Phase II metabolism of hesperetin by individual UDP- glucuronosyltransferases and sulfotransferases and rat and human tissue samples

Article abstract of DOI:10.1124/dmd.109.031047

Phase II metabolism by UDP-glucuronosyltransferases (UGTs) and sulfotransferases (SULTs) is the predominant metabolic pathway during the first-pass metabolism of hesperetin (4′-methoxy-3′,5,7- trihydroxyflavanone). In the present study, we have determined the kinetics for glucuronidation and sulfonation of hesperetin by 12 individual UGT and 12 individual SULT enzymes as well as by human or rat small intestinal, colonic, and hepatic microsomal and cytosolic fractions. Results demonstrate that hesperetin is conjugated at positions 7 and 3′ and that major enzyme-specific differences in kinetics and regioselectivity for the UGT and SULT catalyzed conjugations exist. UGT1A9, UGT1A1, UGT1A7, UGT1A8, and UGT1A3 are the major enzymes catalyzing hesperetin glucuronidation, the latter only producing 7-O-glucuronide, whereas UGT1A7 produced mainly 3′-O- glucuronide. Furthermore, UGT1A6 and UGT2B4 only produce hesperetin 7-O-glucuronide, whereas UGT1A1, UGT1A8, UGT1A9, UGT1A10, UGT2B7, and UGT2B15 conjugate both positions. SULT1A2 and SULT1A1 catalyze preferably and most efficiently the formation of hesperetin 3′-O-sulfate, and SULT1C4 catalyzes preferably and most efficiently the formation of hesperetin 7-O-sulfate. Based on expression levels SULT1A3 and SULT1B1 also will probably play a role in the sulfo-conjugation of hesperetin in vivo. The results help to explain discrepancies in metabolite patterns determined in tissues or systems with different expression of UGTs and SULTs, e.g., hepatic and intestinal fractions or Caco-2 cells. The incubations with rat and human tissue samples support an important role for intestinal cells during first-pass metabolism in the formation of hesperetin 3′-O-glucuronide and 7-O-glucuronide, which appear to be the major hesperetin metabolites found in vivo. Copyright

Full text of DOI:10.1124/dmd.109.031047

0090-9556/10/3804-617–625$20.00
DRUG METABOLISM AND DISPOSITION
Copyright © 2010 by The American Society for Pharmacology and Experimental Therapeutics
DMD 38:617–625, 2010
Vol. 38, No. 4
31047/3569496
Printed in U.S.A.
Phase II Metabolism of Hesperetin by Individual UDP-
Glucuronosyltransferases and Sulfotransferases and Rat and
Human Tissue Samples
Walter Brand, Marelle G. Boersma, Hanneke Bik, Elisabeth F. Hoek-van den Hil,
Jacques Vervoort, Denis Barron, Walter Meinl, Hansruedi Glatt, Gary Williamson,
Peter J. van Bladeren, and Ivonne M. C. M. Rietjens
Division of Toxicology, Wageningen University, Wageningen, The Netherlands (W.B., M.G.B., H.B., E.F.H.v.d.H., P.J.v.B.,
I.M.C.M.R.); Nestle´ Research Center, Nestec Ltd., Lausanne, Switzerland (W.B., D.B., G.W., P.J.v.B.); Biqualys, Wageningen,
The Netherlands (J.V.); Department of Nutritional Toxicology, German Institute of Human Nutrition, Potsdam-Rehbru¨ cke,
Nuthetal, Germany (W.M., H.G.); and School of Food Science and Nutrition, University of Leeds, Leeds,
United Kingdom (G.W.)
Received November 5, 2009; accepted January 7, 2010
ABSTRACT:
Phase II metabolism by UDP-glucuronosyltransferases (UGTs) and UGT1A1, UGT1A8, UGT1A9, UGT1A10, UGT2B7, and UGT2B15 con-
sulfotransferases (SULTs) is the predominant metabolic pathway jugate both positions. SULT1A2 and SULT1A1 catalyze preferably
during the first-pass metabolism of hesperetin (4؅-methoxy-3؅,5,7- and most efficiently the formation of hesperetin 3؅-O-sulfate, and
trihydroxyflavanone). In the present study, we have determined SULT1C4 catalyzes preferably and most efficiently the formation of
the kinetics for glucuronidation and sulfonation of hesperetin by hesperetin 7-O-sulfate. Based on expression levels SULT1A3 and
12 individual UGT and 12 individual SULT enzymes as well as by SULT1B1 also will probably play a role in the sulfo-conjugation of
human or rat small intestinal, colonic, and hepatic microsomal hesperetin in vivo. The results help to explain discrepancies in
and cytosolic fractions. Results demonstrate that hesperetin is metabolite patterns determined in tissues or systems with different
conjugated at positions 7 and 3؅ and that major enzyme-specific expression of UGTs and SULTs, e.g., hepatic and intestinal frac-
differences in kinetics and regioselectivity for the UGT and SULT tions or Caco-2 cells. The incubations with rat and human tissue
catalyzed conjugations exist. UGT1A9, UGT1A1, UGT1A7, UGT1A8, samples support an important role for intestinal cells during first-
and UGT1A3 are the major enzymes catalyzing hesperetin gluc- pass metabolism in the formation of hesperetin 3؅-O-glucuronide
uronidation, the latter only producing 7-O-glucuronide, whereas and 7-O-glucuronide, which appear to be the major hesperetin
UGT1A7 produced mainly 3؅-O-glucuronide. Furthermore, UGT1A6
and UGT2B4 only produce hesperetin 7-O-glucuronide, whereas
metabolites found in vivo.
The flavanone hesperetin (4Ј-methoxy-3Ј,5,7-trihydroxyflavanone) phloridzin hydrolase and/or facilitated transport into the intestinal
(Fig. 1) is the aglycone of hesperidin (hesperetin 7-O-rutinoside),
which is the major flavonoid present in sweet oranges (Citrus sinen-
sis) and orange juice and also occurs in other citrus fruits and some
herbs (Toma´s-Barbera´n and Clifford, 2000). Hesperidin and hespere-
tin have been reported to provide beneficial effects on health, includ-
ing reduced risk of osteoporosis (Horcajada et al., 2008).
Upon ingestion, hesperidin has to be hydrolyzed into hesperetin
aglycone by colonic microbiota before its absorption (Ne´meth et al.,
2003). Enzymatic conversion of hesperidin before consumption to the
monosaccharide hesperetin-7-O-glucoside has been demonstrated to
result in absorption in the small intestine after deglucosylation by
cells by a sugar transporter such as sodium-glucose cotransporter 1
followed by intracellular deglucosylation (Nielsen et al., 2006). In the
intestinal cells or during further first-pass metabolism, hesperetin
aglycone is metabolized by UDP-glucuronosyltransferases (UGTs)
and sulfotransferases (SULTs) into, respectively, glucuronidated and
sulfonated metabolites, which have been detected in human and rat
plasma (Manach et al., 2003; Matsumoto et al., 2004; Mullen et al.,
2008; Brett et al., 2009). The intestinal barrier is believed to play a
dominant role in phase II conjugation during the first-pass metabolism
of hesperetin (Silberberg et al., 2006) and in its limited bioavailability
because of efflux of the metabolites back to the intestinal lumen by
ABC transport proteins (Liu and Hu, 2007; Brand et al., 2008).
UGTs form a gene superfamily and currently a total of 22
different UGT proteins have been detected in human tissues,
belonging to either the UGT1A (UGT1A1, UGT1A3, UGT1A4,
UGT1A5, UGT1A6, UGT1A7, UGT1A8, UGT1A9, and UGT1A10), the
This study was supported by the Nestle´ Research Center, Nestec Ltd. (Lau-
sanne, Switzerland).
Article, publication date, and citation information can be found at
http://dmd.aspetjournals.org.
doi:10.1124/dmd.109.031047.
