Species and tissue differences in serotonin glucuronidation

Article abstract of DOI:10.3109/00498254.2015.1101509

1. Serotonin is a UGT1A6 substrate that is mainly found in the extrahepatic tissues where some UGT1As are expressed. The aim of the present study was to characterize serotonin glucuronidation in various tissues of humans and rodents.2. Serotonin glucuronidation in the human liver and kidney fitted to the Michaelis-Menten model, and the Km values were similar to that of recombinant UGT1A6. However, serotonin glucuronidation in the human intestine fitted to the Hill equation, indicating that it is likely catalyzed not only by UGT1A6, but also by another UGT1A isoform. Serotonin glucuronidation in the rat liver, intestine and kidney fitted well to the Michaelis-Menten model and exhibited monophasic kinetics in the kidney, but biphasic kinetics in the liver and intestine. Furthermore, serotonin glucuronidation in the rat brain fitted best to the Hill equation. Serotonin glucuronidation in the mouse tissues fitted to the Michaelis-Menten model and exhibited monophasic kinetics in the liver and intestine microsomes, but biphasic kinetics in the kidney and brain microsomes.3. In conclusion, we clarified that tissue and species differences exist in serotonin glucuronidation. It is necessary to take these potential differences into account when considering the pharmacodynamics and pharmacokinetics of serotonin.

Full text of DOI:10.3109/00498254.2015.1101509

Xenobiotica
the fate of foreign compounds in biological systems
ISSN: 0049-8254 (Print) 1366-5928 (Online) Journal homepage: http://www.tandfonline.com/loi/ixen20
Species and tissue differences in serotonin
glucuronidation
Yukiko Sakakibara, Miki Katoh, Taisho Kawayanagi & Masayuki Nadai
To cite this article: Yukiko Sakakibara, Miki Katoh, Taisho Kawayanagi & Masayuki Nadai
(2015): Species and tissue differences in serotonin glucuronidation, Xenobiotica, DOI:
10.3109/00498254.2015.1101509
To link to this article: http://dx.doi.org/10.3109/00498254.2015.1101509
Published online: 02 Nov 2015.
Submit your article to this journal
Article views: 21
View related articles
View Crossmark data
Full Terms & Conditions of access and use can be found at
http://www.tandfonline.com/action/journalInformation?journalCode=ixen20
Download by: [Dalhousie University]
Date: 13 November 2015, At: 00:56

http://informahealthcare.com/xen
ISSN: 0049-8254 (print), 1366-5928 (electronic)
Xenobiotica, Early Online: 1–7
2015 Taylor & Francis. DOI: 10.3109/00498254.2015.1101509
!
RESEARCH ARTICLE
Species and tissue differences in serotonin glucuronidation
Yukiko Sakakibara, Miki Katoh, Taisho Kawayanagi, and Masayuki Nadai
Department of Pharmaceutics, Faculty of Pharmacy, Meijo University, Nagoya, Japan
Abstract
Keywords
1. Serotonin is a UGT1A6 substrate that is mainly found in the extrahepatic tissues where some
UGT1As are expressed. The aim of the present study was to characterize serotonin
glucuronidation in various tissues of humans and rodents.
Enzyme kinetic study, serotonin, species
differences, tissue differences, UGT
2. Serotonin glucuronidation in the human liver and kidney fitted to the Michaelis–Menten
model, and the Km values were similar to that of recombinant UGT1A6. However, serotonin
glucuronidation in the human intestine fitted to the Hill equation, indicating that it is likely
catalyzed not only by UGT1A6, but also by another UGT1A isoform. Serotonin
glucuronidation in the rat liver, intestine and kidney fitted well to the Michaelis–Menten
model and exhibited monophasic kinetics in the kidney, but biphasic kinetics in the liver and
intestine. Furthermore, serotonin glucuronidation in the rat brain fitted best to the Hill
equation. Serotonin glucuronidation in the mouse tissues fitted to the Michaelis–Menten
model and exhibited monophasic kinetics in the liver and intestine microsomes, but biphasic
kinetics in the kidney and brain microsomes.
History
Received 12 August 2015
Revised 24 September 2015
Accepted 25 September 2015
Published online 30 October 2015
3. In conclusion, we clarified that tissue and species differences exist in serotonin
glucuronidation. It is necessary to take these potential differences into account when
considering the pharmacodynamics and pharmacokinetics of serotonin.
Introduction
the body. Accordingly, characterization of serotonin glucur-
onidation in the extrahepatic tissues would be valuable.
UGTs have overlapping substrate specificities, and most of
their substrates are glucuronidated by more than one UGT
isoform (Radominska-Pandya et al., 1999). However, unlike
1-naphthol (Ebner & Burchell, 1993; Harding et al., 1988)
and acetaminophen (Court et al., 2001; Fisher et al., 2000),
serotonin has been shown to be a highly selective substrate for
UGT1A6 in the human liver and extrahepatic tissues (King
et al., 1999; Krishnaswamy et al., 2003a).
