Non-bicarbonate Buffer Value Research Articles

Effects of temperature on hardhead minnow (Mylopharodon conocephalus) blood-oxygen equilibria

Habitat perturbations, including dam construction with consequent temperature changes and the introduction of non-native species to California’s mid- to low-elevation streams, have negatively influenced some native fish populations’ historic distribution and abundance. Populations of hardhead, Mylopharodon conocephalus (Cyprinidae), have experienced such population declines, but environmental temperature effects on this large (to 60 cm SL), native species are poorly documented. We measured temperature effects on in vitro blood-oxygen affinity and equilibrium curve shape, key dynamics of the species’ oxygen-transport system, derived from blood collected from wild-caught hardhead. Over an 11–30 °C temperature range, the half-saturation value (P50, an inverse measure of affinity) increased with the temperature from 0.51 to 1.80 kPa for low-PCO2 (“arterial”) treatments and from 2.02 to 2.92 kPa for high-PCO2 (“venous”) treatments. The apparent heat of oxygenation (temperature effect) was higher at temperatures > (absolute value) 19 °C. Therefore, hardhead’s blood has a decreased ability to bind oxygen at its gills at temperatures ≥25 °C, compared to that at temperatures ≤19 °C. The hardhead’s Bohr factors (Ф), non-bicarbonate buffer values (β), nucleoside triphosphate (NTP) concentrations, blood oxygen capacities (CBO2), and mildly sigmoid-shaped oxygen equilibrium curves showed no relationship with temperature. Overall, their blood-oxygen equilibria suggest that hardhead can tolerate moderate hypoxia and temperature variations in its environment and that they have some capacity for sustained, high-aerobic activity.

Respiratory properties of blood in the harbor porpoise,Phocoena phocoena

Harbor porpoises are active divers that exchange O(2) and CO(2) with the environment during a fast single breath upon surfacing. We investigated blood O(2)-transporting properties, buffer characteristics, Cl(-) transport via the erythrocyte anion exchanger (AE1), circulating nitric oxide metabolites and hemoglobin nitrite reduction in harbor porpoises with the aim to evaluate traits that are adaptive for diving behavior. Blood O(2) affinity was higher in harbor porpoises than in similar sized terrestrial mammals, as supported by our parallel recordings of O(2) equilibria in sheep and pig blood. Further, O(2) affinity tended to increase with increasing body mass. A high O(2) affinity favors O(2) extraction from the lungs, but a normal Bohr effect (ΔlogP(50)/ΔpH=-0.46) gradually lowers O(2) affinity during dives (where CO(2) accumulates) to assist O(2) off-loading to perfused tissues. The true plasma non-bicarbonate buffer value was moderately higher than in terrestrial mammals and increased upon deoxygenation. Plasma bicarbonate was also relatively high, contributing to increase the overall buffer capacity. The apparent Cl(-) permeability of harbor porpoise erythrocytes was similar to the human value at 37°C, showing absence of a comparative increase in the velocity of erythrocyte HCO(-)(3)/Cl(-) exchange to aid CO(2) excretion. The Q(10) for AE1-mediated Cl(-) transport in harbor porpoises was lower than in humans and seemed to match the Q(10) for metabolism (Q(10)≈2). Plasma nitrite, plasma nitrate and hemoglobin-mediated nitrite reduction were elevated compared with mammalian standards, suggesting that increased nitric oxide bioavailability and nitrite-derived nitric oxide could play important roles in diving physiology.

