1

Kohji Hirakoba

INTRODUCTION

It is well known that lactic acid (La) production during heavy exercise

( i.e., above lactate threshold ) is increased by promotion of anaerobic

glycolyisis. The hydrogen ion (H+) dissociated from La in exercising muscle

and in blood leads to a decrement of pH (i.e., lactic acidosis) which could

affect on the body functions via both positive and negative ways (Sahlin, 1983).

Briefly, the positive effects of the pH decrement are: (1) increased O2 delivery

(Bohr effect) and (2) increased muscle blood flow due to local vasodilation,

whereas the negative effects are: (1) inhibition of glycolysis and lypolysis

(Fredholm and Hjemdahl, 1978), (2) increased water content of muscle

(Sahlin et al., 1978), (3) decreased lactate efflux from muscle (Hirche et al.,

1975), (4) increased requirement of Ca2+ (Donaldson et al., 1978), (5)

decreased myosin ATP-ase activity (Shalder, 1967), (6) increased protein

binding of Ca2+ in sarcoplasmic reticulum (Nakamura and Schwarts, 1972) and

(7) increased of K+ in the extracellular space (Saltin et al., 1981).

The above-mentioned negative mechanisms have been considered mainly

to inhibit exercise performance capacity via an acceleration of local muscle

fatigue since body functions are very sensitive to changes in pH. It is

Department of Human Sciences, Faculty of Computer Scienceand Systems Engineering, Kyushu Institute of Technology

Buffering Capacity in Human SkeletalMuscle: A Brief Review

Kohji Hirakoba2

hypothesized that breakdown of the whole muscle glycogen store (400 mmol

glucosyl units・kg­1 dry muscle) to lactate would release 800 mmol of protons

and the concomitant accumulation in H+ ion decrease muscle pH to less than 3

without the protective mechanisms (Sahlin, 1994). It has been, however,

reported in human skeletal muscle that actually measured pH values

immediately after high-intensity exercise were from 6.5 to 6.9 (Table 1). From

the finding that the magnitude of pH decrement was lesser than being

expected from La accumulation in exercising muscle, it is supposed that

skeletal muscle possesses an ability to neutralize H+ loaded. This is defined as

buffering capacity, which is a potent protective mechanism to prevent pH

decrement from excessive acidosis. Thus high buffering capacity in skeletal

Table 1. Muscle pH after high-intensity exercise to fatigue in human

Exercise Condition Muscle SampleMuscle lactate Muscle pH

Referencerest fatigue rest fatigue

maximal treadmill sprinting for 30 s vastus lateralis -- -- 7.17 6.57 Allsop et al. 1990

one-leggedmaximal pedaling for 60 s vastus lateralis -- -- pr 6.92po 6.94

6.596.72

Bell & Wegner 1988

treadmill running at 125%VO2maxgastrocnemiusvastus lateralis --

54.3a

47.0a7.037.04

6.886.86 Costill et al. 1983

dynamic exhaustive cycling at～112%VO2max vastus lateralis 6.8 87.3 7.03 6.72 Green at al. 1993

isometric exercise at 60%MVCdynamic exhaustive cycling

quadriceps femoris6.26.7

64.784.6

7.197.20

6.846.73

Mannion et al. 1993

isometric contraction at 60%MVC quadriceps femorissed 3.5tra 3.4

83.959.2

7.127.10

6.616.80

Sahlin & Henriksson1984

isometric contraction at 68%MVCdynamic exhaustive cycling

quadriceps femoris 4.194.0

114.07.08

6.566.60

Sahlin 1978

incrementalexhaustive cycling vastus lateralis --pr 93.0a

po 111.3a 7.09pr 6.65po 6.69

Sharp et al. 1986

52 times of electrical stimulation lasting1.6 s at 20 Hz followed by 1.6 s of rest

quadriceps femoris 3.2 108.9 7.12 6.55 Spriet et al. 1986

Values are expressed as means.Muscle lactate, mmol・kg­1 dry musclel; MVC, maximum voluntary contraction force; VO2max, maximal oxygen uptake;pr, pre-training; po, post-training; sed, sedentrary; tra, traineda Calculated from the conversion factors: 1 mmol・kg­1 dry muscle=0.23 mmol・kg­1 wet muscle=0.3 mmol・kg­1 muscle

water, assuming that water content in muscle is 77 % (Hultman and Sahlin, 1980)

Buffering Capacity in Human Skeletal Muscle: A Brief Review 3

muscle is considered to delay muscle fatigue at the same level of La

accumulation in muscle by attenuating the pH decrement-induced negative

effects, which may be associated with the improvement of performance in short

-term, heavy exercise.

