European Journal of Scientific Research (ISSN: 1450 ... - EuroJournals

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© European Journal of Scientific Research, Vol 7, No 5, 2005
incubated at 37°C and stopped after 60 min by addition of 30 µl of 30% trichloroacetic
acid. Absorbance of the final product of the reaction was measured at 586 nm.
VITAMIN E PREPARATION
α-Tocopherol (ATF) and γ-tocotrienol (GTT) was prepared from 500 mM stock. 45 µl of
500 mM vitamin E (ATF or GTT) was incubated with 60 µl fetal calf serum (FCS) for
overnight at 37 o C. Then, 105 µl of absolute ethanol and 90 µl of complete culture
medium (CCM) were added to make a solution with vitamin E concentration of 75 mM.
Serial dilution was done to obtain the 50, 100, 150, 200 and 300 µM of vitamin E
concentration for the cells treatment.
VITAMIN E UPTAKE
The method for vitamin E determination was as described by Meydani et al. (1987) (21)
with slight modifications. The E47 and C34 cell lines were plated in a separate petri dish.
After 48 hours, the cultured medium was replaced with new culture medium in control
group and the culture medium that contain different concentrations of α-tocopherol and
γ-tocotrienol. After 24-hour of treatment, the cells were collected and washed a few times
with ice cold PBS by centrifugation. The pellets were resuspended in ice cold PBS,
incubated with BHT followed by addition of hexane and mixed. The top layer of the
solution was transferred into other tube and dried using the freeze dryer. The preparation
was done on ice in the dark. Later hexane was added to the dried sample, followed by
mixing and filtering process. HPLC was used to determine the concentration of vitamin E
in each sample. Five different vitamin E isomers which were α-tocopherol, α-tocotrienol,
γ-tocotrienol, γ-tocopherol and δ-tocotrienol were isolated according to the method.
DETERMINATION OF THE LEVEL OF TOTAL MDA AND 4HNE
Lipid peroxidation status was determined by measuring the level of total MDA and
4HNE. 6 x 10 6 cells were plated for 48 hours for each group of treatment. Then, the
culture media was removed and replaced according to treatment (75 µM ethanol and 5
doses of palm oil α-tocopherol and γ-tocotrienol ie. 50, 100, 150, 200 and 300 µM were
used). After 24 hours, cells were collected, washed a few times with PBS and assayed for
the level of lipid peroxidation using Lipid Peroxidation Assay Kit (Calbiochem).
STATISTICAL ANALYSIS
Results refer to mean ± standard deviation and are average of three values per experiment
and each experiment was repeated three times. Student’s t test was used to analyze the
difference between treatment groups and p