SKELETAL MUSCLE INTERSTITIUM AND BLOOD PH AT REST … · since the measurement of interstitial pH in human skeletal muscle has never been performed before, these studies will represent

SKELETAL MUSCLE INTERSTITIUM AND BLOOD PH AT REST AND DURING EXERCISE

IN HUMANS

A thesis submitted for the degree of

Doctor of Philosophy

to

Queensland University of Technology

School of Human Movement Studies

Darrin Street

B.App.Sci. Hons.

2003

Preface

III

PREFACE

The present thesis is divided into three major parts corresponding to the three

experimental studies undertaken: Chapter 3, measurement of dialysate pH

representative of interstitial pH at rest and during dynamic leg exercise,

Chapter 4, effect of alkali ingestion rate on plasma acid-base [H+] and ionic

[K+] status, and Chapter 5, effect of sodium citrate on interstitial pH in human

skeletal muscle. These studies individually look at establishing a new method

for the detection of interstitial pH in humans at rest and during exercise,

developing an optimal ingestion regime of sodium citrate to manipulate blood

pH at rest and finally, by combining these two techniques, ascertaining the

degree to which the interstitial space is alkalised after ingesting sodium

citrate. Chapter 3 further details the in vitro experiments used to evaluate

the accuracy and reliability of the fluorometric method established for

measuring interstitial pH.

Such manipulation of a system can provide valuable information as to how the

system operates and thereby lead to a greater understanding of human

exercise physiology, which is the main concern of this thesis. Specifically,

since the measurement of interstitial pH in human skeletal muscle has never

been performed before, these studies will represent a collective approach to

gaining further knowledge about the movement of H+ between the blood and

interstitial space within skeletal muscle.

Other major sections of this thesis include the literature review, general

discussion, conclusion and future research. Collectively, these sections set

the context for the thesis, provide background for the support and

identification of the three experimental studies, analyses and interpretations

of results collected from all three studies, main findings and future directions

inspired from this work. In the literature review, my aim is to provide

information about the specific physiology pertaining to sodium citrate

ingestion and pH regulation. Consequently, I have divided it into four main

sections: gastro-intestinal absorption and sodium citrate, renal regulation, H+

and K+ regulation at rest and during exercise, and alkalosis at rest and during

exercise. Each of these sections, in turn, discusses the existing research

relevant to the experimental studies and identifies the need for further

Preface

IV

research. The general discussion mainly addresses the key findings as a

whole from Chapters 3, 4 and 5, while the conclusion adds to this information

with the inclusion of future research directions.

Keywords

V

KEYWORDS

Alkali ingestion rate

Alkalosis

Bicarbonate perfusate

Blood pH

Dialysate pH

Ergogenesis

Interstitial pH

Knee-extensor exercise

Microdialysis

pH

Proton

Skeletal muscle

Sodium citrate

Urine pH

Abstract

VII

ABSTRACT

The aims of this thesis were to: 1) develop a new method for the

determination of interstitial pH at rest and during exercise in vivo, 2)

systematically explore the effects of different ingestion regimes of 300 mg.kg-

1 sodium citrate on blood and urine pH at rest, and 3) to combine the new

interstitial pH technique with the findings of the second investigation in an

attempt to provide a greater understanding of H+ movement between the

extracellular compartments.

The purpose of the first study was to develop a method for the continuous

measurement of interstitial pH in vastus lateralis was successfully developed

using microdialysis and 2,7-bis-(2-carboxyethyl)-5-(and-6)-

carboxyfluorescein (BCECF). To avoid the presence of an artificial alkalosis

during exercise, it was necessary to add 25 mM HCO3- to the perfusate. The

outlet of the probe was cut <10 mm from the skin and connected to a

stainless steel tube completing the circuit to a microflow-through cuvette (8

µl) within a fluorescence spectrophotometer. This prevented the loss of

carbon dioxide from the dialysate and any subsequent pH artefact. Interstitial

pH was collected from six subjects before, during and after five minutes of

knee-extensor exercise at three intensities 30, 50, and 70 W. Mean±SEM

interstitial pH at rest was 7.38±0.02. Exercise reduced interstitial pH in an

almost linear fashion. The nadir value for interstitial pH at 30, 50 and 70 W

exercise was 7.27, 7.16 and 7.04, respectively. The lowest pH was obtained

1 min after exercise, irrespective of workload, after which the interstitial pH

recovered in a nearly exponential manner. The mean half time of interstitial

recovery was 5.2 min. The changes in interstitial pH exceeded the changes in

venous blood pH. This study demonstrated that interstitial pH can be

measured using microdialysis and that it is continuously decreased during

muscle activity.

The purpose of the second study was to establish an optimal ingestion regime

for the ingestion of 300 mg.kg-1 of sodium citrate and maximise the alkalotic

effect while minimising any side effects. Increasing the effectiveness of alkali

ingestion may lead to further increases in muscle performance. Ingesting 300

mg.kg-1 sodium citrate at a rate of 300 mg.min-1 was identified as the optimal

Abstract

VIII

ingestion regime to maximise alkalosis at rest, which occurred 3.5 h post-

ingestion. This was determined by monitoring eight human subjects ingesting

300 mg.kg-1 sodium citrate at five different rates, control (no ingestant),

bolus, 300, 600 and 900 mg.kg.min-1 on five days separated by at least 48

hours. Sodium citrate was ingested in capsule form with water ad libitum,

with the exception of bolus, which was combined with 400 ml <25 percent

orange juice and consumed in <1 min. Arterialised blood (mean 71.3±3.5

mmHg) acid-base and electrolyte status was assessed via the withdrawal of

~5 ml of blood every 30 min across an eight hour duration, placed on ice and

analysed within five minutes. No alkalotic difference was found between

ingestion rates (mean 7.445±0.004, 7.438±0.004 and 7.442±0.004 for 300,

600 and 900 mg.min-1, respectively). All experimental ingestion regimes

were associated with elevations in [HCO3-] (29.6, 29.7, 29.8, 29.9 and 26.3

mmol.l-1 for bolus, 300, 600, 900 and control, respectively). The 300

ingestion regime had the greatest impact on [H+], a 0.66 meq.l-1×10-8 change.

Bolus ingestion (3.93±0.08 mmol.l-1) of sodium citrate had no effect on

control (4.06±0.08 mmol.l-1) blood [K+], however, 300 mg.min-1 decreased

blood [K+] (p<0.05). There was no effect of sodium citrate on blood [Cl-], but

after 2.5 h blood [Cl-] was lower than pre-ingestion values (p<0.05). All

ingestion rates of sodium citrate increased (p<0.05) urine pH above control.

This is the first study to investigate the effect of varying ingestion rates on

acid-base status at rest in humans. The results suggest that ingesting sodium

citrate in small doses in quick succession induce a greater blood alkalosis than

the commonly practised bolus protocol.

Using the interstitial pH technique described above and the optimal ingestion

regime (300 mg.min-1) identified above, the final experiment was designed to

assess the influence of sodium citrate ingestion on interstitial pH at both rest

and during exercise. Five subjects ingested 300 mg.kg-1 sodium citrate at

300 mg.min-1 again in capsule form with water ad libitum. Prior to ingestion,

each subject had a cannula placed into their cephalic vein and one

microdialysis probe (CMA-60) inserted into their left thigh, orientated along

the fibres of vastus lateralus. This probe was used for the measurement of

pH as described above. At the end of this period, an exercise protocol

required five subjects to perform light exercise (10 W) for 10 min, before

starting an intense exercise period (~90-95% leg VO2peak) to exhaustion

followed by a 15 min recovery period. Dialysate and blood samples were

Abstract

IX

collected across all periods. Mean±SEM interstitial pH for placebo and

alkalosis were 7.38±0.12 and 7.24±0.16, respectively. Sodium citrate

ingestion was not associated with an interstitial alkalosis. An exercise induced

acidosis was observed in the interstitium during placebo but not during

alkalosis (p<0.05). Mean±SEM venous pH were 7.362±0.003 and

7.398±0.003 for placebo and alkalosis, respectively. Sodium citrate ingestion

was not associated with a venous alkalosis. Sodium citrate ingestion was

associated with an increase in mean±SEM venous [HCO3-] (placebo 25.5±0.2,

alkalosis 28.1±0.2). This increase in the blood bicarbonate buffer system was

not associated with an increase in time to exhaustion (placebo 352±71,

alkalosis 415±171). This was the first study to investigate the effects of

sodium citrate ingestion on interstitial pH. The results of this study

demonstrated that an interstitial alkalosis does not ensue after alkali

ingestion, however, it was associated with the lack of an exercise induced

acidosis suggesting an improved pH regulation during exercise.

Table of Contents

XI

TABLE OF CONTENTS

SKELETAL MUSCLE INTERSTITIUM AND BLOOD PH AT REST AND DURING EXERCISE IN HUMANS ..........................................................................................................................................................I

PREFACE ......................................................................................................................................III KEYWORDS ...................................................................................................................................V ABSTRACT ................................................................................................................................. VII TABLE OF CONTENTS ...................................................................................................................XI LIST OF FIGURES ........................................................................................................................ XV LIST OF TABLES ...................................................................................................................... XVII LIST OF EQUATIONS ..................................................................................................................XIX ABBREVIATIONS .......................................................................................................................XXI

Chemical Formulas:..............................................................................................................XXI Units of Measurement: ........................................................................................................ XXII

STATEMENT OF ORIGINAL AUTHORSHIP .................................................................................. XXV ACKNOWLEDGEMENTS..........................................................................................................XXVII

CHAPTER 1 INTRODUCTION ............................................................................................................ 1

CHAPTER 2 LITERATURE REVIEW................................................................................................. 5 Introduction...............................................................................................................................5 Gastro-Intestinal Absorption and Sodium Citrate.....................................................................5 Renal Regulation.......................................................................................................................8 H+ and K+ regulation at rest and during exercise...................................................................12

Proton - H+ ..........................................................................................................................12 Potassium - K+.....................................................................................................................15

Alkalosis at rest and during exercise ......................................................................................21 CHAPTER 3 MEASUREMENT OF DIALYSATE PH, REPRESENTATIVE OF INTERSTITIAL PH, AT REST AND DURING DYNAMIC LEG EXERCISE ................................................................... 27

Preface ....................................................................................................................................27 Introduction.............................................................................................................................28

PART I – IN VITRO VALIDATION OF MEASURING INTERSTITIAL PH USING MICRODIALYSIS AND BCECF ..........................................................................................................................................29

In vitro microdialysis components and system........................................................................29 Day to day stability of 2,7-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein (BCECF) dye.................................................................................................................................................32 Effect of albumin and stir rate on fluorescence using BCECF dye .........................................33 Influence of 1 cm polyurethane outlet tubing exposed to air...................................................34 Effect of HCl, La- and CO2 titrations on pH calibration curves..............................................35 Effect of dye concentration on pH calibration ........................................................................36 Effect of HCO3

- perfusate on pH calibration...........................................................................37 PART II – EFFECT OF PERFUSATE [HCO3

-] ON INTERSTITIAL PH IN VIVO......................................39 Methods...................................................................................................................................39

Subjects ...............................................................................................................................39 Exercise protocol.................................................................................................................39 Probe insertion and perfusate ..............................................................................................39 Fluorometric measurements and determination of pH ........................................................40

Results .....................................................................................................................................41 Discussion ...............................................................................................................................42

PART III – INTERSTITIAL PH AT REST AND DURING DYNAMIC LEG EXERCISE ...............................43 Methods...................................................................................................................................43

Subjects ...............................................................................................................................43 Exercise protocol.................................................................................................................43

Table of Contents

XII

Probe insertion and perfusate ..............................................................................................43 Fluorometric measurements and determination of pH.........................................................43

Results .....................................................................................................................................44 Interstitial pH at rest and during muscle activity .................................................................44 Recovery from exercise .......................................................................................................44

Discussion ...............................................................................................................................46 Changes in muscle interstitial pH during and after exercise................................................46 Recovery of interstitial pH ..................................................................................................48 Comparison between pH changes in muscle interstitium and blood ...................................48 Comparison between cellular and interstitial pH changes...................................................49 Physiological implications of changes in interstitial pH......................................................50

Conclusion...............................................................................................................................51 CHAPTER 4 EFFECT OF ALKALI INGESTION RATE ON PLASMA ACID-BASE, [H+], AND IONIC, [K+], STATUS...................................................................................................................................53

Introduction .............................................................................................................................53 Methods ...................................................................................................................................54

Subjects ...............................................................................................................................54 Experimental Protocol .........................................................................................................55 Blood collection...................................................................................................................55 Ingestion ..............................................................................................................................55 Measurement and Analysis..................................................................................................56 Statistics...............................................................................................................................56

Results .....................................................................................................................................57 Acid-base.............................................................................................................................57 Strong Ions ..........................................................................................................................62 pO2.......................................................................................................................................71 Urine....................................................................................................................................71

Discussion ...............................................................................................................................72 pO2 status.............................................................................................................................72 Sodium citrate and GI absorption ........................................................................................73 Sodium citrate and acid-base balance..................................................................................75 Ingestion time and [H+] .......................................................................................................76 Sodium citrate and [K+] .......................................................................................................76 Sodium citrate and urinary alkalosis....................................................................................77

Conclusion...............................................................................................................................78 CHAPTER 5 EFFECT OF SODIUM CITRATE ON INTERSTITIAL PH IN HUMAN SKELETAL MUSCLE ..................................................................................................................................79

Introduction .............................................................................................................................79 Methods ...................................................................................................................................81

Subjects ...............................................................................................................................81 Exercise protocol .................................................................................................................81 Experimental exercise intensity...........................................................................................82 Probe insertion.....................................................................................................................82 Perfusate ..............................................................................................................................82 Fluorometric measurements and determination of pH.........................................................83 Blood collection and analysis ..............................................................................................83 Ingestion ..............................................................................................................................84

Results .....................................................................................................................................84 Interstitial pH.......................................................................................................................84 Venous pH and HCO3

-.........................................................................................................87 Venous blood gases pO2 and pCO2 .....................................................................................90 Time to exhaustion ..............................................................................................................91

Discussion ...............................................................................................................................93 Conclusion...............................................................................................................................97

CHAPTER 6 GENERAL DISCUSSION..............................................................................................99

Table of Contents

XIII

Introduction.............................................................................................................................99 The importance of effective pH manipulation .........................................................................99 The importance of interstitial pH ..........................................................................................100 Limitations.............................................................................................................................102

CHAPTER 7 CONCLUSIONS AND FUTURE RESEARCH ......................................................... 103 Conclusions ...........................................................................................................................103 Future Research ....................................................................................................................104

REFERENCES ............................................................................................................................................ 107

List of Figures

XV

LIST OF FIGURES

Figure 1 Primary active transport of H+ into the renal tubule .........................................................9 Figure 2 Secondary active secretion of H+ into the renal tubule ....................................................11 Figure 3 Modified CMA60 microdialysis probe .............................................................................30 Figure 4 The complete fluorometric microdialysis system for the measurement of H+ in vitro......31 Figure 5 Day to day variability of 2,7-bis-(2- carboxyethyl)-5-(and-6)-carboxyfluorescein .........33 Figure 6 Effect of magnetic stir rate and albumin on [H+] measured with BCECF in vitro ..........34 Figure 7 Influence of 1 cm polyurethane tubing exposed to air on [H+] determination in vitro ....35 Figure 8 Titration curves for HCl-CO2 and HCl-La- ......................................................................36 Figure 9 Effect of BCECF dye concentration on pH calibration curves.........................................37 Figure 10 Effect of perfusate [HCO3

-] on pH calibration curves in vitro......................................38 Figure 11 Influence of [HCO3

-] on the estimation of interstitial pH during 10 W dynamic leg exercise............................................................................................................................................41 Figure 12 Interstitial acidification during exercise ........................................................................45 Figure 13 Peak interstitial pH during exercise at different power outputs.....................................46 Figure 14 Intracellular, interstitial and venous pH during knee-extensor exercise........................50 Figure 15 Blood pH 8-h temporal response....................................................................................57 Figure 16 Mean blood pH response pre- and post-ingestion..........................................................58 Figure 17 Blood pH relative change between PRE and 2 h post-ingestion ....................................58 Figure 18 Mean post-ingestion blood [HCO3

-] for all IR ...............................................................59 Figure 19 Mean blood [HCO3

-] temporal responses across 8 h for all IR .....................................60 Figure 20 Mean post-ingestion blood [pCO2] for each IR..............................................................61 Figure 21 Temporal response for blood [pCO2] for each IR..........................................................61 Figure 22 Individual IR temporal responses in blood [H+] for all conditions................................63 Figure 23 Mean (± SEM) blood [H+] for each IR at 3 time points .................................................64 Figure 24 Relative percent changes in blood [H+] .........................................................................64 Figure 25 Mean Blood K+ temporal profiles for all conditions ......................................................66 Figure 26 Mean (± SEM) blood [K+] for each condition at 3 time points ......................................67 Figure 27 Relative percent changes in blood [K+] .........................................................................67 Figure 28 Mean (±SEM) blood [Cl-] response for each condition .................................................68 Figure 29 Mean Cl- temporal response for each condition.............................................................69 Figure 30 Mean (±SEM) blood [Cl-] response at 3 time points......................................................69 Figure 31 Mean (±SEM) blood [Na+] response for all conditions .................................................70 Figure 32 Mean blood [Na+] temporal response for all conditions ...............................................70 Figure 33 Mean (±SEM) blood PO2 response for all conditions ....................................................71 Figure 34 Mean (±SEM) urine pH temporal response for each condition .....................................72 Figure 35 Interstitial pH temporal response..................................................................................85 Figure 36 Overall effect of sodium citrate ingestion on venous pH...............................................87 Figure 37 Venous pH temporal response.......................................................................................88 Figure 38 Overall effect of sodium citrate ingestion on venous [HCO3

-] ......................................89 Figure 39 Venous HCO3

- temporal response .................................................................................89 Figure 40 Venous pO2 status for both conditions placebo and alkalosis .......................................90 Figure 41 Venous pO2 temporal response.......................................................................................90 Figure 42 Venous pCO2 temporal response...................................................................................91 Figure 43 Time to exhaustion for both placebo and alkalosis conditions......................................92

List of Tables

XVII

LIST OF TABLES

Table 1 Ion concentration within plasma, interstitium and muscle.................................................13 Table 2 Blood acid-base and interstitial pH values with ingestion of CaCO3 (placebo) and Na3C8H5O7 (alkalosis).....................................................................................................................86 Table 3 Individual exhaustion times for intense knee-extensor exercise........................................92

List of Equations

XIX

LIST OF EQUATIONS

Equation 1 Calculation of pH .........................................................................................................21 Equation 2 Calculation for electrical neutrality .............................................................................24 Equation 3 Calculation of strong ion difference (SID) ...................................................................24

Abbreviations

XXI

ABBREVIATIONS

ATP – Adenosine Tri-phosphate

ATPase - Adenosine Tri-phosphatase

a-v difference – arterio-venous difference

BCECF – 2,7–bis–(2-carboxyethyl)-5-(and-6-)-carboxyfluorescein

BMI – Body Mass Index

EDL – Extensor Digitorum Longus

Ex - Exercise

Exh - Exhaustion

GI – Gastro-intestinal

Ing - Ingestion

IOC – International Olympic Committee

IR – Ingestion Rate

MVC – Maximum Voluntary Contraction

NAE – Net Acid Excretion

NMR – Magnetic Resonance Spectroscopy

NVA – Non-Volatile Acid

PFK – Phosphofructokinase

RBC – Red Blood Cells

Rec - Recovery

SB – Sodium Bicarbonate

SC – Sodium Citrate

SE – Standard Error

SEM - Standard Error of Mean

SID – Strong Ion Difference

TA – Titratable Acid

VO2peak – Peak Oxygen Consumption per minute

Chemical Formulas: Ca2+ - Calcium

CaCO3 – Calcium Carbonate

Cl- - Chloride

CO2 – Carbon Dioxide

H+ - Hydrogen Ion/Proton

H2CO3 – Carbonic Acid

Abbreviations

XXII

Hb – Haemoglobin

HCl – Hydrogen Chloride

HCO3- - Bicarbonate

K+ - Potassium

KOH – Potassium Hydroxide

La- - Lactate

Mg2+ - Magnesium

Na+ - Sodium

Na3C8H5O7 – Sodium Citrate

NaHCO3- - Sodium Bicarbonate

NaOH – Sodium Hydroxide

NH4 – Ammonia

pCO2 - Partial Pressure of Carbon Dioxide

PCr2- - Phosphocreatine

pH – “Pouvoir Hydrogene” (Hydrogen Concentration)

pO2 - Partial Pressure of Oxygen

PO4 - Phosphate

SO42- - Sulfate

Units of Measurement: µl – microlitre

da – Daltons

g – grams

g.l-1 – grams per litre

h – hour

Hz - Hertz

Kg – Kilograms

M – Molar

m.mol.l-1 - millimoles per litre

meq.h-1 – milli-equivalents per hour

meq.l-1 – milli-equivalents per litre

mg.kg-1 – milligrams per kilogram

mg.min-1 – milligrams per minute

min – minutes

ml – millilitre

mM – millimolar

mmHg – millimetres of mercury

Abbreviations

XXIII

mOsm.l-1 – milliosmolar per litre

mOsmol.kg-1 – milliosmolar per kilogram

mV – millivolt

N – Nolar

neq.l-1 – nano-equivalents per litre

nM - nanomolar

s - seconds

W – Watt

Statement of Original Authorship

XXV

STATEMENT OF ORIGINAL AUTHORSHIP

The work contained in this thesis has not previously been submitted for a

degree or diploma at any other higher education institution. To the best of my

knowledge, this thesis does not contain any material previously published or

written by any other person, except where due reference is made.

_______________________

Candidates Signature

8/8/2003

Acknowledgements

XXVII

ACKNOWLEDGEMENTS

This thesis would not have made it to completion without the assistance of

some great people. I would like to take this opportunity to express my

sincere gratitude to them.

Dr Simon Green I would like to thank my principle supervisor, Dr Simon Green for inspiring me

to embark on a journey that has provided me with many rewards. His

continual encouragement and support throughout my candidature is greatly

appreciated.

Associate Professor Carsten Juel The opportunity to perform the Danish experiments would not have been

possible without the efforts of Associate Professor Carsten Juel. My sincere

appreciation is extended to him for this and his kind hospitality. Tusind tak.

Associate Professor Andrew Hills To my associate supervisor, Associate Professor Andrew Hills, thank you for

your thoughtful comments on the final preparation of this thesis.

Ms Connie Wishart Thank you to Connie for her genuine willing, friendly and caring nature that

made the long days in the laboratory seem so much shorter and all the years

easier to pass.

Volunteers A sincere thank you to all the subjects that volunteered from the exercise

physiology class of Semester 1, 1997. Your efforts were outstanding over an

arduous 10 h day. Also, an earnest thank you for the efforts of my Danish

friends and colleagues who volunteered their time and body’s for my

experiments in Copenhagen.

Acknowledgements

XXVIII

Team AKI A special thank you to the team at the August Krogh Institute that made my

time in Copenhagen so productive and enjoyable. In particular, I would like

to especially thank Associate Professor Jens Bangsbo for his unique

combination of professionalism and comic relief in the laboratory and Mr Jens

Jung Nielsen for his great depth of talent with microdialysis and always

cheerful demeanour.

Institutions Thank you to the institutions that have provided the financial, administrative

and infrastructural support necessary to enable the production of this thesis;

the Copenhagen Muscle Research Centre, August Krogh Institute, Danish

Research Agency and Queensland University of Technology.

Comrades To all my fellow comrades I gratefully thank you for the mateship you have

provided over the years. In particular, to Glenn, Chris, Jarrod, Nuala and

Kate thank you for helping me through the ‘downs’ and being there for the

‘ups’.

Chapter 1 - Introduction

1

CH A P T E R 1 IN T R O D U C T I O N

Before detailing the physiology relating to sodium citrate ingestion and

skeletal muscle function in the literature review, it will be useful to outline in

brief the importance of pH regulation at rest and during exercise. At rest,

muscle pH is slightly alkaline and constantly regulated around 7.10 (Juel,

1998a) to maintain optimal cell functioning. The alkaline pH value at rest

helps to buffer the small amounts of acid produced via basal metabolism,

however, during intense exercise cellular metabolism produces large amounts

of H+ that are released within the cell, thus rapidly decreasing intracellular

pH. Decreased intracellular pH has negative effects on several important

mechanisms within the cell. Low intracellular pH inhibits the rate limiting

enzyme of glycolysis, phosphofructokinase (PFK) (Hermansen, 1981) and

interacts with the contractile proteins during contraction (Mainwood &

Renaud, 1985). Free H+, concomitant with low pH, compete with Ca2+ at the

binding site of troponin, thereby interfering with myofilament cross-bridge

cycling (Donaldson & Hermansen, 1978). In contrast, pyruvate

dehydrogenase activity (Hollidge-Horvat et al., 1999) and La- transport from

within the intracellular compartment are all increased at low pH. These

examples are not definitive or causative links to fatigue, but rather used to

demonstrate the extent to which low pH can effect metabolism within the

muscle cell. Collectively, the above effects of low pH during intense exercise

reduce the cells’ ability to maintain force development by both energetic

and/or chemical/mechanical interference.

To combat the interference of the cellular metabolism H+ load, skeletal muscle

possess a triad of defence systems, physicochemical buffering, consumption

or production of non-volatile acids and transmembrane fluxes of H+ and HCO3-

. When blood flow is occluded from muscle, such as during isometric exercise

at >20 percent MVC, and intracellular PCO2 is constant the physicochemical

system buffers more H+ (Sahlin, 1978a). The physicochemical buffering

system can also be sub-divided into three categories involving phosphates,

proteins, and bicarbonate buffer systems. The combined efforts of all of the

above systems attempt to buffer excess metabolically produced H+ and thus

maintain intracellular [H+] and pH.


2

The muscle cell is separated from the capillary blood supply by the interstitial

space or interstitium. When substances leave the intracellular space they first

pass into the interstitium before entering the blood. Therefore, metabolite

concentration within the interstitium will influence ionic movements between

the intra and extracellular compartments. In addition, circulating interstitium

substances have been identified as potential mediators in important

processes, such as blood flow (Quayle et al., 1997) and sympathetic nerve

activity (Victor et al., 1988). Approximately a decade ago, the first in vivo

measurements of interstitial metabolites within skeletal muscle using the

microdialysis technique were being attempted. Recently, this method has

been successful in measuring a number of interstitial metabolites including,

lactate (Rosdahl et al., 1998), insulin (Sjostrand et al., 1999), glucose

(Maclean et al., 1999) and potassium (Green et al., 1999) at rest and during

exercise. Although it is possible to measure [H+] in muscle and blood, a

method has not been established to measure interstitial pH. One reason for

this has been due to the difficulty in controlling the movement of both HCO3-

and CO2 within the PVC tubing used in Microdialysis. As a result, no

investigations have been performed on pH regulation within skeletal muscle

interstitium in vivo. For this reason, the movement of H+ within the

interstitium is not well understood, the movement of H+ out of skeletal muscle

and into the capillary network, however, has been well documented (Bangsbo

et al., 1993a; Lindinger & Heigenhauser, 1991; Juel et al., 1990).

Efflux of H+ from muscle into the blood decreases blood pH. To maintain a

positive gradient required for continual efflux from the muscle, the additional

H+ must be buffered and removed from the contracting muscle. The same

buffer systems present in muscle are located within the blood, except that the

bicarbonate system is now the most heavily relied upon. This system consists

of two key elements, HCO3- and CO2, which can be regulated by the kidneys

and lungs. The ability to regulate these compounds enables blood pH to be

adjusted in both directions and greatly increases its capacity. Although the

capacity of the blood system is far greater, it remains finite. If high rates of

metabolically produced H+ continue, the electrochemical balance between

intra and extracellular compartments will be disturbed, compromising the

cell’s ability to move H+, resulting in both decreasing blood and muscle pH.

Consequently, the blood buffering systems will only delay increasing [H+]


3

effects on intracellular metabolic processes that reduce the muscle’s ability to

produce force, i.e., muscle performance.

Increasing one or all of the buffer systems’ capacities, either intracellularly or

in the blood, has the potential to further delay the onset of a critical pH level,

thereby increasing the time for muscle to maintain force production. The

ingestion of sodium bicarbonate or sodium citrate results in an increase in the

capacity of the blood bicarbonate system (Kowalchuk et al., 1989; Costill et

al., 1984). In addition, the ingestion of either of these compounds disturbs

the blood acid-base balance causing a decrease in [H+] or increase in pH (Ball

& Maughan, 1997; Jones et al., 1977b), referred to as being in a state of

alkalosis. Theoretically, manipulating blood [H+] and [HCO3-] via inducing

alkalosis will improve muscle function or muscle performance. Numerous

investigations of ingesting sodium bicarbonate or sodium citrate prior to

exercise have led to increases in swimming (Gao et al., 1988), running

(Shave et al., 2001), cycling (Potteiger et al., 1996a) and rowing

(McNaughton & Cedaro, 1991) performances. Even so, there is evidence to

demonstrate the contrary across each of these modes, respectively (Schabort

et al., 2000; Robinson, 1997; Ibanez et al., 1995; Pierce et al., 1992).

As is often the case with orally induced drugs, there exists a dose-response

relationship. That is, there is a minimum quantity of the drug that must be

consumed to manipulate the system to a high enough degree to enable the

observation of a significant effect. Although this work has been performed

(Horswill et al., 1988) (McNaughton, 1992a; McNaughton, 1990) and a

minimum dose identified, 300 mg.kg-1 for both sodium bicarbonate and

sodium citrate, it was tested across one ingestion regime only, namely bolus.

It may be possible that other ingestion regimes have differing effects.

