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1 School of Applied Sciences, University of Glamorgan, Pontypridd, South Wales CF37 1DL; 2 Department of Medicine, Queen's University, Belfast BT12 6BJ; 3 Department of Medicine, University of Liverpool, Liverpool L69 3GA, United Kingdom; and 4 Divison of Physics and 5 Department of Medicine, University of California San Diego, La Jolla, California 92093
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ABSTRACT |
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 TOP
 ABSTRACT
 INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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There is no direct evidence to
support the contention that contracting skeletal muscle and/or
associated vasculature generates free radicals in exercising humans.
The unique combination of isolated quadriceps exercise and the
measurement of femoral arterial and venous free radical concentrations
with the use of electron paramagnetic resonance (EPR) spectroscopy
enabled this assumption to be tested in seven healthy men. Application
of ex vivo spin trapping using 
-phenyl-tert-butylnitrone
(PBN) resulted in the detection of oxygen- or carbon-centered free
radicals (aN = 1.38 ± 0.01 mT and
a
-hydrogen coupling constants, respectively) with
consistently higher EPR signal intensities of the PBN spin adduct
observed in the venous compared with the arterial circulation
(P < 0.05). Incremental exercise further increased the
venoarterial intensity difference [85 ± 58 arbitrary units (AU)
at 24 ± 6% maximal work rate (WRmax) vs. 387 ± 214 AU at 69 ± 7% WRmax; P < 0.05]. When combined with measured changes in femoral venous blood
flow (
), this resulted in a net adduct outflow of 130 ± 118 and 1,146 ± 582 AU/min (P < 0.05), which was
positively associated with leg oxygen uptake (r2 = 0.47, P < 0.05) and
(r2 = 0.47, P < 0.05). These results provide the first evidence for oxygen- or
carbon-centered free radical outflow from an active muscle bed in humans.
spin trapping; blood flow; oxygen uptake
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INTRODUCTION |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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RESEARCHERS HAVE TRADITIONALLY focused on indirect biological "footprints" of free radical-mediated lipid peroxidation confined to the peripheral circulation by using exercise models that typically recruit heterogeneous muscle groups characterized by a substantial isometric component (12). This may have seriously influenced prior interpretations of the source and mechanisms associated with exercise-induced free radical generation.
In light of these observations, the present study applied an electron
paramagnetic resonance (EPR) spin-trapping technique to the single-leg
knee extensor (KE) model to more precisely document free radical
outflow from an isolated muscle bed during incremental exercise. We
hypothesized that free radical outflow would increase commensurate with
single-leg oxygen uptake (
O2).
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METHODS |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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Subjects
Seven apparently healthy men, aged 48 (mean) ± 25 (SD) yr, volunteered to participate in the present study after written, informed consent was obtained according to the ethical requirements of the University of California, San Diego, Human Subjects Committee. All procedures conformed to the code of Ethics of the World Medical Association (Declaration of Helsinki).Experimental Protocol
Preliminary phase. Subjects performed two or three training bouts on the dynamic KE apparatus to ensure familiarity with the equipment and testing environment. The final practice involved subjects doing an incremental KE test to volitional exhaustion while breathing room air for the determination of maximal work rate (WRmax). The initial WR was set at 3-10 W and increased by 3-6 W/min (at a cadence of 60 rpm) according to the subject's predicted exercise capacity, resulting in a WRmax of 37 ± 7 W.
Experimental phase.
Thirty minutes after vascular catheterization according to the
procedures previously outlined by Richardson et al. (17), each subject completed incremental exercise that encompassed 25 and
70% of their previously established WRmax. Each work
intensity was continued for 3 min to achieve steady-state pulmonary
O2, and the following sequence of events
was conducted: 1) sampling of femoral arterial and venous
blood for the direct measurement of free radicals and blood gases,
2) duplicate assessment of femoral vein blood flow ()
via a constant-infusion thermodilution technique (1), and
3) continuous measurement of arterial and venous pressures. These three procedures took ~60-90 s to complete. Resting
measurements were not performed because of the technical difficulties
and inherent variability associated with the measurement of resting
venous flow via the thermodilution technique (personal observations).
Measurement of Free Radicals
Adduct extraction.
Ex vivo spin trapping was incorporated to overcome the technical
difficulties associated with the low background concentration and short
spin-relaxation times of free radicals in human whole blood (4,
7, 13). Venous blood (4.5 ml) was collected into a 6-ml glass
serum-separation tube vacutainer that contained 1.5 ml of the
spin trap, -phenyl-tert-butylnitrone (PBN)
(0.140 mol/l). After centrifugation, the PBN spin adduct was extracted from the serum supernatant with toluene (nitrogen gassed and scanned for artifactual EPR signals, 99.8% HPLC grade; Sigma Chemical, Dorset,
UK). The adduct (200 µl) was pipetted into a 5-mm-OD precision-bore quartz EPR sample tube (Wilmad) that had been flushed with compressed N2. The sample was immediately vacuum degassed (West
Technology, Bristol, UK) by using a freeze (liquid N2)-thaw
procedure at a fixed vacuum of 10
3 Torr (Pirani 14-gauge
detector; Edwards, APG-NW, West Sussex, UK) with the use of a turbo
molecular pump (ACT 200T, Alcatel, Annecy, France) for two cycles
(total degassing time of 9 min). All procedures and chemicals were
performed and stored in the dark to avoid photolytic degradation of the
spin trap.
