The relationship between alpha-tocopherol and phospholipid fatty acids in rat liver subcellular membrane fractions

(1) A method was devised for the subfractionation of normal rat liver and for the subfractionation of the mitochondrial fraction into inner and outer membrane fractions. The purity of the fractions was assessed using marker enzyme measurements. (2) alpha-Tocopherol was measured in all the fractions by a sensitive HPLC technique. Profiles of phospholipid fatty acids were also determined by gas-liquid chromatography in all the fractions, and these values were calculated in terms of the percentage of each fatty acid in the total fatty acid of the fraction, as well as of the mass of each fatty acid per mumol of phospholipid phosphorus. Tocopherol values were expressed as the mass of tocopherol per g wet liver and per mumol of phospholipid phosphorus. (3) The results show that the mitochondrial and microsomal fractions were the major tocopherol-containing fractions, and both the inner and outer mitochondrial fractions contained substantial amounts of alpha-tocopherol. (4) The mitochondrial and microsomal fractions also had the highest levels of polyunsaturated fatty acids (PUFA) in their phospholipid fraction, especially 20:4 and 22:6, which were particularly localised in the inner mitochondrial membrane fraction. The high inner mitochondrial and microsomal PUFA levels were particularly apparent when the sum of all the unsaturated fatty acids with three or more double bonds was calculated. (5) Calculation of molar ratios of of some phospholipid fatty acids to alpha-tocopherol gave values of the order of several thousand to one. (6) It is concluded that the protective effect of each molecule of alpha-tocopherol must be exerted towards a large number of molecules of membrane unsaturated fatty acids simultaneously. This perhaps implies specific localisation of tocopherol in regions of membrane particularly liable to attack, such as might be expected to occur close to respiratory enzymes that can donate electrons to molecular oxygen.