Identification of flavone glucuronide isomers by metal complexation and tandem mass spectrometry: Regioselectivity of uridine 5′-diphosphate- glucuronosyltransferase isozymes in the biotransformation of flavones

Article abstract of DOI:10.1021/jf304853j

Flavone glucuronide isomers of five flavones (chrysin, apigenin, luteolin, baicalein, and scutellarein) were differentiated by collision-induced dissociation of [Co(II) (flavone-H) (4,7-diphenyl-1,10-phenanthroline) ₂]⁺ complexes. The complexes were generated via postcolumn addition of a metal-ligand solution after separation of the glucuronide products generated upon incubation of each flavone with an array of uridine 5′-diphosphate (UDP)-glucuronosyltransferase (UGT) isozymes. Elucidation of the glucuronide isomers allowed a systematic investigation of the regioselectivity of 12 human UGT isozymes, including 8 UGT1A and 4 UGT2B isozymes. Glucuronidation of the 7-OH position was the preferred site for all the flavones except for luteolin, which possessed adjacent hydroxyl groups on the B ring. For all flavones and UGT isozymes, glucuronidation of the 5-OH position was never observed. As confirmed by the metal complexation/MS/MS strategy, glucuronidation of the 6-OH position only occurred for baicalein and scutellarein when incubated with three of the UGT isozymes.

Full text of DOI:10.1021/jf304853j

Article
pubs.acs.org/JAFC
Identification of Flavone Glucuronide Isomers by Metal
Complexation and Tandem Mass Spectrometry: Regioselectivity of
Uridine 5′-Diphosphate−Glucuronosyltransferase Isozymes in the
Biotransformation of Flavones
Scott A. Robotham and Jennifer S. Brodbelt*
Department of Chemistry and Biochemistry, University of Texas at Austin, 1 University Station A5300, Austin, Texas 78712, United
States
S
* Supporting Information
ABSTRACT: Flavone glucuronide isomers of five flavones (chrysin, apigenin, luteolin, baicalein, and scutellarein) were
differentiated by collision-induced dissociation of [Co(II) (flavone-H) (4,7-diphenyl-1,10-phenanthroline)2]+ complexes. The
complexes were generated via postcolumn addition of a metal−ligand solution after separation of the glucuronide products
generated upon incubation of each flavone with an array of uridine 5′-diphosphate (UDP)−glucuronosyltransferase (UGT)
isozymes. Elucidation of the glucuronide isomers allowed a systematic investigation of the regioselectivity of 12 human UGT
isozymes, including 8 UGT1A and 4 UGT2B isozymes. Glucuronidation of the 7-OH position was the preferred site for all the
flavones except for luteolin, which possessed adjacent hydroxyl groups on the B ring. For all flavones and UGT isozymes,
glucuronidation of the 5-OH position was never observed. As confirmed by the metal complexation/MS/MS strategy,
glucuronidation of the 6-OH position only occurred for baicalein and scutellarein when incubated with three of the UGT
isozymes.
KEYWORDS: human UDP−glucuronosyltransferase, flavonoid, regioselectivity, mass spectrometry, metal complexation,
glucuronidation
the uridine 5′-diphosphate (UDP)−glucuronosyltransferase
(UGT) family of enzymes.8 This family is split into three
main subgroups and contains a total of 19 different isoforms
including 9 UGT1As, 3 UGT2As, and 7 UGT2Bs. Currently, it
is known that the UGT1A group and the UGT2B group play a
major role in phase II metabolism; however, little is known
about the function of the UGT2A group.9 With respect to their
biotransformative role, UGT enzymes catalyze the addition of
glucuronic acid to any hydroxyl group, resulting in formation of
O-glucuronide products.8 This rather ubiquitous glucuronida-
tion process makes it particularly difficult to identify the
products with confidence as flavonoids may have multiple
hydroxyl groups. Reports have shown that the addition of
glucuronic acid to a flavone can greatly alter the bioactivity of
flavones. The apparent impact of glucuronidation on bioactivity
has stimulated efforts to unravel the formation and distribution
of the glucuronides as well as the effects of glucuronidation on
the bioactivities of the flavonoids.10,11
INTRODUCTION
■
The biotransformation of flavonoids has been a topic of
increasing research activity over the past decade due to the
interest in mapping the correlation between the beneficial
chemopreventive properties of flavonoids and the structures of
the active compounds in the body.1,2 Moreover, understanding
the bioavailabilities of flavonoids demands consideration of the
metabolism of native flavonoids upon consumption.3−5 In this
context, there have been a number of strategies aimed at
elucidating the structures of the biotransformation products of
flavonoids. This task is challenging due to the number of ways
that flavonoids can be metabolized and the number of isomeric
structures that may defy facile differentiation.6 Although
flavonoids are typically glycosylated in fruits and vegetables,
they are readily enzymatically deglycosylated by β-glucosidases
or lactose phloridzin hydrolases in the small intestine after
ingestion.7 Once the sugar side chain is removed, flavonoids are
most frequently modified by addition of a glucuronic acid or
sulfate group or in some cases by methyl or hydroxyl groups.7,8
These processes are mainly carried out by phase II enzymes
found in the small intestine, kidneys, and liver.8 It is these
conjugated flavonoid species that are absorbed by the body. In
fact, it has been shown that flavonoid aglycones in general have
poor bioavailability. The poor availability has motivated many
investigations of metabolism in order to rationalize how
inactive compounds or ones with poor bioavailability may
exert positive health benefits.6
Flavones, a subclass of flavonoids, are distinguished from
other subgroups of flavonoids by a double bond between the 2
and 3 positions on the C ring and a lack of a hydroxyl group at
the 3 position. The basic structure is shown in Figure 1.
