Inhibition of 20-Hydroxyeicosatetraenoic Acid Synthesis
Using Specific Plant Lignans
In Vitro and Human Studies
Jason H.Y. Wu, Jonathan M. Hodgson, Michael W. Clarke, Adeline P. Indrawan, Anne E. Barden,
Ian B. Puddey, Kevin D. Croft
Downloaded from http://hyper.ahajournals.org/ by guest on June 17, 2017
Abstract—Sesamin, the major lignan found in sesame, has been shown to increase vitamin E levels by inhibiting its
metabolism via the cytochrome P450 isozyme CYP4F2. CYP4F2 and CYP4A11 are the predominant human isoforms
that synthesize 20-hydroxyeicosatetraenoic acid (20-HETE) from arachidonic acid. Considerable evidence suggests that
20-HETE may play a role in the pathogenesis of hypertension. We hypothesized that sesamin could be an inhibitor of
20-HETE synthesis. This study investigated the effects of sesamin on 20-HETE synthesis in vitro and the effect of
sesame supplementation on plasma and urinary 20-HETE concentrations in humans. Human microsomes were used to
investigate the potency and selectivity of sesamin inhibition of 20-HETE synthesis. Sesamin inhibited human renal and
liver microsome 20-HETE synthesis with IC50 ⬍20 ␮mol/L. It was selective toward CYP4F2 (IC50: 1.9 ␮mol/L) and
had reduced activity toward CYP4A11 (IC50: ⬎150 ␮mol/L), as well as cytochrome P epoxygenation of arachidonic
acid (IC50: ⬎50 ␮mol/L). In a randomized, controlled crossover trial, overweight men and women (n⫽33) consumed
25 g/d of sesame (⬇50 mg/d of sesame lignan) or an isocaloric matched control for 5 weeks each. Relative to control,
sesame supplementation resulted in a 28% decrease in plasma and a 32% decrease in urinary 20-HETE (P⬍0.001).
Urinary sodium, potassium, and blood pressure were not affected. This study demonstrates for the first time that sesame
supplementation in humans reduces the plasma and urinary levels of 20-HETE, likely via inhibition of CYP4F2 by
sesame lignans. These results suggest that sesame lignans could be used for the investigation of potential roles of
20-HETE in humans. (Hypertension. 2009;54:1151-1158.)
Key Words: 20-HETE 䡲 cytochrome P450 䡲 sesame 䡲 vitamin E 䡲 cardiovascular disease
T
he mono-oxygenation of arachidonic acid by cytochrome
P450 enzymes is recognized as an important metabolic
pathway.1 The major products generated via this enzymatic
conversion include 20-hydroxyeicosatetraenoic acid (20HETE) and regioisomeric forms of epoxyeicosatrienoic acids
(EETs).2 These arachidonic acid metabolites possess physiological functions that may influence the development of
kidney diseases and hypertension.2 20-HETE, formed via
␻-hydroxylation of arachidonic acid, has been a focus of
recent investigations that suggest that it plays a key role in the
regulation of vascular and renal function. 20-HETE acts as a
vasoconstrictor in renal, cerebral, and mesenteric arteries.3
Furthermore, it has been suggested to mediate pressure
natriuresis by inhibiting sodium reabsorption in the renal
proximal tubule.3 20-HETE, thus, possesses activities that
could be prohypertensive or antihypertensive depending on
its site of production, and this has been supported by animal
models of hypertension.4,5
Sesame seeds are a popular and commonly consumed
food.6 The abundance of unique lignans in sesame seeds has
been suggested to contribute to health benefits. The predominant lignan in sesame is sesamin (Figure 1). One biological
activity of sesamin that has been the target of several recent
investigations is its ability to modulate ␥-tocopherol metabolism. ␥-Tocopherol is a major dietary isoform of vitamin E,
and human intervention studies have demonstrated that a
higher dietary intake of sesame (as seeds or oil) leads to
elevated serum ␥-tocopherol concentrations.6,7 In humans, a
major pathway of ␥-tocopherol metabolism is via conversion
to carboxyethyl hydroxychromans (CEHCs), which are water
soluble metabolites excreted in the urine.8 This metabolic
pathway is initiated by the ␻-hydroxylation of ␥-tocopherol
by cytochrome P (CYP). It has been shown that the major
CYP isoform responsible for ␥-tocopherol hydroxylation is
CYP4F2. Sesamin has been shown in in vitro studies to
inhibit CYP4F2 hydroxylation of ␥-tocopherol.9 This likely
Received July 16, 2009; first decision August 2, 2009; revision accepted August 31, 2009.
