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PubMed1997

Tissue and perfusate pharmacokinetics of melphalan in isolated perfused rat hindlimb.

Melphalan is commonly used as a cytotoxic agent in isolated limb perfusion for locally recurrent malignant melanoma. The time course of melphalan concentrations in perfusate and tissues during a 60-min melphalan perfusion and 30-min drug-free washout in the single-pass perfused rat hindlimb was examined using a physiologically based pharmacokinetic model. The rat hindlimbs were perfused with Krebs-Heinseleit buffer containing 4.7% bovine serum albumin (BSA) or 2.8% dextran 40 at a constant rate of 3.8 ml/min. The concentration of melphalan in perfusate and tissues was determined by high-performance liquid chromatography. The tissue concentrations of melphalan were significantly higher with the perfusate containing dextran than BSA during the 60-min perfusion. During the washout period, the melphalan concentration in the perfusates decreased rapidly in first few minutes, followed by a slower monoexponential decline. The estimated half life (t1/2) for melphalan removal from skin and fat was 59 +/- 2 min for both BSA and dextran perfusates. However, the estimated t1/2 for melphalan removal from muscle was 79 and 96 min for BSA and dextran washout perfusates, respectively. The predicted concentration-time profiles obtained for melphalan with BSA and dextran perfusates appear to correspond closely to the observed data. This study showed that the uptake of melphalan into perfused tissues is impaired by the use of perfusates in which melphalan is highly bound. Melphalan washout from muscle, but not skin and fat, was facilitated by the use of perfusates in which melphalan is highly protein bound.

Adipose Tissue↗
PubMed1994

Blunt injury augments interleukin-6 but not tumor necrosis factor in isolated, perfused rat hindlimbs.

An isolated, perfused hindlimb model in rats was used to examine the immediate inflammatory response after blunt tissue injury. A femur-fracture degloving model was used in isolated rat hindlimbs perfused with a modified Kreb's buffer (pH 7.4) containing albumin, washed human red blood cells (RBCs), amino acids, and glucose at 37 degrees C. Arterial and venous perfusate was sampled at 5, 20, and 80 minutes of perfusion. Initial experiments were conducted in perfusate void of white blood cells (WBCs), group 1 (-inj/-WBC, n = 6) and group 2 (+inj/-WBC). Subsequent experiments were conducted in perfusate containing activated WBCs, group 3 (-inj/+WBC, n = 6) and group 4 (+inj/+WBC, n = 7). Hindlimb muscle was analyzed for adenylate energy charge (EC) and lactate-to-pyruvate ratios (LPR) at the end of each perfusion. This preparation appeared metabolically stable in that oxygen consumption and lactate remained stable during the 80-minute perfusion and muscle EC and LPR indicated aerobic metabolism. Tumor necrosis factor (TNF) and thromboxane B2 (TXB2) were measured in all four groups while prostaglandin F (PGF1 alpha), IL-6, myeloperoxidase, and 8-isoprostane were measured in groups 3 and 4. Initial perfusions in the -WBC hindlimbs indicated no change in TNF release after injury. The TXB2 level increased during perfusion irrespective of injury. The PGF1 alpha was elevated at 80 minutes in both groups 3 and 4, however at 20 minutes PGF1 alpha levels were higher in group 4 compared with group 3. Interestingly, the IL-6 level was significantly elevated at 80 minutes in group 4 but not in group 3.(ABSTRACT TRUNCATED AT 250 WORDS)

6-Ketoprostaglandin F1 alpha↗
PubMed1975

The metabolism of 3-H-cortisone and 3-H-cortisol by the isolated perfused rat and guinea pig lungs.

Isolated perfused rat lungs removed more than 35% of 3-H-cortisone (1 times 10-9M) from the perfusate during one passage through the pulmonary circulation. The cortisone in the lungs was then rapidly converted to cortisol, which was returned to the perfusate. The tritiated steroid taken up was so rapidly washed from the lung, that only 10% remained after a 12 minute perfusion with steroid-free medium. In recirculating experiments, nearly 60% conversion to cortisol occurred over 32 cycles; in addition, there was a slow increase in the percentage of polar compounds in the medium. Similarly, the perfused hindlimbs preparation from the rat converted cortisone to cortisol and returned the cortisol to the perfusate. In contrast, guinea pig isolated perfused lungs had neglible effect on cortisone. Rat lungs demonstrated only a limited ability to convert 3-H-cortisol to cortisone. The results suggest that the lungs may play an important role in maintaining cortisone/cortisol levels in the plasma.

