myocardial tissue iron delocalization and evidence for lipid peroxidation after two hours of...

ORIGINAL CONTRIBUTION ischemia, iron delocalization, lipid peroxidation

Myocardial Tissue Iron Delocalization and Evidence for Lipid Peroxidation After Two Hours of Ischemia

lschemie tissue injury has been proposed to be in part due to oxygen-radical-mediated lipid peroxidation. In vitro studies of such reactions show that they are thermodynamically unfavorable unless catalyzed by transitional metals such as iron in low molecular weight species (LMWS iron), ie, the iron-ADP complex. This study tests for iron delocalization into a LMWS pool during myocardial ischemia and for increased tissue malondialdehyde (MDA), a product of lipid peroxidation. Anesthesia was induced in eight dogs (weighing 20 to 30 kg) with ketamine and maintained by ventilation with 1% halothane. The left anterior descending coronary artery was ligated in four animals, and the circumflex coronary artery was ligated in the other four. Two hours after ligation, the animals were sacrificed by a central venous injection of KCI. Tissue samples were immediately taken from the ischemic zone and from the corresponding nonischemic zone. MDA was determined by the thiobarbituric acid assay. LMWS iron was determined on a tissue ultrafiltrate by the o-phenanthroline assay. Statistical data analysis used the matched-pair two-tailed t test. LMWS iron was 18.3 nM/lO0 mg in ischemic tissue versus 13.1 nM/lO0 mg in nonischemic tissue (t = 4.14; P < .01). MDA was 0.91 nM/lO0 mg in ischemic tissue versus 0.83 nM/lO0 mg in nonischemic tissue (t = 7.27; P < .005). We conclude that there is a significant increase in tissue LMWS iron and in MDA after two hours of regional myocardial ischemia. This iron might be the catalyst for maturatiol~ of tissue in jury during reperfusion as observed by other investigators. Therapeutic iron chelators such as desferoxamine should be examined for tissue protection during reperfusion following ischemia. [Holt S, Gunderson M, Joyce K, Nayini NR, Eyster GF, Garitano AM, Zonia C, Krause GS, Aust SD, White BC: Myocardial tissue iron delocalization and evidence for lipid peroxidation after two hours of ischemia. Ann Emerg Med October 1986; 15:1155-1159.]

INTRODUCTION Myocardial infarction resulting from acute coronary artery occlusion re-

mains the single greatest threat to longevity in our population. Studies in regional myocardial ischemia have focused in part on interventions to increase myocardial resistance to ischemic insults. 1 Such interventions have introduced the concept of tissue protective therapy 2 and rely on a growing knowledge of the biochemistry of ischemic tissue injury.

Ischemic myocardial tissue injury is thought to be in part the result of oxygen-radical-mediated lipid peroxidation. 2 In vitro studies have shown that the initiation of the peroxidation process is thermodynamically unfavorable unless it is catalyzed by transitional metals such as iron. 3 Other potential metal catalysts appear less likely to have a role in biological lipid peroxidation. 4 The active metal is in low molecular weight species (LMWS), ie, the iron-ADP complex.3

Our study was designed to test for iron delocalization into a LMWS pool and for evidence of lipid peroxidation during two hours of severe incomplete regional myocardial ischemia.

MATERIALS AND METHODS Eight mongrel dogs weighing between 20 and 30 kg were anesthetized with

ketamine (7 mg/kg). The animals were intubated and allowed to breathe

Steven Holt, MD* Mark Gunderson, MD* Kathleen Joyce, MD1- Narsimha R Nayini, PhD¢ George F Eyster, DVM§ Ann Marie Garitand I Carolyn Zonia, RN II Gary S Krause, MD # Steven D Aust, PhD :i: Blaine C White, MD# Grand Rapids, Michigan Detroit, Michigan East Lansing, Michigan

From the Department of Emergency Medicine, Butterworth Hospital, Grand Rapids, Michigan;* the Department of Anesthesia, Sinai Hospital, Detroit, Michigan;t and the Department of Biochemistry,:~ the College of Veterinary Medicine,§ the College of Human Medicine,II and Section of Emergency Medicine,# Michigan State University, East Lansing, Michigan.

