Intracellular Acid-base Status Research Articles

Acid-base disturbances and effects on oxygen uptake rates in Nile tilapia (Oreochromis niloticus) following acute and prolonged CO2 exposure

High levels of dissolved carbon dioxide (CO2) occur nightly in earthen ponds characterized by high respiration rates. Exposure to high CO2 conditions (hypercapnia) leads to acidosis in fish, which can be compensated by an accumulation of HCO3− to recover intra- and extracellular pH levels, with a capacity that appears to be species-specific. For Nile tilapia, a freshwater tropical teleost traditionally produced in earthen ponds, little information is available on the tolerance to dissolved levels of CO2 and associated acid-base disturbances. Here, we investigated first the effects of acute and progressively increasing CO2, from normocapnic conditions to 60 mg CO2 L−1, on oxygen uptake rates (MO2). This was followed by exposure to three concentrations of CO2; 10, 30, and 60 mg L−1 (equivalent to pCO2 of 5.4, 16.2, and 32.4 mmHg) against a normocapnic control (pCO2 0.3 mmHg), to investigate acute (1 h) or prolonged (24 h) effects on standard (SMR) and maximum metabolic rates (MMR), haematology, and extra- and intracellular acid-base status in adult Nile tilapia (mean BM 435 ± 16 g ± SE). Acute exposure to hypercapnia led to concentration-dependent decreases in both SMR and MMR. Fish were able to fully or partially recover MMR and metabolic scope (MS) after 24 h, while depression of SMR persisted at all CO2 levels. Acute exposure to CO2 caused intra- and extracellular pH levels to decrease by up to 0.5 units in a concentration-dependent manner. Only the lowest hypercapnic treatment (pCO2 5.4 mmHg) was able to fully recover within 24 h. Changes in haematological variables appeared minor, being restricted to increasing haematocrit, haemoglobin concentration, and mean cell volume in the highest CO2 treatments after 24 h exposure. Although the Nile tilapia is generally considered a species able to tolerate poor water quality, the modest or slow acid-base regulation following hypercapnic exposure suggests sensitivity to hypercapnia.

B-005 Blood pH and Phosphate Tend to Follow an Inversely Correlated Homeostatic Status

Abstract Background Phosphorus containing biomolecules perform an essential biochemical function in maintaining extra and intracellular acid-base status. Maintaining normal concentration (2.5–4.5 mg/dl) of phosphorus is indispensable for optimal cellular function and homeostasis. Serum concentration of phosphorus (mainly inorganic orthophosphates) is an indicator of H2PO4−/HPO42− acid-base buffer condition. Disturbance of pH could be an immediate impact of many respiratory and metabolic morbidities. Extra/intracellular redistribution of phosphates in response to an abnormal pH is an established homeostatic phenomenon. Failure to maintain an appropriate phosphate level to support the extra/intracellular redistribution may result in a pH shock. Therefore, it is important to have a clear understanding on how blood phosphate and pH levels are interconnected which could assist in guiding therapeutic interventions to fix phosphate abnormalities and acid-base disturbances. This study examined the homeostatic correlation between blood pH and phosphate level among hospitalized and ambulatory patients. Methods Serum phosphorus concentration and pH were retrieved from the biochemical profiles and arterial blood gas data of hospitalized and ambulatory patients who presented or admitted our tertiary care level university hospital during the first week of July, 2021. Logarithmic (base ten) values of phosphorus concentration were determined to normalize the distribution of the phosphorus data more uniform. Correlation analyses between phosphorus/log[phosphorus] and pH were performed by GraphPad PRISM version 8. Results A total of 101 corresponding blood gases and phosphate levels were obtained. Both phosphorus and log [phosphorus] values of the patients displayed an inverse correlation with arterial pH (Figure 1a and b). Pearson r values demonstrated a negative correlation between phosphorus/log [phosphorus] and pH (−0.6737 and −0.6450, respectively).Fig. 1. Conclusion Monitoring of phosphate levels provide an early indication of acid base status. Phosphorus level maintains an inverse correlation with pH profile. Therefore, a therapeutic manipulation of phosphorus or pH may have a mutual effect requiring further investigation.

Effects of muscle damage on 31 phosphorus magnetic resonance spectroscopy indices of energetic status and sarcolemma integrity in young mdx mice.

