Canadian Journal of Physiology and Pharmacology
Intracellular pH Regulation and the Acid Delusion
Journal: Canadian Journal of Physiology and Pharmacology
Manuscript ID cjpp-2020-0631
Manuscript Type: Review
Date Submitted by the 29-Oct-2020 Author:
Complete List of Authors: Magder, Sheldon; Mcgill University Health Centre Magder, Alexandr; Royal College of Surgeons in Ireland Samoukovic, Gordan; McGill University Faculty of Medicine, critical care
Is the invited manuscript for consideration in a Special Not applicable (regular submission) Issue: Draft
Keyword: strong ion, acid base, stewart, buffer, sodium hydrogen exchanger
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4 Intracellular pH Regulation and the Acid Delusion
5 Sheldon Magder, Alexandr Magder, Gordan Samoukovic 6 McGill University Health Centre 7 Department of Critical Care 8 687 Pine Av W 9 Montreal, Quebec 10 H3A 1A1 11 12 13 14 15 Address correspondence to: 16 Draft 17 S Magder 18 Royal Victoria Hospital 19 687 Pine Av W 20 Montreal, Quebec 21 H3A 1A1 22 [email protected] 23 24 25 26 27
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29 Abstract
30 The concentration H+ ([H+]) in intracellular fluid (ICF) must be maintained in a narrow range in
31 all species for normal protein functions. Thus, mechanisms regulating ICF are of fundamental
32 biological importance. Studies on the regulation of ICF [H+] have been hampered by use of pH
33 notation, failure to consider the roles played by differences in the concentration of strong ions (
+ - 34 SID), the conservation of mass, the principle of electrical neutrality and that [H ] and [HCO3 ]
35 are dependent variables. This argument is based on the late Peter Stewart’s physical- chemical
36 analysis of [H+] regulation reported in this journal nearly forty years ago. We start by outlining
37 the principles of Stewart’s analysis and then provide a general understanding of its significance 38 for regulation of ICF [H+]. The system mayDraft initially appear complex, but it becomes evident that 39 changes in SID dominanate regulation of [H+]. The primary strong ions are Na+, K+ and Cl-, and
40 a few organic strong anions. The second independent variable, PCO2, can easily be assessed. The
41 third independent variable, the activity of intracellular weak acids ([Atot]), is much more complex
42 but largely plays a modifying role. Attention to these principles potentially will provide new
43 insights into ICF pH regulation.
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45 Key Words: strong ion, acid-base, Stewart, buffer, sodium-hydrogen exchanger
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51 Introduction
52 Close to 40 years ago in this journal the late Peter Stewart laid out the principles of a
53 quantitative physical-chemical approach to the understanding of the determinants of hydrogen
54 ion concentration ([H+], pH, and acid-base in the water-based biological solutions (Stewart
55 1983). His work created a lot of controversy at the time, but over the past decades his approach
56 has become increasing accepted in clinical medicine for analysis of acid-base disturbances in
57 serum (Berend et al. 2014; Gilfix et al. 1993; Jones 1990a, b; Kellum et al. 1995; Magder and
58 Emami 2015). Stewart’s detailed monograph on the subject (Stewart 1981) also has been
59 republished in a new edition (Kellum and Elbers 2009). However, there still is a striking lack of 60 consideration of the principles he laid outDraft for analysis of intracellular fluid (ICF) [H+] regulation, 61 even though ICF volume is twice as large as extracellular volume and it is where changes in [H+]
62 ultimately have their greatest biological implications. As will be seen, the reason why we believe
63 that this is of importance is that when physical-chemical principles are not taken into account,
64 [H+] is considered as an independent variable when it actually is a dependent variable, and the
65 factors that really determine the regulation of ICF [H+] such as the role of strong ions, the
66 importance of electrical neutrality and the behavior of weaker electrolytes are not considered.
67 As a result, many experimental errors occur in experimental reasoning and important
68 mechanisms are missed.
69 Of historical interest, in an early examination of temperature effects on pH in the blood of
70 in the non-endothermic alligator , Austen et al showed in 1927 that changes in the concentrations
71 of Na+, K+ and Cl- occur are part of the process regulating pH in whole blood presumably by
72 shifts between red cells and plasma(Austin et al. 1927). A more recent exception is the work of
73 Roberg (Robergs et al. 2004). He analyzed how the multiple metabolic biochemical changes that
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74 occur inside muscle fibres during exercise alter intracellular [H+] and the limitation of the simple
75 terms lactic acid and lactic acidosis. However, even his work fails to consider the considerable
76 importance of strong elements in intracellular solutions and their potential to move into and out
77 of cells and into intracellular compartments (Demes et al. 2020). He also did not consider the
78 importance of the principle of electrical neutrality and thus the need for a quantitative analysis of
79 all positive and all negative charges. His work, though, does indicate that it is unlikely that
80 complexity of the rapid changes in electrolyte characteristics during a process such as exercise
81 will allow a precise prediction of changes in [H+] in cells. We will come back to his work later,
82 but our goal is less ambitions. In this review, we believe that what is important is to better 83 understanding the factors that can regulateDraft intracellular pH (pHi). Hopefully this will lead to an 84 appreciation of new processes that are critical for the regulation of intracellular [H+] and provide
85 some principles that should be considered in studies of intracellular [H+].
86 The central thesis of this essay, which is based on the work of Peter Stewart(Stewart
87 1978, 1981, 1983), is that it is actual [H+] that we must care about in biological solutions.
88 Emphasis on “acids” and “bases” rather than maintenance of [H+] in a given range, the
89 definitions used for acids and bases, and the use of pH notation, obscure important mechanistic
90 insights into the regulation of [H+]. Key omissions are failure to account for the role of water
91 itself and ignoring the conservation of mass, the principle of electrical neutrality, and most
92 importantly, that all the components of solutions interact and together determine to [H+] and
- 93 [HCO3 ] which are dependent and not independent variables.
94 There are over 1014 cells in the body, each with its own internal environment, and there
95 are even variations in sub-compartments within cells with pH values that vary from 8.0 in
96 mitochondria to 5.0 in lysosomes. Thus, this discussion is about a “generic” cell, and its
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97 “standard” intracellular cytoplasmic fluid (ICF), under quasi-steady state conditions, while
98 appreciating that there are large variations in cytoplasmic composition among cell types and in
99 sub-compartments of the cell (Bal et al. 2012; Demes et al. 2020; Kyne and Crowley 2016;
100 Luby-Phelps 2013). A key principle in a physical chemical analysis is that [H+] concentration is
101 unique for each fluid compartment. It thus is not meaningful to consider total body [H+] balance
102 or even [H+] in a whole cell. The factors that determine [H+] in each compartment can interact
103 with other compartments by moving from one compartment to another by moving down a
104 concentration gradients or by being actively moved (Demes et al. 2020), but the components
105 determining [H+] in each compartment are unique at any given instant. Hopefully, attention to 106 simple basic concepts will lead to more Draftdirected experimental approaches for analyzing [H+] 107 regulation in cells and their sub-compartments. Our objective is to provide a roadmap for use of
108 physical-chemical concepts in future studies on the regulation of ICF [H+].
109 Importance of Hydrogen Ion
110 [H+] is maintained between 6 to 16 x 10-8 mol/L (pH 6.8-7.2) in almost all cells
111 throughout the animal kingdom, from the eggs of sea urchins to the cytoplasm of human cells
112 (Boron 2004; Burggren and Bautista 2019; Janis et al. 2020; Putnam 1998; Roos and Boron
113 1981). Even most bacteria regulate cytoplasmic pH in the 6.5 to 7.0 range (Booth 1985). [H+]1 is
114 extremely low compared to other major elements such as extracellular sodium (Na+) and
115 intracellular potassium (K+), which are in the range of 0.140-0.150 mol/L. To put this low [H+]
116 value into a more tangible context, there is one H+ per 500 million water molecules in a glass of
117 pure water at standard temperature and pH 7.0 (Ball 2001). Despite being very low, [H+] must be
118 maintained in a very tight range for normal cell function. This is because of the high charge
1 [ ] will be used to denote concentration
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119 density of a proton (H+) relative to its mass. This charge density creates a strong electric field
120 gradient, which is capable of altering the tertiary configurations of neighboring organic
121 molecules with consequent effects on a multitude of cellular functions, including intracellular
122 metabolism, cytoskeletal shape and mobility, muscle contraction, cell-cell coupling, membrane
123 conductance, intracellular signaling, cell activation, growth, proliferation, and regulation of cell
124 volume (reviewed in(Janis et al. 2020; O'Neill 1999; Putnam 1998; Robergs et al. 2004; Roos
125 and Boron 1981)).
126 Use of the terms H+ and hydroxyl ion (OH-) needs clarification. Water is always
127 dissociating and re-associating into its component ions, H+ and OH- (Bal et al. 2012). It is 128 generally agreed that H+ and OH- do notDraft actually exist as independent entities, but rather form 129 solvation structures with water. In the case of H+, the hydrated form interconverts from the Eigen
+ + + + - 130 cation, H9O4 = (H20)3H3O , to the Zundel ion, H5O2 = H20-H -OH2 (71), and OH interconverts
- - 131 during the solvation phase between H9O5 and H7O4 (Botti et al. 2003). What actually is
132 measured is the activity of H+ and OH- in these compounds, but it is much more convenient to
133 use H+ and OH- as a simple notation for the activity and chemical potential of these more
134 complex structures.
135 Stewart’s physical-chemical analysis
136 Before considering [H+]regulation in cells, it is necessary to review the determinants of
137 [H+] in a water solution as analyzed in detail by the late Peter Stewart (Kellum and Elbers 2009;
138 Stewart 1981, 1983). Stewart went back to basic equilibrium equations for the components of
139 biological solutions as expressed in standard physical-chemistry textbooks (for example (Tinoco
140 et al. 1995). To begin, it is important to appreciate that discussion of [H+] in biology is
141 essentially always about [H+] in water-based solutions. Stewart, as have others (Ricci 1952),
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142 defined an acid solution in water as one in which [H+] is greater than [OH-], a neutral solution as
143 one in which [H+] equals [OH-], and an alkaline solution as one in which [H+] is less than [OH-].
144 As will be seen, these simple and seemingly obvious definitions have great significance for the
145 behavior of water-based solutions. Importantly, it means that a pH value does not indicate
146 whether a solution is an acidic or basic. Stewart used the approach put forth by Arrhenius in
147 1883, and others later (Bates 1966; MacConnachie 1970; Ricci 1952) define an acid and base.
148 He defined an acid as a substance that when added to a solution brings about an increase in [H+],
149 and a base as a substance that when added to a solution brings about a decrease in [H+]. An acid
150 does this by dissociating in the solution to yield an H+ and an anion, or by associating with an 151 OH- and thereby increasing the dissociationDraft of water, which results in greater [H+] relative to 152 [OH-] as in the Arrhenius’ definition of an acid. A base acts by dissociating to yield a cation and
- + + + 153 an OH or by associating with H and lowering free [H ] (an example of the latter is NH3/NH4 ).
154 As will be seen, by this definition strong negative ions are not a “base” as commonly stated but
155 have an acidifying effect.
156 Stewart identified three independent determinants of [H+] in biological solutions: the
157 strong ion difference (SID), the partial pressure of carbon dioxide (PCO2), and the amount of
158 concentration of weak electrolytes. The first of these, the SID, is the most important, but it is
- 159 neglected in the traditional PCO2/HCO3 approach. Electrolytes are substances that exist as ions
160 in aqueous solutions. Strong electrolytes are almost completely dissociated in aqueous solutions.
