First Hour Of Perfusion Research Articles

Subnormothermic Ex Vivo Porcine Kidney Perfusion Improves Energy Metabolism: Analysis Using 31P Magnetic Resonance Spectroscopic Imaging.

Background.The ideal preservation temperature for donation after circulatory death kidney grafts is unknown. We investigated whether subnormothermic (22 °C) ex vivo kidney machine perfusion could improve kidney metabolism and reduce ischemia-reperfusion injury.Methods.To mimic donation after circulatory death procurement, kidneys from 45-kg pigs underwent 60 min of warm ischemia. Kidneys were then perfused ex vivo for 4 h with Belzer machine perfusion solution UW at 22 °C or at 4 °C before transplantation. Magnetic resonance spectroscopic imaging coupled with LCModel fitting was used to assess energy metabolites. Kidney perfusion was evaluated with dynamic-contrast enhanced MRI. Renal biopsies were collected at various time points for histopathologic analysis.Results.Total adenosine triphosphate content was 4 times higher during ex vivo perfusion at 22 °C than at 4 °C perfusion. At 22 °C, adenosine triphosphate levels increased during the first hours of perfusion but declined afterward. Similarly, phosphomonoesters, containing adenosine monophosphate, were increased at 22 °C and then slowly consumed over time. Compared with 4 °C, ex vivo perfusion at 22 °C improved cortical and medullary perfusion. Finally, kidney perfusion at 22 °C reduced histological lesions after transplantation (injury score: 22 °C: 10.5 ± 3.5; 4 °C: 18 ± 2.25 over 30).Conclusions.Ex vivo kidney perfusion at 22°C improved graft metabolism and protected from ischemia-reperfusion injuries upon transplantation. Future clinical studies will need to define the benefits of subnormothermic perfusion in improving kidney graft function and patient’s survival.

344.3: Extracellular Vesicles Released During Normothermic Machine Perfusion Are Associated With Human Donor Kidney Characteristics

Background: Extracellular Vesicles (EVs) represent stable, tissue specific nano-sized particles that reflect the conditional state of their tissue of origin. Here, the dynamic release and phenotype of kidney EVs was characterized and quantified during Normothermic Machine Perfusion (NMP) of Expanded-Criteria Donor (ECD) kidneys to examine whether EVs could function as a potential biomarker for assessing kidney quality before transplantation. Materials & Methods: Eight discarded ECD kidneys (~13 ± 5 hours of cold ischemia, age 68 ± 7 (mean ± standard deviation), all male) were perfused in a closed system at 37 0C for 6 hours. Perfusates were taken before and at 1, 3 and 6 hours of NMP and examined with Nanoparticle Tracking Analysis (NTA) and Imaging Flow Cytometry (IFCM). For IFCM, perfusates were stained with the tetraspanins CD9, CD63 or CD81 (general EV markers), or a mix of these three markers in combination with CFDA-SE (a non-fluorescent molecule that acquires fluorescent properties after cleavage by intravesicular esterases) to identify, quantify and characterize EVs. Results: Analysis of perfusates with NTA revealed that the majority of nanoparticles present in the perfusates are <300 nm. For CFSE and the mix of tetraspanin double-positive EVs, we observed a ~700/ 740/ 560 fold increase compared to EV levels before perfusion at 1, 3 and 6 hours of NMP, respectively. Analysis of EV concentrations with crude donor characteristics (e.g. age, cold ischemia time (CIT), kidney weight) and NMP viability characteristics (renal flow, renal flow resistance, urine production) revealed that double-positive EV are negatively correlated with CIT whilst positive correlations were found with donor age after the first hour of NMP. Furthermore, tetraspanin CD81 was found to represent the majority (~75%) of the excreted double-positive EV (CD9: ~16%/ CD63 ~8%) (Figure). Conclusion: EVs <300 nm are released by ECD kidneys during NMP with highest excretion levels during the first hour of perfusion. Tetraspanin CD81 is predominantly present on these EVs, and EV concentrations were shown to be correlated with well-established indicators of kidney quality such as donor age and CIT. The characterization of the excreted EVs as well as their correlation with clinical parameters provide a starting point to study their role as potential biomarkers of kidney quality.

