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D A Mickle

Publications and source records attributed to D A Mickle.

At least 19 recordsLinked to original sources
PubMed1992

Analysis of the upstream regulatory region of human ventricular myosin light chain 1 gene.

To explore the mechanisms regulating expression of ventricular myosin light chain 1, the human gene including 5'-flanking DNA was cloned and characterized by Southern blot and restriction mapping. A 2 kb 5'-flanking DNA was sequenced and linked to a chloramphenicol acetyltransferase reporter gene. The constructs then were transfected into cultured human and rat cardiomyocytes as well as rat aortic endothelial cells. Deletion analysis of constructs revealed that the basal promoter sequences, which were located within 62 base pairs of the cap site, could direct high levels of chloramphenicol acetyltransferase gene expression in the cardiomyocytes and endothelial cells. The region between -62 to -312 base pairs strongly repressed the chloramphenicol acetyltransferase gene expression in the cardiomyocytes and endothelial cells. Positive elements were found between -312 and -2000 base pairs of the cap site. These results are indicative, among other possibilities, that the human ventricular myosin light chain 1 gene is turned on in cardiomyocytes by the presence of trans-acting factors that are bound to upstream positive elements and is turned off in non-muscle cells by the presence of repressor-binding proteins. But this mechanism remains to be established.

Amino Acid Sequence↗
PubMed1992

The regulation of glutathione peroxidase gene expression by oxygen tension in cultured human cardiomyocytes.

In earlier studies we have shown that the activity of the antioxidant enzyme glutathione peroxidase is regulated by oxygen tension in cultured tetralogy of Fallot (TOF) ventricular myocytes and in the ventricles of TOF patients having corrective cardiac surgery. The present study was undertaken to determine the mechanism of this regulation. Northern and slot blot analysis was performed using RNA isolated from TOF myocytes cultured at oxygen tensions of 150 and 40 mmHg for 3, 7, 14, 21, and 28 days. As was found for enzyme activities, glutathione peroxidase mRNA levels were lower in the cells cultured at a pO2 of 40 mmHg than at 150 mmHg and could be elevated with an increase in oxygen tension. These results were standardized against house-keeping gene hexosaminidase B which showed no difference in mRNA levels between the two oxygen tensions throughout the time course. Nuclear run-off assays indicated that glutathione peroxidase was regulated by oxygen tension at the transcriptional level, while hexosaminidase B and total mRNA synthesis levels remained unchanged.

Adult↗
PubMed1992

Prolonged hypothermic cardiac storage for transplantation. The effects on myocardial metabolism and mitochondrial function.

Cardiac storage for transplantation is currently limited to 6 hours. To better understand the metabolic changes that occur during hypothermic (4 degrees C) storage, we monitored the morphologic and metabolic changes in the canine myocardium at 0, 12, and 24 hours of storage in University of Wisconsin solution. Attempts to isolate cardiac mitochondria resulted in a progressive decline in the yield (milligrams of mitochondria per gram of heart tissue), which decreased (p less than 0.05) from 9.2 +/- 0.4 at 0 hours (control) to 4.0 +/- 0.3 after 12 hours and further decreased (p less than 0.05) to 1.9 +/- 0.2 after 24 hours of cold storage. Mitochondrial state 3 respiration fell to 64% of control after 12 hours and 28% of control after 24 hours of cold storage (p less than 0.05). Citrate synthetase activity, but not cytochrome C oxidase activity, was significantly depressed after 12 and 24 hours of cold storage. Adenosine triphosphate content decreased to 67% of control after 12 hours and 50% of control after 24 hours. After 12 hours of storage, sufficient adenosine diphosphate and monophosphate were present to permit some restoration of adenosine triphosphate, provided mitochondrial function was normal after transplantation. However, restoration of mitochondrial function and adenosine triphosphate levels sufficient to support myocardial contractility was unlikely after 24 hours of storage. This study suggests that a return of adequate cardiac function after transplantation may be possible after 12 hours of cold storage in University of Wisconsin solution but not after 24 hours of cold storage.