From the School of Medicine and Pharmacology (J.H.Y.W., J.M.H., A.P.I., A.E.B., I.B.P., K.D.C.) and the Department of Core Clinical Pathology and
Biochemistry (M.W.C.), Royal Perth Hospital, University of Western Australia, Perth, Australia.
This study is registered at the Australian New Zealand Clinical Trials Registry (http://www.anzctr.org.au/), registration No. ACTRN 12607000158460.
Correspondence to Jason H.Y. Wu, School of Medicine and Pharmacology, University of Western Australia, PO Box X2213 GPO, Perth, Western
Australia 6847, Australia. E-mail [email protected]
© 2009 American Heart Association, Inc.
Hypertension is available at http://hyper.ahajournals.org
DOI: 10.1161/HYPERTENSIONAHA.109.139352
1151
1152
Hypertension
A Sesamin
November 2009
B Sesamolin
O
O
O
O
C Pinoresinol
H3 CO
OH
O
O
O
O
O
O
O
O
HO
O
O
OCH 3
O
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D Secoisolariciresinol
HO
E Enterodiol
OCH3
OH
OH
F Enterolactone
O
HO
OH
OH
OH
O
HO
OCH3
OH
OH
Figure 1. Chemical structures of plant lignans (A) sesamin, (B) sesamolin, (C) pinoresinol, (D) secoisolariciresinol, and their major metabolic end products (E) enterodiol and (F) enterolactone. The methylenedioxyphenyl moieties of (A) sesamin and (B) sesamolin are highlighted in bold, and this structural feature is not present in the other lignans shown.
leads to a decrease in ␥-tocopherol metabolism, thus providing a potential mechanistic explanation for the observed
increase in ␥ -tocopherol concentration after sesame
supplementation.6,7
The CYP4F subfamily of enzymes has gained recent
recognition for its important role in eicosanoid and drug
metabolism.10 In particular, several recent studies have suggested a potential association between CYP4F2 and hypertension through the production of 20-HETE.11,12 It has been
shown that CYP4F2 is responsible for the majority of
20-HETE synthesis in human liver and kidney, with
CYP4A11 being the other isoform contributing toward 20HETE synthesis.13,14 Previous studies have demonstrated that
sesamin inhibits CYP4F2 catalyzed ␥-tocopherol hydroxylation.8 We, therefore, hypothesized that this lignan may also
inhibit 20-HETE synthesis in humans. In the current study,
we carried out in vitro studies to characterize the potency and
specificity of sesamin toward CYP-catalyzed formation of
20-HETE. We also carried out a human intervention study to
investigate the effect of sesame supplementation on plasma
20-HETE levels and urinary excretion of 20-HETE. We
additionally measured urinary excretion of ␥-CEHC, the
major metabolite of ␥-tocopherol, as an additional surrogate
measure of in vivo CYP4F2 activity.
Subjects and Methods
Sesamin Inhibition of 20-HETE Synthesis:
In Vitro Experiments
Microsome Experiments
The ability of sesamin and structurally related lignans to inhibit
eicosanoid synthesis was examined in isolated microsomes. Pooled
human liver (n⫽50; mixed gender) and kidney (n⫽8; mixed gender)
microsomes were prepared by Xenotech LLC (Lenexa). Microsomes
prepared from baculovirus-infected insect cells that express recombinant CYP4F2 and 4A11 were obtained from BD Gentest. Inhibitions of the conversion of arachidonic acid to oxygenated metabolites
were assayed according to Lasker et al13 Briefly, microsomes (0.1 to
0.5 mg of protein) were preincubated for 5 minutes at 37°C with
arachidonic acid (100 ␮mol/L) and various concentrations of plant
lignans or vehicle (as control) in 200 ␮L of 100 mmol/L of
potassium phosphate buffer (pH 7.4). Reactions were initiated with
1 mmol/L of NADPH. Depending on the microsome system, the
reaction mixture was incubated for between 8 and 40 minutes and
terminated with 20 ␮L of 2 N HCl. The protein concentrations and
incubation times chosen for each type of microsome were shown in
optimization assays to be within the range for which product
formation was linear. The mixture was extracted with 1 mL of ethyl
acetate. The extract was dried under nitrogen and analyzed by liquid
chromatography-mass spectrometry.
