Vitamin-C, Anti-Oxidant, DNA Adducts, 8-Hydroxyguanosine, NK Cell Activity, Apoptosis, Cell Cycle Analysis

In this study, twenty healthy volunteers (12 females and 8 males aged between 24-53) with no history of chronic diseases and with normal hematology, blood chemistry and urinalysis were divided into four groups. Three females and two male subjects were randomly assigned in each group. The first group was assigned placebos and given 500 mg of calcium carbonate per day orally for two weeks. The experimental groups were given 500 mg, or 6-10 mg/kg, 1,000 mg, or 11-20 mg/kg, 5,000 mg or 60-90 mg/kg of ascorbic acid per day for the same period of time. Besides the calcium carbonate and oral supplementation with ascorbic acid, none of the participants were taking vitamins. Blood (30 ml) was drawn from each individual in a yellow top tube containing solution A (Beckton Dickinson, Palo Alto, CA) for baseline studies of intracellular levels of ascorbic acid, DNA adducts, NK cell activity, apoptosis and cell cycle analysis. After the first draw, participants immediately ingested the ascorbic acid capsules. Exactly 24 hours, one week, two weeks and three weeks (two weeks on ascorbic acid and one week on placebo) later, blood was drawn again for the follow-up study. All subjects were followed by weekly laboratory examinations, such as complete hematology with flow cytometry, blood chemistry, and a urinalysis, including liver and kidney function tests. No abnormalities were detected due to oral administration of ascorbic acid.

Preparation of Peripheral Blood Lymphocytes

Lymphocytes were prepared from fresh heparinized peripheral venous blood by Ficoll-Hypaque density gradient centrifugation (Litton Bionetics, Rockville, MD). Cells were washed three times with Hanks' balanced salt solution (HBSS), and resuspended to a concentration of 10x10⁶ cells/mL in a complete medium (CM) that consisted of RPMI-1640 supplemented with 10% fetal calf serum and 1% antibiotics (100 U penicillin and 100 µg/ml streptomycin). Purity of the cells was examined by flow cytometry using CD45/CD14 monoclonal antibodies and was greater than 95%. Cells were used for different assays within an hour of isolation.

Measurement of Intracellular Level of Ascorbic Acid

After Ficoll-Hypaque separation, PBLs were kept on ice and immediately sonicated for 10 seconds in 50 mM H₃PO₄ (pH 1.8) that contained 0.1 mM EDTA to minimize auto-oxidation. The pH of the resulting extract was below 2.0. The extract was spun for 5 min. at 16,000 g at 4° C. An equal volume of 200 mM potassium phosphate (pH 9.0) was added to the supernatant followed by treatment with 10mM 2-Mercaptoethanol at 24° C. The commercial standard of ascorbic acid was subjected to the same conditions as described above. Both the standards and sample were used to determine intracellular levels by using the HPLC system (ESA Chemsford, MA) Model 5600 coularray detector with solvent delivery pump model 580 and analytical cell that makes use of two porous graphite electrodes. The column was B 3043-13 packed with TSK-gel particles of 5µ size. The optimal mobile phase was 0.2M = KH₂P0₄/H₃P0₄ PH 3.0, delivered at 1 ml/min³⁴. The detector's response to each concentration of ascorbic acid was determined by making repeated injections of the standards onto the chromatograph. Day to day variability of the quality control specimens was less than 20%.

Measurement of DNA Adducts

DNA was isolated from PBLs by the method described earlier³⁵ with some modifications and used for measurement of DNA adducts³⁶. To the cell pellet, 250 µl of homogenizing buffer (pH 7.3) containing 0.3 M sucrose, 0.025 M TRIS, and 0.002 M EDTA was added.

Residual RNA was destroyed by adding 50µl RNase (1 mg/ml) to each sample, followed by incubation at 50° C for 30 min. and then heated to 70° C for 10 min. The samples were then sonicated and 250 µl of solution containing 1.0 M LiC1, 2 M urea, 0.04 M sodium citrate, 0.005 M disodium EDTA, 2% sodium dodecyl sulfate (SDS) was added. 50 µl proteinase K (1 mg/ml) was added and incubated at 50° C for approximately 1 hr., vortexing every 15 min. and 250 µl of Chloroform-isoamyl alcohol (24:1) was added to each sample and shaken for 5 minutes, 3 consecutive times to extract the DNA. The aqueous layers were transferred to new tubes and 1/10 volume of 3M sodium acetate and 2 volumes of 95% ethanol were added. The samples were placed in a freezer (-70° C) for 1 hr. and then centrifuged at 14,000 g for 20 minutes. The alcohol was decanted and 1 ml of 70% ethanol was added to wash the DNA pellets. The samples were centrifuged at 14,000 g for 10 minutes and the alcohol was decanted again. Each sample was placed in a centrivap concentrator for 30 min. at 60° C. 250 µl of TE (0.01 M Tris, 0.002 M EDTA pH7.4) was added to solubilize the DNA. The DNA was quantified spectrophotometrically by measuring the absorbance at 260 and 280 nm. DNA digestion was done by adding 25 µl of 0.5 M sodium acetate and 3 µl of 1M magnesium chloride to each sample, incubating at 100° C for 5 min. They were immediately cooled on ice for 5 min. 10 µl of nuclease P₁ (1 mg/ml) was added and then incubated for 1 hr. at 37° C. This step was followed by the addition of 8 µl of 1M TRIS pH 8.5 was added to pH the samples to 7.8. Then 2 µl of alkaline phosphatase (2 units) was added and the samples were incubated for 1 hr. at 37° C. 4 µl of 5.8 M acetic acid was added to precipitate the enzymes. The samples were then centrifuged at 14,000 g using DNA isolation spin cartridges (GibcoBRL, Gaithersburg, MD), and were then ready for injection. The HPLC system of ESA (Chemsford, MA) model 5600 coularray detector with solvent delivery pump model 580 and YMC column (4.6 x 150mm) was used. The mobile phase consisted of 100 mM sodium acetate in 5% methanol pH 5.2. The flow rate was set at 1.0 ml/min and ran for 15 min. Voltages were set at 150 mV, 450 mV for the detection of 2-deoxyguanosine (2dG), and 850 mV for detecting 8-hydroxyguanosine. The 2-Deoxyguanosine and all HPLC grade reagents were purchased from Sigma Chemicals and 8-hydroxy-2'-deoxy-guanine (8OH2dTG) were purchased from Wako Chemicals. Similar badge of standards and quality control materials kept at -8° C were used for all HPLC assay. Day-to-day variability and reproducibility of two different DNA adduct quality control material were less than 20%. If variation in our quality control material as compared to the standards was 20% or higher, data was rejected, columns and cells were washed, recalibrated and assays were repeated.

