DOI 10.1515/hmbci-2013-0044 Horm Mol Biol Clin Invest 2013; 16(3): 129–138
John K. Cutts, Thomas R. Peavy, Doyle R. Moore, Jeevan Prasain, Stephen Barnes and Helen Kim*
Ovariectomy lowers urine levels of unconjugated (+)-catechin, (–)-epicatechin, and their methylated metabolites in rats fed grape seed extract Abstract: Steroid hormones modulate expression of enzymes that metabolize xenobiotics, including dietary supplements. Half of the human population undergoes menopause, yet the effect of this age-related loss of ovarian steroid hormones on the metabolism of dietary supplements has yet to be determined. Grape seed extract (GSE) is a dietary supplement comprised of monomeric and oligomeric catechins and has health benefits in models of age-related diseases. We hypothesized that surgically-induced loss of ovarian hormones would increase methylation, glucuronidation, and/or sulfation of the grape seed polyphenols (+)-catechin and (–)-epicatechin. Fourteen-week-old spontaneously hypertensive rats (SHRs) were ovariectomized (OVX) or sham-OVX. At 17 weeks of age, SHRs were gavaged with vehicle (water) or GSE (300 mg/kg body weight) once daily for 6 days. Urinary excretion of (+)-catechin, (–)-epicatechin, and their metabolites was analyzed by liquid chromatography-mass spectrometry. Although total urinary output of (+)-catechin, (–)-epicatechin, and their methy lated metabolites was unaffected by OVX, the amounts of (+)-catechin, (–)-epicatechin and their m ethylated metabolites that were not conjugated with glucuronic acid or sulfate were lowered by OVX. S pecifically, urine from OVX SHRs administered GSE contained 30% higher proportions (91.8% vs. 62.3%) of glucuronidated (+)-catechin and (–)-epicatechin and glucuronidated methyl (+)-catechin and methyl (–)-epicatechin than urine from sham-OVX SHRs. However, there were no differences in urinary levels of total methylated or sulfated catechins in OVX SHRs. This is the first quantitative characterization of metabolites of grape seed polyphenols in a model of menopause; it indicates that ovariectomy causes either an increase in expression and/or activity of select uridine 5′-diphospho-glucuronosyltransferase(s). Keywords: catechin; glucuronides; grape seed extract; menopause; metabolism.
*Corresponding author: Helen Kim, University of Alabama at Birmingham, Department of Pharmacology and Toxicology, McCallum Building, Room 460, 1918 University Blvd., Birmingham, AL 35294, USA, Phone: (205) 934-3880, Fax: (205) 934-6944, E-mail: [email protected]; and Targeted Metabolomics and Proteomics Laboratory, University of Alabama at Birmingham, Birmingham, AL, USA John K. Cutts and Thomas R. Peavy: Department of Pharmacology and Toxicology, University of Alabama at Birmingham, Birmingham, AL, USA Doyle R. Moore, Jeevan Prasain and Stephen Barnes: Department of Pharmacology and Toxicology, University of Alabama at Birmingham, Birmingham, AL, USA; and Targeted Metabolomics and Proteomics Laboratory, University of Alabama at Birmingham, Birmingham, AL, USA
Introduction Circulating steroidal hormones play important roles in regulating enzymes that metabolize endogenous and exogenous compounds. 17β-Estradiol induces cytochrome P450 (CYP) 1B1 for its own metabolism [1], as well as CYP3A4, but suppresses CYP1A1, CP2C11, CYP2C19 and CYP3A2 [2–4]. Progesterone induces both catechol-O-methyltransferase (COMT) [5] and uridine 5′-diphospho-glucuronosyltransferase (UGT) 1A1 [6]. 17β-Estradiol on the other hand, down-regulates COMT [7]; consistent with this, tamoxifen, an estrogen antagonist, increases activity and expression of COMT [8]. 17β-Estradiol reduces mRNA levels of UGT1A1 [9] and along with dihydrotestosterone reduces the activity of the UGTs that glucuronidate dihydrotestosterone and androsterone [10]. Thus hormones modulate either expression and/or activity of enzymes involved in phase I (CYPs) and II (COMT, UGTs, and sulfotransferases) xenobiotic metabolism. GSE is a dietary supplement that has demonstrated health benefits in mammalian models of diseases, including cancers [11–15], cardiovascular diseases [16, 17], and neurodegenerative diseases [18, 19]. GSE is enriched in proanthocyanidins, which are oligomers of the monomeric flavan-3-ols (+)-catechin and (–)-epicatechin (Figure 1A).
130 Cutts et al.: Ovariectomy lowers unconjugated urinary catechins
B
10.00 10.89
1.2e7
A
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5
8
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OH
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14
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10.94
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10.04
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138.9
1.20e5 1.00e5 8.00e4 6.00e4 2.00e4
160.8 165.1 179.2
115.2127.2 150.8
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123.1
8.0e5 4.0e5 95.1 119.1
147.1 161.0165.1 179.0 151.0
6.00e4
122.8
207.1
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147.3 161.1 95.3 116.4 130.5
165.1 178.9 206.9
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207.2
138.9
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161.2164.9 179.1
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Figure 1 Confirmation of (+)-catechin and (–)-epicatechin in the urine of sham-OVX and OVX SHRs by LC-MS/MS. (A) Chemical structures of (+)-catechin and (–)-epicatechin. Extracted ion chromatograms of m/z 291 from (B) (+)-catechin (tR 10.00) and (–)-epicatechin (tR 10.89) and from (C) urine from an OVX SHR fed GSE. Product ion mass spectra from (D) (+)-catechin and (E) the peak at tR 10.04 min from the urine of an OVX SHR fed GSE. Product ion mass spectra from (F) (–)-epicatechin and (G) the peak at tR 10.94 min from the urine of an OVX SHR fed GSE.
While these monomeric flavanols comprise only 4%–8% of GSE by weight [20], nonetheless they are thought to be the bioactive components of GSE [21, 22]; thus it is important to understand how these compounds are metabolized once ingested. Orally administered (+)-catechin and (–)-epicatechin are absorbed in the small intestine where they are metabolized, as well as in the liver, to methylated, glucuro nidated, and sulfated forms. The methylated forms of (+)-catechin and (–)-epicatechin can also be glucuro nidated or sulfated [23–25]. Each of these metabolic forms of (+)-catechin and (–)-epicatechin has been detected in the blood, urine, and brain of rodents that have ingested GSE [21, 26].
Although beneficial actions of GSE polyphenols have been reported in transgenic mouse models of dementia [18], in healthy rat brain [27], and in ovariectomized (OVX) rats [17], it is unknown whether the same metabolites were active in non-OVX vs. OVX rats. We hypothesized that the loss of ovarian hormones caused by OVX would increase methylation, glucuronidation, and/or sulfation of GSE catechins. We used OVX spontaneously hypertensive rats (SHRs) as a model of menopause to address this hypothesis. The results reported here show that OVX increased glucuronidated catechins in the urine of OVX SHRs and decreased unconjugated catechins relative to sham-OVX SHRs. No differences in methylated or sulfated catechins were detected.
Cutts et al.: Ovariectomy lowers unconjugated urinary catechins 131
Materials and methods Chemicals and reagents (+)-Catechin, (–)-epicatechin, and apigenin were purchased from Indofine Chemical Company, Inc. (Hillsborough, NJ), 3′-O-methyl (–)-epicatechin from Nacalai (San Diego, CA), Helix pomatia β-glucuronidase/sulfatase, 4-methylumbelliferyl sulfate, and phenolphthalein β-D-glucuronide from Sigma-Aldrich (St. Louis, MO). Powdered GSE was provided by Kikkoman Corporation (Chiba, Japan). AIN-93M maintenance purified diet was purchased from TestDiet (Richmond, IN).
Animals, GSE administration, and sample collection Female SHRs were purchased at 12 weeks of age from Charles River (Wilmington, MA). At 13 weeks of age SHRs were taken off the normal rodent chow and put onto AIN-93M diet. They were maintained on this latter diet for the remainder of the study. At 14 weeks of age half of the SHRs underwent bilateral OVX and the other half sham-OVX and were allowed to post-operatively recover for 3 weeks. At 17 weeks of age SHRs were split into four dietary groups: sham-OVX+vehicle (n=5), sham-OVX+GSE (n=4), OVX+vehicle (n=4), and OVX+GSE (n=4). SHRs were gavaged once daily with 0.5 mL of either vehicle (water) or GSE dissolved in water (300 mg/kg body weight) for 6 days. Urine samples were collected over the 24 h after gavage on day 4, and stored at –80°C until analysis. SHRs were euthanized on day 6. The uterus was removed from each animal, weighed, and stored at –80°C. All experimental procedures were approved by the Institutional Animal Care and Use Committee of the University of Alabama at Birmingham.
Extraction of catechins from urine In preparation for analysis by mass spectrometry, urine samples (100 μL) were incubated for 1 h at 37°C with or without 300 U of Helix pomatia β-glucuronidase/sulfatase, after mixing with 300 mM ammonium acetate, pH 5 (50 μL), acetic acid (5 μL), internal standards (6 μL; 0.6 nmoles each of 4-methylumbelliferyl sulfate, phenolphthalein β-D-glucuronide, and apigenin) and water to a total volume of 200 μL. After incubation, samples were treated with ice-cold methanol: 0.5% acetic acid (400 μL) and the precipitated material was removed by centrifugation at 16,000 g for 15 min at 4°C. The supernatant was used for analysis of the catechin metabolites.
Analysis of (+)-catechin, (–)-epicatechin and their metabolites by liquid chromatographytandem mass spectrometry Liquid chromatography-tandem mass spectrometry (LC-MS/MS) was carried out on urine extracts to analyze (+)-catechin, (–)-epicatechin, and their metabolites as described by Prasain et al. [26] using a model SIL-HT refrigerated Shimadzu autosampler, a Prominence HPLC ( Shimadzu Scientific Instruments, Columbia, MD) and an
API 4000 triple quadrupole mass spectrometer (AB Sciex, Concord, Ontario, Canada). Aliquots (10 μL) of urine extracts were injected onto a Phenomenex (Torrance, CA) Synergy Hydro-RP 80R C18 column (250 × 2.0 mm i.d., 4 μm particle size) and peaks were eluted by reversephase LC using a 15 min gradient at a flow rate of 0.2 mL/min. Mobile phase A consisted of 0.1% formic acid and mobile phase B consisted of 0.1% formic acid in methanol. The column was pre-equilibrated with 90% A and 10% B. The gradient applied was from 0 to 7 min, 10%–60% B; from 7 to 8 min, 60%–100% B; from 8 to 9 min, 100% B; from 9 to 10 min, 100%–10% B; and r e-equilibration from 10 to 15 min in 10% B. Multiple reaction monitoring (MRM) analysis in positive ion mode was used to quantify (+)-catechin and (–)-epicatechin using the mass transition m/z 291/139, and methyl (+)-catechin and methyl (–)-epicatechin using the mass transition m/z 305/139. MRM analysis in negative ion mode was used to detect the g lucuronides of (+)-catechin and (–)-epicatechin using the mass transition m/z 465/289, and of methyl (+)-catechin and (–)-epicatechin using the mass transition m/z 479/303. The sulfates of (+)-catechin and (–)-epicatechin using the mass transition m/z 369/289, and of methyl (+)-catechin and (–)-epicatechin using the mass transition m/z 383/303 were also assessed using MRM in negative ion mode. (+)-Catechin, (–)-epicatechin, and 3′-O-methyl (–)-epicatechin standards were spiked into control urine samples (from sham-OVX SHRs gavaged with vehicle), before extraction to generate s tandard curves. The standard curve for 3′-O-methyl (–)-epicatechin was used for quantification of 3′-O-methyl (+)-catechin. The peak that eluted 1 min before 3′-O-methyl (–)-epicatechin was designated as 3′-O-methyl (+)-catechin. The standard curves for (+)-catechin and (–)-epicatechin used concentrations of 5, 1, 0.5, 0.1, and 0.05 μg/ mL and for 3′-O-methyl (–)-epicatechin concentrations of 1, 0.5, 0.1, and 0.05 μg/mL. Correlation coefficients for each of the standard curves for (+)-catechin, (–)-epicatechin, and 3′-O-methyl (–)-epicatechin were > 0.99.
