The Ozone Paradox: Ozone Is a Strong Oxidant as Well as a Medical Drug

Abstract

After five decades characterized by empiricism and several pitfalls, some of the basic mechanisms of action of ozone in pulmonary toxicology and in medicine have been clarified. The present knowledge allows to understand the prolonged inhalation of ozone can be very deleterious first for the lungs and successively for the whole organism. On the other hand, a small ozone dose well calibrated against the potent antioxidant capacity of blood can trigger several useful biochemical mechanisms and reactivate the antioxidant system. In detail, firstly ex vivo and second during the infusion of ozonated blood into the donor, the ozone therapy approach involves blood cells and the endothelium, which by transferring the ozone messengers to billions of cells will generate a therapeutic effect. Thus, in spite of a common prejudice, single ozone doses can be therapeutically used in selected human diseases without any toxicity or side effects. Moreover, the versatility and amplitude of beneficial effect of ozone applications have become evident in orthopedics, cutaneous, and mucosal infections as well as in dentistry.

Full text

The Ozone Paradox: Ozone Is a Strong
Oxidant asWell as a Medical Drug
Velio Bocci,
1
Emma Borrelli,
2
Valter Travagli,
3
and Iacopo Zanardi
3
1
Department of Physiology, University of Siena, Siena, Italy
2
Department of Surgery and Bioengineering, University of Siena, Siena, Italy
3
Department of Pharmaceutical Chemistry and Technology, University of Siena, Siena, Italy
Published online 3 March 2009 in Wiley InterScience (www.interscience.wiley.com).
DOI 10.1002/med.20150
.
Abstract: After five decades characterized by empiricism and several pitfalls, some of the basic me-
chanisms of action of ozone in pulmonary toxicology and in medicine have been clarified. The present
knowledge allows to understand the prolonged inhalation of ozone can be very deleterious first for the
lungs and successively for the whole organism. On the other hand, a small ozone dose well calibrated
against the potent antioxidant capacity of blood can trigger several useful biochemical mechanisms and
reactivate the antioxidant system. In detail, firstly ex vivo and second during the infusion of ozonated
blood into the donor, the ozone therapy approach involves blood cells and the endothelium, which by
transferring the ozone messengers to billions of cells will generate a therapeutic effect. Thus, in spite of a
common prejudice, single ozone doses can be therapeutically used in selected human diseases without
any toxicity or side effects. Moreover, the versatility and amplitude of beneficial effect of ozone ap-
plications have become evident in orthopedics, cutaneous, and mucosal infections as well as in dentistry.
&2009 Wiley Periodicals, Inc. Med Res Rev, 29, No. 4, 646–682, 2009
Key words: oxidative stress; antioxidants; oxidative preconditioning; ozone; ozonated autohemotherapy
1. INTRODUCTION
A. A Brief Historical Review
Christian Friedrich Scho
¨nbein, in 1839, noticed the emergence of a pungent gas with an
‘‘electric smell.’’ According to the Greek language, he called it ‘‘ozone’’ and presented a
lecture entitled ‘‘On the smell at the positive electrode during electrolysis of water’’ at the
Basel Natural Science Society.
1,2
In nature ozone is continuously produced in the strato-
sphere (at 25–30 km from the Earth surface) by UV radiation (o183 nm) by splitting an
atmospheric oxygen molecules into two highly reactive oxygen atoms, in agreement with the
Chapman theory. By an endothermic reaction, each of these atoms combines to intact oxygen
to form the triatomic ozone.
Correspondenc e to: Velio Bocci,Department of Physiology, Universityof Siena,Via Moro 2,53100 Siena,Italy,E-mail: bocci@unisi.it
Medicinal Research Reviews,Vol. 29, No. 4, 646--682, 2009
&2009 Wiley Periodicals, Inc.

It is also produced during the electric discharge of lightning, which catalyzes the for-
mation of ozone from atmospheric oxygen. Ozone has a molecular weight of 48 and it is a
bluish gas with a pungent odor and a solubility in water, about ten-fold higher than oxygen
(49 mL in 100 mL, 0.02 M, at 01C), even though an ample variability is present in the lit-
erature.
3
While it rapidly dissolves in pure water and obeys Henry’s law, in biological water
ozone instantly reacts with inorganic and organic molecules dissolved in water generating a
variety of free radicals. Ozone as a gas spontaneously decomposes with a half-life of 40 min,
at 201C. This means that ozone is a metastable gas with a temperature-dependent half-life,
but it can be stored in liquid form at a temperature below 111.91C with a specific weight of
1.571 g/mL. Methods for generating ozone are based on UV radiation, corona discharge, and
an electrochemical process. Industrial ozone is produced from air but medical ozone must be
generated ex tempore only by using medical oxygen because otherwise the simultaneous
generation of nitric dioxide (NO
2
) will be very toxic.
4
The most recent medical ozone gen-
erator can control the electric voltage from 5 kV up to about 14 kV, the space between the
electrodes able to modulate a gradual increase in ozone concentration and the flow of pure
oxygen usually regulated between 1 and 10 L/min. The final ozone concentration is inversely
proportional to the oxygen flow, hence, per unit time, the higher the oxygen flow, the lower
the ozone concentration. In the final oxygen–ozone mixture, the maximum ozone con-
centration can be only 5%.
2. BEHAVIOR OF OZONE
A. Ozone as an Oxidant
Ozone has a cyclical structure assessed by the absorption at 253.7 nm with a distance among
oxygen atoms of 1.26 A
˚and exists in several mesomeric states in dynamic equilibrium
5
(Fig. 1). Among oxidant agents, it is the third strongest (E1512.076 V), after fluorine and
persulphate. Molecular oxygen, by containing two unpaired electrons, is a diradical but it has
not the reactivity of ozone and, by a stepwise reduction with four electrons, forms water. On
the other hand, ozone having a paired number of electrons in the external orbit is not a
radical molecule, but it is far more reactive than oxygen and generates some of the radical
oxygen species (ROS) produced by oxygen during mitochondrial respiration. Phagocytes
reacting with pathogens
6–8
produce anion superoxide (O
2), H
2
O
2
, and hypochlorous
acid (HClO) catalyzed by mieloperoxidase. Wentworth et al.
9,10
have postulated that in
atherosclerotic patients human endothelium cells may produce ozone, but their findings
remain still doubtful.
11
Moreover, H
2
O
2
is produced by almost all cells by the nicotinamide
adenine dinucleotide phosphate (NADPH)-oxydase isoenzymes, indicating the relevance of
ROS in the normal organism. Interestingly, ozone, in the presence of inorganic and/or
organic compounds immediately reacts and generates a great variety of oxidized molecules,
disappearing in a matter of seconds.
12
Figure 1. Stru cture and meso meric state s of ozone.
THE OZONE PARADOX K647
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B. Ozone as UV screen
In the stratospheric layer, ozone has an average concentration of 10 parts per million (ppm)
and it has the important role to absorb most of the UV radiations, particularly bands B (from
280 to 320 nm) and C (from 100 to 280 nm), which are mutagenic and can enhance skin
carcinogenesis.
13
Unfortunately, during the last decades, short-sighted human activities, by
releasing chlorofluorocarbons in the atmosphere, have led to a decreased ozone concentra-
tion, particularly in the Antarctic, which will take several decades to be restored.
C. Ozone as an Air Pollutant
On the other hand, the tropospheric amount of ozone ought to be about 1 mg/m
3
(0.001 ppm),
ten times lower than our odor perception threshold for ozone about 20 mg/m
3
(0.02 ppm).
However during the last decades, in large cities, ozone levels in summer time can increase up
to dangerous levels ranging from 200 to 900 mg/m
3
. Moreover, additional anthropogenic
emissions of NO, NO
2
, methane, CO, sulphuric compound, and fine particulates have
enhanced the toxicity not only for the respiratory tract but also for the eyes and the skin.
The US Clean Air Act has set an ozone level of 120 mg/m
3
as an 8 hr mean concentration to
protect the health of workers.
14
Evaluation of recent studies
15–18
allows establishing an average
environmental ozone concentration of 90710 mg/m
3
. However, ozone concentration in urban air
can exceed 0.8 ppm in high pollution conditions.
19,20
For 8 hr at rest (a tidal volume of about 10 L/
min and a retention of inspired ozone of no less than 80%), the ozone dose amounts to
0.70–0.77 mg daily. This is likely the minimal ozone intake because physical activity increases the
volume of inhaled air, and, at peak time, the ozone levels can easily augment to 500–900 mg/m
3
,
reducing pulmonary functions and markedly enhancing the risk of cardiovascular deaths.
15,17,18
Ozone levels of 500 mg/m
3
may not seem too high but one must consider that any single air
inhalation implies an ozone dose that immediately reacts with the airway surface fluid and
immediately at the epithelial lining fluid (ELF) generates the ROS and lipid oxidation products
(LOP) minimally quenched by the scarce antioxidant present in a liquid film of about 0.1 mm.
21
As a consequence, the whole respiratory tract against the continuous inhalation of ozone-
contaminated air opposes only the ELF’s volume of about 20–40 mL,
22
which is negligible when
compared to a plasma volume of about 2700 mL. Thus, throughout the day we must consider,
neither simply the ozone concentration nor a single respiratory act, but the ozone cumulative
dose that can easily sum up to 1–2 g ozone in 5 months. While ozone vanishes within the ELF,
23
the generated ROS, LOP, and nitrating species
24–28
damage the epithelial lining. The phos-
phorylation of a protein kinase, by activating the nuclear factor-kB(NF-kB), allows the
synthesis and release of a number of cytokines such as TNFa,IL-1,IL-8,IFNg,andTGFb1.
Moreover, this situation starts a vicious circle because the increased inflow of neutrophils and
activated macrophages into the alveolar space worsens and perpetuates the production of more
ROS including HClO,
8,26
tachykinins, proteases, alkenals, and F
2
-isoprostanes
25,29
able to self-
maintain a chronic inflammation. ROS have a very brief half-life and damage mostly the
pulmonary microenvironment while alkenals and proinflamatory cytokines are absorbed by the
human large expanse (about 70 m
2
) of the bronchial–alveolar space. Recent studies
25,30,31
have
detected 4-hydroxynonenal (4-HNE), isoprostanes, H
2
O
2
, and malondialdehyde (MDA) in the
bronchoalveolar lavage fluid. The interesting study by Last et al.
32
has clearly shown that mice
exposed to 1 ppm for 8 hr during three consecutive nights lose about 14% of their original body
weight, decrease their food consumption by 42%, and enter into a cachectic state. Another
important aspect of the pulmonary ozone toxicity is its reverberation on the whole organism,
especially on the vascular system, heart, liver, brain, and kidneys. The pharmaco-toxicological
behavior of both LOP compounds, ceramide signaling, and proinflammatory cytokines is
characterized by a continuous absorption from the pulmonary area into the blood and, even
648 KBOCCI ET AL.
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though the half-life of these compounds is brief,
28,33–37
the constant endogenous synthesis
insures a constant toxicity explaining the increased morbidity and mortality of population
inhaling polluted air for several months of the year.
D. Ozone as a Biological Cytotoxic Agent
Either normal or neoplastic cells in culture are very sensitive to a constant exposure of ozone
even if the gas has a very low concentration.
38–40
This observation is correct but it has led to the
misleading conclusion that ozone is always cytotoxic. Indeed, we know too well that cells culture
studies are mostly performed with air–CO
2
at pH 7.3 but with a pO
2
of 160 mmHg, i.e. more
than double of cells in vivo. Even more important is the fact that culture media have a sig-
nificantly lower level of antioxidants than plasma, particularly of albumin.
41–45
Indeed, the
usual fetal calf serum is added at a 5–10% concentration that is equivalent to hardly 50% of the
albumin present in the extracellular fluid. Among antioxidants, albumin with its available –SH
reducing group is one of the most protective compounds.
46
Moreover, antioxidant components
are not dinamically replenished in vitro while cells remain exposed to a constant ozone con-
centration. Obviously ozone dissolves in the fluid every second, exhausts the scarce antioxidants,
and generates toxic compounds that cannot undergo either dilution with extracellular fluid or
excretion. This unfavorable situation has been demonstrated when thiobarbituric acid reactive
substances (TBARS), incubated in vitro at 371C and pH 7.3 in human ozonated plasma remain
at a constant level for 9 hr.
47
On the other hand TBARS present in ozonated blood declined
very rapidly with a half-life of 4.271.7 min
48,49
after intravenous infusion in patients with age-
related macular degeneration (ARMD) demonstrating the relevance of critical pharmacological
properties to be extensively discussed in Section 4A. Moreover, the damaging effect of ozone on
saline washed erythrocytes, totally deprived of the plasma protection, has noticeably con-
tributed to consider ozone as a deleterious gas.
3. MAY OZONE BE USED AS A MEDICAL DRUG?
At first sight, the strong oxidizing properties of ozone discard the possibility that this gas may
display some therapeutic effects. However, even today some ozonetherapists advance the
whimsical idea that ozone, by decomposing in the blood, gifts the body its intrinsic energy
accumulated during its synthesis, as shown
3O2þ68;400 cal !2O3
On the 19th century, ozone had been already identified as a potent bactericidal gas and it was
used during World War I for treating German soldiers affected by gaseous gangrene due to
Clostridium anaerobic infections. In two pioneristic studies, Stoker
50,51
reported the first 21
medical cases successfully treated with ozone at the Queen Alexandria Military Hospital. It
remains uncertain how a Swiss dentist, E.A. Fisch (1899–1966)
52
had the first idea to
use ozone as either a gas or ozonated water in his practice. By a twist of fate, a surgeon,
Dr. E Payr (1871–1946) had to be treated for a gangrenous pulpite and remained astonished
by the result achieved with local ozone treatment. He enthusiastically extended its application
to general surgery and at the 59th Congress of the German Surgical Society (Berlin, 1935)
reported ‘‘which other disinfectant would be tolerated better than ozone? The positive results
in 75% of patients, the simplicity, the hygienic conditions and the safety of the method are
some of the many advantages’’.
53
In 1936, a French physician, Dr. P. Aubourg successfully
treated chronic colitis and rectal fistulae by the direct insufflation of oxygen–ozone mixture
into the rectum. It seems that Dr. Payr was the first to inject a small volume of the O
2
–O
3
gas
THE OZONE PARADOX K649
Medicinal Research Reviews DOI 10.1002/med

