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Cardiovascular pharmacology
Effects of ozone therapy on haemostatic and oxidative stress index
in coronary artery disease
Gregorio Martı
´nez-Sa
´nchez
a,
n
, Livan Delgado-Roche
b
, Arquı
´mides Dı
´az-Batista
c
,
Gema Pe
´rez-Davison
d
, Lamberto Re
a,d
a
Medinat srl Clinic, Via Fazioli 22, 60021 Camerano, Italy
b
Center of Studies for Research and Biological Evaluations, Pharmacy and Food Sciences College, University of Havana, Havana, Cuba
c
National Institute of Angiology and Vascular Surgery, Havana, Cuba
d
D.I.S.M.A.R., University of Ancona, Ancona, Italy
article info
Article history:
Received 20 February 2012
Received in revised form
25 June 2012
Accepted 2 July 2012
Available online 13 July 2012
Keywords:
Ozone
Coronary artery diseases
Oxidative stress
Aspirin
Policosanol
abstract
Coronary artery disease (CAD) is the most common cause of sudden death, and death of people over 20
years of age. Because ozone therapy can activate the antioxidant system and improve blood circulation
and oxygen delivery to tissue, the aim of this study was to investigate the therapeutic efficacy of ozone
in patients with CAD, treated with antithrombotic therapy, Aspirin
s
and policosanol. A randomized
controlled clinical trial was performed with 53 patients divided into two groups: one (n¼27) treated
with antithrombotic therapy and other (n¼26) treated with antithrombotic therapy plus rectal
insufflation of O
3
. A parallel group (n¼50) age and gender matched was used as reference for the
experimental variables. The efficacy of the treatments was evaluated by comparing hemostatic indexes
and biochemical markers of oxidative stress in both groups after 20 day of treatment. Ozone treatment
significantly (Po0.001) improved prothrombin time when compared to the antithrombotic therapy
only group, without modifying bleeding time. Combination antithrombotic therapy þO
3
improved the
antioxidant status of patients reducing biomarkers of protein and lipid oxidation, enhancing total
antioxidant status and modulating the level of superoxide dismutase and catalase with a 57% and 32%
reduction in superoxide dismutase and catalase activities respectively, moving the redox environment
to a status of low production of O
2
with an increase in H
2
O
2
detoxification. No side effects were
observed. These results show that medical ozone treatment could be a complementary therapy in the
treatment of CAD and its complications.
&2012 Elsevier B.V. All rights reserved.
1. Introduction
Cardiovascular diseases, comprising coronary artery (CAD) and
cerebro-vascular diseases, are currently the leading cause of
death globally, accounting for 21.9% of total deaths, and are
projected to increase to 26.3% by 2030 (WHO, 2008). The factors
that coalesce to increase the risk of developing atherosclerotic
CAD were demonstrated and have subsequently been shown to be
pervasive across ethnicities and regions all over the world (Yusuf
et al., 2004). These are not new risks, but the ubiquity of smoking,
dyslipidemia, obesity, diabetes, and hypertension has been gra-
dually escalating (Gupta et al., 2008), and is thought to be the
driving influence behind the epidemic of heart disease faced
today (Franco et al., 2011).
Ozone administered in an appropriate dose interval can
modulate many biochemical pathways with the activation of
second messengers (Bocci et al., 2011). Scientific evidence con-
nected the modulation of different biomarkers (e.g. antioxidant
enzymes, nitric oxide pathways, 2,3-diphosphoglycerate) as a
consequence of applying low ozone doses. Those facts support
some of the current clinical applications of ozone (Colombo et al.,
2000;Re et al., 2008;Steppan et al., 2010). In an experimental
model, rectal insufflation (a simple, easy and inexpensive method
of delivering ozone) showed a sustained improvement in ery-
throcytes deformability suggesting its systemic effects (Seda Artis
et al., 2010).
In this work, we considered that the treatment with ozone at
repeated low doses by rectal way plus regular anti-thrombotic
therapy in CAD patients, could improve blood homeostatic index,
reducing the oxidative stress and providing an antioxidant status,
which can compensate for the CAD-derived cardiovascular com-
plications. Our results confirmed this hypothesis and demon-
strated that ozone not only improved blood homeostatic indexes
Contents lists available at SciVerse ScienceDirect
journal homepage: www.elsevier.com/locate/ejphar
European Journal of Pharmacology
0014-2999/$ - see front matter &2012 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.ejphar.2012.07.010
n
Corresponding author. Tel.: þ39 71 731076; fax: þ39 71 731347.
E-mail address: gregorcuba@yahoo.it (G. Martı
´nez-Sa
´nchez).
European Journal of Pharmacology 691 (2012) 156–162
but also compensated for the antioxidant/pro-oxidant balance in
those patients.
2. Materials and methods
2.1. Study design
This randomized parallel controlled clinical trial was approved
by an institutional review board (Scientific and Ethics Committees
of the Institution) in accordance with the principle of the
Declaration of Helsinki (WMA, 2004). All patients gave their
informed consent to being enrolled after receiving adequate
information about the study (characteristics of the study, benefits
and possible side effects). Before enrolling, all participants
attended a training program to familiarize them with the study
objectives and treatment plans. The personnel involved empha-
sized that all participating physicians would treat each patient
according to the randomised scheme of treatment.
Adult patients of both gender and different ethnic origin with a
diagnosis of CAD who were hospitalized in the Institute of
Angiology and Vascular Surgery (La Habana, Cuba) were eligible
to participate in the study. Exclusion criteria were: severe septic
conditions, hypersensitivity to the medication to be used, hepatic
dysfunction, renal failure (serum creatinine level41.32
m
mol/l),
pregnancy, cancer or other serious disease, inability to cooperate
with the requirements of the study, recent history of alcohol or
drug abuse, current therapy with any immunosuppressive agent
or anticonvulsant, concurrent participation in another clinical
study, or current treatment with an investigational drug.
For the calculation of the size of the sample, the G
n
Power
3 system (version 3.1) (Faul et al., 2007) was used. The type
1 error was 5% and the type 2 error was 20%, with a minimal
difference between effect rates not higher than 25%. The target
level of enrollment was determined to be 23 patients per group.
