Mary Ann Williams

Essential fatty acid deficiency and mitochondrial function

Abstract Some biochemical and ultrastructural properties of liver mitochondria from rats fed diets deficient in essential fatty acids (EFA) are described. Within 2 weeks, the proportions of arachidonic and linoleic acids showed marked decreases, and large changes in the fatty acid composition of cardiolipin and phosphatidylcholine, but not phosphatidylethanolamine, occurred. In general, the activites of inner membrane functions, such as respiration, oxidative phosphorylation, and the capacity for energized translocation of mono- and divalent cations, were largely unchanged by EFA deficiency. Ion transport-induced mitochondrial swelling was also unaltered, but mitochondria from EFA-deficient animals were more resistent to lipid peroxidation, and hence to peroxidative swelling. Oscillations of mitochondrial volume in response to ion transport in EFA-deficient preparations were slower, and the amplitude of the oscillatory response upon repeated O 2 pulses was diminished to a greater extent, in mitochondria from EFA-deficient animals. An electron microscope study of rat liver mitochondria revealed the following ultra-structural differences due to EFA deficiency: (1) inner membranes were more irregularly organized in vitro , and (2) the mitochondria were larger in situ , and enlarged more upon isolation than did mitochondria from rats on EFA-supplemented diets. These changes occurred within 4 weeks.

Linolenic acid deficiency.

Linolenic acid deficiency has not been demonstrated clearly in warm blooded animals, yet circumstantial evidence suggests that n−3 fatty acids may have functions in these animals. The fact that several species of fish definitely require dietary n−3 fatty acids indicates that n−3 fatty acids have important and specific functions in these animals and suggests that such functions may also be present in warm blooded animals. It is also true that n−3 fatty acid distribution in tissues of birds and mammals appears to be under strict metabolic control, and that this complex metabolic control mechanism apparently has survived evolutionary pressure for a very long time. So far, attempts to produce linolenic acid deficiency in mammals have not revealed an absolute requirement for n−3 fatty acids. If functions for n−3 fatty acids do exist in warm blooded animals, it seems probable that they may be located in the cerebral cortex or in the retina, because these tissues normally contain high concentrations of n−3 fatty acids.

Perturbations of the dynamics of lipid alkyl chains in membrane systems: Effect on the activity of membrane-bound enzymes

Abstract Cellular and membrane systems that are well defined with respect to lipid compositions were obtained from cultures of unsaturated fatty acid auxotrophs of Saccharomyces cerevisiae supplemented with specific 18-carbon unsaturated fatty acids. Lipophilic spin labels were used to probe the physical state of the lipids in the resulting membrane systems. Arrhenius plots for the O 2 uptake of cells grown on lactate, and for the motion of a spin label in the lipid moieties of these cells display discontinuities at the same temperature. The characteristic temperature which defines the change in slopes in these plots is dependent upon the physical characteristics of the unsaturated fatty acid that supplemented a given culture. It is inferred that the changes in slopes originate from temperature-dependent changes in the physical state of lipid moieties. Adamantane, a hydrophobic quasi-spherical molecule, was used as a liphophilic perturber to cooperatively hinder the axial ordering of phospholipid alkyl chains. Experimental evidence indicates that adamante lowers the transition temperatures detected from the temperature dependence of the motion of spin labels and from the Arrhenius plots of O 2 uptake. Additional experiments were carried out to determine the effect of other perturbing molecules and of mixtures of unsaturated fatty acids on the temperature dependence of spin-label motion. The approximate coincidence of transition temperatures in both O 2 uptake and spin-label experiments infers that membrane-dependent physiological functions are dependent upon the physical state of membrane lipids.

Relation of unsaturated fatty acid composition of rat liver mitochondria to oscillation period, spin label motion, permeability and oxidative phosphorylation