mixture directly into the human cubital vein, giving rise to a procedure that in the 90s,
adopted by charlatans, became so dangerous to be prohibited. After the invention of the first
medical ozone generator by the physicist Joachim Hansler (1908–1981), the physician Hans
Wolff (1927–1980) deserves the credit for having developed the ozonated autohemotherapy
(O
3
-AHT) by insufflating ex vivo the gas into the blood contained in a dispensable ozone-
resistant glass bottle. For almost three decades ozone therapy was used in Germany but the
lack of scientific and clinical studies arose scepticism and prejudice still common today.
Lacking the knowledge of the complexity of biological mechanisms, a distinguished chemist
wrote that ‘‘ozone is toxic, no matter how you deal with it and should not be used in
medicine’’ (personal communication to V.B.).
54
This negative concept may only be changed
by valid scientific and clinical data. It is worthwhile to mention what Timbrell
55
wrote in his
book ‘‘The poison paradox; chemicals as friends and foes.’’ The essential facts are that first it is
the dose that makes a chemical toxic, and second and more important, toxicity results from
the interaction between chemical and biological defenses. Indeed the subtlety and complexity
of biological systems may defy the concept that ozone is always toxic. Interestingly,
Paracelsus (1495–1541) did not know biochemistry but guessed that ‘‘all things are poison
and nothing is without poison, only the dose permits something not to be poisonous.’’
56
4. BIOLOGICAL MECHANISMS ELICITED BY OZONE IN HUMAN BLOOD
As it was mentioned, ozone as a gas equilibrates in 5 min in pure water and, in a closed glass
bottles, its concentration (about 25% of the ozone concentration in the gas mixture) remains
fairly stable for many hours. However, in a physiological environment, it immediately reacts
with antioxidants, polyunsaturated fatty acids (PUFA), proteins, carbohydrates and, if in
excess, with DNA and RNA.
57,58
Thus, ozone leads to the formation of ROS, LOP, and a
variable percentage of oxidized antioxidants.
59,60
A. Reactions with Plasma Components
Blood is an ideal tissue because it is composed of about 55% plasma and cells, especially
erythrocytes, able to cooperate for taming the oxidant properties of ozone. The plasma has a
wealth of hydrophilic reductants, such as ascorbic acid (50 mM), uric acid (400 mM), and a
little amount of reduced glutathione (GSH). These compounds have been measured before
and after ozonation.
61–63
Plasma contains albumin (45 mg/mL) that by virtue of a wealth of
–SH groups, is one of the most important antioxidants also because the plasma pool contains
about 112 g of albumin.
46
Moreover, the presence of proteins such as transferrin and cer-
uloplasmin quenches oxidizing reactions by chelating transition metals (mainly Fe
21
and
Cu
1
). Presence of traces of these metals must be avoided because either in the presence of
hydrogen peroxide, via the Fenton’s reaction, or in the presence of anion superoxide (O
2) via
the Haber–Weiss reaction, they will catalyze the formation of the most reactive hydroxyl
radical

OH.
Fe2þþH2O2ÐFe3þOH þOH
O
2þH2O2ÐOH þOHþO2
Although

OH has a half-life of 1 10
9
sec, it reacts with any other molecule and
produces another radical. Blood cells contain not only the bulk of GSH (1–5 mM) but also
thioredoxin and several lipophilic compounds such as a-tocopherol, retinol, lycopene,
ubiquinol, and a-lipoic acid, which are able to cooperatively reduce oxidized compounds,
thus restoring the initial antioxidant status. Moreover, blood cells contain a variety of en-
650 KBOCCI ET AL.
Medicinal Research Reviews DOI 10.1002/med

