. Panel B, 7-Hydroxycoumarin (top) and 7-
ethoxycoumarin (bottom) are shown with an arrow highlighting the 7-hydroxyl
group, which when modified, leads to a selective decrease in compound fluores-
cence but not absorbance. Panel C, 7-Hydroxywarfarin forms diastereomeric cyclic
hemiacetals when present in organic solvents, like methanol, as observed during
characterization of products synthesized through green chemistry.
The enantiomers of 7-hydroxywarfarin were prepared by
adapting the green chemistry procedure as described by others
[22,23]. 4,7-Dihydroxycoumarin (107 mg, 0.600 mmol), E-4-phe-
nylbut-3-en-2-one (90 mg, 0.616 mmol), (R,R)- or (S,S)-1,2-diphen-
ylethylenediamine (12 mg, 0.057 mmol), 1 mL of tetrahydrofuran
and 285
lL of glacial acetic acid were stirred in a sealed 10 mL
round-bottomed flask at the ambient temperature until a white

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C. Preston Pugh et al. / Archives of Biochemistry and Biophysics 564 (2014) 244–253
solid precipitated (typically this took 2–4 days). The R,R catalyst
produces the R configuration at C-11 in 7-hydroxywarfarin, while
the S,S catalyst yields the S configuration (Fig. 1, Panel C). The
precipitate resulting from either reaction was collected by vacuum
filtration, rinsed with water, and allowed to dry. The yield for each
reaction was typically around 70% for the white solid.
Recrystallization from ethanol/water provided each product with
an enantiomeric excess greater than 99% based on chromato-
graphic analyses.
differentially affect visible spectral properties based on the critical
role the 7-hydroxyl group plays in fluorescence, but not absor-
bance, of coumarin derivatives. This property has been utilized
for measuring the increase in fluorescence upon O-dealkylation
of 7-ethoxycoumarin by P450s [28]. We tested our hypothesis by
assessing the relative fluorescence and absorbance properties of
7-hydroxycoumarin and 7-ethoxycoumarin by HPLC. Specifically,
samples of 50 lM 7-hydroxycoumarin and 7-ethoxycoumarin
were prepared in water from 10 mM stocks in methanol and
injected onto a Waters Breeze HPLC system, compounds were
resolved employing the same method described for analyzing the
microsomal reaction toward 7-hydroxywarfarin in section ‘‘HPLC
analysis of 7-hydroxywarfarin glucuronidation reaction’’. Elution
of the compounds was then monitored by absorbance (370 nm)
and fluorescence (excitation: 370 nm, emission: 450 nm) and the
relative areas of the peaks compared.
We compared the products to R- and S-7-hydroxywarfarin
standards that were prepared by using
a chiral preparative
chromatography of the racemic commercial compound as
described previously [15]. In this study, recrystallized compounds
were injected onto a Waters Breeze HPLC system and resolved
using a 2.1 mm ꢀ 150 mm, 5.0
lM Astec Chirobiotic V column
(Supelco) operated at room temperature (22.0 °C) using an iso-
cratic method with 20% methanol: 80% 0.1% acetic acid/water at
As a complement to those efforts, we analyzed the fractions
by mass spectrometry to determine the parent masses and frag-
mentation patterns for individual metabolites. Samples were
injected onto an 6590 Agilent triple quadrupole mass analyzer
using an isocratic flow of 0.4 mL/min 70% acetonitrile/0.1% for-
mic acid. Ion spectra were acquired in full scan monde monitor-
ing the m/z range of 50–1000 amu. Subsequently product ion
spectra were generated from the theoretical precursor ion m/z
501 monitoring the m/z range 50–600 amu at various collision
energies.
a
flow rate of 1.2 mL/min. Elution of the compounds was
monitored by absorbance at 275 and 325 nm and fluorescence
(excitation: 325 nm; emission: 420 nm) and compared against
individual R- and S-7-hydroxywarfarin standards.
The structure of the compounds were further analyzed by 1H
and 13C NMR as well as high resolution MS. The 1H (400 MHz)
and 13C (100 MHz) NMR spectra were acquired on a JEOL ESC
400 spectrometer with CD3OD as solvent; therefore, hydroxyl
protons are exchanged with deuterium and are not observed.
Assignment of chemical shifts was aided by comparison to pub-
lished data for warfarin itself [24,25], as well as use of DEPT-90,
DEPT-135, and HMQC. Specific assignments for hydrogens or car-
bons refer to the numbering on Fig. 1, Panel C. Solvent conditions
for these studies favored the ring closure of warfarin to the hemi-
acetal and resulting two diastereomers (Fig. 1, Panel C). Data for
13C NMR experiments are expressed in ppm relative to the solvent
absorbance at 49.15 ppm. All data for the NMR studies are summa-
rized in Tables S2 and S3 in Supplemental Data. Melting points
were obtained on an electrothermal apparatus and are uncor-
rected. The mp range was 223–228 °C with decomposition, which
is slightly higher than reported previously (208–210 °C) [26]. Mass
spectra for the synthesized compounds were acquired on a Waters/
Micromass ESI-QTOF-MS spectrometer. The high resolution mass
spectral molecular ion for C19H16O5 was calculated to be 324.33,
which matched the experimentally-determined m/z.
