Improved recovery after cold crystalloid cardioplegia using low-dose glutamate enrichment during reperfusion after aortic unclamping: a study in isolated blood-perfused pig hearts.

Increased glutamate utilization is a part of the metabolic adaptation to oxygen deprivation by the heart. The effect of low-dose L-glutamate (2 mmol/L) during continuous reperfusion after aortic unclamping on postcardioplegic recovery was studied in pig hearts similar in size, anatomy, and function to the human adult heart. After cold crystalloid cardioplegic arrest (CCC) with Bretschneider solution no 3, hearts were excised from pigs weighing 70-80 kgs (heart weight, average +/- SEM: 308 +/- 4 grams), and reperfused in an isolated blood-perfused heart model for 120 minutes. Three groups of hearts were compared. One group of hearts was subjected to 30 minutes of CCC only (30 min group; n = 9), another group of hearts to 90 minutes of CCC and storage (Control group: n = 16), and a third group to 90 minutes of CCC and storage, but with L-glutamate added to the blood reperfusate (2 mmol/L) (Glutamate group: n = 18). In the Control group 14 of 16 hearts (88%) needed electrical defibrillation after start of reperfusion, significantly more (p < 0.05) than the 8 of 18 (44%) in the Glutamate group; the difference between the 30-min (2 of 9 [22%]) and the Glutamate group was not significant (p = 0.48). Developed left-ventricular pressure (DLVP) and positive dP/dtmax (+dP/dtmax) was significantly higher in the Glutamate group than in the Control group during early reperfusion (DLVP: p < 0.05: +dP/dtmax: p < 0.01) and the entire reperfusion (DLVP and +dP/dtmax: p < 0.05), while reperfusion responses in the Glutamate and 30-min groups were not significantly different. Furthermore, myocardial oxygen uptake was significantly higher in the Glutamate group than in the Control group (p < 0.001), but not higher than that in the 30-min group. Decreased lactate release was found in the Glutamate group compared to the Control group during early reperfusion (p < 0.01), and the entire reperfusion (p < 0.001). No differences were found between the Control and Glutamate groups in alanine exchange. Thus, L-glutamate has a beneficial effect in pig hearts on both functional and metabolic recovery after cold crystalloid cardioplegia and storage when present in a concentration even as low as 2 mmol/L during continuous reperfusion after aortic unclamping. A possible mechanism is a glutamate-induced stimulation of the malate-aspartate shuttle leading to increased intramyocardial lactate utilization.

Thrombosis and intrinsic fibrinolysis in percutaneous transluminal angioplasty.

Coagulation and fibrinolysis were investigated in 14 claudicants undergoing percutaneous transluminal angioplasty (PTA) for femoropopliteal artery lesions. Cross-linked fibrin degradation products (XL-FDP), tissue plasminogen activator (t-PA) antigen, fibrinopeptide A (FPA), and plasminogen activator inhibitor-1 (PAI-1) activity were measured in peripheral blood. XL-FDP and t-PA increased, and FPA and PAI-1 decreased significantly after angioplasty. XL-FDP increased from baseline 266 +/- 72 ng/ml to 481 +/- 239 ng/ml (p < 0.0005) 30 min after PTA, indicating mural thrombus formation in spite of the significant fall in FPA influenced by heparin. A groin haematoma developed after PTA in 4/6 patients, who received more than 5600 IU heparin and in 1/8 patients receiving smaller dosages. The alterations in PAI-1 showed no correlation with those of t-PA, whereas heparin had a sparing effect on PAI-1 consumption. These findings may indicate that PAI-1 acts as a thrombin inhibitor following deep vessel wall injury by angioplasty. In two patients, who had signs of rethrombosis on the next day, residual FPA was relatively high, XL-FDP peaked at 3530 +/- 1170 ng/ml, and t-PA increased by 2.6 +/- 1.0 ng/ml. The corresponding values in patients with an uncomplicated course were 406 +/- 89 ng/ml (p < 0.0001) and 0.1 +/- 0.5 ng/ml (p < 0.02). We conclude that thrombin promotes activation of coagulation and fibrinolysis in femoropopliteal PTA. Instability between these counteracting systems resulting in thrombosis is not prevented by conventional heparin administration at dosages causing bleeding complications.

Determination of high energy phosphates and glycogen in cardiac and skeletal muscle biopsies, with special reference to influence of biopsy technique and delayed freezing.