The metabolic fate of oxaliplatin in the biological milieu investigated during in vivo lung perfusion using a unique miniaturized sampling approach based on solid-phase microextraction coupled with liquid chromatography-mass spectrometry.

Adjuvant chemotherapy after pulmonary metastasectomy for colorectal cancer may reduce recurrence and improve survival rates; however, the benefits of this treatment are limited by the significant side effects that accompany it. The development of a novel in vivo lung perfusion (IVLP) platform would permit the localized delivery of high doses of chemotherapeutic drugs to target residual micrometastatic disease. Nonetheless, it is critical to continuously monitor the levels of such drugs during IVLP administration, as lung injury can occur if tissue concentrations are not maintained within the therapeutic window. This paper presents a simple chemical-biopsy approach based on sampling with a small nitinol wire coated with a sorbent of biocompatible morphology and evaluates its applicability for the near-real-time in vivo determination of oxaliplatin (OxPt) in a 72-h porcine IVLP survival model. To this end, the pigs underwent a 3-h left lung IVLP with 3 doses of the tested drug (5, 7.5, and 40 mg/L), which were administered to the perfusion circuit reservoir as a bolus after a full perfusion flow had been established. Along with OxPt levels, the biocompatible solid-phase microextraction (SPME) probes were employed to profile other low-molecular-weight compounds to provide spatial and temporal information about the toxicity of chemotherapy or lung injury. The resultant measurements revealed a rather heterogeneous distribution of OxPt (over the course of IVLP) in the two sampled sections of the lung. In most cases, the OxPt concentration in the lung tissue peaked during the second hour of IVLP, with this trend being more evident in the upper section. In turn, OxPt in supernatant samples represented ∼25% of the entire drug after the first hour of perfusion, which may be attributable to the binding of OxPt to albumin, its sequestration into erythrocytes, or its rapid nonenzymatic biotransformation. Additionally, the Bio-SPME probes also facilitated the extraction of various endogenous molecules for the purpose of screening biochemical pathways affected during IVLP (i.e., lipid and amino acid metabolism, steroidogenesis, or purine metabolism). Overall, the results of this study demonstrate that the minimally invasive SPME-based sampling approach presented in this work can serve as (pre)clinical and precise bedside medical tool.

Sildenafil crosses the placenta at therapeutic levels in a dually perfused human cotyledon model

Sildenafil already is administered during gestation in patients with pulmonary hypertension and is under evaluation as a treatment for several pregnancy complications, such as preeclampsia and intrauterine growth restriction. Animal studies have shown a potential therapeutic effect of the drug in fetuses with congenital diaphragmatic hernia, rescuing peripheral pulmonary vasculature, and airway phenotype. When considering this drug for evaluation in a clinical trial, data on effective human placental drug passage are required. We quantified transplacental passage of sildenafil in the exvivo dually perfused cotyledon model. Six placentas that were collected after term delivery from healthy volunteers were cannulated and perfused dually. Sildenafil citrate was added to the maternal circulation at 2 different concentrations: 500 ng/mL, which represented the maximum tolerated concentration (n=3), and 50 ng/mL, which represented the therapeutic concentration (n=3). Samples were collected from both the fetal and the maternal reservoir at 0, 6, 30, 60, 90, 120, 150, and 180 minutes; the concentrations of sildenafil and its metabolite desmethyl-sildenafil were determined with the use of high performance liquid chromatography. The fetal/maternal concentration ratio was calculated for each timepoint. Transfer clearance was calculated as the rate of maternal to fetal passage/maternal concentration. Sildenafil crossed the placenta at both maximal and therapeutic concentrations. Maternal and fetal levels reached a plateau at 90-120 minutes. Transfer clearance was the highest during the first hour of perfusion: 3.15 mL/min (range, 2.14-3.19 mL/min) for the maximum tolerated concentration and 3.07mL/min (range, 2.75-3.42 mL/min) for the therapeutic concentration (not significant). The fetomaternal concentration ratio significantly increased over time, up to 0.91±0.16 for the maximal concentration and 0.95±0.22 for the therapeutic concentration at the end of the perfusion (not significant). Desmethyl-sildenafil was not detected in any sample. Sildenafil crosses the term placenta at a relatively high rate exvivo, which suggests that there is sufficient placental transfer to reach clinically active fetal drug levels at the currently used maternal doses.