ABBREVIATIONS: UGT, UDP-glucuronosyltransferase; SULT, sulfotransferase; HPLC, high-performance liquid chromatography; DAD, diode
array detector; UDPGA, UDP-glucuronic acid; PAPS, 3Ј-phosphoadenosine 5Ј-phosphosulfate; DMSO, dimethyl sulfoxide.
617

618
BRAND ET AL.
Materials and Methods
Materials. Alamethicin (from Trichoderma viride), hesperetin (pu-
rity Ն95%), L-ascorbic acid, and UDP-glucuronic acid (UDPGA)
were obtained from Sigma-Adlrich (St. Louis, MO), 3Ј-phosphoad-
enosine 5Ј-phosphosulfate (PAPS) was from Fluka (Buchs, Switzer-
land), deuterated acetic acid, dimethyl sulfoxide (DMSO), dipotas-
sium hydrogen phosphate trihydrate, hydrochloric acid, and potassium
dihydrogen phosphate were from Merck (Darmstadt, Germany), ace-
tonitrile and methanol were from Sigma-Aldrich (Steinheim, Ger-
many), Tris was from Invitrogen (Carlsbad, CA), and trifluoroacetic
acid was from Mallinckrodt Baker (Phillipsburg, NJ). Deuterated
methanol-d4 (99.96% d) was obtained from Euriso-Top (Gif-sur-
Yvette, France). Authentic standards of hesperetin 7-O-glucuronide
(purity Ͼ90%), hesperetin 3Ј-O-glucuronide (purity Ͼ90%), and hes-
peretin 7-O-sulfate (purity Ͻ50%) were provided by the Nestle´ Re-
search Center (Lausanne, Switzerland).
UGT Supersomes from cDNA-transfected insect cells expressing
individual human UGTs were obtained from BD Gentest (Woburn,
MA), and their glucuronidation activities toward standard substrates
as described by the supplier were as follows: UGT1A1 (lot 95244)
and UGT1A3 (lot 70200), 817 and 190 pmol minϪ1 mg of proteinϪ1
estradiol 3-glucuronidation activity, respectively; UGT1A4 (lot
95375), 1100 pmol minϪ1 mg of proteinϪ1 trifluoperazine gluc-
uronidation activity; UGT1A6 (lot 70201), UGT1A7 (lot 68106),
UGT1A8 (lot 95862), UGT1A9 (lot 81291), UGT1A10 (lot 96097),
UGT2B4 (lot 93808), UGT2B7 (lot 83494), and UGT2B15 (lot
70203), 5200, 12,000, 630, 7200, 86, 180, 1200, and 3000 pmol
minϪ1 mg of proteinϪ1 7-hydroxy 4-trifluoromethylcoumarin gluc-
uronidation activity, respectively; and UGT2B17 (lot 09302), 1100
pmol minϪ1 mg of proteinϪ1 eugenol glucuronidation activity.
SULTs from cDNA-transfected bacteria expressing quantified con-
centrations of individual human SULT enzymes were prepared as
described elsewhere in detail (Meinl et al., 2006).
Pooled human small intestinal microsomes (batch MIC318012),
pooled rat (male Sprague-Dawley) small intestinal microsomes
(batch MIC323019), pooled human small intestinal cytosol (batch
CYT318004), and pooled rat (male Sprague-Dawley) small intes-
tinal cytosol (batch CYT323008) were obtained from Biopredic
(Rennes, France), without quantified glucuronidation or sulfon-
ation activities. Human single-donor colon microsomes from a
64-year-old male (batch MIC317008), pooled colon microsomes
from rat (male Sprague-Dawley) (batch MIC322003), human sin-
gle-donor colon cytosol from a 64-year-old male (batch
CYT317005), and pooled colon cytosol from rat (male Sprague-
Dawley) (batch CYT322003) were provided by Biopredic, without
quantified glucuronidation or sulfonation activities. Ethical per-
mission for the use of the human tissue extract was obtained by
Biopredic. Pooled human liver microsomes (lot 28831) with 920
pmol minϪ1 mg of proteinϪ1 estradiol 3-glucuronidation activity,
890 pmol minϪ1 mg of proteinϪ1 trifluoperazine glucuronidation
activity, and 2400 pmol minϪ1 mg of proteinϪ1 propofol gluc-
uronidation activity, pooled rat (male Sprague-Dawley) liver mi-
crosomes (lot 83481) without quantified glucuronidation activity,
and pooled human liver cytosol (lot 99925) and pooled rat (male
Sprague-Dawley) liver cytosol (lot 08003) with 320 and 1900 pmol
minϪ1 mg of proteinϪ1 7-hydroxycoumarin sulfotransferase activ-
ity, respectively, as described by the supplier, were provided by
BD Gentest.
FIG. 1. Chemical structure of hesperetin (4Ј-methoxy-3Ј,5,7-trihydroxyflavanone).
UGT2A (UGT2A1, UGT2A2, and UGT2A3), the UGT2B (UGT2B4,
UGT2B7, UGT2B10, UGT2B11, UGT2B15, UGT2B17, and UGT2B28),
the UGT3 (UGT3A1 and UGT3A2), or the UGT8 (UGT8A1) family
(Mackenzie et al., 2005). SULTs form a gene superfamily and a total of 10
different SULT proteins have been detected in human tissues including
SULT1A1, SULT1A2, SULT1A3 (encoded by SULT1A3 and SULT1A4
and therefore also called SULT1A3/4), SULT1B1, SULT1C2, SULT1C4,
SULT1E1, SULT2A1, SULT2B1_v2, and SULT4A1_v2 (Teubner et al.,
2007; Riches et al., 2009). In addition, there are some SULTs that have only
been detected at the mRNA level: SULT2B1_v1, SULT1C3, and SULT6B1
(Meinl et al., 2008a), the latter solely in testis (Freimuth et al., 2004).
SULT1C2, SULT1C4, SULT2B1_v1, SULT2B1_v2, and SULT4A1_v2
are also referred to as SULT1C1, SULT1C2, SULT2B1a, SULT2B1b, and
SULT4A1, respectively, in the literature, not following the nomenclature
proposed by Blanchard et al. (2004).
We previously characterized the metabolism of hesperetin in vitro
using Caco-2 cell monolayers as a model for the small intestinal
barrier and reported that hesperetin is metabolized into 7-O-glucuro-
nide and 7-O-sulfate metabolites (Brand et al., 2008). However,
analysis of metabolites in plasma demonstrated the existence of other
glucuronide and sulfo-conjugates as well (Matsumoto et al., 2004;
Mullen et al., 2008; Brett et al., 2009). Different individual UGTs and
SULTs probably possess different kinetics and regioselectivity for the
conjugation of hesperetin, as has been reported for the glucuronidation
and sulfonation of other flavonoids (Boersma et al., 2002; Otake et al.,
2002; Zhang et al., 2007a; Tang et al., 2009), and, therefore, different
levels of expression of UGTs and SULTs might lead to different
metabolite patterns.
In the present article, we determined the kinetics for the conversion
of hesperetin into glucuronidated and sulfonated metabolites by indi-
vidual UGT and SULT enzymes, respectively. The metabolites
formed were identified by HPLC-DAD in combination with authentic
standards or 1H NMR. The UGT isoforms tested include 12 individual
UGTs reported to be expressed, at least at the mRNA level, in human
intestinal and hepatic cells: UGT1A1, UGT1A3, UGT1A4,
UGT1A6, UGT1A7, UGT1A8, UGT1A9, UGT1A10, UGT2B4,
UGT2B7, UGT2B15, and UGT2B17 (Gregory et al., 2004; Naka-
mura et al., 2008; Izukawa et al., 2009; Ohno and Nakajin, 2009).
