Effect of hyperbaric oxygen and vitamin C and E supplementation on biomarkers of oxidative stress in healthy men.
ABSTRACT The objectives of the present study were to evaluate the effect of normobaric and hyperbaric O2 (HBO) on plasma antioxidants and biomarkers of oxidative stress in plasma and urine and to investigate the effect of a 4-week vitamin C plus E supplementation on HBO-induced oxidative stress. Nineteen healthy men were exposed to HBO (100 % O2; 240 kPa) before and after 4 weeks' supplementation with 500 mg vitamin C plus 165 mg alpha-tocopherol equivalents. Exposure to 21 % O2 at 100 kPa served as intra-individual controls (control). Samples for the analysis of plasma antioxidants and oxidative stress biomarkers were collected before and immediately after each treatment. The present results showed that when compared with 'control', a single exposure to HBO resulted in a decrease of plasma vitamin C (P = 0.027) and an increase of lipid peroxides (P = 0.0008) and urinary 8-oxo-deoxyguanosine (8-oxodG) excretion (P = 0.006). Oxidative stress was not prevented by a 4-week supplementation with vitamins C and E. HBO-induced changes in plasma parameters correlated with basal antioxidant levels. The increase of urinary 8-oxodG after HBO plus supplementation correlated negatively with vitamin E intake (P = 0.023). We concluded that in healthy men HBO caused oxidative stress, which could not be prevented by dietary vitamin C plus E supplementation. The present data support the idea that HBO is a suitable model for oxidative stress in healthy volunteers.
- JNCI Journal of the National Cancer Institute 12/1994; 86(21):1645-8. · 14.34 Impact Factor
- [show abstract] [hide abstract]
ABSTRACT: Observational studies suggest that people who consume more fruits and vegetables containing beta carotene have somewhat lower risks of cancer and cardiovascular disease, and earlier basic research suggested plausible mechanisms. Because large randomized trials of long duration were necessary to test this hypothesis directly, we conducted a trial of beta carotene supplementation. In a randomized, double-blind, placebo-controlled trial of beta carotene (50 mg on alternate days), we enrolled 22,071 male physicians, 40 to 84 years of age, in the United States; 11 percent were current smokers and 39 percent were former smokers at the beginning of the study in 1982. By December 31, 1995, the scheduled end of the study, fewer than 1 percent had been lost to follow-up, and compliance was 78 percent in the group that received beta carotene. Among 11,036 physicians randomly assigned to receive beta carotene and 11,035 assigned to receive placebo, there were virtually no early or late differences in the overall incidence of malignant neoplasms or cardiovascular disease, or in overall mortality. In the beta carotene group, 1273 men had any malignant neoplasm (except nonmelanoma skin cancer), as compared with 1293 in the placebo group (relative risk, 0.98; 95 percent confidence interval, 0.91 to 1.06). There were also no significant differences in the number of cases of lung cancer (82 in the beta carotene group vs. 88 in the placebo group); the number of deaths from cancer (386 vs. 380), deaths from any cause (979 vs. 968), or deaths from cardiovascular disease (338 vs. 313); the number of men with myocardial infarction (468 vs. 489); the number with stroke (367 vs. 382); or the number with any one of the previous three end points (967 vs. 972). Among current and former smokers, there were also no significant early or late differences in any of these end points. In this trial among healthy men, 12 years of supplementation with beta carotene produced neither benefit nor harm in terms of the incidence of malignant neoplasms, cardiovascular disease, or death from all causes.New England Journal of Medicine 06/1996; 334(18):1145-9. · 51.66 Impact Factor
- [show abstract] [hide abstract]
