Supplemental material to this article can be found at:
http://dmd.aspetjournals.org/content/suppl/2011/08/19/dmd.111.041418.DC1
0090-9556/11/3911-2117–2129$25.00
DRUG METABOLISM AND DISPOSITION
Copyright © 2011 by The American Society for Pharmacology and Experimental Therapeutics
DMD 39:2117–2129, 2011
Vol. 39, No. 11
41418/3726310
Printed in U.S.A.
Bovine Serum Albumin Decreases Km Values of Human
UDP-Glucuronosyltransferases 1A9 and 2B7 and Increases Vmax
Values of UGT1A9□S
Nenad Manevski, Paolo Svaluto Moreolo, Jari Yli-Kauhaluoma, and Moshe Finel
Division of Pharmaceutical Chemistry (N.M., J.Y.-K.) and Centre for Drug Research (N.M., P.S.M., M.F.), Faculty of Pharmacy,
University of Helsinki, Helsinki, Finland
Received June 28, 2011; accepted August 18, 2011
ABSTRACT:
namely lowering the Km value without a large effect on the enzyme’s Vmax value. The unexpected BSA effect on UGT1A9 was
independent of the expression system because it was found in a
recombinant enzyme that was expressed in baculovirus-infected
insect cells as well as in the native enzyme in human liver microsomes. Moreover, the effect of BSA on the kinetics of 4-methylumbelliferone glucuronidation by recombinant UGT1A9 was similar
to its effect on entacapone glucuronidation. Contrary to the aglycone substrates, the effect of BSA on the apparent Km of UGT1A9
for the cosubstrate UDP-␣-D-glucuronic acid was nonsignificant.
Our findings call for further investigations of the BSA effects on
different UGTs and the inhibitors that it may remove.
Introduction
various tissues, most notably liver, intestine, and kidney (Ohno and
Nakajin, 2009).
Because of partial overlaps in the substrate specificity of individual
UGTs and the expression of multiple isoforms in each tissue that
expresses these enzymes, in vitro studies on the UGTs and drug
glucuronidation are often performed using recombinant human UGTs
that are either expressed in insect cells (mainly Spodoptera frugiperda
Sf9 cells), or in human embryonic kidney (HEK) 293 cells (Radominska-Pandya et al., 2005). Enzyme kinetic constants (e.g., Km and
Vmax) and inhibition (IC50 or Ki) parameters of drug glucuronidation,
determined from in vitro assays, are commonly used to estimate the
extent of glucuronidation in vivo (Miners et al., 2010). Because there
is currently no good method to extract the UGTs from the membrane
and purify them as fully active enzymes, in vitro glucuronidation
assays are performed with different cell fractions rather than with
highly purified enzymes. In such systems, nonspecific substrate binding to the membrane and different proteins within it, as well as the
presence of inhibitors within the membrane, can lead to erroneous
estimation of UGT activity. The acquired errors may lead to erroneous
estimation of in vivo glucuronidation activity, namely poor in vivo-in
vitro extrapolation, significantly weakening the ability to predict the
pharmacokinetics properties of a drug under development, a problem
that was already faced in the case for many therapeutic drugs that are
Human UDP-glucuronosyltransferases (UGTs) play major roles in
the metabolic elimination of numerous endo- and xenobiotics. They
are membrane enzymes of the endoplasmic reticulum that catalyze
glucuronic acid transfer from the cosubstrate, UDP-␣-D-glucuronic
acid (UDPGA), to nucleophilic groups of chemically diverse substrates. There are 19 functional human UGTs and they are divided into
three subfamilies: 1A, 2A, and 2B (Mackenzie et al., 2005). Closely
related enzymes that use somewhat different nucleotide cosubstrates
have recently been discovered and assigned to subfamily 3A (MacKenzie et al., 2011), but they will not be further considered in this
work. Individual UGT isoforms have distinctive substrate and inhibitor selectivity (Miners et al., 2010) and are differentially expressed in
This study was supported by the Graduate School in Pharmaceutical Research, Academy of Finland (Project Number 120975); the Sigrid Juselius
Foundation; and a Helsinki University Research Foundation grant for young
researchers.
Article, publication date, and citation information can be found at
http://dmd.aspetjournals.org.
doi:10.1124/dmd.111.041418.
□
S The online version of this article (available at http://dmd.aspetjournals.org)
contains supplemental material.
ABBREVIATIONS: UGT, UDP-glucuronosyltransferase; AZT, zidovudine (3⬘-azido-3⬘-deoxythymidine); BSA, bovine serum albumin; 3D, threedimensional; HEK, human embryonic kidney; HLM, human liver microsomes; HPLC, high-performance liquid chromatography; 4-MU, 4-methylumbelliferone; NSBf, nonspecific binding to the filter device; UDPGA, UDP-␣-D-glucuronic acid; UPLC, ultraperformance liquid chromatography;
MeOH, methanol; fu, fraction unbound.
2117
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The human UDP-glucuronosyltransferase (UGT) enzymes UGT1A9
and UGT2B7 play important roles in the hepatic glucuronidation of
many drugs. The presence of bovine serum albumin (BSA) during in
vitro assays was recently reported to lower the Km values of both
these UGTs for their aglycone substrates without affecting the
corresponding Vmax values. Nonetheless, using the specific substrates entacapone and zidovudine (AZT) for UGT1A9 and UGT2B7,
respectively, and using an improved ultrafiltration method for measuring drug binding to BSA and to biological membranes, we found
that the presence of BSA during the glucuronidation reaction leads
to a large increase in the Vmax value of UGT1A9, in addition to
lowering its Km value. On the other hand, in the case of UGT2B7,
our results agree with the previously described effect of BSA,
2118
MANEVSKI ET AL.
Materials and Methods
Compounds and Reagents. 4-Methylumbelliferone (⬎99%, CAS 90-335), UDPGA (triammonium salt, 98 –100%, CAS 63700-19-6), alamethicin
(⬎90%, CAS 27061-78-5), 4-methylumbelliferone-␤-D-glucuronide (4-MU;
ⱖ98%, CAS 6160-80-1), zidovudine (3⬘-azido-3⬘-deoxy-thymidine, ⱖ98%,
CAS 30516-87-1), sodium phosphate monobasic dihydrate (ⱖ99%, CAS
13472-35-0), and BSA (ⱖ96%, CAS 9048-46-8, essentially fatty acid free,
⬍0.004%) were purchased from Sigma-Aldrich (St. Louis, MO). Entacapone
(batch 1044842) was a generous gift from Orion Corporation (Espoo, Finland).
Entacapone-␤-D-glucuronide was synthesized in our laboratory (Luukkanen et
al., 1999). Tween 20 (CAS 9005-64-5) and Tween 80 (CAS 9005-65-6) were
purchased from Acros Organics (Fairlawn, NJ). Magnesium chloride hexahydrate and perchloric acid were obtained from Merck (Darmstadt, Germany).
