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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).

Prolonged ischemia to skeletal muscle as occurs after an acute arterial occlusion results in alterations in adenine nucleotide metabolism. Adenosine triphosphate continues to be used for cellular functions, and an ischemia-induced degradation of phosphorylated adenine nucleotides is initiated. In this experiment we demonstrated the time-dependent aspect of adenine nucleotide depletion during ischemia and the production of large quantities of soluble precursors. In addition, we studied the rate of conversion of xanthine dehydrogenase to xanthine oxidase, a potential source of oxygen-free radicals, after controlled periods of total normothermic ischemia (4 hours and 5 hours) and during the reperfusion phase. During ischemia complete depletion of creatine phosphate occurred in both groups, and adenosine triphosphate fell from 22.1 +/- 1.3 to 10.3 +/- 1.4 mumol/gm dry weight after 4 hours and from 21.6 +/- 0.7 to 3.9 +/- 0.8 mumol/gm dry weight after 5 hours (p less than 0.05). During reperfusion, creatine phosphokinase resynthesis occurred in both groups, but adenosine triphosphate levels were not significantly increased (p greater than 0.05). A washout of lipid soluble products of adenine nucleotide metabolism occurred equally in both groups. The relationship between phosphorylated adenine nucleotides as measured by the energy charge potential fell significantly in both groups (p less than 0.05), but after the shorter period of ischemia (4 hours it returned to normal during early reperfusion but did not after 5 hours of ischemia. There was 21% +/- 4% necrosis after 4 hours and 51% +/- 8% after 5 hours of ischemic stress when assessed at 48 hours. In conclusion, the degree of adenine nucleotide degeneration as determined primarily by the length of the ischemic period, may be the most important determinant of the ultimate extent of skeletal muscle ischemic necrosis that results from an acute interruption of circulation.

The lower extremity may be exposed to prolonged periods of ischemia, resulting in depletion of intracellular energy stores in the affected skeletal muscle. The role of adenine nucleotide reduction and failure of resynthesis on reperfusion in determining the extent of muscle necrosis was investigated in this study, in addition to the possible beneficial effects of the addition of exogenous adenosine triphosphate-magnesium chloride during early reperfusion. The isolated paired canine gracilis muscle model was used. After 4 hours of normothermic ischemia in group I, a perfusate Krebs-Henseleit solution plus the gradual reintroduction of oxygenated blood flow was compared to standard reperfusion. In group II, a similar infusion protocol was used, with the addition of 2 mmol/L adenosine triphosphate-magnesium chloride and compared to normal reperfusion. Adenosine triphosphate-magnesium chloride resulted in the salvage of skeletal muscle, 57% +/- 12% versus 44% +/- 14% (p less than 0.05, n = 6 pairs). Reperfusion with the solution alone increased the resulting necrosis (42% +/- 13% vs 60% +/- 20%, n = 6 pairs). Adenine nucleotide stores were not increased, but oxygen consumption was increased by magnesium chloride-adenosine triphosphate (p less than 0.05, analysis of variance [ANOVA]). A clear relationship was demonstrated between the fall in energy stores, as measured by a change in energy charge potential from preischemia to end ischemia levels, and the extent of resulting necrosis (p less than 0.01). In summary, the addition of 2 mmol/L to an infusion of Krebs-Henseleit solution during reperfusion results in significant salvage of skeletal muscle.(ABSTRACT TRUNCATED AT 250 WORDS)