ABSTRACT: In several epidemiological studies, high intakes of fruits and vegetables have been associated with a lower incidence of cancer. Theoretically, intake of antioxidants by consumption of fruits and vegetables should protect against reactive oxygen species and decrease the formation of oxidative DNA damage. We set up a parallel 24-day dietary placebo-controlled intervention study in which 43 subjects were randomized into three groups receiving an antioxidant-free basal diet and 600 g of fruits and vegetables, or a supplement containing the corresponding amounts of vitamins and minerals, or placebo. Blood and urine samples were collected before, once a week, and 4 weeks after the intervention period. The level of strand breaks, endonuclease III sites, formamidopyrimidine sites, and sensitivity to hydrogen peroxide was assessed in mononuclear blood cells by the comet assay. Excretion of 7-hydro-8-oxo-2'-deoxyguanine was measured in urine. The expressions of oxoguanine glycosylase 1 and excision repair cross complementing 1 DNA repair genes, determined by real-time reverse transcription-PCR of mRNAs, were investigated in leukocytes. Consumption of fruits and vegetables or vitamins and minerals had no effect on oxidative DNA damage measured in mononuclear cell DNA or urine. Hydrogen peroxide sensitivity, detected by the comet assay, did not differ between the groups. Expression of excision repair cross complementing 1 and oxoguanine glycosylase 1 in leukocytes was not related to the diet consumed. Our results show that after 24 days of complete depletion of fruits and vegetables, or daily ingestion of 600 g of fruit and vegetables, or the corresponding amount of vitamins and minerals, the level of oxidative DNA damage was unchanged. This suggests that the inherent antioxidant defense mechanisms are sufficient to protect circulating mononuclear blood cells from reactive oxygen species.Cancer Epidemiology Biomarkers & Prevention 10/2003; 12(10):1016-22. · 4.56 Impact Factor
Effect of hyperbaric oxygen and vitamin C and E supplementation
on biomarkers of oxidative stress in healthy men
Nicolle Bader1*, Anja Bosy-Westphal2, Andreas Koch3, Gerald Rimbach4, Allan Weimann5,
Henrik E. Poulsen5and Manfred J. Mu ¨ller2
1Insititute of Biological Chemistry and Nutrition, University of Hohenheim, Garbenstrasse 28, D-70593 Stuttgart, Germany
2Insititute of Human Nutrition and Food Science, Christian-Albrechts-Universita ¨t zu Kiel, Du ¨sternbrooker Weg 17,
D-24105 Kiel, Germany
3German Naval Medical Institute, Kopperpahler Allee 120, D-24119 Kronshagen, Germany
4Insititute of Human Nutrition and Food Science, Christian-Albrechts-Universita ¨t zu Kiel, Hermann-Rodewald-Strasse 6,
D-24098 Kiel, Germany
5Department of Clinical Pharmacology Q7642, Rigshospitalet, 9 Blegdamsvej, DK-2100 Copenhagen, Denmark
(Received 5 August 2006 – Revised 25 February 2007 – Accepted 15 March 2007)
The objectives of the present study were to evaluate the effect of normobaric and hyperbaric O2(HBO) on plasma antioxidants and biomarkers of
oxidative stress in plasma and urine and to investigate the effect of a 4-week vitamin C plus E supplementation on HBO-induced oxidative stress.
Nineteen healthy men were exposed to HBO (100% O2; 240kPa) before and after 4 weeks’ supplementation with 500mg vitamin C plus 165mg
a-tocopherol equivalents. Exposure to 21% O2at 100kPa served as intra-individual controls (control). Samples for the analysis of plasma anti-
oxidants and oxidative stress biomarkers were collected before and immediately after each treatment. The present results showed that when com-
pared with ‘control’, a single exposure to HBO resulted in a decrease of plasma vitamin C (P¼0·027) and an increase of lipid peroxides
(P¼0·0008) and urinary 8-oxo-deoxyguanosine (8-oxodG) excretion (P¼0·006). Oxidative stress was not prevented by a 4-week supplementation
with vitamins C and E. HBO-induced changes in plasma parameters correlated with basal antioxidant levels. The increase of urinary 8-oxodG after
HBO plus supplementation correlated negatively with vitamin E intake (P¼0·023). We concluded that in healthy men HBO caused oxidative
stress, which could not be prevented by dietary vitamin C plus E supplementation. The present data support the idea that HBO is a suitable
model for oxidative stress in healthy volunteers.
Hyperbaric oxygen: Normobaric oxygen: Oxidative stress: Supplementation: Vitamin C: Vitamin E
The human organism is constantly exposed to oxidants (reac-
tive oxygen species) from both physiological processes and
pathophysiological conditions, foreign compound metabolism
and radiation1. An increased production of reactive oxygen
species together with a failure of the network of enzymic,
endogenous and nutritional antioxidants leads to oxidative
stress. Chronic oxidative stress may be involved in the
development of chronic diseases such as cancer, CHD,
neurodegenerative diseases, diabetes mellitus and cataract,
and has also been suggested as a mechanism of ageing2.
