Depletion of high energy phosphates implicates post-exercise mortality in carp and trout; an in vivo 31P-NMR study

As in vivo 31P-Nuclear Magnetic Resonance spectroscopy is currently the state of the art method to measure continuously intracellular pH (pH(i)) and energy status of muscle tissue, we used this method to study the recovery from exhaustive exercise. The biochemical changes during recovery are not well understood and it was suggested that post-exercise mortality could be caused by low pH(i); other studies however indicate that energy depletion might be more important. To analyse the mechanism of post-exercise recovery pH(i), ATP, P(i), and PCr must be measured at the same time, which is possible using in vivo 31P-NMR. Common carp and rainbow trout of about 100 g were exercised to exhaustion in a swim tunnel. After swimming 10 h at 1.5 body lengths (BL)/s (aerobic control), 50% of the fish were forced to swim at 6 BL/s until exhaustion. Recovery of energy rich phosphates was found to be faster in carp (1.2-1.9 h) than in trout (1.5-2.3 h). The same applied for the recovery from acidosis, which took 1.75 h in carp and 5.75 h in trout. In parallel experiments the energy phosphates and lactate levels were measured in liver, red muscle, and white muscle. Exhaustion caused a significant drop in the energy status of red and white muscle tissue of trout and carp (corroborates NMR data), while no change at all was observed in liver tissue. The lactate levels were increased in the muscle but not in liver and blood. While all experimental animals looked healthy after exhaustion, 40-50% of the carp as well as trout died during the recovery phase. The energy status of those individuals measured by 31P-NMR was much lower than that of the survivors, while in contrast there was no difference in pH(i). Thus, it appears that not acidosis but depletion of high energy phosphates disabled muscle function and therefore may have been the cause of death of the non-survivors.     

Recovery from an activity-induced metabolic acidosis in the American alligator, Alligator mississippiensis

The metabolic acidosis resulting from an intense exercise bout is large in crocodilians. Here we studied recovery from this pH perturbation in the American alligator. Metabolic rate, minute ventilation, arterial pH and gases, and strong ion concentration were measured for 10 h after exhaustion to elucidate the mechanisms and time course of recovery. Exhaustion resulted in a significant increase in lactate, metabolic rate, and ventilation, and a decrease in arterial PCO2), pH and bicarbonate. By 15 min after exhaustion, oxygen consumption returned to rest though carbon dioxide excretion remained elevated for 30 min. Arterial PO2), [Na+], and [K+], increased following exhaustion and recovered by 30 min post-exercise. Minute ventilation, tidal volume, [Cl-], and respiratory exchange ratio returned to resting values by 1 h. The air convection requirement for oxygen was elevated between 15 and 60 min of recovery. Breathing frequency and pH returned to resting values by 2 h of recovery. Lactate levels remained elevated until 6 h post-exercise. Arterial PCO2) and [HCO3-] were depressed until 8 h post-exercise. Compensation during recovery of acid-base balance was achieved by altering ventilation: following the initial metabolic acidosis and titration of bicarbonate, a relative hyperventilation prevented a further decrease in pH.     

The effect of high buffer cardioplegia and secondary cardioplegia on cardiac preservation and postischemic functional recovery: a 31P NMR and functional study in Langendorff perfused pig hearts.

High buffer cardioplegia may provide protection against ischemic damage by reducing the extent of intracellular acidosis. Secondary cardioplegia may improve postischemic recovery by restoration of high energy phosphates, ionic gradients, and intracellular pH. To test these hypotheses, pig hearts were arrested with high buffer (150 mM MOPS) cardioplegia or modified St. Thomas' solution II and then kept ischemic at 12 degrees C for 8 h. High energy phosphates and intracellular pH were followed during the period of ischemia, using 31P nuclear magnetic resonance spectroscopy, and functional recovery was followed during reperfusion. The hearts arrested by high buffer cardioplegia showed significantly higher intracellular pH than hearts preserved with St. Thomas' solution, but there were no significant differences in high energy phosphates. There were no significant differences in functional recovery. We found, however, that secondary cardioplegia abolished ventricular fibrillation, and resulted in improved functional recovery after 8 h of ischemic preservation compared with the hearts reperfused with Krebs-Henseleit solution alone. Our results suggest that despite attenuating the decreases in intracellular pH, high buffer cardioplegia does not improve recovery following 8 h of preservation at 12 degrees C. Secondary cardioplegia reduces the incidence of ventricular fibrillation and improves postischemic functional recovery of the myocardium.     

