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Acute intestinal ischaemia/reperfusion injury (AII/R) is an adaptive physiologic response during critical illness, involving mesenteric vasoconstriction and hypoperfusion. Prevention of AII/R in high risk patient populations would have a significant impact on morbidity and mortality. The purpose of this study was to investigate the protective effects of VSL#3 probiotic treatment in a murine model of AII/R. Adult 129/SvEv mice were subjected to an experimental AII/R model using superior mesenteric artery occlusion. Animals were pre-treated with either three days or two weeks of VSL#3 probiotics. Local tissue injury markers were assessed by levels of myeloperoxidase and activation of nuclear factor kappa B (NFкB). Systemic and local cytokines, including interleukin (IL)-1ß, IL- 10, TNFα, and interferon gamma were measured by ELISA and multiplex fluorescent detection. VSL#3 probiotics reduced local tissue inflammation and injury due to AII/R. A two-week course of VSL#3 was more effective than a shorter three-day course. The reduction in local inflammation from the two-week course of VSL#3 is correlated to a significant reduction in levels of active IL-1ß, and tissue levels of myeloperoxidase. Levels of active NFкB were significantly elevated in the vehicle-fed AII/R mice, corroborating with tissue inflammation, which were attenuated by VSL#3 administrations. VSL#3 did not cause any systemic inflammation or lung injury. VSL#3 probiotics are effective in reducing local tissue injury from AII/R by down-regulating pro-inflammatory mediators and immune cell recruitment. This study highlights a potential role for VSL#3 in management of patients at high risk for AII/R.

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The objective of the present investigation was to evaluate the effects of acidic pH of the perfusate and presence of lipopolysaccharide (LPS) on permeability of rumen and colon mucosal tissues to mannitol and LPS using the Ussing chamber system. Rumen and colon tissues (n = 8), obtained from slaughtered feedlot steers, were tested for changes in permeability to (3)H-mannitol under pH of 4.5, 5.5, and 6.5 for rumen and at 5.5, 6.5, and 7.4 for colon, with or without LPS from Escherichia coli B:055 at 500 microg/mL. The (3)H-Mannitol was added at 10 microL (525.4 GBq/mmol) on the mucosal side of the Ussing chamber to detect changes in permeability, and 4 samples were taken at 20, 25, 30, and 35 min from the serosal side. Permeability of rumen and colon mucosa to (3)H-mannitol increased 6- and 5-fold, respectively, at acidic pH values of 4.5 and 5.5 and in the presence of 500 micro/mL of LPS. In contrast, LPS did not affect rumen and colon permeability at pH that ranged from 5.5 and 7.4. Translocation of LPS across the rumen and colon mucosa of cattle was not pH dependent. The LPS translocated through these tissues if present at the mucosal side. In conclusion, the permeability of rumen and colon tissues to (3)H-mannitol increased in presence of LPS and under acidic pH, whereas LPS permeated through mucosal tissues independently of the pH of the perfusate. Further research is warranted to understand the mechanism(s) by which acidic pH of the rumen digesta and presence of LPS make rumen and colon tissues "leaky".

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A two-layer cold storage method (TLM) allows sufficient oxygen delivery to pancreata during preservation and resuscitates the viability of ischemically damaged pancreata. This study determined the effect of additional preservation of ischemically damaged human pancreata by the TLM before islet isolation. Human pancreata were procured from cadaveric organ donors and preserved by the TLM for 3.2 +/- 0.5 hours (mean +/- SEM) at 4 degrees C after 11.1 +/- 0.9 hours of cold storage in University of Wisconsin solution (UW) (TLM group), or by cold UW alone for 11.0 +/- 0.3 hours (UW group). Islet isolations of all pancreata were performed using the Edmonton protocol. Islet recovery and in vitro function of isolated islets were significantly increased in the TLM group compared with the UW group. In the metabolic assessment of human pancreata, ATP levels were significantly increased after the TLM preservation. This study showed that additional short-term preservation by the TLM resuscitates the viability of ischemically damaged human pancreata before islet isolation, leading to improvements in islet recovery and in vitro function of isolated islets.