Therefore, maintaining the endogenous antioxidant defence
system by supplementing with antioxidants appears worth-
However, current research results concerning the protective
effects of antioxidants on biomarkers of oxidative stress and
on diseases are contradictory3–11. Several intervention studies
of a meta-analysis of nineteen randomised, placebo-controlled
trials with high-dose vitamin E rather than dietary levels
showed a dose-dependent relationship between vitamin E sup-
plementation and all-cause mortality12. Specifically, all-cause
mortality in volunteers with a high risk for a chronic disease
progressively increased for dosages approximately greater
It has been suggested that a preventive effect of antioxidants
may only be seen in a situation of high oxidative stress13and
we therefore based the present study on the following two
concepts. First, a standardised and well-proven model of
oxidative stress (hyperbaric O2; HBO) was used to study the
effects of an enhanced formation of reactive oxygen species
using various parameters of oxidative stress. Second, intra-
and inter-individual variabilities of the biomarkers were
assessed for comparison with the effects of HBO. HBO was
also used to investigate the effect of vitamin C plus E
*Corresponding author: Dr Nicolle Bader, fax þ49 0711 45923386, email firstname.lastname@example.org
Abbreviations: GSH, reduced glutathione; HBO, hyperbaric O2; NBO, normobaric O2; 8-oxodG, 8-oxo-deoxyguanosine; T0, control treatment; T1, 100% oxygen
under normobaric conditions; T2, 100% oxygen under hyperbaric conditions; T3, vitamin supplementation plus 100% oxygen under hyperbaric conditions.
British Journal of Nutrition (2007), 98, 826–833
q The Authors 2007
British Journal of Nutrition
supplementation. Apparently healthy male non-smokers were
examined, and to avoid the potential confounding factor of
diet their antioxidant intake was also monitored.
Materials and methods
The study protocol was approved by the ethical committee of
the Christian-Albrechts University of Kiel (Germany). All vol-
unteers gave their written informed consent.
Nineteen men were recruited by notice board postings (age
range 21–39 years). All volunteers were non-smokers and not
taking any medication or vitamin supplementation. Exclusion
criteria were claustrophobia and previous dives with ‘nitrox’
(breathing gas with a higher proportion of O2 than the
normal 21%). Volunteers were asked to retain their usual life-
style and not to diet during the study period.
The study consisted of four protocols, referred to as T0, T1,
T2 and T3, which were carried out over a period of 5
months (Fig. 1). The pressure chamber system Hydra 2000w
(Haux, Karlsbad, Germany) was used for all treatments. Treat-
ment with normobaric ambient air (100kPa or 1·0bar; 21%
O2) served as the control treatment (T0). At T1 volunteers
were treated with 100% O2under normobaric conditions (nor-
mobaric O2; NBO). At T2 and T3 volunteers were exposed to
HBO (240kPa or 2·4bar; 100% O2). Each treatment consisted
of 131min and was interspersed with 2 £ 10min periods
of breathing ambient air (21% O2). For 28d before T3 volun-
teers were supplemented with a daily dose of 500mg slow-
release vitamin C and 182mg RRR-a-tocopheryl acetate (cor-
responding to 165mg a-tocopherol equivalents; CellaVie;
Ferrosan A/S, Soeborg, Denmark). Compliance was assessed
by counting remaining pills and checking plasma levels of
vitamin C and vitamin E before and after supplementation.
Data from one subject were omitted because of a lack of com-
pliance as evidenced by unchanged vitamin C and vitamin E
levels after supplementation.
Nutrient and fruit and vegetable intake
Seven-day dietary records were analysed using the computer
software Prodi 4.5 (Wissenschaftliche Verlagsgesellschaft
mbH, Stuttgart, Germany) at each study period. Number and
size of daily portions of fruit and vegetables including fruit
and vegetable juices were assessed according to the definitions
of the worldwide campaign ‘5-a-day’.
Venous blood and urine samples were collected before and
immediately after treatments at T0, T1, T2 and T3. Blood
samples were immediately centrifuged at 2500g. Plasma and
whole-blood fractions were stored at 2808C until analyses.
Urine samples were fumigated with N2 and immediately
stored at 2808C until 8-oxo-deoxyguanosine (8-oxodG)
Measurement of antioxidants and antioxidant capacity
Plasma levels of vitamin C, a-tocopherol, retinol, b-carotene
and reduced glutathione (GSH) in whole blood were measured
by commercial HPLC kits supplied by Chromsystems
(Munich, Germany). The preparation of the samples was
done according to the manufacturer’s description. Separation
of antioxidants on a C18 column was followed by UV (vita-
min C, 245nm; a-tocopherol, 295nm; retinol, 325nm; b-car-
otene, 453nm) and fluorescence detection (GSH, 385 and
510nm). A Waters HPLC
Germany) with a pump (model 515; Waters), an autosampler
(model 717plus; Waters), a UV detector (model 2487; Waters)
and a fluorescence detector (model 1100; Hewlett Packard,
Bo ¨blingen, Germany) was used. A commercial ELISA kit
by Immundiagnostik (Bensheim, Germany) was used to
measure plasma antioxidant capacity. Quantification of antiox-
idants was based on the reaction of exogenous peroxide with
antioxidants present in the sample. Unreactive peroxides
were quantified by a peroxidase-catalysed reaction. The reac-
tion was stopped by adding acid to give a colorimetric end-
point that was read spectrophotometrically at 450nm by the
microplate reader Sunrise (Tecan, Crailsheim, Germany).