Physiological responses of Daphnia pulex to acid stress

BackgroundAcidity exerts a determining influence on the composition and diversity of freshwater faunas. While the physiological implications of freshwater acidification have been intensively studied in teleost fish and crayfish, much less is known about the acid-stress physiology of ecologically important groups such as cladoceran zooplankton. This study analyzed the extracellular acid-base state and CO2 partial pressure (PCO2), circulation and ventilation, as well as the respiration rate of Daphnia pulex acclimated to acidic (pH 5.5 and 6.0) and circumneutral (pH 7.8) conditions.ResultsD. pulex had a remarkably high extracellular pH of 8.33 and extracellular PCO2 of 0.56 kPa under normal ambient conditions (pH 7.8 and normocapnia). The hemolymph had a high bicarbonate concentration of 20.9 mM and a total buffer value of 51.5 meq L-1 pH-1. Bicarbonate covered 93% of the total buffer value. Acidic conditions induced a slight acidosis (ΔpH = 0.16–0.23), a 30–65% bicarbonate loss, and elevated systemic activities (tachycardia, hyperventilation, hypermetabolism). pH 6.0 animals partly compensated the bicarbonate loss by increasing the non-bicarbonate buffer value from 2.0 to 5.1 meq L-1 pH-1. The extracellular PCO2 of pH 5.5 animals was significantly reduced to 0.33 kPa, and these animals showed the highest tolerance to a short-term exposure to severe acid stress.ConclusionChronic exposure to acidic conditions had a pervasive impact on Daphnia's physiology including acid-base balance, extracellular PCO2, circulation and ventilation, and energy metabolism. Compensatory changes in extracellular non-bicarbonate buffering capacity and the improved tolerance to severe acid stress indicated the activation of defense mechanisms which may result from gene-expression mediated adjustments in hemolymph buffer proteins and in epithelial properties. Mechanistic analyses of the interdependence between extracellular acid-base balance and CO2 transport raised the question of whether a carbonic anhydrase (CA) is involved in the catalysis of the reaction, which led to the discovery of 31 CA-genes in the genome of D. pulex.

Cationic composition and acid–base state of the extracellular fluid, and specific buffer value of hemoglobin from the branchiopod crustacean Triops cancriformis

Recent insights into the allosteric control of oxygen binding in the extracellular hemoglobin (Hb) of the tadpole shrimp Triops cancriformis raised the question about the physico-chemical properties of the protein's native environment. This study determined the cationic composition and acid-base state of the animal's extracellular fluid. The physiological concentrations of potential cationic effectors (calcium, magnesium) were more than one order of magnitude below the level effective to increase Hb oxygen affinity. The extracellular fluid in the pericardial space had a typical bicarbonate concentration of 7.6 mM but a remarkably high CO(2) partial pressure of 1.36 kPa at pH 7.52 and 20 degrees C. The discrepancy between this high CO(2) partial pressure and the comparably low values for water-breathing decapods could not solely be explained by the hemolymph-sampling procedure but may additionally arise from differences in cardiovascular complexity and efficiency. T. cancriformis hemolymph had a non-bicarbonate buffer value of 2.1 meq L(-1) pH(-1). Hb covered 40-60% of the non-bicarbonate buffering power. The specific buffer value of Hb of 1.1 meq (mmol heme)(-1) pH(-1) suggested a minimum requirement of two titratable histidines per heme-binding domain, which is supported by available information from N-terminal sequencing and expressed sequence tags.

Effects of temperature and carbon dioxide on green sturgeon blood–oxygen equilibria

There are three Northeast Pacific Rivers still supporting spawning populations of green sturgeon, Acipenser medirostris, but all have been modified hydrologically and thermally by dam construction. Age 1- to 3-year-old green sturgeon, progeny of artificially spawned, wild-caught Klamath River adults, were used to assess the effects of temperature and carbon dioxide on critical hematological parameters related to evolutionary adaptations of this species to its physical environment. In vitro measurement of the effect of temperature and carbon dioxide on blood–oxygen affinity and equilibrium curve shape yielded the following data for the respective temperature treatments (11, 15, 19, and 24°C): half-saturation values (P50’s, kPa, a measure of affinity) 1.26, 1.44, 1.63, 1.69 for low-PCO2 treatments and 2.08, 2.41, 2.74, 2.94 for high-PCO2 treatments; Bohr factors −0.322, −0.327, −0.366, −0.536; and non-bicarbonate buffer values (slykes) −6, −3, −5, −8. Temperature sensitivities (ΔH, kJ mol O 2 −1 ) between these respective temperatures were −34.20, −15.24, −6.74 for low-PCO2 treatments and −20.05, −27.00, and −11.55 for the high-PCO2 treatments. These data suggest that juvenile green sturgeon may tolerate moderate environmental hypoxia, moderate aerobic activity, low to moderate hypercapnia, and moderate temperature changes in their environments.