Steady-state kinetic assays for R- and S-7-hydroxywarfarin
glucuronidation by human liver microsomes
We initially carried out control experiments to guide the design
of steady-state studies on the metabolism of R- and S-7-hydroxy-
warfarin. Specifically, we ensured linearity of response as a func-
tion of protein concentration and time. Based on those findings,
the kinetic reactions included 0.5 mg/mL HLM150 incubated with
0, 20, 50, 100, 150, 250, 300, 500, 750, 1000, 1250, 1500, 1750,
and 2000
lM R- and S-7-hydroxywarfarin in the presence of
50 mM Tris–HCl pH 7.4 (under reaction conditions), 25
lg/mL ala-
methacin, 8 mM MgCl2, and 5 mM saccharolactone for 15 min at
37 °C. The reactions were initiated with 2 mM UDPGA and
quenched at 1 h with equal volume of 0.4 N perchloric acid. The
reactions were centrifuged at 2500 RPM for 15 min at 4 °C and
the glucuronide metabolites quantified by HPLC as described in
section ‘‘HPLC analysis of 7-hydroxywarfarin glucuronidation reac-
tion’’. Initial rates at each concentration were determined based on
the completion of four to ten experimental replicates.
HPLC analysis of 7-hydroxywarfarin glucuronidation reaction
Unlike our previous studies [14], we developed a new high per-
formance liquid chromatography (HPLC) method for analyzing glu-
curonides. The samples were injected onto a Waters Breeze HPLC
Analysis of steady-state kinetic profiles for R- and S-7-
hydroxywarfarin glucuronidation
system and resolved using a 4.6 ꢀ 150 mm Zorbax Eclipse 3.5
lM
XDB-C18 column (Agilent) heated to 45 °C using an isocratic
method with 40% methanol: 60% 0.1% acetic acid/water at a flow
rate of 1.2 mL/min. Elution of the compounds was monitored for
10 min by absorbance at 275 and 325 nm and fluorescence (excita-
tion: 325 nm; emission: 420 nm). For our reactions, peak areas of
the two metabolites were normalized to R/S-warfarin and then
quantitated relative to the standard response for 7-hydroxywarfa-
rin due to the lack of authentic glucuronide standards. It has been
shown previously that the addition of the glucuronic acid moiety
does not alter the extinction coefficient from that of the unreacted
substrate [27].
Kinetic profiles were not hyperbolic, but demonstrated
a
decrease in rate at higher substrate concentrations. Thus, the data
were initially fit and analyzed with respect to the hyperbolic
Michaelis–Menten and uncompetitive substrate inhibition equa-
tions using GraphPad Prism version 5.0 (GraphPad Software, Inc.,
La Jolla, CA). A statistical comparison of the quality of the resulting
fits using Akaike Information Criterion revealed a strong prefer-
ence for the uncompetitive substrate inhibition mechanism; how-
ever, there are possibilities other than an uncompetitive
mechanism that can explain substrate inhibition. Consequently,
we further investigated the mechanism and parameters for equi-
librium constants and reaction rates using DynaFit version 3.28
(BioKin, Ltd., Watertown, MA) [20].
Impact of modification at 7-hydroxyl group on spectral properties and
mass
For the reanalysis of the data, we globally fit kinetic profiles to
six different kinetic mechanisms and identified the most statisti-
cally probable one using DynaFit version 3.28 [20]. Specifically,
Glucuronidation of 7-hydroxywarfarin can occur at C4 or C7
hydroxyl groups; we hypothesized that these modifications would

C. Preston Pugh et al. / Archives of Biochemistry and Biophysics 564 (2014) 244–253
247
Vmax
we fit the data to single binding site mechanism reported by
Michaelis–Menten [19] and five different two-site binding mecha-
nisms including those describing substrate inhibition and cooper-
ativity (Fig. S1 and Table S1 in Supplemental Data), as described
previously [21]. These latter mechanisms possess a binary (ES)
complex, ternary (ESS) complex, or both complexes, whereby they
are active with the same or differing maximal rates. Binding events
presented in the model are ordered with respect to stoichiometry,
but the occupancy of each individual binding site was indistin-
guishable in this assay. The most probable mechanism and corre-
sponding parameters for R- and S-7-hydroxywarfarin were
identified using the advanced tools of numerical analysis and
applied statistics, as implemented in the software DynaFit. After
globally fitting reactions to these mechanisms, we generated a cor-
responding Akaike information criterion (AICc) to identify statisti-
cally the most plausible mechanism based on the quality of the fits
[20]. The presentation of data and the corresponding fit to the
appropriate mechanism was achieved through the use of GraphPad
Prism version 5.0.
m
¼
ð2Þ
½hydroxywarfarinꢁð1 þ ½hydroxywarfarinꢁ=KsÞ þ Km
where
m is the initial rate of the reaction, Vmax the maximal rate of
the reaction, Km the Michaelis constant, and Ks the inhibition con-
stant for occupancy of the second site.
3. Results
Identification of steady-state conditions and potential glucuronides
resulting from R- and S-7-hydroxywarfarin metabolism
Initially, we determined conditions demonstrating linear prod-
uct formation as a function of time and enzyme concentration.
Over the course of a four hour incubation, microsomal reactions
for both R- and S-7-hydroxywarfarins yielded two compounds,
whose appearance seemed consistent with products of the reaction
and occurred simultaneously minimizing the likelihood for a sec-
ondary glucuronidation reaction (Fig. 2, Panel A). Consequently,
we labeled them metabolites 1 and 2 based on elution order during
HPLC analyses. The amounts of these metabolites increased as a
function of time and linearly correlated with enzyme concentra-
tion, as shown in supplemental data (Fig. S2 in Supplemental Data).
