Corespondenta Prof V. BOCCI - Pof. Nazarov

The Open letter to professor V.Bocci
Posted by: Eugen Nazarov in News

Dear Professor Bocci,
Allow me to continue the discussion, which took place at the conference in Istanbul. It seems to me
that some important questions, which you put for the consideration to the congress, were not
considered in sufficient details. Allow me to return to them again, in order to express the position of
the Russian school of ozone therapy.
On one of your slides these questions were formulated as follows:
DURING THE 1ST CONGRESS OF OZONE THERAPY, it is hoped:
1. to discuss and clarify the best technological advances,
2. the need of using ozone-resistant materials to avoid toxicity. Plastic bags regularly used for
blood storage are unsuitable as, in the presence of ozone, they release phthalates and plastic
microparticles into blood. Neutral glass bottles are idoneous.
First, about phtalates. More than 90% of phthalates produced in Europe are used to plasticize PVC.
We use many PVC products every day but tend to take many of them for granted. They include
everything from lifesaving medical devices such as medical tubing and blood bags. Phthalates have
been used for more than 40 years in flexible vinyl (PVC) products. Their use has led to
improvements in the health and well being of billions of patients, many of them children,
throughout the world. No plasticizer (materials used to make plastic soft) has ever been subjected to
toxicity and safety testing to the same degree as DEHP. It has been a known fact in the scientific
community for many years that di -(2-ethylhexyl) phthalate, DEHP, migrates from medical devices,
such as blood bags and tubing, in minute amounts. However, not one single piece of validated
scientific evidence shows that these products cause adverse health effects in humans.
Thus, many yearspractice of blood storage in PVC bags showed absence of any toxic effect of
phthalates. Can ozone accelerate the migration of phthalates into the blood? Are there any solidly
established scientific facts of the acceleration of phthalate migration from PVC into the blood under
the action of ozone and ozone- induced formation of PVC microparticles? We will be glad to get
acquainted with such data, if they exist. In any case, while estimating possible risks of the
application of plastic bags of PVC one should take into consideration the following:
1) ozone does not interact with the bag material directly, since the conventional practice of big
autohemotherapy lies in the fact that ozone- oxygen mixture first comes in contact with the blood,
which must neutralize ozone for a split second.
We have made the measurement of ozone concentration in the bag immediately after the completion
of the blood treatment with the ozone- oxygen mixture with the ozone concentration equal to 20
micrograms per milliliter. We have not discovered the content of ozone in the bag, in the sensitivity
of the method of 0.1 micrograms per milliliter. It is obvious that only one of the components of
ozone- oxygen mixture - oxygen comes in contact with the bag walls.
2) There were carried out about 200 000 procedures of big autohemotherapy with the use of PVC
bags in Ukraine, Russia, Turkey, in some EU countries, in Latin America and countries of the
Southeastern Asia. Being the inventor of the method variant of big autohemotherapy based on the
use of the peristaltic pump and PVC- bag of special construction, I collect information about the
excesses and complications, which appear in the practice of doctors, who use instruments and bags
of my construction carefully. Since 2001, when the practice of this variant of big autohemotherapy
have begun, I obtained 1137 reports about the complications while conducting this procedure.
Complications can be divided into two types - during the procedure (862) and in the first twentyfour
hours after the procedure (275). Complications during the procedure were manifested by
vertigo, nausea, decreased arterial pressure, fainting.
As a rule, such patients admitted that this reaction usually appeared in them at the sight of the
blood. The second type of complications was expressed by the increased body temperature of the
patients after the first procedure, sleepiness, weakness. In certain cases, especially after the
complaints of patients on sleepiness, the single dosage of ozone was reduced. These symptoms
disappeared during the course.
We consider these complications to be a routine reaction of patients to the procedures associated
with the blood taking, since this is a usual profile of complications in the donor practice.
In conclusion, it should be noted that on the basis of the positive experience of using polypropylene
in your works on EBOO, and prejudices which some doctors have, especially in the West, we
mastered the production of the plastic bags of 100% polypropylene, which we presented at the
congress. Thus, doctors can select between two types of bags based on PVC and polypropylene,
which are produced by us.
