Anaerobic End Products Research Articles

Effects of hypothermic hypoxia on anaerobic energy metabolism in isolated anuran livers.

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.

Effects of thermal stress and tri(n)butyl tin on anaerobic energy metabolism in Mytilus edulis

The purpose of this study was to understand the effects of tri(n)butyl tin (TBT) on anaerobic energy metabolism and whether TBT causes a lowered ability of Mytilus edulis to withstand emersion. The study design included two levels of anaerobic effect and elevated temperature. Two TBT-dosed groups of mussels (10 μg g −1 dry wt) and two control groups, were thermally stressed at 35 °C for 2 h, one TBT/control pair in air and the second TBT/control pair under nitrogen. Using an HPLC-based method, mono- and dicarboxylic acid metabolites associated with anaerobic energy metabolism were quantified before and after treatment for subsamples of all groups. TBT exposure was found to significantly increase anaerobic mortality at elevated temperatures. The rates of accumulation of the anaerobic end products succinate, lactate and propionate were greatest for the nitrogen-exposed TBT group. As an uncoupler of fumarate reductase in anaerobic energy metabolism, TBT causes lowered ATP production and a stimulation of glycogen breakdown and glycolysis consistent with the increased rate of accumulation of anaerobic end products observed in the TBT group relative to the control group. This effect is most pronounced for the strictly anaerobic nitrogen-exposed TBT group. The increased rate of lactate accumulation in the nitrogen-exposed TBT group reflects an increased glycolytic rate leading to the increased pyruvate levels observed for this group. This lowering of anaerobic metabolic efficiency is consistent with the observed lowered tolerance of emersion in TBT-exposed mussels.

Metabolic Adaptations of a Lower Vertebrate to Long-Term Hypothermic Hypoxia Provide Clues to Successful Clinical Liver Preservation

This study was designed to determine whether the metabolic adaptations developed by frogs to tolerate natural events of hypothermic hypoxia would precondition its liver forex vivoorgan 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°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 μmol/g. Even after 96 h cold storage, ATP remained > 1.0 μmol/g. In contrast, ATP levels in stored rat livers dropped rapidly, and by 4 h ATP was 1.2 μmol/g. In terms of anaerobic endproduct accumulation, lactate levels rose 5.8 μmol/g in frog liver (over 96 h) and by 8.6 μmol/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 μmol/g/h compared to 2.26 μmol/g/h for rat liver; a Q10value of 6.2 was estimated for livers fromR. 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 fromR. castabienasimply 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.

Investigation of a primary requirement of organ preservation solutions: supplemental buffering agents improve hepatic energy production during cold storage.

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. 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. 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. 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.

OXYGEN CONSUMPTION AND VENTILATION IN DECLINING OXYGEN TENSION AND POSTHYPOXIC RECOVERY IN EPIGEAN AND HYPOGEAN CRUSTACEANS

ABSTRACT Respiratory and ventilatory responses to declining O2 tension and posthypoxic recovery were investigated in 3 hypogean and in 2 epigean aquatic crustaceans. The aims of this study were to determine how these species reacted to declining O2 tension, to investigate their changes in ventilation and metabolic rate during progressive hypoxia, and to extend our knowledge on the fate of anaerobic end products during subsequent recovery. Hypogean organisms had normoxic O2 consumption rates 1.7―3.5 times lower than the epigean species. All 5 crustaceans were able to maintain their O2 consumption rates (1) at relatively constant levels and (2) independent of Po2 between normoxia and the critical Po2. Hypogean species also possessed lower critical Po2 than epigean ones, which may indicate that these organisms are better adapted to low O2 content and are better equipped to stay aerobic under hypoxia. For all species, posthypoxic recovery resulted in a high O2 debt. The payment of this debt was smaller in hypogean than in epigean species. The main explanations of the lower 0, debt shown by hypogean organisms are the lower energetic expenditures noticed during hypoxia, partly due to a decrease in locomotory and ventilatory activities.

