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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
i
Well, we knocked the bastard off!
Sir Edmund Hillary, on first climbing Mount Everest
ii
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)
iii
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
iv
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
v
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
vi
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
vii
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
viii
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............................