The SULT isoforms tested include 12 individual SULTs, which
have been detected in human intestinal and hepatic tissues:
SULT1A1, SULT1A2, SULT1A3, SULT1B1, SULT1C2,
SULT1E1, SULT1C4, and SULT2A1 (Teubner et al., 2007; Meinl
et al., 2009; Riches et al., 2009) and in addition SULT1C3,
SULT2B1_v1, SULT2B1_v2, and SULT4A1_v2, which have not
been detected on the protein level in these tissues (Meinl et al.,
2008a,b).
Furthermore, we studied the apparent kinetics of glucuronidation
and sulfonation using, respectively, microsomes and cytosol derived
from tissues from human and rat playing a role during the first-pass
Incubations with UGTs or Rat or Human Microsomes. To
metabolism of hesperetin after ingestion of hesperidin or hesperetin study glucuronidation of hesperetin by individual UGTs or microso-
7-O-glucoside: the small intestine, the colon, and the liver. mal preparations, incubation mixtures (total volume 200 ␮l) were

PHASE II METABOLISM OF HESPERETIN BY UGTs AND SULTs
619
prepared containing 10 mM MgCl2, 25 ␮g/ml alamethicin added from gradient for the analysis of samples from the incubations with cytosol
a 200ϫ concentrated stock solution in methanol (final concentration or SULTs started at 0% acetonitrile in nanopure water containing
0.5% methanol), 0.1, 0.2, or 0.5 mg/ml protein, and 1 mM UDPGA in 0.1% trifluoroacetic acid, increasing to 10% acetonitrile in 5 min, to
50 mM Tris-HCl (pH 7.5) (Boersma et al., 2002). The reaction was 15% in the following 16 min, to 50% in the next 16 min, and to 80%
started by addition of hesperetin from a 200ϫ concentrated stock in 1 min, followed by a cleaning and reequilibration step. The gradient
solution in DMSO (final concentration 0.5% DMSO) and incubated to analyze the samples from the incubations with microsomes or
for 5 min (UGT1A10, UGT2B7, and human and rat liver micro- UGTs started at 0% acetonitrile in nanopure water containing 0.1%
somes), 10 min (UGT1A1, UGT1A3, UGT1A7, UGT1A8, UGT1A9, trifluoroacetic acid, increasing to 25% acetonitrile in 10 min, which
UGT2B15, and human and rat small intestinal microsomes), 15 min condition was kept for 21 min, whereafter the percentage of acetoni-
(human and rat colon microsomes), or 30 min (UGT1A4, UGT1A6, trile was increased to 60% in 7 min and to 80% in 1 min, followed by
UGT2B4, and UGT2B17) at 37°C. The final concentration series was a cleaning and reequilibration step. DAD-UV spectra were recorded
1, 2.5, 5, 10, 15, 25, 35, and 50 ␮M (n ϭ 1–2) or 2.5, 5, 10, 15, 25, between 200 and 420 nm, and chromatograms acquired at 280 nm
and 50 ␮M (n ϭ 1–3) hesperetin for all UGTs tested. The reaction was were used for presentation and quantification.
terminated by addition of 50 ␮l of acetonitrile. Under these conditions
Metabolite Identification and Quantification. Hesperetin 7-O-
metabolite formation was linear in time and with the amount of glucuronide, hesperetin 3Ј-O-glucuronide, and hesperetin 7-O-sulfate
protein added (data not shown). Activity is expressed in nanomoles were identified using authentic standards by their HPLC-DAD reten-
per minute per milligram of protein.
tion times and UV spectra. With use of the HPLC gradient for analysis
Incubations with SULTs or Rat or Human Cytosol. To study of the samples from the glucuronidation reactions, the retention times
sulfonation of hesperetin, incubation mixtures (total volume 100 ␮l) were as follows: hesperetin, 37.2 min (UVmax ϭ 285.9 nm); hespere-
were prepared containing 5 mM MgCl2, 100 ␮M PAPS, and 0.04 to tin 7-O-glucuronide, 17.7 min (UVmax ϭ 285.9 nm); and hesperetin
0.23 mg/ml protein (cytosol), or 0.03 to 0.1 mg/ml protein (individual 3Ј-O-glucuronide, 18.5 min (UVmax ϭ 285.9 nm). With use of the
SULTs) in 50 mM potassium phosphate (pH 7.4). The reaction was HPLC gradient for the analysis of the samples from the sulfonation
started by addition of hesperetin (final concentration series was 1, 2.5, reactions, the retention times were as follows: hesperetin, 36.7 min
5, 10, 15, 25, 35, and 50 ␮M hesperetin) from a 100-fold concentrated (UVmax ϭ 285.9 nm) and hesperetin 7-O-sulfate, 31.6 min (UVmax ϭ
stock solution in DMSO (final concentration 1% DMSO) and incu- 281.2 nm, shoulder at 338 nm). Another metabolite resulting from the
bated for 3 min (SULT1A1, SULT1A2, SULT1C4, and human and rat cytosolic and SULT incubations with PAPS at a retention time of 30.7
liver cytosol), 5 min (SULT1E1 and human small intestinal cytosol), min (UVmax ϭ 290.7 nm) was repeatedly collected during HPLC-
9 min (SULT1A3), 10 min (SULT1B1), 90 min (human colon cy- DAD separation, freeze-dried, and resolved in acidified, deuterated
1
1
tosol), 120 min (SULT1C2 and SULT2A1), 150 min (rat small methanol for H-NMR analysis. H-NMR analysis revealed this me-
intestinal and colon cytosol), or 180 min (SULT1C3, SULT2B1_v1, tabolite to be hesperetin 3Ј-O-sulfate (for details, see Results). Hes-
SULT2B1_v2, and SULT4A1_v2) at 37°C. Because SULT1A1, peretin 7-O-glucuronide and hesperetin 3Ј-O-glucuronide were
SULT1A2, SULT1C4, SULT1E1, and some cytosolic fractions quantified on the basis of a calibration curve made with authentic
showed substrate inhibition at concentrations Ͼ1, Ͼ1, and Ͼ3 ␮M, standards. Hesperetin 7-O-sulfate and hesperetin 3Ј-O-sulfate were
respectively, additional series of 0.1, 0.15, 0.25, 0.35, 0.5, 0.75, 1, and quantified indirectly using the calibration curve for hesperetin, and
1.5 ␮M hesperetin (SULT1A1, SULT1A2, and SULT1C4), or a series multiplication factors were determined by enzymatic hydrolysis of
of 0.1, 0.25, 0.5, 0.75, 1, 1.5, 2.5, and 3.5 ␮M hesperetin (SULT1E1 hesperetin 7-O-sulfate (factor 1.27) and hesperetin 3Ј-O-sulfate (fac-
and cytosolic fractions) were used. The reaction was terminated by tor 0.86) into unconjugated hesperetin, which could be quantified with
addition of 25 ␮l of acetonitrile. Under these conditions metabolite a calibration curve.
formation was linear with time and the amount of protein added (data
1H NMR Analysis. 1H NMR analysis was performed using an
not shown). Activity is expressed in nanomoles per minute per mil- Avance III 600 MHz (Bruker, Ettlingen, Germany) with cryoprobe. A
ligram of protein for the cytosolic fractions and in nanomoles per Noesygppr1d pulse sequence with 3-s delay, 0.1-s mixing time, and
minute per milligram of SULT protein for the individual SULTs 1.8-s acquisition time was used (18,028 Hz sweep width; 64 K data
(Meinl et al., 2006).
points). Spectra were obtained at 25°C. Resonances are reported
Enzyme Kinetics. To determine the kinetics for glucuronidation relative to methanol-d4 at 3.34 ppm.