Quantification was done by external calibration. The prep-
aration of samples was done according to the manufacturer’s
description. Control samples were run together with each
batch of samples. All measurements were done in duplicates.
system (Waters, Eschborn,
Spot urine 8-oxo-deoxyguanosine analysis
Urinary 8-oxodG was measured by a previously described LC-
MS/MS method14. Briefly, frozen urine samples were thawed
and diluted 1:1 with 100mM-lithium acetate buffer containing
8-oxodG labelled with stable isotopes. Samples were heated to
378C for 10min and whirly mixed before centrifugation at
5000g for 10min. A Perkin Elmer Series 200 HPLC equipped
with two pumps, autosampler, solvent cabinet and vacuum
degasser was used (Perkin Elmer, Norwalk, CT, USA). The
HPLC was fully controlled by the mass spectrometer (Sciex
API 3000 triple quadrupole mass spectrometer) with a turbo-
ionspray source (Sciex, Thornill, Canada). The HPLC separ-
ation was performed on a Phenomenex Prodigy ODS HPLC
Fig. 1. Study protocol. NBO, normobaric O2; HBO, hyperbaric O2.
Hyperbaric oxygen and vitamin supplementation 827
British Journal of Nutrition
column (100 £ 2mm; 3mm), protected by a C18 guard
column (4 £ 2mm), both obtained from Phenomenex (Tor-
rance, CA, USA). The measurements were done in duplicates.
Results are expressed as ng/mg creatinine. Creatinine was
measured by the Jaffe ´ method15.
Measurement of lipid peroxidation
Malondialdehyde in plasma was measured by a commercial
HPLC kit (Chromsystems, Munich, Germany). Samples were
prepared accordingly to the manufacturer’s description. The
HPLC system consisted of a pump (model 2150; Pharmacia
Biosystems, Freiburg, Germany), an autosampler (model
AS-100; BioRad, Munich, Germany) and a fluorescence detec-
tor (model 1100; Hewlett Packard, Bo ¨blingen, Germany).
Plasma lipid peroxides were measured photometrically with
a commercial ELISA kit (Immundiagnostik, Bensheim,
Germany) on a plate reader (Sunrise; Tecan, Crailsheim,
Germany). Measurement of lipid peroxides was based on the
reaction of peroxidase with peroxide and a subsequent reac-
tion with tetramethylbenzidine. The reaction was stopped by
adding acid to give a colorimetric endpoint that was read
photometrically at 450nm. Quantification was done by exter-
nal calibration. Control samples were run together with each
batch of duplicate samples.
The power calculation was based on the variability of 8-
oxodG measurements obtained previously, a 0·05 type I
error and a statistical power of 80%. We calculated that a
study size of nineteen participants would enable us to detect
a change of 10% in 8-oxodG excretion. Data were analysed
using Statistica 6 (StatSoft, Tulsa, OK, USA) and are
represented as mean values with their standard errors or
medians and lower and upper quartiles as given in each
table and figure. All data were tested for normality of distri-
bution with the Shapiro–Wilk test. Due to a majority of
non-parametric data, statistical analyses were performed by
Friedman’s test was used for multiple statistical compari-
sons between the changes (pre-treatment v. post-treatment)
of the treatments where subject is regarded as a random
effect with treatment (T0, T1, T2, T3) as a fixed effect. The
same test was used to compare baseline values with subject
as a random effect and time (T0, T1, T2, T3) as a fixed
effect. When the effect of treatment (or time) was significant,
comparisons of treatment means (baseline means) were done
by Wilcoxon’s matched-pair signed-rank test. Spearman’s cor-
relation coefficient was used for relationships between differ-
ent parameters. Intra- and inter-individual CV were calculated
for all parameters assessed using the basal values at T0, T1
and T2. A ‘sensitive’ subject was defined as an individual
whose response to HBO exceeded twice the square root of
the within-subject variance. McNemar’s test was performed
to test significant changes in terms of ‘sensitivity’ in response
between T2 and T3. P values less than 0·05 were considered
Basal anthropometric data, plasma antioxidant concentrations,
biomarkers of oxidative damage and dietary intake of the vol-
unteers at T0, T1, T2 and T3 are given in Table 1. There were
no differences in dietary intake data between T0, T1, T2 and
T3, respectively. These anthropometric data differed signifi-