BUFFERING AND RESPIRATORY COMPENSATION OF LACTIC ACIDOSIS AFTER THE WINGATE TEST

742 The “pH-defense” in the extracellular space after rapid addition of lactic acid by performing 30 s anaerobic exercise was studied in 13 subjects. Acid-base status and lactate concentration in earlobe blood were determined before and up to 30 min after the test. After this time [lactate] was still increased and pH as well as pCO2 decreased.-Δ[lactate]plasma/ΔpH as a measure of total pH defense(bicarbonate and nonbicarbonate buffering, respiratory compensation) increased steadily from 61 ± 14 (SE) to 120 ± 9 mmol/l.Δ[HCO3-]plasma/ΔpH (bicarbonate buffering and respiratory compensation) rose in a parallel manner from 42 ± 10 to 96± 7 mmol/l. The difference between-Δ[lactate]plasma/ΔpH andΔ[HCO3-]plasma/ΔpH amounted to 17 ± 3 mmol/l. The nonbicarbonate buffer value can be used to estimate the effect of hyperventilation and to calculate buffer capacities for constant pCO2 and thus nonrespiratory (nr) pH changes.-Δ[lactate]plasma/ΔpHnr was initially higher than-Δ[lactate]plasma/ΔpH (71 + 8 mmol/l) because of temporarily increasing pCO2, reached a minimum of 57 ± 1 mmol/l after 6 min and than increased again up to the initial values.Δ[HCO3-]plasma/ΔpHnr followed an equal time course but at a lower level (difference 17 mmol/l). Calculation ofΔ[HCO3-]plasma/ΔpHnr from initial values by use of the Henderson-Hasselbalch equation yielded only slightly different values demonstrating that no considerable bicarbonate exchange occurred with the cells. The apparently increasing total pH defense after the Wingate test resulted from a consistently decreased pCO2. The relative importance of this respiratory compensation increases with decreasing lactic acid concentration and thus decreasing total pH changes.

Carbon dioxide transport in alligator blood and its erythrocyte permeability to anions and water.

Deoxygenation of alligator red blood cells (RBCs) caused binding of two HCO3- equivalents per hemoglobin (Hb) tetramer at physiological pH. At lowered pH, some HCO3- binding also occurred to oxygenated Hb. The erythrocytic total CO2 content was large, and Hb-bound HCO3-, free HCO3-, and carbamate contributed about equally in deoxygenated cells. The nonbicarbonate buffer values of RBCs and Hb were high, and the Hb showed a significant fixed acid Haldane effect. Binding of HCO3- on deoxygenation occurred without a change in RBC intracellular pH, revealing equivalence between oxylabile HCO3- and H+ binding. Erythrocyte volume, plasma pH, and plasma HCO3- concentration also varied little with the degree of oxygenation. Diffusional water permeability was higher in oxygenated than deoxygenated RBCs. The RBCs have rapid band 3-mediated Cl- and HCO3- transport, which was not affected by degree of oxygenation, but net fluxes of Cl- and HCO3- via the anion exchanger are small during blood circulation at rest. Most of the CO2 taken up into the blood as it flows through tissue capillaries is carried within the erythrocytes as Hb-bound HCO3- until CO2 is excreted when blood flows through pulmonary capillaries.

Temperature-dependent shift of pHi in fish white muscle: contributions of passive and active processes.