EPR spectroscopy. EPR was performed at 21°C by using an EMX X-band spectrometer (Bruker) and a Bruker ER TM110 cavity operating at 9.7 GHz at 20-mW power, 0.05-mT modulation, 1 × 105 receiver gain, 82-ms time constant, 345.0-mT magnetic field center, and ±5.00-mT scan width for 15 incremental scans. We obtained EPR spectral parameters using commercially available software (Bruker Win EPR system, version 2.11). The average spectral peak-to-peak line height was considered a measure of the relative spin-adduct concentration after conformation of peak-to-peak line-width conformity.
Statistical Analyses
We assessed changes in the concentration of the serum PBN spin adduct as a function of sample site and exercise intensity with the use of a two-factor (site × intensity) repeated-measures ANOVA with Bonferroni corrected paired samples t-tests (see Fig. 2). Paired samples t-tests or Wilcoxon's matched pairs signed ranks tests were also incorporated to assess simple effects of exercise intensity. We analyzed the relationship between two dependent variables by using Pearson's product-moment correlation (see Fig. 3). Significance for all two-tailed tests was established at P < 0.05, and data are expressed as means ± SD.| |
RESULTS |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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General Hemodynamic/Metabolic Responses
Consistent with previous research conducted in our laboratory, incremental KE exercise increased net muscle hydrogen outflow, heart rate, mean arterial and venous pressures, oxygen delivery, andO2. The increase in
O2 was due to an increase in , as peripheral extraction did not change (data not shown).
Nitroxide PBN Spin-Adduct Formation: Qualitative Aspects
Typical EPR spectra of nitroxide PBN spin-adduct (R2NO) formation detected in the femoral arterial and venous circulation of one subject are illustrated in Fig. 1. The species detected exhibited nitrogen and hydrogen nuclear hyperfine splittings of aN = 1.38 ± 0.01 mT and a-hydrogen coupling constants, respectively.
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Quantitative Aspects
Incremental exercise resulted in a marked venoarterial PBN spin-adduct concentration difference because of increased venous concentration (Fig. 2). An exercise-induced change in the venous but not the arterial concentration was also apparent when the adduct was expressed relative to the calculated H+ concentration [arterial: 25% WRmax = 29 ± 4 vs. 29 ± 5 arbitrary units (AU)/H+ at 70% WRmax; not significant and venous: 25% WRmax = 27 ± 4 vs. 55 ± 12 AU/H+ at 70% WRmax; P < 0.05]. When combined with the observed rise in, the PBN
venoarterial spin-adduct concentration difference resulted in a net
adduct outflow that increased from 130 ± 118 AU/min at 25%
WRmax to 1,146 ± 582 AU/min at 70% WRmax
(P < 0.05). When expressed relative to absolute
O2, the PBN venoarterial difference
increased from 516 ± 363 AU · l1 · min1
at 25% WRmax to 1,206 ± 1,051 AU · l1 · min1
at 70% WRmax (P < 0.05).
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Correlation Analyses
Adduct outflow was positively associated with legO2 (Fig.
3) mainly because of an increase in
(r2 = 0.47, P < 0.05) as opposed to arteriovenous oxygen difference that remained
invariant with exercise intensity. The PBN spin-adduct venoarterial
difference was positively associated with the venoarterial difference
for calculated H+ (r2 = 0.38, P < 0.05), and thus, by consequence, adduct outflow
was also related to net muscle H+ outflow
(r2 = 0.45, P < 0.05).
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DISCUSSION |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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The EPR spectroscopic assessment of free radicals in the arterial
and venous circulation of humans performing isolated, single-leg KE
exercise represents an ideal scenario for a controlled examination of
the source and mechanisms associated with exercise-induced free radical
generation. Our study has highlighted two major findings. First, the
nitroxyl adducts presented in Fig. 1 provide clear evidence that this
technique can successfully detect free radicals in the circulation of
exercising humans. Close inspection of the hyperfine splittings are
consistent with published values for a carbon-centered species such as
the alkyl radical or conversely an oxygen-centered alkoxyl or peroxyl
radical. Second, muscle contraction was associated with net adduct
outflow that increased proportionately with muscle
O2 and .