Urine creatinine analysis Creatinine concentrations were measured for each urine sample using an isotope dilution procedure with 2H3-creatinine by LC-MS [28]. Urinary outputs of catechins and their metabolites were corrected for creatinine output and were expressed as μg/mg creatinine.
Statistical analysis A Student’s t-test was used to determine differences between sham-OVX and OVX groups given GSE. p ≤ 0.05 was considered to be statistically significant. Data are presented as mean ± standard error of the mean (SEM).
Results Identification of (+)-catechin and (–)-epicatechin in rat urine by LC-MS/MS LC-MS/MS analysis of (+)-catechin and (–)-epicatechin was performed in positive ion mode by selecting
132 Cutts et al.: Ovariectomy lowers unconjugated urinary catechins their protonated molecular ion (m/z 291). (+)-Catechin and (–)-epicatechin eluted at retention times (tR) of 10.00 min and 10.89 min, respectively (Figure 1B). Figure 1C shows that two peaks with tR 10.04 min and 10.94 min were detected in a urine sample from an OVX SHR fed GSE. Product ions of the peak at tR 10.04 min in the urine sample were consistent with the product ions from authentic (+)-catechin, confirming that the peak that eluted with tR of 10.04 min was (+)-catechin in the urine (Figure 1D and E). Similarly, the product ions of the peak at tR 10.94 min in the urine were consistent with the product ions from (–)-epicatechin, confirming that the peak in the urine sample with the same retention time as authentic (–)-epicatechin was (–)-epicatechin (Figure 1F and G). The product ion spectra were consistent with previously reported (+)-catechin and (–)-epicatechin product ion spectra [29, 30]. Similar results were obtained for peaks detected in urines from sham-OVX SHRs.
Effect of ovariectomy on urinary (+)-catechin and (–)-epicatechin Urine samples from 18-week-old sham-OVX and OVX SHRs given GSE were analyzed by LC-MRM MS for (+)-catechin and (–)-epicatechin. In these experiments
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(+)-catechin and (–)-epicatechin spiked in control urine samples eluted at tR 8.98 min and tR 9.99 min, respectively, using the mass transition m/z 291/139. Urine samples from both sham-OVX+GSE (Figure 2A) and OVX+GSE (Figure 2B) SHRs had two peaks elute at tR of 8.97 min and tR 9.99 min with the mass transition m/z 291/139, confirming that both types of urine samples contained (+)-catechin and (–)-epicatechin. Urine samples from OVX+GSE SHRs contained 18.6 ± 1.0 μg/mg creatinine of nonmetabolized (+)-catechin and 12.8 ± 5.7 μg/mg creatinine of nonmetabolized (–)-epicatechin; whereas urine samples from shamOVX+GSE SHRs contained 61.0 ± 2 1.8 μg/mg creatinine of nonmetabolized (+)-catechin (p = 0.100 compared to OVX+GSE SHRs) and 142.3 ± 57.5 μg/mg creatinine of nonmetabolized (–)-epicatechin (p = 0.066 compared to OVX+GSE SHRs) (Figure 2C). In urine incubated with β-glucuronidase/sulfatase before extraction, OVX+GSE SHRs contained 220.5 ± 34.6 μg/mg creatinine of hydrolyzable (+)-catechin and 226.8 ± 43.6 μg/mg creatinine of hydrolyzable (–)-epicatechin, whereas urine samples from sham-OVX+GSE SHRs contained 291.9 ± 42.2 μg/mg creatinine of hydrolyzable (+)-catechin (p = 0.238) and 330.7 ± 59.7 μg/mg creatinine of hydrolyzable (–)-epicatechin (p = 0.209) (Figure 2D). Urine samples from shamOVX+vehicle and OVX+vehicle SHRs did not contain (+)-catechin or (–)-epicatechin (data not shown).
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Figure 2 Lower levels of GSE-derived urinary (+)-catechin and (–)-epicatechin in OVX SHRs vs. sham-OVX SHRs. MRM ion chromatograms of urines from (A) a sham-OVX SHR and (B) an OVX SHR fed GSE. Quantification of (C) non-metabolized and (D) hydrolyzable (+)-catechin and (–)-epicatechin in urine from sham-OVX (n = 4) and OVX (n = 4) SHRs fed GSE. White bars represent sham-OVX SHRs fed GSE and black bars represent OVX SHRs fed GSE. Data are expressed as the mean ± SEM.
Cutts et al.: Ovariectomy lowers unconjugated urinary catechins 133
Effect of ovariectomy on urinary 3′-O-methyl (+)-catechin and 3′-O-methyl (–)-epicatechin Urine samples from 18-week-old sham-OVX (Figure 3A) and OVX (Figure 3B) SHRs given GSE were analyzed by LC-MRM MS for 3′-O-methyl (+)-catechin and 3′-O-methyl (–)-epicatechin. 3′-O-methyl (–)-epicatechin spiked in control urine samples eluted at tR 10.86 min with the mass transition m/z 305/139. The peak at tR 10.09 min was designated as 3′-O-methyl (+)-catechin. Normalized to the urinary creatinine concentrations, urine samples from OVX+GSE SHRs contained 11.4 ± 3.7 μg/mg creatinine of nonmetabolized 3 ′-O-methyl (+)-catechin and 20.0 ± 4.5 μg/mg creatinine of nonmetabolized 3 ′-O-methyl (–)-epicatechin, whereas urine samples from sham-OVX+GSE SHRs contained 46.2 ± 18.5 μg/mg creatinine of nonmetabolized 3′-O-methyl (+)-catechin (p = 0.115 compared to OVX+GSE SHRs) and 111 ± 35.1 μg of nonmetabolized 3′-O-methyl (–)-epicatechin (p = 0.041 compared to OVX+GSE SHRs) (Figure 3C). In urine incubated with β-glucuronidase/sulfatase before extraction, OVX+GSE SHRs contained 163.6 ± 11.2 μg/mg creatinine of hydrolyzable 3′-O-methyl (+)-catechin and 185.3 ± 17.7 μg/mg creatinine of hydrolyzable 3′-O-methyl (–)-epicatechin, whereas urine samples from sham-OVX+GSE SHRs contained 174.6 ± 9.8 μg/mg creatinine of hydrolyzable
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3′-O-methyl (+)-catechin (p = 0.489) and 200.9 ± 13.4 μg/mg creatinine of hydrolyzable 3′-O-methyl (–)-epicatechin (p = 0.509) (Figure 3D). Urine samples from shamOVX+vehicle and OVX+vehicle SHRs did not contain 3′-O-methyl (+)-catechin or 3′-O-methyl (–)-epicatechin (data not shown). The amount of nonmetabolized (+)-catechin was expressed as a percentage of all flavanol forms in urine samples. All flavanol forms include hydrolyzable (+)- catechin, (–)-epicatechin, 3′-O-methyl (+)-catechin, and 3′-O-methyl (–)-epicatechin from Figures 2D and 3D for sham-OVX and OVX SHRs. This was also done to determine the percentages of nonmetabolized (–)-epicatechin, 3′-O-methyl (+)-catechin, and 3′-O-methyl (–)-epicatechin. The percentages of nonmetabolized (+)-catechin, (–)-epicatechin, 3′-O-methyl (+)-catechin, and 3′-O-methyl (–)-epicatechin in urine samples from OVX+GSE SHRs were 2.4%, 1.6%, 1.6%, and 2.6%, respectively, and the percentage of nonmetabolized (+)-catechin, (–)-epicatechin, 3′-O-methyl (+)-catechin, and 3′-O-methyl (–)-epicatechin in urine samples from sham-OVX+GSE SHRs were 6.2%, 14.7%, 4.9%, and 11.9%, respectively. Based on these calculations, monomeric catechins conjugated with glucuronic acid or sulfate were 62.3% of total monomeric catechins in urines from sham-OVX SHRs; this percentage increased to 91.8% of monomeric catechins in urines from OVX SHRs.
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Figure 3 Lower levels of GSE-derived urinary 3′-O-methyl (+)-catechin and 3′-O-methyl (–)-epicatechin in OVX SHRs vs. sham-OVX SHRs. MRM ion chromatograms of urines from (A) a sham-OVX SHR and (B) an OVX SHR fed GSE. Quantification of (C) nonmetabolized and (D) hydrolyzable 3′-O-methyl (+)-catechin and 3′-O-methyl (–)-epicatechin in urine from sham-OVX (n = 4) and OVX (n = 4) SHRs fed GSE. White bars represent sham-OVX SHRs fed GSE and black bars represent OVX SHRs fed GSE. Data are expressed as the mean ± SEM.
134 Cutts et al.: Ovariectomy lowers unconjugated urinary catechins
Effect of ovariectomy on the glucuronidation of (+)-catechin, (–)-epicatechin, and their methylated metabolites Urine samples from 18-week-old sham-OVX and OVX SHRs gavaged with GSE were analyzed by LC-MRM MS for the glucuronidated forms of (+)-catechin, (–)-epicatechin, 3′-O-methyl (+)-catechin, and 3′-O-methyl (–)-epicatechin. Urine samples incubated without β-glucuronidase/sulfatase (monitored with the mass transition m/z 465/289) had two dominant peaks elute at tR of 8.21 min and 8.92 min and other smaller peaks at tR 7.95 min and 9.83 min (data not shown), confirming the presence of (+)-catechin and (–)-epicatechin glucuronides in the urine. Urine samples incubated without β-glucuronidase/sulfatase (monitored with the mass transition m/z 479/303) had two dominant peaks elute at tR of 9.38 min and tR 9.96 min and another smaller peak at tR 8.73 min (Figure 4A), confirming the presence of 3′-O-methyl (+)-catechin and 3′-O-methyl (–)-epicatechin glucuronides in the urine. Each of these peaks disappeared when the urines were incubated with β-glucuronidase/sulfatase (Figure 4B). All urine samples had phenolphthalein β-D-glucuronide added to confirm hydrolysis of glucuronides by β-glucuronidase/sulfatase. Using the mass transition m/z 317/93, urine samples incubated without β-glucuronidase/sulfatase contained low amounts of phenolphthalein (Figure 4C) whereas
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Using the mass transition m/z 369/289 for (+)-catechin and (–)-epicatechin sulfates, urine samples incubated without β-glucuronidase/sulfatase had low intensity peaks elute at tR 9.52 min, tR 10.10 min, and tR 10.82 min. Using the mass transition m/z 383/303 for 3′-O-methyl (+)-catechin and 3′-O-methyl (–)-epicatechin sulfates, urine samples incubated without β-glucuronidase/sulfatase had two dominant peaks elute at tR of 10.82 min and tR 11.28 min (Figure 5A). However, unlike for the β-glucuronides, the amounts of (+)-catechin and (–)-epicatechin sulfates (data not shown) and 3′-O-methyl (+)-catechin and 3′-O-methyl (–)-epicatechin sulfates were unaffected by incubation with β-glucuronidase/sulfatase (Figure 5B). All urine samples had 4-methylumbelliferyl sulfate added prior to extraction to monitor sulfatase activity in the β-glucuronidase/sulfatase preparation. Using the mass transition m/z 175/119 for 4-methylumbelliferone,
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urine samples incubated with β-glucuronidase/sulfatase contained high amounts of phenolphthalein (Figure 4D) at tR 12.61 confirming the hydrolysis of phenolphthalein β-D-glucuronide.
