Chapter 4 - From Bocci V. OZONE. A NEW MEDICAL DRUG, Springer,
Please use this chapter only for educational purposes
HOW DOES OZONE ACT?
HOW AND WHY CAN WE AVOID OZONE TOXICITY?
This is one of the most important chapters because I believe
that, if the ozone therapist understands how ozone reacts with body
fluids and cells, he can achieve useful therapeutic results. The
patient represents the substrate yielding a number of biochemical,
pharmacological and psycho-neuro immunological reactions and as
such, she/he is an essential part of the process.
represents the bulk (95-98 %) of the gas mixture, by considering the
enormous dilution of the small re-infused oxygenated-ozonated blood
with venous blood, it has a negligible role. While, only thanks to
oxygen we can live, this gas has a negative effect on the long run
because cell respiration allows the formation of reactive oxygen
species (ROS), among which, hydroxyl radical (OH.- ) is one of the
most destructive radical compounds for precious enzymes and DNA.
Almost every one knows that ageing, the metabolic disorders
(atherosclerosis, diabetes, cell degeneration) can be worsened by
ROS and, only in part, we can prevent their damageable effects.
Ironically, even the partial lack of oxygen (hypoxia), observable in
ischemic vascular diseases, represents the cause of death due to
limb ischemia, heart infarction and stroke. Moreover, hypoxia
enhances neoplastic metastatisation and ultimately leads to death.
Ozone, the triatomic oxygen, synthesized in the stratosphere to
protect us from excessive UV radiation, can be precisely produced
with a medical generator but it is up to us to use it proficiently
as a real drug. As ozone is one of the most potent oxidants, we must
learn how to tame it and
the scope of this chapter isto
define its therapeutic coefficient, or
, in simple words
to distinguish the therapeutic from the toxic dose.
When I ask physicians how ozone acts, I receive
odd answers: a favoured one is the esoteric idea that ozone, during
its decomposition to oxygen, will transfer some energy to the body
thus invigorating it, and another is that ozone will be absorbed
and, after entering into the cells, will turn them on. In comparison
to other complementary approaches based on philosophical
postulations, a positive characteristic of ozonetherapy
is that it
can undergo the most objective scientific
investigation carried out with normal biological and clinical
It has been unfortunate that for several decades,
empiricism and the lack of basic studies have delayed an
understanding of the mechanisms of action. Moreover, dangerous, even
deadly infusion of ozone by quacks, a good dose of prejudice and
the inconsistent dogma that “ozone is always toxic”
are responsible for the strong and dull opposition of conventional
medicine to the use of ozonetherapy. However I will persevere in my
endeavour and I feel confident that this wrong belief will change in
the near future.
At the moment my duty is to schematically try
to demonstrate that ozone obeys perfectly well to common physical,
chemical, physiological and pharmacological notions and that its
activities modulating several cellular functions are already known.
First of all, ozone, as any other gas, dissolves in the water
either of the plasma (the liquid part of blood), or into the
extracellular fluids, or into the thin layer of water covering the
skin and particularly the mucosae of the respiratory tract, gut,
vagina, etc. At normal temperature and atmospheric pressure, owing
to its high solubility and depending upon its relative pressure,
some ozone dissolves into the water but, unlike oxygen, DOES NOT
EQUILIBRATE with the ozone remaining in the gas phase. This happens
ozone, being a potent oxidant, REACTS IMMEDIATELY
with a number of molecules present in biological fluids, namely
antioxidants, proteins, carbohydrates and, preferentially,
polyunsaturated fatty acids (PUFAs).
The reaction of ozone with so many molecules
implies two fundamental processes:
I call the first “THE OZONE INITIAL REACTION” because some of
the ozone dose is unavoidably consumed during oxidation of
ascorbic and uric acids, sulphydryl (SH)-groups of proteins and
glycoproteins. Although albumin, ascorbic and uric acids tame the
harsh reactivity of ozone (Halliwell, 1996), they allow this first
reaction that is important because it generates reactive oxygen
species (ROS), which triggers several biochemical pathways in
blood ex vivo (ie, in the glass bottle). ROS are neutralized
within 0.5-1 minute by the antioxidant system.
