The phosphate pool of isolated dog heart during global ischaemia: comparison of two cardioplegic solutions with 31P NMR spectroscopy.

31P NMR spectroscopy was used to study the time course of changes in the concentration of high-energy metabolites and intracellular pH in the dog myocardium during hypothermic ischaemia at 9 degrees C in Bretschneider (HTK-B) and St. Thomas' Hospital (StTH) cardioplegic solutions. It was found that ATP and phosphocreatine degrade slowlier in HTK-B than in StTH, with phosphocreatine depletion occurring within 7.9 +/- 1.4 h in HTK-B and within 6.2 +/- 1.4 h in StTH. The values are virtually identical with the time intervals at which ATP concentration falls below the critical level (60% of initial ATP concentration). In agreement with biochemical analysis, a higher concentration of phosphomonoesters was noted until the 180th minute of ischaemia in HTK-B, a finding suggesting more rapid glycogen degradation in HTK-B. Even though HTK-B contains a high concentration of histidine buffer, higher values of intracellular pH were found during ischaemia in StTH. The effect of extracellular concentration of sodium ions on intracellular pH is discussed.     

Effectiveness of protecting the myocardium against ischemia with a normothermic cardioplegic solution and creatine phosphate

The protective effects of cardioplegic solutions (CS) containing creatine phosphate (CP) were studied in a rat heart model of cardiopulmonary bypass and ischemic cardiac arrest. Isolated rat hearts were subjected to a 3-minute coronary infusion with CS containing CP in normothermic (37 degrees C) and hypothermic (4-6 degrees C) regimes. In the normothermia group, the postischemic functional recovery was 70-75% of the preischemic control value, while the cellular ATP and CP content was reduced but insignificantly. By contrast, in the hypothermia group, the postischemic functional recovery was markedly depressed, with the tissue high-energy phosphate content being appreciably lowered. The data obtained confirm high efficacy of CP-containing cardioplegic solutions administered under normothermia conditions.     

Acid-base buffering in organ preservation solutions as a function of temperature: new parameters for comparing buffer capacity and efficiency

Control of acidity and preventing intracellular acidosis are recognized as critical properties of an effective organ preservation solution. Buffer capacity and efficiency are therefore important for comparing the relative merits of preservation fluids for optimum hypothermic storage, but these parameters are not available for the variety of organ preservation solutions of interest in transplantation today. Moreover, buffer capacity is dependent upon both concentration and pH such that buffer capacity is not easily predicted for a complex solution containing multiple buffer species. Using standard electrometric methods to measure acid dissociation constants, this study was undertaken to determine the maximum and relative buffer capacities of a variety of new and commonly used hypothermic preservation solutions as a function of temperature. The reference data provided by these measurements show that comparative buffer capacity and efficiency vary widely between the commonly used solutions. Moreover, the fluids containing zwitterionic sulfonic acid buffers such as Hepes possess superior buffering for alpha-stat pH regulation in the region of physiological importance.     

Protective effect of a low K+ cardioplegic solution on myocardial Na,K-ATPase activity.

Long duration ischemia in hypothermic conditions followed by reperfusion alters membrane transport function and in particular Na,K-ATPase. We compared the protective effect of two well-described cardioplegic solutions on cardiac Na,K-ATPase activity during reperfusion after hypothermic ischemia. Isolated perfused rat hearts (n = 10) were arrested with CRMBM or UW cardioplegic solutions and submitted to 12 hr of ischemia at 4 degrees C in the same solution followed by 60 min of reperfusion. Functional recovery and Na,K-ATPase activity were measured at the end of reperfusion and compared with control hearts and hearts submitted to severe ischemia (30 min at 37 degrees C) followed by reflow. Na,K-ATPase activity was not altered after 12 hr of ischemia and 1 hr reflow when the CRMBM solution was used for preservation (55 +/- 2 micromolPi/mg prot/hr) compared to control (53 +/- 2 micromol Pi/mg prot/hr) while it was significantly altered with UW solution (44 +/- 2 micromol Pi/mg prot/hr, p < 0.05 vs control and CRMBM). Better preservation of Na,K-ATPase activity with the CRMBM solution was associated with higher functional recovery compared to UW as represented by the recovery of RPP, 52 +/- 12% vs 8 +/- 5%, p < 0.05 and coronary flow (70 +/- 2% vs 50 +/- 8%, p < 0.05). The enhanced protection provided by CRMBM compared to UW may be related to its lower K+ content.     