Energy state, pH, and vasomotor tone during hypoxia in precontracted pulmonary and femoral arteries

To assess effects of smooth muscle energy state and intracellular pH (pH(i)) on pulmonary arterial tone during hypoxia, we measured ATP, phosphocreatine, P(i), and pH(i) by (31)P-NMR spectroscopy and isometric tension in phenylephrine-contracted rings of porcine proximal intrapulmonary arteries. Hypoxia caused early transient contraction followed by relaxation and late sustained contraction. Energy state and pH(i) decreased during relaxation and recovered toward control values during late contraction. Femoral arterial rings had higher energy state and lower pH(i) under baseline conditions and did not exhibit late contraction or recovery of energy state and pH(i) during hypoxia. In pulmonary arteries, glucose-free conditions abolished late hypoxic contraction and recovery of energy state and pH(i), but endothelial denudation abolished only late hypoxic contraction. NaCN had little effect at 0. 1 and 1.0 mM but caused marked vasorelaxation and decreases in energy state and pH(i) at 10 mM. These results suggest that 1) regulation of tone, energy state, and pH(i) differed markedly in pulmonary and femoral arterial smooth muscle, 2) hypoxic relaxation was mediated by decreased energy state or pH(i) due to hypoxic inhibition of oxidative phosphorylation, 3) recovery of energy state and pH(i) in hypoxic pulmonary arteries was due to accelerated glycolysis mediated by mechanisms intrinsic to smooth muscle, and 4) late hypoxic contraction in pulmonary arteries was mediated by endothelial factors that required hypoxic recovery of energy state and pH(i) for transduction in smooth muscle or extracellular glucose for production and release by endothelium.     

The binding of physiologically significant protons to 2,3-diphosphoglycerate

The intrinsic pKa values of protons of 2,3-diphosphoglycerate (DPG) which titrate in the physiologically significant range (i.e., pH 6.8-7.8) have been determined by measuring the changes in chemical shifts of the two phosphate resonances of the molecule as a function of pH using 31P-NMR spectroscopy. While conventional acid-base titration techniques resulted in apparent pKa values of 6.39 and 7.39 for these protons, analysis of the 31P-NMR data by statistical thermodynamic methods yielded intrinsic pKa values of 6.99 +/- 0.07 and 7.28 +/- 0.04, for protons associated with the phosphates bound to carbon-3 (C-3) and carbon-2 (C-2), respectively, with an interaction energy of +0.77 kcal/mol. The free energies for the binding of protons to the C-2 and C-3 phosphates and the associated interaction energies determined by 31P-NMR were used to generate a theoretical titration curve which was essentially identical to that determined by conventional acid-base titration. The physiological implications of this work are briefly discussed.     

In vivo and in vitro 31P-NMR spectroscopy of rat liver treated with halocarbons

Both in vivo and in vitro 31P-NMR spectroscopy were used to demonstrate metabolic changes in rat liver as a function of time after exposure to either carbon tetrachloride (CCl4) or bromotrichloromethane (BrCCl3). The inorganic phosphate resonance, measured in vivo, moves upfield, which is associated with a decrease in cytosolic pH over a 12 or 20 h period (for BrCCl3 or CCl4, respectively). Intoxication by CCl4 or BrCCl3 causes an intracellular acidosis to pH 7.05 or 6.82 (+/- 0.05), respectively. Also, it has been found that halocarbon exposure increases the amounts of phosphomonoesters (PME) detected. High resolution in vitro 31P-NMR spectroscopy studies of perchloric acid extracts of CCl4-treated rat livers indicated a significant increase in the height of the phosphocholine resonance in the PME region 4-5 h after CCl4 exposure.     

Application of 31P-NMR saturation transfer techniques to investigate phospholipid motion and organization in model and biological membranes

The potential of ³¹P-NMR saturation transfer experiments for determining motional characteristics (in the millisecond to second time scale) of phospholipids in model and biological membranes is demonstrated. A technique to separate membrane phospholipid ³¹P-NMR signals from those of small water-soluble phosphates in intact cells in liver tissue is also illustrated.     