Investigations that have followed similar dosing strategies have not found

positive effects of sodium bicarbonate (Tiryaki & Atterbom, 1995) or sodium

citrate ingestion (van Someren et al., 1998). Further, some studies have

shown gastro-intestinal side effects when ingesting sodium bicarbonate

(Webster et al., 1993), and sodium citrate (Shave et al., 2001) to a lesser

extent, in bolus form. Systematic exploration of ingestion regime has yet to

be performed on either of these alkalis. Such investigations are important as

they might reveal that lower doses ingested more slowly invoke similar

physiological effects, but with reduced side effects.


4

Therefore, the aims of this thesis are to: a) develop a new method for

determination in vivo of interstitial pH at rest and during exercise, b)

systematically explore the effects of different ingestion regimes of 300 mg.kg-

1 sodium citrate on blood and urine pH at rest, and c) to combine the new

interstitial pH technique with the findings of the second investigation, in an

attempt to provide a greater understanding of pH within the extracellular

compartment.

Chapter 2 – Literature Review

5

CH A P T E R 2 LI T E R A T U R E RE V I E W

Introduction As outlined in the preface, the objective of this literature review is to provide

information about the specific physiology pertaining to sodium citrate

ingestion and pH regulation in skeletal muscle. The four main sections,

gastro-intestinal absorption and sodium citrate, renal regulation, H+ and K+

regulation at rest and during exercise, and alkalosis at rest and during

exercise, in turn, discuss the existing research relevant to the experimental

studies and identify the need for further research.

Gastro-Intestinal Absorption and Sodium Citrate A potentially large factor that influences the degree to which induced alkalosis

affects the muscle cell is the gastro-intestinal (GI) system. That is, how the

alkali will be introduced to the body will play a role in how it will eventually

affect the muscle cell. The two main ways of introducing an alkali to the body

are orally and intravenously. The most common method used in research of

skeletal muscle function is orally, probably due to the ease of use in the

applied setting. Since this is the case, the rate at which the alkali is

introduced to the GI system will directly affect the time it takes to manipulate

blood pH. Although there has been an abundance of research investigating

sodium bicarbonate metabolism through the GI system (Kuu et al., 1998;

Belangero & Collares, 1992; Busse et al., 1989), little has been performed

with sodium citrate (SC). Therefore, it is not completely understood how SC

is metabolised within the GI system, although it seems logical that when

introduced it dissociates into its constituent ions, sodium and citrate. The GI

metabolism of these ions is very well understood and is considered hereafter.

At a given dose of 0.4 ml.kg-1, SC increases gastric pH from 2.5 to 6.11

(Hauptfleisch & Payne, 1996). The direct mechanism for this process is not

known and further GI investigation is required to ascertain this. It is,

however, known that the level of acidity within the GI system has a direct

effect on the rate of digestion (Guyton, 1991). It is possible that the increase

in pH associated with the ingestion of SC may in itself have a direct effect on

absorption through the GI system. If SC dissociates into its constituent ions,


6

then the mechanisms and factors that contribute to its absorption may differ

from the absorption of the molecule as a whole.

The absorption of Na+ from the GI system takes place in the small intestine.

The active process to transport Na+ out of the small intestine involves two

steps (Schultz, 1984). The first is to move the Na+ into the intestine epithelia

and the second is to move it out into the interstitial space. Although the

objective is to move Na+ from the small intestine lumen, the process begins

by actively transporting Na+ out of the epithelia across the basolateral

membrane and into the interstitial space. In doing so, the epithelia

intracellular [Na+] is reduced to ~50 meq.l-1 and thus creates a steep

electrochemical gradient for Na+ to move out of the chyme (~140 meq.l-1)

and into the epithelia cell (Wright & Loo, 2000). In effect, it replaces the

actively transported Na+.

There are two positive side effects of this process. The first being a

concurrent movement of Cl- and the second a constant movement (in effect,

absorption) of water from the small intestine to the interstitial space (Binder

et al., 2000). Since there is a large electrochemical attraction between Na+

and Cl-, the movement of Na+ in itself, draws Cl- to follow out of the epithelia

and into the interstitium (Liedtke, 1989). Chloride within the small intestine

then has two forces acting upon it to move into the epithelia, a concentration

gradient and the strong electrical attraction of Na+ moving into the cell. The

large increase in ions present in the interstitium creates an osmotic pressure

forcing water to move from within the epithelia cell into the interstitial space,

continuing to the capillary supply of the villi (Schultz & Dubinsky, 2001). The

reduction of water within the epithelia cell creates another gradient between

the lumen of the small intestine, which draws water from the chyme into the

cell. These processes combined create an almost constant flow of Na+, Cl-

and water between the lumen of the small intestine and the capillary blood

supply of the villi (Schultz, 1984).

The second dissociated constituent to be considered is citrate. Absorption

through the GI tract can be influenced by many different factors. The volume

of secreted gastric acid and bile can alter the breakdown of foodstuffs into its

constituents and thus delay the start of absorption processes through the

duodenal wall. These factors do not appear to severely limit the digestion of


7

citrate as Erskine and Hunt (1981) showed that approximately half the

quantity of SC when orally ingested reached the duodenum within three

minutes. Others have suggested that the caloric content of a meal is not an

important limiting factor (Hattner, 1991), however Moore et al. (1981) have

shown a positive relationship between the caloric content of a meal and its

emptying rate from the GI tract. The caloric value of SC is very low, almost

negligible, which would tend to rule out any effect this factor might have on

its absorption. The form of a meal, liquid or solid, appears to have a

significant impact on the emptying rate from the GI tract. Notivol et al.

(1984) demonstrated a liquid meal empties from the duodenum ~35 percent

faster than a solid meal.

Some investigators have attempted to look for relationships between

individual physical characteristics and GI function. However, their

investigations showed that no relationship exists between anthropometric

data and GI function in humans (Notivol et al., 1984; Moore et al., 1981).

Notivol et al. (1984) did find differences between male and female GI

function. In addition to a slower GI function, females also exhibit variance in

gastric emptying corresponding to the phase of their menstrual cycle.

MacDonald (1957) showed an increase in gastric emptying on day 14 that

gradually decreased as the cycle continued. Further, gastric pH increased

during days one to fourteen of the menstrual cycle, but reversed during days

fifteen to twenty-eight.

Vist and Maughan (1995) tested two concentrations of carbohydrate in liquid

form, high (188 g.l-1) and low (40 g.l-1), and found the high concentration

emptied slower from the duodenum. Further, they found that by increasing

liquid osmolality, gastric empting was diminished, although not to the same

extent. The work of Erskine and Hunt (1981), demonstrating that glucose

enters the duodenum at 5%.min-1 compared with 15%.min-1 for citrate,

supported these findings. Citrate entered the duodenum three-fold faster

than glucose. This may have important implications for the commonly

practised administration of citrate in bolus form (Schabort et al., 2000),

whereby the citrate is added to a glucose/carbohydrate solution. By ingesting

citrate in this manner, it may decrease the uptake and any corresponding

effect in the blood. Other factors that alter gastric emptying include circadian

rhythm (Goo et al., 1987) and smoking (Notivol et al., 1984). Although


8

ingesting foodstuffs at 20.00 hrs decreases gastric emptying half time by ~30

percent, circadian rhythm has no effect on the digestion of liquids. In

contrast, people who smoke have an increased gastric emptying rate

compared to those who do not smoke (Notivol et al., 1984).

Renal Regulation Systemic pH is tightly regulated due to large effects of relatively small

disturbances. A key organ involved in the regulation of systemic pH is the

kidney (Tannen, 1980). The kidney controls the amount of acid and base

excreted or reabsorbed to maintain systemic pH stability. Specifically, this is

performed by balancing the amount of HCO3- and H+ secreted into the tubule

for excretion (Hopfer & Liedtke, 1987). When more HCO3- is removed from

the blood and extracellular compartments and secreted via the glomerular

filtrate, more HCO3- will be present in the urine increasing its alkalinity while

reducing the alkalinity of systemic pH. Conversely, an increase in the removal

of H+ from the blood and extracellular compartments and followed by

secretion from the tubular epithelia will result in an increase in the acidity of

the urine while reducing the acidity of systemic pH (Maren, 1988). Thus by

manipulating these two pathways the kidney has the ability to tightly regulate

systemic pH. At normal extracellular pH, 7.4, HCO3- and H+ are secreted at

3.46 and 3.50 mmol.l-1, respectively. Therefore, at rest, slightly more acid is

excreted to accommodate the small amount (0.8 meq.kg.day-1) of acid

continually produced via metabolism, thereby reducing normal resting urine

pH to ~6.0 (Cogan et al., 1990; Oster et al., 1988; Kachadorian & Johnson,

1970).

The regulation of H+ via the kidneys is the result of two mechanisms, primary

active transport and secondary counter transport (Eiam-Ong & Sabatini,

1996). Primary active transport relies on the diffusion of CO2 from the

extracellular compartment into the tubular epithelia (Ilundain, 1992). The

CO2 combines with water to form carbonic acid (H2CO3), which quickly

dissociates into HCO3- and H+. The H+ is then actively moved across the

tubular membrane via an ATP driven process. Once the H+ is in the tubule it

is excreted in the urine. The remaining HCO3- within the epithelia is

reabsorbed into the extracellular space via an HCO3-/Cl- exchanger with the

Cl- passively lost to the urine (Pajor, 1999). The net result being for each H+


9

secreted to the urine, there is a loss of one Cl- and reabsorption of one HCO3-

(Boyarsky et al., 1988) This process is normally responsible for less than 5

percent of the regulation of H+ due to its location in the distal part of the

tubule and a large (up to 900-fold) concentration gradient (see Figure 1)

(Tanner, 1980).

Figure 1 Primary active transport of H+ into the renal tubule

The diffussion of CO2 into the epithelial cell results in the formation of H+, which is transported into the tubule via an ATP-driven process. Associated with this process is the passive movement of Cl- into the tubule. Figure adapted from Guyton (1991).

Secondary active transport again relies on the simple diffusion of CO2 from

the extracellular fluid into the tubular cell and follows the same steps to

dissociate into both HCO3- and H+ (Dunn & Walley, 1991). However, the H+

takes an alternative path and is moved into the tubular lumen via the positive

gradient of Na+ in counter transport (Krapf, 1989). In effect, for each H+

TUBULE EXTRACELLULAR

CI-

H+

CI-

ATP

ADP

CI-

HCO-3 HCO-

3 + H+

H2CO3

H2O +

CO2 CO2


10

removed there is a net reabsorption of Na+. The H+ then combines with

tubular HCO3- to reform H2CO3, which again dissociates into CO2 and water to

be excreted in the urine (Guyton, 1991). Although most segments of the

nephron participate in acid-base balance, regulation of HCO3- occurs mainly

(70-90 percent) via the glomerular filtrate in the proximal tubule of the

kidney (Eiam-Ong & Sabatini, 1996). When the HCO3-:CO2 increases, CO2

movement into the tubular cell is decreased, which in turn leads to a lower

H2CO3 concentration available to dissociate into HCO3- and H+. This reduces

the amount of H+ available to actively transport into the tubular lumen and be

excreted in the urine. Concurrently, in the glomerular filtrate, the excess

HCO3- is being transported into the proximal tubule at a much higher rate,

resulting in an increased concentration of HCO3- in the urine (Pajor, 1999).

The net result is alkaline urine due to both a low [H+] and high [HCO3-]. The

removal of HCO3- decreases the buffer concentration within the extracellular

space and shifts pH in the acid direction, cf. the Henderson Hasselbach

equation. This is further enhanced by the isohydric principle that states, ‘all

buffer systems will move in the acid direction in an attempt to correct

alkalosis’ (see Figure 2)(Guyton, 1991).

Ingestion of 2 mmol.kg-1 NaHCO3- increases plasma HCO3

- by 4 meq and

blood pH by 0.02 units (Cogan et al., 1990). In an attempt to restore this

disturbance to acid-base balance the two mechanisms previously mentioned

result in the following observations. As aforementioned, normal urinary pH is

approximately 6.0, however, under conditions of alkali ingestion the urinary

pH can climb to values as high as 8.0 while attempting to rid the body of

excess base (author’s unpublished observation). Oster et al. (1988) found

that renal net acid production was reduced by 70 percent in an attempt to

reverse the effects of SC ingestion. The process to rectify such imbalances

via the renal system is not fast. In fact, the system is only capable of

excreting about 500 mmol of excess acid or base each day (Dunn & Walley,

1991). Amounts greater than this will result in disturbances that the renal

system will be unable to cope with and major disturbances to extracellular

normality will result. If left untreated, the ensuring alkalosis will cause over

excitability of the neural system resulting in tetany and could be fatal in the

form of tetany of the respiratory muscles (Tanner, 1980).


11

Figure 2 Secondary active secretion of H+ into the renal tubule

Sodium reabsorption in exchange for proton ions secreted, proton and bicarbonate ions combine in the tubule to form carbon dioxide and water. Figure adapted from Guyton (1991).

To further aid the bicarbonate buffer system in combating acid-base

disturbances, the renal system can reabsorb ‘new’ bicarbonate (Maren, 1988).

The mechanism occurs largely in conjunction with the removal of excess H+.

When H+ is finally transported to the tubular lumen it will combine with HCO3-,

collected in the proximal tubule, to again form H2CO3, which then dissociates

into water and CO2. This CO2 rapidly diffuses back into the tubular epithelia,

and under the influence of the enzyme carbonic anhydrase combines with

water to form H2CO3 once again. Dissociation again occurs leaving the two

original components HCO3- and H+. Proton is then moved back into the lumen

via the processes mentioned above, while decreased K+ levels stimulate the

enzyme H,K-ATPase to transport HCO3- out of the cell and back into the

extracellular compartment (Eiam-Ong & Sabatini, 1996). In effect, this

Na+ + HCO-3

Tubule Extracellular Fluid

Na+

H+

H2CO3

CO2 + H2O

(Carbonic anhydrase)

Na+ Na+ - H+Active transport

K+ K+

HCO-3 + H+

Na+

+

HCO3 Counter transport

H2CO3

H2O +

CO2 CO2


12

process re-introduces newly generated HCO3- back into the circulation, thus

helping to maintain the HCO3- buffer system’s integrity.

H+ and K+ regulation at rest and during exercise

Proton - H+ It has been well documented in both animal and human skeletal muscle

investigations that muscle acidity increases with high intensity exercise

(Hermansen, 1979; Sahlin et al., 1978b) (Juel & Pilegaard, 1998; Bangsbo et

al., 1996; Thompson et al., 1992). Many experiments have demonstrated

that muscle acidity has major regulatory effects on numerous mechanisms,

including key energetic pathways (Spriet et al., 1987; Sutton et al., 1981),

membrane transport systems (Juel, 1998b; Juel et al., 1994; Davies, 1990;

Inesi & Hill, 1983), excitation-contraction coupling (Donaldson & Hermansen,

1978; Fabatio & Fabatio, 1978), and blood flow (Aalkjaer & Peng, 1997). In

contrast, there exists some controversy as to whether or not [H+] per se is

responsible for these effects (Spriet, 1991a; Ren & Hultman, 1989). As it

appears that the results are not yet conclusive, it is therefore important to

continue to develop new techniques that will assist in providing a greater

understanding of the intricate regulation of H+.

Approximately 45-75 percent of the body is comprised of fluid. The fluid

within the body can be divided into intracellular and extracellular

compartments. Approximately 55 percent of the total body fluid is contained

within the intracellular compartment. The extracellular space can be

subdivided to consist of blood and interstitial fluid. Each of these

compartments contains approximately 4 and 16 percent of the total body

fluid, respectively. Therefore, the regulation of intracellular [H+] is firstly

influenced by the cytosol and interstitial fluid that are separated by the

sarcolemma. Although, muscle, interstitium and plasma are osmotically and

electrically neutral at rest, selective permeability between these

compartments maintains a unique ionic composition (Table 1) (Kowalchuk &

Scheuermann, 1995). The exact composition of these compartments will be

influenced by the hydration status of the individual as evidenced by disparity

in the literature (Kowalchuk & Scheuermann, 1995).


13

This table is not available on line. Please consult the hardcopy thesis available

at the QUT Library.

Table 1 Ion concentration within plasma, interstitium and muscle

This table shows the ionic concentrations of the major anions and cations within each respective compartment (Aickin & Thomas, 1977)

Intracellular pH at rest is kept slightly alkaline to offset small amounts of acid

produced via basal cellular metabolism (Bangsbo et al., 1993a; Costill et al.,

1983). In addition, H+ continually passively fluxes across the sarcolemma

due to electrochemical forces (Juel, 1998a). Thus to prevent the

accumulation of intracellular H+ at rest, it is necessary for skeletal muscle to

possess mechanisms through which H+ can be transported out of the cell.

Transporters that envelop this ability include the Na+/H+ exchanger (Juel,

1998b) and HCO3- exchangers (Putnam, 1990).

The contribution of these exchangers at rest is not well defined. Several

investigators have attempted to describe the contribution of the Na+/H+

transporter by inhibiting its function with amiloride (Juel, 1995; Kemp et al.,

1994; Grossie et al., 1988). However, the results of these investigations

showed only modest intracellular acidification without the Na+/H+ pathway.

Evidence from animal studies would suggest that up to 20 percent of pH

regulation in mouse slow twitch fibres is managed by a bicarbonate transport

system (Aickin & Thomas, 1977), while more recent investigations using

vesicles from homogenised rat skeletal muscle suggest a capacity similar to

that of the Na+/H+ transporter (Juel, 1995).

During exercise, metabolism is increased. It has been well documented that

this is associated with an increase in intracellular [H+] (Sullivan et al., 1994;


14

Spriet et al., 1989; Greenhaff et al., 1988; Costill et al., 1983). To maintain

optimal cell functioning and relocate the additional H+, there is a high demand

for the transport systems to increase their rate. To assist with the increased

demand additional transporters are involved during exercise. Furthermore,

H+ can passively diffuse across the sarcolemma in the form of undissociated

lactic acid (Juel, 1996). In both animals (Juel & Pilegaard, 1998; Lindinger &

Heigenhauser, 1991; Seabury et al., 1977) and humans (Bangsbo et al.,

1997) the function and capacity of the La-/H+ transporter has been

investigated. The study by Juel (1995) demonstrated the functional

capacities of the three H+ transporters in rat hind limb muscle using giant

sarcolemmal vesicles. His data showed that the La-/H+ system capacity was

approximately eight- and five-fold larger than the Na+/H+ and HCO3- systems,

respectively. This is further supported by the findings of Westerblad and Allen

(1992) that demonstrated no effect on force production and intracellular pH

when the Na+/H+ transport system is blocked with amiloride in mouse skeletal

muscle. Therefore, the La-/H+ co-transporter is of major importance in

exporting H+ out of the cell during exercise. However, there are additional

mechanisms at play including; passive movement, Na+/H+ exchange, HCO3-

systems and diffusion of undissociated lactic acid. Together, these systems

assist in the maintenance of an optimum intracellular pH environment.

At the cessation of intense exercise, intracellular [La-] is high and pH can

decrease to values as low as ~6.56 (range 6.4 – 6.7) (Bangsbo et al., 1996;

Spriet et al., 1987; Costill et al., 1983; Hermansen, 1981; Sahlin et al.,

1978b). While the intracellular [La-] remains high, there exists a large

positive gradient from muscle to interstitium and plasma. The La-/H+ co-

transport system is largely regulated by increases in intracellular [La-] and

relatively insensitive to the increased [H+] (Juel, 1996). However, later in

recovery when the La- gradient is low and the [H+] is approximating resting

values, the Na+/H+ transport system’s sensitivity to these [H+] re-activates it

to finely tune the intracellular [H+] to resting levels (Juel, 1996). Further, it

has been suggested that the HCO3- systems may also contribute to this fine-

tuning process (Putnam & Roos, 1986a).

It should be noted that the regulation of H+ within the interstitium is

remarkably unexplored and as such poorly understood. The importance of

this sub-compartment of the extracellular space has been identified by others


15

that have suggested that interstitial [H+] can influence H+ efflux from the

muscle cell (Roth & Brooks, 1990). Recently, interstitial [H+] has been

measured in humans using microdialysis and pH sensitive electrodes placed in

the dialysate (outflow) (Maclean et al., 2000). The data reported in this study

does not immediately concur with previous investigations of muscle and blood

pH. As mentioned above, there is sound support for the regulation of

intracellular H+ via exchangers and passive diffusion. Further, the same

investigators and others (Lindinger et al., 1992) report increases in blood [H+]

associated with intense exercise. Therefore, it seems reasonable to assume

that the H+ removed from the muscle cell will inevitably end up in the

interstitium and then further to the blood. If this is the case, the interstitium

[H+] would increase (decrease pH). However, this is in direct contrast to the

findings reported by Maclean et al. (2000). They report a mean±SEM resting

interstitial pH of 7.162±0.023 and almost immediately after the initiation of

exercise, a decrease in interstitial [H+]. This exercise-induced interstitial

alkalosis is observed for the full duration of exercise and very sharply returns

to pre-exercise levels at the cessation of exercise. It seems peculiar that

muscle H+ efflux to the blood via the interstitial space during intense exercise

would result in a decline in interstitial [H+]. Further investigations are

required to validate the microdialysis method and clarify H+ regulation within

this extracellular compartment.

Potassium - K+ Arterial K+ is an important factor in many mechanisms of control, including

ventilation (McCoy & Hargreaves, 1992; Busse et al., 1992), circulation

(Fallentin et al., 1992), cardiac arrhythmias (Kjeldsen, 1991) and has been

suggested as a regulator of muscle function (Juel, 1986). It is this last role

that is most interesting and pertinent to this thesis.

The propagation of an action potential is required for the activation of both

neural and skeletal tissue (Fitts & Balog, 1996). In skeletal muscle, the action

potential is initiated by a negative electrical imbalance created across the

sarcolemma. Constantly shifting three Na+ ions out of and two K+ ions into

the muscle cell creates high and low intracellular concentrations of K+ and

Na+, respectively (Fitts & Balog, 1996). Concurrently, a negative electrical

imbalance or membrane potential between intra and extracellular


16

compartments is generated. This active process is mediated by a pump

mechanism driven by ATP hydrolysis and is commonly referred to as the Na-K

pump or Na-K ATPase (Brody & Akera, 1977). To increase the complexity of

the process, specialised K+ channels within the sarcolemma constantly allow

K+ to leak out of the cell, thereby decreasing intracellular [K+] and thus the

membrane potential (Sakobe et al., 1991). In order to maintain the

membrane potential, the Na-K pump must replenish the intracellular [K+] at a

rate equal to the K+ lost through the channels.

Two factors that aid in this process are the total number and location of the

Na-K pumps within a cell. The pump numbers are far in excess of that

required to maintain K+ concentrations. Only 2-6 percent of the pumps need

to be activated to maintain [K+] within the cell (Clausen et al., 1987; Clausen,

1986). The location of the pumps are restricted to certain parts of the

interstitium, decreasing K+ travelling distance, thereby reducing repeated

depolarisation-repolarisation time (Wasserman et al., 1997). The capability of

the Na-K pump has recently been theoretically described using a

mathematical approach (Lindinger et al., 1995). By removing Na-K pump

activity from the K+ regulatory process and using K+ kinetic data from

previous experiments, they calculated the subsequent rise in arterial plasma

[K+] as a result of continual loss through K+ channels. The calculations

demonstrate that cycling at 400 percent VO2peak for 30 s without the use of

the Na-K pump, arterial plasma [K+] would increase from 6.3 (recorded) to 27

mmol.l-1. Such an increase in arterial plasma [K+] would result in

disturbances to the cells’ ability to maintain the membrane potential and

generate action potentials, which would compromise muscle cell contraction

and force production. Further experimental work has demonstrated that

hyperkalemia is due to a failure of the Na-K pump to keep pace with K+ loss

from the muscle cell during contraction (Wasserman et al., 1997; Lindinger et

al., 1995).

Prior to the onset of exercise, the Na-K pump ensures K+ is the main ion

intracellularly and Na+ extracellularly. However, at the onset of exercise, the

intracellular environment is perturbed. Specifically, other non-diffusible anion

concentration is altered and due to the physicochemical laws of ions in

solution, K+ is released from the cell via K+ channels. The increased K+ efflux

via these K+ channels decreases intracellular [K+] thus signalling the pumps to


17

increase activity in an attempt to restore intracellular [K+] (Wasserman et al.,

1997).

Continual muscle activity results in K+ loss from the cell with the majority of

efflux occurring during the repolarisation phase of the muscle action potential

(Marcos & Ribas, 1995). Studies using mice (Juel, 1986) have shown

intracellular K+ loss during stimulation only accounts for half of the associated

depolarisation, suggesting K+ leaked out through the channels and into the

interstitium. Further interstitial measurements showed a concurrent two-fold

increase in [K+]. It has been demonstrated (Clausen, 1986) that intracellular

K+ and interstitial K+ concentrations are regulated by the Na-K pump and K+

channels, and that the amount of efflux into the interstitium is proportional to

the frequency of muscle action potentials (Marcos & Ribas, 1995).

Blood flow into the muscle (arterial) and blood draining the muscle bed

(venous) are both influenced by the release of K+ from the muscle via the

interstitium. Venous blood is the first compartment to be affected due to the

collection of metabolites from the active muscle. Release from muscle is very

rapid and increases femoral vein [K+] during the first 3 minutes of both high

and low intensity exercise (Wasserman et al., 1997). It appears that trained

status has no bearing on the peak venous values obtained during intense

exercise. During cycling to exhaustion in two groups, cyclists and controls

exercising at the same relative intensity but exhibiting markedly different

absolute values (400 and 250 W respectively) elicited indifferent venous K+

changes of 35 and 31 percent, respectively (Marcos & Ribas, 1995). In

contrast, arterial K+ efflux of cyclists was greater compared with control

subjects. A possible explanation for this was an up regulation of the Na-K

pump numbers with training, thereby increasing the amount of K+ reabsorbed

into the muscle cell and reducing the concentration with the plasma (Marcos &

Ribas, 1995). Resting arterial blood [K+] is approximately 4.0 mmol.l-1,

however, during intense exercise this can increase as high as 6.3 mmol.l-1,

which can be influenced by fluid shifts in and out of the plasma. Even so, in

some cases the fluid changes that can occur are too small (~10%) to account

for these large changes (~50%) in plasma [K+] (Wasserman et al., 1997;

Marcos & Ribas, 1995).


18

At the cessation of exercise, the stimulated Na-K pump continues to return K+

into the intracellular space while removing excess Na+ from within the

contracting cell. The Na-K pump is highly effective at performing this as

demonstrated by Juel (1986) in mice and (Lindinger et al., 1995) in humans

where, within 2-3 minutes post exercise intracellular [K+] was only slightly

lower than control, and after 30 s supra-VO2peak exercise approximately 50

percent of lost K+ was returned bringing it to within 90 percent of pre-exercise

values, respectively. The rate of return influx could be modulated by

extracellular [K+] or K+ activated diffusion, intracellular Na+ or circulating

hormones, such as epinephrine (Marcos & Ribas, 1995; Juel et al., 1990).

Nonetheless, it appears K+ clearance post-exercise is more dependent on Na-

K pump activity rather than an increase in blood flow (Marcos & Ribas, 1995).

In evidence of this was a faster clearance of venous K+ in trained cyclists

compared with control subjects (Marcos & Ribas, 1995), since trained muscle

has been shown to have an up regulated number of Na-K pumps (Green et

al., 1999; Green et al., 1993).

Normal function and optimal force production of a muscle cell requires a

membrane electrical potential of approximately –90 mV (Guyton, 1991). As

explained above, the Na-K pump creates this electrical potential by actively

restoring and removing K+ and Na+ within and from the cell. If the Na-K

pump function is compromised, cell contractile function will be impaired

inevitably resulting in a decline in force production. Theoretically, assuming

all other ions remain constant, a decrease in intracellular [K+] from 146 to

117 mmol.l-1 and an increase in interstitial [K+] from 4.5 to 7.0 mmol.l-1

would result in a loss of membrane potential from –92 mV to –75 mV

(Lindinger et al., 1995). Not only does this demonstrate the contribution both

intracellular and interstitial K+ play, 70 and 30 percent respectively, in

maintaining the electrical potential, but also the magnitude of change that is

required to elicit such an effect on the membrane potential. Studies in mice

have demonstrated this relationship whereby a force reduction of 29 and 10

percent in soleus and extensor digitorum longus (EDL) muscle, respectively,

was associated with a measured intracellular K+ loss of 14 and 22 percent,

respectively (Juel, 1986).

There are two ways of determining Na-K pump activity, by measuring arterial-

venous K+ difference at exhaustion and by measuring increases in intracellular


19

[K+] during the first minutes of exercise (Lindinger et al., 1995). Later,

Fraser and McKenna (1998) modified the K+ stimulated method yielding

significant improvements in the reliability of measuring Na-K pump activity.

In vitro work has provided evidence that ouabain is a specific blocker of the

Na-K pump. By simply adding ouabain to a bath, in vitro, intracellular K+ loss

was reduced from 32 to 25 mmol.l-1 (Juel, 1986). Using these techniques to

examine the functional significance of the Na-K pump has demonstrated that,

at the onset of exercise, the Na-K pump is operating but not optimally

(Lindinger et al., 1995; Juel, 1986). Although it has been demonstrated that

only 2-6 percent of the Na-K pump capacity is required to maintain

intracellular [K+] even during intense exercise, a down regulation is possible

but probably unlikely. Even so, Juel et al. (1990) have provided evidence to

support this hypothesis, in that K+ efflux was reduced late in a 3-minute

intense exercise bout. They proposed two explanations for this finding: that

the reduced K+ efflux was mediated by the progressively lowering intracellular

pH, which (based on work by Blatz, 1980) alters the permeability of K+

channels; and/or that the increased Na-K pump efficiency late in exercise was

mediated by an increased plasma [K+], intracellular [Na+] or catecholamines.

Unfortunately, this increased pump efficiency late in exercise is insufficient to

restore intracellular [K+] during exercise to the level required to maintain the

optimal membrane potential.