This short review will focus on the components that contribute to

buffering actions in human skeletal muscle and on the effects of physical

training on skeletal muscle buffering capacity.

A large part of H+ dissociated from La during exercise will be buffered in

both intra- and extracellular buffer actions. The pH regulation (acid-base

balance) in the cell during exercise has been indicated to depend on three

buffering processes (Siesjö and Messeter, 1971).

1) Physco-chemical buffering

a. Muscle protein (protein-bound histidine residutes, histidine related

dipeptides)

dipeptide carnosine (N-b-alanyl-metyl-L-histidine)

dipeptide anserine (N-b-alanyl-3-metyl-L-histidine)

b. Inorganic phosphate (HPO42­+H+→ H2PO4

­)

c. Bicarbonate (HCO3­+H+→ CO2+H2O)

2) Metabolic buffering

a. Phosphocreatine (PCr+ADP+H+→ Cr+ATP)

b. Glutamate (Glutamate+NH3→ Gulutamine)

3) Transmembrane fluxes of H+ or HCO3­ ions

CONSTITUENTS FOR SKELETAL MUSCLE BUFFERING

CAPACITY

Buffering Capacity in Human Skeletal Muscle: A Brief Review

Table 1. Muscle pH after high-intensity exercise to fatigue in human

Exercise Condition Muscle SampleMuscle lactate Muscle pH

Referencerest fatigue rest fatigue

maximal treadmill sprinting for 30 s vastus lateralis -- -- 7.17 6.57 Allsop et al. 1990

one-leggedmaximal pedaling for 60 s vastus lateralis -- -- pr 6.92po 6.94

6.596.72

Bell & Wegner 1988

treadmill running at 125%VO2maxgastrocnemiusvastus lateralis --

54.3a

47.0a7.037.04

6.886.86 Costill et al. 1983

dynamic exhaustive cycling at～112%VO2max vastus lateralis 6.8 87.3 7.03 6.72 Green at al. 1993

isometric exercise at 60%MVCdynamic exhaustive cycling

quadriceps femoris6.26.7

64.784.6

7.197.20

6.846.73

Mannion et al. 1993

isometric contraction at 60%MVC quadriceps femorissed 3.5tra 3.4

83.959.2

7.127.10

6.616.80

Sahlin & Henriksson1984

isometric contraction at 68%MVCdynamic exhaustive cycling

quadriceps femoris 4.194.0

114.07.08

6.566.60

Sahlin 1978

incrementalexhaustive cycling vastus lateralis --pr 93.0a

po 111.3a 7.09pr 6.65po 6.69

Sharp et al. 1986

52 times of electrical stimulation lasting1.6 s at 20 Hz followed by 1.6 s of rest

quadriceps femoris 3.2 108.9 7.12 6.55 Spriet et al. 1986

Values are expressed as means.Muscle lactate, mmol・kg­1 dry musclel; MVC, maximum voluntary contraction force; VO2max, maximal oxygen uptake;pr, pre-training; po, post-training; sed, sedentrary; tra, traineda Calculated from the conversion factors: 1 mmol・kg­1 dry muscle=0.23 mmol・kg­1 wet muscle=0.3 mmol・kg­1 muscle

water, assuming that water content in muscle is 77 % (Hultman and Sahlin, 1980)

Kohji Hirakoba4

It is difficult to quantify the transmembrane of H+/HCO3­ fluxes in vivo

because of rapid ionic equilibration and of small fluxes of H+ or HCO3­ ions

under short-term exercise (Hultman and Sahlin, 1980). Consequently, the

primary buffer systems in skeletal muscle appear to consist of physico-

chemical and metabolic buffering actions. According to Hultman and Sahlin

(1980), about 61 % of H+ ion uptake in muscle during exercise is due to

physico-chemical buffering and the remaining 39 % of the total H+ ion uptake

in muscle is provided by metabolic buffering processes.

Buffer value (β) is usually expressed as the mmols of acid (H+) or base

(OH­) needed to change one pH unit per liter solution (mmol・l­1・pH­1).