Energy status and free fatty acid patterns in tissues of common carp (Cyprinus carpio,L.) and rainbow trout (Oncorhynchus mykiss,L.) during severe oxygen restriction

Common carp and rainbow trout were exposed to a severe level of oxygen restriction up to a near lethal value, to study the occurrence of tissue damage. Rainbow trout lost equilibrium at a PO₂ of 3.2 kPa, whereas carp were able to survive 1.5 hr of anoxia. In both species, the anaerobic metabolism was significantly activated and the energy status (PCr, ATP and energy charge) was significantly depressed in brain, liver, and red and white muscle. No marked release of PUFA to the FFA pool was observed, while membrane leakage was not increased as evidenced by plasma LDH-activity. These results indicate the absence of a marked hydrolysis of membrane lipids. Thus, even after a near lethal exposure to hypoxia or anoxia, no tissue damage occurs in fish liver and skeletal muscles. The changes of the FFA patterns in the skeletal muscles and liver of both species after oxygen deprivation may be related to changes in desaturase activities, a reduction of lipolytic activity and PUFA metabolism.     

In vivo 31P-NMR studies on energy metabolism in and catecholamine effect on rat liver during hypovolemic shock

In vivo 31P-NMR spectroscopy (31P-MRS) was used to study the metabolism of phosphate compounds in rat liver under various conditions. The changes in hepatic concentrations of ATP and inorganic phosphate (Pi) or intracellular pH (pHi) were monitored during hypovolemic shock with or without the infusion of catecholamines. Rapid decreases in the ATP level and pHi with a concomitant increase of Pi were observed upon induction of the hypovolemic shock. Dopamine infusion markedly improved the liver ATP concentration and intracellular acidosis, but epinephrine or norepinephrine were without effects. The present results suggest that dopamine increases abdominal blood flow and improves the energy metabolism in the liver during hypovolemic shock.     

Differences in some properties of lactate dehydrogenase from muscles of the carp Cyprinus carpio and trout Salmo gairdneri

Some catalytic and kinetic properties of lactate dehydrogenase (LDH) isolated from trout and carp skeletal muscles were compared. The specific activity of LDH in the carp muscle was lower by about one third than the activity in the trout muscle. Temperature and pH optima for LDH isolated from the carp muscle were higher than those for the trout muscle LDH. Moreover, in direct reaction, the carp muscle LDH had a higher affinity both for pyruvate and for NADH, i.e., it had lower K _M values. Instead, the trout muscle LDH showed the positive kinetic cooperativity (the Hill coefficient > 1) of the substrate and coenzyme binding sites. Thus, the carp LDH seems to function more effectively under anaerobic conditions and at higher temperatures.     

The role of intermediary metabolism in the maintenance of proton and charge balance during exercise

The purpose of this investigation was to examine the role of intermediary metabolism in the maintenance of proton and charge balance in rainbow trout white muscle during exercise. With increasing power outputs, there was a greater reliance on white fibers and anaerobic processes for energy production. Glycogen content declined from a pre-exercise (pre-ex) level of 23 to less than 1 µmol/g following the exhaustive swim, with its greatest rate of decline occurring during the burst swim. Lactate accumulation reached a maximum of 43 µmol/g during the exhaustive swim. PCr declined from about 20 to less than 2 µmol/g at exhaustion with a concomitant accumulation of Cr. ATP decreased from about 7.3 to 2.7 µmol/g while inorganic phosphate and IMP increased to about 56 and 4.3 µmol/g, respectively. The intramuscular pH fell from 6.97 to 6.93 during the sustained swim, declining further to 6.65 during the burst swim and reaching a minimum of 6.56 at exhaustion. Exercise induced depletions of high energy compounds and accumulations of metabolic end products nearly stabilized the accompaning intracellular perturbations in charge and proton levels. Compensatory shifts in Na⁺, K^– and Cl^– served to negate the residual imbalances such that electrical neutrality, membrane potential and pH were preserved.     