UDP-glucuronosyltransferase (UGT) catalyzes the major
phase II reaction, which is responsible for catalyzing the
conjugation of glucuronic acid with a wide range of xeno- and
endobiotics, including drugs (de Leon, 2003; Tukey &
Strassburg, 2000), hormones (Court, 2005) and neurotrans-
¨
mitters (Itaaho et al., 2009). An essential neurotransmitter,
serotonin, is conjugated by UGT (Krishnaswamy et al.,
2003a). Serotonin is mainly found in the extrahepatic tissues
such as the intestine (Roth & Gordon, 1990), kidney (Hafdi
et al., 1996; Stier & Itskovitz, 1985) and brain (Zheng et al.,
2012), where some UGT1As are expressed at a higher level
than in the liver (Buckley & Klaassen, 2007; Nakamura et al.,
2008; Shelby et al., 2003). A previous study reported that 30%
of injected serotonin was excreted in the urine as serotonin
glucuronide, and the amount increased to 70% when mono-
amine oxidase was inhibited by iproniazid in the mouse
(Wessbach et al., 1960). These results suggested that
glucuronidation represents an important compensatory cata-
bolic pathway of serotonin. Therefore, characterization of the
serotonin kinetics in the extrahepatic tissues is of great value
to understand the pharmacological effects of serotonin in
A previous study reported that serotonin glucuronidation
was reduced in homozygous mutant (J/J) Gunn rat liver
homogenates to about 20% of the activity observed in
normal Wistar rat liver homogenates (Leakey, 1978).
Another study demonstrated that serotonin glucuronidation
in homozygous mutant and heterozygous (J/+) Gunn rats
decreased to 13% and 60%, respectively, compared to the
activities measured in normal Wistar rats (Krishnaswamy
et al., 2003b). These results suggested that serotonin is
primarily catalyzed by Ugt1a isoforms (possibly Ugt1a6) in
rat liver microsomes. Moreover, Ugt2 isoforms may also
contribute to serotonin glucuronidation because of the low
but significant activity observed in the homozygous mutant
Gunn rat liver. In terms of mice, serotonin glucuronidation
was found to be catalyzed by Ugt1a6a and Ugt1a6b with
different enzymatic properties (Uchihashi et al., 2013).
However, no study has comprehensively evaluated the
Address for correspondence: Miki Katoh, Ph.D., Department of
Pharmaceutics, Faculty of Pharmacy, Meijo University, 150
Yagotoyama, Tempaku-ku, Nagoya 468-8503, Japan. Tel: +81
528392680. Fax: +81 528348090. E-mail: mkatoh@meijo-u.ac.jp

2
Y. Sakakibara et al.
Xenobiotica, Early Online: 1–7
species and tissue differences in the kinetics of serotonin
glucuronidation.
Mass/charge (m/z) ion transitions were recorded in the
multiple reaction-monitoring mode, with m/z 352.8/160.0
for serotonin b-D-glucuronide and m/z 357.2/164.1 for sero-
tonin-d4 b-D-glucuronide. The retention time of both sero-
tonin b-D-glucuronide and serotonin-d4 b-D-glucuronide was
4.5 min. The limit of detection for serotonin b-D-glucuronide
was 0.28 pmol. The limit of quantification in the reaction
mixture was 28 nM with a coefficient of variation of less than
10%. In a preliminary study, we confirmed the linearity of the
protein concentrations and incubation times.
Therefore, the aim of the present study was to characterize
serotonin glucuronidation in various tissues of humans and
rodents and to clarify the species and tissue differences in the
kinetics of serotonin glucuronidation.
Materials and methods
Materials
Alamethicin and uridine 50-diphosphoglucuronic acid triso-
dium salt were obtained from Sigma-Aldrich (St. Louis, MO).
(p-Amino diphenyl)methanesulfonyl fluoride and dithiothrei-
tol were purchased from Wako Pure Chemicals (Osaka,
Japan). Serotonin b-D-glucuronide and serotonin-d4
b-D-glucuronide were purchased from Toronto Research
Chemicals (Toronto, Canada). All other chemicals and
solvents were of the highest grade commercially available.
Inhibition study for serotonin glucuronidation in
human intestine microsomes
1-Naphthol, a substrate for UGT1A6 (Court et al., 2001;
Fisher et al., 2000; Uchaipichat et al., 2004), emodin, a
substrate for UGT1A8 and UGT1A10 (Watanabe et al., 2002),
¨¨
estrone, a substrate for UGT1A10 (Kallionpaa et al., 2015),
androstanediol and 5-(40-hydroxyphenyl)-5-phenylhydantoin
(HPPH), substrate for UGT2B15 (Nakajima et al., 2007;
Turgeon et al., 2001), were used as UGT inhibitors to clarify
their inhibitory effects on serotonin glucuronidation in pooled
human intestinal microsomes.