Performance of cold-preserved rat liver Microorgans as the biological component of a simplified prototype model of bioartificial liver.

AIMTo develop a simplified bioartificial liver (BAL) device prototype, suitable to use freshly and preserved liver Microorgans (LMOs) as biological component.METHODSThe system consists of 140 capillary fibers through which goat blood is pumped. The evolution of hematocrit, plasma and extra-fiber fluid osmolality was evaluated without any biological component, to characterize the prototype. LMOs were cut and cold stored 48 h in BG35 and ViaSpan® solutions. Fresh LMOs were used as controls. After preservation, LMOs were loaded into the BAL and an ammonia overload was added. To assess LMOs viability and functionality, samples were taken to determine lactate dehydrogenase (LDH) release and ammonia detoxification capacity.RESULTSThe concentrations of ammonia and glucose, and the fluids osmolalities were matched after the first hour of perfusion, showing a proper exchange between blood and the biological compartment in the minibioreactor. After 120 min of perfusion, LMOs cold preserved in BG35 and ViaSpan® were able to detoxify 52.9% ± 6.5% and 53.6% ± 6.0%, respectively, of the initial ammonia overload. No significant differences were found with Controls (49.3% ± 8.8%, P < 0.05). LDH release was 6.0% ± 2.3% for control LMOs, and 6.2% ± 1.7% and 14.3% ± 1.1% for BG35 and ViaSpan® cold preserved LMOs, respectively (n = 6, P < 0.05).CONCLUSIONThis prototype relied on a simple design and excellent performance. It’s a practical tool to evaluate the detoxification ability of LMOs subjected to different preservation protocols.

Post myocardial infarction left bundle branch block disappearing with levosimendan

A 51 year old man presented with a Killip III acute anterior myocardial infarction due to a very late thrombosis of a stent in the proximal left anterior descending artery. the eKg after being admitted to the coronary care unit following primary pCI showed a new onset left bundle branch block (lBBB) (Figure 1: A). In spite of the initial favorable evolution throughout the first two days under treatment with diuretics and ACe inhibitors, the lBBB persisted. during the third day congestive signs worsened, together with a reduction in diuresis and worsening of the renal function. At that point, treatment with 0.1 mcg/kg/ min levosimendan perfusion was administered for 24 hours. During the first hours of perfusion the LBBB disappeared in the monitor and the eKg at the end of the perfusion showed narrow Qrs with persistent st elevation (Figure 1: B).

(750) - Lineage Tracing of Host and Graft Cells After Lung Transplant and During Club Cell Ablation Induces Allograft Rejection

Best results were achieved with GalTKO.hCD46.hCD55.hEPCR.hCD47. hTFPI lungs when immunosuppression was given (30hrs LS, n= 2). In both cases, animals died due to ventricular fibrillation with functioning xenografts. Macroand microscopically, lung tissue from these experiments appeared grossly normal. Pathologic findings, previously seen in recipient animals, such as progressive ascites, escalating volume and inotrope requirements and native (baboon) lung edema, were not seen in recent experiments. In association with GPIb blockade and donor DDAVP pre-treatment, platelet consumption was < 10% during the first hour of perfusion. Conclusion: These preliminary in vivo results support our hypothesis that effective control of complement and coagulation (mainly by pig genetics) and judicious use of drugs to block other important pathways (non-physiologic vWF/GPIb interaction, thromboxane and histamine release) will be necessary and sufficient to accomplish prolonged life-supporting lung xenograft function, and may translate into other xenogeneic model as well.