OBJECTIVE: The aim was to clarify the influence of biopsy technique and the effects of temporal delay between sampling and freezing on tissue contents of labile metabolites. METHODS: Cardiac and skeletal muscle concentrations of adenine nucleotides, phosphocreatine, creatine, and glycogen in pigs were determined in endomyocardial and transmural myocardial biopsies and in skeletal muscle biopsies obtained with either endomyocardial bioptome or Tru-cut needle. The influence of the temporal delay between biopsy sampling and freezing was evaluated by keeping the biopsies at room temperature for varying intervals up to 300 s before freezing. RESULTS: Skeletal muscle showed higher concentrations of creatine compounds and lower contents of ADP and AMP than cardiac muscle, whereas ATP, total adenine nucleotide pool, and glycogen were similar. Lower phosphocreatine contents were found both in endomyocardial biopsies and in skeletal muscle biopsies obtained with bioptome compared to transmural myocardial biopsies and skeletal muscle biopsies obtained with Tru-cut needle, respectively. Other metabolites were unaffected by the biopsy technique. With extended delays between biopsy sampling and freezing, an increase in skeletal muscle phosphocreatine averaging 26% after 5 min was observed. In the heart, a decrease in glycogen content averaging 42% after 5 min was found. These changes were not related to the biopsy procedure and were not observed within the period usually required to freeze biopsies in experimental as well as clinical settings. CONCLUSIONS: There are essential metabolic differences between cardiac and skeletal muscle. Low endomyocardial phosphocreatine levels are influenced by the biopsy technique, compromising the use of endomyocardial biopsies for establishing myocardial phosphocreatine content. Reliable measurements of adenine nucleotides and glycogen can be obtained with endomyocardial biopsies.

Analytical evaluation of high energy phosphate determination by high performance liquid chromatography in myocardial tissue.

High performance liquid chromatography (HPLC) is an established method for the determination of myocardial high energy phosphates (HEP). Quantification of HEP compounds in small tissue specimens obtained by endomyocardial biopsy technique requires maximal sensitivity without impairment of precision. Employing isocratic ion-pair reversed-phase HPLC, high sensitivity and precision were obtained by running analyses for adenonucleotides and creatine compounds separately at detection wavelengths of 254 and 210 nm, respectively. Further reasons for separate runs were given by the necessity for different sample preparation as remaining perchloric ion after deproteinizing and pH in the samples had various effects on adenonucleotides and creatine compounds. Mechanical homogenization for 20 s in 0.42 mol/l perchloric acid ensured a consistent myocardial HEP extraction. Sample preparation directly following biopsy sampling is preferable since HEP compounds were labile in tissue within days at -80 degrees C even though an initial metabolic inhibition in liquid nitrogen had been induced. Following extraction and neutralization, HEP compounds were stable for up to 3 months at -20 degrees C.

Comparison of non-collagen protein and total creatine as reference for determination of energy stores in endomyocardial biopsies.

OBJECTIVE: The aim was to establish a reliable reference system for biochemical measurements in endomyocardial biopsies. METHODS: Myocardial tissue samples were obtained from pigs before and after cardioplegic arrest and reperfusion. Non-collagen protein content was evaluated as a non-specific reference system and compared with total creatine content representing a specific myocardial reference system. The influence of base strength, extraction temperature, and extraction time on protein yields was determined in tissue precipitates redissolved in NaOH. Interference from protein of collagenous origin was excluded by hydroxyproline determinations. Variability of myocardial ATP content in relation to non-collagen protein and total creatine was compared in endomyocardial biopsies taken before and after cardioplegic arrest and reperfusion. RESULTS: The two methods showed comparable analytical precision. Apart from an interference in 1.0 mol.litre-1 NaOH for extended extraction periods at high temperatures, myocardial protein yields increased with increasing base strength, extraction temperature, and extraction time. During cardioplegic arrest and reperfusion heart weight increased due to oedema. Simultaneously, myocardial non-collagen protein content decreased. No change in total creatine was found during cardioplegic arrest but there was a significant loss of creatine after reperfusion. Comparison of variability in myocardial ATP content with non-collagen protein or total creatine as reference systems revealed no difference. CONCLUSIONS: Determination of non-collagen protein can be optimised with standardised conditions for protein extraction in tissue precipitates. Employment of total creatine as a reference system does not reduce variability of myocardial metabolite determinations in endomyocardial biopsies compared with non-collagen protein. Loss of myocardial creatine may in itself provide additional information about myocardial injury but this makes it unsuitable as a reference system for measuring metabolic changes during reperfusion. Multiple biopsies seem necessary for estimation of myocardial energy stores.

Cross-linked fibrin degradation products (XL-FDP) as marker of early rethrombosis in percutaneous transluminal angioplasty.