The second, well characterized reaction is known as “LIPID
PEROXIDATION” (Pryor et al., 1995). In the hydrophilic plasma
environment, one mole of an olefin (particularly arachidonic acid
present in plasma triglycerides and chylomicrons) and one mole of
ozone give rise to two moles of aldehydes and one mole of hydrogen
peroxide (H2O2). These two reactions,
completed within seconds, use up the total dose of ozone that
generates hydrogen peroxide, an oxidant but not a radical molecule
(usually included in the ROS family) and a variety of aldehydes
known as LIPID OXIDATION PRODUCTS (LOPs).
FROM NOW ON, NOT OZONE, BUT ONLY ROS (MOSTLY HYDROGEN
PEROXIDE) AND LOPs ARE RESPONSIBLE FOR THE SUCCESSIVE AND MULTIPLE
BIOCHEMICAL REACTIONS HAPPENING IN DIFFERENT CELLS ALL OVER THE
Therefore it should be clear that
good deal of ozone is consumed by the antioxidants present in plasma
and only the second reaction is responsible for the late biological
and therapeutic effects
. This should clarify why a very low
ozone dose can be ineffective or equivalent to a placebo.
ROS include several radicals as anion
superoxide (O2.-), nitrogen monoxide (NO.), peroxynitrite
(O=NOO-), the already mentioned hydroxyl radical and other oxidant
compounds such as hydrogen peroxide and hypoclorous acid (HClO).
All of these compounds are potentially cytotoxic
(Fridovich, 1995; Pullar et al, 2000; Hooper et al., 2000)
luckily have a very short half-life (normally a fraction of a
second) and both the plasma and cells have antioxidants able to
neutralize them, if their concentrations do not overwhelm the
LOPs generated after peroxidation of a great variety of PUFAs are
heterogenous and briefly are represented by peroxyl radicals (ROO.),
a variety of hydroperoxides (R-OOH) and a complex mixture of low
molecular weight aldehydic end products, namely malonyldialdeyde (MDA),
and alkenals, among which 4-hydroxy-2,3 transnonenal (4-HNE), is one
of the most cytotoxic. The chemistry and biochemistry of these
compounds has been masterfully described by Esterbauer’s group
(1991). If one thinks about the wealth and chemical heterogeneity of
lipids, glycolipids and phospholipids present in plasma, it becomes
difficult to imagine how many potent, potentially noxious, compounds
can be generated by the lipids reacting with ozone. During one of my
several disputes with American referees, a distinguished scientist
It is grotesque to think that any Western World Drug
Regulating Agency would condone infusing the hodgepodge of ozonized
products to treat diseases, although it is probable that the
products would initiate and/or modulate a wide spectrum of
-immune processes to varying degrees”.
In my opinion,
this referee missed what I believe is the
formidable strength of ozonetherapy: provided that we can control
(by using precise ozone concentrations exactly related to the blood
volume and antioxidant capacity) the amount of LOPs, we can achieve
a multitude of biological effects unthinkable with a single drug
The scheme ought to fix in the reader’s mind this crucial
point and the sequence of events eventually leading to the
therapeutic results: ROS are produced only during the short time
that ozone is present in the glass bottle, ex vivo, and
they yield EARLY biological effects on blood, whereas LOPs, which
are simultaneously produced, have a far longer half-life and, during
the reinfusion of ozonated blood in the donor, they reach the
vascular system and practically all the organs where they trigger
Figure 1. The scheme intends to show that ozone dissolved in
the plasmatic water reacts immediately with a number of biomolecules
and disappears. The compounds generated during the reactions (ROS
and LOPs) represent the “ozone messengers” and are responsible for
the biological and therapeutic effects.
We have come to a critical point: how can we reconcile the
production of toxic compounds with the idea that these compounds
exert important biological and therapeutic effects?
Let us first examine the behaviour and pharmacodynamic of
hydrogen peroxide, which in practical terms is the most important
ROS. As soon as ozone dissolves in the plasmatic water and reacts
the concentration of hydrogen peroxide starts to
increase but, just as rapidly, decreases because this unionized
molecule diffuses quickly into erythrocytes, leukocytes and
platelets, where it triggers several biochemical pathways.