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Lack of a small animal model of the human hepatitis C virus (HCV) has impeded development of antiviral therapies against this epidemic infection. By transplanting normal human hepatocytes into SCID mice carrying a plasminogen activator transgene (Alb-uPA), we generated mice with chimeric human livers. Homozygosity of Alb-uPA was associated with significantly higher levels of human hepatocyte engraftment, and these mice developed prolonged HCV infections with high viral titers after inoculation with infected human serum. Initial increases in total viral load were up to 1950-fold, with replication confirmed by detection of negative-strand viral RNA in transplanted livers. HCV viral proteins were localized to human hepatocyte nodules, and infection was serially passaged through three generations of mice confirming both synthesis and release of infectious viral particles. These chimeric mice represent the first murine model suitable for studying the human hepatitis C virus in vivo.

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BACKGROUND: The inability to diagnose early rejection of an islet allograft has previously proved to be a major impediment to progress in clinical islet transplantation. The need to detect early rejection will become even more relevant as new tolerance-inducing protocols are evaluated in the clinic. We explored three novel approaches toward development of early diagnostic markers of islet rejection after islet allotransplantation. METHODS: (a) Canine islet allograft transplant recipients were immunosuppressed for 1 month, then therapy was withdrawn. Serum glutamic acid decarboxylase antigen (GAD65), an endogenous islet protein, was monitored daily with a CO2 release assay. (b) Rodent islets were genetically engineered to express a unique foreign protein (beta-galactosidase) by using adenoviral vectors, and after allograft transplantation, the viral-specific protein was measured in serum using optical luminescence. (c) Rodents receiving islet allografts were immunosuppressed temporarily, and daily glucose tolerance tests were followed until graft failure occurred. RESULTS: (a) Although serum monitoring of GAD65 antigen demonstrated elevated levels preceding loss of graft function in preliminary studies, the effect was not reproducible in all animals. (b) Genetically engineered rodent islets demonstrated normal insulin kinetics in vitro (insulin stimulation index 2.57+/-0.2 vs. 2.95+/-0.3 for control islets, P=ns), and purified viral protein products had a stable half-life of 8 hr in vivo. After islet allotransplantation, there were two peak elevations in serum viral proteins, confirming that an intra-islet "sentinel signal" could be detected serologically during acute rejection. There was no lead-time ahead of hyperglycemia, however. (c) Daily sequential intravenous glucose tolerance (IVGT) tests demonstrated evidence of allograft dysfunction (decline in KG) with a 2-day lead time to hyperglycemia (2.58+/-0.3 vs. 1.63+/-0.2%/min, respectively, P<0.001), with an accuracy of 89%, sensitivity of 78%, and specificity of 95%. CONCLUSIONS: Of the three diagnostic tests, metabolic assessment with an abbreviated IVGT was the most effective method of demonstrating early islet dysfunction due to rejection.

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We investigated the metabolic effects of buffering agents alpha-amino-4-imidazole-propionic acid (Histidine), N, N-bis(2-hydroxyethyl)glycine (bicine), N, N-bis(2-hydroxyethyl)-2-aminoethanesulfonic acid (BES) on anaerobic energy production (via glycolysis) and conservation of key regulatory enzyme activity, and phosphofructokinase (PFK) throughout prolonged hypothermic hypoxia in porcine hearts. Hearts from 35 to 40 kg pigs were flushed with one of the following five solutions: St. Thomas' Hospital solution (STHS); modified University of Wisconsin (UW) solution; and three solutions containing modified UW plus 90 mM of histidine, bicine, or BES. The hearts were then stored at 4 degrees C for 10 h. After 10 h of hypothermic hypoxia, lactate values were 6.7-12.9 micromol/g higher than control; this reflected an increase in anaerobic end product of 35-67%. The consequences of enhanced anaerobic metabolism were higher ATP, total adenylate, Energy Charge, and ATP/ADP ratios in most of the buffered groups after 4-10 h cold storage; effectiveness of the buffers employed correlated with buffering capacity (BES proved to be the most effective). PFK remained activated throughout most of the 10-h period in hearts stored with buffers and did not undergo the rapid inactivation experienced by hearts stored in STHS. Conservation of PFK integrity with buffering agents was not related to a pH-mediated event; changes in kinetic parameters suggested that this protection was due to an irreversible posttranslational modification, specifically a dephosphorylation event.