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Figure 4 β-Glucuronidase/sulfatase (H. pomatia) hydrolyzes glucuronides of 3′-O-methyl (+)-catechin and 3′-O-methyl (–)-epicatechin. MRM ion chromatograms of 3′-O-methyl (+)-catechin and 3′-O-methyl (–)-epicatechin glucuronides (m/z 479/303) in urine from an OVX SHR incubated (A) without and (B) with β-glucuronidase/sulfatase. MRM ion chromatograms of phenolphthalein (m/z 317/93) in urine from the same OVX SHR incubated (C) without and (D) with β-glucuronidase/sulfatase, showing the appearance of the phenolphthalein released from phenolphthalein β-D-glucuronide.
Cutts et al.: Ovariectomy lowers unconjugated urinary catechins 135
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Time, min
Figure 5 β-Glucuronidase/sulfatase (H. pomatia) does not hydrolyze sulfate esters of 3′-O-methyl (+)-catechin and 3′-O-methyl (–)-epicatechin, although it hydrolyzes 4-methylumbelliferyl sulfate. MRM ion chromatograms of 3′-O-methyl (+)-catechin and 3′-O-methyl (–)-epicatechin sulfates (m/z 383/303) in urine from an OVX SHR incubated (A) without and (B) with the β-glucuronidase/sulfatase preparation show no difference in peak areas. Conversely, MRM ion chromatograms of 4-methylumbelliferone (m/z 175/119) in urine from the same OVX SHR incubated (C) without and (D) with β-glucuronidase/sulfatase show that the preparation has sulfatase activity, albeit with poor specificity for the flavanol sulfate esters.
LC-MRM MS determined that the urine extracts that were incubated without β-glucuronidase/sulfatase contained only trace amounts of 4-methylumbelliferone (Figure 5C), whereas urine samples incubated with the enzyme contained the expected amount of 4-methylumbelliferone (Figure 5D) at tR 12.34. These data confirm that H. pomatia β-glucuronidase/sulfatase did not hydrolyze the sulfate esters of (+)-catechin, (–)-epicatechin, and their methylated metabolites even though it successfully carried out the hydrolysis of 4-methylumbelliferyl sulfate. Therefore, the increase in the amounts of (+)-catechin, (–)-epicatechin, methyl (+)-catechin, and methyl (–)-epicatechin following incubation with β-glucuronidase/sulfatase represents the β-glucuronides of these flavanols and not the sulfates.
Effect of ovariectomy and GSE on body and uterine weights in sham-OVX and OVX SHRs Body weights were taken at 17 weeks of age before SHRs were gavaged with vehicle or GSE. OVX SHRs had higher body weights than sham-OVX SHRs (Table 1). At 18 weeks of age OVX SHRs continued to have higher body weights than sham-OVX SHRs (Table 1). GSE-gavaged SHRs had lower body weights than vehicle-gavaged SHRs. Uterine
weights were taken at 18 weeks of age when SHRs were euthanized. OVX SHRs had lower uterine weights than sham-OVX SHRs (Table 1). GSE did not affect uterine weight.
Discussion In the present study, we showed that OVX altered the metabolism of GSE catechins: specifically, lower levels of unconjugated (+)-catechin, (–)-epicatechin, and their methylated metabolites were quantified in the urines of OVX SHRs vs. sham-OVX SHRs, whereas higher levels of the glucuronides of these flavanols were measured in the urines of OVX versus sham-OVX SHRs. No differences in the levels of methylated or sulfated (+)-catechin or (–)-epicatechin were detected between OVX and shamOVX SHRs. Quantification of flavanols in urine samples from OVX SHRs gavaged with GSE showed 3–5-fold lower amounts of unconjugated (+)-catechin, (–)-epicatechin, 3′-O-methyl (+)-catechin, and 3′-O-methyl (–)-epicatechin excreted over 24 h compared to sham-OVX SHRs. These data represent a trend with p-values from 0.04 to 0.11. Failure to achieve full significance may have been
136 Cutts et al.: Ovariectomy lowers unconjugated urinary catechins Table 1 Body and uterine weights of sham-OVX and OVX SHRs. Group
17 Weeks old body weight, g
18 Weeks old body weight, g
18 Weeks old uterine weight, g
Sham-OVX+vehicle Sham-OVX+GSE OVX+vehicle OVX+GSE
203 ± 1 201 ± 2 237 ± 2 241 ± 3
194 ± 2 181 ± 6 231 ± 1 217 ± 1
0.56 ± 0.06 0.53 ± 0.10 0.10 ± 0.00 0.10 ± 0.00
due to a larger variance in the urine output of catechins and methyl catechins in sham-OVX SHRs than in the OVX SHRs, which may be a result of changes in the concentration of 17β-estradiol during the estrous cycle in rats [31]. There are reports of variation of 17β-estradiol concentration during the estrous cycle altering responsiveness to physiological and external stimuli [32, 33]. Furthermore, Kulkarni et al. (2012) have recently shown that the bioavailability and metabolism of another bioflavonoid – genistein – depends on the stage of the estrous cycle of the rat [34]. Monomeric catechins are methylated by the phase II enzyme COMT. In this study, the total amount of 3′-O-methyl (+)-catechin and 3′-O-methyl (–)-epicatechin excreted in the urine was not different between sham-OVX and OVX SHRs. These findings were not consistent with previous work where OVX increased COMT expression and activity [8]. Follow-up experiments will address this discrepancy. LC-MRM MS of the urine samples incubated without β-glucuronidase/sulfatase detected glucuronidated and sulfated forms of (+)-catechin, (–)-epicatechin, 3′-O-methyl (+)-catechin, and 3′-O-methyl (–)-epicatechin in urines from both OVX and sham-OVX SHRs gavaged with GSE. Some of these conjugated forms of catechin had multiple peaks, which may represent isomers of (+)-catechin and (–)-epicatechin glucuronide/sulfate conjugation on hydroxyl groups at carbon 3′, carbon 5, or carbon 7 [25]. Whereas urine samples that were incubated with β-glucuronidase/sulfatase no longer contained the glucuronidated forms of (+)-catechin, (–)-epicatechin, 3′-O-methyl (+)-catechin, and 3′-O-methyl (–)-epicatechin, the peak areas of the sulfated forms of (+)-catechin, (–)-epicatechin, 3′-O-methyl (+)-catechin, and 3′-O-methyl (–)-epicatechin were unchanged. LC-MRM MS indicated that while both phenolphthalein and 4-methylumbelliferone were released from the internal standards phenolphthalein β-D-glucuronide and 4-methylumbelliferyl sulfate when incubated with H. pomatia β-glucuronidase/ sulfatase, the sulfatase activity of this enzyme preparation did not hydrolyze the sulfate esters of catechins and
methyl catechins. These data confirm the findings of Saha et al. who have recently shown that flavanols, specifically (–)-epicatechin, are poor substrates for many sulfatasecontaining commercial preparations [35]. Accordingly, the increase in levels of the amounts of urinary (+)-catechin, (–)-epicatechin, 3′-O-methyl (+)-catechin, and 3′-O-methyl (–)-epicatechin following incubation with β-glucuronidase/sulfatase represents increased glucuronides of the catechins, consistent with our hypothesis that OVX would increase glucuronidation of catechins, presumably because of dysregulation of UGTs resulting from the loss of estrogens. These data are consistent with studies [9, 10] showing that estrogen attenuates UGT expression and activity. Follow-up studies using a sulfatase that has efficacy in removing the sulfate from catechins might reveal an increase in sulfation due to OVX. At the beginning of the study, 3 weeks after ovariectomy, OVX SHRs had higher body weights compared with sham-OVX SHRs, consistent with the weight gain previously shown to be a consequence of ovariectomy [36, 37]. This difference in body weight between OVX and shamOVX SHRs continued throughout the study. It was noted that SHRs gavaged with GSE had lower body weights compared to SHRs gavaged with vehicle. The SHRs given vehicle lost 3%–4% of their starting body weight whereas the SHRs given GSE lost 10% of their starting body weight. Given the short duration of the study (6 days), the slight loss of body weight could simply have reflected initial adjustment to being gavaged, particularly with the GSE. As expected, OVX SHRs had lower uterine weights compared with sham-OVX SHRs, consistent with the lower uterine weights previously shown to be a consequence of ovariectomy [37]. The administration of GSE did not affect the uterine weight in sham-OVX or OVX SHRs. It has been reported that protein levels of the male specific CYP2C11 and the female specific CYP2C12 in the liver were increased [38] and decreased [39], respectively, following OVX in rats. It will be important in follow-up studies to confirm that CYP2C11 is increased and CYP2C12 is decreased in our OVX rats and to determine whether GSE affects these anticipated effects. Based on the lack of estrogenic activity of GSE on reproductive tissue in this study, we predict that the OVX-induced differences in protein levels for CYP2C11 and CYP2C12 will not be affected by GSE. As glucuronidation is generally considered to be a mechanism whereby the body excretes xenobiotics, the results presented here suggest that postmenopausal women may not achieve equivalent physiological levels as premenopausal women of unconjugated (+)-catechin, (–)-epicatechin, and their methylated metabolites from
Cutts et al.: Ovariectomy lowers unconjugated urinary catechins 137
intake of the same dose of a dietary supplement like GSE. It has recently been shown that 3′-O-methyl (–)-epicatechin is glucuronidated in vitro most effectively by UGT1A9 [40]; follow-up studies will determine whether changes in 17β-estradiol affect the expression and/or activity of UGT1A9. It should be kept in mind that 3′-O-methyl (–)-epicatechin glucuronide added to hippocampal slices enhanced long-term potentiation [21], however it is not known whether the bioactive form in the neurons was the glucuronide or whether it had been de-conjugated. This is the first report of differences in the flavanols (+)-catechin, (–)-epicatechin, 3′-O-methyl (+)-catechin, and 3′-O-methyl (–)-epicatechin in a rodent model of menopause following oral intake of GSE; further experiments will examine the molecular basis and the clinical implications of these differences. Acknowledgements: This work was supported by grant R21AT004083-02 from the National Institutes of Health, National Center for Complementary and Alternative
Medicine to Helen Kim, a predoctoral fellowship awarded to John Cutts by the NIH Office of Dietary Supplements as an additional slot on the University of Alabama at Birmingham Cellular and Molecular Biology T32 predoctoral training program (2T32GM008111, Brad Yoder, PI) and by funds from the Department of Pharmacology and Toxicology at the University of Alabama at Birmingham. The analysis of urine creatinine was carried out by John Rodgers (UAB Department of Genetics and the Heflin Genomics Center), supported by a National Institue of Diabetes and Digestive and Kidney Diseases grant (P30 DK079337, Anupam Agarwal, PI) to the O’Brien UAB-UCSD Acute Kidney Injury Center. Kikkoman Corporation (Chiba, Japan) graciously provided the GSE for this study. The AB Sciex 4000 triple quadrupole mass spectrometer used in this study was purchased with a grant from the UAB Health Services Foundation General Endowment Fund awarded to Stephen Barnes. Received August 16, 2013; accepted October 29, 2013
References 1. Tsuchiya Y, Nakajima M, Kyo S, Kanaya T, Inoue M, Yokoi T. Human CYP1B1 is regulated by estradiol via estrogen receptor. Cancer Res 2004;64:3119–25. 2. Ishii T, Nishimura K, Nishimura M. Administration of xenobiotics with anti-estrogenic effects results in mRNA induction of adult male-specific cytochrome P450 isozymes in the livers of adult female rats. J Pharmacol Sci 2006;101:220–55. 3. Scott LM, Durant P, Leone-Kabler S, Wood CE, Register TC, Townsend A, Cline JM. Effects of prior oral contraceptive use and soy isoflavonoids on estrogen-metabolizing cytochrome P450 enzymes. J Steroid Biochem Mol Biol 2008;112:179–85. 4. Mwinyi J, Cavaco I, Pedersen RS, Persson A, Burkhardt S, Mkrtchian S, Ingelman-Sundberg M. Regulation of CYP2C19 expression by estrogen receptor α: implications for estrogendependent inhibition of drug metabolism. Mol Pharm 2010;78:886–94. 5. Inoue K, Creveling CR. Induction of catechol-O-methyltransferase in the luminal epithelium of rat uterus by progesterone. J Histochem Cytochem 1991;39:823–28. 6. Jeong H, Choi S, Song JW, Chen H, Fischer JH. Regulation of UDP-glucuronosyltransferase (UGT) 1A1 by progesterone and its impact on labetalol elimination. Xenobiotica 2008;38:62–75. 7. Jiang H, Xie T, Ramsden DB, Ho SL. Human catechol-O-methyltransferase down-regulation by estradiol. Neuropharm 2003;45:1011–8. 8. Schendzielorz N, Rysa A, Reenila I, Raasmaja A, Mannisto PT. Complex estrogenic regulation of catechol-O-methyltransferase (COMT) in rats. J Physiol Pharmacol 2011;62:483–90. 9. Strasser SI, Smid SA, Mashford ML, Desmond PV. Sex hormones differentially regulate isoforms of UDP-glucuronosyltransferase. Pharm Res 1997;14:1115–21.
