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Copyright by Eunyoung Park 2007 - The University of Texas at Austin

Copyright by Eunyoung Park 2007 - The University of Texas at Austin

FIGURE 2.2. RETINOL IS

FIGURE 2.2. RETINOL IS NOT METABOLIZED TO BIOACTIVE COMPOUNDS IN HCT- 15 AND SW620 CELLS BUT IS CONVERTED TO ATRA BY HCT-116 AND WIDR CELLS. 33

FIGURE 2.2. RETINOL IS NOT METABOLIZED TO BIOACTIVE COMPOUNDS IN HCT- 15 AND SW620 CELLS BUT IS CONVERTED TO ATRA BY HCT-116 AND WIDR CELLS. Cells were plated and allowed to attach for 24 hr before addition of medium containing 0, 1, or 10 μM retinol. Cells were allowed to grow for 24, 48, 72 or 96 hr before the medium was removed and replaced with new medium containing 5% FCS and 50 nM [ 3 H]-retinol. The concentration of nonradioactive retinol in 5% FCS was ≈ 50 nM (51). Cells were allowed to incubate for another 2, 4, 8 or 16 hr before harvest. The retinoids were extracted from cell and medium samples and separated by HPLC as described (51). Data shown represent radiolabeled retinoids extracted from medium (A) or cells (B) of the indicated cell lines 48 h after treatment with 0 or 10 μmol/L retinol followed by incubation for 8 h with 50 nM [ 3 H]-retinol. The identities of the retinoids were determined by coelution with known nonradiolabeled (cold) standards included in the samples. Changes in absorbance are recorded as changes in voltage by the FloOne software that generated these chromatographs and controls the liquid scintillation counter. Thus, the units for the y-axis of “Standards” panel are volts. The slight difference in elution time between the cold standards and the [ 3 H]-peaks is due to the transit time from the photo diode array to the scintillation counter. [ 3 H]-Retinol extracted from labeling medium at time zero is shown in the left column, one panel from the bottom. F9 murine teratocarcimona cells were treated with 1 μmol/L RA for 48 hr followed by incubation with [ 3 H]-retinol for 16 hr prior to retinoid extraction. The retinoids extracted from F9 murine teratocarcimona cells are included as a positive control for 4-oxoretinol production and are shown in the last panel of the right column. Retinoid standards are shown in the bottom panel of the left column and eluted as follows: 4-oxoretinol, 13.5 min; ATRA, 18.5 min; all-trans retinol, 34.0 min, and AR, 39.8 to 40.5 min. This experiment was repeated twice with similar results. 34

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