Does the increased intracellular concentration of hydrogen
peroxide become toxic for the cell?
Because, at the same time, it undergoes reduction to water
in both plasma and intracellular water, thanks to the presence of
powerful antioxidant enzymes such as catalase, glutathione-peroxidase
(GSH-Px) and free reduced glutathione (GSH). Perhaps for one second,
the plasma-intracellular concentration has been estimated to range
from 1 to 10 micromolars, which avoids any toxicity (Stone and
Collins, 2002). The transitory presence of hydrogen peroxide in the
cytoplasm means that it acts as one of the ozone chemical messengers
its level is critical: it must be above acertain
threshold to be effective but not too high to become noxious. In
performed with human blood exposed to ozone
concentrations ranging from 20 to 80 mcg/ml per ml of blood,
the process of hydrogen peroxide generation, diffusion and reduction
wasfound always extremely transitory
(Bocci et al.,1993a;
Figure 2. The multivaried biological response of the
organism to ozonized blood can be envisaged by considering that
ozonized blood cells and the generated LOPs 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.
ER: erythrocytes, PLAT: platelets, BMC: blood mononuclear
cells, GRAN: granulocytes, CNS: central nervous system, GIT:
gastrointestinal tract, MALT: mucosal associated lymphoid tissue.
Hydrogen peroxide is now widely recognised as an
intracellular signaling molecule able to activate a tyrosine kinase,
which phosphorylates a transcription factor (Nuclear Factor KB, NFKB),
which allows the synthesis of a number of different proteins (Baeuerle
and Henkel, 1994; Barnes and Karin., 1997). Basically hydrogen
peroxide functions by oxidizing cysteines (Rhee et al., 2000), and
we and Others have found that it acts on blood mononuclear cells (Bocci
and Paulesu, 1990; Bocci et al., 1993b; 1998a; Reth, 2002), on
platelets (Bocci et al., 1999a), on endothelial cells (Valacchi and
Bocci, 2000) and on erythrocytes (Bocci, 2002).
ROS entering into the erythrocytes are almost immediately
reduced (hydrogen peroxide to water and lipoperoxides to
hydroperoxides) at the expense of GSH. The enormous mass of
erythrocytes can easily mop up hydrogen peroxide and, within 10-15
minutes, marvellously recycle back oxidized antioxidants in reduced
form (Mendiratta et al., 1998a, b). While
reductase (GSH-Rd) utilises the reduced
nicotinamide adenine dinucleotide phosphate
coenzyme serves as an electron donor for various biochemical
to recycle oxidized glutathione(GSSG) to the
original level of GSH,
the oxidized NADP is reduced after
the activation of the pentose phosphate pathway, of which
glucose-6-phosphate dehydrogenase (G-6PD) is the key enzyme
Thus, glycolysis is accelerated with a consequent increase of ATP
levels. Moreover 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-haemoglobin dissociation curve due
either to a slight decrease of intracellular pH (Bohr effect) or/and
an increase of 2,3-diphosphoglycerate (2,3-DPG) levels.
Figure 3. A summary of the biological effects elicited
during exposure of human blood to oxygen-ozone, ex vivo and
during its reinfusion in the donor.
There is an ample literature regarding the cytotoxicity of LOPs.
These compounds, when tested either in tissue culture, or examined
in the context of the delicate respiratory system, are toxic even at
a concentration of 1 micromolar. Surprisingly, submicromolar
concentrations (0.01-0.5 microM) tested in several cell types can
stimulate proliferation and useful biochemical activities. These
findings lead to believe that toxicity of ozonated lipid products
depends upon their final concentrations and tissue-localization, so
that they can act either as injurious or useful signals (Dianzani,
1998; Parola et al., 1999; Bosch-Morell et al., 1999; Larini et al.,
Blood, in comparison to the lungs, is a much more
ozone-resistant “tissue” and we have never observed any damage.
