Robert Blake Reeves

An imidazole alphastat hypothesis for vertebrate acid-base regulation: Tissue carbon dioxide content and body temperature in bullfrogs☆☆☆

A chemically defined model system consisting of two weak acid-conjugate base buffer pairs, one of which is carbonic acid-bicarbonate and the other the imidazole group of protein histidine residues, will quantitatively account for the change in blood pH and PCO2 temperature observed in closed in vitro blood samples at constant carbon dioxide content (Rosenthal pH-temperature curve). Application of the same equations to open systems in the steady state, poikilotherm vertebrates in vivo, will also quantitatively describe the temperature dependence of blood pH and PCO2, in frogs, toads and turtles. A necessary condition for this application, constant CO2 content of blood over wide body temperature excursions in vivo, is shown to hold for bullfrogs. The regulation of ventilation in the in vivo animal can be viewed as one in which blood PCO2, is regulated to maintain a constant fractional dissociation (alpha) of imidazole groups. Data from striated and cardiac muscle suggest that alpha imidazole values in these compartments are independent of changes in body temperature as well. It is proposed that acid-base regulation in all vertebrates is consistent with primary regulation of alpha imidazole resulting in a stable OH − /H + ratio and the observed change in blood pH with temperature, dpH/dT = −0.015 to 0.020 u/°C.

Intracellular ph in cold-blooded vertebrates as a function of body temperature

Intracellular pH (pHi) was measured in vivo in tissue of frogs (Rana catesbeiana) and turtles (Pseudemys scripta) using the DMO technique. Animals were permitted 3-8 days to come to a new steady-state body temperature (Tb) which ranged 5-32 degrees C. Least squares regression equation for pHi data are: frog blood, 8.184-0.0206 Tb; frog striated muscle, 7.275-0.0152 Tb; turtle blood, 8.092-0.0207Tb; turtle muscle, 7.421-0.0186 Tb; turtle heart, 7.452-0.0122 Tb; turtle liver, 7.753-0.0233 Tb; turtle esophageal smooth muscle, 7.513-0.0141 Tb. Only turtle cardiac muscle deltapHi/deltaT was significantly different from deltapH/deltaT of blood. Results have been interpreted in terms of protein charge state alterations; in the physiological pH range, histidine residues of proteins are the principal dissociable groups (HPr+ = H+ + Pr) affected by pHi and Tb changes. Constancy of protein charge state can be assessed by monitoring alpha imidazole, alphaIM = Pr/(HPr+ + Pr). A uniform pKIM of 6.85 (20degreesC) and a deltaHO of 7 kcal/mol are assumed in calculating alphaIM. Intracellular alphaIM is preserved in the tissues studied as body temperature changes. These results indicate that ectotherm acid-base balance, alphastat control, regulates not only extracellular blood proteins, but also intracellular compartment proteins in such a way as to preserve functions dependent upon protein net charge states.

Model studies of intracellular acid-base temperature responses in ectotherms

Measurements of intracellular pH (pHi) in air-breathing ectotherms have only been made in the steady state; these pHi indicate that protein charge state, measured as alpha imidazole (alphaIM), the fractional dissociation of protein histidine imidazole groups, is preserved when ectotherm tissues change temperature in vivo, with related changes in pHi and PCO2. In partial answer to the question of how such tissues are able to avoid disrupting transients to functions sensitive to protein charge states, model studies were carried out to assess the passive intracellular buffer system response to a combined change in body temperature and CO2 partial pressure as occurs in vivo in these species. The cell compartment was modeled as a closed volume of ternary buffer solution, containing protein imidazole (50 mM/1); phosphate (15 mM/1) and CO2-bicarbonate buffer components, permeable only to CO2 and permitted no change in buffer base. Excursions from a steady-state non-equilibrium pHi were computed to a step-change in temperature/PCO2. Computations for frog (Rana catesbeiana) striated muscle show that the calculated pHi response on the basis of estimated composition and concentration of cell buffer components, moves along the curve describing the steady-state temperature relationship. No transient away from steady-state alphaIM and carbon dioxide content need be postulated. Applications to turtle (Pseudemys scripta) striated muscle are also explored. These calculations show that ectotherm cells may be capable of responding without appreciable time for adaptation to intracellular acid-base state changes incurred by sudden alteration of body temperature in vivo, given the observed adjustments of blood PCO2 with temperature.