The production and prevention of calcium paradox injury in myocardium was studied in a canine model of cardiopulmonary bypass with multidose, moderately hypothermic, crystalloid cardioplegic solution. During 4 1/2 hours of global ischemia, three groups of six dogs each received one of three histidine-buffered cardioplegic solutions (500 ml initially and 250 ml every 30 minutes) at 27 degrees C. Group 1 cardioplegic solution was calcium free, group 2 solution contained a trace amount of calcium chloride (70 mumols /L), and group 3 cardioplegic solution was calcium free but contained diltiazem (150 micrograms/kg body weight). Left ventricular function measured as percent control of developed pressure revealed significantly greater (p less than 0.05) recovery in groups 2 and 3. Triphenyltetrazolium chloride staining showed 35% +/- 9% (mean +/- standard error) of heart mass necrosis in group 1 versus 0% and 0.5% +/- 0.4% in groups 2 and 3, respectively (p less than 0.001). Electron microscopy revealed ultrastructural changes characteristic of calcium paradox injury in group 1 myocardium. Calcium paradox injury was produced in an in vivo model of global myocardial ischemia and multidose cardioplegia despite moderate hypothermia and non-coronary collateral flow. The addition of either trace levels of calcium or diltiazem to the cardioplegic solution was effective in preventing this injury.

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.

Impaired myocardial fatty acid and glucose metabolism following ischemia and cardioplegia may limit the recovery of myocardial oxidative metabolism and ventricular function. Lactate, a simple three carbon compound, can be readily metabolized to pyruvate and is possibly the preferred substrate for aerobic metabolism. Therefore, increasing arterial lactate concentrations may improve myocardial metabolic recovery after ischemia and cardioplegia. Myocardial lactate metabolism and ventricular function were assessed in a canine model of 45 mins of global normothermic ischemia followed by 60 mins of cold potassium cardioplegic arrest. Thirteen dogs received a perioperative infusion of sodium lactate to elevate arterial concentrations (from 6 to 12 mmol/L) and 12 dogs received an equivalent amount of saline. The high arterial lactate concentrations were associated with an increased myocardial lactate consumption and oxidation (as assessed by 14C-labelled lactate) during reperfusion. Myocardial ATP concentrations fell during reperfusion despite improved myocardial oxidation. The recovery of ventricular function (as assessed by a compliant intraventricular balloon) was incomplete and only marginally better with the high arterial lactate concentrations. An infusion of lactate improved myocardial oxidative metabolism following ischemia and cardioplegia. However, the recovery of ventricular function was incomplete perhaps because of inadequate preservation of myocardial ATP.

An exercising in vivo canine hindlimb model was used to assess the pattern of exogenous substrate delivery and uptake as a function of contraction frequency. The limb was stimulated to contract at 0, 4, 8, 12 8, 4, and 0 Hz for 10-min periods. Blood flow was proportional to stimulatory frequency. Oxygen consumption increased to a maximum at 12 Hz and subsequently declined. Glucose, free fatty acid, and lactate uptakes decreased despite increased deliveries and maintained arterial concentrations at 12 Hz. At this point there was a net release of glycerol and lactate from the muscle. Microvascular shunting was not the cause of decreased exogenous substrate uptake at 12 Hz. A switch from exogenous to endogenous muscle substrates occurred with the onset of decreased substrate uptake. The mechanism that facilitates this switch from exogenous to endogenous substrates remains unknown.

Changes in the skeletal muscle adenine nucleotide pool during prolonged periods of normothermic ischemia, followed by reperfusion are a result of an exaggerated breakdown to lipid soluble precursors, the degree of reactive hyperemia, and activities of the salvage and direct pathways for resynthesis. We show that the degree of breakdown of ATP, ADP and AMP, is time dependent, and with restoration of circulation there is washout of these lipid soluble precursors, and no resynthesis of ATP. We demonstrated a relationship between the loss of energy during ischemia, and the degree of resultant necrosis, suggesting that a limit on the effectiveness of therapeutic interventions during reperfusion in reducing the extent of necrosis may exist.