During high intensity exercise, large quantities of La- are produced, massive

effluxes of K+ and small influxes of Na+, Cl- and water occur (Hebestreit et al.,

1996). The large efflux of K+ may be to control intracellular osmolality and

cell volume (Lauf, 1987) or be important in maintaining intracellular

concentrations of ATP (Castle & Haylett, 1987; Spruce et al., 1985). The

increase in extracellular:intracellular concentration of K+ may inhibit

excitation-contraction coupling and reduce the muscles’ ability to produce

tension (Heigenhauser et al., 1990). In addition, this will also reduce the

velocity of the propagation of the action potential to the transverse tubule

(Bigland-Ritchie et al., 1981). Together, these two effects will result in a

slower release of Ca2+ from the sarcoplasmic reticulum (Heigenhauser et al.,

1990).

Under alkalotic conditions, efflux of K+ is smaller while La- and non-volatile

acid (NVA) efflux is greater (Lindinger et al., 1990). The precise mechanism


20

for this reduced K+ loss is not known (Lindinger et al., 1990). A possible

mechanism may be due to an increase in the Na-K pump activity and/or a

decrease in sarcolemmal permeability to K+ per action potential (Juel et al.,

1990; Lindinger et al., 1990). The loss of K+ during muscular contraction is

very closely linked to the accumulation of intracellular La- (Lindinger et al.,

1990).

Potassium shifts may play a role as a limiting factor for muscle function (Juel

et al., 1990). As K+ is reaccumulating in the muscle, La- is continually

released from the muscle during the recovery period (Juel, 1997). The small

shrinkage of erythrocytes found during high intensity exercise is a function of

both the increased plasma [La-], [K+] and water movement into the active

muscles (Sjogaard et al., 1985). Haemoglobin (Hb) concentration is increased

during high intensity exercise and then returns to normal values during

recovery. This is closely related to the changes in [K+] and the change in

K+/Hb ratio is due entirely to volume changes (Juel et al., 1990). This

suggests that red blood cells (RBC) shrink during exercise and return to

normal size during recovery, thereby allowing the potassium content of the

RBC to remain unaltered during the exercise and recovery period. More

support for this is provided by the K+/Hb ratio remaining relatively constant

thus suggesting no net movement of K+ across the cell membrane.

During exercise, 70 percent of gross K+ efflux is immediately reaccumulated,

30 percent is lost to the blood, and one-third of the 30 percent lost to the

blood is accumulated in the other tissues. The presence of a K+ a-v difference

suggests K+ must have moved out of other tissue and into the blood. Since

approximately 10 percent of K+ accumulates in the interstitial space during

intense exercise, it should be considered a possible agent of fatigue (Juel et

al., 1990).

Both respiratory and metabolic acidoses increase muscle La- and K+ efflux,

and reduce the intracellular muscle water volume in the hind limb of the rat

(Lindinger et al., 1990). By increasing K+ efflux from exercising muscle, the

ionic environment created augments the effects on extracellular [H+] and the

ability of the muscle to maintain homeostasis (Heigenhauser et al., 1990).


21

Alkalosis at rest and during exercise It has been well documented that ingestion of an alkali at rest, such as

sodium citrate or sodium bicarbonate, decreases the blood [H+] as

represented by an increased blood pH and further alkalises the blood

(Linossier et al., 1997; Hausswirth et al., 1995; Lambert et al., 1993; Iwaoka

et al., 1989; Brien & McKenzie, 1989). The presence of a blood alkalosis prior

to exercise has been associated with improvements in human performance

(ergogenesis) across a variety of modalilities (Shave et al., 2001; Potteiger et

al., 1996a; McNaughton & Cedaro, 1991; Gao et al., 1988). The precise

mechanism/s of alkalosis on exercise performance, however, has not been

conclusively established. As such, there is evidence to suggest both positive

(McNaughton et al., 1999; McNaughton et al., 1991), neutral (Pierce et al.,

1992; Gaitanos et al., 1991) and negative (Goldfinch et al., 1988; Wilkes et

al., 1983) effects of pre-exercise induced alkalosis on performance. The

negative effects refer to side-effects of alkali ingestion that inhibit a subject to

perform the exercise task. Before detailing the specifics of these

investigations, it is important to briefly clarify the relationship between [H+]

and pH.

The Brønsted-Lowry theory describes an acid as any molecule or ion that can

act as a proton donor. Conversely, any molecule or ion that can act as a

proton acceptor is identified as a base. The expression pH can be defined as

the negative logarithm of the hydrogen ion concentration (Equation 1) and

was invented to provide a more convenient method of expressing

concentration better given in scientific notation.

[ ] [ ]++ =−=

HHpH 1loglog

Equation 1 Calculation of pH

For dilute solutions this value will usually fall between 1 and 14. It is

important to understand that the pH scale is logarithmic and not linear. For

example, a decrease in muscle pH as observed during intense exercise (Allsop

et al., 1990) from 7.1 to 6.5 equates to approximately a five-fold increase in

[H+]. Normal resting plasma, skeletal interstitial and intracellular pH are

approximately 7.4 (Cogan et al., 1990), 7.2 (Maclean et al., 2000) and 7.1


22

(Greenhaff et al., 1987), respectively. At rest all of the body’s fluid

compartments are therefore slightly alkalised.

Since early in the 20th century, it has been hypothesised that a state of

alkalosis would positively influence physical work capacity (Denning et al.,

1931). A number of studies to investigate the effects of alkalosis have

followed (Cox & Jenkins, 1994; Costill et al., 1984; Jones et al., 1977a; Jones

et al., 1977b). Approximately 50 percent show a positive improvement in

performance with alkali loading. The ergogenic effect of alkalosis appears to

be independent of mode of exercise as improvements have been

demonstrated in cycling (Potteiger et al., 1996a), running (Shave et al.,

2001), swimming (Gao et al., 1988) and rowing (McNaughton & Cedaro,

1991). From these studies the most common mode (~60%) investigated was

cycling.

Although there has been no formal investigation into the effect of trained-

status on alkalosis ergogenesis, there is room for limited speculation between

investigations. It would appear there is no effect of trained status as

supported by the observations of performance improvements in sprint trained

(Pierce et al., 1992; Goldfinch et al., 1988), endurance trained (Bird et al.,

1995), active healthy (Iwaoka et al., 1989) and sedentary (Hausswirth et al.,

1995) populations. These statements should be interpreted cautiously, as

there may be other factors mediating the ergogenic effect of alkalosis

independent of trained status.

It has been suggested that to observe an ergogenic effect of alkalosis, the

exercise intensity needs to be highly anaerobic in nature (Heigenhauser &

Jones, 1991). However, recent investigations by McNaughton et al. (1999)

and Potteiger et al. (1996a) have demonstrated improvements in aerobic

cycling exercise performance lasting up to one hour in duration. In contrast,

other investigations have found no such improvements over long durations

(Potteiger et al., 1996b; Kowalchuk et al., 1984). It has been adequately

demonstrated that below 120 seconds exercise duration there is no

improvement in performance with either sodium bicarbonate (McNaughton,

1992b) or sodium citrate (McNaughton & Cedaro, 1992). This has been

further supported by the work of Parry-Billings and MacLaren (1986)

demonstrating that alkalosis had no performance effect on 30 second Wingate


23

tests. Therefore, there is considerable evidence to suggest that duration of

exercise should not be less than 120 seconds, an upper limit, however, is still

under some conjecture.

There exists a positive dose-response relationship for both sodium citrate

(McNaughton, 1990) and sodium bicarbonate (McNaughton, 1992a). Both

these investigations identified a minimum dose (300 mg.kg-1) required to

observe an ergogenic effect of alkalosis on cycling performance. Work by

Horswill et al. (1988) further supported this finding by demonstrating no

effect of 100 or 200 mg.kg-1 sodium bicarbonate on exercise performance.

Increasing the dose of sodium citrate to 400 and 500 mg.kg-1 resulted in a

corresponding increase in performance (McNaughton, 1990). The same

cannot be said for sodium bicarbonate. Further dose increases to 400 and

500 mg.kg-1 led to increases in gastro-intestinal complications in the subjects,

thereby preventing the task being performed (McNaughton, 1992a).

As identified above, there has been much research identifying many factors

that are associated with the ergogenic potential of orally induced alkalosis.

However, investigations into the mechanisms underpinning this phenomenon

have been less prolific. Before discussing these studies in detail, it will be

beneficial to briefly introduce a theoretical concept introduced by Stewart

(1983) in the early 1980’s. The Stewart approach uses physicochemical

principles to describe the behaviour of H+ in physiological aqueous solutions.

These relate to three fundamental laws that govern [H+] in solutions such as

plasma and muscle: conservation of mass, maintenance of electrical

neutrality (Equation 2) and the equilibrium state of weak electrolytes and

water. Specifically, [H+] becomes a dependent variable determined by

independent variables: strong and weak electrolytes and pCO2.. Therefore,

the regulation of intracellular [H+] is a result of the exchange of strong ions

(Na+, K+, Cl-, SO42-, Mg2+, Ca2+, La-, PCr, NH4, ketones) between muscle and

extracellular fluid and intracellular pCO2 regulation (down its concentration

gradient). Strong ions exert their influence on [H+] through the difference

between the sum of strong cations and the sum of strong anions, termed the

strong ion difference (SID) (Equation 3).


24

[ ] [ ] [ ] [ ] [ ] [ ] 0233 =−−−−+ −−−−+ OHCOAHCOHSID

Equation 2 Calculation for electrical neutrality

[ ] [ ] [ ]∑ ∑−= anionsstrongcationsstrongSID

Equation 3 Calculation of strong ion difference (SID)

Two proposed sequential mechanisms through which alkalosis improves

muscle function are 1) an enhanced CO2-HCO3

- blood system to buffer H+

released from muscle during exercise and 2) an attenuated rate and decrease

in intracellular [H+] (Matson & Tran, 1993; Heigenhauser & Jones, 1991).

This theory does not discriminate between the different types of alkalis

ingested, such as sodium citrate, sodium bicarbonate or potassium

bicarbonate. Therefore, it can be deduced that the way in which alkalosis is

induced is not of primary concern, the end result of an increase in [HCO3-]

and [H+], however, is. Although this seems logical, recently, it was

demonstrated that sodium and potassium bicarbonate bring about alkalosis in

distinctly unique ways (Lindinger et al., 1999). An increase in [SID] was the

key determinant of alkalosis using both alkalis, however, the ions that

contributed to the increase in [SID] were different between the alkalis.

Specifically, an increase in plasma [Na+] and an increase in plasma [K+] both

combined with decreases in plasma [Cl-] to invoke plasma alkalosis.

Still, there seems to be some disagreement regarding the metabolism of

sodium citrate and the mechanisms involved that ensue alkalosis (Linossier et

al., 1997). It has been suggested that tri-sodium citrate dissociates into its

constituent ions, sodium and citrate, and is rapidly absorbed into the blood

(Kowalchuk et al., 1989). There are conflicting theories as to how citrate

manipulates plasma [HCO3-] and [H+]. Halperin (1982) suggested plasma

[HCO3-] is increased via the hepatic oxidation of citrate, while in contrast,

Kowalchuk et al. (1989) suggested that as the citrate anion is removed from

the plasma the SID is increased (via the remaining Na+) requiring a decrease

in [H+] and increase in [HCO3-] to maintain electrical neutrality. It is, of

course, possible that a combination of both of these theories contributes to

the increased [HCO3-] associated with alkalosis.


25

In summary, there is good evidence to suggest that ingestion of sodium

bicarbonate, potassium bicarbonate and sodium citrate increase both plasma

[HCO3-] and [H+] in humans. Further, there is considerable evidence to

support the contention that ingestion of 300 mg.kg-1 of an alkali can improve

human performance greater than 120 s and up to one hour in duration.

However, the exact physiological mechanisms mediating the ergogenic effect

of alkalosis, especially when ingesting sodium citrate, remain elusive. Further

research into these mechanisms is thus required.

Chapter 3 – Interstitial pH

27

CH A P T E R 3 ME A S U R E M E N T O F D I A L Y S A T E PH,

R E P R E S E N T A T I V E O F I N T E R S T I T I A L PH, A T R E S T

A N D D U R I N G D Y N A M I C L E G E X E R C I S E

Preface This chapter is centred round the paper authored by Darrin Street1, Jens

Bangsbo2 & Carsten Juel2, “Interstitial pH in human skeletal muscle during

and after dynamic graded exercise”, published in Journal of Physiology

(2001), 537.3, pp. 993-998. 1Department of Human Movement Studies, Queensland

University of Technology, 2August Krogh Institute, University of Copenhagen.

Statement of Joint Authorship This chapter is in part a result of a cooperative effort between myself and the

stated authors. The following outlines the relative contribution/s of each

author:

Darrin Street (Candidate) in vitro validation of method Part I (Duration:11 months) Experimental design Parts I, II and II Recruitment of subjects Parts II and III Equipment and subject preparation Parts II and III Data collection Parts I, II and III Data management Parts I, II and III Data analysis Parts I, II and III (90%) Data presentation Parts I, II and III (90%) Data interpretation Parts I, II and III (75%) Chapter preparation (75%)

Carsten Juel (Associate Professor) Experimental design Part III Supervised in vitro validation of method Assisted in experiment preparation Parts II and III Assisted in data collection Parts II and III Data analysis (10%) Data presentation (10%) Data interpretation (25%) Chapter preparation (20%)

Jens Bangsbo (Associate Professor) Subject preparation Part II and III Assisted in data collection Part III Chapter preparation (5%)


28

Introduction This chapter consists of three parts; Part I details the in vitro experiments

undertaken to test the validity and reliability of measuring pH using

microdialysis and a pH-sensitive dye, Part II details the in vivo experiment

performed to ascertain the effect of perfusate [HCO3-] on interstitial pH

measurement at rest and during exercise, Part III details the culmination of

these previous methodological studies in an in vivo experiment undertaken to

determine interstitial pH in humans at rest, during exercise and recovery

using microdialysis.

During muscle activity, accumulation of lactic acid and CO2 reduces cellular pH

and subsequently interstitial pH due to acid efflux from the muscle cells. It

has been proposed that the changes in interstitial pH during muscle activity

may be an important signal in the regulation of blood flow (Street et al.,

2001). In accordance with this idea, acidosis is probably mediated by

reduced extracellular pH and a subsequent lowering of the intracellular

calcium concentration (Aalkjaer & Peng, 1997). Furthermore, it has been

suggested that changes in pH may modulate vascular K+ channels (Davies,

1990) and thereby influence blood flow (Quayle et al., 1997), and that

acidosis may activate sensory nerve endings located in the muscle

interstitium (Victor et al., 1988). In order to evaluate such modulatory

effects, it is important to describe and quantify the exercise-induced

interstitial pH changes in human muscle.

The exercise-induced changes in muscle pH have, so far, mainly been

described from changes in venous blood pH (Bangsbo et al., 1993a; Juel et

al., 1990; Sjogaard et al., 1985), but the relationship between interstitial pH

and blood pH cannot be easily predicted because venous blood is mixed with

blood draining from active tissue (Radegran & Saltin, 1998). Furthermore,

studies using microdialysis in active muscle have demonstrated that during

muscle activity interstitial concentrations of lactate are higher than venous

lactate concentrations (Maclean et al., 1999), suggesting that the

equilibration across the capillary wall is restricted. It may therefore also be

expected that interstitial to venous pH gradients exist during exercise. In

addition, there is no protein present in the interstitium thereby reducing the

buffer capacity in comparison to blood (Aukland & Reed, 1993). For these


29

reasons, it can be hypothesised that the exercise-induced changes in

interstitial and venous blood pH are different and that the changes in venous

blood pH underestimate the local interstitial pH changes. Although electrodes

have previously been used in an attempt to measure pH directly (Allsop et al.,

1990), this was only performed at rest after exercise and showed an

artificially slow recovery from muscle pH after muscle activity. Thus, it has

not previously been possible to record interstitial pH during exercise in

humans.

Consequently, the objective of the present studies was to develop a method

to determine the changes in interstitial pH in human skeletal muscle during

and after exercise at different intensities. For that purpose, a microdialysis

technique was combined with the use of pH-sensitive fluorescent dye BCECF,

making it possible to perform continuous measurements of interstitial pH both

during and after muscle activity. Further, the first experiment was conducted

in an attempt to clarify some methodological considerations, specifically the

effect of a HCO3- perfusate on [H+] at rest and during exercise in humans.

PART I – IN VITRO VALIDATION OF MEASURING

INTERSTITIAL PH USING MICRODIALYSIS AND BCECF

In vitro microdialysis components and system Figure 3 shows a schematic of a microdialysis probe (CMA-60, CMA

Microdialysis AB, Sweden) that was used for all in vitro experiments.

The major difference between the probe set-up for measuring [H+] and other

metabolites was the removal of the polyurethane outlet tubing and its

replacement with 350 mm of 0.16 mm internal diameter (∅) stainless steel

tubing. The use of stainless steel on the outlet prevented the equilibration of

CO2 with the atmosphere and subsequent loss of CO2 from the dialysate,

which has the potential to influence dialysate [H+]. The total internal volume

of the modified CMA60 probe (H+ probe) was 35.7 µl, although only 3 µl was

exposed to the polyamide membrane where diffusion can occur. It is

important to note that the total volume from the microdialysis membrane to


30

the outlet was 15 µl and, at a perfusion rate of 5 µl.min-1, will result in a 3 min

delay.

The H+ probe was then placed within the following set-up for the

measurement of [H+] in vitro (Figure 4). The H+ probe itself was attached to

a 100 ml glass beaker using slick tape. The probe was orientated

perpendicularly within the centre of the beaker. In addition, a pH meter

(Radiometer ABL505, Copenhagen) probe was placed within the beaker for

reference monitoring of the solution. On the inlet side of the H+ probe, a 1 ml

syringe was attached and placed within a CMA 100 microdialysis pump set at

a flow rate of 5 µl.min-1.

Figure 3 Modified CMA60 microdialysis probe

A microdialysis probe (CMA60, Sweden) is modified for the measurement of [H+] in vitro by the removal of the polyurethane outlet tubing and replaced by 350 mm of stainless steel tubing to prevent CO2 loss.

The stainless steel outlet of the H+ probe was secured and sealed within a

micro flow-through cuvette (Sterna 73-1-FQ1, Great Britain) so that the end

of the tube would be as close as physically possible to the measuring window

Polyurethane Tubing (400 mm)

Stainless Steel Tubing (350 mm)

Polyamide Membrane

Polyurethane ShaftTubing


31

of the cuvette, thereby reducing lag time to analysis. The outlet of the

cuvette was placed into a waste beaker. The cuvette was placed in a

fluorescence spectrophotometer (Hitachi F2000, Japan), which completed the

continuous flow through system for the measurement of H+ in solution.

With the emission wavelength constant at 530 nm, the fluorescence

spectrophotometer continuously switched between the excitation wavelengths

of 440 and 500 nm with a bandpass of 10 nm. The excitation intensity at 440

nm was insensitive to pH, while the excitation intensity at 500 nm was

sensitive to pH. The two excitation intensities created an excitation intensity

ratio of 500/440. The intensity ratio was proportional to pH and insensitive to

any water movements from or into the probe. The temperature in the

fluorescence spectrophotometer was kept constant by circulating

thermostatically controlled water.

Figure 4 The complete fluorometric microdialysis system for the measurement of H+ in vitro

The modified microdialysis probe is immersed in a 100 ml beaker containing a solution of Na+ (154 mM), Cl- (154 mM) and HCO3

- (25 mM). The probe is perfused via micro-pump (CMA100, Sweden) set at 5 µl.min-1 and connected to fluorescent spectrophotometer. A laboratory pH meter (Radiometer ABL505, Copenhagen) is placed within the beaker solution for reference measurement.

Stainless Steel

CMA60 Microdialysis Probe

pH Meter

Fluorescence Spectrophotometer

7.35 Cl-

H+

Na+

HCO3-

Microdialysis Syringe Pump


32

Day to day stability of 2,7-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein (BCECF) dye Since many microdialysis investigations occur across several hours, it was

important to assess the stability of the dye across long durations. On two

occasions separated by 24 hrs, [H+] was measured in a beaker solution (Na+

154 mmol.l-1, Cl- 154 mmol.l-1, HEPES 5 mmol.l-1, NaHCO3- 20 mmol.l-1) by

perfusing a H+ probe with sterile saline and 0.1 mg.ml.l-1 of the pH sensitive

dye, BCECF. The pH of the beaker solution was manipulated from 6.8 to 7.2

and back to the initial setting by the addition of small amounts of 5 M HCl or 6

N KOH. Since the molecular cut-off of the polyamide membrane is 20000

daltons (da), the BCECF was coupled to dextran (molecular mass 70000 da),

thereby preventing any loss of the dye from the perfusate. The perfusate was

placed into a 1 ml syringe, mounted in a microdialysis pump and connected to

the inlet of the H+ probe. Figure 5 shows the results of this in vitro

experiment. Two trials of each experiment were performed each day for a

duration of 10 min with pH initiating at ~6.82, increasing it to ~7.23 after 3

min and decreasing it back to ~6.78 for the remainder of sampling.

Fluorescence was recorded at a frequency of 10 Hz for the full duration of the

test.

There was no difference between the H+ fluorescent signals obtained when

measuring pH on the 2 separate days. Closer inspection of the data revealed

minor fluorescent differences between the days but this was due to different

pH values (~0.04) at the time of sampling, which further demonstrated the

sensitivity of the system. The upward drift in signal on both days was

explained by the open nature of the system and the continual loss of CO2 to

the environment. These results would thus indicate that BCECF is stable in

detecting pH changes across a 24-hour period. Therefore, it was concluded

that H+ measurement using BCECF is not influenced by experiments of long

durations up to 24 hours.


33

Time (min)

Fluo

resc

ent R

atio

0.0

3.0

3.5

4.0

4.5

5.0

5.5

6.0

Figure 5 Day to day variability of 2,7-bis-(2- carboxyethyl)-5-(and-6)-carboxyfluorescein The small drift upwards is explained by the open nature of the system and the continual loss of CO2 to the environment.

Effect of albumin and stir rate on fluorescence using BCECF dye In an attempt to simulate the in vivo effects of proteins surrounding the

polyamide membrane and muscle cells contracting in close proximity to the

membrane, the probe was subjected to a series of high magnetic stirring rates

with the solution saturated with albumin. The high stirring rates resulted in a

large amount of turbulence created within the beaker and, as a result, the

membrane moved vigorously. [H+] was measured under three conditions;

magnetic stirring at 100 rpm and no albumin (100 control), magnetic stirring

at 500 rpm and albumin (500 albumin) and, pulsing the spin rate between

100 and 500 rpm in 30 s intervals and albumin (100/500 albumin). Each

condition was performed for a duration of 10 min with pH initiating at ~6.82,

3 min later increasing to ~7.23 and decreasing it back to ~6.78 for the

remainder of measurement. Fluorescence was recorded at a frequency of 10

Hz for the full duration of the test.

Figure 6 displays the fluorescent data collected during this experiment. Both

stir rate and albumin failed to affect the H+ fluorescent data. The differences

observed were again due to the nature of the in vitro system and subsequent

Day 1

Day 2


34

CO2 loss resulting in an upward drift in pH that was coincident with a

corresponding drift in

Time (min)

Fluo

resc

ent R

atio

0.0

3.0

3.5

4.0

4.5

5.0

5.5

6.0

12 13

Figure 6 Effect of magnetic stir rate and albumin on [H+] measured with BCECF in vitro

fluorescence, and small pH manipulations between experiments. Therefore, it

was concluded that an abundance of protein and dynamic movement of the

membrane was unlikely to interfere with the measurement of H+ using 2,7-

bis-(2- carboxyethyl)-5-(and-6)-carboxyfluorescein in vivo.

Influence of 1 cm polyurethane outlet tubing exposed to air During in vivo H+ measurement using microdialysis, approximately 1 cm of

the probe outlet would emerge from the skin of the thigh and thus be exposed

to the air. Stainless steel tubing was attached to the 1 cm of outlet to

transport the dialysate to the fluorometric spectrophotometer without the loss

of CO2. The internal volume of the outlet was ~0.35 µl, which allowed ~4

seconds for the diffusion of CO2 from the dialysate (assuming a flow rate of 5

µl.min-1). Given the steep CO2 gradient from dialysate to air, the aim of this

experiment was to establish whether there was a significant loss of CO2 from

the exposed polyurethane tubing. Two probes were used, one with a 1 cm

piece of tubing and the other with no tubing emerging from the solution in the

beaker. Then, across a wide pH range (6.8-8.0) four duplicate measurements

were made using both types of tubing. The fluorometric ratios were plotted

100 Control

500 Albumin

100/500 Albumin


35

against the corresponding pH values at the time of measurement with

subsequent linear regression analysis. As shown in Figure 7, 1 cm of exposed

polyurethane tubing had no effect on the [H+] regression curves suggesting

that CO2 loss under these conditions is not a concern.

Fluorescence Ratio

0.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0

Beak

er p

H

0.0

6.0

6.5

7.0

7.5

8.0

8.5

9.0

1 cm y = 5.232+0.5048x0 cm y = 5.384+0.4624x

Figure 7 Influence of 1 cm polyurethane tubing exposed to air on [H+] determination in vitro

Effect of HCl, La- and CO2 titrations on pH calibration curves All acid-base manipulations in vitro were performed via the addition of HCl,

however this acid is not physiological within skeletal muscle. Alterations in

acid-base status during muscular contraction are largely influenced by

metabolically produced La- and CO2, therefore the microdialysis probe was

tested to examine if it performed similarly titrating with both La- and CO2 to

manipulate pH in vitro. The same probe was used for each titration, then

across a wide range of physiological pH values the [H+] was manipulated with

each of the chosen acids. The fluorometric ratios were plotted against the

corresponding pH values at the time of measurement with subsequent linear

regression analysis. Figure 8 shows the titration curves for HCl-La- and HCl-

CO2 comparisons. No difference was found between any of the regression

curves for all titrations. These findings suggest two things; that in vitro

titrations with HCl were applicable to the in vivo model and, since the probe


36

performed similarly with all titrations, its performance in vivo should be

adequate.

Fluorometer Ratio

0 2 3 4 5 6 7 8

Beak

er p

H

0.0

6.0

6.5

7.0

7.5

8.0

8.5

HCl y = 5.371+0.3588xCO2 y = 5.525+0.3291x

Fluorometer Ratio

0 2 3 4 5 6 7

Beak

er p

H

0.0

6.0

6.5

7.0

7.5

8.0

8.5

HCl y = 5.426+0.4244xLa- y = 5.421+0.4326x

Figure 8 Titration curves for HCl-CO2 and HCl-La-

Effect of dye concentration on pH calibration Previously, the effect varied dye concentration would have on pH calibration

was unknown. To examine this question, three concentrations of dye were


37

assessed, low (0.1 mg.ml-1), medium (0.2 mg.ml-1) and high (0.4 mg.ml-1).

Across a wide pH range (6.8-8.0), four duplicate measurements were made

using each dye concentration. The fluorometric ratios were plotted against

the corresponding pH values at the time of measurement with subsequent

linear regression analysis (Figure 9). Interestingly, there was not a

systematic increase or decrease in the calibration curves generated during

this experiment. Although the y-intercepts were similar between

concentrations, the positive slopes were not 0.37, 0.46 and 0.41 for low,

middle and high, respectively. When using the above functions to calculate

pH, the results obtained varied greatly, 6.01 for low to 7.60 for high. This has

obvious implications for the amount of dye used and for establishing which is

the correct or most accurate concentration for in vivo pH determination.

Currently, there is no explanation for these results and further investigation is

required to elucidate possible reasons.

Fluorometer Ratio

0 2 3 4 5 6 7 8

Beak

er p

H

0.0

6.0

6.5

7.0

7.5

8.0

8.5

LowMiddleHigh

Figure 9 Effect of BCECF dye concentration on pH calibration curves

Effect of HCO3- perfusate on pH calibration

Blood consists of approximately 25 mmol.l-1 HCO3-, and evidence suggests

that a similar concentration may exist within the interstitium (Geers & Gros,

2000). Any concentration of HCO3- within the perfusate would alter the

equilibration between the interstitial space and probe membrane. To test the


38

effect of perfusate [HCO3-] on the recovery of H+ in vitro, two HCO3

-

concentrations were used, 0 and 25 mmol.l-1. These two concentrations were

chosen for the following reasons, 0 mmol.l-1 had been used in a recent paper

by Maclean et al. (2000) and 25 mmol.l-1 being similar to the interstitial

concentration would reduce the fractional uptake or loss of HCO3- from the

microdialysis probe, which would in turn reduce concurrent HCO3- effects on

[H+] within the probe. Several measurements were made using each

perfusate [HCO3-] across a wide range of beaker pH values (6.5–7.5). Figure

10 shows the resultant calibration curves for each perfusate and the beaker

pH for reference. The data clearly shows that perfusing a probe without

HCO3- alkalises the pH calibration curve, while 25 mmol.l-1 HCO3

- acidified the

calibration curve. However, further in vivo testing is required to ascertain

what effects this may have at rest and during exercise.

Beaker pH

0.0 6.6 6.8 7.0 7.2 7.4 7.6

Fluo

rom

eter

Rat

io

0

2

3

4

5

6

7

8

Beaker pHNo HCO3-

25mM HCO3-

Figure 10 Effect of perfusate [HCO3-] on pH

calibration curves in vitro


39

PART II – EFFECT OF PERFUSATE [HCO3-] ON INTERSTITIAL

PH IN VIVO

Methods

Subjects Two human subjects were continuously monitored at rest, during 5 min of

one-legged knee extensor exercise at 10 W, and during recovery. The

subjects were aged 24 and 32 years. They were 163 and 178 cm in height

and had a mass of 58 and 85 kg, respectively. Both subjects were active

individuals with no health-related problems. Prior to the start of the

experiment, the subject were informed of any risks and discomforts related to

the experiment. Both subjects signed a written consent form prior to the

experiments. The study was approved by the local ethics committee (August

Krogh Institute, University of Copenhagen) and conformed to the Declaration

of Helsinki.

