Cardiac RDHs activity assay was performed as described by others41 (link), and the details of the experiment were as follows.
100 μg of cardiac microsomal fractions protein was incubated with 3 μM all-trans-retinol (17772, Sigma-Aldrich, St. Louis, MO) solubilized with bovine serum albumin and 1 mM NAD + (NAD98-RO, Sigma-Aldrich, St. Louis, MO) in 0.5 ml of the reaction buffer for 20 min at 37 °C. Reactions were stopped by the addition of an equal volume of ice-cold methanol, and Retinaldehyde were extracted twice with 2 ml of hexane. Hexane layers were dried, and the dry residue was reconstituted in 0.2 ml of acetonitrile. Retinaldehyde were separated by normal-phase HPLC using Spherisorb S3W column (4.6 mm × 100 mm; Waters) and isocratic mobile phase consisting of acetonitrile at 1 ml/min and analyzed.
100 μg of cardiac microsomal fractions protein was incubated with 3 μM all-trans-retinol (17772, Sigma-Aldrich, St. Louis, MO) solubilized with bovine serum albumin and 1 mM NAD + (NAD98-RO, Sigma-Aldrich, St. Louis, MO) in 0.5 ml of the reaction buffer for 20 min at 37 °C. Reactions were stopped by the addition of an equal volume of ice-cold methanol, and Retinaldehyde were extracted twice with 2 ml of hexane. Hexane layers were dried, and the dry residue was reconstituted in 0.2 ml of acetonitrile. Retinaldehyde were separated by normal-phase HPLC using Spherisorb S3W column (4.6 mm × 100 mm; Waters) and isocratic mobile phase consisting of acetonitrile at 1 ml/min and analyzed.
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