cantly between T1 and T2 (P,0·05). At T2 plasma vitamin
C concentration was higher when compared with T0 and T1
and vitamin E concentration increased from T0 to T1
(P,0·05). GSH concentrations at T1 and T2 were signifi-
cantly higher compared with T0 (P,0·05). From the basal
data at T0, T1 and T2 intra- and inter-individual CV were cal-
culated for weight, BMI, plasma antioxidant concentrations,
biomarkers of oxidative damage and nutrient intake, respect-
ively (Table 1). Mean intra- and inter-individual CV ranged
from 1·1 and 12·7% for BMI up to 76·9 and 112·1% for alco-
hol intake, respectively. In general the highest intra-individual
and inter-individual variations were seen in variables of
Effects of control treatment, 100% oxygen under normobaric
conditions and 100% oxygen under hyperbaric conditions
Treatment with ambient air at 100kPa did not lead to changes
(pre-T0 v. post-T0) in the concentrations of plasma antioxi-
dants and biomarkers of oxidative damage (P.0·05). In
contrast, NBO resulted in a significant decrease of plasma
vitamin C concentration when compared with the change at
T0 (change in pre-T0–post-T0 v. change in pre-T1–post-T1;
Table 2). HBO resulted in a significant decrease in plasma
vitamin C concentration and also resulted in an increased urin-
ary 8-oxodG excretion and increased plasma lipid peroxides in
comparison with the control situation (change in pre-T0–post-
T0 v. change in pre-T2–post-T2; Table 2). However, HBO-
induced changes were similar to the changes at NBO
(change in pre-T1–post-T1 v. change in pre-T2–post-T2;
P.0·05). Taking into account the within-subject variation of
the considered parameters, ‘sensitive’ and ‘non-sensitive’
volunteers can be differentiated (i.e. ‘sensitive’ volunteers
are characterised by HBO-induced changes exceeding twice
the square root of the within-subject variance) using lipid per-
oxides and urinary 8-oxodG as suitable biomarkers. From
these data it was calculated that 58% (concerning lipid per-
oxides) and 72% of the volunteers (concerning urinary
8-oxodG) can be considered as ‘sensitive’ individuals at T2,
Effect of vitamin intervention
A 4-week supplementation with vitamin C and vitamin E sig-
nificantly increased vitamin C and E plasma concentrations
(pre-T3) when compared with the concentrations pre-T0,
pre-T1 and pre-T2 (P,0·05; Fig. 2). Pre-T3 levels of GSH
were lower when compared with basal data at T0, T1 and
T2 (P,0·05; Fig. 2). Also basal lipid peroxide levels signifi-
cantly decreased after supplementation with vitamins C and E
(148 v. 85mmol/l; P¼0·002; Table 1).
N. Bader et al.828
British Journal of Nutrition
Table 1. Anthropometrics, plasma antioxidant concentration, biomarkers of oxidative damage and nutrient intake of the study group at pre-T0, pre-T1, pre-T2 and pre-T3*
(Medians and lower and upper quartiles)
T0 (n 19) T1 (n 19)T2 (n 19)T3 (n 18)
CVintra(%)CVinter(%)Median LQ, UQMedian LQ, UQ Median LQ, UQMedian LQ, UQCVintra/CVinter
Plasma antioxidant concentrations
Vitamin C (mmol/l)
Vitamin E (mmol/l)
Vitamin A (mmol/l)
Biomarkers of oxidative damage
Urinary 8-oxodG (ng/mg
Lipid peroxides (mmol/l)
Vitamin C (mg/d)
Vitamin E (mg/d)
Vitamin A (RE mg/d)
Fruit and vegetables
3·7 2·8, 4·53·93·0, 4·6 3·52·2, 3·9 3·72·9, 4·4 17·831·7 0·56
10 8779688, 11811
10 5599701, 11 962
9785 8537, 10 714
10 1409140, 13 448
LQ, lower quartile; UQ, upper quartile; CVintra, intra-individual CV; CVinter, inter-individual CV; AOC, antioxidative capacity, GSH, reduced glutathione, 8-oxodG, 8-oxo-deoxyguanosine; MDA, malondialdehyde; RE, retinol
a,b,cMedian values within a row with unlike superscript letters were significantly different (P,0·05).
*For details of the study protocols, see Materials and methods.
Hyperbaric oxygen and vitamin supplementation
British Journal of Nutrition
Effect of vitamin intervention plus 100% oxygen under
HBO-induced changes of plasma vitamin C and lipid per-
oxides were significantly higher compared with changes at
T0 (change in pre-T0–post-T0 v. change in pre-T3–post-T3;
P,0·05; Table 2). Except for GSH (P¼0·04) HBO-induced
changes at T3 were not statistically different from HBO-
induced changes at T2 (change in pre-T2–post-T2 v. change
in pre-T3–post-T3). There was no correlation between
HBO-induced changes of 8-oxodG and lipid peroxides at T2
and T3, respectively (data not shown).