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BACKGROUND/AIM: During cold liver storage in University of Wisconsin solution, glycolysis is inhibited by declining intracellular pH and a reduction in glycogen phosphorylase activity. The current study investigated the effects of a histidine-buffered, modified University of Wisconsin solution with cyclic-AMP analogue plus phosphodiesterase inhibitors to optimize both pH and PK A-mediated limits on glycolytic energy production. METHODS: In an isolated rodent-liver system, dioctanoyl-cAMP was supplemented with each phosphodiesterase inhibitor (isobutylmethylxanthine, papaverine, Ro 20-1724, dipyridamole). Once the most efficacious combination was determined, a separate group of livers was cold-stored for 24 h and then reperfused at 37 degrees C to examine regeneration of high energy adenylates. RESULTS: Lactate accumulation in the histidine-lactobionate-raffinose group was 8.7 micromol/g; net increases were greater with all four phosphodiesterase inhibitors with dioctanoyl-cAMP; dipyridamole resulted in a maximum increase of 16.7 micromol/g. ATP was consistently higher in all treatment groups with phosphodiesterase inhibitors throughout 24 h; even after 10-24 h, levels with dipyridamole-treatment were 250-280% higher than with University of Wisconsin (p<0.05). Assessment of glycogen phosphorylase activity in the dipyridamole-treatment group indicated that increased glycolytic activity over the first 4 h was a direct consequence of elevated enzyme levels. However, between 4-10 h, phosphofructokinase underwent a phosphorylation, leading to an inhibition at this point in glycolysis. Upon reperfusion, the higher ATP/ADP and ADP/ AMP ratios found with phosphodiesterase inhibitor treatment suggested that adenylate regeneration was superior with dipyridamole+dioctanoyl-cAMP. CONCLUSION: Dipyridamole plus dioctanoyl-cAMP treatment achieved increased glycogenolysis throughout 24 h storage by maintaining glycogen phosphorylase in a phosphorylated (active) state; however, a PK A-mediated phosphorylation (inhibition) of phosphofructokinase resulted in decreased glycolytic ATP production between 4-10 h.

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The ability of brief hypothermic reperfusion (HtR) to restore hepatic energy metabolism following periods of cold hypoxic preservation was studied in isolated rat livers after storage times of 5, 10, and 24 h. In addition, investigations were performed on the effects of HtR used to restore liver oxidative metabolism in the middle of a prolonged (24 h) hypoxic preservation period. A histidine-lactobionate-raffinose solution was used for the initial cold portal flush in all groups. Results showed that cold hypoxia for either 5 or 10 h yielded livers capable of similar recoveries of ATP, energy charge, and total adenine nucleotides, but that HtR after 24 h cold preservation resulted in reduced regeneration of ATP, a lower energy charge, and a fall in tissue adenine nucleotides. When livers were stored for 24 h but subjected to brief HtR after either 5 or 10 h before return to hypoxic storage, improved recoveries of the energy metabolites were seen over those recorded after 24 h hypoxia alone. The fact that these improvements were not due to an improved supply of adenine nucleotide precursors was demonstrated by studying groups which were given HtR with perfusate containing precursors of adenine nucleotides (adenosine, adenine, and inosine) after 24 h cold hypoxia. These data are consistent with the hypothesis that poor metabolic recovery after long-term hepatic cold preservation results more from decreased mitochondrial oxidative phosphorylation than from a lack of precursors for adenine nucleotide resynthesis. In addition, restoring oxidative metabolism at hypothermia for brief periods can to some extent protect final metabolic status after prolonged storage.