Animals↗
PubMed1998

Time course of active and passive liquid and solute movement in the isolated perfused rat lung model.

The isolated perfused rat lung model (IL) is used to study alveolar epithelial transport properties. Most of the previous studies have been done over a short period of time and have not used the same preparation as a control and intervention group. We evaluated whether the IL preparation could be used for a prolonged period of time (5 h) and studied the rates of active Na+ transport, lung liquid clearance, and passive movement of solutes. Active Na+ transport and lung liquid clearance were stable from 1 to 5 h. The passive movement of small solutes (Na+, mannitol) did not change significantly, and albumin movement increased slightly at the fifth hour. Total RNA isolated from IL after 5 h was intact, and the Na+-K+-ATPase activity in alveolar type II cells isolated at the end of 5-h experiments was equal to Na+-K+-ATPase function from freshly isolated alveolar type II cells. Finally, we measured the stimulatory effect of the beta-adrenergic-agonist terbutaline and the inhibitory effect of the Na+-K+-ATPase-antagonist ouabain by using the same animal as a control. Accordingly, the isolated perfused lung model is functionally stable for at least 5 h, and it could be utilized to evaluate the effect of different interventions by using the same preparation.

Animals↗
PubMed1979

Effect of chlorpromazine on hepatic perfusion and bile secretory function in the isolated perfused rat liver.

The hepatotoxicity of CPZ was studied in the isolated perfused rat liver in order to more closely define possible mechanisms of phenothiazine-induced cholestasis. Perfusate concentrations of CPZ were increased from 5 x 10(-6) M to 5 x 10(-4) M until bile secretion was significantly inhibited. Measurements were then made of determinants of bile secretory function, including the magnitude of lobar distribution of perfusate flow, BAIF, and liver plasma membrane enzyme activity, Na+,K+-ATPase, Mg++-ATPase and 5'-nucleotidase. BAIF diminished significantly from control values of 1.76 +/- 0.07 microliter min-1gm-1 of liver to 1.34 +/- 0.15 and 0.80 +/- 0.09 following 2.5 and 5 x 10(-4) M CPZ, respectively. Perfusate flow also diminished from 5.64 +/- 0.44 to 1.24 +/- 0.12 ml min-1 gm-1 of liver 20 min following 5 x 10(-4) M CPZ and was associated with reduced flow to peripheral areas of the hepatic lobes as demonstrated by Tc-HAM. By 30 min, perfusate flow had returned to baseline values. CPZ also transiently diminished the excretion of bile acids in livers receiving a constant infusion of 40 mumol hr-1 sodium taurocholate. Defects in hepatic perfusion could not account entirely for the impairment in BAIF, since comparable mechanical restriction of perfusate flow in controls only diminished BAIF to 1.49 +/- 0.08 microliter min-1gm-1 of liver. CPZ signofocamt;u rediced tje secofoc actovotu pf Mg++-ATPase and 5'-nucleotidase but did not affect Na+,K+-ATPase in liver plasma membrane isolated 20 min after 5 x 10(-4) M CPZ. CPZ also resulted in a profound shift in the recovery of protein in isolated liver plasma membrane fractions from the light (density = 1.16) to heavier (density = 1.18) fractions. These findings, together with previous observations demonstrating alterations in hepatic ultrastructure, indicate that CPZ interacts in a complex manner with hepatocyte plasma and cytoplasmic membrane components and suggest that these drug-membrane interactions independently result in diminished hepatic perfusion, impairment of bile acid excretion, and inhibition of bile acid-independent bile secretion.

Adenosine Triphosphatases↗
PubMed1975

Analysis of regulatory factors for urea synthesis by isolated perfused rat liver. II. Comparison of urea synthesis in livers of rats subjected to different dietary conditions.