Formic acid (98 –100%) was from Riedel-deHaën (Seelze, Germany). Disodium hydrogen phosphate dihydrate was purchased from Fluka (Buchs, Switzerland). Radiolabeled [14C]UDPGA was acquired from PerkinElmer Life and
Analytical Sciences (Waltham, MA). High-performance liquid chromatography (HPLC)-grade solvents were used throughout the study.
Enzyme Sources. Recombinant human UGT2B7 and UGT1A9 were expressed as His-tagged proteins in baculovirus-infected Sf9 insect cells as
described previously (Kurkela et al., 2007). The collected cells were osmotically lysed and the suspension was centrifuged at 41,000g for 2 h. The
resulting pellets were homogenized, suspended in 25 mM Tris-HCl buffer (pH
7.5) and 0.5 mM EDTA and stored in aliquots at ⫺70°C until use (Kurkela et
al., 2003).
Control samples, insect cell membranes without any human UGT, were
prepared by infecting insect cells with baculovirus that does not encode any
human UGT and then treating the cells as described above. Pooled HLM (lot
18888) and recombinant UGT1A9 “supersomes” (lot 81661; expressed in Sf9
insect cells) were purchased from BD Gentest (Woburn, MA). Protein concentrations were determined by the BCA protein assay (Thermo Fisher Scientific, Waltham, MA).
Drug Binding Assays. We developed an ultrafiltration method to measure
the binding of AZT, entacapone, and 4-MU to BSA, control insect cell
membranes, and HLM. The technical component of the assay and basic
calculations to determine the drug fraction that is bound to the device are
described under Nonspecific Binding to the Filter Device and Filter Pretreatment, whereas the results for the different drugs and the determination of the
unbound drug fraction under different conditions are described under Results.
The filter devices for the drug binding assays were Amicon Ultra filters with
10-kDa Ultracel regenerated cellulose membrane, 500 ␮l volume, and they
were purchased from Millipore Corporation (Billerica, MA).
Nonspecific Binding to the Filter Device and Filter Pretreatment. A
sample of the compound in phosphate buffer (500 ␮l solution, 50 mM, pH 7.4)
was transferred to a filter device and centrifuged twice at 2500g, 1 min each
time, to collect two separate 50-␮l filtrate fractions. In total, a maximal volume
of 100 ␮l was allowed to pass through the filter, namely ⱕ20% of the total
loaded volume. The first filtrate sample was removed, and a 30-␮l aliquot from
the second 50-␮l filtrate fraction, as well as a similar sized sample from the
prefiltered solution, were collected, each mixed with 60 ␮l of 4 M perchloric
acid/methanol (MeOH) (1:5 mix) and submitted to ultra-performance liquid
chromatography (UPLC) analysis. The experiments were performed in triplicate, and the nonspecific binding to the filter device was calculated using the
following equation:
NSBf ⫽
[S]prefilter ⫺ [S]filtrate
[S]prefilter
in which [S] is the substrate concentration in the respective solutions.
The nonspecific binding to the filter device (NSBf) was significantly lowered by the following pretreatment: filter device wash twice with 400 ␮l of 1%
Tween 20 and removal of the remaining detergent solution by 5 min of
centrifugation at 5000g and a subsequent wash with 500 ␮l of phosphate buffer
(50 mM, pH 7.4).
The integrity of the filter device membrane was tested after the pretreatment
by filling the device with 450 ␮l of either 2% BSA solution or 1 mg/ml of
control insect cell membranes, centrifuging for 10 min at 5000g, transfer of a
200-␮l aliquot of the resulting filtrate to a 1.5 ml-centrifuge tube, acidification
of the sample with 20 ␮l of 4 M perchloric acid, transferring to ice for 20 min,
centrifuging for 10 min at 16,000g, and visually inspecting the centrifuge tube
for protein precipitates.
Determination of Substrate Binding to BSA, HLM, and Control Insect
Cell Membranes. The substrate of interest was first incubated with BSA,
HLM, or insect cell membranes in phosphate buffer (50 mM, pH 7.4) in a total
volume of 500 ␮l for 60 min at 37°C. The solution was then transferred to the
filter device and centrifuged twice for 1 min at 2500g. In total, a maximal
volume of 100 ␮l was allowed to pass through the filter (ⱕ20% of total
volume). The 30-␮l aliquots from the second filtrate fraction and from the
prefiltered solution were each mixed with 60 ␮l of 4 M perchloric acid/MeOH
(1:5 mix), transferred to ice for 20 min, centrifuged for 10 min at 16,000g, and
then submitted to substrate concentration determination by a UPLC analysis.
The experiments were performed in triplicates.
Drug Glucuronidation Assays. Stock solutions of AZT, entacapone, and
4-MU were prepared in methanol and diluted with methanol to the desired
concentrations immediately before use. Appropriate amounts of these dilutions
were transferred into 1.5-ml centrifuge tubes and the solvent was evaporated in
vacuo at ambient temperature. The solid residues were dissolved in the reaction
mixture containing phosphate buffer (50 mM, pH 7.4), MgCl2 (10 mM), BSA
(0 –2%), and an enzyme source (0.02– 0.2 mg/ml total protein in the membrane, depending on the enzyme source) to a final volume of 100 ␮l.
Downloaded from dmd.aspetjournals.org at ASPET Journals on July 31, 2017
eliminated by glucuronidation (Boase and Miners, 2002; Kilford et al.,
2009; Raungrut et al., 2010). Hence, there is a clear need for better
understanding of factors that affect the outcome of UGT assays in
vitro.
In a series of studies, it was found that adding purified bovine
serum albumin (BSA) to assays with several recombinant human
UGTs that were expressed in HEK293 cells, or to the native enzymes
in human liver microsomes (HLM), can significantly decrease the Km
for drugs that are glucuronidated by UGT1A9 and UGT2B7 without
affecting the reaction Vmax (Uchaipichat et al., 2006; Rowland et al.,
2007, 2008). These authors suggested that long-chain fatty acids (e.g.,
oleic, linoleic, and arachidonic acid) competitively inhibit the UGTs,
and that BSA addition reversed that inhibition by binding the inhibitory fatty acids (Rowland et al., 2007). Because similar inhibition was
not observed in cultured hepatocytes (Engtrakul et al., 2005), the
authors speculate that the inhibitory fatty acids are released during
microsome preparation from either human liver or the cells that were
used for recombinant UGT expression.
Raungrut et al. (2010) have recently studied the effect of BSA on
codeine glucuronidation by the recombinant UGT2B4 and UGT2B7
that were expressed in insect cells. However, thus far, this is the only
study that examined the BSA effect on UGTs that were expressed in
insect cells, even if most of the research on different aspects of the
UGTs is currently conducted using such recombinant enzymes because the commercial UGTs are expressed in insect cells. There are
differences in lipid composition between the HEK293 cells, insect
cells, and HLM (Marheineke et al., 1998), but it is unclear if these
differences are “translated” into differences in the BSA effect on
individual UGTs. Our original goal was to investigate the effect of
BSA on the activities of recombinant UGTs that were expressed in
insect cells. As reported below, the obtained results led us also to
reexamine the earlier reports about the native UGT enzymes in HLM,
particularly the BSA effect on UGT2B7 and UGT1A9. Although the
BSA effect does not appear to be dependent on the expression system,
the new findings should raise general awareness about factors that can
influence UGT assays in vitro and the complexity of the BSA effect.
