Am J Physiol Regul Integr Comp Physiol 289: R895–R901, 2005;
doi:10.1152/ajpregu.00641.2004.
The following is the abstract of the article discussed in the
subsequent letter:
Robergs, Robert A., Farzenah Ghiasvand, and Daryl
Parker. Biochemistry of exercise-induced metabolic acidosis.
Am J Physiol Regul Integr Comp Physiol 287: R502–R516, 2004
doi:10.1152/ajpregu.00641.2004.—The development of acidosis
during intense exercise has traditionally been explained by the in-
creased production of lactic acid, causing the release of a proton and
the formation of the acid salt sodium lactate. On the basis of this
explanation, if the rate of lactate production is high enough, the
cellular proton buffering capacity can be exceeded, resulting in a
decrease in cellular pH. These biochemical events have been termed
lactic acidosis. The lactic acidosis of exercise has been a classic
explanation of the biochemistry of acidosis for more than 80 years.
This belief has led to the interpretation that lactate production causes
acidosis and, in turn, that increased lactate production is one of the
several causes of muscle fatigue during intense exercise. This review
presents clear evidence that there is no biochemical support for lactate
production causing acidosis. Lactate production retards, not causes,
acidosis. Similarly, there is a wealth of research evidence to show that
acidosis is caused by reactions other than lactate production. Every
time ATP is broken down to ADP and Pi, a proton is released. When
the ATP demand of muscle contraction is met by mitochondrial
respiration, there is no proton accumulation in the cell, as protons are
used by the mitochondria for oxidative phosphorylation and to main-
tain the proton gradient in the intermembranous space. It is only when
the exercise intensity increases beyond steady state that there is a need
for greater reliance on ATP regeneration from glycolysis and the
phosphagen system. The ATP that is supplied from these nonmito-
chondrial sources and is eventually used to fuel muscle contraction
increases proton release and causes the acidosis of intense exercise.
Lactate production increases under these cellular conditions to prevent
pyruvate accumulation and supply the NAD⫹ needed for phase 2 of
glycolysis. Thus increased lactate production coincides with cellular
acidosis and remains a good indirect marker for cell metabolic
conditions that induce metabolic acidosis. If muscle did not produce
lactate, acidosis and muscle fatigue would occur more quickly and
exercise performance would be severely impaired.
Lactate accumulation, proton buffering, and pH change in
ischemically exercising muscle
By reviewing theory (6, 11, 12, 14) and reanalyzing data (3, 15,
19, 24, 25), Robergs and colleagues argue (21), among other
things, for a particular approach to the generation and disposal
of protons in exercising skeletal muscle. They rightly insist that
interpreting muscle cell pH changes requires an appropriate
analysis of proton stoichiometry. I would add two points. First,
one may be misled by neglecting, even for illustrative pur-
poses, the pH dependence of this stoichiometry. Second, care-
ful accounting is needed to assess how physicochemical buff-
ering contributes to cellular acid-base balance. To argue this, I
will consider only ischemic exercise (exercise without blood
flow), which has the simplifying advantages that mitochondrial
proton uptake and cellular proton efflux (21) are negligible,
and the only glycolytic substrate is glycogen (although for
completeness, results for glucose will also be given).
What might be called the traditional analysis criticized by
Robergs et al. takes the increase in cytosolic lactate concen-
tration as a measure of the proton load arising from glycogen-
olysis, which is disposed of in two ways (14, 16). The first is
physicochemical buffering [also called static (23) or structural
(21) buffering], predominantly by cytosolic imidazole groups
and inorganic phosphate (1, 2, 16, 23, 26). The second is the
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Letters to the Editor
consumption of protons by the Lohmann reaction (17); this
term refers to the hydrolysis of ATP and its simultaneous
regeneration at the expense of phosphocreatine, catalyzed by
creatine kinase (7, 17, 21) and is stoichiometrically equivalent
to the splitting, or hydrolysis, of phosphocreatine to inorganic
phosphate [all these terms can be found in the literature, but
note that this reaction does not occur (21), being only a name
for the sum of two enzyme-catalyzed processes]. This proton
consumption is sometimes called dynamic (23) or metabolic
(21) buffering. Taking the fall in cytosolic pH (to use a
physical analogy) as the strain resulting from the glycolytic
proton-load stress, the ratio of lactate increase to pH fall has
been used as an apparent buffer capacity, a measure of both
these components of proton handling (23) More properly, the
true physicochemical buffer capacity is the ratio to the pH fall
of the net proton load, that is, the total glycogenolytic proton
load less that consumed by the Lohmann reaction (16).
In criticizing this approach, Robergs et al. (21) note that the
glycogenolytic proton load is generated by the hydrolysis of
ATP rather than ionization of lactic acid (6, 12, 14), while the
reduction of pyruvate to lactate actually consumes protons,
mitigating acidification rather than causing it. Thus the naı̈ve
notion that glycolysis adds lactic acid to the cell is untenable
(21). Robergs et al. develop this criticism to argue for a
different understanding of acid-base balance, one implication
of which is that for biopsy and 31P MRS studies of muscle
energetics, the traditional approach yields gross underestima-
tions of the true muscle buffer capacity (21).
I will argue that while this analysis of proton generation is
broadly correct at resting pH, it is much less so at low pH
values commonly found in exercising muscle. Furthermore,
because this analysis of muscle cell buffering capacity adds
together components of proton generation that are partially
cancelled by processes of proton consumption, it may be
physiologically misleading.
BIOCHEMICAL BACKGROUND
To understand this, we must consider the stoichiometry (Table
1) and then examine its implications for the analysis of cellular
buffering (Table 2). In ischemic exercise ATP is produced by
glycogenolysis to lactate and by the Lohmann reaction. The
effects on cytosolic pH (which can vary from ⬃7 at rest to ⬃6
in intense exercise) depend on the pH-dependent proton stoi-
chiometries of the component processes (1, 2, 6, 7, 12, 14, 16,
17). Table 1 shows (centered) the biochemical equations in a
form that takes account of this pH dependence and cites the
corresponding expressions or figures in Robergs et al. (21)
where, for a simpler exposition, this complication is ignored. (I
follow Robergs et al. in not using the alternative strong ion
difference formalism, which is not very useful when lactate is
the only strong ion measured.) Table 1 also shows algebraic
expressions for the proton stoichiometries, along with the
numerical values stated or implicit in Robergs et al. (21) and as
calculated from the present analysis for three pH values span-
ning the physiological range. Table 2 gives expressions for the
estimation of various versions of calculated buffer capacity.
Figure 1 illustrates the application to a data set (16).
Glycolysis from glycogen to pyruvate (Table 2 in Ref. 21)
generates about one proton per pyruvate at resting pH, increas-
ing as pH falls (Eq. 1b); this is not very different for glycolysis
from glucose (although in the simplified Robergs analysis the
R895
Letters to the Editor
R896
Table 1. Components of proton (H⫹) generation and consumption in ischaemically exercising muscle
Stoichiometry in Robergs et al. (21) and the Present Analysis†