Adenosine Triphosphate↗
PubMed1992

In vitro assessment of the effects of glucose added to the University of Wisconsin solution on myocyte preservation.

BACKGROUND: University of Wisconsin solution (UWS) has been successfully used for liver transplantation and may be beneficial for hypothermic cardiac storage. The addition of glucose may enhance myocardial preservation. METHODS AND RESULTS: Cultured human ventricular myocytes (eight dishes per group) were stored at 0 degree C for 12 hours in either unmodified UWS or UWS with glucose (1, 3, 10, 30, or 100 mmol/l). Cells were assayed for protein by spectrofluorometry and adenine nucleotides by high performance liquid chromatography after storage. Protein recovery, adenosine triphosphate (ATP), adenosine diphosphate (ADP), and total adenine nucleotides (ATP+ADP+AMP) were all depleted after storage (p < 0.0001 by ANOVA). Protein recovery (p < 0.005), ATP (p < 0.05), and ADP (p < 0.05) were increased with glucose administration compared with unmodified UWS. Improvement was maximal using 30 mmol/l (protein, 0 mmol/l = 0.48 +/- 0.14 and 30 mmol/l = 0.65 +/- 0.11 mg per dish; ATP, 0 mmol/l = 3.08 +/- 0.63 and 30 mmol/l = 4.32 +/- 0.90 nmol/mg protein; ADP, 0 mmol/l = 3.76 +/- 0.80 and 30 mmol/l = 4.63 +/- 0.38 nmol/mg protein, mean +/- SD). Total adenine nucleotides tended to increase at any glucose concentration (p = 0.07 by ANOVA) and were significantly better with 30 mmol/l glucose (0 mmol/l = 7.61 +/- 1.58 and 30 mmol/l = 9.62 +/- 1.08 nmol/mg protein). CONCLUSIONS: Increasing the glucose concentration from 0 to 30 mmol/l improved adenine nucleotide and cellular protein preservation in this in vitro assessment.

Adenine Nucleotides↗
PubMed1992

Alternative techniques of cardioplegia.

BACKGROUND: Although normothermic cardioplegia has been used with acceptable clinical results, no studies have previously been performed to determine the metabolic consequences of these various techniques of myocardial protection. Therefore, we have performed a randomized clinical trial to assess the effects of three cardioplegic techniques on myocardial metabolic recovery. METHODS AND RESULTS: Seventy-four patients undergoing coronary artery bypass graft surgery were randomized to receive normothermic antegrade blood cardioplegia (n = 25), normothermic retrograde blood cardioplegia (n = 23), or intermittent cold antegrade blood cardioplegia (n = 26). Myocardial oxygen consumption and lactate production, adenine nucleotides, and adenine nucleotide degradation products were measured during the operation, and cardiac creatine kinase isoenzyme (CK-MB) release was assessed after surgery. Warm antegrade cardioplegia maximized myocardial oxygen consumption during cardioplegic delivery. Postoperative CK-MB release was less after warm antegrade cardioplegia, but the difference was not statistically significant. Warm retrograde cardioplegia resulted in the greatest degree of anaerobic lactate production but did not increase morbidity and mortality. Perioperative myocardial infarctions and postoperative low-output syndrome were most common after cold cardioplegia, but this trend was not statistically significant. During warm antegrade cardioplegia, adenosine triphosphate (ATP) was metabolized to diffusible precursors, which were washed out during cardioplegic infusion. Warm retrograde cardioplegia produced a breakdown of ATP to inosine and hypoxanthine, small molecules that accumulated during the cross-clamp period and were not washed out, perhaps because of inadequate perfusion with retrograde delivery. During cold cardioplegia, ATP was dephosphorylated, and adenosine diphosphate, adenosine monophosphate, and adenosine accumulated. These compounds were not regenerated to ATP but were not washed out of myocytes because they are large anionic molecules. CONCLUSIONS: Intermittent cold cardioplegia inhibited mitochondrial function but prevented the degradation of adenine nucleotides. Warm antegrade cardioplegia had the greatest myocardial oxygen consumption, and warm retrograde cardioplegia had the greatest anaerobic lactate production. There were no differences in clinical outcomes between cardioplegic groups.