Polyadenosine- and adenosine-diphosphoribosylated proteins of hamster liver were quantitatively isolated with the aid of m-aminophenyl boronic acid glutaryl hydrazide polyacrylamide affinity resin by selective adsorption at pH 8.2 and elution at pH 4.0. Polymer-free proteins, DNA, and RNA are readily separated from adenosine-diphosphoribosylated proteins. The total quantity of proteins that is covalently modified by the homopolymer is 14.3 micrograms/mg of DNA or 37.4 micrograms/g of liver in controls and 38.7 micrograms/mg of DNA or 116 micrograms/g of liver in dimethylnitrosamine-treated hamsters. Polymer content increases from 9 to 15 nmol/mg of DNA to 42 to 118 nmol/mg of DNA following treatment with dimethylnitrosamine. Pulse labeling with [14C]ribose results in a parallel doubling in dimethylnitrosamine-treated animals of the specific activities of adenosine- diphosphoribose and NAD+ and of the [14C]ribose content of polyadenosine-diphosphoribose of chain length between 20 and 40, indicating chain elongation of pre-existing larger polymers. Two groups of proteins that are isolated as polyadenosine-diphosphoribose adducts are increased significantly after treatment with dimethylnitrosamine, one minor component of a mass between 100-112 X 10(3) daltons, and a major group exhibiting a mass of 158-162 X 10(3) daltons. Polyadenosine-diphosphoribose synthetase activity of isolated hepatic nuclei is increased by 32-37% after dimethylnitrosamine treatment, and since the change in glycohydrolase activity is negligible relative to the increase in synthetase, the augmentation of polyadenosine-diphosphoribosylated proteins can be explained by the increased synthetase of nuclei. The molecular size distribution of DNA in liver nuclei of control and dimethylnitrosamine-treated hamsters is indistinguishable.

The macromolecular association of immunoreactive naturally occurring polyadenosine diphosphoribose n greater than 4 with histones and non-histone proteins was determined with the aid of an improved method of extraction of polyadenosine diphosphoribose and a combination of radioimmunoassay and molecular filtration. More than 99% of the naturally occurring polyadenosine diphosphoribose n greater than 4 was present in rat liver in covalent association with non-histone proteins. The chain length of the polymer varied between n = 4 and n = 34. Less than 1% of naturally occurring polyadenosine diphosphoribose n greater than 4 was almost evenly distributed between histone fractions f1, f2a, f2b, and f3. Adenosine diphosphoribose polymers of relatively long chain length were also detected in the histone fractions. The covalent association of polyadenosine diphosphoribose with non-histone proteins was demonstrated by affinity chromatography.

Myocardial substrate preferences following cardioplegic arrest for coronary bypass surgery have not been established. Fatty acids are believed to be the major fuel source for aerobic metabolism. Following cardioplegic arrest arterial fatty acid levels are elevated and myocardial fatty acid accumulation without oxidation may contribute to reperfusion injury. Perioperative fatty acid metabolism was evaluated in 18 patients undergoing elective coronary bypass surgery who were randomized to receive either blood (n = 11) or crystalloid (n = 7) cardioplegia. Palmitate labeled with 14carbon was infused perioperatively and arterial and coronary sinus blood samples were obtained to calculate myocardial fatty acid extraction and oxidation before and after cardioplegic arrest. Lactate and glycerol were released from the heart during both blood and crystalloid cardioplegia, suggesting ischemic glycolysis and lipolysis. Myocardial oxygen consumption was depressed and the myocardial consumptions of lactate, glucose, and fatty acids were minimal during the first 60 min after aortic clamp removal in both groups despite high arterial concentrations. Fatty acid oxidation was minimal after blood cardioplegia and was not found after crystalloid cardioplegia. Fatty acids were extracted by the heart, but were not aerobically metabolized following cardioplegic arrest. Myocardial fatty acid accumulation without oxidation may have been deleterious. The inability of the heart to oxidize exogenous fatty acids may reflect altered myocardial exogenous substrate preferences during reperfusion following coronary bypass surgery.

