Regulation of free radical outflow from an isolated muscle bed
in exercising humans
Damian M. Bailey,1,2Ian S. Young,3Jane McEneny,3Lesley Lawrenson,4
Jeannie Kim,4Jeremy Barden,4and Russell S. Richardson4
1Departments of Anesthesiology and Surgery, Colorado Center for Altitude Medicine and
Physiology, University of Colorado Health Sciences Center, Aurora, Colorado 80111;2Hypoxia
Research Unit, Department of Physiology, School of Applied Sciences, University of Glamorgan,
South Wales CF37 1DL;3Department of Medicine, Queens University, Belfast BT12 6BJ, United Kingdom;
and4Department of Medicine, University of California San Diego, La Jolla, California 92093
Submitted 17 February 2004; accepted in final form 13 May 2004
Bailey, Damian M., Ian S. Young, Jane McEneny, Lesley
Lawrenson, Jeannie Kim, Jeremy Barden, and Russell S. Rich-
ardson. Regulation of free radical outflow from an isolated muscle
bed in exercising humans. Am J Physiol Heart Circ Physiol 287:
00148.2004.—Incremental knee extensor (KE) exercise performed at
25, 70, and 100% of single-leg maximal work rate (WRMAX) was
combined with ex vivo electron paramagnetic resonance (EPR) spec-
troscopic detection of ?-phenyl-tert-butylnitrone (PBN) adducts, lipid
hydroperoxides (LH), and associated parameters in five males. Blood
samples were taken from the femoral arterial and venous circulation
that, when combined with measured changes in femoral venous blood
flow, permitted a direct examination of oxidant exchange across a
functionally isolated contracting muscle bed. KE exercise progres-
sively increased the net outflow of LH and PBN adducts (100% ?
70% ? 25% WRMAX, P ? 0.05) consistent with the generation of
secondary, lipid-derived oxygen (O2)-centered alkoxyl and carbon-
centered alkyl radicals. Radical outflow appeared to be more inti-
mately associated with predicted decreases in intracellular PO2(iPO2)
as opposed to measured increases in leg O2 uptake, with greater
outflow recorded between 25 and 70% WRMAX (P ? 0.05 vs.
70–100% WRMAX). This bias was confirmed when radical venoarte-
rial concentration differences were expressed relative to changes in
the convective components of O2 extraction and flow (25–70%
WRMAX P ? 0.05 vs. 70–100% WRMAX, P ? 0.05). Exercise also
resulted in a net outflow of other potentially related redox-reactive
parameters, including hydrogen ions, norepinephrine, myoglobin, lac-
tate dehydrogenase, and uric acid, whereas exchange of lipid/lipopro-
teins, ascorbic acid, and selected lipid-soluble anti-oxidants was
unremarkable. These findings provide direct evidence for an exercise
intensity-dependent increase in free radical outflow across an active
muscle bed that was associated with an increase in sarcolemmal
membrane permeability. In addition to increased mitochondrial elec-
tron flux subsequent to an increase in O2extraction and flow, exercise-
induced free radical generation may also be regulated by changes in
iPO2, hydrogen ion generation, norepinephrine autoxidation, peroxi-
dation of damaged tissue, and xanthine oxidase activation.
electron paramagnetic resonance; spin-trapping; lipid peroxidation;
antioxidants; mitochondrial redox
THE MOLECULAR DETECTION of free radical species during exer-
cise is technically challenging because of their high reactivity
and low steady-state concentration (19). As a consequence,
investigators have traditionally relied on comparatively stable
molecular “footprints” of oxidative damage to lipids, proteins,
and DNA formed downstream of the primary production path-
However, the analytic techniques employed have been
mostly indirect and the subject of much concern (37). Reactive
intermediates exhibit markedly different thermodynamic and
kinetic properties adding to the inconsistencies reported in the
exercise science literature (4). Exercise models also deserve
critical evaluation because they typically recruit heterogenous
muscle groups characterized by different modes of contraction.
Furthermore, blood sampling has usually been confined to the
mixed venous circulation distal to the activated musculature of
interest, which can also prove problematic.
