Division of Comparative Physiology and Biochemistry, Society for Integrative and

Comparative Biology

A Reexamination of the Mechanisms Underlying the Arteriovenous Chloride Shift

Author(s): Edward A. Westen and Henry D. Prange
Source: Physiological and Biochemical Zoology, Vol. 76, No. 5 (September/October 2003), pp.
603-614
Published by: The University of Chicago Press. Sponsored by the Division of Comparative
Physiology and Biochemistry, Society for Integrative and Comparative Biology
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INVITED PERSPECTIVES IN PHYSIOLOGICAL AND BIOCHEMICAL ZOOLOGY
A Reexamination of the Mechanisms Underlying the Arteriovenous
Chloride Shift
Edward A. Westen*
Henry D. Prange
Medical Sciences Program, Indiana University, Bloomington,
Indiana 47405
Accepted 7/22/03
ABSTRACT
The chloride shift is the movement of Cl⫺ from the plasma
into erythrocytes as blood moves from the arterial to the venous
end of systemic capillaries. The traditional explanation for the
chloride shift emphasizes the causative roles of the rise in Pco2
and the exclusive presence of carbonic anhydrase within the
red blood cell. The purpose of this article is, first, to reexamine
two aspects of the chloride shift that we feel are traditionally
underemphasized. They are the role of hemoglobin in causing
the chloride shift and the affect of the chloride shift on the
acid-base status of the blood. Second, we wish to reconcile
more recent work with the traditional understanding of the
chloride shift. The chloride shift has never been modeled from
the perspective of the Stewart strong ion approach. Similarly,
the traditional understanding has generally treated Cl⫺ as a
passive participant in the chloride shift whose role was simply
to replace the lost negative charge of the outward moving
HCO⫺3 . More recent work has suggested that the ingoing Cl⫺
is important for both O2 unloading and acid-base balance of
the blood. We conclude this article with a model of the chloride
shift that uses the Stewart approach and, though harmonious
with the traditional understanding, highlights the importance
of hemoglobin and Cl⫺ in the chloride shift.
Introduction
The “chloride shift” is the name given to the movement of
chloride ions from the plasma into red blood cells as blood
* Corresponding author. Present address: Division of Biology, Wartburg
College, 100 Wartburg Boulevard, Waverly, Iowa 50677-0903; e-mail:
ed.westen@wartburg.edu.
Physiological and Biochemical Zoology 76(5):603–614. 2003. 䉷 2003 by The
University of Chicago. All rights reserved. 1522-2152/2003/7605-2157$15.00
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603
undergoes the transition from arterial to venous gas partial
pressures within systemic capillaries. The traditional description
of the chloride shift emphasizes two major points as causal
factors. The first is the increase in Pco2 that is concomitant
with the transition from arterial to venous conditions, while
the second is the exclusive presence of the enzyme carbonic
anhydrase within the red blood cell relative to plasma.
The chloride shift is functionally significant for several rea-
sons. First, since most of the CO2 carried by the blood is in
the form of HCO⫺3 , the chloride shift is important because it
enhances the carrying capacity of the blood for HCO⫺3 (re-
viewed in Klocke 1988).
A more recently discovered role of the chloride shift in an-
imal physiology is that it modulates loading/unloading of O2
from hemoglobin. Cl⫺ is an allosteric effector of Hb whose
importance is variable throughout the animal kingdom (re-
viewed in Ingermann 1997). While it has not been explored in
all animals, at least the brown bear, two species of bats, and
several ruminants have been found to use Cl⫺ as their primary
modulator of Hb-O2 affinity. Brix et al. (1990) suggested that
the large influx of Cl⫺ during the chloride shift in the brown
bear contributed to O2 unloading via modulation of Hb-O2
affinity. The extent to which the chloride shift is important for
O2 unloading in other species has not been explored.
The purpose of this article is, first, to reexamine two aspects
of the chloride shift that we feel are traditionally underem-
phasized. They are the role of hemoglobin in causing the chlo-
ride shift and the affect of the chloride shift on the acid-base
status of the blood.
Second, we wish to reconcile more recent work with the
traditional understanding of the chloride shift. The chloride
shift has never been modeled from the perspective of the Stew-
art strong ion approach (Stewart 1981). Similarly, the tradi-
tional understanding has generally treated Cl⫺ as a passive par-
ticipant in the chloride shift whose role was simply to replace
the lost negative charge of the outward-moving HCO⫺3 . More
recent work has suggested that the ingoing Cl⫺ is important
for both O2 unloading (Brix et al. 1990) and acid-base balance
of the blood (Prange et al. 2001).
Hemoglobin
Introduction
The conformational change in hemoglobin (Hb) from the re-
laxed (R) to the taut (T) state is one of the most important
604 E. A. Westen and H. D. Prange
factors causing the chloride shift. While O2 is the most obvious
molecule whose binding affinity is altered by the change in
conformational state, so too are the binding affinities for Cl⫺,
H⫹, CO2, and 2,3-BPG. The conformational change from R
state to T state changes the distance between certain amino
acids and exposes other amino acids with the result that T-
state Hb has more binding sites for these other molecules than
does R-state Hb. When these molecules bind to Hb, a change
in the electrochemical environment within the red cell occurs.
Because this change in electrochemical environment causes the
Cl⫺ shift, we feel it is important to provide a brief description
of the binding sites as well as the physiological role of each of
the allosteric effectors.
2,3-BPG (Formerly Known as 2,3-DPG)
2,3-bisphosphoglycerate is a small molecule derived via gly-
colysis. In addition to other functions, 2,3-BPG is a negative
allosteric effector in many species. It decreases O2 affinity by
cross-linking the b chains and thereby stabilizes the T state
(Perutz 1970). In R-state Hb, the 2,3-BPG binding site closes.
The distance between the amino acids that make up the binding
site changes such that 2,3-BPG no longer fits. In arterial blood
where most of the Hb is in the R state, 2,3-BPG is simply free
in the red cell cytoplasm. In venous blood where some of the
Hb is in each conformation, some 2,3-BPG is free, and some
is bound to T-state Hb.
CO2 Binding
The recognition that CO2 binds reversibly with Hb was made
early in the twentieth century (Edsall 1980). Bohr made the
original hypothesis that CO2 would bind to the amine groups
of Hb:
R-NH 2 ⫹ CO2 ↔ R-NHCOO⫺ ⫹ H⫹.
Arnone (1974) demonstrated that the amine groups of valine
1 on both the a and b chains were responsible for CO2 binding.
Estimates of the importance of CO2 binding, also known as
carbamate formation, were based on studies conducted without
knowledge of either the 2,3-BPG effect or the Cl⫺ effect. Val(1)b
is one of the residues involved in 2,3-BPG binding. 2,3-BPG
and CO2 compete for this site, and in the presence of physi-
ological concentrations of 2,3-BPG, very little CO2 binds (Ar-
turson et al. 1974). The original estimates that 20%–30% of
CO2 carriage could be attributed to carbamate formation (Fer-
guson and Roughton 1934) have recently been downgraded to
5% (Arturson et al. 1974). In fact, in our judgment, even 5%
is probably an overestimate because Cl⫺ competes with CO2
for the Val(1)a sites, and no study has measured carbamate
formation in the presence of both physiological [2,3-BPG] and
[Cl⫺].
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H⫹ Binding
The binding of H⫹ to Hb has long been known to influence
both the O2 affinity of Hb (the Bohr effect) and the carriage
of CO2 in the blood (the Haldane effect). Most textbooks dis-
cuss the Bohr and Haldane effects separately as if they were
caused by different mechanisms. In fact, both effects are pri-
marily caused by preferential binding of H⫹ to T-state Hb.
Many studies have been done attempting to identify the
specific amino acids involved in H⫹ binding to Hb (Riggs 1988).
It has been a controversial subject. Despite utilization of the
most advanced techniques available, it is still not possible to
make a conclusive determination of the H⫹ binding sites (Riggs
1988). The following are the few claims that are fairly well
accepted.
Approximately 50% of H⫹ binding is Cl⫺ dependent
(O’Donnell et al. 1979). Binding of H⫹ and Cl⫺ between Val(1)a
and Ser(131)a is well established as the dominant binding site
for the Cl⫺-dependent aspect of H⫹ binding (O’Donnell et al.
1979; Van Beek et al. 1979; Van Beek and DeBruin 1980).
An unknown fraction of T-state H⫹ binding is BPG depen-
dent (Riggs 1988). That is, BPG changes the pK of certain
amino acids in and near the BPG binding pocket such that
they take up H⫹. Much of the remainder of T-state H⫹ binding
is due to His(146)b (Riggs 1988). In spite of its location on
the exterior aspect of the molecule, His(146)b changes position
relative to nearby residues sufficiently to undergo a large pK
change. Estimates of its pK are 8.0 in the T state and 7.1 in
the R state (Kilmartin et al. 1973). These numbers have been
challenged as has the assertion that His(146)b accounts for the
entirety of the Cl⫺-independent aspect of H⫹ binding (Russo
et al. 1980). The prevailing view currently is that many exte-
riorly located histidine residues play a role in the Cl⫺-
independent T-state H⫹ binding but that His(146)b is the most
important (Riggs 1988).
Cl⫺ Binding
Chloride is a strong allosteric effector of Hb. In many mammals,
Cl⫺ is the dominant allosteric effector (Ingermann 1997). In
humans, it has been estimated that 1.8 chloride ions per tet-
ramer are given off in the conversion from T state to R state
(Riggs 1988). However, the exact binding sites for Cl⫺ in the
T state have been difficult to determine (Chiancone et al. 1972,
1975, 1976; O’Donnell et al. 1979; Van Beek et al. 1979; Nigen
et al. 1980). It is well established that Cl⫺ binds with H⫹ between
Val(1)a and Ser(131)a and that this accounts for much of the
Cl⫺-dependent Bohr effect (Chiancone et al. 1972, 1975, 1976;
O’Donnell et al. 1979; Nigen et al. 1980; Van Beek and DeBruin
1980). It is also well known that Cl⫺ binds at Lys(82)b. The
importance of this site is small, however, because it is part of
the BPG binding pocket and, in humans, BPG outcompetes
Cl⫺ for it (Riggs 1988).