161 Examples are sodium (Na+), potassium (K+) and chloride (Cl-). NaCl does not exist in an
162 aqueous solution in any significant amounts, but rather the solution has Na+ and Cl-. Weak
163 electrolytes are substances that only partially dissociate in an aqueous solution because their low
164 dissociation constants allow for the existence of the parent compound as well as the dissociated
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165 charged components. At equilibrium the rate of dissociation equals the rate of recombination.
166 This equilibrium is exponentially related to the standard free energy change per mole of the
167 reaction, which determines how much of each species exists at equilibrium. A fundamental
168 physical-chemical principle of electrical neutrality which indicates that in a macro-solution all
169 positive charges must equal all negative charges. If the equivalent activities of strong positive
170 and negative electrolytes differ, which is called the strong ion difference (SID), the charge
171 difference creates a strong electrical force. This electrical force must be dissipated to restore
172 thermodynamic equilibrium in the solution. This occurs by alterations of the dissociation of
173 partially dissociated substances including weak acids and water itself until a new equilibrium is 174 reached and electrical neutrality is re-established.Draft These distortions of the equilibrium 175 dissociations of weak electrolytes and water determine the final [H+] of the solution. The SID is
176 the major factor in biological solutions affecting [H+], but is not accounted for in the simple
177 Henderson-Hasselbach equation nor in the detailed analysis by Robergs of the contribution of
178 metabolic products to [H+](Robergs et al. 2004). A proper analysis must include an accounting of
179 all positive and negative charges. For practical purposes, strong elements dominate the process
180 and they are in millimolar range. Thus only compounds with charges in this range have a
181 significant contribution. Of historical interest, Henderson appreciated that the strong ion Cl- is a
+ 182 critical element that alters the dissociation value of H2CO3 and the predicted [H ] in blood. He
183 even included it in his analytic diagram. (Henderson 1921).
184 Another key principle is the conservation of mass (Stewart 1981). This requires that the
185 sum of all dissociated components of a substance in an aqueous solution must remain constant
186 unless the substance is added or removed from the solution, or the substance is generated or
187 metabolized within the solution. For the elements in the ICF, such as Na+, K+, or Cl- a change in
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188 their mass only can occur by moving into or out of the cell. In in some cases Na+, Cl- and Ca2+
189 can be “effectively” removed by being sequestered by proteins but quantitatively this usually is
190 small. Additionally, strong organic molecules produced or metabolized by cells, such as lactic
191 acid, keto-acids, almost all amino acids, and acetic acid, are almost completely dissociated at
192 intracellular [H+] and thus behave as strong ions and contribute to the SID. This makes ICF
193 solutions much harder to analyze than plasma in which under normal conditions the only
194 significant weak acids are carbonic acid and albumin. Under disease conditions, keto-acids,
195 phosphoric acid, sulfuric acid, formic acid and some others can contribute strong negative ions to
196 the plasma but these all can be easily measured. 197 The second independent variableDraft determining [H+] is the concentration of weak 198 nonvolatile electrolytes (Stewart 1981). Their effect must be considered based on the
199 concentrations of the associated form, which we will call [HA], the anionic form [A-], and the
- + 200 sum of [HA] and [A ], which we will call [Atot]. Their effect on [H ] of a solution depends upon
201 their dissociation constant of HA and the activity (ie charge) of the dissociated anion, A-.
202 Albumin is the dominant non-volatile weak acid in plasma and its role has been determined
203 empirically and theoretically (Figge et al. 1992; Figge et al. 1991). Unfortunately, the total
204 activity of weak ions in the ICF is very difficult to assess because of the great number of weak
205 ions, which have large concentrations, activities that change with metabolic and catabolic
206 processes, and dissociation constants that change with [H+] itself (Robergs et al. 2004). The
207 substances also have the potential to move into or out cells as well shift between intracellular
208 compartments. Thus, a rigorous analysis of regulation of ICF [H+] can only be done with empiric
209 studies of the content for each cell type under strictly controlled standard conditions, and even
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210 then, as an approximation. Perhaps it will have to be describe as a probability as in quantum
211 chemistry analysis.
212 The third independent variable is CO2 (Stewart 1981). When CO2 dissolves in water it
+ - 213 forms the weak acid H2CO3, which dissociates into H and HCO3 . The characteristics of this
+ 214 system explain why CO2 has such a central role in [H ] regulation. First, CO2 is produced along
215 with H2O as part of the electron sink for the reduction of O2 in aerobic metabolism. There thus is
216 a constant source of CO2 in aerobically functioning cells. Furthermore, even the low PCO2 in air
- + 217 provides sufficient [HCO3 ] to alter [H ] without considering CO2 produced in cells. The PCO2 in
- 218 the atmosphere of only 0.3 mmHg results in [HCO3 ] in pure water at a pH of 7.4 of around 0.19 219 mEq/L. Although this is a small numberDraft compared to the 24 mEq/L in blood, it is almost 200 220 times the concentration of [OH-] in bodily solutions. Without this small amount of CO2 in air,
221 limestone rocks, coral reefs and mollusk shells would disappear by evaporation over time.
222 Second, CO2 is highly diffusible, and with a few possible exceptions (Waisbren et al. 1994),
223 easily passes through cell membranes. This means that total CO2 can be regulated by the
224 circulation and ventilation. When CO2 is kept at a constant value by neural control mechanisms,
225 or by maintaining a constant ambient PCO2 for cells in culture, CO2 in all its forms thus acts as
226 an independent variable in the regulation of [H+] because its mass is conserved. Third, the pKa of
227 the dissociation of dissolved CO2 and production of H2CO3 is 6.35, which is close to the
228 biological set point of 7.4 in plasma and 7.0 to 7.2 inside cells. Without CO2, biological solutions
+ 229 would have a much lower [H ] (higher pH). CO2, thus provides an acidifying effect that counters
230 the alkalinizing effect of a positive SID.
231 As is the case with the analysis of non-volatile weak electrolytes, analysis of the effect of
+ 232 PCO2 on [H ] must consider the total CO2 mass in all its forms in the solution. When the system
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- 233 is “open” and the external PCO2 is held constant, all forms of CO2, including HCO3 and H2CO3,
234 must be in equilibrium with this regulated PCO2 and the mass of CO2 becomes fixed. Addition of
- 235 any form of CO2, including HCO3 to the body or cells in culture surrounded by a fixed ambient
+ 236 PCO2 only transiently alters [H ] of the solution because equilibrium must again occur with the
237 ambient PCO2 and the other components of the solution. In a solution with a negative SID,
- 238 [HCO3 ] is essentially zero because H2CO3 is not dissociated. In a solution with CO2, a positive
- - 239 SID and no weak acids, SID approximates [HCO3 ] except for the very small [OH ]. If a weak
- - 240 acid (HA) also is present, SID is equal to the sum of [A ] and [HCO3 ]. However, when there is
241 no ventilation or no blood flow to regulate the total amount of carbonate species, and the system 242 is closed to the outside, carbonate speciesDraft accumulate as they are produced by normal - 243 metabolism. They also accumulate if HCO3 is added to the solution in the form of NaHCO3. In
244 these situations, the total of all CO2 products acts as an independent variable. This is a very
245 important factor to keep in mind when studying isolated tissues or cells. If CO2 flux is not kept
246 constant as occurs in vivo, the behavior of carbonate species is very different from the in vivo
247 situation.
248 What is an acid?
249 With these physical-chemical principles of biological solutions in mind, we can turn to
250 the meaning of an “acid”. The terms acid and base were in use long before the development of
251 modern concepts of chemistry and the understanding of the existence of molecules and ions. The
252 history is wonderfully reviewed by MacConnachie (MacConnachie 1970) and summarized from
253 his work here. The terms acid and base were initially used to describe physical phenomena such
254 as taste. Sour substances were called acids and the word “acid” is derived from the Latin word
255 meaning sour; in contrast, bases are bitter or bland. Acids turn litmus paper red and bases turn it
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256 blue. The term “base” was introduced by Lavoisier to describe the primary or “basic” elements in
+ 257 families of substances such as NaOH, NaHCO3 and NaCl that can replace H to form a salt.
258 Lavoisier also gave hydrogen its name. Because he realized that H+ is a component of water, he
259 called it “water former”, and considered it a “base” of water. The concept of ionization evolved
260 in the late nineteenth century, and building on the work of Faraday and Clausius, Arrhenius
261 elaborated the concept of charged electrolytes. He also introduced the concept of the hydrogen
262 ion, H+, and defined an acid as a substance that increases [H+] of a solution and a base as a
263 substance that decreases it (Waddell and Bates 1969). A greater amount of acidity has since been
264 used to indicate increased [H+] but it should be appreciated that it does not mean that the solution 265 is acidic as defined by Arrhenius. Draft 266 Sorensen introduced the logarithmic pH terminology at the turn of the twentieth century
267 (Waddell and Bates 1969). It has the advantage of dealing with the large range of possible [H+]
268 and gives a tangible value for the very low [H+] in water. The output voltage signal obtained with
269 “pH meters” varies with a logarithmic function of [H+] and thus the logarithmic scale of pH is
270 reasonable. Thirdly, the mass action analysis of weak electrolytes is related to ratios of the
271 products and therefore described well by logarithms (Davis 1967; Waddell and Bates 1969).
272 Unfortunately, pH terminology also adds much confusion. The formula for pH, which is pH= -
+ 273 log10([H ], includes a reciprocal and a logarithmic transformation, which are both non-linear.
274 This makes it difficult to intuitively relate a change in pH by titration with a strong ion to a
275 change in [H+]. The importance of this is that the changes in [H+] for a change in linear and not
276 logarithmic and thus obscured by the pH notation. Furthermore, normal physiological conditions
277 span a very narrow range of pH, usually from 6.5 to 7.5, with most changes being in the decimal
278 range, so that the full range pH scale does not add much value in biology. It is argued that pH
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279 electrodes actually measure activity of H+ rather than its concentration and that activity is the
280 factor needed for calculating chemical equilibrium reactions, and not the concentration of H+.
281 The importance of using activity rather than the concentration was raised at the time of
282 Arrhenius’ presentation of strong electrolytes and dealt with by Debye and Huckel who
283 introduced the concept of activity coefficients (1966a; 1966b; Davis 1967). Stewart’s analysis,
284 too, is based on activity (Eq/L) rather than concentration (Mol/L), for the importance of activity
285 versus concentration is true for all reactants in the solution (Stewart 1981).
