Distinct temporal relation among oxygen uptake, malondialdehyde formation, and low-level chemiluminescence during microsomal lipid peroxidation

An oxystat system was employed in conjunction with a single-photon counting apparatus for simultaneous monitoring of oxygen uptake, oxidative decomposition of membrane lipids, and occurrence of electronically excited species during microsomal lipid peroxidation. During NADPH/ADP-iron-promoted lipid peroxidation at a steady state oxygen partial pressure (pO2) of 30 mm Hg, complex time relationships among oxygen uptake, malondialdehyde (MDA) formation, and low-level chemiluminescence were observed. While the first two parameters occurred nearly simultaneously, low-level chemiluminescence occurred with a significant delay. A decrease of the steady state pO2 to 3 mm Hg led to significant increases of the lag phases of all three parameters and a further enhancement of the time displacement of low-level chemiluminescence in relation to oxygen uptake and MDA formation. At a pO2 of 0.5 mm Hg, the lowest pO2 maintained during this study, no low-level chemiluminescence was observed while oxygen uptake and MDA formation were still detected. In contrast, during NADPH/CCl4-promoted lipid peroxidation at a pO2 of 0.5 mm Hg a sudden drastic rise of low-level chemiluminescence accompanying oxygen uptake and MDA formation was observed. At pO2 between 0.5 and 3 mm Hg all three parameters occurred nearly concomitantly during the entire incubation. At pO2 levels above 3 mm Hg all three parameters showed principally the same behavior. However, the respective maxima of low-level chemiluminescence were reached with some delay. The present observations support the assumption that the decomposition of membrane lipid peroxyl radicals to MDA and the formation of electronically excited species proceed via different pathways. The time displacement between oxygen uptake and MDA formation, on the one hand, and low-level chemiluminescence, on the other hand, depends on the type of initiating radical system and on the steady state pO2 level. It is suggested that the differences are due to distinct subsets (chemical or spatial) of secondary peroxyl radicals in the membrane.     

The critical steady-state hypoxic conditions in carbon tetrachloride-induced lipid peroxidation in rat liver microsomes

Defined steady-state oxygen partial pressures (PO2) were maintained constant with an oxystat system to study carbon tetrachloride (CCl4)-induced lipid peroxidation and oxygen uptake in rat liver microsomes. The initial rates of oxygen uptake and malondialdehyde formation indicated drastically increasing lipid peroxidation by decreasing PO2, attaining a maximum between 1-10 mmHg (0.1-1.3 kPa). Under these conditions, at the hypoxic end of the physiological PO2 in liver, CCl4 caused a 5-fold increase in the oxygen uptake rate and a 20-fold increase in the malondialdehyde formation rate while, at 80 mmHg (10.7 kPa) the haloalkane caused only an increase of 2- and 4-fold, respectively; in comparison, there was only a slight increase in NADPH-induced lipid peroxidation with increasing PO2. These data clearly demonstrate the critical role of low steady-state PO2 in CCl4-induced lipid peroxidation and support lipid peroxidation as a key factor in CCl4 hepatotoxicity.     

The effect of oxygen and vitamin E on the lifespan of human diploid cells in vitro

Human diploid cells (WI-38) were serially subcultivated at partial pressures of oxygen (Po2) ranging from 5.6 mm Hg to 608 mm Hg. At a Po2 of 5.6 mm Hg, the number of doublings to phase out was less than that of control cells at a Po2 of 137 mm Hg. Cultures grown at Po2's of 24, 49, or 137 mm Hg grew at the same rate and phased out after a similar number of population doublings. Population lifespan was markedly shortened by chronic exposure to elevated Po2's, a phenomenon that was, in part, reversible. d-1-alpha-Tocopherol (10 microgram/ml or 100 microgram/ml) homogenized into the medium at each weekly subcultivation did not extend the lifespan of cells at reduced, ambient, or elevated oxygen tensions. These results indicate that neither oxygen toxicity nor free radical reactions play a significant role in limiting the lifespan of WI-38 cells grown in vitro under ambient oxygen tensions (Po2 137 mm Hg).     