The physiological response of diploid and triploid brook trout to exhaustive exercise

Using triploidy as an experimental model, we examined whether cell size limits the post-exercise recovery process in fish. Because triploids generally possess larger cells, which could affect many physiological and biochemical processes, we hypothesized that triploids would take longer to recover from exhaustive exercise compared to diploids. To test this, we measured plasma lactate, glucose and osmolality, and white muscle energy stores (glycogen, phosphocreatine and ATP) and lactate before and immediately following exhaustive exercise and during recovery at 2 and 4 h post-exercise. In addition, oxygen consumption and ammonia excretion rates were determined before and after exhaustive exercise. Overall, diploid and triploid brook trout showed similar metabolic responses exercise, but plasma osmolality, white muscle lactate, white muscle ATP and post-exercise oxygen consumption rates recovered earlier in triploids compared to diploids. The results of this study suggest that the characteristic larger cell size of triploidy does not limit the physiological response to, or recovery from, exhaustive exercise.     

Glycogen concentrations and endurance capacity of rats with chronic heart failure

The endurance capacities of rats with myocardial infarctions (MI) and of rats having undergone sham operations (SHAM) were tested during a submaximal exercise regimen that consisted of swimming to exhaustion. During this test, a decrement in the endurance capacity of the MI rat was demonstrated as the SHAM rat swam 25% longer than the MI rat (65 +/- 4 vs. 52 +/- 4 min). Glycogen concentrations were measured in the liver and the white gastrocnemius, plantaris, and soleus muscles of SHAM and MI rats that were randomly divided into four subgroups, which consisted of resting control, swim to exhaustion, swim to exhaustion + 24 h recovery, and swim to exhaustion + 24 h recovery + a second swim to exhaustion. The results demonstrated that the glycogen concentrations found in the liver, white gastrocnemius, plantaris, and soleus muscles of the SHAM and MI rats belonging to the resting control groups were similar. After swimming to exhaustion the glycogen concentrations in these tissues were significantly reduced compared with those found in the resting control groups of rats, and after 24 h of recovery the glycogen concentrations in these tissues were again similar to those found in the resting control groups of rats. Since the magnitude of the glycogen depletion in the liver and the white gastrocnemius, plantaris, and soleus muscles was similar in the SHAM and MI rats and because the SHAM rats consistently swam for longer periods of time in each of the experimental groups, it would be logical to assume that the rates of glycogen utilization for the various tissues may have been greater in the MI rat during exercise.(ABSTRACT TRUNCATED AT 250 WORDS)     

Free Mg<Superscript>2+</Superscript> concentration in the calf muscle of glycogen phosphorylase and phosphofructokinase deficiency patients assessed in different metabolic conditions by <Superscript>31</Superscript>P MRS

Background

The increase in cytosolic free Mg²⁺ occurring during exercise and initial recovery in human skeletal muscle is matched by a decrease in cytosolic pH as shown by in vivo phosphorus magnetic resonance spectroscopy (³¹P MRS). To investigate in vivo to what extent the homeostasis of intracellular free Mg²⁺ is linked to pH in human skeletal muscle, we studied patients with metabolic myopathies due to different disorders of glycogen metabolism that share a lack of intracellular acidification during muscle exercise.

Methods

We assessed by ³¹P MRS the cytosolic pH and free magnesium concentration ([Mg²⁺]) in calf muscle during exercise and post-exercise recovery in two patients with McArdle's disease with muscle glycogen phosphorylase deficiency (McArdle), and two brothers both affected by Tarui's disease with muscle phosphofructokinase deficiency (PFK).

Results

All patients displayed a lack of intracellular acidosis during muscle exercise. At rest only one PFK patient showed a [Mg²⁺] higher than the value found in control subjects. During exercise and recovery the McArdle patients did not show any significant change in free [Mg²⁺], while both PFK patients showed decreased free [Mg²⁺] and a remarkable accumulation of phosphomonoesters (PME). During initial recovery both McArdle patients showed a small increase in free [Mg²⁺] while in PFK patients the pattern of free [Mg²⁺] was related to the rate of PME recovery.