10. Guillemette C, Hum DW, Belanger A. Regulation of steroid glucuronosyltransferase activities and transcripts by androgen in the human prostatic cancer LNCaP cell line. Endocrinology 1996;137:2872–9. 11. Akhtar S, Meeran SM, Katiyar N, Katiyar SK. Grape seed proanthocyanidins inhibit the growth of human non-small cell lung cancer xenografts by targeting insulin-like growth factor binding protein-3, tumor cell proliferation, and angiogenic factors. Clin Cancer Res 2009;15:821–31. 12. Meeran SM, Vaid M, Punathil T, Katiyar SK. Dietary grape seed proanthocyanidins 12-O-tetradecanoyl phorbol-13-acetatecaused skin tumor promotion in 7,12-dimethylbenz[a] anthracene-initiated mouse skin, which is associated with the inhibition of inflammatory responses. Carcinogenesis 2009;30:520–8. 13. Velmurugan B, Singh RP, Agarwal R, Agarwal C. Dietary-feeding of grape seed extract prevents azoxymethane-induced colonic aberrant crypt foci formation in fischer 344 rats. Mol Carcinogen 2010;49:641–52. 14. Velmurugan B, Singh RP, Daul N, Agarwal R, Agarwal C. Dietary feeding of grape seed extract prevents intestinal tumorigenesis in APCmin/+ mice. Neoplasia 2010;12:95–102. 15. Gao N, Budhraja A, Cheng S, Yao H, Zhang Z, Shi X. Induction of apoptosis in human leukemia cells by grape seed extract occurs via activation of c-Jun NH2-terminal kinase. Clin Cancer Res 2009;15:140–9. 16. Pataki T, Bak I, Kovacs P, Bagchi D, Das DK, Tosaki A. Grape seed proanthocyanidins improved cardiac recovery during reperfusion after ischemia in isolated rat hearts. Am J Clin Nutr 2002;75:894–9.
138 Cutts et al.: Ovariectomy lowers unconjugated urinary catechins 17. Peng N, Clark JT, Prasain J, Kim H, White CR, Wyss JM. Antihypertensive and cognitive effects of grape polyphenols in estrogen-depleted, female, spontaneously hypertensive rats. Am J Physiol Regul Integr Comp Physiol 2005;289:R771–5. 18. Wang J, Ho L, Zhao W, Ono K, Rosensweig C, Chen L, Humala N, Teplow DB, Pasinetti GM. Grape-derived polyphenolics prevent Aβ oligomerization and attenuate cognitive deterioration in a mouse model of Alzheimer’s disease. J Neurosci 2008;28:6388–92. 19. Wang Y-J, Thomas P, Zhong J-H, Bi F-F, Kosaraju S, Pollard A, Fenech M, Zhou X-F. Consumption of grape seed extract prevents amyloid-β deposition and attenuates inflammation in brain of an Alzheimer’s disease mouse. Neurotox Res 2009;15:3–14. 20. Yamakoshi J, Saito M, Kataoka S, Kikuchi M. Safety evaluation of proanthocyanidin-rich extract from grape seeds. Food Chem Toxicol 2002;40:599–607. 21. Wang J, Ferruzzi MG, Ho L, Blount J, Janle EM, Gong B, Pan Y, Gowda GA, Raftery D, Arrieta-Cruz I, Sharma V, Cooper B, Lobo J, Simon JE, Zhang C, Cheng A, Qian X, Ono K, Teplow DB, Pavlides C, Dixon RA, Pasinetti GM. Brain-targeted proanthocyanidin metabolites for Alzheimer’s disease treatment. J Neuroscience 2012;32:5144–50. 22. Schroeter H, Heiss C, Balzer J, Kleinbongard P, Keen CL, Hollenberg NK, Sies H, Kwik-Uribe C, Schmitz HH, Kelm M. (-)-Epicatechin mediates beneficial effects of flavanol-rich cocoa on vascular function in humans. PNAS 2006;103:1024–9. 23. Donovan JL, Crespy V, Manach C, Morand C, Besson C, Scalbert A, Remesy C. Catechin is metabolized by both the small intestine and liver of rats. J Nutr 2001;131:1753–7. 24. Tsang C, Auger C, Mullen W, Bornet A, Rouanet J-M, Crozier A, Teissedre P-L. The absorption, metabolism and excretion of flavan-3-ols and procyanidins following the ingestion of a grape seed extract by rats. Br J Nutr 2005;94:170–81. 25. Ottaviani JI, Momma TY, Kuhnle GK, Keen CL, Schroeter H. Structurally related (-)-epicatechin metabolites in humans: assessment using de novo chemically synthesized authentic standards. Free Radical Biol Med 2012;52:1403–12. 26. Prasain JK, Peng N, Dai Y, Moore R, Arabshahi A, Wilson L, Barnes S, Wyss JM, Kim H, Watts RL. Liquid chromatography tandem mass spectrometry identification of proanthocyanidins in rat plasma after oral administration of grape seed extract. Phytomedicine 2009;16:233–43. 27. Deshane J, Chaves L, Sarikonda KV, Isbell S, Wilson L, Kirk M, Grubbs C, Barnes S, Meleth S, Kim H. Proteomics analysis of rat brain protein modulations by grape seed extract. J Agric Food Chem 2004;52:7872–83. 28. Takahashi N, Boysen G, Li F, Li Y, Swenberg JA. Tandem mass spectrometry measurements of creatinine in mouse plasma and urine for determining glomerular filtration rate. Kidney Int 2007;71:266–71.
29. Li H-J, Deinzer ML. Tandem mass spectrometry for sequencing proanthocyanidins. Anal Chem 2007;79:1739–48. 30. Cren-Olive C, Deprez S, Lebrun S, Coddeville B, Rolando C. Characterization of methylation site of monomethylflavan-3-ols by liquid chromatography/electrospray ionization tandem mass spectrometry. Rapid Commun Mass Spec 2000;14: 2312–9. 31. Marcondes FK, Miguel KJ, Melo LL, Spadari-Bratfisch RC. Estrous cycle influences the response of female rats in the elevated plus-maze test. Physiol Behav 2001;74:435–40. 32. Lapointe J, Roy M, St-Pierre I, Kimmins S, Gauvreau D, MacLaren LA, Bilodeau J-F. Hormonal and spatial regulation of nitric oxide synthases (NOS) (neuronal NOS, inducible NOS, and endothelial NOS) in the oviducts. Endocrinology 2006;147:5600–10. 33. Znamensky V, Akama KT, McEwen BS, Milner TA. Estrogen levels regulate the subcellular distribution of phosphorylated Akt in hippocampal CA1 dendrites. J Neurosci 2003;23:2340–7. 34. Kulkarni KH, Yang Z, Niu T, Hu M. Effects of estrogen and estrus cycle on pharmacokinetics, absorption, and disposition of genistein in female Sprague-Dawley rats. J Agric Food Chem 2012;60:7949–56. 35. Saha S, Hollands W, Needs PW, Ostertag LM, de Roos B, Duthie GG, Kroon PA. Human O-sulfated metabolites of (-)-epicatechin and methyl-(-)-epicatechin are poor substrates for commercial aryl-sulfatases: implications for studies concerned with quantifying epicatechin bioavailability. Pharmacol Res 2012;65:592–602. 36. Fortepiani LA, Zhang H, Racusen L, Roberts II LJ, Reckelhoff JF. Characterization of an animal model of postmenopausal hypertension in spontaneously hypertensive rats. Hypertension 2003;41:640–5. 37. Lopez-Sepulveda R, Jimenez R, Romero M, Zarzuelo MJ, Sanchez M, Gomez-Guzman M, Vargas F, O’Valle F, Zarzuelo A, Perez-Vizcaino F, Duarte J. Wine polyphenols improve endothelial function in large vessels of female spontaneously hypertensive rats. Hypertension 2008;51:1088–95. 38. Oropeza-Hernandez LF, Sierra-Santoyo A, Cebrian ME, Manno M, Albores A. Ovariectomy modulates the response of some cytochrome P450 isozymes to lindane in the rat. Toxicol Lett 2001;124:91–9. 39. Liao D-Z, Porsch-Hallstrom I, Gustafsson J-A, Blanck A. Persistent sex differences in growth control of early rat liver lesions are programmed during promotion in the resistant hepatocyte model. Hepatology 1996;23:835–9. 40. Blount JW, Ferruzzi M, Raftery D, Pasinetti GM, Dixon RA. Enzymatic synthesis of substituted epicatechins for bioactivity studies in neurological disorders. Biochem Biophys Res Comm 2012;417:457–61.