Expression of Human CD46 Modulates Inflammation Associated With GalTKO Lung Xenograft Injury

Evaluation of lungs from GalTKO.hCD46 pigs, genetically modified to lack the galactose-α(1,3)-galactose epitope (GalTKO) and to express human CD46, a complement regulatory protein, has not previously been described. Physiologic, hematologic and biochemical parameters during perfusion with heparinized fresh human blood were measured for 33 GalTKO.hCD46, GalTKO (n = 16), and WT pig lungs (n = 16), and 12 pig lungs perfused with autologous pig blood. Median GalTKO.hCD46 lung survival was 171 min compared to 120 for GalTKO (p = 0.27) and 10 for WT lungs (p < 0.001). Complement activation, platelet activation and histamine elaboration were significantly reduced during the first 2 h of perfusion in GalTKO.hCD46 lungs compared to GalTKO (ΔC3a at 120' 812 ± 230 vs. 1412 ± 1047, p = 0.02; ΔCD62P at 120' 9.8 ± 7.2 vs. 25.4 ± 18.2, p < 0.01; Δhistamine at 60' 97 ± 62 vs. 189 ± 194, p = 0.03). We conclude that, in addition to significant down-modulation of complement activation, hCD46 expression in GalTKO lungs diminished platelet and coagulation cascade activation, neutrophil sequestration and histamine release. Because GalTKO.hCD46 lung failure kinetics correlated directly with platelet and neutrophil sequestration, coagulation cascade activation and a rise in histamine levels within the first hour of perfusion, further progress will likely depend upon improved control of these pathways, by rationally targeted additional modifications to pigs and pharmacologic interventions.

Hyperinsulinaemia increases vascular resistance and endothelin‐1 expression in the equine digit

Insulin leads to overexpression of endothelin-1 (ET-1) in the endothelium of insulin-resistant rodents. If this is also the case in equine laminar tissue, this could explain the predisposition of insulin-resistant horses to laminitis. To investigate the effect of hyperinsulinaemia on metabolism and vascular resistance of the isolated equine digit in a model of extracorporeal perfusion. Randomised, controlled study with interventional group, with blinded evaluation of histology results. After exsanguination, equine digits (n = 11) and autologous blood were collected at an abattoir. One digit served as a hyperinsulinaemic pilot limb, 5 digits were assigned to the hyperinsulinaemic perfusion (IP) group and 5 to the control perfusion (CP) group. Digits were perfused for 10 h at a defined perfusion rate of 12 ml/min/kg. After the first hour of perfusion (equilibration period), insulin was added to the reservoir of the IP digits. Perfusion pressure, glucose consumption, lactate and lactate dehydrogenase were monitored. Vascular resistance was calculated as perfusion pressure (in millimetres of mercury) in relation to the flow rate (in millilitres per minute). After perfusion, histology samples of the dorsal hoof wall (haematoxylin & eosin or periodic acid-Schiff) were evaluated. Immunohistology with a polyclonal rabbit-derived anti-endothelin antibody was used for detection of ET-1. In the IP group, the mean insulin concentration in the plasma of the perfusate was 142 ± 81 μiu/ml, while insulin concentration was <3 μiu/ml in the CP group. Mean vascular resistance was significantly higher (P<0.01) in the IP group (2.04 ± 1.13 mmHg/ml/min) than in the CP group (1.31 ± 0.55 mmHg/ml/min). Histology of the IP group samples showed significantly more vessels with an open lumen, increased width of the secondary epidermal lamellae and formation of oedema. In the lamellar vessels (veins and arteries) and nerve fibres, ET-1 expression was much more prominent in the IP group than in the CP group samples. Short-term hyperinsulinaemia leads to increased vascular resistance in the equine digit and increased expression of ET-1 in the laminar tissue.

Blockade of leucocyte function-associated antigen-1 (LFA-1) decreases lymphocyte trapping in the normal pulmonary vasculature: studies in the isolated buffer-perfused rat lung.