Flavones are found in various types of fruits and vegetables, as
well many different herbs.12 This subclass of flavonoids has
Received: November 14, 2012
Revised: January 28, 2013
Accepted: January 29, 2013
Published: January 30, 2013
One of the most common conjugates formed during
metabolism are O-glucuronides. Glucuronidation arises from
© 2013 American Chemical Society
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celery (apigenin), Scutellariae radix (baicalein), honey (chrys-
in), peppermint (luteolin), and Scutellaria lateriflora (scutellar-
ein). While other studies have examined UGT isozyme
regioselectivities, there has been little focus on how the base
structure of flavones affects this selectivity.25,42,44,45 The current
systematic study provides insight into the regioselectivity of
UGT isozymes for flavones and also shows the unique affect
that a hydroxyl group at the 6 position of a flavone exerts on
the regioselectivity of UGT isozymes. The metal complexation/
MS/MS strategy is complementary to the UV-shift method26
and sometimes gives confident differentiation of glucuronides
not possible by other methods.
MATERIALS AND METHODS
■
Figure 1. Structures of flavones.
Reagents. All UGT isozymes were purchased from BD Biosciences
(Woburn, MA, USA). Apigenin, baicalein, chrysin, luteolin, and
scutellarein were all purchased from Indofine Chemical Co. (Hill-
sbrough, NJ, USA). UDP−glucuronic acid (UDPGA) trisodium salt,
4,7-diphenyl-1,10-phenanthroline (4,7-dpphen), and cobalt(II) bro-
mide were purchased from Sigma−Aldrich (St. Louis, MO, USA).
High-performance liquid chromatography (HPLC)-grade acetonitrile,
HPLC-grade water, potassium phosphate, and methanol were
purchased from Thermo Fisher Scientific, Inc. (Waltham, MA, USA).
Synthesis of Flavonoid Glucuronides by UGT Enzymes. The
procedure for the glucuronidation reactions was modified from the
protocol reported in Davis et al.40 Each enzyme was divided into 25
μL aliquots and stored at −80 °C until use. The following reaction
procedure was used for each combination of UGT enzyme (UGT1A1,
1A3, 1A4, 1A6, 1A7, 1A8, 1A9, 1A10, 2B4, 2B7, 2B15, and 2B17) and
flavone (apigenin, baicalein, chrysin, luteolin, and scutellarein). All
volumes were delivered using appropriate micropipets. The synthesis
was carried out by adding 2 mM aqueous UDPGA (65 μL), 20 mM
potassium phosphate buffer pH 7.0 (378.75 μL), and 10 mM
methanolic solution of flavones (6.25 μL) to a microcentrifuge tube.
The reaction was initiated by addition of 25 μL of a UGT enzyme (5
mg/mL). This concentration of enzyme was used based on a protocol
reported by Plumb et al.21 The mixture was incubated at 37 °C
overnight. To quench the reaction, 1.5 mL of acetone was added. The
final mixture was centrifuged for 10 min at 16 000g. The supernatant
was removed, and the acetone was evaporated using a Savant DNA120
SpeedVac concentrator (Thermo Electron, Waltham, MA, USA) on
low heat for 1 h 40 min. The remaining mixture was refrigerated until
analysis. The activities of UGT enzymes were previously assessed in
the presence of organic solvents at various concentrations, and it was
reported that there were no significant changes in enzyme activities for
solutions containing up to 2% methanol content.43 Thus, the use of a
minor portion of methanol (∼1.25% of the total volume of solution)
in the present study was not expected to be a major detriment to
enzymatic activity.46 This low concentration of methanol enhanced the
solubility of the flavones and led to more accurate concentrations in
solution.
The reaction conditions used in this study were similar to those
used in refs 25 and 42. Other glucuronidation procedures have been
reported.44,45 For example, previous studies have added magnesium
chloride (0.88 mM), saccharolacton (4.4 mM), and alamethicin (0.022
mg/mL) to the incubates. The two major differences between the
present study and previous studies44,45 include the concentration of
UDPGA added as well as the incubation time. The UDPGA
concentration used in the present study was 260 μM, much less
than the 3.5 mM used in refs 44 and 45. As for the incubation time,
refs 44 and 45 used times between 5 and 60 min, whereas the reactions
proceeded overnight in the present study. These changes in times and
concentrations might cause some differences in the distributions of
products in the present relative to former studies, but it is clear that
the results agree in almost every case where they can be compared,
leading to the conclusion that this change in incubation time does not
affect the types of products formed.
been reported to be very biologically active and may play a role
in countering diabetes mellitus, arteriosclerotic vascular disease
and even breast cancer.13,14 Numerous studies have demon-
strated these positive chemopreventive properties in a variety of
in vitro, in vivo, and case control studies.15−17 As alluded to
above, structural characterization of flavone-O-glucuronides is
difficult. With UGT enzymes able to modify any hydroxyl
group, the formation of different isomers is feasible. For many
years, identification of these glucuronides isomers has been
performed by the comparison of retention times with
synthesized standards of the different isomers. However, this
method is limited by the lack of availability of standards and the
complexity of synthesizing and purifying such compounds.17−24
NMR spectroscopy is arguably the most effective method for
characterization of flavone glucuronide isomers, as demon-
strated by Boutin et al.25 for the structural differentiation of
flavone glucuronides produced from UGT enzymes, but NMR
requires scaled up sample quantities that make it less practical
for broad scale in vivo studies.25 Recently, methods have been
developed to facilitate differentiation of these types of isomers
based on advanced chromatographic methods with tandem
mass spectrometry (MS/MS) and/or use of the UV-shift
method to assign conjugation positions.26−28 Of these methods,
tandem mass spectrometry of flavonoid−metal complexes has
proven to be extremely effective.29−31 For example, we have
shown that using metal complexation is an effective method for
differentiation of isomeric flavonoids and their glucuronides,
including ones in the subclass of flavones.32−39 This metal
complexation strategy has also been adapted for the
identification of flavonoid glucuronide isomers in urine.40,41
Most recently, this metal complexation method was applied to a
large-scale systematic study that allowed detailed insight into
regioselectivity of UGT isozymes for five common flavonoids
including hesperetin, naringenin, isorhamnetin, kaempferol, and
quercetin.42 There has also been considerable progress in
modeling human UGT quantitative structure−activity relation-
ships (QSAR) and prediction of regioselectivity, as summarized
in a recent review.43 This type of QSAR modeling has already
begun to shed light on understanding the complex substrate
selectivity of human UGTs.43
In this present study, we have expanded our investigation of
the selectivity of glucuronidation of the 12 most common UGT
enzymes (1A1, 1A3, 1A4, 1A6, 1A7, 1A8, 1A9, 1A10, 2B4, 2B7,
2B15, and 2B17) for 5 flavones (apigenin, baicalein, chrysin,
luteolin, and scutellarein) for which the glycoside forms are
among the most common found in foods. These flavones are
pervasive in common fruits, vegetables, and herbs, such as
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HPLC-UV Analysis. HPLC of the flavone glucuronides was
undertaken using a Prominence HPLC with a manual injector, a 50
μL loop (Shimadzu, Columbia, MA, USA), and a LCQ Duo
quadrupole ion trap mass spectrometer (Thermo Electron, Waltham,
MA, USA) with electrospray ionization (ESI). The column was a 50
mm × 2.1 mm i.d., 3.5 μm, Symmetry C18 column with a 10 mm × 2.1
mm i.d. guard column of the same material (Waters, Milford, MA).
