To begin, place the extracted mouse tissues into the tissue grinder tube containing 50%ice cold saline and 50%methanol. Insert the pestle into the grinder tube and gently rotate it five times to homogenize the tissue. Immediately transfer the homogenized tissue into a 15 milliliter tube.
Add two milliliters of methanol and one milliliter of 0.1 molar hydroxylamine to the homogenized sample. Allow the mixture to sit for 15 minutes at room temperature. For retinoid extraction, add 10 milliliters of hexane to the homogenate and vortex the tube horizontally for at least 10 seconds.
Centrifuge the homogenate hexane mixture for three minutes at 1000 x g to separate the phases. Using a pipette, transfer the hexane layer into a separate 15 milliliter glass tube and place the sample into a vacuum centrifuge to completely evaporate the hexane from the extracted sample. Resuspend the dried retinoids in the 15 milliliter tube with 100 microliters of hexane and vortex well to ensure all retinoids are dissolved.
Pipette the entire 100 microliters of hexane into the second glass tube and vortex the tube to dissolve the retinoids. Then pipette the entire 100 microliters of hexane into single glass inert for HPLC analysis. Set up the HPLC run using the appropriate conditions.
Identify peaks using retention times and UV spectra for each retinoid of interest based on standards. Using the chromatographic data system, integrate the identified peaks and reference the external standard curve to quantify the analyte. For biological tissue chromatograms, manually integrate the peaks to account for variabilities in retention times.
In the chromatogram of the control mouse eye, 13-cis-retinal, 11-cis-retinal, all-trans-retinal, 11-cis-retinol, and all-trans-retinol were identified. In the chromatogram of the mouse eye treated with hydroxylamine, the retention times were increased. Syn and anti isomers of these retinaldehydes were also present.
In the chromatogram of mouse hepatic tissue, retinyl palmitate and all-trans-retinol were identified as the main forms of vitamin A, and that in mouse blood showed a trans-retinol peak. The UV absorbent spectra confirmed the presence of retinoid isoforms in the chromatogram peaks.