Hepatic Uptake of Solutes from the Preservation Solution during Hypothermic Storage: A H NMR Study in Rat Liver

The hepatic uptake of histidine and carnosine (histidyl-alanine), used as buffer agents in four preservation solutions, was studied during 24-h hypothermic storage of rat livers by use of ¹H nuclear magnetic resonance (NMR) spectroscopy. Results demonstrated that there was a progressive, concentration-linked passive diffusion of histidine into liver tissues throughout the storage period. A similar inward diffusion of carnosine was also noted. Of the carbohydrate osmotic buffers in the preservation solutions, mannitol permeated the liver tissues to a greater degree and more rapidly than raffinose after the flushing with equivalent concentrations and storage at hypothermia. In general, many solutes from preservation solutions will increasingly penetrate the hepatic inter- and intracellular spaces during extended hypothermic preservation and ¹H NMR spectroscopy is one technique that can assist in the identification of these changes.     

Hepatic uptake of solutes from the preservation solution during hypothermic storage: a (1)H NMR study in rat liver.

The hepatic uptake of histidine and carnosine (histidyl-alanine), used as buffer agents in four preservation solutions, was studied during 24-h hypothermic storage of rat livers by use of (1)H nuclear magnetic resonance (NMR) spectroscopy. Results demonstrated that there was a progressive, concentration-linked passive diffusion of histidine into liver tissues throughout the storage period. A similar inward diffusion of carnosine was also noted. Of the carbohydrate osmotic buffers in the preservation solutions, mannitol permeated the liver tissues to a greater degree and more rapidly than raffinose after the flushing with equivalent concentrations and storage at hypothermia. In general, many solutes from preservation solutions will increasingly penetrate the hepatic inter- and intracellular spaces during extended hypothermic preservation and (1)H NMR spectroscopy is one technique that can assist in the identification of these changes.     

Validity of a model of cultured myocardial cells for assessment of cardioplegia

Myocardial protection is usually studied in vitro on perfused heart preparations, but never directly on cultured cardiomyocytes. We evaluated a model of cultured newborn rat cardiomyocytes to study both the cytotoxicity and the protective effect against chemical hypoxia of three cardioplegic solutions (St Thomas' I, Bretschneider, St Thomas' II) under normothermic (37°C) and hypothermic (4°C) conditions. Cytotoxicity was evaluated in 50% and 100% concentrations of the cardioplegic solutions with incubation times from 90 to 360 min. Myocardial protection was studied in 50% cardioplegic solution with metabolic inhibitors. Immediate and late viabilities, after 24 h of recovery in the medium, were evaluated by simultaneous staining with fluorescein diacetate and propidium iodide.At 37°C, the 50% concentration of the three cardioplegic solutions did not modify cell viability. At 37°C, with 360 min of incubation, the 100% concentration of the St Thomas' I and Bretschneider solutions diminished immediate viability (mean ± SD: medium 87% ± 2%; St Thomas' I 58% ± 5%; Bretschneider 37% ± 8%; St Thomas' II 89% ± 3%) as well as late viability (medium 69% ± 2%; St Thomas' I 32% ± 3%; Bretschneider 24% ± 7%; St Thomas' II 65% ± 4%). At 4°C, immediate and late viabilities were unaffected by cardioplegic solutions.At 37°C, after 360 min incubation time, metabolic inhibitors diminished immediate viability to 29% ± 1% and late viability to zero. None of the three cardioplegic solutions used at 50% concentration prevented this effect.At 4°C, immediate viability was not significantly affected by metabolic inhibitors (73% ± 10%), but the use of Bretschneider cardioplegic solution seemed to be detrimental (53% ± 9%). On the other hand, recovery phase after pretreatment with metabolic inhibitors with or without cardioplegic solutions for 360 min significantly diminished late viability (medium 63% ± 7%; metabolic inhibitors 17% ± 8%; St Thomas' I 17% ± 6%; Bretschneider 8% ± 6%; St Thomas' II 15% ± 3%) and again cardioplegia was inefficient. In conclusion, in this in vitro model for the study of cardioplegic solutions, only pure concentrations of the St Thomas' I and Bretschneider solutions under normothermic conditions were cytotoxic. The well-known protective effects of hypothermia against ischemia and reperfusion injury were both reproduced. Therefore, and even though cardioplegia failed to have any protective effect, probably owing to a severe metabolic inhibition, this model may be useful for studying myocardial protection.     