31 Phosphorus magnetic resonance spectroscopy (31 P-MRS) has been shown to detect altered energetic status (e.g. the ratio of inorganic phosphate to phosphocreatine: Pi/PCr), intracellular acid-base status, and free intracellular magnesium ([Mg2+ ]) in dystrophic muscle compared with unaffected muscle; however, the causes of these differences are not well understood. The purposes of this study were to examine 31 P-MRS indices of energetic status and sarcolemma integrity in young mdx mice compared with wild-type and to evaluate the effects of downhill running to induce muscle damage on 31 P-MRS indices in dystrophic muscle. In vivo 31 P-MRS spectra were acquired from the posterior hindlimb muscles in young (4-10weeks of age) mdx (C57BL/10ScSn-DMDmdx) and wild-type (C57BL/10ScSnJ) mice using an 11.1-T MR system. The flux of phosphate from PCr to ATP was estimated by 31 P-MRS saturation transfer experiments. Relative concentrations of high-energy phosphates were measured, and intracellular pH and [Mg2+ ] were calculated. 1 H2 O-T2 was measured using single-voxel 1 H-MRS from the gastrocnemius and soleus using a 4.7-T MR system. Downhill treadmill running was performed in a subset of mice. Young mdx mice were characterized by elevated 1 H2 O-T2 (p < 0.01), Pi/PCr (p = 0.02), PCr to ATP flux (p = 0.04) and histological inflammatory markers (p < 0.05) and reduced (p < 0.01) [Mg2+ ] compared with wild-type. Furthermore, 24 h after downhill running, an increase (p = 0.02) in Pi/PCr was observed in mdx and wild-type mice compared with baseline, and a decrease (p < 0.001) in [Mg2+ ] and a lower (p = 0.048) intracellular [H+ ] in damaged muscle regions of mdx mice were observed, consistent with impaired sarcolemma integrity. Overall, our findings demonstrate that 31 P-MRS markers of energetic status and sarcolemma integrity are altered in young mdx compared with wild-type mice, and these indices are exacerbated following downhill running.

Assessing intracellular pH regulation in H+-ATPase-rich ionocytes in zebrafish larvae using in vivo ratiometric imaging.

The H+-ATPase-rich (HR) cells of zebrafish larvae are a sub-type of ion-transporting cell located on the yolk sac epithelium that are responsible for Na+ uptake and H+ extrusion. Current models of HR cell ion transport mechanisms in zebrafish larvae are well established, but little is known about the involvement of the various ion transport pathways in regulating intracellular acid-base status. Here, a ratiometric imaging technique was developed and validated to monitor intracellular pH (pHi) continuously in larval zebrafish HR cells in vivo Gene knockdown or CRISPR/Cas9 knockout approaches were used to evaluate the roles of the two principal apical membrane acid excretory pathways, the Na+/H+ exchanger (NHE3b; slc9a3.2) and the H+-ATPase (atpv1aa). Additionally, the role of HR cell cytosolic carbonic anhydrase (CAc) was investigated because of its presumed role in providing H+ for Na+/H+ exchange and H+-ATPase. The temporal pattern and extent of intracellular acidification during exposure of fish to 1% CO2 and the extent of post-CO2 alkalisation were altered markedly in fish experiencing knockdown/knockout of CAc, NHE3b or H+-ATPase. Although there were slight differences among the three knockdown/knockout experiments, the typical response was a greater degree of intracellular acidification during CO2 exposure and a reduced capacity to restore pHi to baseline levels post-hypercapnia. The metabolic alkalosis and subsequent acidification associated with 20 mmol l-1 NH4Cl exposure and its washout were largely unaffected by gene knockdown. Overall, the results suggest markedly different mechanisms of intracellular acid-base regulation in zebrafish HR cells depending on the nature of the acid-base disturbance.

Role of acidic pH of intravenous fluids in subsequent development of metabolic acidosis- may not be what it seems.

