Improved lipid saponification for chromatographic quantificationof fatty acids in porcine erythrocytes – an important lipidomicbiomarker of the effectiveness of dietary fat supplementationin pigs as a large animal model for human studies

The objectives of our study were to enhance and evaluate an improved saponification method for the quantification of fatty acids (FA), with a specific focus on highly-unsaturated long-chain polyunsaturated FA (LPUFA) that are easily peroxidised in pig erythrocytes. These erythrocytes serve as a valuable large animal model for human studies. We implemented a modified saponification procedure, involving a shorter initial saponification at 95 °C for 2 min, followed by overnight saponification at 22-25 °C, and subsequent gentle base and acid-catalysed methylation of FA. These analytical procedures proved to be suitable for gas-chromatographic analysis of highly-unsaturated LPUFA, which are prone to peroxidation in processed piglet erythrocytes. The modified initial saponification at 95 °C, followed by overnight saponification at 22-25 °C, and subsequent methylation, were used to assess the suitability of erythrocytes for the determination of FA, tocopherol and malondialdehyde (MDA) concentrations in monogastric mammalian tissues. The content of docosahexaenoic acid (DHA) in erythrocytes can serve as a marker for DHA bioaccumulation in animal tissues and dietary contents of n-3LPUFA (especially DHA). The concentrations of δ-, γ-, α-tocopherols, total cholesterol and MDA increased in piglet erythrocytes during 16 days of exposure to dietary fat. The levels of FA and tocopherols can therefore be used in erythrocytes as indicators of the bioaccumulation yield of these compounds in animal tissues. Additionally, we propose that FA and tocopherol profiles in erythrocytes may be used as indicators of the concentrations of these components in diets and the effectiveness of their bioaccumulation in animal tissues.