Exercise protocol Subjects performed one-legged knee-extensor exercise in a supine position

and were secured via a series of straps, two shoulder, one waist and one

thigh strap, so exercise was restricted to the quadriceps muscle (Bangsbo et

al., 1990). During exercise, the subjects had visual feedback in the form of a

digital display showing the cadence and power output. Subjects were

required to maintain a cadence of 60 rpm for the exercise duration. The 10 W

exercise period was preceded and succeeded by 5 min and 10 min of rest,

respectively. This test was performed on three occasions, corresponding to 3

perfusates without HCO3- (wHCO), 25 mmol.l-1 HCO3

- (25HCO) and 50 mmol.l-

1 HCO3- (50HCO), each separated by 7 days.

Probe insertion and perfusate Before the experiment, the subjects rested in a supine position with the legs

well supported. For each microdialysis probe to be inserted, the subject was

given approximately 1 ml of 20 g.l-1 xylocaine via a 25-gauge needle at the

insertion site. An 18-gauge cannula was first passed through the skin and

fascia to make way for the probe. A second cannula containing the


40

microdialysis probe (CMA-60, CMA Microdialysis AB, Sweden) was then

pushed through the skin and fascia and orientated along the length of the

fibres of the vastus lateralis muscle. The cannula was removed leaving the

microdialysis probe within the muscle. After insertion of the probe, it was

secured with tape and the outlet cut at a maximal length of 10 mm from the

skin. The subjects recovered for 1.5 h after probe insertion before any

measurements were performed. The pH-sensitive fluorescent dye BCECF was

coupled to dextran (molecular mass cut off at 70000 Da), which prevented

any diffusion of dye across the probe membrane (cut off at a molecular mass

of 20000 Da). The dye (0.1 mg ml.l-1) was dissolved in a sterile saline

solution (154 mM Na+). The perfusate was then placed into a sterile 1 ml

syringe equipped with a filter, mounted in a microdialysis pump and

connected to the inlet of the microdialysis probe. The outlet from the probe

was removed, replaced with a steel tube and connected to a micro flow-

through cuvette (total volume 8 µl) in a (Hitachi F-2000, Japan) fluorescence

spectrophotometer. The pump rate was 5 µl.min-1 in all experiments. The

time scales on the figures have been corrected for the delay due to the

volume of tubing and cuvette.

Fluorometric measurements and determination of pH With the emission wavelength constant at 530 nm, the fluorescence



nm was insensitive to pH, but dependent on the amount of dye, whereas the

excitation intensity at 500 nm was also sensitive to pH. Thus, the excitation

intensity ratio 500/440 was proportional to pH and independent of changes in

dye concentration and consequently was insensitive to any water movements

from or into the probe. The temperature in the fluorescence

spectrophotometer was kept constant by circulating thermostatically

controlled water. For calibration, a microdialysis probe was placed in a beaker

with magnetic stirring and connected to a pump and the fluorometer. The

beaker contained saline and bicarbonate, and the pH was monitored with a

laboratory pH meter. The pH in the beaker was changed in a stepwise

manner by adding HCl/NaOH and the excitation ratio was recorded. This was

performed with a separate probe before the experiment (as a backup) and

then after the experiment with the probe used in vivo (if it was still intact). A


41

calibration curve was obtained by plotting the excitation intensity ratio versus

external pH. The constants obtained from a linear regression to the

calibration curve were used to convert fluorescent signals obtained in human

experiments to interstitial pH.

Results The mean pH at rest was 7.42, 7.07 and 6.77 for wHCO, 25HCO, and 50HCO,

respectively. Exercise resulted in an alkalosis of 0.24 pH units without HCO3-,

while 25HCO and 50HCO showed slight acidoses of 0.01 and 0.02 pH units,

respectively. Five minutes of 10 W leg flexion and extension resulted in an

average exercise interstitial pH of 7.66, 7.06 and 6.75 for wHCO, 25HCO and

50HCO, respectively. After 5 minutes recovery, interstitial pH had returned to

7.50, 7.05 and 6.72 for wHCO, 25HCO and 50HCO, respectively. Only 25HCO

and 50HCO had made a full recovery to pre-exercise pH values. Figure 11

shows this data, which have been time adjusted to show real time at the

muscle interstitium.

Time (min)

0 5 10 15 20 25

pH

0.00

6.50

6.75

7.00

7.25

7.50

7.75

8.00without HCO3-

25 mM HCO3-

50 mM HCO3-

Figure 11 Influence of [HCO3-] on the estimation of interstitial pH

during 10 W dynamic leg exercise

Three interstitial pH curves generated from different concentrations of HCO3-

, no HCO3- (blue), 25 mM HCO3

- (red), 50 mM HCO3- (green), before, during

and after 10 W knee-extensor exercise. Exercise started 5 min after the initial recording, marked with a black line.


42

Discussion The main finding from this experiment was that [HCO3

-] within perfusate

profoundly influences the estimated interstitial pH during dynamic leg exercise

using BCECF dye and microdialysis. Specifically, the absence of HCO3- in the

perfusate significantly alkalises the pH value at rest (7.42), during exercise

(7.66), and in recovery (7.50). In contrast, doubling the estimated interstitial

[HCO3-] to 50 mmol.l-1 resulted in an acidosis of the rest (6.77), exercise

(6.75) and recovery (6.72) pH values, respectively. These results suggest

that the total absence or presence of too much perfusate HCO3- leads to an

over or underestimation of interstitial pH, respectively. Further, the temporal

response during exercise under the wHCO condition (Figure 11) shows a

significant increase in pH or alkalosis. This has been demonstrated previously

by Maclean et al. (2000), who also used a perfusate lacking HCO3-. The 10 W

exercise intensity was specifically chosen because of its small effects on

muscle and blood pH. As such, it was expected that 10 W exercise intensity

would have very little effect on interstitial pH. This prior knowledge suggests

that the method involving no HCO3- in the perfusate causes erroneous

interstitial pH results. It is possible that the large gradient of bicarbonate

between interstitium and the probe led to bicarbonate diffusing across the

probe membrane. For most compounds, the equilibration across the probe

membrane is only partial (fractional uptake) and the rate of equilibration is

increased by movement (Juel et al., 2000; Maclean et al., 1999). Therefore,

the apparent alkalisation during exercise could be due to an increased

fractional uptake of bicarbonate induced by movement. It follows then that

since there is approximately 25 mM bicarbonate is present in blood (Bangsbo

et al., 1997) and presumably also in the interstitium (Geers & Gros, 2000) the

addition of 25 mM bicarbonate to the perfusate best reduces the bicarbonate

concentration gradient between probe and interstitial space. It thus appears

that adding bicarbonate to the perfusate is crucial and that there is no

evidence to support an interstitial alkalisation during muscle contraction.


43

PART III – INTERSTITIAL PH AT REST AND DURING

DYNAMIC LEG EXERCISE

Methods

Subjects Six male subjects with a mean age of 31 years (range 24–52 years)

participated in the study. Mean height and weight were 181 cm (175–190

cm) and 82 kg (68–97 kg), respectively. All subjects were active individuals

with no health-related problems. Prior to the start of the experiment, each

subject was informed of any risks and discomforts related to the experiment.

All subjects signed a written consent form prior to experiments. The study

was approved by the local ethics committee (August Krogh Institute,

University of Copenhagen) and conformed to the Declaration of Helsinki.

Exercise protocol The only modification to the exercise protocol that was performed in

experiment I was the addition of a third exercise intensity of 70 W.

Probe insertion and perfusate The method used for placing microdialysis probes in each subject has been

described in detail in the section on experiment I and was not altered for this

experiment. However, in accordance with the findings of experiment I, 25

mmol.l-1 HCO3- was added to the perfusate without pH adjustment.

Fluorometric measurements and determination of pH To ascertain interstitial pH values the same method was used as described

above in experiment I.


44

Results

Interstitial pH at rest and during muscle activity With the addition of 25 mmol.l-1 HCO3

- to the perfusate, mean calculated

interstitial pH at rest was 7.38±0.02. Exercise induced a reduction in muscle

interstitial pH in all six subjects and at all intensities (Figure 12).

Interstitial pH was gradually reduced during the 5 min of exercise. The

decrease in interstitial pH during exercise was nearly linearly related to the

power output. The mean value of the lowest interstitial pH at 30, 50 and 70

W exercise was 7.27 (range 7.18–7.34), 7.16 (7.05–7.24) and 7.04 (6.93–

7.12), respectively. The mean peak acidification during exercise at a power

output of 30, 50 and 70 W was 0.11 (0.06–0.20), 0.22 (0.13–0.34) and 0.34

(0.22–0.41) pH units, respectively. For each subject there was a correlation

between power output and peak acidificat-ion (Figure 13). The large inter-

individual variation in peak acidification was probably due to the large

variation in relative workload.

Recovery from exercise The peak acidification was obtained 1.0 min (0.5–2.0 min) after the cessation

of exercise. Recovery from peak acidification proceeded in an exponential

fashion (Figure 12). The mean half time for recovery of interstitial pH after

70 W exercise was 5.2 (4.1–6.1) min. For most subjects, the pH curve in

recovery after 70 W intersected the curve obtained after 50 W, indicating that

the rate of pH recovery was higher after 70 W than after 50 W exercise.


45

0 5 10 15 20 25 30

Inte

rstit

ial p

H

6.9

7.0

7.1

7.2

7.3

7.4

7.5

0 5 10 15 20 25 306.9

7.0

7.1

7.2

7.3

7.4

7.5

Time (min)

0 5 10 15 20 25 306.9

7.0

7.1

7.2

7.3

7.4

7.5

0 5 10 15 20 25 30

Inte

rstit

ial p

H

6.9

7.0

7.1

7.2

7.3

7.4

7.5

0 5 10 15 20 25 306.9

7.0

7.1

7.2

7.3

7.4

7.5

Time (min)

0 5 10 15 20 25 30

Inte

rstit

ial p

H

6.9

7.0

7.1

7.2

7.3

7.4

7.5

Figure 12 Interstitial acidification during exercise

Individual recordings of interstitial pH during 5 min knee-extensor exercise are shown. The power output was 30 W (green), 50 W (blue) and 70 W (red). Exercise was started 5 min after the onset of the recording (marked with a horizontal bar). One subject, marked *, became exhausted after 2 min of 70 W exercise. Numbers at the top-centre of each graph depict subject age.

*

52 24

26 26

27 29


46

Power output (Watts)0 10 20 30 40 50 60 70

Inte

rstit

ial p

H

0.0

6.9

7.0

7.1

7.2

7.3

7.4

Figure 13 Peak interstitial pH during exercise at different power outputs

For each subject the line connects pH at rest and pH at peak interstitial acidification obtained at a power output of 30, 50 and 70 W. One subject, marked *, became exhausted after 2 min of 70 W exercise.

Discussion This is the first study to continuously measure interstitial pH during and after

muscle activity in humans. At each intensity, interstitial pH gradually reduced

during the entire exercise period. Peak acidification was obtained

approximately 1 min after cessation of exercise, after which interstitial pH

recovered in an exponential manner. It was also demonstrated that

interstitial pH is reduced proportional to power output during muscle exercise.

Changes in muscle interstitial pH during and after exercise The rate of interstitial acidification at the onset of exercise can be evaluated

from the slope of the curves in Figure 12. In most subjects, the slope of

acidification was greater the higher the power output. This finding probably

demonstrates that the rate of acid accumulation in the muscle cells is related

to the power output and that the acid transport across the sarcolemma

membrane is dependent on the concentration gradient from muscle to

interstitium.

*


47

The present study showed that muscle activity induced an interstitial

acidification. In contrast, two studies also using the microdialysis technique

but with the probe attached to a flow-through pH microelectrode have

reported an alkalisation during muscle activity and a fast (1 min) recovery

(Maclean et al., 2000; Maclean et al., 1998). In these studies, no bicarbonate

was added to the perfusate. When bicarbonate was not added to the

perfusate, it was observed, as in previous studies (Maclean et al., 2000;

Maclean et al., 1998), that exercise induced an apparent fast alkalisation,

succeeded by a plateau and a fast recovery (see experiment I). It is likely

that the large gradient of bicarbonate between interstitium and the probe had

caused the bicarbonate to diffuse across the probe membrane. For most

compounds, the equilibration across the probe membrane is only partial

(fractional uptake) and the rate of equilibration is increased by movement

(Juel et al., 2000; Maclean et al., 1999). Therefore, the apparent alkalisation

during exercise in previous experiments could be due to an increased

fractional uptake of bicarbonate induced by movement. Approximately 25 mM

bicarbonate is present in blood (Bangsbo et al., 1997) and presumably also in

the interstitium (Geers & Gros, 2000). In the present study, 25 mM

bicarbonate was thus added to the perfusate, which resulted in a low

bicarbonate concentration gradient between probe and interstitial space. With

bicarbonate in the probe, exercise induced an interstitial acidification,

succeeded by a recovery phase with a time course similar to recovery in the

intracellular space and in blood (see Figure 12). It therefore appears that

adding bicarbonate to the perfusate is crucial and that there is no evidence to

support an interstitial alkalisation during muscle activity.

Extracellular (interstitial) muscle pH has also been measured with a needle-

protected glass pH electrode before and after exercise (Allsop et al., 1990).

With this method, pH after exercise was found to be 6.6. An apparently fast

initial recovery after activity was succeeded by a slow, nearly linear recovery,

which was only partial even after 30 min (Allsop et al., 1990). The low pH

and the slow and only partial recovery could indicate that the measurements

were influenced by fibre damage and/or the existence of a large artificial

space in the muscle created by the needle. In contrast, the present study

showed a complete recovery of interstitial pH 20 min after exercise.


48

Recovery of interstitial pH The lowest interstitial pH was obtained approximately 1 min after exercise. A

similar time course has been found for cellular pH determined by phosphorus

magnetic resonance spectroscopy (NMR) (Bangsbo et al., 1993b; Arnold et

al., 1985). A possible reason for the further acidification after cessation of

exercise is that a large fraction of phosphocreatine resynthesis, which

releases H+, occurs within the first minute after exercise (Bangsbo et al.,

1993b; Arnold et al., 1985).

The recovery of interstitial pH took place with a mean half-time of 5.2 min

calculated from the end of exercise (see Figure 12). This value is similar to

the recovery of muscle pH (intracellular pH) measured in needle biopsies

obtained at different time intervals after exercise (Juel, 1998a; Juel et al.,

1990). These studies have reported half-time values of 4.9 min for muscle

pH, approximately 4 min for H+, and 5 min for lactate (Juel, 1998a; Juel et

al., 1990). Consequently, the time course of changes in interstitial pH after

exercise closely corresponds with the changes in intracellular pH reported in

the literature. The recovery of interstitial pH after intense exercise (70 W)

was faster than after more moderate exercise (50 W) (see Figure 12). In

fact, some of the recovery curves for 70 W crossed the curves for 50 W

approximately 5 min after exercise. This observation could suggest that pH

recovery is blood flow dependent, as blood flow after intense exercise is

higher than after moderate exercise (Radegran & Saltin, 1998).

Comparison between pH changes in muscle interstitium and blood The interstitial pH at rest (7.38±0.02) fell within the range (7.37–7.43) of

femoral venous pH values reported in the literature (Bangsbo et al., 2000;

Bangsbo et al., 1993a; Juel et al., 1990; Sjogaard et al., 1985). In Figure 14,

the exercise-induced interstitial pH values at peak acidification are compared

to femoral venous pH values. The figure includes unpublished values from the

same laboratory as well as femoral venous blood pH values obtained in

comparable knee-extensor exercise studies (Bangsbo et al., 2000; Bangsbo et

al., 1993a; Sjogaard et al., 1985). This figure depicts two distinct interstitial

acidification relationships with venous blood. Low intensity exercise (10–30

W) induced an interstitial acidification similar to venous blood, whereas high


49

intensity exercise (>50 W) resulted in a larger interstitial acidification than in

venous blood. There may be several reasons for this difference. During one-

legged knee-extensor exercise, the femoral vein does not drain only the

active part of the quadriceps muscle, since a fraction of venous blood comes

from inactive tissues and inactive parts of the quadriceps muscle. At an

exercise intensity of 50 W, blood flow may be 5.3 l.min-1 (Juel et al., 1990)

(Bangsbo et al., 1990) of which 1.2 l.min-1 may be perfusing inactive muscle

(Radegran & Saltin, 1998). This results in a decrease in femoral venous blood

pH that is less than the interstitium pH decrease of the active muscle.

Another reason for the larger acidification of the interstitial space during

muscle activity is probably its lower buffer capacity compared to blood. The

lack of protein concentration within the interstitial space makes the

interstitium more exposed to pH fluctuations (Geers & Gros, 2000). A third

possibility is that the higher blood flow rate during high intensity exercise

reduces the mean transit time for the blood passing the capillaries in the

active muscle during exercise (Radegran & Saltin, 1998; Bangsbo et al.,

1990; Juel et al., 1990), which may result in only partial equilibration of H+

between interstitium and blood. The present study does not allow for

discrimination between the possibility that a pH gradient exists across the

capillary wall during exercise and that the pH difference between interstitium

and femoral venous blood is exclusively due to mixing with blood from

inactive tissues.

Comparison between cellular and interstitial pH changes One method to access intracellular pH has been to measure pH in

homogenised needle-biopsy material. This method is based on the fact that

the intracellular space makes up the main fraction of the muscle and that the

homogenate pH therefore mainly represents the intracellular pH (Sjogaard et

al., 1985). The homogenisation method reveals resting intracellular pH

values in the range of 7.04–7.17 (Bangsbo et al., 1996; Bangsbo et al.,

1993a; Juel et al., 1990; Sjogaard et al., 1985). Thus, resting muscle pH is

at least 0.2 pH units lower than interstitial pH (7.38). In Figure 14, the

intracellular pH values during exercise reported in the literature are plotted

together with the interstitial pH values obtained in the present study.


50

It can be seen that cellular pH is 0.2–0.3 pH units lower than interstitial pH

both at rest and immediately after intense exercise. However, if the data in

the figure are converted to [H+], it is evident that the H+ gradient across the

muscle membrane is larger (approximately 100 nM) after intense exercise

than at rest (30 nM). This is probably due to the large intracellular

accumulation of lactic acid during muscle activity (Juel et al., 1990).

Power output (Watts)

0 10 20 30 40 50 60 70

pH

0.0

6.7

6.8

6.9

7.0

7.1

7.2

7.3

7.4

Figure 14 Intracellular, interstitial and venous pH during knee-extensor exercise

, mean (± SEM) peak interstitial pH obtained in the present study. Data connected by a straight line. , femoral venous pH during knee-extensor exercise (authors’ unpublished data); each value represents mean from six subjects. 1, femoral venous pH during exhaustive knee-extensor exercise in three studies (Sjogaard et al., 1985) (Bangsbo et al., 1996) (Bangsbo et al., 2000). The dashed line represents a regression line for the venous data. , intracellular pH obtained with the homogenisation technique (Sjogaard et al., 1985) (Juel et al., 1990) (Bangsbo et al., 1993a) (Bangsbo et al., 1996). Data from the literature represent means of five to six subjects.

Physiological implications of changes in interstitial pH Since the relationship between exercise intensity and interstitial pH displayed

similarities with exercise-related changes in blood flow (Bangsbo et al., 1990;

Juel et al., 1990), it is reasonable to speculate that blood flow during muscle

activity is regulated by changes in interstitial pH. Supporting this hypothesis

is the finding that interstitial pH has a local modulatory effect on smooth

muscle cells inducing vasodilatation, which is probably mediated by a


51

reduction in intracellular calcium (Aalkjaer & Peng, 1997) or changes in the

activity of potassium channels (Quayle et al., 1997; Davies, 1990).

Alternatively, blood flow during knee-extensor exercise increases rapidly and

has reached a steady level after about 1.5 min (Radegran & Saltin, 1998),

whereas interstitial pH progressively decreases during exercise. In addition,

the time courses for recovery of blood flow and interstitial pH after exercise

are different (Bangsbo et al., 1990; Juel et al., 1990). Consequently, it would

seem that other factors are also involved in the regulation of blood flow

during and after exercise. The exercise induced decrease in interstitial pH

may also have other effects. The lower interstitial pH could modulate the

sensory response from the muscle, since interstitial pH has been linked to

sympathetic nerve discharge (Victor et al., 1988). In addition, interstitial pH

is probably also important in modulating transport systems and ion channels

in muscle sarcolemma as well as in vascular cells.

Conclusion The present study demonstrated that interstitial pH is continuously decreasing

during muscle activity. The exercise-induced reduction in interstitial pH was

correlated with power output and was larger at high exercise intensities than

the pH reduction of femoral venous blood.

Chapter 4 – Ingestion Regime

53

CH A P T E R 4 EF F E C T O F A L K A L I I N G E S T I O N

R A T E O N P L A S M A A C I D-B A S E , [H + ] , A N D I O N I C ,

[K + ] , S T A T U S

Introduction Increasing one or all of the buffer systems’ capacities, either intracellularly or

in the blood, has the potential to further delay the onset of a critical pH level,

thereby increasing the time for muscle to maintain force production. The

ingestion of sodium bicarbonate or sodium citrate can increase the capacity of

the blood bicarbonate system (Kowalchuk et al., 1989; Costill et al., 1984).

Associated with this increase in blood [HCO3-] is a decrease in [H+] or

increase in pH (Ball & Maughan, 1997), and is commonly referred to as a

state of alkalosis. Manipulating blood [H+] and [HCO3-] via inducing alkalosis

can improve muscle function and performance. Numerous investigations of

ingesting sodium bicarbonate or sodium citrate prior to exercise have led to

increases in swimming (Gao et al., 1988), running (Shave et al., 2001),

cycling (Potteiger et al., 1996a) and rowing (McNaughton & Cedaro, 1991)

performances. However, there is evidence to demonstrate the contrary

across each of these modes of exercise, respectively (Schabort et al., 2000;

Robinson, 1997; Ibanez et al., 1995; Pierce et al., 1992).

As is often the case with ingested substances, there exists a dose-response

relationship. That is, there is a minimum quantity of the substance that must

be consumed to manipulate the system to a high enough degree to enable the

observation of a significant effect. Although this work has been performed

(McNaughton, 1992a; McNaughton, 1990; Horswill et al., 1988) and a

minimum dose identified, 300 mg.kg-1 for both sodium bicarbonate and

sodium citrate, it was tested across one ingestion regime only, namely bolus.

The bolus regime usually involves subjects consuming the ingestant in less

than 3 min. This rapid infusion of alkali very quickly increases gastric pH from

2.5-6.11 (Hauptfleisch & Payne, 1996), thereby dramatically altering the GI

milieu. Such alterations challenge the body’s regulatory and absorptive

mechanisms. Unfortunately, sometimes the stress is too much for the system

and results in symptoms of nausea and vomiting (Goldfinch et al., 1988;

Wilkes et al., 1983). Although this is a rare occurrence in this type of


54

research, it is a clear message of overloading the system. Under these

circumstances any potential benefits of alkali ingestion are usually negated.

Therefore, this situation needs to be avoided at all costs. Further, it may be

possible that even when these symptoms are not displayed by subjects, the

bolus method is still overloading and not optimising the absorptive

mechanisms within the GI system. The investigations of Tiryaki and Atterbom

(1995) and van Someren et al. (1998) support this view, as they reported no

side-effects but, more importantly, no ergogenic effects either.

Ingestion of an alkali at a slower rate would have the benefit of better

matching the absorption processes within the GI system. This could, in turn,

result in greater increases and decreases in pH, thereby further increasing the

protection against pH during muscular contraction. In addition, decreasing

the amount of alkali within the GI system at any one time would decrease the

likelihood of overload and the chance of subjects experiencing nausea or

vomiting. To date there has been no systematic examination of SB or SC

ingestion regime on blood acid-base status. Such investigations are

important, as they could reveal that lower doses ingested more slowly invoke

greater physiological effects but with reduced side effects.

In an attempt to identify an optimal ingestion rate for SC, four ingestion rates

(bolus, 300, 600 and 900 mg.min-1) were investigated. The first hypothesis

tested was that the bolus ingestion rate was not the optimal rate in humans

at rest, as measured by change in blood pH. In addition, since SC has

previously shown ergogenic properties and is not currently on the IOC banned

substances list, the second hypothesis tested whether SC ingestion would

induce a measurable alkalosis in urine.

Methods

Subjects Eight human subjects participated in the study. The mean age of the subjects

was 21±3 years with mean heights and weights 173.9±3.1 cm and 69.2±4.2

kg, respectively. All subjects were active individuals with no health-related

problems. Prior to the start of the experiment, each subject was informed of

any risks and discomforts related to the experiment. All subjects signed a


55

written consent form prior to the experiments. The study was approved by

the University Research Ethics committee (Queensland University of

Technology) and conformed to the Declaration of Helsinki.

Experimental Protocol During the 24-h period prior to testing, subjects refrained from vigorous

exercise, alcohol and caffeine ingestion. Approximately 3-h prior to testing,

subjects consumed a normal meal consisting largely of carbohydrates,

minimal fats and proteins (e.g., cereal, toast and juice). The meal was

recorded and repeated prior to subsequent tests in an attempt to standardise

the effect of prior food. The total length of experimenting was approximately

9-h, 1-h preparation and 8-h data collection.

Blood collection The laboratory temperature was a constant 210 centigrade (C) and the

humidity was 55±10 percent. To prevent blood flow being compromised and

due to a drafting effect within the laboratory, all subjects were informed to

bring warm clothing to use at their own discretion. After initial weighing, each

subject rested in the supine position for a period of 10-min. Subjects’

forearm cephalic vein was then cannulated with a 23-gauge needle (Becton

Dickinson, Germany) and attached to a 3-way tap (Terumo, Belgium). Ten

millilitres (ml) of blood was removed as discard and a 1-ml sample was

subsequently obtained and immediately placed on ice. To maintain cannula

patency and plasma volume, an equivalent amount of 0.9 percent saline was

re-infused after each sample. After 15 minutes in the supine position, resting

samples were obtained prior to the start of ingestion. Subjects were in the

supine position ten minutes prior to all sampling thereafter. Immediately

after obtaining the resting sample, subjects provided approximately 50 ml of

urine. The order of the ingestion protocols was randomised and separated by

at least 48 hours.

Ingestion The initiation of ingestion followed the resting samples and subjects were

required to ingest 300 mg.kg-1 of tri-sodium citrate (sigma chemicals -

Na3C8H5O7.H2O) at one of four rates, bolus, 300, 600, 900 mg.min-1. In an


56

attempt to disguise the taste and make the ingestion more palatable, the

bolus regime involved the respective quantity of sodium citrate added to 400

ml of low energy (approximately 6 percent orange juice) solution. The

sodium citrate for the remaining ingestion rates was consumed in ‘OO’ size

gelatine capsules with water ad libitum. Each capsule was weighed before

and after being filled with sodium citrate to determine the exact quantity

(mean 1.2 g.capsule-1). The determined quantity of sodium citrate was

divided by the ingestion rate in question to obtain the timing for ingestion of

each capsule. The control condition required the participants to remain in the

laboratory for the full testing duration and provide the same samples as on an

ingestion day. After the ingestion process was finished, the subjects were

encouraged to drink water ad libitum across the duration of testing. No solid

food was ingested by the subjects during testing.

Measurement and Analysis Arterialised blood was achieved by immersing the subject’s hand in

approximately 460 C water for five minutes prior to sampling. Samples were

collected at 30-min intervals across an 8-h duration into 1.5 ml heparinised

syringes (Terumo, Belgium). The sample was immediately analysed

(Radiometer ABL505, Copenhagen) in duplicate for acid-base status (pH,

HCO3-, pCO2) and selected plasma ions (K+, Na+, Cl-). Urine samples were

obtained hourly across 8-h and immediately analysed (Radiometer ABL505,

Copenhagen) for pH.

Statistics All reported values are means±SEM. A two-way analysis of variance with

repeated measures was used to examine differences in dependent variables

with respect to treatment and time. Significant F-values were further

subjected to a Tukey’s post-hoc test to identify differences. Statistical

significance was set at p<0.05.


57

Results

Acid-base Figure 15 shows the blood pH temporal response for all conditions. The mean

pre-ingestion pH was 7.397±0.004 and was not different between conditions.

Blood pH was significantly elevated two hours post-ingestion, mean

7.44±0.004, and stayed elevated for the remaining collection time (p<0.05).

The blood pH response during this time was not different between ingestion

rates. Figure 16 compares the average blood pH response for two time

periods: PRE and 1.5–8 h post-ingestion (POST) for each condition. PRE

values were not different between conditions. Control values were also not

different. As indicated by *, ingestion rates (IR) bolus, 300, 600 and 900

increased POST pH values, 7.445±0.004, 7.438±0.004, 7.442±0.004,

respectively, higher than control, 7.415±0.004 (p<0.05, p=0.06, p<0.05,

respectively). In addition, there was a significant interaction between time

and condition for all IR (p<0.05). Figure 17 shows the relative change in pH

from PRE to two hours post-ingestion for all conditions. There was a small,

0.66 percent, but significantly greater relative change above PRE pH values

for all conditions (p<0.05). There was no difference in relative change

between ingestion rates.

Time (hr)

0 1 2 3 4 5 6 7 8

Blo

od p

H

0.00

7.36

7.38

7.40

7.42

7.44

7.46

7.48

7.50

ControlBolus300600900

Figure 15 Blood pH 8-h temporal response

Mean (±SEM) blood pH temporal responses for all conditions. All IR, indicated by *, increased pH above control from 2-8 h (p<0.05).

*


58

Condition

control bolus 300 600 900

Blo

od p

H

0.00

7.36

7.38

7.40

7.42

7.44

7.46

7.48 PREPOST

Figure 16 Mean blood pH response pre- and post-ingestion

Mean (±SEM) blood pH values for each IR are shown. Comparison of mean blood pH pre-ingestion (PRE) and 1.5-8 h post-ingestion (POST) within condition. PRE values were not different between conditions. Control values were not different. All IR, marked *, increased pH above control POST values (p<0.05, p<0.10 and p<0.05 for 300, 600 and 900, respectively).