Sir, We extend our thanks to Singh et al. for drawing attention toward the controversies surrounding the use of acidic intravenous fluids (IVFs) infusion.[1] They have rightly mentioned the propensity of 5% dextrose (D5) and 0.9% saline (NS) to cause thrombophlebitis and other deleterious effects.[1] But unlike mentioned by the authors, current literature discusses the known and putative reasons for the pH of 5.5 of NS.[2] Dissolved CO2 from atmosphere, the ionic composition of the solution and inherent properties of the packaging material in which the solutions are supplied, are thought to determine it.[2] We believe that the same set of reasons may also explain the acidic pH (4.2) of D5. Free acid activity is indicated by pH but the nondissociated acid molecules in a solution may not be characterized by the pH value.[2,3] Thus solutions with high titratable acid have reservoir of hydrogen ion (H+) irrespective of pH.[3] Citing the work of Gaudry et al. the authors attribute the acidity of IVFs to their method of sterilization, Gaudry et al. in their manuscript mentioned that the titratable acidity of majority of solutions under study (e.g. D5, NS) varied between 0.063 to 9.480 meq acid/l, depending on the method used for preparation and sterilization.[1,4] Gaudry et al. also mention that though many have measured the pH of common IVFs, not much data are available regarding their titratable acidity.[4] The work of Gaudry et al. dates back to 1972 and a PubMed search by us with the term ‘titratable acidity fluid/titratable acidity solution/titratable acidity infusion’ revealed only two studies relevant to this discussion. These studies date back to 1973 and 2005 and only the abstracts could be retrieved by us.[3,5] Though the in vitro pH of D5 and NS is acidic, their effect on H+ balance is probably negligible.[2,4] It may be because of their low titratable acid content.[4] But metabolic acidosis accompanies infusion of these solutions.[1,2,4,6,7] This acidosis was explained by dilution of bicarbonate content in plasma.[2,6,7] Subsequently Stewart's physiochemical approach was used to describe the in vivo effect on acid base status by IVFs.[6,7] According to the later concept, the acid-base effect of an IVF is determined by the strong ion deference (SID) and the total weak acid concentration (ATOT) of the fluid.[6,7] pH falls with decreasing SID, increasing ATOT or CO2TOT(total CO2 content) / PCO2.[6,7] As the IVFs equilibrate with ECF, they alter SID of ECF, as SID of these IVFs defers appreciably from that of plasma.[6,7] SID of D5 and NS is 0, they decrease the SID of plasma and exert an acidifying effect.[6,7] These crystalloids are devoid of any weak acids and decrease ATOT of plasma due to its diluting effect causing alkalosis.[6,7] Whereas some colloids (e.g. albumin, gelatin) itself may contribute toward ATOT of plasma, thus, acidifying it.[6,7] Although CO2TOT/PCO2 also independently influences the pH of a biological system, its contribution in determining in vivo acid base activity of an IVF is generally regarded to be low as the concentration of dissolved CO2 is itself low (about 0.012 mmol/l).[2,6,7] But it should be kept in mind that IVFs with high CO2TOT have impact on intracellular acid base status, more during rapid administration or low perfusion states.[6,7] Contrary to the prediction by Singh et al. the pH of hypertonic saline (3%) is 5.8 and it causes more acidosis than NS because of its higher osmolarity that draws more water from other body fluid compartments.[6,7] Although the pH of Hartmann`s solution is 5, its in vivo SID is 27 meq/l, which is close to the ideal SID of 24 meq/l.[2,6,7] And despite its pH of 5, it is known to prevent infusion-related metabolic acidosis.[2,6,7] In-depth discussion on application of the concept of quantitative physical chemistry on the in vivo effect of acid base homeostasis is available.[6,7] The effect on plasma pH by solutions with same pH may be different and differing pH can have similar acid-base effects.[2,6] Thus, the effect of an IVF on plasma/ECF SID, ATOT and CO2TOT depending on the amount infused should be considered while predicting its possible in vivo effect on acid base status, not merely its in vitro pH.[1,2,3,4,5,6,7]

Effects of recovery time on phosphocreatine kinetics during repeated bouts of heavy-intensity exercise

The purpose of this study was to examine the kinetics of phosphocreatine (PCr) breakdown in repeated bouts of heavy-intensity exercise separated by three different durations of resting recovery. Healthy young adult male subjects (n = 7) performed three protocols involving two identical bouts of heavy-intensity dynamic plantar flexion exercise separated by 3, 6, and 15 min of rest. Muscle high-energy phosphates and intracellular acid-base status were measured using phosphorus-31 magnetic resonance spectroscopy. In addition, the change in concentration of total haemoglobin (Delta[Hb(tot)]) and deoxy-haemoglobin (Delta[HHb]) were monitored using near-infrared spectroscopy. Prior exercise resulted in an elevated (P < 0.05) intracellular hydrogen ion ([H(+)](i)) after 3 min (182 +/- 72 (SD) nM; pH 6.73) and 6 min (112 +/- 19 nM; pH 6.95) but not after 15 min (93 +/- 8 nM; pH 7.03) compared to pre-exercise in Con (90 +/- 3 nM; pHi 7.05). The on-transient time constant (tau) of the PCr primary component was not different amongst the exercise bouts. However, in each of the subsequent bouts the amplitude of the PCr slow component, total PCr breakdown, and rise in [H(+)](i) were reduced (P < 0.05). At exercise onset, Delta[Hb(tot)] was increased (P < 0.05) and the Delta[HHb] kinetic response was slowed (P < 0.05) in the exercise after 3 min, consistent with improved muscle perfusion. In summary, neither the level of acidosis or muscle perfusion at the onset of exercise appeared to be directly related to the time course of the on-transient PCr primary component or the magnitude of the PCr slow component during subsequent bouts of exercise.

