Eric D. Wills

Mechanisms of lipid peroxide formation in tissues Role of metals and haematin proteins in the catalysis of the oxidation of unsaturated fatty acids

Abstract Oxidation of unsaturated fatty acids such as linoleic acid and linolenic acid is catalysed by metals at 37° in the pH range 4.5–7.5 with the formation of peroxides. Co 2+ and Mn 2+ are very active catalysts whilst Cu 2+ , Fe 3+ and Fe 2+ are weakly active. The catalytic activity of Fe 3+ can be strongly stimulated by addition of ascorbic acid or cysteine but both these substances delay oxidation catalysed by Co 2+ or by haematin proteins. The pH optimum for oxidation catalysed by Fe 3+ plus ascorbic acid is 5.5, for Co 2+ catalysis it is 6.5 but haemoglobin-catalysed oxidation is unaffected by pH over the range 4.5 to 8.0. o -Phenanthroline and 8-hydroxyquinoline powerfully inhibit Co 2+ -catalysed oxidation but powerfully stimulate Fe 3+ -catalysed oxidation. Co 2+ -catalysed oxidation is unaffected by most amino acids but is strongly inhibited by histidine, by serum albumin and by some other proteins. It is considered that, in vivo , lipid peroxide formation is likely to be a result of oxidation of unsaturated lipids. catalysed by Fe 3+ and a reducing agent such as ascorbic acid or by heamatin proteins.

The destruction of —SH groups of proteins and amino acids by peroxides of unsaturated fatty acids

Abstract Emulsions of linoleic acid and other unsaturated fatty acids, when incubated at 37 °C in air with cysteine, glutathione and sulphydryl proteins such as papain, cause a rapid destruction of —SH groups. The rate of —SH group destruction is slowed down in a nitrogen atmosphere but considerably increased in oxygen. Sulphydryl groups are destroyed much more rapidly by oxidized linoleic acid emulsions than by fresh emulsions and the rate of destruction is proportional to the peroxide value of the emulsion. During the destruction of —SH groups by fatty acid peroxides the peroxides themselves are destroyed. The exact fate of the —SH groups is uncertain but cysteine has been shown to be converted to a mixture of cystine, cysteic acid and cystine disulphoxide.

Effect of unsaturated fatty acids and their peroxides on enzymes

Abstract Many enzymes of various types, e.g. urease, glyoxalase and papain, have been found to be inhibited by emulsions of oxidised unsaturated fatty acids such as linoleic or linolenic. Some enzymes, such as catalase and D -amino acid oxidase, were unaffected. In most cases, the extent of the inhibition was dependent on the peroxide value of the emulsion, and on the time of contact of enzyme and emulsion. In general, —SH enzymes were found to be more readily inhibited than enzymes not possessing —SH groups and to be protected by sulphydryl compounds, such as glutathione.

The effect of dietary lipids and vitamin E on lipid peroxide formation, cytochrome P-450 and oxidative demethylation in the endoplasmic reticulum.

Abstract The effects of varying the lipid components of the diet have been studied on the cytochrome P-450 content and the rate of oxidative demethylation of aminopyrine in the liver endoplasmic reticulum. The cytochrome P-450 content and rate of oxidative demethylation ( V max ) were lowest when a fat-free diet was fed, increased by addition of 10% lard (containing mainly saturated and mono-unsaturated fatty acids but 6% linoleic acid) and much more by addition of 10% corn oil (containing 50% linoleic acid). Following induction with phenobarbitone the rates of oxidative demethylation and cytochrome P-450 were also greatest in animals fed the corn oil diet and least in animals fed the fat-free diet. Addition of vitamin E (120 mg/kg diet) to the lard diet caused a significant increase in the rate of oxidative demethylation but the synthetic antioxidant 2,6,di- tert -butyl- p -cresol (BHT) was ineffective. The lipid peroxide content of the endoplasmic reticulum and the rate of NADPH stimulated peroxidation were much greater if the corn oil diet was fed than if the fat free diet was fed. Addition of vitamin E reduced the lipid peroxide in the endoplasmic reticulum when a lard diet was fed but BHT was ineffective. It is concluded that polyunsaturated fatty acids, primarily linoleic acid and vitamin E are essential in the diet for the content of cytochrome P-450 and the rate of oxidative demethylation to be a maximum in the endoplasmic reticulum.