This study was designed to determine the mechanisms causing temperature-induced pH shifts in the white muscle of the marine teleost Zoarces viviparus. The white musculature undergoes an intracellular acidification with increasing body temperature at a slope of the pH-temperature relationship equal to -0.016 +/- 0.003 U/degree C. This is in good accordance with the overall relationship between the change in pK and the change in temperature of the intracellular proteins, which was determined to be -0.013 +/- 0.001 U/degree C. Thus the dissociation state of muscle proteins is kept fairly constant in white muscle of Zoarces viviparus. The passive component of the observed pH shift, which is due to the physicochemical response of the intracellular buffers to temperature change, accounts for only 35% of the pH transition. Ventilatory adjustment of intracellular PCO2 does not contribute to the temperature-induced shift of intracellular pH (pHi) in Zoarces viviparus. Therefore, the remaining 65% of pH adjustment must be ascribed to ion exchange mechanisms. The nonbicarbonate buffer value amounted to 34.4 +/- 2.3 meq.pH-1 kg cell water-1 at 12 degrees C and decreased slightly but not significantly with temperature. On the basis of our data we calculated that a removal of 0.52 mmol base equivalents.kg cell water-1.degree C-1 was necessary to shift pHi to its new steady state.

Temperature and CO2Effects on Blood O2Equilibria in Northern Squawfish,Ptychocheilus oregonensis

In vitro blood O2equilibrium curves were constructed at 9, 15, 18, and 21 °C from temperature-acclimated northern squawfish, Ptychocheilus oregonensis. At low [Formula: see text] (<1 mm Hg, 1 mm Hg = 133.32 Pa), P50s generally showed variable increases with temperature from 3.6 mm Hg at 9 °C to 8.7 mm Hg at 21 °C, leading to whole-blood temperature effects (ΔH, kilocalories per mole O2) ranging from a low +4.4 at 15–18 °C to a peak −21.2 at 18–21 °C. High-[Formula: see text] (7.6 mm Hg) conditions decreased blood pH and increased P50s at each temperature (Bohr factor). Bohr factors (Φ) ranged from −0.46 at 21 °C to −0.70 at 18 °C. Considered together, ΔH and Φ values suggest an optimal temperature range of 15–18 °C for hemoglobin O2loading and unloading in northern squawfish. Nonbicarbonate buffer values ranged from −10.04 at 21 °C to −14.13 at 9 °C. Overall, the high O2affinities and hyperbolic blood O2equilibrium curves of northern squawfish resemble those of other large cyprinids (e.g., common carp, Cyprinus carpio, tench, Tinca tinca, Sacramento blackfish, Orthodon microlepidotus) indicating a better ability to tolerate hypoxic environments than sympatric rainbow trout, Oncorhynchus mykiss. High northern squawfish blood O2capacities and Φs suggest high aerobic capacity, especially at temperatures <21 °C.

In vitro Interactions Between Oxygen and Carbon Dioxide Transport in the Blood of the Sea Lamprey (Petromyzon Marinus)

ABSTRACT In vitro experiments were carried out to examine the interactions between oxygen and carbon dioxide transport in the blood of the sea lamprey. Oxygen dissociation curves for whole blood obtained from quiescent lampreys had Hill numbers (NH) ranging from 1.52 to 1.89. The Bohr coefficient for whole blood was −0.17 when extracellular pH (pHe) was considered, but was much greater (−0.63) when red blood cell pH (pHi) was considered. The pHi was largely dependent on haemoglobin oxygen-saturation and the pH gradient across the red blood cell membrane was often reversed when was increased and/or was lowered. The magnitude of the increase in pHi associated with the Haldane effect ranged from 0.169 pH units at 2.9 kPa to 0.453 pH units at a of 0.2 kPa. Deoxygenated red blood cells had a much greater total CO2concentration than oxygenated red blood cells, but the nonbicarbonate buffer value for the red blood cells was unaffected by oxygenation. Plasma was not significantly different under oxygenated or deoxygenated conditions. Partitioning of CO2 carriage in oxygenated and deoxygenated blood supports recent in vivo observations that red blood cell CO2 carriage can account for much of the difference between arterial and venous blood. Together, the results also suggest that oxygen and carbon dioxide transport may not be tightly coupled in the blood of these primitive vertebrates. Finally, red cell sodium concentrations were dependent on oxygen and carbon dioxide tensions in the blood, suggesting that sodium-dependent ion transport processes may contribute to the unique strategy for gas transport in sea lamprey blood.