Assuming that 10% of study patients would be lost to follow-up,
26 patients per group were studied. In the study were included 26
patients per treatment group and data from 50 gender- and age-
matched healthy subjects was used as reference to establish the
normal reference interval of the experimental variables (oxidative
stress bio-markers).
Patients were randomized to two different groups (1) regular
anti-thrombotic therapy; 27 patients were treated with oral
anticoagulant (Aspirin
s
, acetyl salicylic acid 125 mg and Ater-
omixol
s
, policosanol 5 mg) once a day during 20 day, and (2)
ozone plus anti-thrombotic therapy; 26 patients were treated
daily with ozone and anti-thrombotic therapy as in group 1.
Patients of group 2 were treated with 200 ml of O
3
/O
2
containing
40
m
g/ml of ozone once a day during 20 day. Nelaton Robinson
catheter 40 cm was introduced 10/15 cm by rectal way to deliver
the gas and was place in site for 5 min. The patients were
encouraged to empty his or her bladder and bowels before the
procedure. The insufflation was done immediately after a colonic
enema in case of constipation.
Ozone was generated by OZOMED equipment (Center for
Ozone Research, Havana, Cuba), and was administered by rectal
insufflation. Ozone was obtained from medical grade oxygen, and
was used immediately upon generation and represented only
about 3% of the gas mixture (O
2
þO
3
). The ozone concentration
was measured by using a built-in UV spectrophotometer set at
254 nm (Delgado-Roche et al., 2011).
Blood samples for biochemical analysis were obtained after a
12 h overnight fast, at the beginning and 24 h after the last ozone
treatments. The samples were immediately centrifuged at 3000 g,
at 4 1C for 10 min. The serum was collected and aliquots were
stored at 70 1C until analysis (Delgado-Roche et al., 2011).
2.2. Biochemical determinations
All biochemical parameters were determined as previously
described (Delgado-Roche et al., 2011) by spectrophotometric
methods using an Ultrospect Plus Spectrophotometer from Phar-
macia LKB (Sweden). Catalase activity was measured by following
the decomposition of hydrogen peroxide at 240 nm at 10 s
intervals for 1 min (Haining and Legan, 1972). Superoxide dis-
mutase (SOD) activity was measured using kits supplied by
Randox Laboratories Ltd., Ireland (Cat. No. SD125 and No.
RS505). Concentrations of malondialdehyde (MDA) were analyzed
using the LPO-586 kit obtained from Calbiochem (La Jolla, CA,
USA). In the assay, the production of a stable chromophore, after
40 min of incubation at 45 1C, was measured at 586 nm. For
standards, freshly prepared solutions of malondialdehyde bis
[dimethyl acetal] (Sigma, St. Louis, MO, USA) were used and
assayed under identical conditions (Esterbauer and Cheeseman,
1990). Quantification of total hydroperoxides was measured by
Bioxytech H
2
O
2
-560 kit (Oxis International Inc., Portland, OR,
USA) using xylenol orange to form a stable colored complex,
which can be measured at 560 nm. The peroxidation potential
(PP) was measured by inducing lipid peroxidation by adding Cu
þ
(2 mM) to serum (incubated for 24 h at 37 1C), in order to know
the balance between prooxidant-antioxidant factors. The differ-
ence between MDA levels, measured at 0 and 24 h after induction,
for each sample, was calculated (Ozdemirler et al., 1995).
After precipitation of thiol proteins using trichloroacetic acid
10%, reduced glutathione was measured according to the method
of Sedlak and Lindsay (1968) with Ellman’s reagent [5
0
5 dithiobis
(2-nitrobenzoic acid) 10
2
M (Sigma St. Louis, MO, USA)]; absor-
bance was measured at 412 nm. The advanced oxidation protein
products (AOPP) were measured as the oxidation of iodide anion
to diatomic iodine by AOPP (Witko-Sarsat et al., 1998). Briefly, the
technique consists in treating 100
m
l of serum in PBS (1 ml) with
50
m
l of potassium iodide 1.16 M followed by 100
m
l of acetic acid.
The absorbance of the reaction mixture was immediately read at
340 nm. AOPP concentrations were expressed as
m
M of chlora-
mine-T (Sigma St Louis, MO, USA). Ferric Reducing Ability of
Plasma (FRAP) was assayed through the reduction of Fe
3þ
to Fe
2þ
by serum or reference (ascorbic acid). The Fe
2þ
–2,4,6-tripiridil-s-
triazine complex was detected at 593 nm (Benzie and Strain,
1996).
To assay prothrombin time, patient plasma was incubated
before addition of thromboplastin (Dade Thromboplastin C Plus;
Dade Behring, Marburg, Germany) in buffer and time in seconds
to clot formation was determined by using a Sigma Amelung KC4
coagulation instrument. The laboratory reference interval was
10.1–15.2 s (Schmaier, 2008).
Measurement of bleeding time was conducted during the
blood extraction procedures. Briefly, a blood pressure cuff was
inflated around the patient’s upper arm. Two small cuts on the
lower arm were done just deep enough to cause a tiny amount of
bleeding. The blood pressure cuff was immediately deflated.
Blotting paper was touched to the cuts every 20 s until the
bleeding stops. The bleeding time was recorded with a timer.
The laboratory reference interval was 1–3 min (Schmaier, 2008).
2.3. Statistical analysis
The OUTLIERS preliminary test for detection of error values
was initially applied. Afterward, data were analyzed by one-way
analysis of variance (ANOVA) followed by a homogeneity variance
test (Bartlett-Box). In addition, a multiple comparison test was
used (Duncan test). Results are presented as means7standard
deviation. The level of statistical significance used was Po0.05.
G. Martı
´nez-Sa
´nchez et al. / European Journal of Pharmacology 691 (2012) 156–162 157
3. Results
3.1. General characteristics of the patients involved in the study
In relation to the baseline characteristics (Table 1), both
groups were similar at randomization (P40.05). Seventy-five
percent of patients in both groups were older than 60 years and
males were the majority. Their medical history was characterized
mainly by hypertension and ischemic cardiopathy.
