British Journal dHaematology, 1975, 29, 369.
Annotation DETERMINANTS OF INTRACELLULAR pH I N THE ERYTHROCYTE The pH within red blood cells is of fundamental importance. Hydrogen ion concentration serves as a governor for both glycolysis and oxygcn dclivery, the former supplying energy and intermediates to maintain erythrocyte function, thc lattcr being crucial to the integrity of the organism. Under resting conditions, crythrocytes, like most other cclls studied, have an intracellular pH that is lower than the extracellular pH (Waddell & Bates, 1969). In norimal human erythrocytes from venous blood, the intracellular pH has been reported to be between 7.07 and 7.28 with an cxtracellular pH in the range of 7.25-7.39 (Waddell & Bates, 1969). The intracellular pH of the erythrocyte is controlled by a numbcr of forces. One determinant is the surrounding medium. As the pH of the medium changes, the pH within the cell tends to change in the same direction in vitro (Hilpert et al, 1963) or in vivo (Waddell & Bates, 1969; Desforges & Slawsky, 1972). A major factor controlling this is the transfer of hydrogen ion across the membrane according to the Gibbs-Doniian equilibrium (Deuticke et al, 1971). Penetrating anions or hydrogen ion are distributed according to the following: The ratio r =
c1; c1;
cc-
OH, c c -H: H; OH,
Thus, changes in chloride or hydroxyl concentration have the capacity to alter the CO~C'CIItration of H f . It has also been shown that the accumulation of organic phosphatc anions, by far the most concentrated of which within the red cell is 2,3-DPG, leads to a dccrcased value of r (Deuticke et a!, 1971), and hence this has the capacity to raise H t . The effect is self-limiting because of the inhibitory effect of increasing levels of H t on glycolysis (Murphy, 1960) and because accumulation of 2,3-DPG leads to decreased production of z,3-DPG by conipetitive feedback inhibition of diphosphoglycerate mutase (Rose, 1968). The concentration of 2,3-DPG then falls and, because of its effect on r, H t falls and pH, returns to its former level. In clinical states of acidosis and alkalosis, intracellular pH is modified by coiiconiiitant changcs in concentration of 2,3-DPG. Factors which coiitrol these changes, therefore, are important to intracellular metabolism. In metabolic acidosis, 2,3-DPG is low (Chillar ef al, 1971) and, therefore, r is increascd and the H:/Hf ratio decreased, resulting in less intraccllular acidosis than might otherwisc occur. In metabolic alkalosis, on the other hand, when z,3-DPG increases, r decreases and the intracellular ciivironnicnt is less alkalotic than it would be were Y to remain unchanged. Thus, the intraccllular pH is defended against changes in either direction by associated shifts in 2,pDPG concentration. In uraemia, however, this compensation may not be available. Serum phosphate is elevated and this provides a stimulus for glycolysis with subsequent production of 2,3-DPG. ThcreCorrespondence: Professor Jane F. Dcsforges, Departrncnt of Medicine, Tufts University School of Medicine, Boston, Massachusetts, U.S.A.
3 69
370
Annotation
fore, the concentration of this intermediate may be normal or slightly elevated even in the presence of acidosis (Hurt & Chanutin, 1964; Lichtman & Miller, 1970) and one may assume that the effect of ZJ-DPG on the Donnan equilibrium would result in a greater degree of intracellular acidosis than in other acidotic states. Another major determinant of pH in the erythrocyte is the Bohr effect. Carbon dioxide produced in tissues enters red cells by diffusion, and under the influence of carbonic anhydrase, hydrogen ion is released (Riley, 1965). The H f is taken up by reduced haemoglobin to form proton bound haemoglobin. This proton binding by reduced haemoglobin results in an increase in pH of a solution of deoxygenated haemoglobin as compared to an identical solution of oxygenated haemoglobin. This is one aspect of the alkaline Bohr effect (Ranney, 1972). It occurs as a concomitant of the buffering action of haemoglobin. The molecular basis for the alkaline Bohr effect may be explained by the bonds formed by (a) the imidazole group of the C-terminal histidine (146) of the j3-chain with the carboxyl group of aspartate at position 94 of the same fl-chain; and (b) the a-amino group of each a-chain (valine I a ) with the C-terminal carboxyl group of its partner a-chain (arginine 141 a). In oxyhaemoglobin these bonds are not present; their formation in deoxyhaemoglobin consumes protons and is therefore associated with an increase in pH of the solution (Perutz et al, 1969; Perutz, 1970). Furthermore, the imidazole group of histidine 122 a1 which