31P-NMR study of normoxic and anoxic perfused turtle heart during graded CO2 and lactic acidosis

We studied the effects of graded acidosis (both CO2 and lactic acid) and anoxia on intracellular pH (pHi) regulation, high-energy phosphates, and mechanical function of isolated perfused hearts of the turtle (Chrysemys picta bellii) at 20 degrees C using 31P-nuclear magnetic resonance (NMR) spectroscopy. During CO2 acidosis, anoxia had no effect on apparent nonbicarbonate buffer value (d[HCO3-]/dpHi = 71 and 89 mM/pH in normoxia and anoxia, respectively) or on pHi regulation (dpHi/dpHe = 0.52 and 0.43 in normoxia and anoxia, respectively, where pHe is extracellular pH). During normoxic lactic acidosis, dpHi/dpHe was similar to the values observed in CO2 acidosis and averaged 0.55 overall. During anoxic lactic acidosis, however, similar regulation occurred over only a narrow range of pHe, and then dpHi/dpHe increased to greater than 1.0 at pHe less than 7.1. Creatine phosphate (CP), calculated as the area of the NMR peak, fell more in response to normoxic CO2 acidosis than to normoxic lactic acidosis; in anoxia, the fall in CP was further increased but to similar extreme levels (10-20% of control) in both acid perfusions. Cardiac output and maximum rate of pressure development each fell during acidosis in similar fashion in all protocols, and the responses were similar in normoxic and anoxic hearts. Heart rate, in contrast, decreased during acidosis, but this effect was more pronounced when hearts were anoxic. We conclude that the effect of acidosis on cardiac function can depend on the type of acidosis imposed. Based on the heart's insensitivity to anoxia alone, we suggest that anoxia may normally depress function indirectly via its effect on intracellular acid-base state.

Time course of temperature effects on arterial acid-base status in Alligator mississippiensis

Temperature effects on extracellular acid-base status in terrestrial poikilotherms are well known. Comparatively less is known concerning the time course of acid-base responses to prolonged changes in temperature. In this study, two questions were addressed: (1) what is the time course of acid-base responses to a temperature change of 5 days duration in Alligator mississippiensis?; and (2) what are the relative contributions of respiratory vs metabolic (strong ion) processes to the acid-base response? The effects of acute (4–6 h) vs chronic (24–120 h) decreases in ambient temperature from 30 to 20°C were determined on arterial blood gases, acid-base status and plasma electrolyte concentrations ([Na +], [K +] and [Cl −]) in 7 awake alligators. In addition, in vivo and in vitro non-bicarbonate buffer values were determined in 6 anesthetized alligators. Acute decreases in temperature decreased Pa CO 2 (31 to 18 mmHg) and Pa O 2 (91 to 50 mmHg), increased pH (7.48 to 7.66), but had no significant effect on plasma bicarbonate concentration ([HCO 3 −]). Chronic exposure to 20°C had no further effect on any variable. dpH/dT ranged between −0.018 and −0.011 U/°C, over the duration of the experiment. Temperature did not alter [Na +] or [Cl −] at any time, and had only minor effects on [K +]. In vivo (11.5±0.7 mEq/L/pH unit) and in vitro (16.1±0.9 mEq/L/pH unit) buffer values were significantly different, but neither was affected by temperature. Temperature had no effect on the base excess (estimated with either in vitro or in vivo buffer values), nor on estimates of the strong ion difference. We conclude that pH changes during prolonged exposure to decrease temperature occur rapidly and can be explained by changes in Pa CO 2 , CO 2 solubility and pK′, without major contributions from metabolic or strong ion adjustments.