Abstract An investigation was made of the influence of mitochondrial unsaturated fatty acid composition on the following mitochondrial parameters: oscillation period, spin label motion (ESR), permeability, and oxidative phosphorylation. Liver mitochondria from rats fed diets deficient in or supplemented with essential fatty acids showed approximately the same total number of unsaturated fatty acids but changed unsaturation levels. Electron microscopy showed that the morphology of the inner membrane compartment was unchanged. Two differences were correlated with unsaturated fatty acid composition: (1) a slower frequency (or time period) of the oscillatory state of energy-dependent ion transport and (2) a reduction in the motional freedom of each of three spin labels (12NS, 5N10 and 7N14). The increase in oscillation period could arise from a number of rate-limiting processes, including permeability of mitochondria to various anions, cations, and substrate metabolites. However, when the permeability of mitochondria to such substances was tested, no changes were observed in passive or active uptake of these substances or in the efficiency of oxidative phosphorylation under steady-state conditions. Thus, the two parameters, oscillation period and freedom of spin label motion, which are dependent upon large domains of the mitochondrial membranes, are significantly influenced by the change in unsaturated fatty acid composition in essential fatty acid-depleted mitochondria, even though processes such as permeability of ionic materials and oxidative phosphorylation were not measurably affected by these changes in unsaturated fatty acid composition.

Comparison of α-linolenate and oleate in lowering activity of lipogenic enzymes in rat liver: Evidence for a greater effect of dietary linolenate independent of food and carbohydrate intake

Abstract The role of polyunsaturated fatty acids in the regulation of lipogenesis has been the subject of considerable speculation. To determine whether a polyunsaturated fatty acid has a greater effect than a monounsaturated fatty acid in reducing activities of lipogenic enzymes in rats fed a high-carbohydrate, fat-free diet (Muto, Y. and Gibson, D. M. (1970) Biochem. Biophys. Res. Commun. 38, 9–15) or whether the effect of polyunsaturated fatty acids is only indirect (Gozukara, E. M., Frohlich, M. and Holten, D. (1972) Biochim. Biophys. Acta 286, 155–163) through reduction of food and carbohydrate intake, the effects of α-linolenate or oleate supplementation on the activities of lipogenic enzymes in liver were compared under conditions of equalized food intake (pair-feeding). In comparison with oleate supplementation, linolenate supplementation significantly reduced the activities of liver glucose-6-phosphate and 6-phosphogluconate dehydrogenases, fatty acid synthetase, malic enzyme and citrate lyase in fasted-refed rats or in essential fatty acid-deficient rats pair-fed during the supplementation period. These results provide firm evidence for the hypothesis that polyunsaturated fatty acids are more effective than monoenoic or saturated fatty acids in reducing the activity of lipogenic enzymes in liver.

Comparison of C18-, C20- and C22-unsaturated fatty acids in reducing fatty acid synthesis in isolated rat hepatocytes.

C18-, C20- and C22-unsaturated acids were tested for inhibition of fatty acid synthesis in hepatocytes isolated from essential fatty acid-deficient rats. Fatty acid synthesis was measured by incorporation of radioactivity from [1-14C-A1-acetate or 3H2O into fatty acids. C20-polyunsaturated fatty acids included arachidonic acid (20 : 4 (n-6)) and 4 other fatty acids formed from linoleic acid (18 : 2 (n-6)) or linolenic acid (18 : 3 (n-3)). These were (11,14)-icosadienoic acid (20 : 2 (n-6)), (8,11,14)-icosatrienoic acid (20 : 3 (n-6)), (11,14,17)-icosatrienoic acid (20 : 3 (n-3)) and (5,8,11,14,17)-icosapentaenoic acid (20 : 5 (n-3). All of these have essential fatty acid activity. The fatty acid (5,8,11)-icosatrienoic acid (20 : 3 (n-9)) was also tested. This fatty acid is formed from oleic acid (18 : 1 (n-9)) and is not an essential fatty acid or a prostaglandin precursor. C20-unsaturated fatty acids and (22 : 6 (n-3)) were as effective as stearic acid in inhibiting fatty acid synthesis and were more inhibitory than their precursor C18-unsaturated fatty acids. These results are evidence that C20-unsaturated fatty acids of the linoleic and linolenic acid series can act in short-term inhibition as well as in adaptive inhibition of fatty acid synthesis (Bloch, K. and Vance, D. (1977) Annu. Rev. Biochem. 46, 263--298). The effectiveness of (5,8,11)-icosatrienoic acid indicates that short-term inhibition by C20-unsaturated fatty acids is not limited to those fatty acids which have essential fatty acid activity.

