Clinical Science and Molecular Medicine (1917) 53, 459-466.
Intracellular pH and bicarbonate concentration as determined
in biopsy samples from the quadriceps muscle of man at rest
K. SAHLIN, A. ALVESTRAND, J. BERGSTROM A N D E. HULTMAN
Departments of Metabolic Research Laboratory and Nephrology. S:t Eriks sjukhus, and
Department of Clinical Chemistry, Serafmerlasarettet, Stockholm, Sweden
(Received 15 November 1976;accepted 20 June 1977)
Summary
1. A method for measuring intracellular
pH and bicarbonate concentration of human
muscle is described.
2.Muscle biopsies from the quadriceps muscle
of 13 healthy subjects at rest were analysed for
acid-labile carbon dioxide and volume of extraand intra-cellular water. Extracellular water
volume was estimated from the chloride content
and intracellular water volume from the potassium content, or alternatively derived from the
sample weight.
3. The measured total carbon dioxide content
in muscle was 9.84f 1.39 mmol/kg.
4. Assuming a normal membrane potential
(88 mV) and Pco2 of muscle equal to venous
blood, calculated intracellularpH was 7-00k0.06
and intracellular bicarbonate concentration was
10.2 f1.2 mmol/l of water.
Key words: bicarbonate, carbon dioxide, intracellular pH, muscle.
Abbreviations: [Cl-I,, extracellular concentration of chloride (mmol/l of water); [Cl-11,
intracellular concentration of chloride (moll1
of water); Clm, muscle content of chloride
(mmol/kg fat-free dry weight); CI;, plasma
content of chloride (mmol/l of plasma); DMO,
5,5-dimethyl-2,4-oxazolidinedione;
HCO:,, =
plasma content of bicarbonate (mmol/l of
plasma); [HCO>Is = extracellular concentration of bicarbonate (mmol/l of water); [HC0;Ii
Correspondence: Kent Sahlin, Metabolic Research
Laboratory, S:t Eriks sjukhus S-112 82 Stockholm,
Sweden.
459
= intracellular concentration of bicarbonate
(mmol/l of water); (HzO),, (HzO), and HzO,,
muscle content of extracellular water, intracellular water and total water respectively (l/kg
fat-free dry weight); H20,, plasma content of
water (1/1 of plasma); [K+II, intracellular
concentration of potassium (mmolll of water);
Kt, muscle content of potassium (mmol/kg
fat-free dry weight); [Na+Ii, intracellular concentration of sodium (mmol/l of water); Na;,
muscle content of sodium (mmol/kg fat-free
dry weight); pHI, intracellular pH; TCOz,
muscle content of acid-labile carbon dioxide
(mol).
Introduction
Intracellular pH (pH1) in skeletal muscle of
man has previously been determined with
the 5,5-dimethyl-2,4-oxazolidinedione(DMO)
method (Bittar, Watt, Pateras & Parrish, 1962;
Maschio, Bazzato, Bertoglia, Sardini, Mioni,
D’Angelo & Marzo, 1970). Though direct
measurements of pH in homogenized muscle
tissue (Hermansen 8c Osnes, 1972; Sahlin,
Harris & Hultman, 1975; Sahlin, Harris,
NyIind & Hultman, 1976) have given valuable
information on muscle pH after exercise, values
cannot by definition be called pH1. Studies of
intracellular acid-base metabolism of man have
been hampered by methodological difficulties
which are more pronounced when only small
tissue samples are available.
We now describe a method to determine
pH, in muscle biopsy samples, and give the
results from 13 subjects at rest. The principle
of the method is based on the Henderson-
460
K.Sahlin et al.
Hasselbalch equation (S is solubility constant
for CO,):
Total muscle content of CO, is determined by
the method described by PontBn & Siesjo
(1964) and water distribution is estimated by
two methods depending on the chloride space.
Material and methods
Subjects
Thirteen subjects (ten males, three females)
aged between 19 and 41 years participated.