Murine Isolated Heart Model of Myocardial Stunning Associated with Cardioplegic Arrest

The following protocol is of use to evaluate impaired cardiac function or myocardial stunning following moderate ischemic insults. The technique is useful for modeling ischemic injury associated with numerous clinically relevant phenomenon including cardiac surgery with cardioplegic arrest and cardiopulmonary bypass, off-pump CABG, transplant, angina, brief ischemia, etc. The protocol presents a general method to model hypothermic hyperkalemic cardioplegic arrest and reperfusion in rodent hearts focusing on measurement of myocardial contractile function. In brief, a mouse heart is perfused in langendorff mode, instrumented with an intraventricular balloon, and baseline cardiac functional parameters are recorded. Following stabilization, the heart is then subject to brief infusion of a cardioprotective hypothermic cardioplegia solution to initiate diastolic arrest. Cardioplegia is delivered intermittently over 2 hr. The heart is then reperfused and warmed to normothermic temperatures and recovery of myocardial function is monitored. Use of this protocol results in reliable depressed cardiac contractile function free from gross myocardial tissue damage in rodents.     

Preservation of amino acids during long term ischemia and subsequent reflow with supplementation of L-arginine,the nitric oxide precursor,in the rat heart

We investigated whether L-arginine, used in heart preservation to limit endothelial damage, may influence the pool of amino acids during long term ischemia and reflow. Isolated isovolumic rat hearts (n = 23) were submitted to 8 h of hypothermic ischemia after cardioplegic arrest with the Centre de Résonance Magnétique Biologique et Médicale (CRMBM) solution with or without L-arginine (Arg and No Arg groups respectively). Hearts were freeze-clamped after ischemia (n = 11) or submitted to 60 min of reflow (n = 12) and freeze-clamped. Eight hearts were perfused aerobically for 20 min and freeze-clamped (No ischemia group). Addition of L-arginine to the CRMBM solution limited aspartate depletion and decreased lysine level at the end of ischemia. After reflow, L-arginine supplementation increased the pool of glutamate and arginine and limited the depletion of serine, asparagine, glycine and taurine. We conclude that adding L-arginine to the CRMBM cardioplegic solution during long term ischemia preserved the amino acids pool.     

Improvement of preservation with cardioplegia induced by heat stress is mediated by calcitonin gene-related peptide

Previous studies have shown improvement of preservation with cardioplegia by calcitonin gene-related peptide (CGRP)-induced preconditioning. Therefore we examined the hypothesis that endogenous CGRP may be involved in the protection of heat stress against myocardial damages after prolonged cardioplegic arrest in isolated rat heart. Reperfusion after 4 h of hypothermic ischemia caused a decline of cardiac function and an increase of creatine kinase (CK) release. Heat stress induced by pretreatment with whole body hyperthermia (rectal 42 degrees C) for 15 min produced a significant increase in the plasma content of CGRP, an improvement of cardiac function and a decrease in the release of CK. However, after pretreatment with capsaicin (50 mg/kg, s.c.) to deplete CGRP in cardiac sensory nerves, the plasma concentration of CGRP was no longer increased and the cardioprotection afforded by heat stress was abolished. These findings suggest that improvement of preservation with cardioplegia by heat stress may be mediated by endogenous CGRP in the rat.     