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John K. Cutts, Thomas R. Peavy, Doyle R. Moore, Jeevan Prasain, Stephen Barnes and Helen Kim*
Ovariectomy lowers urine levels of unconjugated (+)-catechin, (–)-epicatechin, and their methylated metabolites in rats fed grape seed extract Abstract: Steroid hormones modulate expression of enzymes that metabolize xenobiotics, including dietary supplements. Half of the human population undergoes menopause, yet the effect of this age-related loss of ovarian steroid hormones on the metabolism of dietary supplements has yet to be determined. Grape seed extract (GSE) is a dietary supplement comprised of monomeric and oligomeric catechins and has health benefits in models of age-related diseases. We hypothesized that surgically-induced loss of ovarian hormones would increase methylation, glucuronidation, and/or sulfation of the grape seed polyphenols (+)-catechin and (–)-epicatechin. Fourteen-week-old spontaneously hypertensive rats (SHRs) were ovariectomized (OVX) or sham-OVX. At 17 weeks of age, SHRs were gavaged with vehicle (water) or GSE (300 mg/kg body weight) once daily for 6 days. Urinary excretion of (+)-catechin, (–)-epicatechin, and their metabolites was analyzed by liquid chromatography-mass spectrometry. Although total urinary output of (+)-catechin, (–)-epicatechin, and their methy lated metabolites was unaffected by OVX, the amounts of (+)-catechin, (–)-epicatechin and their m ethylated metabolites that were not conjugated with glucuronic acid or sulfate were lowered by OVX. S pecifically, urine from OVX SHRs administered GSE contained 30% higher proportions (91.8% vs. 62.3%) of glucuronidated (+)-catechin and (–)-epicatechin and glucuronidated methyl (+)-catechin and methyl (–)-epicatechin than urine from sham-OVX SHRs. However, there were no differences in urinary levels of total methylated or sulfated catechins in OVX SHRs. This is the first quantitative characterization of metabolites of grape seed polyphenols in a model of menopause; it indicates that ovariectomy causes either an increase in expression and/or activity of select uridine 5′-diphospho-glucuronosyltransferase(s). Keywords: catechin; glucuronides; grape seed extract; menopause; metabolism.
*Corresponding author: Helen Kim, University of Alabama at Birmingham, Department of Pharmacology and Toxicology, McCallum Building, Room 460, 1918 University Blvd., Birmingham, AL 35294, USA, Phone: (205) 934-3880, Fax: (205) 934-6944, E-mail: [email protected]; and Targeted Metabolomics and Proteomics Laboratory, University of Alabama at Birmingham, Birmingham, AL, USA John K. Cutts and Thomas R. Peavy: Department of Pharmacology and Toxicology, University of Alabama at Birmingham, Birmingham, AL, USA Doyle R. Moore, Jeevan Prasain and Stephen Barnes: Department of Pharmacology and Toxicology, University of Alabama at Birmingham, Birmingham, AL, USA; and Targeted Metabolomics and Proteomics Laboratory, University of Alabama at Birmingham, Birmingham, AL, USA
Introduction Circulating steroidal hormones play important roles in regulating enzymes that metabolize endogenous and exogenous compounds. 17β-Estradiol induces cytochrome P450 (CYP) 1B1 for its own metabolism [1], as well as CYP3A4, but suppresses CYP1A1, CP2C11, CYP2C19 and CYP3A2 [2–4]. Progesterone induces both catechol-O-methyltransferase (COMT) [5] and uridine 5′-diphospho-glucuronosyltransferase (UGT) 1A1 [6]. 17β-Estradiol on the other hand, down-regulates COMT [7]; consistent with this, tamoxifen, an estrogen antagonist, increases activity and expression of COMT [8]. 17β-Estradiol reduces mRNA levels of UGT1A1 [9] and along with dihydrotestosterone reduces the activity of the UGTs that glucuronidate dihydrotestosterone and androsterone [10]. Thus hormones modulate either expression and/or activity of enzymes involved in phase I (CYPs) and II (COMT, UGTs, and sulfotransferases) xenobiotic metabolism. GSE is a dietary supplement that has demonstrated health benefits in mammalian models of diseases, including cancers [11–15], cardiovascular diseases [16, 17], and neurodegenerative diseases [18, 19]. GSE is enriched in proanthocyanidins, which are oligomers of the monomeric flavan-3-ols (+)-catechin and (–)-epicatechin (Figure 1A).
130 Cutts et al.: Ovariectomy lowers unconjugated urinary catechins
B
10.00 10.89
1.2e7
A
OH 3′
HO
OH
HO
4′
OH
0
OH
3
5
8
11 12 10 Time, min
9
OH
OH
(+)-Catechin
(–)-Epicatechin
13
14
13
14
10.94
4.0e6
10.04
3.2e6
OH Intensity
3
7
O
7
C 5
6.0e6 3.0e6
3′
OH
4′
O
7
Intensity
9.0e6
2.4e6 1.6e6 8.0e5 0
F
138.9
1.20e5 1.00e5 8.00e4 6.00e4 2.00e4
160.8 165.1 179.2
115.2127.2 150.8
G Intensity
Intensity
123.1
8.0e5 4.0e5 95.1 119.1
147.1 161.0165.1 179.0 151.0
6.00e4
122.8
207.1
20 40 60 80 100 120 140 160 180 200 220 240 260 280 300 m/z
147.3 161.1 95.3 116.4 130.5
165.1 178.9 206.9
20 40 60 80 100 120 140 160 180 200 220 240 260 280 m/z
1.6e6 1.2e6
8.00e4
2.00e4
207.2
138.9
1.9e6
10 11 12 Time, min
1.00e5
4.00e4
20 40 60 80 100 120 140 160 180 200 220 240 260 280 300 m/z
E
9 139.0
147.1
4.00e4
8
1.20e5
123.1
Intensity
Intensity
D
7
300
138.9
2.0e6 1.6e6 1.2e6
123.0
8.0e5 147.0
4.0e5 95.2
119.3
161.2164.9 179.1
207.2
20 40 60 80 100 120 140 160 180 200 220 240 260 280 m/z
300
Figure 1 Confirmation of (+)-catechin and (–)-epicatechin in the urine of sham-OVX and OVX SHRs by LC-MS/MS. (A) Chemical structures of (+)-catechin and (–)-epicatechin. Extracted ion chromatograms of m/z 291 from (B) (+)-catechin (tR 10.00) and (–)-epicatechin (tR 10.89) and from (C) urine from an OVX SHR fed GSE. Product ion mass spectra from (D) (+)-catechin and (E) the peak at tR 10.04 min from the urine of an OVX SHR fed GSE. Product ion mass spectra from (F) (–)-epicatechin and (G) the peak at tR 10.94 min from the urine of an OVX SHR fed GSE.
While these monomeric flavanols comprise only 4%–8% of GSE by weight [20], nonetheless they are thought to be the bioactive components of GSE [21, 22]; thus it is important to understand how these compounds are metabolized once ingested. Orally administered (+)-catechin and (–)-epicatechin are absorbed in the small intestine where they are metabolized, as well as in the liver, to methylated, glucuro nidated, and sulfated forms. The methylated forms of (+)-catechin and (–)-epicatechin can also be glucuro nidated or sulfated [23–25]. Each of these metabolic forms of (+)-catechin and (–)-epicatechin has been detected in the blood, urine, and brain of rodents that have ingested GSE [21, 26].
Although beneficial actions of GSE polyphenols have been reported in transgenic mouse models of dementia [18], in healthy rat brain [27], and in ovariectomized (OVX) rats [17], it is unknown whether the same metabolites were active in non-OVX vs. OVX rats. We hypothesized that the loss of ovarian hormones caused by OVX would increase methylation, glucuronidation, and/or sulfation of GSE catechins. We used OVX spontaneously hypertensive rats (SHRs) as a model of menopause to address this hypothesis. The results reported here show that OVX increased glucuronidated catechins in the urine of OVX SHRs and decreased unconjugated catechins relative to sham-OVX SHRs. No differences in methylated or sulfated catechins were detected.
Cutts et al.: Ovariectomy lowers unconjugated urinary catechins 131
Materials and methods Chemicals and reagents (+)-Catechin, (–)-epicatechin, and apigenin were purchased from Indofine Chemical Company, Inc. (Hillsborough, NJ), 3′-O-methyl (–)-epicatechin from Nacalai (San Diego, CA), Helix pomatia β-glucuronidase/sulfatase, 4-methylumbelliferyl sulfate, and phenolphthalein β-D-glucuronide from Sigma-Aldrich (St. Louis, MO). Powdered GSE was provided by Kikkoman Corporation (Chiba, Japan). AIN-93M maintenance purified diet was purchased from TestDiet (Richmond, IN).
Animals, GSE administration, and sample collection Female SHRs were purchased at 12 weeks of age from Charles River (Wilmington, MA). At 13 weeks of age SHRs were taken off the normal rodent chow and put onto AIN-93M diet. They were maintained on this latter diet for the remainder of the study. At 14 weeks of age half of the SHRs underwent bilateral OVX and the other half sham-OVX and were allowed to post-operatively recover for 3 weeks. At 17 weeks of age SHRs were split into four dietary groups: sham-OVX+vehicle (n=5), sham-OVX+GSE (n=4), OVX+vehicle (n=4), and OVX+GSE (n=4). SHRs were gavaged once daily with 0.5 mL of either vehicle (water) or GSE dissolved in water (300 mg/kg body weight) for 6 days. Urine samples were collected over the 24 h after gavage on day 4, and stored at –80°C until analysis. SHRs were euthanized on day 6. The uterus was removed from each animal, weighed, and stored at –80°C. All experimental procedures were approved by the Institutional Animal Care and Use Committee of the University of Alabama at Birmingham.
Extraction of catechins from urine In preparation for analysis by mass spectrometry, urine samples (100 μL) were incubated for 1 h at 37°C with or without 300 U of Helix pomatia β-glucuronidase/sulfatase, after mixing with 300 mM ammonium acetate, pH 5 (50 μL), acetic acid (5 μL), internal standards (6 μL; 0.6 nmoles each of 4-methylumbelliferyl sulfate, phenolphthalein β-D-glucuronide, and apigenin) and water to a total volume of 200 μL. After incubation, samples were treated with ice-cold methanol: 0.5% acetic acid (400 μL) and the precipitated material was removed by centrifugation at 16,000 g for 15 min at 4°C. The supernatant was used for analysis of the catechin metabolites.
Analysis of (+)-catechin, (–)-epicatechin and their metabolites by liquid chromatographytandem mass spectrometry Liquid chromatography-tandem mass spectrometry (LC-MS/MS) was carried out on urine extracts to analyze (+)-catechin, (–)-epicatechin, and their metabolites as described by Prasain et al. [26] using a model SIL-HT refrigerated Shimadzu autosampler, a Prominence HPLC ( Shimadzu Scientific Instruments, Columbia, MD) and an
API 4000 triple quadrupole mass spectrometer (AB Sciex, Concord, Ontario, Canada). Aliquots (10 μL) of urine extracts were injected onto a Phenomenex (Torrance, CA) Synergy Hydro-RP 80R C18 column (250 × 2.0 mm i.d., 4 μm particle size) and peaks were eluted by reversephase LC using a 15 min gradient at a flow rate of 0.2 mL/min. Mobile phase A consisted of 0.1% formic acid and mobile phase B consisted of 0.1% formic acid in methanol. The column was pre-equilibrated with 90% A and 10% B. The gradient applied was from 0 to 7 min, 10%–60% B; from 7 to 8 min, 60%–100% B; from 8 to 9 min, 100% B; from 9 to 10 min, 100%–10% B; and r e-equilibration from 10 to 15 min in 10% B. Multiple reaction monitoring (MRM) analysis in positive ion mode was used to quantify (+)-catechin and (–)-epicatechin using the mass transition m/z 291/139, and methyl (+)-catechin and methyl (–)-epicatechin using the mass transition m/z 305/139. MRM analysis in negative ion mode was used to detect the g lucuronides of (+)-catechin and (–)-epicatechin using the mass transition m/z 465/289, and of methyl (+)-catechin and (–)-epicatechin using the mass transition m/z 479/303. The sulfates of (+)-catechin and (–)-epicatechin using the mass transition m/z 369/289, and of methyl (+)-catechin and (–)-epicatechin using the mass transition m/z 383/303 were also assessed using MRM in negative ion mode. (+)-Catechin, (–)-epicatechin, and 3′-O-methyl (–)-epicatechin standards were spiked into control urine samples (from sham-OVX SHRs gavaged with vehicle), before extraction to generate s tandard curves. The standard curve for 3′-O-methyl (–)-epicatechin was used for quantification of 3′-O-methyl (+)-catechin. The peak that eluted 1 min before 3′-O-methyl (–)-epicatechin was designated as 3′-O-methyl (+)-catechin. The standard curves for (+)-catechin and (–)-epicatechin used concentrations of 5, 1, 0.5, 0.1, and 0.05 μg/ mL and for 3′-O-methyl (–)-epicatechin concentrations of 1, 0.5, 0.1, and 0.05 μg/mL. Correlation coefficients for each of the standard curves for (+)-catechin, (–)-epicatechin, and 3′-O-methyl (–)-epicatechin were > 0.99.