The injection volume was 50 μL. The mobile phase was 0.33% formic
acid in water (A) and 0.33% formic acid in acetonitrile (B). The
gradient used began at 15% B and increased to 40% B over 30 min.
(This same setup and conditions are used for the companion LC-MS/
MS analysis.) To evaluate the relative product distributions of different
flavonoid glucuronides for each enzymatic reaction, the peak area for
each resulting product was integrated based on its LC-UV chromato-
graphic profile at 360 nm. The area of each product peak was divided
by the total area of all product peaks in order to calculate the relative
product distributions as percentages. Although absorption coefficients
may vary for different products, in fact, the same products are being
measured relative to each other for any flavone reacting with a series of
UGT enzymes.
Mass Spectrometric Analysis. Samples were first analyzed by
HPLC in the negative ESI mode in order to search for flavonoid
glucuronides. The spray voltage was set at 4.5 kV, the heated capillary
temperature was 200 °C, and the automatic gain control was set to 5 ×
107 ions with a maximum injection time of 500 ms and 5 microscans
averaging. All other parameters were set to obtain optimal signal. The
positive ESI mode was used for LC-MS/MS analysis of the flavone−
metal complexes. The metal complexes were formed by postcolumn
addition of a methanolic solution of 10 mM CoBr2 and 4,7-dpphen,
which was infused at a rate of 20 μL/min controlled by a syringe
pump. The spray voltage for the positive ion mode was set to 5 kV,
and the heated capillary temperature was 200 °C. The automatic gain
control for MS/MS was set to 2 × 107 ions with a maximum injection
time of 500 ms and 5 microscan averaging; the isolation width was set
to 4 Da, and a collision energy of 35% normalized collision energy was
used for collision-induced dissociation (CID). For direct infusion of
metal complex solutions, each flavone or flavone glucuronide was
mixed in a methanolic solution with CoBr2 and 4,7-dpphen in a 1:1:1
ratio. The solutions were made to have a final concentration of 10 μM.
Samples were then infused at a rate of 5 μL/min via a syringe pump.
The rest of the MS parameters were kept the same as those used for
the samples analyzed by LC-MS.
The 7-O-glucuronides also demonstrate the loss of the flavone
aglycone (−Agl) to a lesser extent. In contrast, the metal
complexes of 5-O-glucuronides generally undergo a prominent
loss of the glucuronic acid moiety along with the auxiliary
ligand (−(GlcA + Aux)); additionally, the 5-O-glucuronide
characteristically elutes before the 7-O-glucuronide.40 The 6-O-
glucuronides do not dissociate by elimination of the aglycone
moiety, thus allowing them to be differentiated from the 7-O-
glucuronides. B ring glucuronides dissociate via the loss of the
auxiliary ligand (−Aux) as well as the combined losses of both
the auxiliary ligand and glucuronide moiety (−(GlcA +
Aux)).27 4′-O-Glucuronides elute prior to 3′-O-glucuronides
and after the corresponding 7-O-glucuronides. As summarized
briefly here, these characteristic elution orders and fragmenta-
tion patterns allow facile differentiation of isomeric flavonoid
glucuronides.
With respect to the implementation of this approach, the
flavone glucuronides were derived from the supernatants
obtained after centrifugation of the enzymatic reaction
incubates. The glucuronides were separated and then ionized
by either negative ESI or via postcolumn metal complexation
prior to introduction into the ion trap mass spectrometer. To
pinpoint the elution of the flavone products of interest, specific
m/z values corresponding to each unmodified flavone and its
monoglucuronidated (aglycone +176) and diglucuronidated
(aglycone +176 + 176) products were searched in the total ion
chromatograms. In the present study, no diglucuronidated
products were found. CID of the positively charged flavone
glucuronide−metal complexes, not the deprotonated flavone
glucuronides, yielded the most distinctive fragmentation
patterns that confirmed the identity of each species. Examples
of the MS/MS spectra for some of the metal complexes are
shown in Figure 2 for the luteolin glucuronides produced from
RESULTS AND DISCUSSION
■
After reaction of the flavones in the presence of the
glucuronysyltransferases, identification of the products as
glucuronides is straightforward by LC-MS due to the
characteristic mass shift (+176 Da) due to attachment of the
glucuronyl moiety. However, the flavones with multiple
hydroxyl groups produced one or more glucuronides upon
incubation with each glucuronosyltransferase, yielding isobaric
products. The MS/MS spectra of the resulting isobaric flavone
glucuronides are too similar to allow their differentiation. An
alternative approach utilizing the MS/MS spectra of the metal
complexes [Co(II) (FG−H) (4,7-dpphen)2]+ (where FG
represents a flavone glucuronide) in conjunction with the
absolute or relative chromatographic retention times allowed
differentiation and assignment of the various flavone glucur-
onides, including isomers. A postcolumn complexation method
was used to generate the [Co(II) (FG−H) (4,7-dpphen)2]+
upon elution of each glucuronide, and these complexes gave
distinctive, diagnostic fragmentation patterns upon CID, thus
giving more confident identification than obtained for
deprotonated or protonated flavone glucuronides.27,40,42 For
example, metal complexes incorporating 7-O-glucuronides
exhibit losses of the auxiliary ligand (−Aux), the glucuronic
acid moiety (−GlcA), or both (−(GlcA + Aux)), upon CID.
