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Extracorporeal Blood Oxygenation and Ozonation (EBOO): A Controlled Trial in Patients with Peripheral Artery Disease

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Abstract

Since 1990 our group has been using extracorporeal circulation to ozonate blood by an original method, known as extracorporeal blood oxygenation and ozonation (EBOO), with the aim of amplifying the results observed with ozone autohemotherapy. To verify the hypothesis that EBOO improves the skin lesions typical of peripheral artery disease (PAD) patients. Twenty-eight patients with PAD were randomized to receive EBOO or intravenous prostacyclin in a controlled clinical trial. The primary efficacy parameters were regression of skin lesions and pain,and improvement in quality of life and vascularisation. Patients treated with EBOO showed highly significant regression of skin lesions with respect to patients treated with prostacyclin. Other parameters that were significantly different in the two groups of patients were pain,pruritus, heavy legs and well-being. No significant differences in vascularisation of the lower limbs before and after treatment were found in either group. No side effects or complications were recorded during the 210 EBOO treatments. EBOO was much more effective than prostacyclin for treating skin lesions in PAD patients and also had a positive effect on patient general condition without any apparent change in arterial circulation. This suggests other mechanisms of action of EBOO.
INTRODUCTION
Although inhalation of ozone may cause severe lung
damage, ozone has been found extremely useful for
different therapeutic purposes, such as ozone
autohemotherapy (O
3
AHT) of infectious diseases, in which
it activates the immune system (1-10), and of vascular
diseases, in which oxygen utilisation is improved by
release of growth factors that reduce ischemia (11-13). In
O
3
AHT, small quantities of blood are brought into contact
with ozone and then reinfused. Recent studies to clarify
the mechanism of action have shown that contact between
ozone and blood gives rise to effects that can be exploited
in medicine (14-16). Exposure of human blood to a
New Perspectives
The International Journal of Artificial Organs / Vol. 28 / no. 10, 2005 / pp. 1039-1050
Extracorporeal blood oxygenation and ozonation
(EBOO): A controlled trial in patients with peripheral
artery disease
N. DI PAOLO
1
, V. BOCCI
2
, D.P. SALVO
1
, F. PALASCIANO
3
, M. BIAGIOLI
4
, S. MEINI
5
, F. GALLI
6
, I. CIARI
7
,
F. MACCARI
8
, F. CAPPELLETTI
1
, M. DI PAOLO
9
, E. GAGGIOTTI
1
1
Nephrology and Dialysis Department, University Hospital of Siena, Siena - Italy
2
Institute of General Physiology, University of Siena, Siena - Italy
3
Surgical Department, Vascular Surgery Section, University of Siena, Siena - Italy
4
Department of Internal Medicine, Dermatology Section, University of Perugia, Perugia - Italy
5
Department of Internal Medicine and Immunological Science, University of Perugia, Perugia - Italy
6
Department of Internal Medicine, Section of Applied Biochemistry and Nutritional Sciences, University of Perugia,
Perugia - Italy
7
Department of Internal Medicine, Section of Biochemistry, University of Siena, Siena - Italy
8
Department of Quantitative Methods, University of Siena, Siena - Italy
9
Institute of Legal Medicine, University of Pisa, Pisa - Italy
©
Wichtig Editore, 2005 0391-3988/1039-12 $15.00/0
ABSTRACT: Background: Since 1990 our group has been using extracorporeal circulation to ozonate
blood by an original method, known as extracorporeal blood oxygenation and ozonation (EBOO),
with the aim of amplifying the results observed with ozone autohemotherapy.
Objective: To verify the hypothesis that EBOO improves the skin lesions typical of peripheral artery
disease (PAD) patients.
Methods: Twenty-eight patients with PAD were randomized to receive EBOO or intravenous
prostacyclin in a controlled clinical trial. The primary efficacy parameters were regression of skin
lesions and pain, and improvement in quality of life and vascularisation.
