Determination of rat brain buffering in vivo by 31P-NMR

Buffering capacity of most tissues is composed of both rapid and slow phases, the latter presumably due to active acid extrusion. To examine the time course of brain buffering the brain pH of Sprague-Dawley rats was measured using 31P-nuclear magnetic resonance. The effect on brain pH of 30- or 58-min exposures to 20% CO2 followed by 30- or 38-min recovery periods, respectively, was studied. Brain pH reached its lowest value after a 15-min exposure to elevated CO2, thereafter slowly and steadily increasing. During recovery brain pH rose rapidly in the first 5 min exceeding control brain pH by 0.08 pH units. Brain pH fell during the next 30 min despite increases in blood pH and decreases in blood CO2 tension. Calculated intrinsic brain buffering rose steadily threefold during the last 40 min of CO2 exposure and during the final 30 min of recovery. These data show that in rat brain there is a temporally late buffering process, most likely active acid extrusion, requiring greater than 30 min for full activation and at least 30 min for discontinuation.     

Electrode and cuvette for rapid continuous CO2 tension and pH measurement in blood

An electrode and cuvette system has been developed for the continuous and rapid measurement of either blood CO2 tension or pH. The CO2 electrode consists of a 1.5-mm-diameter flat-tip glass pH electrode covered by a film of carbonic anhydrase solution, over which a 25-micron-thick dimethyl silicone membrane is attached. Porous ceramic filled with 20% polyacrylamide, equilibrated with a salt solution, serves as a salt bridge between a Ag-AgCl reference electrode and the pH electrode surface. The electrode is housed in a four-port cuvette assembly. Blood from a vessel of interest is delivered to the cuvette by means of an occlusive roller pump. The cuvette maintains the electrode and blood at a constant temperature and directs a continuous jet of blood against the electrode surface. The cuvette also allows for easy and frequent calibration of the electrode with either gas or liquid standards. The 90% response time of the CO2 electrode is 3.0 s for liquids and 1.3 s for gases. Removal of the dimethyl silicone membrane and carbonic anhydrase film yields a pH electrode that can continuously measure blood pH with a 90% response time of 1.6 s.     

Effects of temperature change on acid-base regulation in skipjack tuna (Katsuwonus pelamis) blood

The effects of temperature change (in vitro) on acid-base balance of skipjack tuna blood were investigated. By examining the relationship between blood pH and temperature (in vitro) under conditions of constant CO2 tension (open system), it was observed that dpH/dT = -0.013 U/degrees C. This value falls well within the range of in vivo values reported for other ectothermic vertebrates, and is only slightly different than results obtained in vitro under conditions of constant CO2 content (closed system; dpH/dT = -0.0165 U/degrees C). It is concluded that changes in pH following temperature changes can be accounted for solely by the passive, in vitro behaviour of the chemical buffer system found in the blood, so that active regulatory mechanisms of pH adjustment need not be postulated for skipjack tuna.     

Effect of acute hypercapnia on limb muscle contractility in humans

The effect of acute hypercapnia on skeletal muscle contractility and relaxation rate was investigated. The contractile force of fresh and fatigued quadriceps femoris (QF) and adductor pollicis (AP) was studied in normal humans by use of electrical stimulation. Maximum relaxation rate from stimulated contractions was measured for both muscles. Acute hypercapnia led to a rapid substantial reduction of contraction force. The respiratory acidosis after 9% CO2 was breathed for 20 min [mean venous blood pH 7.26 and end-tidal PCO2 (PETCO2) 65.1 Torr] reduced 20- and 100-Hz stimulated contractions of QF to 72.8 +/- 4.4 and 80.0 +/- 5.1% of control values, respectively. After 8 and 9% CO2 were breathed for 12 min, AP forces at 20- and 50-Hz stimulation were also reduced. Twitch tension of AP was reduced by a mean of 25.5% when subjects breathed 9% CO2 for 12 min [mean arterialized venous blood pH (pHav) 7.25 and PETCO2 66 Torr]. Over the range of 5% (pHav 7.38 and PETCO2 47 Torr) to 9% CO2, there was a linear relationship between twitch tension loss and pHav, arterialized venous blood PCO2, and PETCO2. Acute respiratory acidosis (mean PETCO2 61 Torr) increased the severity of low-frequency fatigue after intermittent voluntary contractions of AP. At 20 min of recovery, twitch tension was 63.2 +/- 13.4 and 46.8 +/- 16.4% of control value after exercise breathing air and 8% CO2, respectively. Acute hypercapnia (mean PETCO2 65.1 and 60.5 Torr) did not alter the maximum relaxation rate from tetanic contractions of fresh QF and from twitch tensions of AP.     

