Characterization of iron-mediated peroxidative injury in isolated hepatic lysosomes.

Peroxidative degradation of the lysosomal membrane and the resultant release of hydrolytic enzymes may be responsible for hepatocellular injury in iron toxicity. In this study, highly purified hepatic lysosomes were exposed to iron salts in vitro; the nature of this iron-mediated process of injury and the susceptibility of the lysosomal integrity were studied. Native hepatic lysosomes from rats were isolated by free flow electrophoresis. Incubation of the lysosomes at 37 degrees C with Fe3+-ADP in the presence of ascorbate resulted in rapid generation of malondialdehyde, which approached a plateau at 20 min. Subsequently, the loss of lysosomal latency, determined as an increased percentage free activity of N-acetyl-beta-glucosaminidase, also occurred and reached a maximum loss at 30 min. The half-maximal level of ascorbate, required to promote the Fe3+-ADP mediated lysosomal peroxidation, was approximately 10 microM; high concentrations of ascorbate were inhibitory and half-maximal inhibition was achieved at a concentration of 2 mM. The iron-mediated lysosomal peroxidation was not inhibited by most active oxygen scavengers and appeared to depend solely on the generation of Fe2+ species. When a fresh solution of Fe2+ was incubated with the lysosomes, both the extent of lipid peroxidation and the degree of latency loss increased as a function of increasing Fe2+ concentration. High concentrations of Fe2+ stimulated lysosomal lipid peroxidation instantaneously and reached the highest level within 10 min; whereas the subsequent maximum loss of latency was achieved within 20 min. Both the MDA formation and the loss of latency in either the Fe3+-ADP + ascorbate or the Fe2+ system were effectively prevented by the presence of vitamin A or vitamin E.

Effect of collateral flow on epicardial and endocardial lysosomal hydrolases in acute myocardial ischemia.

Early changes in lysosomal enzymes must occur if their role is significant in irreversible myocardial injury. Therefore, we ligated the anterior descending coronary artery in 14 dogs and after 60 min excised epicardial and endocardial samples from the ischemic and adjacent normal heart. The collateral flow measured with radioactive microspheres in the endocardial samples averaged 19% of control. The muscle was disrupted and fractionated by ultracentrifugation into nuclear pellet (NP), heavy lysosomal pellet (HL), light lysosomal pellet (LL), microsomal pellet (M) and supernate (S). Electron microscopy demonstrated changes characteristic of sichemia in whole tissues and sedimented fractions. Acid phosphatase reaction product was present in residual bodies in the HL fraction and membrane-bound vesicles in the LL fraction and in the intact tissue. Significant decreases in the specific activity of N-acetyl-beta-glucosaminidase and beta-glucuronidase occurred in the endocardial LL fraction, while significant increases in both were found in the ts fraction (P less than 0.05). Losses of acid phosphatase occurred in both LL and S fractions. Moreover, decreases of total N-acetyl-beta-glucosaminidase in the HL fraction and of total beta-glucuronidase and acid phosphatase in the LL fraction were positively correlated (P less than 0.01) with the degree of ischemia measured with radioactive microspheres. Only insignificant enzymatic changes were found when the collateral flow was greater than 40%, and the differences were less significant in epicardial samples where the flow averaged 29%. The early loss of enzymes from the lysosomal fractions in severe ischemia suggests a role for lysosomal hydrolases in the necrosis that follows coronary occlusion.

Cardiac sarcolemma of the hamster. Enrichment of the (Na+ + K+)-ATPase.

Cardiac sarcolemma was prepared from normal hamsters using gentle homogenization, extraction with 0.6 M KCl and continuous density gradient centrifugation. The final fraction exhibited high (Na+ + K+)-ATPase activity (24 micromol/mg per h) and contained minimal contamination from mitochondria, myofibrils and lysosomes.

Characteristics of multiple forms of the acidic triacylglycerol lipase(s) of canine cardiac myocytes.