Results: Patients treated with EBOO showed highly significant regression of skin lesions with respect
to patients treated with prostacyclin. Other parameters that were significantly different in the two
groups of patients were pain, pruritus, heavy legs and well-being. No significant differences in
vascularisation of the lower limbs before and after treatment were found in either group. No side
effects or complications were recorded during the 210 EBOO treatments.
Conclusion: EBOO was much more effective than prostacyclin for treating skin lesions in PAD patients
and also had a positive effect on patient general condition without any apparent change in arterial
circulation. This suggests other mechanisms of action of EBOO. (Int J Artif Organs 2005; 28: 1039-50)
KEY WORDS: Ozone therapy, Extracorporeal circulation, Peripheral arterial disease
mixture of oxygen and ozone is not toxic for blood,
provided exposure times and concentrations are
appropriate (17-20).
Despite the considerable development of O
3
AHT (21-
25), it has never been completely convincing as a therapy
due to its modest results, the difficulty of demonstrating
the expected hematochemical changes in circulating blood
and the impossibility of demonstrating a mechanism of
action. In our opinion, these limits of O
3
AHT may be due to
the fact that only small volumes of blood (200-300
ml/week) can be treated. We therefore looked for a way to
increase these quantities.
We assumed that the theoretical basis of O
3
AHT was
correct and over a period of 13 years developed an
oxygen-ozone Gas Exchange Device (GED) resistant to
the corrosive effects of ozone and capable of producing
efficient gaseous exchange in large quantities of blood in
extracorporeal circulation, in vitro and in vivo (26-28). We
called the technique extracorporeal blood oxygenation and
ozonation (EBOO). All studies in vitro, in animals and
eventually in humans, were approved by the local ethics
committee and conducted according to the Helsinki
guidelines. The results demonstrated that the method is
innocuous (29-32).
The story of these studies has been told elsewhere (33,
34). Since we found therapeutic effects of EBOO in
peripheral arterial disease (PAD) patients in preliminary
studies, we determined to seek confirmation in a clinical
controlled study.
MATERIALS AND METHODS
The present study is a randomized double controlled
trial conducted at the Ozone Study Center of the Azienda
Ospedaliera Universitaria Senese (Siena, Italy). The
protocol was approved by the ethical committee and
patients gave their written informed consent.
Twenty-eight PAD patients were enrolled in the study
and assigned randomly to two groups. Inclusion criteria
were age under 80 years and Fontain stage IV PAD.
Exclusion criteria were systemic diseases except diabetes,
neoplastic disease, hemorrhagic disease, alcoholism or
drug abuse, pregnancy, active chronic hepatitis, HIV
infection, immune complex diseases, psychiatric
disorders, obesity (Body Mass Index (BMI) > 40 kg/m
2
)
and recent (last 3 months) history of myocardial infarction
or peripheral venous thrombosis.
Seven days before treatment, patients underwent a run-
in phase with screening that included routine laboratory
tests, echo-color Doppler examination of the lower limbs,
measurement and photographic documentation of lesions,
impedance measurements, ECG and assessment of
concomitant medication. The diagnosis of PAD was based
on typical history, clinical examination, echo-Doppler-
tomography and if necessary angiography. Lesion surface
area was calculated manually allowing a 5% margin of
error. Lesions were staged using the scale of Leriche.
Treadmill testing of claudication was not possible because
of the severity of PAD, so claudication was evaluated
subjectively, asking patients to assign themselves a score
from 0 to 4: 0 absence of claudication, 1 claudication after
walking more than 100 m, 2 after 50-100 m, 3 after 25-50
m and 4 after less than 25 m. Well-being was assessed
with the WHO questionnaire (35), consisting of five
questions scored 0 to 4. The following tests were done by
autoanalyser: glycemia, total cholesterol, HDL cholesterol,
triglycerides, amylase, total proteins, beta globulin, alpha
globulin, gamma globulin, albumin, hematocrit, leucocytes,
erythrocytes, platelets, hemoglobin, neutrophils,
eosinophils, monocytes, basophils, lymphocytes,
glycosylated hemoglobin, BUN, creatinine, thromboplastin
time, SGPT, SGOT, LDH, haptoglobin, CPK, gamma GT,
bilirubin, fibrinogen, IgG, IgA, IgM, C3, C4, VES (IK) and
PCR.