Phosphocreatine Metabolism and Acidosis during Repeated Bouts of Heavy-Intensity Exercise Separated by 15-min Rest

Prior heavy-intensity exercise (HVY) results in a reduced reliance on substrate-level phosphorylation during a subsequent exercise bout when the bouts are separated by 6-min rest. Proposed mechanisms for this response have been associated with acidosis and lactate accumulation. PURPOSE: To determine if a similar favourable energetic response in the second exercise is evident when the bouts are separated by 15-min of rest. In this condition intracellular acid-base status will have returned to pre-exercise resting levels prior to the second bout. METHODS: Healthy young adult male subjects (n = 7) performed a 6-min bout of HVY isotonic plantar flexion exercise (HVY1) followed by 15-min of rest, then an identical 6-min bout (HVY2). Muscle phosphocreatine (PCr) and intracellular acid-base status were measured using 3-tesla 31P-magnetic resonance spectroscopy. Each subject repeated the protocol three times. PCr was expressed as percent (%) of average pre-HVY1 resting value. RESULTS: Prior to the onset of exercise, resting intracellular hydrogen ion ([H+]i) was not different (P = 0.11) at the onset of exercise in HVY1 [94 nM (SD 4)] and HVY2 [99 nM (SD 8)]. The increase in [H+]i during exercise was reduced ∼ 45% during HVY2, with end-exercise [H+]i lower (P < 0.01) in HVY2 [139 nM (SD 38)] than HVY1 [167 nM (SD 48)]. Resting [PCr] was greater (P = 0.03) in HVY2 [101.8% (SD 2.1)] than HVY1, while at end-exercise, [PCr] was greater (P = 0.02) in HVY2 [55.1% (SD 15.1)] than HVY1 [48.7 % (SD 10.9)]. CONCLUSIONS: The lower accumulation of [H+]i (and presumably lactate accumulation) in HVY2 (delta [H+]i 40 nM) than HVY1 (delta [H+]i 73 nM), along with the decreased PCr breakdown, are consistent with reduced substrate-level phosphorylation. Therefore, these results demonstrate that the benefits of prior heavy-intensity exercise are evident after 15-min of rest, when intracellular acid-base status has returned to pre-exercise resting levels.

Phosphorus-31 MR Spectroscopy in Pediatric Liver Transplant Recipients: A Noninvasive Assessment of Graft Status with Correlation with Liver Function Tests and Liver Biopsy

Noninvasive in vivo hepatic phosphorus-31 MR spectroscopy has recently been shown to provide information about hepatic functional status. We sought to show the correlation of phosphorus-31 MR spectroscopy with blood biochemistry and liver biopsy results in pediatric patients after liver transplantation. Eleven pediatric transplant recipients (eight with good graft function, two with chronic hepatitis, and one with acute rejection) and four healthy control subjects were studied with in vivo 31P MR spectroscopy. Ratios of phosphomonoesters (PME) to total phosphorus (TP), phosphodiester (PDE) to TP, nucleotide triphosphates (NTP), inorganic phosphate (Pi), and intracellular acid-base status (pH) were measured. Liver function test (n = 11) and biopsy (n = 3) results were obtained for correlation with spectroscopic findings. The eight patients with good graft function displayed spectral profiles similar to those of the healthy subjects, and no significant difference in the metabolic ratios of these patients compared with the control subjects was detected. Three patients with abnormal liver function and biopsy-proven hepatic complications showed elevated PME/TP ratios when compared with those of both the control subjects and the group with good graft function. Phosphorus-31 MR spectroscopy is a feasible technique for the noninvasive assessment of host-related complications in pediatric patients after liver transplantation. Our preliminary data suggest that the technique may be integrated with MRI for the investigation of impaired liver function in transplant recipients when neither a biliary complication nor a vascular complication is identified.