Condition


% c

hang

e

0.0

0.2

0.4

0.6

0.8

Figure 17 Blood pH relative change between PRE and 2 h post-ingestion

Mean relative blood pH changes for each IR are shown. Each IR, indicated by *, induced a small (mean 0.66%) but significant relative change in blood pH (p<0.05).

** *

* *

*

*


59

Figure 18 shows the average post-ingestion blood [HCO3-] for each condition.

As indicated by * bolus, 300, 600 and 900 (29.6, 29.7, 29.8 and 29.9 mmol.l-

1, respectively) conditions significantly (p<0.05) elevated blood [HCO3-] above

that for control (26.3 mmol.l-1). There were no statistical differences between

the respective increases in blood [HCO3-] for each ingestion rate of 12.5,

12.9, 13.3 and 13.7 percent. Temporal responses for each condition are

depicted in Figure 19. After 1.5 h of ingestion, all experimental IR had

elevated blood [HCO3-] higher than control (p<0.05). This observation

continued for the next 6.5 h. Interestingly, the most rapid dose, bolus, had a

faster temporal response than the three remaining ingestion rates. Under

bolus, only thirty minutes was required to elevate blood [HCO3-] above control

(p<0.05). Although the response was faster under the bolus condition, the

magnitude of change was slightly less (approximately 0.8 percent or 0.2

mmol.l-1) than the other three rates.

Condition

Control Bolus 300 600 900

HC

O3- (

mm

ol.l-1

)

0

20

22

24

26

28

30

32

34

Figure 18 Mean post-ingestion blood [HCO3-] for all IR

Mean (±SEM) blood [HCO3-] for each IR are shown. Ingestion rates, marked

*, elevated post-ingestion [HCO3-] significantly above control (p<0.05).

* ***


60

Time (h)

0 1 2 3 4 5 6 7 8

HC

O3- (

mm

ol.l-1

)

05

24

26

28

30

32

34

36


Figure 19 Mean blood [HCO3

-] temporal responses across 8 h for all IR

All ingestion rates elevated blood [HCO3-] (SEM removed for clarity) above

control after 1.5 and remained elevated to 8 h, represented by * (p<0.05). Bolus, marked #, had the fastest response elevating [HCO3

-] above control in 30 min (p<0.05).

Average post-ingestion blood pCO2 response for each condition is shown in

Figure 20. Similar to the response of blood [HCO3-], bolus, 300, 600 and 900

(45.4, 43.8, 44.7 and 44.8 percent, respectively) conditions elevated blood

[pCO2] above control (41.5 percent) (p<0.05). Figure 21 shows the temporal

blood pCO2 response for each condition. The pre-ingestion values were not

significantly different from each other. Unfortunately, the between-day

variability obscured the observation of any condition effect on pCO2.

* #


61

Condition


PC

O2 (

mm

Hg)

0

38

40

42

44

46

48

50

Figure 20 Mean post-ingestion blood [pCO2] for each IR

Mean (±SEM) blood [pCO2] for all IR are shown. Each IR, marked *, significantly raised post-ingestion [pCO2] above control (p<0.05).

Time (h)

0 1 2 3 4 5 6 7 8

pCO

2 (m

mH

g)

0

38

40

42

44

46

48

50

52


Figure 21 Temporal response for blood [pCO2] for each IR

Mean blood [pCO2] (SEM removed for clarity) for each condition were not different from each other or control. This was mainly due to large between-day variability.

***

*


62

Strong Ions Figure 22, (a) through (e), shows the mean blood H+ response for each

condition separately. The ↓ symbol identifies the time at which the nadir PRE

values occurred. The number (e.g., 0.50) above the arrow represents the

magnitude of change expressed in meq.l-1×10-8. The fastest IR, bolus,

induced the slowest peak change from PRE values at 6.0 h, whereas the

greatest magnitude of change (0.66 meq.l-1x10-8) was induced by the 300 IR.

Figure 22 (f) shows the average PRE and POST blood [H+] for each condition.

No differences were identified between the PRE values, mean 4.0x10-8 meq.l-

1. As indicated by * all IR reduced POST blood [H+], mean 3.6x10-8 meq.l-1,

lower than POST control values, 4.0×10-8 meq.l-1 (p<0.05), however the

reductions caused by each IR were not different from each other. Figure 23

shows the average blood [H+] at three time points: PRE, 1.5 h post-ingestion

(POST1) and 2.5 – 5.5 h post-ingestion (POST2). Control PRE blood [H+] was

not different between the three time points, mean 4.0×10-8 meq.l-1. Further,

PRE blood [H+] was not different between conditions, mean 3.95×10-8 meq.l-1.

As indicated by *, the 300 IR significantly lowered blood [H+] at POST1,

3.55×10-8 meq.l-1, from control, 3.87×10-8 meq.l-1 (p<0.05). IR 300, 600 and

900 POST2, marked #, [H+] (3.48, 3.58, 3.52×10-8 meq.l-1) were lower than

POST2 control [H+] (3.87×10-8 meq.l-1) (p<0.05). When compared with their

respective PRE values, all IR significantly reduced blood [H+] at both POST1

and POST2 time points (p<0.05). In Figure 24, the relative change for each

time period, POST1 and POST2, can be seen. The relative changes from PRE

values at both time points for control were less than one percent. However,

under the influence of SC ingestion this increased to approximately 10, 14, 10

and 7 percent during POST1 and a further 2, 2, 2 and 7 percent during POST2

for bolus, 300, 600, and 900 mg.min-1 IR, respectively (p<0.05). When

considering the above changes as a 100 percent change, the POST2

component measures approximately 19, 14, 17 and 43 percent of the

decrease in blood [H+] for each IR, respectively.


63

Time (hr)

0 1 2 3 4 5 6 7 8

Bloo

d [H

+ ] (m

eq.l-1

)

0.0

3.0e-8

3.2e-8

3.4e-8

3.6e-8

3.8e-8

4.0e-8

4.2e-8

4.4e-8

Control

Time (hr)

0 1 2 3 4 5 6 7 8

Bloo

d [H

+ ] (m

eq.l-

1 )

0.0

3.0e-8

3.2e-8

3.4e-8

3.6e-8

3.8e-8

4.0e-8

4.2e-8

4.4e-8

Bolus

Time (hr)

0 1 2 3 4 5 6 7 8

Bloo

d [H

+ ] (m

eq.l-

1 )

0.0

3.0e-8

3.2e-8

3.4e-8

3.6e-8

3.8e-8

4.0e-8

4.2e-8

4.4e-8

300

Time (hr)

0 1 2 3 4 5 6 7 8

Bloo

d [H

+ ] (m

eq.l-

1 )

0.0

3.0e-8

3.2e-8

3.4e-8

3.6e-8

3.8e-8

4.0e-8

4.2e-8

4.4e-8

600

Time (hr)

0 1 2 3 4 5 6 7 8

Bloo

d [H

+ ] (m

eq.l-

1 )

0.0

3.0e-8

3.2e-8

3.4e-8

3.6e-8

3.8e-8

4.0e-8

4.2e-8

4.4e-8

900

Condition


Bloo

d [H

+ ] (m

mol

.l-1)

0.0

3.0e-8

3.2e-8

3.4e-8

3.6e-8

3.8e-8

4.0e-8

4.2e-8

4.4e-8

PREPOST

Figure 22 Individual IR temporal responses in blood [H+] for all conditions

Mean (±SEM) blood H+ response for each condition separately (a) – (e). Time at peak change is marked with ↓. The number above ↓ represents magnitude of change in meq.l-1×10-8. Mean (±SEM) PRE and POST values are compared in (f). IR POST blood [H+] was significantly lower than POST control and marked with * (p<0.05).

*

0.50

0.50 0.66

0.45* * *

(a)

(e)

(d)

(b)

(c)

(f)


64

Condition


Bloo

d [H

+ ] (m

mol

.l-1 )

0.0

3.0e-8

3.2e-8

3.4e-8

3.6e-8

3.8e-8

4.0e-8

4.2e-8

4.4e-8

PREPOST1POST2

Figure 23 Mean (± SEM) blood [H+] for each IR at 3 time points

Collection time was divided into 3 periods, pre-ingestion (PRE), 1.5 h post-ingestion (POST1) and 2.5–6 h post-ingestion (POST2). Only 300 mg.min-1 POST1 [H+], marked *, was statistically lower than control POST1 (p<0.05). However, 300, 600 and 900 mg.min-1 POST2 [H+], marked #, were lower than control POST2 [H+] (p<0.05).

Condition


% C

hang

e

0

2

4

6

8

10

12

14

16

18 POST1POST2

Figure 24 Relative percent changes in blood [H+]

Decrease in [H+] expressed as percent change. Total change has been proportioned into the respective changes for POST1 and POST2 periods.

*## #


65

Figure 25, (a) through (e), shows the mean blood K+ response for each

condition separately. Again, the ↓ symbol identifies the time at which the

nadir PRE values occurred. The number (e.g., 0.29) above the arrow

represents the magnitude of change expressed in mmol.l-1. The most rapid

ingestion regime, bolus, induced what seems to be a two-step K+ response.

There was a slight decline from PRE [K+] (4.03±0.10 mmol.l-1) for the first 2.5

hours post ingestion (3.96±0.09 mmol.l-1) and then a further decline that

peaked at 6 hours post ingestion (3.74±0.09 mmol.l-1). The overall decline in

[K+] was only slight at 0.29 mmol.l-1 compared with the other IR. The

remaining IR (Figure 25 c, d and e) resulted in a steady decline in [K+] across

time peaking at 2.5, 4.5 and 5.5 hours post ingestion, respectively. The

maximum decline in [K+] for each IR (bolus, 300, 600 and 900 mg.min-1) was

0.29, 0.44, 0.54 and 0.50 mmol.l-1, respectively. Figure 25 (f) compares both

average pre-ingestion (PRE) and 1.5-8 hours post ingestion (POST) K+ values

for each condition. No differences were identified between PRE values, mean

4.06±0.04 mmol.l-1. As indicated by * 300 (3.68±0.09 mmol.l-1), 600

(3.74±0.09 mmol.l-1) and 900 (3.74±0.09 mmol.l-1) IR reduced blood POST

[K+] significantly lower than their respective PRE (4.08±0.09 mmol.l-1) values

(p<0.05). Although all three rates were lower, the 300 IR POST [K+] was

significantly lower (p=0.11) than either the 600 or 900 IR POST [K+], as

represented by #. Figure 26 shows the average blood [K+] at three time

points: PRE, 1.5 h post ingestion (POST1) and 2.5 – 5.5 h post ingestion

(POST2). At no time were control (mean 4.06±0.08 mmol.l-1) and bolus

(mean 3.93±0.08 mmol.l-1) [K+] different. As represented by *, only the 300

POST1 IR decreased [K+], (3.66±0.08 mmol.l-1), lower than all three control

time points (4.01±0.11, 4.20±0.08, 4.10±0.08 mmol.l-1) (p=0.06). In

addition, 300, 600 and 900 POST2 [K+] were lower than their respective PRE

values, as indicated by # (p<0.05).

Figure 27 shows the relative change in blood [K+] for both time periods,

POST1 and POST2. There was a larger variation, almost five percent,

between control K+ values than what was seen with H+, however, this was not

significant. With the exception of the bolus and 600 IR, a large proportion of

total change from PRE K+ values, 300–83 percent and 900–86 percent,

occurred after 1.5 hours post ingestion. Although large changes occurred

with IR 600 and 900, it would appear this has been exacerbated by PRE K+

values (4.13±0.09 and 4.09±0.09, respectively), which were approximately 3


66

percent higher than the other IR. Even taking this into consideration, the

combination of the 600 IR and longer ingestion time induced a further 28

percent change in [K+].

Time (hr)

0 1 2 3 4 5 6 7 8

[K+ ] (

mm

ol.l-1

)

0.0

3.43.53.63.73.83.94.04.14.24.34.44.5

Control

Time (hr)

0 1 2 3 4 5 6 7 8

[K+ ] (

mm

ol.l-1

)

0.0

3.4

3.5

3.6

3.7

3.8

3.9

4.0

4.1

4.2

4.3

4.4

4.5

Bolus

Time (hr)

0 1 2 3 4 5 6 7 8

[K+ ] (

mm

ol.l-1

)

0.0

3.4

3.5

3.6

3.7

3.8

3.9

4.0

4.1

4.2

4.3

4.4

4.5

300

Time (hr)

0 1 2 3 4 5 6 7 8

[K+ ] (

mm

ol.l-1

)

0.0

3.4

3.5

3.6

3.7

3.8

3.9

4.0

4.1

4.2

4.3

4.4

4.5

600

Time (hr)

0 1 2 3 4 5 6 7 8

[K+ ] (

mm

ol.l-1

)

0.0

3.4

3.5

3.6

3.7

3.8

3.9

4.0

4.1

4.2

4.3

4.4

4.5

900

Condition


[K+ ] (

mm

ol.l-1

)

0.0

3.43.53.63.73.83.94.04.14.24.34.44.5

PREPOST

Figure 25 Mean Blood K+ temporal profiles for all conditions

Mean (±SEM) blood K+ response for each condition separately (a) – (e). Time at peak change is marked with ↓. The number above ↓ represents magnitude of change in mmol.l-1. Mean (±SEM) PRE and POST values are compared in (f). IR 300, 600 and 900, marked *, reduced POST [K+] lower than their respective PRE values (p<0.05). In addition, 300 IR, marked #, [K+] was lower than both 600 and 900 IR (p=0.11).

*#* *

0.54

0.50

0.44

0.29

(a)

(e)

(d)

(b)

(c)

(f)


67

Condition


Bloo

d [K

+ ] (m

mol

.l-1)

0.03.53.63.73.83.94.04.14.24.34.44.5

PREPOST1POST2

Figure 26 Mean (± SEM) blood [K+] for each condition at 3 time points

Collection time was divided into 3 periods, pre-ingestion (PRE), 1.5 h post-ingestion (POST1) and 2.5–6 h post-ingestion (POST2). 300 mg.min-1 POST1 IR, marked *, reduced [K+] below all control values (p=0.06). 300, 600 and 900 POST2 IR, marked #, reduced [K+] lower than their respective PRE values (p<0.05).

Condition


% c

hang

e

-5

0

5

10

15

20

POST1POST2

Figure 27 Relative percent changes in blood [K+]

Decrease in [K+] expressed as percent change. Total change has been proportioned into the respective changes for POST1 and POST2 periods. 83, 28 and 86 percent of the decrease in [K+] occurred during POST2 for 300, 600 and 900 IR, respectively.

*

# # #


68

When comparing 1.5 h post-ingestion and control values (Figure 28), blood

[Cl-] was unaffected by the ingestion of sodium citrate independent of IR.

When exploring the Cl- response temporally (Figure 29) it would seem there

was a negative time effect independent of the treatment (sodium citrate

ingestion). To test this further, the data were separated into three time

periods, pre-ingestion (PRE), 2.5-5.5 h post-ingestion (POST1) and 7.0-8.0 h

post-ingestion (POST2) and then compared. It was revealed that after 2.5 h

of ingestion PRE blood [Cl-], 98.2±0.4 mmol.l-1, significantly declined to

94.9±0.4 mmol.l-1 (p<0.05), however this level was maintained (95.0±0.4

mmol.l-1) across the remaining collection period (Figure 30).

Condition


Cl- (m

mol

.l-1)

0

90

92

94

96

98

100

Figure 28 Mean (±SEM) blood [Cl-] response for each condition

SC ingestion had no effect on mean [Cl-] response for each condition.


69

Time (h)

0 1 2 3 4 5 6 7 8

Cl- (m

mol

.l-1)

0

90

92

94

96

98

100


Figure 29 Mean Cl- temporal response for each condition

No ingestion rate effect on [Cl-], however there appears to be a negative time effect.

Time

PRE POST1 POST2

Cl- (m

mol

.l-1)

0

90

92

94

96

98

100

Figure 30 Mean (±SEM) blood [Cl-] response at 3 time points

Collection time was divided into 3 periods, pre-ingestion (PRE), 2.5–5.5 h post-ingestion (POST1) and 7.0–8.0 h post-ingestion (POST2). A negative time effect is apparent by POST1, marked *, and is maintained through POST2 (p<0.05).

As shown in Figure 31, blood [Na+] was elevated above control values

(130.9±0.5 mmol.l-1) only when sodium citrate was ingested at a rate of 300

mg.min-1 (133.6±0.5 mmol.l-1) (p<0.05).

* *


70

Figure 32 shows the temporal response for each condition. The large

variability within and between conditions greatly affected the ability to

demonstrate differences. Although there were differences between some

conditions, they appeared not to be systematic.

Condition


Na+ (m

mol

.l-1)

0

130

132

134

136

138

140

Figure 31 Mean (±SEM) blood [Na+] response for all conditions

SC ingested at 300 mg.min-1, marked *, increased [Na+] above control values (p<0.05).

Time (h)

0 1 2 3 4 5 6 7 8

Na+ (m

mol

.l-1)

010

130

132

134

136

138


Figure 32 Mean blood [Na+] temporal response for all conditions

The large variability (not shown) within and between IR greatly affected the identification of any differences.

*


71

pO2 To ensure the arterialisation status was consistent across all samples and

therefore had no confounding influence, pO2 was measured in every sample.

The mean [PO2] across all conditions was 71.3±3.5 mmHg. [PO2] was not

different between conditions as shown in Figure 33.

Condition


pO2 (

mm

Hg)

0

50

55

60

65

70

75

80

85

90

Figure 33 Mean (±SEM) blood PO2 response for all conditions

Stable arterialisation was maintained across all conditions, no differences were observed.

Urine The urine pH response was quite marked and is depicted in Figure 34. All

sodium citrate ingestion regimes increased urine pH (p<0.05). This increase

was detectable one hour after the initiation of ingestion. Again, the bolus

regime altered urine pH to the greatest extent earliest, increasing pH by 32

percent compared with 21, 24 and 21 percent for 300, 600 and 900 mg.min-1,

respectively, within the first hour. A further 6, 19, 15 and 17 percent

increase in pH for bolus, 300, 600 and 900 mg.min-1, respectively, brought

each condition to its maximum where they remained up to seven hours post-

ingestion.


72

Time (h)

0 1 2 3 4 5 6 7 8

Urin

e pH

0.00

5.00

6.00

7.00

8.00

9.00

Control Bolus 300600900

Figure 34 Mean (±SEM) urine pH temporal response for each condition

All IR, indicated by *, alkalised urine pH above control (p<0.05). Bolus, 300, 600 and 900 IR increased urine pH by 38, 40, 39 and 38 percent, respectively.

Discussion This is the first study to compare four ingestion rates of 300 mg.kg-1 body

mass of tri-sodium citrate on blood and urine pH in humans at rest. The

present study supports the hypothesis that consuming 300 mg.kg-1 of sodium

citrate in bolus form is not the optimal regime to perturb blood pH at rest.

The greatest blood alkalosis, 7.473 pH units, was achieved by ingesting

sodium citrate at a rate of 300 mg.min-1. Secondly, this study supports the

hypothesis that SC ingestion, independent of IR, induces a clearly measurable

urine alkalosis. The mean urine alkalosis across all experimental conditions

was approximately 40 percent (range 38–40 percent) larger than control.

pO2 status Heating of the hand, by immersing it in 460 C water, increases blood flow via

the dilation of local vasculature. In doing so, low oxygenated venous blood is

mixed with higher oxygenated arterial blood, which is reflected by a [pO2]

approximately midway between venous and arterial blood. This technique

allows the investigator to obtain close-to arterial blood values without the

complications of arterial punctures. To ensure the same level of arterial-

*


73

venous mixing was achieved, the [pO2] was recorded for each sample. The

[pO2], mean 71.3±3.5 mmHg, was not different between any of the conditions

(Figure 33), thus confirming that any differences observed in other measured

ions were not an artefact of the pO2 status.

Sodium citrate and GI absorption In the current study, bolus ingestion was associated with a blood [H+]

decrease from 4.0×10-8 meq.l-1 to 3.57×10-8 meq.l-1 at rest. The observed

decrease in [H+] is greater than reported in the literature (Kowalchuk et al.,

1989), however this can be explained by the timing of these observations.

The lower [H+] in the present study was recorded at 6 h post-ingestion

compared with only 1 h for the above-mentioned study. The 1 h [H+] for both

studies is comparable. Of the other ingestion rates investigated, 300 mg.min-

1 was associated with the largest effect and continued to decrease [H+]

beyond that experienced during the bolus regime to 3.37×10-8 meq.l-1. This

further 45 percent change in blood [H+] may be linked to a number of

explanations.

The first of these may lie in how the alkali is introduced to the body, i.e., via

the GI tract. This method relies upon the components of the GI tract to

transport the alkali through the intestinal wall across the interstitial space and

finally into the blood via the capillary network of the villi. In the current

study, the bolus regime combined the sodium citrate with a low energy

flavoured drink of 400 ml in an attempt to disguise the taste and make

ingestion more palatable. This technique has been previously reported in the

literature (Ibanez et al., 1995; McNaughton & Cedaro, 1992). The 30 g.l-1

carbohydrate concentration contained within the solution probably slows

gastric emptying from the stomach into the duodenum in comparison to the

300 mg.min-1 regime, which has no carbohydrate content. The work of

Erskine and Hunt (1981), which demonstrated that sodium citrate given in 6

small doses entered the duodenum 200 percent faster than 100 g.l-1 of

glucose, may support this theory. Therefore, it appears that small doses of

sodium citrate taken in quick succession enter the small intestine faster than

one large dose. This is especially true when using a bolus dose in combination

with carbohydrate.


74

Carbohydrate content and osmolality can affect absorption through the small

intestine (Vist & Maughan, 1995). Vist and Maughan (1995) demonstrated

that a higher concentration of carbohydrate slowed emptying from the small

intestine. In the present study, the carbohydrate concentration for the bolus

regime and the 300, 600 and 900 mg.min-1 regimes collectively were 30 g.l-1

and 0 g.l-1, respectively. Therefore, it is reasonable to assume that combining

sodium citrate with carbohydrate may slow its absorption through the small

intestine. Based on the assumption that 100 ml of water was consumed with

each sodium citrate capsule, the calculated osmolality for the bolus ingestion

regime and the 300, 600 and 900 mg.min-1 regimes collectively were 171.7

and 4.1 mosmol.kg-1, respectively. As suggested by Vist and Maughan

(1995), the solution osmolality may play an additional role in emptying from

the small intestine. That is, the higher the osmolality, the slower the solution

will be emptied from the intestine. Consequently, the bolus ingestion regime

seems to have two properties inhibiting its movement and uptake through the

stomach and small intestine. These inhibiting properties may mediate a loss

of sodium citrate uptake under the bolus regime resulting in a decreased

effect on blood pH. In contrast, the 300 mg.min-1 ingestion regime creates a

better environment for passage through the GI tract and into the blood, which

is supported by the increased blood pH response.

No subject in the current study exhibited GI upsets, which is consistent with

other studies using sodium citrate (Schabort et al., 2000; Linossier et al.,

1997; Hausswirth et al., 1995). The use of sodium bicarbonate, in contrast,

has often led to GI upsets (McNaughton, 1992a; Goldfinch et al., 1988;

Wilkes et al., 1983). This problem is probably a function of the mechanisms

through which the substances act. While the exact mechanism explaining

how sodium citrate increases pH is not well understood (Potteiger et al.,

1996b), studies using sodium bicarbonate may help to explain this

phenomenon, as it has similar physiological effects to sodium citrate (Spiegal,

1997; Tiryaki & Atterbom, 1995; Parry-Billings & MacLaren, 1986). Citrate is

not an alkali per se (Tiryaki & Atterbom, 1995), rather it produces sodium

bicarbonate via its hepatic oxidation (Halperin, 1982). Consequently, and in

contrast to sodium bicarbonate, the alkalising effect of citrate is not apparent

until after it has left the stomach, which most likely decreases the risk of

nausea and vomiting.


75

Sodium citrate and acid-base balance Sodium citrate ingestion was associated with an elevated blood pH across all

conditions. This finding is consistent with other research (van Someren et al.,

1998; Ball & Maughan, 1997) that has investigated the effects of SC ingestion

at a similar dose. Analysis of the blood pH temporal response showed that all

ingestion rates had similar effects (Figure 15). The two most common

methods for ingesting sodium citrate have been diluting it in a solution of

300-500 ml (Hausswirth et al., 1995; Ibanez et al., 1995; Kowalchuk et al.,

1989) or in gelatine capsules with water ad libitum (Potteiger et al., 1996b).

Typically, the ingestion period is 90 min with only pre-ingestion and pre-

exercise pH values reported. Therefore, it appears that a) there is a lack of

understanding relating to the most beneficial ingestion regime and b) that the

general assumption is that after 90 min the alkali has had enough time to

induce a complete physiological effect and cause an ergogenic response. The

present study demonstrated that ingesting SC at 300 mg.min-1 induced a

greater alkalosis than the bolus regime commonly practiced (7.473 and 7.447

pH units, respectively). Further, at 90 min post-ingestion, pH had only

partially alkalised the blood with peak alkalosis occurring some 60 and 120

min later for bolus and 300 mg.min-1, respectively. Therefore, if the degree

of alkalosis is in part responsible for the ergogenic effect of SC, it should be

consumed at a rate of 300 mg.min-1 and the impending performance should

not be initiated less than 3.5 h post-ingestion.

The metabolic alkali load of 70.4 mmol (mean total ingestion 20.7 g sodium

citrate) was associated with increases in HCO3- and pCO2 concentrations of

3.47 mmol.l-1 and 3.15 mmHg, respectively. These values are similar to

those found in other investigations (Hausswirth et al., 1995; Tiryaki &

Atterbom, 1995; McNaughton, 1990). When comparing the four ingestion

rates, the most rapid ingestion rate, bolus, resulted in a corresponding

increase in blood [HCO3-]. Under these conditions blood [HCO3

-] was elevated

above control after 30 min. In contrast, the blood pH response was not

different from control until 2 h post-ingestion. These inconsistent findings

suggest that different mechanisms are involved in the regulation of each ion.


76

Ingestion time and [H+] The times at which peak [H+] changes occurred were also different between

conditions. Some researchers (Potteiger et al., 1996a; Cox & Jenkins, 1994;

McNaughton, 1990) have assumed that 90 minutes post ingestion was a

sufficient waiting time to obtain the physiological benefits from alkali loading,

while others have suggested longer periods up to 3 h (Potteiger et al., 1996b;

Heigenhauser & Jones, 1991). This study demonstrated that for sodium

citrate ingested in bolus and at 300, 600 and 900 mg.min-1 only a partial

physiological response is achieved at 90 minutes post ingestion (Figure 24).

Peak changes in [H+] ranged from 3.5 h-6.0 h for 300 mg.min-1 and bolus,

respectively. These additional ingestion periods resulted in [H+] decreasing a

further 15–20 percent. The implications of a further reduction in [H+] on

exercise performance are unknown and further research is warranted to

establish this. Nevertheless, McNaughton (1990) showed a positive

relationship between sodium citrate and performance for three doses of

sodium citrate, 300, 400 and 500 mg.kg-1. Associated with these observed

performance increases was a continual decline in blood [H+]. Consequently,

the degree of decreased [H+] induced may be linked to the ergogenic

potential of sodium citrate. Numerous studies have shown no ergogenic

effect of alkalosis (Schabort et al., 2000; Tiryaki & Atterbom, 1995; Cox &

Jenkins, 1994; Kozak-Collins et al., 1994; Gaitanos et al., 1991), even with

significant decreases in blood [H+]. The current study suggests that a longer

ingestion time would have led to a greater alkalosis and possibly the

observation of an ergogenic effect.

Sodium citrate and [K+] This is the first study to examine the effect of SC ingestion on the blood K+

response in humans. An interesting observation was that SC ingestion in

bolus form had no effect on blood [K+]. In contrast, all capsule ingestion

rates (slower) were associated with decreases in blood [K+] approximating 0.5

mmol.l-1. The magnitude of these responses are similar to those recently

reported by Lindinger et al. (1999). Moreover, the ingestion regime they

employed was similar to the 300 mg.min-1 used here. Although their

investigation used sodium bicarbonate to manipulate plasma ion regulation,

other investigations have shown that the physiological effects of sodium

citrate and sodium bicarbonate are similar (Tiryaki & Atterbom, 1995) and


77

therefore comparable. Even though the effects of SC on blood [K+] are not

fully understood, it has been suggested that the NaHCO3- alkalosis decreases

extracellular [K+] via an increased intracellular [Na+] activated Na+-K+-ATPase

activity (Clausen, 1986). Accordingly, the current venous data supports this

potential role with an increased blood [Na+] when ingesting at 300 mg.min-1

(Figure 31). Unfortunately, it is impossible to support or refute the work of

others without corresponding muscle and blood arterial data. Furthermore,

venous blood values often vary due to the rapidity of gas and ion exchange

processes in skeletal muscle (Lindinger et al., 1999) and should therefore be

interpreted carefully. To elucidate the mechanisms underpinning K+

regulation, further research incorporating the above-mentioned techniques is

thus required. Still, the present data does indicate that ingesting SC in

capsules at a rate of 300, 600 or 900 mg.min-1 can manipulate blood [K+].

Sodium citrate and urinary alkalosis The aim of collecting and analysing urine pH hourly was to establish whether

the ingestion of 300 mg.kg-1 sodium citrate was associated with a urinary

alkalosis of a sufficient magnitude to be identified. The mean urine pH at rest

was 6.45±0.65 and is consistent with the literature (Lindinger et al., 2000;

Cogan et al., 1990; Oster et al., 1988; Kachadorian & Johnson, 1970).

