Evaluating Extraction Protocols for Quantifying Hepatic Retinol and Retinyl Esters

Abstract

Accurate quantification of hepatic retinoids is a key step in determination of vitamin A status in animal models. Our objective was to compare published non‐saponification extraction protocols for HPLC analysis of hepatic retinol and retinyl esters. Based on this comparison, we then investigated the effects of: 1) homogenizing rat liver in ethanol versus aqueous buffer; and 2) adding water to facilitate phase separation. The extraction protocol of Tan et al. (J Lipid Res 2014;55:1077‐86) resulted in higher hepatic retinoid concentrations (96.9 ± 1.8 μg retinol equivalents/g) than the methods of Poulaert et al. (Food Chem 2014;159:477‐85) (28.0 ± 2.5 μg/g) and Kim et al. (Methods Mol Biol 2010;652:263‐75) (16.2 ± 1.7 μg/g) (P < 0.0001). The corresponding recovery of spiked retinyl acetate was also higher: 97.9 ± 1.8% versus 66.7 ± 2.1% and 53.8 ± 3.5%, respectively (P < 0.0001). Using the method of Tan et al., lower hepatic retinol equivalent concentrations were obtained when: 1) liver was homogenized in phosphate buffered saline (PBS) (37.5 ± 4.3 μg/g) instead of ethanol (91.6 ± 1.3 μg/g) (P < 0.0001); or 2) water was added in an effort to facilitate phase separation (66.3 ± 0.7 μg/g) (P < 0.0001). In conclusion, homogenizing liver tissue in ethanol instead of aqueous buffers and avoiding the subsequent addition of water result in substantially increased extraction efficiency.