Intracellular [H+]: a determinant of cell volume in skeletal muscle

Intracellular [H⁺]: a determinant of cell volume in skeletal muscle

Studies on the extra- and intracellular acid-base status and its role on metabolic depression in the land snail Helix lucorum (L.) during estivation.

The aim of the present study was to examine the acid-base status of extra- and intracellular fluids and its possible role on the regulation of the metabolic rate of Helix lucorum during prolonged estivation. For this purpose, the rate of oxygen consumption for active and estivating snails was determined. The acid-base status was also examined in the hemolymph and tissues from active and estivating snails acclimated at 25 degrees C. In addition, the buffer values of hemolymph and tissues were determined in order to examine whether there is a change in the snails during estivation. The rate of oxygen consumption decreased significantly within the 1st 10 days of estivation from 122.51+/-10 microl.g(-1).h(-1) to 25.86+/-5.2 microl.g(-1).h(-1), indicating a marked decrease in metabolic rate. P(CO2)increased within the 1st 20 days of estivation from 13.52+/-0.68 mmHg to 25.09+/-2.05 mmHg, while the pH of hemolymph (pH(e)) decreased from 7.72+/-0.04 to 7.44+/-0.06. The level of bicarbonates decreased in the hemolymph of estivating snails, indicating a metabolic acidosis, which was moderate in extracellular fluids. In contrast to pH(e), the intracellular pH (pH(i)) was maintained in the tissues of estivating H. lucorum, indicating a regulation of pH(i) despite the developed hypercapnia. According to the results presented here, it seems that the timing of pH(e) changes does not correlate with the timing of metabolic rate reduction in estivating H. lucorum.

Effects of prolonged acclimation to cold on the extra--and intracellular acid-base status in the land snail Helix lucorum (L.).

The aim of this study was to examine the effect of prolonged acclimation to cold on the acid-base status of extra- and intracellular fluids in the land snail Helix lucorum. For this purpose, acid-base parameters in the hemolymph and tissues were determined. In addition, the buffer values of hemolymph and tissues were determined in order to examine whether they change in the snails during acclimation to cold. According to the results presented, there is an inverse pH-temperature relationship in the hemolymph within the first day of acclimation, which is consistent with alphastat regulation. The Pco2 decreased, and pH in the hemolymph (pH(e)) increased by 0.32 U within the first day of acclimation to cold, which corresponds to a change of 0.013 U degrees C(-1). After the first day of acclimation, Pco2 increased in the hemolymph, resulting in a significant drop in pH(e) by 90 d of acclimation to cold. Acclimation of snails to low temperatures did not change the buffer value of the hemolymph. Also, intracellular pH (pH(i)) and intracellular buffer values remained stable during acclimation to cold for prolonged periods. The latter results in conjunction with those obtained by the in vitro determination of the passive component of intracellular fluids indicate an active regulation of pH(i) in H. lucorum during acclimation to cold.

Effects of anoxia on the extra- and intracellular acid-base status in the land snail helix lucorum (L.): lack of evidence for a relationship between pyruvate kinase down-regulation and acid-base status

The aims of the present study were to describe a possible correlation between the regulation of the key glycolytic enzyme pyruvate kinase and the acid-base status in the haemolymph and in several other tissues of land snails during anoxia. To illustrate whether such a relationship exists, we determined (i) the acid-base variables in the haemolymph and tissues of the land snail Helix lucorum, (ii) the kinetic properties of pyruvate kinase from several tissues and (iii) the levels of the anaerobic end-products d-lactate and succinate in the haemolymph and tissues of aerobic and anoxic Helix lucorum. The results showed that the pH of haemolymph (pHe) decreased significantly over the first 20 h of anoxia and then recovered slowly towards control values. A similar pattern was observed for intracellular pH (pHi), which decreased significantly over the first 16 h of anoxia and slowly returned towards control levels. The reduction and recovery of pHi and pHe seem to reflect the rate of anaerobic metabolism. The main anaerobic end-products, d-lactate and succinate, accumulated rapidly during the initial stages of anoxia and more slowly as anoxia progressed. The decrease in the rate of accumulation of anaerobic end-products during prolonged anoxia was due to the conversion of tissue pyruvate kinase to a less active form. The results demonstrate a correlation between pyruvate kinase down-regulation and the recovery of acid-base status in the haemolymph and the tissues of land snails during anoxia.

Respiratory and metabolic functions of carbonic anhydrase in exercised white muscle of trout.

