Kevin H. Cheeseman

Free radical mechanisms in relation to tissue injury

The present paper is a broad-ranging account of free-radical biochemistry in general, and of free-radical mechanisms of tissue injury in particular. Because it is broad-ranging within tight constraints of length it is necessarily lacking in detail on some issues of relevance; the following reviews can be consulted for additional coverage: Slater (1972, 1978, I&), Pryor (1976-84), Mason (1982), Halliwell & Gutteridge (1984). Free radicals can be defined as molecules or molecular fragments containing a single unpaired electron; this unpaired electron usually gives a considerable degree of chemical reactivity to the free radical: in chemical formulas the unpaired electron is conventionally shown as a ‘superscript dot’, as with the hydroxyl free radical OH’. Free radicals can be produced in the cells and tissues of our bodies by various processes and reactions; Table I divides these into two main sections: (I) formation of free radicals as a result of the impact of radiation, and (2) formation by reduction4xidation (redox) reactions involving the transfer of an electron. For discussion of the mechanisms that result in the production of free radicals, see Pryor (1966) and Slater (1972). Table I also indicates major ways by which free-radical intermediates can be converted to non-radical products by the action of free-radical scavengers; discussion of this important aspect of free-radical biochemistry is at the end of this paper. As already mentioned, free radicals are usually reactive chemically although some important examples of stable free radicals are known, such as diphenylpicrylhydrazyl (DPPH’) and Fremy’s salt (potassium nitrosodidphonate). The

Biokinetics in humans of RRR-alpha-tocopherol: the free phenol, acetate ester, and succinate ester forms of vitamin E.

The bioavailability of RRR-alpha-tocopherol from the oral administration of RRR-alpha-tocopherol itself and its acetate and succinate esters was determined in healthy human subjects. Venous blood samples were withdrawn periodically over a 51-h period following oral administration of a gelatin capsule containing an equimolar mixture of RRR-alpha-tocopherol and RRR-alpha-tocopheryl acetate. In a second study, subjects received a capsule containing an equimolar mixture of RRR-alpha-tocopheryl acetate and RRR-alpha-tocopheryl succinate. In Study 1, RRR-alpha-tocopherol was absorbed at similar rates from both the free phenol, and the acetate ester and maximum plasma levels occurred at 12 h in most subjects. The extent of absorption of RRR-alpha-tocopherol varied considerably between subjects in absolute terms, but the relative absorption from the two forms was remarkably consistent, and a ratio of 1.0 was found for parameters of relative bioavailability in plasma. The concentration of RRR-alpha-tocopherol from each form was maximal at approximately 27 h in red blood cells and, as seen with the plasma data, there was a large interindividual variability. In Study 2, there was no significant difference in the extent of absorption of RRR-alpha-tocopherol from the acetate ester and the succinate ester, although there was an apparently higher initial rate of absorption from the acetate ester.

Studies on the reduction of nitroblue tetrazolium chloride mediated through the action of NADH and phenazine methosulphate.

The general features of the reduction of nitroblue tetrazolium chloride (NBT) by NADH and phenazine methosulphate (PMS) have been studied under aerobic and anaerobic conditions. Under aerobic condition the reduction appears to be mediated through the intermediate formation of the superoxide anion radical O2-.; this reaction is strongly inhibited by superoxide dismutase and by a number of O2-. scavengers such as propyl gallate, (+)-catechin, manganous ions, reduced glutathione and benzoquinone. Cupric ions inhibited the overall reaction by reoxidising reduced PMS. Under anaerobic conditions, superoxide dismutase had only a small inhibitory action and, with the exception of cupric ions, the other substances mentioned above were ineffective as inhibitors. The data presented show that the use of NBT to detect the presence of O2-. is fraught with difficulties due to an equally rapid reduction of NBT by NADH and PMS under anaerobic conditions.

Comparative evaluation of the antioxidant activity of α-tocopherol, α-tocopherol polyethylene glycol 1000 succinate and α-tocopherol succinate in isolated hepatocytes and liver microsomal suspensions

Abstract The antioxidant activity of α-tocopherol polyethylene glycol 1000 succinate (TPGS) and of α-tocopherol succinate (TS) has been examined in isolated hepatocytes and microsomal fractions from rat liver. Both TPGS and TS require esterase activity to yield free α-tocopherol and, hence, antioxidant activity. TPGS and TS consistently exerted a more effective antioxidant protection than an equivalent amount of directly-added free α-tocopherol. The low antioxidant efficiency of directly added free α-tocopherol in such water-based experimental systems as used here seems to be due to its extreme hydrophobicity. TPGS, on the other hand, is an extremely hydrophilic compound that is being examined as a useful source of α-tocopherol in certain clinical situations and is here shown to be a convenient and effective source for experimental studies into lipid peroxidation and antioxidant mechanisms.

Lipid peroxidation and molecular damage to polyunsaturated fatty acids in rat liver. Recognition of two classes of hydroperoxides formed under conditions in vivo.