Figure 2. CID mass spectra of [Co(II) (FG−H) (4,7-dpphen)2]+ for
all UGT1A1−luteolin products: (A) 7-O-glucuronide, m/z 1184; (B)
3′-O-glucuronide, m/z 1184; (C) 4′-O-glucuronide, m/z 1184. −Aux
(loss of auxiliary ligand); −GlcA (loss of glucuronic acid moiety);
−Agl (loss of flavonoid aglycon); −(GlcA + Aux) (loss of glucuronic
acid moiety and auxiliary ligand).
UGT1A1 and in Figure 3A,B for the baicalein glucuronides
produced from UGT1A1. All products identified based on the
unique MS/MS patterns of the metal complexes are
summarized in Table 1 along with the quantitative distribution
of products obtained by integration of the chromatographic
peak areas of each product and unmodified flavone.
The glucuronide products of four additional flavones,
including three with just a single hydroxyl group at the 5, 6,
or 7 position, as well as one flavone with hydroxyl groups at the
6 and 7 positions, were also evaluated in order to provide
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Figure 3. CID mass spectra of [Co(II) (FG−H) (4,7-dpphen)2]+ for all UGT1A8−baicalein products compared to the CID of the baicalein 7-O-
glucuronide standard: (A) 6-O-glucuronide, m/z 1168; (B) 7-O-glucuronide, m/z 1168; (C) 7-O-glucuronide standard, m/z 1168. −Aux (loss of
auxiliary ligand); −GlcA (loss of glucuronic acid moiety); −Agl (loss of flavonoid aglycon); −(GlcA + Aux) (loss of glucuronic acid moiety and
auxiliary ligand).
a
Table 1. Glucuronide Product Distributions
chrysin
chrysin
1A1
1A3
1A4
1A6
1A7
1A8
1A9
1A10
2B4
2B7
2B15
2B17
35
−
65
trace
−
100
100
−
trace
30
−
70
100
−
trace
40
−
60
20
−
80
80
−
20
100
−
−
65
−
35
100
−
trace
100
−
trace
5-O-glucuronide
7-O-glucuronide
apigenin
1A1
1A3
1A4
1A6
1A7
1A8
1A9
1A10
2B4
2B7
2B15
2B17
apigenin
35
−
65
70
−
30
100
−
−
−
1A4
80
−
20
100
−
−
−
1A7
65
−
35
10
−
90
75
−
25
100
−
−
−
2B4
95
−
5
100
−
−
−
2B15
100
−
−
−
2B17
5-O-glucuronide
7-O-glucuronide
4′-O-glucuronide
luteolin
−
1A1
−
1A3
−
1A6
−
1A8
−
1A9
−
1A10
−
2B7
luteolin
40
−
10
20
30
80
−
10
5
100
−
−
−
−
100
−
−
−
−
70
−
10
15
5
55
−
10
5
85
−
5
100
−
−
−
−
70
−
5
100
−
−
−
−
100
−
−
−
−
5-O-glucuronide
7-O-glucuronide
3′-O-glucuronide
4′-O-glucuronide
baicalein
−
40
40
15
20
10
trace
25
trace
5
15
1A1
1A3
1A4
1A6
1A7
1A8
1A9
1A10
2B4
2B7
2B15
2B17
baicalein
5
5
100
−
−
trace
−
−
100
−
−
−
1A7
trace
−
70
trace
−
5
trace
−
20
100
−
−
−
2B4
100
−
−
−
2B7
100
−
−
−
2B15
100
−
−
−
2B17
5-O-glucuronide
6-O-glucuronide
7-O-glucuronide
scutellarein
−
−
95
−
−
95
−
100
30
95
80
1A1
1A3
1A4
1A6
1A8
1A9
1A10
scutellarein
30
−
−
trace
−
−
100
−
−
−
−
5
100
−
−
−
−
trace
−
50
50
−
trace
−
25
75
−
5
100
−
−
−
−
100
−
−
−
−
100
−
−
−
−
100
−
−
−
−
5-O-glucuronide
6-O-glucuronide
7-O-glucuronide
4′-O-glucuronide
−
−
95
−
−
20
75
−
70
100
−
−
a
All values are percentages of total product distribution. A dash is used to indicate the absence of a product. The average standard deviation is 6%.