The anaerobic endproduct lactate has a behavioural and metabolic signalling function in the shore crab

This study presents the first evidence that, in invertebrates, the anaerobic endproduct lactate has an alarm signal function and induces metabolic and behavioural responses as in the anuran Bufo marinus. In support of this function, behavioural hypothermia was demonstrated in the shore crab Carcinus maenas. The animals moved to a cooler environment when exposed to hypoxic conditions. A decrease in preferred temperature of the same magnitude was also found when normoxic animals were injected with an iso-osmotic lactate solution resulting in a haemolymph concentration of approximately 12 mmol l-1. Under normoxic (PO2=>18 kPa) and moderately hypoxic conditions (PO2=12 kPa), injection of this lactate solution also caused a significant increase in the rate of oxygen consumption (100 and 50 % respectively). The increase in the rate of oxygen consumption was smaller and delayed, but lasted longer, under hypoxic conditions compared with normoxic conditions. Low but significant correlations between levels of lactate and levels of adrenaline, octopamine and tryptophan suggest the involvement of biogenic amines in the mediation of the signal.

Temperature induced anaerobiosis in two populations of the polychaete worm Arenicola marina (L.)

Temperature dependent changes in the mode of energy metabolism and in acid-base status were studied in the range from )1.7 to 26 oC in two populations of Are- nicola marina collected in summer as well as in winter from intertidal flats of the North Sea (boreal) and the White Sea (subpolar). Extreme temperatures led to an accumulation of anaerobic end products, indicating the existence of both a low and a high critical temperature, beyond which anaerobic metabolism becomes involved in energy production. In summer animals from the North Sea the high critical temperature was found at tempera- tures above 20 oC, and the low critical temperature below 5 oC. Latitudinal or seasonal cold adaptation lead to a more or less parallel shift of both high and low critical temperature values to lower values. Between critical temperatures intracellular pH declined with rising tem- perature. Slopes varied between )0.012 and )0.022 pH- units/oC. In summer animals from the North Sea, the slope was slightly less than in White Sea animals, but dierences appeared independent of the season. However, slopes were no longer linear beyond critical temperatures. A drop in intracellular pH at low temperatures coincided with the accumulation of volatile fatty acids in the body wall tissue of North Sea animals. A failure of active pHi adjustment is held responsible for the reduced ΔpHi/Δ Ta t temperatures above the high critical temperature. Extra- cellular pH was kept constant over the whole temperature range investigated. The ability of North Sea animals to adapt to temperatures beyond the critical temperature is poor compared to White Sea specimens. The larger range of temperature fluctuations at the White Sea is seen as a reason for the higher adaptational capacity of the sub- polar animals. A hypothesis is proposed that among other mechanisms critical temperature values are set by an adjustment of mitochondrial density and thus, aerobic capacity.

Marenzelleria cf. wireni (Polychaeta: Spionidae) from the Tay estuary. Metabolic response to severe hypoxia and hydrogen sulphide

The appearance of the spionid polychaete Marenzelleria spp. in the Tay estuary (Scotland) was first reported in 1984. Since estuaries are known as environments where abiotic conditions fluctuate widely, a laboratory study was conducted aimed to elucidate how this introduced species deals with low oxygen concentrations and exposure to hydrogen sulphide. Experimental evidence reveals that during severe hypoxia Marenzelleria cf. wireni switched to anaerobic pathways known from other marine invertebrates. Under the influence of sulphide (1 mmol l-1) accumulation of anaerobic end products is more pronounced. It is assumed that in the presence of sulphide, due to its inhibition of aerobic respiration, the worms have to switch to anaerobic energy production directly after the onset of hypoxia. Seasonal differences in the metabolic reaction were found. It was shown that in specimens that were filled with gametes (winter), accumulation of end products of anaerobic metabolism was more pronounced than in immature worms (summer). The amount of succinate, an indicator for anaerobic energy production for instance, was nearly twice as high in the ‘winter specimens’. Since the amount of thiosulphate (a typical sulphide detoxification product) in the tissues is relatively low, it is suggested that Marenzelleria cf. wireni also accumulates other detoxification products. Sulphite, however, also known as a detoxification product, was found only in traces. Field measurements provide evidence that the potential of Marenzelleria cf. wireni to survive low oxygen and high sulphide concentrations is clearly higher than that normally needed in the Tay estuary.

Anaerobic metabolism in the leech (Hirudo medicinalis L.): direct and indirect calorimetry during severe hypoxia.