zymes (SOD, catalase, GSPase, GSH-redox system), which cooperate either simultaneously
or in a sequential way to restore the redox system. The work performed during the last 18
years in our lab has clarified the most important compounds generated ex vivo during the
initial reaction of ozone with some plasma components and how these compounds activate
some biochemical pathways in cells revealed by therapeutic effects after the transfusion of
ozonated blood in the donor.
The biochemical effects displayed by ozone when it comes in contact with blood com-
ponents will be briefly reviewed.
47,63
After having performed thousands of treatments, the
standard procedure is to add 200 mL of a gas mixture composed of medical oxygen (495%)
with ozone (o5%) to 180 mL of blood after the previous addition of 20 mL of 3.8% sodium
citrate at room temperature. The blood–gas volumes are gently mixed in a sterile glass bottle
by rotation, avoiding gas bubbling. Within 5 min, about 1.5 mL of O
2
and 2.4 mL of O
3
dissolve in the blood water but their fate is quite different. Oxygen physically diffuses into
erythrocytes and fully saturates hemoglobin (Hb
4
O
8
) but in spite of the pO
2
as high as
450 mmHg, the therapeutic value of oxygenation is irrelevant because the successive infusion
of oxygenated–ozonated blood (about 15 mL/min) hardly modify the pO
2
(40 mmHg) of
about 5 L/min of the simultaneous venous blood inflow to the heart. On the contrary, ozone
dissolves more readily in plasma water than oxygen, and instantaneously reacts with
hydrosoluble antioxidants and with readily available PUFA bound to albumin.
Several years ago, by using a reliable ozone generator able to deliver precise
ozone concentrations, the first aim was to define if indeed ozone was always deleterious
or if a range of ozone therapeutic concentrations could be determined. The range was
determined between 10 mg/mL gas (0.21 mmol/mL) and 80 mg/mL gas (1.68 mmol/mL)
per mL of anticoagulated blood, corresponding to total ozone doses comprises between
1 and 8 mg for 100 mL blood, respectively. It was crucial to precisely calibrate the ozone
dose (gas volume ozone concentration) against the individual variable antioxidant
capacity of the patient’s blood, thereby on one hand avoiding ozone toxicity and, on
the other hand, allowing the activation of several biochemical pathways on blood cells.
It was proven that during the slow mixing of the blood with the gas phase, all the ozone is
consumed in less than 5 min. Several studies
47,51,59,63–65
have clarified that some albumin
and uric acid behave as sacrificial molecules whereas several antioxidants after oxidation
are rapidly reduced by an efficient recycling system.
66,67
Some ozone reacts with PUFA as
follows
leading to the simultaneous formation of 1 mol of H
2
O
2
(included among ROS) and 2 mol of
LOP.
23,68,69
The fundamental ROS molecule is H
2
O
2
, which is not ionized but is an oxidant able to
act as an ozone messenger responsible for eliciting several biological and therapeutic
effects.
70–75
As it was mentioned, the old concept that H
2
O
2
is always harmful has been widely
revised because, in physiological amounts, it acts as a regulator of signal transduction and
represents a crucial mediator of host defense and immune responses.
74,76–80
While exposure
to oxygen is ineffective, ozone causes the generation of H
2
O
2
and of the chemiluminescent
reaction in both physiological saline and plasma.
47,81
However, while in saline there is a
consistent and prolonged increase in H
2
O
2
, in the ozonated plasma both chemiluminescence
and H
2
O
2
increase immediately but decay very rapidly with a half-life of less than 2 min
THE OZONE PARADOX K651
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suggesting that both antioxidants and traces of enzymes rapidly reduce H
2
O
2
to water.
47
In
ozonated blood the reduction of H
2
O
2
is so fast that it has been experimentally impossible to
measure it. H
2
O
2
is able to easily pass through the cell membrane, but the intracellular
concentration increases only 1/10 of the extracellular one.
72,74,78
Its relative stability allows
measuring it in plasma; in normotensive subjects its concentration is of 2.5 mM.
70,71
In this
case the intracellular concentration of H
2
O
2
will be at the most of 0.25 mM, while the
maximal intracellular concentration that can be generated for signaling purposes during the
ozonation process may reach 0.5–0.7 mM.
47
It appears ubiquitous as it has been detected in
urine and in exhaled air.
71
Depending upon its local concentration and cell-type, H
2
O
2
can
either induce proliferation or cell death.
78,80,82,83
It can regulate vascular tone by causing
constrictions of vascular beds or vasodilatation although it remains uncertain if it acts as an
endothelium-derived hyperpolarizing factor.
84
A very enlightening finding was achieved by evaluating the variation of the total anti-
oxidant status (TAS) as measured by the Rice-Evans and Miller’s method
85
in plasma after
ozonation and 1 min rapid mixing of the liquid–gas phases of either fresh blood or the
respective plasma withdrawn from the same ten donor.
Figure 2 shows that, after ozonation of plasma with either a medium or a high ozone
concentration (0.84 mmol/mL or 1.68 mmol/mL of gas per mL of plasma, respectively), TAS
Figure 2. KineticsofTASlevelsinplasma(top)andinblood(bottom)samplesfromdonors (n510 ; m e an 1SD; unpublished
results). Plasma and blood samples were exposed for 1min either O
2
(control, ) or O
2
--O
3
with ozone concentrations of 4 0 ( &)
and 80 (K)mg/mL.
652 KBOCCI ET AL.
Medicinal Research Reviews DOI 10.1002/med

level progressively decreases at first and then remain stable after 20 min.
47
The decrease was
ozone-dose dependent and varied between 46 and 63%, respectively. Conversely, TAS levels
in blood treated with the same ozone concentrations only decreased from 11 to 33%,
respectively, in the first minute after ozonation. Then they recovered and returned to the
original value within 20 min, irrespective of the two ozone concentrations, indicating the
great capacity of blood to regenerate oxidized antioxidants, namely, dehydroascorbate and
GSH disulfide (GSSG). Indeed, Mendiratta et al.
66,67
have found that dehydroascorbate can
be recycled back to ascorbic acid within 3 min. Similarly, only about 20% of the in-
traerythrocytic GSH has been found oxidized to GSSG within 1 min after ozonation, but
promptly reduced to normal after 20 min.
86
These data were enlightening and showed that
the therapeutic ozonation modifies only temporarily and reversibly the cellular redox
homeostasis. There is now full agreement that ascorbic acid, a-tocopherol, GSH, and lipoic
acid, after oxidation, undergo an orderly reduction by a well-coordinated sequence of elec-
tron donations.
87
LOP production follows peroxidation of PUFA present in the plasma: they are
heterogeneous and can be classified as lipoperoxides (LOO), alkoxyl radicals (LO),
lipohydroperoxides (LOOH), F
2
-isoprostanes, and alkenals, among which 4-hydroxynonenal
(4-HNE), acrolein and MDA. As free radicals and aldehydes are intrinsically deleterious,
only precise and appropriate ozone doses must be used in order to generate them in
very low concentrations. Figure 3 comparatively shows the modifications of plasma
levels of TBARS, hemolysis, TAS, and protein thiols in a typical experiment when 13 human
blood samples were exposed to air, O
2
, or either 40 or 80 mg/mL ozone concentrations.
Plasma TBARS in vitro are far more stable than ROS,
47
but, upon blood reinfusion,
they have a brief half-life owing to a marked dilution in body fluids, excretion (via urine and
bile), metabolism by glutathione-S-transferases (GST) and aldehyde dehydrogenase
(ALDH).
Among the aldehydes, 4-HNE is quantitatively the most important. It is an amphipathic
molecule and reacts with a variety of compounds such as albumin, enzymes, GSH, carnosine,
and phospholipids.
88,89
There is no receptor for 4-HNE but Poli et al.
89
have reported that,
after binding to more than 70 biochemical targets, it exerts some deleterious activity. Luckily,
intracellular concentrations of GSH are high enough to frequently prevent or remove 4-HNE
from adducts with enzymes. Owing to the unexpected stability of 4-HNE when samples of
ozonated human plasma were incubated at 371C for 9 hr, it was postulated that ozone, for its
high solubility in the plasmatic water, steric reasons, and the abundance of albumin mole-
cules prefers to target their bound PUFA. The scheme presented in Figure 4 envisages the
events occurring in the plasma phase. It appears reasonable that during the rapid reaction of
ozone with albumin PUFA in water, the suddenly generated aldehydes, mainly 4-HNE, will
immediately form adducts with contiguous albumin molecules. This hypothesis is now well
supported by recent findings,
90–92
which have shown that human albumin, rich in accessible
nucleophilic residues, can quench up to 11 different 4-HNE molecules, the first being with
Cys34, followed by Lys199 and His146. These important data clarify why ex vivo ozonation
of blood does not harm the vascular system during the infusion of ozonated blood. The
albumin-4-HNE adducts, not only are rapidly diluted in the blood pool but, being trans-
ferred into the extravascular pool, represent only a small aliquot of the whole albumin pool,
containing as much as about 310 g protein. On this basis, it would be worthwhile exploring
whether either the 4-HNE-modified albumin has an abnormal fate or how the aldehyde is
released into other cell compartments, thus becoming able to trigger biochemical mechan-
isms. 4-HNE is the major product of peroxidation of n-6-PUFA, its concentration in normal
plasma varies from 0.07 to 0.15 mM and increases with aging.
93,94
Needless to say that a
constant increase in peroxidation as it happens after ischemia-reperfusion, CCl
4
intoxication,
THE OZONE PARADOX K653
Medicinal Research Reviews DOI 10.1002/med

Figure 3. Thirteen human blood samples were exposed to air (control), or O
2
,orO
2
--O
3
with ozone concentrations of 40 and
80 mg/m L for 1min. WhileTBARS, TAS, and PTG leve ls vary significant ly (po0.01) after ozone exposure, there is a negligible increase
in hemolysis. (Bocci V. How does ozone act? Oxygen--ozone therapy. A critical evaluati on, chap.13. Figure 40. Kluwer Academic
Publishers; 200 2. p 114.With kind permission from Springer Science1Business Media, formerly Kluwer Academic Publishers.)
654 KBOCCI ET AL.
Medicinal Research Reviews DOI 10.1002/med