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This study was designed to determine whether the metabolic adaptations developed by frogs to tolerate natural events of hypothermic hypoxia would precondition its liver for ex vivo organ storage. The metabolic responses of the frog, Rana castabiena, were compared to those of a mammalian system (rat) throughout a prolonged period of organ storage. Livers from rats and frogs were flushed and stored in UW solution at 5 degrees C for periods of 24-96 h. In frog livers, ATP was maintained high and constant over the first 24 h of storage; values ranged from 2.7 to 3.0 micro mol/g. Even after 96 h cold storage, ATP remained > 1.0 micro mol/g. In contrast, ATP levels in stored rat livers dropped rapidly, and by 4 h ATP was 1.2 micro mol/g. In terms of anaerobic endproduct accumulation, lactate levels rose 5.8 micro mol/g in frog liver (over 96 h) and by 8.6 micro mol/g in rat liver (over 24 h). This difference in flux through glycolysis was also reflected in relative rates of carbohydrate catabolism (i.e., glucose + lactate production). The rate of carbohydrate catabolism for frog liver was 0.74 micro mol/g/h compared to 2.26 micro mol/g/h for rat liver; a Q10 value of 6.2 was estimated for livers from R. castabiena. An assessment of glycolytic enzyme activities revealed that key differences in the responsiveness of pyruvate kinase to allosteric modifiers may have been responsible for the marked drop in the rate of anaerobic energy production in frog tissues. Although the concept of depressed metabolism in a lower vertebrate is not new, the data presented in this study demonstrate that a depressed metabolic state can be achieved in isolated livers from R. castabiena simply through cold exposure. With respect to clinical relevance, the results of this study indicate that energetics of stored livers can be maintained effectively through an efficient reduction in energy use in combination with a slow, yet continuous, rate of energy production facilitated by glycolysis.

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BACKGROUND: This study was designed to investigate the effects of a modified University of Wisconsin (UW) solution supplemented with one of four buffering agents (histidine, bicine [N,N-bis(2-hydroxyethyl)glycine], tricine [N-tris(hydroxymethyl)methylglycine], and Tris) on liver metabolism during cold ischemic storage. METHODS: Rat livers were flushed and stored for a maximum period of 24 hr at 4 degrees C, and tissue energetics, substrate, and anaerobic end-products were assessed; the group exhibiting the best results during storage was recovered in a 60-min period of warm reperfusion. Relative buffering capacities of the experimental solutions (measured over physiological pH range, in mM H+/L) were: UW, 4.1; histidine+UW, 9.8; Tris+UW, 19.0; bicine+UW, 22.5; tricine+UW, 26.8. RESULTS: In the UW group, ATP levels dropped rapidly over the first 4 hr; 1.0 micromol/g (40% of initial) remained after 4 hr of storage. By 2 hr, ATP levels in bicine- and tricine-treated groups were 0.5 and 1.1 micromol/g greater than in the UW-stored livers and by 10 hr, ATP in bicine-treated livers was twofold that of the control (UW) group. Total adenylate levels also reflected a superior elevation of cellular energetics; even after 24 hr, quantities were 1.4 and 2.0 micromol/g higher than the UW group in bicine- and histidine-supplemented organs. The increase in energetics occurred as a result of increased flux through the major anaerobic energy-producing pathway, glycolysis. The glycolytic rate was significantly greater at storage times > 10 hr with solutions supplemented with bicine, histidine, and tricine. Final values for net lactate accumulation over the entire 24-hr storage period were: UW, 10.1 micromol/g; histidine, 14.3 micromol/g; bicine, 15.2 micromol/g; tricine, 13.8 micromol/g. Activities of glycogen phosphorylase revealed that the activity of this enzyme dropped by 50% within 2 hr of storage in UW. However, histidine and bicine supplementation resulted in a substantial elevation of phosphorylase "a" over 4 hr and 10 hr, respectively. The best buffer of the four examined in this study was bicine; energetics, glycolytic flux, and patterns of adenylate regeneration upon reperfusion were markedly superior to modified UW solution. CONCLUSION: The results of this study suggest that supplementing the "gold standard" UW solution with an additional buffering agent (in order of efficacy: bicine>tricine>histidine) may improve the metabolic status of livers during clinical organ retrieval/storage.

