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Comparison between the use of whole blood versus a diluted perfusate in regional isolated perfusion by continuous monitoring of transcutaneous oxygen tension: a pilot study.

In 12 successive women (median age, 58 [39-89] years) who were treated with regional isolated perfusion for melanoma of the lower extremities, peroperative continuous monitoring of the transcutaneous oxygen tension (PtcO2) was performed as an indicator for tissue oxygenation and (sub)cutaneous perfusion. Regional perfusion started using whole blood as perfusate with a hematocrit of 40.7 +/- 4.9. After 40 min of drug circulation the perfusate was diluted to a hematocrit of 25.3 +/- 4.9, a value usually applied in perfusion. Flow rates (mean 51.2 +/- 14.4 mL/min/L perfused tissue) were kept at a level that caused no systemic leakage and no more than 10-cm increase above starting venous pressure. Oxygen supply was set at one half of the flow of the perfusate (in mL/min). The mean PtcO2 during the first part of perfusion, in which seven patients could achieve preperfusion levels for at least some of the time, was significantly higher than during the last part, in which no patient reached the preperfusion level (30.8 mm Hg vs 23.5 mm Hg; p = .0019). The mean maximal decrease in PtcO2 after dilution was 21.3 +/- 11.6 mm Hg. Venous blood gases of the perfusate also deteriorated after dilution. Two patients encountered a grade III and two a grade IV toxicity reaction after perfusion. We conclude that increasing the hematocrit of the perfusate to physiologic levels by using whole blood can guarantee a physiologic tissue oxygenation at relatively low flow rates. However, physiologic tissue oxygenation on its own is not enough to prevent toxicity after perfusion.

Adult↗

Interactions of cationic drugs and cardiac glycosides at the hepatic uptake level: studies in the rat in vivo, isolated perfused rat liver, isolated rat hepatocytes and oocytes expressing oatp2.

This paper deals with a crucial mechanism for interaction of basic drugs and cardiac glycosides at the hepatic uptake level. Available literature data is provided and new material is presented to picture the differential transport inhibition of bulky (type2) cationic drugs by a number of cardiac glycosides in rat liver. It is shown that the so called organic anion transporting peptide 2 (oatp2) is the likely interaction site: differential inhibition patterns as observed in oocytes expressing oatp2, could be clearly identified also in isolated rat hepatocytes, isolated perfused rat liver and the rat in vivo. The anticipation of transport interactions at the hepatic clearance level should be based on data on the relative affinities of interacting substrates for the transport systems involved along with knowledge on the pharmacokinetics of these agents as well as the chosen dose regimen in the studied species. This review highlights the importance of multispecific tranporter systems such as OATP, accommodating a broad spectrum of organic compounds of various charge, implying potential transport interactions that can affect body distribution and organ clearance.

Animals↗

Effects of amifostine on perfused isolated rat heart and on acute doxorubicin-induced cardiotoxicity.

PURPOSE: To determine the effects of amifostine on an isolated perfused rat-heart model and its protective activity with regard to cardiotoxic doxorubicin perfusion. METHODS: Langendorff constant-pressure isolated rat-heart preparations were used to analyze the effects of the drugs during a 40-min period of perfusion after a 20-min stabilization interval. The first study was conducted with amifostine alone (controls and 10(-6), 10(-5), and 10(-4) M amifostine; n=6 in each group). The second study was conducted with amifostine and doxorubicin (controls, 2.5 x 10(-5) M doxorubicin, 2.5 x 10(-5) M doxorubicin and 10(-5) M amifostine, and 2.5 x 10(-5) M doxorubicin and 10(-4) M amifostine; n=4 in each group). RESULTS: Amifostine had no significant effect on hemodynamic parameters at 10(-6), 10(-5), and 10(-4) M concentrations. However. amifostine increased the coronary flow expressed as a percentage+/-SEM of the baseline flow as follows: 82+/-4% for controls, 95+/-6% for 10(-6) M amifostine, (P=0.13), 111+/-4% for 10(-5) M amifostine (P < 0.01), and 104+/-3% for 10(-6) M amifostine (P < 0.01). When we commenced an amifostine perfusion 20 min in advance of and then during a 40-min perfusion with doxorubicin, at a cardiotoxic concentration of 2.5 x 10(-5) M the left ventricular pressures (LVDP, expressed as percentages +/-SEM of the baseline LVDP before doxorubicin) were 55+/-3% for the doxorubicin controls, 68+/-2% for doxorubicin with 10(-5) M amifostine (P=0.05), and 80+/-3% for doxorubicin with 10(-4) M amifostine (P < 0.01). Whether this protective effect might be related to the known free-radical-scavenging activity of amifostine remains to be determined. CONCLUSION: On a Langendorff-type model of rat heart, 10(-5) and 10(-4) M amifostine alone induced a coronary dilation and, when associated with a cardiotoxic concentration of 2.5 x 10(-5) M doxorubicin, 10(-5) and 10(-4) M amifostine displayed a cardioprotective effect.