Lipid peroxidation and cell viability in isolated hepatocytes in a redesigned oxystat system: evaluation of the hypothesis that lipid peroxidation, preferentially induced at low oxygen partial pressures, is decisive for CCl4 liver cell injury

An oxystat system is described which is capable of maintaining steady-state oxygen partial pressures (PO2) at levels between 0.1 and 300 mm Hg for hours or even days in incubations of respiring cells. The system was used to study effects of the hepatotoxin carbon tetrachloride (CCl4) on lipid peroxidation and cell viability in isolated hepatocytes from phenobarbital-pretreated rats at various steady-state PO2. At PO2 below 35 mm Hg, with a maximum effect at 7 mm Hg, CCl4 induced an immediate lipid peroxidation, the rate of which slowed down during further incubation. AT PO2 between 35 and 70 mm Hg, CCl4 initially induced only slight lipid peroxidation, while there was a significant increase in lipid peroxidation after approximately 30 min. At PO2 above 100 mm Hg, no lipid peroxidation was induced by CCl4. At PO2 of 70 mm Hg and below, with the maximum effect at 3 mm Hg, CCl4 also induced marked losses of cell viability. Under anaerobic conditions and at PO2 greater than 70 mm Hg, CCl4 was without effect on the viability of the liver cells. Cells isolated from the pericentral area of the liver lobule showed more lipid peroxidation and loss of cell viability than cells from the periportal area of the lobule. These results provide further evidence for the decisive role of lipid peroxidation, preferentially induced at low PO2, in CCl4 liver injury.     

Changes in hydrogen peroxide content and activities of antioxidant enzymes in tomato seedlings exposed to mercury

Thirty-day-old seedlings of tomato (Lycopersicon esculentum Mill.) were treated with various Hg concentrations (0, 10, and 50 μM) for up to 20 days, and the hypothesis that Hg induces oxidative stress leading to the reduction of biomass and chlorophyll content in leaves was examined. The accumulation of Hg in seedlings increased with external Hg concentration and exposure time, and Hg content in roots exposed to 50 μM Hg for 20 days was about 27-fold higher than that in shoots. Furthermore, Hg exposure not only reduced biomass and chlorophyll levels in leaves but also caused an overall increase of endogenous H₂O₂, lipid peroxidation products (malondialdehyde), and antioxidant emzymes activities such as superoxide dismutase, catalase, and peroxidase in leaves and roots. Our results suggest that the suppression of growth and the reduction of chlorophyll levels in tomato seedlings exposed to toxic Hg levels may be caused by an enhanced production of active oxygen species and subsequent high lipid peroxidation.     

Prooxidant activity of β-carotene under 100% oxygen pressure in rat liver microsomes

The effects of the partial pressure of oxygen (pO₂ on antioxidant efficiency of β-carotene in inhibiting 2,2′-azobis(2-amidinopropane) (AAPH)-induced lipid peroxidation are investigated in rat liver microsomal membranes. The rate of peroxyl radicals generated by thermolysis of AAPH at 37°C is markedly higher at 150 than 760 mm Hg pO₂. At 150 mm Hg pO₂ β-carotene acts as an antioxidant, inhibiting 2,2′-azobis(2-amidinopropane) (AAPH)-induced Malondialdehyde (MDA) formation. At 760 mm Hg pO₂, it loses its antioxidant activity and shows a prooxidant effect, increasing lipid peroxidation products, -Tocopherol prevents the prooxidant effect of β-carotene in a dose-dependent manner. Our data provide the first evidence of a prooxidant effect of β-carotene under 100% oxygen pressure in a biological membrane model and point out the existence of cooperative interactions between β-carotene and -tocopherol.     

Loss of latent activity of liver microsomal membrane enzymes evoked by lipid peroxidation. Studies of nucleoside diphosphatase, glucose-6-phosphatase, and UDP glucuronyltransferase