This expression is based on the original study of Van Slyke (1922), and the

unit for β is termed“slyke”．

1 Physico­chemical buffering

In generally, protein has been recognized as a major buffer in skeletal

muscle. Bate-Smith (1938) reported that in mammalian muscle in rigor

protein contribution was 25-40 % of skeletal muscle buffering capacity (βm).

Sahlin (1978) showed that βm in human skeletal muscle (quadriceps femoris)

was 68 slykes and protein buffer value was 15 slykes, which corresponds to

about 40 % of physico-chemical buffering and to 22 % of βm. In human muscle

buffer value of protein has been estimated to be around 15-45 slykes or

～ 40 % of total buffering capacity (Kemp et al., 1993). Concerning histidine

containing dipeptides (mainly carnosine), Harris et al. (1990) measured βm in

Thoroughbred horse and Greyhound dog and man, and found that most of the

variation in βm among the three species could be accounted for by carnosine

5Buffering Capacity in Human Skeletal Muscle: A Brief Review

levels in their skeletal muscles. If we try to calculate the rate of contribution

of carnosine to βm from the data of Harris et al. (1990), it will give the rates of

contribution ranging from 6.7 (man) to 30.6 % (Thoroughbred) of βm. This

calculation reveals a lower rate of carnosine contribution to βm in human

skeletal muscle.

Free inorganic phosphate (Pi) has been indicated to be the second most

important buffer constituent in physico-chemical buffering actions.

Monohydrogen phosphate (HPO42­) readily accepts an extra H+ to become

dihydrogen phosphate (H2PO4­). When muscle pH decreases from 7.0 (rest)

to 6.4 (fatigue), assuming that H+ ion uptake per mmol of Pi is 0.33 mmol,

the buffer value of Pi can be calculated from Pi concentration in skeletal

muscle at rest and the changes of muscle pH (β=0.33 ×[Pi]/∆pH; Hultman

and Sahlin, 1980). The Pi concentration previously reported (Sahlin, 1978;

Sahlin et al., 1997) is 9.4-13.2 mmol・l­1 muscle water (31.3-44.0 mmol・kg­1

dry muscle) so that the contribution of Pi (5.2-7.3 slykes) is only approxi-

mately 8-11 % of βm (68 slykes). Gordon et al. (1991) have described that

the importance of phosphate buffer system is much greater in intracellular

environment although its buffer system plays a small role in exracellular space.

Namely, this implies that the pH (6.9-7.1 at rest; 6.4-6.6 during exercise) in

intracellular fluid is close to the effective pH (pH=6.1-7.7) of phosphate buffer

system.

Maximum buffering capacity in bicarbonate (CO2-HCO3­) buffer system

is obtained when CO2 can diffuse freely in and out of the body fluids. Dynamic

exercise is regarded as an open system where exchanges substances with

blood and static exercise as a closed system where CO2 is trapped (CO2-HCO3­

Kohji Hirakoba6

buffer system is negligible). Since local circulation within the muscle utilizing

during isometrc contractions (static exercise) is occluded due to muscle

tension exerted, the efflux of CO2 from the muscle to blood is very small

compared with dynamic exercise, which would lead to the inhibition of the

CO2-HCO3­ buffering action (HCO3

­+H+ → CO2+H2O ) in the muscle.

According to Sahlin’s study (1978), βm (73 slykes) in dynamic bicycle

exercise was higher than that in static exercise (57 slykes). The difference of

βm between these two types of exercise is approximately equivalent to the

buffering capacity of CO2-HCO3­ system, indicating that the CO2-HCO3

­ buffer

system in skeletal muscle could contribute to 18-20 % of βm.

2 Metabolic buffering

There are two buffer constituents of metabolic buffering processes, i.e.,

phosphocreatine and glutamate. The buffering action of glutamate is negligible,

since glutamate buffering action is very small in human muscle (Sahlin, 1978).

Breakdown of phosphocreatine (PCr) gives an immediate energy source to

resynthesize ATP from ADP and Pi during short-term, heavy exercise and

is also associated with proton consumption (PCr+ADP+H+→ creatine+ATP).