Feeding pure docosahexaenoate or arachidonate decreases plasma triacylglycerol secretion in rats

Essential fatty acid (EFA)-deficient rats were fed highly purified methyl esters of docosahexaenoate (22∶6n−3), arachidonate (20∶4n−6), alpha-linolenate (18∶3n−3) or oleate (18∶1n−9) (100 mg/day, tube fed for 3–10 days), and their plasma triacylglycerol (TG) secretion rates were measured. Secretion rates of TG into plasma were reduced by tube-feeding 22∶6n−3, 20∶4n−6, 18∶3n−3, but not 18∶1n−9, to EFA-deficient rats. A significant reduction occurred after feeding 22∶6n−3 for only three days. Feeding 22∶6n−3 or 18∶3n−3 to EFA-deficient rats for three days also reduced the activities of liver lipogenic enzymes and sharply increased the proportions of 22∶6n−3 and 20∶5n−3 in liver phospholipid fractions. Mechanisms by which these EFA may reduce lipogenesis are discussed.

Surface viscosities of phospholipids alone and with cholesterol in monolayers at the air-water interface

Surface viscosities in lipid monolayers at the air-water interface were measured by the oscillating pendulum method. The logarithms of successive oscillations decreased linearly with time. Surface viscosity is reported here in terms of the rate constant, k, for decay of oscillation. Viscosities were measured as a function of surface pressure at 22±2 C. Lipids investigated included cholesterol, 1-palmitoyl-2-arachidonoyl phosphatidylcholine (PC), 4 other unsaturated PC, 1-palmitoyl-2-stearoyl PC, 1,2-distearoyl PC, 1-palmitoyl-lysophosphatidylcholine, 1-palmitoyl-lysophosphatidylserine, tetrapalmitoyl bisphosphatidic acid, and the dipalmitoyl species of PC, phosphatidylethanolamine (PE), phosphatidyldimethylethanolamine, phosphatidylmonomethylethanolamine, phosphatidylglycerol and phosphatidic acid. Pressure-area curves are presented for the saturated phospholipids. Surface viscosities of most of the phospholipids were high and increased with increasing surface pressure. However, surface viscosiries in monolayers of cholesterol, 1-palmitoyl-2-arachidonoyl PC, lysophosphatidylcholine or lysophosphatidylserine were very low and barely detectable under our experimental conditions. One mol % of cholesterol in monolayers of dipalmitoyl PC greatly reduced the surface viscosity of the film and, in mixed films containing 10% or more of cholesterol, surface viscosity was too low to measure. Cholesterol also reduced surface viscosities in monolayers of the other dipalmitoyl phospholipids. It is suggested that cholesterol functions in lung surfactant by reducing the surface viscosity of its highly saturated phospholipid components.

Vitamin B6 and Amino Acids—Recent Research in Animals

Publisher Summary This chapter focuses on recent studies of certain aspects of vitamin B 6 and amino acid metabolism in animals. All these topics re-emphasize the importance of vitamin B 6 in every aspect of amino acid utilization. With limited amino acid intakes, a high intake of the vitamin can improve the ability of the animal to synthesize and interconvert dietary and tissue amino acids so that the limiting amino acid may be used more efficiently. As the protein intake increases, the intake of nonutilizable amino acids may increase, with the result that the need for vitamin B 6 in amino acid catabolism becomes a more dominant factor. This aspect of vitamin B 6 function appears even more important in view of the evidence that activities of enzymes in amino acid metabolism may vary constantly in relation to protein intake or to other dietary changes. Both activation and synthesis of vitamin B 6 -dependent enzymes may depend upon the vitamin B 6 intake, and the activities of these enzymes may control the pathway by which an amino acid will be metabolized.

Effect of essential fatty acid deficiency on the size and distribution of rat plasma HDL

Rat plasma high density lipoproteins (HDL) are comprised of two major particle size subpopulations, HDL1 (255 Å−140 Å) and HDL2 (140 Å−84 Å), in which the proportion of arachidonate in fatty acids of cholesteryl esters is greater than 50%. To determine whether decreased availability of arachidonate for cholesterol esterification would alter the distribution and/or amounts of the HDL subpopulations, we compared HDL subpopulations in EFA-deficient and control rats. To separate the effects of EFA deficiency and fat deficiency and to evaluate effects of different saturated fats, we used EFA-deficient diets that were fat-free or that contained 5% saturated fat. The control diets were the EFA-deficient diets plus 1% safflower oil. The saturated fats were hydrogenated coconut oil, hydrogenated cottonseed oil and saturated medium-chain triglycerides. All EFA-deficient diets decreased the proportion of the HDL1 subpopulation and the peak diameter of the HDL2 subpopulation. These changes appeared after quite brief EFA depletion in young rats and may be related to the increased liver cholesteryl ester concentrations typical of EFA-deficient rats.