and sulfonation, incubations were performed as described above. The
maximum velocity (Vmax) and Michaelis-Menten constant (Km) for
Results
the formation of the different phase II metabolites of hesperetin were
Identification of Hesperetin Metabolites. Figure 2 depicts part of
determined by fitting the data to the Michaelis-Menten steady-state a chromatogram from the HPLC-DAD analysis of the supernatant of
model v ϭ Vmax/(1 ϩ (Km/[S])), with [S] being the hesperetin con- an incubation of hesperetin with UGT1A9 and UDPGA. Two metab-
centration, using the LSW data analysis toolbox (version 1.1.1; MDL olites were formed and identified as hesperetin 7-O-glucuronide (tR,
Information Systems, San Ramon, CA). For reactions demonstrating 17.7 min; UVmax, 285.9 nm) and hesperetin 3Ј-O-glucuronide (tR,
substrate inhibition, the Vmax, Km, and inhibition constant (Ki) were 18.5 min; UVmax, 285.9 nm) on the basis of analysis of the corre-
determined by fitting the data to the substrate inhibition equation v ϭ sponding authentic metabolite standards. Figure 3 depicts part of a
V
max ϫ [S]/[Km ϩ [S] ϫ (1 ϩ [S]/Ki)] using GraphPad Prism (version chromatogram from the HPLC-DAD analysis of the supernatant from
5.02; GraphPad Software Inc., San Diego, CA). an incubation of hesperetin with SULT1A3 and PAPS. Two metab-
HPLC Analysis. To analyze the formation of hesperetin metabo- olites were formed, one of which was identified as hesperetin 7-O-
lites in the enzymatic incubations, reaction mixtures were centrifuged sulfate (tR, 31.6 min; UVmax, 281.2 nm, shoulder at 338 nm) on the
for 4 min at 16,000g and samples of 50 ␮l of the supernatant were basis of analysis of the corresponding authentic metabolite standard.
injected onto a Alliance 2695 separation module (Waters, Milford, The fraction containing the second metabolite (tR, 30.7 min; UVmax
,
MA) connected to a 2996 DAD (Waters) with an Alltima C18 5-␮m 290.7 nm), which could not be identified with the available authentic
150 ϫ 4.6 mm column with 7.5 ϫ 4.6 mm guard column (Alltech, standards, was collected, freeze-dried, dissolved in acidified metha-
1
Breda, The Netherlands). Elution was at a flow rate of 1 ml/min. The nol, and analyzed by H-NMR.

620
BRAND ET AL.
FIG. 2. Representative section of the HPLC chromatogram of
the supernatant from the incubation of hesperetin with
UGT1A9 and UDPGA showing the hesperetin glucuronide
conjugates. AU, absorbance units.
FIG. 3. Representative section of the HPLC chromatogram of
the supernatant from the incubation of hesperetin with
SULT1A3 and PAPS showing the hesperetin sulfo-conjugates.
AU, absorbance units.
TABLE 1
1H NMR data of hesperetin and the metabolite (identified as hesperetin 3Ј-O-sulfate) formed in the incubation mixture of hesperetin with specific SULT isoforms and
human small intestinal cytosol and PAPS
The differences in chemical shift values of the protons in the metabolite compared with the chemical shift values of the same protons in hesperetin are given in parentheses.
Compound
H6
H8
H3 a
H3 b
H2
H2Ј
H5Ј
H6Ј
Hesperetin
1H NMR chemical shift (ppm)
J (Hz)
Peak splitting
5.91
2.2
d
5.95
2.2
d
3.11
17.0; 12.9
dd
2.74
17.0; 3.0
dd
5.36
12.9; 3.0
dd
6.98
1.7
d
6.96
8.4
d
6.94
1.7; 8.4
dd
Metabolite
1H NMR chemical shift (ppm)
J (Hz)
Peak splitting
5.91
2.1
d
5.95
2.1
d
3.10 (Ϫ0.01)
17.1; 12.9
dd
2.80 (ϩ0.06)
17.1; 3.0
dd
5.41 (ϩ0.05)
12.9; 3.0
dd
7.64 (ϩ0.66)
7.08 (ϩ0.12)
7.27 (ϩ0.33)
2.1; 8.4
dd
2.1
d
8.4
d
d, doublet; dd, doublet of doublets.
Modern NMR instruments with dedicated cryoprobes provide 7 of the hesperetin molecule. Together these data identify the
excellent sensitivity with relatively small amounts of material, unknown metabolite as hesperetin 3Ј-O-sulfate.
provided that this material is of high purity. The sample analyzed
Glucuronidation by Individual UGT Enzymes. Glucuronidation
contained approximately 0.5 nmol (150 ng) of the unknown sul- of hesperetin was characterized using human recombinant UGT
1
fonated metabolite. Table 1 summarizes the H NMR data for this enzymes. The Vmax and Km values obtained for the formation of hes-
unknown sulfonated hesperetin metabolite as well as that of the peretin 7-O-glucuronide and hesperetin 3Ј-O-glucuronide by the
parent compound hesperetin. Comparison of the chemical shift various UGT enzymes are shown in Table 2, as well as the catalytic
values and J values of the corresponding protons in hesperetin and efficiencies (Vmax/Km) derived from these values. The results re-
in the unknown sulfonated hesperetin metabolite reveals changes, veal that hesperetin is most efficiently glucuronidated by
1
especially in the H NMR data of the protons of the B-ring upon UGT1A9. The efficiency of glucuronidation (Vmax/Km) decreases
conjugate formation: a relative shift of ϩ0.66 ppm for H2Ј and a in the order of UGT1A9 Ͼ UGT1A1 Ͼ UGT1A7 Ͼ UGT1A3 Ͼ
relative shift of ϩ0.33 ppm for H6Ј (Table 1). This result indicates UGT1A8 Ͼ UGT1A10 Ͼ UGT2B7 ϭ UGT2B15 Ͼ UGT2B4. The
a modification of the hydroxyl moiety at C3Ј, resulting in a rate of formation of hesperetin 7-O-glucuronide by UGT1A6 was
relatively large change in the chemical shift values of the protons virtually linear with the applied concentration: 0.018 nmol minϪ1
H2Ј and H6Ј at the positions ortho and para with respect to the mg of proteinϪ1 7-O-glucuronide formed per micromole of hes-
modified hydroxyl moiety and is in line with earlier 1H NMR peretin, which excluded determination of the kinetic parameters
studies on metabolites of quercetin (van der Woude et al., 2004). Vmax and Km by fitting the data to the Michaelis-Menten equation.
The signals of the protons H6 and H8 remained unchanged, ex- Higher doses of hesperetin could not be tested because of the
cluding modification of the other hydroxyl-groups at position 5 or limited solubility of hesperetin in aqueous solutions. The enzymes

PHASE II METABOLISM OF HESPERETIN BY UGTs AND SULTs
621
TABLE 2
Vmax and Km values determined from three to four independent curves and the catalytic efficiencies (Vmax/Km) derived from these values, for the glucuronidation of
hesperetin (1 up to 50 ␮M) by individual UGT enzymes
Data are means Ϯ S.E.M.
7-O-Glucuronidation
3Ј-O-Glucuronidation
UGT Isoform
Km
Vmax
Vmax/Km
Km
Vmax
Vmax/Km
␮M
nmol minϪ1 mg proteinϪ1
␮l minϪ1 mg proteinϪ1
␮M
nmol minϪ1 mg proteinϪ1
␮l minϪ1 mg proteinϪ1
UGT1A1
UGT1A3
UGT1A4
UGT1A6
UGT1A7
UGT1A8
UGT1A9
UGT1A10
UGT2B4
UGT2B7
UGT2B15
UGT2B17
4.0 Ϯ 1.3
16.5 Ϯ 4.6
N.D.a
1.35 Ϯ 0.29
3.94 Ϯ 0.74
N.D.a
339
239
1.2 Ϯ 0.5
N.D.
N.D.
0.46 Ϯ 0.06
N.D.
