ATP level in the whole rat liver during cold hypoxia and subsequent rewarming

Cold storage of the whole liver at 4 degrees C in SBS and UW solution allowed to prevent from osmotic swelling of cells, which appeared at early stages of liver storage at 4 degrees C in just saline solutions. This effect of preserving solutions contributes to the preservation of quite high level of intracellular ATP content in liver at the first two stages of hypothermic storage (6 and 18 hrs), which preserves even during following normothermic reperfusion of an organ. A statistical ATP reduction in comparison with the control level (almost twice) can be explained on the one hand by the exhaustion of intracellular substrates of oxidation and on the other hand by their loss for the supporting of homeostasis under cold ischemia and following incubation of liver at 37 degrees C.     

Apoptosis vs. necrosis: glutathione-mediated cell death during rewarming of rat hepatocytes

Hypothermia induces injury in its own right, but the mechanisms involved in the cell damage are still unclear. The aim of this study was to test the effects that glutathione (GSH) depletion induces on cell death in isolated rat hepatocytes, kept at 4 degrees C for 20 h, by modulating intracellular GSH concentration with diethylmaleate and buthionine sulfoximine (DEM and BSO). Untreated hepatocytes showed Annexin V stained cells (AnxV(+)), scarce propidium iodide stained cells (PI(+)) and presented a low level of lactate dehydrogenase (LDH) leakage after 20 h at 4 degrees C and rewarming at 37 degrees C. When DEM and BSO were added before cold storage, we observed a few AnXV(+) cells and an increase in PI(+) cells associated with LDH release in the incubation medium. Conversely, the addition of DEM and BSO only during rewarming caused a marked increase in cell death by apoptosis. Production of reactive oxygen species (ROS) and thiobarbituric acid species (TBARS), associated with a decrease in GSH concentrations, was higher when DEM and BSO were added before cold storage. Cells treated with DEM and BSO before cold storage showed lower ATP energy stores than hepatocytes treated with DEM and BSO only during rewarming. Pretreatment of hepatocytes with deferoxamine protected against apoptotic and necrotic morphology in conditions of GSH depletion. These results suggest that pretreatment of hepatocytes with DEM and BSO before cold storage induces necrosis, while the treatment of hepatocytes only during rewarming increases apoptosis. In both conditions, iron represents a crucial mediator of cell death.     

Selenophosphate as a substrate for mammalian selenocysteine synthase, its stability and toxicity

The mechanism of selenocysteine synthesis on tRNASec in mammals was previously studied by means of HSe- as a Se donor to synthesize selenocysteine. It has been recently established that HSe- in E. coli is activated by ATP to become selenophosphate (SeP). In this study, we provide evidence that [75Se]selenocysteine is produced by bovine selenocysteine synthase from Ser-tRNASec and [75Se]Sep, synthesized from elemental 75Se and Tris(trimethylsilyl)phosphite. We also studied the stability of SeP by NMR measurement. SeP was stable during storage under nitrogen at -80 degrees C for 3 months in 0.2 M Hepes buffer at pH 6.8. However, SeP decomposed at 0 degree C in air (half life 32 hrs) or at 22 degrees C under nitrogen (half life 30 hrs) at pH 6.8. The half lives of SeP at -19 degrees C in air and at 0 degree C under nitrogen at pH 6.8 were 740 and 840 hrs, respectively. At pH 4 under nitrogen at 22 degrees C, the half life was 240 hrs. The half life was only 9.2 hrs at pH 9 under nitrogen at 0 degree C. Thus, SeP was proved to be stable at low temperature, under acidic and anaerobic conditions, but labile under neutral and alkaline conditions. The LD50 of SeP administered i.p. to mice was 37.5 mg/kg body weight.     