Adhesion molecules regulate the migration of lymphocytes in lymphoid and non-lymphoid organs. In the lung, little is known about lymphocyte sticking and migration through the pulmonary vascular endothelium in physiological or pathological situations. Therefore the isolated buffer-perfused rat lung was used to investigate the mobilization of lymphocytes out of the normal lung into the venous effluent and to the bronchoalveolar space. The lymphocyte subset composition was characterized in the venous effluent, the lung tissue and the bronchoalveolar lavage (BAL) using immunocytology. Lymphocytes continuously left the normal lung at a total of 5.0 +/- 0.7 x 106 cells within the first hour of perfusion. The injection of 200 x 106 lymphocytes via the pulmonary trunk increased the venous release of lymphocytes by 170%. To investigate the effect of LFA-1 and CD44 on the adhesion of lymphocytes to the pulmonary endothelium, lymphocytes preincubated with an anti-LFA-1 MoAb, which blocks the interaction of LFA-1 and intercellular adhesion molecule-1 (ICAM-1), or lymphocytes preincubated with an anti-CD44 MoAb, were injected. The injection of LFA-1-blocked lymphocytes led to an increase by 70% of injected cells recovered in the perfusate within the first hour, whereas anti-CD44 treatment of injected lymphocytes had no effect. The LFA-1-blocked lymphocytes showed higher numbers of T and B cells in the effluent. Thus, the present experiments demonstrate that LFA-1 influences the trapping of lymphocytes in the vasculature of the healthy rat lung.

Treatment of postoperative pain for burn patients with intravenous analgesia in continuous perfusion using elastomeric infusors

Introduction: Postoperative pain after surgery with patients suffering from burns tends to be moderate or severe, and its treatment requires a combination of high-strength analgesics (opioids) with others having different action mechanisms according to the concept of multimodal analgesia. Aims: In this article we propose the use of continuous intravenous analgesia with morphine using elastomeric infusors at fixed dose for the treatment of this kind of pain. An evaluation is made of its analgesic efficacy, side effects and level of satisfaction. Material and methods: A study was made of 17 burn patients operated on in our unit who received continuous intravenous analgesia during the postoperative period, with morphine at 1 mg/h, using elastomeric infusors for a period of 24 h. Its analgesic efficacy was analysed using the visual analogical scale (VAS) at different moments; side effects and the level of acceptance by the patient was also evaluated. Results: The results confirm a good analgesic effect after 2 h from starting perfusion (VAS < 3). The side effects reveal a similar or lesser incidence to the use of morphine in bolus or using the PCA system, and in no cases did they require treatment to be halted. The level of acceptance of the procedure by patients was good. Conclusion: This method reveals a high level of analgesic efficacy in the postoperative period with burn patients in this study. However, it is important to note the lower results obtained in the first hours of perfusion, and proposing a heavy initial dose of analgesics when starting perfusion. This is presented as an efficient analgesic method that is easy to apply, has a low cost, and the possibility of extending its indications to ambulatory treatment.

The effect of dichloroacetate and alanine on the metabolic recovery of perfused mouse liver after cold ischemia

Pyruvate dehydrogenase has been thought to be involved in the improved recovery of livers, from fasted donors, reperfused with alanine after cold preservation. The aim of this work was to investigate the effect on perfused mouse liver of dichloroacetate, an activator of this enzyme. Livers from fed and fasted animals were perfused with oxygenated Krebs–Henseleit buffer for 30 min, then stored at 4 °C in University of Wisconsin solution for 48 h. Then reperfusion at 37 °C was performed with Krebs–Henseleit buffer containing 2 mM dichloroacetate for 1 h. (3-13C)Alanine (8 mM) was then added and perfusion was continued for a second hour. 31P-NMR was used to measure nucleoside triphosphate recovery of the livers. At the end of reperfusion, 13C-NMR spectra of perfusates were recorded. Dichloroacetate (DCA) was found to activate pyruvate dehydrogenase in all cases. However, it decreased the functional recovery of livers from both fed and fasted mice. In order to study the effect of alanine on this DCA deleterious effect, we reperfused the livers according to a modified protocol. The first hour of perfusion without alanine was omitted and the organs were reperfused directly for 1 h in the presence of 2 mM dichloroacetate and 8 mM (3-13C)alanine. In this protocol, the deleterious effect of DCA was completely suppressed for livers from fasted mice. These results led to the conclusion that the specific beneficial effect of alanine on livers from fasted livers persists in the presence of DCA and thus cannot be explained solely by the induction of a greater pyruvate dehydrogenase reaction rate. Copyright © 1999 John Wiley & Sons, Ltd.