They may also be instrumental for better understanding of the glucuronidation reaction mechanism and how it may be inhibited as well
as for better predictability of the in vitro assays.
2119
ALBUMIN EFFECT IN UGT2B7 AND UGT1A9
Glucuronidation activities are reported as the average and S.E. of at least
three replicate determinations. Please note that because of the lack of suitable
isoform-specific anti-UGT antibodies, the glucuronidation rates and Vmax
values of the different recombinant UGTs and HLM cannot be compared
directly.
Enzyme Kinetic Analyses. The protein concentrations and incubation
times for the kinetic analyses reactions were selected based on preliminary
assays to ensure that product formation was within the linear range with
respect to protein concentration and time, and that the substrate consumption
during the reaction was less than 10%. The substrate concentration ranges for
AZT, entacapone, and 4-MU enzyme kinetic experiments were 50 to 2000, 5
to 750, and 5 to 500 ␮M, respectively. The UDPGA enzyme kinetic assays
were performed with either 75 ␮M entacapone or 50 ␮M 4-MU as the
aglycone substrate. The incubation times varied from 15 to 60 min.
The enzyme kinetic parameters were obtained by fitting kinetic models to
the experimental data using GraphPad Prism version 5.01 for Windows
(GraphPad Software Inc., San Diego, CA). The best model was selected based
on the corrected Akaike’s information criterion, the calculated r2 values,
residuals graph, parameter S.E. estimates, 95% confidence intervals, and visual
inspection of the Eadie-Hofstee plots. In assays containing BSA, the free
substrate concentrations (fu, or fraction unbound), were corrected according to
the estimated drug binding to BSA under the specific conditions of each
glucuronidation assay. Data were fitted with the following models:
Michaelis-Menten equation
v⫽
V max[S]
K m ⫹ [S]
where v is the initial velocity of the enzyme reaction, Vmax is the maximal
velocity, [S] is the substrate concentration, and Km is the Michaelis-Menten
constant (concentration of substrate at 0.5 of Vmax).
Substrate inhibition equation
V max[S]
冉
v⫽
K m ⫹ [S] 1 ⫹
[S]
Ki
冊
where Ki is the constant describing the substrate inhibition interaction.
Allosteric sigmoidal model (Hill equation)
v⫽
V max[S]h
S h50 ⫹ [S]h
where S50 is the concentration of substrate at 0.5 of Vmax (analogous to Km in
the Michaelis-Menten model) and h is the Hill coefficient.
Two-site biphasic model equation (Korzekwa et al., 1998)
v⫽
V max1[S] ⫹ CLint[S]2
K m1 ⫹ [S]
where Vmax1 and Km1 are estimated from the curved portion of the plot at
lower substrate concentrations. The CLint represents the ratio of Vmax2/Km2
and describes the linear portion of the plot exhibited at higher substrate
concentrations.
Two-sites model equation (Houston and Kenworthy, 2000)
冉
冊
[S] ␤[S]2
⫹
Ks
␣K s2
v⫽
2[S] [S]2
1⫹
⫹
Ks
␣K s2
V max
where Ks is a substrate dissociation constant, ␣ describes the change in
substrate binding affinity for the second enzyme site, and ␤ describes the
change in rate of product formation from the substrate-enzyme-substrate (S 䡠
E 䡠 S) complex compared with the enzyme-substrate (E 䡠 S) complex.
Results
Drug Binding to the Filter Device, BSA and Insect Cell Membranes, and HLM. A prerequisite for correct interpretation of the
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The reaction mixtures for the HLM assays also contained alamethicin at a
final concentration of 5% of the microsomal protein concentrations, and they
were placed on ice for 30 min (Fisher et al., 2000) before continuing as with
the recombinant UGT-containing samples. Alamethicin was not added to the
incubations with recombinant UGTs because it has no significant effect on the
glucuronidation activity of such samples (Zhang et al., 2011).
The reaction mixtures (in the case of HLM, after the preincubation with
alamethicin) were incubated first for 30 min at room temperature, followed by
5 min at 37°C, initiated by the addition of UDPGA to a final concentration of
5 mM, and performed at 37°C (15– 60 min) protected from light. Negative
controls, including without UDPGA, without substrate, or with control
(“empty”) insect cell membranes, were performed for each set of assays. The
4-MU glucuronidation reactions were terminated by the addition of 10 ␮l of
ice-cold perchloric acid (4 M). In the cases of AZT and entacapone, the
reactions were terminated by the addition of 60 ␮l of ice-cold 4 M perchloric
acid/MeOH (1:5 mix). After reaction termination, the tubes were transferred to
ice for 30 min and then centrifuged at 16,000g for 10 min. Aliquots of the
resulting supernatants were transferred to dark glass vials and subjected to
HPLC or UPLC analyses.
Analytical Methods. The HPLC system consisted of an Agilent 1100 series
degasser, binary pump, 100-vial autosampler, thermostated column compartment, multiple wavelengths UV detector, and fluorescence detector (Agilent
Technologies, Santa Clara, CA). The resulting chromatograms were analyzed
with Agilent ChemStation software (revision B.01.01) on Windows XP Professional software (Microsoft, Redmond, WA). For separation and detection of
4-MU-␤-D-glucuronide, we used a Chromolith SpeedROD RP-18e (50 ⫻ 4.6
mm, 3 ␮m; Merck) column (at a column temperature of 40°C and injection
volume of 20 ␮l). The mobile phase consisted of 80% 50 mM phosphate
buffer, pH 3 (A) and 20% methanol (B) at a constant flow rate of 2 ml/min. A
fluorescence detector with an excitation wavelength of 316 nm and emission
wavelength of 82 nm was used for detection. The retention time of 4-MU-␤D-glucuronide under these conditions was 1.45 min. The quantification was
based on a standard curve prepared using an authentic glucuronide standard.
The UPLC system consisted of a Waters Acquity UPLC (Waters, Milford,
MA) system equipped with an Acquity UPLC BEH C18 column (2.1 ⫻ 100
mm, 1.7 ␮m; Waters) and a precolumn (column temperature of 40°C), column
manager, sample manager, binary solvent pump, and photodiode array UV
detector. The UV detector was equipped with high-sensitivity 2.4-␮l flow cell.
The resulting chromatograms were analyzed with Empower 2 software (Build
2154; Waters) on a Windows XP Professional operating system. We developed
UPLC methods to separate and detect zidovudine-␤-D-glucuronide and entacapone-␤-D-glucuronide on the basis of their UV absorbance as well as
zidovudine, entacapone, and 4-MU substrates for analyses of the binding assay
results. The injection volume was 10 ␮l for all samples.
For the separation of AZT-␤-D-glucuronide, the mobile phase consisted of
0.1% formic acid (A) and acetonitrile (B) and the flow rate was 0.6 ml/min.
