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Proton generation and control during anaerobic glycolysis in heart cells
Journal
of Molecular
and Cellular
Cardiology
(1980)
LETTER
Proton
Generation
12, 1483-1486
TO THE
and Control in Heart
EDITOR
during Cells
Anaerobic
Glycolysis
Professor Gevers’ Editorial [3] in the November 1977 issue of this Journal expresses opinions later challenged by Professor Wilkie in his letter of reply [7]. In view of the importance of proton accumulation in ischaemic myocardium it becomes equally important that points raised by these authors and still at issue are resolved. Confusion would appear to stem from the use of the single term “proton generation” which fails to separate two distinct types of chemical process in respect of anaerobic glycolysis. 1. Irreversible Reactions resulting in “de nova protons” (vide @a). 2. Equilibrium Reactions which concern the whole of the multi-component pH-buffer system which resists, as far as buffer capacities allow, pressures placed upon it by, “de nova protons”. Proton release is controlled by these equilibria and should be treated therefore as a process quite separate from the proton generating mechanisms. 1. Cyclic Mechanisms The summation of the nine balanced glycolysis equations (Gevers’ equation, page 2) elegantly and clearly demonstrates the formation of lactate anion without accompanying hydrogen ion production : glucose I would
+ two ADP3much
two ATP4-
prefer
to follow
+ two H,O
in order to emphasise of (1) and (2) gives:
B
B
equation
two ADP3relationship
two lactatesl-
+ two H,O*
immediately + two Pi”-
with + two
between
them.
* Included
(1)
the equation H+ Then
:
(2) the sum
+ two H+
(3)
Wilkie’s concept of lactic “acid” formation is reconciled with ion formation, except for the time and place of proton production.
Gevers’
for true stoichiometry.
0022-2828/80/1214a3+o4 M.C.C.
this latter
an inseparable
glucose Thus, lactate
two lactatesl+two ATP4-
+ two Pis- B
s02.00~0
0 1980 Academic
Press Inc.
(London)
Limited 3L
1484
LETTER
TO
THE
FDITOR
However, this approach by Gevers omits detailed examination of the key reactions (Gevers’ 5th and 6th sequential equations, page 868) in which neutral unionized triose aldehyde group is oxidized to charged carboxylate anion (phosphoglycerate) without the concomitantf formation of a corresponding proton which might be expected by analogy with a simple in vitro oxidation: RCH =o+o neutral aldehyde
B
RCOOH
>
RCOOcarboxylate anion
+ H’ + proton
(4)
This failure to produce a proton at this stage of the cycle, is associated with the intermediates diphosphoglycerate and ATP which “store” the proton in the form of the anhydride bond-an interesting “proton storage” reaction. It is noteworthy that the liberated proton is derived from an irreversible bond-breaking reaction and intrinsically is independent of the degree of ionization of both the ADP and the phosphate liberated in the same reaction. The behaviour of these anions, however, is governed by the pH of the environment which is determined by the buffer system in toto. Under anaerobic conditions these two “de novo carboxylate anions” derived from one molecule of glucose are preserved via pyruvate through to lactate which accumulates as a stable product. Potentially these anions are associated with an equivalent number of ‘rde novo protons” (later to be released from ATP which acts as the storage and transferring depot). In contrast, if aerobic conditions prevail, the carboxylate anion is lost during the formation of acetyl co-enzyme A : two CHsCOCGO-
+ two HSCoA + two NAD+ . two CHsCOSCoA + two NADH
+ two CO,.
(5)
Here the most important factors are loss of two equivalents of de novo carboxylate anion as carbon dioxide and its complete removal from the system, which prevents partial reformation of protons from the CO,/HCO,-/H+ equilibrium. This loss of carboxylate anion is exactly balanced by the ATP produced from triose phosphate. Hence potential protons produced by hydrolysis of this complement of glycolytic ATP are balanced by equivalent loss of potential protons associated with CO, removed from the system. This approach to anaerobic glycolysis adds weight to Gevers’ initial use of a single appropriately ‘charged species (i.e. MgATP2-, Pi2- etc.) formed at a static pH of 8.0, but the proviso must be added that the identically ionized species are re-used in equations restoring ADP to ATP via the cyclic glycolytic pathway. Failure to observe this proviso and failure to separate proton generation from buffer effects can lead to errors as in Gevers’ equations (3) and (4) (page 868)
7 The protons accompaniment
appearing as products of nny NADf oxidation.
in Gevers’
fifth
glycolysis
equation
are the most
usual
f.ETTER
TO
THE
1485
EDITOR
in which ADP, but not Pi, is represented as two differently charged species even though the pK, values for the relevant ionization of ADP and Pi are closely similar. Using Gevers’ representation here, these equations should read : glucose
+ ADP3-
glucose
+ two MgADF’-
which
show a greater
+ ADP2-
increase
+ Pi2- + Pil- + 2Mg2+ -+ two lactates’+ two MgATP2+ Pi2- + PiiF two lactate& + two MgATP2in proton
production
2. Non-cyclic
than the original
+ 2Hf + H+ equations.