N.D.
376
a
—
b
b
—
—
18b
5
N.D.
N.D.
105 Ϯ 67.9
63.3 Ϯ 11.8
5.3 Ϯ 0.8
30.4 Ϯ 8.2
119 Ϯ 43.7
53.9 Ϯ 17.6
34.5 Ϯ 1.6
N.D.c
0.47 Ϯ 0.04
2.33 Ϯ 1.20
2.19 Ϯ 0.10
2.82 Ϯ 0.69
0.42 Ϯ 0.09
1.53 Ϯ 0.25
0.28 Ϯ 0.01
N.D.c
8.7 Ϯ 1.6
19.2 Ϯ 3.9
4.0 Ϯ 0.3
31.2 Ϯ 12.6
N.D.
42.4 Ϯ 13.9
29.9 Ϯ 0.3
N.D.c
2.47 Ϯ 0.63
3.18 Ϯ 1.78
3.89 Ϯ 0.20
0.89 Ϯ 0.29
N.D.
0.88 Ϯ 0.23
1.19 Ϯ 0.02
N.D.c
285
166
981
28
37
411
93
4
28
8
21
40
c
c
—
—
N.D., not detectable.
a UGT1A4 very poorly glucuronidated hesperetin into solely hesperetin 7-O-glucuronide, only measurable at the highest test concentration (50 ␮M), precluding determination of kinetics.
b Conjugation velocity by UGT1A6 of hesperetin into hesperetin 7-O-glucuronide occurred in a linear manner with dose (0.018 nmol minϪ1 mg of proteinϪ1 ␮mol of hesperetinϪ1), precluding
determination of the individual Michaelis-Menten parameters Vmax and Km, but allowing the definition of the catalytic efficiency because the slope of the linear relationship between the rate of
formation as a function of the substrate concentration equals Vmax/Km
.
c UGT2B17 very poorly glucuronidated hesperetin, only measurable at the highest test concentrations (50 ␮M).
UGT1A4 and UGT2B17 demonstrated only very poor glucuronida-
Sulfonation by Individual SULT Enzymes. The Vmax and Km
tion activity toward hesperetin under the conditions used in this values determined for the formation of hesperetin 7-O-sulfate and
study, precluding determination of the kinetics. Figure 4 presents an hesperetin 3Ј-O-sulfate by individual human SULTs are shown in
overview of the regioselectivity of the glucuronidation of hesperetin Table 3, as well as the catalytic efficiencies (Vmax/Km) derived
by the individual UGTs at a concentration of 10 ␮M hesperetin. For from these values. SULT1A1 demonstrated strong substrate inhi-
all UGTs, the regioselectivity at 1 or 50 ␮M hesperetin was similar to bition at hesperetin concentrations Ͼ0.15 ␮M, precluding deter-
that obtained at 10 ␮M. UGT1A3, UGT1A6, and UGT2B4 catalyze mination of kinetic parameters. The high rate of 3Ј-O-sulfonation
glucuronidation specifically at the hydroxyl moiety at C7 of hespere- up to 117 nmol minϪ1 mg of SULT1A1Ϫ1 (at 0.15 ␮M hesperetin)
tin, whereas UGT1A7 almost solely conjugated the hydroxyl moiety indicates that SULT1A1-mediated sulfonation could probably play
at C3Ј. UGT1A1, UGT1A10, and UGT2B7 converted hesperetin into an important role in the conjugation of hesperetin at low concen-
both hesperetin 3Ј-O-glucuronide and hesperetin 7-O-glucronide, trations. SULT1A2, SULT1C4, and SULT1E1 also demonstrated
however preferentially into the latter, whereas UGT1A8, UGT1A9, substrate inhibition at concentrations Ͼ1, Ͼ1, and Ͼ3 ␮M, respec-
and UGT2B15 preferentially conjugated the hydroxyl moiety of hes- tively. The catalytic efficiency of sulfonation (Vmax/Km) of the
peretin at position 3Ј. Overall, relatively more hesperetin 7-O-gluc- SULTs (other than SULT1A1) decreases in the order of
uronide is formed at higher substrate concentrations (Table 2).
SULT1C4 Ͼ SULT1A2 Ͼ SULT1E1 Ͼ SULT1A3 Ͼ SULT1B1 Ͼ
SULT1C2 Ͼ SULT2A1. The isoenzymes SULT1C3, SULT2B1_v1,
SULT2B1_v2, and SULT4A1_v2 did not show any sulfonation
activity toward hesperetin under the conditions used in this study.
SULT1C4 and SULT2A1 selectively catalyzed the sulfonation at
the hydroxyl moiety of position 7 of hesperetin, whereas
SULT1A1, SULT1A2, and SULT1E1 solely conjugated the hy-
droxyl moiety at position 3Ј (Fig. 5). SULT1C2 converted hes-
peretin into both hesperetin 3Ј-O-sulfate and hesperetin 7-O-sulfate,
however preferentially into the latter, whereas SULT1A3 and
SULT1B1 preferentially conjugated the hydroxyl moiety of hespere-
tin at position 3Ј (Fig. 5). For all SULTs, the regioselectivity at 1 or
50 ␮M hesperetin was similar to that obtained at 10 ␮M.
Glucuronidation by Human and Rat Tissue Samples. The ap-
parent Vmax and Km values for the formation of hesperetin 7-O-
glucuonide and hesperetin 3Ј-O-glucuronide by human and rat micro-
somal fractions from different tissues are shown in Table 4, as well as
the apparent catalytic efficiencies (Vmax/Km) derived from these val-
ues. Hesperetin was converted into both glucuronide metabolites by
microsomes from all human and rat tissues tested. Generally, the
affinity was higher (Km lower) for glucuronidation at position 3Ј,
whereas the capacity (Vmax) was higher for glucuronidation at position
7 (Table 4), and, as a result, at higher hesperetin concentrations
FIG. 4. Regioselectivity of the glucuronidation of hesperetin at position 7 (f) or
position 3Ј (Ⅺ) by different UGT enzymes and human and rat microsomes ex-
pressed as percentage of the total amount of hesperetin glucuronides formed at a 10
␮M hesperetin concentration. HLM, human liver microsomes; RLM, rat liver
microsomes; HSIM, human small intestinal microsomes; RSIM, rat small intestinal
microsomes; HCM, human colon microsomes; RCM, rat colon microsomes.

622
BRAND ET AL.
TABLE 3
Vmax and Km values determined from three independent curves and the catalytic efficiencies (Vmax/Km) derived from these values for the sulfonation of hesperetin
(1 up to 50 ␮M unless stated otherwise) by individual SULT enzymes
Data are means Ϯ S.E.M.
7-O-Sulfonation
3Ј-O-Sulfonation
SULT Isoform
Km
Vmax
Vmax/Km
Km
Vmax
Vmax/Km
␮M
nmol minϪ1 mg proteinϪ1
␮l minϪ1 mg proteinϪ1
␮M
nmol minϪ1 mg proteinϪ1
␮l minϪ1 mg proteinϪ1
a
a
SULT1A1
SULT1A2
SULT1A3
SULT1B1
SULT1C2
SULT1C3
SULT1C4
SULT1E1
SULT2A1
SULT2B1_v1
SULT2B1_v2
SULT4A1_v2
N.D.
N.D.
N.D.
N.D.
Ͻ0.15a
0.5 Ϯ 0.2b
13.2 Ϯ 3.0
4.3 Ϯ 0.2
28.3 Ϯ 14.5
N.D.
—
—
454 Ϯ 94.8b
276 Ϯ 24.8
21.5 Ϯ 2.14
1.45 Ϯ 0.46
N.D.
881,553b
20,964
5003
12.5 Ϯ 3.3
3.9 Ϯ 0.5
66.7 Ϯ 16.1
N.D.