286 Although pH 7.0 is often thought of as indicating a neutral solution, this is only true for
287 pure water under standard conditions. In any other solutions, including non-standard water, 288 deviations from pH 7.0 do not indicate whetherDraft the solution is acidic or basic based on the 289 definition of an acid solution as one in which [H+] is greater than [OH-]. For example, pH of a
290 beaker of pure water at 24°C is 7.0, whereas pH of the same beaker at 37° C is 6.86, but the
291 solution is not “acidic” because [H+] still equals [OH-]. The pH is lower because the dissociation
292 constant of water increases with increases in temperature and increases [H+]. The “acid-base”
293 terminology evolved around the rapid change in pH that occurs when titrating a solution with
294 strong ions and the SID changes from a negative value to a positive value (figure 1a). When SID
295 is negative, except for a very small range of SID (i.e. -10-6 mol/l), the addition of the strong acid
296 HCl produces a change in [H+] that is equal to the change in [Cl-] (figure 1b) so that it looks as if
297 H+ has been added and that H+ is an independent variable. We say “small” because SID in plasma
298 and cells is in the 10-3 range, a thousand fold higher. What really was added was a solution that
+ - - 299 had H , Cl , OH and H2O and this solution formed a new equilibrium with the components of the
300 original solution. However, when HCl is added to a solution with a positive SID, the change in
301 [H+] is much smaller than the [H+] added with the [Cl-] and it becomes obvious that H+ is not an
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302 independent variable (figure 1c). The change in [H+] is less than the change in [Cl-] because the
303 electrical charge due to the SID distorts the dissociation equilibrium of water. When a weak acid
304 also is present in a solution with a positive SID, [H+] remains greater than [OH-], and the
305 solution remains acidic, until SID is greater than half the concentration of the sum of all the
306 components of the weak acid, at which point, the solution becomes alkaline meaning that [OH-]
307 is > than [H+]. This is because the negative ion of the weak acid must be balanced by an H+ so
308 that “neutral” occurs at a higher [H+] and lower pH than in pure water. In cells, a “neutral
309 solution” occurs around a pH of 6.2 (assuming a PCO2 of 50 mmHg) and SID around 100
310 mEq/L. Normal intracellular SID is in the range of 130 mEq/L (Stewart 1981). 311 With a few exceptions, such as gastricDraft fluid in the fasting state and lysozymes, under 312 most physiological conditions, all biological fluids, and the cytoplasm of all cells, are alkaline.
313 Observed changes in pH or [H+] thus represent more or less alkalinity. Solutions are never acidic
314 in normal biology by which we mean solutions in which [H+] is greater than [OH-], although this
315 is not always true under experimental conditions. Furthermore, it means that in contrast to the
316 linear increase of [H+] that occurs when an acid is added to a solution with a negative SID, in
317 normal biology, the increase of [H+] with addition of an acid solution with a positive SID is
318 always less than the amount of [H+] added. Thus, the simple behavior of [H+] in negative SID
319 solutions greatly confuses the behavior of [H+] in the alkaline solutions of normal biology, hence
320 our use of the term “acid delusion”. The concept of acid-base balance is useful for taste and
321 titration curves but not in biology where what we want to know is whether there are more or less
322 H+ in the solution. Furthermore, the small changes in [H+] that occur in positive SID solutions
323 are not obvious with the pH notation.
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324 What really is being described in an analysis of acid base-balance is the deviation of [H+]
325 from a reference value. If one uses the analogy of deviations of [Na+] from a standard value as
326 indicating hyponatremia or hypernatremia, and pH 7.4 as the reference value for [H+] in blood,
327 perhaps it would make more sense to call a pH below 7.4 at body temperature
328 hyperhydrogenemia and a pH above 7.4 hypohydrogenemia and then refer to [H+] increasing
329 processes and [H+] decreasing processes. However, even this is not helpful for intracellular [H+]
330 varies among cell types and under different metabolic conditions so that there is no simple
331 “normal” ICF [H+]. We are better off just considering [H+] increasing and [H+] decreasing
332 conditions and “standard” physiological conditions. Rahn and Howell, acknowledging that they 333 were unwittingly re-incarnating a view firstDraft proposed in 1919 by Benjamin Moore, argued that 334 the ratio of OH-/H+ is critical value for the determination of the ionized sites on proteins. In
335 biological solutions the ratio of OH-/H+ is generally between 7 and 8 to 1 which gives an alkaline
336 pH as defined above (Rahn and Howell 1978). The ratio concept nicely explains adaptations in
337 bodily fluids that occur with temperature changes (Howell et al. 1970). In essence, it gives an
338 indication of the dissociation of water and works because in biological solutions OH- usually is
339 much larger than H+. However, its usefulness is limited because a ratio is non-linear.
340 Although the Brønsted and Lowry definitions of acids and bases as proton acceptors or
341 donators generally have been accepted as the preferred terminology (1966a; 1966b), we believe
342 that their use, too, has created much confusion. Singer challenged the Brønsted-Lowry concept
343 many years ago at a consensus conference on acid-base terminology, but his minority viewpoint
344 was rejected (1966a). The use of the terms “acceptor” and “donator” of protons in the definition
345 makes it sound as if H+ actually moves and act as an independent variable. This leads to such
346 terms as “rate of acid formation”, “transportation of H+”, “acid accumulation”, “acid secretion”
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347 and “exchange of H+” (Aronson et al. 1982; Boron 2004; Grinstein et al. 1984; Kinsella and
348 Aronson 2001; Lagadic-Gossmann et al. 1992; Vaughan-Jones and Spitzer 2002) and ignores
349 that [H+] is a dependent variable that is determined by all the elements in the solution and the
+ 350 largest reservoir of H in the body is H2O itself. The Brønsted-Lowry definition was developed
351 to deal with non-aqueous solutions in which ionization of substances is not necessarily the same
352 as in water. For example, HCl does not dissociate in benzene and therefore is not an acid in that
353 solution by Arrhenius’ terminology (1966a). However, as already noted, biological solutions are
354 water based and it is the interaction with water that we want to know (MacConnachie 1970).
355 The consideration of acids and bases as part of “conjugate” pairs in the Brønsted-Lowry 356 definitions also makes it sound as if all Draftcomponents have equal effects in the solution. For - - 357 example OH and HCO3 are called bases (Bates 1966) and it sounds as if they have an
358 “independent” existence, but this misses the sense that their concentrations are very dependent
359 upon the concentration and activity of the cation to which they were initially attached. If that
360 cation is strong, such as Na+, then it is the cation that is the important and “basic” element (as
361 defined first by Lavoisier) because the cation changes SID and transiently distorts electrical
362 neutrality. The terminology becomes even more confusing with a strong anion such as Cl-, which
363 is labeled as a base in the Brønsted-Lowry approach, yet addition of Cl- with H+ or a weak cation
364 narrows the SID and increases [H+] and thus has an acidifying effect, which is what we want to
365 know. It also is not helpful that by this definition H2O can be an acid or a base. The Lewis
366 definition was created to include the effects of metals such as iron in the definition of acids and
367 bases and deals with electrons, which are the true charge carriers in a current. Lewis bases are
368 the same as Brønsted-Lowry bases but this definition of an acid takes us further away from [H+],
369 which is the real interest in biology, and it loses the sense that “acidic” in biology is really a short
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370 hand for greater [H+] than the [OH-], or equally, lower [OH-] than [H+]. The Lewis definition
371 seems to us to be the least preferred for biology.
372 For us, the seemingly more sophisticated definitions of acids and bases miss the flavor of
373 what occurs physically in a water solution, which is that constituents are dissociating and
374 forming solvation structures with water in what are called clathrates. This even includes the
375 water molecule itself which dissociates into [H+] or [OH-]. As pointed out by Ricci, “the ions H+
376 and OH- are water ions and always present in any aqueous solutions”. They pertain to water and
377 not the solute; they never are “contributed” by the solutes” (Ricci 1952). Each of these
378 dissociations is determined by thermodynamic constants and the final [H+] of the solution can be 379 determined by solving the equations forDraft all the equilibrium reactions of constituents of the 380 solution (Stewart 1981).
381 Although [H+] regulation traditionally has been considered the centre of the discussion,
382 the analysis works equally well in the opposite direction based on [OH-] in water-based solutions
383 (Aronson 1985), because the two values are related through the dissociation constant of water.
384 Thus the “hydrogen” electrode could equally be described as a hydroxyl electrode and it would
385 even have a more direct relationship to changes in SID in biological solutions. The use of [H+] is
386 thus a reference choice. The symmetry extends to the discussion of all the key variables in the
387 system. Does H+ move out of the cell and decrease intracellular [H+] or does Na+ move into the
+ - 388 cell and the consequent increase in SID lowers intracellular [H ]? Does HCO3 move into the cell
+ - + - 389 and lower [H ] or does Cl move out which widens SID, which lowers [H ] and increases [HCO3
390 ] by increasing the dissociation of H2CO3? The argument is not simply semantic for it relates to
391 the fundamental physical-chemical principles and what are the dependent and what are the
392 independent variables that are being regulated, in other words, what is added and what is
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393 changed by the addition of a substance. It also directs which variables need to be controlled in
394 experiments.
395 Buffers and Buffer Power
396 The concept of buffers in the traditional analysis of [H+] regulation leads to further
397 confusion. Van Slyke defined buffering power (β) as the amount of strong base (such NaOH)
398 that must be added to a solution to produce a unit change in pH and –β as the amount of strong
399 acid (such as HCl) that must be added to produce a unit change in pH (1966c; Bates 1966; Boron
400 2004). In a solution that has a weak acid, ie [Atot] or weak base [Btot], which are the traditional
401 buffers, titration with a strong acid changes their dissociation equilibrium. A because of the 402 characteristics of the transformation of [HDraft+] into pH. If the ordinate is [H+] instead of pH, the 403 relationship is much simpler (Stewart 1981) (figure 2). In a solution with an [Atot] and a negative
404 SID, HA is not dissociated. When HCl is added to the solution, H+ from the added HCl are
405 matched by the added strong anion, Cl-, and electrical neutrality is satisfied and HA still is not
406 dissociated and the solution behaves as if only strong ions are present. When SID is positive, and
407 the concentration is greater than the total amount of the weak acid [HA], the weak acid is
408 essentially completely dissociated and this solution, too, behaves as if it only has strong ions;
409 [H+] is extremely small and [OH-] increases linearly with increases in SID for there are no other
410 anions to balance the SID and maintain electrical neutrality. The interesting range occurs when
411 SID goes from zero to [HA]. In this range the charge from the increasing SID drives the
412 dissociation of the weak acid. When SID = [Atot]/2, 50% of the weak acid is dissociated, pH of
413 the solution equals the pKa of the weak acid, the concentration of the anion [A-] approximates
414 SID, and the pH versus SID relationship has a plateau. Importantly, these clear relationships
415 only occur if there is one weak acid present. If there is more than one, the plateau is not flat and
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416 the transition is more complex. If a weak base is present [Btot], the same occurs in the negative
417 SID range.
418 When the weak volatile acid of PCO2 is combined with a [Atot], the relationship becomes
419 even more complicated. There are now two substances dissociating with changes in SID; the
420 relative changes are based on their dissociation constants. The non-volatile weak acid [A-] no
- - 421 longer linearly increases with increases in SID and SID now equals [A ] plus [HCO3 ].
422 A further examination of the change in [H+] with a change in SID in a solution with a
+ 423 positive SID and [Atot], indicates that there is a greater change in [H ] for a change in SID in the
424 range of [HA] and therefore there is actually less buffering as defined by a change in SID/change 425 in [H+] when the “buffer” is present (figureDraft 2)! This is because as SID increases, the weak acid 426 effectively becomes stronger (more dissociated). On the other hand, in the range of SID in which
+ 427 SID = [Atot]/2, the weak acid raises the [H ] of the solution and puts the pH of the solution in the
+ 428 range of the pKa of the weak acid (figure 2) (Stewart 1981). Without the [Atot], [H ] would
429 simply be K’w/SID (where K’ is the product of the dissociation constant and concentration of
430 water at a given temperature). From this discussion, it should be apparent that buffers do not
431 reduce changes in [H+] in response to the addition of a strong acid, but instead, when SID is less
+ 432 than [Atot] they set the magnitude of [H ] and pH in the range of the KA or pKa of the weak acid.