The Cl⫺ effect is known to be larger than the sum of the
Val(1)a and Lys(82)b binding sites. No direct method has been
able to determine the location of the other binding sites (Perutz
et al. 1994). Bonaventura and Bonaventura (1978) hypothesized
that Cl⫺ neutralizes positively charged residues in the central
cavity of T-state Hb. The central cavity is wider in T-state Hb
and contains eight pairs of positive charges (Val(1)a, Lys(99)a,
His(103)a, Arg(104)b, His(143)b, Lys(82)b, Val(1)b, His(2)b)
compared with only three pairs of negative charges (Asp(95)a,
Asp(126)a, Glu(101)b; Perutz et al. 1994). However, for the
most part, the positive charges are not near enough to one
another for the binding of Cl⫺ to cross-link them (Shaanan
1983; Safo et al. 2001). Bonaventura’s hypothesis was that rather
than cross-linking the chains, Cl⫺ binding decreased electro-
static repulsion in the center cavity and thereby stabilized the
T state and decreased O2 affinity (Bonaventura and Bonaven-
tura 1978).
Indirect evidence in the form of Hb variants has provided
confirmation of Bonaventura’s hypothesis. Perutz et al. (1994)
measured the Cl⫺ effect in all known Hb variants in which the
excess positive charge in the center cavity was altered. In each
case, replacement of the positively charged residue with a neu-
tral one resulted in a predictable change in the Cl⫺ effect and
led Perutz et al. to the conclusion that Cl⫺ binds to no particular
site in the center cavity but rather resonates between positively
charged residues and helps to alleviate electrostatic repulsion
and thereby stabilizes the T state and decreases O2 affinity.
The importance of Cl⫺ binding to T-state Hb for CO2 car-
riage and the chloride shift depends on the magnitude of the
binding. All previous studies of Cl⫺ binding have been focused
on the effect of chloride on O2 affinity and have, therefore,
measured changes in P50 while disregarding changes in free
[Cl⫺]. Recently, Prange et al. (2001) quantitated the reversible
binding of Cl⫺ to Hb as 1–2 mmol/L between simulated arterial
and venous gas mixtures.
Because the whole blood chloride shift is of similar mag-
nitude and the plasma [H⫹] arteriovenous difference is ap-
proximately 750,000 times smaller (McKenna et al. 1997), it is
clear that reversible binding of Cl⫺ to Hb is of sufficient mag-
nitude to influence the chemistry of the red cell–plasma system.
The Physicochemical System
Introduction
This section provides a brief overview of Stewart’s strong ion
approach to acid-base balance because it is the electrochemical
framework that we will use to model the chloride shift. The
Stewart (1981) approach has gained acceptance among many
physiologists as a useful framework for the study of acid-base
balance in the body (Truchot 1987; Jones 1990; Fencl and Leith
1993; Jennings 1994; Constable 1997). It combines the laws of
mass action, conservation of mass, and electroneutrality into a
summary equation that describes the electrochemical environ-
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Mechanisms of the Chloride Shift 605
ment of a solution. The utility of the independent variable
approach lies in its distinction between those variables that can
be directly or independently controlled and those that are de-
pendent or can be only indirectly controlled.
The three independent variables in blood acid-base are, first,
the strong ion difference ([SID]), which is the difference be-
tween the combined concentrations of the strong (completely
dissociated) cations and anions; second, the Pco2; and third,
the sum of the concentrations of associated and dissociated
nonvolatile weak acids including albumin and other proteins
([Atot]). The dependent variables are [H⫹], [HCO⫺3 ], [CO32⫺],
and [OH⫺]. KA and the dissociation constants for HCO⫺3 , car-
bonate, and water (KC, K3, and K W , respectively) complete the
terms of Equation (1):
K CPco2 KA[Atot]
[SID] p [H⫹] ⫺ ⫺
[H⫹] (KA ⫹ [H⫹])
K 3 K CPco2 K
⫺ ⫹
⫺ W p 0. (1)
[H ] [H⫹]
In biological solutions, [SID] is always positive and, with the
second term, [H⫹], makes up the total positive charge in the
solution. The remaining terms are the weak anion concentra-
tions expressed in terms of their dissociation constants and
independent variables. They are HCO⫺3 , combined nonvolatile
weak acid anions ([A⫺]), carbonate ([CO32⫺]), and hydroxyl
ions ([OH⫺]), respectively. The magnitudes of the [H⫹],
[CO32⫺], and [OH⫺] are vanishingly small relative to the other
terms (Constable 1997) but are presented here for mathematical
completeness.
The following sections will describe the acid-base status of,
first, the plasma and, then, the red blood cell. No study has
ever measured all of the independent variables in both the
plasma and the red cell. Because this is true, the values for the
independent variables and their constituents found in Tables
1–7 of this manuscript are our own compilations based on the
best available data in humans (Stewart 1981; McKenna et al.
1997). The theoretical model describing the chloride shift that
is presented at the end of this manuscript is primarily concerned
with the electrochemical changes that occur as blood undergoes
the arteriovenous transition rather than the absolute magni-
tudes of the variables in either state. As such, we chose values
for the independent variables that could be true in an individual
blood sample so that we can observe the changes in the de-
pendent variables that must occur as a result of changes in the
independent variables. The magnitude of each dependent var-
iable described in Tables 1–7 was calculated on the basis of the
chosen values for the independent variables using Equation (2)
in the computer program Mathcad 2000 (MathSoft).
606 E. A. Westen and H. D. Prange