Acidic lipase activity was extracted by digitonin treatment from particulate fractions prepared from isolated adult canine myocytes. Both methylumbelliferyloleate (MUO) and trioleoylglycerol were hydrolyzed with an apparent Km of 13 and 135 microM, respectively. The primary products of trioleoylglycerol lipolysis were oleic acid and 1,2-dioleoylglycerol. Hydrolysis of either MUO or triacylglycerol was stimulated in vitro by the addition of cardiolipin or Triton X-100. Triton X-100 alone was sufficient for maximal stimulation of MUO hydrolysis, but cardiolipin further stimulated triacylglycerol lipolysis in the presence of an optimal concentration of Triton X-100. Cardiolipin increased the Vmax without altering the Km for trioleoylglycerol. Upon gel filtration chromatography the 4-methylumbelliferyloleate and triacylglycerol lipase activities eluted in regions consistent with molecular weights of approx. 47 000 and 55 000, respectively. Chromatofocusing revealed predominantly one form of acidic 4-methylumbelliferyloleate hydrolase (pI approx. 6.3), whereas acidic triacylglycerol lipase activity eluted continuously in the pH gradient from 7.2 to 4.3 with no clearly predominant peak of activity. Two forms of both 4-methylumbelliferyloleate and triacylglycerol lipase were eluted from columns of carboxymethyl Bio-Gel at pH 5.7; one form of each lipase activity was not bound and another form of each lipase was eluted with 50-60 mM KCl. The non-bound forms of each lipase were indistinguishable from their respective carboxymethyl-bound forms on the basis of pH dependency or kinetically (similar Km). The non-bound and carboxymethyl-bound peaks of lipolytic activity differed in the ratios of 4-methylumbelliferyloleate hydrolase to triacylglycerol lipase activity. The results suggest that the cardiac myocyte contains multiple forms of acidic lipase, and that the catalytic units primarily responsible for the hydrolysis of methylumbelliferyl esters and triacylglycerols may not be identical.

Iron potentiates nitric oxide scavenging by dithiocarbamates in tissue of septic shock mice.

Vanin and co-workers (Kubrina et al., Biochim. Biophys. Acta 1176 (1993) 240-244; Mikoyan et al., Biochim. Biophys. Acta 1269 (1995) 19-24) reported that short term (30 min) iron (Fe) exposure potentiates nitric oxide (NO) production in tissues of septic shock mice, based on increased formation of NO complex by diethyldithiocarbamate (DETC). We have reexamined the effect of Fe administration in mice treated with Escherichia coli lipopolysaccharide (LPS) and have not found any changes in nitrosylhemoglobin (HbNO) or (NOs- + NO3-) levels in blood 30 min after Fe-citrate complex injection. However, Fe-citrate promotes NO complex formation by iron-dependent NO traps: DETC, pyrrolidinedithiocarbamate (PDTC) and N-methyl-D-glucamine dithiocarbamate (MGD), when given simultaneously at 6 h after LPS. Rather than potentiation of NO production, our data support that short-term iron treatment (30 min) enhances in vivo spin trapping ability of dithiocarbamate.

Captopril protects against myocardial injury induced by magnesium deficiency.

We have previously reported that antioxidant drug intervention protects against magnesium deficiency-induced myocardial lesions. In the present study, Golden Syrian male hamsters were fed either a magnesium-deficient diet or a magnesium-supplemented diet. Animals from each group received sulfhydryl-containing angiotensin converting enzyme inhibitors: captopril, epi-captopril (a stereoisomer of captopril), and zofenopril* (arginine blend of zofenopril containing a free SH group); another group of animals received the non-sulfhydryl-containing angiotensin converting enzyme inhibitor enalaprilat. The animals were killed after 14 days, and their hearts were isolated for morphological and morphometric analyses. Hematoxylin and eosin-stained sections were examined by a computer image analysis system for a morphometric determination of the severity of myocardial injury. Captopril reduced both the density of lesions, from 0.32 to 0.08 lesions/(mm2) (p less than 0.01), and the area fraction of lesions, from 7.42 x 10(-4) to 2.03 x 10(-4) lesion area/(mm2) (p less than 0.01), as well as the degree of inflammatory infiltration around the blood vessels. Epi-captopril and zofenopril* were virtually equipotent to captopril, but enalaprilat afforded only slight (nonsignificant) protection. These results indicate that a significant component of the protective effect of captopril in this model was attributable to its sulfhydryl moiety, rather than solely due to the inhibition of the angiotensin converting enzyme. These data further support our previous findings of possible free radical participation in cardiomyopathy due to magnesium deficiency.