approaches the guanidiniuni group of arginine 30 PI in oxyhaemoglobin lies closer to the carboxyl group of aspartate 126 a1 in deoxyhaemoglobin. This re-arrangement likewise consumes hydrogen ions (Perutz, 1970). The mathematical expression of the Bohr effect at a given oxygen saturation is: b log POJA pH = -0.50 (Astrup et a!, 1965). Thus, for example, if PO, decreased from 80 to 60 mmHg then pH is increased by approximately 0.25 of a pH unit, to achieve a given oxygen saturation. There is some evidence that the Bohr effect induced by CO, is greater than that induced by fixed acid or alkali because of the added effect of molecular CO, on 0, dissociation by carbaniate formation (Wranne et a!, 1972). Therefore, in the erythrocytes of patients with respiratory acidosis, for example, one might postulate a greater Bohr effect than in the red cells of patients with metabolic acidosis. 2,3-DPG and, to a lesser degree, ATP, play a role in the Bohr effect as well as in Donnan equilibrium. They bind competitively to reduced haemoglobin (Rorth, 1968; Lo & Schimmel, 1969). Therefore, at a given pOz, their concentrations are a determinant of oxygen affinity. Increased free 2,3-DPG or ATP leads to decreased oxyhaemoglobin at a given PO,. A relative increase in deoxygenated haemoglobin results. This increases the pH according to the Bohr effect. However, according to the Dolman equilibrium, increased organic phosphate ions result in a decreased value of r, with an increase in Hf and a decrease in pHi. They have, then, two opposing actions over pHi. Finally, another determinant of the intracellular pH is the transmembrane potential. For an erythrocyte, this has been calculated as - 10millivolts (mv) assuming the passive distribution of chloride and bicarbonate (Woodbury, 1965, pp 23-24). The outside of the membrane is positive and the inside negative. If hydrogen ion is distributed passively, that is, if it is an ion whose equilibrium potential is equal to the steady membrane potential (Woodbury, 1965, p IZ), then using the Nernst equation (Woodbury, 1965, p 23) :
Annotation
371
(H'k - 10 = -61 log,, (H+)i
Solving this equation: (H+)i/(H+)e= 1.46. Using the reported concentration of intracellular H+ at given extracellular concentrations (Waddell & Bates, 1969), the range of (H+)i/(H+)efor human red cells from venous blood is 1.29-1.62. For arterial blood it is 1.41-2.04. The fact that these valucs approximate 1.46 may indicate that H+ is distributed passively across the erythrocyte membrane. Assuming homogeneity of the interior of red blood cells, an active transport process is unlikely to be involved, as a determinant of intraerythrocyte pH. Thus, multiple and often interlocking factors control the intraerythrocyte pH. It is appairciit that numerous related phenomena provide close rcgulation of intracellular pH and this coincides with the need of living cells to maintain careful control of their internal milieu. ACKNOWLEDGMENT
These studies wcrc supported by U.S. Public Hcalth Scrvice Grant number HL-15157 fr.om the Heart and Lung Institute of the National Institutes of Health.
Blood Research Laboratory, N e w England Medical Center Hospital, and Departnzcnt of Medicine, Ti@ University School of Medicine, Boston, Massachusetts, U.S.A.
JAMES JANE
F.
WARTH
DESFORGES
REFERENCES ASTRUP,P., ENGEL,K., SBVERINGHAUS, J.W. & MUNSON,E. (1965) The influence of temperature and pH on the dissociation curve of oxyhemoglobin of human blood. Scandinavian Journal of Clinical and Laboratory Investigation, 17, 51s. CHILLAR, R.K., SLAWSKY, P. & DESFORGES, J.F. (1971) Rcd cell 2,3-diphosphoglycerate and adenosine triphosphate in patients with shock. British Journal of Haernatology, 21, 183. DESFORGES, J.F. & SLAWSKY, P. (1972) Red cell 2,3diphosphoglycerate and intracellular arterial pH in acidosis and alkalosis. Blood, 40, 740. DEUTICKE, B., BUHM,J. & DIERKESMANN, R. (1971) Maximal elevation of 2,3-diphosphoglycerate concentrations in human erythrocytes: influence on glycolytic metabolism and intracellular pH. P'iigers Archiv, 326, IS. HILPERT,P., FLEISCHMA",R.G., KEMPE,D. & BARTELS, H. (1963) The Bohr effect related to blood and erythrocyte pH. American Journal of Physiology, 205, 337. HURT,G.A. & CHANUTIN, A. (1964) Organic phosphate compounds of erythrocytes from individuals with uremia. Journal of Laboratory and Clinical Medicine, 64, 675. LICHTMAN, M.A. & MILLER, D.R. (1970) Erythrocyte glycolysis, 2,3-diphosphoglycerate and adenosine