Table 1: Plasma blood chemistry values ([UA⫺]) is quite small, the major change to [SID] that occurs
in the transition from arterial to venous gas tensions at rest is
Arterial Venous
the fall in [Cl⫺] following its movement into the red blood cell
⫹
[Na ] 135.0 135.0 (the chloride shift).
[K⫹] 5.0 5.0 Table 1 also shows the concentrations of the dependent var-
[UC⫹] 5.0 5.0 iables. The results, obtained by use of Equation (2), indicate a
[Cl⫺] 104.0 102.0 normal arteriovenous rise in [HCO⫺3 ] from 23.0 to 25.2 mmol/
[HCO⫺3 ] 23.0 25.2 L and a drop in pH from 7.40 to 7.38. The rise in [HCO⫺3 ]
[UA⫺] 1.0 1.0 and fall in pH are secondary to the changes in the independent
[A⫺] 17.0 16.8 variables Pco2 and [SID]. The rise in Pco2 is the necessary
[SID] 40.0 42.0 result of CO2 production in the tissues, while the rise in [SID]
Pco2 40 46 is the result of the shift of chloride from the plasma to the red
[Atot] 20.0 20.0 cell.
pH 7.40 7.38 If we model the hypothetical situation where the chloride
[H⫹] 40 42 shift and the consequent rise in venous [SID] is prevented, then
KA 2.2 # 10⫺7 2.2 # 10⫺7 the pH falls from 7.40 to 7.35 and the [HCO⫺3 ] rises only from
Note. [UC⫹], unmeasured cations; [UA⫺], unmeasured an- 23.0 to 23.6 mmol/L. The conclusion to be drawn from this
ions; [SID], strong ion difference; [Atot], total weak acid con- analysis is that in addition to enhancing HCO⫺3 carriage, a major
centration; [A⫺], net protein charge; r[Cl⫺, HCO⫺3 , H⫹], Donnan role of the chloride shift is mitigation of the fall in pH that
ratio for chloride, bicarbonate, and hydrogen ion. All concen-
would otherwise occur as a result of the arteriovenous rise in
tration units are milliequivalents per liter except [H⫹], which
is nanoequivalents per liter. Pco2 (Bidani 1991).

Plasma
Red Blood Cell
Table 1 shows the constituents of arterial and venous plasma
in normal resting humans. All concentrations are in milli- Table 2 shows the arterial and venous constituents of the red
equivalents per liter except [H⫹], which is nanoequivalents per blood cell. Because the red cell membrane is freely permeable
liter. to CO2, the Pco2 in the RBC is the same as plasma in both
The total weak acid concentration, [Atot], in plasma is pri- arterial and venous conditions. [Atot] is much larger in the
marily made up of albumin and inorganic phosphate (Rossing RBC-ICF than in the plasma. It is primarily made up of Hb
et al. 1986). Other proteins such as immunoglobulins can be and 2,3-BPG. The KA within the red cell is also different than
ignored because of their very small concentrations (Figge et al. that in plasma because of the makeup of Hb as compared with
1991). KA is the weak acid dissociation constant for albumin albumin. Human Hb is made up of 574 amino acids, many of
and inorganic phosphate. It is a simplification to treat albumin which have pK values near enough to the normal red cell pH
and inorganic phosphate as though they have a uniform KA, to be considered individual weak acids. Therefore, the idea of
but the error introduced by this simplification is vanishingly
small (Figge et al. 1991). The value used for the KA of plasma Table 2: Red blood cell blood chemistry values
comes from Stewart (1981). [Atot] in arterial plasma is ap-
Arterial Venous
proximately 20 meq/L (Stewart 1981) and does not change
importantly in the transition to venous gas partial pressures [Na⫹] 20.0 20.0
(McKenna et al. 1997). [K⫹] 103.7 103.7
[Na⫹], [K⫹], and [Cl⫺] are the constituents of [SID] that are [UC⫹] 5.0 5.0
normally measured. Because they do not vary significantly, [Cl⫺] 69.7 70.4
[Mg2⫹] and [Ca2⫹] are typically not measured and are lumped [HCO⫺3 ] 15.4 17.4
into the category “unmeasured cations” ([UC⫹]). Similarly, [UA⫺] 1.0 1.0
[UA⫺] is the category for typically unmeasured anions such as [A⫺] 42.6 39.9
lactate, sulfate, and other organic anions. [SID] is calculated [SID] 58.0 57.3
as Pco2 40 46
[Atot] 60.0 60.0
[SID] p ([Na⫹] ⫹ [K⫹] ⫹ [UC⫹]) ⫺ ([Cl⫺] ⫹ [UA⫺]). (2) pH 7.22 7.21
[H⫹] 60 61
Because cations are largely impermeable to the red cell KA 1.5 # 10⫺7 1.3 # 10⫺7
membrane and, at rest, the unmeasured anion concentration Note. See Table 1 note for definitions of abbreviations.

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 Mechanisms of the Chloride Shift 607

Table 3: Arterial blood chemistry values red cell and plasma separately, the red cell–plasma system takes
on the appearance of a Donnan equilibrium (Nikinmaa 1990).
RBC-ICF Plasma
The net negatively charged species, Hb and 2,3-BPG, are con-
⫹
[Na ] 20.0 135.0 fined to the red cell. Albumin and other plasma proteins are
[K⫹] 103.7 5.0 confined to the plasma. The red cell membrane is also largely
[UC⫹] 5.0 5.0 impermeable to cations.
[Cl⫺] 69.7 104.0 Quantitatively, the most important permeable ions are Cl⫺
[HCO⫺3 ] 15.4 23.0 and HCO⫺3 . We will also discuss H⫹ among the permeable ions,
[UA⫺] 1.0 1.0 not because it is quantitatively important but because [H⫹] is
[A⫺] 42.6 17.0 how acid-base status is generally defined.
[SID] 58.0 40.0 The Donnan ratio, r, describes the proportionality of the
Pco2 40 40 permeable ions at equilibrium as follows:
[Atot] 60.0 20.0
pH 7.22 7.40 CCl⫺RBC ⫺
CHCO3RBC ⫹
CHPlasma
[H⫹] 60 40 rp p p . (3)
CCl⫺Plasma CHCO3Plasma
⫺
CH⫹RBC
KA 1.5 # 10⫺7 2.2 # 10⫺7
Note. r[Cl⫺, HCO⫺3 , H⫹] p 0.67, 0.67, 0.67. See Table 1 note The chloride shift is an ionic redistribution secondary to the
for definitions of abbreviations. transient disequilibrium of the Donnan ratios that occurs dur-
ing the arteriovenous transition. The remainder of this man-
uscript examines the contribution of Hb to causing the tran-
a uniform red cell KA is oversimplified, but, to our knowledge, sient disequilibrium in the Donnan ratios and helping maintain
every physiologically relevant study has used this simplification. acid-base balance in the red cell–plasma system.
A confounding factor is that the apparent KA of Hb changes
during transition from R state to T state. This change in KA is
what causes the Haldane effect. No reliable value for the change The Effect of Acute Changes in Hematocrit on the
in KA of Hb exists because the definitive work on the Haldane Chloride Shift and Acid-Base Balance of the Plasma
effect did not measure [Cl⫺] and, thus, overestimated the
change in KA (Siggaard-Anderson 1971). The arteriovenous val-
ues for KA in Table 2 have been used previously (Prange et al. Introduction
2001) and, in light of the final model presented later, are prob- Hemoglobin (Hb) is an important physiological buffer both
ably good estimates of the true KA. because of the Haldane effect and because of its ability to re-
[SID] is much larger in the RBC-ICF than in the plasma.
This is largely the result of a much smaller [Cl⫺]. As in most
cells, the [Na⫹] is relatively low, and the [K⫹] is high. Because Table 4: Plasma and red cell blood chemistry
of experimental constraints, no one has ever measured the ar- values resulting from rise in partial pressure of
teriovenous change in [SID]. Chloride is known to move into carbon dioxide
the RBC-ICF as part of the chloride shift, but its impact on
RBC-ICF Plasma
the acid-base status of the red cell has rarely been considered
⫹
(Prange et al. 2001). [Na ] 20.0 135.0
Modeling using Equation (2) is instructive. If we imagine [K⫹] 103.7 5.0
that all of the incoming Cl⫺ from the chloride shift is simply [UC⫹] 5.0 5.0
free in the RBC-ICF, then the [SID] will fall 2 mmol/L to 56 [Cl⫺] 69.7 104.0
mmol/L. Using this [SID] with the known changes in Pco2 and [HCO⫺3 ] 16.2F 23.6F
KA and no change in [Atot] yields a pH of 7.17 and a [UA⫺] 1.0 1.0
[HCO⫺3 ] of 16.5 mmol/L. [A⫺] 41.8f 16.4f
However, we know that 1–2 mmol/L of the incoming Cl⫺ [SID] 58.0 40.0
binds to Hb (Prange et al. 2001). If we imagine the exact figure Pco2 46 46
for the binding of Cl⫺ to Hb to be 1.3 mmol/L (the reason for [Atot] 60.0 20.0
this figure will be defended later), then the fall in [SID] is only pH 7.19f 7.35f
to 57.3 mmol/L, and the resulting pH and [HCO⫺3 ] are 7.21 [H⫹] 65 45
and 17.4 mmol/L, respectively. The binding of Cl⫺ to Hb is, KA 1.5 # 10⫺7 2.2 # 10⫺7
therefore, important for maintenance of red cell pH. Note. r[Cl⫺, HCO⫺3 , H⫹] p 0.67, 0.69, 0.69. See Table 1 note
While it is convenient to consider the acid-base status of the for definitions of abbreviations.