A 60 min time point was chosen to avoid excessive substrate
depletion and thus remain within steady-state conditions. Both
metabolites eluted before 7-hydroxywarfarin during HPLC
analyses, indicating a higher polarity as expected for glucuronide
metabolites. Finally, the introduction of UGT-specific inhibitors
blocked formation of the compounds (section ‘‘Multiple
microsomal UGT enzymes contribute to R- and S-7-hydroxywarfa-
rin glucuronidation’’).
Glucuronidation can only occur at two locations of 7-hydroxy-
warfarin yielding molecules with different spectral properties,
and thus we leveraged those differences to predict the structures
for metabolites 1 and 2. The formation of a bond with the C7
hydroxyl group on a coumarin ring does not significantly impact
absorbance yet causes a large drop in fluorescence. This property
is leveraged in P450 catalytic assays whereby O-dealkylation of
substrate to form 7-hydroxycoumarins result in a significant
increase in fluorescence as a marker for product formation [28].
Therefore, we hypothesized that glucuronidation of the C7 hydro-
xyl group and not the one at C4 will decrease 7-hydroxywarfarin
fluorescence selectively, and thus provide evidence for the regio-
specificity of glucuronidation. As a control, we investigated this
effect for 7-hydroxy- and 7-ethoxycoumarin under HPLC condi-
tions used in this study (Fig. 2, Panel B, Table 1). At equal molar
concentrations, the spectral ratio between fluorescence and
absorbance for these compounds was almost 10-fold higher value
for 7-hydroxycoumarin relative to 7-ethoxycoumarin, indicating
selective suppression of coumarin fluorescence upon modification
of the hydroxyl group at C7. Analysis of the 7-hydroxywarfarin
metabolites demonstrated that glucuronidation decreased
compound fluorescence regardless of the site of modification;
however, the effect was more severe for the second metabolite
than the first (Fig. 2, Panel A, Table 1). The magnitude of the differ-
ence in the spectral ratio between metabolites 1 and 2 for both
UGT substrates was about 10-fold as observed for the coumarin
model compounds.
Inhibitor phenotypying of R- and S-7-hydroxywarfarin
glucuronidation by UGTs present in human liver microsomes
For these experiments, 0.5 mg/mL protein (HLM150) was incu-
bated with R- and S-7-hydroxywarfarin (100 lM or 1000 lM),
UDPGA (2 mM), and a chemical inhibitor specific for an individual
UGT in 50 mM Tris–HCl buffer, pH 7.4. The inhibitors used in these
experiments were bilirubin (25
hecogenin (10 M) for UGT1A4 [30], serotonin (200
UGT1A6 [31], and mefenamic acid (25 M) for UGT1A9 and
l
M) for mainly UGT1A1 [29],
l
l
M) for
l
UGT2B7 [32]. The inhibitors were initially dried down with R- or
S-7-hydroxywarfarin before reconstituting the reactions. By
contrast, bilirubin was prepared fresh daily in amber vials as a
2 mM stock solution in DMSO. The inhibitors were incubated with
R- and S-7-hydroxywarfarin and HLM for 15 min prior to initiation
of the reaction. Upon addition of UDPGA to start the reaction, the
samples were incubated at 37 °C for 1 hour and analyzed as
described for steady-state experiments. The statistical significance
of the specific inhibition by each compound compared to the
respective control reaction was calculated using
a one-way
analysis of variance (ANOVA) test as implemented by GraphPad
Prism software, version 5.0 (San Diego, CA). Final values for
inhibitor potency reflected a minimum of four experimental
replicates.
Modeling R- and S-7-hydroxywarfarin metabolic clearance
We obtained a perspective on the potential clinical importance
of these studies by modeling metabolic clearance with in vitro
kinetic parameters [33,34]. We fit our data to mechanistic equa-
tions describing the relationship between 7-hydroxywarfarin
enantiomer concentration and its clearance (rate/[7-hydroxywar-
farin]). The metabolic clearance of 7-hydroxywarfarin based on
the traditional model was predicted using the parameters derived
from fitting the HLM150 data to the Michaelis–Menten equation
and applying the results to Eq. (1) as follows:
Vmax
m
¼
ð1Þ
½hydroxywarfarinꢁ þ Km
As a complement to kinetic, chromatographic, and spectral data,
we subjected the metabolites form the four hour time course
experiment to mass spectral analysis. These studies would not
likely be able to identify the site of glucuronidation as reported
by others [14,15]; however, it would be possible to distinguish
whether the metabolites were either a mono- or di-glucuronide
based on mass. The calculated mass for the mono-glucuronide is
500.45 and that for the di-glucuronide is 676.56. The full mass
where
m
is the initial rate of the reaction, Vmax the maximal rate of
the reaction, and Km the Michaelis constant. For comparison, the
impact of substrate inhibition on the metabolic clearance of
7-hydroxywarfarin by glucuronidation was predicted using Eq. (2)
and parameters derived from fitting the HLM150 kinetic data to a
derivation of the equation describing substrate (uncompetitive)
inhibition as follows:

248
C. Preston Pugh et al. / Archives of Biochemistry and Biophysics 564 (2014) 244–253
Fig. 2. Modification of hydroxyl group alters the relative signal of 7-hydroxycoumarin derivatives. HPLC traces of compounds are overlaid to demonstrate impact of possible
modification of the 7-hydroxyl group on the coumarin ring on relative fluorescence and absorbance properties. Solid line, fluorescence; dotted line, absorbance. Panel A,
50 lM steady state reaction of glucuronide metabolite 1 (1) and glucuronide metabolite 2 (2) (fluorescence excitation at 325 nm and emission 420 nm; absorbance at
325 nm). These compounds were later identified as 7-OHWAR-4-GLUC and 7-OHWAR-7-GLUC, respectively, in section ‘‘Discussion’’. Panel B, 50
l
M 7-hydroxycoumarin (1)
and 7-ethoxycoumarin (2) (fluorescence excitation at 370 nm and emission 450 nm; absorbance at 370 nm).
