Ching K. Chow

Lipid peroxidation and endothelial cell injury: implications in atherosclerosis

Vascular endothelial cells, which play an active role in the physiological processes of vessel tone regulation and vascular permeability, form a border separating deeper layers of the blood vessel wall and cellular interstitial space from the blood and circulating cells. Damage or dysfunction of endothelial cells may reduce the effectiveness of the endothelium to act as a selectively permeable barrier to plasma components, including cholesterol-rich lipoprotein remnants. This may be involved in the etiology of atherosclerosis. Experimental evidence indicates that free radical-mediated lipid peroxidation can induce endothelial cell injury/dysfunction. Reactive oxygen species, including peroxidized lipids capable of initiating cell injury, may be generated within endothelial cells, be present in plasma components, or be derived from neutrophils or other blood-borne cells. Lipid peroxidation could initiate or promote the process of atherosclerotic lesion formation by directly damaging endothelial cells, and by enhancing the adhesion and activation of neutrophils and the susceptibility of platelets to aggregate. Endothelial cell injury by lipid hydroperoxides also could increase the uptake of LDL into the vessel wall. These events and other cellular dysfunctions may individually or collectively initiate and/or help to sustain the development of atherosclerosis.

Measurement of malondialdehyde by high performance liquid chromatography with fluorescence detection

A method has been developed to measure malondial-dehyde (MDA) in biological systems. MDA was reacted with 2-thiobarbituric acid (TBA) in the presence of butylated hydroxytoluene (BHT) to minimize formation of artifacts. Initial separation of the TBA-MDA adduct was accomplished by isobutanol extraction. Further elimination and separation of interfering substances was achieved by high performance liquid chromatography. The mobile phase consisted of a 1∶1 (v/v) mixture of methanol and water with 0.05% (w/v) tetrabutyl ammonium dihydrogen phosphate added as an ion pairing reagent. At a flow rate of 1 ml/min, the TBA-MDA adduct was eluted from a 15-cm, c-18, reversed phase column in approximately 4.9 min. The TBA-MDA adduct was quantitated with a fluorescence detector set at 515 nm excitation and 550 nm emission. Using this method, picomole quantities of MDA can be easily detected in plasma and liver samples.

Vitamin E regulates mitochondrial hydrogen peroxide generation

The mitochondrial electron transport system consumes more than 85% of all oxygen used by the cells, and up to 5% of the oxygen consumed by mitochondria is converted to superoxide, hydrogen peroxide, and other reactive oxygen species (ROS) under normal physiologic conditions. Disruption of mitochondrial ultrastructure is one of the earliest pathologic events during vitamin E depletion. The present studies were undertaken to test whether a direct link exists between vitamin E and the production of hydrogen peroxide in the mitochondria. In the first experiment, mice were fed a vitamin E-deficient or-sufficient diet for 15 weeks, after which the mitochondria from liver and skeletal muscle were isolated to determine the rates of hydrogen peroxide production. Deprivation of vitamin E resulted in an approximately 5-fold increase of mitochondrial hydrogen peroxide production in skeletal muscle and a 1-fold increase in liver when compared with the vitamin E-supplemented group. To determine whether vitamin E can dose-dependently influence the production of hydrogen peroxide, four groups of male and female rats were fed diets containing 0, 20, 200, or 2000 lU/kg vitamin E for 90 d. Results showed that dietary vitamin E dose-dependently attenuated hydrogen peroxide production in mitochondria isolated from liver and skeletal muscle of male and female rats. Female rats, however, were more profoundly affected by dietary vitamin E than male rats in the suppression of mitochondrial hydrogen peroxide production in both organs studied. These results showed that vitamin E can directly regulate hydrogen peroxide production in mitochondria and suggest that the overproduction of mitochondrial ROS is the first event leading to the tissue damage observed in vitamin E-deficiency syndromes. Data further suggested that by regulating mitochondrial production of ROS, vitamin E modulates the expression and activation of signal transduction pathways and other redox-sensitive biologic modifiers, and thereby delays or prevents degenerative tissue changes.

Dietary vitamin E and selenium and toxicity of nitrite and nitrate

Nitrites and nitrates are important antimicrobial and flavoring/coloring agents in meat and fish products. However, nitrites and nitrates may cause methemoglobinemia and other illness, and may react with certain amines to form carcinogenic nitrosamines. The nutritional status of vitamin E and selenium has long been associated with nitrite and nitrate toxicity, although the mechanism involved is not yet clear. Information available recently shows that nitrites and nitrates are both oxidation products and ready sources of nitric oxide (NO*), that NO* reacts rapidly with superoxide to form highly reactive peroxynitrite (ONOO-), and that vitamin E may mediate the generation and availability of superoxide and NO*. Increased formation of ONOO- resulting from nitrite treatment and low intake of vitamin E and selenium may thus be the critical event leading to tissue damage and animal mortality observed previously. The protection against the adverse effects of nitrites/nitrates by vitamin E is attributed to its ability to reduce ONOO- formation, while selenium exerts its protective effects via seleno-enzymes/compounds, which reduce ONOO- formed.

Cigarette Smoking and Oxidative Damage in the Lunga

Cigarette smoke contains a large variety of compounds, including many oxidants and free radicals that are capable of initiating or promotes oxidative damage. Also, oxidative damage may result from reactive oxygen species generated by the increased and activated phagocytes following cigarette smoking. In vitro studies are generally supportive of the hypothesis that cigarette smoke can initiate or promote oxidative damage. However, information obtained from in vivo studies is inconclusive. Contrary to expectations, the levels of lipid peroxidation products were found to be decreased or unchanged in the lungs of chronically smoked rats. Metabolic adaptation, such as accumulation of vitamin E in the lung, and increased activities of superoxide dismutase in alveolar macrophages and pulmonary tissues of chronically smoked animals may enable smoked subjects to counteract oxidative stress and to resist further damage to smoke exposure. However, it is also possible that the metabolic adaptation may be secondary to inflammatory response and injury repair process following smoking exposure. More studies are needed to better understand the role of oxidative damage in the etiology of smoking-related disorders.