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608 E. A. Westen and H. D. Prange

Table 5: Blood chemistry values resulting from Results

reestablishment of equality between the Donnan
Results are shown in Figure 1. The average chloride shift in
ratios
the normal Hct samples was 2.4 Ⳳ 1.0 mmol/L. The chloride
RBC-ICF Plasma shift rose to an average of 4.3 Ⳳ 1.6 mmol/L in the increased
⫹
[Na ] 20.0 135.0 Hct samples. The average change in Hct was 8.25%. The mag-
[K⫹] 103.7 5.0 nitude of the chloride shift was dependent on Hct.
[UC⫹] 5.0 5.0 The arteriovenous rise in [HCO⫺3 ] averaged 4.1 Ⳳ 0.37
[Cl⫺] 70.0F 103.7f mmol/L before Hct increase and 5.7 Ⳳ 0.73 mmol/L afterward.
[HCO⫺3 ] 16.1f 23.8F The magnitude of the arteriovenous rise in [HCO⫺3 ] was also
[UA⫺] 1.0 1.0 dependent on Hct.
[A⫺] 41.6 16.5 The average arteriovenous change in [H⫹] was 7.2 Ⳳ 0.67
[SID] 57.7f 40.3F nmol/L at normal Hct and 5.1 Ⳳ 1.3 nmol/L when Hct was
Pco2 46 46 raised. The size of the arteriovenous [H⫹] change was also
[Atot] 60.0 20.0 dependent on Hct.
pH 7.18 7.36
[H⫹] 66 44
Discussion
KA 1.5 # 10⫺7 2.2 # 10⫺7
Note. r[Cl⫺, HCO⫺3 , H⫹] p 0.67, 0.67, 0.67. See Table 1 note The approximately 8% increase in Hct in this study is smaller
for definitions of abbreviations. than has been documented to occur in blood doping (McArdle
et al. 1996), diving (Lutz and Storey 1997), and altitude accli-
matization (Ward et al. 2000). Nonetheless, it significantly en-
hanced the chloride shift, increased the carriage of [HCO⫺3 ],
versibly bind Cl⫺. Acute increase in [Hb] may, therefore, en- and mitigated the fall in pH between arterial and venous blood.
hance an organism’s ability to withstand changes in acid-base Underlying these effects are the Po2-dependent binding of
status. Acute increases in hematocrit (Hct) and, therefore, [Hb], chloride to Hb and the change in KA of Hb associated with its
occur in a variety of physiological (e.g., altitude acclimatization conformational change (Haldane effect; Prange et al. 2001).
in humans and diving behavior in seals), pathological (e.g., These mechanisms combine to increase the movement of Cl⫺
dehydration), and ergogenic (e.g., blood doping in humans and into the red blood cell, thus increasing the strong ion difference
splenic dumping in racehorses) situations. While each of these of the plasma.
examples is primarily characterized by hypoxia, a secondary The purpose of this experiment was to demonstrate that
characteristic is acid-base upset. We explored the effect of acute acute increases in Hct can have a positive influence on acid-
changes in Hct on [Cl⫺], [HCO⫺3 ], and pH in human blood
equilibrated to simulated arterial and venous gas tensions in
Table 6: Red cell blood chemistry changes
vitro, with the hypothesis that acute increases in Hct would
resulting from conformational change in
enhance the chloride shift, increase the carriage of [HCO⫺3 ],
hemoglobin
and mitigate the fall in pH that occurs from arterial to venous
blood. RBC-ICF Plasma
⫹
[Na ] 20.0 135.0
[K⫹] 103.7 5.0
Material and Methods
[UC⫹] 5.0 5.0
We obtained approximately 20 mL of blood from each of nine [Cl⫺] 68.7f 103.7
human volunteers. Half of the blood was used as drawn. The [HCO⫺3 ] 18.2F 23.8
other half was centrifuged, and approximately 10% of the [UA⫺] 1.0 1.0
plasma was removed. The red cells were then resuspended in [A⫺] 40.8 16.5
the remaining plasma. This procedure increased the Hct of the [SID] 59.0F 40.3
sample by approximately 8% (i.e., from an initial value of 45% Pco2 46 46
to a final value of 53%). Both preparations were equilibrated [Atot] 60.0 20.0
to simulated arterial (15.1% O2, 5.2% CO2, 79.7% N2) and pH 7.24 7.36
venous (3.1% O2, 7.2% CO2, 89.7% N2) gases using a tonometer [H⫹] 58f 44
(Instrumentation Laboratories, IL 237). Following equilibra- KA 1.3 # 10⫺7f 2.2 # 10⫺7
tion, blood gases and electrolytes were measured using a Ra- Note. r[Cl⫺, HCO⫺3 , H⫹] p 0.66, 0.76, 0.76. See Table 1 note
diometer ABL 505 Blood Gas Analyzer. for definitions of abbreviations.