Received for publication May 24, 1985. Revision received November 25, 1985. Accepted for publication February 11, 1986.

Presented at the University Association for Emergency Medicine Annual Meeting in Kansas City, Missouri, May 1985.

Address for reprints: Steven Holt, MD, Department of Emergency Medicine, Butterworth Hospital, 100 Michigan, NE, Grand Rapids, Michigan 49503.

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IRON DELOCALIZATION Holt et al

spontaneously. Anesthesia was maintained with halothane (1% to 2%) in room air. Right external jugular and left femoral artery catheters were placed for monitoring of central venous and arterial pressures. An IV bolus of lidocaine (1 mg/kg) was given followed by a continuous infusion of 2 rag/rain. A left lateral thoracotomy was performed and the pericardium was opened to expose the heart.

Regional myocardial ischemia was induced by suture ligation of the left anterior descending coronary artery in four dogs and the left circumflex coronary artery in the remaining four dogs. This reduces perfusion in the distal tissue supplied by the coronary artery to 10% to 15% normal, s Re- sidual supply is from limited collateral circulation in the ischemic zone. Ligation was accomplished by passage of a single 3-0 silk suture on a needle beneath the coronary artery just distal to the first diagonal branch. The artery was occluded only partially for the first ten minutes in order to avoid ventricular fibrillation (the Harris pro- cedure6). Complete ligation then was carried out. Corresponding ischemic and nonischemic zones were easily identified by intense cyanosis and wall dyskinesis in the ischemic zones.

Halothane anesthesia in room air was continued and maintenance IV fluids were provided. Two hours following ligation, the animals were sacrificed by a central venous injection of KC1 (0.6 mEq/kg). Myocardial tissue samples were taken immediately from both ischemic and nonischemic zones in the same heart and placed in ice- cold Ringer's lactate solution that had been deoxygenated by argon bubbling.

In this study protocol, each heart was sampled twice, once from both ischemic and nonischemic myocardial regions. By this method, each animal served as its own control. Tissue samples then were grouped into ischemic and nonischemic categories with eight samples in each of the two groups.

All reagents were analytical grade and were used without further pu- rification. All solutions were made with distilled water that had been passed over a Chelex-100 ® column (Chelex water) to remove any trace iron. Analytical stock solutions were prepared using chemicals from the following sources: EDTA, (Mallinckrodt, St Louis) thiobarbituric acid (TBA) (Sigma), butylated hydroxytoluene (BHT)(Sigma), o-phenanthroline (Sig-

ma), trichloro-acetic acid (TCA)(Sig- ma), ascorbic acid solution (Sigma), and ammonium acetate (JT Baker Chemical Co, Phillipsburg, NJ).

Assays for malondialdehyde (MDA), a fragmentation product of lipid peroxidation, were conducted on homog- enates of the heart tissue by the method of Buege and Aust. 7 The TCA-TBA- HC1 reagent was prepared in 0.25 N HC1 with 15% w/v TCA, and 0.375% w/v TBA. This solution was warmed to assist in dissolving the TBA. LMWS iron was determined by the method of Brumby and Massey. s

Tissue specimens obtained from the animal model described above were immediately rinsed in cold deoxygenated Chelex water and blotted, and 1 g of tissue was weighed out and ho- mogenized in 10 mL lmM EDTA using a potter-Elvehjam homogenizer for three minutes. To 1 mL homogenate, 2 mL of TCA-TBA-HC1 stock solution was added, and 60 ~1 of 2% BHT in ethanol also was added to prevent further peroxidation of lipids. 9 This suspension was mixed thoroughly and heated for 15 minutes in boiling water. After cooling, the floc- culant precipitate was removed by centrifugation at 1,000 g for ten minutes. The absorbance of the supema- tant was read at 535 nm against a blank that contained all reagents minus the tissue homogenate. The MDA concentration was expressed as nmoles MDA/100 mg tissue using a standard curve.