The next question which you submit for the consideration at the conference was the following:
3. Recently it has become fashionable to use the IV infusion of ozonized saline. In comparison
to the classical ozonized autohemotherapy, this method is quick and inexpensive but is it valid
Ozonized saline must be compared with oxygenated saline and appropriate chemical and
clinical data must be presented. A valid and extensive comparison between clinical results
achieved with either ozonized autohemotherapy or ozonized saline must be presented.
There are two aspects in this question:
1) whether hydrogen peroxide and sodium hypochlorite are formed during treatment of NaCl
solution, which may be the cause for complications in intravenous infusion
2) is the dose of ozone obtained by the patient in this procedure sufficient for full therapy
The affirmative answer is given to the first question in your report. However, lets consider the facts.
Id like to cite the data represented in your report at the congress. It is seen from the diagram that in
treatment of the saline with ozone in the concentration of 50 mg/l for 10 minutes the level of
hydrogen peroxide is approximately 2.5 mcmol/l. Is this large or small Simple calculation shows
that weight unit, concentration of hydrogen peroxide is 85 micrograms per liter or < 0.00001%. On
the other hand, while bubbling the saline solution with ozone- oxygen mixture with this
concentration, we increase the level of dissolved ozone to 4 mg/l. The comparison of the ozone
concentration and hydrogen peroxide shows that ozone concentration exceeds peroxide
concentration 47 times under the conditions described by you. It is obvious that presence of
hydrogen peroxide, and therefore sodium hypochlorite should be disregarded under such conditions.
A question about adequacy of therapy by the method of the intravenous infusion of the ozonized
saline solution seems to me to be more important. Let us calculate the dose of ozone, which the
patient obtains during the procedure of the intravenous infusion of the ozonized saline solution. As
a rule, the ozone concentration in the liquid is equal to 1-3 mg/l used in the Russian method. In this
case the patient is infused 0.2-0.4 liters of the ozonized saline solution. It is easy to calculate that
the patient obtains a dose of 0.2 - 0.6 mg or 0.4 -1.2 mg of ozone, respectively. Thus, a question
about is the Russian method effective is brought to a question - is the dose of ozone of 0.2 - 1.2 mg
sufficient for treatment of forms of diseases traditional for ozone therapy. There is no simple answer
to this question. For some diseases, for example autoimmune ones, this dosage is insufficient. In
case of such diseases big autohemotherapy should be used. At the same time there is an enormous
list of the diseases, for which the Russian method is completely adequate. As an example I will give
the basic results of the dissertation work made at Odessa medical university Use of ozone therapy in
rehabilitation treatment of patients with ischemic heart disease of Dr.A.V.Artiomenko, Odessa,
2004.
Object of the research.
157 patients with stable stenocardia of exertion of 2-3 functional class by criterion class
WHO/ISH,1993, heart deficiency of 2-3 functional class by the classification New York Heart
Association and indications of the endothelial dysfunction became the object of the research .
Control group. 32 patients got standard therapy: digozin 0.25 mg/24 hours, Amlodilin 5-10 mg/24
hours, aspirin 100 mg/24 hours., furosemide 40-80 mg/24 hours. The basic group consisted of 63
patients; ozone therapy was conducted against the background, and consisted of infusion of 150 ml
saline solution in a day started from 1 mg/l, with the following increase to 0.5 mg/l for 3 mg/l and
following decrease to the initial dose, in all 10 procedures. One more group of patients got basic
therapy and placebo in the form of infusion of 150 ml oxygenated saline solution in a day.
In 6 months after finishing the course of ozone therapy the main group was divided into two subgroups.
One of these sub-groups got the repeated course of ozone therapy.
All patients got the standard cycle of clinical-laboratory studies including the spirocycleergometry,
Holter monitoring ECG, dopplerography of brachial artery, Doppler echocardiography, ultrasonic
examination of the heart and others.
Some results of this research are given below.

On the basis of the data analyzed the author came to a conclusion that:
1. Application of ozone therapy in the reconstructive period of treatment of sick people with
IDH increases the antianginal effectiveness of therapy, decreasing frequency to 55.7+-7.8%
and daily quantity of episodes of ischemia of the myocardium to 61.3+-8.2% in comparison
with the group receiving medicamentous therapy.
2. Ozone therapy corrects effectively the endothelidependent dysfunction of patients with
IHD, providing growth of endothelidependent vasodilation by 83%, that is 2.1 times more
than by medicamentous therapy.