Process and Analysis* Component Substrate Robergs pH 7 pH 6.4 pH 6 Equation and Derivation

Glycolysis to pyruvate: Eqs. 1 and 2 and Table 2 in Robergs et al. (21)

X ⫹ 2␻ADPa ⫹ 2␻Pib ⫹ 2NAD⫹ 3 Y ⫹ 2Pyr⫺ ⫹ 2␻ATPc ⫹ 2NADH ⫹ 2␻H2O ⫹ 2[␻(a ⫹ b ⫺ c) ⫹ 2]H⫹
ATP production ⫽ ␻ per pyruvate ⫽ ␻⌬La Eq. 1a
H⫹ production ⫽ [␻(a ⫹ b ⫹ c) ⫹ 2]⌬La H⫹ produced per lactate‡ glycogen (␻ ⫽ 3/2) 0.5 0.91 1.42 1.93 Eq. 1b
glucose (␻ ⫽ 1) 1 1.27 1.62 1.95

Reduction of pyruvate to lactate Fig. 9 in Robergs et al. (21)

Pyr⫺ ⫹ NADH ⫹ H⫹ 3 La⫺ ⫹ NAD⫹
H⫹ consumption ⫽ ⌬La H⫹ consumed per lactate glycogen and glucose 1 1 1 1 Eq. 2

Glycolysis to lactate (i.e., glycolysis to pyruvate) and reduction of pyruvate to lactate: Eqs. 3 and 4 in Robergs et al. (21)
X ⫹ 2␻ADPa ⫹ 2␻Pib 3 Y ⫹ 2La⫺ ⫹ 2␻ATPc ⫹ 2␻H2O ⫹ 2[␻(a ⫹ b ⫺ c) ⫹ 1]H⫹
H⫹ production ⫽ [␻(a ⫹ b ⫺ c) ⫹ 1]⌬La H⫹ produced per lactate glycogen ⫺0.5 ⫺0.09 0.42 0.93 Eq. 3 (from Eqs. 1b and 2)
glucose 0 0.27 0.62 0.95

Hydrolysis of ATP: Fig. 10 and Eq. 7 in Robergs et al. (21)

ATPc ⫹ H2O 3 ADPa ⫹ Pib ⫹ (c ⫺ a ⫺ b)H⫹
H⫹ production: in general ⫽ (c ⫺ a ⫺ b) H⫹ produced per ATP glycogen and glucose 1 0.73 0.38 0.05 Eq. 4a
per ATP
from glycolytic ATP ⫽ ␻(c ⫺ a ⫺ b)⌬La H⫹ produced per lactate glycogen 1.5 1.09 0.58 0.07 Eq. 4b (from Eqs. 1a and 4a)
glucose 1 0.73 0.38 0.05
from ATP via CK ⫽ (c ⫺ a ⫺ b)⌬PCr H⫹ produced per PCr glycogen and glucose 1 0.73 0.38 0.05 Eq. 4c (from Eqs. 4a and 6a)