The effects of lipid peroxidation on latent microsomal enzyme activities were examined in NADPH-reduced microsomes from phenobarbital-pretreated male rats. Lipid peroxidation, stimulated by iron or carbon tetrachloride, was assayed as malondialdehyde formation. Independent of the stimulating agent of lipid peroxidation, latency of microsomal nucleoside diphosphatase activity remained unaffected up to microsomal peroxidation equivalent to the formation of about 12 nmol malondialdehyde/mg microsomal protein. However, above this threshold a close correlation was found between lipid peroxidation and loss of latent enzyme activity. The loss of latency evoked by lipid peroxidation was comparable to the loss of latency attainable by disrupting the microsomal membrane by detergent. Loss of latent enzyme activity produced by lipid peroxidation was also observed for microsomal glucose-6-phosphatase and UDPglucuronyltransferase. In contrast to nucleoside diphosphatase, however, both enzymes were inactivated by lipid peroxidation, as indicated by pronounced decreases of their activities in detergent-treated microsomes. According to the respective optimal oxygen partial pressure (po2) for lipid peroxidation, the iron-mediated effects on enzyme activities were maximal at a po2 of 80 mmHg and the one mediated by carbon tetrachloride at a po2 of 5 mmHg. Under anaerobic conditions no alterations of enzyme activities were detected. These results demonstrate that loss of microsomal latency only occurs when peroxidation of the microsomal membrane has reached a certain extent, and that beyond this threshold lipid peroxidation leads to severe disintegration of the microsomal membrane resulting in a loss of its selective permeability, a damage which should be of pathological consequences for the liver cell. Because of its resistance against lipid peroxidation nucleoside diphosphatase is a well-suited intrinsic microsomal parameter to estimate this effect of lipid peroxidation on the microsomal membrane.     

Analysis of lipid peroxidation mechanisms in human spermatozoa

The mechanisms by which ferrous ion promoters induce malondialdehyde generation by human spermatozoa have been investigated in order to provide a rational basis for the quantification and interpretation of lipid peroxidation assays. Incubation of human spermatozoa with a ferrous ion promoter in the presence of thiobarbituric acid (TBA) led to the generation of the bone fide malondialdehyde-TBA adduct. The importance of iron in the stimulation of lipid peroxidation was emphasized by the ability of Desferal* and EDTA to suppress malondialdehyde generation. Paradoxically, when the concentration of EDTA relative to iron was equimolar or greater, the suppression of malondialdehyde formation was accompanied by the generation of hydroxyl radicals. These results suggested that the addition of promoter did not effect the first-chain initiation of lipid peroxidation but favored an alternative mechanism involving the catalytic decomposition of pre-existing lipid peroxides. This conclusion was reinforced by the inability of reagents that would limit the formation (superoxide dismutase and/or catalase) or availability (mannitol, formate) of hydroxyl radicals, to influence malondialdehyde generation. While hydroxyl radicals were not directly involved in Fe²⁺-promoted malondialdehyde generation, the existence of significant correlations between reactive oxygen species production and the outcome of the TBA assay, suggested that Fenton chemistry might be important in the initiation of peroxidative damage. It is proposed that the impeded propagation of peroxidation initiated by Fenton or Haber Weiss reactions would lead to the accumulation of lipid peroxides in the spermatozoa and it is these peroxides that are induced to decompose during the Fe²⁺-promoted TBA assay, stimulating a lipoperoxidative chain reaction and malondialdehyde formation. © 1993 Wiley-Liss, Inc.     

UV-A induced lipid peroxidation in liposomal membrane

UV-A (365 nm) produced a dose-dependent linear increase of lipid peroxidation, as detected by the assay of malondialdehyde (MDA). MDA formation was inversely related to the UV-A dose rate. Sodium formate and ethylenediaminetetra acetic acid (EDTA) could not inhibit by any significant degree the UV-A induced MDA formation. While butylated hydroxy toluene (BHT) caused about 85% inhibition, sodium azide and L-histidine produced 45-50% inhibition of MDA formation. The involvement of singlet oxygen (1O2) in the UV-A induced lipid peroxidation is discussed.     

The involvement of superoxide and iron ions in the NADPH-dependent lipid peroxidation in human placental mitochondria

Incubation of human term placental mitochondria with Fe2+ and a NADPH-generating system initiated high levels of lipid peroxidation, as measured by the production of malondialdehyde. Malondialdehyde formation was accompanied by a corresponding decrease of the unsaturated fatty acid content. This NADPH-dependent lipid peroxidation was strongly inhibited by superoxide dismutase and singlet oxygen scavengers, markedly stimulated by paraquat, but was not affected by hydroxyl radical scavengers. Catalase enhanced the production of malondialdehyde by placental mitochondria. The effects of catalase and hydroxyl radical scavengers suggest that the initiation of NADPH-dependent lipid peroxidation is not dependent upon the hydroxyl radical produced via an iron-catalyzed Fenton reaction. These studies provide evidence that hydrogen peroxide strongly inhibits NADPH-dependent mitochondrial lipid peroxidation. The inhibitory effect of superoxide dismutase and stimulatory effect of paraquat, which was abolished by the addition of superoxide dismutase, suggests that superoxide may promote NADPH-dependent lipid peroxidation in human placental mitochondria.     