Sodium citrate ingestion was associated with a significant elevation in urine

pH (mean 7.68±0.26), which was evident 1 h post-ingestion and remained

elevated above resting urine pH for a further 7 h. The data collected in this

study offers no explanation for the mechanisms involved in this increase;

however, work done by Oster et al. (1988) may provide some insight. They

compared net acid excretion after chronically loading with both sodium citrate

and sodium bicarbonate for four days. The effects they demonstrated on

urine pH were quite modest in comparison with the current study, but this can

probably be explained by the dose of 2.55 mEq.h-1 (calculated based on 61.2

mEq.day-1) compared with (at least) 70.4 mEq.h-1 here. Nevertheless, they

showed that associated with an alkalotic urine pH, NH4, TA (titratable acid)

and NAE (net acid excretion) were all decreased, while HCO3- excretion was

also increased (Oster et al., 1988). An unexpected result in the current study

was the marked similarity in urine pH response between ingestion rates

despite the marked differences in the rate and magnitude of disturbances

observed in the blood.


78

Conclusion The present study demonstrated that ingesting 300 mg.kg-1 of sodium citrate

at a rate of 300 mg.min-1 induced a greater degree of blood alkalosis than the

commonly practiced bolus regime. In addition, a 90 min ingestion time

resulted in only a partial blood alkalosis with the complete blood alkalosis

achieved at 3.5 and 6.0 h post-ingestion for 300 mg.min-1 and bolus ingestion

rates, respectively. Finally, the ingestion of 300 mg.kg-1 sodium citrate,

independent of ingestion rate, can be easily detected via the analysis of urine

pH.

Chapter 5 – Interstitial Alkalosis

79

CH A P T E R 5 EF F E C T O F S O D I U M C I T R A T E O N

I N T E R S T I T I A L PH I N H U M A N S K E L E T A L M U S C L E

Introduction Ingestion of sodium citrate improves exercise performance across a variety of

modalities (Shave et al., 2001; Potteiger et al., 1996a; McNaughton &

Cedaro, 1991; Gao et al., 1988). Although there has been an abundance of

research in this area, the precise mechanisms underpinning this phenomenon

are still not well understood (Hollidge-Horvat et al., 2000). These studies

have been performed on whole body exercise. There has been little research

performed on the effect of alkali ingestion on small muscle mass. Webster et

al. (1990) showed no effect of sodium bicarbonate ingestion on resistance

exercise performance. Further, there have been no investigations on single

leg quadriceps muscle performance. Due to it well controlled nature, this

latter type of exercise model may be a better model to provide new

information about the mechanisms underpinning the ergogenic properties of

orally induced alkalosis.

Currently, it is known that sodium citrate ingestion is associated with an

increase in blood pH and [HCO3-] (Schabort et al., 2000). It has been

suggested that the increased pH (or decreased [H+]) associated with alkalosis

amplifies the gradient between muscle and blood, thereby augmenting proton

efflux from the muscle (in both H-La and H+ forms) (Tiryaki & Atterbom,

1995; Lindinger et al., 1990; Mainwood & Warsley-Brown, 1975). However,

this theory does not take into account any involvement of the interstitial

compartment that protons pass through to enter the blood. Some studies

have referred to both the blood and interstitial compartments collectively as

extracellular (Brien & McKenzie, 1989; Mainwood & Warsley-Brown, 1975).

While this remains true (they are both outside the muscle cell), these

compartments possess markedly different characteristics and may thus

exhibit different reactions to alkalosis. As there has been limited research

performed on skeletal muscle interstitium during exercise, little is known

about its function and reaction to introduced substances. Recently, however,

Maclean et al. (2000) demonstrated that interstitial pH increases during

isometric exercise. This finding is somewhat difficult to explain, but


80

nonetheless demonstrated that blood and interstitium respond differently

during exercise and therefore should be considered as separate entities. In

addition, it has been shown that the buffer capacities of interstitium and blood

are different (Aukland & Reed, 1993). Although the osmolalities are similar

between blood (306 mosmol.l-1) and interstitium (308 mosmol.l-1), the ionic

composition is not. In particular, the interstitial concentrations of Cl-, HCO3-

and Na+ are all higher than blood. In contrast, there are no proteins found

within the interstitium (Geers & Gros, 2000). Since sodium citrate ingestion

has been linked to increases in blood [HCO3-] (Potteiger et al., 1996a), it may

be possible that the different initial [HCO3-] are influenced in different ways.

Similarly, since the ionic composition is different between these

compartments and alkali ingestion has been shown to influence ionic

composition within blood (Lindinger et al., 2000), it seems reasonable to

assume that induced alkalosis will affect the interstitium dissimilar to blood.

Two factors that mediate changes in pH are HCO3- and pCO2 (Lindinger et al.,

1990). It has been suggested that the sarcolemma is impermeable to HCO3-

(Robin, 1961), and as such metabolic alkalosis has no direct influence on the

intracellular buffer capacity. On the other hand, there appears to be no such

evidence for the movement of HCO3- into the interstitium. Further, pCO2 can

easily diffuse across membranes between compartments (Geers & Gros,

2000; Kowalchuk & Scheuermann, 1995). Since metabolic alkalosis is

associated with elevated blood HCO3-, and it is likely that HCO3

- is capable of

moving between the blood and interstitium, it stands that the effects of

alkalosis may be different within the two compartments. As a result, it is

possible that the suggested augmentation of intracellular H+ efflux associated

with alkalosis is part of a two-tiered process, the first between muscle and

interstitium and the second between interstitium and blood.

Of late, Maclean et al. (2000) have measured interstitial pH during static

quadriceps exercise in humans using microdialysis. However, there were

methodological limitations in their study that resulted in measurements at

minute intervals only. At the time, there was no technique available to

examine changes in interstitial pH during dynamic exercise in humans that

was capable of detailed analysis of short-term exercise. A technique to

accomplish this has been successfully developed and performed as part of this


81

thesis (see Chapter 3). Thus, it is now possible to investigate the role of

interstitial pH during exercise.

As outlined above, the aims of this study were to determine the effects of

metabolic alkalosis on human skeletal muscle interstitial pH at rest and during

intense exercise. The specific hypotheses tested were that sodium citrate

ingestion would increase knee-extensor exercise performance and increase

interstitial pH.

Methods

Subjects Five human subjects participated in this study. The mean age of the subjects

was 31±10 years with mean heights, weights and BMI’s of 180.8±5.4 cm,

81.7±11.1 kg, and 25.0±3.1, respectively. All subjects were active individuals

with no health-related problems. Prior to the start of the experiment, each

subject was informed of any risks and discomforts related to the experiment.

All subjects signed a written consent form prior to the experiments. The

study was approved by the local ethics committee (August Krogh Institute,

University of Copenhagen) and conformed to the Declaration of Helsinki.

Exercise protocol Subjects performed one-legged knee-extensor exercise in a supine position

and were secured via a series of straps, two shoulder, one waist and one

thigh strap, so exercise was restricted to the quadriceps muscle (Bangsbo et

al., 1990). During exercise the subjects had visual feedback in the form of a

digital display showing the cadence and power output. A 10 min warm-up at

an intensity of 10 W was performed prior to the experimental test. The

experimental test required subjects to maintain a cadence of 60 rpm for as

long as possible at a pre-determined exercise intensity. Subjects were given

verbal encouragement to perform, however, the test was terminated when

the cadence was below 60 rpm for more than 15 consecutive seconds.


82

Experimental exercise intensity The experimental exercise intensity was determined via an incremental test

followed by two constant load exercise tests, each separated by 48 h. All

tests were preceded by a 10 min warm-up at an intensity of 10 W followed by

a five minute rest. The incremental test was initiated at 10 W and increased

at a rate of 10 W at the end of each 5 min period until the subject could no

longer maintain a cadence of 60 rpm. Subjects were given verbal

encouragement, however, the test was terminated when the cadence was

below 60 rpm for 15 consecutive seconds. To ascertain the final intensity for

the constant load test, the last incomplete 10 W increment was proportioned

over the 5 min time period. That is, each minute of the stage was equivalent

to a two watt increase. Therefore, if the subjects completed 2 min of an

incremental period, a further 4 W was added to their previously completed

stage. Subjects then returned to the laboratory and performed a constant-

load exercise test at this calculated intensity to ensure the intensity was of a

sufficient magnitude to cause fatigue between 5 and 7 min. This was

repeated on a second occasion for reliability. A second repeat (third test) was

performed in the event that the above objectives were not achieved.

Probe insertion Prior to the experiment, subjects rested in a supine position with their legs

well supported by a chair. Each microdialysis probe was inserted after the

subject was given approximately 1 ml of 20 g.l-1 xylocaine via a 25-gauge

needle at the insertion site. An 18-gauge cannula was first passed through

the skin and fascia to make way for the probe. A second cannula containing

the microdialysis probe (CMA-60, CMA Microdialysis AB, Sweden) was then

pushed through the skin and fascia and orientated along the length of the

fibres of the vastus lateralis muscle. The cannula was removed leaving the

microdialysis probe within the muscle. After insertion of the probe, it was

secured with tape and the outlet cut at a maximal length of 10 mm from the

skin. The subjects recovered for 1.5 h after probe insertion before any

measurements were performed.

Perfusate The pH-sensitive fluorescent dye BCECF was coupled to dextran (molecular

mass cut off at 70000 Da), which prevented any diffusion of dye across the


83

probe membrane (cut off at a molecular mass of 20000 Da). The dye (0.1 mg

ml.l-1) was dissolved in a sterile saline solution (154 mmol.l-1 Na+). The

perfusate was then placed into a sterile 1 ml syringe equipped with a filter,

mounted in a microdialysis pump and connected to the inlet of the

microdialysis probe. The outlet from the probe was removed, replaced with a

stainless steel tube and connected to a micro flow-through cuvette (total

volume 8 µl) in a fluorescence spectrophotometer (Hitachi F-2000, Japan).

The pump rate was 5 µl.min-1 in all experiments. The time scales on the

figures were corrected for the delay due to the volume of tubing and cuvette.

Fluorometric measurements and determination of pH With the emission wavelength constant at 530 nm, the fluorescence



nm was insensitive to pH, but dependent on the amount of dye, whereas the

excitation intensity at 500 nm was also sensitive to pH. Thus, the excitation

intensity ratio 500/440 was proportional to pH and independent of changes in

dye concentration and thereby insensitive to any water movements from or

into the probe. The temperature in the fluorescence spectrophotometer was

kept constant by circulating thermostatically controlled water. For calibration,

a microdialysis probe was placed in a beaker with magnetic stirring and

connected to a pump and the fluorometer. The beaker contained saline (154

mM Na-1) and bicarbonate (25 mM), and the pH was monitored with a

laboratory pH meter. The pH in the beaker was changed in a stepwise

manner by adding HCl/NaOH and the excitation ratio was recorded. A

calibration curve was obtained by plotting the excitation intensity ratio versus

external pH. The constants obtained from a linear regression to the

calibration curve were used to convert fluorescent signals obtained in human

experiments to interstitial pH.

Blood collection and analysis After initial weighing, each subject rested in a seated position for a period of

10-min. Subjects’ cephalic vein was then cannulated with a 23-gauge needle

(Becton Dickinson, Germany) and attached to a 3-way tap (Terumo,

Belgium). Five millilitres of venous blood was removed as discard and then a


84

2-ml venous sample was obtained. The sample was immediately analysed for

acid-base (pH, pCO2, HCO3-) and pO2 status. The sample remained on ice

before a duplicate measurement was performed. To maintain cannula

patency and plasma volume, an equivalent amount of 0.9 percent saline was

re-infused after each sample.

Ingestion The initiation of ingestion followed the resting measurements. The subjects

were required to ingest either 300 mg.kg-1 of tri-sodium citrate (Sigma

Chemicals - Na3C8H5O7.H2O) at a rate of 300 mg.min-1 or calcium carbonate

matched for the number of capsules. Subjects consumed the sodium citrate

in hand-filled ‘OO’ size gelatine capsules with water ad libitum. Each capsule

was weighed before and after being filled to determine the exact quantity of

sodium citrate contained within the capsule (mean 1200 mg.capsule-1). This

mass was divided by 300 to establish the timing for ingestion of each capsule.

After the ingestion process was completed, the subjects were encouraged to

drink water ad libitum across the duration of the experiment. The subjects

consumed no solid food during testing. The warm-up exercise intensity of 10

W was initiated 150 min after the start of ingestion. The order of ingestion

was randomised in a double-blind fashion.

Results Mean±SEM values for blood and interstitial pH, blood gases and bicarbonate at

rest (time 0), during (30-150 min) and after (151-196.4 min) ingestion of

placebo (CaCO3) and alkalosis (Na3C8H5O7) are shown in Table 2.

Interstitial pH Mean resting interstitial pH was 7.38±0.12 and 7.24±0.16 for placebo and

alkalosis, respectively. Sodium citrate ingestion had no alkalising effect on

interstitial pH. Intense exercise was associated with a decrease (p<0.05) in

placebo interstitial pH that commenced after 2 minutes of exercise (Figure

35). Interstitial pH continued to decline throughout the exercise bout and

peaked at three and two minutes after the cessation of exercise, for placebo

and alkalosis, respectively. The magnitude of change in interstitial pH from

rest was 0.59 and 0.29 pH units for placebo and alkalosis, respectively.


85

Placebo interstitial pH was not different from resting values within 7.5 minutes

of recovery.

Time

rest

ing

30in

g 60

ing

90in

g 12

0in

g 15

010

W 1

10 W

210

W 5

10 W

10

10 W

R 1

10 W

R 2

10 W

R 5

10 W

R 1

0ex

0.7

5ex

1.5

ex

2ex

3ex

4ex

5ex

hre

c 1

rec

2re

c 3

rec

4re

c 5

rec

7.5

rec

10re

c 15

Inte

rstit

ial p

H

0.0

6.6

6.7

6.8

6.9

7.0

7.1

7.2

7.3

7.4

7.5

7.6 Placebo Alkalosis

Figure 35 Interstitial pH temporal response

Mean±SEM interstitial pH temporal response for both conditions, placebo (CaCO3) and alkalosis (Na3C8H5O7.H2O). There was no effect of alkalosis on interstitial pH. Intense exercise reduced placebo interstitial pH below (p<0.05) resting placebo values, marked *.

* * *

* * * * * *

*


86

Table 2 Blood acid-base and interstitial pH values with ingestion of CaCO3 (placebo) and Na3C8H5O7 (alkalosis)

pHb HCO3- pCO2 pO2 pHi

Time (min) Placebo

±SEM Alkalosis ±SEM

Placebo ±SEM

Alkalosis ±SEM

Placebo ±SEM

Alkalosis ±SEM

Placebo ±SEM

Alkalosis ±SEM

Placebo ±SEM

Alkalosis ±SEM

Pre-Ingestion 0.00 7.367±0.015 7.367±0.015 26.7±0.6 26.2±0.6 47.9±1.2 46.7±1.2 42.2±4.5 38.5±4.5 7.375±0.118 7.236±0.155

30.00 7.359±0.015 7.362±0.015 26.7±0.6 25.9±0.6 48.2±1.2 47.0±1.2 43.5±4.5 34.9±4.5 7.366±0.118 7.182±0.155 60.00 7.355±0.015 7.366±0.015 26.8±0.6 27.2±0.6 49.0±1.2 49.4±1.2 35.5±4.5 30.4±4.5 7.352±0.118 7.183±0.155 90.00 7.358±0.015 7.372±0.015 27.2±0.6 27.7±06 48.8±1.2 50.6±1.2 39.7±4.5 30.1±4.5 7.331±0.118 7.253±0.155 120.00 7.369±0.015 7.400±0.015 27.2±0.6 28.5±0.6 48.9±1.2 48.6±1.2 41.2±4.5 32.2±4.5 7.311±0.118 7.161±0.155

Ingestion

150.00 7.381±0.015 7.411±0.015 26.5±0.6 29.4±0.6*# 44.3±1.2 48.5±1.2 53.5±4.5 33.6±4.5 7.304±0.118 7.145±0.155

151.00 7.376±0.015 7.417±0.015 27.8±0.8 30.6±0.6*# 48.9±1.7 49.2±1.2 54.7±6.4 33.8±4.5 7.270±0.118 7.152±0.155 152.00 7.374±0.015 7.416±0.015 28.4±0.6 30.8±0.6*# 51.5±1.2 49.7±1.2 37.6±4.5 32.1±4.5 7.282±0.118 7.151±0.155 155.00 7.373±0.015 7.410±0.015 28.6±0.6 31.3±0.6*# 51.7±1.2 51.5±1.2 31.4±4.5 24.2±4.5 7.263±0.118 7.135±0.155

10 W Exercise

160.00 7.372±0.017 7.410±0.017 28.8±0.9 31.3±0.6*# 52.2±1.7 51.7±1.2 32.3±6.4 25.5±4.5 7.307±0.158 7.097±0.159

161.00 7.374±0.015 7.412±0.015 28.1±0.6 30.8±0.6*# 51.1±1.2 51.7±1.2 36.3±4.5 28.3±4.5 7.309±0.158 7.084±0.159 162.00 7.379±0.015 7.415±0.015 28.2±0.6 31.4±0.6*# 51.3±1.2 51.9±1.2 35.8±4.5 28.6±4.5 7.314±0.158 7.080±0.159 165.00 7.374±0.015 7.416±0.015 28.0±0.6 30.9±0.6*# 51.3±1.2 50.4±1.2 35.2±4.5 33.0±4.5 7.252±0.118 7.131±0.155 Recovery

170.00 7.375±0.015 7.418±0.015 28.2±0.6 31.5±0.6*# 50.8±1.2 51.1±1.2 35.9±4.5 30.9±4.5 7.250±0.118 7.124±0.155

170.75 7.379±0.015 7.417±0.015 28.5±0.6 31.1±0.6*# 50.5±1.2 50.6±1.2 40.5±4.5 31.5±4.5 7.248±0.154 7.180±0.118 171.50 7.380±0.015 7.419±0.015 28.4±0.6 31.3±0.6*# 50.3±1.2 50.7±1.2 42.6±4.5 335±4.5 7.246±0.118 7.170±0.118 172.00 7.372±0.015 7.413±0.015 28.4±0.6 31.6±0.6*# 51.1±1.2 51.8±1.2 37.2±4.5 29.6±4.5 6.955±0.118 7.154±0.118 173.00 7.365±0.015 7.406±0.015 27.9±0.6 31.2±0.6*# 50.4±1.2 52.0±1.2 36.9±4.5 29.5±4.5 6.913±0.118 7.123±0.118 174.00 7.368±0.017 7.397±0.017 28.0±0.6 31.0±0.6*# 51.6±1.2 51.6±1.2 30.7±4.5 27.1±4.5 6.875±0.118 7.091±0.118 175.00 7.355±0.017 7.399±0.017 27.5±0.6 30.5±0.6*# 52.1±1.2 51.1±1.2 30.0±4.5 29.0±4.5 6.828±0.118 7.054±0.118

Intense Exercise

181.4±0.9 7.344±0.017 7.382±0.024 25.4±0.9 28.4±0.9 50.3±1.8 49.0±1.8 35.1±6.6 24.0±6.9 6.794±0.118* 6.978±0.118*

182.40 7.341±0.015 7.394±0.015 22.0±0.6* 25.8±06*# 44.1±1.2 44.0±1.2 49.7±4.5 36.6±4.5 6.791±0.118* 6.969±0.118* 183.40 7.339±0.015 7.387±0.015 19.2±0.6* 22.6±0.6*# 38.8±1.2* 39.2±1.2* 69.6±4.5* 61.5±4.5* 6.793±0.118* 6.950±0.118* 184.40 7.334±0.015 7.380±0.017 18.3±0.6* 22.2±0.6*# 37.6±1.2* 39.0±1.2* 78.2±4.5* 64.8±4.6* 6.787±0.118* 6.956±0.118* 185.40 7.334±0.015 7.378±0.017 19.0±0.6* 22.3±0.8*# 39.4±1.2* 38.2±1.7* 61.0±4.5* 70.5±6.4* 6.800±0.118 7.016±0.155 186.40 7.339±0.015 7.377±0.017 18.6±0.6* 22.6±0.6*# 38.8±1.2* 39.9±1.2* 65.6±4.5* 62.4±4.5* 6.799±0.118* 7.007±0.118* 188.90 7.341±0.017 7.388±0.017 19.1±0.6* 23.1±0.6*# 39.1±1.2* 39.8±1.2* 62.0±4.5* 59.5±4.5* 7.023±0.154 7.074±0.118 191.40 7.350±0.017 7.404±0.017 19.6±0.6* 22.8±0.6*# 39.1±1.2* 37.3±1.2* 62.1±4.5* 73.0±4.5* 7.080±0.154 7.128±0.118

Recovery

196.40 7.368±0.017 7.409±0.017 21.2±0.6* 25.3±0.6*# 40.3±1.2* 41.4±1.2* 53.1±4.5 48.6±4.5 7.118±0.154 7.177±0.118

Blood acid-base and interstitial pH changes before (time 0), during (30–150 min) and after (170–196.4 min) ingestion of CaCO3 and Na3C8H5O7. Values are mean±SEM; n=5. HCO3

- concentrations are in mmol.l-1, pCO2 and pO2 concentrations are in mmHg. * significantly different from time 0, p<0.05; # significantly different from Na3C8H5O7, p<0.05.


87

Venous pH and HCO3-

Mean venous pH was 7.362±0.003 and 7.398±0.003 for placebo and alkalosis,

respectively. Sodium citrate ingestion (300 mg.kg-1) was not associated with

a significant elevation in venous blood pH (p=0.19) above placebo (Figure

36). Although the desired statistical power was 0.8, the mean effect size in

combination with a subject pool of five resulted in a calculated statistical

power of 0.18. Nevertheless, inspection of Figure 37 does show an upward

trend in the blood pH response after ingestion of Na3C8H5O7. An exercise-

induced decrease in venous pH was identified (p=0.07) in the placebo trial,

however, this was not evident in the alkalosis trial (Figure 37)

Conditionplacebo alkalosis

Ven

ous

pH

0.00

7.30

7.32

7.34

7.36

7.38

7.40

7.42

7.44

Figure 36 Overall effect of sodium citrate ingestion on venous pH

Mean±SEM venous pH response for both conditions, placebo (CaCO3) and alkalosis (Na3C8H5O7.H2O).


88

Time

rest

ing

30in

g 60

ing

90in

g 12

0in

g 15

010

W 1

10 W

210

W 5

10 W

10

10 W

R 1

10 W

R 2

10 W

R 5

10 W

R 1

0ex

0.7

5ex

1.5

ex

2ex

3ex

4ex

5ex

hre

c 1

rec

2re

c 3

rec

4re

c 5

rec

7.5

rec

10re

c 15

Veno

us p

H

0.00

7.30

7.32

7.34

7.36

7.38

7.40

7.42

7.44Placebo Alkalosis

Figure 37 Venous pH temporal response

Mean±SEM blood pH temporal response for both conditions, placebo (CaCO3) and alkalosis (Na3C8H5O7.H2O).

Mean venous [HCO3-] was 25.5±0.2 and 28.1±0.2 for placebo and alkalosis,

respectively (Figure 38). Ingestion of 300 mg.kg-1 sodium citrate elevated

(p<0.05) venous [HCO3-] above placebo after 120 min of ingestion (Figure

39). The elevated [HCO3-] continued to be present for the remainder of the

experiment. Venous [HCO3-] was reduced (p<0.05) below resting values after

5 minutes of intense exercise in both conditions where it continued to

exhaustion. A further reduction in venous [HCO3-] was observed during

recovery that peaked at the third minute in both conditions. The recovery

period (15 minutes) was of insufficient length for [HCO3-] to return to resting

values in both conditions.


89

Conditionplacebo alkalosis

Ven

ous

[HC

O3- ] (

mm

ol.l-1

)

0.0

25.0

25.5

26.0

26.5

27.0

27.5

28.0

28.5

29.0

Figure 38 Overall effect of sodium citrate ingestion on venous [HCO3-]

Mean±SEM venous [HCO3-] response for both conditions, placebo (CaCO3)

and alkalosis (Na3C8H5O7). Sodium citrate ingestion increased (p<0.05) venous [HCO3

-] above placebo, marked *.

Time

rest

ing

30in

g 60

ing

90in

g 12

0in

g 15

010

W 1

10 W

210

W 5

10 W

10

10 W

R 1

10 W

R 2

10 W

R 5

10 W

R 1

0ex

0.7

5ex

1.5

ex

2ex

3ex

4ex

5ex

hre

c 1

rec

2re

c 3

rec

4re

c 5

rec

7.5

rec

10re

c 15

Veno

us [H

CO

3- ] (m

mol

.l-1)

0

16

18

20

22

24

26

28

30

32

34Placebo Alkalosis

Figure 39 Venous HCO3- temporal response

Mean±SEM venous [HCO3-] for both conditions, placebo (CaCO3) and

alkalosis (Na3C8H5O7.H2O) across time. Sodium citrate ingestion significantly elevated (p<0.05) venous [HCO3

-] above placebo after 120 min, marked *. This observation continued for the remainder of the experiment. After 5 min of intense exercise venous [HCO3-] was significantly reduced below rest values (p<0.05), marked #, and continued during exercise and recovery.

# # # # # #

#

* * * * * *

* * * * * * * * *

*

*

* * * * * *

*

*


90

Venous blood gases pO2 and pCO2 Mean venous [pO2] was 45.2±2.3 and 38.5±2.3 for placebo and alkalosis,

respectively (Figure 40).

Condition

placebo alkalosis

Ven

ous

[pO

2] (m

mH

g)

0

10

20

30

40

50

60

70

80

90

Figure 40 Venous pO2 status for both conditions placebo and alkalosis

Mean±SEM venous [pO2] for both conditions, placebo (CaCO3) and alkalosis (Na3C8H5O7.H2O). Outlier [pO2] are marked . Venous [pO2] was not different between conditions.

Time

rest

ing

30in

g 60

ing

90in

g 12

0in

g 15

010

W 1

10 W

210

W 5

10 W

10

10 W

R 1

10 W

R 2

10 W

R 5

10 W

R 1

0ex

0.7

5ex

1.5

ex

2ex

3ex

4ex

5ex

hre

c 1

rec

2re

c 3

rec

4re

c 5

rec

7.5

rec

10re

c 15

Ven

ous

[pO

2] (m

mH

g)

0

18

27

36

45

54

63

72

81

Placebo Alkalosis

Figure 41 Venous pO2 temporal response

Mean±SEM venous pO2 response for both conditions, placebo (CaCO3) and alkalosis (Na3C8H5O7.H2O) across time. Sodium citrate ingestion had no effect on venous [pO2], however, recovery from intense exercise was associated with an increase (p<0.05) in [pO2] across both conditions, marked *.

*

* *

* *

*


91

There was no effect of alkalosis on venous [pO2] collected from the forearm,

but there was a significant time-effect present during recovery from intense

exercise (p<0.05). Venous [pO2] was elevated above rest, ingestion and light

exercise during intense exercise recovery from 2 to 10 minutes (Figure 41).

Mean venous [pCO2] was 47.2±0.6 and 47.3±0.6 for placebo and alkalosis,

respectively. Alkalosis had no effect on forearm venous [pCO2]. However,

there was a significant (p<0.05) time effect present during recovery (Figure

42). Intense exercise was associated with a decreased venous [pCO2] from

rest (47.3±0.9 mmHg) and peaked after 10 minutes of recovery (38.2±0.9

mmHg).

Time

rest

ing

30in

g 60

ing

90in

g 12

0in

g 15

010

W 1

10 W

210

W 5

10 W

10

10 W

R 1

10 W

R 2

10 W

R 5

10 W

R 1

0ex

0.7

5ex

1.5

ex

2ex

3ex

4ex

5ex

hre

c 1

rec

2re

c 3

rec

4re

c 5

rec

7.5

rec

10re

c 15

Veno

us [p

CO

2] (m

mH

g)

0

36

39

42

45

48

51

54Placebo Alkalosis

Figure 42 Venous pCO2 temporal response

Mean±SEM venous pCO2 response for both conditions, placebo (CaCO3) and alkalosis (Na3C8H5O7.H2O) across time. The ingestion of sodium citrate (300 mg.kg-1) had no influence on [pCO2], however, recovery from intense exercise was associated with a decrease (p<0.05) in [pCO2] below rest values, marked *.

Time to exhaustion The mean time to exhaustion was 352 and 415 seconds for placebo and

alkalosis, respectively (Figure 43). Ingesting 300 mg.kg-1 of sodium citrate

was not associated with an increased time to exhaustion during knee-extensor

*

*

* * *

* * *


92

exercise. Table 3 shows the individual exhaustion times for both conditions.

The individual responses were considerably different between the subjects

ranging from a decrease of 17 percent to an improvement of 63 percent in

exercise time.

Subject Placebo Alkalosis Percent Change 1 390 394 1 2 230 190 -17 3 375 400 7 4 357 422 18 5 410 670 63

Mean±SEM 352±71 415±171 14

Table 3 Individual exhaustion times for intense knee-extensor exercise

Individual subject exhaustion times for both conditions, placebo (CaCO3) and alkalosis (Na3C8H5O7.H2O). Note the large variation in percent change, range –17 to 63 percent. Alkalosis did not improve exhaustion time.

Time to exhaustion

Condition

placebo alkalosis

Tim

e (s

ec)

0

300

325

350

375

400

425

450

475

500

Figure 43 Time to exhaustion for both placebo and alkalosis conditions

Mean±SEM exhaustion times for each condition, placebo and alkalosis. Sodium citrate ingestion (300 mg.kg-1) had no influence on exhaustion time.


93

Discussion This is the first study to investigate the effect of alkalosis, using sodium

citrate ingestion, on interstitial pH at rest and during exercise. The results of

the current study refute the hypothesis that sodium citrate ingestion improves

knee-extensor exercise performance in healthy individuals. The second

hypothesis that sodium citrate ingestion would increase interstitial pH was

also refuted by these findings.