Microsomes from the liver, kidney, intestine and brain
The present study was approved by the Institutional Animal
Care and Use Committee of Meijo University. Microsomes
from the human liver (pooled, 50 donors), intestine (pooled, 7
donors) and kidney (pooled, 4 donors) were purchased from
BD Gentest (Woburn, MA) or KAC (Kyoto, Japan). The
pooled microsomes from rat (n = 10) and mouse (n = 10)
tissues were prepared as described previously (Shiratani et al.,
2008) and stored at -80 ꢀC until analysis. Protein concentra-
tion was determined using Bio-Rad Protein Assay kit (Bio-
Rad, Hercules, CA).
Enzyme kinetic analyses
Kinetic parameters were estimated from the fitted curves
using the KaleidaGraph computer program (Synergy
Software, Reading, PA) designed for nonlinear regression
analysis. The following equations were applied for the
Michaelis–Menten (Equation 1), Hill (Equation 2) or sub-
strate inhibition (Equation 3) kinetics model:
Recombinant UGT isoforms
Vmax ꢂ S
V ¼
V ¼
,
ð1Þ
ð2Þ
Km þ S
Human recombinant UGT1A1, UGT1A6, UGT1A7,
UGT1A8, UGT1A10, UGT2B7 and UGT2B15 supersomes
were obtained from BD Gentest.
Vmax ꢂ Sn
,
Sn50 þ Sn
Serotonin glucuronidation
Vmax ꢂ S
Serotonin glucuronidation was determined using the method
described by Mohamed & Frye (2011) with slight modifica-
tions. The incubation mixture contained 50 mM Tris–HCl
buffer (pH 7.4), 5 mM MgCl2, 25 mg/mL alamethicin, 0.1 mg/
mL microsomes from the liver, intestine and kidney, or
0.5 mg/mL microsomes from the brain, and 0.02–40 mM
serotonin. The reaction mixture was incubated at 37 ꢀC for
60 min. The reaction was terminated by the addition of 20 mL
60% perchloric acid and 17.8 nmol serotonin-d4 b-D-glucur-
onide was added as an internal standard. After the mixture
was centrifuged at 15 000 g for 10 min, 10 mL of the obtained
supernatant was subjected to liquid chromatography–tandem
mass spectrometry (LC–MS/MS). LC was performed using a
Prominence apparatus (Shimadzu, Kyoto, Japan), which was
V ¼
,
ð3Þ
Km þ S þ S2=Ki
where Km is the Michaelis–Menten constant and Vmax is the
maximum velocity. S50 is the substrate concentration showing
the half-Vmax, n is the Hill coefficient and Ki is the substrate
inhibition constant. Intrinsic clearance (CLint) was calculated
as Vmax/Km for the Michaelis–Menten kinetics. For sigmoidal
kinetics, maximum clearance (CLmax) was calculated as
Vmaxꢂ(n - 1)/(S50ꢂn(n - 1)1/n) to estimate the highest clearance
(Houston & Kenworthy, 2000). Data are presented as the
mean of three independent determinations using pooled
microsomes from each tissue.
Results
equipped with
a
Develosil XG-C30 M3 column
Serotonin glucuronidation in microsomes from human
tissues
(2.1 mm ꢁ 150 mm; Nomura Chemical, Aichi, Japan). The
column temperature was 40 ꢀC, and the flow rate was 0.2 mL/
min. The mobile phase consisted of methanol/10 mM ammo-
nium formate (pH 4.5) [5:95 (v/v)]. The LC setup was
connected to an API4000 tandem mass spectrometer (Applied
Biosystems, Fostercity, CA) operated in the positive electro-
spray ionization mode. Turbo gas was maintained at 400 ꢀC.
As shown in Figure 2, serotonin glucuronidation in the human
liver and kidney fitted to the Michaelis–Menten model.
Eadie–Hofstee plots for serotonin glucuronidation in the
human liver and kidney microsomes exhibited monophasic
kinetics, and the Km values were similar in both tissues.

DOI: 10.3109/00498254.2015.1101509
Kinetics of serotonin glucuronidation
3
However, the kinetics of serotonin glucuronidation in the
human intestine fitted to the Hill equation with a coefficient
of 1.30 (Table 1). The values of Vmax (29.67 nmol/min/mg
protein) and CLint (4.66 mL/min/mg protein) for human liver
microsomes were higher than those for the intestine and
kidney (Table 1, Figure 2).