CO uptake kinetics of red cells and CO diffusing capacity

The rate at which CO displaces oxygen from combination with hemoglobin in intact red cells was measured spectrophotometrically in whole blood thin films that minimize unstirred layer extra-cellular diffusion barriers. A step-change was made in CO tension from zero to one of four values (2, 7, 21 and 70 Torr) during a constant background of one of eight O2 tensions (0, 40, 70, 100, 153, 214, 285 and 428 Torr). For PO2 greater than 100 Torr measured red cell initial CO uptake rates were compared with calculated rates at the same PCO-PO2 based on the Gibson-Roughton rate equation (Gibson and Roughton, Proc. R. Soc. B 143: 310-334, 1955) for a well mixed Hb solution. Measured CO uptake rates expressed as initial rate of saturation change (delta S/delta t) quantitatively followed the theoretical rate equation (time in seconds) [sequence: see text] These measurements provide new values for theta CO, the specific conductance of whole blood (ml.min-1.Torr-1; PCO, PO2 in Torr): [sequence: see text] These results signify that in vivo, in normoxia and hyperoxia, red cell CO uptake rate is wholly reaction rate limited and that pulmonary capillary red cell CO diffusion equilibrium is rapidly achieved. The Bohr-Krogh assumption that red cell PCO = 0 during CO uptake is untrue.

Oxygen affinity and equilibrium curve shape in blood of chicken embryos

Oxygen equilibrium curves of blood from 4- to 18-day chicken embryos were investigated at 38 degrees C. Curves were recorded at the PCO2 measured in the air cell of each egg. Since arterial PCO2 is known to closely approximate that of the air cell, these curves reflect in vivo arterial pH. Curves were also recorded at a second, higher PCO2, allowing calculation of the curve at estimated venous PCO2 and of physiological curves by interpolation. Half saturation PO2 of physiological curves increased from 38 Torr at 4 days to 52 Torr at 8 days, and then decreased to 31 Torr at 18 days. Increasing blood oxygen affinity late in development favors oxygen loading at the falling PO2 that exists at the chorioallantoic surface, while higher affinity before 8 days may be related to diffusion resistance of the inner shell membrane early in incubation. In blood from 4- to 6-day embryos, the Hill coefficient, nH, of curves at air cell PCO2 increased from 1.5 at oxygen saturation 0.1 to 6.5 at saturation 0.85. After 6 days, nH steadily increased at low saturation and decreased at high saturation. By 18 days, nH varied only from 2 to 3.4. A biphasic equilibrium curve shape (hump at the low end of the oxygen equilibrium curve) developed in 4- and 5-day embryo blood after a period of storage on ice, and was accentuated if some of the cells were intentionally lysed.

A rapid micro method for obtaining oxygen equilibrium curves on whole blood

A method for recording complete dynamic oxygen equilibrium curves (O2EC) from microliter samples of whole blood is described. The blood sample is compressed into a thin film between two 6-micron thick Teflon membranes in order to promote rapid gas exchange with gas volume surrounding the membranes. Oxygen tension of the gas volume around the blood film is charged at a controlled rate from zero to ca. 100 Torr by a specially designated gas-exchanging cuvette. Saturation of red cell hemoglobin in the film is measured by dual-wavelength spectrophotometry using Soret wavelengths of 430-453 nm. Gas volume oxygen tension is measured with a Teflon membrane covered oxygen cathode. Full saturation is secured by introducing an oxygen mixture whose PO2 exceeds 650 Torr. A single O2EC can be run in about 4 min at 37 degrees; multiple O2EC can be recorded from the same blood film. Each curve is run isocapnically at a preselected carbon dioxide tension. Blood film pH is calculated from the CO2 partial pressure and an independently determined buffer line. Data are presented to show that normal standard curve for man as determined with this blood-film method and those determined by other methods are equivalent.

Oxygen affinity of blood of adult domestic chicken and red jungle fowl.