A hydroxylated analog of the beta-adrenoceptor antagonist, carvedilol, affords exceptional antioxidant protection to postischemic rat hearts.

The antioxidant and cardioprotective effects of the beta-adrenoceptor antagonist, carvedilol, and its hydroxylated analog. BM-910228, were compared using the postischemic rat heart model. Hearts were infused with either agent (0.01, 0.10, or 10 nM final, or drug-free infusate) for 10 min prior to 30 min global ischemia, and also during the initial 15 min of reperfusion. Recovery of postischemic hemodynamic parameters (left ventricular systolic and developed pressures, mean diastolic pressure, cardiac output, coronary flow rate, and cardiac pressure-volume work), and the extent of postischemic tissue lactate dehydrogenase (LDH) loss, lipid hydroperoxide (LOOH) formation, and lipid peroxidation (LPO)-derived free radical production were assessed and compared among the treatment groups. The depressive pharmacological properties (beta- and alpha-blockade) of both agents masked the extent of postischemic hemodynamic recovery, except at the lowest dose (10 pM) of the analog, which provided significant improvements in systolic and developed pressures, and cardiac work. Treatment with both agents provided significant dose-dependent reductions in postischemic LOOH formation and lipid alkoxyl radical production, as determined by electron spin resonance spectroscopy and alpha-phenyl-tert-butylnitrone. (PBN) spin trapping (PBN/alkoxyl adduct hyperfine splitting alpha N = 13.63 G and alpha H = 1.93 G). Although both agents reduced oxidative injury, the hydroxylated analog was clearly the superior antioxidant (equipotent at doses two to three orders of magnitude lower) compared to the parent compound. This was also reflected with respect to three orders of magnitude lower) compared to the parent compound. This was also reflected with respect to drug-mediated improvement in myocardial preservation (reduced LDH release), which paralleled the antioxidant protective effects. Because neither agent displayed significant primary radical scavenging ability at doses (< or = 10 nM), which did provide substantial inhibition of postischemic LOOH and alkoxyl formation, our data suggest that the antioxidant properties of carvedilol and its analog are mediated primarily through a LPO chair-breaking mechanism. Moreover, the significant antioxidant protection afforded by the analog BM-910228 at subnanomolar levels places this agent into an exclusive category reserved for exceptionally potent antioxidants.

Magnesium-deficiency potentiates free radical production associated with postischemic injury to rat hearts: vitamin E affords protection.

Preexisting magnesium deficiency may alter the susceptibility of rat hearts to postischemic oxidative injury (free radicals). This was examined in rats maintained for 3 weeks on a magnesium-deficient (Mg-D) diet with or without concurrent vitamin E treatment (1.2 mg/day, SC). Magnesium-sufficient (Mg-S) rats received the same diet supplemented with 100 mmol Mg/kg feed. Following sacrifice, isolated working hearts were subjected to 30-, 40-, or 60-min global ischemia and 30-min reperfusion. Postischemic production of free radicals was monitored using electron spin resonance (ESR) spectroscopy and spin trapping with alpha-phenyl-N-tert butylnitrone (PBN, 3 mM final); preischemic and postischemic effluent samples were collected and then extracted with toluene. PBN/alkoxyl adduct(s) (PBN/RO.; alpha H = 1.93 G, alpha N = 13.63 G) were the dominant signals detected in untreated Mg-S and Mg-D postischemic hearts, with comparably higher signal intensities observed for the Mg-D group following any ischemic duration. Time courses of postischemic PBN/RO. detection were biphasic for both groups (maxima: 2-4 and 8.5-12.5 min), and linear relationships between the extent of PBN/RO. production and the severity of both mechanical dysfunction and tissue injury were determined. Following each duration of ischemia, Mg-D hearts displayed greater levels of total PBN adduct production (1.7-2.0 times higher) and lower recovery of cardiac function (42-48% less) than Mg-S hearts. Pretreating Mg-D rats with vitamin E prior to imposing 40-min ischemia/reperfusion, led to a 49% reduction in total PBN/RO. production, a 55% lower LDH release and a 2.2-fold improvement in functional recovery, compared to untreated Mg-D hearts. These data suggest that magnesium deficiency predisposes postischemic hearts to enhanced oxidative injury and functional loss, and that antioxidants may offer significant protection against the pro-oxidant influence(s) of magnesium deficiency.