Detection of lipid peroxidation in lung and in bronchoalveolar lavage cells and fluid

Inhalation of toxic materials such as asbestos, silica, 100% oxygen, ozone, or nitrogen dioxide may lead to an increased production of reactive oxygen metabolites which may initiate lipid peroxidation. Measurement of lipid peroxidation in cells and fluid obtained by bronchoalveolar lavage (BAL), as well as in lung tissue, may aid in monitoring the development and extent of pulmonary damage after inhalation of a toxic substance. In this study, we employed a sensitive assay for detection of malondialdehyde (MDA), a breakdown product of lipid peroxidation. By separation of the adduct with thiobarbituric acid, using a reverse phase high pressure liquid chromatographic technique, we accurately and sensitively measured the content of MDA in BAL cells, lavage fluid, and lavaged lung tissue homogenates of rats. The amounts of sample required for detection of MDA were small enough possibly to be applied to use with human specimens; in addition, recovery of added MDA was acceptable with all types of samples. Inclusion of a metal chelator in the preparation of samples appeared necessary to prevent metal-catalyzed propagation of lipid peroxidation during the assay. Overall, the method described here using samples from rats may be applicable to detecting lipid peroxidation in BAL samples from humans.     

Two phases in development of chemiluminescence during lipid peroxidation induced by Fe2+ ions

After cessation of the stirring of reagents in the egg yolk lipoprotein suspension where lipid peroxidation had been initiated by ferrous ions a drop of chemiluminescence (CL) was observed after a lag period, the duration of which increased in the course of stationary-state CL development. The CL intensity was but slightly dependent on oxygen pressure in the gas phase above incubation mixture within oxygen partial pressure limits 760-40 mm Hg. These data suggest that the CL quantum yield is growing during lipid peroxidation development as a result of the formation in the system of some compounds amplifying the CL intensity and/or development of a new CL reaction whose rate depends purely on oxygen and whose photon emitters are different.     

Protective effects of lazaroids against oxygen-free radicals induced lysosomal damage

Lipid peroxidation of membranes by oxygen free radicals has been implicated in various disease states. Different antioxidants and iron chelators have been used to reduce lipid peroxidation. Lazaroids have been used for the acute treatment of central nervous system disorders such as trauma and ischemia wherein lipid peroxidative processes take place.In this study we evaluated the effect of lazaroids (U-785 18F and U-74389F) on the release of acid phosphatase activity and formation of malondialdehyde (MDA) in rat liver lyosomes subjected to exogenously generated oxygen free radicals. There was a significant increase in the acid phosphatase release and MDA formation in the presence of oxygen free radicals. This was prevented by both the lazaroids. In a separate study the effect of lazaroid U-74389F was seen on the zymosan-stimulated polymorphonuclear (PMN) leukocyte-derived chemiluminescence. The PMN leukocyte chemiluminescent activity was attenuated by the lazaroid in a dose-dependent manner. These studies suggest that lazaroids may inhibit lipid peroxidation and stabilize the membrane.     

Lipid peroxidation in isolated hepatocytes.

