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Preliminary Human Study to Determine the Bioavailability of The Right C®
and Another Vitamin C In a Blinded Cross-over Study

This study was conducted at Kilden Helse in Oslo, Norway under the direction of Dr. Roald Strand. The protocol was designed by Dr. Anthony J. Verlangieri, Professor of Pharmacology and Toxicology.

Purpose

To determine the rate of oral absorption of The Right C® and Another Vitamin C by analysis of Total Vitamin C (ascorbic acid, AA) delivered to plasma at 90 minutes post-ingestion.

Study Design

Ten (10) healthy male subjects were randomized into two groups as described below. They were to refrain from taking any vitamin supplements for at least 7 days prior to the start of the study. They were to avoid intake of fruits and vegetables prior to and during the study period. The study required that all subjects fast prior to the administration of the test material.

The study groupings consisted of two groups. Prior to the cross-over, half of the subjects received 1000 mg of Another Vitamin C orally, and the other half received 1000 mg of The Right C®. After a 7 day wash out period, the cross-over treatments began, with the groupings being reversed.

Prior to administration of the test material, a blood sample was collected in a heparin tube (0 time, baseline). Blood samples were then taken at 30 min., 60 min., and 90 min. after administration of the test material.

Plasma AA was analyzed by HPLC as described below. All results are expressed as mg AA/L of plasma.

Materials

Ascorbic acid, dithiothreitol (DTT), tetrasodium EDTA, chloroacetic acid, sodium hydroxide (NaOH), octyl sodium sulfate, and meta-phosphoric acid (MPA) were purchased from Sigma-Aldrich Chemical Co., Milwaukee, WI. High Pressure Liquid Chromatography (HPLC) instrumentation platform included an Alliance 2695 Separations Module (Waters Inc., Milford, MA) with an autosampler, a Waters 2996 Photodiode Array Detector utilizing Empower Pro Software. The column used was a 250 x 4.6 Ultrasphere column (Beckman Instruments, Inc., Fullerton CA) packed with 5-µm octadecylsilane particles. All solvents were degassed with nitrogen prior to use.

Methods

Isocratic elution at a flow rate of 0.5 mL/min at ambient temperature was used, and the mobile phase consisted of a solution of 14.1 g chloroacetic acid, 4.65 g NaOH, 0.85 g of tetrasodium EDTA, and 200 mg of octyl sodium sulfate in 1L of distilled water. The photodiode array detector was set at a wavelength of 265 nm. Standard curve measurements produced a standard curve with R=0.9931. Ascorbic acid had a retention time of 6.1 min +/- 0.2 min.

Stock solutions of MPA (30 g/L) and DTT (1 g/L) were prepared and stored cold. Ascorbic acid standards were prepared and analyzed as follows. Ascorbic acid (2.0 mg) was weighed and added to a solution containing 0.7 mL of MPA solution (30 g/L) and 0.3 mL of DTT solution (1 g/L). This 2.0 mg/mL stock solution was serial diluted to generate final ascorbic acid standards ranging from 1.25 µg/mL to 20 µg/mL. Human blood samples were collected (3.0 mL), centrifuged, and the plasma (0.5 mL) was supplemented with 0.5 mL of MPA (30 g/L) solution for stability, and frozen at -78°C. After the plasma samples were received (in duplicate), each were treated with 0.2 mL of stock MPA solution, and 0.02 mL of stock DTT solution, vortexed, and centrifuged at 2000xG. Final concentrations of MPA and DTT were 17.2 g/L and 0.2 g/L, respectively. The supernatant (1.0 mL) was transferred to a microfuge tube, stored on ice, and further centrifuged at 13200xG for 1 minute to remove residual suspended particles. The clear supernatant plasma solution was immediately transferred to autosampling vials and analyzed for ascorbic acid. Injection volumes used for standards and plasma samples was 10 µL. A dilution factor of 2.24 was used for measuring final ascorbic acid concentrations based on the method described above. Plasma ascorbic acid concentrations were reported in mg/L.

SEE RESULTS

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