Adenosine Triphosphate↗
PubMed1992

Effect of oxygen tension and cardiovascular operations on the myocardial antioxidant enzyme activities in patients with tetralogy of Fallot and aorta-coronary bypass.

Since the chronically cyanotic myocardium appears to be more susceptible to reperfusion injury after cardiac operations than the noncyanotic myocardium, we studied the association between the preoperative arterial oxygen tension and the myocardial superoxide dismutase, catalase, and glutathione peroxidase activities. Fourteen patients with tetralogy of Fallot scheduled for elective operations had baseline arterial blood gas measurements done before operation. During the operation right ventricular biopsy specimens were taken for enzyme analysis immediately before cold blood cardioplegic arrest and 20 minutes after crossclamp removal. The tissue antioxidant enzyme activities of the patients with tetralogy of Fallot were compared with the myocardial results in 15 adults with stable angina pectoris having elective aorta-coronary artery bypass graft operations. Myocardial tissues removed from two patients with hypertrophic obstructive cardiomyopathy who had corrective operations were analyzed for antioxidant activities. There were no changes in myocardial antioxidant enzyme activities during the operation in the patients with tetralogy of Fallot and coronary artery bypass graft. The myocardial superoxide dismutase, catalase, and glutathione peroxidase activities correlated (0.82, 0.68, and 0.89, respectively) significantly (p values were less than 0.01, 0.05, and 0.01, respectively) with the preoperative arterial oxygen tensions in the patients with tetralogy of Fallot. The myocardial glutathione peroxidase activities were at least four times higher in the myocardium of patients with coronary artery bypass graft and hypertrophic obstructive cardiomyopathy than in that of those with tetralogy of Fallot. This study provides putative evidence that the myocardium of patients with tetralogy of Fallot is a risk of oxygen-derived free radical injury during and immediately after corrective cardiovascular operations.

Catalase↗
PubMed1991

Effect of orally administered alpha-tocopheryl acetate on human myocardial alpha-tocopherol levels.

Free radical injury may contribute to the delayed postoperative recovery of myocardial metabolism and ventricular function after elective coronary artery revascularization. This clinical study was designed to evaluate, in stable angina patients having aortocoronary bypass surgery, whether orally administered alpha-tocopheryl acetate was effective in increasing myocardial alpha-tocopherol levels and the effect of cardioplegic arrest followed by reperfusion on the myocardial alpha-tocopherol levels. Twenty-four patients with stable angina pectoris for elective revascularization received preoperatively the natural stereoisomer of alpha-tocopheryl acetate labelled with deuterium (D3) and six patients were used as controls. Since four patients who received 300 mg of D3-alpha-tocopheryl acetate preoperatively for 1 and 2 days did not have significant increases in their myocardial total or D3-tocopherol levels, the remaining 20 patients received 100 mg (n = 6), 300 mg (n = 8), or 900 mg (n = 6) of D3-alpha-tocopheryl acetate for 14 consecutive preoperative days. The left ventricular deuterated and nondeuterated alpha-tocopherol levels were measured by gas chromatography/mass spectrometry. Although there was a decrease (p less than 0.05) in myocardial alpha-tocopherol levels with the onset of reperfusion (cross-clamp removal), the myocardial tocopherol levels were not statistically different from preoperative levels by 20 minutes of reperfusion. At least 300 mg of alpha-tocopherol must be taken orally for 14 consecutive days to double the myocardial alpha-tocopherol levels.