Urine creatinine analysis Creatinine concentrations were measured for each urine sample using an isotope dilution procedure with 2H3-creatinine by LC-MS [28]. Urinary outputs of catechins and their metabolites were corrected for creatinine output and were expressed as μg/mg creatinine.
Statistical analysis A Student’s t-test was used to determine differences between sham-OVX and OVX groups given GSE. p ≤ 0.05 was considered to be statistically significant. Data are presented as mean ± standard error of the mean (SEM).
Results Identification of (+)-catechin and (–)-epicatechin in rat urine by LC-MS/MS LC-MS/MS analysis of (+)-catechin and (–)-epicatechin was performed in positive ion mode by selecting
132 Cutts et al.: Ovariectomy lowers unconjugated urinary catechins their protonated molecular ion (m/z 291). (+)-Catechin and (–)-epicatechin eluted at retention times (tR) of 10.00 min and 10.89 min, respectively (Figure 1B). Figure 1C shows that two peaks with tR 10.04 min and 10.94 min were detected in a urine sample from an OVX SHR fed GSE. Product ions of the peak at tR 10.04 min in the urine sample were consistent with the product ions from authentic (+)-catechin, confirming that the peak that eluted with tR of 10.04 min was (+)-catechin in the urine (Figure 1D and E). Similarly, the product ions of the peak at tR 10.94 min in the urine were consistent with the product ions from (–)-epicatechin, confirming that the peak in the urine sample with the same retention time as authentic (–)-epicatechin was (–)-epicatechin (Figure 1F and G). The product ion spectra were consistent with previously reported (+)-catechin and (–)-epicatechin product ion spectra [29, 30]. Similar results were obtained for peaks detected in urines from sham-OVX SHRs.
Effect of ovariectomy on urinary (+)-catechin and (–)-epicatechin Urine samples from 18-week-old sham-OVX and OVX SHRs given GSE were analyzed by LC-MRM MS for (+)-catechin and (–)-epicatechin. In these experiments
Intensity
1.6e5 1.2e5
8.97
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B
6
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10 9 Time, min
11
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1.9e5 1.6e5
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Non-metabolized (epi)catechin, µg/mg creatinine
C
9.99
1.9e5
1.2e5 8.0e4 4.0e4
8.97
9.99
0 6
7
8
9 10 Time, min
11
12
13
Hydrolyzable (epi)catechin, µg/mg creatinine
A
(+)-catechin and (–)-epicatechin spiked in control urine samples eluted at tR 8.98 min and tR 9.99 min, respectively, using the mass transition m/z 291/139. Urine samples from both sham-OVX+GSE (Figure 2A) and OVX+GSE (Figure 2B) SHRs had two peaks elute at tR of 8.97 min and tR 9.99 min with the mass transition m/z 291/139, confirming that both types of urine samples contained (+)-catechin and (–)-epicatechin. Urine samples from OVX+GSE SHRs contained 18.6 ± 1.0 μg/mg creatinine of nonmetabolized (+)-catechin and 12.8 ± 5.7 μg/mg creatinine of nonmetabolized (–)-epicatechin; whereas urine samples from shamOVX+GSE SHRs contained 61.0 ± 2 1.8 μg/mg creatinine of nonmetabolized (+)-catechin (p = 0.100 compared to OVX+GSE SHRs) and 142.3 ± 57.5 μg/mg creatinine of nonmetabolized (–)-epicatechin (p = 0.066 compared to OVX+GSE SHRs) (Figure 2C). In urine incubated with β-glucuronidase/sulfatase before extraction, OVX+GSE SHRs contained 220.5 ± 34.6 μg/mg creatinine of hydrolyzable (+)-catechin and 226.8 ± 43.6 μg/mg creatinine of hydrolyzable (–)-epicatechin, whereas urine samples from sham-OVX+GSE SHRs contained 291.9 ± 42.2 μg/mg creatinine of hydrolyzable (+)-catechin (p = 0.238) and 330.7 ± 59.7 μg/mg creatinine of hydrolyzable (–)-epicatechin (p = 0.209) (Figure 2D). Urine samples from shamOVX+vehicle and OVX+vehicle SHRs did not contain (+)-catechin or (–)-epicatechin (data not shown).
250 200 150 100 50 0
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Figure 2 Lower levels of GSE-derived urinary (+)-catechin and (–)-epicatechin in OVX SHRs vs. sham-OVX SHRs. MRM ion chromatograms of urines from (A) a sham-OVX SHR and (B) an OVX SHR fed GSE. Quantification of (C) non-metabolized and (D) hydrolyzable (+)-catechin and (–)-epicatechin in urine from sham-OVX (n = 4) and OVX (n = 4) SHRs fed GSE. White bars represent sham-OVX SHRs fed GSE and black bars represent OVX SHRs fed GSE. Data are expressed as the mean ± SEM.
Cutts et al.: Ovariectomy lowers unconjugated urinary catechins 133
Effect of ovariectomy on urinary 3′-O-methyl (+)-catechin and 3′-O-methyl (–)-epicatechin Urine samples from 18-week-old sham-OVX (Figure 3A) and OVX (Figure 3B) SHRs given GSE were analyzed by LC-MRM MS for 3′-O-methyl (+)-catechin and 3′-O-methyl (–)-epicatechin. 3′-O-methyl (–)-epicatechin spiked in control urine samples eluted at tR 10.86 min with the mass transition m/z 305/139. The peak at tR 10.09 min was designated as 3′-O-methyl (+)-catechin. Normalized to the urinary creatinine concentrations, urine samples from OVX+GSE SHRs contained 11.4 ± 3.7 μg/mg creatinine of nonmetabolized 3 ′-O-methyl (+)-catechin and 20.0 ± 4.5 μg/mg creatinine of nonmetabolized 3 ′-O-methyl (–)-epicatechin, whereas urine samples from sham-OVX+GSE SHRs contained 46.2 ± 18.5 μg/mg creatinine of nonmetabolized 3′-O-methyl (+)-catechin (p = 0.115 compared to OVX+GSE SHRs) and 111 ± 35.1 μg of nonmetabolized 3′-O-methyl (–)-epicatechin (p = 0.041 compared to OVX+GSE SHRs) (Figure 3C). In urine incubated with β-glucuronidase/sulfatase before extraction, OVX+GSE SHRs contained 163.6 ± 11.2 μg/mg creatinine of hydrolyzable 3′-O-methyl (+)-catechin and 185.3 ± 17.7 μg/mg creatinine of hydrolyzable 3′-O-methyl (–)-epicatechin, whereas urine samples from sham-OVX+GSE SHRs contained 174.6 ± 9.8 μg/mg creatinine of hydrolyzable
C
10.87
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Non-metabolized methyl (epi)catechin, µg/mg creatinine
A
3′-O-methyl (+)-catechin (p = 0.489) and 200.9 ± 13.4 μg/mg creatinine of hydrolyzable 3′-O-methyl (–)-epicatechin (p = 0.509) (Figure 3D). Urine samples from shamOVX+vehicle and OVX+vehicle SHRs did not contain 3′-O-methyl (+)-catechin or 3′-O-methyl (–)-epicatechin (data not shown). The amount of nonmetabolized (+)-catechin was expressed as a percentage of all flavanol forms in urine samples. All flavanol forms include hydrolyzable (+)- catechin, (–)-epicatechin, 3′-O-methyl (+)-catechin, and 3′-O-methyl (–)-epicatechin from Figures 2D and 3D for sham-OVX and OVX SHRs. This was also done to determine the percentages of nonmetabolized (–)-epicatechin, 3′-O-methyl (+)-catechin, and 3′-O-methyl (–)-epicatechin. The percentages of nonmetabolized (+)-catechin, (–)-epicatechin, 3′-O-methyl (+)-catechin, and 3′-O-methyl (–)-epicatechin in urine samples from OVX+GSE SHRs were 2.4%, 1.6%, 1.6%, and 2.6%, respectively, and the percentage of nonmetabolized (+)-catechin, (–)-epicatechin, 3′-O-methyl (+)-catechin, and 3′-O-methyl (–)-epicatechin in urine samples from sham-OVX+GSE SHRs were 6.2%, 14.7%, 4.9%, and 11.9%, respectively. Based on these calculations, monomeric catechins conjugated with glucuronic acid or sulfate were 62.3% of total monomeric catechins in urines from sham-OVX SHRs; this percentage increased to 91.8% of monomeric catechins in urines from OVX SHRs.
10.08 10.87 9.40 11.25 7
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Figure 3 Lower levels of GSE-derived urinary 3′-O-methyl (+)-catechin and 3′-O-methyl (–)-epicatechin in OVX SHRs vs. sham-OVX SHRs. MRM ion chromatograms of urines from (A) a sham-OVX SHR and (B) an OVX SHR fed GSE. Quantification of (C) nonmetabolized and (D) hydrolyzable 3′-O-methyl (+)-catechin and 3′-O-methyl (–)-epicatechin in urine from sham-OVX (n = 4) and OVX (n = 4) SHRs fed GSE. White bars represent sham-OVX SHRs fed GSE and black bars represent OVX SHRs fed GSE. Data are expressed as the mean ± SEM.
134 Cutts et al.: Ovariectomy lowers unconjugated urinary catechins
Effect of ovariectomy on the glucuronidation of (+)-catechin, (–)-epicatechin, and their methylated metabolites Urine samples from 18-week-old sham-OVX and OVX SHRs gavaged with GSE were analyzed by LC-MRM MS for the glucuronidated forms of (+)-catechin, (–)-epicatechin, 3′-O-methyl (+)-catechin, and 3′-O-methyl (–)-epicatechin. Urine samples incubated without β-glucuronidase/sulfatase (monitored with the mass transition m/z 465/289) had two dominant peaks elute at tR of 8.21 min and 8.92 min and other smaller peaks at tR 7.95 min and 9.83 min (data not shown), confirming the presence of (+)-catechin and (–)-epicatechin glucuronides in the urine. Urine samples incubated without β-glucuronidase/sulfatase (monitored with the mass transition m/z 479/303) had two dominant peaks elute at tR of 9.38 min and tR 9.96 min and another smaller peak at tR 8.73 min (Figure 4A), confirming the presence of 3′-O-methyl (+)-catechin and 3′-O-methyl (–)-epicatechin glucuronides in the urine. Each of these peaks disappeared when the urines were incubated with β-glucuronidase/sulfatase (Figure 4B). All urine samples had phenolphthalein β-D-glucuronide added to confirm hydrolysis of glucuronides by β-glucuronidase/sulfatase. Using the mass transition m/z 317/93, urine samples incubated without β-glucuronidase/sulfatase contained low amounts of phenolphthalein (Figure 4C) whereas
9.38
3.0e5
8.73
Using the mass transition m/z 369/289 for (+)-catechin and (–)-epicatechin sulfates, urine samples incubated without β-glucuronidase/sulfatase had low intensity peaks elute at tR 9.52 min, tR 10.10 min, and tR 10.82 min. Using the mass transition m/z 383/303 for 3′-O-methyl (+)-catechin and 3′-O-methyl (–)-epicatechin sulfates, urine samples incubated without β-glucuronidase/sulfatase had two dominant peaks elute at tR of 10.82 min and tR 11.28 min (Figure 5A). However, unlike for the β-glucuronides, the amounts of (+)-catechin and (–)-epicatechin sulfates (data not shown) and 3′-O-methyl (+)-catechin and 3′-O-methyl (–)-epicatechin sulfates were unaffected by incubation with β-glucuronidase/sulfatase (Figure 5B). All urine samples had 4-methylumbelliferyl sulfate added prior to extraction to monitor sulfatase activity in the β-glucuronidase/sulfatase preparation. Using the mass transition m/z 175/119 for 4-methylumbelliferone,
1.20e5 9.00e4 6.00e4
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urine samples incubated with β-glucuronidase/sulfatase contained high amounts of phenolphthalein (Figure 4D) at tR 12.61 confirming the hydrolysis of phenolphthalein β-D-glucuronide.