Mechanisms
Quite different considerations apply to that portion of ATP lost from the adenine mononucleotide pool during ischaemia. Here the state of ionization of cumulative products (mainly Pi) cannot be disregarded. However, it is still instructive to examine the simplified overall equation expressing ATP loss in terms of proton generation : MgATP2-
+ three H,O
------+
Inosine
+ three
Pi2+NH,
+ 2H+
+ Mg2+
(6)
NGH+ in which two anhydride bonds upon fission release two protons accompanied by three phosphate anions. The importance of accounting for base (ammonia production) is illustrated here. These buffer anions (HPOa2-, ADP3and others) are buffer bases (proton acceptors) and as such require recognition in their control of net proton release. Both Gevers and Wilkie have dealt comprehensively with the proportions of these various ionic forms present at a specified pH. Even under the most favourable conditions, i.e. short period of ischaemia/small pH fall, the one remaining proton shared amongst three phosphate anions leaves little proton residue, particularly since the net loss of ATP under such conditions is very small. Just as Gevers has examined Mg-chelation as a possible factor affecting proton release (here is a complex Mg-dependent pH buffer system) [6] so attention might be directed toward divalent cations reducing the availability of phosphate. Therefore, the progressively insoluble series MgHPO,, [6] CaHPO,, [5] and Mg(NH,)PO, [4] should not be neglected as possible sequestering agents for both cation and anion. While hydrolysis resulting in ATP or phosphocreatine (PC) loss may be small in comparison with proton generation accompanying lactate ion formation, these effects should not be ignored. Thus, Garlick et al. [Z], calculated (de nova) 3~2
1486
LETTER
TO THE
EDITOR
proton generation in order to estimate the decrease in pHi of isolated rat heart during 10 min of global ischaemia by assuming each glycogen, ATP and PC molecule lost generated respectively +2, + 1, and -1 protons. In this context virtually all the ATP lost is degraded to inorganic phosphate. After 10 min the proton yield is $2 from anhydride bond fission, - 1 from ammonia formation and - (3 x 0.8) = -2.4 from absorption of protons by liberated H,PO,lto give the equilibrium mixture close to Pi’(80%) + Pi2- (200/A) existing at the final 1.4 proton/molecule of ATP lost from pHi 6.2. The net yield is therefore the mononucleotide pool. In a similar manner phosphocreatine provides the same equilibrium mixture of Pil- and Pi2- to give a proton yield of -0.8 for each molecule of PC hydrolyzed. Clearly, due consideration should be given to new phosphate liberated during ischaemia and not part of the original intracellular buffer system. It is hoped that these considerations may stimulate further discusssion on this all important aspect of myocardial ischaemia. R.N. Department
SEELYE
of Pathology, School of Medicine, University of Auckland, Private Bag, Auckland, New Zealand
REFERENCES 1. CLARKE, H.-B., CUSWORTH, D. C. & SUTTON, S. P. Thermodynamic quantities for the dissociation equilibria of biologically important compounds. Biochemistry Journal 58, 146-154 (1954). P. B., RADDA, G. K. & SEELEY, P. J. Studies of acidosis in the ischaemic 2. GARLICK, heart by phosphorus nuclear magnetic resonance. Biochemical Journal 184, 547-554 (1979). 3. GEVERS, W. Generation of protons by metabolic processes in heart cells. Journal of Molecular and Cellldar Cardiology 9, 867-874 (1977), 4. Handbook of Chemistry and Physics 55th Edition. Data on solubility products B 232. Ohio: C.R.C. Press (1975). W. & KATZ, A. M. Mechanism of early “pump” failure of the ischaemic 5. KUBLER, heart: possible role of adenosine triphosphate depletion and inorganic phosphate accumulations. American Journal of Cardiology 40, 467-47 1 (1977). 6. PHILLIPS, R. C., GEORGE, P. & RUTMAN, R. Thermodynamic data for the hydrolysis of adenosine triphosphate as a function of pH, Mg2+ ion concentration and ionic strength. Journal of Biological Chemistry 244, 3330-3342 (1969). D. R. Generation of protons by metabolic processes other than glycolysis in 7. WILKIE, muscle cells. A critical view. Journal of Molecular and Cellular Cardiolopv 11, 325-330 (1979).