Intracellular lipid peroxidation was initiated by the addition of ADP-complexed ferric iron to isolated rat hepatocytes and the reaction monitored by the thiobarbituric acid method or by measurement of the formation of conjugated dienes. Both the production of malondialdehyde (thiobarbituric-acid-reacting substances) and of conjugated dienes was dependent, on the ADP-Fe-3+ concentration in a dose-related fashion. Malondialdehyde formation stopped spontaneously within 20 min after the initiation of the reaction and the plateau reached was also related to the ADP-Fe-3+ concentration. Control experiments revealed that more than 90% of the malondialdehyde accumulating during the incubation period could be ascribed to intracellular production. The cellular NADPH/NADP+ ratio was always high and only slightly decreased upon ADP-Fe-3+-induced lipid peroxidation which, however, was associated with a marked decrease in the cellular glutathione concentration. The rate of accumulation of malondialdehyde as well as the final level reached during ADP-Fe-3+-initiated lipid peroxidation was increased by the addition of chloral hydrate. This apparent stimulatory effect could, however, be ascribed to the inhibition of the mitochondrial oxidation of the malondialdehyde formed during cellular lipid peroxidation, thus allowing more malondialdehyde to accumulate during the process. ADP-Fe-3+-induced cellular lipid peroxidation was associated with a decrease in the concentration of glutathione. Also, lowering of the intracellular glutathione level by the addition of diethyl maleate or by simply preincubating the hepatocytes (up to 50 min) promoted the ADP-Fe-3+ malondialdehyde production and formation of conjugated dienes. Furthermore, when cellular glutathione concentration had been lowered by preincubation of the hepatocytes, significant malondialdehyde production could be observed even at ADP-Fe-3+ concentrations which were too low to induce measurable lipid peroxidation in fresh hepatocytes. It is thus concluded that glutathione has an important role in the cell defence against lipid peroxidation and suggested that the isolated hepatocytes provide a suitable experimental model system for the characterization of this and other possible cellular defence mechanisms and how they are affected by the nutritional status of the donor animal.     

Lactoperoxidase-catalyzed lipid peroxidation of microsomal and artificial membranes

Lactoperoxidase, in the presence of H₂O₂, I^?, and rat liver microsomes, will peroxidize membrane lipids, as evidence by malondialdehyde formation. Fe³⁺ assists in the formation of malondialdehyde. Fe³⁺ can be added at the end of the reaction period as well as at the beginning with equal effectiveness, suggesting that it only acts to assist in the conversion of lipid peroxides, previously formed by lactoperoxidase, to malondialdehyde. The addition of EDTA to the microsomal reaction mixture results in a 40% decrease in malondialdehyde formation. The antioxidant butylated hydroxytoluene will completely block the formation of malondialdehyde. Malondialdehyde formation is not dependent upon the production of superoxide, singlet oxygen, or hydroxyl radicals. Peroxidation of membrane lipids by this system is equally effective in both intact microsomes and in liposomes, indicating that iodination of microsomal protein is not required for lipid peroxidation to occur.     

The mechanism of liver microsomal lipid peroxidation.

In the presence of Fe-3+ and complexing anions, the peroxidation of unsaturated liver microsomal lipid in both intact microsomes and in a model system containing extracted microsomal lipid can be promoted by either NADPH and NADPH : cytochrome c reductase or by xanthine and xanthine oxidase. Erythrocuprein effectively inhibits the activity promoted by xanthine and xanthine oxidase but produces much less inhibition of NADPH-dependent peroxidation. The singlet-oxygen trapping agent, 1, 3-diphenylisobenzofuran, had no effect on NADPH-dependent peroxidation but strongly inhibited the peroxidation promoted by xanthine and xanthine oxidase. NADPH-dependent lipid peroxidation was also shown to be unaffected by hydroxyl radical scavengers.. The addition of catalase had no effect on NADPH-dependent lipid peroxidation, but it significantly increased the rate of malondialdehyde formation in the reaction promoted by xanthine and xanthine oxidase. The results demonstrate that NADPH-dependent lipid peroxidation is promoted by a reaction mechanism which does not involve either superoxide, singlet oxygen, HOOH, or the hydroxyl radical. It is concluded that NADPH-dependent lipid peroxidation is initiated by the reduction of Fe-3+ followed by the decomposition of hydroperoxides to generate alkoxyl radicals. The initiation reaction may involve some form of the perferryl ion or other metal ion species generated during oxidation of Fe-2+ by oxygen.     

Mechanism of cobalt (II) ion inhibition of iron-supported phospholipid peroxidation

Co2+ inhibited nonenzymatic iron chelate-dependent lipid peroxidation in dispersed lipids, such as ascorbate-supported lipid peroxidation, but not iron-independent lipid peroxidation. Histidine partially abolished the Co2+ inhibition of the iron-dependent lipid peroxidation. The affinity of iron for phosphatidylcholine liposomes in Fe(2+)-PPi-supported systems was enhanced by the addition of an anionic lipid, phosphatidylserine, and Co2+ competitively inhibited the peroxidation, while the inhibiting ability of Co2+ as well as the peroxidizing ability of Fe(2+)-PPi on liposomes to which other phospholipids, phosphatidylethanolamine, or phosphatidylinositol had been added was reduced. Co2+ inhibited microsomal NADPH-supported lipid peroxidation monitored in terms of malondialdehyde production and the peroxidation monitored in terms of oxygen consumption. The inhibitory action of Co2+ was not associated with iron reduction or NADPH oxidation in microsomes, suggesting that Co2+ does not affect the microsomal electron transport system responsible for lipid peroxidation. Fe(2+)-PPi-supported peroxidation of microsomal lipid liposomes was markedly inhibited by Co2+.     