Amifostine↗

Leukocytic .O2- and cardiac dysfunctions in isolated perfused rat hearts.

Superoxide radicals are supposed to contribute to myocardial reperfusion injury. Their origin, however, is still a matter of debate. Polymorphonuclear leukocytes (PMNL) have been discussed as a major postischaemic .O2- source [Lucchesi and Mullane (1986) Ann Rev Pharmacol Toxicol 26: 201-224]. We studied the role of .O2- derived from human polymorphonuclear leukocytes in reperfused and normoxic perfused isolated rat hearts. During reperfusion PMNL exerted deleterious effects on different parameters (e.g. contractility, coronary flow) of isolated rat hearts. Under normoxic perfusion conditions stimulation of PMNL with N-formyl-L-methionyl-L-leucyl-L-phenylalanine (FMLP) caused bradycardia and a transient vasoconstriction. Superoxide dismutase (SOD) administration did not influence any of the PMNL effects mentioned, suggesting that leukocytic .O2- was not involved in PMNL-induced cardiac dysfunctions.

Animals↗

Effect of dextran on the stability of isolated, perfused rat heart.

Stability of cardiac function and myocardial intracellular ionic composition was examined in the Krebs-perfused isolated rat working heart. Hearts perfused by a conventional Krebs solution showed stable contraction for 2 h, but during this period, intracellular ionic environment (Na, K, Ca) substantially changed. Addition of dextran to the perfusate inhibited, though not completely, this time-dependent deterioration and made the heart less vulnerable to the hypoxia-reoxygenation.

Animals↗

[Effects of endothelin antagonists on isolated perfused murine livers in the early phase of warm ischemia-reperfusion injury under propofol anesthesia].

We investigated the effects of selective and non-selective endothelin (ET) antagonists on warm ischemia-reperfusion injury of the early phase in the murine liver under propofol anesthesia. We examined portal pressures, O2 consumptions and liver enzymes to estimate injuries using perfused isolated murine liver model. Experiment 1: we compared the perfusate only group, the soy oil (as vehicle) group, and the propofol group without ischemic loading to determine whether propofol and its vehicles themselves have any effect. Experiment 2: we determined the effects of ET antagonists on reperfusion injury after warm ischemia of 40 minute's cutoff of perfusate under propofol anesthesia and compared five groups up to 90 min after reperfusion; the propofol only group (control; 4 mg.l-1), the BQ-485 group (20 microM), the BQ-788 group (50 microM), the bosentan group (40 microM), and the BQ-485 + BQ-788 group. The soy oil group showed significantly more liver damage, and the propofol group showed no damage. In addition, propofol did not alter the effect of ET antagonists on reperfusion injury. Clinical dose of propofol did not seem to alter the effects of ET antagonists on the murine liver in the early phase of ischemia-reperfusion injury.

Anesthesia, Intravenous↗

Comparative cardiac effects of terlipressin, vasopressin, and norepinephrine on an isolated perfused rabbit heart.

