Total Adenylate Concentrations Research Articles

Magnesium Signaling in Plants.

Free magnesium (Mg2+) is a signal of the adenylate (ATP+ADP+AMP) status in the cells. It results from the equilibrium of adenylate kinase (AK), which uses Mg-chelated and Mg-free adenylates as substrates in both directions of its reaction. The AK-mediated primary control of intracellular [Mg2+] is finely interwoven with the operation of membrane-bound adenylate- and Mg2+-translocators, which in a given compartment control the supply of free adenylates and Mg2+ for the AK-mediated equilibration. As a result, [Mg2+] itself varies both between and within the compartments, depending on their energetic status and environmental clues. Other key nucleotide-utilizing/producing enzymes (e.g., nucleoside diphosphate kinase) may also be involved in fine-tuning of the intracellular [Mg2+]. Changes in [Mg2+] regulate activities of myriads of Mg-utilizing/requiring enzymes, affecting metabolism under both normal and stress conditions, and impacting photosynthetic performance, respiration, phloem loading and other processes. In compartments controlled by AK equilibrium (cytosol, chloroplasts, mitochondria, nucleus), the intracellular [Mg2+] can be calculated from total adenylate contents, based on the dependence of the apparent equilibrium constant of AK on [Mg2+]. Magnesium signaling, reflecting cellular adenylate status, is likely widespread in all eukaryotic and prokaryotic organisms, due simply to the omnipresent nature of AK and to its involvement in adenylate equilibration.

The effect of prefreeze rejuvenation on postthaw storage of red blood cells in AS-3 and SAGM.

We investigated whether improving the metabolic status of red blood cell concentrates before freezing could extend the postthaw shelf life beyond 14 days while still meeting the requirements for hemolysis (0.8%) and total adenylate (>82% of original values). At Day 8 after collection, four leukoreduced red blood cell concentrates in saline-adenine-glucose-mannitol (SAGM) were pooled, mixed, and split (n = 4). Of these concentrates, two were rejuvenated in Rejuvesol. In addition, two leukoreduced red blood cell concentrates in phosphate-adenine-glucose-guanosine-gluconate-mannitol (PAGGGM) were pooled, mixed, and split at Day 8 after collection (n = 4). All concentrates were glycerolized, frozen, and stored for at least 2 weeks at -80°C. After thawing and deglycerolization, from each pair, one red blood cell concentrate was resuspended in SAGM, and one was suspended in AS-3. During postthaw storage at 2 to 6°C for 35 days, all concentrates were sampled weekly and analyzed for hematologic, metabolic, and morphologic parameters. Both Rejuvesol and PAGGGM treatment produced increased adenosine triphosphate and total adenylate and 2,3-diphosphoglycerate levels compared with untreated red blood cell concentrates. Regardless of prefreeze Rejuvesol or PAGGGM treatment, postthaw hemolysis remained below 0.8% during 7 days in SAGM and during 35 days in AS-3. At Day 35 of postthaw storage in AS-3, total adenylate in nonrejuvenated red blood cell concentrates had decreased to 72% of the original values; whereas, in prefreeze Rejuvesol-treated and PAGGGM-treated concentrates, adenylate values were still were at 101% and 98%, respectively. Based on maximum allowable hemolysis of 0.8% and total adenylate content greater than 82% of the original value, thawed, prefreeze Rejuvesol-treated or PAGGGM-treated red blood cell concentrates can be stored for 35 days at 2 to 6ºC in AS-3.

Effects of hypoxic stress on energy metabolism in red sea bream, Pagrus major, 1: Stress response and energy status in various tissues of red sea bream, Pagrus major, subjected to hypoxic exposure

Young red sea bream, Pagrus major, were exposed to a gradual decrease in partial pres- sure of oxygen (PO2) down to 20 mmHg for 5 h, and then sustained under 20 mmHg PO2 for an additional 3 h at 19.9oC. Fish respiratory frequency slowly increased and attained a peak level at approximately 35 mmHg PO2, and then gradually decreased. Fish sank to the tank bottom about an hour after PO2 fell below 20 mmHg, and respiration began to arrest after another hour. Hematocrit value increased and mean cellular hemoglobin content (MCHC) decreased with increasing hypoxia load. Plasma cortisol and glucose levels were significantly augmented when respiratory frequency decreased. ATP, total adenylate (TA) concentration and energy charge (EC) in the hepatopancreas, in addition to TA in the kidney, were considerably diminished by hypoxia. However, this was not observed in the gill and brain of all fishes prior to respiratory arrest. In the hepatopancreas, levels of ATP, TA and EC notably declined as respiratory frequency decreased. These results show that the cortisol stress response to hypoxia is significantly induced with the decreased respiratory fre- quency. Decreased energy status in the hepatopancreas appears to be important and occurs with stress response as a metabolic depression in the whole body.