UV absorbance at 267 nm was used for detection. The gradient in this method
was as follows: 0 to 1 min of 5% B, 1 to 4 min of 5 to 30% B, 4.5 to 5 min
of 30 to 80% B, and 5 to 6 min of 5% B. The AZT-␤-D-glucuronide retention
time was 2.40 min. The quantification of AZT-␤-D-glucuronide was based on
a standard curve constructed using the UV absorption of zidovudine.
For the separation of entacapone-␤-D-glucuronide, the mobile phase consisted of 50 mM phosphate buffer, pH 3 (A), and acetonitrile (B), and the flow
rate was 0.5 ml/min. UV absorbance at 309 nm was used for detection, and the
quantification was done using a standard curve made with an authentic glucuronide standard. The gradient in this method was as follows: 0 to 3 min of 20
to 30% B, 3 to 3.2 min of 30 to 80% B, 3.2 to 4 min of 80% B, 4 to 4.1 min
of 80 to 20% B, and 4.1 to 6 min of 20% B. The entacapone-␤-D-glucuronide
retention time was 2.18 min.
A single UPLC method was developed for separation and analysis of
AZT, entacapone, and 4-MU in drug binding assays. The mobile phase
consisted of 50 mM phosphate buffer, pH 3 (A), and acetonitrile (B), and
the flow rate was 0.6 ml/min throughout. UV absorbance at 267, 309, and
321 nm was used for detection of AZT, entacapone, and 4-MU, respectively. The run was isocratic with 35% B for 1.5 min. The retention times
of AZT, entacapone, and 4-MU and were 0.72, 0.87, and 1.32 min,
respectively. The quantification was based on standard curves prepared
with the respective compounds.
2120
MANEVSKI ET AL.
We further examined the effect of drug concentration on its NSBf
and found that for entacapone (the most lipophilic compound among
the tested drugs in this study) it is saturable in nature and exponentially decreases with increasing entacapone concentration (Fig. 1A,
inset). To obtain a good estimation of the NSBf of entacapone at any
given entacapone concentration, the determined binding values were
fitted to the following exponential decay (empirical) equation:
NSBf ⫽ (NSBf-max ⫺ NSBf-min)e ⫺ k[S] ⫹ NSBf-min
where NSBf-max is the maximal measured NSBf, NSBf-min is the
minimal measured NSBf at the plateau region, k is the exponential
decay rate constant, and [S] is the concentration of entacapone.
Unlike entacapone, the NSBf of 4-MU was essentially concentration
independent. Because of this, for calculating its fu, we took the mean
value of the obtained data points (fu ⫽ 7%). The NSBf of AZT to
pretreated filter devices was negligible.
After the clarification of the NSBf and its dependence on drug
concentration, we turned to the determination of drug binding to BSA.
A Entacapone binding to 0.1% BSA
1.0
0
4
0.4
06
0.6
NSB f (entacapone)
fu (Entacapo
one)
0.8
0.4
0.2
Entacapone nonspecific binding
g
to filter (NSBf)
0.3
0.2
0.1
0.0
0
0.0
0
100
200
200
400
600
[Entacapone], µM
300 400 500
[Entacapone], µM
600
800
700
800
B 4-MU binding to 0.1 and 1% BSA
1.0
0.5
NSBf (4-M
MU)
fu (4-MU))
0.8
0.6
4-MU nonspecific binding
to filter (NSBf)
0.4
03
0.3
0.2
0.1
0.4
0.0
0
100
200 300
[4-MU], µM
400
500
0.2
0.0
fu at 0.1% BSA
fu at 1% BSA
0
100
200
300
[4-MU], µM
400
500
FIG. 1. Binding of entacapone to 0.1% BSA (A) and binding of
4-MU to 0.1 and 1% BSA (B). The NSBf of entacapone and 4-MU
is presented as figure insets. The results are presented as fu and
represent mean ⫾ S.E. (n ⫽ 3). The entacapone binding data were
fitted to a empirical hyperbolical equation; the entacapone NSBf
data were fitted with an empirical exponential decay equation;
4-MU binding data to 0.1 and 1% BSA were fitted with empirical
exponential association equation and linear equation, respectively;
and the 4-MU NSBf data were fitted with linear equation (see
Materials and Methods for all details).
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BSA effect on enzyme kinetics is being able to determine the concentration of free substrate (the drug) in the presence of BSA, the
so-called fraction unbound (fu). Rowland et al. (2007) have used an
equilibrium dialysis device for this, and in the current study, in the
absence of such an instrument, we developed an ultrafiltration
method. To obtain values of fu that are as accurate as possible, we took
into consideration the amount of drug that binds to the filter device
even in the absence of BSA, the so-called nonspecific binding (NSBf).
The method is detailed in the Materials and Methods, and examples
of the obtained results with an untreated filter device for 20 ␮M AZT,
entacapone, and 4-MU solutions are NSBf values of 20, 99, and 40%,
respectively. In an effort to decrease the NSBf, we tested several ways
to decrease that nonspecific binding by a suitable filter pretreatment.
The best results were achieved by a double wash with a solution of the
mild detergent Tween 20, 1% final concentration, followed by phosphate buffer rinse, as described under Materials and Methods. This
pretreatment reduced the NSBf of 20 ␮M AZT, entacapone, and 4-MU
solutions to the acceptable values of 1, 27, and 7%, respectively.
ALBUMIN EFFECT IN UGT2B7 AND UGT1A9
To avoid impractically high fu values, particularly for entacapone, we
tested the effects on enzyme kinetics of lower BSA concentrations
than 2%, the value used in the previous studies (Rowland et al., 2007,
2008). It turned out that the presence of as low as 0.1% BSA in the
reaction mixture was sufficient to yield the stimulatory effect (see
below) and, therefore, for entacapone we mainly used 0.1% BSA. In
the case of 4-MU, the kinetic experiments were performed in the
presence of either 0.1 or 1% BSA, whereas 1% BSA was used for the
AZT studies to make them more comparable to the previously published studies.
The binding of AZT, entacapone, and 4-MU to BSA and the
different biological membranes (enzyme sources) was studied in the
concentration ranges of 5 to 2000, 5 to 750, and 5 to 500 ␮M,
respectively. The fu was calculated by the following equation:
fu ⫽
[S]filtrate
[S]prefilter(1 ⫺ NSBf)
fu ⫽
f u-max[S]
K s ⫹ [S]
where fu-max is the maximal fraction unbound achieved in the
presence of an unlimited amount of entacapone and Ks is the
concentration of entacapone at half fu-max. The binding of entacapone to 1% BSA was also tested, but because it was excessive and
the estimated fu was well below 1% at low entacapone concentrations (data not shown), all subsequent experiments on the effect of
BSA on entacapone glucuronidation were performed in the presence of 0.1% BSA.
The fu of 4-MU in the presence of 0.1% BSA increased curvilinearly with increasing 4-MU concentration, and the results were
well described by the following empirical equation of exponential
association:
f u ⫽ f u-min ⫹ (f u-max ⫺ f u-min)(1 ⫺ e ⫺k[S])
In this equation, fu-min is the minimal measured fraction unbound,
fu-max is the maximal fu, k is an association rate constant, and [S] is the
concentration of 4-MU. The fu of 4-MU at 1% BSA increased linearly
with increasing 4-MU concentration.