433 When the solution has a weak base, ie [Btot] the behavior is similar to that of a weak acid but in
434 the opposite direction. It is important to appreciate that “buffering” is a property of the whole
435 solution and is depends upon the interaction of all components. When there is a mixture of weak
436 acids and bases, as is the case in most biological solutions, titration curves do not have a simple
437 break that identifies an equilibrium point. Furthermore, the “buffering power” that is calculated
438 for intact cells likely includes movements of strong ions and PCO2 across the membrane and
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439 these may dominate the buffering mechanisms in the ICF [H+]. To truly calculate buffering
440 power of a solution with strong ions, CO2, and an [Atot], one needs an equation that deals with
441 the interaction of all components of the solution and that there be no loss or gain of ions. The
442 equation can then be differentiated with two of the three independent variables held constant and
443 the third varied to obtain the buffering effect of changes in SID, change in PCO2 or change in
444 [Atot] and [Btot](Stewart 1981). This is essentially impossible in a living cell. It is much more
445 meaningful to assess the effects of changes in the components, that is, SID, PCO2 and [Atot] and
+ 446 possibly [Btot] on [H ].
447 Potential errors from the incorrect assessment of buffers power inside cells. 448 The failure to consider the behaviorDraft of weak ions and “buffering” limits mechanistic 449 analysis and potentially produces quantitative errors. The widely used NH4Cl pulsing technique
450 to decrease intracellular pH and to calculate “buffering power” is a good example (Boron 1977).
451 It is argued that when the extracellular milieu of cells is pulsed with NH4Cl, the NH4Cl
+ - + + 452 dissociates into NH4 and Cl and some of the NH4 dissociates further into NH3 and H . NH3
453 freely crosses cell membranes and enters the cytoplasm. Based on intracellular pH (pHi) and the
+ + 454 pKa of NH3/NH4 , in most studies, more than 98% of NH3/NH4 inside and outside the cell is in
+ + 455 the form of NH4 . It is argued that this occurs in the cell by NH3 taking up H and thereby
456 reducing intracellular [H+] (increases pHi) (Boron 1977; Boyarsky et al. 1988) and indeed, a
457 rapid rise in (pHi) is observed (figure 3 is an example taken from Boyarsky et al (Boyarsky et al.
458 1988)). It is further argued that when NH3 inside the cell is in equilibrium with extracellular
+ + 459 NH3, NH4 enters the cell and creates a small fall in pHi by “releasing” H . However,
+ 460 dissociation of NH4 is dependent upon the pH of a solution, which is dependent upon the
461 components of the solution that set the SID. The independent variable for the response to NH3/
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+ + 462 NH4 is the total amount of [NH4 ] and [NH3] and not their individual concentrations just as is
+ + + 463 the case for [Atot]. At normal biological [H ] most of the NH3/NH4 is in the form of NH4 which
+ 464 acts as a strong cation both inside and outside the cell. When NH3/NH4 is in the form of NH4Cl
+ 465 it has no effect on extracellular pH because the strong ion NH4 is balanced by an equal amount
- 466 of the strong ion Cl (ie zero effect on SID). However, when NH3 diffuses into the intracellular
- + 467 space without Cl and again forms NH4 it adds a strong cation and increases the ICF SID. This
468 is an alkalizing effect. Normal ICF SID is in the range of 130 mEq/L, so that the pulse of 50
469 mmol of NH4Cl used in some experiments (Boron 1977) produces a very large increase in
470 intracellular SID. After the initial alkalization, an increase in [H+] (fall in pH) is observed, which
+ + 471 is said to be due to continued influx of NHDraft4 and release of H . Besides requiring an explanation + 472 for why the ionized form of NH4 did not cross the cell membrane initially, the total
+ 473 NH3/NH4 would be increased which has an alkalinizing effect. There thus must have been a
474 decrease in SID, which is an acidifying effect, to explain the fall in pHi. This could occur
475 because there was an efflux of strong cations such as [Na+] or [K+], an influx of a strong anion
476 such as [Cl-] or influx of other weak acids from the media. Cl- is a likely candidate, at least in
+ 477 some studies, because it is added with the NH4 and would be moving down a large
478 concentration gradient created by its addition with NH4Cl to the extracellular space. When the
+ 479 NH3 is washed out of the extracellular environment, ICF NH4 must fall too. As would be
480 expected with the loss of a strong cation, ICF [H+] markedly increases, but this also indicates that
+ 481 ICF strong anions increased or strong cations decreased during the NH3 pulse. Removal of Na
482 from the extracellular space or blocking the Na+/H+ exchanger block recovery indicating that
483 there likely is an important role for either Na+ or K+ in the extracellular fluid at this stage as
484 shown in figures 3 and 5, (Zhao et al. 1995). Since extracellular [Cl-] is increased as part of
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485 NH4Cl pulse, it too could play a role. However, although an increase in ICF [H+] late in the
486 pulse still occurs when extracellular Cl- is substituted by glucuronate (Lagadic-Gossmann et al.
487 1992). This might be because the glucuronate, which behaves as a strong ion at physiological
+ 488 pH, also moves into the cell (figure 4). This would reduce the NH4 induced increase in SID in
- 489 the same way that Cl does. As a final consideration, when NH3 diffuses into the cell, unmatched
490 Cl- is left in the extracellular, space which would acidify the solution and could have triggered
491 other membrane effects. The key point from this discussion is that none of these effects are
492 known because there was a failure consider the implications of the physical-chemical principles
493 and the effects on [H+] of the reagents used in the experiment.
+ + 494 It is further argued that since onlyDraft a small amount of H is free, for each NH4 formed in + 495 the cytoplasm, the H taken up by NH3 must come from intracellular buffers and therefore their
+ + 496 buffering capacity can be calculated from delta[NH4 ]i/delta pHi. For this purpose, [NH4 ]i s
497 calculated from the extracellular [NH3] and intracellular pH, the assumption being that
498 intracellular and extracellular [NH3] are equal, which only is true if the composition of the SID
499 on either side did not change. What this analysis is missing is that [H+] is regulated in the cell by
500 the intracellular SID and [Atot] and both of these must remain constant if one is assessing
501 intracellular “buffering” but from the above analysis it is likely that SID or [Atot] changed.
+ 502 Secondly, as discussed above, NH4 effectively acts as a strong cation and increases the SID so
503 that its effect is identical to what would occur if intracellular [Na+] were increased or [Cl-]
504 decreased by the same amount and therefore this was likely “buffering” due to a change in SID.
505 When this response was compared to titration with a strong cation, such as Na+, it is not
506 surprising that the stoichiometry comes out 1:1 for these are chemically equivalent (Grinstein et
507 al. 1984). Furthermore, this “buffer power” only deals with the SID component. To evaluate the
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- 508 buffering effect of [Atot] and PCO2/HCO3 , a separate titration would be required for changes in
509 [HA] at a constant SID and constant PCO2 which is likely impossible in a living cell. Of course,
510 dissociation of water also is plays a role and it is important that the cell does not shrink or
511 expand because a change in water would change the concentration of all ICF substances and it is
512 concentration charge differences that counts for the effect on [H+].
513 When the “rate of acid extrusion” from the cell is calculated from the change in pH
514 multiplied by “buffer power”, which was really a SID titration (Grinstein et al. 1984) as
+ 515 calculated by the NH4Cl pulse technique, the number of H that are purported to cross a cell
516 membrane is in the mMol/L range, whereas total body H+ is only about 1.6 uMols in a 75 Kg 517 man. On the other hand, this result is understandableDraft when one realizes that SID is in mMol/L 518 and the effect of a change in SID is really what was calculated.
519 Another example of potential misinterpretation of the response to NH3Cl loading is a
520 report in the journal Nature that claimed to have identified cells that have pH neutral transport of
+ 521 NH4 in renal tubular cells and apparent impermeability to NH3 (Kikeri et al. 1989). Application
+ 522 of NH4Cl to the apical side of these cells resulted in a rise in [H ] instead of the usual
+ 523 alkalization (figures 3 to 5). The author’s argued that the cells are impermeable to NH3 but NH4
524 moved in through the Na+/K+/2Cl- co-transporter and then “donated” H+ which caused the
+ 525 acidification. First, basic physical chemistry indicates that whether NH4 or NH3 moved into the
+ 526 cell the effect would be identical because the dissociation of NH3/NH4 depends upon the pH and
+ + 527 SID. Thus the ICF NH3/NH4 would still be primarily in the form of NH4 , which has an
+ 528 alkalinizing effect. Second, movement of NH4 into the cells was inferred from the change in
529 pHi and not actually measured. The basic chemistry would indicate that it far more likely that the
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530 change occurred from either an outward movement of Na+ or K+ or, more likely, an influx of Cl-
531 through the co-transporter that they identified as being responsible.
532 Concentration of intracellular components.
+ 533 To determine the potential role of SID, PCO2 and [Atot] in the regulation of [H ] in the
534 ICF one must have some idea of the relative concentrations of these components and the
- 535 dissociation constants of PCO2/HCO3 and the HA. However, these values are not easily
536 obtained in cytoplasmic fluids and only a few studies have tried to obtain all the values needed to
537 allow calculation of the interacting components (Heigenhauser and Lindinger 1988; Lindinger
538 1995; Lindinger and Heigenhauser 1987; Lindinger et al. 2005). Furthermore, intracellular 539 values vary among cell types and even differDraft in compartments of the intracellular space. The 540 composition of electrolytes in cells, the water content, and osmolality all can change rapidly, and
541 these changes can alter the rate constants involved in the equilibrium equations and activity
542 coefficients of the elements (Lindinger et al. 2005; Robergs et al. 2004). Finally, parts of the cell
543 may have structured water (pollack 2001) which will affect the dissociation constant of water, a
544 primary determinant of [H+]. It might thus seem to be a hopeless task to try to analyze pHi.
545 However, it still is possible to understand the principles that govern intracellular [H+] and to
546 predict mechanisms that likely take place to make changes in, or defend, intracellular [H+] as
547 well as to avoid potential artifacts created by the experimental approach. A basic one that needs
548 to be studied more carefully is the movements of the strong ions Na+, K+] and Cl-.
549 Components of the ICF of a generic cell are given in table 1 and figure 6 and are
550 compared to those of generic interstitial fluid (ISF) and plasma. K+ is the dominant cation. The
551 concentration of Mg2+ is much larger than in plasma. [Na+] and [Cl-] are much lower than in the
552 extracellular fluid and SID is more than three times that of plasma. The high SID likely evolved
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553 because [HA ] is tenfold higher in the generic cell than in plasma because of all the complex
554 peptides and proteins inside the cell and plays a more important role in regulating [H+] in ICF
555 than in plasma.
556 Disturbances and Regulation of Intracellular [H+]
557 If cells were passively permeable to H+, at an extracellular pH of 7.4 and a
558 transmembrane potential of -60 mV relative to the outside, the Nernst equation predicts an
559 intracellular pH of 6.4, but this is does not occur. It thus has been argued that there must be an
560 active transport mechanisms to regulate [H+] (Aronson et al. 1982) (Putnam 1998). However,
561 this use of the Nerst equation fails to treat the system as an integrated whole and to consider the
562 physical-chemical determinants of [H+].Draft Measurements of inner and outer cellular pH are
563 indicators of the dissociation of water in their respective solutions. These values are set by the
564 SID, PCO2 and the properties and quantity of the [HA] of the solutions on either side of the
565 membrane. This violates the assumption in the Nernst equation that “H+ passively distributes
566 across the membrane (at equilibrium)” (Putnam 1998). The concentration of H2O molecules in
567 water is very high, 55.5 mol/L, and provide an almost infinite source of protons. [H+] of pure
568 water at body temperature is actually higher than [H+] of most biological solutions. The
569 dissociation and re-association of water occurs at 106 /s (Lindinger et al. 2005) and it is thus very
570 unlikely that an actual H+ diffuse through any distance in a water based systems. Instead, the
571 charge associated with H+ in the macro-solution is distributed by the Grotthuss mechanism
572 without an H+ actually having to move (Cukierman 2006).