The subjects were not especially well trained,
though some of them regularly participated in
some form of physical activity. In every case
the nature and purpose of the study were explained to the subjects before their voluntary
consent was obtained.
Subjects were investigated in the morning
after an overnight rest. Before samples were
taken they rested for at least 20 min, and in the
last 5 min they were supine. Muscle tissue was
obtained from the quadriceps muscle with the
needle-biopsy technique (Bergstrom, 1962). Two
to four biopsies were taken from each subject.
The first of these biopsies (sample A) was used
for determination of muscle content of water
(HzO,), potassium (Kk),sodium (Nat) and
chloride (Cl;); the other(s) (sample B) for
measurements of total content of acid-labile
COz (TCO,), K+ and C1-. Immediately after
the last muscle biopsy, blood samples were
taken from an antecubital vein and femoral
artery for determination of Pco, and pH.
The plasma content of protein and electrolytes
were determined in venous blood.
Analytical methods
Blood samples. Routine chemical analyses
using an Autochemist system (Auto Chem
Instrument AB, Lidingo, Sweden) were used
for determination of protein and electrolytes
in plasma. Acid-base variables were analysed
in an automatic system (Radiometer, Copenhagen; type ABL1) with pH and Pco, electrodes.
Determination of water,potassium, sodium and
cfiloride content in muscle sample A . The muscle
sample was dissected free from all visible
connective tissue, and if possible divided into
two to three pieces, which were repeatedly
weighed on an electrobalance (Cahn G-2).
The wet weight was obtained by extrapolation
to zero time and the water content after drying
at 90°C. The dried muscle samples were freed
from fat by extraction with light petroleum
and extracted with HN03.Sodium and potassium were determined by atomic absorption
photometry and chloride by titration with
AgNOJ, as previously described (Bergstrom Lk
Hultman, 1974).
Determination of potassium, chloride and
total COz content in muscle sample B. The
muscle samples were frozen in the needles with
liquid Nt.The needles were thereafter sealed
with tape to prevent contamination with
atmospheric COz and stored in liquid Nt
until further treatment.
TCOz was determined by a modification of a
method described by Pont6n & Siesjo (1964), in
which CO, is liberated from tissue with acid and
absorbed in Ba(OH), solution, which is subsequently titrated with acid.
The diffusion chambers were weighed with
stoppers and magnetic stirrers before and after
addition of 0.5 ml of HzS04 (1 mol/I). The
exact volume of added acid was obtained by
dividing the difference in weight by the density
of the solution (1.06 g/ml). Samples (stored in
liquid Nz) and equipment were transferred to
a glove-box, which was freed from CO, by
purging with N n gas; 200 pl of Ba(OH), (6
mmol/l) was added to one branch tube of the
diffusion chambers with a semi-automatic
pipette (Eppendorf) .
The muscle sample was taken out of the
needle, dissected free from any obvious blood,
ground to a fine powder under liquid Nz and
added to the pre-cooled empty branch tube of
the diffusion chamber. The vessel was rapidly
closed with the stoppers and slightly tipped in
order to pour the acid over the sample. The
muscle powder did not thaw until mixed with
the acid. After processing all the samples and
blanks (usually one blank per two samples)
the vessels were again weighed. The weight of
the muscle powder was obtained by subtracting
the mean increase in weight of the blank vessels
from the increase in weight of the sample vessel.
Samples weighing below 15 mg were discarded.
Though experiments with standard solutions
Intracellular p H in human muscle
of carbonate did show that 95% of the liberated
C02 was absorbed in the Ba(OH)2 solution
within 15 min a total diffusion time of 2 h was
allowed, during which the contents of the
branch tubes were stirred. As the diffusion
chambers were purged with Nz, the Ba(OH)2
solution was titrated with HCl (2.5 mmolil)
with thymolphthalein as indicator. The exact
concentration of HCl was determined by comparison with phthalic acid solutions and the
amount of carbon dioxide was calculated:
pmol of COz = A(V, - V,)/2, where A is the
concentration of HCI (mmol/l), V, and V, are
the volumes (pl) of acid required for titrating
blank and sample respectively.