89.9 Ϯ 7.43
3.54 Ϯ 0.66
18.8 Ϯ 6.83
N.D.
7178
899
282
51
0.1 Ϯ 0.0c
N.D.
87.4 Ϯ 8.13c
N.D.
1,117,263c
127
N.D.
N.D.
2.5 Ϯ 1.0d
N.D.
538 Ϯ 134d
N.D.
219,242d
80.2 Ϯ 21.0
N.D.
10.2 Ϯ 1.96
N.D.
N.D.
N.D.
N.D.
N.D.
N.D.
N.D.
N.D.
N.D.
N.D.
N.D.
N.D., not detectable.
a SULT1A1 demonstrated strong substrate inhibition at concentrations Ͼ0.1 ␮M, precluding determination of kinetic parameters.
b SULT1A2 showed substrate inhibition at concentrations Ͼ1 ␮M hesperetin; Ki ϭ 1.9 ␮M.
c SULT1C4 showed substrate inhibition at concentrations Ͼ1 ␮M hesperetin; Ki ϭ 23.5 ␮M.
d SULT1E1 showed substrate inhibition at concentrations Ͼ3 ␮M hesperetin; Ki ϭ 12.9 ␮M.
hibition at hesperetin concentrations Ͼ0.25 ␮M, whereas the sample
of human liver cytosol demonstrated very little 7-O-sulfate formation
(Table 4).
Discussion
In the present study, the kinetics for the conjugation of hes-
peretin by individual UGT and SULT enzymes and rat or human
microsomes and cytosol from small intestine, colon, and liver was
characterized. Hesperetin was conjugated at the C7 and C3Ј hy-
droxyl moieties. It is interesting to note that conjugation at the C5
hydroxyl moiety was not catalyzed. This phenomenon can be
explained by the strong intramolecular hydrogen bond between this
hydroxyl moiety and the C4 carbonyl moiety preventing the phase
II conjugation (Exarchou et al., 2002). Of all UGTs tested,
UGT1A9, UGT1A1, UGT1A7, UGT1A3, and UGT1A8 demon-
strated the highest catalytic efficiencies (Table 2). These UGTs
have been reported to efficiently catalyze the glucuronidation of
other flavonoids as well, as was recently reviewed Zhang et al.
(2007b), although the relative efficiency of different UGTs seems
to be highly dependent on the flavonoid structure involved. Hes-
peretin, as other flavonoids, seems not to be a suitable substrate for
UGT1A4 (Walle et al., 2000; Boersma et al., 2002; Tang et al.,
FIG. 5. Regioselectivity of the sulfonation of hesperetin at position 7 (f) or
position 3Ј (Ⅺ) by different SULT enzymes and human and rat cytosol expressed as
percentage of the total amount of hesperetin sulfates formed at a 10 ␮M hesperetin
concentration. HLC, human liver cytosol; RLC, rat liver cytosol; HSIC, human
small intestinal cytosol; HCC, human colon cytosol; RCC, rat colon cytosol.
relatively more hesperetin 7-O-glucuronide than hesperetin 3Ј-gluc- 2009). The regioselectivity of hesperetin glucuronidation varied
uronide is formed. enzyme specifically (Fig. 4). Differences in the regioselectivity of
Sulfonation by Human and Rat Tissue Samples. The apparent the flavonoid conjugation by different individual UGTs has also
Vmax and Km values determined for the formation of hesperetin been reported for the conjugation of other flavonoids (Boersma et
7-O-sulfate and hesperetin 3Ј-O-sulfate by human and rat cytosol al., 2002; Otake et al., 2002; Zhang et al., 2007a; Tang et al.,
from different tissues are shown in Table 4, as well as the apparent 2009), the regioselectivity being dependent on the isoenzyme
catalytic efficiencies (Vmax/Km) derived from these values. Hesperetin involved, the flavonoid converted, and the substrate concentration.
was predominantly converted into hesperetin 3Ј-O-sulfate by human For instance, luteolin (3Ј,4Ј,5,7-tetrahydroxyflavone) was almost
small intestinal cytosol with a Km of 0.6 ␮M and a capacity of 0.79 solely (98% of HPLC chromatogram peak area) converted into a
nmol minϪ1 mg of proteinϪ1, whereas rat small intestinal cytosol did 7-O-glucuronide metabolite by UGT1A6, whereas quercetin
not show sulfonation activity toward hesperetin (Table 4). Rat as well (3,3Ј,4Ј,5,7-tetrahydroxyflavone), bearing one extra hydroxyl moi-
as human colonic cytosol showed low catalytic efficiencies. Liver ety, was metabolized into its 4Ј-O-glucuronide (32%), 7-O-
cytosol of both species converted hesperetin into hesperetin 3Ј-O- glucuronide (30%), 3Ј-O-glucuronide (22%), and 3-O-glucuronide
sulfate already at low concentrations and demonstrated substrate in- (16%) by UGT1A6 (Boersma et al., 2002).
hibition at hesperetin concentrations Ͼ0.25 ␮M. It is remarkable that
Of the SULT enzymes (apart from SULT1A1), SULT1A2,
rat liver cytosol also demonstrated efficient conversion of hesperetin SULT1C4, and to a lesser extent SULT1E1 and SULT1A3 dem-
into hesperetin 7-O-sulfate at low concentrations, with substrate in- onstrated the highest catalytic efficiencies for the sulfonation of

PHASE II METABOLISM OF HESPERETIN BY UGTs AND SULTs
623
hesperetin (Table 3). SULT1A1, SULT1A3, and SULT1E1 have
been reported to sulfonate other flavonoids as well (Otake et al.,
2002; Nakano et al., 2004; Ung and Nagar, 2007). In the present
study, SULT1A1, SULT1A2, and SULT1E1 solely catalyzed the
formation of hesperetin 3Ј-O-sulfate, whereas SULT1C4 and
SULT2A1 solely catalyzed the formation of hesperetin 7-O-sulfate
(Fig. 5). The regioselectivity of flavonoid sulfonation appears to be
dependent on the SULT isoenzyme as well as on the flavonoid
studied: daidzein (4Ј,7-dihydroxyisoflavone) and genistein (4Ј,5,7-
trihydroxyisoflavone) were reported to be predominantly sulfated
by SULT1A1 at position 7 rather than at position 4Ј, whereas the
hydroxyl moieties at both positions were sulfonated with similar
efficiency by SULT1E1 (Nakano et al., 2004). At low concentra-
tions of hesperetin, SULT1A2, SULT1C4, SULT1E1 (Table 3),
and especially SULT1A1 demonstrated substrate inhibition. This
property of SULTs in the conjugation of flavonoids at low con-
centrations is also reported for the SULT1A1-mediated sulfonation
of daidzein (Ͼ1.5 ␮M) and genistein (Ͼ2 ␮M) (Nakano et al.,
2004) and for the SULT1E1-mediated sulfonation of quercetin and
chrysin (Ung and Nagar, 2007).
␮
␮
␮
␮
Incubations of hesperetin with human and rat microsomal frac-
tions in the presence of UDPGA resulted in formation of hesperetin
7-O-glucuronide and hesperetin 3Ј-O-glucuronide (Table 4). Based
on the kinetics of the individual human UGTs (Table 2) and the
data on UGT mRNA expression levels (Gregory et al., 2004;
Nakamura et al., 2008; Izukawa et al., 2009; Ohno and Nakajin,
2009), the 7-O-glucuronidation of hesperetin in human tissue frac-
tions is probably catalyzed by UGT1A1 and UGT1A9. The 3Ј-O-
glucuronidation of hesperetin by human microsomes is probably
catalyzed by UGT1A9 and UGT1A1, whereas in the human small
intestinal and colonic microsomes UGT1A7 and UGT1A8 may
contribute as well. However, one should keep in mind that selec-
tivity profiles by single UGTs in complete systems, in which
protein-protein interactions may occur, may be different from
those in in vitro model systems (Fujiwara et al., 2007). Taking the
catalytic efficiencies (Table 2) and the mRNA expression levels of
the rat ortholog UGTs into account (Shelby et al., 2003), it can be
foreseen that, in rat liver and rat intestinal microsomes, rat
UGT1A1 and rat UGT1A7 are probably responsible for the gluc-
uronidation of hesperetin.