Immediately after the run-in week, patients in the EBOO
group began two treatments per week for 7 weeks and
those in the ivP group began their specific therapy.
Lesions were photographed every 10 days for 3 months.
The duration of the experiment was 7 weeks, after which
the lesions were assessed and data was gathered on
subjective symptoms, general condition, impedance,
laboratory parameters and echo-Doppler. Forty-five days
after the end of the experiment, this assessment was
repeated.
EBOO technique
For extracorporeal circulation we currently use a Bellco
(Modena) apparatus consisting of a blood pump with
standard alarms and pressometers. Arterial and venous
lines are connected to a GED (Dideco, Mirandola)(surface
area 0.6 m
2
). In the blood circuit, the pump maintains a
constant flow of 75 ml/s. Ozone is produced by an
Ozonline International (Medica, Bologna) device which
enables mixing with oxygen from 0.5 to 10 µg/mL at a
1040
Ozone therapy in peripheral artery disease
pressure of 0.2 bar; a specific photometer (Ozonosan 590,
Iffezheim, Germany) enables control of the quantity of
ozone used. As in previous studies in vitro, in animals and
in humans, we used ozone concentrations between 0.5 to
1 µg/mL with 98-95% oxygen. The gas leaving the GED is
conveyed to an inactivation system (Catalyzer Sonder
Zubehor, Ozonosan, Iffezheim, Germany) which uses
palladium salts activated at 60°C by electric heater, so that
no ozone is released into the atmosphere. For
environmental protection, ozone detectors (Ozon Sensor,
Mod. C. 307 X) were fitted in the treatment room as well
as aspirators incorporating an ozone inactivation system
(Ozonline Air Nov Mini/ 578, Medica, Bologna) that cuts in
if ozone is accidentally released. The return line is fitted
with a bubble disperser and automatic level detector. For
further details, see our previous papers (26-34).
Treatments were conducted after overnight fasting. The
cubital veins of both arms were used or a catheter placed
in the jugular vein. Anti-clotting was obtained by injecting
heparin as a bolus (10,000 IU) at the start of treatment.
Once the extracorporeal circuit was stable, the ozone-
oxygen mixture was allowed into the compartment and
treatment began. In an hour, 4500 mL of blood was
treated. Each patient did 14 treatments in the seven-week
period.
It is technically impossible to measure ozone directly in
the blood or assay ROS in ozonated plasma because of
their very brief half-life (fractions of a second) (1). To
monitor the efficiency of the EBOO method, we assayed
serum protein thiol groups (PTG) and thiobarbituric acid
reactive substances (TBARS) as described elsewhere
(29). This is an indirect method of monitoring the oxidising
effect of ozone in the body through terminal products and
biochemical modifications of the plasma antioxidant
system (35, 36). Assay of these substances was carried
out during EBOO from the blood lines, immediately before
and after the GED, 30 minutes after the start of the second
treatment. All patients took vitamin C (0.5 g/day) and
acetylcysteine (600 mg/day) to ensure optimal anti-oxidant
capacity (1, 37).
Prostacyclin treatment
After a week of run-in, patients in this group were
treated intravenously with 0.5 ng/kg/m’ prostacyclin
(trometamol salt, Endoprost 50, Italfarmaco) for 28 days
using 0.1 mg Endoprost solution diluted in 500 mL saline
solution. Infusion lasted about 6 hours.