All values are rounded to the nearest 5%. Trace indicates a value that falls below 2.5%.
additional confirmatory evidence about the relative retention
times of flavones modified at the 5, 6, or 7 position. The
additional flavones are listed in Figure 1, and the MS/MS
patterns of the [Co(II) (FG−H) (4,7-dpphen)2]+ are in the
Supporting Information. Interestingly, the fragmentation
patterns of the glucuronide products generated from the
simple 6-hydroxy and 7-hydroxy flavones do not display the
identical multiple pathways noted for the flavones that possess
multiple hydroxyl groups. For example, the only pathway for
the glucuronidated 6-hydroxyflavone is the loss of the auxiliary
ligand (−Aux), and the most dominant fragmentation pathway
for the glucuronidated 7-hydroxyflavone is the loss of the
glucuronic acid group (−GlcA). This notable simplification of
the fragmentation patterns is not surprising. The streamlined
monohydroxyl flavones do not have multiple metal coordina-
tion sites like the other multihydroxyl flavones, and thus, the
array of possible metal-chelation structures that lead to
diagnostic fragment ions is correspondingly reduced, thus
yielding simpler MS/MS patterns. This point is clearly
demonstrated by comparison of the MS/MS patterns of the
glucuronide of 6-hydroxyflavone, 7-hydroxyflavone, and the two
glucuronides of 6,7-dihydroxyflavone (Figure 4). Whereas the
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A single product was observed for baicalein when modified in
the presence of UGT1A1, 1A3, and 1A6, and two glucuronides
were produced upon reactions with UGT1A8, 1A9, and 1A10.
The product formed from the 1A1, 1A3, and 1A6 reactions
showed characteristic losses of Aux, GlcA, and (GlcA + Aux).
These are also the same fragments observed for the first eluting
product of the 1A8, 1A9, and 1A10 reactions. The (GlcA +
Aux) ion is the dominant fragment ion in the spectra, in
addition to less abundant ions representing losses of Aux and
GlcA. This MS/MS pattern is the same fragmentation pattern
seen for the 7-O-glucuronide of the 6,7-dihydroxyflavone
discussed earlier. The second product from the reactions of
baicalein with UGT1A8, 1A9, and 1A10 showed prominent
fragments attributed to the loss of Aux or the loss of (Aux +
GlcA) that matches the dissociation pattern of the 6-O-
glucuronide product from the 6,7-dihydroxyflavone reaction.
The structural assignment of these two bacalein glucuronides
was confirmed by comparing these fragmentation patterns to
that obtained for a commercially available reference compound,
baicalein 7-O-glucuronide. Upon CID, the latter exhibited the
loss of Aux or GlcA as well as a dominant fragment
corresponding to the loss of (Aux + GlcA) (Figure 3C). On
the basis of this evidence as well as the retention time of
baicalein 7-O-glucuronide (Figure 5A) relative to the retention
Figure 4. CID mass spectra of [Co(II) (FG−H) (4,7-dpphen)2]+ for
all UGT1A9−6,7-dihydroxyflavone products: (A) 6-O-glucuronide, m/
z 1152; (B) 7-O- glucuronide, m/z 1152. −Aux (loss of auxiliary
ligand); −GlcA (loss of glucuronic acid moiety); −Agl (loss of
flavonoid aglycon); −(GlcA + Aux) (loss of glucuronic acid moiety
and auxiliary ligand).
fragmentation patterns for 7-O-glucuronide from 7-hydroxy-
flavone show only a single product, the 7-O-glucuronide formed
from 6,7-dihydroxyflavone shown in Figure 4A exhibits a richer
series of diagnostic fragment ions (i.e., loss of glucuronic acid
moiety, loss of aglyone, loss of auxiliary ligand, and loss of both
glucuronic acid and auxiliary ligand) that allow ready
differentiation of the 6-O- and 7-O-glucuronides. In short, the
relative retention times but not the MS/MS patterns of the
simplest monohydroxyl flavones are useful for supporting the
assignment of glucuronide products of the multihydroxylated
flavones. With respect to the retention times, the 7-O-
glucuronides consistently elute sooner than the 6-O-glucur-
onides, thus providing important confirmatory evidence.
The 5-hydroxyflavone formed one detectable glucuronide
product upon incubation with the UGT isozymes, as indicated
based on observation of a presumed deprotonated glucuronide
upon LC-MS. However, this 5-O-glucuronide did not form
stable metal complexes. Glucuronidation at the 5-O position
inhibits metal coordination between that position and the
nearby keto group, thus explaining the lack of metal complexes.
Glucuronidation at the 5-O position was not found for any of
the multihydroxylated flavones described in this study,
suggesting that the 5-O position is the least favorable when
other sites are available.
Figure 5. Selective ion chomatogram (m/z 445) for (A) baicalein 7-O-
glucuronide and (B) UGT1A8−baicalein products.
Identification of Flavone Glucuronides. Two flavones,
chrysin and apigenin, produced at most one characteristic
monoglucuronide when reacted in the presence of each UGT
isozyme. The reactions with apigenin resulted in the same
monoglucuronide product for UGT1A1, 1A3, 1A6, 1A8, 1A9,
1A10, and UG2B7 but no products for UGT1A4, 1A7, 2B4,
2B15, and 2B17. The sole product exhibited losses of GlcA,
Agl, and Aux, a pattern that is characteristic of 7-O-glucuronide
products. On the other hand, chrysin formed a single product
for all glucuronosyltransferases except for UGT2B4 that
resulted in no products. The single monoglucuronidated
product from chrysin dissociated by pathways characteristic of
a 7-O-glucuronide (losses of GlcA, Agl, and Aux).
Luteolin, with three hydroxyl groups, generated three
different products upon reaction in the presence of UGT1A1,
1A3, 1A7, 1A8, 1A9, 1A10, and 2B7. The first eluting product
dissociated by losses of GlcA, Agl, and Aux, all which are
consistent with a 7-O-glucuronide product. The next two
products each displayed losses of Aux, (Aux + GlcA), and GlcA
upon CID. Since 4′-O-glucuronide products typically elute
before 3′-O-glucuronide products, the two species are identified
as the 4′-O and 3′-O products, respectively. (Figure 2).
times of the two baicalein−glucuronide products (see Figure
5B), it is clear that the single product formed by bacalein upon
reaction with UGT1A1, 1A3, and 1A6 and the first eluting
product upon reaction of bacalein with UGT 1A8, 1A9, and
1A10 corresponds to glucuronidation of the 7-O position. This
also allows the confident identification of the second product as
a 6-O-glucuronide based on its greater retention time relative to
the 7-O-glucuronide, as described earlier for the model flavones.