Mass Spectrometry Analysis
Eicosanoids generated from in vitro microsomal experiments were
analyzed by high-performance liquid chromatography. Thermo
Wu et al
A
B
Human renal microsomes
Sesame Supplementation Reduces 20-HETE in Humans
Human liver microsomes
80
80
40
20
0
% inhibition
100
80
% inhibition
100
% Inhibition
100
60
60
40
20
10
20
30
40
50
Inhibitor Concentration (µM)
-20
60
40
20
0
0
0
1153
10
20
30
40
50
10
-20
Sesamin concentration (µM)
20
30
40
50
Sesamolin concentration (µM)
Sesamin
Enterolactone
Secoisolariciresinol
20-HETE
20-HETE
Sesamolin
Enterodiol
Pinoresinol
EET + DHET
EET + DHET
Figure 2. Effect of plant and mammalian lignans on the formation of 20-HETE in (A) human renal and (B) human liver microsomes.
Results are expressed as the percentage of inhibition vs control, and each point represents a mean⫾SEM from 3 independent
experiments.
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Fisher Accela Pump and autosampler interfaced to a triplequadrupole mass spectrometer (Thermo-Fisher TSQ Quantum Ultra). Analytes were eluted using acetonitrile/water/formic acid
(37.00:63.00:0.02; solvent A) and acetonitrile/isopropanol (50:50;
solvent B) in a gradient as follows (0 minutes, 0% B; 6.0 minutes,
33% B; 15.0 minutes, 33% B; 15.5 minutes, 100% B; 17.5 minutes,
100% B; 17.6 minutes, 0% B; and 19.6 minutes, 0% B). Negative
ions were generated using Heated Electrospray Ionization. The
autotune function of the instrument was used to optimize the source
conditions and ion optics for maximum sensitivity using eicosanoid
standards (Cayman Chemical) before sample analysis. Quantitative
analyses were obtained by the stable isotope dilution method using
ion transitions, as described.15 Data from the in vitro microsome
inhibition assays are expressed as the percentage of the control
activity. Curve fitting and IC50 estimation were carried out by
nonlinear regression using Prism (version 4, GraphPad Software).
Human Intervention Study
Study Population
Overweight (body mass index: ⬎25 kg/m2) men and postmenopausal
women were recruited from the general population. Details of the
study population and study design have been published previously.16
Briefly, volunteers were included if they had ⱖ1 of the risk factors
for the metabolic syndrome, as defined by the American Heart
Association Adult Treatment Panel III criteria,17 or if they had
elevated low-density lipoprotein cholesterol (⬎3.4 mmol/L). All of
the volunteers provided written informed consent, and the study was
approved by the University of Western Australia Human Ethics
Committee. Thirty-three volunteers completed the study.
Study Design
This was a randomized, placebo-controlled crossover study.16 After
an initial 4-week washout period, half of the volunteers replaced
their usual breakfast with a sesame breakfast bar and the other half
with a placebo breakfast bar for 5 weeks. At the end of the first
treatment period, volunteers had another 4 weeks of washout (during
which time the volunteers reverted to their usual breakfast intake).
The volunteers then commenced a second 5-week treatment period
where they replaced their breakfast with the alternative bar. The
order of treatment was randomized via permutated block randomization, using computer-generated random numbers. Volunteers
were advised to maintain their lifestyle, including diet and physical
activity, throughout the study period. Volunteers visited the study
center on the morning before the start of each treatment period, as
well as the morning after the end of each treatment period, where
fasting blood and 24-hour urine samples were collected. They were
also fitted with an ambulatory blood pressure monitor, as described
previously,16 which was worn during awake hours.
Supplements
The composition of the bars has been published previously (Figure S1,
please see the online Data Supplement at http://hyper.ahajournals.org).16
During the sesame supplementation, the volunteers received 60 g of
each bar per day, with the sesame bar providing 26.2 g of sesame
seeds per day, and the placebo bars matched for energy, macronutrient, vitamin E, and sodium composition. Sesamin content of the
seeds used in this study (151 mg/100 g of seeds) was comparable to
the mean value reported previously for 65 natural sesame seed
samples (163 mg/100 g).18
Biochemical Measurements
All of the biochemical measurements were carried out in a blinded
fashion. Plasma unesterified 20-HETE, urinary 20-HETE, and vitamin E metabolites were measured using gas chromatography-mass
spectrometry, as described previously.19,20 Internal standards were
added to plasma and urine samples before extraction. Plasma
arachidonic acid was measured by gas chromatography,21 and
plasma tocopherols were measured by high-performance liquid
chromatography with electrochemical detection following published
methods.19
Statistical Analysis
Data are shown for participants who completed the study (n⫽33).