Inhibition study for serotonin glucuronidation in
human intestine microsomes
To elucidate the UGT isoforms responsible for serotonin
glucuronidation in human intestine, inhibition study was
performed using UGT substrates (Figure 5). Serotonin
glucuronidation in human intestinal microsomes decreased
by 34% in the presence of 10 mM 1-naphthol (UGT1A6).
Serotonin glucurotnidation in human intestine decreased to
40% in the presence of 100 mM emodin (UGT1A8 and
UGT1A10). Serotonin glucuronidation decreased to less than
20% in the presence of 1000 mM HPPH (UGT2B15).
Serotonin glucuronidation by human recombinant
UGT isoforms
Serotonin glucuronidation was determined at a concentration
of 4 mM serotonin using recombinant UGT isoforms,
including UGT1A1, UGT1A6, UGT1A7, UGT1A8,
UGT1A10, UGT2B7 and UGT2B15 (Figure 3), which are
mainly expressed in the human intestine. UGT1A6 resulted in
the highest level of serotonin glucuronidation (1092 pmol/
min/mg protein). The other isoforms also catalyzed serotonin
glucuronidation, but these activities were much lower than
that of UGT1A6. Furthermore, enzyme kinetic studies were
performed using these recombinant human UGT isoforms
(Table 2 and Figure 4) except for UGT1A1 and UGT2B7,
whose glucuronidation levels were not high enough to detect
for kinetics studies, especially at lower serotonin concentra-
tions. Serotonin glucuronidation by UGT1A6, UGT1A7 and
UGT1A10 fitted to the Michaelis–Menten model, whereas
that by UGT1A8 and UGT2B15 fitted to the substrate
inhibition model.
Serotonin glucuronidation in microsomes from rat
tissues
Serotonin glucuronidation in the rat liver, intestine and kidney
fitted to the Michaelis–Menten model. Eadie–Hofstee plots
exhibited biphasic kinetics for serotonin glucuronidation in
the rat liver and intestine microsomes, and the Km values were
similar in both tissues. By contrast, the rat kidney microsomes
exhibited monophasic kinetics, and the Km value (3.53 mM)
differed from those in the rat liver and intestine microsomes.
However, serotonin glucuronidation in the rat brain fitted to
the Hill equation with a coefficient of 1.49. The value of Vmax
for rat kidney microsomes was highest among all rat tissues
examined. Similarly, the value of CLint for the rat liver
microsomes was highest among all rat tissue microsomes
(Figure 6).
Serotonin glucuronidation in microsomes from mouse
tissues
HO2C
NH2
O
HO
HO
NH2
O
HO
Serotonin glucuronidation in all four mice tissues examined
fitted to the Michaelis–Menten model. Eadie–Hofstee plots
for serotonin glucuronidation in the mouse liver and intestine
microsomes exhibited monophasic kinetics. The values of Km
for the mouse liver and intestine microsomes were 1.47 and
N
H
HO
Serotonin glucuronide
N
H
Serotonin
Figure 1. Serotonin glucuronidation.
Table 1. Kinetics parameters of serotonin glucuronidation in the human, rat and mouse.
CLint (CLmax
(mL/min/mg)
)
Species
Human
Tissue
Affinity
Km (S50) (mM)
Vmax (nmol/min/mg)
na
Liver
6.37 0.31
4.47 0.61b
6.13 1.10
0.30 0.01
0.83 0.02
0.31 0.01
0.73 0.03
3.53 0.21
1.29 0.06b
1.47 0.13
2.83 0.13
0.05 0.00
0.91 0.02
1.28 0.07
3.60 0.08
29.67 0.58
0.76 0.06
5.00 0.36
45.26 0.54
61.81 0.31
15.49 0.24
20.85 0.16
153.33 5.77
0.08 0.02
68.00 2.50
67.00 1.41
0.49 0.02
2.45 0.01
14.40 0.44d
25.24 0.30d
4.66 0.16
0.17 0.01c
0.83 0.10
Intestine
Kidney
Liver
1.30
Rat
High
Low
High
Low
151.27 4.15
72.09 0.10
50.48 1.98
28.38 1.12
43.45 1.90
0.07 0.01c
48.57 2.36
23.65 0.59
9.92 0.25
Intestine
Kidney
Brain
Liver
Intestine
Kidney
1.49
Mouse
High
Low
High
Low
0.54 0.03
11.25 0.60e
7.01 0.00e
Brain
Each value represents the mean SD.
aHill coefficient.
bS50
.
cCLmax
.
dpmol/min/mg.
enL/min/mg.

4
Y. Sakakibara et al.
Xenobiotica, Early Online: 1–7
Intestine
Figure 2. Kinetic study of serotonin glucur-
onidation by recombinant human UGT iso-
forms. Serotonin glucuronidation was
measured with various serotonin concentra-
tions ranging from 0.1 to 40 mM. Eadie–
Hofstee plots are shown as insets in each
graph. The points represent the means of
three independent determinations.