Electron paramagnetic resonance (EPR) spectroscopy is
considered the most sensitive, specific, and direct molecular
technique for the detection and subsequent identification of
free radicals sine qua non, although its application to the
exercise environment has been limited (39). We (8) recently
applied an ex vivo EPR spin-trapping technique to the
functionally isolated single-leg knee extensor (KE) model to
overcome some of these experimental limitations in an
attempt to more accurately define the source and mecha-
nisms associated with exercise-induced free radical genera-
tion. Through the insertion of a femoral arterial-venous
catheter and simultaneous measurement of leg blood flow
(LBF), we (8) documented, for the first time, direct analytic
evidence for oxygen- and carbon-centered free radical out-
flow across a skeletal muscle bed during submaximal exer-
cise. A schematic overview of the experimental techniques
and exercise models traditionally employed and the meth-
odological advances introduced by our laboratory is pre-
sented in Fig. 1.
However, whereas preliminary findings demonstrated a ten-
tative association between radical outflow and hemodynamic
parameters, it is equally plausible that alternative mechanisms
may have contributed to the downstream generation of lipid-
derived radicals. Potential mechanisms include altered lipid-
substrate and hydrogen ion (H?) exchange, catecholamine
auto-oxidation, molecular peroxidation of damaged skeletal
tissue, and xanthine oxidase activation. Furthermore, to our
knowledge, there are no published reports that have simulta-
Address for reprint requests and other correspondence: D. M. Bailey, Depts.
of Anesthesiology and Surgery, Colorado Center for Altitude Medicine and
Physiology, Univ. of Colorado Health Sciences Center, PO Box 6508, Mail
Stop F524, Aurora, CO 80111 (E-mail: email@example.com).
The costs of publication of this article were defrayed in part by the payment
of page charges. The article must therefore be hereby marked “advertisement”
in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Am J Physiol Heart Circ Physiol 287: H1689–H1699, 2004.
First published May 20, 2004; 10.1152/ajpheart.00148.2004.
0363-6135/04 $5.00 Copyright © 2004 the American Physiological Societyhttp://www.ajpheart.orgH1689
neously examined the exchange of aqueous and lipid-soluble
antioxidants to provide a more complete understanding of the
dynamic interaction between pro-oxidant challenge and anti-
oxidant defense during muscular work.
Using proton magnetic resonance spectroscopy to detect
changes in myoglobin saturation using an identical experimen-
tal paradigm, Richardson et al. (49, 50) have consistently
demonstrated a marked decrease in intracellular PO2 (iPO2)
between the low-to-moderate intensity domains [25–70% max-
imal work rate (WRMAX)]. However, no changes were ob-
served between the moderate-to-high intensity domains (70–
100% WRMAX) subsequent to increased muscle O2diffusional
conductance, allowing an undeterred rise in muscle O2 flux.
Thus the inclusion of an additional exercise increment in the
present study afforded an opportunity to disassociate changes
in O2flux from changes in iPO2and, for the first time, examine
their respective contributions to radical exchange.
On the basis of existing knowledge, three experimental
hypotheses were tested. First, that incremental exercise would
be associated with an incremental increase in the net outflow
(defined as the product of a positive venoarterial concentration
difference and LBF) of lipid hydroperoxides (LH) and ?-phe-
nyl-tert-butylnitrone (PBN) adducts despite a net uptake or
consumption of antioxidants. Second, a decrease in iPO2would
further compound radical outflow initiated by an increase in
muscle O2 flux (measured as O2 uptake, V˙O2). Thus we
anticipated comparatively greater radical outflow during the
low-to-moderate exercise intensity transition compared with
the moderate-to-high transition. Finally, we expected a com-
parable outflow in other redox-reactive parameters, including
blood lipids, H?, catecholamines, biomarkers of tissue dam-
age, and uric acid thus providing additional insight into poten-
tial sources and mechanisms associated with exercise-induced
free radical generation.
MATERIALS AND METHODS
The study was designed with 90% power at the P ? 0.05 level to
detect biologically significant changes in oxidant exchange across the
working leg based on a calculated critical difference of 121% for PBN
adducts and previous findings (8). Five healthy men, aged 47
(mean) ? 22 (SD) yr (range: 22–67 yr) subsequently provided written
informed consent according to the ethical requirements of the Uni-
versity of California San Diego, Human Protection Program. The
heterogenous sample facilitated an examination of mechanisms per-
tinent to exercise-induced free radical generation across a broad
spectrum of submaximal single-leg V˙O2 values. It was considered
unethical to recruit additional subjects owing to the invasive nature
Fig. 1. Comparison of traditional and current
(present study) methods employed for the assess-
ment of exercise-induced oxidative stress in hu-
mans. Bracketted numbers refer to the number of
published reports in the human exercise literature
from 1950 to the present day (taken from Med-
line, National Institutes of Health).
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