HPLC/UV quantitation of retinal, retinol, and retinyl esters in serum and tissues
Section snippets
Materials
Solvents were purchased from Fisher Scientific. O-Ethylhydroxylamine and retinoids, except 9-cis-retinol (9cROL), were purchased from Sigma–Aldrich. Retinoid standards were prepared on the day of use. Concentrations were verified spectrophotometrically using ε values [42].
9cROL was prepared by reducing 9-cis-retinal (9cRAL) with excess NaBH4 in 95% tetrahydrofuran/methanol. Acetone was added to destroy excess NaBH4. The reaction mixture was evaporated to dryness and resuspended in 50%
Results and discussion
We previously developed a quantitative RA assay with attomol detection limits [40], [41]. Here we discuss methods for RE/ROL and RAL quantification. The diversity in chemical properties and endogenous retinoid concentration ranges (see Fig. 1) render it impractical to perform accurate quantification of RE/ROL/RAL/RA in a single chromatographic run. The vastly different concentrations of RE and ROL in some tissues requires two different volume injections for RE and ROL to be quantifiable in
Acknowledgments
This work was supported by National Institutes of Health (NIH) grants DK36870, AG13566, and DK46839 as well as an NIH Kirschstein Individual Fellowship (F32 DK066924 to M.A.K.).
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