The individual effect of vitamin C and E supplementation on
HBO-induced changes in biomarkers of oxidative damage,
urinary 8-oxodG excretion and lipid peroxides is shown in
Fig. 3. The benefit of the supplementation was limited to
17% of the volunteers in the case of lipid peroxides and
12% in the case of urinary 8-oxodG excretion, respectively
(Fig. 3). Those volunteers showed an increase in lipid per-
oxides and urinary 8-oxodG at T2 above their individual
within-subject variance, whereas the HBO-induced changes
at T3 were below the within-subject variance. By contrast,
28 and 23% of the volunteers were affected by an increase
in the concentrations of lipid peroxides and urinary 8-oxodG
excretion due to HBO after vitamin supplementation (Fig. 3),
while their response to HBO at T2 was below the within-sub-
ject variance. Most of the volunteers (65 and 55%) did not
show any difference between their response to HBO at T2
and T3. In terms of ‘sensitivity’ there was no significant
Table 2. Changes (pre- v. post-treatment) of plasma antioxidant concentrations and biomarkers of oxidative
damage at T0, T1, T2 and T3†
(Mean values with their standard errors)
Changes pre- v. post-treatment (%)
Plasma antioxidant concentrations
Biomarkers of oxidative damage
AOC, antioxidative capacity; GSH, reduced glutathione; 8-oxodG, 8-oxo-deoxyguanosine; MDA, malondialdehyde.
*Mean change was significantly from the change pre- v. post-treatment at T0 (P,0·05; Wilcoxon test).
†For details of the study protocols, see Materials and methods.
Fig. 2. Changes of blood antioxidants after 4 weeks of supplementation with
vitamin C (500mg/d) and vitamin E (165mg a-tocopherol equivalents/d) pre-
T0 (V), pre-T1 (O) and pre-T2 (†) in comparison with pre-T3 (100%). For
details of the study protocols, see Fig. 1 and Materials and methods. Data
were analysed with Wilcoxon’s test and are shown as medians, with 25–75
percentiles represented by vertical bars. AOC, antioxidative capacity; GSH,
reduced glutathione. *Median value was significantly different to that pre-T3
Fig. 3. Distribution of the volunteers (n 18) according to the effect of hyper-
baric O2 (HBO) after supplementation with vitamins C and E (T3) on the
extent of oxidative damage to DNA and lipids when compared with HBO
without vitamin supplementation (T2). For details of the study protocols, see
Fig. 1 and Materials and methods. Sensitive subjects (S) were those where
HBO-induced changes exceeded twice the square root of the within-subject
variance. Non-sensitive subjects (NS) were those where HBO-induced
changes did not exceed twice the square root of the within-subject variance.
( ), Enhancement of damage (NS in T2 became S in T3); ( ), no benefit of
supplementation (S in T2 remained S in T3); ( ), no influence of supplemen-
tation (NS in T2 remained NS in T3); (A), benefit of supplementation (S in T2
became NS in T3); 8-oxodG, 8-oxo-deoxyguanosine.
N. Bader et al.830
British Journal of Nutrition
change in HBO response between T2 and T3 for both
parameters as calculated by McNemar’s test (8-oxodG,
P¼0·68; lipid peroxides, P¼0·72).
At T3 a negative correlation was found for the HBO-
induced changes in GSH (pre-T3 v. post-T3) and pre-T3
GSH concentrations (r 20·72; P¼0·001), i.e. those volunteers
with high pre-T3 GSH showed smaller HBO-induced
changes than those with low pre-T3 GSH concentrations.
Similarly, HBO-induced changes in antioxidant capacity
(pre-T2 v. post-T2) correlated negatively with antioxidant
status (r 20·60; P¼0·009), i.e. those with low antioxidant
capacity had the largest change following HBO exposure. Fur-
thermore, the change in 8-oxodG excretion at T2 correlated
P¼0·013). This means that those volunteers with a low
GSH status had the largest change following HBO exposure.
Nutrient and antioxidant intake
The increase of urinary 8-oxodG excretion (pre-T3 v. post-T3)
showed a negative correlation with vitamin E intake from diet
(r 20·53; P¼0·023) and from the vitamin supplement
(r 20·53; P¼0·023). Change in GSH (pre-T3 v. post-T3)
was negatively correlated with intake of fruit and vegetables
(r 20·50; P¼0·043). In contrast, at T2 HBO-induced changes
of plasma antioxidants and biomarkers of oxidative damage
(pre-T2 v. post-T2) were not affected by diet (P.0·05).