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Figure 4 β-Glucuronidase/sulfatase (H. pomatia) hydrolyzes glucuronides of 3′-O-methyl (+)-catechin and 3′-O-methyl (–)-epicatechin. MRM ion chromatograms of 3′-O-methyl (+)-catechin and 3′-O-methyl (–)-epicatechin glucuronides (m/z 479/303) in urine from an OVX SHR incubated (A) without and (B) with β-glucuronidase/sulfatase. MRM ion chromatograms of phenolphthalein (m/z 317/93) in urine from the same OVX SHR incubated (C) without and (D) with β-glucuronidase/sulfatase, showing the appearance of the phenolphthalein released from phenolphthalein β-D-glucuronide.
Cutts et al.: Ovariectomy lowers unconjugated urinary catechins 135
A
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Figure 5 β-Glucuronidase/sulfatase (H. pomatia) does not hydrolyze sulfate esters of 3′-O-methyl (+)-catechin and 3′-O-methyl (–)-epicatechin, although it hydrolyzes 4-methylumbelliferyl sulfate. MRM ion chromatograms of 3′-O-methyl (+)-catechin and 3′-O-methyl (–)-epicatechin sulfates (m/z 383/303) in urine from an OVX SHR incubated (A) without and (B) with the β-glucuronidase/sulfatase preparation show no difference in peak areas. Conversely, MRM ion chromatograms of 4-methylumbelliferone (m/z 175/119) in urine from the same OVX SHR incubated (C) without and (D) with β-glucuronidase/sulfatase show that the preparation has sulfatase activity, albeit with poor specificity for the flavanol sulfate esters.
LC-MRM MS determined that the urine extracts that were incubated without β-glucuronidase/sulfatase contained only trace amounts of 4-methylumbelliferone (Figure 5C), whereas urine samples incubated with the enzyme contained the expected amount of 4-methylumbelliferone (Figure 5D) at tR 12.34. These data confirm that H. pomatia β-glucuronidase/sulfatase did not hydrolyze the sulfate esters of (+)-catechin, (–)-epicatechin, and their methylated metabolites even though it successfully carried out the hydrolysis of 4-methylumbelliferyl sulfate. Therefore, the increase in the amounts of (+)-catechin, (–)-epicatechin, methyl (+)-catechin, and methyl (–)-epicatechin following incubation with β-glucuronidase/sulfatase represents the β-glucuronides of these flavanols and not the sulfates.
Effect of ovariectomy and GSE on body and uterine weights in sham-OVX and OVX SHRs Body weights were taken at 17 weeks of age before SHRs were gavaged with vehicle or GSE. OVX SHRs had higher body weights than sham-OVX SHRs (Table 1). At 18 weeks of age OVX SHRs continued to have higher body weights than sham-OVX SHRs (Table 1). GSE-gavaged SHRs had lower body weights than vehicle-gavaged SHRs. Uterine
weights were taken at 18 weeks of age when SHRs were euthanized. OVX SHRs had lower uterine weights than sham-OVX SHRs (Table 1). GSE did not affect uterine weight.
Discussion In the present study, we showed that OVX altered the metabolism of GSE catechins: specifically, lower levels of unconjugated (+)-catechin, (–)-epicatechin, and their methylated metabolites were quantified in the urines of OVX SHRs vs. sham-OVX SHRs, whereas higher levels of the glucuronides of these flavanols were measured in the urines of OVX versus sham-OVX SHRs. No differences in the levels of methylated or sulfated (+)-catechin or (–)-epicatechin were detected between OVX and shamOVX SHRs. Quantification of flavanols in urine samples from OVX SHRs gavaged with GSE showed 3–5-fold lower amounts of unconjugated (+)-catechin, (–)-epicatechin, 3′-O-methyl (+)-catechin, and 3′-O-methyl (–)-epicatechin excreted over 24 h compared to sham-OVX SHRs. These data represent a trend with p-values from 0.04 to 0.11. Failure to achieve full significance may have been
136 Cutts et al.: Ovariectomy lowers unconjugated urinary catechins Table 1 Body and uterine weights of sham-OVX and OVX SHRs. Group
17 Weeks old body weight, g
18 Weeks old body weight, g
18 Weeks old uterine weight, g
Sham-OVX+vehicle Sham-OVX+GSE OVX+vehicle OVX+GSE
203 ± 1 201 ± 2 237 ± 2 241 ± 3
194 ± 2 181 ± 6 231 ± 1 217 ± 1
0.56 ± 0.06 0.53 ± 0.10 0.10 ± 0.00 0.10 ± 0.00
due to a larger variance in the urine output of catechins and methyl catechins in sham-OVX SHRs than in the OVX SHRs, which may be a result of changes in the concentration of 17β-estradiol during the estrous cycle in rats [31]. There are reports of variation of 17β-estradiol concentration during the estrous cycle altering responsiveness to physiological and external stimuli [32, 33]. Furthermore, Kulkarni et al. (2012) have recently shown that the bioavailability and metabolism of another bioflavonoid – genistein – depends on the stage of the estrous cycle of the rat [34]. Monomeric catechins are methylated by the phase II enzyme COMT. In this study, the total amount of 3′-O-methyl (+)-catechin and 3′-O-methyl (–)-epicatechin excreted in the urine was not different between sham-OVX and OVX SHRs. These findings were not consistent with previous work where OVX increased COMT expression and activity [8]. Follow-up experiments will address this discrepancy. LC-MRM MS of the urine samples incubated without β-glucuronidase/sulfatase detected glucuronidated and sulfated forms of (+)-catechin, (–)-epicatechin, 3′-O-methyl (+)-catechin, and 3′-O-methyl (–)-epicatechin in urines from both OVX and sham-OVX SHRs gavaged with GSE. Some of these conjugated forms of catechin had multiple peaks, which may represent isomers of (+)-catechin and (–)-epicatechin glucuronide/sulfate conjugation on hydroxyl groups at carbon 3′, carbon 5, or carbon 7 [25]. Whereas urine samples that were incubated with β-glucuronidase/sulfatase no longer contained the glucuronidated forms of (+)-catechin, (–)-epicatechin, 3′-O-methyl (+)-catechin, and 3′-O-methyl (–)-epicatechin, the peak areas of the sulfated forms of (+)-catechin, (–)-epicatechin, 3′-O-methyl (+)-catechin, and 3′-O-methyl (–)-epicatechin were unchanged. LC-MRM MS indicated that while both phenolphthalein and 4-methylumbelliferone were released from the internal standards phenolphthalein β-D-glucuronide and 4-methylumbelliferyl sulfate when incubated with H. pomatia β-glucuronidase/ sulfatase, the sulfatase activity of this enzyme preparation did not hydrolyze the sulfate esters of catechins and
methyl catechins. These data confirm the findings of Saha et al. who have recently shown that flavanols, specifically (–)-epicatechin, are poor substrates for many sulfatasecontaining commercial preparations [35]. Accordingly, the increase in levels of the amounts of urinary (+)-catechin, (–)-epicatechin, 3′-O-methyl (+)-catechin, and 3′-O-methyl (–)-epicatechin following incubation with β-glucuronidase/sulfatase represents increased glucuronides of the catechins, consistent with our hypothesis that OVX would increase glucuronidation of catechins, presumably because of dysregulation of UGTs resulting from the loss of estrogens. These data are consistent with studies [9, 10] showing that estrogen attenuates UGT expression and activity. Follow-up studies using a sulfatase that has efficacy in removing the sulfate from catechins might reveal an increase in sulfation due to OVX. At the beginning of the study, 3 weeks after ovariectomy, OVX SHRs had higher body weights compared with sham-OVX SHRs, consistent with the weight gain previously shown to be a consequence of ovariectomy [36, 37]. This difference in body weight between OVX and shamOVX SHRs continued throughout the study. It was noted that SHRs gavaged with GSE had lower body weights compared to SHRs gavaged with vehicle. The SHRs given vehicle lost 3%–4% of their starting body weight whereas the SHRs given GSE lost 10% of their starting body weight. Given the short duration of the study (6 days), the slight loss of body weight could simply have reflected initial adjustment to being gavaged, particularly with the GSE. As expected, OVX SHRs had lower uterine weights compared with sham-OVX SHRs, consistent with the lower uterine weights previously shown to be a consequence of ovariectomy [37]. The administration of GSE did not affect the uterine weight in sham-OVX or OVX SHRs. It has been reported that protein levels of the male specific CYP2C11 and the female specific CYP2C12 in the liver were increased [38] and decreased [39], respectively, following OVX in rats. It will be important in follow-up studies to confirm that CYP2C11 is increased and CYP2C12 is decreased in our OVX rats and to determine whether GSE affects these anticipated effects. Based on the lack of estrogenic activity of GSE on reproductive tissue in this study, we predict that the OVX-induced differences in protein levels for CYP2C11 and CYP2C12 will not be affected by GSE. As glucuronidation is generally considered to be a mechanism whereby the body excretes xenobiotics, the results presented here suggest that postmenopausal women may not achieve equivalent physiological levels as premenopausal women of unconjugated (+)-catechin, (–)-epicatechin, and their methylated metabolites from
Cutts et al.: Ovariectomy lowers unconjugated urinary catechins 137
intake of the same dose of a dietary supplement like GSE. It has recently been shown that 3′-O-methyl (–)-epicatechin is glucuronidated in vitro most effectively by UGT1A9 [40]; follow-up studies will determine whether changes in 17β-estradiol affect the expression and/or activity of UGT1A9. It should be kept in mind that 3′-O-methyl (–)-epicatechin glucuronide added to hippocampal slices enhanced long-term potentiation [21], however it is not known whether the bioactive form in the neurons was the glucuronide or whether it had been de-conjugated. This is the first report of differences in the flavanols (+)-catechin, (–)-epicatechin, 3′-O-methyl (+)-catechin, and 3′-O-methyl (–)-epicatechin in a rodent model of menopause following oral intake of GSE; further experiments will examine the molecular basis and the clinical implications of these differences. Acknowledgements: This work was supported by grant R21AT004083-02 from the National Institutes of Health, National Center for Complementary and Alternative
Medicine to Helen Kim, a predoctoral fellowship awarded to John Cutts by the NIH Office of Dietary Supplements as an additional slot on the University of Alabama at Birmingham Cellular and Molecular Biology T32 predoctoral training program (2T32GM008111, Brad Yoder, PI) and by funds from the Department of Pharmacology and Toxicology at the University of Alabama at Birmingham. The analysis of urine creatinine was carried out by John Rodgers (UAB Department of Genetics and the Heflin Genomics Center), supported by a National Institue of Diabetes and Digestive and Kidney Diseases grant (P30 DK079337, Anupam Agarwal, PI) to the O’Brien UAB-UCSD Acute Kidney Injury Center. Kikkoman Corporation (Chiba, Japan) graciously provided the GSE for this study. The AB Sciex 4000 triple quadrupole mass spectrometer used in this study was purchased with a grant from the UAB Health Services Foundation General Endowment Fund awarded to Stephen Barnes. Received August 16, 2013; accepted October 29, 2013
References 1. Tsuchiya Y, Nakajima M, Kyo S, Kanaya T, Inoue M, Yokoi T. Human CYP1B1 is regulated by estradiol via estrogen receptor. Cancer Res 2004;64:3119–25. 2. Ishii T, Nishimura K, Nishimura M. Administration of xenobiotics with anti-estrogenic effects results in mRNA induction of adult male-specific cytochrome P450 isozymes in the livers of adult female rats. J Pharmacol Sci 2006;101:220–55. 3. Scott LM, Durant P, Leone-Kabler S, Wood CE, Register TC, Townsend A, Cline JM. Effects of prior oral contraceptive use and soy isoflavonoids on estrogen-metabolizing cytochrome P450 enzymes. J Steroid Biochem Mol Biol 2008;112:179–85. 4. Mwinyi J, Cavaco I, Pedersen RS, Persson A, Burkhardt S, Mkrtchian S, Ingelman-Sundberg M. Regulation of CYP2C19 expression by estrogen receptor α: implications for estrogendependent inhibition of drug metabolism. Mol Pharm 2010;78:886–94. 5. Inoue K, Creveling CR. Induction of catechol-O-methyltransferase in the luminal epithelium of rat uterus by progesterone. J Histochem Cytochem 1991;39:823–28. 6. Jeong H, Choi S, Song JW, Chen H, Fischer JH. Regulation of UDP-glucuronosyltransferase (UGT) 1A1 by progesterone and its impact on labetalol elimination. Xenobiotica 2008;38:62–75. 7. Jiang H, Xie T, Ramsden DB, Ho SL. Human catechol-O-methyltransferase down-regulation by estradiol. Neuropharm 2003;45:1011–8. 8. Schendzielorz N, Rysa A, Reenila I, Raasmaja A, Mannisto PT. Complex estrogenic regulation of catechol-O-methyltransferase (COMT) in rats. J Physiol Pharmacol 2011;62:483–90. 9. Strasser SI, Smid SA, Mashford ML, Desmond PV. Sex hormones differentially regulate isoforms of UDP-glucuronosyltransferase. Pharm Res 1997;14:1115–21.