The remaining 9 mL of homogenate was passed through a 30,000 molecular weight ultrafiltration membrane (PM-30, 25 mm, Amicon). To an aliquot of 1.5 mL of filtrate, 0.5 mL of 20% TCA was added, thoroughly mixed, and allowed to stand for ten minutes. Then 0.15 mL of 0.1% o-phenanthroline was added to 0.76 mL of the TCA-treated sample mixture and mixed. To this solution, 50 ~1 of 1% ascorbic acid and 40 ~1 of saturated ammonium acetate were added and mixed. The final solution was allowed to stand for ten minutes, and the absorbance was read at 510 nm against a blank that contained all the reagents minus the filtrate. The absorbance was converted to iron concentration using a standard curve, and the results were expressed as nmol/LMWS iron/ 100 mg tissue.

Da ta f rom i s c h e m i c and nonischemic groups were examined by the matched-pair two-tailed t test

Annals of Emergency Medicine

for paired observations. The null hy. pothesis was that no difference in LMWS iron or MDA can be demon. strated between ischemic and nonis. chemic myocardium. The method ot matched-pair testing examines the distribution of differences between pairs of data when each pair of values is derived from the same subject as in our study. In this study, we reversed the region of myocardial ischemia (ie, anterior wall in four dogs, lateral wall in four dogs) so that if a statistically significant difference was found in LMWS iron and MDA in ischemic and nonischemic zones, this could not be accounted for by any "normal" re. gional differences that might exist.

RESULTS Data from all eight animals for

LMWS iron and MDA are shown (Ta. ble). Values are represented as nm/100 mg myocardium.

Mean LMWS-iron was 18.3 nm/100 mg of ischemic myocardium compared with 13.1 nm/100 mg of nonischemic myocardium. This represents a significant increase in LMWS iron in the ischemic zone (t = 4.14; P < .01). Standard deviat ion of differences between data pairs was 1.27.

Mean MDA levels were 0.91 nm/100 mg ischemic myocardium compared with 0.83 nm/100 mg of nonischemic myocardium. This represents a significant increase in MDA in the ischemic zone (t = 7.27; P < .005). Standard de. viation of differences between data pairs was 0.011.

DISCUSSION Our study demonstrated a signifi.

cant increase in LMWS iron in ischemic myocardium following two hours of regional ischemia. This was accompanied by a small but significant rise in tissue MDA, a product of lipid peroxidation. 7 This delocalized iron may represent the transitional metal catalyst that mus t become available for the initiation of lipid peroxidation. 4

There is considerable evidence that low levels of lipid peroxidation occur con t inuous ly in normal subjects. Thus background lipid peroxidation is an analytic problem. There are nume~" ous products of lipid peroxidation; 7 however, most products are either very transient or inappropriate for sin" gle-tissue analysis of material from/n vivo experiments. Ethane and pe~" tane, which are thought to be break"

44/1156 15:10 October 1986

TABLE. Myocardial assays in nm/lO0 mg myocardium

Dog LMWS Iron MDA Ischemic Nonischemic Ischemic Nonischemic

1 6.15 3.70 1.12 1.06

2 22.31 14.23 0.86 0.81

3 25.44 15.35 0.80 0.76

4 16.53 10.61 0.90 0.79

5 13.59 11.20 0.95 0.70

6 23.30 24.10 1.25 1.17

7 21.90 14.90 0.82 0.77

8 17.35 10.36 0.58 0.52

,R 18.32 13.06 0.91 0.83

down products of lipid peroxidation, r are always present in expired air from animals or human beings.lO, u The general involvement of body tissues in the evolution of these gases, however, abrogates their potential utility in regional single-tissue experiments. Lipid hydroperoxides can be measured by a modified MDA assayj 12 but this does not improve on direct measurement of tissue MDA. Conjugated diene formation also can be measured, 13 but these species are readily reactive with oxy- genZ and are very transient. For these reasons, MDA remains an appropriate benchmark assay for pi lot studies such as ours.