The effect of dichloroacetate and alanine on the metabolic recovery of perfused mouse liver after cold ischemia.

Pyruvate dehydrogenase has been thought to be involved in the improved recovery of livers, from fasted donors, reperfused with alanine after cold preservation. The aim of this work was to investigate the effect on perfused mouse liver of dichloroacetate, an activator of this enzyme. Livers from fed and fasted animals were perfused with oxygenated Krebs-Henseleit buffer for 30 min, then stored at 4 degrees C in University of Wisconsin solution for 48 h. Then reperfusion at 37 degrees C was performed with Krebs-Henseleit buffer containing 2 mM dichloroacetate for 1 h. (3-(13)C)Alanine (8 mM) was then added and perfusion was continued for a second hour. (31)P-NMR was used to measure nucleoside triphosphate recovery of the livers. At the end of reperfusion, (13)C-NMR spectra of perfusates were recorded. Dichloroacetate (DCA) was found to activate pyruvate dehydrogenase in all cases. However, it decreased the functional recovery of livers from both fed and fasted mice. In order to study the effect of alanine on this DCA deleterious effect, we reperfused the livers according to a modified protocol. The first hour of perfusion without alanine was omitted and the organs were reperfused directly for 1 h in the presence of 2 mM dichloroacetate and 8 mM (3-(13)C)alanine. In this protocol, the deleterious effect of DCA was completely suppressed for livers from fasted mice. These results led to the conclusion that the specific beneficial effect of alanine on livers from fasted livers persists in the presence of DCA and thus cannot be explained solely by the induction of a greater pyruvate dehydrogenase reaction rate.

Study of the Biotransformation of Benfluron Using the Isolated Perfused Rat Liver

The isolated perfused rat liver method (IPRL) was used to find, isolate and identify further metabolites of Phase I and Phase II biotransformation of the potential cytostatic agent benfluron with special regard to the conjugation processes. Its pharmacokinetic profile during the perfusion was also estimated. The rat liver was isolated from the body and perfused in vitro using a recirculating perfusion system. Benfluron was added to the reservoir as a bolus in doses of 200, 100, 30 mg/kg of body weigh and 1 mg/perfusate volume and also as a continual infusion in a dose of 0.1 mg/min in separate series of experiments. The following metabolites formed during Phase I biotransformation were found in the perfusion liquid as well as in the bile: benfluron N-oxide, 9-hydroxy benfluron, demethylated 9-hydroxy benfluron, demethylated benfluron, and reduced benfluron. The major Phase II metabolite found in the bile samples was the glucuronide of 9-hydroxy benfluron. The pharmacokinetic profile of benfluron in IPRL indicated its main disposition and metabolic pathway, i.e. its rapid extraction from perfusate by the liver (t1/2 alpha = 3.76 min), 9-hydroxylation followed up O-glucuronidation and excretion to the bile. It was revealed that 12% of the total dose of the parent compound was excreted to the bile in the form of conjugates during the first hour of perfusion, 32% during 1.5 hour, and 70% during 2 hours after the administration of benfluron. The conjugates with glucuronic acid represented 96-98% of all metabolites found in the bile.

Beneficial effect of alanine on metabolic recovery of fasted livers submitted to cold ischemia.