Conclusion

i) homeostasis of free [Mg²⁺] in human skeletal muscle is strongly linked to pH as shown by patients' [Mg²⁺] pattern during exercise;ii) the pattern of [Mg²⁺] during exercise and post-exercise recovery in both PFK patients suggests that [Mg²⁺] is influenced by the accumulation of the phosphorylated monosaccharide intermediates of glycogenolysis, as shown by the increased PME peak signal.iii) ³¹P MRS is a suitable tool for the in vivo assessment of free cytosolic [Mg²⁺] in human skeletal muscle in different metabolic conditions;

     

Efficacy of recombinant human Hb by 31P-NMR during isovolemic total exchange transfusion.

The ability of recombinant human Hb (rHb1.1), which is being developed as an oxygen therapeutic, to support metabolism was measured by in vivo 31P-NMR surface coil spectroscopy of the rat abdomen in control animals and in animals subjected to isovolemic exchange transfusion to hematocrit of <3% with human serum albumin or 5 g/dl rHb1.1. No significant changes in metabolite levels were observed in control animals for up to 6 h. The albumin-exchange experiments, however, resulted in a more than eightfold increase in Pi and a 50% drop in phosphocreatine and ATP within 40 min. The tissue pH dropped from 7.4 to 6.8. The decrease in high-energy phosphates obeyed Michaelis-Menten kinetics, with a Michaelis-Menten constant of 3% as the hematocrit at which a 50% drop in high-energy phosphates was observed. Exchange transfusion with rHb1.1 resulted in no significant drop in high-energy phosphates, no rise in Pi, and no change in tissue pH from 7.35 +/- 0.15 for up to 5 h after exchange. By these criteria, rHb1.1 at a plasma Hb concentration of approximately 5 g/dl after total exchange transfusion was able to sustain energy metabolism of gut tissue at levels indistinguishable from control rats with a threefold higher total Hb level in erythrocytes.     

High-energy phosphate metabolites in loach (Cobitis biwae) during urethane anesthesia

1. Changes in the metabolism of high-energy phosphates in the loach were studied under urethane anesthesia using in vivo³¹P-NMR.2. Little change was observed in creatine phosphate (PCr) and β-ATP concentration after the administration of urethane, but the inorganic phosphate (Pi) concentration decreased with a resultant tilt of the intracellular pH to the acid side.3. Recovery to the initial energy level state occurred about 2 hr after discontinuation of anesthesia.4. Urethane anesthesia is considered to change the intracellular ionic environment in the muscle of the loaches.     

Human muscle fatigue after glycogen depletion: a 31P magnetic resonance study.

To differentiate the effects of high energy phosphates, pH, and [H2PO4-] on skeletal muscle fatigue, intracellular acidosis during handgrip exercise was attenuated by prolonged submaximal exercise. Healthy human subjects (n = 6) performed 5-min bouts of maximal rhythmic handgrip (RHG) before (CONTROL) and after prolonged (60-min) handgrip exercise (ATTEN-EX) designed to attenuate lactic acidosis in active muscle by partially depleting muscle glycogen. Concentrations of free intracellular phosphocreatine ([PCr]), adenosine triphosphate ([ATP]), and orthophosphate ([P(i)]) and pH were measured by 31P nuclear magnetic resonance spectroscopy and used to calculate adenosine diphosphate [ADP], [H2PO4-], and [HPO4(2-)]. Handgrip force output was measured with a dynamometer, and fatigue was determined by loss of maximal contractile force. After ATTEN-EX, the normal exercise-induced muscle acidosis was reduced. At peak CONTROL RHG, pH fell to 6.3 +/- 0.1 (SE) and muscle fatigue was correlated with [PCr] (r = 0.83), [P(i)] (r = 0.82), and [H2PO4-] (r = 0.81); [ADP] was 22.0 +/- 5.7 mumol/kg. At peak RHG after ATTEN-EX, pH was 6.9 +/- 0.1 and [ADP] was 116.1 +/- 18.2 mumol/kg, although [PCr] and [P(i)] were not different from CONTROL RHG (P greater than 0.05). After ATTEN-EX, fatigue correlated most closely with [ADP] (r = 0.84). The data indicate that skeletal muscle fatigue 1) is multifactorial, 2) can occur without decreased pH or increased [H2PO4-], and 3) is correlated with [ADP] after exercise-induced glycogen depletion.     