Statistical analysis
Normal distribution of the sample was evaluated by the
non parametric version of the Chi-square test and non
parametric tests were also used for the other analyses due to
the small populations size. The tests were: Sign test for
normal distribution of the data of the two groups, the sign test
separately on the two samples to test the hypothesis no
effect of treatment on lesion surface area and subjective
symptoms, the Mann-Whitney-Wilcoxon test on the pooled
sample to test the hypothesis equal effect of the two
treatments on lesion staging, subjective symptoms,
impedance parameters and laboratory variables that seemed
most appropriate for distinguishing between treatments. The
median was used instead of the mean because of the small
sample size and the use of categoric variables.
RESULTS
Twenty-eight patients entered the one-week run-in
phase, 15 of whom were randomized to receive EBOO
treatment and 13 to receive i.v. prostacyclin.
Table I shows the demographic and clinical
characteristics of EBOO and ivP treated groups (gender,
age, weight, concomitant diseases, concomitant therapies
and site of lesions). The two groups were homogeneous
according to the non parametric version of the chi-square
test for goodness-of-fit of k samples (k>1).
EBOO group
Extracorporeal circulation proceeded normally using the
cubital veins (14 patients). The jugular vein was used in
only one patient for the whole period of treatment. In three
dialysis patients, the arteriovenous fistula was used.
Ozonation of blood, measured indirectly by assay of
serum TBARS and PTG, before and after the GED, 30 min
after the start of the second session of EBOO, showed
good passage of ozone into the blood (Tab. II).
The patients did not record any side-effects or
subjectively unpleasant sensations, common in other
treatments using extracorporeal circulation, such as
hemodialysis. After treatment, patients reported a feeling
of well-being and euphoria that lasted several hours. The
15 patients underwent a total of 210 treatments. No side
effects were reported in periods between treatments and
there were no drop-outs.
Di Paolo et al
1041
Prostacyclin (ivP) treatment group
Of the 13 patients undergoing ivP, only ten completed
the cycle of therapy. Two dropped out due to headache
and one due to diarrhea. Only the data of patients who
completed therapy was used for statistical analysis.
The evolution of skin lesions is reported in Figures 1
and 2.
Figure 1 shows the first quartile, median and third
quartile of the distribution of lesion areas. A considerable
reduction in lesion area was evident in patients treated
with EBOO and a slighter improvement in those treated
with ivP. The hypothesis no effect of treatment was tested
separately for the data of the two groups by the sign test.
The hypothesis was only rejected in the EBOO group, and
significance was less than 0.01.
Figure 2 shows the trend of lesion staging according to
Leriche. The graph shows a sharp decrease in median
lesion stage in the EBOO group (from 4 to 1) whereas
median lesion stage (4) of patients treated with ivP did not
change. This suggests that EBOO has a much greater
effect on lesion stage than prostacyclin therapy. This effect
was confirmed by the Mann-Whitney-Wilcoxon test, testing
the hypothesis no effect of treatment. The hypothesis was
rejected with a significance less than 0.01.
The effects of the two therapies on subjective symptoms
1042
Ozone therapy in peripheral artery disease
TABLE I - COMPARISON OF DEMOGRAPHIC AND CLINICAL CHARACTERISTICS OF THE TWO GROUPS OF PAD
PATIENTS
EBOO ivP Significance
n% n%
Sex ns
M 6 40.0 5 50.0
F 9 60.0 5 50.0
Age (years) ns
<65 2 13.3 2 20.0
66-75 4 26.7 3 30.0
>75 9 60.0 5 50.0
Weight (kg) ns
<60 2 13.3 1 10.0
61-70 7 46.7 4 40.0
>70 6 40.0 5 50.0
Concomitant disease ns
diabetes 11 73.3 7 70.0
hypertension 9 60.0 6 60.0
cronic renal failure 7 46.7 5 50.0
Concomitant therapy ns
antiaggregans or anticlotting 6 40.0 4 40.0
antidiabetics 12 80.0 7 70.0
hypotensive agents 11 73.3 7 70.0
Site of lesions ns
lower limbs 14 93.3 9 90.0
upper limbs 1 6.7 1 10.0
fingers 3 20.0 2 20.0
Fig. 1 - Trend of lesion surface area.