The thrombotic response to percutaneous transluminal angioplasty (PTA) was investigated in 31 patients treated for 1-10 cm femoropopliteal (n = 28) and tibial (n = 3) artery obstructions by measurement of cross-linked fibrin degradation products (XL-FDP) in peripheral blood samples drawn before and 30 min after PTA. XL-FDP increased from 400 +/- 147 ng/ml to 700 +/- 445 ng/ml (median +/- S.E., p = 0.0005). XL-FDP rose from 320 +/- 110 ng/ml to 540 +/- 102 ng/ml in 23 patients, whose ankle/brachial systolic blood pressure index (ABI) increased > 0.15 after PTA, whereas XL-FDP increased from 850 +/- 450 ng/ml to 2620 +/- 1472 ng/ml in eight patients, who failed to increase ABI in spite of preceding recanalisation. XL-FDP increased by more than 1000 ng/ml in 1/23 (4.3%) patients with uncomplicated PTA and in 6/8 (75%) patients with haemodynamic failure (p = 0.0005). Using a XL-FDP increase of 1000 ng/ml as cut-off, estimates of positive and negative predictive values (95% confidence limits) for early failure of PTA were 85.7% (42.1-99.6%) and 91.7% (73.0-99.0%), respectively. We conclude from this pilot study that femorotibial PTA produces a hypercoagulable state which may result in failure of early patency due to rethrombosis. We suggest for the first time XL-FDP as a marker of early rethrombosis in PTA, and report a sequential XL-FDP assay which may be useful for identification of high-risk patients requiring thrombolytic therapy after PTA for maintenance of early vascular patency.

Myocardial loss of glutamate after cold chemical cardioplegia and storage in isolated blood-perfused pig hearts.

Metabolic adaptation of the ischemic human heart includes release of lactate, augmented uptake of glucose and glutamate, together with increased release of citrate and alanine. In the present study exchanges of these metabolites were examined in relation to left ventricular function (LVF) in pig hearts during reperfusion after hypothermic cardioplegic-induced global ischemia and storage. Three groups of pig hearts were studied. Group I consisted of 11 hearts subjected to 9 minutes of warm ischemia prior to cold chemical cardioplegia with Bretschneider's cardioplegic solution (CCC), and hypothermic storage (HS), for a total of 180 minutes. Groups II and III, 8 hearts in each, were subjected to 90 and 180 minutes of CCC and HS, without precardioplegic warm ischemia. All hearts were reperfused in an isolated blood-perfused Langendorff model. Myocardial oxygen uptake and LVF were two-fold depressed in Group I compared to Groups II and III during the first 25 minutes of reperfusion. An increased uptake of glucose (p < 0.05) and augmented release of lactate (p < 0.01) and citrate (p < 0.001) were found during the reperfusion period in the hearts subjected to precardioplegic warm ischemia, indicating an increased total ischemic burden compared to Groups II and III. No significant changes in LVF or myocardial metabolism were noted between Groups II and III during reperfusion. In all three heart groups a substantial release or loss of glutamate was found at start of reperfusion, although in the preischemic state prior to cardioplegia pig hearts were found to extract glutamate from the circulation to an extent similar to that of the human heart.(ABSTRACT TRUNCATED AT 250 WORDS)

Influence of pre-existing ischemia on recovery from chemical cardioplegia. A study on pig hearts in an isolated blood-perfused model.

The impact of prior cardiac ischemia on recovery from chemical cardioplegia was investigated in pig hearts. Group I hearts were subjected to 9-min normothermic ischemia before the start of chemical cardioplegia. After 180 min of induced cardiac arrest, all hearts were reperfused and monitored for 120 min in a blood-perfused Langendorff model. Consistent with left ventricular performance, myocardial oxygen uptake was significantly lower in group I than in the other hearts during the first 60 min of reperfusion. Lactate elimination was significantly higher in group I at the start of reperfusion, but showed no intergroup difference after 25 min. Nor was intergroup difference found in left ventricular end-diastolic pressure, total myocardial flow or glucose extraction fraction during reperfusion. The mitochondrial ultrastructure was identical in the two groups before chemical cardioplegia. During cardioplegia it deteriorated in group I but normalized in group II. During reperfusion these circumstances were reversed. Although precardioplegic ischemia thus significantly impaired left ventricular performance during early recovery, with corresponding effects on metabolism and ultrastructure, stable performance during reperfusion indicated that the ischemic injury did not worsen.

Recovery after cold cardioplegic arrest of isolated blood-perfused hearts excised from non-anesthetized pigs.

An isolated blood-perfused pig heart model has been established in order to evaluate the recovery of hearts obtained from slaughterhouse domestic pigs avoiding anesthesia and direct experiments on animals. Eleven hearts subjected to 9 min of normothermic ischemia were infused with cold modified Bretschneider solution. After 180 min of cardioplegic-induced global ischemia (including 9 min of normothermic ischemia) 8 hearts were reperfused for 120 min. Left ventricular function (measured isovolumetrically by means of a balloon, and expressed as developed left ventricular pressure, positive and negative dP/dt) was stable during the whole reperfusion period. Lactate production was abolished after 25 min of reperfusion, while there was a small glucose extraction during the whole reperfusion period. Slight deterioration of the mitochondria was found during the induced cardiac arrest, however, reversing during the reperfusion. Thus, due to the stability of left ventricular function, improved metabolism and ultrastructure during the reperfusion period, the model with no use of laboratory animals, and without any influence of anesthesia, seems to be suitable for testing the pure effect on the performance of the left ventricle of drugs and substrates added to the reperfusate during the reperfusion period.