Table 1
Spectral properties for 7-hydroxycoumarin derivatives.
Compound
Amount (
lM)
Spectral properties
Absorbancea
Fluorescenceb
Fluorescence
Absorbance
7-Hydroxycoumarin
7-Ethoxycoumarin
R-7-hydroxywarfarin
Metabolite 1d
50
50
10
1.48 ꢀ 103
1.30 ꢀ 103
5.64 ꢀ 104
3.78 ꢀ 103
1.37 ꢀ 105
5.29 ꢀ 104
2.45 ꢀ 103
7.89 ꢀ 104
1.24 ꢀ 106
9.60 ꢀ 104
4.75 ꢀ 106
9.68 ꢀ 104
3.69 ꢀ 105
4.48 ꢀ 106
4.83 ꢀ 104
2.25 ꢀ 105
837
73.8
84.2
25.6
2.70
84.7
19.7
2.84
c
⁄
c
Metabolite 2e
⁄
S-7-Hydroxywarfarin
10
Metabolite 1d
⁄
c
Metabolite 2e
⁄
c
a
b
c
Area of absorbance peak measured at 370 nm and 325 nm for coumarin and hydroxywarfarin derivatives respectively.
Area of fluorescence emission peak measured following excitation at 370 nm and 325 nm for coumarin and hydroxywarfarin derivatives respectively.
Product areas observed for the reaction containing 500 M substrate.
Later identified as 7-HOWAR-4-GLUC (see section ‘‘Discussion’’).
Later identified as 7-HOWAR-7-GLUC (see section ‘‘Discussion’’).
l
d
e
spectrum for both metabolites showed signals consistent with the
parent mass of a mono-glucuronide in positive and negative ion
mode (including sodium adducts) (Fig. S3 in Supplemental Data),
and subsequent MS/MS experiments revealed product ions of m/z
443, 655, 267, 179, and 147, which match those reportedly previ-
ously by others for the mono-glucuronide [14,15].
second one (R-7-OHWAR-7-GLUC) involved Michaelis–Menten
kinetics (Table 2). Nevertheless, uncompetitive inhibition is only
one of many possible kinetic mechanisms resulting in substrate
inhibition.
We employed DynaFit [20], a more powerful software package,
and re-analyzed the data comparing the suitability of data fits to
the Michaelis–Menten (single site) mechanism or multiple two-
site mechanisms in which the UGT responsible for the reaction
possesses catalytic and cooperative binding sites (Fig. S1 and
Table S1 in Supplemental Data). Based on Akaike Information Cri-
terion, the most probable mechanism for both S-hydroxywarfarin
metabolites was a two-site ES active model in which catalytically
active ES complex forms as a function of S-hydroxywarfarin con-
centration Table 3. The most probable mechanisms for R-hydroxy-
warfarin glucuronidation into two metabolites involved different
mechanisms of action. The first metabolite (R-7-OHWAR-4-GLUC)
Substrate inhibition during microsomal glucuronidation of R- and S-7-
hydroxywarfarin
Microsomal glucuronidation reactions revealed that after an ini-
tialincrease, theinitial rate actuallysignificantlydecreased at higher
substrate concentrations (Fig. 3). An initial fit of the data revealed a
strong statistical preference for the uncompetitive substrate inhibi-
tion mechanism over that for the Michaelis–Menten mechanism for
both S-7-hydroxywarfarin metabolites, later identified as S-7-
hydroxywarfarin b-
D-4-glucuronide (S-7-OHWAR-4-GLUC) and
was preferentially formed through a two-site cooperative
S-7-hydroxywarfarin b-
D
-7-glucuronide (S-7-OHWAR-7-GLUC),
mechanism in which catalytically active ES and ESS complexes
formed as a function of R-7-hydroxywarfarin, whereas generation
of the second metabolite (R-7-OHWAR-7-GLUC) was more likely
through a single-site mechanism following Michaelis–Menten
respectively (see section ‘‘Discussion’’). The first metabolite
(R-7-OHWAR-4-GLUC) for R-7-hydroxywarfarin was also formed
through a substrate inhibition mechanism, while generation of the

C. Preston Pugh et al. / Archives of Biochemistry and Biophysics 564 (2014) 244–253
249
Fig. 3. Steady-state kinetics for glucuronidation of R- and S-7-hydroxywarfarin by HLM150. For the kinetic reactions, 0.5 mg/mL protein for HLM150 was incubated with
0–2000 M R- and S-7-hydroxywarfarin for 60 min at 37 °C. Panel A and B represent the kinetic profiles for S-7-hydroxywarfarin glucuronidation into 7-OHWAR-4-GLUC and
l
7-OHWAR-7-GLUC, respectively. Panel C and D represent the glucuronidation of R-7-hydroxywarfarin steady-state kinetics for glucuronidation at the C4 and C7 hydroxyl
groups, respectively. The reported values represent the average from 4 to 10 replicates for each individual experiment. Solid line depicts fit of data to the preferred
mechanism indicated in Table 3, i.e. Michaelis–Menten for the major metabolic pathway for R-7-hydroxywarfarin and substrate inhibition for the other reactions. For
comparative purposes, dashed line depicts fit of data to Michaelis–Menten equation when substrate inhibition was the preferred mechanism.