It has been proposed that ischemic tissue injury is in part due to lipid peroxidation mediated by oxygen radicals generated during ischemia and early reperfusion.3, 4 Several investigators have examined the possibility that reactive oxygen species (such as superoxide anion, hydroxyl radical, or hydrogen peroxide) might be responsible for myocardial injury associated with either global or regional myocardial ischemia. A growing body of research data now strongly suggests a role for oxygen free radicals in peroxidative myocardial injury in the setting of both induced global and regional myocardial ischemia.

A major recent insight into the mechanism of ischemic myocardial injury is the recognition that there is marked exacerbation of myocardial injury by membrane peroxidation during early reperfusion of previously ischemic tissue.14 In this context of "reperfusion injury," attention has been directed to oxygen free radicals and their role in myocardial tissue injury.

Several lines of evidence indicate a major role for oxygen free radicals in reperfusion injury. Many studies have examined the potential tissue-protective effects of enzymes known to inac- tivate reactive oxygen species. Super- oxide dismutase (SOD) converts the superoxide anion (0 2 - ) to hydrogen peroxide (H202). H20 2 can be converted to water and oxygen by catalase (CAT). In 1982 Shlafer and colleagues is studied administrat ion of SOD and CAT in an isolated perfused rabbit heart model of ischemia. They observed that in hearts made globally ischemic for two hours, administration of these enzymes significantly improved left ventr icular recovery when compared to hearts not receiving enzyme treatment. Werns and coworkers 16 studied the effects of SOD and CAT separately during regional myocardial ischemia and reperfusion in the dog. These investigators found that infarct size was significantly reduced by SOD but not by CAT This suggests that 02, but not H202, may play a role in myocardial injury due to ischemia and reperfusion.

Other evidence that oxygen free radicals play a role in ischemia/reperfusion injury was provided by Stewart and associates, lz This group used a cardioplegic solution containing SOD and the hydroxyl radical scavenger mannitoL This was administered during 60 minutes of hypothermic global ischemia in canine hearts, followed by 45 minutes of reperfusion. All parameters of left ventricular function were significantly better in the enzyme- treated hearts in comparison to the non-enzyme- t rea ted controls~ In a study by Casale, 18 myocardial tissue


injury was determined by microscopic examination of cellular and subcellu- lar structures. Protection was demonstrated in isolated rabbit hearts treated with SOD and CAT either just prior to reflow or at the onset of ischemia.

Further evidence implicating oxygen radicals in myocardial ischemia/ reperfusion injury was provided by Mitsos and associates, 19 who demonstrated a reduction in infarct size in dogs receiving N-2-mercaptopropionyl glycine, a free-radical scavenger, during regional myocardial ischemia followed by reperfusion. A similar reduction in infarct size was observed by Horneffler and colleagues 20 when reperfusion of ischemic pig hearts was accompanied by early administration of SOD and CAT Investigators also have observed tissue-protective effects of allopurinol in ischemia/reperfusion models.21, 22 Al lopur ino l i nh ib i t s xanthine oxidase. 23 During ischemia, xanthine dehydrogenase is converted to xanthine oxidase. 24 Xanthine oxidase uses molecular oxygen as an elec- tron acceptor, thus forming a superoxide rad ica l for each m o l e c u l e of hypoxanthine metabolized. The conversion of xanthine dehydrogenase to xanthine oxidase and the accumula- tion of hypoxanthine during ischemia 2a therefore results in the production of 0 2 - by this enzyme during reperfusion. Allopurinol, by inhibiting xanthine oxidase, then would prevent the generation of superoxide radicals.