Glycolysis to lactate coupled with ATP hydrolysis: Eqs. 5 and 6 in Robergs et al. (21)
X⫺ 3 Y ⫹ 2La⫺ ⫹ 2H⫹
H⫹ production ⫽ ⌬La H⫹ produced per lactate glycogen and glucose 1 1 1 1 Eq. 5a (from Eqs. 3 and 4b)
⫹
Fraction of this H derived from ATP glycogen 1.5 1.09 0.58 0.07 0.05 Eq. 5b (from Eqs. 4b and 5a)
hydrolysis ⫽ ␻(c ⫺ a ⫺ b)
glucose 1 0.73 0.38 0.05

ATP regeneration by phosphocreatine, catalysed by CK: Fig. 4 in Robergs et al. (21)

PCrg ⫹ ADPa ⫹ (c ⫺ a ⫺ g)H⫹ 3 Cr ⫹ ATPc
ATP production ⫽ 1 ATP per PCr Eq. 6a
H⫹ consumption ⫽ (c ⫺ a ⫺ g)⌬PCr H⫹ consumed per ATP glycogen and glucose 1 0.93 0.82 0.70 Eq. 6b

Lohmann reaction: ATP hydrolysis plus ATP regeneration: Figs. 4 and 11 in Robergs et al. (21)
PCrg ⫹ (b ⫺ g)H⫹ ⫹ H2O 3 Pib ⫹ Cr
H⫹ consumption ⫽ (b ⫺ g)⌬PCr H⫹ consumed per ATP glycogen and glucose 0 0.20 0.44 0.65 Eq. 7 (from Eqs. 4c and 6b)
Pyr, pyruvate; La, lactate; PCr, phosphocreatine; Cr, creatine; ADP, adenosine diphosphate. In the biochemical equations (centered) ATP, ADP, PCr, and Pi
refer to the sum of all relevant chemical species, and the superscripts a, b, c, g are the generally nonintegral average charges of ADP, Pi, ATP and PCr (Cr is
uncharged), respectively. In the algebraic equations, concentration brackets are omitted for simplicity; ⌬La denotes an increase and ⌬PCr and ⌬pH a decrease
during exercise, and the stoichiometric coefficients a, b, c, g are simply the charges in the biochemical equations. The proton stoichiometry of these equations
follows from the electroneutrality condition. The conclusions here follow directly from this, relatively independent of the numerical values of the charges. The
equations apply to both glycolytic substrates: for glycogen (the predominant substrate in ischemic exercise), the symbols X and Y refer to glycogenn and
glycogenn⫺1 and the ATP stoichiometric factor ␻ ⫽ 3/2; when X refers to glucose, Y is nothing and ␻ ⫽ 1. ␤ is the physicochemical cytosolic buffer capacity.
Eqs. 1–7 in Table 1 are based mainly on Refs. 12, 14, 17, Eqs. 8 –12 in Table 2 mainly on Refs. 1, 23, 26; see Ref. 16 for more details. Equations 13–15 in
Table 2 from Ref. 21 are translated into the present terminology using the analysis presented here. One complication ignored in these expressions, although not
in the illustrative calculations in Fig. 1, is that the pH dependence of both stoichiometric coefficients and buffer capacity requires that, for example, (b ⫺ g)⌬PCr
and ␤⌬pH should be (b ⫺ g)dPCr and ␤dpH, or in practice their finite-sum equivalents, ⌺[(b ⫺ g)␦PCr] and ⌺[␤␦pH]. *Reference is made to the numbered
equations and figures in Robergs et al. (21), where for simplicity, pH effects on stoichiometry are ignored, which are explicit here. Equations 1– 4 (21) ignore
the charges on ADP, ATP, Pi, lactate, and pyruvate, and Figs 4, 10, and 11 in (21) show only ADP3⫺, ATP4⫺, Pi2⫺ and PCr2⫺ (see footnote†). †These
stoichiometric coefficients are calculated using the analysis of Kushmerick (17), in which ␣, ⫺␤, and ⫺␥ are equivalent to c ⫺ a ⫺ b, c ⫺ a ⫺ g and b ⫺ g
here (this ␤ is not that used here for buffer capacity); these coefficients can be calculated empirically from pH using ␥ ⫽ 27.239 ⫺ 13.593pH ⫹ 2.1440(pH)2 ⫺
0.10887(pH)3, ␤ ⫽ ⫺11.869 ⫹ 5.6685pH ⫺ 0.92213(pH)2 ⫹ 0.047950(pH)3 and ␣ ⫽ 39.108 ⫺ 19.262pH ⫹ 3.0662(pH)2 ⫺ 0.15682(pH)3 (17). Values are
given for pH 7 (rest), pH 6.5 (end of exercise in Figure 1) and pH 6 (an extreme pH which can be reached in intense exercise); they are near-linear with pH
over this range. Values of the coefficients implicit in Robergs’ analysis are calculated by setting a ⫽ ⫺3, b ⫽ g ⫽ ⫺2, c ⫽ ⫺4, so that c ⫺ a ⫺ b ⫽ c ⫺ a ⫺
g ⫽ 1 and b ⫺ g ⫽ 0 (see footnote*). Glycogen and glucose substrate are distinguished where necessary. The conclusions presented here are not very sensitive
to the exact values chosen for these coefficients. ‡In Eq. 1 the product is pyruvate, but the expression is given for protons per lactate, because what is usually
measured (24, 25) or inferred (16) is accumulation of lactate, which in ischemic exercise is very close to lactate ⫹ pyruvate.