This LC-MS/MS strategy also allows confident assignment
of the products of the scutellarein glucuronidation reactions.
Similar to bacalein, scutellarein also formed a single glucuronide
when incubated with UGT1A1, 1A3, and 1A6 and two
products when incubated with UGT1A8, 1A9, and 1A10. On
the basis of the MS/MS patterns and relative retention times,
the single product from the UGT1A1, 1A3, and 1A6 reactions
and the first eluting species from reaction of UGT1A8, 1A9,
and 1A10 is attributed to a 7-O-glucuronide product. The
second eluting product of the UGT1A8, 1A9, and 1A10
reactions shows losses of both Aux and (Aux + GlcA),
analogous to the pattern seen for the 6-O modification of
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Article
baicalein, so this product can be identified as a modification of
the 6-O position of scutellarein
the 3′ position, as luteolin is the only flavone that exhibited
glucuronidation of the B ring. For baicalein and scutellarein,
three of the UGT1A isozymes (1A8, 1A9, and 1A10) resulted
in the formation of 6-O-glucuronides, enabling the fragmenta-
tion rules for the metal complexation/MS/MS strategy to be
expanded. Consistent with our previous results for flavonols,
the planar structure of the flavones decreases their glucur-
onidation by the UGT2B isozymes.
Selectivity Trends. 7-Hydroxyflavone, chrysin, apigenin,
and luteolin afford an interesting series for comparison of how
additional hydroxyl groups affect glucuronidation site selectivity
because each of these flavones has an increasing number of
hydroxyl groups, starting with the standard 7-OH, then adding
the 5-OH, then adding the 4′-OH, and then adding the 3′-OH
for each flavone in the series. Singh et al.45 investigated the
selectivity of UGT1A1, 1A3, 1A6, 1A7, 1A8, 1A9, 1A10, and
2B7 with chrysin and apigenin by using a UV-shift method.45
They reported that apigenin and chrysin were glucuronidated
solely at the 7-O position, along with minor modification of the
5-O position of apigenin for UGT1A6 and 1A10. Our results
agree with these findings with the exception that we observed
no glucuronidation reactions with UGT1A7 and no glucur-
onidation at the 5-O position for apigenin. Comparing the
results obtained for chrysin and apigenin, it appears that a
hydroxyl group at the 4′ position of a flavone has little to no
effect on the selectivity of the UGT isozymes as there is
virtually no difference in the product distributions for chrysin
and apigenin for all the UGT isozymes. Luteolin provides a
unique opportunity to observe how UGT selectivity changes
upon addition of another hydroxyl group at the 3′ position.
Interestingly, the addition of the second hydroxyl to the B ring
of flavone results in modification of both B-ring sites for all
active UGT isoforms.
ASSOCIATED CONTENT
■
S
* Supporting Information
MS/MS spectra for chrysin, apigenin, scutellarein, 6-hydroxy-
flavone, and 7- hydroxyflavone glucuronides complexed with
cobalt(II) and 4,7-dpphen. This material is available free of
charge via the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
■
Corresponding Author
*Phone: (512) 471-0028; fax: (512) 471-8696; e-mail:
jbrodbelt@cm.utexas.edu.
Funding Sources
Funding from the NIH (R03 CA133924-02) and the Welch
Foundation (1155) is gratefully acknowledged.
Notes
The authors declare no competing financial interest.
Baicalein and scutellarein each have three potential
glucuronidation sites on the A ring (5-OH, 6-OH, and 7-
OH) and are also the only two flavones that have the 6-OH
group. Chen et al.18 reported that no glucuronidation of
baicalein occurred for reactions with UGT1A3 and UGT1A9.18
In the current study, we found baicalein does in fact form
abundant monoglucuronide products in the presence of all
UGT1A isozymes except for UGT1A4 and 1A7 and exhibits no
reaction with any of the UGT2B isozymes. Scutellarein showed
similar activity to baicalein with all UGT isozymes. The site
selectivity for bacalein and scutellarein are similar, suggesting
that the extra hydroxyl group at the 4′ position for scutellarein
is a nonreactive site. For baicalein and scutellarein, incubation
with UGT 1A8, 1A9, and 1A10 results in the formation of 6-O-
glucuronides.
ABBREVIATIONS USED
■
CID, collision-induced dissociation; UGT, UDP−glucuronosyl-
transferase; UDPGA, UDP−glucuronic acid; 4,7-dpphen, 4,7-
diphenyl-1,10-phenanthroline
REFERENCES
■
(1) Ross, J. A.; Kasum, C. M. Dietary flavonoides: bioavailability,
metabolic effects, and safety. Annu. Rev. Nutr. 2002, 22, 19−34.
(2) Kaur, C.; Kapoor, H. C. Antioxidants in fruits and vegetables −
the millennium’s health. Int. J. Food Sci. Technol. 2001, 36, 703−725.
(3) Manach, C.; Williamson, G.; Morand, C.; Scalbert, A.; Remesy, C.
Bioavailability and bioefficacy of polyphenols in humans. I. Review of
97 bioavailability studies. Am. J. Clin. Nutr. 2005, 81, 230S−242.
(4) Rice-Evans, C. Flavonoid antioxidants. Curr. Med. Chem. 2001, 8,
797.
All flavones show limited reactivity with the UGT2B
isozymes. This same trend was noticed previously for
glucuronidation of flavonols in our earlier study in which it
was hypothesized that the planar nature of these compounds
restricted their modification by the UGT2B isozymes. This
outcome contrasts with the ample glucuronidation observed for
flavanones (a class of flavonoids that lack the 2−3 double bond,
thus allowing greater conformational flexibility of the C ring).42
This study provides insight into the regioselectivity of 12
UGT isozymes for 5 naturally occurring flavones and
demonstrates the differentiation of glucuronide isomers that
is essential for bioavailability and biotransformation studies.