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 Mechanisms of the Chloride Shift 609

Table 7: Venous blood chemistry values roles of Pco2 and Hb conformation on the chloride shift and
(2) to determine the magnitude of the chloride shift in cow
RBC-ICF Plasma
blood.
⫹
[Na ] 20.0 135.0
[K⫹] 103.7 5.0
[UC⫹] 5.0 5.0
Material and Methods
[Cl⫺] 70.4F 102.0f
[HCO⫺3 ] 17.4F 25.2F Approximately 20 mL of blood was obtained from the jugular
[UA⫺] 1.0 1.0 vein of each of nine healthy Black Angus cows. The blood was
[A⫺] 39.9f 16.8f kept on ice for 1 h while it was transported to the lab.
[SID] 57.3f 42.0F To separate the effects of Pco2 and conformation of Hb, we
Pco2 46F 46F equilibrated the blood to each of three gas mixtures using a
[Atot] 60.0 20.0 tonometer. Gas mixture A (15% O2, 5% CO2, balance N2) rep-
pH 7.21f 7.38f resented the arterial condition and resulted in low Pco2, high
[H⫹] 61F 42F O2 saturation (∼100%) blood. We used O2 saturation as an
KA 1.3 # 10⫺7f 2.2 # 10⫺7 indicator of Hb conformation, where a high saturation indi-
Note. r[Cl⫺, HCO⫺3 , H⫹] p 0.69, 0.69, 0.69F. See Table 1 note cated mostly R-state Hb. Gas mixture B (3% O2, 7% CO2,
for definitions of abbreviations. balance N2), which represented the venous condition, resulted
in high Pco2, lower O2 saturation (∼40%) blood. Gas mixture
base balance by way of the reversible binding of Cl⫺ to Hb and C (15% O2, 7% CO2, balance N2) was designed to result in
its influence on the chloride shift. Application of these obser- high Pco2 and high O2 saturation (∼100%) blood. By com-
vations to the whole organism leads to several interesting ques- paring the plasma [Cl⫺] between blood equilibrated to gas mix-
tions. Does enhanced ability to remove chloride from the ture A and gas mixture C we were able to isolate the effect of
plasma play a significant role in the ergogenic benefit of blood Pco2 on Cl⫺ movement. By comparing the plasma [Cl⫺] be-
doping and splenic dumping? Also, how do acute changes in tween blood equilibrated to gas mixture B and gas mixture C,
Hct alter acid-base balance in animals whose Hb either binds we were able to isolate the effect of Hb conformation. Finally,
more chloride or has a stronger Haldane effect than humans? by comparing the plasma [Cl⫺] of blood equilibrated to gas
mixture A and gas mixture B, we were able to determine the
Independent Effects of Pco2 and Hemoglobin magnitude of the normal arteriovenous chloride shift, which
Conformation on the Chloride Shift also represented the combined effects of Pco2 and Hb confor-
mation on the chloride shift.
Introduction
The fact that both a rise in Pco2 and a conformational change
in Hb occur as blood undergoes the arteriovenous transition
suggests that both factors influence the chloride shift. One of
the aims of this experiment was to evaluate the individual roles
of Pco2 and conformational state of Hb on the chloride shift.
To study this phenomenon, we used cow blood. The O2
affinity of cow hemoglobin is primarily modulated by chloride
rather than 2,3-BPG (Perutz and Imai 1980; Fronticelli et al.
1984). While the large chloride shift in bears is thought to be
the result of a high magnitude of Cl⫺ binding to Hb (Brix et
al. 1990), it is not known whether the same applies to cows.
We were unable to find any reference to the magnitude of the
chloride shift in cows. Further, the magnitude of Cl⫺ binding
to Hb in cows is a matter of controversy (Fronticelli et al. 1984,
1988; Perutz et al. 1993). Therefore, a secondary aim of this
study was to measure the chloride shift in cow blood. A chloride
shift similar to bear blood (20–30 mmol/L; Brix et al. 1990)
would indicate a high magnitude of Cl⫺ binding to Hb, while
Figure 1. Effect of acute changes in hematocrit on arteriovenous
a chloride shift similar to human blood (∼2 mmol/L) would changes in chloride concentration (chloride shift) in mmol/L, plasma
indicate a smaller degree of chloride binding. Thus, the two bicarbonate ion concentration ([HCO⫺3 ]) in mmol/L, and plasma hy-
aims of this experiment were (1) to determine the separate drogen ion concentration ([H⫹]) in nmol/L.

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610 E. A. Westen and H. D. Prange

Results
Results are shown in Figure 2. The average Pco2 of the blood
equilibrated to gas mixtures A, B, and C was 36.0 mmHg, 49.0
mmHg, and 47.8 mmHg, respectively. The average Hb-O2 sat-
uration of blood equilibrated to mixtures A, B, and, C was
99%, 38%, and 99%, respectively. The difference in plasma
[Cl⫺] between blood equilibrated to gas mixtures A and B
represented the normal chloride shift. This value averaged
1.7 Ⳳ 0.4 mmol/L. The difference in plasma [Cl⫺] between
blood equilibrated to gas mixtures A and C represented the
Pco2-dependent aspect of the chloride shift. This value averaged
0.4 Ⳳ 0.5 mmol/L. The difference in plasma [Cl⫺] between
blood equilibrated to gas mixtures B and C represented the Hb
conformation dependent aspect of the chloride shift. This value
averaged 1.3 Ⳳ 0.5 mmol/L. These results indicate that the mag-
nitude of the chloride shift in cow blood is approximately equal
to that in human blood and that it is more dependent on Hb
conformation than on Pco2.
Discussion
We found that the chloride shift was of similar magnitude in
cows (1.7 mmol/L) to that in humans (∼2.0 mmol/L). Perutz
et al. (1993) found that the allosteric effect of chloride was the
same in cows and humans. Cows, they concluded, have an
inherently low-affinity Hb that is modulated by chloride. Hu-
mans, in contrast, have an inherently high-affinity Hb whose
affinity is reduced by both chloride and 2,3-BPG. Perutz et al.’s
(1993) conclusions regarding cow blood are in contrast to the
conclusions of Fronticelli et al. (1984, 1988), who concluded
that the chloride effect is greater in cows than in humans.
Fronticelli et al. (1988) propose a mechanism for the chloride
effect in cows that requires a greater magnitude of chloride
binding. Perutz et al.’s (1993) conclusion requires a magnitude
of chloride binding that is similar to humans. Our results on
the chloride shift in cows are in accord with the proposal of
Perutz et al. (1993).
We found that approximately 75% of the chloride shift was
caused by a change in Hb conformation, while just 25% was
caused by a rise in Pco2. Pco2 has long been considered the
most important factor in causing the chloride shift. Our find-
ings suggest that the role of Hb has been underemphasized in
the traditional description of the chloride shift.
Modeling the Arteriovenous Change in Blood Chemistry
Introduction
This section consolidates the information presented into a
model for the change in blood chemistry that occurs when
blood undergoes the arteriovenous transition. To be an accurate
model, it must take all of the following considerations into
Figure 2. Independent effects of CO2 and O2 on the chloride shift.
Total v-a represents the difference in plasma chloride concentration
in blood equilibrated to simulated arterial (gas mixture A) and sim-
ulated venous (gas mixture B) gas mixtures. CO2 effect refers to the
difference in plasma chloride concentration when blood is equilibrated
to high (gas mixture C) and low (gas mixture A) partial pressure of
carbon dioxide. Oxygen effect refers to the difference in plasma chlo-
ride concentration when blood is equilibrated to high (gas mixture A)
and low (gas mixture B) partial pressures of oxygen.
account: (1) the normal arteriovenous chloride shift is ap-
proximately 2 mmol/L; (2) the rise in Pco2 alone accounts for
less than 50% of the chloride shift; (3) the conformational
change in hemoglobin alters the chemistry of the system by
enhancing Cl⫺ binding and H⫹ binding (the Haldane effect)
and accounts for more than 50% of the chloride shift; (4) at
equilibrium, the constraints of the independent variable ap-
proach will hold true for the red cell and the plasma individ-
ually; and (5) at equilibrium, the Donnan ratios for Cl⫺,
HCO⫺3 , and H⫹ will be equal to each other.
The model that follows is broken into five steps. Each step
is described via a table and a discussion. Steps 1 and 5 describe,
respectively, the arterial and venous conditions at equilibrium.
The middle steps describe the intermediate, nonequilibrium
conditions that result from the rise in Pco2 and conformational
change of Hb. These two phenomena occur simultaneously, so
it is only for the purpose of illustrating their individual effects
that we have put them in an order as though they occurred in
a separate time frame. Arbitrarily, we have decided to describe
the changes resulting from the rise in Pco2 in steps 2 and 3,
while steps 4 and 5 describe the changes that result from the
conformation change of Hb.
Step 1: The Arterial Condition
Table 3 describes the blood chemistry values for arterial blood.
These values will be used as the initial conditions from which