Concomitant treatments were those used to control hyperten-
sion (captopril in 31%, nifedipine in 10% and nitropental in 2% of
patients respectively) with no significant difference in propor-
tions between groups. The prevalence of risk factors for cardio-
vascular diseases as hypercholesterolemia and obesity was
monitored in patients. No side effects were observed in patients
enrolled in the study.
3.2. Homeostatic indicators
At the beginning of the treatments prothrombin time and
bleeding time were in the normal reference interval values for all
the patients enrolled in the study, with no significant statistical
differences between the groups. At the end of the treatments
(20 day), prothrombin time was significantly (Po0.001) raised in
both group. The increment was significantly (Po0.001) higher in
the group treated with antithrombotic therapy þO
3
when com-
pared to the antithrombotic therapy group. However values of
bleeding time were non-modified at the end of the study in any
group (antithrombotic therapy or antithrombotic therapyþO
3
).
3.3. Biomarkers of antioxidant-prooxidant balance
The AOPP and MDA concentrations, the end products of
protein and lipid peroxidation respectively, were higher at the
beginning of the treatment in both groups than in the reference
normal subjects. At the end a significant decrease (Po0.05) was
achieved for both markers in the antithrombotic therapy þO
3
group compared to the group antithrombotic therapy. Even the
improvement of those biomarkers in antithrombotic therapy þO
3
group values remain significantly (Po0.05) higher than the
normal reference interval (Table 3). At the beginning of the study,
TH levels were high in both groups (antithrombotic therapy
142.977.7
m
M and antithrombotic therapyþO
3
155.478.6
m
M),
significantly higher (Po0.05) than the reference values
(103.7717.7
m
M). At the end of treatment values in both groups
Table 1
Baseline characteristics of CAD patients.
Characteristics Group antithrombotic therapy
(n¼27)
Group antithrombotic therapyþO
3
(n¼26)
n%n%
Age (years) 50–60 5 18.51 8 30.76
61–70 10 37.03 13 50.00
71–80 8 29.62 4 15.38
480 4 14.81 1 3.84
Gender Female 8 29.62 9 34.61
Male 19 70.37 17 65.38
Previous history Hypertension
a
21 77.77 20 76.92
Myocardial stroke 3 11.11 2 7.69
Ischemic cardiopathy 25 92.59 24 92.30
Risk factors Hypertension 19 70.37 20 76.92
Hypercholesterolemia
b
8 29.62 9 34.61
Obesity
c
10 37.03 13 50.00
Smoking 15 55.55 13 50.00
Complementary diagnosis criteria TC (mM) 7.0570.90 6.8571.33
BMI (kg/m
2
) 32.1577.31 34.21 79.63
No significant statistical differences between groups (P40.05) for these variables were achieved. CAD, coronary artery disease. TC: total
cholesterol. Anti-thrombotic therapy (acetyl salicylic acid 125 mg and policosanol 5 mg). Antithrombotic therapy þO
3
, anti-thrombotic
therapy plus ozone therapy (200 ml of O
3
/O
2
40
m
g/ml) BMI, body mass index: weight(kg)/height(m
2
).
a
Hypertension was defined as elevation of systolic ( 4140 mmHg) and/or diastolic ( 490 mmHg) blood pressure.
b
Hypercholesterolemia: increase in total cholesterol 46.7 mM.
c
BMI430.
Table 2
Hemostatic indexes, during the course of the study.
Parameter Start (X7S.D.) End (X7S.D.)
Prothrombin time (s) Antithrombotic therapy, n¼27 11.070.4 18.474.6
a
Antithrombotic therapyþO
3
,n¼26 11.270.6 24.6 76.6
a,b
Bleeding time (min) Antithrombotic therapy, n¼27 1.470.1 1.6 70.2
Antithrombotic therapyþO
3
,n¼26 1.570.1 1.7 70.2
Start and end, beginning and end of treatment (after 20 day) with antithrombotic therapy (acetyl salicylic acid 125 mg and policosanol
5 mg) or antithrombotic therapy þO
3
: antithrombotic therapy plus ozone therapy (200 ml of O
3
/O
2
40
m
g/ml). Data are means7S.D.
For prothrombin time the laboratory reference interval was 10.1–15.2 s and for bleeding time 1–3 min.
a
Po0.001 vs. compared to start point between the same group.
b
Po0.001 vs. prothrombin time in group antithrombotic therapy at the end of the treatment.
G. Martı
´nez-Sa
´nchez et al. / European Journal of Pharmacology 691 (2012) 156–162158
(antithrombotic therapy and antithrombotic therapyþO
3
) were
not significantly different from reference values.
Variables of total antioxidant activities in lipid (PP) or hydro-
soluble fractions (FRAP) at the beginning of the study in both groups
were significantly different from reference values. However, at the
end of the study, a significant (Po0.05) decrease in PP was achieved
in antithrombotic therapyþO
3
, which tend to reach normal values.
FRAP was improved in both treatment groups but final values did
not reach the reference normal values.
After or before treatment, the level of reduced glutathione was
lower in both treatment groups when compared to the reference
group. However, after the treatment the concentration of GSH
was higher in the antithrombotic therapy þO
3
group (with sig-
nificant (Po0.05) differences from the initial levels) than in
antithrombotic therapy patients.
Enzymatic activities of catalase and SOD were significantly
(Po0.05) increased in both groups before treatment when
compared to the reference control group, but maintained the
same ratio (catalase/SOD) compared to the reference. After treat-
ment, a reduction in 67.2% of catalase activities, without mod-
ification of SOD activities, was observed in antithrombotic
therapy group with a consequent significant (Po0.05) reduction
in catalase/SOD ratio compared to the reference group. In con-
trast, in antithrombotic therapyþO
3
group a reduction of 57.1% in
SOD and 32.2% in catalase activities was noted, corresponding
with a significant (Po0.05) increment of the catalase/SOD ratio
compared to the reference group.
4. Discussion
Cardiovascular diseases is a major factor in mortality rates
around the world and contributes to more than one-third of
deaths in the USA (Roger et al., 2011). It has been predicted that
atherosclerosis will be the primary cause of death in the world
by 2020 (Scott, 2002). Consequently, developing a treatment
regimen that can slow or even reverse the atherosclerotic process
is imperative. In this context ozone therapy, simultaneously
applied with regular drugs, may represent a promising approach
for correcting oxidative stress and improving the uncertain
prognosis of many patients (Bocci et al., 2009;Mason, 2011).