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Many lower vertebrates (reptilian and amphibian species) are capable of surviving natural episodes of hypoxia and hypothermia. It is by specific metabolic adaptations that anurans are able to tolerate prolonged exposure to harsh environmental stresses. In this study, it was hypothesized that livers from an aquatic frog would possess an inherent metabolic ability to sustain high levels of ATP in an isolated organ system, providing insight into a metabolic system that is well-adapted for low temperature in vitro organ storage. Frogs of the species, R. pipiens were acclimated at 20 degrees C and at 5 degrees C. Livers were preserved using a clinical preservation solution after flushing. Livers from 20 degrees C-acclimated frogs were stored at 20 degrees C and 5 degrees C and livers from 5 degrees C-acclimated frogs were stored at 5 degrees C. The results indicated that hepatic adenylate status was maintained for 96 h during 5 degrees C storage, but not longer than 4-10 h during 20 degrees C storage. In livers from 5 degrees C-acclimated animals subjected to 5 degrees C storage, ATP was maintained at 100% throughout the 96-h period. Warm acclimation (20 degrees C) and 20 degrees C storage resulted in poorer maintenance of ATP; energy charge values dropped to 0.50 within 2 h and by 24 h, only 24% of control ATP remained. Lactate levels remained less than 25 mumol/g dry weight in all 5 degrees C-stored livers; 20 degrees C-stored livers exhibited greater accumulation of this anaerobic endproduct (lactate reached 45-50 mumol/g by 10 h). The data imply that hepatic adenylate status is largely dependent on exposure to hypothermic hypoxia and although small amounts of ATP were accounted for by anaerobic glycolysis, there must have been either a substantial reduction in cellular energy-utilization or an efficient use of low oxygen tensions.

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We have studied biochemical markers of energy metabolism and glycolysis by enzyme analyses and 1H NMR spectroscopy in livers of the freshwater turtle, Pseudemys scripta, after vascular flush and cold storage. Values for hepatic ATP content and energy charge remained unchanged for 24 h and showed only small declines between 24 and 48 h of cold hypoxia. Lactate and glucose levels increased over the 48-h period, demonstrating, respectively, progressive glycolysis and glycogenolysis. These observations are in contrast to those made in mammalian liver, where ATP levels fall precipitously during the first few hours of cold hypoxia and glycolysis is inhibited. Additional changes suggested by 1H NMR spectroscopy may indicate a role for other metabolic pathways. Isolated organs of species such as Pseudemys may be useful models for studying the biochemical basis of resistance to cold hypoxic damage.

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Rat livers were flushed with different preservation solutions and stored at 4 degrees C for 24 h before being reperfused with a synthetic air-equilibrated, water-based solution. Four solutions were tested using this isolated rat liver model: Marshall's hypertonic citrate (HC); modified University of Wisconsin solution (Mod UW); a histidine-based solution (HIS); and a histidine-lactobionate-raffinose-based solution (HLR). After storage, livers were perfused at 4 degrees C for a period of 2 h and biopsy samples taken, at different time points, to investigate energy metabolism. Livers stored in HLR and HIS had higher 24-h storage levels of ATP (0.41 and 0.24 mumol/g respectively; P < 0.05) than those stored in Mod UW and HC. On reperfusion, all groups regenerated ATP by 1-2 h. However, significantly greater levels of ATP regeneration occurred in livers stored in the HLR (1.6 +/- 0.08 mumol/g) and Mod UW (1.3 +/- 0.18) than HC (0.58 +/- 0.19) and HIS (0.96 +/- 0.12); P < 0.05. Energy charge (1) (EC) recovered in all groups but was significantly higher in HLR and Mod UW (0.79 and 0.68, respectively; P < 0.05) than HC and HIS. These represent 95% (HLR) and 80% (Mod UW) of values observed in FIL. During the reperfusion period, total adenine nucleotide levels (TA) did not vary significantly within each storage group, except in the HIS solution. However, TAs were greater with livers stored for 24 h in HLR (2.5 +/- 0.25) and Mod UW (2.7 +/- 0.20) than those the other two storage groups (P < 0.05 in each case). This study has demonstrated that it was possible to resuscitate liver energetics after prolonged hypothermic ischemia by a period of cold reperfusion, and the method can differentiate between preservation solutions. The livers stored in each solution showed varying degrees of success in regeneration of ATP and EC, demonstrating that oxygen was not a limiting factor when using an air-equilibrated perfusate. The solutions providing the better preservation conditions gave the greater resuscitation of liver energetics (Mod UW and HLR). Overall, livers stored in HLR had the greatest resuscitation of energy metabolism, which correlates with survival data from other studies (29-31, 33).

