Jay H. Kramer

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.

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 Coronary sinus blood samples were withdrawn at 1, 3, 5, 10, 15, 20, and 25 min in post-cross-clamp and immediately mixed with isosmotic alpha-phenyl-tert-butylnitrone (PBN) and then centrifuged to obtain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.

Role of free radicals and substance P in magnesium deficiency

In the United States the literature contains only sporadic references to clinical disorders of Mg-deficiency, compared to more recent interest in the benefits of magnesium infusion in myocardial infarction and other acute clinical conditions [1,2]. In Europe the clinical interest in Mg-deficiency was pioneered by Durlach in his book entitled Le Magnesium en Prutique Clinique; the English edition was entitled Magnesium in Clinical Practice [3]. In the conclusion of his book, Durlach stated: “This ion which is present in all the cells is involved in many different pathologies. Integrating a search for the disorders of magnesium metabolism in daily diagnostic processes allows determination of the indications and precise methods of magnesium therapy.” In the United States, Seelig authored a text in 1980 entitled Magnesium Dejicicncy in the Parhogerzesis sf Disease [4] and reviewed the literature concerning magnesium requirements in human nutrition and the association of magnesium deficiency with cardiovascular disease [s]. That same year Wacker published an excellent book entitled Mugnesium und Mm in which he emphasized the clinical relevance of magnesium 161. Six decades ago MacCollum [7l studied the effects of Mg-deficiency on development, reproduction, neuromuscular and humoral abnormalities in animals. In 1959 Bajusz and Selye published a paper describing the influence of electrolytes in the process of myocardial injury [8]. More recently, Lehr focused attention on magnesium and the process of cardiac necrosis [9], or cardiomyopathic lesions which had been described earlier [lo]. B.T. Altura and B.M. Altura published a series of papers which postulated

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.

Postischemic free radical production in the venous blood of the regionally ischemic swine heart. Effect of deferoxamine.

Background We tested the hypothesis that secondarily produced free radicals can be detected venous coronary effluent without the need for direct exposure of postischemic tissue to the spin trapping agent a-phenyl-tert-butylnitrone (PBN). Methods and Results The left anterior descending coronary artery (LAD) of pigs was ligated for 15, 30, 40, or 60 minutes, and the tissue was subsequently reperfused for 60 minutes. Venous effluent (6.5 ml) from the risk area was withdrawn sequentially at 1.5-minute intervals during reperfusion. The effluent blood was immediately infused (4.5 ml/min) with an isotonic saline solution containing 120 mM PBN. Preischemic control effluent samples were collected in an identical fashion. Plasma from each sample was extracted in organic solvent and subsequently analyzed by electron spin resonance (ESR) spectroscopy. Another group of pigs received an infusion of the metal chelator deferoxamine mesylate (25 mg/kg/hr) into the right atrium starting 1 hour before the 40-minute ligation and continuing throughout ligation and reperfusion. We were able to demonstrate the postischemic production of ESR signals for PBN adduct(s) from untreated hearts having spectral characteristics similar to an alkoxyl adduct (PBN-RO.; hyperfine splitting constants for β-hydrogen [aH] =2.0–2.25 G; nitrogen [aN] = 13.5–13.75 G). The reperfusion time course of PBN adduct production had a unique pattern: 1) multiple low-level bursts during. the initial 15 minutes of reperfusion, and 2) a prominent PBN adduct signal during a relatively late time (20–25 minutes) of reperfusion. Total postischemic PBN adduct production rose with increasing duration (15–60 minutes) of ischemia and was associated with a progressive elevation of total lactate dehydrogenase in the effluent. Infusion of deferoxamine markedly diminished PBN adduct production as well as total release of lactate dehydrogenase. Conclusions These data suggest the potential feasibility of using an ex vivo ESR spin trapping technique in blood-perfused models of cardiovascular injury and that chelatable free iron contributes to the production of alkoxyl radicals.