Natural Killer Cell Count

NK cell subset enumeration was carried out by using lymphocytes from ascorbate-treated and non-treated individuals and FACScan (Becton Dickinson, Palo Alto, CA). Mononuclear cell populations were determined by two-color direct immunofluorescence and the use of whole-blood staining technique with the appropriate monoclonal antibody and flow cytometry³⁷. The fluorescein isothiocyanate (FITC) or phycoerythrin (PE)-conjugated monoclonal antibodies (Becton Dickinson) CD56 PE and CD3-FITC were selected for determination of the total NK cells and NKHT3⁺/NKHT3^- cells. To monitor lymphocyte markers, bit maps were set on the lymphocyte population of the forward-angle light scatter versus a 90° light scatter histogram. The percentage of positively stained cells for each marker pair, as well as the percentage of doubly stained cells, was determined. Estimates of the absolute numbers of lymphocyte positive for the respective surface markers were determined by multiplying the peripheral lymphocyte cell count by the percentage of cells positive for each surface marker.

Chromium⁵¹-release Assay for Measuring NK Activity

A standard 4-hr ⁵¹Cr-release assay was employed²³. Briefly, 1 X 10^{4 51}Cr-labelled K562 tumor target cells in 0.1mL complete medium were added to different wells of a microtiter plate. Effector cells were then pipetted into four wells to give effector:target (E:T) ratios of 6:1, 12:1, 24:1 and 48:1. After a 4-hr. incubation at 37° C, the plates were centrifuged at 1400 rpm for 5 min. and 0.1mL of supernatant from each well was collected and placed in a gamma counter. The percentages of isotope released were calculated by the following formula:

One feature of LU, in contrast with using the percentage of lysis at a single E:T ratio, is that four values at four distinct ratios are used, generating more information and, therefore, greater precision³⁸.

Detection of apoptosis by flow cytometry

The rate of apoptosis was determined by flow cytometry, utilizing the Apo-BRDU kit as described by the manufacturer (Phoenix Diagnostics, San Diego, CA). Briefly, the large number of DNA strand breaks in apoptotic cells results in a multitude of 3'-hydroxy termini DNA ends. In the presence of terminal deoxynucleotidyl transferase (TdT), the 3'-termini of DNA can be labeled with fluorescein tagged deoxyuridine triphosphate (F-dUTP). PBLs from different groups (1 to 2x10⁶ cells) were cultured in RPMI-1640 supplemented with 10% fetal calf serum (FCS) and 2% penicillin-streptomycin in 12-well plastic plates. The plates were incubated for 12 hrs at 37° C in a humidified 5% C0₂ incubator. The cells were harvested, centrifuged at 1000 x g for 5 min. at room temperature and washed twice with PBS (5 ml per wash). Paraformaldehyde (1% w/v in PSB) was added to the cells, followed by a 15 min. incubation on ice. The cells were pelleted and washed twice with PBS as described above. The cells were fixed with ice-cold 70% ethanol and incubated at 20° C overnight. The fixed cells, as well as positive and negative controls, were labeled by adding 50 µl of DNA labeling solution containing 10 µl TdT reaction buffer, 0.75 µl TdT enzyme 8 µl of Br-duTP and 32.25 µl of distilled water. The positive control was a lymphocytic cell line treated with camptothecin³⁹. Cells were incubated with the DNA labeling solution for 60 min. at 37° C. At the end of the incubation time cells were washed and resuspended in 0.1 ml of fluoresceinPRB-1 antibody solution in the dark for 30 min. at R.T. After addition of 0.5 ml of propidium iodide/RNase A solution and 30 min. incubation cells were analyzed by flow cytometry. The day-to-day variability of the assay was less than 20%.

Cell Cycle Analysis

Isolated PBLs were processed and ethanol-fixed as described above. The fixed cells were prepared for cell cycle analysis. Briefly, cells were resuspended in 100 µl of RNase A (Sigma)-PBS solution (180 µg RNaseA/ml) and incubated at room temperature for 30 min. The RNase A solution was replaced with 1 ml of PI solution (50 µg/ml in PBS with 0.1% Triton-X-100). PI staining was performed for 15 min. at room temperature in the dark. Fluorescence measurements were performed with FACScan (Becton-Dickinson) using the 488 nm line on an argon laser operating at 15 mW. The data were collected into a two-parameter histogram showing light scatter versus red fluorescence and were gated to eliminate any particles that were not the correct size for intact cells.³⁹. Similar to apoptosis, sample-to-sample and day-to-day variation of this assay was less than 20%.

Test results are for physician use only.