In the present study HBO exposure was used as a stress model
to study the intra-individual and inter-individual consequences
of an increased formation of reactive oxygen species on
plasma antioxidant concentration as well as biomarkers of oxi-
dative stress in healthy men, and the effects of 4 weeks’ sup-
plementation with vitamins C and E.
When compared with the control situation, a single 2h
exposure to HBO decreased plasma vitamin C and resulted
in a significant formation of lipid peroxides and urinary
8-oxodG excretion in comparison with the control situation
The changes observed in plasma vitamin C were also seen
after exposure to 100% O2at normobaric conditions (NBO),
but, however, without the changes in DNA and lipid oxidation.
It thus appears that both HBO and NBO cause oxidative stress,
but that HBO also includes intracellular oxidative stress as
evidenced by oxidation of macromolecules. Our findings are
in line with the findings of Oter et al.16demonstrating a
directly proportional relationship between oxidative damage
and HBO exposure pressure starting from normobaric 100%
HBO has been successfully used for the treatment of a var-
iety of clinical conditions related to hypoxia, since HBO
favourably leads to an increase of dissolved O2in the blood.
Narkowicz et al.17were the first supplying evidence for the
occurrence of reactive oxygen species in men exposed to
HBO. A linear relationship between the formation of H2O2
and the O2pressure was shown in rats exposed to HBO18.
In a recent experimental study the exposure to NBO also
increased thiobarbituric acid-reactive substance levels in rat
lung, brain and blood16. Since plasma vitamin C decreased
in the present study, the equilibration between oxidants and
antioxidants in the organism seemed to be affected by NBO
alone. However, indices of oxidative damage did not increase
after exposure to NBO. The present data support the idea that
HBO is a suitable stress model in healthy volunteers. This is in
line with previous data19,20. When compared with other stress
models (for example, smoking, exhausting exercise and
chronic diseases), HBO provides the advantage that oxidative
stress can be controlled quantitatively.
Supplementation with vitamins C and E at five and thirteen
times the recommended daily intakes increased plasma vita-
mins C and E by about 30–40% (Fig. 2) but did not prevent
HBO-induced oxidative damage (Table 2). The present study
was the first investigating the effect of a combination of vita-
mins C and E on HBO-induced oxidative stress in healthy
human subjects. In the present study pharmacological doses
of antioxidant vitamins were administered, whereas other
authors have used endogenous substances with an antioxidant
capacity. The supplementation of human subjects with
a-lipoic acid reduced HBO-induced lipid and DNA oxi-
dation21. In addition a new formulation consisting of wheat
gliadin chemically combined with a vegetal preparation of
superoxide dismutase prevented the formation of F2-isopros-
tanes and DNA strand breaks due to HBO in human sub-
It is important to point out that antioxidant supplements are
not always safe. Although toxicity of consumed antioxidants is
very low and only occurs at very high intake levels23, the
degree of benefit or harm of antioxidant supplementation
depends also on genetic susceptibilities. Besides that it is by
no means clear how antioxidant supplements interact with
each other and with other dietary constituents affecting the
in vivo redox balance. There are a few studies showing that
individual genetic polymorphisms related to the activity of
metabolic and detoxification enzymes influence the effects
of antioxidants. For example, it was shown that differences
in base excision repair capacity which is due to polymorph-
isms in the XRCC1 gene may modulate the effect of dietary
antioxidant intake on prostate cancer risk24. Antioxidants
and oxidants are able to activate certain genes and signalling
pathways by modulating the redox state of the cell. It was
also shown in cultured cells that vitamin C might affect
gene expression and this seems to be mediated by its redox
To our knowledge there are no studies investigating the
influence of antioxidant intake and basal antioxidants on
NBO- and HBO-induced oxidative damage. We found that
NBO-induced changes were not affected by the intake of anti-
oxidants or by the basal antioxidant status. HBO-induced
changes of antioxidant capacity showed a negative association
with basal concentration. This indicates a greater decrease in
plasma antioxidants in human subjects with lower basal
levels, i.e. individuals with lower antioxidant status are more
vulnerable to oxidative stress. Accordingly, a low basal
GSH concentration at T2 resulted in more damage to DNA
as indicated by a higher urinary 8-oxodG excretion. However,
these associations were not seen at T3. Since basal GSH con-
centrations were markedly smaller at T3 we expected an even
stronger association to urinary 8-oxodG excretion at T3. It can
only be supposed that the real effects were masked by the
effects of the excessive antioxidant supplementation.