Abnormal platelet aggregation has direct consequences in CAD
that can be avoided by antithrombotic agent. As shown in Table 2
a combination of Aspirin
s
and policosanol significant increase the
prothrombin time without modification of the bleeding time, in
line with previous results (Arruzazabala et al., 1997). Low dose
regimens (50–325 mg) of the antiplatelet agent Aspirin
s
(acet-
ylsalicylic acid) are a standard treatment for the secondary
prevention of cardiovascular outcomes (Campbell et al., 2007).
Meta-analysis of randomized controlled trials has shown that low
doses of Aspirin
s
is protective in most types of patients that are
at increased risk of occlusive vascular events (Collaboration,
2002). Guidelines recommend long term use of low dose aspirin
(75–150 mg/day) as an effective antiplatelet regimen for patients
with cardiovascular diseases, unless contraindicated (Graham
et al., 2007).
A randomized, double-blind, placebo-controlled study com-
pared the effects of policosanol, Aspirin
s
and combination
therapy on platelet aggregation. Meanwhile, Aspirin
s
signifi-
cantly reduced platelet aggregation induced by collagen (61.4%)
and epinephrine (21.9%) but not ADP-induced aggregation. Com-
bined therapy significantly inhibited aggregation induced by all
the agonists reaching the highest reductions of platelet aggrega-
tion induced by collagen (71.3%) and epinephrine (57.5%). It was
demonstrated that policosanol was as effective as Aspirin
s
.
Moreover, combination therapies have shown some advantages
compared to the respective monotherapies (Arruzazabala et al.,
1997). The rationale for using two or more types of antiplatelet
agents is that platelets can be activated by various pathways.
Aspirin
s
targets cyclooxygenase-related activation pathway,
while policosanol acts by other mechanisms (Noa et al., 2007).
According to the present results, combined therapy (Aspirin
s
plus
Table 3
Biomarkers of oxidative damage and antioxidant-pro/oxidant balance.
Groups Reference Antithrombotic therapy Antithrombotic therapyþO
3
Biomarkers (n¼50) (n¼27) (n¼26)
AOPP (
m
M of chloramine) Time 0 9.19 70.64
a
23.1275.31
b
23.9070.84
b
20 day – 24.2577.18
b
18.2673.61
c
MDA (
m
M) Time 0 3.8070.07
a
13.7173.01
b
12.0470.44
b
20 day – 10.8272.52
b
8.3873.63
c
TH (
m
M) Time 0 103.78717.71
a
142.9677.71
b
155.4378.61
b
20 day – 98.08712.29
a
104.65713.27
a
PP (
m
M) Time 0 9.1770.82
a
20.4178.16
b
16.2573.62
b
20 day – 19.0077.03
b
6.1672.51
a
FRAP (
m
M) Time 0 1017.17206.0
a
146.2762.3
b
191.30757.0
b
20 day – 386.07104. 3
c
471.97115.8
d
GSH (mM) Time 0 2.56 70.31
a
1.5070.23
b
1.1870.23
b
20 day – 1.0870.45
b
1.5570.63
c
SOD (U/ml) Time 0 11.3571.97
a
40.48710.12
b
37.7577.13
b
20 day – 35.27714.21
b
16.2778.39
c
CAT (U/l) Time 0 231.80711.33
a
880.47250.1
b
839.57165.3
b
20 day 288.0799.6
a
543.47172.3
c
CAT/SOD Time 0 0.020470.0057
a
0.022070.0306
b
0.0213 70.0152
b
20 day – 0.0081 70.0070
c
0.033370.0205
d
Antithrombotic therapy (acetyl salicylic acid 125 mg and policosanol 5 mg); antithrombotic therapy þO
3
, antithrombotic therapy plus ozone therapy (200 ml of O
3
/O
2
40
m
g/ml); AOPP, advanced oxidation protein products; CAT, catalase; GSH, reduced glutathione; FRAP, ferric reducing ability of plasma; MDA, malondialdehyde; PP,
peroxidation potential; SOD, superoxide dismutase; TH, total hydroperoxides. Data are means 7S.D. Means having different superscript letters indicate significant
difference (Po0.05) comparing reference, initial and final time of each groups values between the same set.
G. Martı
´nez-Sa
´nchez et al. / European Journal of Pharmacology 691 (2012) 156–162 159
policosanol) can induce an antithrombotic response, however it only
partially modifies some markers of the oxidative stress system
(normalized catalase and TH and improved FRAP) (Table 3).
The significant (Po0.001) increment in prothrombin time
observed in antithrombotic therapyþO
3
compared to antithrom-
botic therapy (Table 2), could be as consequence of the up-
regulation of adenosine A
2
receptors, induced by conditions such
as oxidative stress (Johnston-Cox and Ravid, 2011). It was
demonstrated that ozone oxidative preconditioning can activate
adenosine A
1
receptors (Leon Fernandez et al., 2008). If the same
mechanisms happen also for adenosine A
2
receptor in platelet the
increment of adenosine signaling mechanisms can act stimulating
platelets adenylate cyclase and increasing platelet cyclic-3
0
,5
0
-
adenosine monophosphate levels. Throughout this mechanism,
platelet aggregation is inhibited in response to various stimuli
such as platelet activating factors, collagen, and adenosine
diphosphate.
Clinical practice has consolidated the use of ozone therapy in
the treatment of peripheral occlusive arterial disease (POAD). It
has been demonstrated that ozonized autohemotransfusion may
be useful to improve both the poor rheological properties of the
blood and the oxygen delivery to tissues in patients suffering
from POAD (Giunta et al., 2001;Turczynski et al., 1991;Verrazzo
et al., 1995). Even if the mechanism of action is not fully under-
stood so far, it seems evident that ozone does not affect the blood
coagulation parameters (Biedunkiewicz et al., 2006). However, it
is now proven that low doses of ozone can modulate the
antioxidant system and improve the redox status (Bocci et al.,
2011). As shown in Table 2, ozone potentiates the anticoagulant
effect of Aspirin
s
plus policosanol, without modification of the
bleeding time. In addition, the combination antithrombotic
therapyþO
3
improves the antioxidant status of the patients
(Table 3).