triphosphate concentration in uremic subjects: relationship to extracellular phosphate concentration. Journal of Laboratory and Clinical Medicine, 76, 267. Lo, H.H. & SCHIMMEL, P.R. (1969) Interaction of human hemoglobin with adenine iiucleotides. Journal ofBiological Chsmistry, 244, 5084. MURPHY,J.R. (1960) Erythrocyte metabolism. 11. Glucose metabolism and pathways. Journal of Laboratory and Clinical Medicine, 55, 286. PERUTZ,M.F. (1970) Sterochemistry of cooperative effects in haemoglobin. Nature, 228, 726. PERUTZ,M.F., MUIRHEAD,H., MAZZARELLA. L., CROWTHER, R.A., GREER,J. & KILMARTIN, J.V. (1969) Identification of residues responsible for the alkaline Bohr effect in hacmoglobin. Nature, 222, 1240. RANNEY,H.M. (1972) Transport function o f the erythrocyte. Hematology (Ed. by W. H. Williams, E. Beutler, A. J. Erslev and R. W. Rundlcs), p 148. McGraw-Hill, New York. RILEY, R.L. (1965) Gas exchange and transport. Physiology andBiophysics (Ed. by T. C. Ruch and H. D. Patton), 19th edn, p 768. Saundcrs, Philadelphia. RORTH,M. (1968) Effects of some organic phosphate compounds on the oxyhemoglobin dissoci.ation curve in human erythrolysate. Scandinavian journal of Clinical and Laboratory Investigation, 22, 208.
3 72
Annotation
ROSE,Z.B. (1968)The purification and properties of diphosphoglycerate mutase from human erythrocytes. Journal of Biological Chemistry, 243, 4810. WADDELL, W.J.& BATES, R.G. (1969) Intracellular pH. Physiological Reviews, 49, 285. WOODBURY, J.W. (1965) The cell membrane: Ionic and potential gradients and active transport.
Physiology and Biophysics (Ed. by T. C. Ruch and H. D. Patton), 19th edn, pp 12-25. Saunders, Philadelphia. WRANNE, B., WOODSON, R.D. 81 DETTER, J.C. (1972) Bohr effect: interaction between H,' COz, and ZJ-DPG in fresh and stored blood. Journal of Applied Physiology, 32, 749.
Annotation DETERMINANTS OF INTRACELLULAR pH I N THE ERYTHROCYTE The pH within red blood cells is of fundamental importance. Hydrogen ion concentration serves as a governor for both glycolysis and oxygcn dclivery, the former supplying energy and intermediates to maintain erythrocyte function, thc lattcr being crucial to the integrity of the organism. Under resting conditions, crythrocytes, like most other cclls studied, have an intracellular pH that is lower than the extracellular pH (Waddell & Bates, 1969). In norimal human erythrocytes from venous blood, the intracellular pH has been reported to be between 7.07 and 7.28 with an cxtracellular pH in the range of 7.25-7.39 (Waddell & Bates, 1969). The intracellular pH of the erythrocyte is controlled by a numbcr of forces. One determinant is the surrounding medium. As the pH of the medium changes, the pH within the cell tends to change in the same direction in vitro (Hilpert et al, 1963) or in vivo (Waddell & Bates, 1969; Desforges & Slawsky, 1972). A major factor controlling this is the transfer of hydrogen ion across the membrane according to the Gibbs-Doniian equilibrium (Deuticke et al, 1971). Penetrating anions or hydrogen ion are distributed according to the following: The ratio r =
c1; c1;
cc-
OH, c c -H: H; OH,
Thus, changes in chloride or hydroxyl concentration have the capacity to alter the CO~C'CIItration of H f . It has also been shown that the accumulation of organic phosphatc anions, by far the most concentrated of which within the red cell is 2,3-DPG, leads to a dccrcased value of r (Deuticke et a!, 1971), and hence this has the capacity to raise H t . The effect is self-limiting because of the inhibitory effect of increasing levels of H t on glycolysis (Murphy, 1960) and because accumulation of 2,3-DPG leads to decreased production of z,3-DPG by conipetitive feedback inhibition of diphosphoglycerate mutase (Rose, 1968). The concentration of 2,3-DPG then falls and, because of its effect on r, H t falls and pH, returns to its former level. In clinical states of acidosis and alkalosis, intracellular pH is modified by coiiconiiitant changcs in concentration of 2,3-DPG. Factors which coiitrol these changes, therefore, are important to intracellular metabolism. In metabolic acidosis, 2,3-DPG is low (Chillar ef al, 1971) and, therefore, r is increascd and the H:/Hf ratio decreased, resulting in less intraccllular acidosis than might otherwisc occur. In metabolic alkalosis, on the other hand, when z,3-DPG increases, r decreases and the intracellular ciivironnicnt is less alkalotic than it would be were Y to remain unchanged. Thus, the intraccllular pH is defended against changes in either direction by associated shifts in 2,pDPG concentration. In uraemia, however, this compensation may not be available. Serum phosphate is elevated and this provides a stimulus for glycolysis with subsequent production of 2,3-DPG. ThcreCorrespondence: Professor Jane F. Dcsforges, Departrncnt of Medicine, Tufts University School of Medicine, Boston, Massachusetts, U.S.A.
