Synergistic effect of acidification and hypoxia: in vivo 31P-NMR and respirometric study in fishes.

We used 31P nuclear magnetic resonance (31P-NMR) spectroscopy to measure intracellular pH (pHi) and high-energy phosphate levels of white muscle of the fish tilapia (Oreochromis mossambicus) during exposure to the stressors hypoxia and water acidification separately and simultaneously. The protocol for the graded hypoxia load was 100, 40, 30, 20, 10, 5, and 3% air saturation for 1 h at each level. For environmental acidosis the pH of the water was lowered from 7.6 to 4.0 over 6 h, with time of exposure approximately 11 h. All protocols were followed by 6 h of reoxygenation at 20 degrees C. We also measured total oxygen consumption of the animal. The results of this in vivo study revealed that environmental acidification had no effect on oxygen consumption, pHi, or phosphocreatine depletion. Hypoxia caused moderate changes in these parameters and fast and complete recovery during reoxygenation. In contrast, the combination of environmental acidosis and hypoxia resulted in 50% mortality, increased depletion of the phosphocreatine pool, and a retarded recovery of the pHi during reoxygenation compared with the group with hypoxia as a single stressor. The combination of environmental acidification and hypoxia has a more profound effect and works synergistically compared with the conditions imposed separately. To investigate whether an adaptation response occurred during chronic exposure to environmental acidification, animals were exposed for 6 wk to pH 4.0 before experimentation. The pHi in the white muscle dropped from 7.2 (control group) to 6.9 during this period, whereas no effect was found in the phosphorylated compounds and oxygen consumption. Therefore, it is concluded that no adaptation response occurs in animals exposed to long-term environmental acidosis.

Fish muscle energy metabolism measured during hypoxia and recovery: an in vivo 31P-NMR study.

Three fish species were exposed to graded hypoxia levels and allowed to recover. Levels of high-energy phosphate compounds in epaxial white muscle were monitored by in vivo 31P nuclear magnetic resonance (NMR) spectroscopy. Furthermore, O2 consumption of the animals was measured. With increasing hypoxia load, metabolic parameters started to change in the following order: phosphocreatine (PCr)-to-Pi ratio (decrease), O2 consumption (decrease), [PCr] (decrease), intracellular pH (pHi; decrease), Pi (increase), free ADP concentration ([ADP]free; increase), [ATP] (decrease). PCr levels fell with the PO2. After each increment, the [PCr] reached a stable plateau value while, in some cases, a recovery was observed. This recovery could be explained because the balance between anaerobic and aerobic metabolism is continuously fluctuating during hypoxia as a consequence of changes in the activity of the fish. Consequently the [ADP]free are fluctuating, resulting in an activation of the creatine kinase reaction and the anaerobic glycolysis. In all three species, anaerobic glycolysis was activated, but in contrast to anoxia exposure, metabolic suppression was absent. The changes of [ADP]free and [H+] (which influences the position of the creatine kinase equilibrium) are species dependent. Species differences observed in the other parameters were small. It is concluded that the pattern of the activation of anaerobic metabolism under deep hypoxia is different from that under anoxia.

Reversed-phase ion-paired HPLC of purine nucleotides from skeletal muscle, heart and brain of the goldfish, Carassius auratus L.--I. Development of the analytical method.

1. A reversed-phase ion-paired liquid chromatographic (HPLC) method was developed to measure AMP, ADP, ATP, IMP, NAD+ and NADP+ levels in white muscle, heart and brain of anoxic goldfish. 2. Mobile phase parameters of the HPLC method (concentrations of buffer, organic modifier and counter-ion, and pH) were varied to establish the optimal conditions for separation of the compounds of interest. 3. The analytical method was evaluated by calculating some relevant chromatographic parameters (reproducibility and linearity). 4. The HPLC method showed sufficient selectivity, high sensitivity and reproducibility, and excellent linearity.

Reversed-phase ion-paired HPLC of purine nucleotides from skeletal muscle, heart and brain of the goldfish, Carassius auratus L.--II. Influence of environmental anoxia on metabolite levels.