Data are expressed as mean⫾SD or mean⫾SEM. Pearson correlation coefficient was calculated to quantify association between
variables. Nonnormally distributed data were log-transformed before
analysis and are presented as geometric mean (95% CI). All of the
outcome data from the clinical study were analyzed using linear
mixed-models (PROC MIXED, version 9.1; SAS Institute Inc).
Subjects were included as a random factor nested under treatment
sequence within a linear mixed model. The following factors were
always included as fixed effects in the model: baseline values,
treatment, period, and treatment sequence. Differences were considered significant when P⬍0.05.
Results
In Vitro Inhibition of Eicosanoid Synthesis by
Sesame Lignans
We initially examined the ability of sesame lignans to inhibit
20-HETE synthesis in human renal microsomes. We found
that sesame lignans inhibited 20-HETE synthesis in a dosedependent manner, with IC50 values of 5.31 ␮mol/L and
3.39 ␮mol/L for sesamin and sesamolin (another sesame
lignan with similar structure to sesamin), respectively (Figure
2A). Because sesame lignans are precursors to the mammalian lignans, enterolactone and enterodiol, we also examined
1154
Hypertension
November 2009
CYP4F2
% inhibition
100
CYP4A11
100
80
80
60
60
40
40
20
20
0
0
0
5
10
15
20
25
0
Sesamin concentration (µM)
50
100
150
Sesamin concentration (µM)
Figure 3. Effect of sesamin on the formation of 20-HETE in microsomes prepared from baculovirus-infected insect cells that express
recombinant CYP4F2 and 4A11. Results are expressed as the percentage of inhibition vs control, and each point represents a
mean⫾SEM from 3 independent experiments.
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the inhibitory activities of these compounds. In contrast to
their precursors, mammalian lignans had comparatively much
weaker inhibitory activity (Figure 2A, at 50 ␮mol/L, enterolactone and enterodiol inhibited 20-HETE production by
27⫾4.3% and 19⫾1.6%, respectively). To investigate the
structural features of sesame lignans, which may be related to
their inhibitory activity, we further examined 2 other structurally related dietary lignans found in flaxseeds, pinoresinol
and secoisolariciresinol. As shown in Figure 2A, both compounds exhibited minimal activity for the inhibition of
20-HETE synthesis.
We also investigated the inhibitory activity of sesame
lignans in human liver microsomes, because both major
20-HETE producing CYPs (4F2 and 4A11) are also expressed in the human liver.14 Furthermore, human liver
microsomes generate EET, the other major class of eicosanoids generated by CYP enzymes.22 This allowed for the
investigation of whether sesame lignans demonstrate differential inhibitory activity toward the ␻-hydroxylation and
epoxidation pathways. Both sesamin and sesamolin dosedependently inhibited 20-HETE synthesis in human liver
microsomes (IC50 values of 17.3 ␮mol/L and 9.6 ␮mol/L,
respectively; Figure 2B). Mixed human liver microsomes
produced regioisomeric 14,15-, 11,12-, and 8,9-EET, as well
as their respective dihydroxyeicosatrienoic acid. Total epoxygenase activity was calculated as the sum of all EETs and
dihydroxyeicosatrienoic acid. We found that sesame lignans
showed weaker inhibitory activity toward EET synthesis (at
50 ␮mol/L, sesamin and sesamolin inhibited epoxygenase
activity by 47⫾2.1% and 35⫾4.5%, ie, IC50 ⱖ50 ␮mol/L).
Finally, we determined the specificity of sesamin toward
the 2 major human 20-HETE CYP synthases, CYP4F2 and
CYP4A11. We focused on sesamin, because it is the major
lignan found in sesame. As shown in Figure 3, sesamin
strongly inhibited 20-HETE synthesis in microsomes expressing human CYP4F2 (IC50: 1.87 ␮mol/L). Conversely, it
only weakly inhibited CYP4A11-catalyzed 20-HETE production with IC50 ⬎150 ␮mol/L (Figure 3).