3 69
370
Annotation
fore, the concentration of this intermediate may be normal or slightly elevated even in the presence of acidosis (Hurt & Chanutin, 1964; Lichtman & Miller, 1970) and one may assume that the effect of ZJ-DPG on the Donnan equilibrium would result in a greater degree of intracellular acidosis than in other acidotic states. Another major determinant of pH in the erythrocyte is the Bohr effect. Carbon dioxide produced in tissues enters red cells by diffusion, and under the influence of carbonic anhydrase, hydrogen ion is released (Riley, 1965). The H f is taken up by reduced haemoglobin to form proton bound haemoglobin. This proton binding by reduced haemoglobin results in an increase in pH of a solution of deoxygenated haemoglobin as compared to an identical solution of oxygenated haemoglobin. This is one aspect of the alkaline Bohr effect (Ranney, 1972). It occurs as a concomitant of the buffering action of haemoglobin. The molecular basis for the alkaline Bohr effect may be explained by the bonds formed by (a) the imidazole group of the C-terminal histidine (146) of the j3-chain with the carboxyl group of aspartate at position 94 of the same fl-chain; and (b) the a-amino group of each a-chain (valine I a ) with the C-terminal carboxyl group of its partner a-chain (arginine 141 a). In oxyhaemoglobin these bonds are not present; their formation in deoxyhaemoglobin consumes protons and is therefore associated with an increase in pH of the solution (Perutz et al, 1969; Perutz, 1970). Furthermore, the imidazole group of histidine 122 a1 which approaches the guanidiniuni group of arginine 30 PI in oxyhaemoglobin lies closer to the carboxyl group of aspartate 126 a1 in deoxyhaemoglobin. This re-arrangement likewise consumes hydrogen ions (Perutz, 1970). The mathematical expression of the Bohr effect at a given oxygen saturation is: b log POJA pH = -0.50 (Astrup et a!, 1965). Thus, for example, if PO, decreased from 80 to 60 mmHg then pH is increased by approximately 0.25 of a pH unit, to achieve a given oxygen saturation. There is some evidence that the Bohr effect induced by CO, is greater than that induced by fixed acid or alkali because of the added effect of molecular CO, on 0, dissociation by carbaniate formation (Wranne et a!, 1972). Therefore, in the erythrocytes of patients with respiratory acidosis, for example, one might postulate a greater Bohr effect than in the red cells of patients with metabolic acidosis. 2,3-DPG and, to a lesser degree, ATP, play a role in the Bohr effect as well as in Donnan equilibrium. They bind competitively to reduced haemoglobin (Rorth, 1968; Lo & Schimmel, 1969). Therefore, at a given pOz, their concentrations are a determinant of oxygen affinity. Increased free 2,3-DPG or ATP leads to decreased oxyhaemoglobin at a given PO,. A relative increase in deoxygenated haemoglobin results. This increases the pH according to the Bohr effect. However, according to the Dolman equilibrium, increased organic phosphate ions result in a decreased value of r, with an increase in Hf and a decrease in pHi. They have, then, two opposing actions over pHi. Finally, another determinant of the intracellular pH is the transmembrane potential. For an erythrocyte, this has been calculated as - 10millivolts (mv) assuming the passive distribution of chloride and bicarbonate (Woodbury, 1965, pp 23-24). The outside of the membrane is positive and the inside negative. If hydrogen ion is distributed passively, that is, if it is an ion whose equilibrium potential is equal to the steady membrane potential (Woodbury, 1965, p IZ), then using the Nernst equation (Woodbury, 1965, p 23) :
Annotation
371
(H'k - 10 = -61 log,, (H+)i
Solving this equation: (H+)i/(H+)e= 1.46. Using the reported concentration of intracellular H+ at given extracellular concentrations (Waddell & Bates, 1969), the range of (H+)i/(H+)efor human red cells from venous blood is 1.29-1.62. For arterial blood it is 1.41-2.04. The fact that these valucs approximate 1.46 may indicate that H+ is distributed passively across the erythrocyte membrane. Assuming homogeneity of the interior of red blood cells, an active transport process is unlikely to be involved, as a determinant of intraerythrocyte pH. Thus, multiple and often interlocking factors control the intraerythrocyte pH. It is appairciit that numerous related phenomena provide close rcgulation of intracellular pH and this coincides with the need of living cells to maintain careful control of their internal milieu. ACKNOWLEDGMENT
These studies wcrc supported by U.S. Public Hcalth Scrvice Grant number HL-15157 fr.om the Heart and Lung Institute of the National Institutes of Health.
