1.6: Alphastat Hypothesis

1.6.1 Beyond Davis : the Alphastat Hypothesis

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:

What is the ideal intracellular pH?

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.

First Hypothesis: pH(ICF) = pN

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:

Is the actual intracellular pH as predicted?

According to Rahn, measurements confirmed that the mean intracellular pH of man is 6.8 at 37°C which is indeed the pH of neutrality (pN) at that temperature!

Before going further we need to understand:

What is meant by 'neutrality'?

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:

\(pN = 0.05 \times pKw\) where pKw is the ion product for water⁴

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 37°C) as there is no reason to believe that humans at 37°C 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:

Does intracellular pH change with temperature in order to remain equal to pN at each temperature? (And if so: How does this happen?)

Measurements of intracellular pH in skeletal muscles have been carried out in several ectothermic animals which have been acclimatised at temperatures ranging from 5°C to 31°C. 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 (eg ΔH = 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.

Alphastat Hypothesis The degree of ionisation (alpha) of the imidazole groups of intracellular proteins remains constant despite change in temperature.

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 (eg 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.

Shouldn't neutral pH be 7.0? Many people have a belief that a pH of 7.0 is the 'neutral pH' and consequently have trouble understanding how the change in pN with temperature can be possible. By definition, neutrality is when the solution has [H^+] = [OH^-]. At a temperature of 25°C, this condition does indeed occur in pure water when pH is 7.0 and this is the basis of the common high-school teaching. But, as indicated in the calculations in Section 10.4, this condition of [H^+] = [OH^-] is based on the amount that water dissociates and occurs when pH = 0.5 x pKw. This pH (the pN) is dependent only on the ion product of water pKw' (which indicates how much water dissociates); the value of this term is very temperature dependent. At a higher temperature, water still dissociates to produce equal amounts of H^+ and OH^- (the neutral condition) but there is more dissociation so the [H^+] is higher (i.e. pH is lower). At 37°C, pN is 6.8

Alpha-stat versus pH-stat

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 20°C or 25°C or whatever it is.

Blood gas results: To temperature correct or not? The pH-stat approach is also implicitly the approach used by anyone who temperature corrects blood gas results to the patient's temperature but interprets the values against the reference range relevant to 37°C. No reference range is available for temperatures other than 37°C but the pH-stat approach is that the reference range for 37°C is valid at all temperatures. The alpha-stat approach is to never temperature correct blood gas results. Do not report the patient's temperature on the request form, or if doing the gases yourself, only enter the temperature as 37°C no matter what the patient's actual temperature. The results from the blood gas machine must then be those as measured in the machine at 37°C. The reference range for 37°C is obviously the correct one to apply when assessing these results. You should be careful because if you or a colleague indicate the patient's actual temperature on the blood-gas request form, the lab technician will enter this temperature into the blood-gas machine and the printed report will have the values calculated for this patient temperature (i.e. the 'corrected' values). Note that whatever the actual patient temperature, the machine is always thermostatted to 37°C and all measurements are consequently performed at 37°C. For other patient temperatures, the computer in the machine uses various correction formulae to calculate what the values for the parameters would be at the patient's actual temperature. The pH correction used in most machines is the Rosenthal correction factor. The manual for the blood gas machine has a complete listing of the formulae it uses for all calculated values.

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 (eg 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.

Summary: The two reasons why pH is so important for metabolism Effect on small molecules: Intracellular trapping of intermediary metabolites (iethe Davis hypothesis) Effect on large molecules (proteins): Maintaining optimal protein function both intracellularly and extracellularly. Consequently, the body regulates pH very tightly

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.

Example: Alphastat Management during Induced Hypothermia

As an example, consider the management of a patient who is cooled during open heart surgery.

A patient is cooled to 20°C for cardiac surgery while on cardiac bypass. Imagine an arterial sample was drawn and analysed at 20°C and showed pH 7.65 and pCO₂ 18 mmHg. Now if this same sample was analysed at 37°C then at that temperature, the values would be pH 7.4 and pCO₂ 40 mmHg. So which value do you want reported to you?

The values for 37°C can be interpreted against the known reference values for 37°C 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 20°C but without having to know what it is.

The values for 20°C could also be interpreted against the reference values for 37°C. [Actually the blood gas machine measures at 37°C then applies the correction formulae and reports what the values would be if measured at 20°C]. This is the pH-stat approach (ie the idea is that the pH must be kept at the semi-magical 7.4 value at every temperature).

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.

Summary of important aspects of Chapter One

• The approach discussed in the majority of this book is the traditional approach to acid-base physiology as this is still almost the only approach discussed in physiology texts. An alternative approach is the Stewart quantitative approach which is derived from basic physicochemical principles - though now well supported by evidence this approach is more difficult to use in everyday clinical practice - this approach is discussed in Chapter 10.
• The Bronsted-Lowry acid-base theory is normally used in biology.
  Definitions: - An acid is a proton donor & a base is a proton acceptor
• Hydrogen ions (ie protons) do not exist free in solution but are linked to adjacent water molecules by hydrogen bonds. Because of this interaction it is the activity (or effective concentration) of hydrogen ions rather than the actual concentration that is important for biological effects
• pH is the quantity used to assess the acidity or alkalinity of a solution. It is defined as the negative log of the hydrogen ion activity. It is measured using an ion-selective glass electrode
• pH is typically 7.4 in plasma ([H⁺] about 40 nmol/l) but lower values of pH are found intracellularly.
• [H⁺] in the body is tightly regulated. The physiological advantages principally involve providing conditions for optimal intracellular function, particularly:
  - intracellular trapping of metabolite intermediates is maximised at an intracellular pH of neutrality
  - activity of all proteins (incl enzymes) is optimised because their net charge is kept constant
• In the body, the intracellular pH changes with temperature such that the intracellular pH remains at or close to pH of neutrality. This is achieved by appropriate temperature-induced changes in the pK of the imidazole group of histidine. The idea that the degree of dissociation (known as alpha) of imidazole remains constant despite changes in temperature is known as the alpha-stat hypothesis. This has implications for clinical practice (e.g. management of hypothermia during cardiopulmonary bypass; and not temperature-correcting values in ABG reports.)

References

• Reeves R. An imidazole alphastat hypothesis for vertebrate acid-base regulation: tissue carbon dioxide content and body temperature in bullfrogs. Respir. Physiol. 14,219 -236.
• Rahn H Body temperature and acid-base regulation Pneumonologie. 1974;151(2):87-94
• Davis B On the importance of being ionized Arch Biochem Biophys 1958; 78: 497-509
• Austin J and Cullen G Hydrogen ion concentration of the blood in health and disease Medicine 1925; 4: 275-343. (NB: In this article these authors introduced the symbol pN to represent 'pH of neutrality' - see page 299)