To evaluate the temporal relationship and potential correlation between intramuscular phosphagen levels, lipid oxidation, and extent of muscle injury, a canine gracilis muscle model was used to study the consequences of a global ischemic episode for up to 7 h duration with reperfusion for 4 h. In this model the contralateral gracilis muscle was prepared identically to the test side but was not subjected to ischemia and thus served as a control. Blood flow, oxygen consumption, and lactate and glycerol release were measured before and after 2- and 7-h ischemic stress periods. The intramuscular metabolites, glycogen, lactate, phosphocreatine, and ATP, as well as free fatty acid conjugated dienes, were measured before, during, and after the ischemic insult. A 2-h ischemic insult resulted in minimal ultrastructural damage and complete regeneration of intramuscular phosphagens and glycogen on reperfusion with complete normalization of lipid oxidation products. In contrast, a 7-h ischemic insult resulted in profound injury at the ultrastructural level with an inability to restore intramuscular phosphagens and glycogen on reperfusion. This severe muscle injury correlated with a 2.5-fold increase in lipid oxidation products (free fatty acid conjugated dienes) and a decline in ATP levels below 5 mumol/g dry wt on reperfusion. Our results emphasize the prolonged glycolytic activity of skeletal muscle during global ischemia and document the increased production of oxygen free radical-mediated lipid oxidation products in irreversibly injured muscle.

Measurement of oxalate in urine has been automated for use with the Cobas Fara centrifugal analyzer. No sample pretreatment other than (a critical) pH adjustment is required. Between-run CVs were less than 4%. Results were linearly related to oxalate concentration to 1000 mumol/L. Ascorbic acid ingestion, up to 5 g of ascorbate daily, caused no demonstrable interference with the assay. This practical, automated method for assaying urinary oxalate is substantially faster than other chemical or enzymatic methods.

Extensive skeletal muscle necrosis may occur after prolonged ischemia to the lower extremity, with serious consequences both locally and systemically. The extent of necrosis is a combination of cellular damage that occurs during both the period of ischemia and the period of reperfusion. The purpose of this study was to reduce the extent of reperfusion-induced muscle necrosis by therapeutic interventions administered only during the initial period of reperfusion. Indeed, the pretreatment of patients who have an acute arterial occlusion is rarely possible and only interventions applicable to the reperfusion phase would be clinically relevant. By perfusing the isolated gracilis muscle in a controlled manner with reduced oxygen concentrations alone and in combination with free radical scavengers, we were able to reduce the extent of muscle necrosis. By means of controlled oxygen delivery alone, muscle necrosis was reduced from 87% +/- 8% in the control muscle to 67% +/- 9% (p less than 0.05) in the treated muscle. The combination of reduced oxygen delivery and free radical scavengers reduced necrosis from 78% +/- 8% in the control muscle to 53% +/- 7% (p less than 0.01) on the experimental side. We conclude that controlled oxygen delivery and free radical scavengers can reduce skeletal muscle necrosis occurring after prolonged normothermic ischemia.

In addition to the determination of the presenting symptom of patients with peripheral vascular occlusive disease, evaluation of these patients may include the noninvasive measurements of ankle/arm pressure ratio, limb blood flow, and treadmill testing to evaluate the severity of the reduction in blood flow. We have included metabolic studies to assess the effect of this reduced blood flow in patients with stable intermittent claudication (n = 20), and with end-stage ischemia (night and rest pain) (n = 11), and in a control group without vascular disease (n = 8). No correlations were found between the resting limb blood flow, ankle/arm pressure ratios, maximum walking distance, and stated walking distance for the patients with stable claudication. Although the oxygen consumption was reduced only in the patients with end-stage ischemia, the percent oxygen extraction was increased to the same level in the patients with stable claudication and those with end-stage ischemia. Intramuscular stores of high-energy phosphates and glycogen were maintained in all groups with the lactate/pyruvate ratio increased only in the patients with end-stage ischemia. The complex interrelationships between the rate and distribution of blood flow with exercise and enzyme adaptation in patients with vascular disease make current resting hemodynamic and metabolic evaluations a poor reflection of the severity of the clinical condition within each patient group. Therefore laboratory testing may offer no advantage over clinical presentation in the overall evaluation of these patients.