573 PART 2
574 General schema for the regulation of intracellular [H+]
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575 Based on the physical chemical principles, a general schema for the regulation of
+ 576 intracellular [H ] must be based on changes in SID, PCO2 and [Atot] and [Btot] (figure 7). Of
577 these, SID is the primary regulator and when one considers the available strong electrolytes, the
578 only three that have concentrations sufficiently large to be important most often physiologically
579 are [Na+], [K+], and [Cl-]. An increase in intracellular [Na+] or [K+] increases the SID and
580 decreases [H+], whereas a decrease in intracellular [Na+] or [K+] narrow SID and increase [H+].
581 An increase in intracellular [Cl-] narrows the SID and increases [H+], whereas a decrease in
582 intracellular [Cl-] widens SID and decreases [H+]. It then becomes quite simple to predict the
583 dominant processes that likely regulate intracellular [H+]. Extracellular [Na+] is very high 584 compared to intracellular [Na+] so that oneDraft would expect that the major role of Na+ in [H+] 585 regulation is to move into the cell and decrease an elevated [H+]. This is consistent with what is
586 recognized in the traditional approach where it is noted that “acid extrusion”, in other words a
587 decrease in intracellular [H+], requires Na+ (Lagadic-Gossmann et al. 1992) (figure 3 and 4). A
588 rise in intracellular [Na+] will increase osmolality, which will increase intracellular water and
589 dilute all components of ICF. This decreases SID and will raise intracellular [H+] and somewhat
590 offset the increase in [H+] from an inward flux of [Na+]. Movement of Na+ out of the cell will
591 result in an increase in ICF [H+], but this effect is limited by the low baseline intracellular [Na+].
592 Extracellular [Cl-] also is high and intracellular [Cl-] is low so that the primary role of Cl- in [H+]
593 regulation is likely to move into the cell and to increase [H+]. This is consistent with the
594 recognition in the traditional approach that movement of Cl- into the cell is an “acid loader”
595 (Vaughan-Jones 1979a, b). As is the case with Na+, movement of Cl- out of the cell reduces
596 intracellular [H+] (figure 4), but as with Na+, this effect, too, is limited by the low intracellular
597 [Cl-]. The effect of changes in K+ will be the same as that of Na+ but in the opposite direction for
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598 intracellular [K+] is high and extracellular [K+] is low, so that the primary effect occurs with
599 outward movement which increases ICF [H+] and would be expected to more likely be a
600 consequence of changes in metabolic function such as increased metabolism in exercising
601 muscle (Lindinger 1995) rather than a general regulatory process because of the importance of
602 transmembrane [K+] for cell membrane potential.
603 The presence of a positive SID rather than a negative SID in most biological solutions
604 could have an evolutionary advantage. The high charge concentration of H+ means that it has a
605 much greater effect on the tertiary structure of surrounding proteins than OH-. The smaller
606 change in [H+] than in [OH-] that occurs when SID is positive thus adds stability to protein
607 structures when transmembrane ion concentrationsDraft change.
- + 608 The PCO2/HCO3 system has a number of key roles in [H ] regulation. The presence of
+ 609 H2CO3 raises [H ] of a solution above what it would be if there were just a positive SID and it
610 creates a plateau in the titration curve as discussed under buffers. PCO2 is of particular
611 importance for this process because it is diffusible and complex organisms can regulate
612 extracellular PCO2. This is not the case in single cells and primitive organisms in which the
613 PCO2 around them is determined by the environment (Burggren and Bautista 2019; Heisler
614 1984), but then again they do not have the large fluctuations in metabolic activity and the
615 accompanying PCO2 production that occurs in aerobically active organisms.
616 The presence of [Atot] and [Btot] contributes further to the “set point” of relatively stable
617 [H+] values. It is quite likely that changes in the composition of the molecules that determine
618 intracellular [Atot] and [Btot] contribute to long term regulation, but this has not been studied in
619 any great detail to our knowledge.
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620 Where does an increase in H+ “come” from in the cell?
621 Biochemistry and physiology texts and articles frequently refer to the production of H+
622 by metabolic processes and create balance sheets of production and clearance of H+ (for example
623 ref (Hochachka and Mommsen)). However, these analyses ignore the dependence of [H+] on
+ 624 SID, PCO2 and [Atot] and that in a solution with a positive SID, change in H is less than the
625 amount of free H+ added (Brooks 2020; Robergs et al. 2004). They also ignore the contributions
626 of H+ from readjustments of the equilibrium of water and weak acids and bases (“buffers”).
627 Production of a strong organic anion in a metabolic process narrows the SID and increases [H+].
628 Examples include lactate, acetate, phosphocreatine and amino acids, which typically have pKa’s
+ 629 in the mid 3-4 range. Production of a strongDraft organic cation in a metabolic process, such as NH4 ,
630 widens the SID and decrease [H+], Breakdown of strong cations or anions does the opposite.
631 Metabolic production of a weak acid contribute to the effective [Atot], which alters the range of
632 the plateau of change in [H+] with a change in SID (that is the “buffering set point” or effective
633 KA of the [HA]). Examples include phosphates, ATP, and ADP. If strong or weak cations are
634 produced, the opposite occurs. The final effect of the addition of a weak anion or strong cation
+ 635 on [H ] depends upon how much of it is produced (that is, its contribution to [Atot]), its
636 dissociation constant, the charge of the ionic component and its clearance. We next discuss the
+ 637 changes in SID, PCO2 and [Atot] that alter ICF [H ].
638 Change in SID: A change in SID has more than double the effect of an equivalent change in
+ 639 [Atot] and thus SID is the primary regulator of intracellular [H ]. Furthermore, because the SID
640 of the ICF is almost three times that plasma, the ICF is more sensitive to changes in SID.
641 Changes in SID can occur from increases or decreases in strong electrolytes or strong organic
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642 ions but there is a difference in their effects. Except for some minor release from stores, ions of
643 elements cannot be “created” or “destroyed” but rather move into or out of the IC compartment.
644 Organic ions, too, can be moved into or out of the ICF, but they also can be produced or
645 metabolized in the ICF. There are also constraints on elemental electrolytes. For example, [K+]
646 sets the transmembrane potential of cells, which is especially important for intracellular
647 signaling, and [Na+] is linked to calcium fluxes as well as excitability. A potentially very
648 interesting element is Ca2+ which can help deal with the handling of metabolically produced
649 PCO2 in species that are transfering between land and being underwater where it is more difficult
650 to excrete CO2. It has been shown that there can be large shifts of HCO3- with Ca2+ into the 651 bony structures that serve this purpose suchDraft as the shell of a tortoise or the mantel of dinosaurs 652 (Janis et al. 2020).
653 Given the importance of [H+] for the regulation of cellular metabolic processes, it is not
654 surprising that many mechanisms have evolved through evolution to achieve this end. One of the
655 best studied is the so called Na+/H+ exchanger (NHE) (Counillon and Pouyss‚gur 2000; Grinstein
656 et al. 1984; Orlowski and Grinstein 1997). This family of highly regulated phosphoproteins is
657 present in all mammalian tissues in both the cytoplasm and inner mitochondrial membrane. The
658 description of these proteins as “acid extruders” is an example of the clash between a physical
659 chemical analysis and classical approach. The following discussion may seem to be just like
660 looking at an image in a mirror for both images seem to represent reality, but they are in opposite
661 directions. However, as is true of a mirror, one image is a real substance and the other is just a
662 reflection. As their name indicates, these exchangers are purported to exchange Na+ for H+ in a
663 one to one ratio (Grinstein et al. 1984; Moolenaar et al. 1981; Orlowski and Grinstein 1997). To
664 begin, this is an unlikely event considering the more than 106 fold difference between [H+] and
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665 [Na+] (at pH of 7.4 [H+]= 40 x 10 -9 Eq/L and [Na+] 140 x 10-3 Eq/L). Indeed, there has never
666 been a study in which a radiolabeled H+ has been shown to cross the cell membrane. What
667 actually is measured is an increase in pH in the cytoplasm which is what would be expected if
668 intracellular [Na+] increased and increased intracellular SID. A pH measurement with an
669 electrode actually is calibrated by changing the SID of the solution bathing an electrode and so
670 are the pH induced color changes with colorimetric dyes. The change in output measured with
671 dyes or a pH electrode is proportional to the change in [H+] when SID is negative for the
672 calibrations, but [H+] does not actually determine the reaction; the SID determines the
673 dissociation of the weak acid or base if a dye is used, or a change in [Cl-] if a silver electrode is 674 used for pH measurement. It is arguedDraft that an H+ must be exchanged with Na+ because the 675 exchanger is electrically neutral (O'Neill 1999). However electrical neutrality could be
676 maintained without actual movement of H+ if the movement of Na+ creates a change in [H+]
677 across the membrane by the Grotthuss mechanism and readjustment of all elements in the
678 solution. There even could be an accompanying “wave” of OH- in the water surrounding Na+ or
679 by a Grotthuss-type mechanism across the membrane for OH-(Cukierman 2006). Aronson
680 pointed out in an early review on the Na+-H+ exchanger that it could equally be called a cation-
681 hydroxyl co-transporter (Aronson 1985), which based on the 1:1 relationship of [OH-] to SID at
682 physiological pH, makes more sense. It has been noted that there is non-linearity in the rate of
683 Na+ extrusion versus H+ inflow by the exchanger, which is attributed to allosteric changes in the
684 exchanger (Aronson et al. 1982). However, the vesicles used in these experiment had a minimal
685 amount of weak electrolytes so that at pH below 7.0 the solution likely had a [H+] greater than or
686 close to [OH-] and in this transition zone [H+] increases much more rapidly with decreases in
687 SID. When SID is negative, changes in [H+] linearly match changes in SID.
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688 The crystal structure of the E. coli Na+- H+ antiporter, a member of the CPA2 family of
689 cation/proton antiporters has been established and a mechanism was proposed for the exchange
690 of Na+ and H+ based on the structure (Taglicht et al. 1993). However, the mechanism was forced
691 into the classic paradigm of [H+] regulation and did not consider the physical chemistry
692 properties. The action of this antiporter is different from the NHE exchanger. The fluid
693 surrounding the cells in the body is isotonic relative to their cytoplasm and the dominant action
694 of the NHE is likely to increase intracellular [Na+] to decrease an increase in intracellular [H+].
695 In contrast, the antiporter evolved in bacteria to allow tolerance to a high salt alkaline
696 environment; its primary purpose seems to be to regulate intracellular [Na+] which, besides being 697 important for regulating pHi, is critical forDraft intracellular volume regulation. Its primary function 698 is thus likely to extruding Na+ from the cytoplasm to keep intracellular [Na+] from increasing but
699 at the same this also will increase intracellular [H+]. The antiporter is turned off at low pHi which
700 makes sense when one appreciates that a low pHi indicates that ICF SID is lower than normal
701 and extruding more intracellular Na+ would further reduce SID and further lower the pHi. When
702 pHi is high, Na+ and the SID are likely high and the system extrudes Na+ which lowers pHi.