The acid-treated muscle sample was centrifuged with the sulphuric acid. The supernatant
was analysed for K+ and C1- and the total
content obtained by multiplication with the
water volume in the HZSO4extract. The water
volume was obtained by correcting the added
volume of sulphuric acid (added before the
start of analysis) for evaporation during titration and for the added muscle water.
461
eqn. (5), where C1; and H20, were obtained
from analyses.
C1G = (HzO, -(HzO)e) [Cl-lr
+ 0320). [Cl-le (4)
(H2O)t = HzO,-(HzO).
(5)
[K+J and [Na+Il were calculated from eqn.
(6) and eqn. (7), where KA, Naf and Na+
were obtained from analyses.
[K+]I= KfI(Hz0)i
(6)
Extra- and intra-cellular bicarbonate concentration and p H i . The amount of intraand extra-cellular water in muscle sample B
was obtained irom the total content of potassium and chloride in the HzS04extract (eqn. 8
and eqn. 9, where K+,, C1-l and Cl-. were
calculated as described above).
H201 = K+/[K+],
(8)
Calculation
Intracellular electrolyte concentrations. It was
assumed that
[CI-]. = C1;/(0.96
*
H20J
(1)
where 0.96 is the Donnan factor. Clp was
obtained from analyses and H20, was calculated from the regression formula (eqn. 2)
by Eisenman, MacKenzie, & Peters (1936):
HzO, = 0.984-7.18 *
(g of proteinll of
plasma)
(2)
Chloride is distributed over the cell membrane according to the Nernst equation (Wilde,
1945; Conway, 1957):
where E = membrane potential, R = gas law
constant, T = absolute temperature and F =
Faraday’s constant.
Assuming a normal membrane potential,
E = 88 mV (Bolte, Riecker & Rohl, 1963;
Cunningham, Carter, Rector & Seldin, 1971);
[Cl-1, = 26[CI-],. (H20). and (HzO), in
sample A were calculated from eqn. (4) and
The distribution of water can also be obtained
from the sample weight and Cl- content.
Though in this case no extract muscle sample
for determination of [K+Ilisnecessary, a normal
muscle water content must be assumed.
The bicarbonate content of venous plasma
was calculated from the Henderson-Hasselbalch equation and [HCO;], was obtained
by correcting this for a Donnan factor (0.96)
and for the plasma water content. The Pco2 of
muscle was assumed to be the same as in
in venous blood. No carbamino compounds
are believed to exist in muscle (Butler, Poole &
Waddell, 1967) and [HCO;II can therefore be
calculated by deducting the amount of extracellular HCO; and the amount of intra- and
extra-cellular C 0 2 and H2C03from TCOz:
[HCO;], =
(TC02-H20e [HCO3]e(HzOt +H20e) P C O *~SI)/H201 (10)
where Siis the solubility constant for C 0 2 in
muscle.
K. Sahlin et al.
462
(0).
Sz = 0.236 mmol of COz kg-l of water kPa-'
(Siesjo, 1962b). The solubility of C 0 2 in tissues
(SI)is dependent upon the protein concentration and has been determined to be 0.266 and
0.271 mmol of C 0 2 kg-' of water kPa-' in
tissue homogenates of blood cells (van Slyke,
Sendroy, Hastings & Neill, 1928) and brain
(Siesjo, 1962b) respectively. The necessity of
using two different solubility constants for C o t
derives from the fact that tissues are multiphase
systems (for a full discussion see Siesjo, 1962a).
Values of S , , Sz and pK. were corrected to
the normal muscle temperature at rest, 35°C
(Harris, Hultman, Kaijser & Nordesjo, 1975),
according to Bartels & Wribitsky (1960) and
Siggaard-Andersen (1962) respectively: S1=
0.284 mmol of COz kg-' of water kPa-I;
Sz = 0.247 mmol of COz kg-' of water kPa- I ;
pK, = 6.14. Values are given throughout as
meankl SD.
where S2 is the solubility constant of COz in
the intracellular water phase.