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 Letters to the Editor
R897
Table 2. Analysis of H⫹ buffering in ischaemically exercising muscle
Processes and Equations Equation and Derivation

Physicochemical version of traditional analysis

Net H⫹ load to cytosolic buffers ⫽ ⌬[La] ⫺ (b ⫺ g)⌬PCr ⫽ (glycolytic H⫹ load) ⫺ (Lohmann-consumed H⫹)
In the absence of glycolysis*; net H⫹ load ⫽ (b ⫺ g)⌬PCr, which is negative Eq. 8 (from Eqs. 5a and 7)
H⫹ buffered ⫽ ␤⌬pH Eq. 9
f Estimated physicochemical buffer capacity* ⫽ (net H⫹ load)/⌬pH ⫽ ␤ Eq. 10 (from Eq. 9)
Metabolic version of traditional analysis†
Metabolic H⫹ load ⫽ H⫹ production by glycolysis coupled with ATP hydrolysis ⫽ ⌬La Eq. 5a
H⫹ consumption ⫽ buffered ⫹ consumed by ATP regeneration from phosphocreatine ⫽ ␤⌬pH ⫹ (b ⫺ g)⌬PCr Eq. 11 (from Eqs. 7 and 9)
f Apparent buffer capacity ⫽ (glycolytic H⫹ load)/(pH fall)‡ ⫽ ⌬[La]/⌬pH Eq. 12a (from Eqs. 5a and 11)
f Ratio to true ␤ ⫽ 1 ⫹ (b ⫺ g)⌬PCr/(␤⌬pH) ⫽ 1 ⫹ (Lohmann-consumed H⫹)/(buffered H⫹) Eq. 12b (from Eqs. 9 and 12a)
Total muscle buffering analysis‡ of Robergs et al. (21)
H⫹ production ⫽ H⫹ from ATP hydrolysis ⫹ H⫹ from pyruvate synthesis ⫽ (c ⫺ a ⫺ b)[⌬PCr ⫹ ␻⌬La] ⫹
[␻(a ⫹ b ⫺ c) ⫹ 2]⌬La ⫽ 2⌬La ⫹ (c ⫺ a ⫺ b)⌬PCr Eq. 13 (from Eqs. 1b, 4b, and 4c)
H⫹ consumption ⫽ H⫹ buffered ⫹ H⫹ consumed by lactate reduction ⫹ H⫹ consumed by phosphocreatine
breakdown ⫽ ␤⌬pH ⫹ ⌬La ⫹ (c ⫺ a ⫺ g)⌬PCr Eq. 14 (from Eqs. 2, 6b, and 9)
f Proposed total buffer capacity ⫽ (total H⫹ production)/(pH fall) Eq. 15a (from Eqs. 9 and 13)
f Ratio to ␤ ⫽ 2 ⫹ [(b ⫹ c ⫺ a ⫺ 2g)⌬PCr/(␤⌬pH)] ⫽ 2 ⫹ [1 ⫹ (c ⫺ a ⫺ g)/(b ⫺ g)](Lohmann-consumed
H⫹)/(buffered H⫹) Eq. 15b (from Eqs. 9 and 15a)
See Table 1 for terminology, abbreviations, and biochemical background, and for the literature sources of the equations in this table; see the body of Table
1 for Eqs. 1– 7. *For example, in very early exercise (1, 4; see Fig 1C). †More complex expressions can take account of changes in other metabolites at lower
concentrations than lactate (13, 23, 25). ‡The relationship between these estimates of buffer capacity depends on the ratio of Lohmann-consumed to buffered
protons (Eq. 12b). This is ⫺1, when glycolysis is negligible, for example, in very early exercise where pH rises (see Fig 1C). It is undefined for pH ⬇ 0, then
achieves a value in later exercise that depends on metabolic regulation (see text), reaching about 0.4 in Fig. 1 (16), at which point the apparent buffer capacity
of Eq. 12a (23) is 40% higher than true physicochemical buffer capacity (Fig 1B), while the total buffer capacity of Eq. 15a (21) is about 3 times higher (Fig
1A).