Mean±SEM resting interstitial pH for all experiments was 7.37±0.01. This is

similar to that reported in the previous study in Chapter 3 (7.38±0.02 pH

units) and falls between venous pH values reported in the literature (7.37-

7.43) (Bangsbo et al., 2000; Sjogaard et al., 1985). In contrast, the

interstitial pH values from the current study are higher than those reported by

Maclean et al. (2000). In their study, dialysate pH was measured using pH

electrodes placed in the tubing on the outlet side of a microdialysis probe

during static exercise, resting dialysate pH was reported as 7.162±0.023.

Together, these studies form the collective sum of research that has

investigated interstitial pH during exercise in humans. Based on such limited

data, it is difficult to provide explanations of the differences observed between

investigations. However, there were important methodological differences

between these studies, which may provide some explanation. The first key

difference between the work of Maclean et al. (2000) was the removal of the

polyurethane outlet tubing and replacing it with stainless steel tubing. By

doing this, CO2 loss from the outlet side was prevented thereby controlling

any CO2 artefact on the measurement of dialysate pH. Secondly, and as

identified in Chapter 3, the addition of 25 mmol.l-1 HCO3- to the perfusate

without pH adjustment proved crucial in accurately determining interstitial pH

at rest and during exercise. However, to fully appreciate the relative

contributions these two factors have on the measurement of dialysate pH,

subsequent investigations need to be designed with these aims specifically in

mind.

Ingestion of sodium citrate (300 mg.kg-1) had no effect on blood pH at rest.

The mean±SEM venous blood pH response depicted in Figure 36 showed an

increase of 0.036 pH units. The magnitude of this response is lower than

previously demonstrated in Chapter 4 (0.052 pH units). The combination of

the lower response with a smaller sample size (n=5) may explain why


94

statistical significance was not achieved. Using these results the sample size

required to obtain significance at the 0.05 level was calculate at 12. The

smaller effects found in the current study are probably explained by the

method of blood collection. Venous samples were collected in the current

study while arterialised samples were obtained in the former. Resting venous

pH was 7.367±0.015 while resting arterialised pH was 7.397±0.003. These

differences can be explained by a larger [pCO2] in venous (47.3±1.2)

compared to arterialised (44.7±0.4) blood. Therefore, it is possible that the

reduced change in venous pH observed in the current study was being

partially obscured by the type of sampling and thus may be disguising the

sodium citrate effect on pH. Inspection of the data reported in the literature

revealed a large disparity of results. (McNaughton, 1990) reported mean

resting arterialised capillary venous pH values of approximately 7.15, in

contrast (Parry-Billings & MacLaren, 1986) reported mean resting capillary

venous pH values of approximately 7.41. These data in combination with the

varied ingestion rates, doses and blood sampling methods make it near

impossible to provide valid comparisons with the current data.

Sodium citrate ingestion failed to induce alkalosis within the interstitium at

rest. Mean±SEM interstitial pH was 7.304±0.118 and 7.145±0.155 for placebo

and alkalosis, respectively. The physicochemical approach to acid-base

control suggests, H+ and HCO3- are influenced by concentrations of pCO2,

weak and strong (fully dissociated) electrolytes (Stewart, 1983). Strong

electrolytes are represented by the strong ion difference (SID), which is the

difference between the sum of strong anions and cations (SID = ∑[strong

cations] - ∑[strong anions]) within the compartment. The major strong

electrolytes contributing to [SID+] in plasma are Na+, K+, Ca2+, Mg2+, Cl-,

SO42- and La-. In muscle the addition of PCr2- to the calculation is required

(Kowalchuk & Scheuermann, 1995). Presently, it is not possible to determine

all of these ion concentrations within the interstitial fluid using microdialysis or

any other techniques at rest and during exercise. Therefore, to explain the

findings of this study via this theory would be purely speculative.

Furthermore, attempting to make calculations based on this theory without all

of ion concentrations can lead to incorrect conclusions. Even ions in small

concentrations, such as Mg2+, can affect the final SID concentration and SID

has the largest effect on [H+] (Roger Fedde & Pieschl, 1995). If ions removed

from the calculation are in similar concentrations and of opposite polarity, the


95

error can be almost cancelled. Nevertheless, accuracy is compromised (Roger

Fedde & Pieschl, 1995). Therefore, it is beyond the realm of this investigation

to attempt to explain the results using the Stewart approach.

The metabolism of sodium citrate is not very well understood (Potteiger et al.,

1996b), however, it has been suggested that HCO3- is produced via its hepatic

oxidation and then added to the plasma (Halperin, 1982). Increasing plasma

[HCO3-] will reduce [H+] or increase pH via the Hendersen-Hasselbach

relationship. Therefore, it is reasonable to assume that this relationship

applies when inducing alkalosis via sodium citrate. Fluid compartments of the

body must obey the laws of electrical neutrality and conservation of mass

(Stewart, 1983). Blood and interstitial fluid are approximately osmotically

and electrically the same. However, since the interstitial fluid is protein free,

the ionic compositions differ somewhat between the two compartments

(Kowalchuk & Scheuermann, 1995). In particular, interstitial fluid has a

higher [Cl-], lower [Na+], [K+] and [SID] (Kowalchuk & Scheuermann, 1995).

The increased [HCO3-] will create a positive gradient between plasma and

interstitium, and since HCO3- can diffuse across the capillary wall, it should

theoretically lead to an increase in interstitial [HCO3-] until equilibrium is

attained. Currently, it is not possible to measure this during exercise in

humans. However, if HCO3- diffuses into the interstitium, it would decrease

[H+] and should thus be represented by a lowered pH. This was not

demonstrated in the current study. One possible explanation for this may be

linked to the HCO3- driving force between the plasma and interstitium.

Sodium citrate ingestion increased plasma [HCO3-] from 26.2 mmol.l-1 to

approximately 31.0 mmol.l-1. Since interstitial [HCO3-] is approximately 25

mmol.l-1 at rest (Geers & Gros, 2000), it may be that this gradient is

insufficient to induce movement into the interstitium. Even so, during intense

exercise (when efflux of H+ from the muscle cell to interstitium is high

(Pilegaard et al., 1999; Bangsbo et al., 1997; Bangsbo et al., 1993a) and

HCO3- is in buffering demand), both the electrical and concentration forces are

increased drawing HCO3- out of the plasma and into the interstitium. The

additional [HCO3-] could potentially buffer more H+, thereby delaying any

increase in [H+] or decrease in interstitial pH. This may explain the lack of an

exercise-induced acidosis observed during the alkalosis exercise period.


96

Another possible explanation could be related to both the Na+/H+ and HCO3-

/Cl- exchangers. These exchangers are approximately 20 percent responsible

for pH regulation at rest in skeletal muscle, but very little is known about their

degree of involvement during exercise (Juel, 1998a). One possibility, is that

under conditions of alkalosis these exchangers are upregulated due to

changes in their respective interstitial ionic components. It has been

suggested that alkalosis may be mediated via an increase in blood [Na+]

(Lindinger et al., 2000). This increased [Na+] may increase interstitial [Na+]

that may upregulate the Na+/H+ exchanger. Similarly, the increase in blood

[HCO3-] may lead to increases in interstitial [HCO3

-], which may have an

upregulatory effect on the HCO3-/Cl- exchanger. Although the data from the

current study does not indirectly support this change in interstitial [HCO3-],

via no change in interstitial pH, another electro-neutral bicarbonate exchanger

identified from animal studies may play a role (Putnam et al., 1986b).

However, very little is known about this exchangers function or importance.

New techniques need to be developed to quantify these ionic concentrations

and exchanger properties to increase the understanding of the function,

capacity and importance of these systems.

The current study did not support the hypothesis that ingestion of sodium

citrate (300 mg.kg-1) results in ergogenesis during intense knee-extensor

exercise. The mean±SEM time to exhaustion was 352±71 and 415±171

seconds for placebo and alkalosis, respectively. While this is consistent with

the findings of several others (Tiryaki & Atterbom, 1995; Ibanez et al., 1995;

Cox & Jenkins, 1994; Kowalchuk et al., 1989; Parry-Billings & MacLaren,

1986), it is worth noting the individual results as depicted in Table 3. The

mean increase in performance was 14 percent which was in contrast to the

findings of Spriet et al. (1991b), where they found only a modest 0.3 percent

mean effect. The range of percent change in performance was much larger

than anticipated (-17 to 63%). This, in itself, is probably the main reason for

the lack of statistical support for a overall ergogenic effect. To statistically

show an ergogenic effect of sodium citrate ingestion with the current

performance results a subject pool of 46 would be required. Even so, it is

difficult to overlook the large 18 and 63 percent improvement in performance

in two of the five subjects. Although it is beyond the scope of this

investigation to attempt to identify any characteristics that may separate


97

these two subjects from the rest, the next logical step would be to establish

the reasons for these individuals’ unique response.

Conclusion In conclusion, this is the first study to investigate the effects of alkalosis on

interstitial pH. The study demonstrated that sodium citrate ingestion (300

mg.kg-1) does not induce interstitial alkalosis, nor does it improve exhaustive

short-term knee-extensor performance. However, the results do suggest that

metabolic alkalosis is associated with an improved interstitial pH regulation,

as demonstrated by the lack of exercise-induced acidosis. Finally, future

research is required to investigate the mechanisms through which this

improved pH regulation may be mediated.

Chapter 6 – General Discussion

99

CH A P T E R 6 GE N E R A L D I S C U S S I O N

Introduction The previous individual discussions have focussed more or less exclusively on

the results and problems of the respective chapters. In contrast, this chapter

focuses on the experimental chapters as a whole in accordance with the

central research problem/s as defined in the preface and general introduction.

Summing up, the experiments contained in this thesis were designed to 1)

develop a new method to determine interstitial pH at rest and during exercise

in vivo, 2) discover a more effective pH-manipulating tool via the

identification of an optimal regime of sodium citrate ingestion, and finally, 3)

to combine the results of the above-mentioned experiments in an attempt to

add to the limited existing knowledge on interstitial pH. The central research

question of this thesis was thus to establish a technique for the manipulation

and detection of interstitial pH. As such, this is the focus of the discussion in

this chapter.

The importance of effective pH manipulation A key component or consideration when attempting to manipulate pH via

orally ingested substances is to prevent any negative side-effects. There

have been several studies that have reported symptoms of nausea and/or

vomiting after the ingestion of sodium bicarbonate (McNaughton & Cedaro,

1992; Goldfinch et al., 1988; Wilkes et al., 1983) or sodium citrate (Shave et

al., 2001; Potteiger et al., 1996a) in doses ranging from 300-500 mg.kg-1.

Although there have been investigations to the contrary where none of these

symptoms were evident (McNaughton, 1992a; Bouissou et al., 1988; Parry-

Billings & MacLaren, 1986), it would be logical to develop a regime that

minimises any potential risk of discomfort. Furthermore, in developing such a

regime, an important factor to obtain is the maximum benefit from the

substance introduced. Chapter 4 was designed to accommodate these two

criteria; through systematic investigation, an attempt was made to identify an

optimal ingestion regime for 300 mg.kg-1 sodium citrate.

Chapter 4 showed that ingestion of 300 mg.kg-1 SC was not associated with

nausea or vomiting independent of the four ingestion rates examined. The


100

maximum blood alkalosis observed was not associated with the bolus

ingestion regime most commonly used by researchers (Ibanez et al., 1995;

McNaughton & Cedaro, 1991). Of the three capsular ingestion rates

investigated, 300 mg.min-1 at the above-mentioned dose of SC was

associated with the greatest blood alkalosis. Since the method of orally

inducing alkalosis involves the GI system, it stands that to obtain maximum

absorption the ingestant should match (as closely as possible) the physical

capabilities of the GI system. This is not the case with the bolus ingestion

regime, and as such it is not surprising that the effect is less than that of the

capsular regimes. In addition to the way in which the alkali is consumed, and

important to the magnitude of effect, is the absorption time. Chapter 4 was

the first study to perform serial measurements of the effect of SC ingestion on

blood pH across 8 hours. This unique component of the study allowed for the

determination of the time it would take to reach the maximum alkalotic effect.

Some researchers have used an ingestion period of 90 minutes (Cox &

Jenkins, 1994), while others have suggested up to 3 h are required for

maximum benefits (Heigenhauser & Jones, 1991). In fact, Chapter 4 showed

that only a partial (75-80%) alkalosis was achieved 1.5 h post-ingestion, and

that a 6 h ingestion period was required to reach peak alkalosis. In contrast,

the 300 mg.min-1 rate reduced this period to 3.5 h.

The importance of interstitial pH Although little is known about the role of interstitial pH during exercise, it has

been suggested that it may be involved in the regulation of local blood flow

(Quayle et al., 1997). Furthermore, it has been suggested that interstitial pH

may affect the sensory response from muscle. This suggestion is drawn from

the findings of Victor et al. (1998), linking interstitial pH to sympathetic nerve

activity. In order to support these suggestions, it is necessary to quantify

changes within the interstitium. There are methods available to measure [H+]

in muscle and blood, however, due to technical difficulties, this had not been

the case for interstitial [H+]. Thus, until a method was successfully developed

to this end, no investigations could be performed to elucidate changes in

interstitial pH within skeletal muscle in vivo. Chapter 3 describes the

development of such a method. Furthermore, the results from this chapter

refute the suggestion that an interstitial alkalosis is present during exercise

(Maclean et al., 2000). Finally, using this method, interstitial pH was


101

successfully measured for the first time. The data from Chapter 3 supports

the contention that interstitial pH is involved in, but not solely responsible for,

the regulation of blood flow. The data revealed a second positive relationship

supporting interstitial pH’s involvement in the regulation of blood flow: the

first being between blood flow and power output (Radegran & Saltin, 1998),

and the second being between interstitial acidification and power output. In

addition, the data also revealed that interstitial pH is not the sole determinant

of blood flow. In support for this is the dissociation found between blood flow

and interstitial pH changes. Specifically, in chapter 3, interstitial pH

continually declined during five-minute knee-extensor exercise. In contrast,

Radegran & Saltin (1998) have shown that blood flow reaches a steady state

after approximately 1.5 minutes.

Using the method developed in Chapter 3 and the optimal ingestion regime

established in Chapter 4, Chapter 5 examined the manipulative potential of

sodium citrate on the interstitium at rest and during exercise. It was

expected that SC would be associated with an interstitial alkalosis at rest,

however the data from Chapter 5 does not support this. Two previous

investigations have postulated two distinctly different suggestions as to the

link between SC metabolism and an increase in plasma [HCO3-] (Kowalchuk et

al., 1989; Halperin, 1982). Nevertheless, the end result of increased [HCO3-]

is the same, which was further supported by the data in Chapter 5: both

plasma and interstitium are osmotically and electrically similar and therefore

the increase of a solute in plasma should result in an increased interstitial

concentration. It is reasonable to assume that if extra bicarbonate did move

into the interstitium, an increase in pH would occur similar to that found in

the blood. However, the data in Chapter 5 does once again not support this.

There appears to be no evidence to explain why the extra bicarbonate does

not equilibrate between the plasma and interstitium. One possible

explanation may lie within the equilibration driving force: SC ingestion

increased blood [HCO3-] from 26.2-31.0 mmol.l-1. In accordance, it has been

suggested that interstitial [HCO3-] is approximately 25 mmol.l-1 (Geers &

Gros, 2000), which may be insufficient to induce movement into the

interstitium.

Although there was no interstitial alkalosis induced prior to exercise, the data

from Chapter 5 suggests an improved pH regulation during exercise.


102

Evidence for this is the lack of an exercise-induced acidosis after the ingestion

of SC. This was an unexpected finding as alkali ingestion induces an alkalosis

(Chapter 4) followed by an exercise-induced acidosis blood response. It was

thought that a similar process would occur in the interstitium when, in fact,

there was no alkalosis followed by acidosis. This is an important finding as it

demonstrates that the two extracellular compartments perform differently.

Therefore, the data presented in Chapter 5 indicates that, where possible, the

two extracellular compartments should be treated as separate entities.

Limitations A major limitation in the measurement of interstitial pH using microdialysis

and BCECF is the demand on equipment. A fluorometer is required for each

probe within the thigh, which in a typical laboratory would limit the use to one

probe. An inherent problem with detecting interstitial pH via the use of only

one probe is the uncertainty with which it represents the whole muscle

interstitium. An additional disadvantage of using only one probe is that if is it

broken during intense contractions, it is impossible to perform accurate

determination of the calibration coefficients and thus a second separate probe

must be used. Further, to quantify the contribution of interstitial pH to

skeletal muscle function, other interstitial metabolites need to be measured.

These were omitted from the current investigations as no methods have been

established for the measurement of the interstitial ions, HCO3-, Ca2+, Mg2+,

and Cl-. When calculating the osmolalities of the different ingestion rates

(Chapter 4), the amount of water consumed with each capsule was not

measured, which may have influenced the results.

Chapter 7 – Conclusions and Future Research

103

CH A P T E R 7 CO N C L U S I O N S A N D F U T U R E

R E S E A R C H

Conclusions Through three collective original investigations, the present thesis has

demonstrated that interstitial pH can be accurately determined during

exercise in humans using microdialysis and the pH-sensitive dye BCECF. The

main advantage of this method is that data can be collected in real-time with

high resolution (10 Hz). The ability to generate data that accurately describes

short-term high intensity exercise where pH regulation is greatly challenged is

of the utmost scientific importance. When applying this newly developed

method, the examination of 5 min intense exercise revealed an exercise-

induced acidosis that was positively correlated with power output and greater

than a concomitant venous acidosis.

The second of the present investigations was designed to establish an optimal

protocol for the ingestion of an alkali (sodium citrate) to maximise the

disturbance to blood pH. This study demonstrated that consumption of an

alkali is an important factor to consider when attempting to maximise blood

alkalosis. In particular, the slowest ingestion rate examined, 300 mg.min-1,

was associated with the greatest change in blood pH from resting values.

This finding is particularly important for the applied setting, where induced

alkalosis has been shown to improve human performance. Although the exact

mechanistic link between alkalosis and ergogenesis has not been elucidated, it

appears that the ensuing alkalosis is in some way responsible. Therefore, a

maximisation of the alkalosis would probably serve to improve previously

observed effects on performance. Furthermore, inducing alkalosis can be a

powerful tool for exploring physiological phenomena potentially mediated via

acid-base changes.

The final study was designed to broaden the collective knowledge pertaining

to pH regulation by combining and applying the knowledge from the previous

two investigations. Specifically, this study explored the possibility of inducing

an interstitial alkalosis via ingestion of sodium citrate. Although this was not

achieved, it was demonstrated that sodium citrate ingestion was not


104

associated with an exercise-induced acidosis during intense knee-extensor

activity. This finding suggests that pH regulation is improved during exercise.

Future Research Although there has been an abundance of research investigating the effects of

alkali ingestion on human performance (Pfefferle & Wilkinson, 1988;

Robertson et al., 1987; McKenzie et al., 1986; Katz et al., 1984; McCartney et

al., 1983), the same level of interest has not been present for the metabolism

of alkalis. In particular, the exact mechanisms underlying the movement of

SC from the GI tract through to the observation of an alkalotic effect in the

blood have not been systematically explored. Two theories have been formed

to explain the association between blood alkalosis and SC ingestion: the

hepatic oxidation of citrate (Halperin, 1982) and an increase in [SID] as a

result of increased [citrate-] (Kowalchuk et al., 1989). Recently, Lindinger et

al. (1999) investigated the plasma and muscle ionic movements associated

with the ingestion of sodium and potassium bicarbonate. Central to their

findings were the contributions of sodium and potassium movements to the

corresponding alkaloses. From the data collected in Chapter 3, SC ingestion

at a rate of 300 mg.min-1 was associated with similar increases in sodium

concentration observed by Lindinger et al. (1999) in the SB condition. Due to

the disparity of the above-mentioned investigations, any conclusions

regarding the exact mechanisms of alkalosis would be pre-emptive. It is still

unknown to what degree each of these are responsible for the effect of

alkalosis, or if there is any degree of interaction between them. Thus new

studies need to be designed to quantify the contributions of each of these

mechanisms.

It has been suggested that alkalosis induced via the ingestion of SB is

mediated by decreases in extracellular [K+]. It was further suggested that

this decrease is brought about via an increased intracellular [Na+] activated

Na+-K+-ATPase activity (Lindinger et al., 1999; Clausen, 1986). The venous

data in Chapter 3 demonstrated an increased blood [Na+] when ingesting SC

at 300 mg.min-1, which may, in turn, support a similar mechanism of action to

SB when using SC. In order to support or refute these hypotheses, however,

corresponding muscle, interstitial and blood data are required. Accordingly,

to clarify the effect of alkalosis on potassium regulation, it is necessary to


105

perform complex and invasive experiments capable of obtaining the required

data.

The results of Chapter 4 have demonstrated that ingesting sodium citrate at

300 mg.min-1 has a greater effect on blood ionic status at rest. To examine if

this greater ionic effect translates to a greater effect on performance, new

studies can now be designed incorporating this ingestion regime and test its

effect on whole body performance. These studies would provide valuable

information for the coach and athlete in the applied setting by ascertaining

the value of this protocol to their needs. Further, Chapter 4 identified that the

popular bolus regime in combination with a 1.5 h ingestion time resulted in a

partial blood alkalosis. At present, it is unknown what performance effect can

be gained from the additional alkalosis obtained by an increased ingestion

time. It seems reasonable to assume that a greater alkalosis would result in

greater performance gains, requiring the longer ingestion time. Associated

with a longer ingestion time in the applied setting is the possible effect of food

ingestion during this time. It seems plausible that the ingestion of food

during ingestion may alter or interfere with the effects of induced-alkalosis.

There have been no investigations into these possibilities, such investigations

would provide further clarification on the efficacy of sodium citrate ingestion

in the applied setting.

As previously mentioned, there has been limited research performed on

skeletal muscle interstitium during exercise. This is largely due to technical

difficulties in developing reliable methods for the accurate detection of

candidates thought to be involved in regulatory processes. As a result little is

known about the role of the interstitium in muscle cell function. There is

evidence to suggest that interstitial pH may be involved in the regulation of

blood flow (Quayle et al., 1997; Davies, 1990). Data from Chapter 5

demonstrated that sodium citrate ingestion was associated with the lack of an

exercise-induced acidosis. Although there was no performance effect of

alkalosis, the data shows that a similar amount of muscular work was

performed without a significant disturbance to interstitial pH, suggesting an

improved interstitial pH regulation during exercise. It is beyond the scope of

these investigations to elaborate on the mechanisms behind this. However,

using the interstitial pH technique developed as part of this thesis in

combination with thermodilution techniques for the determination of blood


106

flow, it is now possible to perform an investigation that can determine if

interstitial pH is involved in the local regulation of blood flow during exercise

in humans.

References

107

RE F E R E N C E S

1. AALKJAER, C. & PENG, H.L. (1997). pH and smooth muscle. Acta Physiol.Scand. 161, 557-566.

2. AICKIN, C.C. & THOMAS, R.C. (1977). An investigation of the ionic mechanism of intracellular pH regulation in mouse soleus muscle fibres. J.Physiol. 273, 295-316.

3. ALLSOP, P., CHEETHAM, M., BROOKS, S., HALL, G.M. & WILLIAMS, C. (1990). Continuous intramuscular pH measurement during the recovery from brief, maximal exercise in man. Eur.J.Appl.Physiol.Occup.Physiol. 59, 465-470.

4. ARNOLD, D.L., TAYLOR, D.J. & RADDA, G.K. (1985). Investigation of human mitochondrial myopathies by phosphorus magnetic resonance spectroscopy. Ann.Neurol. 18, 189-196.

5. AUKLAND, K. & REED, R.K. (1993). Interstitial-lymphatic mechanisms in the control of extracellular fluid volume. Physiol.Rev. 73, 1-78.

6. BALL, D. & MAUGHAN, R.J. (1997). The effect of sodium citrate ingestion on the metabolic response to intense exercise following diet manipulation in man. Exp.Physiol. 82, 1041-1056.

7. BANGSBO, J., GOLLNICK, P.D., GRAHAM, T.E., JUEL, C., KIENS, B., MIZUNO, M. & SALTIN, B. (1990). Anaerobic energy production and O2 deficit-debt relationship during exhaustive exercise in humans. J.Physiol. 422, 539-559.

8. BANGSBO, J., JOHANSEN, L., GRAHAM, T. & SALTIN, B. (1993a). Lactate and H+ effluxes from human skeletal muscles during intense, dynamic exercise. J.Physiol. 462, 115-133.

9. BANGSBO, J., JOHANSEN, L., QUISTORFF, B. & SALTIN, B. (1993b). NMR and analytic biochemical evaluation of CrP and nucleotides in the human calf during muscle contraction. J.Appl.Physiol. 74, 2034-2039.

10. BANGSBO, J., JUEL, C., HELLSTEN, Y. & SALTIN, B. (1997). Dissociation between lactate and proton exchange in muscle during intense exercise in man. J.Physiol. 504, 489-499.

11. BANGSBO, J., KRUSTRUP, P., GONZALEZ-ALONSO, J., BOUSHEL, R. & SALTIN, B. (2000). Muscle oxygen kinetics at onset of intense dynamic exercise in humans. Am.J.Physiol.Regul.Integr.Comp.Physiol. 279, R899-R906

References

108

12. BANGSBO, J., MADSEN, K., KIENS, B. & RICHTER, E.A. (1996). Effect of muscle acidity on muscle metabolism and fatigue during intense exercise in man. J.Physiol. 495 , 587-596.

13. BELANGERO, V.M.S. & COLLARES, E.F. (1992). Gastric emptying and metabolic acidosis. II - Gastric retention of a sodium bicarbonate solution utilizing and experimental model in rats. Arq Gastoenterol 29(1), 23-27.

14. BIGLAND-RITCHIE, B., DONOVAN, F. & ROUSSOS, C.S. (1981). Conduction velocity and EMG power spectrum changes in fatigue of sustained maximal efforts. J.Appl.Physiol 51, 1300-1305.

15. BINDER, H.J., RANJENDRAN, V.M., & GIEBEL, J.P. (2000). Cl-dependent Na-H exchange. A novel colonic crypt transport mechanism. Ann. N.Y. Acad. Sci. 915, 43-53.

16. BIRD, S.R., WILES, J. & ROBBINS, J. (1995). The effect of sodium bicarbonate ingestion on 1500-m racing time. Journal of Sports Sciences 13, 399-403.

17. BLATZ, A.L. (1980). Chemical modifiers and low internal pH block inward-rectifier K channels. Fed Proc 39, 2073

18. BOUISSOU, P., DEFER, G., GUEZENNEC, C.Y., ESTRADE, P.Y. & SERRURIER, B. (1988). Metabolic and blood catecholamine responses to exercise during alkalosis. Medicine and Science in Sports and Exercise 20(3), 228-232.

19. BOYARSKY, G., GANZ, M.B., STERZEL, R.B., & BORON, W.F. (1988). pH regulation in single glomerular mesangial cells. II. Na+-dependent and –independent Cl--HCO3

- exchangers. Am. J. Physiol. 255, C857-C869.

20. BRIEN, D.M. & MCKENZIE, D.C. (1989). The effect of induced alkalosis and acidosis on plasma lactate and work output in elite oarsmen. Eur.J.Appl.Physiol.Occup.Physiol. 58, 797-802.

21. BRODY, T.M. & AKERA, T. (1977). Relations among Na+,K+-ATPase activity, sodium pump activity, transmembrane sodium movement, and cardiac contractility. Fed. Proc. 36, 2219-2224.

22. BUSSE, M.W., MAASSEN, N. & BONING, D. (1989). The calculation of the osmotic volumes of distribution of hypertonic sodium bicarbonate and other hypertonic solutions: a theoretical approach. Journal of Medicine 20, 143-161.

23. BUSSE, M.W., SCHOLZ, J. & MAASSEN, N. (1992). Plasma potassium and ventilation during incremental exercise in humans: modulation by

References

109

sodium bicarbonate and substrate availability. Eur.J.Appl.Physiol.Occup.Physiol. 65, 340-346.

24. CASTLE, N.A. & HAYLETT, D.G. (1987). Effect of channel blockers on potassium efflux from metabolically exhausted frog skeletal muscle. J.Physiol London 383, 31-43.

25. CLAUSEN, T. (1986). Regulation of active Na+ -K+ transport in skeletal muscle. Physiological Reviews 66, 542-580.

26. CLAUSEN, T., EVERTS, M.E. & KJELDSEN, K. (1987). Quantification of the maximum capacity for active sodium-potassium transport in rat skeletal muscle. J.Physiol. 388, 163-181.

27. COGAN, M.G., CARNEIRO, A.V., TATSUNO, J., COLMAN, J., KRAPF, R., MORRIS, R.C.J. & SEBASTIAN, A. (1990). Normal diet NaCl variation can affect the renal set-point for plasma pH-(HCO3-) maintenance. J.Am.Soc.Nephrol. 1, 193-199.

28. COSTILL, D.L., BARNETT, A., SHARP, R., FINK, W.J. & KATZ, A. (1983). Leg muscle pH following sprint running. Med.Sci.Sports Exerc. 15, 325-329.

29. COSTILL, D.L., VERSTAPPEN, F., KUIPERS, H., JANSSEN, E. & FINK, W. (1984). Acid-base balance during repeated bouts of exercise: influence of HCO3. Int.J.Sports Med. 5, 228-231.

30. COX, G. & JENKINS, D.G. (1994). The physiological and ventilatory responses to repeated 60 s sprints following sodium citrate ingestion. J.Sports Sci. 12, 469-475.

31. DAVIES, N.W. (1990). Modulation of ATP-sensitive K+ channels in skeletal muscle by intracellular protons. Nature 343, 375-377.

32. DENNING, H., TALBOT, J.H., EDWARDS, H.T. & DILL, D.B. (1931). Effects of acidosis and alkalosis upon the capacity for work. Journal of Clinical Investigations 9, 601-613.

33. DONALDSON, S.K.B. & HERMANSEN, L. (1978). Differential, direct effects of H+ on Ca2+-activated force of skinned fibers from the soleus, cardiac and adductor magnus muscles of rabbits. Pflugers Archives 376, 55-65.