It has been shown that the pK. for the COz
system is the same in muscle homogenate solutions as in 0.16 mmol/l NaCl solution (Danielsson, Chu & Hastings, 1939). The pK, in the
intracellular fluid and the solubility of COz in
intracellular water have in the present study
therefore been assumed to be equal to that in
0.16 mmol/l NaCI :pK, = 6.13 (Siesjo, 1962a);
Results
The method for determination of tissue COz
content was tested on standard solutions of
carbonate (2.5 mmol/l), prepared from dried
sodium carbonate added to previously boiled
distilled water. By adding different volumes of
this solution to the diffusion chambers, C O ,
contents in the range 0.12-1-0 rmol were
analysed. The linearity and the precision of the
P
0
0.2
0.4
0.8
0.6
1.0
No2C03 added (prnol)
FIG.1. Relationship between added amount of NaZCO3
and COz determined. Values are the mean of three
determinations, of which all were within the symbols
TABLE
1. Blood constituents
Pcoz and pH were measured in blood from an antecubital vein (v) and femoral artery (a).
Subject
M.C.
A.A.
O.D.
L.W.
A.K.
R.J.
E.L.K.
M.H.
P.D.
P.L.
S.A.
R.C.
W.H.
Mean
SD
Sex
M
M
M
F
M
M
F
F
M
M
M
M
M
PH
V
a
1-41
1.41
7.39
7.39
7.36
7.38
1.38
7-44
7.41
1.42
7.41
1.43
1.43
1-48
7-38
7-44
5.59
5-33
5.72
4.81
6.61
6.27
5.47
5-83
5.22
7.40
5.46
1.42
7.43
1.42
1.43
1.37
1-40
7.39
1.41
7.36
7.38
7.39
k0.02
Protein
Pcoz
k0.03
a
(g/l of plasma)
I0
I0
5.19
6.91
6.22
481
416
5.07
4.16
5.15
5.01
3-13
457
3.99
5.13
4.90
5.39
5.73
5.75
k0.61
4.75
k0.59
V
CI,
(mmol/l of plasma)
70
106
106
101
1 LO
101
101
106
106
106
104
103
100
104
71
+4
105
4-3
69
66
78
73
72
12
75
13
64
72
Nap
-
141
142
145
143
141
138
137
138
139
140
138
138
140
140
*2
Intracelhlar p H in human muscle
463
TABLE
2. Derived values of water content, intracellular p H ( p H , ) and bicarbonate concentration ( [ H C 0 J I I )in muscle
H z O ~was calculated from the Kt content and HzO, from the C1- content. pH1 was based (A) on HzO, values
calculated from K + content or (B) on HzOI values calculated from the sample weight assuming 77% total water
content. Mean value+ SD was calculated from the mean value for each subject. SD. was obtained by using an asymmetrical hierarchical model on those values, where two or three analyses were made on the same subject and is an
estimation of the square root of variance due to the analytical error in determination of acid-labile COZ,HzOl and
HzOe.
Subject
M.C.
Leg
L+R
L+R
A.A.
.
O.D.
L.W.
AX.
R.J.
E.L.K.
M.H.
R
L
L
R
L
L
R
R
R
L+R
L+R
L
L
L
L
P.D.
P.L.
S.A.
R.C.
W.H.