Administration, Oral↗
PubMed1991

The limits of cardiac preservation with University of Wisconsin solution.

Previous studies from this institution have suggested that University of Wisconsin solution is preferred for prolonged cardiac storage and preserves high-energy phosphates better than other storage fluids. University of Wisconsin solution contains adenosine (5 mmol/L), which may maintain the concentration of myocardial adenine nucleotides. Cultures of human adult myocytes were grown from left ventricular biopsy specimens obtained from patients undergoing coronary bypass procedures. Cells (seven to nine dishes per group) were rinsed of culture medium and stored at 0 degrees C in University of Wisconsin solution. Cells were analyzed for adenine nucleotide content after 1, 6, 12, and 24 hours of storage by high-performance liquid chromatography (units = nmol/microgram DNA) and compared with control samples (0 hour). Adenosine concentration increased from 0.03 +/- 0.02 (mean +/- standard deviation) to 1.77 +/- 1.03 by 1 hour (p less than 0.0001, analysis of variance) and remained increased thereafter. Adenosine was largely degraded to inosine (0 hours, 0.03 +/- 0.03; 6 hours, 0.88 +/- 0.56; p less than 0.001) and hypoxanthine (0 hours, 0.01 +/- 0.01; 6 hours, 0.15 +/- 0.09; p = 0.004). Measured levels of xanthine and uric acid were extremely low at all time intervals. Adenosine triphosphate levels were maintained at 1 hour (0 hours, 0.64 +/- 0.38; 1 hour, 0.67 +/- 0.45) but declined thereafter (6 hours, 0.21 +/- 0.21; 12 hours, 0.11 +/- 0.09; 24 hours, 0.04 +/- 0.03; p less than 0.0001). Levels of adenosine diphosphate (p = 0.007) and adenosine monophosphate (p less than 0.05) decreased to approximately 25% of original values by 24 hours.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenine Nucleotides↗
PubMed1991

Radiochemical quantitation of conjugated dienes during ischemia and reperfusion in the rat liver.

Conjugated dienes (CD) are putative chemical imprints of oxyradical damage. Employing a highly selective assay for dienes based on their condensation with 14C-tetracyanoethylene (14C-TCNE), and a 14C-TCNE reagent with 60 times higher specific radioactivity than that used by Waller and Recknagel (1978), we have described the profile of phospholipid CD during global ischemia and reperfusion of the rat liver. During 70 min of ischemia, the hepatic CD appeared to increase moderately, but not statistically significantly, relative to that in sham-operated controls (with mean CD = 0.0397 +/- 0.0040 nmoles CD/nmole phosphate, for n = 7). In subsequent reperfusion, CD increased 2.6-3.3 fold higher than in sham controls during the first 10-15 min, declining thereafter to ischemia-like levels by 30 min of reflow. Our data demonstrate that oxyradicals are generated mostly during reperfusion of the post-ischemic rat liver, and that the refined TCNE method can quantitate tissue CD sensitively and relatively specifically.

Alkenes↗
PubMed1991

Prolonged preservation with University of Wisconsin Solution.

Previous studies from this institution using human cell cultures have suggested that University of Wisconsin Solution may be preferred for prolonged cardiac storage. University of Wisconsin Solution (UWS) contains adenosine (5 mmole/liter) which could maintain adenine nucleotides better than other storage fluids. Human cardiomyocytes were isolated from left ventricular biopsies. Cells (seven to nine dishes/group) were rinsed of culture media and placed in one of four solutions: Stanford cardioplegia, phosphate-buffered saline, modified EuroCollins', or UWS. Metabolites were assessed using high-performance liquid chromatography (units = nmole/micrograms DNA) after 24 hr of storage at 0 degrees C and compared to baseline controls (BASE). Adenosine triphosphate (P less than 0.0001, ANOVA), adenosine diphosphate (P less than 0.0001), and adenosine monophosphate (P less than 0.01) decreased with each solution compared to BASE but were maintained best with UWS (P less than 0.05). Adenosine increased in the UWS cells only (BASE, 0.029 +/- 0.118; UWS, 1.836 +/- 1.110; P less than 0.0001, ANOVA). Adenosine in the UWS cells was largely degraded to inosine (UWS, 1.013 +/- 0.779; BASE, 0.034 +/- 0.032; P less than 0.0001) and hypoxanthine (UWS, 0.124 +/- 0.091; BASE, 0.005 +/- 0.005; P less than 0.001). University of Wisconsin Solution does preserve adenine nucleotides better than other storage fluids and may improve the clinical results of cardiac transplantation.