ADP-iron overload, and chronic inflammation typical of some infections disease, diabetes,
atherosclerosis, cancer, and degenerative pathologies causes a marked increase in 4-HNE
levels, especially in the affected tissues. However, aerobic organisms, for accommodating the
toxicity of aldehydic compounds, have simultaneously developed detoxifying systems
37,95–99
and their evaluation is relevant because the infusion of the ozonated blood into the donor
patient implies an amount of an albumin-4-HNE adduct.
The following three processes schematically indicated in Figure 5 clarifies why 4-HNE is
not a risk:
(1) Dilution: The highest concentration of 4-HNE measured after exposing 180 mL of
human blood to the highest ozone amount (16 mg) is less than 1 mM in the plasma.
During the 20 min intravenous infusion, the aldehyde will be promptly diluted in a
total plasma-extracellular fluid volume of about 11 L, causing a transitory increase in
the plasma level up to about 0.1 mM.
(2) Detoxification: Metabolism of 4-HNE is extremely fast either because small amounts
of aldehydes interact with billions of cells endowed with several detoxifying enzymes
such as ALDH, aldose reductase, and GST or the formation of an adduct with
GSH.
36,37,98–100
Several authors
96,101,102
have determined a metabolic rate so high to
conclude that ‘‘even with very high lipid peroxidation rates, 4-HNE cannot accumulate
in an unlimited way’’.
89
These data are in agreement with our results in six patients
when we could assess a half-life of infused TBARS of 4.271.7 min.
48,49
On the
contrary when the same preparation in ozonated plasma was incubated (at 1371C, pH
7.3) in acellular medium, TBARS levels hardly declined during the next 9 hr.
47
Figure 4. The scheme helps to imagine the multiplicity of sub strate reacting with ozone dis solved in plasmatic water. Small
circles, triangles, and squares symboly ze hydrosoluble antioxidants present in 100 mL of human blood (uric acid 4.5 mg/dL,
ascorbic acid1.5mg/dL, glucose 80 mg/dL, etcy). Large albumin molecules (4,000 mg/dL) exposing --SH groups form a cloud
over the ce ll membrane and protect it. Mole cules such as transferrin and ceruloplasmin bind Fe
31
and Cu
1
and prevent form ation
of OH

. The exogenous addition of 4--8 mg of ozone to100 mL of blood is transitory and controlled by antioxidants. In contrast, the
endogenous production of ROS is continuous and barely quenched by intracellular antioxidants.
THE OZONE PARADOX K655
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(3) Excretion: Partially metabolized LOP are eliminated into both bile after hepatic
detoxification and urine after renal excretion. In the rat, 4-HNE was detected in the
urine as mercapturic acid conjugates.
35,98,103,104
In normal conditions, owing to the efficiency of these processes, only submicromolar
concentrations of LOP can reach organs such as bone marrow, endocrine glands, and even
hypothalamic areas deprived of the blood–brain barrier where, via a variety of kinases and
even a possible receptor for F
2
-isoprostanes, may act as a signaling event of an ongoing acute
oxidative stress
105–110
(Fig. 5). As a first conclusion it is clear that the ozonation process
either happening in blood ex vivo or in an intramuscular site represents an acute, albeit small,
oxidative stress. However, this process is acceptable only if the ozone is precisely calibrated
against the antioxidant capacity of either blood or the injected tissue. Moreover, the ozone
dose must never lower the antioxidant capacity more than 30% with a process lasting only a
few minutes during which ozone reacts and disappears after leaving its messengers. Thus, the
process of blood ozonation ex vivo has been characterized by the formation of ROS and LOP
mainly acting in two phases. Among ROS, H
2
O
2
is the earliest messenger rising and
disappearing within 1 min in the plasma, while LOP during drug infusion in the donor reach
the vascular systems, act on endothelial cells, and eventually reach parenchymal cells. Their
pharmacodynamics minimize their potential toxicity thus making LOP as late and effective
messengers.
B. The Effect of Ozone Messengers Onto Blood Cells
There are two questions to be clarified: first, does ozone directly activate the cells? Our
methodological approach and experimental results exclude this possibility because when
blood is gently mixed ex vivo with O
2
–O
3
, ozone dissolves rapidly in the water of plasma and
there it immediately reacts with antioxidants and PUFA. Blood cell membrane phospholipids
surrounded by a cloud of albumin molecules do not come in contact with ozone molecules
because the calculated ozone dose is rapidly exausted (Fig. 4). This dangerous interference
has been excluded by either a negligible hemolysis, or a change of the hematocrit value, or
leakage of K
1
and lactate dehydrogenase, or a change of osmotic fragility, or of electro-
phoretic mobility, or increased methemoglobin.
47,54,65,111,112
Levels (mg/dL) of fibrinogen,
cholesterol, triglycerids, HDL, and LDL in plasma are not modified even using the excessive
Figure 5. The multivariate biological response of the organism to ozonated blood can be envisaged by considering that
ozonated blood cells and the g enerated LOP interact with a number of organs. Some of these represent real targets (liver in chronic
hepatitis, vascular system for vasculopathies), while other organs are probably involved in restoring normal homeostasis. Gastro-
intestinal tract (GIT); mucosal associated lymphoid tissue (MALT).
656 KBOCCI ET AL.
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ozone concentration of 160 mg/mL per mL of blood.
112
Equally important is the stability of
enzymes such as SOD, GSH-Pase, GSH-RD, and G6PDH in the erythrocytes.
112
Moreover,
Shinriki et al.
65
after isolating the erythrocytic membranes after blood ozonation within the
therapeutic range did neither detect a decrease in aa-tocopherol nor an increase in MDA.
It is unfortunate that in the past other authors
57,68,113–117
have reported that erythrocytes
isolated from plasma, after three washings with saline and suspension in protein-free saline,
undergo structural changes and intense hemolysis when exposed to ozone. These misleading
and unphysiological data have greatly contributed to emphasize the ozone cytotoxicity,
which obviously was enhanced by removing plasma antioxidants.
116
Moreover, the critical
protective effect of plasma antioxidants has been emphasized in two recent studies.
118,119
These results were particularly evident on saline-washed blood mononuclear cells (BMC)
with a marked decrease in mitochondrial functions.
118
Our thinking is well supported by
other data
47,120,121
as well as recent results (Fig. 6) obtained after excessive ozonation of
samples of normal human blood either collected in heparin or in sodium citrate. Interest-
ingly, heparinized samples were far more susceptible to ozone most likely because of the
remaining physiological Ca
21
level: in fact, a further addition of 2.5–5 mM Ca
21
enhanced
the hemolysis up to 40%.
Second, how ozone messengers activate blood cells? Initially, the sudden formation of an
H
2
O
2
gradient between the ozonated plasma and the intracellular fluid causes the rapid
passage of about 10% H
2
O
2
into the blood cell cytoplasms and represents the triggering
stimulus: depending upon the cell type, different biochemical pathways can be concurrently
activated in erythrocytes, leukocytes, and platelets resulting in numerous biological effects.
The rapid reduction of H
2
O
2
to water is operated by the high concentration of intracellular
GSH, CAT, and GSPase but, nonetheless, H
2
O
2
must be above the threshold concentration
for activating several biochemical pathways as follows.
The mass of erythrocytes mops up the bulk of H
2
O
2
: GSH is promptly oxidized to GSSG
and the cell, extremely sensitive to the reduction of the GSH/GSSG ratio, immediately
corrects the unbalance by either extruding GSSG, or reducing it with GSH-Rd at the
expenses of ascorbate or of the reduced NADPH, which serves as a crucial electron donor.
Next, the oxidized NADP is promptly reduced after the activation of the pentose phosphate
Figure 6. Kinetics of hemolysis in relation to ozone concentration (mg/mL per mL of blood). Blood of five donors was treated
with CPD () or with 30 U/mL heparin (K)(mean1SD). (Bocci V. What happens in the intracellular environment after blood ozo-
nation? Oxygen --ozone therapy. A critical evaluation, chap. 14. Figure 43. Kluwer Academic Publishers; 2 00 2. p 123. With kind
permission from Springer Science1Business Media, formerly Kluwer Academic Publishers).
THE OZONE PARADOX K657
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pathway, of which glucose-6-phosphate dehydrogenase (G6PDH) is the key enzyme. In
patients with ARMD, after 13 O
3
-AHT, a small increase in ATP formation has been de-
termined but whether this is due to the activation of the pentose cycle or to an increase in
phosphofructokinase activity or to both remains to be clarified. The reinfused erythrocytes,
for a brief period, enhance the delivery of oxygen into ischemic tissues because of a shift to
the right of the oxygen–hemoglobin dissociation curve, due either to a slight decrease in
intracellular pH (Bohr effect) or/and an increase in 2,3-diphosphoglycerate (2,3-DPG) levels
as shown in Figure 7 (unpublished data). Obviously, an increase in this metabolite has a great
significance because it enhances a shift to the right of the oxygenated hemoglobin, hence an
increase oxygen delivery to hypoxic tissues. However, Figure 7 shows that the increase has
been noted only in three patients where the initial levels were rather low. Thus, this
observation needs to be explored in a large number of patients and it will be also necessary to
clarify the activation of 2,3-bisphosphoglycerate mutase. Needless to say that one auto-
hemotherapeutic treatment has a minimal effect and we need to ozonate at least 3–4 L of
blood within a period of 30–60 days.
In another small group of five ARMD’s patients after 15–17 O
3
-AHT, an increase in
some antioxidant enzymes has been determined (Fig. 8). This result has been reported also by
other authors
122,123
and it is likely that LOP act as repeated stimuli on the endothelium and
bone marrow and cause the adaptation to the ozone stress during erythrogenesis. Whether
the enzymatic levels remain sustained for several months during the maintenance therapy
need to be evaluated.
Another relevant finding was that in four patients with ARMD, after a cycle of 13 O
3
-AHT
treatments (in which ca. 3.8 L of blood were ozonated within 7 weeks), isopycnic centrifugation of
blood separated old (heavy) and young (light) erythrocytes (RBC), which showed a marked
increase in G6PDH in the young erythrocytic fraction generated during the course of ozone
therapy (Table I). Whether the enzymatic levels remain sustained with time need to be evaluated.
G6PDH activity, expressed as nmol/hr/mg hemoglobin, in total red blood cells was either 357791
or 406740, before and after the ozone therapy, respectively. While the enzymatic increase in the
Figure 7. 2,3-DPG level variations infour patientsp erformed before treatment (Basal), after 6--7 treatments (Intermediate), and
at the end of treatments (Final). Insert shows the statistical dispersion (mean 7SD) of the data (unpublished results).
658 KBOCCI ET AL.
Medicinal Research Reviews DOI 10.1002/med