are reported in Figures 3-5. The variables were scored
from 0 to 4. Figure 3 shows the medians reported by the
EBOO group before and after treatment. The greatest
changes were observed in the following variables:
claudication, pain, pruritus, heavy legs, weakness, joint
pain (which improved sharply) and general well-being
(which improved). To confirm this evidence, the sign test
was used to test the hypothesis same median before and
Di Paolo et al
1043
TABLE II - MEDIAN AND 1ST AND 3RD QUARTILES OF SERUM CONCENTRATIONS OF TBARS AND PTG
(micromol) MEASURED IN 15 PATIENTS BEFORE AND AFTER THE GED, 30 MIN AFTER THE START OF
THE SECOND SESSION OF EBOO
Before GED After GED p
Q1 Median Q3 Q1 Median Q3
TBARS 2.22 2.78 3.15 3.17 3.78 4.01 <0.001
PTG 495 532 535 399 400 419 <0.001
Fig. 2 - Lesion staging. Lesions were staged according to the Leriche
scale before and after treatment, assigning a score from 0 to 4. The
graph shows the median of these measurements in the two treatment
groups, before and after therapy.
Fig. 3 - Median scores of subjective symptoms before and after
EBOO.
Fig. 4 - Median scores of subjective symptoms before and after ivP. Fig. 5 - Median of differences in subjective symptoms before and
after EBOO and ivP treatment.
after treatment. The variables for which the hypothesis
was rejected are indicated in the figure (* 0.01<p<0.05; **
p<0.01). Figure 4 shows that the group treated with ivP
experienced non significant or no changes in subjective
symptoms.
Figure 5 shows the changes in subjective symptoms in
the groups treated with EBOO and prostacyclin. EBOO
was clearly much more effective than ivP, especially for
the variables mentioned above. When we tested the
difference in effect by the Mann-Whitney-Wilcoxon test,
this evidence was confirmed. Significant differences are
indicated * for 0.01<p<0.05 and ** for p<0.01.
Impedance data measurement
Impedance data is reported in Table III.
The only variable that showed a clear difference
between the two treatments was extracellular water, which
dropped by more than 5% in the EBOO group but
increased by more than 1% in the ivP group (Tab. III). It
was also the only significant variable according to the
Mann-Whitney-Wilcoxon test (p=0.038).
Thirty-seven variables were measured in both groups.
The Mann-Whitney-Wilcoxon test was used to test the
hypothesis no effect of EBOO treatment with respect to
ivP. Figure 6 shows the median percentage changes in
the five significant variables with respect to pre-treatment
values in both groups. Note, however, that serum
concentrations of alpha globulin and BUN had marginal
p-values between 0.05 and 0.10. This means that there
was only weak empirical evidence for rejecting the
hypothesis (Tab. IV).
Peripheral artery flow
Echo-color Doppler examination of flow in the anterior,
posterior and interosseous tibial arteries in both groups,
scored from 0 to 4 before and after treatment, failed to
show any differences subsequent to treatment and so
statistical analysis was not done. Note, however, that in
the EBOO group, the three medians had a score of 2
whereas in the ivP group the median was 2.5 for the
anterior and interosseous and 2 for the posterior tibial
artery.