Adenine Nucleotides↗
PubMed1991

Optimal delivery of blood cardioplegia.

A prospective randomized controlled trial was performed to determine optimal flow rates and hemoglobin concentrations for continuous normothermic blood cardioplegia and to compare warm heart surgery with standard intermittent cold blood cardioplegia. Thirty-five patients received intermittent cold blood cardioplegia, low hemoglobin low flow, low hemoglobin high flow, high hemoglobin low flow, or high hemoglobin high flow warm blood cardioplegia (seven patients per group: low hemoglobin, 50 g/l; high hemoglobin, 80 g/l; low flow, less than 80 ml/min; high flow, greater than 80 ml/min). Hypothermia resulted in a significantly greater accumulation of ADP and AMP during cross clamp, consistent with impaired mitochondrial function. Low hemoglobin low flow warm blood cardioplegia increased myocardial oxygen consumption and coronary sinus blood flow after cross clamp release, and also decreased lactate consumption. Postoperative myocardial performance and diastolic compliance were reduced in low hemoglobin low flow warm patients, and diastolic compliance was increased with high hemoglobin high flow warm blood cardioplegia when compared with cold patients. In this study, continuous normothermic cardioplegia was safe when delivered at 80 ml/min or greater, with a hemoglobin concentration of at least 80 g/l, affording myocardial metabolic and functional recovery comparable to that found after intermittent cold blood cardioplegia.

Blood↗
PubMed1991

Prolonged hypothermic cardiac storage with University of Wisconsin solution. An assessment with human cell cultures.

Hypothermic storage of cardiac allografts is routinely used for transplantation but is associated with an increased mortality when ischemic times are greater than 4 hours. The ideal storage conditions (solution and temperature) could extend the current limits of cold ischemia. Human endothelial cells and ventricular myocytes were studied to screen various solutions and temperatures for organ preservation. Four solutions (modified Euro-Collins, phosphate-buffered saline, Stanford cardioplegia, and University of Wisconsin) were evaluated. Endothelial cells were evaluated after prolonged hypothermic storage consisting of 0 degree, 4 degrees, and 8 degrees C for 36 hours, and ventricular myocytes were stored at 0 degree and 8 degrees C for 24 hours. Cell viability was determined by morphology (10 dishes per group), and trypan blue exclusion (5 dishes per group) in addition to a cell adhesion assay (endothelial cells 5 dishes per group) and adenine nucleotide analysis with high-performance liquid chromatography techniques (ventricular myocytes 5 dishes per group). Endothelial cell morphology was best preserved by University of Wisconsin solution (p less than 0.001, chi 2) and at 0 degree C (p less than 0.01, chi 2). Endothelial cells stored with University of Wisconsin solution excluded trypan blue better (1.0% +/- 0.5% cells stained, p less than 0.001. Analysis of variance [ANOVA]). Cell adhesion was poorly protected with Stanford cardioplegia (p less than 0.001, ANOVA). Myocyte morphology was preserved best with University of Wisconsin solution at 0 degree C (p less than 0.001, chi 2). According to trypan blue staining, Euro-Collins and University of Wisconsin solutions were superior to Stanford cardioplegia or phosphate-buffered solutions (p less than 0.001, ANOVA). Temperature did not influence the trypan blue results. Adenosine triphosphate was maintained best with University of Wisconsin solution at 0 degree C (p less than 0.01, ANOVA). Myocytes were more sensitive to the effects of prolonged storage compared with endothelial cells by morphologic criteria and trypan blue staining characteristics, irrespective of the shorter preservation times. University of Wisconsin solution was the most effective solution tested. Colder temperatures (0 degree to 4 degrees C) provided better protection than 8 degrees C. Myocytes were more sensitive to prolonged preservation than endothelial cells. Furthermore, the technique used appears helpful as a model of prolonged hypothermic storage and could be expanded to assess other interventions.