34. DUNN, J.F. & WALLEY, G.J. (1991). Renal regulation in hypertension. Biochem. Soc. Trans. 19, 421S.

35. EIAM-ONG, S. & SABATINI, S. (1996). Biochemical mechanisms and regulation of hydrogen transport in renal tubules. Mineral and Electrolyte Metabolism 22, 366-381.

References

110

36. ERSKINE, L. & HUNT, J.N. (1981). The gastric emptying of small volumes given in quick succession. Journal of Physiology 313, 335-341.

37. FABATIO, A. & FABATIO, F. (1978). Effects of pH on the myofilaments and the sarcoplasmic reticulum of skinned cells from cardiac and skeletal muscles. Journal of Physiology 276, 233-235.

38. FALLENTIN, N., JENSEN, B.R., BYSTROM, S. & SJØGAARD, G. (1992). Role of potassium in the reflex regulation of blood pressure during static exercise in man. Journal of Physiology 451, 643-651.

39. FITTS, R.H. & BALOG, E.M. (1996). Effect of intracellular and extracellular ion changes on E-C coupling and skeletal muscle. Acta Physiol. Scand. 156, 169-181.

40. FRASER, S.F. & MCKENNA, M.J. (1998). Measurement of Na+, K+-ATPase activity in human skeletal muscle. Analytical Biochemistry 258, 63-67.

41. GAITANOS, G.C., NEVILL, M.E., BROOKS, S. & WILLIAMS, C. (1991). Repeated bouts of sprint running after induced alkalosis. J.Sports Sci. 9, 355-370.

42. GAO, J., COSTILL, D.L., HORSWILL, C.A. & PARK, S.H. (1988). Sodium bicarbonate ingestion improves performance in interval swimming. European Journal of Applied Physiology and Occupational Physiology 58, 171-174.

43. GEERS, C. & GROS, G. (2000). Carbon dioxide transport and carbonic anhydrase in blood and muscle. Physiol.Rev. 80, 681-715.

44. GOLDFINCH, J., MC, N.L. & DAVIES, P. (1988). Induced metabolic alkalosis and its effects on 400-m racing time. Eur.J.Appl.Physiol.Occup.Physiol. 57, 45-48.

45. GOO, R.H., MOORE, J.G., GREENBERG, E. & ALZRAKI, N.P. (1987). Circadian variation in gastric emptying of meals in man. Gastroenterology 92(5), 1408-1408.

46. GREEN, H., DAHLY, A., SHOEMAKER, K., GOREHAM, C., BOMBARDIER, E. & BALL-BURNETT, M. (1999). Serial effects of high-resistance and prolonged endurance training on Na+-K+ pump concentration and enzymatic activities in human vastus lateralis. !Lost Data 165, 177-184.

47. GREEN, H.J., CHIN, E.R., BALL-BURNETT, M. & RANNEY, D. (1993). Increases in human skeletal muscle Na(+)-K(+)-ATPase concentration with short-term training. Am.J.Physiol 264, C1538-C1541

References

111

48. GREEN, S., BULOW, J. & SALTIN, B. (1999). Microdialysis and the measurement of muscle interstitial K+ during rest and exercise in humans. J.Appl.Physiol. 87, 460-464.

49. GREENHAFF, P.L., GLEESON, M. & MAUGHAN, R.J. (1987). The effects of dietary manipulation on blood acid-base status and the performance of high intensity exercise. Eur.J.Appl.Physiol.Occup.Physiol. 56, 331-337.

50. GREENHAFF, P.L., GLEESON, M. & MAUGHAN, R.J. (1988). The effects of diet on muscle pH and metabolism during high intensity exercise. Eur.J.Appl.Physiol.Occup.Physiol. 57, 531-539.

51. GROSSIE, J., COLLINS, C. & JULIAN, M. (1988). Bicarbonate and fast-twitch muscle: evidence for a major role in pH regulation. J.Membr.Biol. 105, 265-272.

52. GUYTON, A.C. (1991). Textbook of medical physiology. Philadelphia: W.B. Saunders Company.

53. HALPERIN, M.L. (1982). Metabolism and acid-base physiology. Artifical Organs 6, 357-361.

54. HATTNER, R.S. (1991). Determining gastric emptying rate. The Journal of Nuclear Medicine 32(10), 2024-2025.

55. HAUPTFLEISCH, J.J. & PAYNE, K.A. (1996). An oral sodium citrate-citric acid non-particulate buffer in humans. British Journal of Anaesthesia 77, 642-644.

56. HAUSSWIRTH, C., BIGARD, A.X., LEPERS, R., BERTHELOT, M. & GUEZENNEC, C.Y. (1995). Sodium citrate ingestion and muscle performance in acute hypobaric hypoxia. Eur.J.Appl.Physiol.Occup.Physiol. 71, 362-368.

57. HEBESTREIT, H., MEYER, F., HTAY-HTAY, HEIGENHAUSER, G.J.F. & BAR-OR, O. (1996). Plasma metabolites, volume and electrolytes following 30-s high-intensity exercise in boys and men. European Journal of Applied Physiology and Occupational Physiology 72, 563-569.

58. HEIGENHAUSER, G.J.F. & JONES, N.L. (1991). Bicarbonate Loading. In Perspectives in exercise science and sports medicine, eds. LAMB, D.R. & WILLIAMS, M.H., pp. 183-212. Wm. C. Brown.

59. HEIGENHAUSER, G.J.F., JONES, N.L., KOWALCHUK, J.M. & LINDINGER, M.I. (1990). The role of the physicochemical systems in plasma in acid-base control in exercise. In Biochemistry of Exercise VII, Anonymouspp. 359-374. Illinois: Human Kinetics.

References

112

60. HERMANSEN, L. (1979). Effect of acidosis on skeletal muscle performance during maximal exercise in man. Bull.Eur.Physiopathol.Respir. 15, 229-241.

61. HERMANSEN, L. (1981). Effect of metabolic changes on force generation in skeletal muscle during maximal exercise. Ciba.Found.Symp. 82, 75-88.

62. HOLLIDGE-HORVAT, M.G., PAROLIN, M.L., WONG, D., JONES, N.L. & HEIGENHAUSER, G.J. (1999). Effect of induced metabolic acidosis on human skeletal muscle metabolism during exercise. Am.J.Physiol. 277, E647-E658

63. HOLLIDGE-HORVAT, M.G., PAROLIN, M.L., WONG, D., JONES, N.L. & HEIGENHAUSER, G.J. (2000). Effect of induced metabolic alkalosis on human skeletal muscle metabolism during exercise. Am.J.Physiol.Endocrinol.Metab. 278, E316-E329

64. HOPFER, U. & LIEDTKE, C.M. (1987). Proton and bicarbonate transport mechanisms in the intestine. Annu. Rev. Physiol. 49, 51-67.

65. HORSWILL, C.A., COSTILL, D.L., FINK, W.J., FLYNN, M.G., KIRWAN, J.P., MITCHELL, J.B. & HOUMARD, J.A. (1988). Influence of sodium bicarbonate on sprint performance: relationship to dosage. Med.Sci.Sports Exerc. 20, 566-569.

66. IBANEZ, J., PULLINEN, T., GOROSTIAGA, E., POSTIGO, A. & MERO, a. (1995). Blood lactate and ammonia in short-term anaerobic work following induced alkalosis. J.Sports Med.Phys.Fitness. 35, 187-193.

67. ILUNDAIN, A. (1992). Intracellular pH regulation in intestinal and renal epithelial cells. Comp. Biochem. Physiol. Comp. Physiol. 101, 413-424.

68. INESI, G. & HILL, T.L. (1983). Calcium and proton dependence of sarcoplasmic reticulum ATPase. Biophys.J. 44, 271-280.

69. IWAOKA, K., OKAGAWA, S., MUTOH, Y. & MIYASHITA, M. (1989). Effects of bicarbonate ingestion on the respiratory compensation threshold and maximal exercise performance. Jpn.J.Physiol. 39, 255-265.

70. JONES, N.L., SUTTON, J.R., TAYLOR, R. & TOEWS, C.J. (1977a). Effect of pH on cardiorespiratory and metabolic responses to exercise. J.Appl.Physiol 43, 959-964.

71. JONES, N.L., SUTTON, J.R., TAYLOR, R. & TOEWS, C.J. (1977b). Effect of pH on cardiorespiratory and metabolic responses to exercise. J.Appl.Physiol 43, 959-964.

References

113

72. JUEL, C. (1986). Potassium and sodium shifts during in vitro isometric muscle contraction, and the time course of the ion-gradient recovery. Pflugers Archives 406, 458-463.

73. JUEL, C. (1995). Regulation of cellular pH in skeletal muscle fiber types, studied with sarcolemmal giant vesicles obtained from rat muscles. Biochim.Biophys.Acta 1265, 127-132.

74. JUEL, C. (1996). Lactate/proton co-transport in skeletal muscle: regulation and importance for pH homeostasis. Acta Physiol.Scand. 156, 369-374.

75. JUEL, C. (1997). Lactate-proton cotransport in skeletal muscle. Physiol.Rev. 77, 321-358.

76. JUEL, C. (1998a). Muscle pH regulation: role of training. Acta Physiol.Scand. 162, 359-366.

77. JUEL, C. (1998b). Skeletal muscle Na+/H+ exchange in rats: pH dependency and the effect of training. Acta Physiol.Scand. 164, 135-140.

78. JUEL, C., BANGSBO, J., GRAHAM, T. & SALTIN, B. (1990). Lactate and potassium fluxes from human skeletal muscle during and after intense, dynamic, knee extensor exercise. Acta Physiol.Scand. 140, 147-159.

79. JUEL, C., KRISTIANSEN, S., PILEGAARD, H., WOJTASZEWSKI, J. & RICHTER, E.A. (1994). Kinetics of lactate transport in sarcolemmal giant vesicles obtained from human skeletal muscle. J.Appl.Physiol. 76, 1031-1036.

80. JUEL, C. & PILEGAARD, H. (1998). Lactate/H+ transport kinetics in rat skeletal muscle related to fibre type and changes in transport capacity. Pflugers Arch. 436, 560-564.

81. JUEL, C., PILEGAARD, H., NIELSEN, J.J. & BANGSBO, J. (2000). Interstitial K(+) in human skeletal muscle during and after dynamic graded exercise determined by microdialysis. Am.J.Physiol.Regul.Integr.Comp.Physiol.2000.Feb.;278.(2.):R400.-6. 278, R400-R406

82. KACHADORIAN, W.A. & JOHNSON, R.E. (1970). Renal responses to various rates of exercise. Journal of Applied Physiology 28, 748-752.

83. KATZ, A., COSTILL, D.L., KING, D.S., HARGREAVES, M. & FINK, W.J. (1984). Maximal exercise tolerance after induced alkalosis. Int.J.Sports Med. 5, 107-110.

References

114

84. KEMP, G.J., THOMPSON, C.H., SANDERSON, A.L. & RADDA, G.K. (1994). pH control in rat skeletal muscle during exercise, recovery from exercise, and acute respiratory acidosis. Magn.Reson.Med. 31, 103-109.

85. KJELDSEN, K. (1991). Muscle Na,K-pump dysfunction may expose the heart to dangerous K levels during exercise. Canadian Journal of Sport Science 16, 33-39.

86. KOWALCHUK, J.M., HEIGENHAUSER, G.J.F. & JONES, N.L. (1984). Effect of pH on metabolic and cardiorespiratory responses during progressive exercise. Journal of Applied Physiology 57(5), 1558-1563.

87. KOWALCHUK, J.M., MALTAIS, S.A., YAMAJI, K. & HUGHSON, R.L. (1989). The effect of citrate loading on exercise performance, acid-base balance and metabolism. Eur.J.Appl.Physiol.Occup.Physiol. 58, 858-864.

88. KOWALCHUK, J.M. & SCHEUERMANN, B.W. (1995). Acid-base balance: origin of plasma [H+] during exercise. Canadian Journal of Applied Physiology 20(3), 341-356.

89. KOZAK-COLLINS, K., BURKE, E.R. & SCHOENE, R.B. (1994). Sodium bicarbonate ingestion does not improve performance in women cyclists. Med.Sci.Sports Exerc. 26, 1510-1515.

90. KRAPF, R. (1989) Physiology and molecular biology of the renal Na/H antiporter. Klin. Wochenschr. 67, 847-851.

91. KUU, W.Y., CHILAMKURTI, R. & CHEN, C. (1998). Effects of relative humidity and temperature on moisture sorption and stability of sodium bicarbonate powder. International Journal of Pharmaceutics 166, 167-175.

92. LAMBERT, C.P., GREENHAFF, P.L., BALL, D. & MAUGHAN, R.J. (1993). Influence of sodium bicarbonate ingestion on plasma ammonia accumulation during incremental exercise in man. Eur.J.Appl.Physiol.Occup.Physiol. 66, 49-54.

93. LAUF, P.K. (1987). Physiology and biophysics of chloride and cation transport. Fed Proc 46, 2377-2394.

94. LIEDTKE, C.M. (1989). Regulation of chloride transport in epithelia. Annu. Rev. Physiol. 51, 143-160.

95. LINDINGER, M.I., FRANKLIN, T.W., LANDS, L.C., PEDERSEN, P.K., WELSH, D.G. & HEIGENHAUSER, G.J. (1999). Role of skeletal muscle

References

115

in plasma ion and acid-base regulation after NaHCO3 and KHCO3 loading in humans. Am.J.Physiol. 276, R32-R43

96. LINDINGER, M.I., FRANKLIN, T.W., LANDS, L.C., PEDERSEN, P.K., WELSH, D.G. & HEIGENHAUSER, G.J. (2000). NaHCO(3) and KHCO(3) ingestion rapidly increases renal electrolyte excretion in humans. J.Appl.Physiol. 88, 540-550.

97. LINDINGER, M.I. & HEIGENHAUSER, G.J. (1991). The roles of ion fluxes in skeletal muscle fatigue. Can.J.Physiol.Pharmacol. 69, 246-253.

98. LINDINGER, M.I., HEIGENHAUSER, G.J., MCKELVIE, R.S. & JONES, N.L. (1992). Blood ion regulation during repeated maximal exercise and recovery in humans. Am.J.Physiol. 262, R126-R136

99. LINDINGER, M.I., HEIGENHAUSER, G.J. & SPRIET, L.L. (1990). Effects of alkalosis on muscle ions at rest and with intense exercise. Can.J.Physiol.Pharmacol. 68, 820-829.

100. LINDINGER, M.I., MCKELVIE, R.S. & HEIGENHAUSER, G.J. (1995). K+ and Lac- distribution in humans during and after high-intensity exercise: role in muscle fatigue attenuation? J.Appl.Physiol. 78, 765-777.

101. LINOSSIER, M.T., DORMOIS, D., BREGERE, P., GEYSSANT, A. & DENIS, C. (1997). Effect of sodium citrate on performance and metabolism of human skeletal muscle during supramaximal cycling exercise. Eur.J.Appl.Physiol.Occup.Physiol. 76, 48-54.

102. MACDONALD, I. (1957) Gastric activity during the menstrual cycle. Gastroenterology 30, 602-607.

103. MACLEAN, D.A., BANGSBO, J. & SALTIN, B. (1999). Muscle interstitial glucose and lactate levels during dynamic exercise in humans determined by microdialysis. J.Appl.Physiol. 87, 1483-1490.

104. MACLEAN, D.A., IMADOJEMU, V.A. & SINOWAY, L.I. (2000). Interstitial pH, K(+), lactate, and phosphate determined with MSNA during exercise in humans. Am.J.Physiol.Regul.Integr.Comp.Physiol.2000.Mar.;278.(3.):R563.-71. 278, R563-R571

105. MACLEAN, D.A., LANOUE, K.F., GRAY, K.S. & SINOWAY, L.I. (1998). Effects of hindlimb contraction on pressor and muscle interstitial metabolite responses in the cat. J.Appl.Physiol. 85, 1583-1592.

References

116

106. MAINWOOD, G.W. & RENAUD, J.M. (1985). The effect of acid-base balance on fatigue of skeletal muscle. Can.J.Physiol.Pharmacol. 63, 403-416.

107. MAINWOOD, G.W. & WARSLEY-BROWN, P. (1975). The effects of extracellular pH and buffer concentration on the efflux of lactate from frog sartorius muscle. Journal of Physiology 250, 1-22.

108. MARCOS, E. & RIBAS, J. (1995). Kinetics of plasma potassium concentrations during exhaustive exercise in trained and untrained men. European Journal of Applied Physiology and Occupational Physiology 71, 207-214.

109. MAREN, T.H. (1988). The kinetics of HCO3- synthesis related to fluid

secretion, pH control, and CO2 elimination. Annu. Rev. Physiol. 50, 695-717.

110. MATSON, L.G. & TRAN, Z.V. (1993). Effects of sodium bicarbonate ingestion on anaerobic performance: a meta-analytic review. Int.J.Sport.Nutr. 3, 2-28.

111. MCCARTNEY, N., HEIGENHAUSER, G.J. & JONES, N.L. (1983). Effects of pH on maximal power output and fatigue during short-term dynamic exercise. J.Appl.Physiol. 55, 225-229.

112. MCCOY, M. & HARGREAVES, M. (1992). Potassium and ventilation during incremental exercise in trained and untrained men. Journal of Applied Physiology 74(4), 1287-1290.

113. MCKENZIE, D.C., COUTTS, K.D., STIRLING, D.R., HOEBEN, H.H. & KUZARA, G. (1986). Maximal work production following two levels of artificially induced metabolic alkalosis. Journal of Sports Sciences 4, 35-38.

114. MCNAUGHTON, L.R. (1990). Sodium citrate and anaerobic performance: implications of dosage. Eur.J.Appl.Physiol.Occup.Physiol. 61, 392-397.

115. MCNAUGHTON, L.R. (1992a). Bicarbonate ingestion: effects of dosage on 60 s cycle ergometry. J.Sports Sci. 10, 415-423.

116. MCNAUGHTON, L.R. (1992b). Sodium bicarbonate ingestion and its effects on anaerobic exercise of various durations. J.Sports Sci. 10, 425-435.

117. MCNAUGHTON, L.R. & CEDARO, R. (1991). The effect of sodium bicarbonate on rowing ergometer performance in elite rowers. The Australian Journal of Science and Medicine in Sport 23(3), 66-69.

References

117

118. MCNAUGHTON, L. & CEDARO, R. (1992). Sodium citrate ingestion and its effects on maximal anaerobic exercise of different durations. Eur.J.Appl.Physiol.Occup.Physiol. 64, 36-41.

119. MCNAUGHTON, L., CURTIN, R., GOODMAN, G., PERRY, D., TURNER, B. & SHOWELL, C. (1991). Anaerobic work and power output during cycle ergometer exercise: effects of bicarbonate loading. J.Sports Sci. 9, 151-160.

120. MCNAUGHTON, L., DALTON, B. & PALMER, G. (1999). Sodium bicarbonate can be used as an ergogenic aid in high-intensity, competitive cycle ergometry of 1 h duration. Eur.J.Appl.Physiol.Occup.Physiol. 80, 64-69.

121. MOORE, J.G., CHRISTIAN, P.E. & COLEMAN, R.E. (1981). Gastric emptying of varying meal weight and composition in man. Digestive Diseases and Sciences 26(1), 16-22.

122. NOTIVOL, R., CARRIO, I., CANO, L., ESTORCH, M. & VILARDELL, F. (1984). Gastric emptying of solid and liquid meals in healthy young subjects. Scandinavian Journal of Gastroenterology 19, 1107-1113.

123. OSTER, J.R., STEMMER, C.L., PEREZ, G.O. & VAAMONDE, C.A. (1988). Comparison of the effects of sodium bicarbonate versus sodium citrate on renal acid excretion. Miner.Electrolyte Metab. 14, 97-102.

124. PAJOR, A.M. (1999). Citrate transport by the kidney and intestine. Semin. Nephrol. 19, 195-200.

125. PARRY-BILLINGS, M. & MACLAREN, D.P. (1986). The effect of sodium bicarbonate and sodium citrate ingestion on anaerobic power during intermittent exercise. Eur.J.Appl.Physiol.Occup.Physiol. 55, 524-529.

126. PFEFFERLE, K.P. & WILKINSON, J.G. (1988). Induced alkalosis and supramaximal cycling in trained and untrained men. Medicine and Science in Sports and Exercise 20, S25

127. PIERCE, E.F., EASTMAN, N.W., HAMMER, W.H. & LYNN, T.D. (1992). Effect of induced alkalosis on swimming time trials. J.Sports Sci. 10, 255-259.

128. PILEGAARD, H., DOMINO, K., NOLAND, T., JUEL, C., HELLSTEN, Y., HALESTRAP, A.P. & BANGSBO, J. (1999). Effect of high-intensity exercise training on lactate/H+ transport capacity in human skeletal muscle. Am.J.Physiol 276, E255-E261

129. POTTEIGER, J.A., NICKEL, G.L., WEBSTER, M.J., HAUB, M.D. & PALMER, R.J. (1996a). Sodium citrate ingestion enhances 30 km cycling performance. Int.J.Sports Med. 17 , 7-11.

References

118

130. POTTEIGER, J.A., WEBSTER, M.J., NICKEL, G.L., HAUB, M.D. & PALMER, R.J. (1996b). The effects of buffer ingestion on metabolic factors related to distance running performance. Eur.J.Appl.Physiol.Occup.Physiol. 72, 365-371.

131. PUTNAM, R.W. (1990). pH regulatory transport systems in a smooth muscle-like cell line. Am.J.Physiol. 258, C470-C479

132. PUTNAM, R.W. & ROOS, A. (1986a). Effect of calcium and other divalent cations on intracellular pH regulation of frog skeletal muscle. J.Physiol. 381, 221-239.

133. PUTNAM, R.W., ROOS, A. & WILDING, T.J. (1986b). Properties of the intracellular pH-regulating systems of frog skeletal muscle. J.Physiol. 381, 205-219.

134. QUAYLE, J.M., NELSON, M.T. & STANDEN, N.B. (1997). ATP-sensitive and inwardly rectifying potassium channels in smooth muscle. Physiol.Rev. 77, 1165-1232.

135. RADEGRAN, G. & SALTIN, B. (1998). Muscle blood flow at onset of dynamic exercise in humans. Am.J.Physiol. 274, H314-H322

136. REN, J.M. & HULTMAN, E. (1989). Regulation of glycogenolysis in human skeletal muscle. J.Appl.Physiol 67, 2243-2248.

137. ROBERTSON, R.J., FALKEL, J.E., DRASH, A.L., SWANK, A.M., METZ, K.F., SPUNGEN, S.A. & LEBOEUF, J.R. (1987). Effect of induced alkalosis on physical work capacity during arm and leg exercise. Ergonomics 30, 19-31.

138. ROBIN, E.D. (1961). Of men and mitochondria: intracellular and sub-cellular acid-base relations. New England Journal of Medicine 265, 780-785.

139. ROGER FEDDE, M. & PIESCHL, R.L. (1995). Extreme derangements of acid-base balance in exercise: advantages and limitations of the stewart analysis. Canadian Journal of Applied Physiology 20(3), 369-379.

140. ROSDAHL, H., HAMRIN, K., UNGERSTEDT, U. & HENRIKSSON, J. (1998). Metabolite levels in human skeletal muscle and adipose tissue studied with microdialysis at low perfusion flow. Am.J.Physiol. 274, E936-E945

141. ROTH, D.A. & BROOKS, G.A. (1990). Lactate and pyruvate transport is dominated by a pH gradient-sensitive carrier in rat skeletal muscle sarcolemmal vesicles. Archives of Biochemisty and Biophysics 279(2), 386-394.

References

119

142. SAHLIN, K. (1978a). Intracellular pH and energy metabolism in skeletal muscle of man. With special reference to exercise. Acta Physiol.Scand.Suppl. 455, 1-56.

143. SAHLIN, K., ALVESTRAND, A., BRANDT, R. & HULTMAN, E. (1978b). Intracellular pH and bicarbonate concentration in human muscle during recovery from exercise. J.Appl.Physiol. 45, 474-480.

144. SCHABORT, E.J., WILSON, G. & NOAKES, T.D. (2000). Dose-related elevations in venous pH with citrate ingestion do not alter 40-km cycling time-trial performance. Eur.J.Appl.Physiol. 83, 320-327.

145. SCHULTZ, S.G. (1984). A cellular model for active sodium absorption by mammalian colon. Annu. Rev. Physiol. 46, 435-451.

146. SCHULTZ, S.G. & DUBINSKY, W.P. (2001). Sodium absorption, volume control and potassium channels: in tribute to a great biologist. J. Membr. Biol. 184, 255-261.

147. SEABURY, J.J., ADAMS, W.C. & RAMEY, M.R. (1977). Influence of pedalling rate and power output on energy expenditure during bicycle ergometry. Ergonomics 20(5), 491-498.

148. SHAVE, R., WHYTE, G., SIEMANN, A. & DOGGART, L. (2001). The effects of sodium citrate ingestion on 3,000-meter time-trial performance. J.Strength.Cond.Res. 15, 230-234.

149. SJOGAARD, G., ADAMS, R.P. & SALTIN, B. (1985). Water and ion shifts in skeletal muscle of humans with intense dynamic knee extension. Am.J.Physiol 248, R190-R196

150. SJOSTRAND, M., HOLMANG, A. & LONNROTH, P. (1999). Measurement of interstitial insulin in human muscle. Am.J.Physiol. 276, E151-E154

151. SOKABE, M., KASAI, M., NOMURA, K., & NARUSE, K. (1991). Electrophysiological analysis of structural aspects of voltage-dependent SR K+ channel. Comp. Biochem. Physiol. 98, 23-30.

152. SPRIET, L.L. (1991a). Phosphofructokinase activity and acidosis during short-term tetanic contractions. Can.J.Physiol.Pharmacol. 69, 298-304.

153. SPRIET, L.L., CAMPBELL, C.B. & DYCK, D.J. (1991b). Effect of aging on the buffering capacity of fast-twitch skeletal muscle. Mech.Ageing Dev. 59, 243-252.

154. SPRIET, L.L., LINDINGER, M.I., MCKELVIE, R.S., HEIGENHAUSER, G.J. & JONES, N.L. (1989). Muscle glycogenolysis and H+ concentration during maximal intermittent cycling. J.Appl.Physiol. 66, 8-13.

References

120

155. SPRIET, L.L., SODERLUND, K., BERGSTROM, M. & HULTMAN, E. (1987). Skeletal muscle glycogenolysis, glycolysis, and pH during electrical stimulation in men. J.Appl.Physiol. 62, 616-621.

156. SPRUCE, A.E., STANDEN, N.B. & STANFIELD, P.R. (1985). Voltage-dependent ATP-sensitive potassium channels of skeletal muscle membrane. Nature London 361, 736-738.

157. STEWART, P.A. (1983). Modern quantitative acid-base chemistry. Can.J.Physiol.Pharmacol. 61, 1444-1461.

158. SULLIVAN, M.J., SALTIN, B., NEGRO-VILAR, R., DUSCHA, B.D. & CHARLES, H.C. (1994). Skeletal muscle pH assessed by biochemical and 31P-MRS methods during exercise and recovery in men. J.Appl.Physiol. 77, 2194-2200.

159. SUTTON, J.R., JONES, N.L. & TOEWS, C.J. (1981). Effect of PH on muscle glycolysis during exercise. Clin.Sci.(Lond.) 61, 331-338.

160. TANNER, R.L. (1980). Control of acid excretion by the kidney. Annu. Rev. Med. 31, 35-49.

161. TANNER, G.A. (1984). Renal regulation of acid-base balance: ammonia excretion. Physiologist 27, 95-97.

162. THOMPSON, L.V., BALOG, E.M. & FITTS, R.H. (1992). Muscle fatigue in frog semitendinosus: role of intracellular pH. American Journal of Physiology 262 (Cell Phys 31), C1507-C1512

163. TIRYAKI, G.R. & ATTERBOM, H.A. (1995). The effects of sodium bicarbonate and sodium citrate on 600 m running time of trained females. J.Sports Med.Phys.Fitness. 35, 194-198.

164. VAN SOMEREN, K., FULCHER, K., MCCARTHY, J., MOORE, J., HORGAN, G. & LANGFORD, R. (1998). An investigation into the effects of sodium citrate ingestion on high-intensity exercise performance. Int.J.Sport.Nutr. 8, 356-363.

165. VICTOR, R.G., BERTOCCI, L.A., PRYOR, S.L. & NUNNALLY, R.L. (1988). Sympathetic nerve discharge is coupled to muscle cell pH during exercise in humans. J.Clin.Invest. 82, 1301-1305.

166. VIST, G.E. & MAUGHAN, R.J. (1995). The effect of osmolality and carbohyrate content on the rate of gastric emptying of liquids in man. Journal of Physiology 486.2 , 523-531.

167. WASSERMAN, K., STRINGER, W.W., CASABURI, R. & ZHANG, Y.Y. (1997). Mechanism of the exercise hyperkalemia: an alternate hypothesis. J.Appl.Physiol. 83, 631-643.

References

121

168. WEBSTER, M.J., WEBSTER, M.N., CRAWFORD, R.E. & GLADDEN, L.B. (1993). Effect of sodium bicarbonate ingestion on exhaustive resistance exercise performance. Med.Sci.Sports Exerc. 25, 960-965.

169. WESTERBLAD, H. & ALLEN, D.G. (1992). Changes of intracellular pH due to repetitive stimulation of single fibres from mouse skeletal muscle. J.Physiol. 449, 49-71.

170. WILKES, D., GLEDHILL, N. & SMYTH, R. (1983). Effect of acute induced metabolic alkalosis on 800-m racing time. Medicine and Science in Sports and Exercise 15, 277-280.

171. WRIGHT, E.M. & LOO, D.D (2000) Coupling between Na+, sugar, and water transport across the intestine. Ann. N.Y. Acad. Sci. 915, 54-66.

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SKELETAL MUSCLE INTERSTITIUM AND BLOOD PH AT REST … · since the measurement of interstitial pH in human skeletal muscle has never been performed before, these studies will represent