Capacities for urea synthesis and amino acid patterns in the perfused livers isolated from rats fed low and high-protein diets were compared. Urea formation with amjonium chlorode as the nitrogen source in perfused livers isolated from rats fed on a 70% casein diet was rapid and the efficiency of conversion of ammonia to urea was 97.9%. However, that in livers isolated from rats fed on a 5% casein diet was much slower and the efficiency of conversion of ammonia to urea was only 36.1%. The ratios of the rate of urea formation from ammonium chloride to activity of ornithine transcarbamylase [EC 2.1.3.3.] in the perfused livers of rats fed on 5 and 70% casein diets were calculated. The ratio of the former condition was much lower than that of the latter. The ratios reached nearly the same level by the addition of ornithine and N-acetylglutamate, the addition of which to the perfusate caused marked elevation of the ratios in both cases. In the perfused livers from rats fed on a 5% casein diet a considerable portion of the ammonia added to the perfusate was fixed into an amino ro an amide group of amino acids such as alamin, aspartate, and glutamine. On the other hand, in the perfused livers from rats fed on a 70% casein diet most of the ammonia added was converted to urea. The regulation of urea synthesis and the relation between anabolism and catabolism of amino acids in rat livers subjected to different dietary conditions were compared.

Amino Acids↗
PubMed1980

Angiotensin II and norepinephrine after indomethacin in isolated perfused canine kidneys. Tachyphylaxis vs. modulator effect of prostaglandins.

High levels of radioimmunoassayable PGE2 were measured in the perfusate of isolated kidneys. Indomethacin inhibited PGE2 release in this system. Small reductions in the pressor effects of norepinephrine (NE) were associated with increasing perfusate levels of PGE2; a large increase in the pressor effect of NE followed additions of indomethacin and reductions in perfusate PGE2 levels. A marked reduction in pressor responsiveness to angiotensin II (AII) was measured in the isolated kidney which could not be prevented or reversed by indomethacin. It is believed that tachyphylaxis was responsible for the marked reduction in pressor responsiveness to AII and that this is independent of alterations in prostaglandin metabolism. However prostaglandins appeared to modulate the pressor effects of AII as they did NE in the isolated perfused kidney.

Angiotensin II↗
PubMed1996

Development of the model of rat isolated perfused heart for the evaluation of anthracycline cardiotoxicity and its circumvention.

1. In order to develop a predictive model for the preclinical evaluation of anthracycline cardiotoxicity and the means of preventing it, we have studied the functional parameters of perfused hearts isolated from rats receiving repeated doses of several anthracyclines. 2. The anthracyclines studied were doxorubicin, epirubicin, pirarubicin and daunorubicin, and we also studied a liposomal formulation of daunorubicin (DaunoXome) and the co-administration of dexrazoxane (ICRF-187) and doxorubicin. 3. Anthracyclines were administered i.p. at equimolar doses corresponding to 3 mg kg-1 per injection of doxorubicin, every other day for a total of six doses. Dexrazoxane was used at the dose of 30 mg kg-1 per injection and was administered either 30 min before or 30 min after doxorubicin. We evaluated any general toxicity towards the animals as well as alterations of left ventricular contractility and relaxation ex vivo. 4. Epirubicin and daunorubicin were significantly less cardiotoxic than doxorubicin, and neither pirarubicin nor DaunoXome caused significant alterations in cardiac function. There was a direct relationship between the decrease in cardiac contractility or relaxation and anthracycline accumulation in the heart, evaluated after the same treatment schedule. 5. Dexrazoxane induced a significant protection against doxorubicin-induced cardiac toxicity when administered 30 min before doxorubicin, whereas this protection was ineffective when administered 30 min after doxorubicin. Direct perfusion of DaunoXome in isolated hearts of untreated animals resulted in a 12-fold reduction of the accumulation of daunorubicin in heart tissue as compared to the perfusion of free daunorubicin, and did not cause alterations in cardiac function at a dosage for which free daunorubicin induced major alterations. 6. The isolated perfused rat heart appears to be a valuable model for screening of new anthracyclines and of strategies for circumventing anthracycline cardiotoxicity.