UVA诱发的1O2和O2-·产生与脂质过氧化水平关系的研究

紫外A(UVA,320 nm-400 nm)诱发的脂质过氧化反应是通过活性氧(ROS)介导的。在UVA照射之后,单线态氧(1O2)和超氧阴离子(O2-.)是细胞内最初产生的ROS,它们进一步生成过氧化氢(H2O2),羟自由基(.OH)等其它自由基。为了探讨UVA照射后最早生成的1O2和O2-.与细胞氧化损伤后果的关系,我们采用一种特异性检测1O2和O2-.的高灵敏度化学发光探针MCLA(2-methyl-6-(4-methoxyphenyl)-3,7-dihydroimid-azo[1,2-α]pyrazin-3-one hydrochloride)检测人外周血淋巴细胞经UVA照射后的化学发光变化。发现不同剂量UVA照射后,细胞MCLA化学发光变化和MDA浓度变化一致。结果表明UVA照射后1O2和O2-.的水平与由此引发的脂质过氧化损伤存在正相关关系。因此,MCLA化学发光方法可望作为一种检测UVA诱发脂质过氧化水平的简单快速方法。     

Inverse relationship of ethane or n-pentane and malondialdehyde formed during lipid peroxidation in rat liver microsomes with different oxygen concentrations

When we incubated rat liver microsomes with ferrous ions and an NADPH-regenerating system, ethane and n-pentane formation increased correspondingly with decreasing concentrations of oxygen in the atmosphere above the incubation, whereas malondialdehyde increased with increasing oxygen concentrations up to a plateau. At very low oxygen concentrations - 100% helium as atmosphere, but presumably traces of oxygen were present in the microsomes - ethane and n-pentane formation were maximal and dependent on the concentrations of ferrous ions, in the case of ethane, a peak being reached at about 20 microM Fe2+, whereas n-pentane continuously increased with increasing concentrations of Fe2+. It is suggested that the inverse relationship of ethane or n-pentane and malondialdehyde is due to two different reaction sequences of microsomal lipid peroxidation with different oxygen sensitivities.     

Oxygen radical-mediated lipid peroxidation and inhibition of Ca2+-ATPase activity of cardiac sarcoplasmic reticulum

Oxygen radicals have been implicated as important mediators of myocardial ischemic and reperfusion injury. A major product of oxygen radical formation is the highly reactive hydroxyl radical via a biological Fenton reaction. The sarcoplasmic reticulum is one of the major target organelles injured by this process. Using a oxygen radical generating system consisting of dihydroxyfumarate and Fe3+-ADP, we studied lipid peroxidation and Ca2+-ATPase of cardiac sarcoplasmic reticulum. Incubation of sarcoplasmic reticulum with dihydroxyfumarate plus Fe3+-ADP significantly inhibited enzyme activity. Addition of superoxide dismutase, superoxide dismutase plus catalase (15 micrograms/ml) or iron chelator, deferoxamine (1.25-1000 microM) protected Ca2+-ATPase activity. Time course studies showed that this system inhibited enzyme activity in 7.5 to 10 min. Similar exposure of sarcoplasmic reticulum to dihydroxyfumarate plus Fe3+-ADP stimulated malondialdehyde formation. This effect was inhibited by superoxide dismutase, catalase, singlet oxygen, and hydroxyl radical scavengers. EPR spin-trapping with 5,5-dimethyl-1-pyrroline-N-oxide verified production of the hydroxyl radical. The combination of dihydroxyfumarate and Fe3+-ADP resulted in a spectrum of hydroxyl radical spin trap adduct, which was abolished by ethanol, catalase, mannitol, and superoxide dismutase. The results demonstrate the role of oxygen radicals in causing inactivation of Ca2+-ATPase and inhibition of lipid peroxidation of the sarcoplasmic reticulum which could possibly be one of the important mechanisms of oxygen radical-mediated myocardial injury.