THE pH OF INTRACELLULAR WATER* Thomas C. Butler, William J. Waddell, Doris T. Poole

Department of Pharmacology, University of North Carolina School of Medicine,

Chapel Hill, N . C .

The water contained within cellular membranes comprises nearly half of the total body weight of a mammal, and it is approximately double the amount of the water situated outside cells. Of the various fluid compartments of the body, only plasma and a few minor compartments are accessible for direct and unequivocal chemical analysis. There is a striking contrast between the rather adequate knowledge of the chemistry of plasma and the much less adequate knowledge of the chemistry of the larger intracellular compartment.

One of the chemical characteristics of the intracellular water that is of the greatest interest is its pH value. It is to be emphasized that pH is a practical scale of acidity defined in terms of standards, and that it does not furnish information necessary for the calculation of absolute hydrogen ion activity. Furthermore, a t physiological values of p H , the concentration of the free hydrogen ion is too low to play any significant role in physical processes. Failure to appreciate these facts has beguiled some workers, through a misguided obsession with the hydrogen ion, to the deplorable and unjustifia- ble practice of calculating hydrogen ion concentrations from measured pH values. The significance of pH is that it is an index of the chemical potential of protons, not only the protons existing free but also the dissociable protons incorporated in proton donor molecules. Even when free protons are present in negligible amounts, those in proton donor molecules may be in abundance. pH can be considered an expression of the ease with which protons may be released from proton donors and as an index to the ratios of proton donors to proton acceptors. Since numerous biochemical reactions involve the transfer of protons or are influenced by the availability of protons it is evident that no adequate study of such reactions can be made without knowledge of the chemical potential of the proton.

Many assumptions have been made concerning the pH of intracellular water and many attempts have been made to measure it, but actual knowledge of the intracellular pH of mammalian cells has been meager and unsatis- factory. The various methods that have been used to measure intracellular pH have been reviewed by Caldwell (1956). These methods include measure- ments on preparations of broken cells, measurements on fluid withdrawn from cells, observations with visible indicators, measurements with micro-

*The investigations from this laboratory described in this paper were supported in part by Public Health Service Research Grant, No. NB 00384, from the National Institute of Neurological Diseases and Blindness.

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electrodes introduced into cells, and measurements of intracellular and extracellular concentrations of weak organic acids and bases. Some of these methods have quite limited applicability, and the validity of the results obtained with most of them is subject to question.

The method probably least subject to theoretical objections and potentially having the widest range of applicability is that based on the distribution of weak organic acids and bases. This method is based on the assumption that cellular membranes are freely permeable to the undissociated forms of weak organic acids and bases. There is a great deal of evidence that cellular membranes in general, while apparently impermeable to the ionic species of acids and bases, are freely permeable to the undissociated forms.

Let us consider two solutions of different pH separated by such a membrane. If a weak organic acid is introduced into the system, the con- centration of the undissociated form will a t equilibrium be equal on the two sides. If this concentration is designated as unity, the concentration of the ionic form is 1 0 ’ p H - p K ’ l and this concentration will not be equal on the two sides of the membrane unless the pH is equal on the two sides. The total concentration that would be determined analytically, i.e. the sum of undis- sociated and dissociated forms, will be higher on the side on which ionization is more extensive, viz. the side of higher pH. In the case of a weak base, the more extensive ionization and the higher total concentration would be on the side of the lower pH. If we can determine analytically the total concentration of the acid or base on both sides of the membrane and if we can measure pH on one side, we can calculate the pH on the other side. Analytical deter- mination of an organic compound in intracellular water is generally more feasible and not subject to the same difficulties and objections as a direct pH determination. This principle thus affords us a method for an indirect calculation of intracellular pH. Waddell and Butler (1959) give equations by which intracellular pH may be calculated from measurable quantities: the pK’ of the indicator compound, the concentrations of the indicator compound in a tissue sample and in the extracellular phase, the intracellular and extracellular water of the tissue sample, and the pH of the extracellular phase.

If the intracellular pH is lower than the extracellular pH, as is the case for muscle, a basic compound will be a more sensitive indicator than an acid. However, it might be expected that the electrostatic interaction between the cationic form of a basic compound and the anionic centers of proteins and other large molecules might significantly affect the distribution of a base. Probably, then, suitable indicators will be found only among acids.