assumed stoichiometric coefficients differ by a factor of 2).
Next, reduction of pyruvate to lactate consumes one proton,
independent of pH (Eq. 2). At resting pH, because this disposes
of protons roughly equivalent to the protons generated earlier
in glycolysis, both glycogenolysis and glycolysis to lactate (Eq.
3) generate few protons (21) [in the simplified Robergs anal-
ysis, glycolysis from glucose to lactate produces none, while
glycogenolysis consumes one proton per glucosyl (21)]. The
ATP generated by glycolysis does not accumulate, being si-
multaneously hydrolyzed by ATPases, resulting in the gener-
ation of almost one proton per ATP at resting pH, decreasing
as pH falls (Eq. 4a). Although two of the products of ATP
hydrolysis, inorganic phosphate and ADP, are recycled by
glycolysis, the protons accumulate to acidify the cell (12, 21).
The result is that glycogenolysis or glycolysis to lactate cou-
pled to simultaneous ATP hydrolysis generates exactly one
proton per lactate, independent of pH (Eq. 5a) (12).
It is the main premise of Robergs et al. (21) that the principal
source of the proton arising from coupled glycolysis is ATP
hydrolysis rather than lactic acid synthesis per se. Table 1
shows that this is true at resting pH (Eq. 5b); however, as pH
falls, this proportion is reversed as proton production by ATP
hydrolysis decreases (6, 12, 14); until pH 6, most of the
glycolytic protons actually do come from glycolysis to lactate
(Eq. 5b). Thus this claim (21) is a significant oversimplification
as a result of neglecting the correct values of the stoichiometric
coefficients. Note that this argument is quite robust. To calcu-
late the protons produced by glycogenolysis coupled to ATP
hydrolysis (Eq. 5a), we simply add the protons produced by
glycogenolysis to lactate (Eq. 3) to those produced by the
balancing hydrolysis of the ATP so formed (Eq. 4b); the
pH-dependent proton stoichiometry cancels out, so the result is
one proton per lactate at all pH values, independent of sub-
strate, and also using the coefficients assumed by Robergs et al.
(21) (Table 1). The fraction of glycolytic protons supplied by
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coupled ATP hydrolysis (Eq. 5b) is therefore numerically
equal to the proton load from hydrolysis of glycolytic ATP
(Eq. 4b): only in the Robergs analysis for glucose is this
fraction unity (21) (Table 1).
Now, we must introduce creatine kinase. In muscle, over a
wide range of ATP turnover, the regeneration of ATP at the
expense of phosphocreatine (Eq. 6a) buffers ATP against
temporary imbalance between ATP use and glycogenolytic
ATP production (9). This, the Lohmann reaction (see previous
section), consumes a number of protons equal to the difference
in charge between phosphocreatine and inorganic phosphate
(Eq. 7). On the simplest analysis in which the phosphocreatine
has charge ⫺2, this is just the monoanion fraction of inorganic
phosphate, 1/[1⫹10(pH⫺6.8)] ⬇ 0.4 at resting pH (1, 14, 16, 26),
but more recent analysis, taking account of hitherto unconsid-
ered potassium binding, suggests a smaller value of ⬃0.2 at
rest, increasing as pH falls (7, 17). There is some disagreement
(16) about which value should be used (compare e.g., Refs. 1,
4, 10, 20, and 22), but this is not important to the present
argument. However, it is a feature of the illustrative stoichi-
ometry in the Robergs analysis (21), where this whole ap-
proach is rejected, that the implied value of the Lohmann
coefficient is zero (see Table 1, †footnote).
“ TRADITIONAL” APPROACHES TO INTRACELLULAR
PROTON BUFFERING
This is summarized in Table 2. A standard interpretation (7, 14,
16, 23, 25, 26) is that glycogenolysis generates protons, which
are buffered by passive processes and consumed by the Loh-
mann reaction (Eq. 7). What the Lohmann reaction leaves
behind is the net proton load (Eq. 8) that produces a pH change
depending on the physicochemical buffer capacity (Eq. 9) (16);
this can, therefore, be estimated as their ratio (Eq. 10), for
comparison with ex vivo measurements (1, 5, 16, 17). Follow-
ing Sahlin’s terminology for the components of buffering (23),
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Letters to the Editor
R898
I call this the physicochemical approach. The underlying con-
cept is of a net proton-generator (or, in early exercise, proton-
consumer) comprising glycolysis, ATPase and creatine kinase
added to a cytosol containing only passive buffers. The argu-
ment is spelled out by Kemp and colleagues (16).
An alternative version of this approach is to regard glyco-
genolysis as a source of protons (Eq. 5a) that are dealt with
both by physicochemical buffering and metabolically by the
Lohmann reaction (Eq. 11). The ratio of the glycogenolytic
proton load (i.e., the lactate increase) to the pH fall (Eq. 12a)
can conveniently be taken as a functional or apparent buffer
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capacity (23, 25), which exceeds the true physicochemical
buffer capacity (Eq. 12b) (there is some ambiguity about this
relationship in the older experimental literature). Following
Robergs’ terminology (21) for the components of buffering, I
call this the metabolic approach. The underlying concept is of
a net proton-generator comprising glycolysis plus ATPase
added to a cytosol containing passive buffers and the net
proton-consuming creatine kinase system. Thus the conceptual
difference between the apparent and true buffer capacities is
where the boundary is drawn between the generation and
disposal of protons, whether creatine kinase is lumped with the
289 • SEPTEMBER 2005 • www.ajpregu.org