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This study examined the effects of dibutyryl-cyclic adenosine monophosphate (db-cAMP) and okadaic acid (a specific inhibitor of protein phosphatases 1 and 2A) as additives to a cold storage solution. The effects on levels of glycogen phosphorylase, the resultant effects on flux through the glycolytic pathway, and the consequences of these changes on adenylate (ATP, ADP, and AMP) levels in rat liver during a 24-hr period of cold hypoxia were studied. The rapid transition to anaerobic metabolism was reflected in the increases in lactate levels for all groups. Total lactate accumulation in control livers (flushed and stored with a histidine-lactobionate-raffinose solution) was 9.8 micromol/g. The one notable difference between the control and experimental groups was the total lactate increase in one of the groups treated with db-cAMP; lactate accumulation was 16.0 micromol/g. There was a preferential maintenance of ATP that correlated with the increased flux through glycolysis observed with db-cAMP treatment; levels were 0.4-0.6 micromol/g higher than control group values between 2 and 10 hr of storage. In the control group, levels of glycogen phosphorylase in the active 'a' form began to decrease within 1 hr of exposure to cold hypoxic storage. Values dropped from 86% to 78% within the first 1 hr and by 10 hr, % 'a' was 57%. The separate addition of db-cAMP and okadaic acid resulted in a sustained maintenance of phosphorylase % 'a' throughout the entire cold hypoxic storage period; % 'a' values at 10 hr ranged from 75% to 81%. The major finding of this study was the clear and distinct correlation between phosphorylase % 'a' and total lactate accumulation (index of flux through glycolysis). This relationship was statistically significant after only 1 hr of storage, with a correlation coefficient of r=0.52 (P<0.025); however, the correlation became stronger as the time of storage progressed (by 10 hr, r=0.72; P<0.001). According to the relationship established, the maximum theoretical limit for lactate accumulation with 100% phosphorylase 'a' is approximately 30 micromol/g lactate. This finding suggests that glycogen phosphorylase and not necessarily glycogen content is one major determinant in maintaining anaerobic metabolism and energy production during cold liver storage. Hence, previous experiments that investigated the effects of nutritional status and glycogen content on tissue viability after experimental transplantation need to be reassessed.

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Lactate-edited 1H NMR difference spectra have been acquired from intact rat liver tissue following flushing and preservation in ice. A peak, initially at 1.26 ppm, was seen to increase in the liver tissue with preservation time. This peak was assigned to lactate, despite the fact that its chemical shift was initially shifted by approximately -0.1 ppm relative to an externally added standard. The assignment was based on the following: (a) the peak increased over a 24-h ischemic storage period; (b) it was coupled to a signal 2.78 +/- 0.02 ppm upfield; and (c) a parallel increase in lactate was noted in perchloric acid extracts of tissue from the same liver. An additional peak, assigned to alanine, was also observed during storage and was also shifted by approximately -0.1 ppm. Inclusion of dimethyl sulfoxide, which readily permeates liver tissue, demonstrated that this chemical shift alteration was a tissue-specific effect. These results demonstrate that 1H NMR spectroscopy of intact liver tissue during hypothermic ischemia is possible, though chemical shift assignments should be made with caution.