Clinical experience indicates that the risk of reparative cardiac operations is increased in the neonatal period relative to that in older infants and children. Age-related differences in the susceptibility to myocardial ischemic dysfunction were evaluated by comparison of left ventricular function and metabolism in neonatal (mean age = 7 days) and weanling (mean age = 96 days) piglets. Six animals in each group supported on cardiopulmonary bypass were subjected to (1) 120 minutes of hypothermic crystalloid cardioplegic arrest (CP-120) and (2) 15 minutes of normothermic ischemic arrest (NA-15) after a 60 minute interval of reperfusion. Left ventricular systolic and diastolic function was measured after each intervention via endocardially implanted ultrasonic dimension crystals in a septolateral minor-axis position. In both groups, systolic dysfunction was evidenced by an increase in the dimension-axis intercept (p = 0.001), but not the slope of the end-systolic pressure-dimension relation. Left ventricular end-diastolic stiffness, expressed as left ventricular end-diastolic pressure versus Lagrangian strain, increased to a similar degree in both groups (p = 0.001). Adenosine triphosphate levels declined significantly (p = 0.001) in both groups in response to the ischemic interventions with no evident intergroup differences. Lactate levels increased significantly during the course of the experiment (p = 0.04); however, the increases were greater (p = 0.009) at all intervals in the neonatal group. This study demonstrates age-related metabolic differences in response to ischemia consistent with a greater dependence on glycolysis in neonatal myocardia. However, the fact that discriminating age-related differences in left ventricular function were not evident suggests that factors other than young age per se account for the increased surgical mortality in the neonatal period.

Systemic flow rates (Q) during nonpulsatile hypothermic cardiopulmonary bypass (CPB) that are consistent with preservation of cerebral function have not to our knowledge been objectively defined. The effect of a sequential reduction in flow rates on cerebral cortical metabolism and function was evaluated in 6 mongrel dogs during hypothermic (25 degrees C) CPB. Cerebral function was assessed using somatosensory cortical evoked potentials (SSEP); cerebral metabolism was assessed by adenosine triphosphate (ATP) and lactate content of snap-frozen gray matter biopsies taken from the hemisphere contralateral to that monitored for SSEP. A progressive decline in ATP levels was observed during flow reduction with virtually complete depletion of ATP at 0.25 L min-1 m-2(p = .0003). The significant (p = .028) dependence of cortical ATP levels on perfusion pressure was no longer evident after adjusting for the effects of flow rate. Lactate levels increased during flow reduction (p = .028), especially at flow rates less than 0.5 L min-1 m-2. Somatosensory neural transmission remained intact until flow was reduced to 0.25 L min-1 m-2 in 5 animals and until total circulatory arrest in 1, at which time loss of the signal occurred. In addition, 5 patients were subjected to brief periods of low-flow CPB (Q = 1.0 L min-1 m-2) at 21 degrees to 25 degrees C. SSEPs remained intact during flow reduction, and postoperative neurologic evaluation was normal in all patients. We conclude that, in the absence of cerebral vascular disease, the flow rate threshold for incurring functional cerebral injury during hypothermic (25 degrees C) nonpulsatile CPB is less than 1.0 L min-1 m-2.(ABSTRACT TRUNCATED AT 250 WORDS)

Myocardial exogenous substrate preference was studied under conditions of increased plasma lactate concentration before and after a severe (halving of tissue ATP concentration, sixfold increase in tissue lactate concentration) but reversible (less than 1% necrosis on reperfusion) global ischaemic stress produced by continuous hypothermic electromechanical arrest of the heart of four hours' duration by aortic cross clamping and multidose potassium cardioplegia. Fatty acid oxidation was studied using 1-14C-palmitate under steady state conditions and under similar isovolumic fixed pressure conditions with the heart at a constant rate using a left ventricular intracavitary balloon. Exogenous free fatty acid oxidation during the pre-ischaemic period with an increased lactate concentration (3.9-5.8 mmol . litre-1) was 0.62(0.21) mumol . min-1 X 100 g-1 (mean (SEM)). This represented a mean of 32% of the total carbon dioxide produced in contrast to a post-ischaemia free fatty oxidation rate of 2.67(0.87) mumol . min-1 X 100 g-1, in the presence of even further increased plasma lactate concentrations (8.47-11.17 mmol . litre-1), representing a mean of 82% of the total carbon dioxide output. These data suggest that the substrate preference of the myocardium, under conditions of increased plasma lactate concentration, shifts to greater oxidation of exogenous free fatty acids after ischaemic stress.