3. Ozone therapy increases tolerance to physical work, decreasing the functional class of
stenocardia and heart deficiency for sure and fast.
4. Ozone therapy at the out-patient stage of rehabilitations of patients with IHD improves
pharmacoeconomic effectiveness of treatment, decreasing necessity of extra antianginal
therapy by 61.5% and frequency of repeated hospitalizations by 58.2%
I assume that this work answers your question, taking into account that oxygenated saline solution
was used as the placebo. We have tens of documented scientific research made in strict accordance
with the modern criteria of the probative medicine, which have no doubts as to the effectiveness of
the method of infusion of the ozonized saline of solution. More than twenty-year practice of
application of this method in a number of the countries of Asia, Europe and Latin America is
evidence of it.
What is a basic difference in the method of infusion of the ozonized saline solution from the method
of big autohemotherapy, which allows to achieve a significant therapeutic effect in considerably
lower dosages of ozone I believe that the reason for this lies in the fact that substantially larger
volume of the blood is treated in the Russian method. In fact, if the velocity of blood flow in the
cubital vein is 50 ml/min, then 1500 ml of the blood is treated for 30 minutes of infusion, which is
15 times more than in big autohemotherapy. Probably, this circumstance is decisive in the Russian
method.
In conclusion I propose you to participate in the organization of the joint project on objective and
independent assessment of the comparative effectiveness of the methods of big autohemotherapy
and infusion of the ozonized saline solution. I propose to select the form of nosologies and the place
of test conduction together.
I thank you for attention, which you paid to my letter. I hope that I managed to elucidate our
position to you.
Accept my sincere assurances in respect and admiration by your works and by the contribution,
which you have made in development of ozone therapy in the world.

President of the Ukrainian Association of ozone therapists
DrSci, Professor Eugeny Nazarov

THE OPEN LETTER TO PROF. E NAZAROV (ANSWER)

Only a few days ago a colleague sent us your letter and, in order to eliminate doubts worrying
several ozonetherapists, we are glad to answer the issues raised in your letter. This is now more
important than ever because, after two decades of intensive work spent on clarifying the
mechanisms of action of ozone in Biology and Medicine, there are now some methodological
approaches invented for minimizing work that are going to jeopardize the future and acceptance of
ozonetherapy within the realm of orthodox medicine. We are still struggling to prove the validity of
this approach with the Food and Drug Administration (USA), that is the fundamental first step that
possibly will allow the acceptance by National Health Authorities in many countries. If the
Russian's proposal of simplified and quick methods will take roots, ozonetherapy, not only will be
never accepted but it will degenerate in a practice only useful for quacks , who already are too
abundant.
From the outset, we want to declare to be only research scientists without any interest in whatever
commercial relationship with ozonetherapy.
Topic: The need of using ozone-resistant materials to avoid toxicity.
Only well- proven ozone-resistant materials must be used to avoid toxic effects in patients.
Contrary to your statement, there is plenty of evidence published in the best international journals
that various plastic microparticles, phthalates and/or other additives are released into blood
components during blood storage in plastic (PVC) autotransfusion bags even during short exposure,
without the presence of oxygen-ozone (O2-O3). While a minimal contamination has been tolerated,
no other substance, absolutely no ozone, should be insufflated into the bag. It is necessary to
remember that up to 1990 the ozonation was carried out in neutral glass bottles that are ozone
resistant.
Unfortunately, later on O3-autohemotherapy (O3-AHT) has never undergone the necessary
standardization so that several variants of the original procedure have been used generating an
enormous confusion.
A critical examination of the various methodologies used in the last decade for carrying out O3-
AHT in Italy and Germany has pointed out serious pitfalls that are potentially risky for the patient.
In Italy another worrisome problem has been the widespread use of plastic (PVC) autotransfusion
bags that, while suitable for storing blood, release various plastic microparticles and phthalates into
the blood even without a short exposure to O2-O3 (Valeri et al., 1973; Thomas et al., 1978; Callahan
et al., 1982; Estep et al., 1984; Labow et al., 1986; Quinn et al., 1986; Whysner et al., 1996; Latini,
2000; Stahlhut et al., 2007; Swan, 2008; Meeker et al., 2009; Jung et al., 2010). As it has been noted
in patients undergoing dialysis, the mutagenic and toxic activity of these compounds is a matter of
grave concern (Lawrence, 1978; Divincenzo et al., 1985). Particularly in the last decade there is
mounting evidence that DEHP, by mimicking human hormones, may disrupt the endocrine system
leading to developmental problems or behave as a mutagenic substance (Lyche et al. 2009). In
January 2006, the European Union placed a ban on six types of phthalate softeners, including
DEHP used in toys (Directive 2005/84/EC).