Increasing evidence has highlighted the roles of oxidative stress
and inflammation in the promotion of atherosclerotic cardiovascular
diseases (Rao and Kiran, 2011). Recent pathological studies have
elucidated specific mediators that appear to link these pathways to
the progression and rupture of the atherosclerotic plaque in the
artery wall (Uno and Nicholls, 2010). Although the symptoms and
signs of CAD are noted in the advanced state of disease, most
individuals with CAD show no evidence of disease for decades. Over
time, vulnerable plaque rupture could activate blood clotting and
consequently an arterial stenosis, which in turn could promote heart
attack, stroke, POAD and major debilitating events (Rao and Kiran,
2011). In addition to the influence on blood coagulation parameters,
modulation of oxidative stress by ozone can be preventive for a
cardiovascular event.
AOPP levels were down-regulated in antithrombotic therapyþO
3
compared to the antithrombotic therapy group. AOPP is a novel
marker of oxidative stress correlating tightly with the degree of
monocyte activation, dityrosine, advanced glycation end products,
neopterin and inflammatory cytokines (i.e.TNF-
a
and its soluble
receptor) levels (Witko-Sarsat et al., 1999). The chlorinated oxidants
are closely related with the mechanism of generation of AOPP.
Moreover, AOPP-induced reactive oxygen species (ROS) production
in endothelial cells is partially mediated by NADPH oxidase activa-
tion. AOPP appeared to act as true inflammatory mediators since
they are able to trigger the oxidative burst and the synthesis of
cytokines (Martinez-Sanchez et al., 2005b). The effect of ozone
regulating AOPP is an indirect index connecting with the control
of the inflammatory process.
Improvement of MDA and PP in the antithrombotic therapyþO
3
group is indicative of the paradoxical antioxidant effect of ozone
therapy, and it is of paramount importance due to the connection of
lipid oxidation with CAD (Rao and Kiran, 2011). Improvement of TH
in antithrombotic therapy and antithrombotic therapyþO
3
groups
after treatment should be connected with the antioxidant effect of
acetyl salicylic acid (Baltazar et al., 2011) or policosanol (Castano
et al., 2002). However, improvement in GSH was only reached in
antithrombotic therapyþO
3
group. The FRAP, indicator of total
antioxidant capacity (Huang et al., 2005), was increased only in
the antithrombotic therapy þO
3
group. FRAP parameter is indicative
of the reducing ability of the sample involving the complex
integration of all compounds with antioxidant activity (Benzie and
Strain, 1996), its rise could be due to the increment of GSH and other
hydro soluble antioxidants. The preconditioning actions of ozone
(Chen et al., 2008;Rodriguez et al., 2009;Stadlbauer et al., 2008), as
well as the improvement in the antioxidant defense systems and the
reduction in ROS, favors an appropriate redox balance, suggesting
that CAD patients could be better protected from an eventual
cardiovascular accident.
It is important to know that the biological effect of rectal
insufflation of O
3
has been demonstrated extensively both experi-
mentally (Gonzalez et al., 2004;Guanche et al., 2010;Seda Artis
et al., 2010) and clinicaly (Calunga et al., 2011;Hernandez Rosales
et al., 2005;Martinez-Sanchez et al., 2005a;Romero Valdes et al.,
1993;Zaky et al., 2011a,2011b). Furthermore, preclinical studies
demonstrated its low toxicity (Diaz-Llera et al., 2009;Guanche
et al., 2010). For this reason, the application of O
3
by rectal way
has been now extended to treat many diseases. Because of the
oxidative preconditioning effect of ozone therapy, a cycle of 20
treatments will be enough to sustain the effect for approximately
3 months, depending on the oxidative stress status of the patients
(Schwartz et al., 2011).
Etiologic inference about the role of oxidative stress on
cardiovascular diseases are complicated by the complexity of
the relation between oxidative stress and antioxidant enzyme
activity (Flores-Mateo et al., 2009). SOD is a scavenger of O
2
,
transforming it in H
2
O
2
. But if H
2
O
2
is a ROS, then SOD activities
cannot be interpreted as a single antioxidant, but as a part of the
antioxidant system. Thus a better understanding of the role of
SOD and catalase could be easily evaluated from the analysis of
the ratio catalase/SOD (Martinez-Sanchez et al., 2005a). According
to the present results (Table 2), CAD patients without treatment
continue to maintain catalase/SOD homeostasis, but experienced
an increment of 3.42 or 3.64 fold in SOD and catalase activities
respectively. After treatment two different scenarios appeared. In
the antithrombotic therapy group, levels of SOD remain high with
normalization of catalase activities compared to non CAD sub-
jects. This scenario implied high level of O
2
production with
normal level of H
2
O
2
detoxification capacity. This fact may be due
to the lowering effect of acetyl salicylic acid in catalase levels
(Kowalczyk et al., 2006). In contrast, in antithrombotic
therapyþO
3
a reduction of 57% and 32% of SOD and catalase
activities respectively moves the system to a status of low
production of O
2
with an increment in H
2
O
2
detoxification.
Those findings suggest a better status for antithrombotic
therapyþO
3
patients. It is well known that in atherosclerotic
plaques, O
2
seems to play an important role in the local
inflammatory response, in migration and proliferation of vascular
smooth muscle and in endothelial dysfunction. Several of these
effects might be mediated by an increased oxidative stress,
reducing the bioavailability of NO and promoting an uncoupled
of nitric oxide sintase (NOS) activity (Miller et al., 2008). In
addition, determining the role of H
2
O
2
in the pathophysiology of
cardiovascular diseases has proven to be difficult. One reason is
that altering the expression of enzymes related to the catabolism
of H
2
O
2
is likely to alter the expression of other genes (e.g.,
through epidermal growth factor receptor transactivation). Over-
expression of CuZnSOD paradoxically accelerates the develop-
ment of atherosclerosis in apolipoprotein E
/
mice and can be
‘‘rescued’’ by overexpression of catalase (Yang et al., 2004). Taking
G. Martı
´nez-Sa
´nchez et al. / European Journal of Pharmacology 691 (2012) 156–162160
these elements into consideration suggests that H
2
O
2
might play
an important role in the development of atherosclerosis. The
modulation of SOD and catalase enzyme by ozone was noted in
other clinical trials (Martinez-Sanchez et al., 2005a) and probably
involves gene expression modulation (Cardile et al., 1995).