L
L
L
L
L
R
R
Amount Acid-labile
tissueof
COZ
(mg)
(pmol)
15.4
240
45.4
33-3
27.4
47.3
42.9
46.3
33.0
17.8
31.7
29.4
58.7
33.4
48.2
34.2
28-5
16.3
42.5
47.2
60.4
47.7
32.4
44.0
0171
0-259
0452
0361
0332
0.610
0.471
0.528
0.254
0.150
0.270
0,303
0.581
0.326
0.493
0-314
0.216
0.137
0.304
0.442
0.599
0.530
0-376
0.388
Hi01
H~01 HzO,
(pl)
(p1)
HzO.+Hz01
9.7
14.0
30.6
22.7
17.9
28.9
26.8
31.9
19.7
1.3
1.9
3.3
3.3
4-3
6.8
4.0
3.0
1 -9
1 -4
2.6
2.5
4.2
2.4
4.3
23
1-6
1.2
29
3.5
3.8
43
24
3.1
0.88
11.8
20.0
18.0
38.8
21.8
32.6
20.2
15.6
9.0
22.3
28-3
38.4
32.3
21-9
26.5
Mean
SD
SDa
method are shown in Fig. 1. The recovery was
98*8k1-8% (n = 13), when 0.500 pmol of
sodium carbonate was added.
Acid-base variables in blood, and plasma content of protein, Na+ and C1- are presented in
Table 1. The muscle content of water, K + , Na+
and C1- were 3.30 f0.16 litres, 463 f.19 mmol,
84 k 18 mmol and 63 f.17 mmol per kg fat-free
dry weight of muscle (n = 13) respectively. The
calculated [Ktll and [NatIl ([K+ll = 161.1 f
5.9 mmol/l of water; “a+]* = 8.2 k2.9 mmolll
of water; n = 13) were in all subjects within
the range (mean f2 SD) previously described
in normal subjects (Bergstrom, 1962).
088
0.90
0.87
[HCO 3 11
(rnmol/l of water)
11.6
12.6
10.0
10.2
0.8 1
10.0
0.81
12.4
11.4
12.1
8.9
8.2
0.87
0.91
091
089
0.88
8-8
0.88
103
090
0.90
0.88
090
0.91
0.88
090
0.91
9-7
10.3
9.9
10.4
9-2
9-7
8.5
10.4
11.3
11.1
11.4
9.0
*0.89
0.02
+ 1.2
0.88
090
0-91
0-88
10.2
f.0 4
pH1
A
B
7.06
7.10
7.02
7.03
7.02
7.08
7.05
7.08
7.00
6.97
7-00
6.94
6.94
702
7.03
7.02
7.01
7.03
7.05
7.07
7.01
7.06
6.92
6.95
6-96
688
6.91
6-99
7.00
6.92
6.82
6.86
6.82
6.96
7.04
7.08
6.95
6.84
7.00
7.00
6.94
6.97
6-96
7.03
7.09
7.08
6-97
6.91
7.00
+ 0.06
+ 0.02
6.95
2 0.08
f.0.03
In Table 2 calculated values of water distribution, [HCOS]~and pHI are presented.
Previous results obtained in this laboratory of
muscle samples dissected free from connective
tissue and fat have shown that the HzOl
constitutes on average about 88% of the total
water content (Bergstrom & Hultman, 1974).
Though the results in Table 2 have been obtained
by a different procedure, most of them are close
to this value. In Table 2 two values of pH1 per
sample are presented. They are based on different estimations of HZO, one calculated from
K+ content and the other calculated from the
weight of the sample. Both methods use the
K. Sahlin et al.
464
0.6
1
wt.01 muscle (mq)
FIG.2. Relationship between sample weight and muscle
content of acid-labile CO,. y = -0~022+0~0106x;
r = 0.92; n =: 24.
C1- space as an estimation of the extracellular
space. Though the two values of pH in most
cases are very close to each other, values based
on sample weight measurement sometimes give
a lower pH. This discrepancy cannot be explained with certainty but an overestimation of
the sample weight is suspected.
The variance in [HC0sll and pH1 due to
analytical errors in the determination of
CO, and water distribution was tested by performing analyses on two or three samples from
the same subject. From Table 2 it appears that
the variance in these analytical steps is small
compared with the variance between individuals.
The muscle content of acid-labile COz is
related to the sample weight in Fig. 2. Total
CO, content in muscle, calculated from data
in Table 2, was 9.84 k 1-39mmol/kg wet muscle
(n = 13).