Animals↗
PubMed1996

Effect of chronic alcohol consumption by rats on tumor necrosis factor-alpha and interleukin-6 clearance in vivo and by the isolated, perfused liver.

The effects of chronic (16-week) alcohol consumption by rats on [125I] tumor necrosis factor (TNF)-alpha and [125I]interleukin (IL)-6 plasma clearance and organ distribution in vivo and uptake and metabolism by the isolated, perfused liver were studied. Alcohol was administered to rats in a liquid diet for 16 weeks, and caused a decreased (48%) plasma clearance rate of IL-6 and converted the plasma clearance kinetics of the cytokine from a biphasic exponential in normal rats to a monophasic exponential decay. Alcohol feeding significantly increased (101%) plasma clearance of TNF-alpha, which followed a biphasic exponential decay and decreased the T1/2 for both the alpha (67%) and beta (76%) elimination components. The distribution of both cytokines in trichloroacetic acid precipitable and non-precipitable fractions of liver, spleen, stomach, small intestine (ileum), lung, kidney, and blood was also studied. The only effect of alcohol treatment was a significant decrease in IL-6 uptake and metabolism by the small intestine. Perfused livers, isolated from alcohol-fed rats, took up and metabolized larger amounts of IL-6 than did livers isolated from pair-fed rats. TNF-alpha uptake and metabolism by the isolated, perfused liver were not affected by chronic alcohol consumption. Regardless of the animal treatment, the isolated perfused liver took up and metabolized significantly larger (17-fold) amounts of TNF-alpha than IL-6, in spite of identical concentrations of cytokines (6 nM) in the perfusion medium. The data presented in this study along with our previous results demonstrating the effects of alcohol consumption on TNF-alpha and IL-6 receptors on various liver cells suggest that the effects of chronic alcohol treatment on cytokine clearance cannot be ascribed to changes in the receptors for the two cytokines. Also, no correlation was found between the effects of alcohol treatment on plasma cytokine clearance and uptake and metabolism of cytokines by the isolated, perfused liver. Experimental data and theoretical considerations suggest that cytokine receptor recycling may play an important role in mediating alcohol effects on cytokine clearance.

Alcohol Drinking↗
PubMed1982

Hepatic drug transport in the rat. A comparison between isolated hepatocytes, the isolated perfused liver and the liver in vivo.

The hepatic transport of three different drugs, the organic anion dibromosulphophthalein, the organic cation d-tubocurarine and the uncharged compound ouabain was studied in vivo in the isolated perfused rat liver and isolated hepatocytes. The respective clearances by uptake were determined for the various substrates and corrected for differences in hepatic blood flow and extracellular protein binding in the three liver preparations. The corrected uptake values in the intact organ, in vivo and in the isolated perfused liver were highly comparable; for dibromosulphophthalein a clearance of 2.1 ml/min per 10(6) hepatocytes was found in vivo, whereas in perfusion a value of 2.4 ml/min per 10(6) cells was calculated. For d-tubocurarine, the values were 34 x 10 (-4) and 55 x 10(-4) ml/min per 10(6) cells obtained in vivo and in the isolated perfused organ, respectively. With ouabain as the substrate, the in vivo clearance amounted to 5.1 x 10(-2), whereas in the isolated perfused liver a value of 4.8 x 10(-2) ml/min per 10(6) cells was calculated. The clearance by uptake obtained for dibromosulphophthalein was ouabain in the isolated hepatocytes appeared to be a factor of 2-3 lower than in the intact organ. In the case of d-tubocurarine however the clearance was identical to that in vivo and the isolated perfused liver. The rate of secretion from isolated hepatocytes was, for dibromosulphophthalein identical to, and for d-tubocurarine and ouabain lower than that in the intact organ, especially as compared with the in vivo preparation. It is concluded that transport function is well preserved in the isolated perfused liver and isolated hepatocytes. For certain substrates freshly isolated hepatocytes may exhibit a somewhat lower uptake and/or secretion rate, in spite of a good cell quality as judged by generally accepted criteria for cell viability. Whether this is due to changes in membrane composition (not detected by our viability tests) or a selection of a subpopulation of hepatocytes, is discussed.U

Animals↗
PubMed1985

Aging in vivo and neuraminidase treatment of rat erythrocytes and their sequestration by the isolated perfused rat liver.