Reactions of captopril and epicaptopril with transition metal ions and hydroxyl radicals: an EPR spectroscopy study.

In the present study, using the technique of EPR spin trapping with DMPO a spin trap, we demonstrated formation of thiyl radicals from thiol-containing angiotensin converting enzyme (ACE) inhibitor captopril (CAP) and from its stereoisomer epicaptopril (EPICAP), a non-ACE inhibitor, in the process of .OH radical scavenging. Splitting constants of DMPO/thiyl radical adducts were identical for both thiols and were aN = 15.3 G, and aH = 16.2 G. Bimolecular rate constants for the reaction of CAP and EPICAP with .OH radicals were close to a diffusion-controlled rate (approximately 2 x 10(10) M-1s-1). Our data also show that both CAP and EPICAP reduce Fe(III) ions and that their respective thiyl radicals are formed in this reaction. In the presence of Fe(III), H2O2, and CAP, or EPICAP, .OH radicals were produced by a thiol-driven Fenton mechanism. Copper(II) ions were also reduced by these thiols, but no thiyl radicals could be detected in these reactions, and no .OH or other Fenton oxidants were observed in the presence of H2O2. Our data show direct evidence that thiol groups of CAP and EPICAP are involved in scavenging of .OH radicals. The direct .OH radical scavenging, together with the reductive "repair" of other sites of .OH radical attack, may contribute to the known protective effect of CAP against ischemia/reperfusion-induced arrhythmias. The formation of reactive thiyl radicals in the reactions of the studied compounds with .OH radicals and with Fe(III) ions may play a role in some of the known adverse effects of CAP.

Magnesium deficiency in vitro enhances free radical-induced intracellular oxidation and cytotoxicity in endothelial cells.

The effect of magnesium (Mg)-deficient culture on endothelial cell susceptibility to oxidative stress was examined. Bovine endothelial cells were cultured in either control sufficient (0.8 mM) or deficient (0.4 mM) levels of MgCl2. Oxygen radicals were produced extracellularly by the addition of dihydroxyfumarate and Fe(3+)-ADP. Isolated Mg-deficient endothelial cells produced 2- to 3-fold higher levels of thiobarbituric acid (TBA)-reactive materials when incubated with this free radical system. Additional studies were performed using digitized video microscopy and 2',7'-dichlorofluorescein diacetate (DCFDA) as an intracellular indicator for oxidative events at the single cell level. In response to the exogenous oxidative stress, endothelial cells exhibited a time-dependent increase in fluorescence, suggestive of intracellular lipid peroxidation. The increase in cellular fluorescence began within 1 min of free radical addition; the Mg-deficient cells exhibited a more rapid increase in fluorescence than that of Mg-sufficient cells. In separate experiments, cellular viability was assessed using the Trypan blue exclusion assay. Mg deficiency increased cytotoxicity of the added oxyradicals, but the loss of cellular viability began to occur only after 15 min of free radical exposure, lagging behind the detection of intracellular oxidation products. These results suggest that increased oxidative endothelial cell injury may contribute to vascular injury during Mg deficiency.

Iron attenuates nitric oxide level and iNOS expression in endotoxin-treated mice.