Lipid Peroxidation‐Derived Free Radical Production and Postischemic Myocardial Reperfusion Injurya

ESR spin trapping techniques, which monitor LPO-derived secondary free radical production, provide a quantitative index of irreversible oxidative injury in in vivo and in vitro models of postischemic stress. Secondary radical detection, in conjunction with other indices of injury, also offers a powerful approach for assessing protection against reperfusion-mediated oxidative injury in the presence of anti-radical intervention (Fig. 11). Monitoring secondary radical production has allowed us to demonstrate (1) a direct relationship between ischemic duration and the subsequent postischemic production of alkoxyl radicals from heart tissue; (2) changes in total secondary radical levels directly parallel the severity of mechanical dysfunction and/or tissue injury in postischemic tissue models; (3) interventions that interrupt LPO-derived secondary radical production by different mechanisms also attenuate reperfusion-mediated functional and/or tissue injury; and (4) secondary radical detection provides a standard measure of injury independent of the oxidative stress model used (A-R cells; buffer- and blood-perfused tissues; and global or regional ischemia) or animal species employed (rat, hamster, swine, dog, and human models). The lack of species-specificity for this index suggests the potential for clinical application, especially in light of our recent report demonstrating detection of secondary alkoxyl and alkyl radicals during the postoperative period after human open heart surgery.

A hydroxylated analog of the β-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.

Dietary magnesium intake influences circulating pro-inflammatory neuropeptide levels and loss of myocardial tolerance to postischemic stress.

Severe dietary Mg restriction (Mg9, 9% of recommended daily allowance [RDA], plasma Mg = 0.25 mM) induces a proinflammatory neurogenic response in rats (substance P [SP]), and the associated increases in oxidative stress in vivo and cardiac susceptibility to ischemla/reperfusion (I/R) injury were previously shown to be attenuated by SP receptor blockade and antioxidant treatment. The present study assessed if less severe dietary Mg restriction modulates the extent of both the neurogenic/oxidative responses in vivo and I/R injury in vitro. Male Sprague-Dawley rats maintained on Mg40 (40% RDA, plasma Mg = 0.6 mM) or Mg100 (100% RDA, plasma Mg = 0.8 mM) diets were assessed for plasma SP levels (CHEM-ELISA) during the first 3 weeks and were compared with the Mg9 group; red blood cell (RBC) glutathione and plasma malondialdehyde levels were compared at 3 weeks in Mg9, Mg20 (plasma Mg = 0.4 mM), Mg40, and Mg100 rats; and 40-min global ischemia/30-min reperfuslon hearts from 7-week-old Mg20, Mg40, and Mg100 rats were compared with respect to functional recovery (cardiac work, and diastolic, systolic, and developed pressures), tissue LDH release, and free radical production (ESR spectroscopy and α-phenyl-N-tert butyinitrone [PBN; 3 mM] spin trapping). The Mg40 diet induced smaller elevations in plasma SP (50% lower) compared with Mg9, but with a nearly identical time course. RBC glutathione and plasma malondialdehyde levels revealed a direct relationship between the severity of oxidative stress and hypomagnesemia. The dominant lipid free radical species detected in all I/R groups was the alkoxyl radical (PBN/alkoxyl: αH = 1.93 G, αN = 13.63 G); however, Mg40 and Mg20 hearts exhibited 2.7- and 3.9-fold higher alkoxyl levels, 40% and 65% greater LDH release, and lower functional recovery (Mg20 < Mg40) compared with Mg100. Our data suggest that varying dietary Mg intake directly influences the magnitude of the neurogenic/oxidative responses in vivo and the resultant myocardial tolerance to I/R Stress.

Cytokines, Neuropeptides, and Reperfusion Injury during Magnesium Deficiency a

In summary, hypomagnesemia enhances reperfusion injury. We postulate that neurogenic inflammation, which occurs very early during hypomagnesemia, predisposes the myocardium to reperfusion injury by depleting endogenous antioxidants and recruiting inflammatory cells, which can participate in enhanced free radical production during postischemic reperfusion. Vitamin E supplements can prevent the occurrence of this enhanced injury possibly through the restoration of endogenous antioxidant defenses.