In human subjects reperfusion injury is implicated by observations in the early postreperfusion phase in patients who have undergone in t racoronary thrombolysis for evolving myocardial infarctions. The evolution of electro- cardiographic signs of necrosis in such patients is clearly reperfusion dependent. zs-29 Reperfusion dramat ica l ly accelerates the evolution of ECG signs of t ransmural myocardial infarction and is associated with accelerated and increased release of CPK. 26 Some authors suggest this is a "washout" phe- nomenon. 26 However, increased CPK release during reperfusion is inhibited by allopurinol and desferoxamine. 3°

It seems clear that during global or regional myocardial ischemia, the bio- chemical milieu wi thin myocardial cells must be altered in such a way as to become favorable for injurious bio- chemical reactions to ensue on reperfusion and reoxygenation.

Oxygen-radical-mediated lipid peroxidation is a process that disrupts

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IRON DELOCALIZATION Holt et al

cellular and organelle membranes and ultimately leads to cell necrosis. Per- oxidation of phospholipid membranes takes place by a chain reaction mecha- nism37 The first step is an initiation reaction in which a reactive oxygen species removes a divinyl hydrogen atom from a polyunsaturated fatty acid (PUFA), thereby forming a lipid alkyl free radical that rearranges to a conjugated diene structure. This is followed by propagation that proceeds by a two-step process. The new lipid alkyl free radical reacts with 0 2 to form a highly reactive peroxyl radical:

L* + 0 2 ........ LOO* Then this lipid peroxyl radical can

attack a divinyl hydrogen on an adja- cent PUFA, forming a lipid hydro- peroxide and a new lipid alkyl free radical as follows:

LOO* + LH ........ L* + LOOH Lipid hydroperoxides break down to

f ragmenta t ion products including MDA, ethane, and pentane. The process proceeds as a chain reaction in the phospholipid membrane until two lipid radicals react with each other to form stable products or until "scavenger" molecules such as vitamin E or glutathione reduce them to nonreac- tive species. Note that 0 2 is inti- mately associated with propagation; this may well explain the seemingly paradoxical acceleration of tissue injury during reperfusion.

It is the initiation reaction, ie, the abstract ion of a divinyl hydrogen atom from a PUFA, that is the rate- limiting step in this membrane injury process. 7 From the perspective of the potential for membrane protective therapy, this initiation step invites special a t tent ion. Ini t ial ly it was thought that superoxide anion or hydroxyl radical was responsible for ini- tiating the peroxidation chain. 3 How- ever, thermodynamic studies have shown that superoxide anion has in- sufficient reactivity to initiate lipid peroxidation,3, 4 and there is now strong evidence that hydroxyl radical also is not the species involved in initiation of lipid peroxidation. 4 Rather, the presence of a transitional metal catalyst such as iron is required to produce species that can initiate and promote lipid peroxidation. 4 LMWS ferrous iron, such as the ADP-Fe2+ complex, readily reacts with oxygen to generate active oxidation species. Such iron-oxygen complexes can initiate lipid peroxidation directly.

In vitro studies of the lipid perox-

idation process demonstrate clearly that the addition of ferrous iron to the reaction medium dramatically accelerates peroxidation. 4 In normal tissue, the concentration of free or LMWS iron capable of catalyzing peroxidation is minuscu le or perhaps nonexis- tent. 31 In fact, the sequestration of re- dox active metals (such as iron) in un- reactive states such as ferritin may be one of the major cellular protective mechanisms against oxidative cytoly- sis. Therefore, the hypothesis that myocardial ischemia results in condi- tions favorable for peroxidative membrane injury to proceed during reperfusion requires that two critical ques- tions be answered: Can ferrous iron become available during myocardial ischemia to catalyze lipid peroxidation; and can products of lipid peroxidation be found in myocardial tissue postischemia?