Protective effects of a modified apelin-12 and dinitrosyl iron complexes in experimental cardioplegic ischemia and reperfusion

The maintenance of nitric oxide (NO) bioavailability has been recognized as an important component of myocardial protection during cardiac surgery. This study was designed to evaluate the efficacy of using two NO-donating compounds in cardioplegia and reperfusion: (i) a modified peptide apelin-12 (MA12) that activates endothelial NO synthase (eNOS) and (ii) dinitrosyl iron complexes with reduced glutathione (DNIC-GS), a natural NO vehicle. Isolated perfused working rat hearts were subjected to normothermic global ischemia and reperfusion. St. Thomas’ Hospital cardioplegic solution (STH) containing 140 μM MA12 or 100 μM DNIC-GS was used. In separate series, 140 μM MA12 or 100 μM DNIC-GS was administered at early reperfusion. Metabolic state of the hearts was evaluated by myocardial content of high-energy phosphates and lactate. Lactate dehydrogenase (LDH) activity in myocardial effluent was used as an index of cell membrane damage. Cardioplegia with MA12 or DNIC-GS improved recovery of coronary flow and cardiac function, and reduced LDH leakage in perfusate compared with STH without additives. Cardioplegic arrest with MA12 significantly enhanced preservation of high-energy phosphates and decreased accumulation of lactate in reperfused hearts. The overall protective effect of cardioplegia with MA12 was significantly greater than with DNIC-GS. The administration of MA12 or DNIC-GS at early reperfusion also increased metabolic and functional recovery of reperfused hearts. In this case, recovery of cardiac contractile and pump function indices was significantly higher if reperfusion was performed with DNIC-GS. The results show that MA12 and DNIC-GS are promising adjunct agents for protection of the heart during cardioplegic arrest and reperfusion.     

Effects of dimethyl sulfoxide on the globally ischemic heart: Possible general relevance to hypothermic organ preservation

Isolated perfused rabbit hearts were made globally ischemic for 2 hr, then reperfused. For 5 min before and after ischemia hearts were perfused with hypothermic (20 or 27 °C), hypoxic, substrate-free cardioplegic solutions, some of which contained 70 mM dimethyl sulfoxide. Postischemic ventricular pressure development, spontaneous heart rate, coronary flow, lactate dehydrogenase release, tissue Ca²⁺ content, and in vitro mitochondrial oxidative phosphorylation were used to evaluate the protective effects of the various solutions. Aside from the expected observations that cold cardioplegia lessens ischemic damage, we found that dimethyl sulfoxide gave no indication that it exacerbated ischemic damage or lessened the protection afforded by cardioplegia. We also found that, compared to values measured in comparable drug-free treated hearts, dimethyl sulfoxide significantly improved mitochondrial State 3 respiratory rates, respiratory control, and oxidative phosphorylation rates, and essentially prevented mitochondrial changes due to ischemia and reperfusion. We propose that dimethyl sulfoxide may act as a “scavenger” of cytotoxic free radicals, many of which are known to be generated by mitochondria during reoxygenation. Since hypoxia, ischemia, and reoxygenation are common accompaniments of most organ preservation protocols, we suggest that low concentrations of dimethyl sulfoxide might serve as a useful adjunct to organ preservation in the nonfrozen state, when cryoprotective concentrations are not needed.     

Hypothermic preservation of the rat heart. Comparison of immersion and low-flow perfusion methods: contribution of 31P-NMR spectroscopy