Human Supplementation Study
Participant Characteristics
Subject baseline profiles are shown in Table 1. Five subjects
were taking antihypertensive medication, and 4 had elevated
fasting glucose levels. None of the volunteers were taking
hypoglycemic medication. We were careful to ensure that
each subject’s medication status remained unchanged
throughout the study. None of the subjects reported any
adverse effects from the supplement bars. Subject compliance
was 99.8⫾0.7% and 98.3⫾3.3% for sesame and placebo bars,
respectively, on the basis of counting the number of bars
returned at the end of each treatment period. As reported
previously, sesame treatment did not lead to any changes to
blood lipids, blood pressure, glucose, or circulating inflammatory and oxidative stress markers.16
Tocopherol Metabolism
Compared with placebo, sesame supplementation led to an
⬇20% increase in serum ␥-tocopherol (P⫽0.009; Table 2).
This result was unaltered by correction for serum lipid
concentrations. To examine whether sesame supplementation
inhibited the metabolism of ␥-tocopherol via the CYP pathway, we measured the excretion of water-soluble metabolites
in the urine. Sesame supplementation led to a significant
Table 1.
Participant Characteristics at First Baseline
Characteristic
All Subjects (n⫽33)
Age, y
55.1⫾8.7
Body mass index, kg/m2
31.0⫾3.8
Sex, male/female
17/16
Cholesterol, mmol/L
Total
5.92⫾0.81
Low-density lipoprotein
3.79⫾0.74
High-density lipoprotein
1.37⫾0.40
Triglycerides, mmol/L
Systolic blood pressure, mm Hg*
1.67⫾0.65
134.0⫾12.2
Diastolic blood pressure, mm Hg*
82.0⫾7.7
Glucose, mmol/L
4.83⫾0.56
Elevated clinic blood pressure, %†
26
Volunteer with metabolic syndrome, %†
21
Data are presented as mean⫾SD or percentage of subjects with the
characteristic unless otherwise described.
*Mean awake blood pressure was measured by an ambulatory blood
pressure monitor.
†Metabolic syndrome characteristics are defined according to the American
Heart Association Adult Treatment Panel III criteria.17
Wu et al
Table 2.
Sesame Supplementation Reduces 20-HETE in Humans
1155
Serum ␥-Tocopherol and Urine ␥-CEHC
Sesame
Variable
␥-Tocopherol, ␮mol/L
Placebo
Before
After
Before
After
P*
2.40⫾0.69
2.80⫾0.93
2.28⫾0.72
2.25⫾0.81
0.009
␥-Tocopherol/lipids, ␮mol/mmol†
0.33⫾0.09
0.38⫾0.10
0.30⫾0.09
0.31⫾0.10
0.009
␥-CEHC/creatinine, ␮mol/mmol‡§
0.52 (0.42 to 0.63)
0.36 (0.31 to 0.42)
0.50 (0.41 to 0.60)
0.67 (0.53 to 0.84)
0.001
Creatinine, mmol/L§
5.59 (4.62 to 6.75)
5.95 (4.97 to 7.13)
5.68 (4.66 to 6.92)
6.18 (5.00 to 7.64)
0.578
Data are presented as mean⫾SD unless otherwise described.
*Data show the comparison of after-treatment means by mixed-model analysis.
†Serum ␥-tocopherol is expressed normalized to lipids, which are calculated as total cholesterol plus triglycerides.
‡Urinary ␥-CEHCs are expressed normalized to creatinine excretion.
§Data were log-transformed before statistical analysis and are presented as geometric mean (95% CI).
reduction in urinary ␥-CEHC excretion by 30% (P⫽0.001
compared with placebo treatment; Table 2).
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Plasma and Urinary Levels of 20-HETE, Urinary
Electrolytes, and Blood Pressure
Sesame supplementation led to a significant reduction in
plasma circulating unesterified 20-HETE by 28% (P⫽0.001
compared with placebo treatment; Table 3). This observation
was not altered when the 20-HETE level was adjusted by its
precursor, arachidonic acid. Sesame supplementation did not
affect plasma arachidonic acid concentration (Table 3). Furthermore, sesame supplementation led to a 32% reduction in
urinary excretion of 20-HETE (P⬍0.001 compared with
placebo treatment; Table 3). Urinary excretion of sodium and
potassium was not affect after sesame supplementation and
neither was ambulatory systolic blood pressure or diastolic
blood pressure (Table 3).