1. The type and the amounts of some (di)nucleotides in white skeletal muscle, heart and brain of anoxic goldfish were established with a reversed-phase ion-paired HPLC method. 2. Significant changes in the levels of these metabolites, as compared to controls, were observed. The most consistent change is the increase of IMP and of IMP-load (IMP/ATP + ADP + AMP) in the three tissues during anoxia. 3. Adenylate energy charges are maintained at a high level in the anoxic white muscle and the anoxic heart. Comparison of these results with those from conventional enzymatic methods for the quantification of (di)nucleotides showed, except for IMP, no significant differences.

Direct observation of the phosphate acceptor and phosphagen pool sizes in vivo.

Earthworms were subjected to environmental anoxia (200 min) and electrical stimulation (5 min, 6 V, 50 Hz). The levels of high-energy phosphate compounds and the intracellular pH were monitored during anoxia, muscular contraction, and recovery by in vivo phosphorus-31 nuclear magnetic resonance (31P-NMR). Several key metabolites were determined by enzymatic analysis and reversed-phase high-performance liquid chromatography of perchlorate extracts. Because lombricine, the phosphagen phosphate acceptor of earthworms, is a phosphodiester, 31P-NMR permitted direct analysis of the extent of phosphorylation of the lombricine pool in vivo. Total lombricine, lombricine phosphate, ATP, and H+ were measured by NMR, and free ADP was calculated from the lombricine kinase equilibrium constant. The ADP concentration was not significantly changed by anoxia, but it rose threefold after stimulation. L-Lactate accumulated on stimulation, whereas multiple end products (L-lactate, alanine, succinate, and glutamine) were formed during environmental anaerobiosis.

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.

Fish muscle energy metabolism measured by in vivo 31P-NMR during anoxia and recovery.

By means of in vivo 31P nuclear magnetic resonance (NMR) we measured energy stores and intracellular pH at 10-min intervals in the myotome of unanesthetized carp and goldfish before, during, and after a period of anoxia (1 h for carp and 4 h for goldfish). The fish were mounted in a modified bioprobe, and their gills were irrigated with a constant flow of aerated or anoxic water. Anoxia caused a steep decline of phosphocreatine and intracellular pH in carp muscle. After the phosphocreatine stores had been exhausted by greater than 85%, [ATP] fell, whereas IMP and phosphodiesters accumulated. In goldfish muscle, initial changes followed the same pattern, but after 20 min a steady state of high-energy phosphates was reached and the development of acidosis was dampened. The resistance of goldfish to anoxia is due to metabolic suppression and a switch from lactate to ethanol and CO2 as the anaerobic end products. In both species, recovery was complete within 3 h. The fast pH recovery seems to be mainly caused by H+ and lactic acid efflux.

Reversed-phase liquid chromatographic analysis of o-phthaldialdehyde-derivatized free amino-acids in two types of goldfish muscles.

o-Phthaldialdehyde-derivatized free amino-acids from muscle tissues of goldfish (Carassius auratus L.) were determined using reversed-phase liquid chromatography (3-microns Ultrasphere ODS) with gradient elution and UV detection of the analyte. An off-line, pre-column derivatization procedure was applied. A full analysis of the derivatives was completed within 40 min. The method showed good selectivity, sensitivity and reproducibility. The developed procedure was employed to quantify free amino-acids in white and red muscle of non-fasted, normoxic goldfish. The differences in the amounts of the individual amino-acids in white and red muscle are given. The rôle of two of the quantitatively most important free amino-acids in white and red muscle of goldfish, histidine and taurine is briefly discussed.

Rates of ion movement from plasma to endolymph in the dogfish.

The rates of movement of Na+, Rb+, Cl- and HCO3- from plasma to endolymph were studied in the elasmobranch fish, Squalus acanthias, by use of the appropriate isotopes. Rb+ was used as a marker for K+. The half-times to equilibrium for Na+, Rb+ and Cl- were about 100 hours; for HCO3- it was 6 hours. The equilibrium ratios, endolymph/plasma, are Na+ 0.87, K+ 26, Cl- 1.37, HCO3- 1.47. Carbonic anhydrase inhibition decreased the rate of HCO3- accumulation, suggesting that the process is actually the formation of endolymphatic HCO3- from plasma or tissue CO2. Increase in plasma pCO2 elevates endolymph HCO3- concentration. The secretory tissue contains carbonic anhydrase and Na-K-ATPase. These and other data suggest that a dominant feature of endolymph chemistry may be HCO3- formation linked in some fashion with K+ transport, through rates catalyzed by these two enzymes.