The formation and CID analysis of metal complexes of the type
([Co(II) (FG−H) (4,7-dpphen)2]+) via postcolumn addition
of a metal−ligand solution was a key analytical strategy that
allowed differentiation of isobaric products, most of which give
identical MS/MS fragmentation patterns for the conventional
deprotonated species. For example, this approach allows
differentiation of 6-O- and 7-O-glucuronides and complements
the UV-shift strategy used by others.44,45 UGT isozyme
selectivity is affected by the presence of a hydroxyl group at
(5) Kupeli, E.; Sahin, F. P.; Yesilada, E.; Calis, I.; Ezer, N. In vivo anti-
inflammatory and antinociceptive activity evaluation of phenolic
compounds from Sideritis stricta. J. Biosci. A 2007, 62, 519−525.
(6) Prior, R. L.; Wu, X.; Gu, L. Flavonoid metabolism and challenges
to understanding mechanisms of health effects. J. Sci. Food Agric. 2006,
86, 2487−2491.
(7) Williamson, G.; Day, A. J.; Plumb, G. W.; Couteau, D. Human
metabolic pathways of dietary flavonoids and cinnamates. Biochem. Soc.
Trans. 2000, 28, 16−22.
(8) King, C. D.; Rios, G. R.; Green, M. D.; Tephly, T. R. UDP-
glucuronosyltransferases. Curr. Drug Metab. 2000, 1, 143.
(9) Wong, Y. C.; Zhang, L.; Lin, G.; Zuo, Z. Structure−activity
relationships of the glucuronidation of flavonoids by human
glucuronosyltransferases. Expert Opin. Drug Metab. Toxicol. 2009, 5,
1399−1419.
(10) Kroon, P. A.; Clifford, M. N.; Crozier, A.; Day, A. J.; Donovan, J.
L.; Manach, C.; Williamson, G. How should we assess the effects of
exposure to dietary polyphenols in vitro. Am. J. Clin. Nutr. 2004, 80,
15−21.
(11) Atmani, D.; Chaher, N.; Atmani, D.; Berboucha, M.; Debbache,
N.; Boudaoud, H. Flavonoids in human health: from structure to
biological activity. Curr. Nutr. Food Sci. 2009, 5, 225−237.
1462
dx.doi.org/10.1021/jf304853j | J. Agric. Food Chem. 2013, 61, 1457−1463

Journal of Agricultural and Food Chemistry
Article
(12) Chao, P.-D.; Hsiu, S.-L.; Hou, Y.-C. Flavonoids in herbs:
biological fates and potential interactions with xenobiotics. J. Food
Drug Anal. 2002, 10, 219−228.
(13) Cermak, R. Effect of dietary flavonoids on pathways involved in
drug metabolism. Expert Opin. Drug Metab. Toxicol. 2008, 4, 17−35.
(14) Ullmannova, V.; Popescu, N. C. Inhibition of cell proliferation,
induction of apoptosis, reactivation of DLC1, and modulation of other
gene expression by dietary flavone in breast cancer cell lines. Cancer
Detect. Prev. 2007, 31, 110−118.
(31) Pikulski, M.; Brodbelt, J. S. Differentiation of flavonoid glycoside
isomers by using metal complexation and electrospray ionization mass
spectrometry. J. Am. Soc. Mass Spectrom. 2003, 14, 1437−1453.
(32) Zhang, J.; Satterfield, M. B.; Brodbelt, J. S.; Britz, S. J.;
Clevidence, B.; Novotny, J. A. Structural characterization and
detection of kale flavonoids by electrospray ionization mass
spectrometry. Anal. Chem. 2003, 75, 6401−6407.
(33) Zhang, J.; Brodbelt, J. S. Screening flavonoid metabolites of
naringin and narirutin in urine after human consumption of grapefruit
juice by LC-MS and LC-MS/MS. Analyst 2004, 129, 1227−1233.
(34) Zhang, J.; Brodbelt, J. S. Gas-phase hydrogen/deuterium
exchange and conformations of deprotonated flavonoids and gas-
phase acidities of flavonoids. J. Am. Chem. Soc. 2004, 126, 5906−5919.
(35) Zhang, J.; Wang, J.; Brodbelt, J. S. Characterization of flavonoids
by aluminum complexation and collisionally activated dissociation. J.
Mass Spectrom. 2005, 40, 350−363.
(15) Graf, B. A.; Milbury, P. E.; Blumberg, J. B. Flavonols, flavones,
flavanones, and human health: epidemiological evidence. J. Med. Food
2005, 8, 281−290.
(16) Knekt, P.; Kumpulainen, J.; Jarvinen, R.; Rissanen, H.;
̈
Heliovaara, M.; Reunanen, A.; Hakulinen, T.; Aromaa, A. Flavonoid
̈
intake and risk of chronic diseases. Am. J. Clin. Nutr. 2002, 76, 560−
568.
(36) Zhang, J.; Brodbelt, J. S.; Wang, J. Threshold dissociation and
molecular modeling of transition metal complexes of flavonoids. J. Am.
Soc. Mass Spectrom. 2005, 16, 139−151.
(17) Chen, Y. K.; Chen, S. Q.; Li, X.; Zeng, S. Quantitative
regioselectivity of glucuronidation of quercetin by recombinant UDP-
glucuronosyltransferases 1A9 and 1A3 using enzymatic kinetic
parameters. Xenobiotica 2005, 35, 943−954.
(18) Chen, Y.; Xie, S.; Chen, S.; Zeng, S. Glucuronidation of
flavonoids by recombinant UGT1A3 and UGT1A9. Biochem.
Pharmacol. 2008, 76, 416−425.
(19) Hong, Y.-J.; Mitchell, A. E. Metabolic Profiling of flavonol
metabolites in human urine by liquid chromatography and tandem
mass spectrometry. J. Agric. Food Chem. 2004, 52, 6794−6801.