Intracellular ATP and total adenylate concentrations are critical predictors of reovirus productivity from Vero cells

The productivity of reovirus type-3 Dearing was studied in cultures of Vero cells in serum-free media. Viral productivity was dependent upon the metabolic state of the cells rather than the phase of growth at which the cells were infected. Cells at different energy states were established by 24-h incubation in nutrient-depleted media. This resulted in variable intracellular nucleotide concentrations but high cellular viability was maintained. Of the nucleotides analyzed at the time of infection only the intracellular [ATP] and total adenylate nucleotides were positively correlated with viral productivity. The correlated data followed a sigmoidal plot with an equation defined by polynomial regression analysis. Apparent threshold values of 3.2 fmol/cell and 3.3 fmol/cell were established for ATP and total adenylate, respectively, at which the viral production was 50% the maximal value. Cultures with lower ATP and total adenylate levels at the time of infection resulted in as much as a 95% reduction in overall viral titer compared to the control. The adenylate energy charge (AEC) showed a negative correlation with viral production with an AEC value >0.97 resulting in low virus productivity. Intracellular ATP or total adenylate concentration at the point of infection may be used as a predictor of viral yield in bioprocesses designed for virus/vaccine production.

Characterization of the photosynthetic apparatus in cortical bark chlorenchyma of Scots pine

Winter-induced inhibition of photosynthesis in Scots pine (Pinus sylvestris L.) needles is accompanied by a 65% reduction of the maximum photochemical efficiency of photosystem II (PSII), measured as Fv/Fm, but relatively stable photosystem I (PSI) activity. In contrast, the photochemical efficiency of PSII in bark chlorenchyma of Scots pine twigs was shown to be well preserved, while PSI capacity was severely decreased. Low-temperature (77 K) chlorophyll fluorescence measurements also revealed lower relative fluorescence intensity emitted from PSI in bark chlorenchyma compared to needles regardless of the growing season. Nondenaturating SDS-PAGE analysis of the chlorophyll-protein complexes also revealed much lower abundance of LHCI and the CPI band related to light harvesting and the core complex of PSI, respectively, in bark chlorenchyma. These changes were associated with a 38% reduction in the total amount of chlorophyll in the bark chlorenchyma relative to winter needles, but the Chl a/b ratio and carotenoid composition were similar in the two tissues. As distinct from winter pine needles exhibiting ATP/ADP ratio of 11.3, the total adenylate content in winter bark chlorenchyma was 2.5-fold higher and the estimated ATP/ADP ratio was 20.7. The photochemical efficiency of PSII in needles attached to the twig recovered significantly faster (28-30 h) then in detached needles. Fluorescence quenching analysis revealed a high reduction state of Q(A) and the PQ-pool in the green bark tissue. The role of bark chlorenchyma and its photochemical performance during the recovery of photosynthesis from winter stress in Scots pine is discussed.

Stress response and energy metabolism in various tissues of Nile tilapia Oreochromis niloticus exposed to hypoxic conditions

: Nile tilapia were exposed to severe hypoxic conditions. The respiratory frequency (RF) of the fish reached a maximum level at approximately 10 mmHg PO2 and decreased gradually thereafter. The fish sank to the bottom and subsequently their respiration began to stop. The hypoxic conditions increased the hematocrit value. As RF decreased, plasma cortisol and glucose levels climbed drastically. Just before respiratory arrest, the ATP and the total adenylate (TA) concentrations were significantly depressed in the liver, kidney and ordinary muscle but not in the heart or gill. As RF decreased, ATP and TA in both the liver and kidney decreased rapidly. Cytochrome oxidase activity increased significantly in the brain, heart, gill and ordinary muscle until respiratory frequency peaked. However, from the peak RF, this activity decreased in the liver and kidney. Lactic acid levels in both ordinary muscle and liver increased markedly when the fish sank. These results indicate that the stress response to hypoxic conditions is induced markedly with the decreased RF. It is also suggested that the decreased energy status in the liver and kidney of tilapia occurs at the same stage as the metabolic depression in the whole body, without increases in the aerobic and anaerobic metabolisms.

