Clinical Science (2004) 107, 589–600 (Printed in Great Britain)
Evidence against redox regulation of energy
homoeostasis in humans at high altitude
Damian M. BAILEY∗ , Philip N. AINSLIE†, Simon K. JACKSON‡,
Russell S. RICHARDSON§ and Mohammed GHATEI
∗
Colorado Center for Altitude Medicine and Physiology, Departments of Anesthesiology and Surgery, University of Colorado
Health Sciences Center, Denver, CO 80262, U.S.A., †Cardiovascular/Respiratory Research Group, Department of Physiology
and Biophysics, University of Calgary, Alberta, Canada T2N 4N1, ‡Department of Medical Microbiology, University of Wales
College of Medicine, Cardiff, CF14 4XN U.K., §Department of Medicine, University of California at San Diego, La Jolla,
CA 92093, U.S.A., and Gastroenterology Laboratory, Department of Medicine, Royal Postgraduate School,
Hammersmith Hospital, London W12 ONN, U.K.
A
B
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The present study examined if free radicals and associated inflammatory sequelae influenced
metabolic biomarkers involved in the neuro-endocrinological regulation of energy homoeostasis
at high altitude. Sixteen mountaineers (11 males/five females) were matched for physical fitness and
caloric intake and assigned in a double-blind manner to either antioxidant (n = 8) or placebo (n = 8)
supplementation, which was enforced for 7 days at sea level and during an 11-day ascent to 4780 m.
Enteral prophylaxis incorporated a daily bolus dose of 1 g of L-ascorbate, 400 international units
of D,L-α-tocopherol acetate and 600 mg of α-lipoic acid. EPR (electron paramagnetic resonance)
spectroscopic detection of PBN (α-phenyl-tert-butylnitrone) adducts confirmed an increase in the
venous concentration of carbon-centred radicals at high altitude in the placebo group, whereas a
decrease was observed in the antioxidant group (P < 0.05 compared with that at sea level). EPR
detection of DMSO/A − (DMSO-supplemented ascorbate free radical) demonstrated that the
increase in carbon-centred radicals at high altitude was associated with a decrease in ascorbate
(r2 = 0.63; P < 0.05). Ascent to high altitude (pooled placebo + antioxidant groups) also increased
the expression of pro-inflammatory cytokines (P < 0.05 compared with that at sea level) and biomarkers of skeletal tissue damage (P < 0.05). Despite a general decrease in leptin, insulin and
glucose at high altitude (pooled placebo + antioxidant groups; P < 0.05 compared with that at sea
level), persistent anorexia resulted in a selective loss of body fat (P < 0.05). In conclusion, antioxidant prophylaxis decreased the concentration of carbon-centred radicals at high altitude (P <
0.05 compared with the placebo group), but did not influence markers of inflammation, appetiterelated peptides, ad libitum nutrient intake or body composition. Thus free radicals do not appear
to be involved in the inflammatory response and subsequent control of eating behaviour at high
altitude.
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Key words: antioxidant, appetite, EPR spectroscopy, free radical, inflammation, weight loss.
Abbreviations: A䊉− , ascorbate free radical; AMS, acute mountain sickness; CPK, phosphocreatine kinase; cTnI, cardiac troponin
I; CV, coefficient of variation; DMSO/A䊉− , DMSO-supplemented A䊉− ; G, Gauss; GLP-1, glucagon-like peptide 1; HOMA,
homoeostasis model assessment; HOMA-βCF, HOMA of β-cell function; HOMA-IR, HOMA of insulin resistance; IL-6,
interleukin-6; LDH, lactate dehydrogenase; NEFA, non-esterified fatty acids; OH䊉− , hydroxyl radical; OOH, hydroperoxide;
PBN, α-phenyl-tert-butylnitrone; PUFA, polyunsaturated fatty acid; RO䊉 , alkoxyl radical; RC䊉 , alkyl radical; Sao2 , arterial oxygen
saturation; TNF-α, tumour necrosis factor-α; V̇o2 max, maximal oxygen uptake.
Correspondence: Dr Damian M. Bailey (email [email protected]).
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INTRODUCTION
A loss of body fat in healthy subjects residing at sea
level stimulates the neuro-endocrinological activation of
orexigenic pathways that initiate compensatory mechanisms to increase caloric intake [1]. However, an uncoupling of peripheral–central mechanisms seems to occur
during a prolonged ascent to high altitude, since anorexia
persists despite a loss of torso adipose tissue [2].
Recent evidence from our laboratory has suggested
a contributory role for the satiety neuropeptide cholecystokinin, which was markedly elevated following an
active ascent to 5100 m [2]. High-altitude anorexia has
subsequently been associated with increased free-radicalmediated skeletal tissue damage [2,3] and expression of
pro-inflammatory cytokines [4]. Furthermore, a followup study conducted at Everest base camp (5180 m)
demonstrated that oral prophylaxis with dietary antioxidant vitamins offset hypophagia by delaying mealinduced satiety [5].
These findings highlight a potential functional link
between free-radical-mediated inflammatory sequelae
and the peripheral release of catabolic signalling molecules known to influence ad libitum feeding behaviour
at high altitude. This is conceivable, since free radicals
up-regulate expression of specific cytokines [6] that can
induce feeding inhibition via activation of hypothalamic-,
gastrointestinal-, metabolic- and endocrine-dependent
mechanisms [7].
Therefore the present study manipulated redox status
at high altitude using the same experimental approach
to that employed previously [5], but with the addition
of serial blood sampling to examine metabolic and
functional changes in ad libitum eating behaviour. We
specifically employed only moderately active subjects,
who we anticipated would be especially susceptible to
eccentric tissue damage during the physically demanding
trek to base camp. We hypothesized that oral prophylaxis
with the same cocktail of aqueous/lipid phase dietary
antioxidant vitamins employed previously [5] would reduce the EPR signal intensity of spin-trapped free
radicals, tissue damage and subsequent activation of proinflammatory anorexigenic cytokines at high altitude.