Adenosine Diphosphate↗
PubMed1991

Trolox protects rat hepatocytes against oxyradical damage and the ischemic rat liver from reperfusion injury.

Trolox, a hydrophilic analog of vitamin E, was reported to scavenge peroxyl radicals from artificial systems better than its parent compound. Here we examined the possible cytoprotective effect of Trolox in cultured hepatocytes and in the rat liver. In cultured rat hepatocytes, 0.5 to 16 mmol/L Trolox (with optimum between 1 to 2 mmol/L) was observed to prolong the survival of cells exposed to oxyradicals generated with xanthine oxidase-hypoxanthine. The protection by 1 mmol/L Trolox surpassed that provided by either ascorbate, mannitol, superoxide dismutase and/or catalase--each at a level giving its maximal protection in the same system. In both a global and partial model of hepatic ischemia-reperfusion in rats, infusion of Trolox (7.5 to 10 mumol/kg body weight) just before reflow reduced by greater than 80% the liver necrosis sustained in untreated (no Trolox) control rats. Such organ salvage was apparently accompanied by approximately 50% reduction in the amount of hepatic conjugated dienes, which were quantified by a highly specific radiochemical assay. Since conjugated dienes are presumed to be good "markers" of oxyradical damage, our data may have provided a semiquantitative link between free radical-induced necrosis and its chemical imprint in vivo. The data also indicated a relatively rapid and potent antioxidant-like action by Trolox on rat hepatocytes and on the postischemic reperfused rat liver.

Animals↗
PubMed1990

Water-soluble antioxidant specificity against free radical injury using cultured human ventricular myocytes and fibroblasts and saphenous vein endothelial cells.

To better understand the protective effect of water-soluble antioxidants against free radical injury to the reperfused ischemic myocardium, we studied the antioxidant effectiveness of superoxide dismutase (SOD), catalase, ascorbic acid, and Trolox, a water-soluble analogue of alpha-tocopherol, in protecting cultured adult human ventricular myocytes and fibroblasts and saphenous vein endothelial cells from hypoxanthine-xanthine oxidase generated free radicals. The cells were cultured at oxygen tension to 150 and 40 mmHg. Passage P2 to P4 cells were injured by a hypoxanthine-xanthine oxidase free radical generation system. The time when all the cells became shriveled divided by the cell count expressed in terms of 100,000 cells was used to compare cellular susceptibilities to free radical injury and the relative effectiveness of the antioxidants. Fibroblasts were more resistant to free radical injury than myocytes which were more resistant than endothelial cells, when all three cell types were cultured at the same oxygen tension. Trolox and ascorbic acid were effective antioxidants for myocytes while SOD and catalase were ineffective. SOD and catalase were more effective than ascorbic acid as antioxidants for endothelial cells and fibroblasts, while Trolox was ineffective. In summary, we have shown that each cultured cell type has a different susceptibility to free radical damage and that antioxidants are not effective for all cell types.

Antioxidants↗
PubMed1990

European versus North American cardioplegia: comparison of Bretschneider's and Roe's cardioplegic solutions in a canine model of cardiopulmonary bypass.