whole erythrocyte population was understandably small, it was found markedly enhanced from
5507157 to 7487182 in very young (light) erythrocytes before and after ozone therapy, respec-
tively. In the so-called old erythrocytes, which practically include the bulk of cells (20–120 days
old), G6PDH obviously increased only from 3107127 up to 435787 nmol/hr/mgHb. It is ne-
cessary to mention that the percentage of either young or old erythrocytes remained practically
constant throughout the treatments (unpublished data). As a consequence, a patient with chronic
limb ischemia (Phase II) undergoing ozone therapy shows a clinical improvement due to the
formation of successive cohorts of erythrocytes progressively more capable of delivering oxygen to
his ischemic tissues.
Although ozone is one of the most potent disinfectants, it has been shown
124,125
that
ozone cannot inactivate bacteria, viruses, and fungi in vivo because, paradoxically, the pa-
thogens are well protected, particularly inside the cells, by the powerful antioxidant system.
Thus, the favorable effect of ozone therapy in some infectious diseases has been interpreted
as due to ozone acting as a mild enhancer of the immune system, by activating neutrophils
and stimulating the synthesis of some cytokines.
64,76,77,79,86,126,127
Once again the crucial
messenger is H
2
O
2
that after entering into the cytoplasm of BMC, by oxidizing selected
cysteines, activates a tyrosine kinase, able to phosphorylate the transcription factor NF-kB.
The release of an heterodimer, via effector genes, causes the synthesis of several proteins,
among which, the acute-phase reactants, adhesion molecules, and numerous pro-in-
flammatory cytokines. This process, checked by a phosphatase or inhibited by cytoplasmic
antioxidants, is very transitory. The release of several cytokines from ozonated blood upon in
Figure 8. Increase in antioxidant enzymes in ARMD patients after 2 0--24 O
3
-AHTs performed during7--8 weeks (n510, mea n
1SD; unpublished resul ts).
Table I. Evaluation of G6PDH Activity in Total, Young and Old Red Blood Cells (RBC) in Blood
Samples from Four Patients With Age-Related Macular Degeneration Before and After an Ozone
Therapy Cycle of 13 Treatments (Unpublished Results)
G6PDH activity
a
Total RBC Young RBC Old RBC
Before treatment (n54) 356.8790.7 550.37157.5 310.77127.3
After treatment (n54) 406.2740.4 784.27181.9 438.8786.7
a
G6PDH activity expressed as nmol/hr/mg hemoglobin in whole ery throcyte population and in young and old
fractions b efore an d after 13 O
2
/O
3
treatments. Results represent mean value7SD.
THE OZONE PARADOX K659
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vitro incubation has been measured since 1990.
128
Once the ozonated leukocytes return into
the circulation, they home in lymphoid microenvironments and successively release cytokines
acting in a paracrine fashion on neighboring cells with a possible reactivation of a depressed
immune system. This process, described as the physiological cytokine response,
129
is a part of
the innate immune system and helps us to survive in a hostile environment. One of our most
interesting result has consisted in observing the variable individual production of IL-8 by
blood donors in 13 blood ozonated samples.
130
Figure 9 shows that the different release of
IL-8 by medium and high ozone concentrations indicates the presence of high, medium, and
no responders. The result was interpreted as due to both genetic factors and variable levels of
plasma antioxidants.
During ozonation of blood, particularly if it is anticoagulated with heparin, an ozone-
dose-dependent increase in activation of platelets has been noted
131,132
with a consequent
release of typical growth factors, which will enhance the healing of chronic ulcers in ischemic
patients (Fig. 10). Whenever possible, albeit with caution, the use of heparin as an antic-
oagulant is preferable to sodium citrate because, by not chelating plasmatic Ca
21
, reinforces
biochemical and electric events.
Finally, during the reinfusion of the ozonated blood into the donor, the vast expanse of
the endothelial cells is activated by albumin-LOP resulting in an increased production of NO,
plasma S-nitrosothiols, and S-nitrosohemoglobin.
133–136
Figure 11 shows the in vitro pro-
duction of nitrite by human vascular endothelial cells after addition of human ozonated
serum. Production of NO was markedly enhanced by the addition of L-arginine (20 mM)
and was potentiated by O
3
, while it was inhibited in the presence of the NO inhibitor N-o-
nitro-L-arginine-methyl ester (L-NAME). While NO has a half-life of less than 1 sec, protein-
Figure 9. Effe ct of 1 min ex pos ure of e ith er O
2
or O
3
(40 an d 80 mg/mL) on the production of IL- 8 after 8 hr incubat ion of 13
blood samples. Average values are reported in the lower panel after subtraction of control values.

Significant difference (po0.01)
compared with samples treated with O
2
. The variable production of IL-8 among donors is notewor thy, particularly the lack of
production of donors no. 3 and 12 likel y due to a highTAS level. (Bocci V. What happens in the intracellular environment after blood
ozonation? Oxygen--ozone therapy. A critical evaluation, chap.14. Figure 53. Kluwer Academic Publishers; 2002. p 134. With kind
permission from Springer Science1Business Media, formerly Kluwer Academic Publishers).
660 KBOCCI ET AL.
Medicinal Research Reviews DOI 10.1002/med

Figure 10. Releas e of factors from human pl atelets dur ing 1, 2, and 4 hr in cubation. The sa me PRP sampl es c ollecte d e ither in
heparin or ACD were not expose d (control), or exposed to O
2
alone or to O
2
--O
3
at concentrations of 20, 4 0, and 8 0 mg/mL for
30 se cbefore incubation. Statistical significance is indicated by (