Oxidative stress as a potential pathogenic mechanism in an animal model of Duchenne muscular dystrophy

Dystrophin-deficiency results in degeneration of most, but not all, skeletal muscles. The mechanisms responsible for degeneration of limb muscle and sparing of extraocular muscle are not known. To address the notion that muscle pathology may be free radical-mediated, we evaluated antioxidant enzyme activities and lipid peroxidation products (TBARS) content in mdx and control mice. TBARS content and the activities of total superoxide dismutase, selenium dependent glutathione peroxidase, glucose-6-phosphate dehydrogenase and catalase were consistently higher in both affected and spared muscles of mdx mice. These data suggest that oxidative stress may be constitutively present in mdx muscle, but may not be the principal pathogenic mechanism. To further test the hypothesis of oxidative stress involvement in dystrophinopathies, control strain and mdx mice were subjected to chronic hyperoxia. The pattern of antioxidant enzyme activities and TBARS content from hyperoxic control strain mice was similar to that of normoxic mdx mice, suggesting that a similar level of oxidative stress was induced. In conclusion, this study has provided indirect evidence for oxidative stress in dystrophin-deficient muscle.

Copper: toxicological relevance and mechanisms

Abstract Copper (Cu) is a vital mineral essential for many biological processes. The vast majority of all Cu in healthy humans is associated with enzyme prosthetic groups or bound to proteins. Cu homeostasis is tightly regulated through a complex system of Cu transporters and chaperone proteins. Excess or toxicity of Cu, which is associated with the pathogenesis of hepatic disorder, neurodegenerative changes and other disease conditions, can occur when Cu homeostasis is disrupted. The capacity to initiate oxidative damage is most commonly attributed to Cu-induced cellular toxicity. Recently, altered cellular events, including lipid metabolism, gene expression, alpha-synuclein aggregation, activation of acidic sphingomyelinase and release of ceramide, and temporal and spatial distribution of Cu in hepatocytes, as well as Cu-protein interaction in the nerve system, have been suggested to play a role in Cu toxicity. However, whether these changes are independent of, or secondary to, an altered cellular redox state of Cu remain to be elucidated.

An improved method for the measurement of malondialdehyde in biological samples.

An improved method was developed for measuring malondialdehyde (MDA) as its thiobarbituric acid (TBA) complex. Samples were initially incubated with 1% potassium iodide and 0.1% butylated hydroxytoluene at 50°C for 20 min, and then with 0.4% TBA at 60°C for 60 min. The MDA-TBA complex formed was extracted with isobutyl alcohol and measured by high-performance liquid chromatography with fluorescence detection. The improved method allows for a more specific determination of MDA present in biological samples.

Effect of dietary carnosine on plasma and tissue antioxidant concentrations and on lipid oxidation in rat skeletal muscle

The effect of dietary carnosine supplementation on plasma and tissue carnosine and α-tocopherol concentrations and on the formation of thiobarbituric acid reactive substances (TBARS) in rat skeletal muscle homo-genates was evaluated. Plasma, heart, liver and hind leg muscle was obtained from rats fed basal semipurified diets or basal diets containing carnosine (0.0875%), α-tocopheryl acetate (50 ppm), or carnosine (0.0875%) plusα-tocopheryl acetate (50 ppm). Dietary carnosine supplementation did not increase carnosine concentrations in heart, liver and skeletal muscle. Dietary supplementation with both carnosine and α-tocopherol increased carnosine concentrations in liver 1.56-, 1.51- and 1.51-fold as compared with diets lacking carnosine, α-tocopherol or both carnosine and α-tocopherol, respectively. Dietary supplementation with both carnosine and α-tocopherol also increased α-tocopherol concentrations in heart and liver 1.38-fold and 1.68-fold, respectively, as compared to supplementation with α-tocopherol alone. Dietary supplementation with carnosine, α-tocopherol or both car-nosine and α-tocopherol was effective in decreasing the formation of TBARS in rat skeletal muscle homogenate, with dietary α-tocopherol and α-tocopherol plus carnosine being more effective than dietary carnosine alone. The data suggest that dietary supplementation with carnosine and α-tocopherol modulates some tissue carnosine and α-tocopherol concentrations and the formation of TBARS in rat skeletal muscle homogenates.

Extraocular, limb and diaphragm muscle group-specific antioxidant enzyme activity patterns in control and mdx mice

The mechanisms primarily responsible for the degenerative processes occurring in dystrophic skeletal muscle remain unresolved. The identification of the mechanisms that lead to the complete sparing of extraocular muscle in dystrophinopathies is of particular interest. A number of studies have provided evidence to suggest that the muscle pathology that characterizes muscular dystrophy may be, in part, free radical mediated. In the present study, we examined the antioxidant enzyme status of extraocular, diaphragm and gastrocnemius muscles in control strain and mdx mice. Our results revealed that in the control strain, both extraocular and diaphragm muscles had higher copper/zinc superoxide dismutase, manganese superoxide dismutase and selenium dependent glutathione peroxidase activities as compared to the gastrocnemius. Furthermore, the diaphragm had higher glutathione reductase activity as compared to the gastrocnemius. These findings indicate that the highly aerobic extraocular and diaphragm muscles have higher antioxidant enzyme capacity than the gastrocnemius, a muscle more dependent on anaerobic energy metabolism. Changes in the antioxidant enzyme status of the mdx mouse correlated, in part, with the degree of histopathological involvement of the three muscle groups assessed.