Liver
30
25
20
15
10
5
800
600
400
200
0
800
80
600
400
200
0
60
40
20
0
0
1
2 3
V/S
4
5
0
20 40 60 80 100120
V/S
0
0
10 20 30 40 50
S (mM)
0
10 20 30 40 50
S (mM)
Kidney
5
4
3
2
1
0
5
4
3
2
1
0
0
0.2 0.4 0.6 0.8 0.1
V/S
0
10 20 30 40 50
S (mM)
1200
1000
isoforms to serotonin in rats and mice are higher than those
in humans.
Serotonin glucuronidation in the human liver and kidney
fitted to the Michaelis–Menten model, and Eadie–Hofstee
plots exhibited monophasic kinetics, with Km values similar
to those obtained in a previous study (liver: 5.2–8.8 mM;
kidney: 6.5 mM) (Krishnaswamy et al., 2003a). However,
serotonin glucuronidation in the human intestine fitted to the
Hill equation. Serotonin glucuronidation in the human
intestine exhibited different kinetics from that in the kidney
or liver. A previous study reported that serotonin was mainly
catalyzed by UGT1A6, but UGT1A7, UGT1A8, UGT1A10,
and UGT2B7 also showed quantifiable activities
(Krishnaswamy et al., 2003a; Kurkela et al., 2007), which is
similar to the results of the present study. Besides these UGT
isoforms, UGT1A1 and UGT2B15 also catalyzed serotonin
glucuronidation in the present study. The difference in these
results is most likely owing to the enhanced detection
sensitivity and the difference of the UGT content in the
enzyme sources. In the inhibition study for serotonin
glucuronidation in human intestinal microsomes, 1-naphthol
(UGT1A6) prominently inhibited serotonin glucuronidation.
Since serotonin glucuronidation also decreased upon the
addition of emodin and HPPH, serotonin glucuronidation in
the human intestinal microsomes appears to be catalyzed not
only by UGT1A6, but also by other UGT isoforms that are
expressed in the intestine with different affinities to serotonin;
indeed, the expression level of UGT1A6 protein in the human
intestine is much lower than that in the liver or the kidney
(Krishnaswamy et al., 2003a). However, in our kinetic study,
we could not identify the main recombinant UGT isoform that
catalyzes serotonin glucuronidation in the human intestine,
because none of the UGT isoforms evaluated fit to the Hill
equation.
800
600
5
4
3
2
1
0
Figure 3. Serotonin glucuronidation by recombinant human UGT
isoforms. Serotonin glucuronidation was measured at a serotonin
concentration of 4 mM. Each column represents the mean of duplicate
determinations.
2.83 mM, respectively. However, Eadie–Hofstee plots for
serotonin glucuronidation in the mouse kidney and brain
microsomes were biphasic, and the Km values for serotonin
glucuronidation differed between the mouse kidney and brain.
The value of CLint for the mouse liver microsomes was
highest among the microsomes of all four mice tissues
examined (Figure 7).
Discussion
In this study, we conducted kinetic evaluations of serotonin
glucuronidation to elucidate whether tissue and species
differences exist. The values of CLint for serotonin glucur-
onidation in human tissues were lower than those in the rat
and mouse tissues (Table 1). Moreover, the Km values in
both the rat and mouse tissues were lower than those in
human tissues, suggesting that the affinities of UGT

DOI: 10.3109/00498254.2015.1101509
Kinetics of serotonin glucuronidation
5
Table 2. Kinetics parameters of serotonin glucuronidation in recombinant human UGT isoforms.
Isoform
Km (mM)
Vmax (pmol/min/mg)
CLint (mL/min/mg)
Ki (mM)
UGT1A6
UGT1A7
UGT1A8
UGT1A10
UGT2B15
8.07 0.95
10.10 2.65
8.33 2.81
7.80 0.66
3.87 0.31
3466.70 251.66
7.10 1.00
4.47 1.36
431.32 19.80
0.72 0.08
0.54 0.05
0.87 0.03
0.64 0.02
14.30 6.56
6.80 0.36
2.47 0.15
Each value represents the mean SD.
UGT1A8
UGT1A7
UGT1A6
3000
2.0
1.5
1.0
0.5
6
4
2
4000
2000
8
6
4
2
0
2.0
1.5
1.0
0.5
0.0
3000
2000
1000
1000
0
0
100 200 300 400 500
V/S
0.0
0.2
0.4
0.6
0.0
0.2
0.4
0.6
V/S
V/S
0.0
0
0
0
10 20 30 40 50
S (mM)
0
10 20 30 40 50
S (mM)
0
40 50
10 20 30
S (mM)
UGT2B15
UGT1A10
3
2
1
8
6
4
2
3
2
1
8
6
4
2
0
0
0.0 0.1 0.2 0.3 0.4 0.5
V/S
0.0
0.2 0.4 0.6 0.8
V/S
0
0
50
0
10
30
20
S (mM)
40
0
10 20 30 40 50
S (mM)
Figure 4. Kinetic study of serotonin glucuronidation in human microsomes. Serotonin glucuronidation was measured in the human liver, intestine and
kidney microsomes with serotonin concentrations ranging from 0.02 to 40 mM. Eadie–Hofstee plots are shown as insets in each graph. The points
represent the means of three independent determinations.