Heat Dissipation, Gas Exchange and Acid-Base Status in the Land Snail Oreoheux During Short-Term Estivation

ABSTRACT Within 4 days following entry into estivation, heat dissipation and oxygen consumption by the land snail Oreohelix spp. decreased by 83% compared to standard non-estivating rates. During both non-estivating and estivating conditions, the quantity of heat dissipated per mole of O2 consumed was indicative of a completely aerobic metabolism. This calorimetric-respirometric (C/R) ratio was –461 ±12 kJ mol−1O2 (S.E.M., N=5) under standard non-estivating conditions and –464±26kJmol−1O2 (N=4) during estivation. Respiratory exchange ratios reflected a primary dependence upon carbohydrate as a metabolic substrate during both states. Carbon dioxide retention occurred during the first 36h of estivation, resulting in an increase in hemolymph and a decrease in pH. The respiratory acidosis during short-term estivation was not compensated by elevation of hemolymph [HCO3−] above levels predicted from the in vitro non-bicarbonate buffer value of hemolymph. A brief period of rapid CO2 release, which caused hemolymph and pH to return to pre-estivation values, preceded the increase in O2 consumption during arousal. Exposure of nonestivating snails to 4.67 kPa (1 kPa=7.5mmHg) caused a rapid and fully reversible 50% suppression of respiration rate. The temporal nature of CO2 retention and release during entry into and arousal from estivation, and the suppression of O2 consumption by artificial hypercapnia, support the hypothesis that elevated or the resultant acidosis may contribute to metabolic suppression during estivation by land snails.

Determination of intracellular buffer values after metabolic inhibition by fluoride and nitrilotriacetic acid

Non-bicarbonate buffer values have been analysed in homogenates of squid mantle muscle and toad gastronemius muscle using the P CO 2 equilibration technique. Homogenate metabolism was inhibited by the addition of potassium fluoride and nitrilotriacetic acid. After and initial change in the concentration of organic phosphates a new metabolic equilibrium was achieved which was insensitive to pH changes during P CO 2 variations. Inaccurate measurements of homogenate buffer values could, thereby, be prevented. The accumulation of inorganic phosphate during the initial metabolic shift caused an increase in homogenate buffer values. Levels of unbound inorganic phosphate, which are effective in buffering, were estimated by enzymatic analysis in homogenate ultrafiltrates. Based on the evaluation of apparent dissociation constants pK′, and buffer values for fluoride and inorganic phosphate, the non-bicarbonate, non-phosphate i, (i:inorganic) buffer value of the tissue was calculated. The level of free inorganic phosphate in the resting tissue was considered for an estimate of the intracellular non-bicarbonate buffer value under control conditions.

Buffering characteristics of eel blood at the extreme conditions in the swimbladder

CO 2 binding in whole blood and true plasma of the eel was estimated by measuring CO 2 content and pH in blood aliquots equilibrated with various P CO 2 values over a wide range expected to occur in the swimbladder. Bicarbonate concentration, [HCO 3 −], was calculated using the CO 2 solubility coefficient, which was measured to average 50 μmol·L −1·Torr −1 (20°C). Buffer lines of non-bicarbonate buffers were obtained in plots of [HCPO 3 −] against pH, and non-bicarbonate buffer values, β NB, were obtained by curve. fitting. In the pH range 6.6–8.2, the buffer line for oxygenated whole blood was sigmoid, while that for deoxygenated blood increased its slope monotonously with increasing pH. The β NB for oxygenated blood displayed a maximum of about 8.6 mmol·L −1pH −1 at pH = 7.4 and dropped down to below 1 mmol·L −1·pH −1 at higher and lower pH. Similar shapes of the buffer lines were obtaned in true plasma; the [HCO 3 −] levels and β NB values were, however, somewhat higher than in whole blood. These data are useful fo assess the back-diffusion of CO 2 and HCO 3 − in the rete mirabile of the fish swimbladder and to estimate the effects CO 2 back-diffusion exerts on the counter-current enhancement of O 2 in the rete.