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 Mechanisms of the Chloride Shift 611

to compare the changes that occur during the arteriovenous
transition (steps 2–5). The values below are the same as the
arterial values presented in Tables 1 and 2 earlier.
[SID] and [Atot] are both much higher in the red cell, while
[Cl⫺] and [HCO⫺3 ] are higher in the plasma. The Pco2 of arterial
blood is generally around 40 mmHg (Guyton and Hall 1996).
The Donnan ratios for Cl⫺, HCO⫺3 , and H⫹ are 0.67 in arterial
blood at rest (Funder and Wieth 1966).
Step 2: The Effect of the Rise in Pco2 on Each Compartment
Separately
Table 4 describes the changes that occur separately in the plasma
and the red cell as a result of the arteriovenous rise in Pco2,
from 40 to 46 mmHg. [HCO⫺3 ] rises slightly in the red cell and
in the plasma. There is a fall in pH in both compartments.
Most of the hydrogen ions produced by the hydration of CO2
to HCO⫺3 are buffered by albumin in the plasma and Hb in
the RBC-ICF, resulting in a fall in [A⫺] that is equal to the rise
in [HCO⫺3 ]. The total rise in [HCO⫺3 ] is only 1.4 meq/L, much
less than the known arteriovenous HCO⫺3 increase. This dem-
onstrates that the rise in Pco2 does not cause most of the rise
in [HCO⫺3 ]. Because of its effect on [HCO⫺3 ], the rise in Pco2
does cause a slight inequality in the Donnan ratios. In the next
step, we will observe the redistribution of Cl⫺, HCO⫺3 , and H⫹
that must occur in order to reestablish equality in the Donnan
ratios.
Step 3: The Chloride Shift Resulting from the Rise in Pco2
Table 5 describes the movement of Cl⫺, HCO⫺3 , and H⫹ that
must occur to reestablish equality in the Donnan ratios after
the rise in Pco2 led to their disequilibrium. The rise in Pco2
causes a 0.3-meq/L movement of chloride from the plasma into
the red cell (i.e., a chloride shift). The chloride shift causes a
0.3-meq/L change in the [SID] of each compartment, which,
in turn, causes the concentrations of all of the dependent ions
to change. The change in [HCO⫺3 ] is not 0.3 meq/L in either
compartment. This may seem strange in light of the fact that
the band 3 transporter is thought to be a one-for-one ion
exchanger. It must be borne in mind, however, that HCO⫺3 is
a dependent ion, which means that while 0.3 meq/L of
HCO⫺3 may have moved across the membrane, the form in
which it exists in its new compartment is dependent on the
electrochemical environment within that compartment. By
comparing step 3 to step 2, it is clear that the chloride shift
that results from the rise in Pco2 changes the total [HCO⫺3 ] by
only 0.1 meq/L. It is also important to note that the rise in
Pco2 caused only a 0.3 meq/L chloride shift. The total arte-
riovenous chloride shift is approximately 2.0 meq/L. This result
is in accordance with the findings presented in the previous
section in which the rise in Pco2 caused less than 50% of the
chloride shift. In the next step, we will observe the effect of
the conformational change in Hb on the chemistry of the red
blood cell.
Step 4: The Effect of the Conformational Change of Hb on the
RBC-ICF
Table 6 describes the changes that occur in the red cell as a
result of the conformational change of Hb from R state to T
state. There are two important ways in which the conforma-
tional change affects the chemistry of the red cell. One is the
enhanced binding of Cl⫺ to Hb, and the other is the enhanced
binding of H⫹. The enhanced ability of Hb to bind H⫹ is
represented by a change in the weak acid dissociation constant
(KA) and is the primary cause of the Haldane effect.
The binding of Cl⫺ to Hb is evidenced by the reduction in
free [Cl⫺] in the red cell in step 4 as compared with step 3.
The binding of Cl⫺ to Hb and, therefore, the fall in [Cl⫺], is
1.3 meq/L in this model. This is a value that we chose because
it is the only value for the binding of Cl⫺ to Hb that results
in an intracellular [Cl⫺] that meets all five constraints set out
in the introduction to “Modeling the Arteriovenous Change in
Blood Chemistry.” Further, Prange et al. (2001) reported the
reversible binding of Cl⫺ to Hb in Hb solutions to be between
1 and 2 meq/L.
The fall in [Cl⫺] in the red cell that results from Cl⫺ binding
to Hb causes an increase in the [SID]. The increased [SID]
combines with the change in KA to cause a rise in the
[HCO⫺3 ] and fall in the [H⫹] of the red cell. The rise in
[HCO⫺3 ] and fall in [H⫹] are quite large as compared with the
changes that occurred from the rise in Pco2 discussed in step
2.
The binding of Cl⫺ to Hb reduces the Donnan ratio for Cl⫺
slightly. However, the binding of Cl⫺ and the change in KA
combine to cause a large increase in the Donnan ratios for
HCO⫺3 and H⫹. Step 5 describes the movement of Cl⫺ and
HCO⫺3 that must occur in order to reestablish equality among
the Donnan ratios after the inequality caused by the confor-
mational change of Hb.
Step 5: The Movement of Cl⫺ and HCO3⫺ as a Result of the
Conformational Change of Hb
Table 7 describes the movement of Cl⫺ and HCO⫺3 that result
from the conformational change of Hb. Arrows in Table 7 refer
to a comparison with Table 3.
In order to reestablish equality among the Donnan ratios,
1.7 meq/L of Cl⫺ moves into the red cell. The resulting change
in [SID] in each compartment causes the observed changes in
[HCO⫺3 ] and [H⫹]. As with the Pco2-induced chloride shift
discussed in step 3, this chloride shift of 1.7 meq/L does not
cause a change in the [HCO⫺3 ] of 1.7 meq/L in either
compartment.