Mineralization of plant-incorporated residues of 14C-isoproturon in arable soils originating from different farming systems

14C-isoproturon residues were incorporated in wheat plants by growing seedlings for 18 days in quartz sand with nutrient solution which was treated with ring-labeled 14C-isoproturon, resulting in 14C-concentration equivalent to 15.4 nmol isoproturon per g dry shoot mass. The residues were characterized by extraction and HPLC-analysis, and were shown to consist of unchanged isoproturon, soluble metabolites (monodemethyl-isoproturon, didemethyl-isoproturon, 1-OH-isoproturon, 2-OH-isoproturon, 2-OH-monodemethyl-isoproturon, 2-OH-didemethyl-isoproturon, isopropenyl-isoproturon and unidentified metabolites), as well as nonextractable residues. Dried plant samples containing these residues were mixed with soil samples originating from different farming systems, and mineralization to 14CO 2 was determined in a closed aerated laboratory system. In addition, the microbial biomass and bioactivity of soils were estimated by determination of substrate-induced heat output, basal heat output, metabolic heat quotient, total adenylate content and adenylate energy charge. Significant positive correlations between 14CO 2 production or adenylate content and microbial biomass were observed in three soils; 14CO 2 production and total microbial biomass were highest in soil samples from organic farming. Soil samples from a former hops plantation contaminated with copper from previous fungicide applications did not fit this correlation, but exhibited a higher mineralization capacity per unit of microbial biomass. Our results indicate that general soil microbial parameters in many cases are insufficient to describe the influence of biotic factors on the fate of pesticides in soil.

Implications of adenylate kinase-governed equilibrium of adenylates on contents of free magnesium in plant cells and compartments.

On the basis of the equilibrium of adenylate kinase (AK; EC 2.7.4.3), which interconverts MgATP and free AMP with MgADP and free ADP, an approach has been worked out to calculate concentrations of free magnesium (Mg(2+)), based on concentrations of total ATP, ADP and AMP in plant tissues and in individual subcellular compartments. Based on reported total adenylate contents, [Mg(2+)] in plant tissues and organelles varies significantly depending on light and dark regimes, plant age and developmental stage. In steady-state conditions, [Mg(2+)] in chloroplasts is similar in light and darkness (in the millimolar range), whereas in the cytosol it is very low in the light and increases to about 0.4 mM in darkness. During the dark-to-light transition (photosynthetic induction), the [Mg(2+)] in chloroplasts falls to low values (0.2 mM or less), corresponding to a delay in photosynthetic oxygen evolution. This delay is considered to result from lower activities of Mg-dependent enzymes in the Calvin cycle. In mitochondria, the changes in [Mg(2+)] are similar but smoother. On the other hand, when the transition from light to darkness is considered, an initial increase in [Mg(2+)] occurs in both chloroplasts and mitochondria, which may be of importance for the control of key regulatory enzymes (e.g. mitochondrial malic enzyme and pyruvate dehydrogenase complex) and for processes connected with light-enhanced dark respiration. A rationale is presented for a possible role of [MgATP]/[MgADP] ratio (rather than [ATP(total)]/[ADP(total)]) as an important component of metabolic cellular control. It is postulated that assays of total adenylates may provide an accurate measure of [Mg(2+)] in plant tissues/cells and subcellular compartments, given that the adenylates are equilibrated by AK.

Implications of adenylate kinase-governed equilibrium of adenylates on contents of free magnesium in plant cells and compartments

On the basis of the equilibrium of adenylate kinase (AK; EC 2.7.4.3), which interconverts MgATP and free AMP with MgADP and free ADP, an approach has been worked out to calculate concentrations of free magnesium (Mg2+), based on concentrations of total ATP, ADP and AMP in plant tissues and in individual subcellular compartments. Based on reported total adenylate contents, [Mg2+] in plant tissues and organelles varies significantly depending on light and dark regimes, plant age and developmental stage. In steady-state conditions, [Mg2+] in chloroplasts is similar in light and darkness (in the millimolar range), whereas in the cytosol it is very low in the light and increases to about 0.4mM in darkness. During the dark-to-light transition (photosynthetic induction), the [Mg2+] in chloroplasts falls to low values (0.2mM or less), corresponding to a delay in photosynthetic oxygen evolution. This delay is considered to result from lower activities of Mg-dependent enzymes in the Calvin cycle. In mitochondria, the changes in [Mg2+] are similar but smoother. On the other hand, when the transition from light to darkness is considered, an initial increase in [Mg2+] occurs in both chloroplasts and mitochondria, which may be of importance for the control of key regulatory enzymes (e.g. mitochondrial malic enzyme and pyruvate dehydrogenase complex) and for processes connected with light-enhanced dark respiration. A rationale is presented for a possible role of [MgATP]/[MgADP] ratio (rather than [ATPtotal]/[ADPtotal]) as an important component of metabolic cellular control. It is postulated that assays of total adenylates may provide an accurate measure of [Mg2+] in plant tissues/cells and subcellular compartments, given that the adenylates are equilibrated by AK.