The binding of AZT to either 0.1 or 1% BSA was negligible
(ⱕ1%), a result that is in good agreement with the previously reported
binding properties of this drug (Rowland et al., 2007).
The dependence of entacapone and 4-MU binding to BSA may also
be presented in the form of a binding plot (supplemental text and
Supplemental Fig. 1). These analyses revealed that the binding of
entacapone and, to a lesser extent, of 4-MU is biphasic, suggesting
that two or more binding sites on albumin may be involved. Because
of this finding, we further tested the binding of entacapone to 0.1%
BSA in the presence of 4-MU. However, the results indicated that the
fu of entacapone is only modestly increased in the presence of 4-MU,
suggesting minor binding competition between the two substrates
(Supplemental Fig. 2).
The binding of AZT, entacapone, and 4-MU to the biological
membranes that carry the tested UGTs was tested using HLM and
control insect cell membranes. The results indicated that up to 0.2
mg/ml (total protein), the highest protein concentration used in this work,
the binding of the three tested drugs is very low. However, it was noted
that binding of entacapone to higher than 0.5 mg/ml enzyme source was
considerable and should be taken into account if such high concentrations
of an enzyme source are used (data not shown).
We also studied drug binding to BSA in the presence of either
insect cell membranes or HLM. It was surprising to note that the
results indicated that the presence of high amounts of an enzyme
source lowers entacapone binding to 0.1% BSA, whereas 4-MU
binding to either 0.1 or 1% BSA was not significantly affected by the
presence of insect cell membranes. Although the effect of membrane
presence on entacapone binding to BSA was not large, it might
be significant when more membranes (enzyme source) are added to
the reaction mixture. We thus interpolated an empirical three-dimensional (3D) function over the experimental data points (Fig. 2B) that
enables estimation of entacapone binding to 0.1% BSA at any given
concentration of entacapone and enzyme source used during an in
vitro assay. Scatchard plots for the binding of entacapone for either
BSA or insect cell membranes, as well as to different combinations of
the two, were also drawn from the data presented in Fig. 2. They show
that although the character of entacapone binding to BSA remains
biphasic, the addition of enzyme source decreases the apparent affinity of entacapone for albumin in a concentration-dependent manner
(Supplemental Fig. 3).
We also tested if other reaction components, such as MgCl2 and
UDPGA, affect drug binding. The results indicated that neither MgCl2
nor UDPGA significantly changes the binding of all of the tested
compounds to BSA or the enzyme sources.
Enzyme Kinetics of AZT Glucuronidation with HLM and
UGT2B7. After all of the needed experiments and analyses that are
described above, we turned to the UGTs and the effect of the presence
of BSA on their kinetics. The studies on the effect of 1% BSA on the
AZT glucuronidation kinetics by HLM and recombinant UGT2B7,
expressed in insect cells, are shown in Fig. 3, and the derived kinetic
parameters are presented in Table 1. AZT glucuronidation by HLM
and UGT2B7 is best described by the Michaelis-Menten model, and
the addition of 1% BSA significantly decreased the Km values for both
enzyme sources, without affecting the respective Vmax values (the
latter values differ from each other because a different enzyme source
was used, and we do not have a good way to determine the UGT2B7
concentration within each membrane sample). A further increase of
the BSA concentration to 2% did not significantly affect the kinetic
parameters. The obtained kinetic parameter values and the effect of
BSA addition on them are in good agreement with previous results
with locally made HLMs and recombinant UGT2B7 that was expressed in a different system than the one used in this study, HEK293
cells (Uchaipichat et al., 2006; Rowland et al., 2007).
Enzyme Kinetics of Entacapone with HLM and UGT1A9. Entacapone is a nearly selective UGT1A9 substrate in the liver (Lautala et
al., 2000), and we used it as a model compound for studying the effect of
BSA on either recombinant UGT1A9 or the native enzyme in HLMs.
Because the preliminary results were significantly different from the
previously published finding for this enzyme (Rowland et al., 2008), we
performed these studies using two different preparations of recombinant
UGT1A9, the one from our laboratory that carry a short C-terminal fusion
peptide (Kurkela et al., 2003) and the commercial UGT1A9 Supersomes
that lack such a fusion peptide. It may be added here that the concentration of active UGT1A9 in the Supersomes sample appears to be significantly higher than in our recombinant UGT1A9 sample, but, as shown
below, the differences between the two preparations do not much affect
their kinetics.
Downloaded from dmd.aspetjournals.org at ASPET Journals on July 31, 2017
where [S]filtrate is the drug concentration in filtrate, [S]prefilter is its
concentration in the prefiltered solution, and NSBf is the nonspecific
binding to the filter in the absence of BSA and/or an enzyme source.
The fu of entacapone in the presence of 0.1% BSA increased
hyperbolically with increasing entacapone concentration and, therefore, the obtained values were fitted to the following empirical equation that appears to provide a good description of the results:
2121
2122
MANEVSKI ET AL.
A Entacapone binding to 0.1% BSA and membrane
10
1.0
1.0
0.6
fu (Entacapone)
apone)
fu (Entaca
0.8
0.4
0.2
0.8
0.6
0.4
0.2
0.0
0.00
0.0
0.05
0.10
0.15
0.20
[Control membrane], mg/mL
100
200
300 400 500
[Entacapone], µM
600
700
B Entacapone binding to 0.1% BSA and membrane
(3D plot)
f u (Entacapone)
1.0
FIG. 2. Binding of entacapone to 0.1% BSA without Sf9 control
membrane (F), and in the presence of 0.032 (Œ), 0.080 (), and
0.16 (⽧) mg/ml of Sf9 control membrane (A). The results are
presented as fu and represent mean of 3 determinations. The S.E.
was very small and for the sake of clarity, in this condensed figure
the S.E. bars were left out. The data were fitted to an empirical
hyperbolical equation. The correlation between measured fu and Sf9
control membrane concentration at different entacapone concentrations is presented as the inset in panel A. An identical set of data are
presented in the form of a 3D scatter (B). An empirical 3D function
was fitted to data points (see supplemental material for all details).
0.8
0.6
0.4
0.2
0.0
[C
on
tro 0.10
l m 0.05
m em
g/
m bra 0.00
L
ne
],
600
0
800
400
M
], µ
200
e
n
po
a ca
t
n
[E
The clear and unexpected result from the experiments with all three
different UGT1A9 samples was that in addition to decreasing the
reaction Km value, the presence of 0.1% BSA in the reaction mixture
led to a large increase in the three respective Vmax values (Fig. 4;
Table 1). Mild substrate inhibition was observed at higher entacapone
concentrations, and the obtained Ki values were 75- to 1500-fold
higher than the respective Km values (Table 1). Because of the large
differences between the Km values and the respective Ki values, the
obtained Vmax values were not significantly influenced by the Km/Ki
ratio and were not largely underestimated as they would have been if
the Km/Ki ratio was closer to 1. The presence of BSA also somewhat
changes the apparent enzyme kinetic model of entacapone glucuronidation from mild substrate inhibition to partial substrate inhibition.
