raffinose and Hypoxia

In order to explore the method to prepare hypoxia UW solution and the stability and preservation of hypoxia UW solution, UW solution was purged by argon or air for 15 min or 60 at a flow rate of 0.8 or 2 L/min, and the oxygen partial pressure of UW solution was detected. The hypoxia UW solution was exposed to the air or sealed up to preserve by using different methods, and the changes of oxygen partial pressure was tested. The results showed that oxygen partial presure of 50 mL UW solution, purged by argon for 15 min at a flow rate of 2 L/min, was declined from 242+/-6 mmHg to 83+/-10 mmHg. After exposure to the air, oxygen partial pressure of hypoxia UW solution was gradually increased to 160+/-7 mmHg at 48 h. After sealed up by the centrifuge tube and plastic bad filled with argon, oxygen partial pressure of hypoxia UW solution was stable, about 88+/-13 mmHg at 72 h. It was concluded that oxygen of UW solution could be purged by argon efficiently. Sealed up by the centrifuge tube and plastic bag filled with argon, oxygen partial pressure of UW solution could be stabilized.

Topics: Adenosine; Allopurinol; Animals; Glutathione; Hypoxia; Insulin; Organ Preservation; Organ Preservation Solutions; Oxygen; Partial Pressure; Raffinose

Various cryopreservation techniques have been investigated to elongate preservation time, however, most have failed to be clinically induced because of damage due to ice crystal formation. Subzero nonfreezing conditions could theoretically reduce organ metabolism without damage due to ice crystal formation. We evaluated the superiority of subzero nonfreezing storage compared with conventional hypothermic storage using isolated rat hepatocytes stored in University of Wisconsin (UW) solution without cryoprotectants.. Hepatocytes of Wistar rats isolated by collagenase digestion were suspended in UW solution and divided into the following three groups: subzero nonfreezing group (-4 degrees C), zero nonfreezing group (0 degrees C), and control group (4 degrees C). They were stored for 48 hr at the temperatures indicated. After 24 and 48 hr of storage, we carried out a trypan blue exclusion test and a 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) assay, and measured lactate dehydrogenase release, lactic acid, ATP content, and the ability of hepatocytes to synthesize urea. After 48 hr of storage, morphological differences between the control group and the subzero nonfreezing group were investigated by scanning and transmission electron microscopy.. Significant improvements of the trypan blue exclusion test and ATP contents in the subzero nonfreezing group were observed. Lactic acid production was also significantly suppressed in the subzero nonfreezing group compared with that in the control group. The MTT assay value was significantly better at -4 degrees C than at 4 degrees C. The rate of urea synthesis at -4 degrees C was higher than that at 4 degrees C. Electron microscopy revealed that subzero nonfreezing delayed the lethal bleb-forming process of stored hepatocytes, which was followed by mitochondrial swelling, compared with the control group.. Subzero nonfreezing storage (-4 degrees C) in UW solution could provide better preservability for isolated rat hepatocytes with protection against hypoxic cell injury compared with conventional hypothermic storage (4 degrees C).

Topics: Adenosine; Adenosine Triphosphate; Allopurinol; Animals; Cell Separation; Cryopreservation; Glutathione; Hypoxia; Insulin; Lactic Acid; Liver; Male; Microscopy, Electron, Scanning; Organ Preservation Solutions; Raffinose; Rats; Rats, Wistar; Tetrazolium Salts; Thiazoles; Trypan Blue

The etiology of primary graft nonfunction and dysfunction is unknown but most likely involves Kupffer cell-dependent reperfusion injury. However, the donor operation and surgical technique may also have an effect on the outcome after transplantation. Because liver manipulation during harvest cannot be prevented completely with standard procedures, its effect on survival was assessed here.. Donor livers were harvested from female Sprague-Dawley rats (200-230 g). Briefly, after minimal dissection during the first 12 min, livers were either manipulated gently or left alone for 13 subsequent minutes. At 25 min, all livers were perfused with cold University of Wisconsin solution via the portal vein, and transplantation was performed after cold storage (1 hr). In some rats, Kupffer cells were destroyed with gadolinium chloride or inactivated with dietary glycine before harvest. Survival, proteolytic activity in the rinse effluent, serum transaminases, trypan blue distribution to index microcirculation, and histology were compared.. In the nonmanipulated group, survival was 100% after transplantation; however, gentle manipulation decreased survival by 70%. Further, manipulation elevated transaminases fivefold and caused about 200% necrosis. At harvest, proteolytic activity and the time for trypan blue to distribute homogeneously were elevated three- to eightfold by manipulation. Gadolinium chloride and glycine prevented the effects of manipulation on all parameters studied.. These data indicate for the first time that brief, gentle manipulation of the donor liver has a marked detrimental effect on survival by priming or activating Kupffer cells. This may represent an important early event in pathogenesis, because Kupffer cells play an important role in primary graft nonfunction.