Until recently nearly all the work based on this principle has utilized carbonic acid as the indicator compound. This was used as early as 1922 by Warburg to measure the pH of erythrocytes and was used in 1928 by Fenn to measure the pH of frog muscle. In 1959 Waddell and Butler, in a consideration

Butler et al.: The p H of Intracellular Water 75

of the theoretical basis of the use of distribution of acids and bases to cal- culate intracellular pH, suggested that 5,5-dimethyl-2,4-oxazolidinedione (DMO) has to an almost ideal degree the attributes to be desired in an indicator of intracellular p H and should have some advantages over carbonic acid. It is an acid with a pK’ of 6.13 a t 37°C. and ionic strength of 0.16. With apK’ this low, it is almost as sensitive an indicator as is possible for an acid. It has very low toxicity. It is not bound to protein and does not enter fat. It is not metabolized. When the urine is acid, it is very slowly excreted. It can be determined analytically by an ultraviolet spectrophotometric method. DMO can readily be prepared labeled with CI4 or H3; and since it i s not metabolized, the measurement of the radioactive compound is not compli- cated by the presence of metabolic products.

If the interior of the cell is inhomogeneous with respect to p H or if the different cells of a tissue sample differ among themselves, the calculated p H is not a mean value in the mathematical sense. This “overall” pH can be given no simple mathematical interpretation except that it is a value lying between the p H of the most acid region and that of the least acid region in the cells. Despite this limitation to its interpretation, the overall p H value does furnish valuable information concerning conditions in the cells. Further- more, i t is the changes in this value that occur under various conditions that are especially meaningful. It is hoped that techniques can ultimately be developed for autoradiography with DMO-H3 that will permit study of p H gradients within the cell.

The DMO method for measurement of intracellular p H should theoreti- cally be applicable to any type of cell. However, with some tissues it may not be feasible to measure extracellular water or to measure the p H of the extracellular phase in contact with the cells. Although the method has been applied to brain, the fact that brain contains more than one type of cell renders the interpretation of the results questionable. Furthermore, uncertainty as to the extracellular water of brain introduces considerable uncertainty into the calculations.

The first application of the DMO technique to the measurement of intracellular pH was the study by Waddell and Butler (1959) of skeletal muscle of the dog. Subsequently a number of other workers have used DMO for the measurement of intracellular p H in various types of cells. Some of the tissues and cells that have been studied include the perfused turtle heart (Waddell & Hardman, 1960), skeletal muscle of the potassium deficient rat (Irvine et al., 1961), the rat diaphragm in uitro (Miller et al., 1963), dog brain (Kibler et al., 1964), normal and leukemic leukocytes (Block & Rall, 1962), erythrocytes (Thomason, 1963; Sanslone & Muntwyler, 1964), and the Ehrlich ascites tumor cell (Poole et al., 1964). An interesting application is the measurement of an overall p H value for the whole body (Robin et al., 1961; Manfredi, 1963). Here the obvious inhomogeneity of the intracellular water of the whole body with regard to p H is disregarded in the hope of

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finding a value variations of which under physiological and pathological conditions might yield useful information.

In all of the in uiuo studies cited above, unlabeled DMO was used, and i t was determined by a n ultraviolet spectrophotometric method. I n the in uitro studies of Block and Rall, Miller et al., and Poole et al., CI4-labeled DMO was used.

The values for pH of muscle as determined by the DMO method are in general agreement with older determinations made by the carbon dioxide method. Direct comparison of the two methods by Miller et al. showed no large differences. Agreement between the values of pH calculated from the distribution of DMO and from the distribution of carbon dioxide supports confidence in the validity of the measurements made by both methods.

The various measurements of intracellular pH that have been made by the DMO method will not be reviewed here, and only certain aspects of the results will be commented upon. The pH of the intracellular water of muscle, brain, the body as a whole, and the Ehrlich ascites tumor cell is affected to a greater degree by variation of extracellular pH occasioned by change of carbon dioxide tension than by variation of extracellular pH at constant carbon dioxide tension. This is a consequence of the free permeability of all cellular membranes to carbon dioxide. However, change of extracellular p H does affect intracellular p H independently of change of carbon dioxide tension. This is seen in the experiments of Waddell and Butler (1959) with dog muscle, but it can be seen more clearly in in uitro experiments in which the extracellular pH can be varied over a wider range, such as the experiments of Waddell and Hardman (1960) with the turtle heart and those of Poole et al. (1964) with the Ehrlich ascites tumor cells. That intracellular pH is not necessarily correlated with extracellular pH in any predictable manner has been impressively demonstrated by work in our laboratory with the Ehrlich ascites tumor cell. In the glycolyzing cell, intra- cellular pH may be rising when extracellular pH is falling, and glucose in the presence of an inhibitor of glycolysis may cause a fall of intracellular pH without change of extracellular pH. Such results emphasize the importance of study of intracellular pH, not merely of extracellular pH, if any adequate insight into the metabolic events in the cell is to be attained.

Despite some possible uncertainties and reservations in its interpreta- tion, we believe that the calculation of intracellular pH from the distribution of DMO has potentialities for increasing knowledge in an area in which knowledge has hitherto been meager and in which fallacious speculation has been abundant. I t should be useful in many fundamental studies of physiological and biochemical mechanisms in normal and pathological conditions.

Butler et al.: The pH of Intracellular Water

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