The Absence of Rapid Chloride/Bicarbonate Exchange in Lamprey Erythrocytes: Implications for CO2 Transport and Ion Distributions Between Plasma and Erythrocytes in the Blood of Petromyzon Marinus

ABSTRACT Carbon dioxide transport and ion distributions were examined in the blood of the lamprey, Petromyzon marinus. Over the range studied, the erythrocytes had the highest total CO2 content, followed by whole blood and true plasma. The nonbicarbonate buffer values were —37·0mequivl-1pHunit-1 for erythrocytes, —3·3mequivl-1pH unit-1 for whole blood and —0·1mequivl-1 pH unit-1 for true plasma. These results are in sharp contrast to the models of carbon dioxide transport in the blood of other vertebrates and are consistent with the view that chloride/bicarbonate exchange is virtually absent in agnathan erythrocytes. Protons are passively distributed in Petromyzon blood. However, the distribution ratio for chloride between plasma and erythrocytes was strikingly different from the distribution ratio for protons. In the absence of rapid chloride/bicarbonate exchange, the erythrocyte volume is relatively constant over the physiological pH range. A model is presented to explain carbon dioxide transport in lamprey blood which does not involve a rapid chloride/bicarbonate exchange mechanism on the erythrocyte membrane.

L-Lactate affects CO 2 transport in crustacean haemolymph

1. 1. A comparative study of carbon dioxide capacitance and buffer capacity of the haemolymph of the decapod crustaceans Ocypode saratan, Carcinus maenas and Palaemon elegans has been carried out. 2. 2. The relationship between the total carbon dioxide content of the blood ( C CO 2) and the pH of the haemolymph was examined. Values for [HCO 3 −] obtained by subtraction of the dissolved CO 2 from C CO 2 were compared with those derived using a constant p K1 value according to the Henderson-Hasselbalch equation. 3. 3. In Carcinus, but not in Palaemon the use of a constant p K1 over-estimated the non-bicarbonate buffer value and under-estimated CO 2 capacitance at high pH. 4. 4. Results obtained for Ocypode were similar to those obtained for Carcinus haemolymph except that increasing the l-lactate concentration in the haemolymph resulted in a marked reduction of the buffer value as estimated by the Henderson-Hasselbalch equation. This was in contrast to the constant buffer value calculated using measured values. 5. 5. Differences between the calculated and observed buffer value of the haemolymph could be attributed to a pH dependence of p K1. 6. 6. The possibility that haemocyanin-CO 2 binding contributes to the CO 2 capacitance of the haemolymph in a manner which is not predicted by the Henderson-Hasselbalch equation and that this component is dependent on pH and possibly on the lactate concentration is discussed.

The effect of prolonged hypoxia on striated muscle pH regulation.

The extracellular acid-base status of mammals exposed to low inspired PO2 for prolonged periods of time has been studied extensively (Bouverot, 1985). However, less is known regarding the intracellular acid-base status during prolonged hypoxia. In a previous study we observed that the intracellular pH of striated muscles of rats subjected to 3 weeks of hypobaric hypoxia was not significantly different from that of normoxic rats (Gonzalez and Clancy, 1986b). However, when the cells were acid-loaded by rendering the animals hypercapnic, the decreases in intracellular pH of tibialis anterior, quadriceps and diaphragm were significantly less than in hypercapnic normoxic rats. The magnitude of cell pH regulation, as measured by the apparent non-bicarbonate buffer value, ranged from 3 (tibialis anterior) to 12 (diaphragm) times greater in the hypobaric hypoxic than the normoxic animals. This augmentation of striated muscle pH regulation during hypercapnia in hypobaric hypoxic rats could result from increased physicochemical buffering and/or increased efflux of proton equivalents. The question arises as to whether augmentation of these cellular pH- regulating mechanisms resulted from intrinsic changes in the cell interior and/or the sarcolemma engendered by the prolonged hypoxia or whether extracellular factors which enhance these cellular pH-regulating mechanisms were increased to a greater extent in the hypercapnic hypoxic rats than in the hypercapnic normoxic rats. To resolve this question cell pH regulation of diaphragms from prolonged hypoxic and normoxic rats was studied under in vitro conditions. Under these conditions the contribution of extracellular factors to cell pH regulation was the same in diaphragms from hypoxic and normoxic rats. Therefore, any difference in cell pH regulation should be attributable to intrinsic changes in one or more of the cell pH-regulating mechanisms.