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612 E. A. Westen and H. D. Prange
The Venous Condition
Table 7 not only describes the movement of Cl⫺ and HCO⫺3
that occur in response to the conformational change in Hb,
but it also represents the venous equilibrium condition. By
comparing the values from step 1 (the arterial condition) with
those of step 5 (the venous condition), we can observe all of
the blood chemistry changes relevant to acid-base balance and
CO2 carriage that occur as blood undergoes the arteriovenous
transition.
[Cl⫺] and [SID]. The arteriovenous chloride shift in this model
is 2 meq/L and results in a 2-meq/L fall in plasma [Cl⫺]. Red
cell [Cl⫺] does not increase by 2 meq/L because much of the
incoming Cl⫺ (1.3 meq/L) binds to Hb. Secondary to changes
in [Cl⫺] are the changes in [SID]. [SID] rises by 2 meq/L in
the plasma and falls by 0.7 meq/L in the red cell.
[HCO3⫺]. The combined plasma and red cell arteriovenous rise
in [HCO⫺3 ] is 4.2 meq/L. Only 1.5 meq/L of this total is due
to the rise in Pco2. The remainder is caused by the confor-
mational change of Hb. The [HCO⫺3 ] increase is approximately
equal in each compartment, though by comparing Table 4 and
Table 6 to Table 3, it can be seen that the total rise in
[HCO⫺3 ] in each compartment separately is only 3.5 meq/L.
The difference between the total rise in [HCO⫺3 ] and the rise
in each compartment treated separately represents the en-
hancement of HCO⫺3 carriage by the chloride shift. The chloride
shift enhances carriage of HCO⫺3 by 0.7 meq/L. On the basis
of similar findings in which he inhibited the chloride shift by
inhibiting the band 3 transporter, Bidani (1991) has argued
that the chloride shift may play only a significant role in
HCO⫺3 carriage during exercise.
[A⫺]. In the plasma, [A⫺] is primarily made up of the weak
acid albumin. [A⫺] falls slightly in the plasma during the ar-
teriovenous transition because some of the hydrogen ions pro-
duced in the hydration of CO2 bind to albumin, thus reducing
its net negative charge. Because it is in low concentration and
because its KA does not change during gas transport, albumin
has a limited ability to buffer a change in [H⫹]. In fact, mod-
eling the arteriovenous transition in the hypothetical situation
in which there is no albumin ([Atot] p 0 meq/L) yields an
arteriovenous difference in [H⫹] of 2 neq/L, which is the same
as the real situation in which albumin is present. Therefore,
even though albumin helps to determine the initial pH of the
plasma, it is relatively unimportant in terms of buffering the
arteriovenous change in pH.
In the red cell, [A⫺] is the sum of the net negative charges
on Hb and 2,3-BPG. [A⫺] falls by 2.7 meq/L in the red cell
during the arteriovenous transition. Because [HCO⫺3 ] rises by
2 meq/L in the red cell and [H⫹] rises only 1 neq/L, effectively
not at all, it can be concluded that the 2 meq/L of H⫹ that
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were produced with HCO⫺3 in the hydration of CO2 were all
buffered by Hb. This accounts for 2 meq/L of the 2.7 meq/L
fall in [A⫺].
The other 0.7-meq/L fall in [A⫺] is due to the fall in free
[2,3-BPG]. In the arterial condition, virtually all of the 2,3-
BPG is free in the red cell. Because it has a net negative charge
but is still a weak acid, it contributes to [A⫺]. In the venous
condition, some of the 2,3-BPG is bound to T-state Hb. While
it is bound, 2,3-BPG no longer contributes to [A⫺]. Binding
of 2,3-BPG does not change the net negative charge of Hb
because it binds to positively charged residues that were hidden
when Hb was in the R state. Therefore, binding of 2,3-BPG to
Hb decreases the contribution to [A⫺] of 2,3-BPG but does
not change the contribution of Hb. The overall effect of 2,3-
BPG binding on [A⫺] is a 0.7-meq/L decrease.
[H⫹]. [H⫹] rises very slightly in the plasma. The fact that it is
a small rise rather than a large one is due to the increase in
[SID] secondary to the shift of Cl⫺ into the red cell. By ob-
serving step 2, we can see that the rise in [H⫹] in the plasma
would be more than twice as great if the rise in Pco2 were not
followed by a shift of Cl⫺ into the red cell. This finding is in
accord with Bidani’s conclusion that the primary importance
of the chloride shift is minimizing the change in acid-base
balance that occurs during arteriovenous blood gas transport
(Bidani 1991).
In the red cell, the [H⫹] also rises slightly. The fact that the
rise is so slight may be surprising considering the rise in Pco2
and the fall in [SID]. However, the fall in [SID] secondary to
inward movement of Cl⫺ from the plasma is considerably less-
ened by the fact that much of the incoming Cl⫺ binds to Hb
(1.3 of the 2 meq/L that enters). Modeling the hypothetical
situation in which none of the incoming Cl⫺ binds to Hb results
in a 2-meq/L fall in [SID] and a rise in [H⫹] to 66 neq/L. The
actual rise in [H⫹] to 61 from 60 neq/L is several times less
than the rise would be if no Cl⫺ bound to Hb. Just as the
chloride shift is important for acid-base balance in the plasma,
binding of Cl⫺ to Hb is critical for acid-base balance of the red
cell.
The other factor that prevents a larger increase in [H⫹] in
the red cell is the change in KA associated with the confor-
mational change of Hb. As shown in step 3, the rise in Pco2
causes a large increase in intracellular [H⫹]. The increase is
entirely reversed when the change in KA is included (step 4).
The conformational change in Hb causes both enhanced Cl⫺
binding and the change in KA (Haldane effect). Modeling the
hypothetical situation in which none of the incoming Cl⫺ binds
to Hb and the KA of Hb does not change results in a [H⫹] of
70 neq/L and a fall in [HCO⫺3 ] from 15.4 meq/L in the arterial
condition to 15.2 meq/L in the venous condition in spite of
the rise in Pco2. Clearly, the conformational change of Hb is
critical for maintenance of acid-base balance and carriage of
HCO⫺3 in the red cell.