BACKGROUND: Terlipressin, a synthetic analog of arginine-vasopressin (AVP), has been proposed as an effective vasopressive therapy in catecholamine-resistant vasodilatory shock. Although beneficial effects of terlipressin on systemic arterial pressure have been clearly demonstrated, its intrinsic effects on coronary circulation and myocardial performances remain unknown. METHODS: The authors compared the coronary and myocardial effects of terlipressin (1-100 nM, n = 10), AVP (10-1000 pM, n = 10), and norepinephrine (1-100 nM, n = 10) on an erythrocyte-perfused isolated rabbit heart. The cardiac effects of terlipressin were also assessed in erythrocyte-perfused hearts in which the myocardial oxygen delivery was maintained constant and buffer-perfused hearts. Finally, the cardiac effects of terlipressin and AVP were studied in hearts pretreated by [d(CH2)5Tyr(Me)]AVP (0.1 microM), a selective V1a receptor antagonist. RESULTS: Norepinephrine induced a biphasic coronary effect associated with a concentration-dependent increase in myocardial performances. AVP and terlipressin significantly decreased coronary blood flow and impaired myocardial performances from 30 pM and 30 nM, respectively (P < 0.05). The cardiac side-effects of terlipressin were confirmed in buffer-perfused hearts but the maintenance of a constant myocardial oxygen delivery constant abolished its effects on myocardial performances. The cardiac effects induced by terlipressin and AVP were nearly completely abolished on hearts pretreated by [d(CH2)5Tyr(Me)]AVP. CONCLUSIONS: On isolated rabbit heart, terlipressin induced a coronary vasopressor effect and in turn myocardial depression only at supratherapeutic concentrations (> or =30 nM). Its effects are mainly mediated via V1a receptors. However, these potential negative side effects on the heart were less pronounced than were those of AVP.

Animals↗

Metabolic control of contractile performance in isolated perfused rat heart. Analysis of experimental data by reaction:diffusion mathematical model.

The intracellular mechanisms of regulation of energy fluxes and respiration in contracting heart cells were studied. For this, we investigated the workload dependencies of the rate of oxygen consumption and metabolic parameters in Langendorff-perfused isolated rat hearts.(31)P NMR spectroscopy was used to study the metabolic changes during transition from perfusion with glucose to that with pyruvate with and without active creatine kinase system. The experimental results showed that transition from perfusion with glucose to that with pyruvate increased the phosphocreatine content and stability of its level at increased workloads. Inhibition of creatine kinase reaction by 15-min infusion of iodoacetamide decreased the maximal developed tension and respiration rates by a factor of two.(31)P NMR data were analyzed by a mathematical model of compartmentalized energy transfer, which is independent from the restrictions of the classical concept of creatine kinase equilibrium. The analysis of experimental data by this model shows that metabolic stability-constant levels of phosphocreatine, ATP and inorganic phosphate-at increased energy fluxes is an inherent property of the compartmentalized system. This explains the observed substrate specificity by changes in mitochondrial membrane potential. The decreased maximal respiration rate and maximal work output of the heart with inhibited creatine kinase is well explained by the rise in myoplasmic ADP concentration. This activates the adenylate kinase reaction in the myofibrillar space and in the mitochondria to fulfil the energy transfer and signal transmission functions, usually performed by creatine kinase. The activity of this system, however, is not sufficient to maintain high enough energy fluxes. Therefore, there is a kinetic explanation for the decreased maximal respiration rate of the heart with inhibited creatine kinase: i.e. a kinetically induced switch from an efficient energy transfer pathway (PCr-CK system) to a non-efficient one (myokinase pathway) within the energy transfer network of the cell under conditions of low apparent affinity of mitochondria to ADP in vivo. This may result in a significant decrease in the thermodynamic affinity of compartmentalized ATPase systems and finally in heart failure.

Animals↗

Effects of a lipoxygenase inhibitor on digoxin-induced cardiac arrhythmias in the isolated perfused guinea-pig heart.