The stoichiometrical uptake (α) of H+ per mmol PCr depleted during exercise

increases with the decrease of muscle pH within the physiological pH range,

as described in an equation: α=1/[1+10(pH-pKa)]. Since hydrolysis of 1 mmol

PCr can theoretically remove H+ ion between 0.38 and 0.83 mmol over the

range pH 7.0-6.0, the buffer value of PCr during exercise can be calculated

using a following equation: β=α × ∆[PCr]/∆pH (Adams et al., 1990). Maximal

proton uptake of PCr at fatigue (α=0.70 at muscle pH=6.4; PCr content=78

7Buffering Capacity in Human Skeletal Muscle: A Brief Review

mmol・kg­1 dry muscle, Sahlin et al., 1997) is expected to be about 55 mmol

・kg­1 dry muscle (β=27 slykes), resulting in the contribution of 40 % to

βm. It has been reported that the content of PCr is higher in type II fibers

with high glycolytic capacity than in type I fibers with low glycolytic and high

oxidative capacity ( Hultman and Sahlin, 1980; Nevill et al., 1996 ) .

Consequently it is possible that metabolic buffering may vary between subject

to subject, depending on muscle fiber type composition and/or PCr content

(Weston et al., 1996).

1 Methods for estimating skeletal muscle buffering capacity m

The methods for estimating βm are: (1) Titrimetric determination of

muscle homogenate (in vitro method, βmvitro) and (2) Calculating the ratio

(∆La/∆pH) of the changes in muscle La and pH due to a exercise stress test

(in vivo method, βmvivo). In HCl titrimetric determination, freeze-dried muscle

is homogenized at a dilution of 20-30 mg dry muscle per ml (50-33 µl per

mg dry muscle) of homogenizing solution containing 145 mM KCl, 10 mM

NaCl, 5 mM IAA ( iodoacetic acid is used to inhibit glycolysis during

homogenization), and pH=7.0. Thereafter the titration is performed with a

serial addition of 2 µl aliquots of HCl (10 mmol・l­1) over the range pH 7.2

-6.2 at a temperature of 37 ℃. βmvitro is calculated from the fitted titration

curve, which means the number of mmoles of H+ required to change the pH of

1 kg of dry muscle from 7.1 to 6.5 (Figure 1). This pH range represents

the changes in muscle pH from rest to fatigue in high-intensity exercise as

ESTIMATIION OF SKELETAL MUSCLE BUFFERING

CAPACITY

Kohji Hirakoba8

listed in Table 1. In contrast, βmvivo is estimated from the changes in muscle

La and pH before (rest) and immediately after exercise to fatigue and is

calculated from a following equation: βmvivo=(Lawork-Larest)/(pHrest-pHwork).

Thus it seems likely that minimal determination errors of La and pH would

lead to greater error in buffer value by calculation from the two points of La

and pH before and after exercise (Mannion et al., 1993). In addition, in vivo

method needs to take muscle sample twice for one trial.

Figure 1. Typical HCl titration curve for human muscle homogenate (Hirakoba, Tonkonogi& Sahlin, unpublished work). The titration was performed with the serial additionof 2 µl aliquots of HCl (10 mM). The number of mmoles of H+ corresponded tothe pH 7.1 and 6.5 were calculated from the interpolation of the titration curve.

9Buffering Capacity in Human Skeletal Muscle: A Brief Review

2 Comparison of mvitro and mvivo

According to the theory on acid-base balance in muscle, βmvitro gives only

physico-chemical buffering capacity. In addition, it has been reported that

buffering capacity in wet muscle was 15-20 % higher than that in dry muscle

and this difference of βmvitro between the two muscle samples resulted from

the loss of HCO3­ under freeze-drying process of muscle (MaCutecheon et

al., 1987, Marlin and Harris, 1991). The value of physico-chemical buffering

capacity in dry muscle without bicarbonate buffering would be lower compared

with the theoretical value. βmvivo reflects total muscle buffering capacity (both

physico-chemical and metabolic buffering actions). Therefore, βmvivo is thought

to be higher than βmvitro since βmvitro measured by the titration technique of

muscle homogenate does not include metabolic buffering processes.