We additionally examined the correlation between the
changes in urinary 20-HETE excretion with the changes in
␥-CEHC excretion (Figure S2). During sesame supplementation, there was a significant correlation between the change in
urinary 20-HETE and ␥-CEHC excretion (Pearson correlation coefficient: 0.507; P⫽0.006), whereas there was no
association during the placebo treatment (Pearson correlation
coefficient: ⫺0.136; P⫽0.499).
Table 3.
Discussion
20-HETE influences cardiovascular and renal function and
has been suggested to play a major role in the development of
hypertension. The recent development of selective inhibitors
of 20-HETE formation has enabled improved elucidation of
20-HETE bioactivities in animal models of hypertension.4,5
However, to the best of our knowledge, there are currently no
such analogous specific inhibitors suitable for administration
to humans. This motivated our investigation into the potential
of sesame-derived lignans for the inhibition of 20-HETE
synthesis.
Sesamin and sesamolin inhibited human renal and liver
microsome 20-HETE synthesis. Although the estimated inhibitory potency of these lignans (IC50 in the low micromolar
range) is relatively weak compared with synthetic inhibitors,
such as N-Hydroxy-N⬘-(4-butyl-2-methylphenyl)-formamidine
(HET0016; IC50 in the nanomolar range),23 our human study
suggests that in vivo concentrations of sesame lignans are
sufficient to reduce levels of circulating and excreted 20HETE after supplementation. Our data also demonstrate that
conversion of sesame lignans to the mammalian lignans
resulted in a substantial loss in inhibitory activity, highlighting that metabolic transformation of micronutrients can have
profound effects on their biological activities. The lack of
Plasma, Urinary 20-HETE, and Urinary Electrolytes
Sesame
Variable
Plasma arachidonic acid, mmol/L
Placebo
Before
After
Before
After
P*
0.27⫾0.06
0.27⫾0.05
0.28⫾0.06
0.27⫾0.06
0.783
Plasma 20-HETE, pmol/L†
832 (714 to 969)
596 (525 to 677)
830 (720 to 958)
768 (663 to 890)
0.001
Plasma 20-HETE/AA, pmol/␮mol†‡
3.13 (2.66 to 3.68)
2.25 (1.98 to 2.55)
3.10 (2.68 to 3.58)
2.96 (2.52 to 3.48)
0.002
Urine 20-HETE/creatinine, pmol/mmol†§
148 (125 to 175)
101 (84 to 120)
149 (125 to 176)
153 (129 to 182)
0.001
Urine sodium/creatinine, mmol/mmol§
12.2⫾3.9
12.4⫾4.4
12.3⫾4.1
11.9⫾4.3
0.556
6.8⫾2.4
6.3⫾2.2
7.2⫾2.7
6.2⫾2.3
0.227
SBP㛳
132.8⫾12.2
132.1⫾11.9
134.2⫾11.9
133.4⫾13.7
0.835
DBP㛳
80.7⫾8.1
81.0⫾7.2
82.0⫾7.5
80.8⫾8.4
0.223
Urine potassium/creatinine, mmol/mmol§
AA indicates arachidonic acid; SBP, systolic blood pressure; DBP, diastolic blood pressure. Data are presented as mean⫾SD except where indicated.
*Data show a comparison of after-treatment means by mixed-model analysis.
†Data were log-transformed before statistical analysis and are presented as geometric mean (95% CI).
‡Data show serum 20-HETE expressed normalized to arachidonic acid.
§Urinary 20-HETE, sodium, and potassium are expressed normalized to creatinine excretion.
㛳Mean awake blood pressure was measured by ambulatory blood pressure monitor.
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20-HETE inhibition by structurally related lignans, such as
secoisolariciresinol, is consistent with literature that the
methylenedioxyphenyl moiety of sesamin is the key structural feature enabling the inhibition of CYP enzymes (Figure
1).24 Although recent intervention studies have suggested that
flaxseeds (rich in secoisolariciresinol) and sesame seeds (rich
in sesamin) are similar in their potential to increase mammalian lignans after supplementation,25 our data suggest important differences in bioactivities between these precursor
compounds in the lignan family.