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The current study was undertaken to investigate energy metabolism during hypoxia in the cold in livers from euthermic and hibernating Columbian ground squirrels. We hypothesized that the hibernating Columbian ground squirrel would be able to maintain liver energetics for a considerably longer time than euthermic animals. Particular reference was made to the function of glycolysis, which is the only mechanism for energy production under hypothermic ischemia. The transition from aerobic to anaerobic metabolism was apparent in both euthermic and hibernating animals as lactate levels rose within 1-3 h; total lactate accumulation was 2.5 micromol/g in both groups. In euthermic squirrels, liver ATP and ADP decreased considerably over the first 3-h storage; values dropped by 55% and 34%, respectively. Conversely, as the drain on high energy phosphate pools progressed, there was an increase in low energy adenylate, AMP. Between 10 and 24 h of storage, increases in AMP accounted for approximately 25-30% of total ATP + ADP decrease. The remainder of the drop in adenylates was accounted for by considerable decreases in total adenylate (TA) contents; by 24 h TA contents had decreased by 2.0 micromol/g. Livers from hibernating squirrels exhibited similar patterns of adenylate change and were not significantly higher than their euthermic counterparts. With respect to regulatory control of glycolysis, livers from euthermic squirrels exhibited no regulatory control at phosphofructokinase (PFK) or pyruvate kinase (PK). Livers from hibernating animals, however, showed an activation at PFK by 10 h of cold storage; levels of hexose phosphates, glucose-6-phosphate + fructose 6-phosphate (G6P + F6P), dropped and fructose 1, 6-biphosphate (F1,6P2), increased. Changes in metabolite levels (phosphoenolpyruvate and pyruvate) associated with another key suspect regulatory enzyme, PK, indicated no role in regulatory control of glycolysis during the 24-h period. The apparent increase in PFK responsiveness to declining energy stores may be a futile activation since there was no accompanying increase in anaerobic end product, lactate, and no maintenance of energetics.

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BACKGROUND/AIMS/METHODS: In this study, we investigated the hepatoprotective effects of three storage solutions containing glycine (180 mM), glycylglycine (180 mM), and a mixture of 20 amino acids (combined concentration of 180 mM) on energy metabolism and levels of glucose and lactate (as an index of glycolytic flux) in rat livers. All effects were compared to those of livers flushed/stored with a modified University of Wisconsin solution. RESULTS: Glycine-treatment showed no improvement in liver energetics (ATP, ADP, AMP) and lactate accumulation; this solution had the lowest buffering capacity of the four tested (approximately 30% of the University of Wisconsin solution). The glycylglycine solution had the highest buffering capacity of the four solutions tested (including University of Wisconsin solution). Complete titration of the glycine-, combined amino acids-, and University of Wisconsin solutions (from 8.0 to pH = 6.0) resulted in a minor decrease in glycylglycine buffer pH; pH dropped by 0.2 pH units. In glycylglycine-treated livers, energetics showed an improvement over the first 1 h cold storage; ATP and 'energy charge' values remained high and ADP levels (and consequently total adenylate contents) were 0.7-2.4 micro mol/g greater than livers stored in University of Wisconsin solution. A 2-fold increase in lactate accumulation suggested that the improvement in liver energetics for the glycylglycine buffer was due to maintained flux through glycolysis brought about by enhanced buffering capacity. The solution containing a combination of amino acids exhibited maximum maintenance of liver energetics via increased glycolytic flux, despite its slightly inferior buffering capacity (85% of University of Wisconsin solution). ATP levels were maintained over the first 2 h storage and ADP levels (and consequently, total adenylate contents) were 1.2-2.1 micro mol/g greater than University of Wisconsin solution-treated livers during the entire 24 h storage period. Energy charge values for livers treated with the combination of amino acids were also significantly higher than with glycine-, glycylglycine- and University of Wisconsin solution-treatment; even at 24 h, energy charge was 0.36 (comparable to only 4 h storage in University of Wisconsin solution). CONCLUSIONS: Our data suggest that a combination of amino acids may be required for maximum protection of the liver, and furthermore there may be several independent mechanisms, including buffering capacity, responsible for cytoprotection of the liver during cold storage.