(20) Lehtonen, H.-M.; Lehtinen, O.; Suomela, J.-P.; Viitanen, M.;
(37) Zhang, J.; Brodbelt, J. S. Silver complexation and tandem mass
spectrometry for differentiation of isomeric flavonoid diglycosides.
Anal. Chem. 2005, 77, 1761−1770.
(38) Davis, B. D.; Brodbelt, J. S. LC−MSn methods for saccharide
characterization of monoglycosyl flavonoids using postcolumn
manganese complexation. Anal. Chem. 2005, 77, 1883−1890.
(39) Pikulski, M.; Aguilar, A.; Brodbelt, J. S. Tunable transition
metal-ligand complexation for enhanced elucidation of flavonoid
diglycosides by electrospray ionization mass spectrometry. J. Am. Soc.
Mass Spectrom. 2007, 18, 422−431.
Kallio, H. Flavonol glycosides of sea buckthorn (Hippophae rhamnoides
̈
(40) Davis, B. D.; Brodbelt, J. S. Regioselectivity of human UDP-
glucuronosyl-transferase 1A1 in the synthesis of flavonoid glucuronides
determined by metal complexation and tandem mass spectrometry. J.
Am. Soc. Mass Spectrom. 2008, 19, 246−256.
(41) Brett, G. M.; Hollands, W.; Needs, P. W.; Teucher, B.; Dainty, J.
R.; Davis, B. D.; Brodbelt, J. S.; Kroon, P. A. 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. 2009, 664−675.
(42) Robotham, S. A.; Brodbelt, J. S. Regioselectivity of human UDP-
glucuronosyltransferase isozymes in flavonoid biotransformation by
metal complexation and tandem mass spectrometry. Biochem.
Pharmacol. 2011, 82, 1764−1770.
(43) Dong, D.; Ako, R.; Hu, M.; Wu, B. Understanding substrate
selectivity of human UDP-glucuronosyltransferases through QSAR
modeling and analysis of homologous enzymes. Xenobiotica 2012, 42,
808−820.
(44) Wu, B.; Xu, B.; Hu, M. Regioselective glucuronidation of
flavonols by six human UGT1A isoforms. Pharm. Res. 2011, 28, 1905−
1918.
(45) Singh, R.; Wu, B.; Tang, L.; Hu, M. Uridine diphosphate
glucuronosyltransferase isoform-dependent regiospecificity of glucur-
onidation of glavonoids. J. Agric. Food Chem. 2011, 59, 7452−7464.
(46) Easterbrook, J.; Lu, C.; Sakai, Y.; Li, A. P. Effects of organic
solvents on the activities of cytochrome P450 isoforms, UDP-
dependent glucuronyl transferase, and phenol sulfotransferase in
human hepatocytes. Drug Metab. Dispos. 2001, 29, 141−144.
ssp. sinensis) and lingonberry (Vaccinium vitis-idaea) are bioavailable in
humans and monoglucuronidated for excretion. J. Agric. Food Chem.
2009, 58, 620−627.
(21) Plumb, G. W.; O’Leary, K.; Day, A. J.; Williamson, G. Enzymatic
synthesis of quercetin glucosides and glucuronides. In Methods in
Polyphenol Analysis; The Royal Society of Chemistry: Cambridge, U.K.,
2003.
(22) Xie, S.; Chen, Y.; Chen, S.; Zeng, S. Structure-metabolism
relationships for the glucuronidation of flavonoids by UGT1A3 and
UGT1A9. J. Pharm. Pharmacol. 2011, 63, 297−304.
(23) Xie, S.-G.; You, L.-Y.; Zeng, S. Phase II metabolism of
flavonoids mediated by human glucuronosyltransferase: an advanced
research. Zhongguo Yaolixue Yu Dulixue Zazhi 2007, 21, 438−443.
(24) Mullen, W.; Boitier, A.; Stewart, A. J.; Crozier, A. Flavonoid
metabolites in human plasma and urine after the consumption of red
onions: analysis by liquid chromatography with photodiode array and
full scan tandem mass spectrometric detection. J. Chromatogr., A 2004,
1058, 163−168.
(25) Boutin, J. A.; Meunier, F.; Lambert, P. H.; Hennig, P.; Bertin,
D.; Serkiz, B.; Volland, J. P. In vivo and in vitro glucuronidation of the
flavonoid diosmetin in rats. Drug Metab. Dispos. 1993, 21, 1157−1166.
(26) Singh, R.; Wu, B.; Tang, L.; Liu, Z.; Hu, M. Identification of the
position of mono-O-glucuronide of flavones and flavonols by analyzing
shift in online uv spectrum (λmax) generated from an online diode
array detector. J. Agric. Food Chem. 2010, 58, 9384−9395.
(27) Davis, B. D.; Needs, P. W.; Kroon, P. A.; Brodbelt, J. S.
Identification of isomeric flavonoid glucuronides in urine and plasma
by metal complexation and LC-ESI-MS/MS. J. Mass Spectrom. 2006,
41, 911−920.
(28) Tang, L.; Zhou, J.; Yang, C.-H.; Xia, B.-J.; Hu, M.; Liu, Z.-Q.
Systematic studies of sulfation and flucuronidation of 12 flavonoids in
the mouse liver S9 fraction reveal both unique and shared positional
preferences. J. Agric. Food Chem. 2012, 60, 3223−3233.
(29) Satterfield, M.; Brodbelt, J. S. Enhanced detection of flavonoids
by metal complexation and electrospray ionization mass spectrometry.
Anal. Chem. 2000, 72, 5898−5906.
(30) Satterfield, M.; Brodbelt, J. S. Structural characterization of
flavonoid glycosides by collisionally activated dissociation of metal
complexes. J. Am. Soc. Mass Spectrom. 2001, 12, 537−549.
1463
dx.doi.org/10.1021/jf304853j | J. Agric. Food Chem. 2013, 61, 1457−1463