The isolated perfused rat liver as a model for phagocytosis leaves the specific arrangement of macrophages in the original tissue structure intact. Therefore phagocytosis may occur under terms closely approaching physiological conditions. Separation of rat erythrocytes according to their density in 13% young, 75% mature and 12% old cells permits the establishment of differences in the mean cellular volume (MCV), mean cellular hemoglobin concentration (MCHC) and content of N-acetylneuraminic acid per red blood cell (NANA/RBC). Perfusion of these fractions does not result in significant differences of sequestration of the isolated liver. Enzymatic release of 90% of membrane-bound sialic acid causes a significant increase of 20% in the elimination of erythrocytes from the perfusate. Treatment with neuraminidase is a trigger for sequestration either by adherence or by complete phagocytosis. Incubation of neuraminidase-treated erythrocytes with plasma does not increase the removal rate of cells during perfusion.

Animals↗
PubMed1993

Activated polymorphonuclear leukocytes increase low-level chemiluminescence of isolated perfused rat lungs.

Low-level chemiluminescence was measured in isolated perfused rat lungs subjected to different types of oxidative stress: perfusion with tert-butyl hydroperoxide (t-BOOH) or stimulation of polymorphonuclear cells (PMN). The time required for t-BOOH-dependent lung chemiluminescence to return to background levels was proportional to the concentration of t-BOOH. From the half times of the decay at different t-BOOH concentrations, we estimated that the lungs metabolize organic peroxides at a rate of 0.045 mM/min. Use of a high dose of t-BOOH (3 mM)or pretreatment of lungs with 1,3-bis(2-chloroethyl)-nitrosourea (100 micrograms/ml) to inhibit glutathione reductase produced chemiluminescence that was much greater and did not decay. Stimulation of 5 x 10(7) PMN with 1 micrograms of phorbol myristate acetate resulted in significant increases in chemiluminescence that occurred in the absence of a significant lung weight gain or measurable lipid peroxidation. Perfusion of isolated lungs with superoxide dismutase (100 U/ml) completely inhibited the chemiluminescence response to PMN activation, whereas treatment with 100 microM U-74389F, a lipid-soluble antioxidant, also significantly decreased PMN-dependent chemiluminescence. Neither catalase (2,000 U/ml) nor 100 microM U-78518F, a water-soluble antioxidant, decreased chemiluminescence after PMN activation. These results indicate that low-level chemiluminescence is a sensitive indicator of oxidative stress in the isolated perfused rat lung and provides a tool for devising and characterizing the effectiveness of antioxidant interventions.

Animals↗
PubMed1982

The effect of burn injury on the heart in the whole body and on the extracorporeally perfused isolated heart.

Cardiac performance during the initial stage of burn shock in dogs was studied in the whole body and compared with the function of the isolated hearts, perfused from donor animals similarly burned. Continuous recording of the functional indices using electro-magnetic flowmetry and ECG with on-line processing of the data enabled us to establish the following facts. Progressive decrease of cardiac output secondary to the depression of myocardial contractility against the background of stable inflow takes place as early as 2-5 minutes after massive burning in the whole body. No significant changes of the functional indices of the isolated hearts, perfused from the perfusion donors similarly burned, were observed in the second group of experiments. The underlying mechanisms of the above phenomena are discussed.

Animals↗
PubMed1991

Effect of plasma from patients with essential hypertension on vascular resistance in the isolated perfused rat kidney.