Anaerobic metabolism in the limnic annelid Hirudo medicinalis L. was investigated by direct and indirect calorimetry. During long-term severe hypoxia, the rate of heat dissipation was reduced up to 13% of the aerobic rate. At the same time, the rate of ATP turnover was reduced to about 30% of the aerobic rate, indicating that metabolic depression is an important mechanism to ensure survival of the leech during environmental anaerobiosis. Heat dissipation during hypoxia was monitored under two experimental conditions, favouring either concomitant hypocapnia (continuous N2 bubbling) or hypercapnia (self-induced hypoxia). The reduction in heat dissipation during hypocapnic hypoxia was less pronounced than during hypercapnic hypoxia, indicating that the different experimental conditions may influence anaerobic metabolism and the extent of metabolic depression. Biochemical analysis of known anaerobic substrates and endproducts provided the basis for indirect calorimetry during self-induced hypoxia. From changes in metabolites, the expected heat dissipation was calculated for initial (0-8 ,h) and long-term severe hypoxia (8-72 h). During the initial period, the calculated heat dissipation fully accounted for direct calorimetric determination. During long-term hypoxia, only 71% of the measured heat production could be explained from biochemical analysis of metabolites. Therefore, an additional unknown endproduct cannot be excluded, especially when anaerobic ammonia production and analysis of the carbohydrate balance are considered.

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.

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.

Effects of environmental parameters on the formation and turnover of acetate by forest soils

The capacity to form acetate from endogenous matter was a common property of diverse forest soils when incubated under anaerobic conditions. At 15 to 20(deg)C, acetate synthesis occurred without appreciable delay when forest soils were incubated as buffered suspensions or in microcosms at various percentages of their maximum water holding capacity. Rates for acetate formation with soil suspensions ranged from 35 to 220 (mu)g of acetate per g (dry weight) of soil per 24 h, and maximal acetate concentrations obtained in soil suspensions were two- to threefold greater than those obtained with soil microcosms at the average water holding capacity of the soil. Cellobiose degradation in soil suspensions yielded H(inf2) as a transient product. Under anaerobic conditions, supplemental H(inf2) and CO(inf2) were directed towards the acetogenic synthesis of acetate, and enrichments yielded a syringate-H(inf2)-consuming acetogenic consortium. At in situ temperatures, acetate was a relatively stable anaerobic end product; however, extended incubation periods induced acetoclastic methanogenesis and sulfate reduction. Higher mesophilic and thermophilic temperatures greatly enhanced the capacity of soils to form methane. Although methanogenic and sulfate-reducing activities under in situ-relevant conditions were negligible, these findings nonetheless demonstrated the occurrence of methanogens and sulfate-reducing bacteria in these aerated terrestrial soils. In contrast to the protracted stability of acetate under anaerobic conditions at 15 to 20(deg)C with unsupplemented soils, acetate formed by forest soils was rapidly consumed in the presence of oxygen and nitrate, and substrate-product stoichiometries indicated that acetate turnover was coupled to oxygen-dependent respiration and denitrification. The collective results suggest that acetate formed under anaerobic conditions might constitute a trophic link between anaerobic and aerobic processes in forest soils.

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.

Stimicrolation of Glycolysis by Histidine Buffers in Mammalian Liver during Cold Hypoxia

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 μmol/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 μmol/g, 4.3-5.5 μmol/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.

Acclimatization to intertidal conditions modifies the physiological response to prolonged air exposure inMytilus edulis

Both the tolerance of air exposure and the metabolic means by which mussles,Mytilus edulis, face air exposure can be modified by intertidal acclimatization. Short-term intertidal acclimatization enhanced the tolerance of air exposure most in early summer. Prevention of air breathing increased the mortality of wild and non-acclimatized cultured (i.e., control) mussels, but only slightly enhanced that of intertidally acclimatized mussels, suggesting their increased dependence upon anaerobic metabolism. In June, anaerobic end products accumulated in the adductor muscle of cultured but not of wild mussels. Intertidal acclimatization shifted the pattern and rates of metabolite accumulation. Control mussels accumulated succinate and alanine in the adductor muscle while intertidally acclimatized mussels accumulated strombine + alanopine, albeit at far lower levels. Pyruvate kinase (PK) from adductor muscle of intertidally acclimatized mussels was gradually activated during prolonged air exposure, while that of control and wild mussels was more inhibited. In July and August, during prolonged air exposure, the PK from the most viable mussles generally was the most inhibited. Prevention of air breathing did not markedly change the time course of PK inhibition during air exposure. Cultured mussels used in the present study were obtained from growers in Quebec, and wild mussels were collected from the Baie des Chaleurs (Quebec, PQ) in 1988.

Recovery after anaerobic metabolism in the leech (Hirudo medicinalis L.).