The diene conjugates formed during the autoxidation of microsomal lipid extracts, and in endoplasmic reticulum in vivo after exposing rats to CCl4 have been examined by second derivative absorption spectrophotometry. Within a few minutes after administering CCl4 to a rat there is a pronounced signal in microsomal lipid extracts that is ascribed to the cis-trans diene conjugates of microsomal polyunsaturated fatty acids. Somewhat later a second signal becomes evident that is ascribed to trans-trans isomers. The appearance of the trans-trans isomer is very strongly suppressed by prior administration of vitamin E to the rat. It is concluded that the relative contents of cis-trans and trans-trans dienes in lipid extracts of tissue reflect the tissue contents of hydrogen donors as already established for model experiments with polyunsaturated fatty acids in vitro.

Lipid peroxidation in regenerating rat liver

Rats entrained to a strictly regulated lighting and feeding schedule have been subjected to partial hepatectomy or a sham operation. In the partially hepatectomised animals the period of liver regeneration is characterised by regular bursts of thymidine kinase activity. Liver microsomes from rats, at times corresponding to maximum thymidine kinase activity, have much reduced rates of lipid peroxidation compared to control preparations: this is due in part to increased levels of lipid‐soluble antioxidant at times of maximal DNA synthesis. This temporal relationship between thymidine kinase and lipid peroxidation is consistent with the view that lipid peroxidation is decreased prior to cell division.

The role of lipid peroxidation in CCl4-induced damage to liver microsomal enzymes: Comparative studies in vitro using microsomes and isolated liver cells

The question as to whether CCl4 decreases the activities of glucose-6-phosphatase and cytochrome P-450 in liver endoplasmic reticulum mainly through its action in stimulating lipid peroxidation has been investigated using Promethazine to block lipid peroxidation. The investigation, moreover, has compared the effects of CCl4, with and without Promethazine, on isolated rat hepatocytes with corresponding effects on rat liver microsomal suspensions. Our data give no support for the view that products of lipid peroxidation are the main cause of the decrease in cytochrome P-450 observed in CCl4-intoxication. However, our present results are consistent with lipid peroxidation being a major contributory factor to the decrease in glucose-6-phosphatase activity observed in CCl4-induced liver injury.

Vitamin E protects proteins against free radical damage in lipid environments

The fragmentation of the membrane protein monoamine oxidase in submitochondrial particles was induced by defined free radicals during radiolysis and by a system dependent on hydrogen peroxide and a transition metal. By injection of alpha-tocopherol in vivo, the levels of this physiological antioxidant in the mitochondrial preparations could be elevated more than ten-fold. In both radical-generating systems the presence of high levels of alpha-tocopherol in the membrane substantially retarded the protein fragmentation, in parallel with lipid peroxidation. It is suggested that membrane-bound proteins are damaged during lipid peroxidation and that alpha-tocopherol protects cells against both types of damage.

Measurement of n-Alkanals and hydroxyalkenals in biological samples☆

A modified method was developed to measure nM levels of a range of n-alkanals and hydroxyalkenals in biological samples such as blood plasma and tissue homogenates and also in Folch lipid extracts of these samples. Butylated hydroxytoluene (BHT) and desferrioxamine (Desferal) were added to samples to prevent artifactual peroxidation. Aldehydes were reacted with 1,3-cyclohexanedione (CHD), cleaned up by solid-phase extraction on a Sep-Pak C18 cartridge and the fluorescent decahydroacridine derivatives resolved by reverse-phase high-performance liquid chromatography (HPLC) with gradient elution. A wider range of aldehydes was detected in lipid extracts of plasma and liver homogenate compared to whole (unextracted) samples. Human plasma contained nM levels of acetaldehyde, propanal, butanal, pentanal, hexanal, and heptanal. 4-Hydroxynonenal (0.93 nmol/g) and alkanals with two to six carbons (up to 7.36 nmol/g) were detected in rat liver. Recovery of aldehydes added to whole plasma or to lipid extracts of plasma was dependent on carbon chain length, varying from 95% for acetaldehyde to 8% for decanal. Recovery from biological samples was significantly less than that of standards taken through the Sep-Pak clean-up procedure, suggesting that aldehydes can bind to plasma protein and lipid components.

Nonesterified fatty acids inhibit iron-dependent lipid peroxidation

The effect of various fatty acids on lipid peroxidation of liver microsomes induced by different methods in vitro was studied using oxygen uptake and malonaldehyde (MDA) production. It was observed that fatty acids with a single double bond are effective inhibitors of peroxidation. Stereo and positional isomers of oleic acid were equally effective as oleic acid. There was an absolute requirement for a free carboxyl group, since methyl esters of fatty acids and long-chain saturated and unsaturated hydrocarbons could not inhibit peroxidation. Saturated fatty acids with a chain length of 12-16 carbon atoms showed inhibition, whereas more than 18 carbon atoms reduced the inhibitory capacity. Fatty acids of lower chain length such as capric and caprylic acids did not show inhibition. Fatty acid inhibition was partially reversed by increasing the concentration of iron in the system. Peroxidation induced by methods which were independent of iron was not inhibited by fatty acids. It was observed that intestinal microsomes which were resistant to peroxidation due to the presence of nonesterified fatty acids in their membrane lipids were able to peroxidise by methods which do not require iron. These results suggest that certain fatty acids inhibit peroxidation by chelating available free iron. In addition, they may also be involved in competing with the esterified fatty acids in the membrane lipids which are the substrates for peroxidation.