Culture pH, CO2 tension, and cell division in Euglena gracilis Z.

Growth characteristics of Euglena gracilis Z as functions of culture pH, CO2 tension, temperature, and lighting regime were investigated. The results are consistent with the possibility that cell division is preceded by a lowered intracellular pH. Also consistent with this possibility is the finding that division rhythmicity can be induced by periodic changes in CO2 tension. It is suggested that the rhythmicity is induced by changes in intracellular pH produced by carbonic acid.     

The effect of acid--base changes on skeletal muscle twitch tension.

The effects of acid--base alterations produced by changing bicarbonate (metabolic type), carbon dioxide tension (respiratory type), or both bicarbonate and carbon dioxide tension (compensated type) on skeletal muscle twitch tension, intracellular pH, and intracellular potassium were studied in vitro. Hemidiaphragm muscles from normal rats and rats fed a potassium-deficient diet were used. Decreasing the extracellular pH by decreasing bicarbonate or increasing CO2 in the bathing fluid produced a decrease in intracellular pH, intracellular K+, and muscle twitch tension. However, at a constant extracellular pH, an increase in CO2 (compensated by an increase in bicarbonate) produced an increase in intracellular K+ and twitch tension in spite of a decrease in intracellular pH. The effect on twitch tension of the hemidiaphragms showed a rapid onset, was reversible, persisted until the buffer composition was changed, and was independent of synaptic transmission. It is concluded that the twitch tension of the skeletal muscle decrease with a decrease in intracellular K+. The muscle tension also decreases with an increase in the ratio of intracellular and extracellular H+ concentration. However, there is no consistent relationship between muscle tension and extracellular or intracellular pH. The muscle tension of the diaphragms taken from K+-deficient rats is more sensitive to variations in CO2, PH, and bicarbonate concentration of the medium than that of the control rat diaphragms.     

Effect of external factors on phototaxis of Chlamydomonas reinhardtii

1. The effects of carbon dioxide, oxygen and pH on the inversion intensity of phototaxis of Chlamydomonas reinhardtii have been investigated. 2. With decreasing with CO2 tension the inversion intensity is decreased. 3. The gassing with CO2 can be substituted by hydrogencarbonate only to a small extent (20%). On the other hand, the effect of decreasing CO2 tension can be prevented also only in part by adjusting the pH to about 6.5-7.0. Thus the effect of CO2 on the inversion intensity of phototaxis is obviously a composite of a true CO2 effect and an effect of the concomitant pH change. 4. Oxygen has only a slight effect. In presence of oxygen (air) the phototactic reaction values are somewhat lower than in its absence. 5. Under certain conditions circadian rhythms seem to be initiated by changing oxygen as well as CO2 tensions. 6. Based on these results some contradictory results of older investigations are discussed.     

Exercise-induced hypercapnia in the horse

The effects of exercise intensity and duration on blood gases in thoroughbred horses were studied to characterize the apparent exercise-induced failure in pulmonary gas exchange that occurs in these animals. In response to 2 min of exercise, arterial CO2 tension (PaCO2) decreased in mild and moderate exercise, returned to normocapnic levels in moderate to heavy exercise, and rose 5-10 Torr above resting values during very heavy exercise when CO2 production (VCO2) exceeded 20 times the resting value, and mixed venous CO2 tension approximated 140 Torr. Exercise-induced hypoxemia occurred at the onset of heavy exercise and was associated with the absence of a hyperventilatory response and an alveolar-arterial PO2 difference that increased four to six times above rest with very heavy exercise. PaCO2 was related to VCO2 but not fb, as changes in breathing frequency (fb) of 8-20 breaths/min at comparable VCO2 did not affect PaCO2. Prolonging very heavy exercise from 2 to 4 min caused a severe metabolic acidosis (arterial pH less than 7.15) and hypoxemia was maintained; however, CO2 was no longer retained, as PaCO2 gradually fell to below resting levels, due to an increased tidal volume at constant fb. We conclude that a truly compensatory hyperventilation to very heavy exercise in the horse is not achieved because of the excessive volumes and flow rates required by their extraordinarily high VCO2 and VO2. On the other hand, the frank CO2 retention during short-term high-intensity exercise occurs even though the horse is not apparently mechanically obligated to tolerate it.     