100
80
60
40
20
0
with a potential partial contribution from Ugt2 isoforms
(Krishnaswamy et al., 2003b). Therefore, serotonin glucur-
onidation in rats could be catalyzed by several isoforms that
have different affinities for serotonin. The value of Vmax in the
rat kidney was higher than that in the liver, unlike the results
observed in humans or mice. This result is consistent with a
previous study showing that 1-naphthol glucuronidation in the
rat kidney was higher than that in the liver (Ito et al., 2005).
The difference in the expression of Ugt isoforms in each tissue
may contribute to the observed differences between the tissues
in the kinetic analysis of serotonin glucuronidation. It has been
reported that the mRNA expression levels of Ugt1a and Ugt2b
isoforms varied among different rat tissues (Shelby et al.,
2003). However, further studies are needed to clarify the Ugt
isoforms that are primarily responsible for catalyzing sero-
tonin glucuronidation in each rat tissue.
Estrone (UGT1A10)
Androstanediol (UGT2B15)
HPPH (UGT2B15)
Emodin (UGT1A8 and 1A10)
1-Naphtol (UGT1A6)
1
10
100
1000
UGT substrate (µM)
Figure 5. Inhibition study for serotonin glucuronidation in human
intestine microsomes. Serotonin glucuronidation was determined at
4 mM serotonin in the presence of some UGT substrates. Each data point
represents the mean of triplicate determinations.
The Eadie–Hofstee plot exhibited monophasic kinetics in
the rat kidney, but biphasic kinetics in the rat liver and
intestine. These results suggested that serotonin glucuronida-
tion is catalyzed by more than two common isoforms in the rat
liver and intestine. Moreover, serotonin glucuronidation in the
rat brain fitted to the Hill equation, which suggests that the Ugt
isoforms catalyzing serotonin glucuronidation in the rat brain
might differ from those in the rat liver, intestine and kidney.
The previous report using Gunn rats indicated that serotonin
glucuronidation was primarily catalyzed by Ugt1a isoforms,
The Eadie–Hofstee plots for serotonin glucuronidation in
the mouse liver and intestine microsomes exhibited mono-
phasic kinetics. However, those in the mouse kidney and brain
microsomes were biphasic, and the Km values were different.
These results suggested that serotonin glucuronidation is
catalyzed by more than two isoforms in the mouse kidney and
brain. It has been reported that Ugt1a6a and Ugt1a6b are
the main isoforms responsible for catalyzing serotonin

6
Y. Sakakibara et al.
Xenobiotica, Early Online: 1–7
Liver
Intestine
80
60
40
20
0
Figure. 6 Kinetic study of serotonin glucur-
onidation in rat microsomes. Serotonin glu-
curonidation was measured in the rat liver,
intestine, kidney and brain microsomes with
serotonin concentrations ranging from 0.02 to
40 mM. Eadie–Hofstee plots are shown as
insets in each graph. The points represent the
means of three independent determinations.
25
20
15
10
5
25
20
80
60
40
20
0
15
10
5
0
0
10 20 30 40 50 60
V/S
0
50 100 150 200
V/S
0
25
15 20 25
10 15 20
S (mM)
0
5
10
0
5
S (mM)
Brain
150
120
Kidney
100
80
60
40
20
0
100
150
120
90
90
60
80
60
40
20
0
60
30
30
0
0
0
10 20 30 40 50
V/S
0
10 20 30 40 50
V/S
0
0
6
5
10 15
S (mM)
20 25
1
2
3
4
5
S (mM)
Liver
Intestine
Figure 7. Kinetic study of serotonin glucur-
onidation in mouse microsomes. Serotonin
glucuronidation was measured in the rat liver,
intestine, kidney and brain microsomes with
serotonin concentrations ranging from 0.02 to
40 mM. Eadie–Hofstee plots are shown as
insets in each graph. The points represent the
means of three independent determinations.