We also examined the effect of sesame lignans on the
production of EET. These metabolites have been shown to
possess renal vasodilatory and natriuretic activities and are
suggested to have antihypertensive effects.26 We found that
sesame lignans exhibited low inhibitory activity toward EET
synthesis with IC50 values that are ⱖ3-fold higher compared
with the 20-HETE pathway, suggesting relative selectivity
toward 20-HETE inhibition. With regard to the major 20HETE-producing CYP, we showed that in vitro sesamin was
a highly selective inhibitor for CYP4F2 and had relatively
minimal inhibitory effect on CYP4A11. This is in contrast to
recently developed synthetic 20-HETE–selective inhibitors,
such as TS-011 and HET0016, which have similar inhibitory
activity toward CYP4F2 and 4A11.27,28 This suggests that
sesamin may be a useful agent for the delineation of respective
contributions made by the CYP4F2 and CYP4A11-20-HETE
pathways to various physiological states, by allowing a relatively
selective inhibition of CYP4F2-20-HETE synthesis.
Previous studies have related a decrease in alcohol and an
increase in salt intake (major changes to salt balance achieved
by saline loading and depletion over 48 hours) to a 30%
decrease and a 40% increase in urine 20-HETE excretion,
respectively.29,30 A comparable reduction (30%) in urine
20-HETE was observed in the present study using whole
sesame supplementation (containing ⬇40 mg of sesamin and
10 mg of sesamolin per day), and this was unlikely because of
confounding factors. We have reported previously that macronutrient and alcohol intake did not differ between the
sesame and placebo treatment periods.16 Although alterations
in dietary salt intake were possible, this would have been
relatively minor compared with salt loading and depletion
studies. The observed decrease in 20-HETE is, therefore,
most likely explained by a direct inhibitory effect of sesame
lignans of the CYP4F2 enzyme, although we cannot exclude
the possibility that sesamin and sesamolin could have also
inhibited CYP4A11. The inhibition of the CYP4F2 pathway
is further supported by the observed correlation between
changes in urinary 20-HETE and ␥-CEHC excretions, because CYP4F2 has been suggested to also play a major role
in ␥-tocopherol metabolism.9 Our results also agree with
previous studies showing that sesame supplementation increases the concentration of ␥-tocopherol.6,7 Despite previous
reports of possible antioxidant activities of ␥-tocopherol,31
levels of plasma F2-isoprostanes (a validated biomarker of
oxidative stress) did not change in our study.16 It is possible
that greater increases in ␥-tocopherol are required to achieve
a reduction in oxidative stress.
CYP4F2 is strongly expressed in the proximal tubular
epithelia in the renal cortex and outer medulla, where 20-
HETE production can influence electrolyte and water reabsorption.13 When interpreted in conjunction with the recent
genetic association studies, which linked polymorphisms in
the CYP4F2 gene to increased urine 20-HETE excretion,
blood pressure, and prevalence of hypertension,32,33 it is
plausible that CYP4F2-generated 20-HETE may play a role
in human renal salt handling and blood pressure regulation.
Interestingly, previous animal studies have also suggested
natriuretic properties for the ␥-tocopherol metabolite
␥-CEHC.34,35 We did not observe any changes in urinary
sodium and potassium excretion in the current study. This
was not unexpected, given the study subjects were ambulatory and no attempts were made to control their salt balance,
and urinary electrolytes measurements were made in 24-hour
urine collections. Future studies with specific designs are
needed to assess whether 20-HETE inhibition after sesame
supplementation is associated with alterations in natriuresis in
humans.
As reported previously, blood pressure was also unchanged
after sesame supplementation in our study.16 Supplementation with purified sesamin has been shown to reduce blood
pressure in animal models of hypertension,36,37 possibly
related to the ability of sesamin to reduce vascular superoxide
production. In Japanese subjects with moderate hypertension,
supplementation with 60 mg/d of sesamin for 4 weeks also
reduced blood pressure.38 In these studies, the effect of
sesamin supplementation on the 20-HETE pathway was not
examined, but our study describes a biological activity of
sesame lignans that could provide a potential explanation for
the observations. In animal models of hypertension, administration of specific 20-HETE inhibitors, such as HET0016,
led to ⱖ90% reduction in urine levels of 20-HETE, with
concomitant changes in blood pressure.4,39 It is possible that
sesame supplementation in our human study did not result in
a sufficient reduction in levels of 20-HETE to affect blood
pressure. Furthermore, because 20-HETE possesses prohypertensive or antihypertensive effects depending on its site of
synthesis,3 systemic inhibition of 20-HETE levels after sesame supplementation could have resulted in counteracting
effects on BP regulation. We also cannot exclude the possibility that sesame supplementation reduces levels of EET,
which may again oppose the physiological effects of decreasing 20-HETE. Additional studies need to investigate whether
greater inhibition of 20-HETE synthesis may be achieved
using higher doses of sesame seeds or purified sesamin and
the possible effect that this has on blood pressure, as well as
additional in vivo markers of vascular function, such as
flow-mediated vasodilation.