Table 2
Preliminary analysis of kinetic mechanism describing R- and S-7-hydroxywarfarin glucuronidation.a
Substrate
Metabolitesb
Model
Model
Parameters for preferred model
c
Preference
Vmax1
Km
(lM)
Ki (lM)
R-7-HOWAR
S-7-OHWAR
7-HOWAR-4-GLUC
7-HOWAR-7-GLUC
Substrate inhibition
Michaelis–Menten
70.7%
75.2%
35.4 (30.4–40.4)
1090 (1040–1140)
60.8 (36.9–84.8)
122 (100–145)
2640 (1360–3920)
–
7-HOWAR-4-GLUC
7-HOWAR-7-GLUC
Substrate inhibition
Substrate inhibition
>99.99%
99.7%
19.7 (17.19–22.2)
1470 (790–2150)
111 (77.7–145)
929 (362–1500)
2840 (1650–4030)
1310 (259–2370)
a
The nonsymmetrical 95% confidence intervals for parameters are shown in parentheses.
Glucuronidation resulted in conjugation of the hydroxyl group at either the C4 or C7 position resulting in the formation of 7-HOWAR-4-GLUC or 7-HOWAR-7-GLUC,
b
respectively, as described in section ‘‘Discussion’’.
c
Units are nmol/min/mg protein.
Table 3
Preferred model mechanism and parameters for steady-state glucuronidation of R- and S-7-hydroxywarfarin by HLM150.a
Substrate
Metabolitesb
Model
Parameters for preferred model
c
c
Vmax1
Vmax2
Ks
(lM)
Kss (lM)
R-7-HOWAR
S-7-OHWAR
7-HOWAR-4-GLUC
7-HOWAR-7-GLUC
Substrate inhibition
Michaelis–Menten
58.0 (49.5–66.1)
1090 (1050–1140)
19.0 (16.3–21.4)
–
145 (112–183)
121 (101–144)
145 (112–183)
–
7-HOWAR-4-GLUC
7-HOWAR-7-GLUC
Substrate inhibition
Substrate inhibition
19.7 (17.6–22.4)
1470 (1020–2700)
–
–
110 (83.0–149)
927 (559–1960)
2850 (1950–4400)
1310 (538–2810)
a
The nonsymmetrical 95% confidence intervals for parameters are shown in parentheses.
Glucuronidation resulted in conjugation of the hydroxyl group at either the C4 or C7 position resulting in the formation of 7-HOWAR-4-GLUC or 7-HOWAR-7-GLUC,
b
respectively, as described in section ‘‘Discussion’’.
c
Units are nmol/min/mg protein.
kinetics. The parameters and corresponding 95% confidence inter-
vals for all reactions are summarized in Table 3. Similarly, both
pathways for glucuronidation of S-7-hydroxywarfarin were best
explained through a two-site cooperative mechanism in which
catalytically active ES and ESS complexes formed as a function of
S-7-hydroxywarfarin.

250
C. Preston Pugh et al. / Archives of Biochemistry and Biophysics 564 (2014) 244–253
Fig. 4. R- and S-7-Hydroxywarfarin clearance involving either Michaelis–Menten (dashed line) or substrate inhibition (solid line) kinetics. GraphPad Prism software (San
Diego, CA) was used to simulate clearance curves for R- and S-7-hydroxywarfarin using equations adapted by Houston and Kenworthy [34] as described in section ‘‘Materials
and methods’’. Panels A and B indicate the clearance of R-7-hydroxywarfarin as a solid line for both metabolites, i.e. R-7-OHWAR-4-GLUC and R-7-OHWAR-7-GLUC. Panels C
and D indicate the clearance of S-7-hydroxywarfarin as a solid line for both metabolites, i.e. S-7-OHWAR-4-GLUC and S-7-OHWAR-7-GLUC. The dashed line in Panels A, C, and
D shows the predicted clearance of the compounds if the metabolic process conformed to the Michaelis–Menten mechanism to highlight the overestimation of
7-hydroxywarfarin clearance by traditional Michaelis–Menten kinetic models.
Impact of substrate inhibition on metabolic clearance of R- and S-7-
hydroxywarfarin
roles for hepatic UGT1A1 and 1A6, as well as 1A9 and/or UGT2B7,
and rule out UGT1A4 in glucuronidation of R- and S-7-
hydroxywarfarin.
We employed the kinetic parameters from fits of the micro-
somal data to either the Michaelis–Menten or substrate inhibition
mechanisms to assess their effects on 7-hydroxywarfarin meta-
bolic clearance. The parameters for the traditional uncompetitive
equation (Table 2) were used to simulate substrate inhibition as
reflected in Eq. (2) (solid line in Fig. 4, Panels A, C, and D). The
dashed line indicates a hypothetical plot for metabolite clearance
through a process dependent on the Michaelis–Menten mecha-
nism for comparative purposes. The resulting log plot demon-
strates that substrate inhibition results in significantly lower
clearance rates at low substrate concentrations such that the clear-
ance of the metabolites is overestimated. At higher 7-hydroxywar-
farin levels, the difference between the clearance models becomes
insignificant. The exception to this observation is R-7-OHWAR-7-
GLUC, the major metabolite derived form the reaction with
R-7-hydroxywarfarin.