1. The effects of a lipoxygenase inhibitor, BW A4C, on digoxin-induced arrhythmias and cardiac dynamics (contractile force, perfusion pressure, heart rate) were investigated in Langendorff-perfused isolated guinea-pig hearts. In the control group, arrhythmias were induced by 25 micrograms/ml digoxin at a perfusion rate of 0.5 ml/min. In the treated groups, BW A4C (1 and 0.3 microM) perfused continuously from 15 min prior to digoxin until cardiac arrest occurred. Digoxin exposure (microgram/g wet weight of heart) for the occurrence of arrhythmias and cardiac arrest were the parameters evaluated to assess cardiotoxicity. 2. Digoxin caused a marked increase in leukotriene B4 release in the coronary effluent, and was collected during tachyarrhythmias. BW A4C markedly inhibited the digoxin-induced elevation of LTB4. 3. BW A4C (1 and 0.3 microM) did not prevent the onset of ventricular fibrillation and ventricular tachycardia despite a slight delay in the occurrence of ventricular fibrillation and cardiac arrest at the 0.3 microM concentration. 4. Contractile force increased significantly after digoxin infusion which was concomitant with the time of onset of arrhythmias. In the presence of BW A4C, the contractile force increased, but not significantly. Perfusion pressure increased initially after digoxin infusion in the absence and the presence of BW A4C, but not significantly. 5. These findings show that the lipoxygenase inhibitor lacked any protective action on digoxin-induced arrhythmias despite its effective suppression of digoxin-induced elevation of LTB4 in coronary effluent.

Animals↗

Lipogenesis from amino acids in perfused isolated dog skin.

Lipogenesis from amino acids has been studied in isolated perfused dog skin. Uniformly labeled alanine-(14)C, glycine-(14)C, isoleucine-(14)C, leucine-(14)C, phenylalanine-(14)C, and valine-(14)C are all incorporated into the cutaneous lipids, with significant incorporation into most of the isolated lipid fractions. Efficiency of lipogenesis has been expressed relative to the extent of incorporation of acetate under the same experimental conditions. This efficiency was highest for the three branched-chain amino acids. The accuracy, uses, and limitations of the perfusion technique for the study of cutaneous lipogenesis have been evaluated.

Acetic Acid↗

Ca2+ antagonists do not protect isolated perfused rat hepatocytes from anoxic injury.

Ca2+ antagonists were studied during anoxia in perfused isolated rat hepatocytes. Cytosolic free calcium (Ca2+i) was measured with aequorin. Anoxia was induced for 2 h by saturating the perfusate with 95% N2/5+ CO2. Anoxia increased Ca2+i in two distinct phases reaching a maximum of 1.5 microM. The increase in Ca2+i was caused by Ca2+ influx from the extracellular fluids because the main Ca2+i surge was totally abolished in Ca(2+)-free media. LDH release increased 6-fold during the second hour of anoxia, but when Ca2+ was removed from the perfusate during the anoxic period, LDH rose only 2.7-fold. Ca2+ antagonists (10(-7) to 10(-5) M) did not prevent the increase in Ca2+i and the rise in LDH release. On the contrary, high concentrations (10(-6) and 10(-5) M) of the blockers nifedipine and diltiazem significantly increased anoxic cell injury. The observation that the increase in LDH and the rise in Ca2+i were not suppressed by Ca2+ antagonists suggests that (i) Ca2+ antagonists protect the whole liver from anoxic injury by acting on cells other than parenchymal cells; (ii) the influx of Ca2+ responsible for the massive increase in hepatocyte Ca2+i evoked by anoxia did not take place through voltage-sensitive Ca2+ channels but must have occurred via the Na(+)-Ca2+ antiporter operating in the reverse mode (Ca2+ influx vs. Na+ efflux), and (iii) high concentrations of Ca2+ antagonists may be deleterious to the parenchymal cells of the liver.

Animals↗

Reduction of dimesna to mesna by the isolated perfused rat liver.