EFFECTS OF HIGH­INTENSITY EXERCISE TRAINING ON m

βm could be considered as an important factor to improvement of

performances in short-term events. Recently, Green et al. (1996) found a

significant, positive correlation (r=0.81) between βm and anaerobic ATP yield

in well-trained male cyclists, suggesting that the higher βm enhances ATP

resynthesis rate at high-intensity exercise. Sahlin and Henriksson (1984)

reported that trained men who carried out anaerobic high-intensity training

had a higher βm than untrained men did. Parkhouse et al. (1985) also

observed that deproteinized muscle buffering capacity in sprinters and rowers

showed higher values compared to marathoners and untrained subjects. From

the cross-sectional comparisons, the βm is anticipated to be increased by high

-intensity exercise training. This possibility has been supported by the

Figure 1. Typical HCl titration curve for human muscle homogenate (Hirakoba, Tonkonogi& Sahlin, unpublished work). The titration was performed with the serial additionof 2 µl aliquots of HCl (10 mM). The number of mmoles of H+ corresponded tothe pH 7.1 and 6.5 were calculated from the interpolation of the titration curve.

Kohji Hirakoba10

longitudinal studies of Bell and Wenger (1988), Sharp at al. (1986) and

Weston et al. (1997) in human (Table 2) and of Troup et al. (1986) and

Weston et al. (1996) in animal. Mizuno et al. (1990) showed a significant,

positive correlation (r=0.83) between relative increases in βm and in short

-term running performance as a result of two weeks of training at high altitude

(2,700 m above sea level), suggesting that physical training under hypoxic

condition would be useful to the increased βm.

On the contrary, Mannion et al. (1995) have recently indicated that βm is

not a major limiting factor to the performance of high-intensity exercise, which

is consistent with the results that high-intensity training had little effect on βm

in spite of the enhanced performances of high-intensity anaerobic exercise

(Mannion et al., 1994, Nevill et al. 1989). However, Nevill et al. (1989)

estimated the changes of βmvivo and βmvitro with high-intensity training, and

Table 2. Changes in muscle buffering capacity before and after high-intensity training in human

Values are expressed as means.Muscle buffering capacity, mmol・kg­1 dry muscle・pH­1; TE, training at 4.19 rad・s­1; TS, training at 1.05 rad・s­1

a Calculated from the conversion factors as given in table 1b Calculated form the difference of mean values between pre- and post-trainingc Significantly increased from pre-training value

Type of Ttaining Muscle Sample MethodMuscle Buffering

Capacity % increaseb Reference

pre post

one-leggedsprint trainingfor 7 wk vastus lateralis in vitro 217a 251a +15.7c Bell & Wegner 1988

sprint training for 8 wk vastus lateralis in vitro 178a 170a ­4.5 Bevan et al. 1986

isokinetic knee extensiontraining for 16 wk

quadriceps femoris in vitroTF 159TS 154

160165

+0.6+7.1

Mannion et al. 1994

sprint training for 8 wk vastus lateralisin vitroin vitro

293a

225a422a

237a+44.0+ 5.3

Nevill et al. 1989

sprint training for 8 wk vastus lateralis in vitro 194a 265a +36.6c Sharp et al. 1986

interval training for 4 wk vastus lateralis in vitro 207 240 +15.9c Weston et al. 1997

11Buffering Capacity in Human Skeletal Muscle: A Brief Review

found that βmvivo showed a tendency to be increased despite the fact that βmvitro

was unchanged after high-intensity training. As mentioned earlier, βmvitro

includes only physico-chemical buffering but excludes metabolic buffering

actions and transmembrane fluxes of H+ or HCO3­ ions during exercise

(Mannion et al., 1993). Hirakoba et al. (1992) reported that the contribution

of bicarbonate buffer system to total buffering actions would be increased by

high-intensity training, presumably owing to the greater transmembrane fluxes

of H+ or HCO3­ ions in post-training status compared with pre-training. If,

for example, physico-chemical buffering was unchanged but metabolic buffering

and transmenbrane fluxes of H+ or HCO3­ ions were increased by training,

it could be thought that although βmvitro maintains a constant value, an increase

in βmvivo occurs after training. Therefore, it is conceivable that conflicting

results could be induced when estimating βm by either in vivo or in vitro.

Sahlin (1994) has also stated that the reason for this discrepancy between

studies is unclear but may be due to methodological differences. Another

possible explanation for this is that βm is significantly related to the cross

-sectional area occupied by type II fibers (Parkhouse et al., 1985, Sewell et al.,

1991, Weston et al., 1996). Although human vastus lateralis is often used to

estimate βm, this muscle is acknowledged to have very heterogeneous fiber

type composition with large inter-individual variation (Johnson et al., 1973).

