University of Montana University of Montana
ScholarWorks at University of Montana ScholarWorks at University of Montana
Integrative Physiology and Athletic Training
Faculty Publications
Integrative Physiology and Athletic Training
2-2013
Blood Oxidative-Stress Markers During a High-Altitude Trek Blood Oxidative-Stress Markers During a High-Altitude Trek
Lindsey E. Miller
Graham R. McGinnis
Brian Kliszczewicz
Dustin Slivka
Walter Hailes
See next page for additional authors
Follow this and additional works at: https://scholarworks.umt.edu/hhp_pubs
Part of the Medicine and Health Sciences Commons
Let us know how access to this document bene>ts you.
Recommended Citation Recommended Citation
Miller, Lindsey E.; McGinnis, Graham R.; Kliszczewicz, Brian; Slivka, Dustin; Hailes, Walter; Cuddy, John;
Dumke, Charles; Ruby, Brent; and Quindry, John C., "Blood Oxidative-Stress Markers During a High-Altitude
Trek" (2013).
Integrative Physiology and Athletic Training Faculty Publications
. 2.
https://scholarworks.umt.edu/hhp_pubs/2
This Article is brought to you for free and open access by the Integrative Physiology and Athletic Training at
ScholarWorks at University of Montana. It has been accepted for inclusion in Integrative Physiology and Athletic
Training Faculty Publications by an authorized administrator of ScholarWorks at University of Montana. For more
information, please contact scholarworks@mso.umt.edu.
65
International Journal of Sport Nutrition and Exercise Metabolism, 2013, 23, 65 -72
© 2013 Human Kinetics, Inc.
Miller, McGinnis, Kliszczewicz, and Quindry are with the
Dept. of Kinesiology, Auburn University, Auburn, AL. Slivka is
with the School of Health Physical Education and Recreation,
University of Nebraska at Omaha, Omaha, NE. Hailes, Cuddy,
Dumke, and Ruby are with the Montana Center for Work
Physiology and Exercise Metabolism, University of Montana,
Missoula, MT.
Blood Oxidative-Stress Markers
During a High-Altitude Trek
Lindsey E. Miller, Graham R. McGinnis, Brian Kliszczewicz, Dustin Slivka, Walter Hailes,
John Cuddy, Charles Dumke, Brent Ruby, and John C. Quindry
Oxidative stress occurs as a result of altitude-induced hypobaric hypoxia and physical exercise. The effect of
exercise on oxidative stress under hypobaric hypoxia is not well understood. Purpose: To determine the effect
of high-altitude exercise on blood oxidative stress. Nine male participants completed a 2-d trek up and down Mt
Rainer, in North America, at a peak altitude of 4,393 m. Day 1 consisted of steady-pace climbing for 6.25 hr
to a nal elevation of 3,000 m. The 4,393-m summit was reached on Day 2 in approximately 5 hr. Climb–rest
intervals varied but were consistent between participants, with approximately 14 hr of total time including
rest periods. Blood samples were assayed for biomarkers of oxidative stress and antioxidant potential at the
following time points: Pre (before the trek), 3Kup (at ascent to 3,000 m), 3Kdown (at 3,000 m on the descent),
and Post (posttrek at base elevation). Blood serum variables included ferric-reducing antioxidant potential
(FRAP), Trolox equivalent antioxidant capacity (TEAC), protein carbonyls (PC), and lipid hydroperoxides.
Serum FRAP was elevated at 3Kup and 3Kdown compared with Pre and Post values (p = .004, 8% and 11%
increase from Pre). Serum TEAC values were increased at 3Kdown and Post (p = .032, 10% and 18% increase
from Pre). Serum PC were elevated at 3Kup and 3Kdown time points (p = .034, 194% and 138% increase from
Pre), while lipid hydroperoxides were elevated Post only (p = .004, 257% increase from Pre). Conclusions:
Findings indicate that high-altitude trekking is associated with increased blood oxidative stress.
Keywords: exercise, hypoxia, reactive oxygen species
Increased production of reactive oxygen species
(ROS) is a well-established response to acute exercise
(Powers, Nelson, & Hudson, 2011). ROS influence
important biological functions including metabolic con-
trol, cell signaling, tissue repair, and adaptation responses
to exercise stress (Powers et al., 2011). Despite these ben-
ets, overaccumulation of ROS results in muscle-tissue
damage and exacerbates inammation (Alessio, Gold-
farb, & Cutler, 1988; Dillard, Litov, Savin, Dumelin, &
Tappel, 1978). Detrimental effects on health and normal
body functioning due to ROS are collectively described
as oxidative stress (Powers et al., 2011). Exercise-induced
oxidative stress generally occurs as a function of increas-
ing exercise intensity and duration (Alessio et al., 1988)
in response to both aerobic- and anaerobic-type exercise
(Alessio et al., 2000; Bloomer, Fry, Falvo, & Moore,
2007; Bloomer, Goldfarb, Wideman, McKenzie, &
Consitt, 2005; Quindry, Stone, King, & Broeder, 2003).