Mesna is administered with ifosfamide and cyclophosphamide to reduce the incidence of hemorrhagic cystitis. In the present model of mesna metabolism and disposition, mesna is rapidly and irreversibly oxidized to dimesna in the plasma, passes unchanged through the liver, and is then reduced by the kidney and excreted. Our detection of a high ratio of mesna to dimesna in the plasma of clinical samples led us to reinvestigate the hepatic metabolism of mesna and dimesna. We perfused isolated rat livers from female Sprague Dawley rats with protein-free buffered solution containing dimesna at concentrations observed during therapy. In single-pass perfusions, each liver was perfused with up to three dimesna concentrations during consecutive 20-min periods. Recirculating perfusions were used to study single supratherapeutic concentrations of dimesna or mesna. Mesna and dimesna concentrations were measured by specific chromatographic procedures. Dimesna reduction, adjusted by the effluent flow rate and liver weight (0.4-58.5 nmol/min/g liver), correlated closely by linear regression (r = 0.98; n = 36) to the perfused dimesna concentration (4.2-249 microM), indicating a clearance of 0.20 ml/min/g liver. The concentration of dimesna that entered the liver closely matched the summed concentration of mesna and dimesna emerging in the effluent perfusate (single-pass experiments: slope, 0.98; intercept, -0.30; r = 1.00; n = 31). Only trace amounts of unidentified thiols were detected in the bile during recirculation of perfusates with 1 mM mesna or 250 microM dimesna. The effluent mesna concentration correlated inversely with the flow rate, which was consistent with a low extraction ratio in the perfusion model. These data suggested that the dimesna reduction rate was limited by hepatic uptake. Dimesna reduction was decreased by agents that deplete glutathione. Pretreatment of rats with up to 100 mg/kg ifosfamide did not impair hepatic dimesna reduction. In control experiments, dimesna was not reduced during recirculation through the apparatus without a liver. Mesna was oxidized to dimesna during oxygenation of the perfusate in the reservoir, but mesna injected directly into the perfusate just before entry into the liver passed unchanged into the effluent. Extrapolation of the dimesna clearance data from the perfusion model to humans suggests that hepatic dimesna reduction may counterbalance the rapid oxidation of mesna in plasma. The proposed equilibrium is consistent with clinical observations and suggests a new model for mesna metabolism and disposition.

Animals↗

Cortisol metabolism and its inhibition by glycyrrhetinic acid in the isolated perfused human placental lobule.

We have previously reported the placental metabolism of prednisolone to prednisone, 20alpha- and beta-dihydroprednisone and 20beta-dihydroprednisolone. In this study, the disposition of cortisol was investigated in vitro in the dual perfused, isolated human placental lobule after the addition of cortisol (1.2 micromol, n = 3 and 12 micromol, n = 4) to the maternal compartment. Analysis of 5 h maternal and fetal perfusate samples by high performance liquid chromatography-electrospray-tandem mass spectrometry (HPLC-ESI-MS/MS) revealed that cortisol was mainly metabolized to cortisone, but a significant production of 20alpha-dihydrocortisone, 20beta-dihydrocortisone, 20alpha-dihydrocortisol and 20beta-dihydrocortisol was also detected. Saturability of metabolism but not transfer was demonstrated. Metabolism was eliminated by co-perfusion with the potent 11beta-hydroxysteroid dehydrogenase (11beta-HSD) enzyme inhibitor 18beta-glycyrrhetinic acid (GA). The disposition of GA was analysed using HPLC-atmospheric pressure chemical ionisation-MS/MS (HPLC-APCI-MS/MS). GA was found to transfer from the maternal to the fetal circulations without detectable metabolism during 6 h of perfusion.

11-beta-Hydroxysteroid Dehydrogenases↗

Effects of some autacoids on breathing and perfusion flow in the isolated perfused rat lung.

The effects of three autacoids [5-hydroxytryptamine (5-HT), bradykinin and acetylcholine (Ach)) on breathing and pulmonary circulation were studied in the isolated perfused and ventilated rat lung after intravascular injection or intrabronchial instillation. 5-HT, bradykinin and Ach all caused a dose related reduction in breathing, after injection into the pulmonary artery, with Ach being the most potent. With regard to effects on the pulmonary circulation, a dose-related reduction in perfusate flow was noted with bradykinin and 5-HT with bradykinin being about 10 times more potent than 5-HT. Ach did not reduce or increase perfusate flow. After intrabronchial instillation only 5-HT caused reduced perfusate flow. Effects on breathing were difficult to evaluate with this route of administration. The isolated perfused and ventilated lung seems to be a usable model to simultaneously study effects on respiration and pulmonary circulation.

Acetylcholine↗

Response of the isolated perfused stomach of the dog to electrical vagal stimulation.

The left vagal trunk of blood-perfused, isolated, canine stomach was continously stimulated electrically for periods of 30 min at 7 v and 5-msec duration; the frequency of stimulation was varied without interruption from 0.5 to 10 Hz. Plasma immunoreactive gastrin (IRG), total gastric blood flow (GBF), and gastric acid output (GAO) were monitored continuously. When the vagus was stimulated, gastrin concentrations and GBF increased rapidly, while GAO rose at a slower rate. Two Hertz appeared to be the optimal stimulus frequency for increasing plasma IGR levels, while 10 Hz was most effective for increasing GBF. Increasing the frequency from 0 to 0.5 Hz produced the maximum observed change in GAO; additional increments to 2 or 10 Hz produced no consistent additional increase in GAO. The results indicate that plasma IRG concentration and GBF increase in response to various frequencies of vagal stimulation. Vagal stimulation increases the output of immunoreactive gastrin, as well as acid, from the isolated canine stomach.