Comparison of rat heart preservation by simple storage in a cardioplegic solution at 4 degrees C (6 hr for group I; 15 hr for group II) and by hypothermic low-flow perfusion of the same solution (0.3 ml min-1, 15 hr: group III) was performed by measuring biochemical and functional parameters and by collecting 31P-NMR spectroscopy data. When compared to control values, adenine nucleotide levels remained unchanged in group I hearts, while glycogen was 45% hydrolyzed and lactate level increased by 700%. Extension of heart immersion to 15 hr (group II) led to breakdown of ATP (-77%), of the sum of adenine nucleotides (-27%), and of glycogen (-77%), whereas lactate accumulation reached 900% of the control value. Functional recovery, measured at the end of a 60-min reperfusion was less than 10% in group II hearts when compared to group I hearts. This dramatic development was completely avoided by hypothermic low-flow perfusion (group III). 31P-NMR data showed that phosphocreatine was completely degraded in all groups of preserved hearts. Low-flow perfusion limited cellular acidosis. The ATP/Pi (Pi = inorganic phosphate) ratio calculated from NMR data was lower for group II hearts (0.04 +/- 0.01, n = 6) than for group I hearts (0.29 +/- 0.12; n = 6) or group III hearts (0.19 +/- 0.09; n = 6) and could constitute a convenient bioenergetic index to predict the capability of the heart to recover satisfactory contractility following a preservation period.     

Ischaemia‐related cell damage in extracorporeal preserved tissue – new findings with a novel perfusion model

Tissue undergoing free transfer in transplant or reconstructive surgery always is at high risk of ischaemia‐related cell damage. This study aims at assessing different procedures using an extracorporeal perfusion and oxygenation system to investigate the expression of hypoxia inducible factor (HIF)‐1‐α as marker for hypoxia and of the pro‐apoptotic protein Caspase‐3 in skeletal muscle to elucidate potential improvements in tissue conservation. Twenty‐four porcine rectus abdominis muscles were assigned to five different groups and examined after they had been extracorporeally preserved for 60 min. time. Group I was left untreated (control), group II was perfused with a cardioplegic solution, group III was flushed with 10 ml of a cardioplegic solution and then left untreated. Group IV and V were perfused and oxygenated with either an isotone crystalloid solution or a cardioplegic solution. Among others, immunohistochemistry (Caspase‐3 and HIF‐1‐α) of muscle samples was performed. Furthermore, oxygen partial pressure in the perfusate at the arterial and venous branch was measured. Expression of Caspase‐3 after 60 min. was reduced in all groups compared to the control group. Furthermore, all groups (except group III) expressed less HIF‐1‐α than the control group. Oxygenation leads to higher oxygen levels at the venous branch compared to groups without oxygenation. Using an extracorporeal perfusion and oxygenation system cell damage could be reduced as indicated by stabilized expressions of Caspase‐3 and HIF‐1‐α for 60 min. of tissue preservation. Complete depletion of oxygen at the venous branch can be prevented by oxygenation of the perfusate with ambient air.     

Hypothermic preservation of hepatocytes

This paper presents a review of recent research on the hypothermic storage of hepatocytes. The first focus is on the diversity of methodologies currently employed in this area. The cell damage caused by hypothermic preservation and its possible mechanism are then investigated on both morphological and molecular biology. Later, the gene expressions on a mRNA level or enzyme level after hypothermic preservation are further discussed. Finally, the improvement of hypothermic storage by preconditioning, such as by increasing temperature, is explored.     

Protective Effect of Creatine Elevation against Ischaemia Reperfusion Injury Is Retained in the Presence of Co-Morbidities and during Cardioplegia

Aims

Ischaemic heart disease is most prevalent in the ageing population and often exists with other comorbidities; however the majority of laboratory research uses young, healthy animal models. Several recent workshops and focus meetings have highlighted the importance of using clinically relevant models to help aid translation to realistic patient populations. We have previously shown that mice over-expressing the creatine transporter (CrT-OE) have elevated intracellular creatine levels and are protected against ischaemia-reperfusion injury. Here we test whether elevating intracellular creatine levels retains a cardioprotective effect in the presence of common comorbidities and whether it is additive to protection afforded by hypothermic cardioplegia.