Qualitative Aspects
The hyperfine splitting constants of spectral signals associated with the PBN spin adducts (Fig. 1) are similar to those reported by other investigators who have applied identical spin traps and extraction solvents to peripheral blood (2, 8-10, 18) and are consistent with published values for either a carbon-centered species such as the alkyl radical and/or an oxygen-centered alkoxyl or peroxyl radical (5, 7, 20). The trapping of PBN-peroxyl radicals is quite unlikely however, despite a comparatively long half-life of 7 s (15), since they typically display smaller aN and aIf we are indeed trapping the alkoxyl radical, then we are clearly detecting species formed distal to the instrumented vasculature; thus the potential for confounding ex vivo redox reactions during incubation of the spin trap with whole blood (4, 13) warrants consideration. However, there are several lines of evidence to suggest that the ex vivo technique employed in the present study represents oxidative events that principally occur in vivo. First, we have not identified major differences in the signal intensity of the venous PBN spin adduct when blood has been incubated for a variety of times ranging from 20 (minimum incubation time required during centrifugation) to 40 min (between addition of whole blood to the spin trap and subsequent organic extraction) after exhaustive exercise in healthy men (n = 3; Bailey, unpublished observations). Second, all samples were treated identically, and clear treatment effects (changes in signal intensity as a function of exercise intensity and sample site) were apparent.
We therefore suggest that the signals observed reflect the oxidation
products of a continuous cascade involving the metal-catalyzed decomposition of lipid hydroperoxides generated as a consequence of
free radical-mediated damage to membrane phospholipids in vivo. Recent
research conducted in our laboratories add some support to this
contention. We have consistently demonstrated an association between
the peripheral concentration of lipid hydroperoxide and signal
intensity of the PBN spin adduct (3). Furthermore, in vitro oxidation of the polyunsaturated fatty acids, linoleic (18:2) and
-linolenic acid (18:3), yields coupling constants
(aN= 1.38 mT, a
Quantitative Aspects
The measurement of resting femoral venous with the use of
the thermodilution technique is technically challenging and to an
extent limited because of poor reproducibility. Although highly variable, we have since recorded flows of 350 ml/min in resting subjects, which is considerably lower than the 1,600 ml/min observed at
the lowest exercise intensity in the present study. When combined with
preliminary observations of an albeit slight venoarterial PBN
spin-adduct difference, the expected radical outflow would be
considerably less than that recorded during exercise. The documentation of radical outflow during the rest to exercise transition would certainly be of interest in future studies, but this was not the primary focus of the present investigation.
It is important to note that, by experimental design, an increase in exercise intensity from 25 to 70% WRmax (attainable without the recruitment of auxiliary stabilizing muscles) did not influence the arterial inflow of free radicals, whereas a clear increase was observed in the venous outflow. This finding suggests that mechanisms other than the recirculation of existing free radicals, which are capable of causing molecular damage, were contributing to release from the muscle bed.
In vitro studies have estimated that between 1 and 2% of total
electron flux can undergo univalent reduction at the NADH dehydrogenase (19) and/or ubiquinone cytochrome-bc segment of
complex III (16) in the mitochondria to form the
superoxide anion, the stoichiometric precursor to hydrogen peroxide
(6). Thus a mass action effect initiated by an increase in
O2 and the subsequent rise in
mitochondrial electron flux may have contributed to free radical
generation by skeletal muscle as our findings tentatively suggest. The
association between the venoarterial difference for H+ and
the PBN spin adduct adds further support to the proposed link between
free radical release and muscle metabolism. Normalization of the
venoarterial differences for the PBN spin adduct relative to leg
O2 indicates that, from low to moderate
exercise intensity, there is more than a doubling of free radical
formation by the muscle bed. This may represent enhanced electron
"leakage" at the higher exercise intensities and/or that other
sources were contributing to outflow.
A more detailed examination of the individual components of convective
O2 transport identified that the primary factor associated with outflow was and not the peripheral extraction of
O2 by muscle, which remained essentially invariant with
increasing exercise intensity. is a well established
physiological stimulus for vascular endothelial O2- and
N2-centered free radical release (11) and, in
addition to the previously mentioned increase in leg
O2, may also have contributed to the
signals observed in the present study. Clearly, our findings exclude
the possibility of an exercise-induced decrease in pH and subsequent
release of redox reactive transition metals in proteins as a major
contributory mechanism. However, potential contributions from
other pro-oxidant sources including phagocytes, catecholamines,
soluble oxidases, and peroxisomal oxidative enzymes cannot, at
present, be dismissed. Further research is required to
isolate the precise mechanisms that regulate free radical release by
the skeletal muscle bed in the exercising human.
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ACKNOWLEDGEMENTS |
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We express our gratitude to Dr. Richardson's research team and acknowledge the kind support of Drs. Peter Wagner, George Feher, Chris Rowlands, and the late Martyn Symons. Finally, we are indebted to the volunteers whose cheerful participation made this study possible.
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FOOTNOTES |
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Address for reprint requests and other correspondence: D. M. Bailey, School of Applied Sciences, Univ. of Glamorgan, Pontypridd, South Wales CF37 1DL, UK (E-mail: dbailey1{at}glam.ac.uk).
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.
10.1152/japplphysiol.01024.2002
Received 7 November 2002; accepted in final form 7 December 2002.
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REFERENCES |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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