Discussion
The first kinetic analysis of hepatic microsomal metabolism of
R- and S-7-hydroxywarfarin revealed important features of the
reaction. Both compounds underwent glucuronidation at the C4
and C7 hydroxyl group. In the absence of authentic standards, we
were able to putatively identify these metabolites based on elution
properties along with fluoresce and mass spectral characteristics.
Their formation by microsomal UGTs demonstrated regio- and
enantioselectivity and resulted in either Michaelis–Menten or sub-
strate inhibition kinetics. Glucuronidation at the C7 hydroxyl
group occurred more readily than at the C4 hydroxyl group, and
the reaction was overall more efficient for R-7-hydroxywarfarin
due to higher affinity and rates of turnover for the substrate. The
use of those mechanisms and parameters to model in vivo clear-
ance of the 7-hydroxywarfarins demonstrated that the contribu-
tions of substrate inhibition would lead to underestimation of
their metabolic clearance at low 7-hydroxywarfarin levels than
that predicted by the traditional Michaelis–Menten kinetics. Lastly,
these processes were driven by multiple UGTs indicating
redundancy in glucuronidation pathways and ultimately metabolic
clearance of R- and S-7-hydroxywarfarin.
Multiple microsomal UGT enzymes contribute to R-and S-7-
hydroxywarfarin glucuronidation
Glucuronidation of 7-hydroxywarfarin to either metabolite by
human liver microsomes was decreased in the presence of rela-
tively specific inhibitors toward individual UGTs. The presence of
bilirubin, serotonin, and mefenamic acid but not hecogenin inhib-
ited activity toward 100
potency of this effect was lost at a higher substrate concentration
(1000 M, data not shown). These patterns were not impacted by
chirality of the substrates and were conserved for both
metabolites, such that the metabolic pathways may be the same.
Nevertheless, the effects of these inhibitors on reactions implicate
l
M substrate by about 50% (Fig. 5). The
Preliminary experiments revealed that hepatic UGTs target both
hydroxyl groups of R- and S-7-hydroxywarfarin for glucuronida-
tion. Based on HPLC analyses, two new peaks formed over the
course of the reaction simultaneously and eluted before the sub-
strates. This observation is consistent with the creation of polar
glucuronides, which were labeled metabolites 1 and 2. It is possible
l

C. Preston Pugh et al. / Archives of Biochemistry and Biophysics 564 (2014) 244–253
251
Fig. 5. Inhibitor phenotyping of steady-state glucuronidation of R- and S-7-hydroxywarfarin by HLM150. Reactions carried out with 0.25 mg/mL protein, 100 lM
7-hydroxywarfarin, and 2 mM UDPGA in the presence of specific inhibitors for specific UGT isoforms at 37 °C and pH 7.4. Panels A and B depict selective inhibition of R-7-
hydroxywarfarin to R-7-OHWAR-4-GLUC and R-7-OHWAR-7-GLUC, respectively. Panels C and D depict selective inhibition of S-7-hydroxywarfarin to S-7-OHWAR-4-GLUC
and S-7-OHWAR-7-GLUC, respectively. Reaction activities were normalized to the control rate and reported as a percent, e.g. 100% in the absence of inhibition. The reported
activities represent the average from a minimum of 4 individual experiments and are shown as the mean standard deviation. Asterisks indicate statistical significance of
inhibition compared to control (⁄⁄⁄p < 0.001).
that the same glucuronide conjugate at the C7 position, yet differ in
whether the compound exists in open and closed structures
[25,35], as shown in Fig. 1, Panels A and C, due to the presence
of organic solvent (methanol) in the mobile phase. This experimen-
tal artifact is not likely, given the absence of this effect with
7-hydroxywarfarin substrate (or other warfarin derivative). More-
over, the ratio between the peak areas was not the same as
expected for an equilibrium process and. Alternatively, these UGT
products may reflect the creation of two monoglucuronides or a
mono- and di-glucuronide. The formation of a di-glucuronide
would require the initial accumulation of the monoglucuronide
to drive the reaction, yet no lag in the time course was observed.
Moreover, the addition of a second glucuronic acid moiety to the
monoglucuronide would significantly increase the polarity of the
molecule making metabolite 1 the only probable di-glucuronide;
however, the fluorescence properties of metabolite 1 was consis-
tent with modification only at the C4 hydroxyl group. Lastly, mass
spectral analysis of the metabolite yielded identical masses and
fragmentation patterns consistent with mono-glucuronides of
hydroxywarfarins as reported previously reported by others
[14,15]. Taken together, these findings suggest that both R- and
S-7-hydroxywarfarin undergoes glucuronidation into two
monoglucuronides by UGT action present in liver microsomes.
Glucuronidation of the C4 or C7 hydroxyl groups would result
in metabolites with different spectral properties and thus provide
a basis for their identification in the absence of authentic stan-
dards. Coumarin rings are inherently fluorescent. That property is
significantly increased when a free C7 hydroxyl group is present
on the molecule, but the effect is selective. Absorbance properties
are minimally affected by the presence of a free or modified
7-hydroxyl group, as demonstrated by our HPLC analysis of
7-hydroxy- and 7-ethoxycoumarin. The same principle can be
applied to predicting the structure of the 7-hydroxywarfarin glucu-
ronides. Modification of the C7 hydroxyl group would lead to a
much lower fluorescence signal relative to absorbance than that
expected for the C4 hydroxyl group. Analysis of the glucuronida-
tion reaction showed that metabolite 2 yielded a much lower rel-
ative fluorescent signal than metabolite 1. In fact, the magnitude
of this difference (ꢂ10-fold) is similar to that observed between
7-hydroxy- and 7-ethoxycoumarin. Consequently, the two mono-
glucuronides, metabolites 1 and 2, generated from the hepatic
microsomal reaction toward R- and S-7-hydroxywarfarin are likely
7-hydroxywarfarin b-
D
-4-glucuronide (7-OHWAR-4-GLUC) and
7-hydroxywarfarin b-
D-7-glucuronide (7-OHWAR-7-GLUC).
