Reeves1 and Rahn2 extended the conclusions reached by Davis3 by considering the dissociation constants (pK) for these metabolic intermediates. They found that the pK for all the acid intermediates was less than 4.6 and the pK of all the basic intermediates was greater than 9.2 . The degree of dissociation of all these compounds at a pH around neutrality was 1.0 (ie fully ionised). The intermediates are all charged and trapped within the lipid cell membrane.
They suggested looking at acid-base physiology from the point of view of the intracellular environment instead of the usual clinical extracellular approach. They first posed the following question:
The work of Davis and the findings concerning pK values suggested that the ideal state for intermediary metabolism is the state of neutrality because maximal ionisation with consequent intracellular trapping of metabolic intermediates occurs at this pH.
If theoretically it is clear that the ideal ICF pH should be the pH of neutrality (pN)see 4, then the next step is to ask the question:
According to Rahn, measurements confirmed that the mean intracellular pH of man is 6.8 at 37C which is indeed the pH of neutrality (pN) at that temperature!
Before going further we need to understand:
Neutrality is defined, for aqueous systems, as the state when [H+] = [OH-]. (This definition derives from the Arrhenius acid-base theory and it is noted in passing that a criticism of the Bronsted-Lowry theory is that it has no definition of neutrality.)
By the Law of Mass Action applied to the dissociation of water (see Section 10.4), then:
pN = 0.5 x pKw (where pKw is the ion product for water)4
Consideration of this equation is important as it provides us with a way to test the Davis, Reeves and Rahn hypothesis that intracellular pH equals pN (with consequent biological advantage of intracellular trapping of metabolic intermediates. The clue is that pKw'is very temperature dependent.
So pN is temperature dependent and if the hypothesis (ICF pH = pN) is correct then intracellular pH should change with change in temperature to maintain the predicted relationship.
An intracellular pH at about pN must surely apply to other animals (with body temperatures other than 37C) as there is no reason to believe that humans at 37C alone should be in a unique position. If this predicted change with temperature does occur, it would lend very strong support to the theory. So the next question is:
Measurements of intracellular pH in skeletal muscles have been carried out in several ectothermic animals which have been acclimatised at temperatures ranging from 5C to 31C. These all show the expected pH change: intracellular pH is maintained at about pN with change in temperature!!
It has been calculated that for the body to have this temperature-pH relationship requires certain things. There must be a buffer system with a pK which is approximately one-half that of water (because a buffer is most effective close to its pK) and which changes its pK so that it maintains this relationship as temperature changes. The buffer must be present in sufficient concentration and have certain chemical properties (e.g. delta H = 7 kcals per mole). For this system to work optimally, it also requires a constant CO2 content.
Experimental work has shown that protein buffering, largely due to the imidazole group of histidine is responsible for maintaining this temperature-pH relationship (aided by phosphate and bicarbonate buffering). Of all the protein-dissociable groups that are available, it is only the imidazole of histidine that has the correct pK and whose pK changes with temperature in the appropriate way.
The imidazole has a degree of dissociation (referred to as alpha) of 0.55 in the intracellular compartment and this remains constant despite changes in temperature (ie the pK is changing with change in temperature). This theory about the constancy of the imidazole alpha value as proposed by Reeves and Rahn has been termed the imidazole alphastat hypothesis.