Effects of Freeze-Thaw and Photobleaching on the Ultraviolet Resonance Raman Spectra of Human Colon Biopsies

We studied the effects of sample freezing and thawing, laser fluence, temperature, and oxygen on the ultraviolet resonance Raman (UVRR) spectra of human colon biopsies at 251 nm excitation. We show that the total adenylate content of the tissue decreases as a result of freezing and subsequent thawing to room temperature. We also show that photobleaching is the result of oxidative as well as thermal damage. Photobleaching consisted of a decrease in the intensity of the purine spectral bands and broadening and intensity increase of the aromatic amino acid bands. Both bio- and photodegradation of the tissue can be minimized by keeping the biopsy samples refrigerated during testing and the laser fluence under 0.2 mJ/μm2. Unlike amino acid photobleaching, purine photobleaching was also greatly reduced by placing the samples in an argon atmosphere. Sample storage could be achieved by refrigerating the fresh specimens and testing them within 30 h of surgical resection, without freezing. Alternatively, the fresh specimens could be snap-frozen in liquid nitrogen, then thawed and tested for up to 30 min. Altogether, these studies can be utilized to guide future experimental design and data interpretation during ultraviolet spectroscopy of biological tissues.

Fatty acid patterns of phospholipids and lipopolysaccharides in the characterisation of microbial communities in soil: a review

This review discusses the analysis of whole-community phospholipid fatty acid (PLFA) profiles and the composition of lipopolysaccharides in order to assess the microbial biomass and the community structure in soils. For the determination of soil microbial biomass a good correlation was obtained between the total amount of PLFAs and the microbial biomass measured with methods commonly used for determinations such as total adenylate content and substrate-induced respiration. Generally, after the application of multivariate statistical analyses, whole-community fatty acid profiles indicate which communities are similar or different. However, in most cases, the organisms accounting for similarity or difference cannot be determined, and therefore artefacts could not be excluded. The fatty acids used to determine the biomass vary from those which determine the community structure. Specific attention has to be paid when choosing extraction methods in order to avoid the liberation of fatty acids from non-living organic material and deposits, and to exclude the non-target selection of lipids from living organisms, as well. By excluding the fatty acids which were presumed to be common and widespread prior to multivariate statistical analysis, estimates were improved considerably. Results from principal component analysis showed that determining the levels of fatty acids present in both low and high concentrations is essential in order to correctly identify microorganisms and accurately classify them into taxonomically defined groups. The PLFA technique has been used to elucidate different strategies employed by microorganisms to adapt to changed environmental conditions under wide ranges of soil types, management practices, climatic origins and different perturbations. It has been proposed that the classification of PLFAs into a number of chemically different subgroups should simplify the evaluating procedure and improve the assessment of soil microbial communities, since then only the subgroups assumed to be involved in key processes would be investigated.

Effects of Hypoxia and Toxicant Exposure on Adenylate Energy Charge and Cytosolic ADP Concentrations in Abalone

Many studies have used adenylate energy charge (AEC) as an index of an organism's metabolic state under conditions of imposed stress, either through natural or xenobiotic stressors. AEC is a linear measure of the ratio of ATP concentration to total adenylate concentration, which ranges in value from 1 in the fully charged state to 0. Paradoxically, high values of AEC are often associated with high toxicant exposures, and low AEC values with low exposures. These discrepancies may be caused by the inability of AEC measurements to adequately evaluate cytosolic adenylate concentrations, which are the critical parameters in enzymatic regulation. Consequently, the goal of this study was to compare AEC values, as measured by high performance liquid chromatography (HPLC), to free adenosine diphosphate (ADP free) concentrations, as measured using the arginine kinase equilibrium reaction and in vivo 31P-NMR, in red abalone ( Haliotis rufescens) in response to either hypoxia or toxicant (pentachlorophenol, or sodium azide) exposure. AEC values remain essentially constant when compared with control animals during periods of stress exposure and recovery. In contrast, calculated ADP free concentrations are approximately a third of those determined by HPLC and nearly double in response to stress exposure. The physiologic importance of this response is demonstrated by increases in ATP formation via arginine kinase. These results are discussed in light of the pertinent mammalian literature.