ATP-generating enzymes of glycolysis or with the proton-
buffers of the cytosol.
The importance of these distinctions is that the balance
between proton consumption and proton buffering depends on
metabolic regulation (23), specifically, the relationship be-
tween glycogen phosphorylase flux and the concentration of its
substrate, phosphate (16). Because of the stoichiometric
Lohmann-reaction relationship between phosphocreatine and
phosphate, this can be seen as a negative feedback mechanism,
whereby phosphate is an error signal responding to the time-
integrated mismatch between ATP usage and glycogenolytic
ATP production (16). Although its role in the regulation of
glycogenolysis is controversial (7, 8, 18), this phosphate de-
pendence of phosphorylase is important in the control of pH,
and therefore in the relationship (Eq. 12b) between apparent
(Eq. 12a) and true buffer capacity (Eq. 10). One reason for the
early popularity (23) of the apparent buffer capacity was that
the system operates so that the relationship between pH and
lactate is remarkably linear and invariant [see Fig. 3 in (21)]:
the constraints on this, and its implications for metabolic
control, are discussed elsewhere (16).
PROPOSED NEW APPROACH TO INTRACELLULAR
PROTON BUFFERING
This is also summarized in Table 2. Robergs et al. (21) argue
that the traditional analysis is wrong because the protons
produced during glycogenolysis arise from ATP hydrolysis
(Eq. 4a) not lactic acid. However, we have seen that the
premise is only approximately true and progressively less so as
pH falls (Eq. 5b). Furthermore, the traditional analysis makes
no necessary assumption about the ultimate source of the
protons in Eq. 5a; references in the literature to protons arising
from lactic acid can safely be replaced by the more conceptu-
ally accurate protons accompanying lactate.
More fundamentally, Robergs et al. argue for a new analysis
of proton generation and consumption, with implications for
the quantification of muscle buffer capacity. Their argument
(21) is supported (in their Fig. 15) by the observation that
efflux of protons during nonischemic exercise exceeds that of
lactate (15) and by calculations comparing the two approaches
Letters to the Editor
R899
applied to published data from exercising human quadriceps
(3, 19, 24, 25). In particular, they argue (in their discussion of
their Figs. 16 and 17) that 1) given the known cytosolic buffer
capacity, the proton balance equations make sense only for the
new model; and 2) the true buffer capacity in vivo is much
greater than the traditional view allows (21). However, com-
parison of estimates of buffer capacity inferred in vivo with
those obtained by titration of muscle homogenate in vitro is by
no means straightforward (1, 16, 17), and cannot therefore
settle this question. Insufficient detail is given of these calcu-
lations (21) to test them directly against the analysis above (see
Appendix). However, the main issue is more fundamental, and
can be argued on theoretical grounds.
Robergs et al. suggest that total proton generation (Eq. 13)
has two components (Fig. 1A). The first is proton generation by
ATP hydrolysis (Eq. 4a), in which one can distinguish the
protons generated by hydrolysis of ATP produced from phos-
phocreatine via creatine kinase (Eq. 4c) from those generated
from ATP produced by glycogenolysis (Eq. 4b). The second
main component of proton generation (21) is glycogenolysis to
pyruvate (Eq. 1b). Total proton generation is independent of
substrate (Eq. 13). Robergs et al. (21) divide total proton
consumption (Eq. 14) into three components. The first (Fig.
1A) is the traditional passively buffered component (Eq. 9).
The second represents the buffering of glycolytic protons by
lactate formation (Eq. 2). The third represents protons con-
sumed in phosphocreatine breakdown; as the acid-base effects
of ATP hydrolysis matching phosphocreatine breakdown are
already taken into account in Eq. 9, this must be the unidirec-
tional reaction (Eq. 6b) rather than the net Lohmann reaction
(Eq. 7). Robergs et al. argue that the correct, or at least most
physiologically meaningful, calculation of buffer capacity is
the ratio of total proton production to pH fall (Eq. 15a). This
gives a value (Fig. 1A) much larger (Eq. 15b) than both the
traditional physicochemical calculation (Eq. 9) and indeed
larger than the traditional concept (the metabolic version, in the
present terminology) of apparent buffer capacity (Eq. 15a;
Fig. 1B).