Conclusion
The chloride shift is the result of reestablishment of equal Don-
nan ratios for Cl⫺ and HCO⫺3 . The primary cause of the tem-
porary disequilibrium is the conformational change of Hb from
the R state to the T state that occurs during the arteriovenous
transition. A secondary cause is the rise in Pco2 that is also
concomitant with arteriovenous blood gas transport. The con-
formational change causes Hb to bind more Cl⫺ and H⫹, which
causes a fall in intracellular [Cl⫺] and rise in intracellular
[HCO⫺3 ]. Together with the small rise in [HCO⫺3 ] attributable
to the increased Pco2, the fall in [Cl⫺] and rise in [HCO⫺3 ] due
to the conformational change result in a fall in the Donnan
ratio for Cl⫺ and a rise in the Donnan ratio for HCO⫺3 . The
approximately 2-meq/L chloride shift that follows occurs as the
result of the disequilibrium between the Donnan ratios for Cl⫺
and HCO⫺3 .
A major role of the chloride shift is mitigation of the change
in pH that occurs during gas transport. Changes in Hct,
whether natural or artificial, may provide an ergogenic benefit
via enhancement of an organism’s ability to withstand an acid-
base insult.
We conclude that the role of Hb in causing the chloride shift
and aiding in maintenance of blood acid-base balance has been
underemphasized in traditional descriptions of the chloride
shift. Renewed emphasis on the importance of Hb may be
beneficial to the understanding of CO2 carriage and acid-base
balance in a variety of comparative settings, for example, in
animals with either different Hb’s or variable [Hb] or in animals
subjected to abnormal inspired gas concentrations.
Acknowledgments
We would like to thank N. Marshall for technical and biblio-
graphic assistance. This work was supported in part by National
Institute of General Medical Sciences grant GM-057372 to
H.D.P.
Literature Cited
Arnone A. 1974. X-ray studies of the interaction of CO2 with
human deoxyhemoglobin. Nature 247:143–145.
Arturson G., L. Garby, B. Wranne, and B. Zaar. 1974. Effect of
2,3-diphosphoglycerate on the oxygen affinity and on the
proton- and carbamino-linked oxygen affinity of hemoglobin
in human whole blood. Acta Physiol Scand 92:332–340.
Bidani A. 1991. Analysis of abnormalities of capillary CO2
exchange in vivo. J Appl Physiol 70:1686–1699.
Bonaventura C. and J. Bonaventura. 1978. Anionic control of
hemoglobin function. Pp. 641–661 in W. S. Caughey, ed.
Biochemical and Clinical Aspects of Hemoglobin Abnor-
malities. Academic Press, New York.
Brix O., B. Thomsen, M. Nuutinen, A. Hakala, J. Pudas, and
This content downloaded from 23.235.32.0 on Mon, 7 Dec 2015 13:12:17 PM
All use subject to JSTOR Terms and Conditions
Mechanisms of the Chloride Shift 613
B. Giardina. 1990. The chloride shift may facilitate oxygen
loading and unloading to/from the hemoglobin from the
brown bear (Ursus arctos L.). Comp Biochem Physiol 95B:
865–868.
Chiancone E., J. Norne, S. Forsen, E. Antonini, and J. Wyman.
1972. Nuclear magnetic resonance quadrupole relaxation
studies of chloride binding to human oxy- and deoxyhae-
moglobin. J Mol Biol 70:675–688.
Chiancone E., J. Norne, S. Forsen, J. Bonaventura, M. Brunori,
E. Antonini, and J. Wyman. 1975. Identification of chloride
binding sites in hemoglobin by nuclear-magnetic-resonance
quadrupole-relaxation studies of hemoglobin digests. Eur J
Biochem 55:385–390.
Chiancone E., J. Norne, S. Forsen, A. Mansouri, and K. Win-
terhalter. 1976. Anion binding to proteins: NMR quadrupole
relaxation study of chloride binding to various human he-
moglobins. FEBS Lett 63:309–312.
Constable P. 1997. A simplified strong ion model for acid-base
equilibria: application to horse plasma. J Appl Physiol 83:
297–311.
Edsall J. 1980. Hemoglobin and the origins of the concept of
allosterism. Fed Proc 39:226–235.
Fencl V. and D. Leith. 1993. Stewart’s quantitative acid-base
chemistry: applications in biology and medicine. Respir
Physiol 91:1–16.
Ferguson J. and F. Roughton. 1934. The direct chemical esti-
mation of carbamino compounds of CO2 with hemoglobin.
J Physiol 83:68–86.
Figge J., T. Rossing, and V. Fencl. 1991. The role of serum
proteins in acid-base equilibria. J Lab Clin Med 117:453–
467.
Fronticelli C., E. Bucci, and C. Orth. 1984. Solvent regulation
of oxygen affinity in hemoglobin: sensitivity of bovine he-
moglobin to chloride ions. J Biol Chem 259:10841–10844.
Fronticelli C., E. Bucci, and A. Razynska. 1988. Modulation of
oxygen affinity in hemoglobin by solvent compounds: in-
teraction of bovine hemoglobin with 2,3-diphosphoglycerate
and monoatomic anions. J Mol Biol 202:343–348.
Funder J. and J. Wieth. 1966. Chloride and hydrogen ion dis-
tribution between human red cells and plasma. Acta Physiol
Scand 68:234–245.
Guyton A. and J. Hall. 1996. Textbook of Medical Physiology.
9th ed. Saunders, Philadelphia.
Ingermann R. 1997. Vertebrate hemoglobins. Pp. 357–408 in
W. Dantzler, ed. Handbook of Physiology. 13. Comparative
Physiology. Vol. 1. Oxford University Press, New York.
Jennings D. 1994. The physicochemistry of [H⫹] and respira-
tory control: roles of Pco2, strong ions, and their hormonal
regulators. Can J Physiol Pharmacol 72:1499–1512.
Jones N. 1990. A quantitative physicochemical approach to
acid-base physiology. Clin Biochem 23:189–195.
Kilmartin J., J. Breen, G. Roberts, and C. Ho. 1973. Direct
614 E. A. Westen and H. D. Prange
measurement of the pK values of an alkaline Bohr group in
human hemoglobin. Proc Natl Acad Sci USA 70:1246–1249.
Klocke R. 1988. Velocity of CO2 exchange in the blood. Annu
Rev Physiol 50:625–637.
Lutz P. and K. Storey. 1997. Adaptations to variations in oxygen
tension by vertebrates and invertebrates. Pp. 1479–1522 in
W. Dantzler, ed. Handbook of Physiology. 13. Comparative
Physiology. Vol. 1. Oxford University Press, New York.
McArdle W., F. Katch, and V. Katch. 1996. Exercise Physiology.
4th ed. Williams & Wilkins, Baltimore.
McKenna M., G. Heigenhauser, R. McKelvie, J. MacDougall,
and N. Jones. 1997. Sprint training enhances ionic regulation
during intense exercise in men. J Physiol 501:687–702.
Nigen A., J. Manning, and J. Alben. 1980. Oxygen-linked bind-
ing sites for inorganic anions to hemoglobin. J Biol Chem
255:5525–5529.
Nikinmaa M. 1990. Vertebrate Red Blood Cells: Adaptations
of Function to Respiratory Requirements. Springer, Berlin.
O’Donnell S., R. Mandaro, T. Schuster, and A. Arnone. 1979.
X-ray diffraction and solution studies of specifically carb-
amylated human hemoglobin A. J Biol Chem 254:12204–
12208.
Perutz M. 1970. Stereochemistry of cooperative effects in hae-
moglobin. Nature 228:726–734.
Perutz M., G. Fermi, C. Poyart, J. Pagnier, and J. Kister. 1993.
A novel allosteric mechanism in haemoglobin: structure of
bovine deoxyhaemoglobin: absence of specific chloride-
binding sites and origin of the chloride-linked Bohr effect
in bovine and human haemoglobin. J Mol Biol 233:536–545.
Perutz M. and K. Imai. 1980. Regulation of oxygen affinity of
mammalian haemoglobins. J Mol Biol 136:421–426.
Perutz M., D. Shih, and D. Williamson. 1994. The chloride
effect in human haemoglobin: a new kind of allosteric mech-
anism. J Mol Biol 239:555–560.
Prange H., J. Shoemaker, E. Westen, D. Horstkotte, and B.
This content downloaded from 23.235.32.0 on Mon, 7 Dec 2015 13:12:17 PM
All use subject to JSTOR Terms and Conditions
Pinshow. 2001. Physiological consequences of oxygen-
dependent chloride binding to hemoglobin. J Appl Physiol
91:33–38.
Riggs A. 1988. The Bohr effect. Annu Rev Physiol 50:181–204.
Rossing T., N. Maffeo, and V. Fencl. 1986. Acid-base effects of
altering plasma protein concentration in human blood in
vitro. J Appl Physiol 61:2260–2265.
Russo I., N. Ho, and C. Ho. 1980. Role of the b146 histidyl
residue in the alkaline Bohr effect of hemoglobin. Biochem-
istry 19:1043–1052.
Safo M., C. Moure, J. Burnett, G. Joshi, and D. Abraham. 2001.
High-resolution crystal structure of deoxy hemoglobin com-
plexed with a potent allosteric effector. Protein Sci 10:951.
Shaanan B. 1983. Structure of human oxyhaemoglobin at 2.1A
resolution. J Mol Biol 171:31.
Siggaard-Andersen O. 1971. Oxygen-linked hydrogen ion bind-
ing of human hemoglobin: effects of carbon dioxide and 2,3-
diphosphoglycerate. I. Studies on Erythrolysate. Scand J Clin
Lab Invest 27:351–360.
Stewart P. 1981. How to Understand Acid-Base: A Quantitative
Acid-Base Primer for Biology and Medicine. Elsevier, New
York.
Truchot J. 1987. Comparative Aspects of Extracellular Acid-
Base Balance. Springer, Berlin.
Van Beek G. and S. DeBruin. 1980. Identification of the residues
involved in the oxygen linked chloride-ion binding sites in
human deoxyhemoglobin and oxyhemoglobin. Eur J
Biochem 105:353–360.
Van Beek G., E. Zuiderweg, and S. DeBruin. 1979. The binding
of chloride ions to ligated and unligated human hemoglobin
and its influence on the Bohr effect. Eur J Biochem 99:379–
383.
Ward M., J. Milledge, and J. West. 2000. High Altitude Medicine
and Physiology. 3d ed. Arnold, London.