The latter means that on the basis of the expectation from the substrate
inhibition model, the inclusion of BSA led to a less-than-expected
decrease in the glucuronidation rate in the presence of high entacapone concentrations. Therefore, in addition to the empirical substrate
inhibition equation, we fitted the experimental data to a mechanistic
two-site model equation (Houston and Kenworthy, 2000). This model
assumes the existence of two identical substrate binding sites and can
be used for sigmoidal and substrate inhibition kinetics. But because no
autoactivation was observed, we constrained parameter ␣ to 1 and
used parameter ␤ to describe the changes in the rate of product
formation from the S 䡠 E 䡠 S complex in comparison to its formation
from an E 䡠 S complex (Table 1).
Enzyme Kinetics of 4-MU Glucuronidation by UGT1A9. To
further explore the effect of BSA on the Vmax of UGT1A9 in entacapone glucuronidation (Fig. 4; Table 1) and find out if this is only a
Downloaded from dmd.aspetjournals.org at ASPET Journals on July 31, 2017
0
2123
ALBUMIN EFFECT IN UGT2B7 AND UGT1A9
03
0.3
0.5
0.2
V, nmol/min/mg
V, nmol/mi n/mg
A AZT, HLM, no BSA and 1% BSA
0.5
1% BSA
no BSA
0.4
0.1
0.4
0.3
0.2
0.1
00
0.0
0.000
0.0
500
1000
[AZT], µM
0.002 0.003
V/[AZT]
1500
0.004
2000
03
0.3
0.5
V, nmol/min/mg
V, nmol/min
n/mg
B AZT,, UGT2B7,, no BSA and 1% BSA
0.5
1% BSA
no BSA
0.4
FIG. 3. Enzyme kinetics of AZT glucuronidation by HLM (A) and
UGT2B7 (B) without BSA and in the presence of 1% BSA. The
points represent an average of three samples ⫾ S.E. Glucuronidation rates are presented as actual (measured) rates in nmol 䡠 min⫺1 䡠
mg⫺1 recombinant protein. The derived kinetic constants are presented in Table 1. The data were fitted to the Michaelis-Menten
equation. The Eadie-Hofstee transforms of the data are presented as
insets.
0.2
0.1
0.4
0.3
0.2
0.1
0.0
0.000
0.0
0
500
1000
[AZT], µM
0.001
V/[AZT]
1500
peculiarity of this substrate, we examined the effect of BSA addition
on the glucuronidation of 4-MU by UGT1A9. Because 4-MU is not a
UGT1A9-specific substrate, the 4-MU glucuronidation assays with
UGT1A9 were limited to the recombinant enzymes. One reason for
the selection of 4-MU as the second test substrate for UGT1A9 is that
its use enables a direct comparison of our results with the previous
study on the effect of BSA on UGT1A9 (Rowland et al., 2008). The
examination of the two different samples of recombinant UGT1A9
that we tested (the locally made and the commercial sample) most
clearly showed that the addition of BSA to either 0.1 or 1% resulted
in a significant Km decrease and a concomitant increase in the Vmax
values, with a slightly larger effect on the Vmax by 1% BSA than by
0.1% (Fig. 5; Table 1). A detailed kinetic analysis revealed that 4-MU
glucuronidation by UGT1A9 also follows the substrate inhibition
equation in the absence and the presence of BSA. It is important to
note that, as in the case of entacapone, the Ki values of UGT1A9 for
4-MU were significantly higher than the corresponding Km values,
allowing for correct determination of Vmax from the experimental data
(Table 1).
0.002
2000
Enzyme Kinetics for the Cosubstrate UDPGA with UGT1A9.
We also explored the possibility that the presence of BSA affects the
kinetics of UGT1A9 with the cosubstrate UDPGA. In these assays, we
used either entacapone or 4-MU as the aglycone substrate, and the
results show that, regardless of the aglycone used, the reaction was
best described by the Michaelis-Menten model (Fig. 6; Table 1). The
inclusion of 0.1% BSA when entacapone was the aglycone substrate
resulted in a Vmax increase without a Km change (Fig. 6A). However,
when 4-MU was the aglycone substrate, the addition of 0.1% BSA led
to a Km decrease and a Vmax increase (Fig. 6B; Table 1). The reason
for these differences is currently unclear and should be examined in
the future.
Discussion
Drug glucuronidation rates and kinetics that are determined using in
vitro assays tend to underestimate the in vivo rates (Boase and Miners,
2002; Miners et al., 2006). Rowland et al. (2007, 2008) have found
that the addition of BSA significantly enhances the activity of
UGT2B7 and UGT1A9 by decreasing their Km for the aglycone
Downloaded from dmd.aspetjournals.org at ASPET Journals on July 31, 2017
0
0.001
TABLE 1
Enzyme Source
UGT1A9
(UDPGA kinetics)
UGT1A9 Supersomes
UGT1A9
nmol 䡠 min⫺1 䡠 mg⫺1g
0.41 ⫾ 0.01
0.36 ⫾ 0.01
7.13 ⫾ 0.38
15.73 ⫾ 1.38
8.35 ⫾ 0.31
18.78 ⫾ 1.29
5.83 ⫾ 0.16
12.44 ⫾ 0.60
29.24 ⫾ 2.02
1.92 ⫾ 0.06
5.12 ⫾ 0.17
1.34 ⫾ 0.04
6.19 ⫾ 0.21
16.26 ⫾ 0.95
␮M
Ks ⫽ 66.27 ⫾ 11.25
18.63 ⫾ 2.05
Ks ⫽ 50.03 ⫾ 6.84
257.7 ⫾ 27.8
13.74 ⫾ 1.95
Ks ⫽ 39.12 ⫾ 4.97
11.77 ⫾ 1.25
Ks ⫽ 43.67 ⫾ 3.51
180.1 ⫾ 21.6
9.03 ⫾ 0.96
Ks ⫽ 32.06 ⫾ 3.76
Vmax
586.1 ⫾ 23.6
381.2 ⫾ 13.9
25.56 ⫾ 3.90
Km
No BSA (Control)
␮M
Km
SI (0.94)
Ki ⫽ 780 ⫾ 118