Effects of iron overload on lipid peroxide formation and oxidative demethylation by the liver endoplasmic reticulum

Abstract Injections of large doses of iron in the form of Imferon into mice (0.125 mg/g body weight) is followed by an increase in the non-haem iron and total iron content of the liver microsomal fraction over a few days. As a result of iron injection, the rate of lipid peroxidation in microsomal suspensions incubated in presence of NADPH increased by approximately 14 per cent. Much larger increases in the rate of peroxidation, of about 75 per cent, occurred however if the microsomes were incubated in presence of ascorbate. Rates of oxidative demethylation of aminopyrine or of p-chloro-N-methyl aniline were reduced 1 day after iron injection by 6–11 per cent and the reduced rate was maintained for 12 days. Non-haem iron in the liver microsomal fraction is believed to be present in at least two forms, as ferritin and as a component of an electron transport chain catalysing lipid peroxide formation in presence of NADPH. A small proportion of the injected iron is converted into the electron transport component which causes a small increase in the rate of NADPH induced peroxidation. Peroxidation in presence of ascorbate is increased to a much larger extent because ascorbate can utilise iron in the normally stored form, ferritin, to catalyse lipid peroxidation. Lipid peroxidation, enhanced by iron overload, is believed to lead to a breakdown of membranes of the endoplasmic reticulum which causes a decrease in the capacity to carry out oxidative demethylation and related oxidative metabolism dependent on cytochrome P-450 and its associated electron transport chain.

The Effect of Ionizing Radiation on the Fatty Acid Composition of Natural Fats and on Lipid Peroxide Formation

The effects of irradiation doses of 200-1000 krad on the fatty acid compositions of saturated and unsaturated natural food fats have been studied. Lard, coconut oil, corn oil, methyl linoleate and herring oil have been analysed before and after irradiation for lipid peroxide content and fatty acid composition. The effects of storage under varied conditions after irradiation have also been investigated. Irradiation doses of 200-1000 krad had little effect on the fatty acid compositions of saturated fats (lard and coconut oil) or of fats with a high antioxidant content (corn oil) but caused destruction of 98 per cent of the highly unsaturated acids (18: 4,20 :5,22 : 6) and 46 per cent of the diene acids (18:2) in herring oil. The destruction of the polyunsaturated fatty acids increased with increasing storage temperature and storage time. The destruction of polyunsaturated fatty acids is accompanied by an increase in lipid peroxide formation. It is considered that changes in fatty acid composition in natural foods after irradiation are important in consideration of the use of irradiation for food preservation.

The effect of dietary fats on the composition of the liver endoplasmic reticulum and oxidative drug metabolism