80
60
40
20
0
75
50
25
0
75
50
25
0
80
60
40
20
0
25
0
5
10 15 20
V/S
0
20
40
V/S
60
25
10 15 20
S (mM)
10 15 20 25
S (mM)
0
5
0
5
Brain
Kidney
2.5
2.0
1.5
1.0
0.5
25
20
15
10
5
2.5
30
25
20
15
10
5
2.0
1.5
1.0
0.5
0.0
0
0
2
4
6
V/S
8
10
0
3
6
9
12
V/S
0
0.0
25
10 15 20 25
S (mM)
0
5
10 15 20
S (mM)
0
5
glucuronidation with Km values of 2.6 and 11.0 mM, respect-
ively (Uchihashi et al., 2013). In the present study, the Km
values for serotonin glucuronidation in the mouse kidney and
brain were lower than those reported previously. These results
indicated that serotonin glucuronidation in the mouse may be
not only catalyzed by Ugt1a6a and Ugt1a6b, but also by other
Ugt isoforms. Although different Ugt isoforms were found to
be expressed in the liver and intestine (Buckely & Klaassen,
2009), the specific Ugt isoforms involved in serotonin
glucuronidation remain unclear. Therefore, the expression
levels in various tissues should be elucidated to determine the
cause of the tissue differences observed in the kinetic analysis
of serotonin glucuronidation.
Conclusion
In conclusion, we clarified that species and tissue differences
exist in serotonin glucuronidation. The present study thus

DOI: 10.3109/00498254.2015.1101509
Kinetics of serotonin glucuronidation
7
human UDP-glucuronosyltransferase (UGT) 1A6. Drug Metab Dispos
31:133–9.
Krishnaswamy S, Duan SX, Von Moltke LL, et al. (2003b). Serotonin (5-
hydroxytryptamine) glucuronidation in vitro: assay development,
human liver microsome activities and species differences.
Xenobiotica 33:169–80.
Kurkela M, Patana AS, Mackenzie PI, et al. (2007). Interactions with
other human UDP-glucuronosyltransferases attenuate the conse-
quences of the Y485D mutation on the activity and substrate affinity
of UGT1A6. Pharmacogenet Genomics 17:115–26.
provides useful information for in vivo UGT studies using
serotonin as the UGT probe. Moreover, this study is expected
to contribute to advancing understanding of the pharmaco-
kinetics of serotonin as an endogenous substrate of UGT and
the physiological significance of UGT. It is necessary to take
these potential differences into account when considering
serotonin pharmacodynamics and pharmacokinetics.
Leakey JE. (1978). An improved assay technique for uridine diphosphate
glucuronosyltransferase activity towards 5-hydroxytryptamine and
some properties of the enzyme. Biochem J 175:1119–24.
Mohamed ME, Frye RF. (2011). Inhibitory effects of commonly used
herbal extracts on UDP-glucuronosyltransferase 1A4, 1A6, and 1A9
enzyme activities. Drug Metab Dispos 39:1522–8.
Declaration of interest
This work was supported partly by JSPS KAKENHI Grant
Number 25460200. The authors report no declarations of
interest.
Nakamura A, Nakajima M, Yamanaka H, et al. (2008). Expression of
UGT1A and UGT2B mRNA in human normal tissues and various cell
lines. Drug Metab Dispos 36:1461–4.
References
Nakajima M, Yamanaka H, Fujiwara R, et al. (2007). Stereoselective
glucuronidation of 5-(40-hydroxyphenyl)-5-phenylhydantoin by
human UDP-glucuronosyltransferase (UGT) 1A1, UGT1A9, and
UGT2B15: effects of UGT-UGT interactions. Drug Metab Dispos
35:1679–86.
Radominska-Pandya A, Czernik PJ, Little JM, et al. (1999). Structural
and functional studies of UDP-glucuronosyltransferases. Drug Metab
Rev 31:817–99.
Roth KA, Gordon JI. (1990). Spatial differentiation of the intestinal
epithelium: analysis of enteroendocrine cells containing immunor-
eactive serotonin, secretin, and substance P in normal and transgenic
mice. Proc Natl Acad Sci USA 87:6408–12.
Shelby MK, Cherrington NJ, Vansell NR, Klaassen CD. (2003). Tissue
mRNA expression of the rat UDP-glucuronosyltransferase gene
family. Drug Metab Dispos 31:326–33.
Shiratani H, Katoh M, Nakajima M, Yokoi T. (2008). Species differences
in UDP-glucuronosyltransferase activities in mice and rats. Drug
Metab Dispos 36:1745–52.
Stier Jr CT, Itskovitz HD. (1985). Formation of serotonin by rat kidneys
in vivo. Proc Soc Exp Biol Med 180:550–7.
Tukey RH, Strassburg CP. (2000). Human UDP-glucuronosyltrans-
ferases: metabolism, expression, and disease. Annu Rev Pharmacol
Toxicol 40:581–616.
Buckley DB, Klaassen CD. (2007). Tissue- and gender-specific mRNA
expression of UDP-glucuronosyltransferases (UGTs) in mice. Drug
Metab Dispos 35:121–7.