However, some investigations examining lower intensity
long-duration fatiguing exercise protocols have failed to
nd a signicant increase in oxidative markers (Quindry
et al., 2008; Quindry et al., 2003). Despite many advances
in understanding exercise-induced oxidative stress, many
unknowns remain. This noteworthy fact is particularly
true about human-based applied physiology research. One
such area pertains to exercise-induced oxidative stress
experienced during exercise at high elevations where
absolute work output is limited by oxygen availability
(Wagner, 2000).
In regard to exercise at extreme altitudes, it has been
proposed that hypoxia decreases antioxidant capacity,
therefore resulting in greater oxidative stress and muscle
fatigue (Moller, Loft, Lundby, & Olsen, 2001; Vasankari,
Kujala, Rusko, Sarna, & Ahotupa, 1997). These ndings
are especially relevant to numerous individuals who travel
to high altitudes for work and recreation. Comparisons
between the responses to exercise at sea level versus
altitude are not easily made due to alterations in relative
exercise intensity versus absolute exercise work. Fac-
tors other than low oxygen availability during altitude
exposure may affect the occurrence of oxidative stress
and muscle fatigue. For instance, physiological stress
www.IJSNEM-Journal.com
ORIGINAL RESEARCH
66 Miller et al.
from cold exposure, ultraviolet rays, and acute mountain
sickness may affect the altitude-induced oxidative-stress
response and warrant further investigation (Askew, 2002).
Based on this rationale, the purpose of this investi-
gation was to examine exercise-induced oxidative stress
in response to high-altitude aerobic exercise in human
participants undertaking a 2-day mountaineering trek on
Mt. Rainier, located in North America, at a peak altitude
of 4,393 m. Oxidative stress experienced during exercise
performed for health and tness stimulates benecial
adaptations, while excessive oxidative-stress levels
experienced during extreme exercise may be harmful.
Therefore, this study examined blood oxidative stress in
a scenario of high-altitude exercise. An oxidative-stress
biomarker panel was examined in the current study to
quantify exercise-induced redox changes in the blood
serum. The panel included two markers of serum antioxi-
dant potential, as well as lipid- and protein-based markers
of oxidative damage (Buettner, 1993; Cao & Prior, 1998).
Methods
Subjects and Study Design
Study approval was granted by the University of Mon-
tana’s internal review board before initiation of the study,
and written informed consent was obtained before data
collection. Nine nonsmoking men ages 18–55 years who
were attempting a 2-day trek to the summit of Mt. Rainier
(4,393 m) and back to base elevation (~1,000 m) were
recruited. All participants trekked with the assistance of
professional mountain guides. Participants reported to
the University of Montana Human Performance Mobile
Laboratory located 6 miles from the Mt. Rainier park
entrance in Ashford, WA. At this time, they were briefed
on the purpose and methods of the study and completed a
Physical Activity Readiness Questionnaire. Body weight
was recorded the evening before the climb was begun and
on return from the mountain using an electronic scale
(Befour, Inc., Cedarburg, WI) as an indicator of poor
hydration and other potentially harmful complications
that might have occurred. The ascent schedule was iden-
tical for all participants. Participants took a 1-hr shuttle
ride from base camp (1,000 m) to the 1,600-m start site.
Day 1 consisted of ve climbing intervals divided into 60
min of steady-pace climbing and 15 min rest, conclud-
ing at a nal elevation of 3,000 m (10,000 ft) for a total
climbing time of 6 hr and 15 min. The summit at 4,393
m (14,410 ft) was reached on the second day. The time to
summit was 5 hr, while the return trek (to 1,600 m) was
approximately 9 hr. Climb and rest intervals varied on
Day 2 but were consistent between participants, with a
total climb time of 14 hr. At the conclusion of the climb,
the participants took a 1-hr shuttle ride back to base camp
(from approximately 1,600 m to 1,000 m). Details of the
trek ascent and descent schedule are presented in Table
1 and Figure 1.