We hypothesized further that ‘downstream’ changes in
the peripheral concentration of putative regulators of
energy homoeostasis would increase appetite and thus,
by consequence, offset the weight loss traditionally
experienced at high altitude.
METHODS
Subjects
Sixteen moderately active Caucasians (11 males and five
females) with a sea-level V̇o2 max (maximal oxygen up−1
−1
take) of 3.24 +
− 0.51 litres/min (45 +
− 8 ml · kg · min ),
established as described previously [3], volunteered for
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the present study following approval by the local Research Ethics Committee (Bro Taff, Cardiff, U.K.).
All procedures were conducted according to the code of
Ethics of the World Medical Association (Declaration
of Helsinki). Subjects were all permanent lowland residents with no previous history of altitude-related
illness. None of the subjects were habitual users of antioxidant vitamins or specialized food supplements and
were thus instructed to maintain their normal dietary
and lifestyle habits throughout the investigative period.
They were also asked to refrain from taking any prophylactic medication against AMS (acute mountain sickness),
such as analgesics, acetazolamide, dexamethasone and
nifedipine, during the ascent to high altitude.
Study design
Subjects were matched retrospectively as closely as possible for sea-level V̇o2 max, caloric intake (following an
initial dietary evaluation) and gender to ensure comparable metabolic responses during the high-altitude
sojourn. They were subsequently assigned in a doubleblind manner in a balanced fashion to one of two
groups: antioxidant [n = 8 (two females, six males;
−1
V̇o2 max = 3.21 +
− 0.49 litres/min or 46 +
− 10 ml · kg ·
−1
min ] and placebo [n = 8 (three females and five males);
−1
V̇o2 max = 3.27 +
− 0.56 litres/min or 44 +
− 5 ml · kg ·
−1
min ].
Intervention
Antioxidant or placebo supplementation commenced at
sea level 7 days prior to departure to India, for 4 days in
Delhi and during a 7 day ascent to base camp located at
4780 m in the Lombarh valley, approx. 200 km west of
Kashmir. Both groups were instructed to consume four
capsules immediately after breakfast at 08:00 hours to
facilitate assimilation.
The antioxidant group was instructed to ingest four
vegetable-based capsules/day that each contained 250 mg
of l-ascorbate, 100 international units of the esterified
ester d,l-α-tocopherol acetate and 150 mg of α-lipoic
acid.
The placebo group ingested four capsules of identical
external appearance, taste and smell each of which contained an equal quantity of plant cellulose extract.
Ascent to base camp
Subjects rested for 4 days in Delhi (approx. 1000 m)
before starting the 7-day trek to base camp. Each active
ascent day involved 3–4 h of trekking, resulting in an
average daily ascent rate of 426 +
− 299 m (calculated as the
final or sleeping altitude minus the starting or waking
altitude) that also incorporated substantial periods of
descent.
Diet
Subjects ingested food and water ad libitum, and there
was a wide choice of freshly prepared palatable food
Free radicals and appetite
items consisting of salads, rice, pasta, beans, fish, bread,
vegetables and chicken during the high-altitude trek.
Metabolic measurements
Overnight fasted blood samples were obtained from an
antecubital forearm vein using the VacutainerTM method
between 10:00 and 11:00 hours following 30 min of
supine rest and 2–3 h after supplementation. Samples
were obtained at sea level prior to supplementation
3 weeks before the expedition departure and during the
first morning following arrival at base camp (day 7 of
the trek). A manually operated centrifuge was utilized
throughout the study, and the serum or plasma supernatant was immediately immersed in liquid nitrogen
following both sea-level and high-altitude sampling.
Frozen high-altitude samples were transported back to
the author’s laboratory based at sea level in the U.K. for
subsequent analysis. Storage time prior to analysis was
identical for sea level and high-altitude samples to limit
any inconsistencies that may have arisen as a consequence
of metabolic decay.
Measurement of free radicals
Venous blood was collected into an SST vacutainer that
contained 190 mmol/l PBN (α-phenyl-tert-butylnitrone;
dissolved in 0.9 % NaCl) as described previously [8,9].
After centrifugation, the PBN adduct was extracted
from the serum supernatant with toluene (nitrogengassed and scanned for artifactual EPR signals; 99.8 %,
HPLC-grade; Sigma, Poole, Dorset, U.K.) and frozen
in liquid nitrogen prior to EPR spectroscopy. The
extraction efficiency for PBN was approx. 85–90 %, as
confirmed by UV spectroscopy. Following return to
the author’s laboratory in the U.K., the adduct (200 µl)
was pipetted into a 5 mm (outer diameter) precisionbore quartz EPR sample tube (Wilmad LabGlass, Buena,
NJ, U.S.A.) that had been flushed with compressed
nitrogen. The sample was immediately vacuum degassed (West Technology, Bristol, U.K.) using a freeze
(liquid nitrogen)–thaw procedure at a fixed vacuum
of 10−3 Torr (Pirani 14-gauge detector; Edwards,
APG-NW, West Sussex, U.K.) using a turbo molecular
pump (ACT 200T; Alcatel, Annecy, France) for two
cycles (total degassing time of 9 min). All procedures and
chemicals were performed/stored in the dark to avoid
photolytic degradation of the spin-trap.
Complementary experiments were performed in an
attempt to tentatively identify the origin of adducts detected in human serum. Toluene extracts were recovered
from an aqueous Fenton reaction mixture containing
3.0 mmol/l H2 O2 , 0.3 mmol/l Fe2+ and 190 mmol/l
PBN designed to generate an ‘authentic’ PBN-hydroxyl
(OH − ) adduct. Furthermore, we assessed the possibility
that adducts were generated from analogues of lipid
hydroperoxides shown previously [10] to increase as a
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function of physical exercise and inspiratory hypoxia.