Dynamics of the changes in homogenate respiratory activity after rat whole liver storage at 4 degrees C

Homogenate respiratory activity was studied after different storage terms of the whole rat liver at 4 degrees C in sucrose-based solution and following normothermic reperfusion. Preservation of homogenate respiratory activity in all metabolic states after normothermic reperfusion of the control liver (60 min, 4 degrees C) is shown. Further storage (6 and 24 hrs) of isolated liver under the mentioned above conditions strengthens the substrate respiration of homogenate both after storage and after normothermic reperfusion. At the same time oxidative phosphorylation does not practically change. No change was noted in respiratory activity in the states 3, 4ADP and 3DNP after 24 hrs of liver storage in respect of a previous term. Following normothermic liver reperfusion contributes to a statistically true reduction of mentioned parameters of respiration, that correlates with a decrease in the degree of respiration and phosphorylation coupled of the studied system.     

The effect of pH on the viability of hypothermically stored rat hepatocytes

Hepatocytes isolated from the rat liver were stored for up to 72 hr at 4 degrees C in a tissue culture medium (Liebovitz-15) at different pH values to determine how pH affects hepatocyte viability. This is a model to simulate cold storage of livers for transplantation and determine the optimal pH for maintenance of liver cell function. The cells were stored in the absence of oxygen. At the end of cold storage the percentage of the total cellular LDH released into the extracellular medium was used as a measure of hepatocyte viability. Also, lactate dehydrogenase (LDH) release was determined in hepatocytes incubated at normothermia (37 degrees C) for 90 min following 72 hr of cold storage. The results demonstrate that hepatocytes tolerate a wide range of pH values in the storage medium and that only about 10% of the total LDH was released from hepatocytes stored up to 72 hr at pH's from 5.0 to 8.0. Normothermic incubation, however, demonstrated that the pH of the storage medium affected viability. After 48 hr of storage only hepatocytes stored at pH values from 7.0 to 8.0 remained viable (LDH release similar to that of freshly incubated hepatocytes = 28 +/- 7.2%). After 72 hr of storage and 90 min of normothermic incubation, hepatocytes incubated at all pH values studied were nonviable (greater than 60% release of LDH). These results suggest that the optimal pH for storage of hepatocytes at 4 degrees C is near neutrality (7.0 to 7.4).     

Ischemic preconditioning in a rodent hepatocyte model of liver hypothermic preservation injury

Ischemic preconditioning (IPC) is a phenomenon of protection in various tissues from normothermic ischemic injury by previous exposure to short cycles of ischemia-reperfusion. The ability of IPC to protect hepatocytes from a model of hypothermic transplant preservation injury was tested in this study. Rat hepatocytes were subjected to 30min of warm ischemia (37 degrees C) followed by 24 or 48h of hypothermic (4 degrees C) storage in UW solution and subsequent re-oxygenation at normothermia for 1h. Studies were performed with untreated control cells and cells treated with IPC (10min anoxia followed by 10min re-oxygenation, 1 cycle). Hepatocytes exposed to IPC prior to warm ischemia released significantly less LDH and had higher ATP concentrations, relative to untreated ischemic hepatocytes. IPC significantly reduced LDH release after 24h of cold storage before reperfusion and after 48h of cold storage and after 60min of warm re-oxygenation, relative to the corresponding untreated hepatocytes. ATP levels were also significantly higher when IPC was used prior to the warm and cold ischemia-re-oxygenation protocols. In parallel studies, IPC increased new protein synthesis and lactate after cold storage and reperfusion compared to untreated cells but no differences in the patterns of protein banding were detected on electrophoresis between the groups. In conclusion, IPC significantly improves hepatocyte viability and energy metabolism in a model of hypothermic preservation injury preceded by normothermic ischemia. These protective effects on viability may be related to enhanced protein and ATP synthesis at reperfusion.     

The hepatic galactosyl receptor system: two different ligand dissociation pathways are mediated by distinct receptor populations

After internalization of 125I-asialo-orosomucoid (ASOR) by isolated rat hepatocytes, ligand dissociates by two kinetically distinct pathways (Oka and Weigel, J. Biol. Chem. 257, 10,253, 1983). These slow and fast dissociation pathways correspond to two functionally different subpopulations of cell surface galactosyl receptors designated, respectively, State 1 and State 2 receptors. Freshly isolated cells or cells equilibrated below 24 degrees C express only State 1 receptors. Cells equilibrated at 37 degrees C express both State 1 and State 2 receptors. Ligand dissociation after internalization of surface-bound 125I-ASOR was measured using the permeabilizing detergent, digitonin. The slow dissociation pathway was mediated by State 1 receptors and was the only pathway expressed by cells which were freshly isolated or had been equilibrated at 24 degrees C. State 2 receptors are expressed at temperatures above about 20 degrees C, and both the fast and slow dissociation pathways occurred in cells equilibrated at 37 degrees C. State 2 receptors therefore mediate the rapid dissociation pathway. Dissociation and subsequent degradation of specifically bound ligand routed in either pathway were complete, respectively, within 3 and 6 hrs.     