) for inte rgroup analy sis and ( 1) for intragroup analysis. (Bo cci V.
What happens in the intracellu lar environment after blood ozonation? Ox ygen--ozone therapy. A critical evaluation,chap.14.Figure
65. Kluwer Academic Publishers ; 20 02. p 158. With kind permission from Springer Science1Business Media, formerly Kluwer
Academic Publishers).
Figure 11. Production of nitrite by HUVECs, measured after 24 hr incubation, after addition of normal human serum either
oxygenated or ozonated (at 4 0 and 80 mg /mL). Effects of addition of L-arginine and L-NAME. The data are presented as the mean1
SD of six different experiments. (Bocci V. What happens in the intracellular environment after blood ozonation? Oxygen--ozone
therapy. A critical evaluati on, chap. 14. Figure 68. Kluwer Academic Publishers; 2002. p 165. With kind permission from Springer
Science1Business Media, formerly Kluwer Academic Publishers).
THE OZONE PARADOX K661
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bound NO can exert vasodilatation also at distant ischemic vascular sites with relevant
therapeutic effect. There is little doubt that the therapeutic advantage observed in many
patients with peripheral obstructive arterial disease (POAD) is due to multiple factors such as
an increased release of oxygen due to vasodilation by trace amounts of NO and CO, and an
increased availability of growth factors from platelets.
All of these data emphasize that submicromolar LOP levels can be stimulatory and ben-
eficial,
137
while it is well established that micromolar levels can be toxic.
89
This conclusion
reinforces the concept that optimal ozone concentrations are critical for achieving a therapeutic
result: too low concentrations are practically useless (at best elicit a placebo effect), too high
may elicit a negative effect (malaise, fatigue), so that they must be just above the threshold level
to yield an acute, absolutely transitory oxidative stress capable of triggering biological effects
without toxicity. There is no doubt that the process of blood ozonation must be precisely
controlled with a calculated ozone dosage: at this condition it is not deleterious and actually
capable of eliciting a multitude of useful biological responses and, possibly, reversing a chronic
oxidative stress due to ageing, chronic infections, and the several diseases grouped within the
metabolic syndrome. Indeed the ozonotherapeutic act has been interpreted as a safe ‘‘ther-
apeutic shock’’ able to restore homeostasis.
138
These aspects are critical and imply two draw-
backs: first, if the ozone generator is not well calibrated or periodically checked, it may release
erroneous and dangerous ozone amounts and, second, if the ozonetherapist does not fully
understand the ozonation process, he may do some mistakes and jeopardize the approach.
Other aspects regarding the future of ozone therapy will be evaluated in Section 9.
5. IS OZONE ABLE TO INDUCE AN ADAPTATION TO CHRONIC OXIDATIVE
STRESS?
That ozone, one of the most potent oxidizer, may induce an antioxidant response capable of
reversing a chronic oxidative stress at first sight seems a paradoxical concept. However, this
concept has become common in the animal and vegetal kingdoms.
147–150
Any change of the
external or internal environment disturbs cell homeostasis, but if the stress is tolerable, or
carefully calibrated in intensity, the cell or the organism can adapt to it and survive. If it is
excessive or the cell is already damaged, the cell programmes its own death. Stresses include
hyperthermia, hyperoxia, ischemia, hypoglycemia, pH modifications, radiation, very likely
mental and hormonal derangement, and chronic infections, which imply an excessive ROS
and LOP production. Obviously, ozone has to be included and the phenomenon of ozone
tolerance is now well known. The concept of ‘‘ischemic preconditioning’’ for the heart, which
after undergoing a brief, nonlethal period of ischemia can become resistant to infarction from
a subsequent ischemic insult was pioneered by Murry et al.
151
‘‘Oxidative preconditioning’’
has been also well demonstrated.
152–157
Therefore, it is of interest that small amounts of ROS
and LOP can elicit the upregulation of antioxidant enzymes on the basis of the phenomenon
described under the term of ‘‘hormesis.’’
158–162
On the basis of this phenomenon that says
‘‘the exposure of an organism to a low level of an agent, harmful at high levels, induces an
adaptive and beneficial response,’’
159,160,163
it has been postulated that LOP, by acting as
long-distance messengers, can transmit to all organs the information of an acute oxidative
stress.
54
The bone marrow is particularly relevant because it can upregulate antioxidant
enzymes during erythrogenesis and may allow the release of staminal cells for possibly
regenerating infarcted organs.
The oxidative preconditioning or, as we prefer, the adaptation to the chronic oxidative
stress has been now demonstrated experimentally.
40,45,48
The increased synthesis of enzymes
such as SOD, GSPase, GSH-Rd, and CAT has been repeatedly determined in experimental
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animals and in patients (reviewed in 57). Iles and Liu
164
have demonstrated the 4-HNE, by
inducing the expression of g-glutamate cysteine ligase, causes an intracellular increase in
GSH, which plays a key role in antioxidant defence. Furthermore LOP induce oxidative
stress proteins, one of which is heme-oxygenase I (HO-1 or HSP-32) that, after breaking
down the heme molecule, delivers very useful compounds such as CO and bilirubin.
165–171
Bilirubin is a significant lipophilic antioxidant and a trace of CO cooperates with
NO in regulating vasodilation by activating cyclic GMP. Fe
21
is promptly chelated by the
upregulated synthesis of ferritin.
172
The induction of HO-1 after an oxidative stress
has been described in thousands of papers as one of the most important antioxidant
defence and protective enzyme. Both mild ozone inhalation and ozonated plasma
induce HSP-70.
170,173
When ozone is judiciously used in small doses, can become a
useful drug able to correct an otherwise irreversible state of oxidative stress. There are
serious pathologies such as chronic infections, neurodegenerative, and autoimmune
diseases in which a vicious imbalance between overproduced oxidants and depleted
antioxidant defenses become established and lead to death. How modern medicine correct
this imbalance? Several therapeutic approaches among which administration of antioxidants
with addition of N-acetylcysteine have been often reported
174–176
but they are only partly
successful.
The ozone treatment is now envisaged as a transitory and miniaturized oxidative stress
resulting in a sort of therapeutic ‘‘shock’’ for the ailing organism. Ozone acting as a prodrug,
realizes this shock because generates a number of messengers able to reach all cells in the
organism (Fig. 5).
Submicromolar levels of LOP act as key mediators and in still responsive cells may
activate a sequence of biochemical mechanisms able to reactivate gene expression leading to a
renewed synthesis of HSP and antioxidant enzymes. If the disease has gone too far, cells
become anergic and are unable to respond to the treatment. Indeed, we have observed that
after intensive chemotherapy, preterminal cancer patients do not improve with ozone ther-
apy. That is also the reason why we always start using low ozone concentrations just above
the threshold level to better achieve the ozone tolerance and in-line with the old concept
‘‘start low, go slow.’’ Moreover, the stimulation of the endocrine and central nervous systems
may help to understand why most of the reactive patients during prolonged ozone therapy
report a feeling of euphoria and wellness probably due to an improved metabolism as well as
to an enhanced hormonal or neurotransmitters release.
6. WHICH ARE THE ROUTES OF OZONE ADMINISTRATION?
Table II shows that ozone can be administered with great flexibility but it should never be
injected intravenously as a gas because of the risk of provoking oxygen embolism, given the
fact that the gas mixture contains always no less than 95% oxygen. So far the most advanced
and reliable approach has been the O
3
-AHT because, on the basis of the patient’s body
weight, a predetermined volume of blood (200–250 mL) to which has been added either
sodium citrate 3.8% (119mL blood) or heparin (20 IU/mL of blood) can be exposed to an
equal volume of gas (O
2
–O
3
) in a stoichiometric fashion, with the ozone concentration pre-
cisely determined by using an ozone-resistant, disposable 500 mL glass bottle under vacuum.
This simple, inexpensive (all the necessary disposable material costs about 12 US$)
procedure has already yielded therapeutic results in vascular diseases superior to those
achieved by conventional medicine (discussed in Section 7A). Moreover, the therapeutic
modalities, until now restricted to major AHT and to the empirical and imprecise rectal
insufflation of gas,
139,177,178
have been extended: they include the quasi-total body exposure
THE OZONE PARADOX K663
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to O
2
–O
3140,179
and the extracorporeal blood circulation against O
2
–O
3
.
141
The latter
procedure is rather invasive because blood collected from a vein circulates through an
ozone-resistant gas exchanger
180,181
and, with the help of a peristaltic pump, returns to the
circulation via a contralateral vein. On the other hand, the partial cutaneous exposure to
oxygen–ozone does not need any venous puncture and, owing to the vast expanse of the skin,
allows a generalized and beneficial effect. Clearly, today we can select the most suitable
method for different pathologies, their stage, and the patient’s condition. A discussion on its
own is needed for the minor AHT, which basically consists of withdrawing 5 mL of blood to
be immediately and vigorously mixed for 1 min with an equal volume of O
2
–O
3
at an ozone
concentration ranging between 80 and 100 mg/mL of gas per mL of blood already extensively
described.
142
The slightly oxidized blood, including the foam, is promptly injected into the
gluteus muscle without the need of any anesthetic. As an unspecific immunomodulatory
approach, it has been widely used during the last two decades for successfully treating
herpetic infections.
143
The slight hemolysis (2%) is purposefully required because the heme released in the
gluteal muscle will stimulate the synthesis of HO-1.
165,171
7. WHICH DISEASES ARE SUITABLY TREATED WITH OZONE THERAPY
On the basis of the mechanisms of action, ozone therapy can induce the following biological
responses: (a) it improves blood circulation and oxygen delivery to ischemic tissue owing to
the concerted effect of NO and CO and an increase in intraerythrocytic 2,3-DPG level; (b) by
improving oxygen delivery, it enhances the general metabolism; (c) it upregulates the cellular
antioxidant enzymes and induces HO-1 and HSP-70; (d) it induces a mild activation of the
immune system and enhances the release of growth factors; (e) it has an excellent disinfectant
activity when topically used, while this is negligible in the circulation owing to blood anti-
oxidant capacity; (f) it does not procure acute or late side effects;
182
(g) it procures a sur-
prising wellness probably by stimulating the neuro-endocrine system. It does seem that
ozone, by acting on many targets, can indirectly help in recovering functional activities gone
astray because of a chronic disease and, if this interpretation is correct, ozone therapy acts as
a biological response modifier. Although ozone therapy is now used in many countries, it is
Table II. Routes of Ozone Administration
Parenteral Topical or locoregional
Intra-arterial (IA)
a
Intramuscular (IM) Nasal
b
Subcutaneous (SC) Tubal
b
Intraperitoneal (Ipe) Auricular
Intrapleural (IPL) Oral
b
Intra-articular (IPL) Vaginal
(a) Periarticular Urethral and intrabladder
(b) Myofascial Rectal
Intradiscal (ID) Cutaneous
Intraforaminal (IF) Dental
Intralesional (Iles)
c
a
No longer used for limb ischemia. Hepatic metastasis could be embolized via the hepatic artery.
b
To be performed during 30--40 sec apnea.
c
Intratumoral or via a fistula.
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mostly used by private physicians and the performance of large clinical trials has been
severely hampered by lack of sponsors, disinterest of pharmaceutical as well as health
authorities, and prejudice by clinical scientists. However, a number of studies have been
performed with the following results:
A. Peripheral Obstructive Arterial Diseases
Even a modest obstruction of limb arteries due to atherosclerosis, diabetes, or Buerger’s
disease (thromboangiitis obliterans) leads to a progressive reduction of blood flow to the feet.
Tissue ischemia and any minor trauma facilitate the formation of an ulcer, which will not
heal because oxygen, nutrients, and growth factors indispensable for the repair process are
lacking. This pathology is the best suited to be treated with O
3
-AHT. According to Fontaine-
Leriche classification, patient at either stage II (intermittent claudication and transitory
pain), or stage III (continuous pain, cyanosis, and possibly initial ulcers) achieve the best
results. Stage IV includes incipient necrosis of toes and unbearable pain leads to surgical
amputation that can be avoided with O
3
-AHT in about 50% of cases.
183–185
In comparison
to pentoxyfilline and prostanoids (the gold standard of orthodox treatment), O
3
-AHT has
proved more effective and without side effects in ischemic vascular disease. In a small trial, 28
patients were randomized to either receive their own ozonated blood or an IV infusion of
prostacyclin.
186
All patients continued conventional treatment with statins, antihypertensive,
and antiplatelet aggregation drugs. Ozone therapy proved more effective than prostacyclin in
terms of pain reduction and improvement in the quality of life, but no significant difference
was seen in vascularization of the lower limbs in either group, most likely due to the short
duration of treatment (14 treatments in 7 weeks). More prolonged treatments lead to a
satisfactory healing of ulcers.
187
Previous studies
122,188–194
have shown the validity of
O
3
-AHT in this complex pathology, but it is a mistake to stop therapy too early in these
patients because O
3
-AHT, as with other conventional drugs, must be continued, albeit less
frequently, for life. An improved schedule on a trial in progress consists of two O
3
-AHT
(225 mL blood plus 25 mL 3.8% sodium citrate solution), given weekly for at least 4 months.
Topical therapy performed with ozonated olive oil is extremely useful when initial dry
gangrene or ulcers are present. The frequency of O
3
-AHT depends upon the stage of the
disease and regarding the III and IV stages it can be done every day in the attempt to prevent
amputation. How well O
3
-AHT works it appears evident by the fact that the nocturnal
excruciating pain disappears after the first two to three treatments, indicating the improve-
ment of blood flow in the ischemic tissue and the lack of ‘‘stealing’’ blood away from
underperfused muscle.
On January 2008, the Lancet published a double-blind, placebo controlled study
(ACCLAIM trial) in 2,426 patients with New York Heart Association (NYHA) functional
classes II–IV chronic heart failure (CHF).
195
Beside standard medication, the experimental
group during a period of some 24 weeks, underwent about 25 intragluteal injections each
patient receiving 10 mL of its own blood heavily oxidized with ozone associated with UV
irradiation and heating at 42.51C. It is unbelivable that 10 mL of blood were oxidized with as
many as 75 mg of ozone, a dose that kills all cells and denature plasma proteins. This
procedure, which is a sort of minor O
3
-AHT,
196
had been invented with the aim to produce
immunosuppressive compounds able to counteract the pathophysiological mechanisms
responsible for the progression of CHF. Results have been disappointing because no dif-
ference in the composite endpoint of death for cardiovascular reasons between the control
and the experimental group were noted. A few researchers
197–200
have criticized the approach
that had been also a failure in the previous Simpadico trial in patients with chronic limb
ischemia.
201
Actually this trial was stopped because of the risk of inducing neoplasia. This
THE OZONE PARADOX K665
Medicinal Research Reviews DOI 10.1002/med