Spin-trapping was subsequently performed at 37 ◦ C
in 4.5 ml of aqueous reaction mixture containing a
final concentration of 8.3 µmol/l FeSO4 · 7H2 O, 0.13 %
cumene-OOH (hydroperoxide) and 190 mmol/l PBN.
In order to detect DMSO/A − [DMSO-supplemented
−
A (ascorbate free radical]), DMSO (0.5 ml) was added
to thawed serum (0.5 ml) and vortex-mixing for 10 s
prior to addition (200 µl) to a glass Pasteur pipette within
60 s.
EPR spectroscopic analyses were conducted at 21 ◦ C
using an EMX X-band EPR spectrometer fitted with an
ER TM110 cavity (Bruker, Karlsruhe, Germany). Operating conditions during measurement of the PBN spinadducts and DMSO/A − respectively, were as follows:
modulation amplitude of 0.5 Gauss [(G; where 1 G =
10−4 T (telsa)] and 2 G, 1 × 105 /1 × 105 receiver gain, time
constant of 82 and 41 ms, magnetic field centre of 3465 G
and 3477 G and scan width of +
− 50 G and +
− 15 G for
ten incremental scans. After identical baseline correction
and filtering, each of the spectral peak-to-trough line
heights were normalized relative to the square root of
line width in G and the mean considered a measure of the
relative spin adduct or DMSO/A − concentration following conformation of peak-to-trough line-width conformity and double integration on a random selection of
samples.
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Assessment of tissue damage
Serum CPK (phosphocreatine kinase) and LDH (lactate
dehydrogenase) activity were determined via reflectance
spectrophotometry using a Vitros 750 analyser (OrthoClinical Diagnostics, Raritan, NJ, U.S.A.). Both the intraand inter-assay CVs (coefficients of variation) for both
analytes were < 5.0 %.
Serum myoglobin and cTnI (cardiac troponin I)
were measured using an automated chemiluminescence
immunoassay (ACS 180; Bayer-Chiron Immunodiagnostics, Fullerton, CA, U.S.A.). The intra-assay CV for
myoglobin at a variety of concentrations ranging from
58–685 µg/l was calculated at 3.5 % (n = 192 samples)
and the inter-assay CV was 1.3 % (n = 192 samples). The
intra-assay CV for cTnI at a variety of concentrations
ranging from 2.5–046.5 µg/l was calculated at 3.5 %
(n = 270 samples) and the inter-assay CV was 3.5 % (n =
270 samples).
The plasma concentration of TNF-α (tumour necrosis
factor-α) and IL-6 (interleukin-6) was determined using
commercial assays (Quantikine, R&D Systems,
Minneapolis, MN, U.S.A.). Intra-assay and inter-assay
CVs were < 5.0 %.
Metabolic regulators of energy
homoeostasis
The plasma concentration of the regulatory proteins
leptin, GLP-1 (glucagon-like peptide 1) and insulin were
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measured using established in-house RIAs. Intra-assay
and inter-assay CVs were < 5.0 %.
Plasma NEFA (non-esterified fatty acids) were measured enzymically (Behring Diagnostics, La Jolla, CA,
U.S.A.). The intra- and inter-assay CVs were 1.6 % and
5.0 % respectively.
Plasma glucose was analysed by the glucose/HK
method (Boehringer Manheim, Mannheim, Germany).
The intra and inter-assay CVs were 2.0 % and 5.0 %
respectively.
Prediction of insulin resistance
and β-cell function
A mathematical model [HOMA (homoeostasis model
assessment)] was incorporated to predict changes in
insulin resistance (HOMA-IR) and β-cell function
(HOMA-βCF) at sea level and high altitude. This model
predicts the relative contributions of insulin resistance
and β-cell deficiency that would account for the observed
fasting plasma concentrations of insulin and glucose
[11]. Briefly, these predictions were calculated using the
following equations;
HOMA-IR (arbitrary units) = insulin (munits/l)
× [glucose concentration (mmol/l)/22.5]
HOMA-βCF (%) = 20 × insulin/(glucose − 3.5).
Additional parameters
All physiological measurements were conducted at sea
level 3 weeks prior to the expedition departure and during
the first morning following arrival at base camp.
The physical symptoms associated with the neurological syndrome AMS were assessed immediately on waking (approx. 07:00 hours) using the Lake Louise Consensus scoring system [12] as reported by Maggiorini
et al. [13]. AMS was defined as a cumulative self-assessment and clinical score of 3 points in the presence
of a headache.
We incorporated mucosal petechiometry and pressure
algometry to assess the functional consequences of potential changes in microvascular permeability that may be
compromised by free-radical-mediated oxidative damage. Mucosal petechiometry (count) was performed according to methods established previously [14]. Briefly,
a subatmospheric pressure of approx. 200 mmHg was
applied to two adjacent sites of the buccal mucous membrane of the lower lip for 60 s using the barrel of a 2 ml
syringe (1 cm diameter). The sum of petechiae produced
at each respective site was counted with the aid of a
magnifying glass (× 10 magnification) and the results
averaged. Pressure algometry (pain threshold) was conducted as described recently [3]. The muscle belly and
distal region of eight sites (bicep belly, vastus lateralis,
quadriceps femoris, gastrocnemius of the left and right
leg) were located by palpation and marked with a per
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2004 The Biochemical Society
manent pen. A pressure algometer (Force DialTM FDK2;
Wagner Instruments, Greenwich, CT, U.S.A.) that
consisted of a round-ended metal probe with a 10 mm
diameter rubber tip was randomly applied to each
site of both legs with the subject prone. Force was
gradually increased up to a maximum of 30 kg. Following
ten practice trials on alternative sites, the subject was
instructed to verbally indicate when the stimulus became
‘uncomfortable’ and the force was subsequently recorded. If no indication of discomfort was reported up
to 30 kg, soreness was not considered to be present at
that specific site. The pain threshold was quantified as
the summed forces divided by the number of sites with
soreness (maximum of eight sites).