703 Sensing seems to be due to alteration in the protonation state of the entrance of the cytoplasmic
704 anionic passage. This narrows to a section with two aspartates that does not allow passage from
705 the cell of fully hydrated Na+. However, partially hydrated Na+ could potentially still pass, and
706 “drag” an OH-. At “acidic” pH (pH = 6.5 and [H+] = 3.16 x 10-7 Eq/L) the exchanger is down
707 regulated and only part of the cation binding site is exposed to the cation passage and the
708 periplasmic side is blocked by an ionic barrier that does not allow ICF Na+ to exit. At “alkaline”
709 ICF, the pH-sensing component transmits a signal, which produces a conformational change in
710 two helices of the structure. This exposes a Na+ binding site made up of the two aspartates to the
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711 cytoplasm and removes an ionic blockade on the periplasmic side that allows Na+ to exit. The
712 charge imbalance produced by binding of Na+ to the active site triggers a rotational movement of
713 the helices such that the binding site is now exposed to the periplasmic side and sealed from the
714 cytoplasm. On release of the Na+, the aspartates are said to re-protonated and a conformational
715 change re-exposes them to the cytoplasmic side. The electrochemical gradient determines the
716 direction of the exchange. A number of comments need to be made. It seems likely that the
717 negative charge in the passage would facilitate the passage of Na+ through the channel, but Na+
718 would not actually have to bind to the aspartate. Even more so, H+ at the entry site would be in a
719 state of equilibrium with the surface of the components of the channel, and more importantly, 720 with the surrounding large volume of waterDraft and the SID in that solution must determine the 721 dissociation of water and the local dissociation of aspartic acid. The authors argue that two H+
722 exchange for each Na+ but given the six fold difference in their concentrations, actual versus
723 “effective’ change seems highly unlikely. The empiric evidence for this 2:1 exchange is based on
724 a study that compared the rate of change in Na+ concentration in liposomes loaded with the
725 antiporter with the rate of H+ extrusion with florescent dyes (Taglicht et al. 1993). The net
726 “proton” loss was estimated to be around 7 umol/min/cuvette. Given that at a pH of 7.2 there are
727 only 7.9 x 10-8 mol/L H+ in the whole body, in other words 100 fold less than is argued are
728 extruded in a minute, these numbers are unrealistic. The values for this conclusion derive from
729 calibration with nigericin, which allows K+ to be extruded. Nigericin likely changes ICF SID to a
730 negative number which means that changes in SID equal changes in [H+]. However during the
731 experimental conditions the pH was 7.5 which means that the SID must have been positive and
732 changes in SID no longer match changes in [H+]. A second problem is the use of the slopes of
733 fluorescence probes which are likely not linear, to determine the rate of change of [H+].
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734 A similar argument against the structural explanation for the mechanism of Na+/ H+
735 exchange also applies to the argument that the crystal structure of aquaporin-1 explains the
736 “longstanding puzzle in physiology-how membranes can be freely permeable to water but
737 impermeable to protons” (Murata et al. 2000). The authors found that the pore of the aqueous
738 pathway narrows to about 3 Å and thus would exclude a proton. However, if it can exclude a
739 proton, it must also exclude a Na+, which is really what would have to enter the cell and lower
740 ICF [H+].
741 Na+-organic anion transporters are regulators of intracellular [H+] that mediate the influx
742 of Na+ with organic ions such as acetate or lactate. In the classical approach it is argued that
743 lactate and acetate are weak bases and thereforeDraft bind intracellular H+ which alkalinizes the
744 intracellular environment (Putnam 1998). First, both lactate and acetate act as strong anions and
745 narrow the SID at physiological pH for they are almost totally dissociated at biological values of
746 pH both intracellularly and extracellularly. Endogenously produced lactate and acetate are
747 always made in the equivalent of the hydrogenated form that will increase ICF [H+]. However,
748 when organic anions are infused or added to media, they are usually are provided in the form of a
749 salt with a strong cation such as Na+ so that SID does not change and they do not reduce ICF
750 [H+] (ie have an acidifying effect). Similarly, if a Na+-organic transporter moves equal amounts
751 of Na+ and an organic anion, SID and [H+] do not change. However, lactate can be metabolized
752 in cells. This leaves an unbalanced Na+ which will then increase the SID, an alkalinizing effect.
753 A similar process can account for the alkalinizing effect produced by pulsing cells with “acetate”
754 (Leem and Vaughan-Jones 1998).
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755 As already discussed, besides increasing or decreasing strong cationic electrolytes, [H+]
756 can be regulated by increasing or decreasing a strong anion and Cl- is the major player. Recently
757 there has been increasing attention to cell membrane regulators of [Cl-]. These include chloride
758 channels (Devuyst and Guggino 2002) and exchangers (Chipperfield and Harper 2000). The
759 exchangers are believed to act by exchanging Cl - with an organic ion such as formate (Cardenas
760 et al. 1998), as well as by a yet poorly defined “pump 3" for Cl-. The action of the exchanger
761 would be the converse of the Na+ organic anion transporter. Extrusion of Cl- from the cell in
762 exchange for a formate would not initially change SID or [H+], but when the formate is
763 metabolized the ICF SID is widened which reduces ICF [H+]. Exchangers also could work in the 764 same way as the Na+/H+ exchanger; in thisDraft case movement of Cl- into the cell would narrow 765 intracellular SID and increase intracellular [H+] while electrical neutrality is maintained by
766 dragging H+ through a Grotthuss mechanism or exchange with OH-.
767 An interesting group of [H+] regulators are what are called the Vacuolar (H+)-ATPases.
768 These are responsible for acidification of intracellular vacuoles in which high [H+] activates the
769 release of ligands from receptors (Stevens and Forgac 1997). Schema for their action show that
770 these ATPases shuttle H+ into vacuoles, but also indicate that this process requires “activity of a
771 parallel chloride channel …”. From the physical-chemical point of view, inward flux of Cl- likely
772 is the real factor for this would increase vacuolar [H+] by narrowing its SID, an acidifying effect.
773 If vacuolar SID becomes a negative value in these vacuoles, changes in [Cl-] would be matched
774 1:1 with the change in [H+]. Another type of ATPases is the P type of which there are five
775 classes (Kuhlbrandt 2004). Type IIc includes the well-studied Na+/K+ATPase that expels three
776 Na+ in exchange for two imported K+ per cycle and creates the resting membrane potential.
777 However, this charge difference also will lower ICF SID due to the net loss of a cation without
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778 loss of an anion and will acidify the ICF unless an anion also is removed. The H+/K+-ATPase is
779 a type IIc ATPase and is responsible for acidifying gastric juice (Kuhlbrandt 2004; Sachs et al.
780 1995). Acidification of the gastric contents requires an increase in [Cl-] relative to strong cations
781 so that the SID actually becomes negative. As part of stomach acidification there is conductance
782 of K+ and Cl- . It is argued that this ATPase exchanges K+ in the cellular efflux (extracellular
783 space) for cytoplasmic H+ (Sachs et al. 1995). However, if the ATPase simply resulted in the
784 return of K+ to the parietal cell without Cl-, and Na+ in the stomach fluid is kept low,
785 acidification of gastric contents would be achieved. The removal of Na+ from the stomach cavity
786 increases [Na+] in the venous drainage of the stomach and causes what is recognized as the 787 alkaline “tide”. However, the effect on theDraft final [H+] balance of plasma is small because added 788 Na+ is still relatively small compared to the amount of Na+ already in the large total extracellular
789 volume (Stewart 1981).
790 It is well recognized that intracellular pH is linked to cell volume and the Na+/ H+
791 exchanger and chloride channels respond to changes in [H+] or changes in intracellular volume
792 (Devuyst and Guggino 2002; Shrode et al. 1970). This makes sense from the physical-chemical
793 point of view. A decrease in intracellular water, for example from an extracellular osmotic stress,
794 narrows ICF SID because the same number of strong ions are present in less volume and the
795 reduction of volume has a proportionally greater effect on molecules with initially smaller
796 concentrations such as Cl- relative to K+. The decrease in SID has an acidifying effect.
797 Changes in PCO2 Under quasi steady state conditions in vivo, CO2 concentration is
798 relatively constant as CO2 diffuses to the blood down a concentration gradient from cells to the
799 lungs. Centrally controlled ventilation maintains a relatively constant arterial PCO2 and thus a
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800 constant total mass of CO2. However, the proportion of CO2 in various forms such as H2CO3,
- 2- 801 HCO3 and CO3 (but not dissolved CO2) is dependent primarily upon the SID of the solutions of
802 each compartment, and to some extent, on the concentration of weak non-volatile acids. Once
803 again, this is because the charge associated with SID distorts the standard equilibrium of H2CO3
804 and its products in a solution, but it does not affect the PCO2 or CO2 (dissolved), which are
- 805 regulated by production and clearance. A comparison of [HCO3 ] in ICF, interstitial fluid (ISF)
- 806 and blood plasma illustrates how SID and [Atot] affect [HCO3 ] (figure 6). PCO2 of the ICF is
- 807 close to that of the interstitial space and greater than that of plasma, whereas [HCO3 ] in the ICF
808 is 12 mEq/L but 31mEq/L in ISF. This makes it look like it is moving up its concentration 809 gradient! This can be understood by appreciatingDraft that the SID must be balanced by negative - - - 810 charges which only can come from HCO3 , A (the anionic component of [Atot]), and OH from
811 water. Although ICF SID is high (approximately 130 mEq/L) compared to that of the ISF (~ 31
812 mEq/L), ICF [A-] also is very high and accounts for 118 mEq/L of the negative charges and
- 813 HCO3 only needs to contribute 12 mEq/L On the other hand, the ISF has little protein to balance
- - 814 the SID and hence [HCO3 ] ≈ SID. The OH component is negligible. Under the conditions in
+ - 815 the ISF, increasing PCO2 increases [H ] but does not significantly change [HCO3 ] because the
+ - - 816 change in [H ] are orders of magnitude smaller and not evident in [HCO3 ]. [HCO3 ] only
817 changes with changes in SID (which also changes [H+]) and the increase in [H+]) does not
- 818 change the SID. Furthermore, if SID of the ISF were negative, [HCO3 ] would be unmeasurable
819 because there would already be an excess of strong anions to balance the negative SID. The pH
- 820 of plasma is the same as that of the ISF, but [HCO3 ] in plasma is lower than that of the ISF even
821 though SID of plasma is greater than that of the ISF. This occurs because the plasma has an ATOT
822 that contributes to the negative charge needed to balance the SID.
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823 These physical-chemical properties of PCO2 have important implications for
824 experimental studies. First, one must insure that neither CO2 production nor clearance change
825 during the measurements because it is total CO2 content that is important and not the
826 components. Second, living cells continuously produce CO2, which means that total CO2 varies
827 with the metabolic rate. As already discussed, this means that despite claims in method sections
828 of papers, “bicarbonate” is not actually added in experiments on isolated tissues or cells in
829 culture in which PCO2 in the ambient air is kept constant by continuous flow of air with a fixed
830 PCO2 because the total CO2 is fixed (Boron 1977; Boron and De Weer 1976; Boyarsky et al.
831 1988; Lagadic-Gossmann et al. 1992; Leem and Vaughan-Jones 1998). After a transient change,
- 832 added HCO3 re-equilibrates with ambientDraft PCO2 so that the added CO2 is cleared. The reason for 833 the quotation marks around “bicarbonate” is because what most often really was added was a salt
- + + 834 of HCO3 such as NaHCO3 or KHCO3 and the critical part is the Na or K which change the SID
- 835 and the change in SID changes the [HCO3 ] of the solution. The ions used to create a given
- 836 [HCO3 ] in the supernatant of cells or organelles in vitro can have profound effects on the ICF
837 responses for these ions can move across the cell membrane and alter ICF SID. This is especially
838 important when very high concentrations are used. For example, in an experiment that created
- + 839 solutions for “out-of-equilibrium” CO2 /HCO3 , [K ] was 549 mEq/L and the anion, gluconate
840 was 507 mEq/L (Zhao et al. 1995); these values are five-fold higher than normal strong ions.