Mineralization of [14C] glyphosate and its plant-associated residues in arable soils originating from different farming systems

The biomineralization of [14C]glyphosate, both in the free state and as 14C-residues associated with soybean cell-wall material, was studied in soil samples from four different agricultural farming systems. After 26 days, [14C]carbon dioxide production from free glyphosate accounted for 34–51% of the applied radiocarbon, and 45–55% was recovered from plant-associated residues. For three soils, the cumulative [14C]carbon dioxide production from free glyphosate was positively correlated with soil microbial biomass, determined by substrate-induced heat output measurement and by total adenylate content. The fourth soil, originating from a former hop plantation, and containing high concentrations of copper from long-term fungicide applications, did not fit this correlation but showed a significantly higher [14C]carbon dioxide production per unit of microbial biomass. Although the cumulative [14C]carbon dioxide production from plant-associated 14C-residues after 26 days was as high as from the free compound, it was not correlated with the soil microbial biomass. This indicates that the biodegradation of plant-associated herbicide residues, in contrast to that of the free compound, involves different degradation processes. These encompass either additional steps to degrade the plant matrix, presumably performed by different soil organisms, or fewer degradation steps since the plant-associated herbicide residues are likely to consist mainly of easily degradable metabolites. Moreover, the bioavailability of plant-associated pesticide residues seems to be dominated by the type and strength of their fixation in the plant matrix. ©1997 SCI

Mineralization of [14C] glyphosate and its plant‐associated residues in arable soils originating from different farming systems

The biomineralization of [14C]glyphosate, both in the free state and as 14C-residues associated with soybean cell-wall material, was studied in soil samples from four different agricultural farming systems. After 26 days, [14C]carbon dioxide production from free glyphosate accounted for 34–51% of the applied radiocarbon, and 45–55% was recovered from plant-associated residues. For three soils, the cumulative [14C]carbon dioxide production from free glyphosate was positively correlated with soil microbial biomass, determined by substrate-induced heat output measurement and by total adenylate content. The fourth soil, originating from a former hop plantation, and containing high concentrations of copper from long-term fungicide applications, did not fit this correlation but showed a significantly higher [14C]carbon dioxide production per unit of microbial biomass. Although the cumulative [14C]carbon dioxide production from plant-associated 14C-residues after 26 days was as high as from the free compound, it was not correlated with the soil microbial biomass. This indicates that the biodegradation of plant-associated herbicide residues, in contrast to that of the free compound, involves different degradation processes. These encompass either additional steps to degrade the plant matrix, presumably performed by different soil organisms, or fewer degradation steps since the plant-associated herbicide residues are likely to consist mainly of easily degradable metabolites. Moreover, the bioavailability of plant-associated pesticide residues seems to be dominated by the type and strength of their fixation in the plant matrix. ©1997 SCI

Some aspects of the extreme anoxia tolerance of the sweet flag,Acorus calamus L.

Acorus calamus L. is a neophyte in Europe with remarkable properties. Among other things, it is the most anoxial tolerant species and a competitive invader at eutrophic sites. The following overview presents the most recent work on these subjects. Carbohydrates of the rhizomes sustain anaerobic ATP production for very long periods. Ethanolic fermentation naturally occurs in winter and produces rather low, but sufficient amounts of ATP for survival, as shown by adenylate energy charge and total adenylate content. Fermentation energy is mainly used for the synthesis and preservation of essential macromolecules, such as proteins and membrane lipids. The extent of these processes is unique. Moreover, ammonia and sulphide uptake is maintained during the cold season. Both ions are detoxified to alanine and thiols which are translocated into the rhizome, where the nitrogen of alanine is used to form arginine. Overwintering leaves contain asparagine instead of arginine. Recycled nitrogen compounds from the rapidly degrading summer leaves return into the rhizomes. Therefore, the nitrogen nutrition consists of an external and internal cycle. The abundance of carbohydrates and nitrogen compounds allows spring shoot growth earlier than other species. These strategies could contribute markedly to the competitive power ofA. calamus at its natural site.