The effect of temperature on the contractile response of cardiac muscle from two frog species, Rana temporaria and Xenopus laevis, at two different calcium concentrations-II. At pH 7.0 and pH 6.6

1. Mechanical parameters were recorded from paced ventricular cardiac muscle strips of two amphibian species. Rana temporaria and xenopus laevis. 2. The strips contracted at pH 6.6 and 7.0 with either 1.6 or 3.0 mM Ca2+ while the temperature was changed from 5 to 30 degree C. 3. In the hearts of R. temporaria, but not in those of X. laevis, in increased Ca2+ concentration at low pH could compensate for the effect of lowered pH. 4. It is concluded that during high CO2 tension and/or low pH the myocardium of X. laevis was more temperature dependent, less Ca2+ dependent in the Ca2+ range examined and less sensitive to the pH and/or the CO2 tension during physiological temperature and Ca2+ concentration, than the cardiac muscle of R. temporaria.     

Acetazolamide teratogenesis: interaction of maternal metabolic and respiratory acidosis in the induction of ectrodactyly in C57BL/6J mice

Exposure of C57BL/6J mice to 20% CO2 for 8 hours on day 10 of gestation has been shown to produce right-sided postaxial forelimb ectrodactyly in 23% of the offspring. Carbon dioxide exposure produces a dramatic increase in maternal plasma CO2 accompanied by an inevitable decrease in plasma pH, both of which appear to be involved in the induction of ectrodactyly. However, the low incidence of ectrodactyly associated with NH4Cl-induced metabolic acidosis suggests that the primary teratogenic factor in respiratory acidosis is elevated CO2 tension. This conclusion is supported by the observation that moderation of maternal plasma pH in the face of sustained elevated PCO2 fails to reduce the incidence of ectrodactyly; moreover, there is a strong correlation between maternal serum CO2 content and the incidence of ectrodactyly.     

Antagonistic interaction between oxygenation-linked lactate and CO2 binding to human hemoglobin

Oxygen binding to hemoglobin (Hb) depends on allosteric effectors (CO(2), lactate and protons) that may increase drastically in concentration during exercise. The effectors share common binding sites on the Hb molecules, predicting mutual interaction in their effects on Hb (de)oxygenation. We analysed the effects of lactate and CO(2), separately and in combination, on O(2) binding of purified human Hb at 37 degrees C and physiological pH and chloride values. We demonstrate pH-dependent, inhibitory interactions between lactate binding and CO(2) binding (carbamate formation); at pH 7.4, physiological CO(2) tension ( approximately 43 mm Hg) reduced lactate binding more markedly ( approximately 75%), than lactate (50 mM) inhibited carbamate formation ( approximately 25%). In contrast to previous studies on blood and Hb solutions, we moreover find that added lactate neither 'reverses' oxylabile carbamate formation (resulting in lower carbamate levels in deoxyHb than in oxyHb) nor exerts greater allosteric effects on Hb-O(2) affinity than equal increases in chloride ion concentrations.     

The effects of pH on the sodium-withdrawal contractures evoked from frog atrial trabeculae

Contractures can be evoked from frog atrial trabeculae by raising extracellular pH in Na-free fluid after the spontaneous relaxatin of the tension evoked by removing extracellular Na+. These alkalinity contractures are unaffected by local anaesthetics but are absent following a brief perfusion with Ca-free fluid but are partially inhibited by local anaesthetics. Perfusion by solution containing a high Pco2 fails to induce contractures. The relationship between [Na]0 and tension is altered by changing extracellular pH; acidic fluids below pH 6.0 reduce tension and alkaline fluids increase tension over the whole range. NH4Cl has the same effect as alkaline fluids while CO2-containing fluid has no effect. These results can be interpreted if the effects of variation of extracellular pH, on Na-withdrawal contractures, are mediated by changes in intracellular pH which in turn affect the Ca-sensitivity of the contractile proteins.     

CO2 induced acute respiratory acidosis and brain tissue intracellular pH: a 31P NMR study in swine

High concentration carbon dioxide (CO(2)) is used to promote pre-slaughter anaesthesia in swine and poultry, as well as short-lasting surgical anaesthesia and euthanasia in laboratory animals. Questions related to animal welfare have been raised, as CO(2) anaesthesia does not set in momentarily. Carbon dioxide promotes anaesthesia by lowering the intracellular pH in the brain cells, but the dynamics of the changes in response to a high concentration of CO(2) is not known. Based on (31)P NMR spectroscopy, we describe CO(2)-induced changes in intracellular pH in the brains of five pigs inhaling 90% CO(2) in ambient air for a period of 60 s, and compare the results to changes in arterial blood pH, P(CO2), O(2) saturation and HCO(3)(-) concentration. The intracellular pH paralleled the arterial pH and P(CO2) during inhalation of CO(2); and it is suggested that the acute reaction to CO(2) inhalation mainly reflects respiratory acidosis, and not metabolic regulation as for example transmembrane fluxes of H(+)/HCO(3)(-). The intracellular pH decreased to approximately 6.7 within the 60 s inhalation period, and the situation was metabolically reversible after the end of CO(2) inhalation. The fast decrease in intracellular pH supports the conclusion that high concentration CO(2) leads to anaesthesia soon after the start of inhalation.     