Perspectives
This study demonstrates for the first time that sesame
supplementation in humans can lead to decreased plasma
levels and urinary excretion of 20-HETE. We further
provide evidence that this bioactivity is likely attributed to
direct inhibition of CYP4F2, one of the major 20-HETE–
producing enzymes in humans, by sesame lignans. Although urine and plasma 20-HETE levels reduced by
⬇30%, there were no changes in sodium excretion or
blood pressure in this study involving overweight subjects.
Wu et al
Sesame Supplementation Reduces 20-HETE in Humans
A major hindrance in the investigation of 20-HETE functionality in humans has been the lack of suitable inhibitors,
and previous investigations have relied heavily on assessing correlations between urinary 20-HETE excretion and
clinical parameters. Sesame is a natural product that is safe
for human consumption. We propose that supplementation
with sesame (or purified sesame lignan) could be incorporated into future human studies designed specifically to
investigate the role of 20-HETE in renal function and its
vascular properties. In particular, using sesamin to selectively inhibit CYP4F2-20-HETE synthesis in targeted populations (eg, those with CYP4F2 functional polymorphisms and/or
untreated hypertension) may yield further insights into the
biology of this important pathway.
Acknowledgments
Downloaded from http://hyper.ahajournals.org/ by guest on June 17, 2017
We thank Nichole Carlyon and Lyn McCahon for their assistance in
coordinating the study and performing the plasma arachidonic acid
measurements, respectively. We also thank Dr Natalie Ward for
reviewing the article and providing feedback. We gratefully acknowledge all of the volunteers for taking part in this study.
Sources of Funding
This study was funded by the National Health and Medical
Research Council of Australia (project grant 403957) and a Royal
Perth Hospital Medical Research Foundation Research Grant.
J.H.Y.W. acknowledges the assistance of a University of Western
Australia Faculty of Medicine, Dentistry, and Health Sciences
Fellowship.
Disclosures
None.
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Inhibition of 20-Hydroxyeicosatetraenoic Acid Synthesis Using Specific Plant Lignans: In
Vitro and Human Studies
Jason H.Y. Wu, Jonathan M. Hodgson, Michael W. Clarke, Adeline P. Indrawan, Anne E.
Barden, Ian B. Puddey and Kevin D. Croft
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Hypertension. 2009;54:1151-1158; originally published online September 28, 2009;
doi: 10.1161/HYPERTENSIONAHA.109.139352
Hypertension is published by the American Heart Association, 7272 Greenville Avenue, Dallas, TX 75231
Copyright © 2009 American Heart Association, Inc. All rights reserved.
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S1. Contents of nutrients in sesame and placebo bars
Placebo Bar
Sesame Bar
Unit/60g
Total energy (kJ)
1213
1171
Protein (g)
7.2
6.9
Total Fat (g)
15.4
14.9
Carbohydrate (g)
30.3
29.3
Dietary Fibre (g)
3.2
3.0
Sodium (g)
0.48
0.44
α-tocopherol (mg)
5.6
5.6
γ-tocopherol (mg)
4.4
4.4
Sesamin (mg)
-
39.5
Sesamolin (mg)
-
12.2
Change in γ-CEHC excretion
(pmol/mmol creatinine)
A. Sesame treatment *
0.4
0
-200
-100
0
100
200
-0.4
-0.8
-1.2
Change in 20-HETE excretion (pmol/mmol creatinine)
Change in γ-CEHC excretion
(pmol/mmol creatinine)
B. Placebo treatment
0.8
0.4
0
-300
-200
-100
0
100
200
300
-0.4
Change in 20-HETE excretion (pmol/mmol creatinine)
S2.
Scatter-plot between changes in urine 20-HETE excretion and
changes in γ-CEHC excretion following sesame and placebo
supplementation. Both 20-HETE and γ-CEHC concentrations are
adjusted by creatinine excretion and expressed as pmol/mmol.
Pearson correlation coefficients were used to quantify relations
between the variables. * Estimated r = 0.507 in the sesame group (P
= 0.006) whereas in the placebo group r = -0.136, (P = 0.499).