, when we reinfuseozonated blood, what is the fate of
We have often measured the kinetic of their
disappearance from blood and their half-life in six patients with
age-related macular degeneration (ARMD) was equivalent to 4.2±1.7
min. On the other hand, if the same ozonated blood samples were
incubated in vitro, levels of LOPs hardly declined during the next
two hours, a result clarifying their toxicity in static cell
cultures. As far as cholesteryl ester hydroperoxide is concerned,
Yamamoto (2000) has emphasized the role of the enzymatic degradation
and hepatic uptake.
Thus LOPs toxicity in vivo is most
likely irrelevant for the following reasons:
DILUTION (up to 150-200 folds) of these compounds in blood and
body fluids rapidly lowers their initial concentration to
pharmacological, but not toxic levels. Obviously the ozone dose
must be within the therapeutic range.
NEUTRALISATION of LOPs due to the antioxidant capacity in body
fluids and cells.
DETOXIFICATION of LOPs (scarcely observable in vitro) due to
the interaction with billions of cells endowed with detoxifying
enzymes such as aldehyde- and alcohol-dehydrogenases, aldose
reductase and GSH-transferases (GSH-T).
EXCRETION of LOPs into the urine and bile after hepatic
detoxification and renal excretion.
BIOACTIVITY without toxicity. As already mentioned,
submicromolar concentrations of LOPs can act as physiological
messengers able to reactivate a biological system gone awry.
From a pharmacokinetic point of view, trace amounts of LOPs, can
reach all organs and particularly the bone marrow and the Central
Nervous System (Figure 2).
LOPs are extremely important in
informing specific cell receptors of a minimal and calculated
oxidative stress eliciting the adaptive response.
to erythrocytes, LOPs can influence the erythroblastic lineage,
allowing the generation of cells with improved biochemical
characteristics. These “supergifted erythrocytes” as I called them,
due to a higher content of 2,3-DPG and antioxidant enzymes, during
the following four months, are able to deliver more oxygen into
ischemic tissues. The consequence of repeated treatments, obviously
depending upon the volume of ozonated blood, the ozone concentration
and the schedule is that, after a few initial treatments, a cohort
(about 0.8 % of the pool) of “supergifted erythrocytes” will enter
daily into the circulation and, relentlessly, will substitute old
erythrocytes generated before the therapy. This means that, during
prolonged ozonetherapy, the erythrocyte population will include not
only cells with different ages but, most importantly, erythrocytes
with different biochemical and functional capabilities. In the
course of ozone therapy, we have already measured a marked increase
of G-6PD and other antioxidant enzymes in young erythrocytes. (Bocci,
2004). The process of cell activation is very dynamic and don’t last
for ever because blood cells have a definite life-time and a limited
biochemical memory; therefore, the therapeutic advantage MUST BE
MAINTAINED WITH LESS FREQUENT TREATMENTS.
zone toxicity to blood, biological fluids and internal
organs can be totally avoided when the ozone dose reduces only in
part and transitorily the multiform and potent antioxidant capacity.
The antioxidant system has evolved during the last two billions
years as an essential defence against oxygen: it is made up of
scavengers components, namely albumin, vitamins C and E, uric acid,
bilirubin, cysteine, ubiquinol, alpha-lipoic acid and of
intracellular antioxidants, such as GSH, thioredoxin and enzymes (superoxide
dismutase, SOD; GSH-Px, GSH-Rd, GSH-T, catalase, etc.,) and proteins
such as transferrin and caeruloplasmin, able to chelate free iron
and copper that, otherwise, can favour the formation of hydroxyl
The wealth and the variety of extracellular and
thoroughly described by Chow and
Kaneko (1979), Halliwell (1994; 1999a, b; 2001), Frei (1999),
Holmgren, (1989), Di Mascio et al., (1989), Jang et al., (1997),
Packer et al., (1997), Bustamante et al., (1998) and Chae et al.,
are able to explain how bland amounts of ozone can
be tamed with the results of stimulating several biological systems
without deleterious effects.
Until this key point is understood,
the dogma of ozone toxicity will continue to linger.