Topics: Adenosine; Allopurinol; Animals; Cell Death; Dissection; Female; Gadolinium; Glutathione; Glycine; Graft Survival; Hepatectomy; Hypoxia; Insulin; Kupffer Cells; Liver; Liver Circulation; Liver Function Tests; Liver Transplantation; Microcirculation; Organ Preservation; Organ Preservation Solutions; Raffinose; Rats; Rats, Sprague-Dawley; Reperfusion

We propose the use of 1H nuclear magnetic resonance (NMR) spectroscopy to investigate metabolite fluxes in the mammalian liver during cold hypoxia. Rat livers were flushed with one of four different preservation solutions and stored on ice in the same solution. The preservation solutions were: Marshall's hypertonic citrate (HC); carnosine modified HC (HC-C); modified University of Wisconsin (mod UW); and Bretschneider's histidine--typtophan--ketoglutarate (HTK). Liver biopsies were taken before and at 1, 2.5. 4, 24, and 48 h after storage, and freeze-clamped. The liver was extracted with perchloric acid and analyzed by 1H NMR spectroscopy. Components of the individual preservation solutions, such as citrate, histidine, mannitol, and raffinose, were detected in the extracts. Lactate was increased over the first 4 h in all stored livers, but only continued to increase in those stored in HC-C and HTK, reaching significantly high levels of 15 and 14 mumol/g, respectively, by 48 h storage (P < 0.05 and P < 0.01, respectively). Levels of succinate and fumarate in all livers were generally unchanged in the first 0-4 h of storage. However, after 4 h of storage, succinate levels rose in the HC and HC-C livers, while remaining unchanged in mod UW and HTK livers. The presence of citrate in the preservation solutions appeared to enhance the late hepatic synthesis of succinate. Fumarate levels were significantly decreased by 48 h of cold storage, indicating continued fumarate consumption at low temperatures. Despite cold hypoxic conditions, some carbon-substrate cycling appears to continue in mammalian liver via pathways other than glycolysis, and citrate from the preservation solution appears to influence this.

Topics: Adenosine; Allopurinol; Animals; Citric Acid; Cold Temperature; Cryopreservation; Glutathione; Hypertonic Solutions; Hypoxia; Insulin; Liver; Magnetic Resonance Spectroscopy; Male; Organ Preservation Solutions; Raffinose; Rats; Rats, Sprague-Dawley

Topics: Adenosine; Aerobiosis; Allopurinol; Anaerobiosis; Animals; Glutathione; Hypoxia; Insulin; Ischemia; Liver; Male; Microscopy, Fluorescence; NAD; Organ Preservation Solutions; Oxidation-Reduction; Oxygen; Oxygen Consumption; Raffinose; Rats; Rats, Wistar

To evaluate the effect of University of Wisconsin solution on endothelium-dependent relaxation and contraction of human hepatic arteries in vitro.. Human hepatic arteries were harvested from 24 patients with hepatocellular carcinoma who had undergone hepatectomy.. A tertiary care center.. Human hepatic arteries (n = 6 in each group) were harvested during resection for hepatocellular carcinoma. The arteries in group 1 (i.e., the control group) were immediately studied without preservation. The arteries in group 2 were preserved in cold (4 degrees C) physiological solution for 1 hour, while the arteries in groups 3 and 4 were preserved in University of Wisconsin solution for 1 and 16 hours, respectively. Segments of control and preserved hepatic arteries with or without endothelium were then suspended in organ chambers to measure the isometric force.. The relaxation of segments of the hepatic arteries with endothelium in response to acetylcholine and adenosine diphosphate was significantly (P < .05) greater than that of segments without endothelium. The maximal relaxation of hepatic arterial segments with endothelium in groups 3 and 4 in response to acetylcholine was notably different from that of segments in groups 1 and 2. The maximal relaxation of hepatic arterial segments with endothelium in groups 3 and 4 in response to adenosine diphosphate was notably different from that of segments in groups 1 and 2. Perfusate hypoxia (mean +/- SD PO2, 30 +/- 5 mm Hg) caused the endothelium-dependent contraction of the arteries (the median initial tension in groups 1, 2, 3, and 4 was 251%, 233%, 276%, and 260%, respectively; P > .05).. The endothelium-dependent relaxation of human hepatic arteries in response to acetylcholine and adenosine diphosphate was notably attenuated by University of Wisconsin solution. The impaired endothelium-dependent relaxation by University of Wisconsin solution and the prominent endothelium-dependent contraction of human hepatic arteries would favor vasospasm and thrombosis after hepatic transplantation.