Glutathione synthesis during the rewarming of rat hepatocytes preserved in the University of Wisconsin solution

In this study, we used isolated rat hepatocytes to investigate the effect of nucleoside content of the preserved cells on the ability to synthesize glutathione (GSH) during the rewarming process. We cold-stored hepatocytes in University of Wisconsin (UW) solution (72 h, 0 degrees C, N(2)) without nucleosides and with the addition of 5 mM adenosine or 10 mM ATP. After 72 h of cold storage, we determined the GSH synthesis rate and the ATP content of the cells. We found a GSH synthesis rate similar to that of freshly isolated hepatocytes only in the group of cells cold-stored with 10 mM ATP. When we tested the cellular ATP concentrations, we found that controls and preserved cells with 10 mM ATP showed a similar value of ATP during the rewarming step. Our results suggested that the incorporation of ATP in the UW solution increased the ATP content and the rate of GSH synthesis of cold-stored hepatocytes during rewarming.     

Reconstitution of galactosyl receptor inactivation in permeabilized rat hepatocytes is ATP-dependent.

A subpopulation of galactosyl receptors (GalRs) on isolated rat hepatocytes undergo a reversible inactivation and reactivation process during constitutive recycling (McAbee, D. D., and Weigel, P. H. (1988) Biochemistry 27, 2061-2069). Here, we report the reconstitution of this GalR inactivation in digitonin-permeabilized rat hepatocytes. Permeabilization of freshly isolated cells at 4 degrees C with 0.002% (w/v digitonin releases cytosol containing 35-40% of the total cellular protein, 10-15% of a lysosomal marker, and 5-10% of an early endosomal marker. Incubation of permeabilized cells with cytosol at 37 degrees C results in a time-dependent reduction of total 125I-asialoorosomucoid binding activity, which proceeds with first order kinetics (t 1/2 = 11.3 min). Only half of the total cellular GalRs are affected; maximal GalR activity loss, obtained by 30 min, is 50.5 +/- 9.5% (n = 21) of the control (4 degrees C) value. Increasing the digitonin concentration up to 0.055% does not increase the extent of inactivation. Permeabilized cells with reduced GalR activity were assessed for GalR protein content by Western blot analysis and by binding of anti-GalR antibody. The results show that the reduced 125I-asialoorosomucoid binding is due to GalR inactivation rather than receptor protein degradation. GalR inactivation does not occur in the absence of cytosol or in the presence of dialyzed cytosol. The cytosol also loses its GalR inactivating ability in the presence of an ATP-depleting system. GalR inactivation in the absence of cytosol is achieved by incubating permeabilized washed cells at 37 degrees C with ATP but not with ADP, AMP, or other NTPs. The rate and extent of inactivation are dependent on the ATP concentration. Half-maximal and maximal GalR inactivation are obtained at 0.3 and 3.0 mM ATP, respectively. In the presence of cytosol, permeabilized hepatocytes could replenish cytosolic ATP by oxidative phosphorylation. As a result, similar levels of GalR inactivation were obtained with 500-fold lower ATP concentrations. We conclude that ATP is the only cytosolic component necessary for GalR inactivation in permeabilized rat hepatocytes.     