Medicinal leeches (Hirudo medicinalis L.) responded to self-induced hypoxia (72 h) with typical anaerobic metabolism characterized by a decrease in adenylate energy charge, utilization of the substrates glycogen and malate, and accumulation of the main anaerobic end-products succinate and propionate. Propionate was also excreted into the medium. Ammonia excretion was suppressed. Aerobic recovery resulted in a profound O2 debt. Resynthesis of ATP was completed within 30 min. Disposal of succinate and restoring of malate required 2-3 h, and clearance of propionate and recharging of glycogen 6-12 h. Ammonia excretion did not exceed normoxic rates and excretion of propionate during recovery accounted for only 10% of total propionate accumulated during hypoxia. It is postulated that the clearance of succinate and propionate involves oxidation but also resynthesis of malate and glycogen. During hypoxia and recovery blood osmolality remained constant. The Na+ and Cl- ion concentrations in blood, the decrease of which was nearly equimolar during hypoxia, were re-established following different time-courses. Na+ concentration returned to normoxic levels after 2-3 h. The delayed increase in Cl- concentration, however, correlating with 6-12 h necessary to clear blood propionate, is interpreted as an anion regulating effect.

Anaerobic metabolism, hypoxia and hydrogen sulphide in the brackish water isopodSaduria entomon(L.)

Abstract The Baltic brackish water isopod Saduria entomon was simultaneously exposed to hydrogen sulphide (150 µmol 1−1 ) and hypoxia (4.0-6.7 kPa) for up to 100 hours. In contrast to haemoglobin-carrying species, the haemocyanin of S. entomon functions as oxyhaemocyanin also in the presence of hydrogen sulphide. The synthesis of new haemocyanin is, however, prevented. The hydrogen sulphide is oxidised in the midgut gland to S2O3 2− and SO3 2− Both lactate and alanine accumulate in the presence of hydrogen sulphide. When exposed to hypoxia and hydrogen sulphide anaerobic end products start to accumulate already at a water oxygen tension of at least 4.0–6.7 kPa, this means that hydrogen sulphide is correlated with an anaerobic metabolism.

Metabolic Rates at Different Oxygen Levels Determined by Direct and Indirect Calorimetry in the Oxyconformer Sipunculus Nudus

ABSTRACT Oxygen uptake and the mode of energy production in Sipunculus nudus L. were determined at different oxygen levels by means of direct and indirect calorimetry. Oxygen consumption declined linearly with decreasing ambient . A similar decrease in heat production was observed down to a of 8.66 kPa. At lower oxygen tensions, a discrepancy between aerobic and total heat production indicated the onset of anaerobic metabolism. The occurrence of the critical between 8.66 and 2.66 kPa was confirmed by estimation of anaerobic end products in the body wall musculature. The contributions of aerobic and anaerobic metabolism to total ATP production were determined at a of 2.66 kPa and were found to be 48 and 52 %, respectively. Measured heat dissipation under extreme hypoxia ( nominally zero) (21.5±3.5mJh-1g-1) agreed with the enthalpy changes calculated from the rates of formation of anaerobic end products (17.9±4.7mJh-1g-1). For the sake of redox balance maintenance, saturation of fatty acids was assumed; this would be accompanied by an additional heat production of 3.3mJh-1g-1, so that the total calculated enthalpy change amounted to 21.2mJh-1g-1.

Urate Does not Accumulate in the Haemolymph of Exercised Blue Crabs, Calunectes Sapidus

ABSTRACT L-Lactate, the only known anaerobic end-product in decapod crustaceans (Gade, 1983), increases haemocyanin oxygen-affinity (Truchot, 1980; Mangum, 1983; Bridges and Morris, 1986). In exercised blue crabs, Callinectes sapidus, the decrease in haemocyanin oxygen-affinity induced by metabolic acidosis via the Bohr shift was substantially balanced by the opposing effect of L-lactate (Booth et al. 1982). Since this study, Morris et al. (1985) have discovered that urate also enhances haemocyanin oxygen-affinity in the crayfish Austropotamobius pallipes. The importance of urate in regulating oxygen transport during environmental hypoxia has recently been stressed in the crab Carcinus maenas and the prawn Penaeus japonicus (Lallier and Truchot, 1989a,b). Since Callinectes sapidus haemocyanin exhibits a urate effect (De Fur et al. 1990), urate may also play a role during functional anaerobiosis.