Derivation of CO2 output from oscillations in arterial pH

Theory predicts that the rate of rise of the oscillation in arterial CO2 partial pressure (PaCO2) is linearly dependent on CO2 flux from venous blood to alveolar gas. We have measured, in the anesthetized cat, CO2 output (VCO2) and oscillations in arterial pH. The pH signal was differentiated to give the maximum rate of fall of pH on the downstroke of the oscillation (dpH/dt decreases max). Since oscillations in pH are due to oscillations in arterial PCO2, dpH/dt decreases max was considered to be equivalent to the maximum rate of rise of the PCO2 oscillation. VCO2 was increased by ventilating the intestines with CO2 and by the intra-arterial infusion of 2,4-dinitrophenol. VCO2 was decreased by filling the intestines with isotonic tris(hydroxymethyl)methylamine buffer. The maximum range of VCO2 covered was 7.8-51 ml/min, and the mean range was from 13.6 +/- 1.3 to 29.7 +/- 1.6 (SE) ml/min. Although CO2 loading produced a small rise and CO2 unloading a small fall in mean PaCO2, the changes were not statistically significant, so that overall the response was close to isocapnia. Over the limited range of VCO2 studied there was a highly significant linear association between dpH/dt decreases max and VCO2 which supports the contention that the slope of the upstroke of the PaCO2 oscillation is determined by the CO2 flux from mixed venous blood to alveolar gas. As such this slope is a potential chemical signal linking ventilation to CO2 production.     

Changes in Renal Tubular and Glomerular Functions and Biological Acid–Base Balance after Soil Replacement in Cd-Polluted Rice Paddies Calculated with a General Linear Mixed Model

Using a general linear mixed model, we conducted a 10-year follow-up investigation of 29 persons requiring observation in the cadmium (Cd)-polluted Kakehashi River basin to determine serial changes in biological parameters after removal of Cd-polluted soil present in rice paddies. In particular, we investigated changes in urinary Cd, urinary total protein, biological acid-base balance based on arterial blood pH, carbon dioxide tension (Pa(CO2)), base excess (BE), serum chloride ion (SCl(-)), and renal glomerular function based on serum creatinine (SCr) and creatinine clearance (C(Cr)). In both sexes, urinary Cd concentrations decreased and total protein concentrations increased with increasing number of years elapsed, with the partial regression coefficients statistically significant in the women. Partial regression coefficients showed positive values for SCl(-), with statistical significance in both sexes. The value for Pa(CO2) was significantly negative in men. Given the serial changes in arterial blood pH, Pa(CO2), BE, SCl(-), it is suggested that the biological acid-base balance will progress to metabolic acidosis with hyperchloremia. Moreover, glomerular dysfunction as indicated by an increase in SCr and a decrease in C(Cr) will continue to progress even after soil replacement. Interventions such as soil replacement appear to be too late to prevent progressive renal failure in Cd-exposed populations.     

Colloid osmotic pressure changes in human whole blood and separated plasma in vitro with changes in CO2 content and pH

Colloid osmotic pressure (COP) and pH were measured on the true plasma of human blood from five subjects tonometered with different concentrations of carbon dioxide. Measurements were also made on their separated plasma. COP (mmHg) of true plasma obtained from tonometered whole blood varied in proportion to the bicarbonate concentration (mEq/l): COP = 0.056 [HCO3-] + 23.3. In separated plasma, as CO2 concentration increased, COP decreased as pH decreased: COP = 1.99 (pH) + 11.0. When the change in COP due to the change in pH was subtracted from the observed change of COP due to CO2 exposure of whole blood, the difference was the change of COP due to the shift of fluid between plasma and red cells: COP adjusted for pH = 0.131 [HCO3-] + 21.5. The COP values of tonometered whole blood and separated plasma are taken to be equal at a pH of 7.40 (at the mixed venous point). The change in COP, adjusted for pH, for a given change in pCO2 is in keeping with the amount of fluid shift calculated from the measured changes in hematocrit and plasma protein concentration. An error in a previous paper (Kakiuchi et al., J. appl. Physiol. 44, 474-478, 1978) had led to an overestimation of the COP change from the exposure of whole blood to CO2 in vitro.     