Forty-five days after the end of treatment, none of the
parameters were significantly different from those obtained
1044
Ozone therapy in peripheral artery disease
TABLE III - MEDIAN PERCENTAGE CHANGES IN IMPEDANCE PARAMETERS WITH RESPECT TO PRE-
TREATMENT VALUES
Median ivp minus Alternative Mann-Whitney- p-value significance
median EBOO hypothesis Wilcoxon statistics
Fat mass 0.10 D < > 0 -0.39 0.697 Ns
Lean mass -0.44 D < > 0 -0.25 0.803 Ns
Muscle mass 0.00 D < > 0 -0.33 0.741 Ns
Intracellular water -0.93 D < > 0 -0.55 0.582 Ns
Extracellular water 6.76 D < > 0 2.08 0.038 *
Total water 0.48 D < > 0 1.55 0.121 Ns
Basal metabolism -0.16 D < > 0 -0.61 0.542 Ns
Phase angle -1.84 D < > 0 -0.44 0.330 Ns
* p < 0.0330
TABLE IV - LABORATORY TESTS: MANN-WHITNEY-WILCOXON TEST OF EFFECT OF TREATMENT MEDIAN
PERCENTAGE CHANGES IN SIGNIFICANT PARAMETERS WITH RESPECT TO PRE-TREATMENT
VALUES
Median ivP Alternative Mann-Whitney- p-value
minus median EBOO hypothesis Wilcoxon statistics
Triglycerides 3.16 D < 0 1.86 0.031
Alpha globulin 2.23 D < > 0 1.91 0.057
BUN 12.22 D < 0 1.36 0.087
Fibrinogen 6.42 D < > 0 2.20 0.027
VES (K index) 13.43 D < 0 1.69 0.046
immediately after treatment or significantly different
between groups. In four EBOO patients, lesions regressed
completely (Figs. 7-10).
DISCUSSION
On contact with blood, ozone dissolves in plasma and
instantly decomposes in a cascade of reactive oxygen
species (ROS), such as hydrogen peroxide (H
2
O
2
),
superoxide anion (O
2
•¯) and hydroxyl radical (OH•) (1).
The half-life of these species is too brief to monitor them.
During peroxidation of plasma lipids, late effectors known
as lipid oxidation products (LOPs) also form. ROS are
normally produced during cell respiration by mitochondria
and during bacterial phagocytosis by leucocytes. Animals
and humans defend themselves from continuous invasion
by pathogenic agents by production of hydrogen peroxide
and hypochlorite (1).
ROS have their own toxicity, however, and aerobic
organisms have evolved an antioxidant system of plasma
substances, such as uric acid, ascorbic acid, albumin,
reduce glutathione (GSH), vitamin E and bilirubin, and
intracellular enzymes, such as superoxide dismutase
(SOD), catalase (T), glutathione peroxidase (GSH-Px),
glutathione reductase (GSH R), glutathione transferase
(GSH T), maintained at optimal levels by enzymes and the
pentose cycle (via NADPH) (1, 14).
Most of the dose of ozone that comes into contact with
blood is partly reduced by water soluble antioxidants and
partly transformed into ROS and LOPs, which are
quenched by the antioxidant system of the body before
they can damage blood cells. A first pharmacological effect
of ozone is due to the slight excess of ROS acting as
chemical messengers for membrane receptors and
various biological functions (1), while LOPs act on
practically all cells after blood reinfusion.
On contact with blood, ozone therefore causes a
transient imbalance between oxidants and antioxidants, in
the form of an acute, exogenous oxidative stress. With
appropriate exposure times and ozone doses, the
oxidative stress may be calculated exactly so as to be
transient with respect to the endogenous toxicity of ROS
produced over a lifetime. This calculated imbalance
activates messengers that trigger biological effects,
without exceeding the capacity of the antioxidant system
(32). Ozone therefore acts like a drug with a precise
therapeutic window: it is not toxic if administered in the
therapeutic range, but may be ineffective due to total
quenching by antioxidants if the dose is too low (18, 34).
On the basis of these principles, ozone has been used
Di Paolo et al
1045
Fig. 6 - Results of laboratory tests: variables found to be significantly
different before and after treatment.
Fig. 7 - Lesions on a foot of a patient before and after EBOO.
in O
3
AHT for four decades with encouraging results,
despite scepticism and much concern about its toxicity.