Functional coupling of glycolysis and phosphocreatine utilization in anoxic fish muscle. An in vivo 31P NMR study

Three fish species with different strategies for anoxic survival (goldfish, tilapia, and common carp) were exposed to environmental anoxia (4, 3, and 1 h, respectively). The concentrations of high energy phosphate compounds and inorganic phosphate, besides the intracellular pH in the epaxial muscle were measured during anoxia and recovery by in vivo 31P NMR spectroscopy. The concentration of free ADP was calculated from the equilibrium constant of creatine kinase. During anoxia the patterns of phosphocreatine utilization and tissue acidification are remarkedly similar. Free ADP rises rapidly during the initial period of oxygen deficiency and reaches a plateau in goldfish and tilapia, while it keeps rising in the common carp. At elevated levels of free ADP, the creatine kinase reaction and anaerobic glycolysis are functionally coupled by H+ as a common intermediate. The coupling between both processes disappears upon reoxygenation, when mitochondrial respiration induces a rapid drop of [free ADP]. The removal of ADP shifts the creatine kinase equilibrium toward phosphocreatine synthesis despite the low pH.     

Influence of hyperosmotic shrinkage and β-adrenergic stimulation on red blood cell volume regulation and oxygen binding properties in rainbow trout and carp

Whole blood from rainbow trout and carp was subjected to hyperosmotic shock and subsequent beta-adrenergic stimulation (isoprenaline) at different oxygen tension ( PO(2)) and carbon dioxide tension ( PCO(2)) levels with the aim to evaluate changes in red blood cell (RBC) volume, pH and ion concentrations and their ultimate effect on blood O(2) transport characteristics. Hyperosmolality (addition of NaCl) induced RBC shrinkage, which was followed by a regulatory volume increase (RVI) that was larger at low than at high PO(2)and more complete in carp than in trout. Carp RBC showed practically full volume recovery within 140 min at low PO(2)and partial recovery at high PO(2), whereas RVI was partial under all PO(2)and PCO(2)conditions in trout. The RVI increased intracellular [Na(+)], water content, and, in carp, also pH (pHi), suggesting activation of Na(+)/H(+) exchange. In trout RBCs, activation of RVI was rapid but succeeded by deactivation. In carp RBCs, activation of Na(+) influx was slower but it continued, allowing full volume recovery. Shrinkage of the RBCs was associated with only minor decreases in blood oxygen saturation and oxygen affinity in both species. Thus, the oxygen affinity decrease expected on the basis of the increased concentration of intracellular haemoglobin and organic phosphates was small, and it appeared to some extent countered during RVI by an oxygen affinity increase via increased pHi. Addition of isoprenaline increased RBC volume and pHi and increased Hb oxygen saturation. The beta-adrenergic response was stronger at low compared to high PO(2) and at high compared to low PCO(2). The PO(2) dependency was largest in carp, whereas the PCO(2) (pH) dependency was more expressed in trout. The adrenergic response of trout RBCs was similar under isoosmotic and hyperosmotic conditions. In carp RBCs, the response was absent at high PO(2) under isoosmotic conditions, but interestingly it could be induced under hyperosmotic conditions. The data suggest that the RBC shrinkage occurring in fish moving from freshwater to seawater has minimal impact on blood O(2) binding properties.     

Effect of phosphoenolpyruvate on energy metabolism of ischemic liver in anesthetized rats--31P-MRS study.

The effect of phosphoenolpyruvate (PEP) on energy metabolism of ischemic liver was examined in anesthetized rats. In vivo 31P-NMR spectroscopy (31P-MRS) was used to monitor cellular energy metabolism. Hepatic ischemia was induced by temporarily clamping the portal vein for 60 minutes. The liver adenosine triphosphate (ATP) levels decreased remarkably during ischemia, and they gradually increased after ischemia but did not return to pre-operative levels. PEP effectively increased the levels of ATP. The ATP levels of the PEP-treated rats were significantly higher than those of the control rats, and also intracellular acidosis was improved during post-ischemic reperfusion. These findings suggest that PEP may have a cytoprotective effect and improve the energy metabolism in the ischemic liver.