10. Guillemette C, Hum DW, Belanger A. Regulation of steroid glucuronosyltransferase activities and transcripts by androgen in the human prostatic cancer LNCaP cell line. Endocrinology 1996;137:2872–9. 11. Akhtar S, Meeran SM, Katiyar N, Katiyar SK. Grape seed proanthocyanidins inhibit the growth of human non-small cell lung cancer xenografts by targeting insulin-like growth factor binding protein-3, tumor cell proliferation, and angiogenic factors. Clin Cancer Res 2009;15:821–31. 12. Meeran SM, Vaid M, Punathil T, Katiyar SK. Dietary grape seed proanthocyanidins 12-O-tetradecanoyl phorbol-13-acetatecaused skin tumor promotion in 7,12-dimethylbenz[a] anthracene-initiated mouse skin, which is associated with the inhibition of inflammatory responses. Carcinogenesis 2009;30:520–8. 13. Velmurugan B, Singh RP, Agarwal R, Agarwal C. Dietary-feeding of grape seed extract prevents azoxymethane-induced colonic aberrant crypt foci formation in fischer 344 rats. Mol Carcinogen 2010;49:641–52. 14. Velmurugan B, Singh RP, Daul N, Agarwal R, Agarwal C. Dietary feeding of grape seed extract prevents intestinal tumorigenesis in APCmin/+ mice. Neoplasia 2010;12:95–102. 15. Gao N, Budhraja A, Cheng S, Yao H, Zhang Z, Shi X. Induction of apoptosis in human leukemia cells by grape seed extract occurs via activation of c-Jun NH2-terminal kinase. Clin Cancer Res 2009;15:140–9. 16. Pataki T, Bak I, Kovacs P, Bagchi D, Das DK, Tosaki A. Grape seed proanthocyanidins improved cardiac recovery during reperfusion after ischemia in isolated rat hearts. Am J Clin Nutr 2002;75:894–9.
138 Cutts et al.: Ovariectomy lowers unconjugated urinary catechins 17. Peng N, Clark JT, Prasain J, Kim H, White CR, Wyss JM. Antihypertensive and cognitive effects of grape polyphenols in estrogen-depleted, female, spontaneously hypertensive rats. Am J Physiol Regul Integr Comp Physiol 2005;289:R771–5. 18. Wang J, Ho L, Zhao W, Ono K, Rosensweig C, Chen L, Humala N, Teplow DB, Pasinetti GM. Grape-derived polyphenolics prevent Aβ oligomerization and attenuate cognitive deterioration in a mouse model of Alzheimer’s disease. J Neurosci 2008;28:6388–92. 19. Wang Y-J, Thomas P, Zhong J-H, Bi F-F, Kosaraju S, Pollard A, Fenech M, Zhou X-F. Consumption of grape seed extract prevents amyloid-β deposition and attenuates inflammation in brain of an Alzheimer’s disease mouse. Neurotox Res 2009;15:3–14. 20. Yamakoshi J, Saito M, Kataoka S, Kikuchi M. Safety evaluation of proanthocyanidin-rich extract from grape seeds. Food Chem Toxicol 2002;40:599–607. 21. Wang J, Ferruzzi MG, Ho L, Blount J, Janle EM, Gong B, Pan Y, Gowda GA, Raftery D, Arrieta-Cruz I, Sharma V, Cooper B, Lobo J, Simon JE, Zhang C, Cheng A, Qian X, Ono K, Teplow DB, Pavlides C, Dixon RA, Pasinetti GM. Brain-targeted proanthocyanidin metabolites for Alzheimer’s disease treatment. J Neuroscience 2012;32:5144–50. 22. Schroeter H, Heiss C, Balzer J, Kleinbongard P, Keen CL, Hollenberg NK, Sies H, Kwik-Uribe C, Schmitz HH, Kelm M. (-)-Epicatechin mediates beneficial effects of flavanol-rich cocoa on vascular function in humans. PNAS 2006;103:1024–9. 23. Donovan JL, Crespy V, Manach C, Morand C, Besson C, Scalbert A, Remesy C. Catechin is metabolized by both the small intestine and liver of rats. J Nutr 2001;131:1753–7. 24. Tsang C, Auger C, Mullen W, Bornet A, Rouanet J-M, Crozier A, Teissedre P-L. The absorption, metabolism and excretion of flavan-3-ols and procyanidins following the ingestion of a grape seed extract by rats. Br J Nutr 2005;94:170–81. 25. Ottaviani JI, Momma TY, Kuhnle GK, Keen CL, Schroeter H. Structurally related (-)-epicatechin metabolites in humans: assessment using de novo chemically synthesized authentic standards. Free Radical Biol Med 2012;52:1403–12. 26. Prasain JK, Peng N, Dai Y, Moore R, Arabshahi A, Wilson L, Barnes S, Wyss JM, Kim H, Watts RL. Liquid chromatography tandem mass spectrometry identification of proanthocyanidins in rat plasma after oral administration of grape seed extract. Phytomedicine 2009;16:233–43. 27. Deshane J, Chaves L, Sarikonda KV, Isbell S, Wilson L, Kirk M, Grubbs C, Barnes S, Meleth S, Kim H. Proteomics analysis of rat brain protein modulations by grape seed extract. J Agric Food Chem 2004;52:7872–83. 28. Takahashi N, Boysen G, Li F, Li Y, Swenberg JA. Tandem mass spectrometry measurements of creatinine in mouse plasma and urine for determining glomerular filtration rate. Kidney Int 2007;71:266–71.
29. Li H-J, Deinzer ML. Tandem mass spectrometry for sequencing proanthocyanidins. Anal Chem 2007;79:1739–48. 30. Cren-Olive C, Deprez S, Lebrun S, Coddeville B, Rolando C. Characterization of methylation site of monomethylflavan-3-ols by liquid chromatography/electrospray ionization tandem mass spectrometry. Rapid Commun Mass Spec 2000;14: 2312–9. 31. Marcondes FK, Miguel KJ, Melo LL, Spadari-Bratfisch RC. Estrous cycle influences the response of female rats in the elevated plus-maze test. Physiol Behav 2001;74:435–40. 32. Lapointe J, Roy M, St-Pierre I, Kimmins S, Gauvreau D, MacLaren LA, Bilodeau J-F. Hormonal and spatial regulation of nitric oxide synthases (NOS) (neuronal NOS, inducible NOS, and endothelial NOS) in the oviducts. Endocrinology 2006;147:5600–10. 33. Znamensky V, Akama KT, McEwen BS, Milner TA. Estrogen levels regulate the subcellular distribution of phosphorylated Akt in hippocampal CA1 dendrites. J Neurosci 2003;23:2340–7. 34. Kulkarni KH, Yang Z, Niu T, Hu M. Effects of estrogen and estrus cycle on pharmacokinetics, absorption, and disposition of genistein in female Sprague-Dawley rats. J Agric Food Chem 2012;60:7949–56. 35. Saha S, Hollands W, Needs PW, Ostertag LM, de Roos B, Duthie GG, Kroon PA. Human O-sulfated metabolites of (-)-epicatechin and methyl-(-)-epicatechin are poor substrates for commercial aryl-sulfatases: implications for studies concerned with quantifying epicatechin bioavailability. Pharmacol Res 2012;65:592–602. 36. Fortepiani LA, Zhang H, Racusen L, Roberts II LJ, Reckelhoff JF. Characterization of an animal model of postmenopausal hypertension in spontaneously hypertensive rats. Hypertension 2003;41:640–5. 37. Lopez-Sepulveda R, Jimenez R, Romero M, Zarzuelo MJ, Sanchez M, Gomez-Guzman M, Vargas F, O’Valle F, Zarzuelo A, Perez-Vizcaino F, Duarte J. Wine polyphenols improve endothelial function in large vessels of female spontaneously hypertensive rats. Hypertension 2008;51:1088–95. 38. Oropeza-Hernandez LF, Sierra-Santoyo A, Cebrian ME, Manno M, Albores A. Ovariectomy modulates the response of some cytochrome P450 isozymes to lindane in the rat. Toxicol Lett 2001;124:91–9. 39. Liao D-Z, Porsch-Hallstrom I, Gustafsson J-A, Blanck A. Persistent sex differences in growth control of early rat liver lesions are programmed during promotion in the resistant hepatocyte model. Hepatology 1996;23:835–9. 40. Blount JW, Ferruzzi M, Raftery D, Pasinetti GM, Dixon RA. Enzymatic synthesis of substituted epicatechins for bioactivity studies in neurological disorders. Biochem Biophys Res Comm 2012;417:457–61.
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