1. The dependence of the rate of oxidative demethylation in the liver endoplasmic reticulum on the fatty acid composition of the endoplasmic reticulum has been studied by varying the lipid content of the diet. 2. The rate of oxidative demethylation was markedly dependent on the percentage of linoleic acid (18:2) incorporated into the membrane. Feeding diets containing (g/kg) 100 coconut oil, 100 lard or 100 maize oil caused respectively the incorporation of 7.6, 10.3 and 25.1% linoleic acid (18:2) and a demethylation rate 3.26, 3.15 and 5.03 nmol formaldehyde/min per mg protein. Feeding 100 g herring oil/kg diet caused incorporation of only 5.1% C18:2 but also 27.2% omega 3 unsaturated fatty acids, including 8.7% eicosapentaenoic acid (20:5) and 17.0% docosahexaenoic acid (22.6) and caused a very high rate of oxidative demethylation (6.53 nmol formaldehyde/min per mg protein). 3. Destruction of the polyunsaturated fatty acids in herring oil by irradiation with 400 krad caused incorporation of a smaller quantity of 3 omega unsaturated acids into the endoplasmic reticulum and decreased the rate of oxidative demethylation (4.83 nmol formaldehyde/min per mg protein). 4. The inductive effects of phenobarbitone on oxidative demethylation were partially dependent on changes in the fatty acid composition of the endoplasmic reticulum. Phenobarbitone (100 mg/kg) increased the percentage of C18:2 from 25.1 to 29.4% in rats given a maize-oil diet, increased the percentage of C20:5 from 8.7 to 10.3% in rats given a herring-oil diet and decreased the percentage of arachidonic acid (20:4) and C22.6 in rats given a lard, maize-oil, herring-oil or irradiated-herring-oil diet. 5. Intraperitoneal alpha-tocopherol (50 mg/kg) increased the percentage of C20:4 from 11.1 to 13.1% in rats given a lard diet and from 5.9 to 7.3% in rats given a herring-oil diet. 6. It is concluded that dietary C18:2 is an important factor in the regulation of the rate of oxidative demethylation in the liver endoplasmic reticulum but this may be replaced effectively by dietary C20:5 omega 3 and C22:6 omega 3 acids. Oxidative demethylation is regulated by changes in the fatty acid composition of the membranes of the liver endoplasmic reticulum.

EFFECTS OF IRRADIATION ON SUB-CELLULAR COMPONENTS. I. LIPID PEROXIDE FORMATION IN THE ENDOPLASMIC RETICULUM.

SummarySuspensions of microsomes prepared from rat liver form lipid peroxide when irradiated in vitro with electron doses within the range 5–100 krads. Peroxide formation is minimal immediately after irradiation but increases markedly if the microsomes are incubated at 4°, 20° or 37°c. Dilute microsomal suspensions form much more peroxide than concentrated suspensions. The rate of peroxidation stimulated by NADPH is further enhanced by irradiation.During peroxide formation, extensive degradation of the microsomal lipid occurs with the release of malonaldehyde, or related di-aldehyde, into the aqueous medium.Vitamin E or glutathione (mM) are good protectors against radiation-induced peroxidations. O-phenanthroline (0·5 mM) completely abolishes peroxidation in untreated microsomes, but after doses greater than 50 krads o-phenanthroline has less effect on the rate of peroxidation. ADP contaminated with low concentrations of inorganic iron stimulates peroxidation in untreated microsomes, but has much less eff...

Effects of Irradiation on Sub-cellular Components: II. Hydroxylation in the Microsomal Fraction

SummaryThe oxidative demethylation of aminopyrine and of p-chloromethylaniline or the hydroxylation of aniline by suspensions of rat-liver microsomes is inhibited after irradiation with doses of electrons or γ-rays in vitro within the range 5–100 krads.There is little fall in activity immediately after irradiation, but after incubation of the microsome suspensions at 4°, 20° and 37°c, substantial decreases in the rates result. The decrease of capacity to demethylate or hydroxylate oxidatively is accompanied by an increase in the lipid peroxide content of the microsomal suspensions. It is considered that peroxidation induced by irradiation causes disintegration of the lipid membranes essential for the integrity of the electron transport system responsible for this oxidative metabolism.

Chapter 1 – Ultrastructure of the mammalian cell

Publisher Summary The chapter focuses on cells, their ultrastructures, and methods to study their functions. Historically, it was proved that all animals and plants were composed of small units, the cells. Many of these appeared identical and tissues of animals appeared to be constructed of very large numbers of these small unit cells in a fashion similar to the utilization of bricks in construction of a building. Cells also possess properties that enable them to recognize cells of identical type and to distinguish foreign cells or those of another tissue. This property, which is probably dependent on the membrane glycoproteins, is clearly of great importance during embryonic development and during tissue growth and repair. All cells are essentially rigid structures but modern photographic techniques have shown that many cells are in a constantly dynamic state, that is, flexible and often changing their shape. It is also clear that all tissues contain different types of cells that may be distinguished by different structures within the cells and sometimes even by different metabolic activities.