Buckley DB, Klaassen CD. (2009). Mechanism of gender-divergent
UDP-glucuronosyltransferase mRNA expression in mouse liver and
kidney. Drug Metab Dispos 37:834–40.
Court MH. (2005). Isoform-selective probe substrates for in vitro studies
of human UDP-glucuronosyltransferases. Meth Enzymol 400:104–16.
Court MH, Duan SX, von Moltke LL, et al. (2001). Interindividual
variability in acetaminophen glucuronidation by human liver micro-
somes: identification of relevant acetaminophen UDP-glucuronosyl-
transferase isoforms. J Pharmacol Exp Ther 299:998–1006.
de Leon J. (2003). Glucuronidation enzymes, genes and psychiatry. Int J
Neuropsychopharmacol 6:57–72.
Ebner T, Burchell B. (1993). Substrate specificities of two stably
expressed human liver UDP-glucuronosyltransferases of the UGT1
gene family. Drug Metab Dispos 21:50–5.
Fisher MB, Vandenbranden M, Findlay K, et al. (2000). Tissue
distribution and interindividual variation in human UDP-glucurono-
syltransferase activity: relationship between UGT1A1 promoter
genotype and variability in a liver bank. Pharmacogenetics 10:727–39.
Hafdi Z, Couette S, Comoy E, et al. (1996). Locally formed 5-
hydroxytryptamine stimulates phosphate transport in cultured opos-
sum kidney cells and in rat kidney. Biochem J 320:615–21.
Harding D, Fournel-Gigleux S, Jackson MR, Burchell B. (1988). Cloning
and substrate specificity of a human phenol UDP-glucuronosyltrans-
ferase expressed in COS-7 cells. Proc Natl Acad Sci USA 85:8381–5.
Houston JB, Kenworthy KE. (2000). In vitro–in vivo scaling of CYP
kinetic data not consistent with the classical Michaelis–Menten
model. Drug Metab Dispos 28:246–54.
¨
Itaaho K, Court MH, Uutela P, et al. (2009). Dopamine is a low-affinity
and high-specificity substrate for the human UDP-glucuronosyltrans-
ferase 1A10. Drug Metab Dispos 37:768–75.
Ito Y, Yokota H, Wang R, et al. (2005). Species differences in the
metabolism of di(2-ethylhexyl) phthalate (DEHP) in several organs of
mice, rats, and marmosets. Arch Toxicol 79:147–54.
´
Turgeon D, Carrier JS, Levesque E, et al. (2001). Relative enzymatic
activity, protein stability, and tissue distribution of human steroid-
metabolizing UGT2B subfamily members. Endocrinology 142:
778–87.
Uchaipichat V, Mackenzie PI, Guo XH, et al. (2004). Human udp-
glucuronosyltransferases: isoform selectivity and kinetics of 4-methy-
lumbelliferone and 1-naphthol glucuronidation, effects of organic
solvents, and inhibition by diclofenac and probenecid. Drug Metab
Dispos 32:413–23.
Uchihashi S, Nishikawa M, Sakaki T, Ikushiro S. (2013). Comparison of
serotonin glucuronidation activity of UDP-glucuronosyltransferase
1a6a (Ugt1a6a) and Ugt1a6b: evidence for the preferential expression
of Ugt1a6a in the mouse brain. Drug Metab Pharmacokinet 28:260–4.
Watanabe Y, Nakajima M, Yokoi T. (2002). Troglitazone glucuronida-
tion in human liver and intestine microsomes: high catalytic activity of
UGT1A8 and UGT1A10. Drug Metab Dispos 30:1462–9.
Weissbach H, Lovenberg W, Redfield BG, Udenfriend S. (1960). In vivo
metabolism of serotonin and tryptamine: effect of monoamine oxidase
inhibition. J Pharmacol Exp Ther 131:26–30.
¨¨
¨
Kallionpaa RA, Jarvinen E, Finel M. (2015). Glucuronidation of estrone
and 16a-hydroxyestrone by human UGT enzymes: the key roles of
UGT1A10 and UGT2B7. J Steroid Biochem Mol Biol 154:104–11.
King CD, Rios GR, Assouline JA, Tephly TR. (1999). Expression of
UDP-glucuronosyltransferases (UGTs) 2B7 and 1A6 in the human
brain and identification of 5-hydroxytryptamine as a substrate. Arch
Biochem Biophys 365:156–62.
Zheng X, Kang A, Dai C, et al. (2012). Quantitative analysis of
neurochemical panel in rat brain and plasma by liquid chromatog-
raphy–tandem mass spectrometry. Anal Chem 84:10044–51.
Krishnaswamy S, Duan SX, Von Moltke LL, et al. (2003a). Validation of
serotonin (5-hydroxtryptamine) as an in vitro substrate probe for