Mitochondria-targeted plastoquinone derivative SkQ<Subscript>1</Subscript> decreases ischemia-reperfusion injury during liver hypothermic storage for transplantation

The ability of the mitochondria-targeted plastoquinone derivative 10-(6′-plastoquinonyl)decyl triphenylphosphonium (SkQ₁) to decrease ischemia-reperfusion injury in isolated liver during hypothermic storage (HS) was studied. Rat liver was stored for 24 h at 4°C without or in the presence of 1 μM SkQ₁ with following reperfusion for 60 min at 37°C. The presence in the storage medium of SkQ₁ significantly decreased spontaneous production of reactive oxygen species and intensity of lipid peroxidation in the liver during HS and reperfusion. The GSH level after HS in solution with SkQ₁ was reliably higher, but reperfusion leveled this effect. At all stages of experiment the presence of SkQ₁ did not prevent the decrease of antioxidant enzyme activities such as catalase, GSH peroxidase, GSH reductase, and glucose-6-phosphate dehydrogenase. The addition of SkQ₁ to the storage medium improved energetic function of the liver, as was revealed in increased respiratory control index of mitochondria and ATP level. SkQ₁ exhibited positive effect on the liver secretory function and morphology after HS as revealed in enhanced bile flow rate during reperfusion and partial recovery of organ architectonics and state of liver sinusoids and hepatocytes. The data point to promising application of mitochondria-targeted antioxidants for correction of the ischemia-reperfusion injury of isolated liver during long-term cold storage before transplantation.     

An AMP nucleosidase gene knockout in Escherichia coli elevates intracellular ATP levels and increases cold tolerance

Disparate psychrophiles (e.g. glacier ice worms, bacteria, algae and fungi) elevate steady-state intracellular ATP levels as temperatures decline, which has been interpreted as a compensatory mechanism to offset reductions in molecular motion and Gibb's free energy of ATP hydrolysis. In this study, we sought to manipulate steady-state ATP levels in the mesophilic bacterium, Escherichia coli, to investigate the relationship between cold temperature survivability and elevated intracellular ATP. Based on known energetic pathways and feedback loops, we targeted the AMP nucleotidase (amn) gene, which is thought to encode the primary AMP degradative enzyme in prokaryotes. By knocking out amn in wild-type E. coli DY330 cells using recombineering methodology, we generated a mutant (AMNk) that elevated intracellular ATP levels by more than 30% across its viable temperature range. As temperature was lowered, the relative ATP disparity between AMNk and DY330 cells increased to approximately 66% at 10 degrees C, and was approximately 100% after storage at 0 degrees C for 5-7 days. AMNk cells stored at 0 degrees C for 7 days displayed approximately fivefold higher cell viability than wild-type DY330 cells treated in the same manner.     

Cardiac hypothermia: 31P and 1H NMR spectroscopic studies of the effect of buffer on preservation of human heart atrial appendages.

31P and 1H nuclear magnetic resonance spectroscopy has been used to follow noninvasively the time course of energetic metabolite levels in human heart atrial appendages preserved under various temperatures and buffer conditions. From sample harvest up to the normal 5-h time limit for heart preservation, ATP levels in human atrial appendages are much better maintained in 0.9% saline and PIPES-buffered preservation solutions at 12 degrees C than at 4 degrees C. Furthermore, preservation at 12 degrees C can be improved considerably by using high extracellular buffer concentrations. The increased buffer concentration allows better maintenance of the intracellular pH and leads to a faster glycolytic rate as measured by lactate production. At 4 degrees C, ATP levels decline rapidly during the first 5 h but reached a stable plateau, which is well maintained over 15-20 h. At this temperature, the rate of lactate production is similar at all buffer concentrations (20, 60, and 100 mM PIPES). As a consequence of these observations, we postulate that the mechanisms of ATP production and utilization at 4 degrees C and at 12 degrees C are different. At 4 degrees C, the rate of glycolysis is temperature limited whereas at 12 degrees C, low intracellular pH inhibits glycolysis.     