In summary, combined treatment using Aspirin
s
, policosanol
and ozone in patients with CAD improved homeostatic and
antioxidant indexes. Integrative therapy with ozone could be a
future opportunity for correcting the chronic oxidative stress
caused by antithrombotic therapy and improving the uncertain
prognosis of CAD patients. In addition, this pilot study, establish
the foundation for a longer time study in order to determine:
(1) clinical implications of antiagregants and ozonetherapy com-
bination on CAD, (2) clinical implications of antiagregant and
ozonetherapy combination on risk factors and (3) possible low-
ering dose of antiagregant.
Acknowledgments
We gratefully acknowledge the support of the staff from the
Laboratory of Biochemistry of the Center of Studies for Research
and Biological Evaluations, Pharmacy and Food Sciences College,
University of Havana, Havana, Cuba. We are grateful and thank
Mr. Lou Dietrich for revising the English manuscript.
References
Arruzazabala, M.L., Valdes, S., Mas, R., Carbajal, D., Fernandez, L., 1997. Comparative
study of policosanol, aspirin and the combination therapy policosanol-aspirin
on platelet aggregation in healthy volunteers. Pharmacol. Res. 36, 293–297.
Baltazar, M.T., Dinis-Oliveira, R.J., Duarte, J.A., Bastos, M.L., Carvalho, F., 2011.
Antioxidant properties and associated mechanisms of salicylates. Curr. Med.
Chem. 18, 3252–3264.
Benzie, I.F., Strain, J.J., 1996. The ferric reducing ability of plasma (FRAP) as a
measure of ‘‘antioxidant power’’: the FRAP assay. Anal. Biochem. 239, 70–76.
Biedunkiewicz, B., Lizakowski, S., Tylicki, L., Skiboeska, A., Nieweglowski, T.,
Chamienia, A., Debska-Slizien, A., Rutkowski, B., 2006. Blood coagulation
unaffected by ozonated autohemotherapy in patients on maintenance hemo-
dialysis. Arch. Med. Res. 37, 1034–1037.
Bocci, V., Travagli, V., Zanardi, I., 2009. May oxygen-ozone therapy improves
cardiovascular disorders? Cardiovasc. Hematol. Disord. Drug Targets 9, 78–85.
Bocci, V.A., Zanardi, I., Travagli, V., 2011. Ozone acting on human blood yields a
hormetic dose-response relationship. J. Transl. Med. 9, 66.
Calunga, J., Paz, Y., Mene
´ndez, S., Martı
´nez, A., Herna
´ndez, A., 2011. Rectal ozone
therapy for patients with pulmonary emphysema. Rev. Med. Chile 139,
439–447.
Campbell, C.L., Smyth, S., Montalescot, G., Steinhubl, S.R., 2007. Aspirin dose for the
prevention of cardiovascular disease: a systematic review. J. Am. Med. Assoc.
297, 2018–2024.
Cardile, V., Jiang, X., Russo, A., Casella, F., Renis, M., Bindoni, M., 1995. Effects of
ozone on some biological activities of cells in vitro. Cell Biol. Toxicol. 11,
11–21.
Castano, G., Menendez, R., Mas, R., Amor, A., Fernandez, J.L., Gonzalez, R.L.,
Lezcay, M., Alvarez, E., 2002. Effects of policosanol and lovastatin on lipid
profile and lipid peroxidation in patients with dyslipidemia associated with
type 2 diabetes mellitus. Int. J. Clin. Pharmacol. Res. 22, 89–99.
Chen, H., Xing, B., Liu, X., Zhan, B., Zhou, J., Zhu, H., Chen, Z., 2008. Ozone oxidative
preconditioning inhibits inflammation and apoptosis in a rat model of renal
ischemia/reperfusion injury. Eur. J. Pharmacol. 581, 306–314.
Collaboration, A.T, 2002. Collaborative meta-analysis of randomised trials of
antiplatelet therapy for prevention of death, myocardial infarction, and stroke
in high risk patients. Br. Med. J. 324, 71–86.
Colombo, R., D’Angelo, F., Arzini, A., Abbritti, F., Boccalon, L., 2000. Arterial
occlusive disease: pharmacological therapy and ozone therapy. Gazz. Med.
Ital. Arch. Sci. Med. 159, 53–57.
Delgado-Roche, L., Martı
´nez-Sa
´nchez, G, Dı
´az-Batista, A., Re, L., 2011. Effects of
ozone therapy on oxidative stress biomarkers in coronary artery disease
patients. Int. J. Ozone Res. 10, 99–104.
Diaz-Llera, S., Gonza
´lez-Herna
´ndez, Y., Mesa, J.E.G., Martı
´nez-Sa
´nchez, G., Re, L.,
2009. Induction of DNA primary damage in peripheral blood leukocytes and
exfoliated colorectal epithelial cells in rats treated with O
3
/O
2
mix. Int. J.
Ozone Ther. 8, 217–221.
Esterbauer, H., Cheeseman, K.H., 1990. Determination of aldehydic lipid peroxida-
tion products: malonaldehyde and 4-hydroxynonenal. Methods Enzymol. 186,
407–421.
Faul, F., Erdfelder, E., Lang, A.G., Buchner, A., 2007. G
n
Power 3: a flexible statistical
power analysis program for the social, behavioral, and biomedical sciences.
Behav. Res. Methods 39, 175–191.
Flores-Mateo, G., Carrillo-Santisteve, P., Elosua, R., Guallar, E., Marrugat, J., Bleys, J.,
Covas, M.I., 2009. Antioxidant enzyme activity and coronary heart disease:
meta-analyses of observational studies. Am. J. Epidemiol. 170, 135–147.
Franco, M., Cooper, R.S., Bilal, U., Fuster, V., 2011. Challenges and opportunities for
cardiovascular disease prevention. Am. J. Med. 124, 95–102.
