Animals and animal husbandry
All animal care and use in this study were performed in accordance with standards set forth in the Guide for Care and Use of Laboratory Animals (National Research Council, 1996), by the U.S. Department of Agriculture through the Animal Welfare Act (7 USC 2131, 1985), and Animal Welfare Standards incorporated in Title 9, Part 3 of the Code of Federal Regulations, 1991.
Male CD ([Crl:CD(SD) IGS BR]; Charles River Laboratories, Portage, MI) rats were received at 6–7 weeks of age and were held in quarantine for 1 week prior to use in the study. During the quarantine period, rats were observed daily for survival and general health status. Prior to randomization into experimental groups, each animal underwent a detailed physical examination to demonstrate its suitability for use as a test animal.
Throughout the study, rats were housed individually in stainless steel cages in a windowless room that was maintained within a temperature range of approximately 18–23°C and a humidity range of approximately 50–80%. Fluorescent lighting in the animal room was provided on a daily cycle of 12 h of light followed by 12 h of darkness. At all times during the quarantine and dosing periods, rats were permitted free access to Certified Rodent Diet #5002 (PMI Nutrition International, Brentwood, MO) and City of Chicago drinking water (administered by an automatic watering system).
Test articles and dosing formulations
Resveratrol (Sochinaz SA, Vionnaz, Switzerland) and pterostilbene (H&Y International Group Ltd., Hangzhou, China) were provided by the Division of Cancer Prevention, National Cancer Institute. The purity of each agent was >99%, as determined by HPLC.
Dosing formulations of resveratrol and pterostilbene were prepared for intravenous administration in a vehicle of DMSO:PEG-300 (15:85; v/v)]; a dosing volume of 2.5 mL/kg body weight was used for intravenous injections. Oral dosing formulations were prepared in a vehicle of 0.5% (w/v) aqueous methylcellulose containing 0.2% (w/v) Tween 80; a dosing volume of 10 mL/kg body weight was used for gavage administration. All vehicle components were purchased from Sigma–Aldrich (St. Louis, MO).
Study design and conduct
At the end of the quarantine period, rats were randomly assigned to one of ten dosing groups using a computerized body weight stratification procedure that produced similar group mean body weight values. Body weights for the animals assigned to the study ranged from 201 to 241 g. The study design is summarized in .
Detailed clinical and physical observations, including body weight collection, were performed on each surviving animal once during the quarantine period, and on Days 1, 8, and 14 of the treatment period. Food consumption was measured individually for each study animal in the repeat-dose groups on Days 8 and 14 of the treatment period.
In groups receiving a single dose of resveratrol or pterostilbene, blood samples were collected from three animals per group at 10 time points (oral dose groups) or 11 time points (intravenous dose groups); blood was collected at time 0 (predose), 5 min (iv groups only), 15 min, 30 min and 1, 2, 4, 6, 8, 12, and 24 h after dose administration. Animals were anesthetized with 70% CO2/30% air, and blood samples (approximately 1 mL) were collected via retro-orbital sinus puncture. The same schedule of blood collection was used after the final dose in animals receiving resveratrol or pterostilbene daily for 14 days.
Blood samples were collected in Vacutainer tubes (Fisher Scientific, Pittsburgh, PA) containing ethylenediaminetetraacetic acid (EDTA). Tubes were inverted several times to mix and were then placed on ice until centrifuged to separate plasma within 1 h time. After centrifugation, plasma was transferred into storage tubes (0.5 mL), which were stored frozen (approximately −70°C) until analyzed.
Plasma levels of resveratrol and pterostilbene were measured using a tandem mass spectrometer (API 3000; Applied Biosystems/MDS Sciex, Foster City, CA) equipped with a high performance liquid chromatograph (Agilent 1200; Agilent Technologies, Wilmington, DE). For resveratrol or pterostilbene determination, a 100 μL aliquot of plasma was mixed with 1 mL of acetonitrile (ACN; Sigma–Aldrich, St. Louis, MO). For resveratrol analysis, an internal standard resveratrol-13C6 (Toronto Research Chemicals, Inc., Ontario, Canada) was added at a concentration of 25 ng/mL. After vortex mixing for 1 min, the sample was centrifuged at 7,000 RPM at 4°C for 10 min to remove precipitated proteins; the supernatant was transferred to a clean tube and dried under nitrogen at room temperature (approximately 25°C). After the evaporation was completed, the residue was reconstituted in 100 μL of methanol with 5 min of sonication, added to 400 μL of water, vortex mixed, and centrifuged again. The resulting supernatant was transferred to a sample vial for instrumental analysis. All sample preparations were conducted under yellow light and using opaque plastic ware to avoid light exposure of the agent.