Fig. 1. Application to ischemic exercise. Illustration of the application of the analyses in Table 2 to data from a 31P MRS study of ischemic forearm exercise
(16). It shows, for each analysis, the summed processes of proton generation and proton consumption: Here, these are equal by definition, as comparing in vivo
and ex vivo estimates of buffer capacity is not the point [see elsewhere for this, and for the technical issues (1, 16, 17)]. Using data from whole 3 min of exercise,
where ⌬pH ⬇ 0.5 and ⬃60% of ATP comes from glycogenolysis, shows the analysis of Robergs et al. (A) and the traditional analysis in both its versions (B);
the identical x-axes show how much larger proton generation/consumption terms is in the Robergs’ analysis. Various buffer capacities are shown next to the
analysis from which they derive, in units of mmol (l cell water)⫺1 (pH unit)⫺1, or slykes (23). (C) shows the results of both analyses for the first exercise data
point, where lactate generation has not begun and ⌬pH ⬇ ⫺0.02 (i.e., a small pH increase); note the different x-axis to (A) and (B), as the quantities are much
smaller. At this point the contribution of lactate to proton production (on the traditional view) and of pyruvate synthesis to proton production and pyruvate
reduction to proton consumption (21) are zero. On the traditional view the buffered and consumed components are equal and opposite (as pH rises), and the
glycolytic proton load is zero; the net proton load to the cytosolic buffers is therefore negative. The estimated buffer capacity (Eq. 9) is the negative ratio of the
(positive) consumed protons to the (negative) pH fall. In the Robergs analysis, total proton production is always positive, whether or not lactate accumulates (Eq.
13) in the absence of lactate accumulation, this proton load is solely from hydrolysis of the ATP generated via creatine kinase (Eq.s. 4 and 13), and such a positive
proton generation cannot explain the rise in pH; the total buffer capacity (Eq. 15a) therefore appears negative. In fact, this proton generation is exceeded by proton
consumption associated with flux through the creatine kinase reaction in the direction of phosphocreatine breakdown (Eq. 6b), the difference between the two
being the net proton consumption by the Lohmann reaction (Eq. 7), which in the traditional view readily explains the pH rise. The difference between the
traditional estimate of physicochemical ␤ at the end (B) and start of exercise (C) is due partly to the contribution of phosphate at the end of exercise and partly
to the effect of lower pH on both phosphate and the other passive buffers of the cell (16). Note that if the calculations in (A) used the values assumed by Robergs
et al. (Table 1A) rather than the correct stoichiometric coefficients derived from (17), the total quantity of protons and thus the total buffer capacity would be
little affected (only 6% higher); however, the implicit proportion of total proton production by ATP hydrolysis (Eq. 5a) would be 1.5 [as glycogenolysis to lactate
is a net consumer of protons at resting pH (21)] rather than the correct value of 1.1 decreasing to 0.6 as pH falls (Table 1A), and the overall fraction of total
proton production (Eq. 13) due to ATP hydrolysis would consequently appear to be 80%, twice the correct figure of 40%. The use of the approximate coefficients
misrepresents the balance between these components. An implication of the simplified Robergs stoichiometry assumptions is that the Lohmann reaction (Eq. 7)
consumes no protons, which would clearly have a large effect on (C, top).