Our study demonstrated that there is indeed an increase in iron available in LMWS in myocardium following two hours of regional ischemia. This is accompanied by a small but significant rise in tissue MDA. While this study made no attempt to identify the source of this "delocalized" iron, in vi tro studies suggest that a likely source is the release of iron from ferritin. Iron is ubiqui tous in mam- malian tissue, and the heart contains 96 -+ 35 uM iron/g tissue ash. 32 The basic mechanism for the release of iron from ferritin requires that the storage form of insoluble ferric (Fe3 + ) iron be reduced to the ferrous (Fe2+) state. 4 The development of a strong reducing and acidotic environment during severe incomplete ischemia would favor such a reduction of ferric iron and result in increasing cellular concentrations of LMWS iron.

An interesting finding recently reported by Thomas and associates 33 is the ability of superoxide anion to me- diate the reductive release of iron from ferritin. This finding, coupled with the fact that superoxide pos- sesses relatively little reactivity to- ward most organic compounds and in- sufficient reactivity to directly initiate lipid peroxidation, suggests a novel role for superoxide in promot ing tissue injury, that is, the reductive release of iron from ferritin. This then provides an alternative explanation for the deleterious effects of superoxide and the apparent protective effects of superoxide dismutase when administered to ischemic myocardium prior

to or during reperfusion. Because of the nature of the bio-

chemical reactions in the peroxidation chain and the difficulty of measuring the unstable intermediates, it is hard to assess directly the occurrence of peroxidation. Evidence that such reactions are of importance is implied in the studies we have reviewed, where. by inactivation or scavenging of the reactive oxygen intermediates affords tissue protection. Other studies have demonst ra ted signif icant rises in tissue MDA levels following reperfw sion of ischemic tissues. Komara and colleagues 28 demonstrated a three-fold increase in LMWS iron associated with a 30% increase in tissue MDA and increased conjugated dienes in the brains of dogs two hours after reperfusion following a 15-minute cardiac arrest. Slightly increased levels of MDA and unusual iron ligands 34 have been reported in left ventricular tissue after 45 minutes of ischemia.

Our study demonstrated a small but statistically significant rise in tissue MDA in the regional ischemic zone following two hours of coronary artery ligation. We believe that the residual 10% to 15% perfusion by collateral circulation into the ischemic zone s promotes the reductive release of iron and is responsible for this low-grade peroxidation process. While not examined in our study, reperfusion of previously ischemic myocardium results in increases in tissue MDA by 70%.35

The finding of iron delocalization in regional myocardial ischemia associated with evidence for peroxidative membrane injury demands that further studies be performed to examine the potential tissue protective effects of iron chelators during reperfusion. Desferoxamine is a clinically available pharmaceutical with a history as a very safe drug. Iron bound in the feri- oxamine complex becomes chem- ically inert and iron-dependent lipid peroxidation cannot occur in the presence of stoichiometrically adequate amounts of desferoxamine. 36 Komara and colleagues 37 have shown that increases in brain MDA levels during postischemic reperfusion are amelio- rated by treatment with desferoxamine. Myers and coworkers 3o showed that administration of desferoxamine to isolated rabbit hearts during ischemia suppressed the creatinine kinase released induced by reperfusion and improved selected parameters of myocardial function.

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C O N C L U S I O N Our s tudy d e m o n s t r a t e d in vivo

that regional myocardial ischemia results in the delocalization of normal iron stores into a LMWS iron pool. In vitro s tudies have shown that l ipid peroxidation reactions with superoxide are thermodynamica l ly unfavorable unless a transitional metal catalyst such as iron becomes available.

We conclude that two hours of regional myocardial ischemia results in the a v a i l a b i l i t y of low m o l e c u l a r weight forms of iron that can promote the in i t ia t ion of phospholipid peroxidation. This delocalization of iron is associated with the production of malondialdehyde, a product of lipid peroxidation. Therapeutic iron-chelating agents such as desferoxamine should be studied for tissue protection during myocardial ischemia and reperfusion.

The authors express special appreciation to Ms Amanda K Stressman for her as- sistance in the preparation of this man- uscript.

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