Methods and Results

CrT-OE mice and wild-type controls were subjected to transverse aortic constriction for two weeks to induce compensated left ventricular hypertrophy (LVH). Hearts were retrogradely perfused in Langendorff mode for 15 minutes, followed by 20 minutes ischaemia and 30 minutes reperfusion. CrT-OE hearts exhibited significantly improved functional recovery (Rate pressure product) during reperfusion compared to WT littermates (76% of baseline vs. 59%, respectively, P = 0.02). Aged CrT-OE mouse hearts (78±5 weeks) also had enhanced recovery following 15 minutes ischaemia (104% of baseline vs. 67%, P = 0.0007). The cardioprotective effect of hypothermic high K⁺ cardioplegic arrest, as used during cardiac surgery and donor heart transplant, was further enhanced in prolonged ischaemia (90 minutes) in CrT-OE Langendorff perfused mouse hearts (76% of baseline vs. 55% of baseline as seen in WT hearts, P = 0.02).

Conclusions

These observations in clinically relevant models further support the development of modulators of intracellular creatine content as a translatable strategy for cardiac protection against ischaemia-reperfusion injury.     

Sustained postischemic cardiodepression following magnesium-diltiazem cardioplegia

Magnesium-diltiazem cardioplegia was evaluated in the intact, perfused rat heart to determine whether the joint administration of these agents would adversely affect myocardial contractile and high-energy phosphate recovery following intermittent, normothermic global ischemic arrest. Sequential metabolic and functional analyses were performed on isolated perfused rat hearts during each phase of the experimental protocol: control (10 min), normoxic cardioplegia (10 min), intermittent global ischemic arrest (two 15-min periods separated by 2 min infusion of the normoxic cardioplegic perfusate), and normoxic postischemic control reperfusion (60 min). Four different cardioplegic solutions were evaluated: 30 mM KCl, 30 mM KCl with 2 mg diltiazem/liter, 20 mM MgCl2, and 20 mM MgCl2 with 2 mg diltiazem/liter. Myocardial phosphatic metabolite levels and intracellular pH were analyzed nondestructively in the intact hearts by phosphorus-31 NMR spectroscopy. Corresponding measurements of peak left intraventricular pressure, rate of peak pressure development (dP/dt), and contraction frequency were performed at the midpoint during each 5-min interval of 31P NMR signal averaging. Magnesium plus diltiazem-treated hearts were distinguished from all other groups by a marked delay in postischemic functional recovery consisting of a prolonged depression in contractility (34% of control, P less than 0.01) that persisted throughout the first 50 min of postischemic reperfusion. Diltiazem in combination with magnesium cardioplegia was detrimental to postischemic functional recovery, despite a rapid restoration of high-energy phosphate stores. The apparent adverse interactive effects of excess magnesium and diltiazem suggest that elective ischemic arrest with magnesium cardioplegia in combination with diltiazem may be contraindicated clinically. The mechanistic basis and drug specificity of this response require further clarification. The present findings appear to exclude ATP and PCr production, and structural causes as the basis for the observed aberrant functional recovery from global ischemia of magnesium plus diltiazem-arrested hearts.     

Regulation of intracellular pH in the myocardium; relevance to pathology

Intracellular pH affects the contractile function of the heart, metabolic reactions, ion exchange and calcium homoeostasis. Numerous studies have concluded that a fall of extracellular pH, by whatever mechanism, causes a fall of contractility by alteration of intracellular pH. Measurement of cytosolic intracellular pH using microelectrodes has confirmed that earlier deduction. Acidosis reduces the slow calcium current and the release of calcium from the sarcoplasmic reticumul but, because the cytosolic calcium does not fall, the major site of action of hydrogen ions appears to be on the calcium sensitivity of the contractile proteins. In man acidosis can be detected 15 s after the occlusion of a coronary artery and is a major mechanism for the simultaneous loss of contractility in ischaemia. A transient alkalosis is not detected in man but has been reported in isolated heart preparations where ATP consumption is low.An imposed mild respiratory acidosis during hypoxia increases the subsequent recovery of mechanical function on reoxygenation whereas a severe acidosis can be harmful. Acidosis in ischaemic may be advantageous due to a cardioplegic effect, inhibition of transsarcolemmal calcium fluxes or a reduction of mitochondrial calcium overload. Calcium uptake on reperfusion or reoxygenation has been linked to an inward movement of sodium in exchange for hydrogen ions on reperfusion and subsequent sodium-calcium exchange. Such a mechanism in its simplest form cannot account for the similar uptake of calcium on reoxygenation and reperfusion. Acidosis is a cause of early contractile failure in ischaemia but the role of acidosis in causing cell necrosis is not established.     