of Molecular
and Cellular
Cardiology
(1980)
LETTER
Proton
Generation
12, 1483-1486
TO THE
and Control in Heart
EDITOR
during Cells
Anaerobic
Glycolysis
Professor Gevers’ Editorial [3] in the November 1977 issue of this Journal expresses opinions later challenged by Professor Wilkie in his letter of reply [7]. In view of the importance of proton accumulation in ischaemic myocardium it becomes equally important that points raised by these authors and still at issue are resolved. Confusion would appear to stem from the use of the single term “proton generation” which fails to separate two distinct types of chemical process in respect of anaerobic glycolysis. 1. Irreversible Reactions resulting in “de nova protons” (vide @a). 2. Equilibrium Reactions which concern the whole of the multi-component pH-buffer system which resists, as far as buffer capacities allow, pressures placed upon it by, “de nova protons”. Proton release is controlled by these equilibria and should be treated therefore as a process quite separate from the proton generating mechanisms. 1. Cyclic Mechanisms The summation of the nine balanced glycolysis equations (Gevers’ equation, page 2) elegantly and clearly demonstrates the formation of lactate anion without accompanying hydrogen ion production : glucose I would
+ two ADP3much
two ATP4-
prefer
to follow
+ two H,O
in order to emphasise of (1) and (2) gives:
B
B
equation
two ADP3relationship
two lactatesl-
+ two H,O*
immediately + two Pi”-
with + two
between
them.
* Included
(1)
the equation H+ Then
:
(2) the sum
+ two H+
(3)
Wilkie’s concept of lactic “acid” formation is reconciled with ion formation, except for the time and place of proton production.
Gevers’
for true stoichiometry.
0022-2828/80/1214a3+o4 M.C.C.
this latter
an inseparable
glucose Thus, lactate
two lactatesl+two ATP4-
+ two Pis- B
s02.00~0
0 1980 Academic
Press Inc.
(London)
Limited 3L
1484
LETTER
TO
THE
FDITOR
However, this approach by Gevers omits detailed examination of the key reactions (Gevers’ 5th and 6th sequential equations, page 868) in which neutral unionized triose aldehyde group is oxidized to charged carboxylate anion (phosphoglycerate) without the concomitantf formation of a corresponding proton which might be expected by analogy with a simple in vitro oxidation: RCH =o+o neutral aldehyde
B
RCOOH
>
RCOOcarboxylate anion
+ H’ + proton
(4)
This failure to produce a proton at this stage of the cycle, is associated with the intermediates diphosphoglycerate and ATP which “store” the proton in the form of the anhydride bond-an interesting “proton storage” reaction. It is noteworthy that the liberated proton is derived from an irreversible bond-breaking reaction and intrinsically is independent of the degree of ionization of both the ADP and the phosphate liberated in the same reaction. The behaviour of these anions, however, is governed by the pH of the environment which is determined by the buffer system in toto. Under anaerobic conditions these two “de novo carboxylate anions” derived from one molecule of glucose are preserved via pyruvate through to lactate which accumulates as a stable product. Potentially these anions are associated with an equivalent number of ‘rde novo protons” (later to be released from ATP which acts as the storage and transferring depot). In contrast, if aerobic conditions prevail, the carboxylate anion is lost during the formation of acetyl co-enzyme A : two CHsCOCGO-
+ two HSCoA + two NAD+ . two CHsCOSCoA + two NADH
+ two CO,.
(5)
Here the most important factors are loss of two equivalents of de novo carboxylate anion as carbon dioxide and its complete removal from the system, which prevents partial reformation of protons from the CO,/HCO,-/H+ equilibrium. This loss of carboxylate anion is exactly balanced by the ATP produced from triose phosphate. Hence potential protons produced by hydrolysis of this complement of glycolytic ATP are balanced by equivalent loss of potential protons associated with CO, removed from the system. This approach to anaerobic glycolysis adds weight to Gevers’ initial use of a single appropriately ‘charged species (i.e. MgATP2-, Pi2- etc.) formed at a static pH of 8.0, but the proviso must be added that the identically ionized species are re-used in equations restoring ADP to ATP via the cyclic glycolytic pathway. Failure to observe this proviso and failure to separate proton generation from buffer effects can lead to errors as in Gevers’ equations (3) and (4) (page 868)
7 The protons accompaniment
appearing as products of nny NADf oxidation.
in Gevers’
fifth
glycolysis
equation
are the most
usual
f.ETTER
TO
THE
1485
EDITOR
in which ADP, but not Pi, is represented as two differently charged species even though the pK, values for the relevant ionization of ADP and Pi are closely similar. Using Gevers’ representation here, these equations should read : glucose
+ ADP3-
glucose
+ two MgADF’-
which
show a greater
+ ADP2-
increase
+ Pi2- + Pil- + 2Mg2+ -+ two lactates’+ two MgATP2+ Pi2- + PiiF two lactate& + two MgATP2in proton
production
2. Non-cyclic
than the original
+ 2Hf + H+ equations.