The aim of this study was to investigate the possible beneficial effect on perfused mouse liver of alanine as an exogenous substrate for gluconeogenesis. Livers from fed and fasted animals were perfused with oxygenated Krebs' Henseleit buffer for 30 min, then stored at 4 degrees C in University of Wisconsin solution for 48 h. Then reperfusion at 37 degrees C was performed according to two protocols. In the first one, reperfusion with alanine-free Krebs' Henseleit buffer was used for 1 h. 8 mM (3-(13)C) alanine was then added and perfusion was prolonged for a second hour. In the second one, the first hour of perfusion was omitted and the organs were reperfused directly for an hour in the presence of 8 mM (3-(13)C)alanine. 31P NMR was used to measure the NTP recovery of the livers. At the end of the reperfusions, 13C and 1H NMR spectra of perfusates and of glutamine extracted from these perfusates by HPLC were recorded. These data were analysed according to a model of liver metabolism assuming that the only substrate of the liver was (3-(13)C)alanine and endogenous substrates were metabolizable only through pyruvate. It was found that in the absence of initial alanine at reperfusion, livers from fasted mice recovered less NTP than those of fed ones (40 +/- 4% vs 60 +/- 5%, p <0.01), but not if this substrate is present at the beginning of reperfusion (61 +/- 5% vs 60 +/- 5%). This was confirmed by the amount of labelled metabolites produced. However, the dilution of 13C labelled metabolites by unlabelled ones did not indicate a larger concentration of endogenous substrates in livers from fed mice. The conclusion reached was that the lower pyruvate dehydrogenase activity of livers from fasted mice relatively to that from fed mice could be compensated for by the greater pyruvate concentration provided by alanine for the initial production of NTP after cold ischemia and warm reperfusion.

Adhesion of coagulase-negative staphylococci and adsorption of plasma proteins to heparinized polymer surfaces

Protease treatment of cells of coagulase-negative staphylococci reduced the adhesion of bacteria to heparinized polyethylene preadsorbed with serum. Fibronectin (Fn), fibrinogen (Fg), vitronectin, complement factor C3c, plasminogen, laminin and to a low extent albumin were detected on tridodecylmethylammonium chloride (TDMAC)-heparinized polyvinyl chloride (PVC) catheters extirpated from the circulation of patients. Using a perfusion model we show that during the first hours of perfusion with human plasma, Fn and Vn dominate, whereas after 22 h of perfusion Fg is the dominant protein. Field emission scanning electron microscopy and atomic force microscopy studies on TDMAC-heparinized catheters as well as on end-point attached heparinized PVC catheters indicate that quantitatively more Fg than Fn is exposed on these surfaces after prolonged exposure (>22 h) to human plasma. Biomaterials (1994) 15, (10) 805–814

Dependence of oxygen uptake on ambient PO2 in isolated perfused frog skin.

Rates of O2 uptake across isolated perfused skin of bullfrogs (Rana catesbeiana) were measured in relation to blood flow at three levels of ambient O2 tension: normoxia (O2 tension = 152 torr), hypoxia (12% O2, 87 torr) and hyperoxia (42% O2, 306 torr). At bulk perfusion rates ranging from 3.4 to 10.1 microliters.cm-2 x min-1, O2 uptake was positively correlated with hemoglobin delivery rate in both normoxia and hyperoxia, but was independent of delivery rate in hypoxia. Mean O2 uptake in normoxia was 3.8 nmol O2 x cm-2 x min-1 at a delivery rate of 9.8 nmol.cm-2 x min-1 and 6.5 nmol O2 x cm-2 x min-1 at a delivery rate of 28.3 nmol.cm-2 x min-1. At any given bulk perfusion rate, oxygen uptake averaged about 49% lower in hypoxia than in normoxia, decreasing in proportion to the reduction of O2 tension difference between medium and blood. In hyperoxia, O2 uptake did not increase proportionally with the difference in O2 tension between blood and medium, averaging only 50% higher at a 2.4-fold greater O2 tension difference. Cutaneous diffusing capacity for O2 averaged 0.041 nmol O2 x cm-2 x torr-1 x min-1 during the first hour of perfusion in normoxia, and was not affected by reduction of ambient O2 tension. The results indicate that cutaneous O2 uptake in hypoxia is highly diffusion limited, and consequently, increases in cutaneous perfusion can not effectively compensate for reduction of ambient O2 tension. In hyperoxia, O2 uptake may be substantially perfusion limited because of reduced blood O2 capacitance at high O2 saturations.