␮
␮
␮
␮
␮
␮
␮
Incubations with the human cytosolic fractions demonstrated pref-
erential sulfonation of position 3Ј of hesperetin (Table 4). Although
SULT1A2, SULT1C4, and SULT1E1 demonstrate high catalytic ef-
ficiencies based on expression levels (Teubner et al., 2007; Riches et
al., 2009), they are minor SULT isoforms in the intestine and liver. It
is concluded that SULT1A1 in particular is involved in the sulfonation
of hesperetin in the human liver, whereas SULT1B1 and SULT1A3
will preferably contribute to the intestinal sulfonation of hesperetin.
Our incubations with human cytosol demonstrating predominant for-
mation of hesperetin 3Ј-O-sulfate, the major metabolite formed by
SULT1A1 as well as by SULT1B1 and SULT1A3, support such a
notion. Hesperetin was not sulfonated by rat small intestinal cytosol,
which corresponds with the negligible SULT expression in the small
intestine of rats (Meinl et al., 2009). The hepatic expression of the rat
ortholog of SULT1C4, a form not detected in human liver (Sakakibara
et al., 1998), probably explains the formation of hesperetin 7-O-
sulfate by rat liver cytosol.
␮
␮
␮
When both SULTs and UGTs play a role at the same time, such
as in the in vivo situation, the existence of mixed conjugates has
been reported as well. In a study in which human volunteers were
given up to 1 liter of orange juice providing 444 mg/l hesperidin,
the circulating forms of hesperetin in the plasma consisted of

624
BRAND ET AL.
Freimuth RR, Wiepert M, Chute CG, Wieben ED, and Weinshilboum RM (2004) Human
cytosolic sulfotransferase database mining: identification of seven novel genes and pseudo-
genes. Pharmacogenomics J 4:54–65.
Fujiwara R, Nakajima M, Yamanaka H, Katoh M, and Yokoi T (2007) Interactions between
human UGT1A1, UGT1A4, and UGT1A6 affect their enzymatic activities. Drug Metab
Dispos 35:1781–1787.
Gregory PA, Lewinsky RH, Gardner-Stephen DA, and Mackenzie PI (2004) Regulation of
UDP glucuronosyltransferases in the gastrointestinal tract. Toxicol Appl Pharmacol 199:
354–363.
Horcajada MN, Habauzit V, Trzeciakiewicz A, Morand C, Gil-Izquierdo A, Mardon J, Lebecque
P, Davicco MJ, Chee WS, Coxam V, et al. (2008) Hesperidin inhibits ovariectomized-induced
osteopenia and shows differential effects on bone mass and strength in young and adult intact
rats. J Appl Physiol 104:648–654.
Izukawa T, Nakajima M, Fujiwara R, Yamanaka H, Fukami T, Takamiya M, Aoki Y,
Ikushiro S, Sakaki T, and Yokoi T (2009) Quantitative analysis of UDP-glucuronosyl-
transferase (UGT) 1A and UGT2B expression levels in human livers. Drug Metab Dispos
37:1759–1768.
Liu X, Tam VH, and Hu M (2007) Disposition of flavonoids via enteric recycling: determination
of the UDP-glucuronosyltransferase isoforms responsible for the metabolism of flavonoids in
intact Caco-2 TC7 cells using siRNA. Mol Pharm 4:873–882.
glucuronides (87%) and sulfoglucuronides (13%) as determined
after specific enzymatic hydrolysis (Manach et al., 2003). In an-
other study in which human volunteers were given 250 ml of
orange juice containing 410 mg/l hesperidin, only hesperetin gluc-
uronides were detected in the plasma; however, substantial
amounts of hesperetin sulfoglucuronides were detected in the urine
as indicated by liquid chromatography-tandem mass spectrometry
(Mullen et al., 2008). The authors argued that the kidney may be
involved in postabsorption phase II metabolism, which could be
explained by the expression of SULT1A1 in the kidneys (Meinl et
al., 2006; Riches et al., 2009), the enzyme for which we found a
high affinity toward hesperetin resulting in sulfonation at very low
concentrations. In a third study in which human volunteers were
given oranges or orange juice providing, respectively, 161 or 145
mg of hesperidin, hesperetin 7-O-glucuronide and 3Ј-O-
glucuronide were detected in blood and plasma, as well as hes-
peretin 3Ј-O-sulfate, as qualified by liquid chromatography-
tandem mass spectrometry and metal complexation techniques
(Brett et al., 2009). The absence of hesperetin 7-O-sulfate in these
human volunteers is supported by the sulfonation kinetics found in
the present study.
Liu Z and Hu M (2007) Natural polyphenol disposition via coupled metabolic pathways. Expert
Opin Drug Metab Toxicol 3:389–406.
Mackenzie PI, Bock KW, Burchell B, Guillemette C, Ikushiro S, Iyanagi T, Miners JO, Owens
IS, and Nebert DW (2005) Nomenclature update for the mammalian UDP glycosyltransferase
(UGT) gene superfamily. Pharmacogenet Genomics 15:677–685.
Manach C, Morand C, Gil-Izquierdo A, Bouteloup-Demange C, and Re´me´sy C (2003) Bioavail-
ability in humans of the flavanones hesperidin and narirutin after the ingestion of two doses
of orange juice. Eur J Clin Nutr 57:235–242.
Matsumoto H, Ikoma Y, Sugiura M, Yano M, and Hasegawa Y (2004) Identification and
quantification of the conjugated metabolites derived from orally administered hesperidin in rat
plasma. J Agric Food Chem 52:6653–6659.
We recently analyzed the metabolism of hesperetin in vitro using
Caco-2 cell monolayers as a model of the intestinal barrier. After
incubations of hesperetin with Caco-2 cell monolayers, formation
of hesperetin 7-O-glucuronide and 7-O-sulfate was observed,
whereas no metabolites of hesperetin conjugated at position 3Ј
were detected. These observations could be explained by the
relatively strong expression of SULT1C4 (Tamura et al., 2001;
Meinl et al., 2008b) and UGT1A6 (Paine and Fisher, 2000), both
enzymes specifically catalyzing the conjugation at position 7,
compared with other SULT or UGT forms in Caco-2 cells. More-
over, small interfering RNA-mediated UGT1A6 silencing in this
cell line heavily decreased the glucuronidation of the flavonoid
apigenin, which demonstrates an important role for UGT1A6 in the
glucuronidation by Caco-2 cells of a structurally related compound
(Liu et al., 2007).
In conclusion, the results of the present study show that indi-
vidual UGTs and SULTs demonstrate marked regioselective kinet-
ics for conjugation of hesperetin. As a result, variations in expres-
sion levels of these UGTs and SULTs give rise to different
metabolite patterns in different biological systems. Because differ-
ent flavonoid conjugates may have different physiological and/or
biological properties, this regioselective conjugation by different
UGT and SULT enzymes should not be ignored in flavonoid
research. Finally, given the high catalytic efficiency and expres-
sion levels of UGTs in intestinal tissue, it can be concluded that
first-pass metabolism within the intestinal cells contributes signif-
icantly to the formation of hesperetin 3Ј-O-glucuronide and 7-O-
glucuronide, the major hesperetin metabolites found in vivo.
Meinl W, Donath C, Schneider H, Sommer Y, and Glatt H (2008a) SULT1C3, an orphan
sequence of the human genome, encodes an enzyme activating various promutagens. Food
Chem Toxicol 46:1249–1256.