The effect of exogenous Fe-citrate complex (Fe doses of 120 and 240 micromol/kg) on nitric oxide (NO) production in vivo has been studied in blood and liver tissue of endotoxin-treated mice. Fe-citrate complex was administered to mice subcutaneously at the same time with intravenous injection of Escherichia coli lipopolysaccharide (LPS). Iron-dependent decrease in NO2-/NO3- and nitrosyl hemoglobin levels in blood of animals was detected at 6 h after LPS administration, suggesting systemic attenuation of NO generation. NO production in the liver tissue of LPS-treated mice was decreased after Fe administration judging from the amount of mononitrosyl-iron complexes formed in the tissue by diethyldithiocarbamate. The iNOS protein determination in the liver tissue of LPS-treated mice demonstrated iron-dependent inhibition of iNOS expression. We have found previously that exogenous iron does not affect systemic NO level when it is given at 6 h after LPS injection, i.e. after iNOS expression. This is a first report demonstrating iron-dependent iNOS down-regulation in endotoxin-treated mice.

Propranolol preserves ultrastructure in adult cardiocytes exposed to anoxia/reoxygenation: a morphometric analysis.

The protective effect of d,l-propranolol was studied using freshly isolated canine ventricular cardiocytes (1.5 x 10(6)/mL) exposed to 30 min anoxia (95% N2/5% CO2) and 0, 3, 20, and 45 min of reoxygenation (95% O2/5% CO2). In addition to preventing lipid peroxide formation, propranolol maintained cellular viability, and minimized ultrastructural alterations. In the absence of propranolol, the outer mitochondria become swollen and rounded up within the first few minutes of reoxygenation. The perinuclear mitochondrial area increased only slightly. We observed that the cellular injury process proceeded differentially from the exterior to the interior, with a mitochondrial area increase and outer membrane rupture. Sarcolemmal damage was also observed with prevalent blebbing and membrane loss. The Z-lines became wider and more diffuse with reoxygenation. Injury to the nuclear double membrane was observed. Incubation with propranolol showed significant protection during postanoxia reoxygenation. In contrast, the more water soluble beta-blocker atenolol only exhibited slight protection. In addition, d-propranolol (the non beta-blocking isomer) and the antioxidant enzymes, SOD and catalase, showed significant protection. These data support previous findings concerning the antioxidant properties of propranolol which appear to be independent of beta-receptor blockade.

Spin-trapping evidence that graded myocardial ischemia alters post-ischemic superoxide production.

The spin trapping agent 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) was used to investigate oxy-radical production in post-ischemic rat hearts previously exposed to 20, 30, or 40 minutes of global ischemia. A hydroxyl spin adduct (DMPO-OH) was identified in coronary effluent during the initial seconds of reperfusion by Electron Spin Resonance (ESR) Spectroscopy. The intensity of the ESR signal in post-ischemic effluent increased as ischemic duration was prolonged; however, regardless of the duration of ischemia, maximal spin adduct detection occurred 3 minutes after initiation of reperfusion. Superoxide dismutase inhibited the formation of DMPO-OH, suggesting that superoxide anion was initially generated and is the principle source for the production on the hydroxyl adduct. Our investigations indicate that superoxide anion is produced during the early moments of reperfusion and that its production in the post-ischemic heart is related to the severity of ischemia.

Identification of free radicals in myocardial ischemia/reperfusion by spin trapping with nitrone DMPO.

The spin trapping ESR technique was applied to investigate oxygen-derived radicals in ischemic and post-ischemic rat hearts. Using 5,5'-dimethyl-l-pyrroline-N-oxide, carbon-centered radicals were identified during ischemia and oxy-radical adducts (superoxide anion radical, O.-2 and hydroxyl radicals, .OH) in post-ischemic rat heart. The formation of these spin adducts was inhibited by superoxide dismutase, suggesting that superoxide plays a role in the adducts' formation. The results demonstrate that oxygen derived free radicals are important byproducts of abnormal oxidative metabolism during myocardial ischemic and reperfusion injuries.

