exercise induced hemolysis, inflammation and hepcidin activity exercise induced hemolysis,...

Exercise Induced Hemolysis, Inflammation

and Hepcidin Activity in Endurance Trained

Runners

Peter Daniel Peeling

Bachelor of Science (Honours)

This Thesis is presented for the degree of Doctor of Philosophy at

The University of Western Australia

School of Sport Science, Exercise and Health

2009

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Well, we knocked the bastard off!

Sir Edmund Hillary, on first climbing Mount Everest

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Statement of Candidate Contribution

The work involved in designing and conducting the studies described in this thesis has

been completed primarily by Peter Peeling (the candidate). The thesis outline and

experimental design of the studies was developed and planned in consultation with

Professor Brian Dawson, Dr Grant Landers and Dr Carmel Goodman (the candidates

supervisors). All participant recruitment and management was carried out entirely by

the candidate, along with the organisation, implementation and performance of every

experimental trial. The assistance of Dr Dorine Swinkels and Erwin Wiegerinck from

the Department of Clinical Chemistry (Radboud University, The Netherlands) was

required to perform the urine analysis of hepcidin, since this laboratory is one of only

two in the world to measure this hormone. The assistance of Associate Professor Debbie

Trinder from the School of Medicine and Pharmacology (UWA) was required to gain

grant funding and to set up this collaboration with the hepcidin analysis team in the

Netherlands.

Signed:

Peter Peeling

(Candidate)

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Executive Summary

Iron is a trace mineral used by the body in many physiological processes that are

essential to athletic performance. Commonly, the body‘s iron stores are compromised

by exercise via several well established mechanisms. One such mechanism is the

destruction of red blood cells (hemolysis), in response to the mechanical stress and

circulatory strain of exercise. Although it appears that a force-dependent relationship

between the heel-strike of the running gait and ground contact exists, the effects of the

intensity trained at and the ground surface type trained upon have not been documented.

Similarly, the effects of a cumulative training stress (i.e. multiple daily sessions) has not

been examined. In addition to hemolysis, exercise also invokes an inflammatory

response that results in an up-regulation of the cytokine interleukin-6 (IL-6). This

cytokine is the primary mediator of hepcidin expression, a liver-produced hormone that

regulates iron metabolism in the gut and in macrophages. The influence of exercise on

hepcidin expression is relatively unknown, and as such it is possible that this hormone

may be a mitigating factor implicated in athletic-induced iron deficiency.

Therefore, the purpose of this thesis was to investigate the effect of different training

frequencies, intensities and ground surfaces on the hemolytic response. In addition, the

impact of exercise-induced inflammation on hepcidin expression in the 24 h post-

exercise was investigated, with the aim of determining whether this hormone may be a

potential new mechanism associated with athletic-induced iron deficiency. Finally, an

interaction between hemolysis and hepcidin activity was examined to investigate their

potential combined effect on iron status in the 24 h post-exercise.

Paper one of this thesis investigated the time course of inflammatory response, urinary

hepcidin production and iron status changes in athletes during the initial 24 h after

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endurance exercise. Eleven moderately trained athletes (6 male and 5 female)

participated in three laboratory testing sessions, including a graded-exercise test for

determination of peak oxygen consumption ( V O2peak) and peak heart rate (HRpeak), a 60

min trial of running (RUN), including 15 min at 75-80% HRpeak and 45 min at 85-90%

HRpeak, and a 60 min trial of seated rest (REST). Throughout the RUN and REST trials,

venous blood and urine samples were collected pre- and immediately post-trial, and

then at 3, 6 and 24 h of recovery. Serum samples were analysed for IL-6, C-Reactive

Protein (CRP), serum iron and serum ferritin. Urine samples were analysed for urinary

hepcidin activity.

Blood data revealed that three participants were classified as stage 1 iron deficient, and

consequently were removed from the analysis. For the remaining eight healthy iron

status subjects, the immediate post-RUN levels of IL-6 and 24 h post-RUN levels of

CRP were significantly increased from baseline, and also greater than measured in the

REST trial (p

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and hepcidin activity experienced during running. For this study, ten highly endurance

trained male athletes were required to complete a graded exercise test, a continuous 10

km run on both a grass (GRASS) and bitumen road surface (ROAD) at 75-80% peak

V O2 running velocity, and a 10 x 1 km intervals running session (INT) on grass at 90-

95% of the peak V O2 running velocity. During each session, venous blood and urine

samples were collected pre and immediately post-exercise, and at 3 and 24 h of

recovery. Serum samples were analysed for circulating levels of IL-6, free hemoglobin

(Hb), haptoglobin (Hp), serum iron and ferritin. Urine samples were analysed for

changes in hepcidin activity.

After running, IL-6, free Hb and Hp levels were significantly greater than pre-exercise

values in all three conditions (p

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implemented of a one session training period (T1), comprising the 10 x 1 km intervals

session only (T1INT). Venous blood and urine samples were collected pre- and

immediately post-exercise, and at 3 and 24 h of recovery. Samples were analysed for

circulating levels of IL-6, free Hb, Hp, serum iron, ferritin and urinary hepcidin activity.

At the conclusion of both the T1 and T2 interval runs, the free Hb and serum Hp were

significantly increased (p

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Table of Contents

Statement of Candidate Contribution......................................................................... ii

Executive Summary................................................................................................... iii

Table of Contents....................................................................................................... vii

List of Tables............................................................................................................. xii

List of Figures............................................................................................................ xiv

List of Abbreviations.................................................................................................. xvi

Acknowledgements.................................................................................................... xviii

Publications Arising from this Thesis........................................................................ xx

Grant Funding............................................................................................................ xxi

Chapter 1 Introduction

1.0 Introduction...................................................................................................2

1.1 Statement of the Problem............................................................................... 3

1.2 Aims............................................................................................................... 4

1.3 Research Hypotheses..................................................................................... 5

1.4 Organisation and Structure of the Thesis....................................................... 6

1.5 Significance of the Study............................................................................... 6

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Chapter 2 Athletic Induced Iron Deficiency: New Insights into the Role

of Inflammation, Cytokines and Hormones

2.0 Abstract.......................................................................................................... 9

2.1 Introduction.................................................................................................... 10

2.2 Iron Metabolism............................................................................................. 10

2.3 Iron Absorption, Transport and Storage........................................................ 11

2.4 Iron Deficiency and Athletic Performance.................................................... 12

2.5 Avenues of Iron Loss During Exercise.......................................................... 18

2.5.1 Gastrointestinal Bleeding................................................................... 19

2.5.2 Hematuria........................................................................................... 19

2.5.3 Sweating............................................................................................. 20

2.5.4 Hemolysis............................