Electrical stimulation of a trout saline-perfused trunk preparation resulted in metabolic and respiratory responses comparable to those occurring after exhaustive exercise in vivo. Recovery of intracellular acid-base status and glycogen resynthesis were faster than in vivo. Intracellular carbonic anhydrase (ICF CA) blockade elevated intracellular PCO2 relative to untreated postexercise controls, whereas extracellular CA (ECF CA) blockade did not, in contrast to previous work with muscle at rest. ECF CA blockade had only a transient effect on postexercise CO2 and ammonia efflux. The relatively small pool of membrane-associated CA appears to be overwhelmed by exercise-induced CO2 production in muscle. Transmembrane ammonia efflux appears to shift from diffusion primarily as NH3 at rest, which is facilitated by ECF CA, to movement predominantly as NH+4 after exercise, which is independent of CA. The postponed recovery of intracellular pH caused by either or both ECF and ICF CA inhibition was consistent with reduced metabolic acid and lactate excretion from muscle. Creatine phosphate resynthesis was delayed by CA inhibition, whereas ATP replenishment was not affected. Delayed glycogen recovery indicates that HCO-3-dependent pathway(s) may be involved in glyconeogenesis.

Copper exposure impairs intra- and extracellular acid-base regulation during hypercapnia in the fresh water rainbow trout (Oncorhynchus mykiss).

In order to evaluate the impact of water-borne copper on acid-base regulation in fresh water rainbow trout, chronically cannulated fish were exposed to copper (0.6 mg 1(-1)), hypercapnia (water PCO2 of 6 mmHg) or a combination of copper and hypercapnia, while a fourth untreated group served as the control. Blood samples obtained at 0 h, 4 h and 24 h were analysed for acid-base status, ion concentrations and respiratory parameters. Tissue samples from caudal skeletal muscle, liver and gill filaments were examined for intracellular acid-base status, ion- and water contents, and copper concentration. Exposure to copper alone elicited a small extracellular metabolic alkalosis, no changes in arterial PO2, and a minor decrease in plasma ion concentrations. Hypercapnia alone increased arterial PCO2 from approximately 2 mmHg to 7.2 mmHg, but the extracellular respiratory acidosis present at 4 h was almost completely compensated at 24 h due to an increase in plasma bicarbonate concentration [HCO3-] from 8.1 mM to 24.4 mM. Combined exposure to hypercapnia and copper resulted in a slightly larger acidosis at 4 h, and the fish failed to restore extracellular pH at 24 h, because plasma [HCO3-] only increased to 16.3 mM. Fish exposed to hypercapnia and copper also showed a delayed recovery of intracellular pH in skeletal muscle, compared to fish exposure to hypercapnia only. Thus, copper exposure impaired both extracellular and intracellular acid-base regulation during hypercapnia. When seen in connection with only minor effects of copper on osmoregulatory and respiratory parameters, the reduced ability to regulate acid-base suggests that acid-base regulation may be one of the most copper-sensitive branchial functions.

Metabolic and energy correlates of intracellular pH in progressive fatigue of squid (L. brevis) mantle muscle.

Squid (Lolliguncula brevis) were exercised at increasing swimming speeds to allow us to analyze the correlated changes in intracellular metabolic, acid-base, and energy status of the mantle musculature. Beyond a critical swimming velocity of 1.5 mantle lengths/s, an intracellular acidosis developed that was caused by an initial base loss from the cells, the onset of respiratory acidification, and, predominantly, octopine formation. The acidosis was correlated with decreasing levels of phospho-L-arginine and, thus, supported ATP buffering at the expense of the phosphagen. Monohydrogenphosphate, the actual substrate of glycogen phosphorylase accumulated, enabling glycogen degradation, despite progressive acidosis. In addition to octopine, succinate, and glycerophosphate accumulation, the onset of acidosis characterizes the critical velocity and indicates the transition to a non-steady-state time-limited situation. Accordingly, swimming above the critical velocity caused cellular energy levels (in vivo Gibbs free energy change of ATP hydrolysis) to fall. A minimal value was reached at about -45 kJ/mol. Model calculations demonstrate that changes in free Mg2+ levels only minimally affect ATP free energy, but minimum levels are relevant in maintaining functional concentrations of Mg(2+)-complexed adenylates. Model calculations also reveal that phosphagen breakdown enabled L. brevis to reach swimming speeds about three times higher than the critical velocity. Comparison of two offshore squid species (Loligo pealei and Illex illecebrosus) with the estuarine squid L.brevis indicates that the latter uses a strategy to delay the exploitation of high-energy phosphates and protect energy levels at higher than the minimum levels (-42 kJ/mol) characterizing fatigue in the other species. A more economical use of anaerobic resources and an early reduction in performance may enable L. brevis to tolerate more extreme environmental conditions in shallow estuarine waters and even hypoxic environments and to prevent a fatal depletion of energy stores.