Spin trapping of oxygen and carbon-centered free radicals in ischemic canine myocardium.

Oxygen free radical injury has been postulated to occur during myocardial ischemia. We have used Electron Spin Resonance and Spin Trapping techniques to directly demonstrate the production of carbon-centered (R.) and oxygen-centered lipid radical (RO.) in ischemic canine heart. In addition, venous effluent from the ischemic region showed that conjugated dienes (lipid peroxidation products) increased with ischemic duration. Our results suggest that the formation of the oxygen-centered and carbon-centered lipid radical species during ischemia are a consequence of oxy-radical peroxidation of myocardial membrane lipids.

Detection of alkoxyl and carbon-centered free radicals in coronary sinus blood from patients undergoing elective cardioplegia.

Confirmation of the involvement of free radicals in postischemic injury in human heart has been elusive. The present study was performed to determine the presence of free radicals in coronary sinus blood from patients undergoing elective open heart surgery and cardioplegia. Six patients who were scheduled for nonurgent elective open heart surgery were used in this study. Coronary sinus blood samples were withdrawn at 1, 3, 5, 10, 15, 20, and 25 min in post-cross-clamp and immediately mixed with isosmotic alpha-phenyl-tert-butylnitrone (PBN) and then centrifuged to obtain plasma. Plasma samples were extracted with toluene and analyzed using electron spin resonance (ESR) spectroscopy. We observed ESR spectra consistent with the formation of alkoxyl and carbon-centered radical adducts of PBN (aN = 13.6 G, a beta H = 1.9 G, and aN = 14.1 G, a beta H = 4.2 G) in six of six patients. We obtained complete free radical production time courses during reperfusion from five patients, and all demonstrated a biphasic profile with an initial burst from 5 to 10 min followed by a second maxima at 25 min. Total PBN-adduct production during reperfusion increased in patients subjected to longer aortic cross-clamp times (global ischemia). These data demonstrate that postcardioplegia free radical production is detectable in coronary sinus blood using an ex vivo spin-trapping technique and that the extent of formation may be related to the severity of ischemia.

Protective effects of calcium channel blockers against free radical-impaired endothelial cell proliferation.

We have shown previously that the lipophilic calcium channel blockers exhibit membrane antioxidant activity. In the present study, when attached bovine aortic endothelial cells were exposed for 20 min to a low concentration of oxy-radicals generated from dihydroxyfumarate + Fe-ADP, no loss of glutathione or viability was detected; however, cell number, determined 48 hr later by the tetrazolium salt MTT assay, decreased to 45% of controls. Treatment of the cells for 1 hr with the calcium blockers (2-20 microM) prior to free radical exposure protected against the impaired cell growth in a concentration-dependent manner. The order of potency was nicardipine > or = nifedipine > or = verapamil > diltiazem, which appears to parallel their antioxidant potency. In addition, (+)-nicardipine, and its pharmacologically inactive isomer, (-)-nicardipine, were similarly effective. We conclude that it was the antioxidant activity of the calcium channel blockers that preserved the cell growth capacity against free-radical damage; such protective effects may contribute to their antiatherogenic effects.

Protective effects of sulfhydryl-containing angiotensin converting enzyme inhibitors against free radical injury in endothelial cells.