[-What is the pKa of imidazole residues in proteins? There is no standard value for this in proteins. The pKa of each imidazole residue in a protein is affected by the local environment in the protein so the different histidine residues have different pKa values at any one temperature. This means that only an average or 'effective' pKa can be determined, and this will be different in different proteins. According to the diagram on page 57 in the book "Acid-Base Regulation and Body Temperature" (1985) (edited by Rahn H and Prakash O) the pKa of histidine is about 6.8 at 37C in animals. The diagram also shows it as equalling pN at different temperatures. As an actual example, a study of transferrin in 1991 (Yue KT et al) found its histidine residues to have an 'average value' of 6.56. The key points though are that the pKa is close to ICF pH (and pN) at each temperature so that despite the temperature changes, a constant degree of ionization (alpha) of the imidazole groups is maintained.-]
The other necessary condition for maintaining imidazole alpha constant is that the CO2 content in blood must be kept constant at different body temperatures. This means that ventilation must be regulated to maintain the imidazole alpha in the blood. It has been found experimentally that this regulation to maintain imidazole alpha constant in blood will result in imidazole alpha being maintained in other compartments (e.g. intracellular fluid) as well. The respiratory control that adjusts ventilation probably involves proteins whose activity is altered in an appropriate direction by an alphastat mechanism. Adjustment of ECF pCO2 is necessary as this maintains a constant relative alkalinity of the ECF relative to the ICF so there is constancy of the gradient for H+ across the cell membrane. In reality this does not mean that ventilation has to increase markedly with decrease in temperature because the reduced metabolic rate will automatically result in decreased CO2 production.
The alternative theory is the pH-stat hypothesis: this argues that the pH should be kept constant despite changes in temperature. This is the same as saying that ECF pH should be kept at 7.4 whether the temperature is 20C or 25C or whatever it is.
This controversy over whether the alpha-stat or the pH-stat theory is "correct" does have practical anaesthetic relevance in patients who are rendered hypothermic (e.g. while on cardiopulmonary bypass). What is the pH level to aim for in these patients? It seems that the alpha-stat theory is now widely accepted. This is probably related to the intellectual attraction of the theoretical arguments because major differences in outcome between groups of patients managed by the pH-stat or the alpha-stat technique have not been clear. Cells are capable of functioning despite the presence of a certain level of perturbation. Clinical studies have concentrated on which approach is best for the heart (myocardial outcome) and/or which approach is best for the brain (neurological outcome). The pH-stat aim to maintain a pH of 7.4 at the lower temperatures of hypothermic cardiac bypass is achieved by having a pCO2 level which is higher than that required for alpha-stat management. This means that from the alphastat point of view, pH-stat management results in a respiratory acidosis at the lower temperature. One effect is that the cerebral blood flow is higher at a given temperature with pH-stat management than it is with alphastat management. (See section 1.6.3)
The alphastat hypothesis is about maintaining alpha which means that the net charge on all proteins is kept constant despite changes in temperature. This ensures that all proteins can function optimally despite temperature changes. The importance of pH is not just about intracellular trapping of metabolic intermediates (small molecule effect) but also about protein function (large molecule effect). This affects all proteins, though enzymes usually figure prominently as examples. So, to answer the question about why pH is so important in metabolism involves these two reasons.
A final point: According to chemists, the situation concerning pH and temperature is actually quite complex: for example, the thermodynamic basis of pH measurement includes a term for the 'ground state potential' which must be arbitrarily defined at every temperature. This means that the absolute value of measured potential at any particular temperature cannot be precisely determined and thus that pH values obtained at different temperatures, strictly speaking, cannot be compared. This really is not a concern to the clinician.
As a example, consider the management of a patient who is cooled during open heart surgery.
A patient is cooled to 20C for cardiac surgery while on cardiac bypass. Imagine an arterial sample was drawn and analysed at 20C and showed pH 7.65 and pCO2 18 mmHg. Now if this same sample was analysed at 37C then the values would be pH 7.4 and pCO2 40 mmHg. So which value do you want reported to you?
The values for 37C can be interpreted against the known reference values for 37C and they would be considered to be normal. This is the alphastat approach and is equivalent to assessing the results against the appropriate reference range for 20C but without having to know what it is.
Alternatively, the values for 20C could be interpreted against the reference values for 37C. This is the pH-stat approach. The problem is partly a mistaken belief that the pH must be kept at 7.4 value at every temperature, and partly because the reference range of arterial pH is not known for any value other than at 37C.
[To avoid confusion, note that ALL blood gas machines measure all parameters at 37C then apply correction formulae to calculate what the values would be at the temperature of the subject.]
By the pH-stat approach then, it would be decided that this patient had a significant respiratory alkalosis and measures would be taken to correct this.
Clearly the two approaches can result in quite different therapies being applied.