The reader can appreciate the complexity of this system in Table 2
Table 2. The antioxidants system
Gluthatione redox system
Reducing equivalents via NADPH and NADH
Glucose, Cysteine, Cysteamine, taurine, Tryptophane,
The interaction among antioxidants, enzymes and the
metabolic system is very important as it allows their rapid
regeneration and the maintenance of a normal antioxidant status.
The following scheme, drawn by Prof. L. Packer, beautifully
illustrates the cooperation among various antioxidant system in
order to neutralize a lipoperoxide radical ROO.
(shown on the left hand side) to a less reactive hydroperoxide, ROOH.
The reducing activity is continuously generated by cellular
metabolism via the continuous reduction of NAD(P)+ to NAD(P)H.
It suffices here to say that, during the transient exposure of
blood to appropriate concentrations of ozone, the antioxidant
reservoir decreases between 2-25 % in relation to ozone doses
between 10-80 mcg/ml of gas per ml of blood.
It is important
to add that this partial depletion is corrected in less than 20 min
thanks to the recycling of dehydroascorbic acid, GSSG, alpha-tocopheryl
radical to the reduced compounds.
CONCLUSIONS:What happens when human blood is exposed to
a therapeutic dose of oxygen-ozone?
Both gases dissolve in the water of plasma depending
upon their solubility, partial pressure and temperature. While
oxygen readily equilibrates between the gas and the blood phases,
the ten-fold more soluble ozone cannot equilibrate because IT REACTS
with biomolecules (PUFA, antioxidants) present in the plasma. The
reaction yields hydrogen peroxide (among other possible ROS) and
lipid oxidation products (LOPs). The sudden rise in plasma of the
concentration of hydrogen peroxide generates a gradient, which
causes its rapid transfer into blood cells where, in a few seconds,
it activates several biochemical processes and simultaneously
undergoes reduction to water by the efficient intracellular
antioxidant system (GSH, catalase, GSH-Px).This critical step
corresponds to a controlled, acute and transient oxidative stress
necessary for biological activation, without concomitant toxicity,
provided that the ozone dose is compatible with the blood
While ROS are responsible for immediate
LOPs are important
as late effectors, when the blood, ozonated ex vivo,
returns into the circulation upon reinfusion (
Figures 2 and
LOPs can reach any organ, particularly the bone marrow
where, after binding to receptors in submicromolar concentrations,
elicit the adaptation to the repeated acute oxidative stress,
which is the hallmark of ozonated autohemotherapy. Upon prolonged
therapy, LOPs activity will culminate in the upregulation of
antioxidant enzymes, appearance of oxidative stress proteins (haeme-oxygenase
I as a typical marker) and probable release of stem cells, which
represent crucial factors explaining some of the extraordinary
effects of ozonetherapy
It must be emphasized that BLOOD EXPOSED TO OZONE
UNDERGOES A TRANSITORY OXIDATIVE STRESS necessary to activate
biological functions without detrimental effects. The stress must be
adequate (not subliminal) to activate physiological mechanisms, BUT
NOT EXCESSIVE to overwhelm the intracellular antioxidant system and
cause damage. Thus, an excessive ozone dose or incompetence in
manipulating this gas can be deleterious. On the other hand, very
low ozone doses (below the threshold), are fully neutralised by the
wealth of plasma antioxidants and can produce only a placebo effect.
The concept that ozonetherapy is endowed with an acute
oxidative stress bothers the opponents of this approach because they
consider it as a damage inflicted to the patients, possibly already
under a chronic oxidative stress. THEY DO NOT BELIEVE THAT
OZONETHERAPY INDUCES A MULTIVARIED THERAPEUTIC RESPONSE ALREADY WELL
DOCUMENTED IN SOME DISEASES. Moreover THEY DO NOT DISTINGUISH
THE CHRONIC OXIDATIVE STRESS (COS)
DUE TO AN ENDOGENOUS AND
WITH THE SMALL AND TRANSIENT
OXIDATIVE STRESSES that we can precisely perform EX VIVO with the
The THERAPEUTIC RESPONSE achieved after these
repeated oxidative stresses can be envisaged as a PRECONDITIONING
EFFECT eventually able to reequilibrate the redox system altered by
This article is for your educational purposes only.
For medical treatment and advice be sure to consult with your physician.