The effects of SH-containing (captopril, epi-captopril, and the free-SH form of zofenopril) and non-SH-containing (enalaprilat and lisinopril) angiotension converting enzyme (ACE) inhibitors on free radical injury in cultured endothelial cells were studied. When cultured endothelial cells were exposed to a superoxide and hydroxyl radical generating system (dihydroxyfumarate + Fe3(+)-ADP) for 30 min, lipid peroxidation [malondialdehyde (MDA) formation] occurred, and cellular viability (trypan blue exclusion) decreased to 41%; concomitantly, plasma membrane blebbing, assessed by scanning electron microscopy, occurred in 65% of the cells. Preincubation of the cells with each of the SH-agents before free radical addition resulted in an equipotent concentration-dependent (10-200 microM) inhibition (15-60%) of MDA formation; both losses in cellular viability and percent blebbed cells were reduced significantly (P less than 0.05) by concentrations as low as 10 microM of each SH-agent. However, neither of the non-SH agents up to 200 microM produced any major effect. When the effects on hydroxyl radical formation in the system were assessed by ESR spin-trapping with 5,5-dimethyl-1-pyrroline-N-oxide (DMPO), concentrations of 10 and 50 microM of the SH-agents reduced the intensity of the DMPO-OH adducts 20 and 50% respectively. Similar results were observed when the hydroxyl radical was generated from the Fenton-reagents (Fe2+ + H2O2), suggesting direct hydroxyl radical scavenging. Thus, these results demonstrate that the SH-containing ACE agents are capable of protecting the endothelial cells against free radical induced lipid peroxidation and cell injury; the mechanism may be due to direct hydroxyl radical scavenging.

Potent inhibitory activities of hydrophobic aci-reductones (2-hydroxytetronic acid analogs) against membrane and human low-density lipoprotein oxidation.

The effects of selected aci-reductones, which are hydrophobic ascorbate-related analogs including 4-chlorophenyl-2-hydroxytetronic acid (Cpd A), 4-(1,1'-biphenyl)-2-hydroxytetronic acid (Cpd B), and 4-(4'-chloro-1,1'-biphenyl)-2-hydroxytetronic acid (Cpd C), on membrane and low density lipoprotein (LDL) oxidation were assessed. Hepatic microsomal lipid peroxidation was induced by the ascorbate + Fe(II) chemical system. All three agents inhibited membrane lipid peroxidation in a concentration-dependent manner with the order of potency: Trolox (vitamin E) < or = Cpd A << Cpd B < Cpd C; based on the EC50 values, Cpd B and Cpd C were 11- and 19-fold, respectively, more potent than Trolox. In contrast to ascorbic acid, all three agents did not display any membrane prooxidative effect in the presence of iron. When human LDL was incubated with 10 microM of Cu(II), LDL oxidation, determined by the formation of thiobarbituric acid reactive substances, followed a typical sigmoidal curve with an initial lag phase. Preincubation of the LDL samples with low micromolar concentrations (1 and 3 microM) of each agents for 30 min before the addition of copper resulted in significant delays of the lag time of LDL oxidation, and the effectiveness of Cpd B and Cpd C was more prominent than that mediated by either Trolox or probucol. Since clinical evidence strongly supports the hypothesis that atherogenesis is initiated by LDL oxidation, the results suggest that these aryl tetronic acid analogs may serve as promising candidates for future therapeutic use as anti-atherogenic agents.

Hypomagnesemia and isoproterenol cardiomyopathies: Protection by probucol.

Chronic magnesium deficiency is associated with injury of heart muscle, blood vessels, and neuronal tissue. Despite its clinical significance, the mechanism of magnesium deficiency-induced damage remains unclear. The myocardial necrosis induced by injecting catecholamines, which is augmented by magnesium deficiency, is thought to involve a free radical component through catecholamine autoxidation. α-tocopherol was shown to ameliorate the myocardial necrosis induced by magnesium-deficiency (Freedman et al. BBRC 1990;170:1102-1106), suggesting a role for free radicals in this process. If free-radical injury plays a role in magnesium deficiency, then the catecholamine-associated free-radical production may explain the synergistic myocardial injury between catecholamine and magnesium deficiency. To test the hypothesis that free-radical damage may play a role in magnesium deficiency-induced myocardial necrosis, we investigated the protective effects of probucol, a hypolipidemic agent with antioxidant properties. Hamsters were fed a Mg-deficient diet for 14 days with or without probucol. At the end of this period, some animals were sacrificed, while others were injected with isoproterenol and killed 48 hours later. All hearts were processed for morphometric analysis of the lesions. Changes in serum lipids were also determined. Probucol reduced the size and number of both the isoproterenol- and magnesium deficiency-induced cardiac lesions. Our results suggest that it is probucol's antioxidant property and not its hypolipidemic action that is responsible for this protection.