approach has been discussed here because, being based on an irrational concept, may
undermine the progress of the real O
3
-AHT that utilizes the minimal amount of ozone just
sufficient for triggering useful biological activities.
Millions of people suffer from chronic limb, brain, and heart ischemia, which represent
the major cause of death worldwide. This has a huge socio-economic impact, particularly in
the developing world. If only orthodox medicine will accept O
3
-AHT as an adjunct to
standard medication, a great leap forward will be noted.
B. Age-Related Macular Degeneration
In the UK alone, some 200,000 patients affected by the ‘‘dry’’ (atrophic) form of ARMD are
suitable for treatment with O
3
-AHT,
202
but all over the world there are about 30 million
people searching for a therapy. Nonetheless, ophthalmologists can only prescribe anti-
oxidants and zinc, which are minimally effective.
203,204
Since 1995, almost 1,000 patients with
the dry form of ARMD have been treated with O
3
-AHT at our polyclinic and three-quarters
have shown an improvement of one to two lines on the visual acuity chart.
144,205
Usually 15–18 treatments, at an initial ozone concentration of 20 mg/mL of gas per mL
blood, slowly upgraded to 60 mg/mL (twice weekly), followed by two monthly session as a
maintenance therapy, allows to maintain the improvement. Although uncontrolled, this
study emphasizes that O
3
-AHT is the only treatment able to dramatically improve the pa-
tient’s quality of life. In this disease there is progressive degeneration and death of the fovea
centralis photoreceptors and of the pigmented retinal epithelium (PRE) as a consequence of
several factors, one of which is chronic hypoxia. Although O
3
-AHT induces a pleiotropic
response, the main advantage is due to an increased delivery of oxygen to the retina, which is
the bodily tissue with the highest oxygen consumption. It is worth noting that O
3
-AHT is
useless, even harmful, in the exudative form of ARMD and in multigenic and progressive
disorders (e.g., retinitis pigmentosa and recessive Stargardt’s disease).
206
The exudative form,
characterized by an aberrant choroidal vascular growth and a vascular hyperpermeability
beneath the retina and the PRE, is caused by worsened ischemia, which negatively stimulates
the release of the vascular endothelial growth factor. It must be emphasized that O
3
-AHT (in
the dry form) not only improves visual activity but at least, in part, renders the patient
capable of autonomous life.
C. Chronic Infectious Diseases
Ozone is regarded as the best topical disinfectant because bacteria, viruses, fungi, and pro-
tozoa, when free in water, are readily oxidized.
207,208
Disappointingly, destruction of free
pathogens in plasma by ozone either ex vivo or in vivo is greatly hampered by soluble
antioxidants such as albumin, ascorbic acid, and uric acid and they are virtually unassailable
when there are intracellular located.
124,125
However, ozone therapy still deserves attention
because, by improving metabolism and operating as a mild cytokine inducer,
64
it can have a
beneficial influence on infectious diseases. Thus, there remains a place for the application of
O
3
-AHT as an adjuvant in chronic viral infections (e.g., HIV, HCV, HSV), in combination
with highly active anti-retroviral therapy (HAART), pegylated interferon-aplus either
lamivudine or ribavirin and the acyclovir.
On the other hand, bacterial septicaemia must be treated with the most suitable anti-
biotics to prevent toxaemia and multisystem organ dysfunction. Particularly important is the
topical application of either (i) ozone as a gas mixture (about 4% ozone and 96%
oxygen);
209,210
or (ii) as ozonated water; or (iii) ozonated oils (where ozone is firmly stabilized
as a triozonide)
208,211–214
for the treatment of bacterial, viral, and fungal infections, aphthous
ulcers, burns, abscesses, and osteomyelitis. Topical therapy is most effective when combined
666 KBOCCI ET AL.
Medicinal Research Reviews DOI 10.1002/med

with O
3
-AHT owing to the improved oxygenation of hypoxic tissues. Radiodermatitis
215
and
wound healing have been enhanced because ozonated solutions display a cleansing effect, act
as a disinfectant, and stimulate tissue reconstruction. A recent review reports that the high
rates of diabetes in many parts of the world make foot ulcers a major and increasing public-
health problem. Foot ulcers cause substantial morbidity, impair quality of life, engender high
treatment costs (about US$17,500–27,987) and are the most important risk factor for lower-
extremity amputation.
216
Although the constant use of rectal–colon insufflation of O
2
–O
3
is
not the optimal approach, it seems to improve the prognosis of diabetes by combining topical
therapy with ozonated oil and O
3
-AHT.
217
This study needs to be confirmed. Ozonated olive
oil is an amazing preparation because combines antibacterial activity with healing properties
due to the slow release of oxygen in hypoxic tissues and the stimulation of fibroblasts
proliferation.
212,213
Chronic ulcers and/or putrid wounds are one of the most distressing and
difficult medical problems with which to deal and are caused by ischemia, diabetes,
immunosuppression, and malnutrition. During the past decade the use of ozone derivatives
in such cases has proved very beneficial,
143
but so far official medicine has not yet discovered
this excellent preparation far more effective than ointments containing often ineffective an-
tibiotics and corticosteroids, which delays healing. With the current increase in health-care
costs, O
3
-AHT and ozonated oils deserve attention because they reduce hospital assistance
and are inexpensive.
D. Pulmonary Diseases
Lung diseases, such as chronic obstructive pulmonary disease (COPD), will soon become the
fourth most common cause of death, which, with emphysema and asthma, make significant
incapacity. Using corticosteroids, long-acting b
2
-agonists, and antibiotics, orthodox medicine
has certainly proved helpful,
218
but it cannot change the course of COPD. However, in a
series of elderly patients affected by macular degeneration and either emphysema or COPD,
a remarkable improvement has been observed by combining ozone therapy
219
(using the
schedule adopted for vasculopathies) with the best conventional treatments. It is unfortunate
that so far a randomized study evaluating orthodox therapy with or without O
3
-AHT has not
been performed.
E. The versatility of Ozone Application in Orthopaedics and Dentistry
The application of ozone in low back pain has proved very effective. It can be administered
directly (intradiscal),
220–224
or indirectly, via intramuscular administration into the para-
vertebral muscles. This latter type of administration has been assimilated to a ‘‘chemical
acupuncture.’’
145
During the last 6 years, more than 30,000 patients with hernial disc have
been treated in Italy with a success rate varying from 62 to 80%. The value of this approach,
minimally invasive and without risk, has been already recognized in several countries, from
China to Spain and South America. As shown also in another study on pain-related disorders
due to sport injury (232 subjects) and inflammatory disorders (770 subjects)
225
it appears that
ozone exerts a multiplicity of effects, such as the activation of the anti-nociceptive system,
and it has anti-inflammatory action due to lipid peroxidation products, with the consequent
inhibition of cyclooxygenase-2 (COX-2).
226,227
Finally, ozone has proved very useful in dentistry for eliminating infections and blocking
primary root carious lesions.
228,229
The interested reader will appreciate the notable book
‘‘Ozone: the revolution in dentistry.’’
230
After almost 80 years the intuition of Dr. Fisch could
not receive a more enthusiastic appreciation by Prof. Lynch.
THE OZONE PARADOX K667
Medicinal Research Reviews DOI 10.1002/med