Blood Research Laboratory, N e w England Medical Center Hospital, and Departnzcnt of Medicine, Ti@ University School of Medicine, Boston, Massachusetts, U.S.A.
JAMES JANE
F.
WARTH
DESFORGES
REFERENCES ASTRUP,P., ENGEL,K., SBVERINGHAUS, J.W. & MUNSON,E. (1965) The influence of temperature and pH on the dissociation curve of oxyhemoglobin of human blood. Scandinavian Journal of Clinical and Laboratory Investigation, 17, 51s. CHILLAR, R.K., SLAWSKY, P. & DESFORGES, J.F. (1971) Rcd cell 2,3-diphosphoglycerate and adenosine triphosphate in patients with shock. British Journal of Haernatology, 21, 183. DESFORGES, J.F. & SLAWSKY, P. (1972) Red cell 2,3diphosphoglycerate and intracellular arterial pH in acidosis and alkalosis. Blood, 40, 740. DEUTICKE, B., BUHM,J. & DIERKESMANN, R. (1971) Maximal elevation of 2,3-diphosphoglycerate concentrations in human erythrocytes: influence on glycolytic metabolism and intracellular pH. P'iigers Archiv, 326, IS. HILPERT,P., FLEISCHMA",R.G., KEMPE,D. & BARTELS, H. (1963) The Bohr effect related to blood and erythrocyte pH. American Journal of Physiology, 205, 337. HURT,G.A. & CHANUTIN, A. (1964) Organic phosphate compounds of erythrocytes from individuals with uremia. Journal of Laboratory and Clinical Medicine, 64, 675. LICHTMAN, M.A. & MILLER, D.R. (1970) Erythrocyte glycolysis, 2,3-diphosphoglycerate and adenosine
triphosphate concentration in uremic subjects: relationship to extracellular phosphate concentration. Journal of Laboratory and Clinical Medicine, 76, 267. Lo, H.H. & SCHIMMEL, P.R. (1969) Interaction of human hemoglobin with adenine iiucleotides. Journal ofBiological Chsmistry, 244, 5084. MURPHY,J.R. (1960) Erythrocyte metabolism. 11. Glucose metabolism and pathways. Journal of Laboratory and Clinical Medicine, 55, 286. PERUTZ,M.F. (1970) Sterochemistry of cooperative effects in haemoglobin. Nature, 228, 726. PERUTZ,M.F., MUIRHEAD,H., MAZZARELLA. L., CROWTHER, R.A., GREER,J. & KILMARTIN, J.V. (1969) Identification of residues responsible for the alkaline Bohr effect in hacmoglobin. Nature, 222, 1240. RANNEY,H.M. (1972) Transport function o f the erythrocyte. Hematology (Ed. by W. H. Williams, E. Beutler, A. J. Erslev and R. W. Rundlcs), p 148. McGraw-Hill, New York. RILEY, R.L. (1965) Gas exchange and transport. Physiology andBiophysics (Ed. by T. C. Ruch and H. D. Patton), 19th edn, p 768. Saundcrs, Philadelphia. RORTH,M. (1968) Effects of some organic phosphate compounds on the oxyhemoglobin dissoci.ation curve in human erythrolysate. Scandinavian journal of Clinical and Laboratory Investigation, 22, 208.
3 72
Annotation
ROSE,Z.B. (1968)The purification and properties of diphosphoglycerate mutase from human erythrocytes. Journal of Biological Chemistry, 243, 4810. WADDELL, W.J.& BATES, R.G. (1969) Intracellular pH. Physiological Reviews, 49, 285. WOODBURY, J.W. (1965) The cell membrane: Ionic and potential gradients and active transport.
Physiology and Biophysics (Ed. by T. C. Ruch and H. D. Patton), 19th edn, pp 12-25. Saunders, Philadelphia. WRANNE, B., WOODSON, R.D. 81 DETTER, J.C. (1972) Bohr effect: interaction between H,' COz, and ZJ-DPG in fresh and stored blood. Journal of Applied Physiology, 32, 749.