Mechanisms
Quite different considerations apply to that portion of ATP lost from the adenine mononucleotide pool during ischaemia. Here the state of ionization of cumulative products (mainly Pi) cannot be disregarded. However, it is still instructive to examine the simplified overall equation expressing ATP loss in terms of proton generation : MgATP2-
+ three H,O
------+
Inosine
+ three
Pi2+NH,
+ 2H+
+ Mg2+
(6)
NGH+ in which two anhydride bonds upon fission release two protons accompanied by three phosphate anions. The importance of accounting for base (ammonia production) is illustrated here. These buffer anions (HPOa2-, ADP3and others) are buffer bases (proton acceptors) and as such require recognition in their control of net proton release. Both Gevers and Wilkie have dealt comprehensively with the proportions of these various ionic forms present at a specified pH. Even under the most favourable conditions, i.e. short period of ischaemia/small pH fall, the one remaining proton shared amongst three phosphate anions leaves little proton residue, particularly since the net loss of ATP under such conditions is very small. Just as Gevers has examined Mg-chelation as a possible factor affecting proton release (here is a complex Mg-dependent pH buffer system) [6] so attention might be directed toward divalent cations reducing the availability of phosphate. Therefore, the progressively insoluble series MgHPO,, [6] CaHPO,, [5] and Mg(NH,)PO, [4] should not be neglected as possible sequestering agents for both cation and anion. While hydrolysis resulting in ATP or phosphocreatine (PC) loss may be small in comparison with proton generation accompanying lactate ion formation, these effects should not be ignored. Thus, Garlick et al. [Z], calculated (de nova) 3~2
1486
LETTER
TO THE
EDITOR
proton generation in order to estimate the decrease in pHi of isolated rat heart during 10 min of global ischaemia by assuming each glycogen, ATP and PC molecule lost generated respectively +2, + 1, and -1 protons. In this context virtually all the ATP lost is degraded to inorganic phosphate. After 10 min the proton yield is $2 from anhydride bond fission, - 1 from ammonia formation and - (3 x 0.8) = -2.4 from absorption of protons by liberated H,PO,lto give the equilibrium mixture close to Pi’(80%) + Pi2- (200/A) existing at the final 1.4 proton/molecule of ATP lost from pHi 6.2. The net yield is therefore the mononucleotide pool. In a similar manner phosphocreatine provides the same equilibrium mixture of Pil- and Pi2- to give a proton yield of -0.8 for each molecule of PC hydrolyzed. Clearly, due consideration should be given to new phosphate liberated during ischaemia and not part of the original intracellular buffer system. It is hoped that these considerations may stimulate further discusssion on this all important aspect of myocardial ischaemia. R.N. Department
SEELYE
of Pathology, School of Medicine, University of Auckland, Private Bag, Auckland, New Zealand
REFERENCES 1. CLARKE, H.-B., CUSWORTH, D. C. & SUTTON, S. P. Thermodynamic quantities for the dissociation equilibria of biologically important compounds. Biochemistry Journal 58, 146-154 (1954). P. B., RADDA, G. K. & SEELEY, P. J. Studies of acidosis in the ischaemic 2. GARLICK, heart by phosphorus nuclear magnetic resonance. Biochemical Journal 184, 547-554 (1979). 3. GEVERS, W. Generation of protons by metabolic processes in heart cells. Journal of Molecular and Cellldar Cardiology 9, 867-874 (1977), 4. Handbook of Chemistry and Physics 55th Edition. Data on solubility products B 232. Ohio: C.R.C. Press (1975). W. & KATZ, A. M. Mechanism of early “pump” failure of the ischaemic 5. KUBLER, heart: possible role of adenosine triphosphate depletion and inorganic phosphate accumulations. American Journal of Cardiology 40, 467-47 1 (1977). 6. PHILLIPS, R. C., GEORGE, P. & RUTMAN, R. Thermodynamic data for the hydrolysis of adenosine triphosphate as a function of pH, Mg2+ ion concentration and ionic strength. Journal of Biological Chemistry 244, 3330-3342 (1969). D. R. Generation of protons by metabolic processes other than glycolysis in 7. WILKIE, muscle cells. A critical view. Journal of Molecular and Cellular Cardiolopv 11, 325-330 (1979).