Extracellular and intracellular acid-base status in the freshwater crayfish, Austropotamobius pallipes between 1 and 12(o)C

Freshwater crayfish, Austropotamobius pallipes, caught in the winter when water temperature was 5 &deg;C, were acclimated in the laboratory to 5 &deg;C for 1 week and to 1 and 12 &deg;C for 1 month before haemolymph and tissue samples were taken for acid&shy;base analysis. In vivo, haemolymph (extracellular) pH increased by 0.14 units between 5 and 1 &deg;C but remained unchanged between 5 and 12 &deg;C, giving an overall deltapHe/deltat value of -0.009 pH units &deg;C-1 and an [H+] value of +3.3 nmol l-1. Haemolymph withdrawn from crayfish acclimated to 1 &deg;C and warmed anaerobically in vitro to 12 &deg;C had a deltapHe/deltat value of -0.010 pH units &deg;C-1. This was depressed compared with the values of -0.015 and -0.016 pH units &deg;C-1 obtained from haemolymph withdrawn from crayfish held at 5 and 12 &deg;C, respectively, but changes in H+ concentration were similar in all three groups. There was little change in intracellular pH with rise in temperature in either claw muscle (deltapHi/deltat=-0.003 pH units &deg;C-1) or hepatopancreas (deltapHi/deltat=-0.006 pH units &deg;C-1). In the heart, deltapHi/deltat paralleled that in the haemolymph (-0.010 pH units &deg;C-1), but the largest change was found in the abdominal muscle (-0.025 pH units &deg;C-1). In all tissues apart from claw muscle, there was a metabolic acidosis, with an increase in metabolic acid load as temperature rose from 1 to 12 &deg;C. The depressed in vivo deltapHe/deltat value and the differences in deltapHi/deltat between the tissues were attributed to active regulation of pHi, possibly to control enzyme function in the intracellular compartments. Tissues that are functionally important in the winter months, such as abdominal muscle, followed alphastat regulation, whereas tissues that are less active in the winter, such as claw muscle and hepatopancreas, maintained pHi irrespective of temperature, resulting in a relatively acidotic pHi, which may serve to depress the activity of metabolic enzymes.

Interactions of Anaerobic Propionate Formation and Acid-Base Status inArenicola marina:An Analysis of Propionyl-CoA Carboxylase

The contribution of propionyl-CoA carboxylase (PCC) to the control of anaerobic metabolism by acid-base parameters (pH, Pco₂, and [HCO₃⁻]) was investigated with a purified enzyme preparation and isolated mitochondria from the body wall musculature of Arenicola marina. The enzyme catalyzes the rate-limiting step in anaerobic propionate formation, namely, the carboxylation of methylmalonyl-CoA with concomitant formation of ATP and base equivalents (= HCO₃⁻). Propionyl-CoA carboxylase is likely not saturated with its substrates methylmalonyl-CoA, ADP, and Pi under in vivo conditions, and propionate formation is therefore activated by a decreasing energy charge of the cell (i. e., increasing ADP and Pi concentrations). The effects of the individual acid-base parameters pH, Pco₂, and [HCO₃⁻] on PCC activity have been determined. Stimulation of PCC by both high proton and low bicarbonate concentrations reflects an amplified control of propionate formation by the intracellular acid-base status. Nonrespiratory acidosis enhances the rate of decarboxylation of methylmalonyl-CoA, leading to a release of base equivalents. This mechanism has a strong stabilizing effect on the intracellular pH during long-term anaerobiosis. Without bicarbonate production by PCC, an additional pH drop of about 0. 03 pH units per hour of anaerobiosis would be observed in A. marina. Our data support the hypothesis that, besides ionic transport mechanisms, metabolism itself contributes to cellular pH regulation.