1. Isolated perfused rat kidneys were used to study the effects of plasma fractions obtained by gel filtration from essential hypertensive patients (n = 40) and from normotensive subjects (n = 36) on resistance vessels. Perfusion pressure was recorded at a constant flow. 2. Plasma fractions were obtained by gel filtration and contained substances with a molecular mass in the range 1000-1500 Da. The plasma fractions from hypertensive patients used in this study had been shown to increase blood pressure after intravenous injection in rats. 3. In the isolated rat kidneys, the hypertensive fractions increased perfusion pressure by 20 +/- 17 mmHg (mean +/- SD, range 5-58 mmHg, n = 40). The analogous fractions from normotensive subjects did not change perfusion pressure significantly. 4. In Ca2(+)-free medium containing 2 mmol/l ethyleneglycol bis-(aminoethyl ether)tetra-acetate, the change in perfusion pressure induced by active plasma fractions was reduced by 95.2 +/- 6.3%. Addition of nifedipine to the perfusion medium reduced, but did not abolish, the pressure response of the kidneys. 5. In solutions containing phentolamine or saralasin, vasoconstriction was not reduced. 6. Thus in the active fractions from hypertensive plasma, a vasopressor agent with direct action on resistance vessels can be demonstrated. This substance probably acts by increasing Ca2+ influx in vascular smooth muscle cells.

Adult↗
PubMed1988

Inhibitory effect of nicotine on benzo(a)pyrene elimination and marked pulmonary metabolism of nicotine in isolated perfused rat lung.

Concomitant administration of nicotine to the perfusion medium of an isolated perfused rat lung decreased the pulmonary elimination of benzo(a)pyrene entering the lung by vascular access. This inhibitory effect of nicotine was observed at a concentration of 0.5 microM nicotine in the perfusion medium. The elimination half-life of benzo(a)pyrene given as a bolus to establish an initial concentration of 1 microM in the perfusate was increased by a factor of about 3 after the addition of nicotine at 60 min of perfusion. A comparably high increase of the benzo(a)pyrene steady-state concentration was observed during constant infusion experiments when nicotine as the interfering substrate was infused parallelly after a control period of 60 min. In perfused rat lungs the rate of metabolism of nicotine given as a bolus (initial concentration of 0.5 microM) was comparable to the rate in isolated rat livers for 5,6-benzoflavone, oil or saline pretreated animals. Induction of cytochrome P-450 by pretreatment of the rats with phenobarbital caused an eightfold increase of the nicotine clearance in isolated livers, whereas the pulmonary nicotine clearance was almost doubled. Overall, expressed per g organ weight, the isolated perfused rat lung exhibited a marked capacity to metabolize nicotine while even the maximally induced phenobarbital-stimulated rat liver reached only 60% of the corresponding clearance values (per g organ) of the isolated rat lung. For the 5,6-benzoflavone and control treatments, values of 14%-21% were estimated for the liver compared with lung.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1983

Effects of hematocrit on oxygenation of the isolated perfused rat liver.

The isolated perfused rat liver is used ubiquitously for metabolic and endocrine studies of hepatic function, yet few data are available regarding the inadequacy of the oxygenation of such preparations. Moreover, the isolated rat liver is usually deprived of its arterial supply and perfused via the hepatic portal vein with low-hematocrit or cell-free solutions. To investigate the efficacy of the oxygen supply, we determined the effect of hematocrit on the relation between oxygen consumption and perfusate flow. We then attempted to define a hematocrit at which hepatic oxygenation was maximal. Livers of male rats anesthesized with pentobarbital sodium were perfused via the portal vein with fresh canine red blood cells suspended in Krebs-Ringer-bicarbonate buffer. Perfusions were carried out at various flow rates, and the relation between perfusate flow and oxygen uptake was determined. At flow rates above 100 ml X min-1 X 100 g liver-1, oxygen uptake was independent of flow but below that value was flow limited, regardless of whether the hematocrit was 10, 20, or 40%. To determine the optimal hematocrit for hepatic oxygen uptake, hepatic portal venous and hepatic venous pressures were held at 10 and 0 mmHg, respectively. The hematocrit was lowered in steps from 80 to 10%. Blood flow increased exponentially as hematocrit fell while oxygen uptake increased to a maximum at approximately 20%. It is concluded that an hematocrit of approximately 20% provides the optimal combination of blood flow and oxygen-carrying capacity while maintaining physiological perfusion pressures, e.g., 10 mmHg.

Animals↗