Metabolic Effects of Cold Storage on Livers from Euthermic and Hibernating Columbian Ground Squirrels

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 μmol/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 μmol/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.

Protective properties of amino acids in liver preservation: effects of glycine and a combination of amino acids on anaerobic metabolism and energetics

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 μmol/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 levles (and consequently, total adenylate contents) were 1.2–2.1 μmol/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.

The Importance of Calcium-Related Effects on Energetics at Hypothermia: Effects of Membrane-Channel Antagonists on Energy Metabolism of Rat Liver

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 μmol/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 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 μmol/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 μmol/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 μmg/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.

An underlying mechanism for improved liver preservation with a combined histidine-lactobionate-raffinose flush solution.

In previous experimental liver transplant studies, it was possible to extend cold ischaemic time (CIT) by using a flush/storage solution combining histidine, lactobionate and raffinose (HLR). In this study, energy metabolism, glycolytic substrate (glucose) and anaerobic end-product (lactate) were examined in rat liver over 24 h of cold storage to determine the mechanism of action of the HLR solution. In livers subjected to simple flush and storage with the HLR solution, levels of ATP and ADP were considerably higher than livers stored with modified UW throughout 24 h of storage; at 4 h of storage, ATP and ADP levels were 1.1 and 3.1 mumol/g for HLR solution versus 0.18 and 0.81 mumol/g for UW solution. Total adenylate contents (TA = ATP + ADP + AMP) also remained 1-2 mumol/g higher in HLR-treated livers than those preserved in UW; TA values ranged from 3.8 to 5.7 mumol/g. Glucose increased to 20-35 mumol/g by 10-24 h of storage (similar to the UW group). Lactate rose to almost twice that in livers stored in UW; total lactate accumulation was approximately 10.0 mumol/g. This study demonstrated that the combined HLR solution is able to prolong the maximum 'safe' CIT by increasing anaerobic metabolism and consequently preserving liver energetics. The second part of the experiment examined the effect of continuous perfusion (with/without O2) over the 1st h of cold ischaemia. Under current methods of liver flushing and excision, the 1st h of cold storage may be the critical time of metabolic 'adjustment' since most of the pH and ATP changes occur during this period.(ABSTRACT TRUNCATED AT 250 WORDS)

An underlying mechanism for improved liver preservation with a combined histidine-lactobionate-raffinose flush solution

Abstract In previous experimental liver transplant studies, it was possible to extend cold ischaemic time (CIT) by using a flush/storage solution combining histidine, lactobionate and raffinose (HLR). In this study, energy metabolism, glycolytic substrate (glucose) and anaerobic end-product (lactate) were examined in rat liver over 24 h of cold storage to determine the mechanism of action of the HLR solution. In livers subjected to simple flush and storage with the HLR solution, levels of ATP and ADP were considerably higher than livers stored with modified UW throughout 24 h of storage; at 4 h of storage, ATP and ADP levels were 1.1 and 3.1 μmol/g for HLR solution versus 0.18 and 0.81 μmol/g for UW solution. Total adenylate contents (TA = ATP + ADP + AMP) also remained 1–2 μmol/g higher in HLR-treated livers than those preserved in UW; TA values ranged from 3.8 to 5.7 μmol/g. Glucose increased to 20–35 μmol/g by 10–24 h of storage (similar to the UW group). Lactate rose to almost twice that in livers stored in UW; total lactate accumulation was approximately 10.0 μmol/g. This study demonstrated that the combined HLR solution is able to prolong the maximum ‘safe’ CIT by increasing anaerobic metabolism and consequently preserving liver energetics. The second part of the experiment examined the effect of continuous perfusion (with/without O2) over the 1st h of cold ischaemia. Under current methods of liver flushing and excision, the 1st h of cold storage may be the critical time of metabolic ‘adjustment’ since most of the pH and ATP changes occur during this period. Therefore, we tested the hypothesis that the combination of a simple flush with an additional brief 1-h perfusion period prior to storage would enhance the maintenance of hepatic energetics. There was no beneficial effect of 1 h of perfusion without O2 compared to simple HLR flush and storage. However, perfusion with O2 resulted in prolonged maintenance of high energy adenylates and total adenylates; at 10 h of storage ATP was 1.0, ADP 3.3, and TA 5.7 μmol/g. However, any improvement in ultimate viability following long-term storage of the livers in these two groups needs to be tested in an animal transplant model.