However, the clinical applications and validation of O
3
AHT
have so far been largely insufficient. The recent
development of a new and more effective therapeutic
approach to ozone therapy, namely extracorporeal blood
oxygenation and ozonation (EBOO), first tested in vitro
and then in vivo in sheep and humans (more than 1200
treatments performed in 82 patients), enables treatment of
up to 4800 mL of heparinised blood per hour with a
mixture of oxygen and ozone (0.5-1 µg/mL oxygen) in
extracorporeal circulation without technical or clinical
problems. Only 250 mL of blood could be treated by
O
3
AHT. The EBOO technique is also easily adapted for
use in hemodialysis. The standard therapeutic cycle is 14
one-hour treatment sessions in 7 weeks. During a session
of EBOO, the interaction of ozone with blood components
causes a 4.5-fold increase in concentrations of
thiobarbituric acid reactants and a proportional decrease
in plasma protein thiols, without any appreciable
hemolysis of erythrocytes. On the basis of preliminary in
vitro and in vivo evidence, these simple laboratory
parameters could be a useful complement in routine
monitoring of biological compliance to treatment (34).
Considered an alternative therapy, O
3
AHT has been
increasingly used in the last few years and has been found
useful in various diseases:
- it activates the immune system in infectious diseases
(21, 25, 38-48);
- it improves oxygen utilization and stimulates release of
growth factors that reduce ischemia in vascular disease
(10-12, 49, 50);
- it activates the immune system and may improve the
quality of life in cancer patients (25, 49, 51, 52).
Despite increasing use of O
3
AHT, the technique has not
yet been fully accepted in scientific circles because its results
have been modest, presumably due to the fact that only small
quantities of blood could be treated (250 mL per treatment).
1046
Ozone therapy in peripheral artery disease
Fig. 8 - Complete healing of vast ulcerating area on the left leg of
another patient after EBOO.
Fig. 9 - Great improvement in severe lesions in a uremic patient with
PAD after EBOO.
Our group considered the theoretical basis of O
3
AHT to
be valid and over the last 13 years developed an oxygen-
ozone exchanger resistant to the corrosive effects of
ozone and capable of providing efficient gaseous
exchange in vitro and in vivo. Successful extracorporeal
ozonation of large quantities of blood in sheep has
demonstrated that the exchanger overcomes the limits of
O
3
AHT. In sheep, EBOO was found to be quite atoxic;
indeed, we were unable to establish an LD
50
, even using
doses one hundred times those used in humans (26-34).
The technique of EBOO is quite simple, especially for staff
trained in extracorporeal circulation.
Peripheral arterial disease is treated medically and
surgically, however few therapies for skin lesions,
especially extensive ones, have proven beneficial (53-57).
Intravenous prostacyclin therapy is known to have positive
effects on patients with PAD, though not all results have
been univocal (58-63). In our previous non controlled trial,
we observed very beneficial clinical effects of EBOO on
PAD, especially the skin lesions, even deep and extensive
ones (26, 29). We obtained this clinical indication from the
many articles published on the effects of O
3
AHT (12, 22,
24, 37, 40, 49).
The present controlled study confirmed the superiority
of EBOO with respect to i.v. prostacyclin therapy in the
treatment of skin lesions in patients with PAD. Prostacyclin
therapy improved pain but had little effect on lesion area,
whereas EBOO improved lesions in all patients, obtaining
complete regression in some. Many other positive findings
emerged from statistical analysis of our data. Claudication,
pain, pruritus, heavy legs, weakness and joint pain
improved significantly more with EBOO than with ivP. As
an index of general condition, impedance measurements
showed significant improvement in only one parameter
with respect to ivP. That parameter was extracellular water
or water retention (a feature of PAD is major edema of the
lower limbs). Of the laboratory parameters tested, only
plasma concentrations of triglycerides, alpha-globulin,
BUN, fibrinogen and VES (K index) showed significant
differences between groups. The decrease in triglycerides
was unexpected, because we had not previously found
any change in this parameter, whereas the reduction in
VES indicated a decrease in dysmetabolic and septic
phenomena. The reduction in BUN may be interpreted as
an improvement in protein anabolism. Good results were
also obtained in three dialysis patients, notoriously more
difficult to treat than other patients (64).