Physiological Responses of the Rock Crab Cancer irroratus (Say) to Environmental Hyperoxia. I. Acid-Base Regulation

Hemolymph acid-base and ion levels and acidic equivalent exchange with the experimental water were monitored in rock crabs (Cancer irroratus Say) exposed to control normoxia (PIo2 ≃ 150 torr), 48 h of hyperoxia (Po2 ≃ 512 torr), and 24 h of subsequent normoxic recovery. Hemolymph CO₂ binding was assessed in vitro on oxygenated and deoxygenated samples. Settled normoxic crabs had the following acid-base status: pH—8.01; Pco2—2.1 torr, and [HCO₃⁻]-9.3 meq·liter⁻¹ and appeared to be in proton balance with the experimental water. Within 6 h of exposure to hyperoxia, the hemolymph exhibited a significant acidosis (0.15 pH units) that was due to respiratory CO2 (Pco2 increased by 50%). This acid-base imbalance was restored between 6 and 24 h by a 50% increase in [HCO₃⁻]. This coincided with a 13-fold increase in excretion of acidic equivalents (primarily titratable) into the experimental water that continued for up to 10 h. Numerically, there was excellent correspondence between acid efflux and the magnitude of the extracellular proton load. Although crabs continued to hypoventilate, preexperimental Pco2 was reestablished by 24 h, suggesting that the original hypercapnia had been due to a perfusion limitation. Between 24 and 48 h, circulating [HCO₃⁻] was also restored to resting levels accompanied by an equivalent net base excretion into the bathing medium. When normoxia was reinstated, hemolymph exhibited a reduction in Pco2 plus a further reduction in [HCO₃⁻] at constant Pco2, making pH alkalotic. The base efflux that persisted into the recovery period significantly exceeded that required to restore extracellular acid-base balance. It is suggested that this base load originated intracellularly and that the reduced cardiac output during hyperoxia limited its removal into the hemolymph. Circulating lactate exhibited a similar washout effect during recovery. Hemolymph of C. irroratus had a low nonbicarbonate buffer value (β) corresponding to reduced protein concentration. However, the Haldane effect, which theoretically could account for the release of 2.13 mmol CO2 per mmol O2 bound, was larger than observed in other decapods.

Blood osmolality in vitro: dependence on base addition, buffer value, and temperature.

Blood osmolality (Osm) increases with PCO2 because of CO2 absorption. The influences of NaOH addition, equilibration temperature, and hemoglobin concentration on these respiratory changes of Osm were measured by freezing-point determination in true plasma. Addition of NaOH increases Osm by 2 mosmol X kg H2O-1 X mmol base-1 X l at constant PCO2 due to the osmotic effects of Na+ and produced bicarbonate. Respiratory compensation of the pH change further increases Osm. This contrasts to the respiratory compensation of the osmolar disturbance caused by fixed acid. Raising the equilibration temperature reduces Osm by 0.5 mosmol X kg H2O-1 X degrees C-1 at constant pH mainly caused by a lower absorption coefficient for CO2 and changed pK value for H2CO3. The slope of the linear regression lines between Osm and pH during CO2 equilibration increases with hemoglobin; the value of the quotient delta Osm/delta pH depends directly on the nonbicarbonate buffer value. The use of this quotient for the estimation of the mean nonbicarbonate buffer value of the whole body is suggested. The osmotic effects of therapeutic base infusion should be regarded with caution.