Biphasic effect of oleic acid on hepatic cholesterogenesis

Livers isolated from adult male rats were perfused in vitro with oleic acid (0.6mM) as a complex with bovine serum albumin. Albumin alone was infused in control experiments. Oleic acid exerted a biphasic effect on incorporation of 3H 2O into cholesterol, which was inhibitory during the first hour of perfusion, but exhibited a net stimulatory effect over a four hour period. No differences were observed in total activity or apparent phosphorylation state of HMG-CoA reductase after one hour of perfusion, with or without addition of oleic acid, implying that some other step limits the rate of cholesterol synthesis during this interval. After four hours of perfusion, HMG-CoA reductase activity was higher in livers perfused with oleic acid than in those perfused in its absence, in agreement with the observed differences in rates of cholesterol synthesis.

Polarity of hepatic glutathione and glutathione S-conjugate efflux, and intraorgan mercapturic acid formation in the skate

Mechanisms of hepatic glutathione and glutathione S-conjugate efflux were investigated in isolated hepatocytes and perfused liver of the little skate ( Raja erinacea). Glutathione was released by isolated skate hepatocytes at a rate of 0.12 ± 0.03 nmol · hr −1 · (mg protein) −1. In the perfused liver, glutathione concentrations in bile were high (∼0.7mM) compared to hepatic tissue levels (0.61 ± 0.11 μmol · g −1). During the first hour of perfusion, the biliary glutathione excretion rate was 3 nmol · hr −1 · (g liver) −1, whereas glutathione accumulated in the recirculating perfusate at a rate of only 1.5nmol · hr −1 · (g liver) −1. Release of glutathione by isolated hepatocytes and perfused liver was not affected by the addition of acivicin, an inhibitor of γ-glutamyltransferase (EC 2.3.2.2), to cell suspension medium or liver perfusate. 1-Chloro-2,4-dinitrobenzene (CDNB) was taken up by isolated hepatocytes, conjugated to glutathione, and released as 5-(2,4-dinitrophenyl) (DNP)-glutathione. After infusion of 0.5 μmol CDNB in perfused liver, S-DNP-glutathione was concentrated in bile (0.5 mM) and was associated with choleresis. S-DNP-Conjugates of cysteinylglycine, cysteine and N-acetylcysteine, were also found in bile, suggesting intrahepatic breakdown of S-DNP-glutathione and subsequent acetylation of the resulting cysteine conjugate to form the mercapturic acid, S-DNP- N-acetylcysteine. This mercapturic acid accounted for 31% of the total S-DNP-conjugates collected in bile. In contrast, neither S-DNP-glutathione nor other S-DNP-conjugates were detected in the perfusate (< 0.5 μM). These findings demonstrate that biliary excretion is the predominant route for efflux of glutathione and a glutathione S-conjugate from skate liver. The results also identify an intrahepatic pathway for mercapturic acid biosynthesis facilitated by biliary glutathione S-conjugate excretion.

Lithium and Free Water Clearances in the .Isolated Perfused Kidney

To determine whether the lithium clearance (CLi) method offers a distinct advantage in the assessment of distal nephron function in the isolated perfused rat kidney (IPRK), estimates of distal Na+ reabsorption (dTNa+) based on the CLi (dTNA (Li)) were compared to those based on free water clearance (dTNa+(H2O)). In the steady state, dTNa+ (Li) was as high as 5-fold greater than (dTNa+(H2O)). The gap was even larger during the first hour of perfusion. These differences indicate that the Cu method is far superior and more accurate than the CH2O method, suggesting that it may be an adequate means for estimating dTNa+ in the IPRK. The CLi, observations also suggest that even in the absence of ADH a significant amount of solute-free water is reabsorbed by the collecting ducts of the IPRK.