Discussion
Intracellular pH (7.00 k 0.06 ;n = 13), measured
in this study for the first time in human muscle
by the COz method, is in the same range as
direct measurements made in muscle homogenates (pH = 6.93, 7.08: cf. Hermansen &
Osnes, 1972; Sahlin et aI., 1975, 1976), though
slightly higher than those obtained with the
DMO method (pH, = 6.9, 6.92: Bittar et al.,
1962; Maschio et al., 1970). These differences
between methods, which are also apparent
from animal studies (Waddell & Bates, 1969),
do not invalidate the methods but emphasize
that absolute values of pH, should be interpreted with caution.
The value of [HCOTJ in resting skeletal
muscle of man presented here, 10.2& 1.2mmol/l
of water, is to our knowledge the first one
published. It is in the same range as values
from animal studies, where a similar technique
(Wallace & Hastings, 1942; Nichols, 1958;
Eckel, Botschner & Wood, 1959; Hudson &
Relman, 1962) or where micro-electrodes have
been used (Khuri, Bogharian & Agulian, 1974).
Calculation of [HCO3IIand pH, is dependent
upon the transmembrane potential. We assumed
this to be normal, since none of the subjects
had signs of severe illness known to affect the
membrane potential. If values are recalculated
at a membrane potential of 60 mV, an extreme
value which has been measured in some severely
ill patients (Cunningham et al., 1971), pHI
would become 7-05+0.05 and [HC0<l1 11.52
1.2 mmol/l of water.
We assumed the COz tension in the muscle
cell to be the same as in venous blood. Direct
measurements of Pco, in muscle in a basal
state seem to justify this assumption (Brantigan,
Ziegler, Hynes, Miyazawa & Smith, 1974).
No differencein Pco, is expected at rest between
blood from the femoral vein and an antecubital
vein (Saltin, Blomqvist, Mitchell, Johnson,
Wildenthal & Chapman, 1968). When regional
differences in venous COz tension are expected
(i.e. muscle exercise and disturbed regional
circulation), Pco, must be measured in blood
from the femoral vein. An error in Pco, of
5 % will change calculated pHI values about
0.02 unit and [HC0<I1about 0.2 mmol.
With the needle-biopsy technique for obtaining muscle samples the time between removing
the sample and freezing is about 5-10 s. From
estimations of high-energy phosphate utilization
in skeletal muscle of man at rest, 0.51 mmol of
-P min-' kg-I of muscle (Harris et al., 1975),
it can be calculated that production of CO,
during 1 min in resting muscle is only about
0.8% of the total content. From these estimations it is clear that, under normal conditions,
the metabolism of CO, during the sampling
period is negligible.
With this method pH, can be derived from
just one muscle sample. In this case it is, however, necessary to assume a normal water
content of muscle and calculate H2O1 from
the weight of the sample. This can introduce
systematic errors, if subjects with altered water
balance are studied. It is recommended that
Intracellular p H in human muscle
two muscle samples are taken, and that the
extra one is used for the calculation of [K+II.
In this case HIOI is derived from the K+
content, which can be determined more precisely than the sample weight.
The most frequently used method to determine pH1 of muscle is the DMO method
introduced by Waddell & Butler (1959). Both
DMO and carbonic acid are weak acids and are
distributed over the cell membrane according
to the pH of either side. DMO is neither
metabolized nor volatile and is therefore easier
to analyse than acid-labile CO,. When the
DMO method is used, it is necessary to infuse
DMO into the blood and wait until it is
equilibrated between the intra- and extracellular space. As the equilibration time is
considered to be as long as 1 h (Waddell &
Butler, 1959), no rapid acid-base changes can
be studied. With our COz method the equilibration time is minimized.
The C0,-HCO:, system is one of the most
important determinants of acid-base balance
in the body, and this analysis will also provide
information on the role of this system. In spite
of the analytical complexity we believe that the
CO, method will be an important tool for
studies of intracellular acid-base metabolism
in man.
Acknowledgments
This investigation was supported by grants
from the Swedish Medical Research Council
(projects nos. B77-19X-1002-12C and B77-19X2647-09C).
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