Our strategy for assigning possible structures to these glucuron-
ides provides an opportunity to speculate on the identity of metab-
olites generated by recombinant UGTs. In previous studies [14,15],
recombinant hepatic UGT1A1 and extrahepatic UGT1A10 metabo-
lized R-7-hydroxywarfarin into distinctly different products based
on chromatographic properties and patterns of mass spectral sig-
nals. This information allowed the authors to conclude that the
enzymes generated monoglucuronides in the reaction but the spe-
cific site of modification could not be determined. Nevertheless,
those monoglucuronides were resolved chromatographically using

252
C. Preston Pugh et al. / Archives of Biochemistry and Biophysics 564 (2014) 244–253
the same HPLC column and mobile phase, as employed in our study,
and thus, the elution order of the metabolites would be conserved
between the studies. Analysis of the recombinant enzyme reactions
showed that the UGT1A10 product eluted before the UGT1A1 prod-
uct like metabolites 1 and 2 in our study. Based on this elution
order, UGT1A10 glucuronidates R-7-hydroxywarfarin to form
7-OHWAR-4-GLUC, while the UGT1A1 reaction produces
7-OHWAR-7-GLUC. Through this re-analysis of data employing
recombinant enzymes [14,15], we advance an understanding of
the stereoselectivity of possible hepatic (UGT1A1) and extrahepatic
(UGT1A10) metabolic pathways for R- and S-7-hydroxywarfarin
catalyzed by individual UGTs.
binding events for the same enzyme or different enzymes that col-
lectively contribute to formation of the 7-OHWAR-4-GLUC. In
either case, these metabolic pathways are far more inefficient than
glucuronidation at the C7 hydroxyl group for R- and S-7-hydroxy-
warfarin, such that the latter pathways dominate the metabolic
clearance of the warfarin metabolite.
We modeled the overall in vivo metabolic clearance of R- and
S-7-hydroxywarfarin taking into consideration the specific
mechanisms as described by others [33,34]. The major metabolic
pathway for R-7-hydroxywarfarin conformed to the traditional
Michaelis–Menten model for clearance. However, the other
metabolic pathways involved substrate inhibition mechanism that
predicted a more efficient clearance of the compounds at lower
concentrations. In other words, the traditional model would
underestimate 7-hydroxywarfarin metabolic clearance in those
cases. An important driver in this process is the concentration or
plasma level of 7-hydroxywarfarin in warfarin patients, which
have been reported to range from 6 to 50 nM for R-7-hydroxywar-
farin and 188 to 1140 nM for S-7-hydroxywarfarin [5,7,39]. The
amount accessible for glucuronidation is actually 20-fold lower
than these reported ranges, because only 4.49% of R-7-hydroxy-
warfarin and 4.27% of S-7-hydroxywarfarin are not bound to
plasma proteins [40]. Consequently, plasma levels of 7-hydoxy-
warfarin available for glucuronidation are sub-nanomolar for
R-7-hydroxywarfarin and mid to low nanomolar for S-7-hydroxy-
warfarin. These levels are at least a 1000-fold lower than the bind-
ing constants for the reaction reported in this study, and thus they
would not be sufficient to saturate the capacity of the liver to
glucuronidate R- and S-7-hydroxywarfarin under in vivo condi-
tions. Furthermore, the process of glucuronidation is not efficiently
coupled with the formation of 7-hydroxywarfarin by cytochromes
P450, because hydroxywarfarin metabolites accumulate in the
plasma. Their elimination may ultimately require glucuronidation
(or possibly sulfonation) and in fact, nearly all hydroxywarfarins
including those in this study are excreted in the urine as conju-
gates based on the analysis of a limited number of patient samples
[8,3].
The major microsomal pathway for hepatic glucuronidation of
R- and S-warfarin involves preferential conjugation at the C7 and
not the C4 hydroxyl group (Fig. 1). The C7 hydroxyl group is
located on the edge of a flat coumarin ring distal from the chiral
center possessing bulky substituents. This arrangement makes
the C7 hydroxyl group more accessible to glucuronidation and
likely explains the regiospecificity of the reaction. The chiral center
does influence the reaction mechanism and corresponding param-
eters though. Metabolism of R-7-hydroxywarfarin yields a hyper-
bolic kinetic profile as predicted by Michaelis–Menten kinetic for
a single active site enzyme [18]. By contrast, metabolism of
S-7-hydroxywarfarin demonstrated a kinetic profile consistent
with substrate inhibition. These types of non-hyperbolic kinetic
profile may occur when initial steady-state conditions are not
met during the course of the experiment. We eliminated those pos-
sibilities by measuring initial rates in the linear response range for
product formation as a function of time and protein concentration.
Moreover, product accumulation was not significant during the
course of the reaction (<20% of substrate concentration), suggest-
ing that the possibility of UDP inhibition of the reaction was
minimal [36]. This validation of the kinetic profile confirms the
observation of substrate inhibition for S-7-hydroxywarfarin
glucuronidation but not the mechanism underlying its occurrence.