Changes in UsnRNA biosynthesis during rat liver regeneration

Partial hepatectomy (P.H.) induces a partially synchronized growth response of liver under normal regulation of growth. In this phase changes in cellular morphology, radial distribution pattern of cells and other biological as well as major biochemical changes are well documented [24]. Here, we have shown that the cellular content of UsnRNAs altered during this proliferative phase as well. The level of spliceosomal UsnRNAs (U1, U2, U4–U6) gradually decreased by 30–50% upto 48 hrs of P.H. followed by gradual increase to reach the normal level within one month of P.H. The U3 snRNA level on the other hand, was nearly equal to that in normal liver at 48 hrs of P.H. but in 24 and 72 hrs of P.H. its level was high (4 fold) in contrast to that in other UsnRNAs. Thus, it is clear from our data that the level of all the six UsnRNAs decreased during 48 hrs of P.H. compared to that after first 24 hrs. This has been correlated in the kinetics of UsnRNAs' synthesis (in terms of labelling) in isolated hepatocytes, where the rate of labelling of all the six UsnRNAs increased 20–30% in 24 hrs regenerating hepatocytes (R.H.) followed by sharp decrease by 30–50% within next 24 hrs, compared to that in the normal hepatocytes. But from 72 hrs onwards in R.H. the rate of labelling of all the six UsnRNAs again increased by 30–50% (compared to that in normal hepatocytes) followed by decrease of their labelling-rate to reach the normal level in R.H. within one month of P.H. Thus, it may be concluded that the changes in UsnRNAs' level during the proliferative phase of liver regeneration may be either due to the alteration in the rate of synthesis (in terms of labelling) or along with it differential turn over rate; this phenomenon may have some consequences with the regenerative process of liver.This paper was published in Molecular and Cellular Biochemistry131:67–73, 1994. Kluwer Academic Publishers regret the publication of the only partly corrected version.     

Photosynthetic regeneration of ATP using a strain of thermophilic blue-green algae

Photosynthetic ATP accumulation was shown in the presence of exogenous ADP plus orthophosphate on illumination to the intact cells of a strain of thermophilic blue-green algae isolated from Matsue hot springs, Mastigocladus sp. Kinetic studies of various effectors on the ATP accumulation proved that the ATP synthesis depends mainly on the cyclic photophosphorylation system around photosystem I (PS-I) in the algal cells. The temperature and pH optima for the accumulation were found at 45 degrees C and pH 7.5. Maximum yield was obtained with light intensity higher than 15 mW/cm(2). Borate ion exerted pronounced enhancement on the ATP synthesis. With a continuous reactor at a flow rate of 1 ml/hr at 45 degrees C and pH 7.5, efficient photoconversion of ADP (2mM, at substrate reservoir) to ATP (1mM, at product outlet) has been maintained for a period of 2.5 days, though the efficiency has decreased in a further 2-day period to the level of 0.5mM ATP/9.5 h of residence time.     

Phospholipid molecular species, beta-oxidation, desaturation and elongation of fatty acids in Atlantic salmon hepatocytes: effects of temperature and 3-thia fatty acids

We have investigated the effects of a 3-thia fatty acid (TTA) and of temperature on the fatty acid (FA) metabolism of Atlantic salmon (Salmo salar). One experiment investigated the activity of the peroxisomal beta-oxidation enzyme, acyl-CoA oxidase (ACO), and the incorporation of TTA into phospholipid (PL) molecular species. Salmon hepatocytes in culture were incubated either without TTA (control(spades)) or with 0.8 mM TTA (TTA(spades)) in a short term (48 h) temperature study at 5 degrees C and at 12 degrees C. TTA was incorporated into the four PL classes studied: phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylinositol (PI) and phosphatidylserine (PS). TTA was preferentially esterified with 18:1, 16:1, 20:4 and 22:6 in the PLs. Hepatocytes incubated with TTA had higher ACO activity at 5 degrees C than at 12 degrees C. In a second experiment salmon were fed a diet based on fish meal-fish oil without any TTA added (control) or a fish meal-fish oil diet supplemented with 0.6% TTA for 8 weeks at 12 degrees C and 20 weeks at 5 degrees C. At the end of the feeding trial, hepatocytes from fish acclimated to high or low temperatures were isolated from both dietary groups and incubated with either [1-(14)C]18:1 n-9 or [1-(14)C]20:4 n-3 at 5 degrees C or 12 degrees C. Radiolabelled 18:1 n-9 was mainly esterified into neutral lipids (NL), whereas [1-(14)C]20:4 n-3 was mainly esterified into PL at both temperatures. The rate of elongation of [1-(14)C]18:1 n-9 to 20:1 n-9 was twice as high in hepatocytes from fish fed the control diet than it was in hepatocytes from fish fed the TTA diet, at both temperatures. The amount of [1-(14)C]20:4 n-3 converted to 22:6 n-3 was approximately the same in hepatocytes from the two dietary groups, but there was a tendency to higher production of 22:6 n-3 at the lower temperature. Oxidation of [1-(14)C]18:1 n-9 to acid soluble products (ASP) and CO(2) was approximately 10-fold greater in hepatocytes kept at 5 degrees C than in those kept at 12 degrees C and the main oxidation products formed were acetate, oxaloacetate and malate.     