Although blood cardioplegia provides excellent protection, myocardial metabolic recovery is delayed. To evaluate the benefits of a terminal warm cardioplegic infusion after cold blood cardioplegia, we performed a prospective randomized trial in 20 patients undergoing elective coronary bypass grafting. Eleven patients received cold blood cardioplegia and nine patients received cold blood cardioplegia and warm blood cardioplegia before cross-clamp removal (hot shot). The hot shot provided oxygen and removed excess lactate from the arrested heart. After the hot shot lactate was extracted by the heart and tissue adenosine triphosphate and glycogen concentrations were preserved. Atrial pacing and volume loading 3 and 4 hours postoperatively decreased myocardial lactate extraction after cold blood cardioplegia but increased lactate extraction after the hot shot. Left atrial pressures were higher at similar end-diastolic volumes (by nuclear ventriculography), which suggested decreased diastolic compliance after cold blood cardioplegia. Terminal warm blood cardioplegia accelerated myocardial metabolic recovery, preserved high-energy phosphates, improved the metabolic response to postoperative hemodynamic stresses, and reduced left atrial pressures.

The myocardial oxidation of fatty acids and glucose, the predominant substrates for aerobic metabolism, is impaired after cardioplegic arrest for coronary revascularization. Because lactate can be readily metabolized to pyruvate, it may be the preferred substrate for aerobic metabolism after cardioplegic arrest when arterial concentrations are elevated. Nineteen patients undergoing elective coronary revascularization with blood cardioplegia were randomized to receive LOW (nine patients, no exogenous lactate) or HIGH (10 patients, a perioperative infusion of Ringer's lactate) arterial lactate concentrations. Coronary sinus catheterization and lactate labeled with carbon 14 permitted calculation of myocardial oxygen consumption and lactate oxidation which were significantly increased during reperfusion in the group with HIGH arterial lactate concentrations. Atrial pacing at 110 beats/min on cardiopulmonary bypass resulted in myocardial lactate production (suggesting ischemic anaerobic metabolism) in the LOW lactate group, but atrial pacing increased lactate consumption and oxidation in the HIGH lactate group (suggesting increased aerobic metabolism). Systolic function (the relation between end-systolic pressure and volume) as assessed by nuclear ventriculography 3 hours postoperatively was significantly better (p less than 0.05 by analysis of covariance) in the HIGH lactate group. Postoperative myocardial creatine kinase release was significantly lower in the HIGH lactate group, which suggested less perioperative ischemic injury. Lactate was the preferred substrate for myocardial oxidative metabolism after cardioplegic arrest, and the higher arterial lactate concentrations improved myocardial metabolic and functional recovery and reduced perioperative ischemic injury.

This study was designed to test the hypothesis that asanguineous reperfusion with a standard crystalloid cardioplegic solution results in improved myocardial salvage after a period of global ischemia. Four groups of 6 dogs each were placed on cardiopulmonary bypass. Control group A (work only) performed two hours of controlled work by contracting against a saline-filled left intraventricular balloon. Control group B (ischemia only) underwent 45 minutes of global normothermic ischemia before simple blood reperfusion while supported on bypass. Groups C and D were subjected to ischemia and reperfusion as in group B, followed by controlled work stress as in group A. Group D, however, received 500 mL of St. Thomas' Hospital solution immediately before blood reperfusion. Morphological analysis showed no significant injury in groups A and B, whereas group C had 11.4% +/- 2.4% necrosis of heart mass versus 2.5% +/- 1.1% in group D (p less than 0.001). Biochemical data from left ventricular biopsies showed no significant differences between groups B, C, and D. Functional analyses showed deterioration of diastolic compliance in group C (p less than 0.05), although a significant difference in systolic functional indexes could not be detected. Myocardial protection and salvage was improved by initial reperfusion with an asanguineous cardioplegic solution versus reperfusion with blood alone.