Freshly prepared resveratrol and pterostilbene standard curves were analyzed along with samples on each day of analysis. Instrument calibrators and quality control (QC) samples were prepared by adding 10 μL of a stock resveratrol or pterostilbene solution (in a methanol/water mixture [v/v 50:50] to 100 μL of rat plasma (Bioreclamation Inc., Westbury, NY). Target calibrator concentrations were 5, 10, 20, 50, 100, 200, 500, and 1,000 ng/mL. QC samples were prepared at approximately 12, 400, and 800 ng/mL. Calibrators and QC samples were processed for analysis using the procedure described earlier. The concentration of conjugated metabolites for both agents was estimated using the calibration curve for each parent compound.
Chromatography was performed using a Luna 3μ C18, 30 × 2.0 mm column (Phenomenex, Torrance, CA) maintained at a temperature of 25°C. A flow rate of 0.25 mL/min was used. Mobile phase (MP) A consisted of 5 mM ammonium acetate in water:isopropanol (98:2, v/v); and MP B consisted of methanol:isopropanol (98:2, v/v). The MP gradient was as follows: after injection, initial conditions with MP A at 90% were held for 0.5 min, decreased to 5% in 3.5 min and held constant for 5 min, returning to initial conditions for another 3 min of re-equilibration time. Retention times for the target analytes were: resveratrol and resveratrol-13C6, 5.2 min; resveratrol glucuronide, 4.1 min; resveratrol sulfate, 4.7 min; pterostilbene, 6.3 min; pterostilbene glucuronide, 5.4 min; and pterostilbene sulfate, 5.5 min. Total run time was 12 min.
A turbo ion spray interface was used as the ion source operating in negative ion mode. Acquisition was performed in multiple reaction monitoring mode using the following ions: resveratrol, 227.0 (Q1) → 185.0 (Q3) Dalton; resveratrol-13C6, 233.0 → 191.0; resveratrol glucuronide, 403.0 → 227.0; resveratrol sulfate, 307.0 → 227.0; pterostilbene, 255.1 → 197.5; pterostilbene glucuronide, 431.1 → 255.1; and pterostilbene sulfate, 335.1 → 255.1. Ion spray voltage was −3,000 V; ion source temperature was 450°C; and collision energy was −30, −35, or 45 V.
The selectivity of the method was assessed by analyzing plasma extracts from different lots for the presence of analytical interferences and comparing the results to those obtained from spiking the blank plasma sources with analytes at the lower limit of quantitation (LLOQ; 5 ng/mL). Linearity was assessed using the internal standard (resveratrol) or external standard method (pterostilbene) and up to eight calibrators with analyte concentrations in the 5–1,000 ng/mL range. The curves were built from peak areas using least-squares linear regression with (1/x) weighting factor. The weighting factor was chosen based on goodness-of-fit criteria including coefficient of determination (r2), the back-calculated concentration of individual calibrators, and minimizing intercept value. Precision and accuracy of the method were determined from QC sample results. Within-run precision and accuracy were assessed from the results from a single day, while between-run precision and accuracy were determined from the results from multiple runs.
No significant peaks interfering with the quantitation of the agents were detected in the chromatograms of blank plasma. Calibration curves for both agents were linear from 5 to 1,000 ng/mL. The r2 values were greater than 0.995. The back-calculated concentration of individual calibrators used to determine the calibration curve ranged from 90 to 100% of the true value. The method’s precision (CV%) was less than 10% and within-run accuracy ranged from 92 to 109% for both agents. Between-run accuracy ranged from 95 to 105% and 101 to 103% for resveratrol and pterostilbene, respectively.
Reference standards for glucuronide and sulfate metabolites were not commercially available for pterostilbene. For this reason, calibration standards were not used to quantitate conjugated metabolites of either agent. Specific enzymatic hydrolysis of the conjugated metabolites was attempted, but resulted in the degradation of pterostilbene. As such, this approach was not considered to be viable for metabolite quantitation.
The analytical results obtained for metabolites of both agents are estimates obtained using the corresponding calibration curve for each parent compound; as such, these data carry a larger experimental uncertainty than do those generated for the parent compound.
Mean plasma concentration–time profiles of resveratrol and pterostilbene in the rats at scheduled (nominal) sampling times were analyzed by noncompartmental pharmacokinetic methods using WinNonlin® Professional Edition software, Version 5.0.1 (Pharsight Corporation, Mountain View, CA). Key pharmacokinetic parameters, including Tmax,, Cmax, AUC0–t, AUC0–inf, and t½, were calculated for both routes of administration. Additional parameters, including C0, CL, and Vss, were calculated for the intravenous route only, and %F was calculated for the oral dosing route only. AUC from time zero to the last measured concentration was estimated by the linear trapezoidal rule up to Cmax, followed by the log trapezoidal rule for the remainder of the curve. AUC extrapolated to infinity is defined as AUC0–inf = AUC0–t + Ct/λz, where λz is the disposition rate constant estimated using log-linear regression during the terminal elimination phase and Ct is the last measureable plasma concentration.