Tyrosine phosphorylation of the asialoglycoprotein receptor

The asialoglycoprotein (ASGP) receptor undergoes constitutive endocytosis through the coated pit/coated vesicle pathway in hepatocytes. Studies on HepG2 cells have shown that the receptor is phosphorylated at serine under control conditions and following protein kinase C stimulation. This study examined whether the ASGP receptor could also serve as a substrate for a tyrosine kinase in HepG2 cells. 32P labeling was performed in membrane preparations, in permeabilized cells at 4 degrees C, and in intact cells at 37 degrees C. The phosphorylated ASGP receptor was isolated by immunoprecipitation, hydrolyzed in 6 N HCl at 110 degrees C, and analyzed by two-dimensional high voltage electrophoresis. The receptor isolated from a membrane preparation incubated in vitro with [gamma-32P]ATP incorporated radiolabel predominantly (greater than 90%) into phosphotyrosine. ASGP receptor phosphorylation at both tyrosine and serine was detected in intact cells incubated with phosphatase inhibitors for 60 min at 37 degrees C. The presence of both phenylarsine oxide (20 microM) and sodium orthovanadate (200 microM) was required for tyrosine phosphorylation. Use of these inhibitors together resulted in a 16.4-fold increase in phosphorylation of the immunoprecipitated ASGP receptor, whereas phosphorylation of total HepG2 membrane proteins was not significantly augmented by this procedure. Selective proteolytic digestion of ASGP receptors in isolated vesicles demonstrated that the phosphorylation site identified in these studies is located at tyrosine 5 in the cytoplasmic tail.     

Recycling of the asialoglycoprotein receptor in isolated rat hepatocytes. ATP depletion blocks receptor recycling but not a single round of endocytosis

Continuous endocytosis of 125I-asialo-orosomucoid (ASOR) mediated by the galactosyl receptor in rat hepatocytes is a cyclic process. 125I-ASOR-receptor complexes are internalized, processed, and the ligand is degraded while the receptor is returned to the cell surface for reutilization. Since a true cycle has a thermodynamic requirement for the input of external energy, we examined the effects of changes in intracellular ATP levels on the function of the receptor cycle. Hepatocytes were depleted of ATP to various extents prior to endocytosis by incubating cells at 15 degrees C in the presence of 2 mM NaF and 0-20 mM NaN3. A luciferase-luciferin bioluminescence assay was used to quantitate the amount of cellular ATP. ATP-depleted cells were allowed to bind 125I-ASOR at 0 degrees C, washed through discontinuous Percoll gradients, and only viable cells were isolated and incubated at 37 degrees C to initiate a synchronous single round of endocytosis. The extent of internalization of this surface-bound 125I-ASOR was unaffected by an ATP depletion to less than 1% of the control level. The rate of internalization of surface-bound ligand was unaffected until the ATP levels decreased to 30% or less; at greater than 98% ATP depletion the initial rate decreased by a maximum of 55% and the kinetics became biphasic. In contrast, continuous endocytosis in the presence of excess ASOR was inhibited by only a 25% decline in cellular ATP content and demonstrated a very sharp threshold response to changing ATP levels. Continuous endocytosis, which requires receptor recycling, was completely inhibited when the total cellular ATP level decreased by only 40%. We conclude that the internalization phase of endocytosis is not dependent on ATP but that the processing and/or externalization phases of the complete receptor cycle are either directly or indirectly dependent on ATP and very sensitive to changes in cellular ATP content.     