Urine osmolality was measured on the morning of
arrival at base camp via freezing-point depression using
a micro-osmometer (Model 3MO plus; Advanced Instruments, Norwoor, MA, U.S.A.).
Upon waking, after voiding and defecation, nude body
mass (kg) was determined using a calibrated electronic
scale (Seca; Cardiokinetics, Salford, U.K.). Harpenden
skinfold calipers (John Bull; British Indicators, Woking,
Surrey, U.K.) and a flexible metallic tape measure
(Holtain, Crymych, U.K.) were used to assess selected
skinfolds (bicep, tricep, subscapular, suprailiac and midthigh) and girths (relaxed bicep, mid-thigh, calf and
forearm) to specifically compare torso and limb body
composition. Absolute changes in body composition (fat
and fat-free mass) were determined from changes in body
mass and the percentage body fat was estimated using
established equations [15]. Circumferences of the open
hand, foot and ankle (left and right sides) were performed
as a surrogate measure of peripheral oedema at high altitude. Three repeated measurements were performed by
the same trained investigator for each site and the mean
value calculated. Previous research in our laboratory has
identified an intra-investigator CVs of 3 % and < 1 %
for these skinfolds and girths respectively (results not
shown).
Sao2 (arterial oxygen saturation) was assessed indirectly using an earlobe pulse oximeter (model 8800,
Nonin Medical, Minneapolis, MN, U.S.A.).
Eating behaviour
Each subject was interviewed by a physiologist and
instructed to complete an appetite rating before and immediately after the breakfast, dinner and evening meals
were consumed ad libitum. Ratings consisted of questions
on hunger, desire to eat, satiety, nausea and thirst, which
were subjectively assessed using 100-mm visual analogue
scales, anchored with hedonic descriptors such as ‘not at
all’ (0 mm) to ‘extremely’ (100 mm).
Dietary assessments were conducted using a selfreport questionnaire that was completed by each subject
during daily interviews with a nutritionist to ensure
Free radicals and appetite
Figure 1 Typical changes in the EPR spectroscopic signal of ex vivo trapped PBN adducts in two separate subjects
Blank spectrum based on degassed toluene + PBN (excludes serum). Computer simulation incorporated hyperfine coupling constants of a N = 13.7 G and a βH = 1.9 G
(90 % of total signal) and a N = 14.0 G and a βH = 4.0 G (10 % of total signal). Ordinates for all spectra are scaled identically.
accuracy of completion and compliance. Dietary analyses
were performed 7 days prior to the start of sea-level
testing and during the 7 day ascent to base camp. Caloric
intake and composition was subsequently determined
using conventional food tables.
Statistics
Following application of repeated Shapiro–Wilk W tests
and confirmation of distribution normality, a two-way
mixed ANOVA with a between- (group: antioxidant
compared with placebo) and within- (location: sea
level compared with high altitude) subjects factor was
subsequently incorporated to assess the effects of antioxidant prophylaxis on selected dependent variables (see
Tables 1–3, 5 and 6). Changes in appetite ratings
(see Table 4) were analysed with a three-way ANOVA
[group × location × timing (pre- or post-meal)]. When an
interaction effect was indicated, within-group comparisons were determined using Bonferroni-corrected (when
appropriate) paired samples Student t tests at each level
of the between-subjects factor. A one-way ANOVA
followed by an a posteriori Tukey honestly significant
difference test was incorporated for between-group
comparisons. Non-parametric equivalents (if data were
not normally distributed; P < 0.05) included Wilcoxon
matched pairs-signed ranks and Mann–Whitney U tests.
The relationship between selected dependent variables
was assessed using a Pearson product moment correlation
(see Figure 3). Significance for all two-tailed tests was
established at an α level of P < 0.05, and data are expressed
as means +
− S.D.
RESULTS
Compliance and blinding
One female discontinued placebo supplementation and
was subsequently excluded from overall analyses (final
n = 7), although this did not influence the initial matching of sea level V̇o2 max between treatment groups (3.21 +
−
−1
−1
0.49 litres/min or 46 +
− 10 ml · kg · min in the antioxidant group compared with 3.38 +
− 0.51 litres/min or
−1
−1
46 +
− 4 ml · kg · min in the placebo; P > 0.05). The
remaining 15 subjects consumed all of the capsules prescribed (72 per subject for the entire study) and there
were no associated side effects reported. A questionnaire
indicated that six out of 15 subjects (two in the antioxidant
group and four in placebo group) guessed the correct
intervention thus confirming the effectiveness of the
blinding protocol.
Free radical metabolism
Figure 1 displays typical EPR spectra of PBN adducts
detected at sea level and high altitude before and after
supplementation in two separate individuals. Compared
with the sea-level control condition, antioxidants decreased spectral amplitude at high altitude, whereas an
increase was observed in the placebo group. The dominant signal exhibited hyperfine coupling constants of
aN = 13.7 G and aβH = 1.9 G. Visual inspection of spectra
revealed the probable presence of a second initially unidentified adduct, albeit of low signal intensity. Computer
simulation tentatively confirmed that this adduct contributed to approx. 10 % of the total signal recovered in
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Figure 2 Corresponding changes in the EPR spectroscopic signal of DMSO-A䊉− for the same subjects depicted in Figure 1
Blank spectrum based on DMSO only (excludes serum). Ordinates for all spectra are scaled identically.
Table 1 EPR spectroscopic detection of free radicals at high altitude
Values are means +
− S.D. All dependent variables were normally distributed. Main effects indicate pooled differences (P < 0.05) for either location (high altitude
compared with sea level) or group (antioxidant compared with placebo) as indicated. Difference ∗ within and †between groups for a given location (P < 0.05). AU,
arbitrary units.