Topics: Adenosine; Allopurinol; Dose-Response Relationship, Drug; Endothelium, Vascular; Glutathione; Hepatic Artery; Humans; Hypoxia; In Vitro Techniques; Insulin; Muscle Contraction; Organ Preservation Solutions; Raffinose; Tissue Preservation

Topics: Adenosine; Adenosine Triphosphate; Allopurinol; Animals; Energy Metabolism; Glutathione; Histidine; Hypoxia; Insulin; Lactates; Liver; Organ Preservation; Organ Preservation Solutions; Phosphofructokinase-1; Raffinose; Rats; Rats, Sprague-Dawley; Time Factors

We compared the ability of human red blood cells (RBC) and a cell-free oxygen carrier to maintain isolated perfused kidney function under moderately hypoxic conditions. Recirculating perfusate was gassed initially with 93% air-7% CO2, and, after 30 min, the gas was changed to 12 O2-7 CO2-81% N2. Oxygen content of the perfusate was increased with RBC (30 g/l Hbg) or highly purified human hemoglobin Ao (HbAo) polymerized with O-raffinose (o-R-poly-Hb, 30 g/l Hbg). For comparison, kidneys were perfused with 60 g/l of bovine serum albumin (BSA) alone. The effects of unmodified hemoglobin were examined by adding 5 g/l of nonpolymerized HbAo to the BSA perfusate after 20 min. The effect of increasing oxygen delivery without hemoglobin was examined by switching to 93% O2 after 20 min during some BSA perfusions (BSA-HiO2). Vascular resistance decreased progressively in o-R-poly-Hb- and BSA-HiO2-perfused kidneys but remained constant in other experiments. Nitro-L-arginine methyl ester (L-NAME) prevented vasodilation and increased the filtration fraction of o-R-poly-Hb-perfused kidneys with no change in other functions. L-NAME also prevented the formation of methemoglobin. After a 70-min perfusion with BSA, Na reabsorption was 82 +/- 3% (means +/- SD), and inulin clearance [glomerular filtration rate (GFR)] was 0.66 +/- 0.33 ml.min-1.g-1. RBC increased reabsorption to 95% (85-98%) (median, 25th-75th percentile) but did not alter GFR (0.52 +/- 0.26 ml.min-1.g-1). o-R-poly-Hb increased Na reabsorption proportionately more than GFR, so that, while GFR was doubled to 1.04 +/- 0.40 ml.min-1.g-1, Na reabsorption increased to 98% (92-99.5%). HbAo increased GFR to 1.07 +/- 0.44 ml.min-1.g-1 and increased reabsorption to 89 +/- 6%.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Absorption; Animals; Erythrocytes; Glomerular Filtration Rate; Hemoglobins; Humans; Hypoxia; In Vitro Techniques; Kidney; Male; Perfusion; Phosphates; Proteinuria; Raffinose; Rats; Rats, Wistar

Topics: Acetylcholine; Adenosine; Adenosine Diphosphate; Allopurinol; Animals; Coronary Vessels; Dinoprost; Dogs; Endothelium, Vascular; Glutathione; Hypoxia; In Vitro Techniques; Insulin; Isoproterenol; Muscle Contraction; Muscle Relaxation; Muscle, Smooth, Vascular; Nitroprusside; Organ Preservation; Organ Preservation Solutions; Potassium; Raffinose