Respiratory properties of blood from adult domestic chicken (White Leghorn) and red jungle fowl (Gallus gallus, ancestor of domestic breeds) at 41 degrees C were investigated. Oxygen affinity was the same in blood of chicken and jungle fowl (P0.5 46.7 Torr at pH 7.5, 41 degrees C, PCO2 about 30 Torr). The Hill coefficient, nH, increased from 2 at oxygen saturation 0.1 to a maximum of 4.11 at saturation 0.8. Leghorn fixed acid and CO2 Bohr coefficients were -0.51 and -0.53, with little variation over the saturation range 0.15-0.95, indicating negligible specific CO2 effect. An nH value of greater than 4 may indicate polymerization of deoxyhemoglobin, comparable to that which occurs in sickle cell hemoglobin. A biphasic equilibrium curve shape (hump at the low end of the oxygen equilibrium curve) was noted in blood having some degree of hemolysis. Factors that may have contributed to the differences between previous investigations of chicken oxygen affinity are discussed.

Respiratory properties of blood of the gray seal,Halichoerus grypus

Summary1.This study examined the O2 and CO2 transport and acid-base properties of blood of the gray seal. Phocid seals use theblood as an oxygen store for aerobic metabolism during diving. Particular objectives were to determine whether CO2 exerts a specific effect on blood oxygen affinity, and whether the Bohr coefficient varies between different levels of oxygen saturation.2.Hematocrit (0.50), blood hemoglobin concentration (12.8 mM heme) and cell hemoglobin concentration (25.4 mM) were, high compared to terrestrial mammals but similar to other seals (Table 2). Isoelectric focusing resolved the same four hemoglobin components in six individuals, with the most important components constituting 60% and 20% of the total. Oxygen affinity (P50=27.1 Torr at pH 7.4, 37.5°C) and equilibrium curve shape (nH=2.59) were similar to other phocid seals and terrestrial mammals (Tables 1,4). The Bohr coefficient for CO2 induced pH change (−0.51) was only slightly higher than that for fixed acid incuded pH change (−0.47), implying little oxygen-linked carbamino formation (Table 1). The bohr coefficients were at the upper end of the range found in terrestrial mammals, and did not vary with oxygen saturation (Table 1).3.Blood buffer value was high (31.5 mM/pH), in accord with the high hemoglobin concentration (Table 3). The Haldane effect (−0.32 mol CO2 per mol O2, Fig. 2) was similar to those in blood of other seals and dog, but higher than that in human blood. Hydrogen ion uptake by hemoglobin upon deoxygenation (0.40 mol/mol O2) was greater than in human blood, in proportion to the larger fixed acid Bohr coefficient in the seal.4.Blood of the gray seal, like that of other phocid seals, is distinguished from that of terrestrial mammals primarily by high hemoglobin concentration and oxygen capacity, which increase blood oxygen stores available for use during diving. Other properties are similar to those of terrestrial mammals or differ only as a consequence of the high hemoglobin concentration.

Kinetics of the root effect and of O2 exchange in whole blood of the eel

Oxygen transfer kinetics in blood of the eel (Anguilla rostrata, A. anguilla) were measured spectrophotometrically in thin blood layers covered by Gore-Tex membranes, which allowed fast changes of the gas phase at the blood surface (Heidelberger and Reeves, 1990 J. Appl. Physiol. 68: 1854-1864). The following main results were obtained for A. rostrata (similar values were measured for A. anguilla): (1) step change in PO2 of the gas phase between 0 and 37 kPa at low PCO2 (0.19 kPa, blood pH, 8.1; 20 degrees C) yielded mean half times (t(on)) for O2 uptake of 7.1 msec, and for O2 release (t(off)), of 42.8 msec. Similar values were obtained at high PCO2 (19 kPa; blood pH, 6.9), indicating O2 kinetics to be independent of pH and PCO2; (2) decreasing the high PO2 from 37 to 14 kPa significantly prolonged oxygen uptake kinetics, but release kinetics were unaltered; (3) changing PCO2 from 0.19 to 19 kPa at constant high PO2 (37 kPa) resulted in a reduction of hemoglobin oxygen saturation (SO2) (Root-off reaction), with t(off) averaging 44.8 msec; likewise, changing PCO2 from 19 to 0.19 kPa increased SO2 with t(on) averaging 64.8 msec (Root-on reaction). As these half times comprise reactions at the hemoglobin molecule and conversion between CO2 and H(+)/HCO3-, the Root effect kinetics of the hemoglobin molecule appear to be even faster. It is concluded that the O2 exchange kinetics of eel blood are comparable with those of human blood.(ABSTRACT TRUNCATED AT 250 WORDS)