Group . . .
Stage of supplementation . . .
Location . . .
Free radicals
√
PBN-spin adduct (AU/ G)
Main effect for group/group × location interaction
√
DMSO-A − (AU/ G)
Main effect for group/group × location interaction
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all samples and coupling constants were subsequently
determined as aN = 14.0 G and aβH = 4.0 G.
Virtually identical spectral characteristics were obtained from Fenton-generated PBN-OH − (aN = 13.5 G
and aβH = 1.6 G), and adducts generated during the metalcatalysed auto-oxidation of cumene-OOH (aN = 13.8 G
and aβH = 2.0 G).
Figure 2 displays doublets characteristic of the A −
(aβH4 = 1.8 G) for the same two individuals depicted in
Figure 1. Compared with the sea-level control condition,
antioxidants increased DMSO/A − at high altitude,
whereas a decrease was observed in the placebo group
(Table 1). An inverse relationship (r2 = 0.63, P < 0.05)
was observed between the change (high altitude minus
sea level) in DMSO/A − and PBN adduct concentration
(Figure 3).
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Placebo (n = 7)
Antioxidant (n = 8)
PRE
Sea level
POST
High altitude
PRE
Sea level
POST
High altitude
6089 +
− 1672
∗
9765 +
− 2518
6794 +
− 2246
∗
1424 +
− 217 †
3078 +
− 431
∗
2254 +
− 592
3261 +
− 379
∗
4454 +
− 297 †
in myoglobin, TNF-α and IL-6 at high altitude (Table 2).
A positive correlation was observed between the pooled
change (high altitude minus sea level) in myoglobin
and TNF-α (r2 = 0.42, P < 0.05). Pain threshold was
not affected by high altitude or antioxidants, whereas
petechiae count was elevated in the placebo group due to
an increase at high altitude (Table 2).
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Tissue damage and inflammation
Antioxidants did not influence selected biomarkers of
tissue damage or inflammation, despite a general increase
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Urine osmolality
Urine osmolality measured at high altitude was 454 +
− 141
and 409 +
171
mosM/kg
of
body
weight
for
placebo
and
−
antioxidant groups respectively (P > 0.05).
Metabolic regulators of energy
homoeostasis
Antioxidants did not influence signalling molecules
associated with the regulation of energy homoeostasis,
despite marked changes at high altitude (Table 3). A
general decrease in leptin was observed due primarily to a
loss of torso adiposity. HOMA-IR decreased markedly at
Free radicals and appetite
Figure 3 Relationship between changes (pooled high-altitude minus sea-level values) in DMSO/A䊉− and PBN adduct
concentration
All dependent variables were normally distributed and expressed in arbitrary units (AU) normalized to the square root (sqrt) of the mean line width in G.
Table 2 Membrane permeability and inflammation
Values are means +
− S.D. All dependent variables, except total CPK and cTnI, were normally distributed. Main effect indicates pooled difference (P < 0.05) for group
(antioxidant compared with placebo) and location (high altitude compared with sea level). †Difference between groups for a given location (P < 0.05).
Group . . .
Stage of supplementation . . .
Location . . .
Molecular damage
Total CPK (units/l)
Myoglobin (ng/ml)
Main effect for location
LDH (ng/ml)
cTnI (ng/ml)
Functional damage
Pain threshold (kg)
Petechiae count (n)
Main effect for group and location/group × location interaction
Pro-inflammatory cytokines
TNF-α (pg/ml)
Main effect for location
IL-6 (pg/ml)
Main effect for location
Placebo (n = 7)
Antioxidant (n = 8)
PRE
Sea level
POST
High altitude
PRE
Sea level
POST
High altitude
188 +
− 189
42 +
− 16
377 +
− 123
71 +
− 30
129 +
− 59
37 +
−7
211 +
− 74
47 +
− 23
437 +
− 45
0.02 +
− 0.03
465 +
− 67
0.01 +
− 0.01
422 +
− 58
0.02 +
− 0.01
450 +
− 64
0.04 +
− 0.04
8.2 +
− 3.1
22 +
−5
9.8 +
− 3.3
28 +
−9
8.4 +
− 3.7
19 +
− 10
9.9 +
− 3.4
16 +
− 5†
3.6 +
− 1.6
7.9 +
− 3.5
3.1 +
− 1.5
6.6 +
− 3.5
2.0 +
− 3.7
15.3 +
− 14.5
2.4 +
− 2.4
11.3 +
− 5.6
high altitude due to the combined decrease in insulin and
glucose, whereas no changes were observed in predicted
HOMA-βCF. In contrast, high-altitude exposure did not
influence GLP-1 or NEFA.
Appetite ratings and dietary intake
Compared with the findings at sea level, hunger ratings
and the desire to eat decreased at high altitude and were
associated with an increase in satiety, nausea and thirst
(Table 4). Caloric intake was subsequently lower due to
a selective decrease in fat intake, although there was a
tendency for carbohydrate and protein intake to increase
(Table 5). However, appetite ratings and dietary intake
were not influenced by antioxidants. Micronutrient intake (independent of supplemented vitamins) was not different as a function of location or group (results not
shown).
Anthropometry
A decrease in body mass was observed at high altitude due
to a reduction in body fat that was not influenced by
antioxidants (Table 6). All remaining anthropometric
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Table 3 Metabolic regulation of energy homoeostasis
Values are means +
− S.D. All dependent variables, except adjusted leptin concentrations and HOMA-βCF, were normally distributed. Adjusted leptin refers to concentration
normalized relative to fat mass (FM). Main effect indicates pooled difference for location (high altitude compared with sea level; P < 0.05). AU, arbitrary units.
Placebo (n = 7)
Group . . .
Stage of supplementation . . . PRE
Location . . .