Both cold and warm ischemia occur during liver transplantation. Hypothermia and Wisconsin solution preserve adenine nucleotide energy status, which is crucial to hepatic function and viability. The volatile anesthetic isoflurane has been shown to preserve energy status in anoxic isolated hepatocytes in warm Krebs solution. The present study examined isoflurane effects on energy status during incubation also in Wisconsin or Krebs-plus-adenosine solution at 37 degrees or 4 degrees. Hepatocytes were isolated from rat liver after perfusion with Krebs + collagenase. In 25-mL flasks, 12.5 million cells in 2.5 mL of Krebs, Krebs plus 5 mmol/L adenosine, or Wisconsin solution were incubated under an atmosphere of O2/CO2 or N2/CO2 (19:1) +/- isoflurane (3 volumes% = 2ED50), for 30 minutes at 37 degrees C or 4 degrees C. Adenine nucleotides were measured by high-performance liquid chromatography (HPLC), lactate enzymatically. During warm (37 degrees) anoxia, Wisconsin solution preserved energy status; Krebs plus adenosine did not. Isoflurane further protected energy status in all three solutions. Hypothermia (4 degrees) alone greatly decreased anoxic loss of energy status in all solutions. In Wisconsin solution only, energy status tended to be higher in anoxic than in oxygenated cells and was further enhanced by isoflurane, with corresponding increases in lactate. During 30 minutes of either warm or cold anoxia, isoflurane and Wisconsin solution each helped preserve adenine nucleotide energy status in isolated hepatocytes, at least in part through enhanced glycolysis.

Topics: Adenine Nucleotides; Adenosine; Allopurinol; Anesthetics, Inhalation; Animals; Cell Hypoxia; Chromatography, High Pressure Liquid; Disease Models, Animal; Energy Metabolism; Glutathione; Hypothermia, Induced; Hypoxia; Insulin; Isoflurane; Isotonic Solutions; Liver; Male; Organ Preservation Solutions; Raffinose; Rats; Rats, Sprague-Dawley

University of Wisconsin (UW) solution has been used almost routinely in the preservation of the hepatic, pancreatic, renal, and cardiac allografts. However, its effect on vascular endothelium is unknown. Experiments were designed to evaluate its effect on canine coronary endothelium. Canine coronary arteries (n = 8 in each group) were preserved in cold (4 degrees C) UW solution (group 1) and physiological solution (group 2) for 6 hr immediately after harvesting. Segments of preserved and control (group 3) coronary arteries with or without endothelium were then suspended in organ chambers to measure isometric force. Perfusate hypoxia (pO2 30 +/- 5 mmHg) caused endothelium-dependent contraction in the arteries of all 3 groups. However, vascular segments with endothelium of group 1 exhibited hypoxic contractions (107 +/- 26% of the initial tension contracted by prostaglandin F2 alpha 2 x 10(-6) mol/L, P < 0.05) that were significantly greater than those of the group 2 and group 3 segments with endothelium (25 +/- 5% and 20 +/- 4%). The hypoxic contraction in arteries of group 1 could be attenuated by NG-monomethyl-L-arginine (L-NMMA), the blocker of endothelial cell synthesis of the nitric oxide from L-arginine. The action of L-NMMA could be reversed by L-arginine but not D-arginine. Endothelium-dependent relaxation of coronary endothelium to acetylcholine and adenosine diphosphate and endothelium-independent relaxation and contraction of coronary smooth muscle were not altered by the UW solution. After preservation with the UW solution, endothelium-dependent contraction of the canine coronary arteries, occurs by L-arginine-dependent pathway, is enhanced. This augmentation by the UW solution would favor vasospasm after transplantation.

Topics: Acetylcholine; Adenosine; Allopurinol; Animals; Arginine; Cell Communication; Coronary Vessels; Dinoprost; Dogs; Endothelium, Vascular; Female; Glutathione; Hypoxia; Insulin; Isoproterenol; Male; Nitric Oxide; omega-N-Methylarginine; Organ Preservation; Organ Preservation Solutions; Potassium; Raffinose; Time Factors; Vasoconstriction; Vasodilation