1H-NMR measurement of fractional dissociation of imidazole in intact animals

The alphastat hypothesis states that intracellular acid-base status is regulated to maintain constancy of the fractional dissociation of intracellular protein and enzyme imidazole-histidine (alpha-imidazole). A major drawback of this theory has been the lack of a means to directly measure alpha-imidazole in intact animals. We developed a method for directly measuring alpha-imidazole in intact unanesthetized animals using 1H-nuclear magnetic resonance spectroscopy (NMR). We measured carnosine alpha-imidazole of white skeletal muscle from intact unanesthetized newts at three body temperatures (10, 20, and 30 degrees C). alpha-Imidazole remained constant, approximately 0.56, with alterations in body temperature, whereas intracellular pH (pHi) changed significantly (-0.015 U/degrees C), affirming the validity of the imidazole alphastat hypothesis for this tissue. This method was also used to determine the pK values of the imidazole moiety of carnosine and the imidazole moiety alone over a temperature (T) range 4-40 degrees C. The pK values of carnosine differed from those of imidazole, but the delta pK/delta T was the same. pHi was also determined using 31P-NMR and found to be the same as that calculated from carnosine alpha-imidazole values. Therefore Pi and carnosine share a similar pHi environment. We describe a novel technique to directly measure alpha-imidazole in intact tissue.

Control and consequences of adrenergic activation of red blood cell Na+/H+ exchange on blood oxygen and carbon dioxide transport in fish.

The catecholamines, adrenaline and noradrenaline, are released into the circulation of fish during a variety of physical and environmental disturbances that share the common feature of a requirement for enhanced blood oxygen transport. Indeed, the dominant factor controlling the mobilization of catecholamines from chromaffin tissue is a depression of blood oxygen content usually coinciding with a reduction of hemoglobin-O2 (Hb-O2) binding to 50-60% saturation. The elevation of plasma catecholamine levels, under such conditions, activates a beta-adrenergic cyclic AMP-dependent Na+/H+ exchanger on the red blood cell (rbc) membrane. The adrenergic responsiveness AMP-dependent Na+/H+ exchanger on the red blood cell (rbc) membrane. The adrenergic responsiveness of the rbc Na+/H+ exchanger to catecholamines varies both within and between species. Such inter- and intra-specific differences may reflect, in part, the availability of cell surface beta-adrenoceptors that are functionally coupled to adenylate cyclase. The activation of rbc Na+/H+ exchange and the accompanying profound adjustments of intracellular and extracellular acid-base status, nucleoside triphosphate (NTP) levels, and cooperativity of Hb-O2 binding have important consequences on both O2 and CO2 transfer and transport in the blood that vary markedly at the sites of oxygenation (the gill) and deoxygenation (the tissues) thereby enabling simultaneous amelioration of O2 loading and unloading. At the gill, oxygen transfer is enhanced owing to increases in Hb-O2 affinity and capacity while at the tissues, oxygen delivery is facilitated by a reduction of Hb-O2 affinity. This reduction in affinity at the tissues is a consequence of the combined effects of increased cooperativity of Hb-O2 binding and a rise in venous PCO2 (PvCO2) caused by the titration of HCO3- by H+ extruded by the rbc Na+/H+ exchanger. This elevation of PvCO2 may contribute to the rise in arterial PCO2 (PaCO2) observed after adrenergic activation of rbc Na+/H+ exchange that is caused primarily by impairment of rbc CO2 excretion related to modification of the intracellular acid-base status.

Recovery From Acute Haemolymph Acidosis in Unfed Locusts: I. Acid Transfer to the Alimentary Lumen is the Dominant Mechanism

ABSTRACT Organismal homeostasis requires regulation of extracellular acid-base status; however, the mechanisms by which insects regulate haemolymph pH are poorly known. We evaluated the recovery of desert locusts Schistocerca gregaria Forskål from acute acid loads, initiated by HCl injections into the haemolymph (0.5 pH unit decrease). Haemolymph pH, and [HCO3−] recovered in 8–24h, providing the first unequivocal evidence that insects regulate extracellular pH. There were no changes in the concentrations of the primary haemolymph buffer compounds (protein, inorganic phosphate) during recovery. Within 1 h, the tracheal system effectively eliminated the carbon dioxide derived from bicarbonate buffering. During the remainder of the recovery, haemolymph was similar to control values; there was no respiratory compensation for decreased haemolymph pH. Approximately 75 % of the acid equivalents removed from the haemolymph during the recovery process were transferred to the lumens of the crop and midgut. Transfer of acid equivalents to the alimentary lumen provides unfed locusts with a mechanism of haemolymph pH regulation that does not compromise intracellular acid-base status or increase ventilatory water loss.