TSI (0.88)
␤ ⫽ 0.22 ⫾ 0.03
SI (0.94)
Ki ⫽ 1494 ⫾ 454
TSI (0.95)
␤ ⫽ 0.29 ⫾ 0.04
SI (0.93)
Ki ⫽ 984 ⫾ 111
TSI (0.91)
␤ ⫽ 0.19 ⫾ 0.02
MM (0.94)
Ks ⫽ 15.31 ⫾ 1.40
2.94 ⫾ 0.32
105.8 ⫾ 8.7
Ks ⫽ 18.31 ⫾ 1.41
2.63 ⫾ 0.43
Ks ⫽ 16.49 ⫾ 1.31
5.41 ⫾ 0.40
MM (0.99)
MM (0.99)
SI (0.90)
1.26 ⫾ 0.16
Ki ⫽ 1919 ⫾ 547
Ks ⫽ 6.11 ⫾ 0.34
TSI (0.88)
␤ ⫽ 0.303 ⫾ 0.050
SI (0.94)
2.37 ⫾ 0.26
Ki ⫽ 1789 ⫾ 334
Ks ⫽ 8.85 ⫾ 0.51
TSI (0.90)
␤ ⫽ 0.30 ⫾ 0.03
MM (0.96)
247.4 ⫾ 12.9
Kinetic Model
(r2)/Ki (␮M) or ␤
37.71 ⫾ 1.53
12.25 ⫾ 0.35
2.66 ⫾ 0.04
9.10 ⫾ 0.35
3.69 ⫾ 0.11
80.12 ⫾ 3.14
33.41 ⫾ 0.66
15.06 ⫾ 0.20
44.97 ⫾ 1.30
16.95 ⫾ 0.43
36.6 ⫾ 0.96
12.49 ⫾ 0.29
nmol 䡠 min⫺1 䡠 mg⫺1
Vmax
0.1% BSA
0.46 ⫾ 0.01
0.45 ⫾ 0.01
SI (0.94)
Ki ⫽ 348 ⫾ 35
TSI (0.85)
␤ ⫽ 0.15 ⫾ 0.02
13.60 ⫾ 0.18
35.86 ⫾ 1.05
1.93 ⫾ 0.08
Ks ⫽ 6.81 ⫾ 0.40
13.45 ⫾ 0.30
4.77 ⫾ 0.13
nmol 䡠 min⫺1 䡠 mg⫺1
␮M
Vmax
1% BSA
76.71 ⫾ 1.53
134.5 ⫾ 7.74
Km
SI (0.99)
Ki ⫽ 1301 ⫾ 191
TSI (0.99)
␤ ⫽ 0.32 ⫾ 0.02
SI (0.96)
2.87 ⫾ 0.25
Ki ⫽ 328 ⫾ 30
Ks ⫽ 10.95 ⫾ 0.65
TSI (0.88)
␤ ⫽ 0.15 ⫾ 0.02
MM (0.96)
SI (0.87)
Ki ⫽ 1898 ⫾ 404
TSI (0.97)
␤ ⫽ 0.27 ⫾ 0.01
SI (0.94)
Ki ⫽ 2116 ⫾ 456
TSI (0.98)
␤ ⫽ 0.30 ⫾ 0.01
MM (0.99)
Kinetic Model
(r2)/Ki (␮M) or ␤
Downloaded from dmd.aspetjournals.org at ASPET Journals on July 31, 2017
MM, Michaelis-Menten; SI, substrate inhibition; TSI, two sites substrate inhibition.
4-MU
UGT1A9
(UDPGA kinetics)
UGT1A9 Supersomes
UGT1A9
Zidovudine HLM
UGT2B7
Entacapone HLM
Substrate
Enzyme Kinetic Parameters
The values represent a best-fit result ⫾ S.E. The reaction velocity is given in actual rates for all of the enzymes. See Materials and Methods for additional details.
AZT, entacapone, and 4-MU glucuronidation kinetic type and parameters
SI (0.99)
Ki ⫽ 256 ⫾ 18
TSI (0.98)
␤ ⫽ 0.24 ⫾ 0.01
SI (0.98)
Ki ⫽ 113 ⫾ 9
TSI (0.96)
␤ ⫽ 0.09 ⫾ 0.01
MM (0.99)
MM (0.99)
Kinetic Model
(r2)/Ki (␮M) or ␤
2124
MANEVSKI ET AL.
ALBUMIN EFFECT IN UGT2B7 AND UGT1A9
2125
A Entacapone, HLM, no BSA and 0.1% BSA
14.0
10.0
8.0
6.0
4.0
V, nmol/min/mg
V, nmol/min/mg
12.0
2.0
12.0
8.0
4.0
0.1% BSA
no BSA
0.0
0.0
0
100
200
2
3
4
V/[Entacapone]
5
300 400 500
[Entacapone], µM
600
700
800
B Entacapone, UGT1A9, no BSA and 0.1% BSA
20.0
V, nmol/min/mg
V, nmol/min/mg
15.0
10.0
15.0
10.0
5.0
0.0
0
1
2
3
4
V/[Entacapone]
5
5.0
0.1% BSA
no BSA
0.0
0
100
200
300 400 500
[Entacapone], µM
600
700
800
C Entacapone, UGT1A9 Supersomes,
no BSA and 0.1% BSA
30.0
V, nmol/min/mg
V, nmol/min/mg
30.0
20.0
20.0
10.0
0.0
0
10.0
2
4
6
8
V/[Entacapone]
10
0.1% BSA
no BSA
0.0
0
100
200
300
[Entacapone], µM
400
500
FIG. 4. Enzyme kinetics of entacapone glucuronidation by HLM
(A), in-house-produced UGT1A9 (B), and commercial UGT1A9
(C) without BSA and in the presence of 0.1% BSA. The points
represent an average of three samples ⫾ S.E. The concentrations of
entacapone were corrected for nonspecific binding. Glucuronidation rates are presented as actual (measured) rates in nmol 䡠 min⫺1 䡠
mg⫺1 recombinant protein. The derived kinetic constants are presented in Table 1. The data were fitted to the two-site equation (see
Materials and Methods for details). The Eadie-Hofstee transforms
of the data are presented as insets.
Downloaded from dmd.aspetjournals.org at ASPET Journals on July 31, 2017
0
1
2126
MANEVSKI ET AL.
V, nmol/min/mg
V, nmol/min//mg
A 4-MU, UGT1A9, no BSA, 0.1
1, and 1% BSA
4
4
3
3
2
1
0
0.0
2
0.5
1.0
1.5
V/[4-MU]
1
no BSA
0.1% BSA
1% BSA
0
100
200
300
[4-MU
U], µM
400
500
V, nmol/min/mg
V, nmol/miin/mg
B 4-MU, UGT1A9 Supersomes
s no BSA,
s,
BSA 0
0.1
1 and 1% BSA
15
10
10
FIG. 5. Enzyme kinetics of 4-MU glucuronidation by in-houseproduced UGT1A9 (A) and commercial UGT1A9 (B) without BSA
and in the presence of 0.1 and 1% BSA. The concentrations of
4-MU were corrected for nonspecific binding. The points represent
an average of three samples ⫾ S.E. Glucuronidation rates are presented as actual (measured) rates in nmol 䡠 min⫺1 䡠 mg⫺1 recombinant protein. The derived kinetic constants are presented in Table 1.
The data were fitted to the substrate inhibition equation. The EadieHofstee transforms of the data are presented as insets.
5
0
0
2
4
6
V/[4-MU]
5
no BSA
0 1% BSA
0.1%
1% BSA
0
0
100
200
300
[4-MU], µM
400
substrate without affecting the Vmax values. They offered an interesting explanation for this “BSA effect”—removal of a long-chain fatty
acid that competitively inhibits the UGTs. At the start of the study
presented here, we wondered if the proposed inhibitory fatty acids are
also present in the recombinant UGTs that we often use for glucuronidation studies—in-house-produced recombinant UGTs that are
expressed in insect cells and carry a C-terminal fusion peptide
(Kurkela et al., 2003, 2007). However, the results took us to different
directions, mainly to validate the initial observation that the BSA
effect in UGT1A9 is different and more complex than in UGT2B7.