Sea level
Leptin (pmol/l)
Main effect for location
Adjusted leptin (pmol/l · kg−1 of FM)
GLP-1 (pmol/l)
NEFA (mmol/l)
Insulin (pmol/l)
Main effect for location
Glucose (mmol/l)
Main effect for location
HOMA-IR (AU)
Main effect for location
HOMA-βCF (%)
Antioxidant (n = 8)
POST
High altitude
PRE
Sea level
POST
High altitude
2.6 +
− 1.2
2.1 +
− 0.8
3.0 +
− 1.6
2.4 +
− 1.1
0.1 +
− 0.1
20.7 +
− 9.0
0.18 +
− 0.06
92.4 +
− 38.9
0.1 +
− 0.1
27.1 +
− 13.6
0.22 +
− 0.10
72.4 +
− 47.3
0.2 +
− 0.1
27.4 +
− 9.7
0.23 +
− 0.10
85.5 +
− 22.7
0.2 +
− 0.1
30.0 +
− 7.9
0.34 +
− 0.15
58.0 +
− 37.6
4.4 +
− 0.5
4.2 +
− 0.7
4.5 +
− 0.3
4.1 +
− 0.3
2.6 +
− 1.2
2.0 +
− 1.5
2.4 +
− 0.7
1.5 +
− 1.0
487 +
− 543
191 +
− 203
252 +
− 82
332 +
− 213
Table 4 Appetite ratings
Values are means +
− S.D. expressed in mm based on visual analogue scales. All dependent variables were normally distributed. Antioxidant group, n = 8; placebo
group, n = 7. Pre- and post-meal ratings each represent the mean of three recordings taken at the breakfast, dinner and evening meals. Main effects indicate pooled
differences (P < 0.05) for either location (high altitude compared with sea level) or timing (pre-compared with post-meal) as indicated.
Location . . .
Sea level (pre-supplementation)
High altitude (post-supplementation)
Timing . . .
Pre-meal
Pre-meal
Group . . .
Placebo
Post-meal
Post-meal
Antioxidant
Placebo
Antioxidant
Placebo
Antioxidant
56 +
− 10
14 +
−7
19 +
− 17
40 +
− 11
38 +
− 14
6+
−4
5+
−7
59 +
− 13
18 +
−9
23 +
− 15
46 +
− 17
51 +
−9
13 +
−6
20 +
− 12
33 +
− 12
46 +
− 15
61 +
− 27
51 +
− 18
48 +
− 12
76 +
−8
74 +
− 14
21 +
− 14
10 +
−6
19 +
− 14
26 +
− 18
27 +
− 19
35 +
− 18
26 +
− 17
48 +
− 16
23 +
−7
28 +
− 16
57 +
−9
62 +
− 10
57 +
− 21
42 +
− 19
variables, including girths and circumferences, did not
change.
S aO2
Hunger
60 +
−6
Main effects for location and timing
Desire to eat
54 +
− 11
Main effects for location and timing
Satiety
31 +
− 11
Main effects for location and timing
Nausea
12 +
−7
Main effects for location
Thirst
55 +
− 10
Main effects for location and timing
Interaction effects for location × timing/location × timing × group
AMS
Antioxidants did not influence the incidence or severity
of AMS at high altitude (incidence, four in the antioxidant
group compared with one in the placebo group; P >
0.05; and severity, 2.9 +
− 1.4 points in the antioxidant group compared with 2.6 +
− 0.5 points in the placebo
group; P > 0.05). A comparative examination of the
physiological characteristics of AMS was not considered
appropriate due to power constraints.
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Placebo
Antioxidant
Antioxidants exerted no effect on resting Sao2 at high
altitude (82 +
− 3 % in the antioxidant group compared
with 84 +
4
%
in the placebo group; P > 0.05).
−
DISCUSSION
The present study adopted a balanced double-blind
placebo-controlled design to examine if free radicals and
associated inflammatory sequelae influenced the neuroendocrinological regulation of energy homoeostasis at
Free radicals and appetite
Table 5 Dietary intake
Values are means +
− S.D. All dependent variables were normally distributed. Main effect indicates pooled difference for location (high altitude compared with sea level;
P < 0.05).
Group . . .
Stage of supplementation . . .
Location . . .
Caloric intake (MJ/day)
Main effect for location
Carbohydrates (%)
Main effect for location
Total fat (%)
Protein (%)
Main effect for location
Placebo (n = 7)
Antioxidant (n = 8)
PRE
Sea level
POST
High altitude
PRE
Sea level
POST
High altitude
9.9 +
− 3.8
8.6 +
− 2.8
10.6 +
− 1.6
11.1 +
− 2.3
49 +
−4
48 +
−4
53 +
−3
56 +
−7
38 +
−4
13 +
−2
35 +
−4
16 +
−5
35 +
−4
11 +
−4
33 +
−5
9+
−2
Table 6 Anthropometrics
Values are means +
− S.D. All dependent variables were normally distributed. Trunk skinfolds were calculated as the sum () of subscapular and suprailliac sites; limb
skinfolds were determined as the sum of bicep, tricep and mid-thigh sites; girths were determined as the sum of bicep, tricep, calf and forearm girths; circumferences were calculated as the sum of hand, foot and ankle circumferences (mean of left + right sides). Main effect indicates pooled difference for location
(high altitude compared with sea level; P < 0.05).
Placebo (n = 7)
Group . . .
Stage of supplementation . . . PRE
Location . . .