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Letters to the Editor
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However, by setting the total proton generation of Robergs
et al. (Eq. 13) equal to their total proton consumption (Eq. 14),
we obtain precisely the traditional expression (Eq. 8). Thus
whatever detailed calculations underlie Figs. 16 and 17 in
Robergs et al. (21), these calculations cannot influence whether
or not the implied value of cytosolic buffer capacity matches
that obtained by titration of muscle homogenate in vitro.
COMPARISON OF THESE TWO APPROACHES
The conceptual difference between these approaches is a mat-
ter of labeling the components of proton balance (compare
Figs. 1A and 1B) but with real consequences for the concept of
cellular buffer capacity. Robergs et al. take a broad view of the
proton load, including all the protons produced by ATP hydro-
lysis (Eq. 4a) and by glycogenolysis to pyruvate (Eq. 1b), and
also of proton consumption, including the protons accounted
for by reduction of pyruvate to lactate (Eq. 2) and by ATP
synthesis at the expense of phosphocreatine (Eq. 6b). However,
much of this proton generation is cancelled by proton con-
sumption, so only a fraction remains to be buffered in the
cytosol.
The key point is what proton buffering means. To tradition-
alists, it is physicochemical buffering (Eq. 9), sometimes
expanded to include proton consumption in the Lohmann
reaction (Eq. 7; 23) [although I prefer to distinguish these
(16)]. The traditional view takes account of both the protons
consumed by phosphorylation of ADP by phosphocreatine (Eq.
6) and the protons produced by the hydrolysis of the ATP
supplied at the expense of phosphocreatine (Eq. 4c) and com-
bines them into the net proton consumption by the Lohmann
reaction (Eq. 7). What remains after this is subtracted from the
glycogenolytic proton load in the physicochemical version of
this approach is the net proton load, which is buffered in the
cytosol (16); in the metabolic version, the ratio of total proton
load to pH fall is a measure of the combined effect of physi-
cochemical and metabolic buffering (16, 23), which may or
may not be useful (see below). In both versions of the tradi-
tional approach, metabolic buffering means the Lohmann re-
action.
In the proposed new view, by contrast, the concept of
metabolic buffering is expanded (21) to include two processes
of proton consumption: reduction of pyruvate to lactate (Eq. 2)
and ATP regeneration at the expense of phosphocreatine (Eq.
6b). However, these are partially cancelled out by two pro-
cesses of proton generation: hydrolysis of ATP arising from
phosphocreatine breakdown (Eq. 4c) and glycolysis to pyru-
vate (Eq. 1b). Thus the ratio of this expanded total proton load
(Eq. 13) to pH fall, proposed as total muscle buffer capacity
(21), has no obvious physiological interpretation (Eq. 15b). It
has a complicated relationship to physicochemical cytosolic
buffer capacity: when acidification is substantial, total muscle
buffer capacity considerably exceeds physiochemical cytosolic
buffer capacity (Fig. 1A); in the special case of no lactate
accumulation (Fig. 1C), which poses no problem to the tradi-
tional approach (4), total cellular buffering capacity is nega-
tive, a reductio ad absurdum.
The apparent buffer capacity (Eq. 12a), which Robergs et al.
criticize has similar defects: it too overestimates physicochem-
ical buffer capacity, although in a more algebraically straight-
forward way (Eq. 12b), which is related to the metabolically
important relationship between glycogen phosphorylase flux,
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phosphate concentration, and creatine kinase (16), insofar as
this influences the relative proportion of protons buffered and
consumed. It has the technical advantage of being equal to the
true physicochemical buffer capacity in the hypothetical ab-
sence of phosphocreatine breakdown but the disadvantage of
being zero in the absence of lactate production. This concept
might be seen as carrying useful information about metabolic
regulation (16), or else as fatally flawed (21), its experimental
simplicity outweighed by conceptual obscurity.
The relative proportion of protons buffered and consumed
features also in the relationship between total and physico-
chemical buffer capacities (Eq. 15b) but in a way that makes
the total buffer capacity exactly twice the true buffer capacity
in the absence of phosphocreatine breakdown and negative in
the absence of lactate production. I suggest that total buffer
capacity is no more useful than apparent buffer capacity and
physiologically more obscure.
CONCLUSION
Robergs et al. have usefully called attention to some ambigu-
ities and confusions in the literature on extra- and intracellular
acidification by glycolysis. In the application to exercising
muscle, however, they have reached a potentially misleading
conclusion about muscle cell buffer capacity. The traditional
view criticized by Robergs et al. (21) certainly appears to
neglect the roles of lactate synthesis in consuming the protons
formed earlier in glycogenolysis and of ATP hydrolysis as the
main source of the protons that accompany lactate. However,
their presentation, which ignores for simplicity the pH depen-
dence of the stoichiometry (6, 12, 14, 26), does not show how
the importance of this point decreases as cell pH falls during
exercise and that it does not in any case affect the logic of the
traditional calculations. Additionally, Robergs et al. (21) make
what is essentially a labeling error in the analysis of cellular
proton buffering. Focusing attention on the individual reac-
tions, they do not take account of how some of the processes of
proton generation are canceled out by those of proton con-
sumption. Thus their total muscle buffer capacity does not
seem to identify a useful property of muscle physiology.
APPENDIX
There are some possible inconsistencies in Figs. 16 and 17 of
Robergs et al. (21), and the relationship to the experimental
papers cited (3, 19, 24, 25) is not entirely clear. For example,
the 125 mmol proton/kg muscle used as the denominator to
estimate total muscle buffer capacity (Eq. 15 here) as 208
slykes (evidently mmol/kg wet wt) appears inconsistent with
the ⬃100 mmol proton/kg muscle shown in Fig. 17. The 72
mmol proton/kg muscle used to calculate the metabolic buff-
ering of 120 slykes appears to be the sum of the components
labeled Pi, CrP and lactate in Fig. 17, which I take to be the
protons buffered by the inorganic phosphate component of ␤
and the protons consumed by phosphocreatine hydrolysis
(Eq. 6b) and the reduction of pyruvate to lactate (Eq. 2),
respectively. However, the first of these should presumably
be considered structural or static buffering: even though
phosphate increases during exercise, the proton-binding
effect of phosphate requires a fall in pH (to drive the ionic
equilibrium Pi2⫺ ⫹ H⫹ 7 HPi⫺ to the right) but no enzyme
action; in this it differs from the indubitably metabolic
Lohmann reaction, which requires enzyme activity but not a
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change in pH; indeed, it can consume protons when pH is
rising (Fig. 1C) and the static buffers are giving protons up.
The remaining structural (physicochemical) buffer capacity
of 88 (⫽208 ⫺ 120) slykes is not, as claimed, close to the
structural buffer capacity reported in (23); the 42 slykes they
cite in Fig. 16 (21) as a value obtained by homogenate
titration appears to be a value predicted from measured and
estimated cytosolic concentrations (23) and is anyway at the
low end of the published range of homogenate-titration
values (16, 23), which are often overestimates (1). Because
of these uncertainties, in this letter, I have taken a theoret-
ical approach supported by illustrative data from another
source.
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Graham Kemp
Division of Metabolic and Cellular Medicine,
School of Clinical Sciences
Faculty of Medicine,
University of Liverpool,
Liverpool L69 3GA, UK
gkemp@liv.ac.uk
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