Giunta, R., Coppola, A., Luongo, C., Sammartino, A., Guastafierro, S., Grassia, A.,
Giunta, L., Mascolo, L., Tirelli, A., Coppola, L., 2001. Ozonized autohemotrans-
fusion improves hemorheological parameters and oxygen delivery to
tissues in patients with peripheral occlusive arterial disease. Ann. Hematol.
80, 745–748.
Gonzalez, R., Borrego, A., Zamora, Z., Romay, C., Hernandez, F., Menendez, S.,
Montero, T., Rojas, E., 2004. Reversion by ozone treatment of acute nephro-
toxicity induced by cisplatin in rats. Mediators Inflamm. 13, 307–312.
Graham, I., Atar, D., Borch-Johnsen, K., Boysen, G., Burell, G., Cifkova, R., Dallonge-
ville, J., De Backer, G., Ebrahim, S., Gjelsvik, B., Herrmann-Lingen, C., Hoes, A.,
Humphries, S., Knapton, M., Perk, J., Priori, S.G., Pyorala, K., Reiner, Z.,
Ruilope, L., Sans-Menendez, S., Scholte op Reimer, W., Weissberg, P.,
Wood, D., Yarnell, J., Zamorano, J.L., Walma, E., Fitzgerald, T., Cooney, M.T.,
Dudina, A., 2007. European guidelines on cardiovascular disease prevention in
clinical practice: executive summary: Fourth Joint Task Force of the European
Society of Cardiology and Other Societies on Cardiovascular Disease Preven-
tion in Clinical Practice (Constituted by representatives of nine societies and
by invited experts). Eur. Heart J. 28, 2375–2414.
Guanche, D., Zamora, Z., Hernandez, F., Mena, K., Alonso, Y., Roda, M., Gonzales, M.,
Gonzales, R., 2010. Effect of ozone/oxygen mixture on systemic oxidative
stress and organic damage. Toxicol. Mech. Methods 20, 25–30.
Gupta, R., Joshi, P., Mohan, V., Reddy, K.S., Yusuf, S., 2008. Epidemiology and
causation of coronary heart disease and stroke in India. Heart 94, 16–26.
Haining, J.L., Legan, J.S., 1972. Improved assay for catalase based upon steady-state
substrate concentration. Anal. Biochem. 45, 469–479.
Hernandez Rosales, F.A., Calunga Fernandez, J.L., Turrent Figueras, J., Menendez
Cepero, S., Montenegro Perdomo, A., 2005. Ozone therapy effects on biomar-
kers and lung function in asthma. Arch. Med. Res. 36, 549–554.
Huang, D., Ou, B., Prior, R.L., 2005. The chemistry behind antioxidant capacity
assays. J. Agric. Food Chem. 53, 1841–1856.
Johnston-Cox, H.A., Ravid, K., 2011. Adenosine and blood platelets. Purinergic
Signal 7, 357–365.
Kowalczyk, E., Kopff, A., Blaszczyk, J., Fijalkowski, P., Kopff, M., 2006. The influence
of selected nonsteroidal anti-inflammatory drugs on antioxidative enzymes
activity. Pol. Arch. Med. Wewn. 115, 112–117.
Leon Fernandez, O.S., Ajamieh, H.H., Berlanga, J., Menendez, S., Viebahn-Hansler, R.,
Re, L., Carmona, A.M., 2008. Ozone oxidative preconditioning is mediated by A1
adenosine receptors in a rat model of liver ischemia/ reperfusion.Transplant Int.
21, 39–48.
Martinez-Sanchez, G., Al-Dalain, S.M., Menendez, S., Re, L., Giuliani, A., Candelario-
Jalil, E., Alvarez, H., Fernandez-Montequin, J.I., Leon, O.S., 2005a. Therapeutic
efficacy of ozone in patients with diabetic foot. Eur. J. Pharmacol. 523,
151–161.
Martinez-Sanchez, G., Giuliani, A., Perez-Davison, G., Leon-Fernandez, O.S., 2005b.
Oxidized proteins and their contribution to redox homeostasis. Redox Rep. 10,
175–185.
Mason, R.P., 2011. Optimal therapeutic strategy for treating patients with
hypertension and atherosclerosis: focus on olmesartan medoxomil. Vasc.
Health Risk Manage. 7, 405–416.
Miller, J.D., Chu, Y., Brooks, R.M., Richenbacher, W.E., Pena-Silva, R., Heistad, D.D.,
2008. Dysregulation of antioxidant mechanisms contributes to increased
oxidative stress in calcific aortic valvular stenosis in humans. J. Am. Coll.
Cardiol. 52, 843–850.
Noa, M., Mas, R., Lariot, C., 2007. Protective effect of policosanol on endothelium
and intimal thickness induced by forceps in rabbits. J. Med. Food 10,
452–459.
Ozdemirler, G., Mehmetcik, G., Oztezcan, S., Toker, G., Sivas, A., Uysal, M., 1995.
Peroxidation potential and antioxidant activity of serum in patients with
diabetes mellitus and myocard infarction. Horm. Metab. Res. 27, 194–196.
Rao, V., Kiran, R., 2011. Evaluation of correlation between oxidative stress and
abnormal lipid profile in coronary artery disease. J. Cardiovasc. Dis. Res. 2,
57–60.
Re, L., Mawsouf, M.N., Menendez, S., Leon, O.S., Martı
´nez-Sa
´nchez, G., Hernandez, F.,
2008. Ozone therapy: clinical and basic evidence of its therapeutic potential.
Arch. Med. Res. 39, 17–26.
Rodriguez, Z.Z., Guanche, D., Alvarez, R.G., Rosales, F.H., Alonso, Y., Schulz, S., 2009.
Preconditioning with ozone/oxygen mixture induces reversion of some indi-
cators of oxidative stress and prevents organic damage in rats with fecal
peritonitis. Inflamm. Res.