Sea level
Body mass (kg)
Main effect for location
Fat mass (kg)
Main effect for location
Fat free mass (kg)
trunk skinfolds (cm)
Main effect for location
limb skinfolds (cm)
girths (cm)
circumferences (cm)
Antioxidant (n = 8)
POST
High altitude
PRE
Sea level
POST
High altitude
74.2 +
− 10.3
70.6 +
− 8.8
71.5 +
− 10.5
68.0 +
− 9.0
18.9 +
− 3.6
16.4 +
− 3.6
16.5 +
− 7.7
14.5 +
− 5.0
55.3 +
− 8.7
39.5 +
− 15.8
54.2 +
− 8.3
31.0 +
− 11.9
55.0 +
− 9.1
30.2 +
− 10.0
53.5 +
− 8.5
27.7 +
− 8.8
32.4 +
− 8.2
137.6 +
− 12.7
66.2 +
− 6.6
30.6 +
− 7.8
139.8 +
− 12.3
65.0 +
− 5.8
29.6 +
− 19.8
134.9 +
− 14.1
62.3 +
− 6.3
24.1 +
− 5.1
135.1 +
− 14.8
63.2 +
− 7.2
high altitude. Our results have revealed several important
findings. Physical ascent to high altitude was associated
with a clear increase in the venous concentration of
ex vivo spin-trapped carbon-centred radicals and markers of skeletal tissue damage and inflammation that
were associated with a depletion in ascorbate. Although
a general decrease in leptin, insulin and glucose was
observed at high altitude, anorexia was shown to persist,
resulting in a selective loss of body fat. Finally, whereas
dietary manipulation with antioxidant vitamins proved
an effective prophylactic against free-radical generation
at high altitude, it did not exert any downstream
functional effects on appetite or ad libitum nutrient intake
as assessed indirectly using a questionnaire-interview
approach. These findings do not support our original
hypotheses and challenge the notion that free radicals are
involved in the inflammatory response and subsequent
control of eating behaviour at high altitude.
Free radicals: identity and origins
To our knowledge, this is the first study to have applied an
ex vivo EPR spin-trapping technique to reveal an increase
in the venous concentration of carbon-centred radicals at
high altitude. However, it is important to emphasize that,
although EPR is considered the most direct, specific and
sensitive analytical technique for the molecular detection
and subsequent identification of free radical species [16],
the spin-trapping approach employed in the present study
still relies on ex vivo detection of a resonance-stabilized
non-damaging moiety formed clearly downstream of the
primary oxidant production pathway, which we assume
reflects oxidative events initiated in vivo [9,16].
Specific attempts were made to determine the origin of
the trapped species, although any definitive assignment
is clearly unrealistic in light of the complex nature of the
biological system under investigation. Consistent with
previous observations [17,18], the stability and spectral
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characteristics of the primary adduct were consistent
with the trapping of PBN-OH − ; an assignment that was
subsequently confirmed following generation of ‘authentic’ PBN-OH − using an in vitro Fenton reaction
system and verified previously using GC–MS [18]. The
primary and secondary adducts were also consistent
with published values for an oxygen-centred alkoxyl
(RO ) and alkyl (RC ) species respectively, using similar
extraction solvents [19].
We and others [8,20,21] have suggested that RO
detected using this technique may have evolved during
the metal-catalysed (Fe2+ ) reductive decomposition of
extracellular hydroperoxides formed distal to primary
radical (perhaps OH − )-mediated attack of membrane
phospholipids. The fact that identical splitting constants
were obtained during the metal-catalysed auto-oxidation
of cumene-OOH adds further support to this contention,
although our failure to directly assess extracellular concentrations of free catalytic iron in human serum was
unfortunate and is a clear limitation.
The minor signal ascribed to RC may therefore represent longer-lived short-chain ethyl, pentyl or pentenyl
radicals formed during β-scission of RO [22]. Additional
evidence suggests further that the adducts were lipidderived, since they were clearly soluble in non-aqueous
solvents and the spectra displayed consistently lower
signal intensities associated with the high-field lines
characteristic of spin inhibition, which may indicate the
binding of a longer length biopolymer radical such as a
PUFA (polyunsaturated fatty acid) carbon side-chain. It
should be noted that, even though these represent secondary species, they are still thermodynamically capable
(EO ≈ + 1000–1300 mV, where EO is the difference
in standard one electron reduction potential) of oxidizing PUFA-OOH to further compound peroxidative
damage.
The magnitude of the increase in free radical generation
at high altitude was comparable with that typically
observed during the rest to maximal exercise transition
at sea level [9] and thus represents a considerable oxidant
load further confirming its suitability as a ‘redox-reactive’
model. Whether the increase in free radicals was a consequence of increased release or impaired disposal could
not be established, since exchange measurements were not
ethically permissible in such a remote location. Furthermore and since it was not our primary focus, we did
not establish if radicals were a consequence of increased
physical activity and/or inspiratory hypoxia, stimuli that
have been identified previously [10] as major contributors
to the oxidant burden of high altitude. The stability
of NEFA tentatively suggests that the signals detected
were independent of altered lipid-substrate delivery,
an important consideration since our ex vivo trapping
technique primarily detects second or third generation
species formed during the reductive cleavage of organic
peroxides by Fe2+ /Fe(III) [8,9,20,21].
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In contrast with our recent study [5], antioxidant
prophylaxis with the same combination of hydrophilic
and lipophilic vitamins did not influence AMS, despite a
predicted 87 % reduction in radical concentration. This
finding challenges our original contention that neurological symptoms ascribed to AMS are caused by vasogenic oedema subsequent to radical-mediated damage
to the blood–brain barrier [4,5,9]. However, potential
dose response and other pharmacokinetic issues need to
be considered in light of the shorter duration of supplementation employed in the present study (18 days
prior to AMS assessment compared with 31 days in the
former) and low-risk setting.
The addition of a molar excess of DMSO has been
shown to improve EPR detection by promoting oneelectron reversible oxidation of ascorbate to the stabilized
A − and thus reflects changes in peripheral ascorbate
concentration as opposed to de novo radical-induced
A − generation [23]. Although recognizing the reducing
potential of α-tocopherol and α-lipoic acid, changes
in DMSO/A − suggest that ascorbate was involved in
terminating carbon-centred radical propagation.