Roe's and Bretschneider's crystalloid cardioplegic solutions were compared in a canine model of total cardiopulmonary bypass with 4.5 hours of hypothermic (27 degrees C) ischemic arrest and 60 minutes of reperfusion. Bretschneider's solution (Group I, six dogs) preserved tissue adenosine triphosphate (ATP) near control levels and maintained coronary effluent pH near 7.0 throughout the ischemic interval, while Roe's solution (Group II, six dogs) allowed progressive acidosis and depletion of ATP (P less than 0.005 versus control). Group I had supranormal left ventricular function during reperfusion (greater than 100% of pre-arrest function) but Group II regained only 40-75% of pre-arrest function. Group I had 2.82% +/- 3.61% necrosis of heart mass and Group II 9.33% +/- 8.26 (P less than 0.10). We conclude that Bretschneider's solution provided better myocardial protection than Roe's solution. The development of acidosis in the Roe group suggests that the more effective buffering of Bretschneider's solution with histidine is the probable basis for its superiority.

Animals↗
PubMed1990

The cytoprotective effect of Trolox demonstrated with three types of human cells.

Trolox, a hydrophilic analogue of alpha-tocopherol, was reported to scavenge peroxyl radicals better than vitamin E in sodium dodecyl sulfate micelles and in liposomes. However, it was not known if Trolox protects human cells against oxyradical damage or if it acts as an antioxidant there. Here we demonstrate that Trolox prolonged substantially the survival of human ventricular myocytes and hepatocyte against oxyradicals generated with xanthine oxidase plus hypoxanthine, and prevented lysis of red cells exposed to an azo-initiator (2,2'-azo-bis(2-amidinopropane) HCl). Note that Trolox did not inhibit xanthine oxidase. In each cell type, the protection by Trolox was dose dependent and surpassed those given by such water-soluble antioxidants as ascorbic acid, superoxide dismutase, and (or) catalase, each examined at or near its optimal level in the same system. Using hepatocytes as a model, we further observed that Trolox reduced markedly the quantity of phospholipid conjugated dienes (a chemical imprint of oxyradical damage) in cells despite their exposure to oxyradicals. These data suggested that Trolox behaves as an antioxidant in cells as illustrated in hepatocytes.

Antioxidants↗
PubMed1990

Subcellular distribution of peroxidized lipids in myocardial reperfusion injury.

Previous studies in our laboratory have demonstrated the peroxidation of myocardial phospholipid in a canine model of reversible global normothermic ischemia and reperfusion while on cardiopulmonary bypass. The present study examines the distribution of phospholipid peroxidation products in three major cellular organelle fractions of myocardium prepared by established centrifugal fractionation procedures (sarcolemma, sarcoplasmic reticulum, and mitochondria). These organelles were isolated from control (nonischemic) and ischemic-reperfused myocardium harvested during early reperfusion (5 min), when previous studies indicated maximal peroxidative injury in whole myocardial biopsies. Utilizing a more rapid analytic procedure for measuring phospholipid containing the conjugated diene chromophore in the polyunsaturated fatty acyl substituents, we were able to establish the fidelity of this procedure by comparing the results obtained with it to the previous more laborious analytic procedure (involving phospholipid hydrolysis with phospholipase A2 and subsequent derivatization for high-pressure liquid chromatography followed by gas chromatographic-mass spectrometric analysis). Analysis of phospholipid extracts from organelle fractions for evidence of peroxidative conjugated diene formation revealed that sarcolemmal membranes had the highest content of oxidized phospholipid containing the conjugated diene chromophore (mean 2.2 +/- 1.2 nmol phospholipid-conjugated diene/mumol phospholipid phosphorus, P less than 0.02 compared with control). Both sarcoplasmic reticulum and mitochondrial membranes were also peroxidized but to a much smaller extent (mean 0.4 +/- 0.2 and 0.3 +/- 0.25 nmol phospholipid conjugated diene/mumol phospholipid phosphorus).

Animals↗