Prolonged ivP administration over a period of 28 days
did not improve objective or subjective measures of PAD
symptoms in this trial, demonstrating the difficulty of
treating stage IV PAD with current therapies (53-59).
It is difficult to interpret this clear improvement in PAD
patients treated with EBOO without any evident
concomitant change in peripheral artery circulation. The
following points, well demonstrated in vitro and in vivo (1),
may be considered, as all of them may help improve the
severe clinical manifestations of Leriche stage IV PAD
patients. Ozone:
- increases oxygenation of ischemic tissues;
- stimulates healing of ulcers;
- decreases blood viscosity;
- activates the immune system;
- acts as a powerful anaboliser;
- increases production of cell growth factors;
- stimulates leucocyte synthesis of many proteins
(interferons, interleukins, prostacyclins).
- acts as a powerful antimicrobic agent.
Di Paolo et al
1047
Fig. 10 - Clear improvement in ischemic lesions of upper limbs in
uremic diabetic patient after treatment with EBOO.
It is logical to suppose that these effects may combine
to produce the therapeutic effects of ozone observed in
PAD.
We failed to find any evidence of toxic effects of EBOO
on the body. On the other hand, ivP has known side-
effects which were the reason for three drop-outs in our
study. EBOO was perfectly well tolerated, despite the fact
that it involves extracorporeal circulation. In no case was it
necessary to interrupt treatment and no side-effects of
extracorporeal circulation or ozone administration were
found. With our experience in nephrology, this was
surprising, as extracorporeal circulation has many side-
effects in dialysis patients. The dialytic patients felt
perfectly well after EBOO, whereas after dialysis they
invariably complain of weakness and various malaises.
The present findings are in line with those of a recent
controlled study by Biedunkiewicz et al (24) who found
that O
3
AHT was effective in treating PAD. Our study also
has points in common with the work of Torre-Amione et al.
(16, 52) which, with a different method, showed an
immune modulating effect of ozone on coronary artery
disease, with significant improvement in clinical
symptoms.
The therapeutic limits of O
3
AHT have been surpassed
by EBOO which makes it possible to ozonate large
quantities of blood, obtaining clinical results relatively
quickly. EBOO patients were generally enthusiastic about
the results. All patients in this trial responded to EBOO
with significant improvement, as we had already found in
non controlled studies (26, 27, 29, 32).
To confirm these results, it is now necessary to extend
controlled research to a larger sample of PAD patients and
simultaneously test EBOO in other vascular diseases,
such as coronary artery disease.
ACKNOWLEDGEMENTS
We thank Dr. Iolanda Semplici, director of our university
hospital, for believing in our ozone project.
We are also grateful to our nurses Daniela Parenti and Monica
Borgogni for their willing collaboration.
We thank Dideco (Mirandola, Modena) and Ozonline
International (Medica, Bologna) for support and technical
assistance.
Address for correspondence:
Prof. Nicola Di Paolo, MD
Department of Nephrology and Dialysis
University Hospital of Siena
Viale Bracci, 1
53100 Siena, Italy
e-mail: n.dipaolo@ao-siena.toscana.it
1048
Ozone therapy in peripheral artery disease
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Ozone therapy in peripheral artery disease
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... Furthermore, in animals, the authors found that EBOO is "completely atoxic" over a wide time period and a wide dose range (20-60 μg/mL, with no clinical side effects during or after treatment. 20 In humans, EBOO clearly has clinical efficacy, which is superior to prostacyclin alone in the treatment of peripheral arterial disease 19 and necrotizing fasciitis. 21 These human studies used a very low concentration of ozone at 0.5-1.0 ...
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