Hyperbaric oxygen and vitamin supplementation831
British Journal of Nutrition
While the intake of antioxidants did not seem to have any
influence on changes due to NBO and HBO, two correlations
were found to be of significance due to HBO plus vitamin sup-
plementation. First, a higher intake of vitamin E correlated
with a lower 8-oxodG concentration in urine. The source of
vitamin E (diet or supplement) made no difference. Second,
the negative correlation between fruit and vegetable intake
and HBO-induced changes in GSH concentration suggests
that a higher intake of fruit and vegetables ‘saved’ the con-
sumption of GSH. Discrepancies in the results at T2 and T3,
respectively, might be explained by the vitamin intervention
before HBO at T3, potential genetic differences between
individuals and thus their susceptibility as well as by the
intra-individual differences (Table 1) resulting from the longi-
tudinal protocol of the present study. The chosen study design
has a lack of strength since there is no parallel arm and there is
no sufficient evidence that differences between the treatments
may derive from chronic prolonged oxidative stress as a result
of the acute stress.
In the steady state, urinary excretion of 8-oxodG in prin-
ciple reflects the rate of oxidative damage, whereas the level
of lesions from surrogate cells should reflect the balance
between damage and repair. Although the volunteers were
asked to avoid physical, mental and environmental stress
before treatments, increased 8-oxodG concentrations in urine
might also be due to the repair of pre-existing damage.
The present study was on healthy and young volunteers and
the findings may not be representative of any nutritional or
health compromise. Moreover, intervention data showed that
supplementation-induced changes of plasma vitamin C and
E levels were also characterised by a high variation inter-indi-
vidually ranging from ‘non-responders’ up to increases by 133
and 107%, respectively. However, the broad range of
response to supplementation was not correlated to the HBO-
induced formation of biomarkers of oxidative stress. Although
the compliance was assessed to be 92%25, devious non-com-
pliant volunteers which influence the assessment result might
not be identified. Besides that, individual response to vitamin
supplementation depends mainly on metabolic rate, tissue dis-
tribution and other related factors, where genetic differences
may have an impact. In order to explain discrepancies in the
individual response, and thus their susceptibility, future
research might be focused on genetic differences.
The homeostasis of the antioxidative network might mainly
explain the decline of the two parameters as described for vita-
min C and uric acid26. Besides type and dosage of the sup-
plementation in the present study, pro-oxidative activities of
the supplementation might also be responsible for the lack in
prevention of the HBO-induced oxidative damage. This is sup-
ported by the decrease in GSH after supplementation. After
tive in the prevention of HBO-induced damage, since it is poss-
iblethatvarioustypes ofantioxidants exert differenteffects that
of the biomarkers of oxidative stress and total antioxidant
capacity are insufficiently validated in vivo and therefore not
suitable for such investigations27. As an example, antioxidant
capacity has failed to demonstrate an effect of supplementation
with antioxidants or antioxidant-rich foods in human subjects
and is therefore considered as a biomarker with limited value
in vivo28. Since we did not use the parameter of antioxidant
capacity in isolation but with other well-validated biomarkers
of oxidative stress, there is nothing to be said against its usage
in the present study.
A low fruit and vegetable intake is one of the three most
important behavioural and environmental risk factors of
death cause in low- and middle-income countries29. Whilst
the risk factor exists in these countries it is not relevant in a
wealthy population. With respect to that finding, the lack in
the present study of finding a positive effect of antioxidant
intervention might be covered by factors resulting from a
high body fat mass and a good nutritional status which is
characteristic for high-income countries. Since the volunteers
of the present study had a good nutritional status and were
not underweight, this may add to our effects.
In summary, 131min of breathing O2 under pressure of
240kPa induces oxidative stress in healthy volunteers as evi-
denced by an increase in urinary 8-oxodG excretion and
plasma lipid peroxides with concomitant decreases in plasma
vitamin C. Oxidative stress was not prevented by 4 weeks’
supplementation with vitamins C and E. There were high
intra- and inter-individual variances in biomarkers tested.
Intra- and inter-individual differences of HBO-induced oxi-
dative stress were neither explained by antioxidant intake
nor by basal antioxidative status of the volunteers.
The work was funded by the Graduiertenkolleg 820 ‘Natural
antioxidants – their occurrence in plants, food, animals and
humans’ of the German Research Foundation (DFG). The
vitamin C and E supplement CellaVie was a gift from Ferro-
san A/S (Denmark). The authors thank Ferrosan A/S, the staff
of the German Naval Medical Institute Kronshagen, and most
of all the volunteers who made the present study possible.
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