Meinl W, Ebert B, Glatt H, and Lampen A (2008b) Sulfotransferase forms expressed in human
intestinal Caco-2 and TC7 cells at varying stages of differentiation and role in benzo[a]pyrene
metabolism. Drug Metab Dispos 36:276–283.
Meinl W, Pabel U, Osterloh-Quiroz M, Hengstler JG, and Glatt H (2006) Human sulphotrans-
ferases are involved in the activation of aristolochic acids and are expressed in renal target
tissue. Int J Cancer 118:1090–1097.
Meinl W, Sczesny S, Brigelius-Flohe´ R, Blaut M, and Glatt H (2009) Impact of gut microbiota
on intestinal and hepatic levels of phase 2 xenobiotic-metabolizing enzymes in the rat. Drug
Metab Dispos 37:1179–1186.
Mullen W, Archeveque MA, Edwards CA, Matsumoto H, and Crozier A (2008) Bioavailability
and metabolism of orange juice flavanones in humans: impact of a full-fat yogurt. J Agric
Food Chem 56:11157–11164.
Nakamura A, Nakajima M, Yamanaka H, Fujiwara R, and Yokoi T (2008) Expression of UGT1A
and UGT2B mRNA in human normal tissues and various cell lines. Drug Metab Dispos
36:1461–1464.
Nakano H, Ogura K, Takahashi E, Harada T, Nishiyama T, Muro K, Hiratsuka A, Kadota S,
and Watabe T (2004) Regioselective monosulfation and disulfation of the phytoestrogens
daidzein and genistein by human liver sulfotransferases. Drug Metab Pharmacokinet
19:216–226.
Ne´meth K, Plumb GW, Berrin JG, Juge N, Jacob R, Naim HY, Williamson G, Swallow DM, and
Kroon PA (2003) Deglycosylation by small intestinal epithelial cell beta-glucosidases is a
critical step in the absorption and metabolism of dietary flavonoid glycosides in humans. Eur
J Nutr 42:29–42.
Nielsen IL, Chee WS, Poulsen L, Offord-Cavin E, Rasmussen SE, Frederiksen H, Enslen M,
Barron D, Horcajada MN, and Williamson G (2006) Bioavailability is improved by enzymatic
modification of the citrus flavonoid hesperidin in humans: a randomized, double-blind,
crossover trial. J Nutr 136:404–408.
Ohno
S and Nakajin S (2009) Determination of mRNA expression of human UDP-
glucuronosyltransferases and application for localization in various human tissues by real-time
reverse transcriptase-polymerase chain reaction. Drug Metab Dispos 37:32–40.
Otake Y, Hsieh F, and Walle T (2002) Glucuronidation versus oxidation of the flavonoid
galangin by human liver microsomes and hepatocytes. Drug Metab Dispos 30:576–581.
Paine MF and Fisher MB (2000) Immunochemical identification of UGT isoforms in human
small bowel and in Caco-2 cell monolayers. Biochem Biophys Res Commun 273:1053–
1057.
Riches Z, Stanley EL, Bloomer JC, and Coughtrie MW (2009) Quantitative evaluation of the
expression and activity of five major sulfotransferases (SULTs) in human tissues: the SULT
“pie.” Drug Metab Dispos 37:2255–2261.
References
Sakakibara Y, Yanagisawa K, Katafuchi J, Ringer DP, Takami Y, Nakayama T, Suiko M, and
Liu MC (1998) Molecular cloning, expression, and characterization of novel human SULT1C
sulfotransferases that catalyze the sulfonation of N-hydroxy-2-acetylaminofluorene. J Biol
Chem 273:33929–33935.
Blanchard RL, Freimuth RR, Buck J, Weinshilboum RM, and Coughtrie MW (2004) A proposed
nomenclature system for the cytosolic sulfotransferase (SULT) superfamily. Pharmacogenet-
ics 14:199–211.
Shelby MK, Cherrington NJ, Vansell NR, and Klaassen CD (2003) Tissue mRNA expression of
the rat UDP-glucuronosyltransferase gene family. Drug Metab Dispos 31:326–333.
Silberberg M, Morand C, Mathevon T, Besson C, Manach C, Scalbert A, and Remesy C (2006)
The bioavailability of polyphenols is highly governed by the capacity of the intestine and of
the liver to secrete conjugated metabolites. Eur J Nutr 45:88–96.
Tamura HO, Taniguchi K, Hayashi E, Hiyoshi Y, and Nagai F (2001) Expression profiling of
sulfotransferases in human cell lines derived from extra-hepatic tissues. Biol Pharm Bull
24:1258–1262.
Tang L, Singh R, Liu Z, and Hu M (2009) Structure and concentration changes affect charac-
terization of UGT isoform-specific metabolism of isoflavones. Mol Pharm 6:1466–1482.
Teubner W, Meinl W, Florian S, Kretzschmar M, and Glatt H (2007) Identification and
localization of soluble sulfotransferases in the human gastrointestinal tract. Biochem J 404:
207–215.
Boersma MG, van der Woude H, Bogaards J, Boeren S, Vervoort J, Cnubben NH, van Iersel ML,
van Bladeren PJ, and Rietjens IM (2002) Regioselectivity of phase II metabolism of luteolin
and quercetin by UDP-glucuronosyl transferases. Chem Res Toxicol 15:662–670.
Brand W, van der Wel PA, Rein MJ, Barron D, Williamson G, van Bladeren PJ, and Rietjens IM
(2008) Metabolism and transport of the citrus flavonoid hesperetin in Caco-2 cell monolayers.
Drug Metab Dispos 36:1794–1802.
Brett GM, Hollands W, Needs PW, Teucher B, Dainty JR, Davis BD, Brodbelt JS, and Kroon
PA (2009) Absorption, metabolism and excretion of flavanones from single portions of orange
fruit and juice and effects of anthropometric variables and contraceptive pill use on flavanone
excretion. Br J Nutr 101:664–675.
Exarchou V, Nenadis N, Tsimidou M, Gerothanassis IP, Troganis A, and Boskou D (2002)
Antioxidant activities and phenolic composition of extracts from Greek oregano, Greek sage,
and summer savory. J Agric Food Chem 50:5294–5299.

PHASE II METABOLISM OF HESPERETIN BY UGTs AND SULTs
625
Toma´s-Barbera´n FA, and Clifford MN (2000) Flavanones, chalcones and dihydrochalcones—
nature, occurrence and dietary burden. J Sci Food Agric 80:1073–1080.
Ung D and Nagar S (2007) Variable sulfation of dietary polyphenols by recombinant human
sulfotransferase (SULT) 1A1 genetic variants and SULT1E1. Drug Metab Dispos 35:740–
746.
Zhang L, Lin G, and Zuo Z (2007a) Involvement of UDP-glucuronosyltransferases in the
extensive liver and intestinal first-pass metabolism of flavonoid baicalein. Pharm Res
24:81–89.
Zhang L, Zuo Z, and Lin G (2007b) Intestinal and hepatic glucuronidation of flavonoids. Mol
Pharm 4:833–845.
van der Woude H, Boersma MG, Vervoort J, and Rietjens IM (2004) Identification of 14
quercetin phase II mono- and mixed conjugates and their formation by rat and human phase
II in vitro model systems. Chem Res Toxicol 17:1520–1530.
Walle T, Otake Y, Galijatovic A, Ritter JK, and Walle UK (2000) Induction of UDP-
glucuronosyltransferase UGT1A1 by the flavonoid chrysin in the human hepatoma cell line
hep G2. Drug Metab Dispos 28:1077–1082.
Address correspondence to: Walter Brand, Division of Toxicology, Wagenin-
gen University, P.O. Box 8000, 6700 EA Wageningen, The Netherlands. E-mail:
walter.brand@wur.nl