8. IS OZONE THERAPY A BAD COPY OF HYPERBARIC OXYGEN THERAPY?
It is often thought that ozone therapy tries to simulate the advantages of the much better
known hyperbaric oxygen therapy (HOT)
231–233
and therefore it seems useful to clarify that
these two approaches are both theoretically and practically different.
In the former, the drug is represented by ozone and, while we have described its
initial reaction and the cascade of active messengers, it has also been pointed out that
oxygenation of blood is not its primary intent. Conversely, by breathing almost pure
oxygen at 2.6 bar into the hyperbaric chamber, the volume of dissolved oxygen in the
plasma increases up to about 5 mL/dL, that is enough to satisfy ischemic tissues even
if the absence of fully oxygenated hemoglobin. HOT is only transitorily effective because
after 2 hr of therapy, hypoxia resumes in ischemic tissues and therefore the therapeutic
effect is temporary. However, HOT has an exclusive role in CO-poisoning, air embolism,
decompression sickness, and perhaps clostridial myonecrosis while ozone therapy is
far more effective and practical to perform in POAD, heart ischemia, ARMD, diabetic
foot, chronic ulcers, and bedsores. Thus, both approaches are relevant but each one
has its selected field of application and the difference should be understood for the sake of the
patient.
146
9. CONCLUSIONS
The history of medicine remind us that in the past the application of several important
approaches has been delayed owing to prejudice, lack of knowledge, or of sponsors and often
by commercial competition. Ozone is inexpensive and therefore ozone therapy does
not make an exception in spite of the fact that all chemical, biochemical, physiological, and
pharmacological mechanisms elicited by ozone as primum movens are in the realm of or-
thodox medicine. One wonders if now with the advent of molecular medicine and gene
therapy, ozone therapy is obsolete or worthwhile being pursued. Our many treated
patients answer for us by saying that it is very beneficial. The compliance is excellent and the
patients, as soon as the therapeutic effect declines, ask for a new cycle, showing the
benefit and lack of side effects. It has been unfortunate that, in the past, the direct
intravenous injection of the gas, now prohibited, the use of primordial ozone generators
and misuse of ozone by incompetent quacks has generated serious doubts about its
validity. Moreover, pulmonary toxicity due to prolonged inhalation of polluted air and
many nonphysiological studies, performed in saline washed erythrocytes unprotected by the
potent plasma antioxidants, have generated the dogma that ozone is always toxic and should
not be used in medicine. This concept cannot be generalized because it does not take
into account the profound difference between the endogenous chronic oxidative stress,
due to aging or to a chronic disease, and the calculated, extremely brief, and well-calibrated
oxidative stress induced on blood by using a precise and small ozone dose. When the ap-
propriate ozone dose reacts with biomolecules it yields a number of compounds that in spite
of their intrinsic toxicity, thanks for their pharmacodynamic, stimulate important bio-
chemical pathways. Indeed, the medical effect depends upon a critical balance between an
appropriate small dose of ozone and an almost infinite reacting variables such as the mul-
tiplicity of antioxidants, the life-time of ROS and LOP, their in vivo pharmacokinetic, and
most important the variability of the biological response depending upon on enzyme re-
activity and the stage of the disease.
Since the discovery of NO as a physiological messenger, other gaseous molecules such as
CO, H
2
S, and H
2
,
234–236
in spite of being known as potentially toxic molecules, if used
668 KBOCCI ET AL.
Medicinal Research Reviews DOI 10.1002/med

judiciously are now considered as possible therapeutic agents. Any drug, depending upon its
dosage can be either therapeutic or toxic. A striking example is represented by a vital
compound such as glucose, its normal concentration in the plasma ranges between 0.7 and
1.0 mg/mL. However, when this concentration falls below 0.4 mg/mL, the consequent hy-
poglycemic coma can be deadly. On the other hand, if the glucose concentration remains
constantly above 1.3 mg/mL, it induces the metabolic syndrome, which is well exemplified by
the current diabetic epidemic. Finally, oxygen at 21% concentration in air (and an arterial
pO
2
of about 99 mmHg) allows us to live for almost 80 years but it is deadly if we breathe
pure oxygen for a few days. Thus, while a further discussion regarding ozone toxicity in
medicine appears futile, it is important to examine if, indeed ozone therapy will be able to
acquire a right place among the medical armamentarium. In the last decade, ozone therapy
has attracted great attention in less-developed countries, while it remains partly prohibited in
USA and poorly regarded in other developed countries. What can be done to change this
severe outlook? Today we have a comprehensive framework for understanding the bio-
chemical mechanisms and the biological effects of ozone and we have at least in part the
capability of recommending ozone therapy in selected diseases either as a first choice or even
better in combination with orthodox therapy. Thus, first, we must continue to organize
specialized courses for physicians for avoiding conceptual or technical pitfalls. Second, while
it is important to continue specific biologic studies, it is imperative to perform controlled and
extensive clinical trials to prove beyond any doubt the value of ozone therapy at least in
vascular diseases. Unless this is done, there is no future for ozone therapy within official
medicine. The stumbling block is represented by lack of sponsors, disinterest of the phar-
maceutical industry, and negligence of health authorities. As ozone therapy is a very cheap
treatment, especially if it will be performed in all hospitals on a daily basis, it will markedly
reduce both medical cost and invalidity. Almost needless to say that ozone therapy, like
orthodox medicine, cannot ‘‘cure’’ several human diseases such as ARMD, atherosclerosis,
and metabolic diseases. However, the maintenance therapy associated with conventional
medication could improve the life of many patients. By considering the huge cost of reliable
controlled and randomized clinical trials, unless health authorities give a financial support,
ozone therapy will remain in limbo and in the hands of private physicians who can only
report anecdotal and yet useless data. Only scientifically well-demonstrated therapeutic
advantages will be able to dissipate prejudice and allow oxygen–ozone therapy to become a
world wide useful medicinal treatment.
10. ABBREVIATIONS
4-HNE 4-hydroxynonenal
ALDH aldehyde dehydrogenase
ARMD age-related macular degeneration
ASF airway surface fluid
ATP adenosine triphosphate
BMC blood mononuclear cells
CAT catalase
CCl
4
carbon tetrachloride
CGMP cyclic guanosine monophosphate
CHF chronic heart failure
CNS central nervous system
CO carbon monoxide
THE OZONE PARADOX K669
Medicinal Research Reviews DOI 10.1002/med

COPD chronic obstructive pulmonary disease
COX-2 cyclooxygenase-2
DPG 2,3-diphosphoglycerate
ELF epithelial lining fluid
G6PHD glucose-6-phosphate dehydrogenase
GSH glutathione
GSH-Rd glutathione reductase
GSPase glutathione peroxidase
GSSG oxidized gluthathione
GST glutathione-S-transferases
HAART highly active anti-retroviral therapy
HClO hypochloric acid
HCV hepatitis C virus
HIV human immunodeficiency virus
HO-1 heme oxygenase-1
HOT hyperbaric oxygen therapy
H
2
O
2
hydrogen peroxide
HSP heat stress proteins
HSV herpes simplex viruses
HUVEC human vascular endothelial cells
IFNginterferon gamma
IL-1 interleukin-1
IL-8 interleukin-8
LDH lactate dehydrogenase
L-NAME n-omega-nitro-L-arginine methyl ester
LOP lipid oxidation products
MA mercapturic acid
MDA malondialdehyde
NADPH nicotinamide adenine dinucleotide phosphate
NF-kB nuclear factor-kB
NO nitric oxide
N
2
O nitric dioxide
O
2anion superoxide

OH hydroxyl radical
O
3
-AHT ozonated autohemotherapy
PDGF platelet-derived growth factor
POAD peripheral obstructive arterial disease
ppm parts per million
PUFA polyunsaturated fatty acids
RBC red blood cells
ROS reactive oxygen species
PRE pigmented retinal epithelium
SOD superoxide dismutase
TAS total antioxidant status
TBARS thiobarbituric acid reactive substances
TGFb1 transforming growth factor b1
TNFatumor necrosis factor alpha
Trx thioredoxin
UV ultraviolet radiation
VEGF vascular endothelial growth factor
670 KBOCCI ET AL.
Medicinal Research Reviews DOI 10.1002/med

ACKNOWLEDGMENTS
One of us (V.B.) is grateful to the University of Siena for the permission to continue to work
in the Department of Physiology as Emeritus Professor of Physiology. We are grateful and
thank Mrs. Helen Carter for revising the English manuscript.
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Velio Bocci, M.D., Ph.D., in Physiology. He is an Emeritus Professor of Physiology at the
University of Siena, Italy. His main research fields are plasma proteins characterization and
labelling; metabolism and pharmacokinetics of interferons and cytokines; biological and medical
evaluation of oxygen– ozone therapy.
Emma Borrelli, M.D., Ph.D., in Cardiovascular Pathophysiology. She is the Director of the
Laboratory of Cardiovascular pathology in the Department of Surgery and Bioengineering of
the University of Siena, Italy. Her research interest deals with cardiovascular pathology and the
application of ozone therapy.
Valter Travagli is an Associate Professor of Pharmaceutical Technology at the University of
Siena, Italy. His main research fields are the interactions of biological macromolecules with
degrading agents and the inner Quality concept for drugs, based on manufacturing processes.
Iacopo Zanardi, Ph.D., in Pharmaceutical Sciences, Department of Pharmaceutical Chemistry
and Technology, University of Siena, Italy. His main research fields are the chemico-physical
evaluation of biological macromolecules and ozone therapy.
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