Curcumin Plasma Bioavailability and Total Curcumin Analysis
Small et al: Supplemental Materials
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SUPPLEMENTAL MATERIALS
Small et al: Memory and Brain Amyloid and Tau Effects of a Bioavailable for of Curcumin in Non-Demented Adults: A Double-Blind, Placebo-Controlled 18-month Trial
Curcumin Plasma Bioavailability and Total Curcumin Analysis
Bioavailability of three forms of curcumin. To determine plasma curcumin bioavailability of Theracurmin®, we compared levels following a one-time consumption of 3 capsules of Theracurmin® (containing 31 mg of curcumin per capsule), 2 capsules of Meriva® (51 mg per capsule) and 1 capsule of GNC Herbal Plus® Standardized Turmeric (433 mg). The curcumin content listed is based on results from our laboratory analysis. Meriva® contained curcumin in the form of a curcumin-phosphatidyl choline complex. GNC Herbal Plus® Standardized Turmeric was supplied by GNC – General Nutrition Center (Pittsburgh, PA); curcumin phospholipid complex (Meriva®) was supplied by Indena SpA (Milan, Italy); and Theracurmin® was supplied by Theravalues Corporation (Tokyo, Japan). The bioavailability study was carried out in accordance with the guidelines of the Office for Protection of Research Subjects of the University of California, Los Angeles, and the clinical protocol was approved by the internal review board (IRB) of the University of California, Los Angeles. Ten healthy volunteers signed an informed consent form and HIPPA release and were randomized to a specific sequence of three different curcumin supplements. For visit one, volunteers arrived at the Center for Human Nutrition, and blood was drawn from an intravenous catheter before (T=0) and 0.5, 1, 2, 4 and 6 hours after consuming one of the three curcumin supplements. After a one week wash out of eating a low curcumin diet, volunteers repeated the procedure two more times to consume one of the other two curcumin supplements (visits 2 and 3). Blood was centrifuged at 1500 x g for 10 min at 10°C and plasma aliquots stored at 80 °C until analysis of total curcumin (glucuronide, sulfate).
HPLC sample preparation for plasma curcumin analysis. 50 L of 0.58 M acetic acid solution were added to 500 L of plasma and subsequently incubated with beta-glucuronidase/sulfatase (G1512, Sigma-Aldrich, St Louis, MO) at 37 C for 60 minutes. The sample was extracted with 500 L of 95% ethyl acetate and 5% methanol, vortexed for 5 minutes, followed by centrifuging for 5 minutes at 20600g, and the supernatant was collected. The sample was extracted two more times using the same procedure, and three extracts from the same sample were combined and dried in a speedvac (Savant). The residue was reconstituted with 200 L of methanol:water (1:1), mixed well, and centrifuged at 20600g for 10 minutes. The supernatant was subjected to HPLC analysis.
HPLC condition. Sample analysis was performed using a Waters Alliance 2690/95 HPLC instrument with 2996 PDA detector and an auto sampler (Waters Corp, Milford, MA). Separation was achieved with an Agilent Zorbax C18 column (4.6x150 mm, 5m) with a C18 guard (4.6x20 mm, 5m)(Agilent, Santa Clara, CA) at 35°C. The mobile phase consisted of two components: 1) 0.1% phosphoric acid/H2O; and 2) acetonitrile. Initial conditions were 70% progressing to 15% in 30 min. The flow rate was set as 1.0 ml/min. Curcumin was detected at 420 nm.
Scanning Procedures
FDDNP was prepared at high specific activities (>37 GBq/µmol).34 All scans (Biograph TruePoint PET/CT scanner; Siemens) were performed with participants in a supine position and with the imaging plane parallel to the orbitomeatal line. A bolus of FDDNP (320-550 MBq) was injected via an indwelling venous catheter, and consecutive dynamic PET scans were performed for 1 hour. Scans were decay corrected and reconstructed using filtered back-projection (Gaussian filter, 3 mm full-width at half-maximum) with scatter and measured attenuation correction. The resulting images contained 109 contiguous sections with slice thickness of 2.027 mm.
FDDNP-PET scans obtained for post-curcumin supplementation (or placebo) were rigidly co-registered to the baseline PET scans using SPM12 ( Bilateral regions of interest were manually traced on the early-summed (0 to 6 minutes postinjection) FDDNP PET images for the parietal, medial temporal (limbic regions, including hippocampus, parahippocampal areas, and entorhinal cortex), lateral temporal, posterior cingulate, anterior cingulate, amygdala, hypothalamus, occipital, motor, and frontal regions, as previously described15,16 and applied to both baseline and post-curcumin-supplement (or placebo) FDDNP-PET scans. Rules for region-of-interest drawing were based on the identification of gyral and sulcal landmarks with respect to the atlas of Talairach and Tournoux.35 The FDDNP binding data were quantified using Logan graphical analysis with the motor strip as the reference region.36 The slope of the linear portion of the Logan plot is the distribution volume of the tracer in a region of interest relative to that in the reference region (motor cortex). Each regional relative distribution volume or binding value was expressed as the mean of the left and right regions calculated from the relative distribution volume parametric images generated using a multilinear formulation of Logan analysis.37 All PET scans were read and regions of interest drawn by individuals blinded to clinical assessments and treatment group.