Values are expressed as means.Muscle buffering capacity, mmol・kg­1 dry muscle・pH­1; TE, training at 4.19 rad・s­1; TS, training at 1.05 rad・s­1

a Calculated from the conversion factors as given in table 1b Calculated form the difference of mean values between pre- and post-trainingc Significantly increased from pre-training value

Type of Ttaining Muscle Sample MethodMuscle Buffering

Capacity % increaseb Reference

pre post

one-leggedsprint trainingfor 7 wk vastus lateralis in vitro 217a 251a +15.7c Bell & Wegner 1988

sprint training for 8 wk vastus lateralis in vitro 178a 170a ­4.5 Bevan et al. 1986

isokinetic knee extensiontraining for 16 wk

quadriceps femoris in vitroTF 159TS 154

160165

+0.6+7.1

Mannion et al. 1994

sprint training for 8 wk vastus lateralisin vitroin vitro

293a

225a422a

237a+44.0+ 5.3

Nevill et al. 1989

sprint training for 8 wk vastus lateralis in vitro 194a 265a +36.6c Sharp et al. 1986

interval training for 4 wk vastus lateralis in vitro 207 240 +15.9c Weston et al. 1997

Kohji Hirakoba12

There have been no direct studies to clarify the mechanisms by which

βm is increased. Many proteins in intracellular fluid act as buffers and its

incorporation into skeletal muscle with training may result in a higher ratio of

protein to weight of muscle. This response to training would account partly for

the increased βm. Parkhouse et al. (1985) pointed out that the high βm in

anaerobically trained athletes may be associated with elevated carnosine levels

in their skeletal muscles, because a significant, positive correlation (r=0.69)

was found between βm and carnosine levels. Moreover, Harris et al. (1990)

have suggested that the higher βm in horse and dog, compared with man,

is predominantly due to higher muscle contents of histidine containing

dipeptides in these species. This idea has been supported by the study of

Sewell et al. (1991) in which non-carnosine muscle buffering capacity (mea-

sured as the difference between βm and carnosine buffering capacity) was

constant at all fiber compositions in horse and pony. However, these results in

the cross-sectional studies are indirect evidences to verify the mechanisms

of the increased βm due to high-intensity training. There have been found no

changes in carnosine levels in human muscle after eight weeks of anaerobic

sprint training (Bevan et al., 1986) and 16 weeks of isokinetic training of knee

extensors (Mannion et al., 1994). Sahlin and Henriksson (1984) indicated

that even if a maximal buffering power of carnosine was assumed, its

contribution was only 2-4 slykes or < 7% of buffering capacity in human

muscle. Therefore, another explanations should be sought for the adaptive

MECHANISMS RELATED TO INCREASED m WITH HIGH­

INTENSITY EXERCISE TRAINING

13Buffering Capacity in Human Skeletal Muscle: A Brief Review

mechanisms related to the increased βm observed after training in man.

CONCLUSIONS

It is inferred that the results of βm between studies may be different,

depending on the methods for estimating βm (in vivo or in vitro methods).

It is important to establish the components of buffering being measured by

either in vivo or in vitro methods as indicated by Mannion et al. (1993) and the

methods for estimating βm should be carefully chosen according to the purpose

of the study, particularly when determining the longitudinal changes in βm

after training.

There have been no direct assessments on the adaptive mechanisms of

the increased βm due to high-intensity training and it is unclear about the

mechanisms by which the training-induced increase in βm occurs. However,

several previous studies have indicated that the higher βm found

predominantly in sprint-trained athletes may be a critical factor of anaerobic

exercise performances accompanied by La accumulation and suggested that

proton buffering proteins (carnosine) levels could be responsible for the in-

creased βm due to training. It is possible that the higher carnosine content in

skeletal muscle and the higher βm may be due to exposure to prolonged

periods of hypoxia and acidosis with high-intensity exercise training, but there

is only indirect evidence to support this possibility. It is necessary to prove

the obvious relation of cause and effect regarding the increased βm with

training. Consequently, more detail studies should be carried out to clarify

as to which buffer constituent contributes to the increased βm observed with

high-intensity training.

Kohji Hirakoba14

ACKNOWLEDGEMENTS

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15Buffering Capacity in Human Skeletal Muscle: A Brief Review

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Kohji Hirakoba16

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