Myocardial protection by propolis during prolonged hypothermic preservation

Oxidative stress is involved in the pathogenesis of ischemia-reperfusion during myocardial transplantation. Therefore, graft preservation solutions may be improved by supplementation with antioxidants to minimize graft dysfunction caused by cold ischemic injury. Propolis is a polyphenol-rich substance which has an important antioxidant activity. The protective effect of propolis against oxidative stress induced by prolonged cold preservation of heart was investigated. Mice were subjected to a hypothermic model of ischemia in which hearts were preserved for 24 h at 4 °C in Krebs-Hensleit (KH) solution in the absence or presence of propolis concentrations (50, 150 and 250 μg/ml). Levels of released Lactate dehydrogenase (LDH), Creatine phosphokinase (CPK) and Troponine-I (Trop I) were assessed in the preservation solution and histological assessement of heart ischemia injuries was performed. Oxidative stress biomarkers malondialdehyde (MDA) and advanced oxidation protein products (AOPP) and antioxidant enzymes superoxide dismutase (SOD) and catalase (CAT) were assessed in cardiac tissue. Mitochondria were isolated from stored hearts and production of reactive oxygen species (ROS) was tested. Propolis supplementation protected efficiently hearts during preservation by reducing significantly levels of lipids and proteins oxidation and restoring activities of antioxidant enzymes. Also, propolis preserved tissue integrity altered by hypothermic ischemia in a concentration-dependent manner. Propolis reduced significantly the rate of H₂O₂ produced by mitochondrial respiration, the best antioxidant effect being obtained at the highest propolis concentration (250 μg/ml). Algerian propolis is a non-temperature sensitive scavenger that protects heart from oxidative damage induced by prolonged hypothermic ischemia.     

The nucleotide metabolism in lactate perfused hearts under ischaemic and reperfused conditions

It was examined whether lactate influences postischaemic hemodynamic recovery as a function of the duration of ischaemia and whether changes in high-energy phosphate metabolism under ischaemic and reperfused conditions could be held responsible for impairment of cardiac function. To this end, isolated working rat hearts were perfused with either glucose (11 mM), glucose (11 mM) plus lactate (5 mM) or glucose (11 mM) plus pyruvate (5 mM). The extent of ischaemic injury was varied by changing the intervals of ischaemia, i.e. 15, 30 and 45 min. Perfusion by lactate evoked marked depression of functional recovery after 30 min of ischaemia. Perfusion by pyruvate resulted in marked decline of cardiac function after 45 min of ischaemia, while in glucose perfused hearts hemodynamic performance was still recovered to some extent after 45 min of ischaemia. Hence, lactate accelerates postischaemic hemodynamic impairment compared to glucose and pyruvate. The marked decline in functional recovery of the lactate perfused hearts cannot be ascribed to the extent of degradation of high-energy phosphates during ischaemia as compared to glucose and pyruvate perfused hearts. Glycolytic ATP formation (evaluated by the rate of lactate production) can neither be responsible for loss of cardiac function in the lactate perfused hearts. Moreover, failure of reenergization during reperfusion, the amount of nucleosides and oxypurines lost or the level of high-energy phosphates at the end of reperfusion cannot explain lactate-induced impairment. Alternatively, the accumulation of endogenous lactate may have contributed to ischaemic damage in the lactate perfused hearts after 30 min of ischaemia as it was higher in the lactate than in the glucose or pyruvate perfused hearts. It cannot be excluded that possible beneficial effects of the elevated glycolytic ATP formation during 15 to 30 min of ischaemia in the lactate perfused hearts are counterbalanced by the detrimental effects of lactate accumulation.