Roger, V.L., Go, A.S., Lloyd-Jones, D.M., Adams, R.J., Berry, J.D., Brown, T.M.,
Carnethon, M.R., Dai, S., de Simone, G., Ford, E.S., Fox, C.S., Fullerton, H.J.,
Gillespie, C., Greenlund, K.J., Hailpern, S.M., Heit, J.A., Ho, P.M., Howard, V.J.,
Kissela, B.M., Kittner, S.J., Lackland, D.T., Lichtman, J.H., Lisabeth, L.D.,
Makuc, D.M., Marcus, G.M., Marelli, A., Matchar, D.B., McDermott, M.M.,
Meigs, J.B., Moy, C.S., Mozaffarian, D., Mussolino, M.E., Nichol, G., Paynter, N.P.,
Rosamond, W.D., Sorlie, P.D., Stafford, R.S., Turan, T.N., Turner, M.B., Wong, N.D.,
Wylie-Rosett, J., 2011. Heart disease and stroke statistics—2011 update: a
report from the American Heart Association. Circulation 123, e18–e209.
G. Martı
´nez-Sa
´nchez et al. / European Journal of Pharmacology 691 (2012) 156–162 161
Romero Valdes, A., Blanco Gonzalez, R., Menendez Cepero, S., Gomez Moraleda, M.,
Ley Pozo, J., 1993. Arteriosclerosis obliterans and ozone therapy. Its adminis-
tration by different routes. Angiologia 45, 177–179.
Schmaier, A.H. (Ed.), 5 ed. Churchill Livingstone Elsevier, Philadelphia.
Schwartz, A., Martinez-Sanchez, G., Re, L., 2011. Guia para el uso me
´dico del ozono.
Fundamentos terape
´uticos e indicaciones, Madrid.
Scott, J., 2002. The pathogenesis of atherosclerosis and new opportunities for
treatment and prevention. J. Neural Transm. Suppl. 63, 1–17.
Seda Artis, A., Aydogan, S., Gokhan Sahin, M., 2010. The effects of colorectally
insufflated oxygen-ozone on red blood cell rheology in rabbits. Clin. Hemor-
heol. Microcirc. 45, 329–336.
Sedlak, J., Lindsay, R.H., 1968. Estimation of total, protein-bound, and nonprotein
sulfhydryl groups in tissue with Ellman’s reagent. Anal. Biochem. 25, 192–205.
Stadlbauer, T.H., Eisele, A., Heidt, M.C., Tillmanns, H.H., Schulz, S., 2008. Precondi-
tioning with ozone abrogates acute rejection and prolongs cardiac allograft
survival in rats. Transplant Proc. 40, 974–977.
Steppan, J., Meaders, T., Muto, M., Murphy, K.J., 2010. A metaanalysis of the
effectiveness and safety of ozone treatments for herniated lumbar discs.
J. Vasc. Interv. Radiol. 21, 534–548.
Turczynski, B.,Sroczynski, J., Antoszewski,Z., Matyszczyk,B., Krupa, G., Mlynarski, J.,
Strugala, M., 1991. Ozone therapy and viscosity of blood and plasma, distance
of intermittent claudication and certain biochemical components in patients
with diabetes type II and ischemia of the lower extremities. Pol. Tyg. Lek. 46,
708–710.
Uno, K., Nicholls, S.J., 2010. Biomarkers of inflammation and oxidative stress in
atherosclerosis. Biomark Med. 4, 361–373.
Verrazzo, G., Coppola, L., Luongo, C., Sammartino, A., Giunta, R., Grassia, A., Ragone,
R., Tirelli, A., 1995. Hyperbaric oxygen, oxygen-ozone therapy, and rheologic
parameters of blood in patients with peripheral occlusive arterial disease.
Undersea Hyperb. Med. 22, 17–22.
WHO, 2008. Department of Measurement & Health Information Systems of the
Information, Evidence and Research Cluster. In: Press, W. (Ed.), Department of
Measurement & Health Information Systems of the Information, Evidence and
Research Cluster. World Health Organization, Geneva, pp. 29–31.
Witko-Sarsat, V., Friedlander, M., Nguyen Khoa, T., Capeillere-Blandin, C.,
Nguyen, A.T., Canteloup, S., Dayer, J.M., Jungers, P., Drueke, T., Descamps-
Latscha, B., 1998. Advanced oxidation protein products as novel mediators of
inflammation and monocyte activation in chronic renal failure. J. Immunol.
161, 2524–2532.
Witko-Sarsat, V., Nguyen-Khoa, T., Jungers, P., Drueke, T.B., Descamps-Latscha, B.,
1999. Advanced oxidation protein products as a novel molecular basis of
oxidative stress in uraemia. Nephrol. Dial. Transplant. 14 (1), 76–78.
WMA, 2004. World Medical Association Declaration of Helsinki. Ethical Principles
for Medical Research Involving Human Subjects. J. Int. Bioethique, Adopted by
the 18th WMA General Assembly, Helsinki, Finland, June 1964, pp. 124–129.
Yang, H., Roberts, L.J., Shi, M.J., Zhou, L.C., Ballard, B.R., Richardson, A., Guo, Z.M.,
2004. Retardation of atherosclerosis by overexpression of catalase or both Cu/
Zn-superoxide dismutase and catalase in mice lacking apolipoprotein E. Circ.
Res. 95, 1075–1081.
Yusuf, S., Hawken, S., Ounpuu, S., Dans, T., Avezum, A., Lanas, F., McQueen, M.,
Budaj, A., Pais, P., Varigos, J., Lisheng, L., 2004. Effect of potentially modifiable
risk factors associated with myocardial infarction in 52 countries (the INTER-
HEART study): case-control study. Lancet 364, 937–952.
Zaky, S., Fouad, E., HI, H.K., 2011a. The effect of rectal ozone on the portal vein oxy-
genation and pharmacokinetics of propranolol in liver cirrhosis (a preliminary
human study). Br. J. Clin. Pharmacol. 71, 411–415.
Zaky, S., Kamel, S.E., Hassan, M.S., Sallam, N.A., Shahata, M.A., Helal, S.R.,
Mahmoud, H., 2011b. Preliminary results of ozone therapy as a possible
treatment for patients with chronic hepatitis C. J. Altern. Complement. Med.
17, 259–263.
G. Martı
´nez-Sa
´nchez et al. / European Journal of Pharmacology 691 (2012) 156–162162