Measurement temperature plays a pivotal role in the distribution of erythrocyte deformability after LPS

Reductions in red blood cell membrane deformability (RBC(D)) may perturb microcirculatory blood flow and impair tissue O(2)-availability. We investigated the effect of assay temperature on the distribution of RBC(D) in endotoxin (LPS) incubated and control RBCs. Fresh blood from healthy rats was incubated with and without the presence of LPS for 6 hrs. An index of red blood cell membrane deformability, delta, was measured via the micropipette aspiration technique at 25 degrees C and 37 degrees C at 0, 2 and 6 hrs of incubation. The ATP content of RBC was measured by the luciferin-luciferase technique. At 25 degrees C, LPS caused a significant decrease in mean delta after 2 and 6 hours incubation compared to controls (-10.0%, p=0.03 and -24.0%, p=0.03, respectively) characterized by a left shift in the distribution (skewness: -1.4). However, at 37 degrees C a significant decrease in delta was only detected after 6 hrs of LPS incubation (-13.8%, p=0.01, compared to -5.1%, p=0.7 at 2 hours) and lacked the left shifted distribution (skewness: 0.2). No significant difference in ATP content of RBCs was observed between groups. We have shown that LPS incubation results in a significant decrease in RBC(D) and that room temperature measurement of physical membrane properties may exaggerate the differences between normal and perturbed RBCs.     

Effect of dihydroxyacetone on metabolic parameters and survival of resuspended erythrocytes after storage at 25 degrees and 4 degrees]

Erythrocyte concentrates from CPD blood were resuspended after separating the leukocyte-thrombocyte layer and stored at 4 degrees C and 25 degrees C. The solutions for resuspension differed in the content of substrate, pH, condition of sterilization and the resuspension volume used, 1 or 4 volumes of cell concentrate to 1 volume of resuspension solution. At 4 degrees C, there was no effect of DHA on the the 2.3 bisphosphoglycerate content (P2G content). The decreased activity of triokinase at low temperature, phosphate deficiency and a partial disintegration of DHA during the autoclave as well as the difference of the commercial DHA preparations are discussed as underlying causes. At 25 degrees C DHA delayed the P2G decrease during storage. The enhanced triokinase activity above 15 degrees C is principally considered to be the cause for it. High rates of haemolysis, rapid ATP decrease, survival rates of 56% after a storage of three weeks for erythrocyte resuspensions with DHA in used compositions render a clinical application impossible at present. It could be well suitable as a substrate for resynthesizing P2G. An addition of ascorbate + nicotinamid + adenin at a storage temperature of 4 degrees C had no influence on the P2G content. A resuspension solution on the basis of xylitol with additions of sorbitol, bicarbonate, inorganic phosphate, pyruvate, adenine and guanosine showed the best properties in the content of P2G and ATP, lactate formation and survival rate during a storage at 4 degrees C.     

The effect of inosine, inorganic phosphates and pyruvate on erythrocyte glycolysis metabolites under conditions of preservation]

Human erythrocytes were stored as resuspensions in solutions containing citrate (Z), inosine + citrate (I), inosine + phosphate (IP), and inosine + phosphate + pyruvate (IPP). The storage was made at + 4 degrees C for 6 weeks; the initial pH-value amounted to 7.4 at + 4 degrees C. The cellular concentrations of 2.3 DPG, ATP, G6P, FDP and DOAP + GAP were determined. The following results were obtained: 1. During the storage in stored Z-blood the 2.3 DPG concentration will fall below 10% of its initial value; it will remain nearly unchanged in stored I-blood and will increase to 170% in stored IP-blood, to 270% of its initial value in stored IPP-blood. 2. The ATP concentration of cells will fall to about 50% of its initial value at the beginning of the storage of all stored blood. After that it will only increase to about 80% of its initial value in stored IP- and IPP-blood. 3. During the storage the G6P concentration will increase to the highest degree in stored IPP-blood and if high pyruvate concentrations are not present, it will have a reciprocal behaviour towards the FDP and triosephosphate level. The results were discussed in view of the regulation of glycolysis under storage conditions.