The corresponding metabolic mechanism rests on the meaning
of the second binding event (Kss). The simplest and most common
explanation is the presence of two binding sites for the enzyme.
The first binding event results in a binary substrate-enzyme com-
plex that follows traditional Michaelis–Menten kinetics; however,
at higher substrate concentrations, a second binding event occurs,
whereby the activity of the enzyme decreases or is eliminated
altogether. This proposed mechanism has been used to explain
multiple UGT reactions [37] including UGT1A10 metabolism of
8-hydroxywarfarins [15]. Alternatively, at higher 7-hydroxywarfa-
rin concentrations, substrate may bind a different UGT present in
the microsomal fraction. The formation of that complex could alter
protein–protein interactions among the UGTs that govern activity
as reported for UGT1A1, 1A6, 1A9, and 2B7 (reviewed in [38]). In
this case, a less active UGT complex may supplant a more produc-
tive one at higher substrate concentrations. The distinction
between these possibilities would require further study. Regard-
less, R-7-hydroxywarfarin was more efficiently metabolized than
the S enantiomer to form the 7-OHWAR-7-GLUC due to higher
substrate affinities and rates of turnover.
Inhibitor phenotyping studies described herein demonstrated
that none of the hepatic UGTs dominated 7-hydroxywarfarin
metabolism. Multiple UGT1A enzymes as well as the possibility
of UGT2B7 contributed to glucuronidation of both hydroxywarfa-
rin enantiomers and the corresponding two glucuronides metabo-
lites. For comparative purposes, only kinetic parameters for
recombinant UGT1A1 have been reported for glucuronidation at
the C7 hydroxyl group [15]. The Km for the UGT1A1 reaction
toward R-7-hydroxywarfarin (130
one observed for the microsomal reaction (121
the values were very different for the S-7-hydroxywarfarin, i.e.
230 M versus 927 M, respectively. It is conceivable then that
lM) was comparable to the
lM); however,
l
l
UGT1A1 may play an important role in the metabolic clearance
of the R-7-hydroxywarfarin, but not the S enantiomer. Neverthe-
less, the demonstration of multiple UGTs metabolizing 7-hydroxy-
warfarin is plausible. These UGTs show significant activity toward
4-methylumbelliferone [30], i.e. 4-methyl-7-hydroxycoumarin,
which is isosteric for the portion of 7-hydroxywarfarin mainly tar-
geted for glucuronidation. Moreover, others have shown that
microsomal glucuronidation of R- and S-7-hydroxywarfarin corre-
lates with the activities of multiple UGT1A and 2B enzymes UGTs
[15]. The contributions of multiple UGTs to these metabolic reac-
tions indicate significant redundancy in glucuronidation pathways
such that variations in individual UGT activities may be compen-
sated by other UGT activities. This mechanism could explain failure
of genome-wide association studies [41–43] to identify any UGT
polymorphisms being linked to warfarin dose–responses in
patients, such that the clinical relevance of glucuronidation
remains unclear.
Hepatic glucuronidation at the C4 hydroxyl group was a minor
pathway for both 7-hydroxywarfarins. Presumably, the bulky sub-
stituents on the nearby chiral center sterically occlude access to
the site for glucuronidation to occur (Fig. 1). The orientation of
the substituents did impact metabolism as evidenced by a much
higher turnover rate for R-7-hydroxywarfarin relative to the S
enantiomer. The overall kinetic profile in both cases replicated
the substrate inhibition observed for glucuronidation of
S-7-hydroxywarfarin at the C7 hydroxyl group. In following, high
substrate concentrations induce
a shift in observed enzyme
activity for these pathways reflecting the contribution of two

C. Preston Pugh et al. / Archives of Biochemistry and Biophysics 564 (2014) 244–253
253
In sum, we report for the first time the mechanism and param-
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Financial support for undergraduate research was provided in
part by the Summer Undergraduate Research Fellowship program
sponsored by the Biochemistry and Molecular Biology Department
at the University of Arkansas for Medical Sciences (CPP and DLP),
the UAMS Summer Undergraduate Research Program to Increase
Diversity in Research under award number R25HL108825 from
the NIH National Heart, Lung and Blood Institute (National Heart,
Lung, and Blood Institute) (DLP), and the Arkansas INBRE program,
supported by Grant funding from the NIH National Institute of
General Medical Sciences (NIGMS) (P2003429, formerly
P20RR016460) (CPP). In addition, financial support was provided
to GPM through a Grant-in-Aid from the SouthWest Affiliate of
the American Heart Association (13GRNT16960043) and a pilot
study Grants provided by the NIH (UL1 TR000039) awarded to
the University of Arkansas for Medical Sciences awarded to GB
and GPM. The content of this publication is solely the responsibil-
ity of the authors and does not necessarily represent the official
views of the University of Arkansas for Medical Sciences, the Amer-
ican Heart Association, or National Institutes of Health.
TEG gratefully acknowledges funding from the Hendrix College
Odyssey and Rwanda Presidential Scholars Programs, as well as
from John and Laura Byrd. Funding was provided by the U.S.
National Science Foundation for the NMR spectrometer (NSF MRI
Grant to Hendrix College, Award No. 1040470). Nick Heathscott,
Doug Hammon, Jake Leffert, Kenyon Plummer, Robert Rurangwa,
and Aline Umuhire-Juru contributed to the early stages of the
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