841 When reviewing experiments, it is essential to examine the ions used to create the pH or the
- 842 accompanying [HCO3 ] in syringes or media because these ions can subsequently alter the SID
- + 843 in the cell and determine the final [HCO3 ] and [H ] at a given ambient PCO2.
844 A complicating factor in the analysis of the actions of PCO2 is that the activation energy
- 845 for the reaction is very high so that the reaction of CO2 with water or OH is very slow (Cardenas
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846 et al. 1998; Jones 2008; McMurtrie et al. 2004; Vaughan-Jones and Spitzer 2002). The half time
847 for equilibrium is as long as 30 seconds at body temperature unless the enzyme carbonic
848 anhydrase (CA) is present to catalyze the reaction. This has significant effects (Kowalchuk et al.
849 1994) on CO2 transport under conditions in which there is an abrupt increase in CO2 production
- 850 because most of CO2 transported in the blood is in the form of HCO3 (Cardenas et al. 1998;
- 851 Jones 2008). It also results in differences in the measurement of PCO2 /HCO3 in vivo and in
852 vitro for changes can continue to occur in the samples used for measurement. It has been
853 proposed that the location of carbonic anhydrase in the cell may have important implications for
854 the regulation of metabolism in metabolically active cells (Sterling et al. 2001). Over longer 855 time periods in an open system with steadyDraft CO2 production, highly diffusible PCO2 quickly 856 moves down its concentration gradient and equilibrates with the extracellular environment.
857 However, when there are rapid and large changes in CO2 production in metabolically active
858 cells, such as in cardiac and skeletal muscle cells, there can be regional differences in the cell
859 (Swietach et al. 2007; Vaughan-Jones et al. 2009) and transient out of equilibrium states have
860 been used experimentally to try to dissect out regulatory mechanisms that are not evident in the
861 steady state analysis (Leem and Vaughan-Jones 1998; Swietach et al. 2003; Zhao et al. 1995).
- 862 Since HCO3 is a dependent variable, it does not make much sense to use such
- - + - 863 terminology such as HCO3 /Cl exchanger (Chen and Boron 1991) or Na /HCO3 co-transporter.
864 The logic is the same as discussed previously for the “exchange” of Na+ and H+, although in the
- - 865 case of HCO3 and Cl , at least the magnitudes are similar, which makes the exchange seem more
- 866 feasible. Once again, it is worth considering what is measured. To obtain [HCO3 ], pH is
- 867 measured and PCO2 inside and outside the membrane are assumed to be equal. HCO3 itself is
868 not measured but rather is calculated, and it would be impossible to actually show movement of
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869 it across the cell for it is constantly associating and dissociating in the cell depending on the local
870 SID and [Atot] and the diffusion of CO2.
+ - 871 Neglect of the importance of SID and the dependent nature of [H ] and [HCO3 ] has
872 impacted the understanding of the pathophysiology of cystic fibrosis. The primary genetic
873 abnormality in this disease is in a transmembrane conductance regulator of Cl-. Recently, there
- - 874 has been increasing interest in the role of a so-called Cl /HCO3 exchange (anion exchangers or
- 875 AE) in this process. For example, an editorial in Nature called [HCO3 ] “The Neglected Ion”
876 (Quinton 2001). Perhaps a better title should have been “The Misunderstood Ion”! As discussed
877 above, when PCO2 is fixed experimentally or physiologically by the circulation and ventilation,
- 878 [HCO3 ] is determined by SID and [Atot]Draft of the solution. In the experiments, AE activity was
- 879 evaluated based on the change in pHi and the predicted [HCO3 ] based on the Henderson-
- 880 Hasselbach equation. However, as discussed above in the comparison of [HCO3 ] in the ICF and
- 881 ISF, this is true only if there is no change in [Atot] or SID. However, the Cl efflux from the cell
882 widens ICF SID and that is what decreases intracellular [H+] (increase pHi) and increases
- - 883 [HCO3 ]. It cannot be assumed that this will change the HCO3 in the extracellular environment
- - 884 or that HCO3 is “extruded” from the cell, for at a constant PCO2, the [HCO3 ] in the extracellular
885 space depends only upon the SID in that solution (assuming negligible [Atot] [Atot] outside the
886 cell) (figure 6). This, too, will not be true if an [Atot] is added to a cell culture for it will alter the
- - 887 change in [HCO3 ]. The only way that the cell can increase extracellular [HCO3 ] is by removing
888 Cl- from the extracellular space or by extruding Na+ or K+ which will widen extracellular SID.
889 Thus, there needs to be careful accounting of the three major strong ions for they are the major
890 determinants of pH changes as well as consideration of an [Atot] added to media of cells in
891 culture. Studies actually contain evidence that these ions do play key roles in CFTR activity. For
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892 example, in one study, a CFTR mutant was shown to be able to reduce intracellular Cl- without a
893 change in pHi. The only explanation for this striking lack of change in [H+] is that a strong
894 positive ion such as Na+ or K+ must have been excreted by the cell at the same time so that there
895 was no change in the ICF SID; otherwise pHi should have increased.
896 Changes in Weak acids The contribution of weak acids to acid-base balance in the ICF is
897 far more complex than in plasma. When analyzing their effect the key factor is the concentration
898 of the charge of [A-] and the pH of the solution because that is what determines how much they
899 can balance the SID of the solution. That value is hard to obtain. It is not sufficient to just
900 identify the pKa of the various weak acids; the concentration of the charge from the ionic
901 component must be known as was performedDraft by Figge et al on albumin (Figge et al. 1992; Figge
902 et al. 1991). The [Atot] in the ICF is ten-fold higher than that of the plasma (Stewart 1981) and it
903 is made up of many different molecules with different pKa’s and different charge effects
904 (Robergs 2017; Robergs et al. 2004). To make matters more complex, the properties and
905 magnitude of [Atot] remains relatively constant in plasma, at least over the short term, but this is
906 not the case inside cells. Molecules are continuously being produced and degraded and the
907 physical chemical properties of the products differ from those of their parents. For example,
908 ADP has a lower pKa than ATP (Rawn 1989) and therefore accumulation of ADP relative to
909 ATP will increase [H+]. It is quite possible that creation and metabolism of weak ions is one of
910 the ways that cells regulate ICF [H+]. By producing compounds with a given pKa, cells could
911 “set” their stable pH range at an appropriate level for their metabolic activity as discussed under
912 “buffers”.
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913 Strong and weak ions in the adjustments of ICF SID
+ - - 914 Whereas the movement of a strong ion changes the SID and alters [H ], [OH ], [HCO3 ],
915 and [A-], there still must be an accounting of all positive and negative charges. Thus, movement
916 of a strong ion without a countering strong ion must in some way be associated with movement
917 of another weak ion to maintain electrical neutrality. In the case of an influx of Na+ or K+ this
- - + 918 could occur by dragging along an OH or an HCO3 , or by H moving in the opposite direction by
+ - - 919 the Grotthuss mechanism (Cukierman 2006). However, the actual [H ] and [OH ] [HCO3 ] on
920 either side of the membrane, as well as all the other weak ions will readjust their concentrations
921 according to the new SID on either side of the membrane so that the final concentration of the
922 presumed weak ion will be different fromDraft what actually moved but the result will be identical
923 and impossible to differentiate no matter which of these three ions moved. This is true even
- -3 - + -8 924 though final [HCO3 ] is in the range of 10 Eq/L, whereas [OH ] and [H ] are in the range of 10
925 to 10-6 Eq/L. In the dynamic state, with all the reactions going through readjustments of
926 equilibrium and actual quantities of the ions varying by large amounts, the same final result can
927 occur without noticeable movement of weak ions. CO2, though, has characteristics which make
928 it different from other components of the solution. CO2 (dissolved) is not itself an ion and can
929 rapidly move across cell membranes which allows rapid adjustments (Vaughan-Jones 1979b).
- + 930 Second, although movement of CO2 / HCO3 behave the same in steady state as movement of H
931 and OH-, this system can behave less predictably than expected over short time periods because
932 of the need for carbonic anyhydrase (CA) to catalyse the normally slow conversion of CO2
933 (dissolved) to H2CO3.
934 Lessons learned about ICF [H+] adjustments during short heavy exercise
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935 A short burst (30 seconds) of heavy exercise provides a well-studied example of
936 integrated ICF [H+] regulation under a physiological stress. At the end of this burst of exercise,
937 intracellular [H+] increased from 86 nEq/L at rest to 420 nEq/l (Lindinger 1995). Of this
938 increase in [H+], 62% was explained by a narrowing of the SID, which occurred because of
939 leakage of K+ from the cell (32% of total increase in [H+]), and by a marked production of the
940 strong anion, lactate (47% of total change in [H+]). The change was modified by a reduction of
+ 941 phosphocreatine, which reduced the SID (-16% of the total change in [H ]). The ICF PCO2
942 increased from 45 mmHg at rest to 100 mmHg, but this only increased [H+] by 40 nEq/L and the
- - 943 calculated [HCO3 ] decreased from 12.2 mEq/L to 5.6 mEq/L. The [A ] increased from 140 to 944 170mEq/L due to increased creatinine andDraft phosphate which was offset by decreased ATP and the 945 net effect was an increase of 93 nEq/L of H+ which was 19% of the total change in [H+]. The
-7 946 change in protein composition increased the effective KA of [Atot] from 1.64 to 1.98 x 10 Eq/L,
947 which contributed 7% of the change in [H+]. The calculated ratio of [OH-] to [H+], even with the
948 marked rise in [H+], was still positive, indicating that the muscle cytoplasm was still an alkaline
949 solution.
950 Conclusions
951 The use in the traditional approach to acid-base balance of only the Henderson-
952 Hasselbalch equation for the dissociation of carbon dioxide species, and the pH terminology,
953 have obscured many important events in the regulation of [H+] in biological solutions. In our
954 proposed road-map for future investigation of the regulation of ICF [H+] (figure 7), the physical
955 chemical principles of water based solutions as discussed in this chapter must always be
956 considered. First, [H+] in biological solutions is almost always less than [OH-] rendering the
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957 environment alkaline, even with the most severe increases in [H+] in disease states. What we are
958 interested in biology is not the developments of an acid solution through “acid” production but
959 rather regulation of normal [H+]. Organisms evolved to regulate [H+] because its strong charge
960 density effects the activity of surrounding complex molecules. Altering [H+] up or down can
961 have both negative and positive physiological effects depending upon the metabolic pathway.
+ - 962 Second, [H ] and [HCO3 ] in a solution are dependent variables that are regulated by three
+ 963 independent variables which are the SID, PCO2 and [Atot] of the solutions. [H ] in the ICF is not
964 affected by the [H+] of outside solutions unless the independent components move into or out of
965 the solution of interest. The [Atot] of the ICF is a very complex component that will vary highly 966 from cell type to cell type and with changesDraft in metabolic activity. Three variables determine the 967 ionic effect of [Atot]; the total amount of the substance, its dissociation constant, and the
968 concentration of the ionic activity of the dissociated component ([A-]). All these can change with
969 changes in the steady-state metabolic activity. This may make it seem that it will not be possible
970 to truly asses the regulation of ICF [H+] (pHi).However, some general principles can allow a
971 better characterization of processes involved.