However, before doing this, we developed the needed methods for
measuring drug binding to BSA and different biological membranes.
Many drugs bind nonspecifically to macromolecules; therefore,
determining the fraction of the added drug that is free under the
experimental conditions (the fu) during in vitro assays is important
because only the unbound fraction interacts with the target enzyme
(Grime and Riley, 2006; Varshney et al., 2010). We measured drug
binding to BSA and two enzyme sources, insect cell membranes and
HLM, by a newly developed ultrafiltration assay. Because ultrafiltra-
500
tion systems often “suffer” from high NSBf values (Lee et al., 2003;
Taylor and Harker, 2006), we took care to minimize, determine, and
take into account the nonspecific binding to the filter device, regardless of whether the binding was to the filter itself or to the walls of the
tube. Our results concerning AZT binding to BSA, negligible binding,
are the same as in a previous study that used a dialysis system
(Rowland et al., 2007). On the other hand, our finding that 4-MU
binding to BSA is concentration dependent to some extent, particularly at 4-MU concentrations less than 100 ␮M 4-MU and in the
presence of 0.1% BSA (Fig. 1B), is not in full agreement with the
published studies/results (Rowland et al., 2008). A possible reason for
this is that the substrate concentration dependence of 4-MU is mainly
visible at substrate concentrations less than 50 ␮M in the presence of
0.1% BSA (Fig. 1B), whereas the lowest 4-MU concentration tested
in the previous study (in the presence of 0.1, 1, and 2% BSA) was 50
␮M (Rowland et al., 2007).
Entacapone binds strongly to the filter device and to BSA (Figs.
1A and 2). Our analysis clearly demonstrates that, at least for some
drugs, the binding is concentration dependent, and therefore the
Downloaded from dmd.aspetjournals.org at ASPET Journals on July 31, 2017
0
ALBUMIN EFFECT IN UGT2B7 AND UGT1A9
2127
V, nmoll/min/mg
V, nmol/min/m
mg
A E
Entacapone,
t
UGT1A9 (UD
DPGA ki
kinetics),
ti )
no BSA and 0.1% BSA
15.0
no BSA
0.1% BSA
15.0
10.0
10.0
5.0
0.0
0.00
0.02
0.04
[UDPGA], µM
0.06
5.0
0
1000
2000
0
3000
[UD
DPGA], µM
4000
5000
B 4-MU, UGT1A9 (UDPGA k
kinetics),
kinetics)
no BSA and 0.1% BSA
3
V, n
nmol/min/mg
mg
V, nmol/min/m
2.5
FIG. 6. Enzyme kinetics of UDPGA using entacapone (A) and
4-MU (B) as aglycone substrates without BSA and in the presence
of 0.1% BSA. The points represent an average of three samples ⫾ S.E. Glucuronidation rates are presented as actual (measured) rates in nmol 䡠 min⫺1 䡠 mg⫺1 recombinant protein. The
derived kinetic constants are presented in Table 1. The data were
fitted to the Michaelis-Menten equation. The Eadie-Hofstee transforms of the data are presented as insets.
2.0
2
1
0
0.000
1.5
0.005
0.010
0.015
0.020
V/[UDPGA]
1.0
05
0.5
0.0
no BSA
0.1% BSA
0
1000
2000
0
3000
[UDPGA], µM
4000
correct fu value for each point on the kinetic curve should be taken
into account. This outcome differs from the simpler view that
emerged from the earlier reports of Rowland et al. (2007, 2008),
but our findings are very clear and indicate that the substrate
dependence of binding to BSA, and perhaps to plasma also, should
be evaluated for each drug under study. A further small complication in the case of entacapone binding, and perhaps other highly
lipophilic compounds, is possible displacement from BSA by the
enzyme source such as insect cell membranes (Fig. 2). This displacement was more significant at substrate concentrations less
than 200 ␮M and in the presence of higher enzyme source concentrations (Fig. 2), and although it was not significant in our case,
it might turn out to be meaningful with other drugs that exhibit
similar features but that serve as poor substrates to the tested UGT,
perhaps leading to the addition of excessive amounts of recombinant enzyme. In such cases, a 3D binding curve, similar to the one
we present in Fig. 2B, may be essential to estimate the fu at
different points of the experiment.
5000
The current results on the effect of BSA addition on AZT glucuronidation by UGT2B7, either in its native state within HLM or as a
recombinant enzyme that was expressed in insect cell membranes and
carries a short C-terminal fusion peptide (Fig. 3), are very similar to
the earlier results for HLM and recombinant UGT2B7 that was
expressed in HEK293 cells (Rowland et al., 2007). This finding
strongly suggests that, at least with respect to the putative competitive
inhibitor that is removed by the addition of BSA to the reaction
mixture, there is no significant difference between the recombinant
UGTs, regardless of whether they were expressed in HEK293 or
insect cells, to the native UGT in HLM. Likewise, our results on the
BSA effect on UGT1A9 do not give any reason to suspect that
significant differences in enzyme kinetics and the interactions of
UGT1A9 with its aglycone substrates exist between the native and the
recombinant enzyme that was expressed in insect cells, with or without a C-terminal fusion peptide (Figs. 4 and 5). On the other hand,
there is a major difference between our results with UGT1A9 and
those from a previous study (Rowland et al., 2008).
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0.0
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MANEVSKI ET AL.
B
E·AX·I
I1
E·AX·B·I
(dead-end complex)
I2
B
E·AX
E·AX·B
E·A
E·A·BX
AX
E
A
samples for in vitro glucuronidation assays. Such a mixture will
probably include inhibitors with different affinities to different individual UGTs and thereby might also explain the small change of the
kinetic model for UGT1A9 that was caused by BSA addition (Fig. 4;
Table 1).
In summary, our results show that addition of BSA enhances the in
vitro activities of UGT2B7 and UGT1A9, regardless of whether a
native enzyme in HLM was used, or a recombinant UGT with or
without a C-terminal fusion peptide. Drug binding to BSA differs in
the extent and dependence on drug concentration, indicating that it
should be determined carefully in each case. The BSA effect is (even)
more complex than described previously, but its full clarification may
lead us to a better and deeper understanding of the full kinetic
mechanism of drug glucuronidation by the UGTs.
Acknowledgments
B
BX
We thank Johanna Mosorin for skillful technical assistance.
E·A·B
(dead-end complex)
FIG. 7. The proposed mechanism for inhibitor interactions with a compulsory ordered
bi-bi mechanism of UGT catalysis (see Discussion for details). E, enzyme (UGTs); AX,
UDP-␣-D-glucuronic acid; A, UDP; B, substrate (aglycone); I, inhibitor.
Participated in research design: Manevski and Finel.
Conducted experiments: Manevski and Morelo.
Performed data analysis: Manevski, Morelo, and Finel.
Wrote or contributed to the writing of the manuscript: Manevski, YliKauhaluoma, and Finel.
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