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Vascular tissue damage, inflammation
and energy homoeostasis
Consistent with previous observations, high-altitude
exposure increased sarcolemmal membrane permeability
[3], microvascular fragility [5] and subsequent expression
of pro-inflammatory cytokines [4,24]. The stability of
cTnI suggested that molecular damage was exclusive to
the skeletal and microvasculature, but not myocardial
tissue in light of its diagnostic specificity as a cardiac
protein [25].
Antioxidant prophylaxis prevented the increase in
petechiae count at high altitude suggesting, although
somewhat tentatively, that free radicals were the initiating
cause of microvascular damage, an observation intimated
in a previous study [5]. In contrast, antioxidants did not
protect against the skeletal tissue damage or associated
inflammatory response at high altitude, thus excluding
a chemical role for free radicals and suggesting that the
initiating stimulus may have been mechanical in origin.
Our subjects were not particularly well trained and thus
probably more susceptible to mechanical tissue damage
inflicted during the eccentric phases of descents encountered during the approach trek to base camp. This may
have contributed to at least part of the oxidant load
experienced at high altitude, since damaged tissue peroxidizes more rapidly than healthy tissue [26].
The possibility that the IL-6 response at high altitude
was unrelated to structural tissue damage or a systemic
acute-phase response cannot be dismissed, since plasma
concentrations are also subject to regulation via blood/
glucose/sympathoadrenal pathways [27]. It was unfortunate that we did not assess α- and β-adrenergic
Free radicals and appetite
contributions to explore further the intimate links
between the sympathetic nervous system, IL-6 response
and energy homoeostasis [28].
However, our primary interest in these phenomena
relates to the proposed interaction between inflammatory
sequelae and the endogenous activation of anorexigenic
pathways [7] potentially involved in the anorexia and
early satiety experienced at high altitude. The symphonic
interplay between multiple pro-inflammatory cytokines
has been shown to induce anorexia by acting at central
nervous system sites directly [29], or by triggering the
peripheral release of humoral mediators considered putative catabolic signalling molecules, such as the adiposity-related hormones, leptin and insulin [7]. The latter
response could not be confirmed in the present study,
since we observed either no change or a decrease in
appetite-related peptides and regulatory proteins, despite
clear evidence for an inflammatory response.
The transduction of afferent input initiated by the
decrease in leptin and insulin would have been expected
to increase the neuronal activation of anabolic effector
pathways to promote hyperphagia and initiate metabolic
responses that promote the deposition of body fat [30].
However, despite adequate availability of palatable foods,
subjects were overtly anorectic which resulted in a
selective loss of body fat at high altitude. It is unlikely
that fluid loss, typically ascribed to increased energy
expenditure at high altitudes [31] accounted for a major
portion of the reduction in body mass observed, since
urine osmolality data indicated that subjects were reasonably well-hydrated assuming that a urine osmolality
of 200–600 mosM/kg of body weight is representative of
a euhydrated state [32]; a likely consequence of subjects being actively encouraged to consume fluids by
investigators. Thus the present findings highlight a functional redundancy in peripheral hormone signalling subsequent to up-regulated cytokine expression, a finding
that clearly warrants future consideration and emphasizes
the potential interpretive limitations associated with the
traditional examination of hormonal markers of appetite
at high altitude.
Despite the unavoidable difficulties associated with
field-based experimentation, the indirect techniques employed in the present study have their limitations. Owing
to the deficiencies of anthropometric models for estimating changes in body composition [33], future studies
should incorporate imaging techniques for the identification of subcutaneous, intra-abdominal and intramuscular
fat depots to more precisely compartmentalize body fat
loss. The limitations associated with self-report questionnaires and failure to directly examine individual
components of energy balance, namely basal and activity
energy expenditure using the doubly labelled water
technique and fecal or urinary losses [34,35], are duly
acknowledged and are to be encouraged in future
studies.
In light of these limitations and assuming that an
energy expenditure of 14.6 MJ is required to metabolize
0.45 kg of adipose tissue [36], the loss of body fat equated
to a cumulative energy deficit of 65 MJ and 81 MJ in
the antioxidant and placebo groups respectively. This
negative energy balance was probably attributable to the
combined effects of increased basal metabolic rate [31],
energy expenditure during the physically demanding trek
to high altitude that served to preserve lean tissue mass
and early satiety subsequent to nausea, a finding that
is consistent with previous investigations [37]. Clearly,
these changes could not be attributed to differences
in physical fitness or gender, since both groups were
matched for these parameters at sea level; important considerations since subtle differences in relative exercise
intensity and subsequent substrate utilization during
ascent to high altitude may have influenced data interpretation.
In conclusion, the present findings demonstrate that
carbon-centred radicals are not involved in the inflammatory response and subsequent hypophagia experienced
at high altitude. The apparent disassociation between
radical generation and inflammation suggests that alternative treatments, including the dietary manipulation of
cytokine production with anti-inflammatory agents such
as ω − 3 PUFAs or ω − 9 MUFAs (mono-unsaturated
fatty acids) [38] may provide more effective prophylaxis
against high-altitude-induced anorexia. Direct assessment of individual components of energy balance,
inclusion of energy sufficient macro/micronutrient controls to complement the ad libitum approach adopted in
the present study and examination of radical exchange
across a selected muscle bed to disassociate release from
disposal mechanisms are to be encouraged in future
studies.
ACKNOWLEDGMENTS
We would like thank to Dr Dave Hullin and Dr Carel
LeRoux for stimulating discussions. Finally, we gratefully
acknowledge the subjects whose cheerful participation
ultimately made this study possible, despite the rigours
of a high-altitude ascent. We remember the late Dr ‘Jack’
T. Reeves who remains a constant source of inspiration.
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Received 17 March 2004/29 June 2004; accepted 26 August 2004
Published as Immediate Publication 26 August 2004, DOI 10.1042/CS20040085
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