Free radical reactions have been suggested to be important events in the mechanism of myocardial injury during ischemia and reperfusion. Most of the in vivo evidence implicating free radical mediated events in the etiology of myocardial injury has been based on intervention studies which document the efficacy of oxygen free radical scavengers in improving function or tissue salvage. We have assayed free fatty acid oxidation products (hydroxy conjugated dienes) derived from myocardial phospholipid as chemical markers of oxidative injury. Biopsies, harvested from canine myocardium subjected to cardiopulmonary bypass with no aortic crossclamp (control), from pre-ischemic, end ischemic (at the end of 45 min of global normothermic ischemia induced by aortic crossclamp) and at 5, 10, 15, 30 and 60 mins of reperfusion were analyzed for hydroxy conjugated dienes using HPLC with structural confirmation by GC-MS. All biopsies were taken from non-necrotic myocardium as indicated by gross tetrazolium staining of myocardial cross sections. Trace levels of hydroxy conjugated dienes could be detected in the preischemic biopsies or control biopsies harvested from hearts subjected to cardiopulmonary bypass with no ischemia. At the end of 45 min ischemia, however, a significant increase in 18:2 and 20:4 hydroxy isomers was detected and confirmed by GC-MS (P less than 0.05 vs. control). After 5 mins of reperfusion a further significant increase in hydroxy conjugated dienes was noted with 18:2, 20:4 and 22:6 isomers being identified (P less than 0.01 vs. end ischemia). By 30 min of reperfusion the concentration of phospholipid oxidation products had returned to pre-ischemic levels. This study presents the first chemically rigorous in vivo evidence for the formation of products of phospholipid oxidation (hydroxy conjugated dienes) during myocardial ischemia and reperfusion and supports the concept of oxygen free radical mediated lipid peroxidation. This study emphasizes the formation of phospholipid oxidation products during ischemia and particularly during the early phase of reperfusion and illustrates the transient nature of these products in myocardial phospholipid.

Recent studies have suggested that oxygen-derived free radicals are involved in the reperfusion injury of ischemic skeletal muscle. Although postischemic necrosis and increased vascular permeability have been attenuated with the addition of free radical scavengers, no unequivocal chemical evidence for free radical injury in skeletal muscle is available. The purpose of this study was to identify products of free radical-mediated membrane injury by isolation of lipid oxidation products (hydroxy-conjugated dienes) from postischemic skeletal muscle. The bilateral canine gracilis muscle model was used, and in each pair one muscle was exposed to 3 and the other to 5 h of normothermic ischemia. Muscle biopsies were taken before and at end ischemia, as well as during the first 3 h of reperfusion. Phospholipids were extracted from the muscle biopsies and the fatty acids hydrolyzed from the 2 position. After methylation, the oxidized fatty acid esters were separated by high-performance liquid chromatography. Hydroxy diene peaks absorbing at 235 nm were collected and subjected to gas chromatography-mass spectrometry (GC-MS) for positive structural identification. No significant increase in the level of conjugated dienes occurred during ischemia. Significant increases, however, were detected during the period of reperfusion, although the time when peak levels were achieved varied between animals. The cumulative sum of dienes produced during reperfusion in both 3- and 5-h muscles was significantly increased over pre- and end-ischemic values. The hydroxy-conjugated diene isomers of 18:2 and 20:4 were positively identified in reperfusion biopsies by GC-MS. These studies provide chemical evidence of free radical-mediated lipid oxidation during reperfusion of ischemic skeletal muscle.

Phenobarbital (PB) in doses previously shown to be optimal for induction of hepatic microsomal enzymes had no effect on the cholesterol content of whole serum or of serum lipoprotein fractions in the guinea pig, but reduced high density lipoprotein cholesterol (with increase in very low density lipoprotein cholesterol) in the rabbit, and increased high density lipoprotein cholesterol (as well as total and low density lipoprotein cholesterol) in the rat. These results, when taken together with previous data demonstrating that PB increases serum triglyceride in the rabbit but lowers it in the rat, suggest that the drug may be atherogenic in the rabbit but not in the rat which, in any case, is highly resistant to atheroma.