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During normothermic metabolism, the active pumping of Ca2+ across the cell membrane, mitochondria, and specialized sequestration organelles accounts for a large proportion of total energy expenditure in the cell. This study was designed to determine the effects of Ca2+ channel antagonists (chlorpromazine, verapamil, nifedipine, prenylamine, and nisoldipine) on energy metabolism and levels of glycolytic substrate (glucose) and anaerobic endproduct (lactate) during cold ischemia in rat livers. We hypothesized that if the passive channels were blocked during cold ischemia, then the ATP requirement of active ion pumping would be reduced and ATP levels and energy charge ratios would remain higher throughout the ischemic period; thus, viability of the liver would also be increased after prolonged ischemia. The most positive effect on energy metabolism was observed in the chlorpromazine-treated livers, followed by verapamil treatment. In the chlorpromazine treatment, total adenylate (TA) contents were 0.5-1.0 mumol/g (P < 0.05) higher than the sham group for most of the 24-h time course. Energy charge (EC) ratios were 0.05-0.07 higher than the sham values up to 4-10 h ischemia. Verapamil treatment was less effective, but still exhibited positive effects on TA levels at several time points (20 min, 10 h, and 24 h) throughout the entire 24-h period. In both of these groups, TA values by 24 h ischemia were similar to levels at 10 h in the sham group (3.1 mumol/g), thus showing a considerable effect in maintaining adenylate levels. Despite similar pharmacological antagonist activities, ATP levels in the nifedipine, prenylamine, and nisoldipine treatment groups were 1.0-1.5 mumol/g (P < 0.05) less than the corresponding sham group (without Ca2+ antagonists) over the first 1 h ischemia. The decreases in high energy adenylate levels were reflected in lower EC ratios in these three groups; values were 0.06-0.17 (P < 0.05) lower than corresponding sham values. Finally, it was an unexpected finding that the sham injection (0.5 mg/kg ethanol+PEG400) resulted in the sustained elevation of ATP, total adenylates, and EC values over the first h; EC ratios remained at initial (t = 0) values (EC = 0.71 +/- 0.01) up to 1 h.

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This study was designed to address the reasons why glycolysis in mammalian liver is unable to function more efficiently during periods of cold hypoxia. Our hypothesis was that control of intracellular pH, by use of amino acid buffers with high pKa values, would allow prolonged flux through glycolysis and better maintenance of liver high-energy adenine nucleotide pool. The effects of two concentrations of histidine (90 and 180 mM) and one of carnosine (90 mM), a histidyl dipeptide, on energy metabolism and levels of glycolytic substrate (glucose) and anaerobic endproduct (lactate) were investigated during cold hypoxia using rat livers to model the mammalian system. The transition to anaerobic metabolism was apparent by an immediate rise in lactate levels upon entry into cold hypoxia. By 10-14 h hypoxia, contents of the endproduct had increased by 10, 13.5, and 14.5 mumol/g in buffers containing 90 and 180 mM histidine and 90 mM carnosine, respectively. As well, ATP, total adenylate contents, and "energy charge" ratios exhibited a rapid decline from initial values of 2.3-3.3 mumol/g, 4.3-5.5 mumol/g, and 0.64-0.75, respectively, over the first 2-4 h of cold hypoxia. With respect to efficacy, the 180 mM histidine buffer exhibited the most positive maintenance of adenylate levels, followed closely by 90 mM carnosine, and finally 90 mM histidine as the least effective of the three buffers. Nevertheless, all three buffers examined in this study showed positive effects compared to similarly treated livers stored in a solution of minor buffering capacity (a citrate-based solution) over the same time period. The data support the hypothesis that glycolytic flux and cellular energetics can be maintained by the inclusion of efficient buffering agents during periods of cold hypoxia.

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