Apparent diffusion limitations on branchial CO2 transfer are revealed by severe experimental anaemia in brown bullhead (Ameiurus nebulosus)

In this study, the rapid (within 2 h) effects of acute anaemia on blood gas and acid-base status, as well as cardiorespiratory variables, were examined in brown bullhead (Ameiurus nebulosus). Anaemia was induced by blood withdrawal coupled to volume replacement with saline. Lowering haematocrit from the control value of 23.5+/-1.0% (mean+/-S.E.M.; N=37) to 5.9+/-0.3% (N=37) resulted in a significant increase (by 2.63+/-0.51 torr; N=7) in arterial CO(2) tension (PaCO(2)) over the subsequent 2-h period in the absence of a change in arterial O(2) tension (PaO(2)). Treatment with bovine carbonic anhydrase (CA) reduced the extent of the PaCO(2) increase to the point where it was not statistically significant. In both control and CA-treated fish, arterial pH decreased during acute anaemia; the acidosis was of mixed respiratory and metabolic origin in control fish and primarily metabolic in CA-treated fish. Inducing anaemia caused increases in both cardiac output (V*b) and heart rate that were similar in control and CA-treated fish. Experimental elevation of V*b equivalent to that observed during anaemia, but in the absence of lowered haematocrit, increased PaCO(2) significantly by 1.49+/-0.74 to 1.64+/-0.78 torr (N=5) without affecting PaO(2). These findings suggest that CO(2) excretion in bullhead, as in rainbow trout, is effectively diffusion-limited, and that approximately half of the increase in PaCO(2) measured during the initial 2 h of anaemia results from the impact of increased blood flow (hence decreased gill transit time) in a diffusion-limited system.     

The CO2/pH ventilatory drive in fish.

That ventilation in fish is driven by O2 has long been accepted. The O2 ventilatory drive reflects the much lower capacitance of water for O2 than for CO2, and is mediated by O2 receptors that are distributed throughout the gill arches and that monitor both internal and external O2 levels. In recent years, however, evidence has amassed in support of the existence of a ventilatory drive in fish that is keyed to CO2 and/or pH. While ventilatory responses to CO2/pH may be mediated in part by the O2 drive through CO2/pH-induced changes in blood O2 status, CO2/pH also appear to stimulate ventilation directly. The receptors involved in this pathway are as yet unknown, but the experimental evidence available to date supports the involvement of branchial CO2-sensitive chemoreceptors with an external orientation. Internally-oriented CO2-sensitive chemoreceptors may also be involved, although evidence on this point remains equivocal. In the present paper, the evidence for a CO2/pH-keyed ventilatory drive in fish will be reviewed.     

Carbon dioxide pressure-concentration relationship in arterial and mixed venous blood during exercise.

To calculate cardiac output by the indirect Fick principle, CO(2) concentrations (CCO(2)) of mixed venous (Cv(CO(2))) and arterial blood are commonly estimated from PCO(2), based on the assumption that the CO(2) pressure-concentration relationship (PCO(2)-CCO(2)) is influenced more by changes in Hb concentration and blood oxyhemoglobin saturation than by changes in pH. The purpose of the study was to measure and assess the relative importance of these variables, both in arterial and mixed venous blood, during rest and increasing levels of exercise to maximum (Max) in five healthy men. Although the mean mixed venous PCO(2) rose from 47 Torr at rest to 59 Torr at the lactic acidosis threshold (LAT) and further to 78 Torr at Max, the Cv(CO(2)) rose from 22.8 mM at rest to 25.5 mM at LAT but then fell to 23.9 mM at Max. Meanwhile, the mixed venous pH fell from 7.36 at rest to 7.30 at LAT and to 7.13 at Max. Thus, as work rate increases above the LAT, changes in pH, reflecting changes in buffer base, account for the major changes in the PCO(2)-CCO(2) relationship, causing Cv(CO(2)) to decrease, despite increasing mixed venous PCO(2). Furthermore, whereas the increase in the arteriovenous CCO(2) difference of 2.2 mM below LAT is mainly due to the increase in Cv(CO(2)), the further increase in the arteriovenous CCO(2) difference of 4.6 mM above LAT is due to a striking fall in arterial CCO(2) from 21.4 to 15.2 mM. We conclude that changes in buffer base and pH dominate the PCO(2)-CCO(2) relationship during exercise, with changes in Hb and blood oxyhemoglobin saturation exerting much less influence.