University of Wisconsin cold-storage solution (UW solution) has markedly improved organ preservation for liver transplantation. However, the efficacy of this solution in preserving hepatocyte viability during warm ischemia is undefined; hence, the effects of UW solution on warm hypoxic injury in the isolated perfused rat liver were examined.. Livers were perfused using a modified protocol that included a period of hypoxic perfusion with isosmotic Krebs' solution at the end of each experiment. Hepatic injury was evaluated by aspartate aminotransferase (AST) release into the perfusate and the trypan blue perfusion technique.. Although UW solution appeared to decrease hepatic injury during hypoxic perfusion, as reflected by low AST release, perfusion with UW solution led to hepatocyte shrinkage and cessation of bile flow even under oxygenated conditions. UW solution did not protect against warm hypoxic injury, as assessed by AST release into the perfusate (182 +/- 15 U/mL, mean +/- SD) or trypan blue staining of the dead hepatocyte nuclei (56% +/- 5%). However, the addition of fructose to UW solution resulted in a significant decrease in AST release (66 +/- 15 U/mL) and parenchymal cell death (39% +/- 7%).. These data suggest that the addition of fructose or other gluconeogenic substrates may complement the overall hepatoprotective effects of UW solution, particularly during periods of warm hypoxia.

Topics: Adenosine; Alanine Transaminase; Allopurinol; Animals; Cell Survival; Fructose; Glutathione; Hypoxia; In Vitro Techniques; Insulin; Liver; Male; Organ Preservation; Organ Preservation Solutions; Perfusion; Raffinose; Rats; Rats, Sprague-Dawley

The critical injury to liver during cold preservation is believed to occur to the sinusoidal endothelium. In this study the viability of cultured sinusoidal endothelial cells from rat liver was assessed during storage in University of Wisconsin solution at 4 degrees C. The vast majority of cells (83 +/- 12%) died within 24 hr of storage. Addition of KCN (1 mM) to the solution to simulate hypoxia markedly increased survival: only 3 +/- 2% of cells had lost viability after 24 hr in the presence of cyanide. Further experiments showed that other inhibitors of mitochondrial ATP formation (antimycin A 1 microM, rotenone 1 microM, oligomycin 10 microM, carbonyl cyanide m-chlorophenylhydrazone 1 microM) were protective as well, whereas glucose (10 mM) greatly diminished the protective effect of cyanide (loss of viability 38 +/- 7% after 24 hr). ATP measurements confirmed the correlation between the energy state of the cells and cell death: ATP levels after 6 hr of incubation were 19.9 +/- 4.0 nmol/10(6) cells in UW solution, 13.7 +/- 2.9 nmol/10(6) cells in UW + glucose, 6.9 +/- 1.9 nmol/10(6) cells in UW + KCN + glucose and 1.9 +/- 1.5 nmol/10(6) cells in UW + KCN. In contrast to the protective effect observed in UW solution, addition of KCN to Krebs-Henseleit buffer led to increased endothelial cell damage upon cold storage. We therefore conclude that in UW solution damage to the sinusoidal endothelium is energy-dependent.

Topics: Adenosine; Adenosine Triphosphate; Aerobiosis; Allopurinol; Animals; Antimycin A; Cell Survival; Cryopreservation; Cyanides; Endothelium; Glucose; Glutathione; Hypoxia; Insulin; Liver; Male; Organ Preservation Solutions; Raffinose; Rats; Rats, Wistar; Reperfusion Injury; Rotenone; Solutions; Time Factors; Tissue Preservation

Topics: Adenine Nucleotides; Adenosine; Allopurinol; Animals; Cold Temperature; Energy Metabolism; Glutathione; Hypoxia; Insulin; Kinetics; Liver; Magnetic Resonance Spectroscopy; Organ Preservation; Organ Preservation Solutions; Perfusion; Phosphorus; Raffinose; Rats; Solutions; Time Factors

Three mutants with altered response of the hemoprotein level to glucose repression and anoxia were characterized biochemically. Two mutants show complete resistance of catalase activity to glucose repression and a lower degree of resistance of cytochrome c oxidase and of some other enzymes. Liquid nitrogen spectra of these mutants grown in high glucose medium are typical for those of glusoce-derepressed spectra of wild-type cells. The third mutant is hypersensitive to glucose repression and shows only traces of catalase T activity when grown in high-glucose media. In these conditions its spectrum is almost devoid of typical peaks of cytochromes. When grown on ethanol or raffinose it forms catalase and shows a typical derepressed spectrum of cytochromes. The regulatory mechanism impaired in the mutants is not known. It seems likely that the regulation of the heme pathway in the mutants does not differ from that of the wild-type.

Topics: Catalase; Culture Media; Electron Transport Complex IV; Ethanol; Gene Expression Regulation, Fungal; Glucose; Hemeproteins; Hypoxia; Mutation; Raffinose; Saccharomyces cerevisiae; Spectrum Analysis