Animals↗

The inhibitory action of oxygen radical scavengers on proteinuria and glomerular heparan sulphate loss in the isolated perfused kidney.

The perfused isolated kidney is a partial ischemic system that is characterised by glomerular proteinuria and release of glomerular heparan sulfate. Metabolic changes associated with the levels of glutathione, xanthine oxidase and glyceraldehyde 3-dehydrogenase indicated that oxygen radical metabolites were being produced during the perfusion. We have demonstrated that a mixture of oxygen metabolite scavengers containing mannitol, superoxide dismutase and catalase included in the perfusion medium significantly reduced protein excretion. Similar results were obtained with the administration of allopurinol to the rat 24h prior to kidney removal and allopurinol in the perfusion medium. [35S]Heparan sulfate loss from the glomerulus was totally inhibited by the scavenger mixture. These results suggest that reactive oxygen metabolites may be involved in damage to renal capillaries, specifically to heparan sulfate proteoglycan, which leads to proteinuria as a result of partial ischemia produced during perfusion.

Animals↗

Transcapillary movement of cationized ferritin in the isolated perfused rat lung.

We have identified the ultrastructural localization of anionic sites on the luminal surface of the pulmonary microvascular endothelium by perfusing isolated rat lungs with polycationized ferritin (CF). The ligand decorated preferentially the luminal plasmalemma, coated pits, intercellular clefts, and about half of the plasmalemmal vesicles open to the lumen. Decoration of the plasmalemma was not uniform particularly in the nonvesiculated regions of the endothelium. Perfusion of the lung with high salt solution completely abolished binding of CF to the plasmalemma, coated pits, and intercellular clefts but did not significantly decrease binding of CF to the diaphragm of the luminal plasmalemmal vesicles. This indicates the presence of highly charged anionic sites on these regions of the endothelium. CF was taken up by vesicles and discharged on the capillary membrane. Perfusion of the lungs at 4 degrees C completely abolished transport of CF across the endothelium but did not modify the pattern of binding to the luminal endothelial surface. These findings are regarded as evidence for a functional subspecialization of plasmalemmal vesicles in the pulmonary microvascular endothelium.

Animals↗

Lymphokine-activated killer cells with interleukin-2: dose toxicity and localization in isolated perfused rat lungs.

Lymphokine-activated killer (LAK) cells combined with recombinant interleukin-2 (rIL-2) can produce tumor regression in murine models and in patients with pulmonary metastatic disease. However, the dose escalations of rIL-2 required for optimal therapeutic effect often result in increased vascular permeability ("vascular leak syndrome") and other toxic systemic consequences. To avoid systemic distribution, lung perfusion was used to administer LAK and rIL-2 locally. Preliminary to using these agents to treat tumor-bearing lungs, we used a nonblood-perfused isolated rat lung model to study the localization of radiolabeled rIL-2 and LAK and to characterize effects on normal lung tissue of increasing dosages and exposure times of rIL-2 and LAK cells, individually and combined. Lung function or permeability was assessed by measuring lung weight gain and pulmonary arterial pressure during the perfusion, extravascular lung water by double indicator dilution techniques, and wet weight to dry weight ratio. After perfusion for 1 hour using 200,000 U (1,300 U/ml) rIL-2, injury was detected as visible pulmonary edema, weight gain and increases in wet to dry weight ratio, and extravascular lung water; no injury was detected at lower, clinically appropriate dosages. When 1 X 10(8) LAK cells combined with 100,000 U rIL-2 (666 U/ml) were perfused for up to 2 hours, no injury was ascertained. Uptake and distribution of the radiolabeled rIL-2 or LAK was uniform to all lung lobes and corresponded to the decrease of 12% of the rIL-2 or 50% of the LAK from the perfusate after 1-hour perfusion.

Animals↗