972 Quantitatively, the dominant determination of [H+] is the SID, and the dominant
973 determinant of the SID is the concentration of the elements [Na+],[K+] and [Cl-] in the ICF. Thus,
974 the first step of in evaluating ICF [H+] must include quantification of the concentrations of these
975 elements, both inside and outside cells, and how they change with the intervention. It has long
976 been possible to study ICF [Na+] and [K+], but sensitive ICF indicators of [Cl-] only have
977 become available more recently and will make this type of analysis now more feasible (Arosio et
978 al. 2010). In cell culture studies, the concentrations of elements and other strong ions added to
979 the supernatant also must always be carefully considered. The importance of these factors can
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980 then be studied by using standard inhibitors of exchangers and blocker of channels as have
981 always been done in the past. Substitution of the elements also can be done as in classical
982 experiments to determine specificity of the role of the element.
983 The second variable is PCO2. In life this PCO2 is a controlled variable, and unless
984 studying the specific effect of an increase in PCO2, it must be carefully controlled in all
- 985 experiments. It must be remembered that adding NaHCO3 is not adding an actual [HCO3 ],
- 986 because [HCO3 ] equilibrates with PCO2 which is blown off to keep plasma (or the cell culture
987 supernatant) at the controlled value. What is added is Na+ and the added Na+ changes the SID. If
988 the effect of a change in PCO2 on ICF is studied, the other two variables, ie SID and [Atot], must
989 remain relatively constant. This likely isDraft difficult to achieve because changes in [H+] change the
990 activity of exchangers, pumps and channels as well at the dissociation of the constituents of
991 [Atot].
992 The third factor, [Atot] and [Btot] is the biggest challenge. In normal plasma, [Atot] is
993 dominated by albumin and thus easily can be assessed (Figge et al. 1992). Unfortunately,
994 numerous factors in the ICF contribute to the [Atot] and their concentrations constantly change, as
995 do their dissociation constants as part of ongoing metabolic processes. However, some
996 simplifications are possible. To be significant, their ionic component needs to be in the mMol
997 range. HA that have pKa in the range of 6 to 8 such as imidazoles, histidine and biphosphates are
998 especially important because as long as their collective mass is greater than that of the SID, they
999 set the range of changes in ICF [H+] around their pKa because this value is close to normal ICF
+ 1000 [H ](Burton 2002; Dolan et al. 2019). It is unlikely that the effect of each component of the [Atot]
1001 effect can be quantified in the ICF but their cumulative effects potentially can be inferred by first
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1002 determining how much of the measured change in [H+] can be accounted for quantitatively by
1003 the measured changes in SID and PCO2, and then determining how much charge is still
1004 unaccounted for. What is left is the [Atot] effect. A variant of this approach has been used to
1005 assess pathological anions in plasma including a lactate acidosis (Magder and Emami 2015).
1006 There then can be a detailed quantitative analysis of the likely components in [Atot] that changed
1007 as in the analyses of Robergs (Robergs et al. 2004). The key factor is to always account for any
1008 changes in the three independent variables. Stewart’s physical-chemical approach is all about
1009 accounting.
1010 By ignoring the importance of strong ions, the properties of PCO2, [Atot], and the role of
1011 water in the seemingly simpler “classical”Draft approach to [H+] regulation, investigators have likely
1012 missed true regulatory variables, especially the movements of elements, failed to appreciate
1013 factors related to the intrinsic contents of cells themselves, and failed to appreciate how the
1014 solutions and conditions used in experiments alter the experimental observations. When the
1015 physical-chemical variables are considered some apparent mysteries begin to disappear.
1016
1017
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Table 1
Quantity Interstitial Blood Generic
Fluid Plasma ICF
[Na+] (mEq/L) 137 143 10
[K+] (mEq/L) 3 4 155
[Mg2+] (mEq/L) 2 2 10
[Ca2+] (mEq/L) 1 1 -
[Cl-] (mEq/L) 111 107 10 Other strong anions (mEq/L) Draft1 1 35
PCO2 (mmHg) 50 40 50
SID (mEq/L) 31 42 130
HA (mMol/L) – 20 200
- HCO3 (mEq/L) 31 25 16 pH 7.4 7.4 7
[H+] (Eq/L) 4.2 x 10-8 4.1 x 10-8 1.0 x 10-7
[OH-] (Eq/L) 1.1 * 10-6 1.1 * 10-6 4.3 x 10-7
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Figure Legends
Figure 1
The effect of the addition of a concentrated acid solution to salt solutions with SID of zero (a), negative value (b) or positive value (c)
Figure 1 a
Addition of acid solution to a neutral solution. The SID of the beaker at the bottom left contains a solution with equal concentrations of Na+ and Cl- and therefore SID = 0. HCl is added to the beaker to increase the final concentration of Cl- by 0.2 x 10-6 Mol. The SID decreases to -0.2 x 10-6 Mol. and the change in [H+] matches the amounts of H+ and Cl- added. The arrow marks the change in [H+] in the graph of [H+] and [OH-] against SID in the upper right.
Figure 1 b
Addition of acid solution to a solution with a negative SID. The SID of the beaker at the bottom left contains a solution with a concentrationDraft of Na+ that is less than that of Cl- and the SID = -0.2 x 10-6 Mol. The addition of enough HCl to increase the final concentration of Cl- by 0.2 x 10-6 Mol. (assume that the HCl solution is concentrated to minimize effects of changes of the concentration of Na+ and Cl- in the initial solution) reduces the SID to -0.4 x 10-6 Mol. As in 1a the change in [H+] matches the amount of H+ added. The arrow marks the change in [H+] in the graph of [H+] and [OH-] against SID in the upper right.
Figure 1 c
Addition of acid solution to a solution with a positive SID. The SID of the beaker at the bottom left contains a solution with a concentration of Na+ which is greater than Cl- and the SID = 0.4 x 10-6 Mol. The addition of enough HCl to change the final concentration of Cl- by 0.2 x 10-6 Mol. (assume that the HCl solution is concentrated to minimize effects of changes of the concentration of Na+ and Cl- in the initial solution) reduces the SID to 0.2 x 10-6 Mol. but the change in [H+] is much smaller and hardly noticeable on the graph. The arrow marks the change in [H+] in the graph of [H+] and [OH-] against SID in the upper right.
Figure 2
pH titration curve against SID. The dotted line is the sigmoidal relationship of pH to change in SID in a solution that only has strong ions (salt) at 37° C. There is an abrupt change in pH at SID
= 0. The bi-sigmoidal curve shows what happens when a weak acid ([Atot] = 0.02 Eq/L) is added to the solution. After the transition to positive SID there is a gradual change in pH until SID] exceeds the concentration of the weak acid (HA) at which point pH again abruptly rises. In the range of SID from just above 0 to .02 Eq/L the slope of the change in pH for change in SID is
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actually greater than slope with no weak acid. However, the weak acid creates a second plateau
(thickened region marked by the arrow) to the curve at a lower pH, which is related to the KA = 2 -7 x 10 Eq/L of the weak acid.
Figure 3. Example of NH4Cl loading of a mesangial cell
[Na+] ranging from 0 to 145 mEq/L were added extracellularly. (figure is used with permission from from Boyarsky et al (Boyarsky et al. 1988)). The interpretation here is based on the physical-chemical approach. The cells were in HEPES buffer and although grown in the presence of 5% CO2 , it was not stated whether the superfusate was bubbled with PCO2 during the experiments. pHi (intracellular pH) rises from a-b with the addition of 20 mM NH4+ which partially dissociates to NH3, enters the cell, and raises pHi by binding H+ to form the strong cation, NH4+ and NH3/ NH4+ reaches a new equilibrium. pHi gradually falls from b-c which the authors attributed to entry of NH4+ into the cell and acidification of the ICF by release of H+. As discussed in the text, it is more likely that ICF SID decreased by an outward movement of Na+ or K+ or an influx of Cl-. From c to d, NH4Cl was washed out and pHi fell to a lower value than at the start likely due to the reduction inDraft ICF SID from b to c. The re-introduction of Na+ gradually raised pHi back to the initial level, likely through the Na+/ H+ exchanger and restoration of the starting SID.
Figure 4 Role of CO2 in amiloride insensitive regulation of pHi
This is a similar preparation as in figure 3 but in this experiment the authors compared solutions without or with CO2 to demonstrate its role in amiloride insensitive pHi regulation (used with permission from figure 2 in Lagadic-Gossmann et a (Lagadic-Gossmann et al. 1992). Recovery from the fall in pHi following NH4Cl loading still occurred without added CO2 and the effect was blocked when amiloride was present to inhibit the Na+/H+ exchanger. Restoring CO2 allowed pHi to return to baseline although it is noteworthy that baseline pHi was high in the ICF. It should also be appreciated that even in the experiments with “no” CO2 there likely still was small amounts present in the supernatant as well as in the cells for cells were exposed to air and were metabolically active and therefore were producing CO2 . Furthermore, the addition of “bicarbonate” also meant that there was an addition of Na+ which would widen the SID if it entered the cells, and would have increased pHi.
Figure 5 - Example of NH4Cl loading in ventricular myocyte from guinea pig with CO2 and Cl removed
The figure is the same preparation as in Figure 4 and is from Lagadic-Gossmann et al, (used with permission from figure 5 (Lagadic-Gossmann et al. 1992)). There is again a rise in pHi with
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NH4Cl loading, a short period of a return to baseline (fall in pHi) and an overshoot of baseline pHi on withdrawal of the NH4Cl. In contrast to figure 3, there is a spontaneous return to the baseline value. The difference in this case is the presence of CO2 which likely diffused out of the cell in association with amiloride insensitive Na+ inflow or possibly even Cl- outflow. The authors indicate that the besides bubbling the perfusate with 5% CO2 , the solution was buffered with 23mM of HCO3- but as discussed in the text, if PCO2 was fixed, this added nothing to the solution except 23 mM more Na+. However, it appears in the paper that the 5% CO2 was not maintained during the experiment in which case there would have been a progressive loss of CO2 during the experiment. The potential effect of Cl- movement is shown in the right part of the figure where Cl- was removed from the supernatant and pHi rose consistent with the expected increase in SID. The subsequent response to NH4Cl loading and withdrawal is muted with less of a final fall of pHi suggesting a role for Cl- in the process. This also could have been due to gradual loss of CO2.
Figure 6. Draft Gamblegram of intracellular fluid (ICF), interstitial fluid (ICF) and plasma. The bars show the values of the cations and anions in each and the sum of these must be equal. Note that the ICF has the same PCO2 as the ISF but a much lower [HCO3-]. On the other hand the pH is the same in the ISF and plasma yet the [HCO3-] is larger in the ISF. This illustrates the interaction of SID and [A-] in the determination of [HCO3-].
Figure 7.
Schema of ways that intracellular [H+] can be increased. The same processes in the opposite direction will lower [H+]
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© The Author(s) or their Institution(s) Page 61 of 67 Canadian Journal of Physiology and Pharmacology
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figure 1c
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2
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figure 3
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figure 4
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figure 5
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figure 6
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© The Author(s) or their Institution(s) Page 67 of 67 Canadian Journal of Physiology and Pharmacology
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figure 7
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© The Author(s) or their Institution(s)