Blood Serum Collection
Blood samples were collected and assayed for blood
serum biomarkers of oxidative stress and antioxidant
potential at the following time points: Pre (the evening
before the trek), 3Kup (at ascent to 3,000 m), 3Kdown
(at 3,000 m on the descent), and Post (posttrek at base
elevation of 1,000 m, after the 1-hr shuttle ride). Five
milliliters of venous blood were collected from an ante-
cubital vein and allowed to clot on ice for 20 min. Blood
was then immediately centrifuged at 3,400 rpm for 20
min in a portable centrifuge (centri A13, Jouan Inc.,
Winchester, VA). Serum was separated and immediately
frozen on dry ice. Serum was kept temporarily on dry
ice for 5 days and stored at –80 °C until assay. All blood
samples were collected under nonfasted conditions. Diet
was not controlled between subjects throughout the trek
for logistical reasons, but all subjects were encouraged
to eat and drink during the rest intervals.
Table 1 Trek Climb and Rest Schedule
Day Stage
Approximate blood-
sampling time
Trek
duration
Rest
duration
Total trek time
per stage
Altitude
achieved
Total trek time
per day
0 Pre 7 p.m.
1 1 60 min 15 min 1.25 hr 3,000 m 6.25 hr
2 60 min 15 min 1.25 hr
3 60 min 15 min 1.25 hr
4 60 min 15 min 1.25 hr
5 3 p.m. 60 min 15 min 1.25 hr
2 1 varying varying 5 hr 4,393 m 14 hr
2 11 a.m. varying varying 4 hr 3,000 m
3 4:30 a.m. varying varying 5 hr 1,000 m
Blood Oxidative Stress and Altitude 67
Biochemical Assays for Serum
Antioxidant Capacity and Potential
Serum nonenzymatic antioxidant capacity (NEAC) was
measured by the serum Trolox equivalent antioxidant
capacity (TEAC) assay modied from the methods of Re
et al. (1999) and Villano, Fernandez-Pachon, Troncoso,
and Garcia-Parrilla (2004). The TEAC assay is based on
the principle that oxidized 2,2′–azinobis-(3-ethylbenzo-
thiazoline-6-sulfonic acid) is reduced by antioxidants
present in the blood serum (Re et al., 1999; Villano et
al., 2004). Serum antioxidant potential was quantied
via the ferric-reducing antioxidant potential (FRAP)
assay according to methodology modied from Benzie
and Strain (1996). The assay principle is based on the
ion reduction of ferric compounds to ferrous, forming
tripyridyltriazine complex with the use of 0- to 1,000-
μM ascorbic acid standards (Benzie & Strain, 1996).
Antioxidant capacity is a direct method of quantifying
the NEAC of serum utilizing the ability of serum to
inhibit oxidation of an indicator substance, while anti-
oxidant potential is an indirect method in which the
total reductive properties of serum are quantied by
the addition of a known oxidant (Bartosz, 2010). Both
TEAC and FRAP result in the formation of colorimetric
solutions that can be assessed spectrophotometrically
(Benzie & Strain, 1996; Re et al., 1999; Villano et al.,
2004).
Biochemical Assays for Serum
Biomarkers of Oxidative Damage
Serum protein carbonyls and lipid hydroperoxides were
used to quantify oxidative damage in samples collected
during the trek. Serum protein carbonyl concentration
was quantied as a marker of protein oxidation. Total
protein content was rst determined based on the methods
of Bradford (1976) to normalize total protein content in
individual samples before the protein carbonyl assay.
Serum samples were assayed using a commercially avail-
able ELISA kit for protein carbonyl content (Biocell,
Papatoetoe New Zealand) according to assay instructions
and reported as μM/mg protein. Lipid peroxidation was
measured via ferrous oxidation-xylenol orange assay
modied from Nourooz-Zadeh et al. (1994) and reported
as μmol/L serum. The assay principle is based on the
oxidation of ferrous ions to ferric ions by lipid hydro-
peroxides and the formation of a colored complex that
was quantied spectrophotometrically (Nourooz-Zadeh,
Tajaddini-Sarmadi, & Wolff, 1994).
Statistical Analysis
A one-way repeated-measures ANOVA was used to
assess differences between time points with paired t tests
to determine between-samples time differences when
appropriate. For key dependent variables, a Mauchly’s
test conrmed that there were no violations in spheric-
ity. Statistical tests were performed using SPSS version
19.0 (Chicago, IL). Signicance was set a priori at p ≤
.05 for ANOVA.
Results
Subject Characteristics
All participants completed the trek within a 30-min time
window of each other. Participant body weights were not
signicantly different between Pre (76.0 ± 4.0 kg) and
Post (74.8 ± 3.9 kg) measurements (p = .830).
Figure 1 — Trek timeline. Syringe icons indicate blood-collection time points.