All of the following data (see Appendix 1) were published in Bocci's book: Oxygen-Ozone therapy.
A critical evaluation, Kluwer Academic Publishers. Dordrecht, The Netherlands. 2002. Chapter 39,
pp: 375-380.
We have tested several samples of plastic bags largely used in Italy for storing blood and inflowoutflow
tubing in polyvinyl chloride-di(2-ethylhexyl)phthalate (PVC-DEHP). All of these bags are
authorized by the Ministry of Health to store blood but not to be insufflated with O2-O3. Bags are
made of PVC for a maximum content of 55% while for achieving a good elasticity additional
materials amount to about 45%. With small differences the composition is the following:
a) about 40% of DEHP
b) about 1% of Zinc 2 ethyl ethylhexanoate
c) about 1% Calcium or Zinc stearate
d) about 1% N,N'-diacyl ethylenediamine
e) 5 - 10% of epoxidized soybean oil or similar.
While all bags are sterile and suitable for storing blood, they are NOT chemically inert when a
strong oxidant mixture (about 250 mL) composed of about 96% O2 and 4% O3 is insufflated into
the bag. Particularly DEHP and butyl-glycobutyl phthalate (BGBP) are immediately released and
bound extensively to plasma lipids. The plasma is likely to yield a higher content of DEHP than
physiological saline. In line with the criteria expressed by the European Pharmacopea (1997), in
1999, we carried out an investigation by using sterile physiological saline that is considered the
optimal "medical device" for evaluating release and size of plastic particles (2, 5, 10, 20 and 25 μm
size), phthalates and other compounds. It is obvious that evaluation of contaminants could not be
performed in blood. Samples were numbered and all the following tests have been carried out in a
blind fashion. The code was open after the final results were available.
In conclusion it is hard to believe that you are not aware of phthalate or other additives toxicity that
can harm patients receiving blood or even worse saline treated with O2-O3 in medical PVC-bags
normally used only for blood storage. Thus, while hoping that this letter may be helpful, my best
advice is to abolish altogether the use of plastic bags and adopt glass bottles.
You have mentioned some 1137 reports of complications some of which are most likely have been
caused by plastic particles and solubilized materials infused with blood in sensitive patients.
Moreover the fate of plastic particles infused with blood taken up by phagocytosis remains a serious
problem with possible late carcinogenic consequences. From 2003 up to 2007, after having
supervised thousands of ozonated autohemotherapy, by both using a careful O3 dosing upgrading
and the only necessary sodium citrate, no side effects have been noted. On the other hand before
1999, by unfortunately using plastic bags, especially in women, we noticed some similar side
effects.
We are finally glad to read that you have now "mastered the production of bags of 100%
polypropylene" that you have presented at the recent Istabul Congress (1st International
Ozonetherapy Congress, 4th-6th December 2009, Istanbul - Turkey). Thus you have come to admit
the use of polypropylene container as ozone-resistant that can be safely sold. Needless to say why
not to use the usual safe glass bottles?
Topic: It has now become fashionable to use the IV infusion of ozonated saline
As a physician, having practised for years in a charity clinic, I vividly remember how busy an
expert dialysis technician and myself were in performing no more than a dozen O3-AHTs in a
afternoon. Thus, I can well understand that in a large clinic where daily there are a hundred patients
to be treated, you cannot entertain the hope to perform the classical O3-AHT and therefore one has
to compromise with an uncertain and semiquantitative procedure like the IV infusion of ozonated
saline or, as in the case of Cubans, to apply the rectal O3 insufflation to all patients, which, for
several reasons, is an even more unreliable approach.
Almost needless to say that the classical O3-AHT, owing to the precise volume of blood, the precise
volume of O3 of which one knows the exact concentration, hence the real dose, makes it an
unsurpassed method. In this case the O3 instantly reacts with several blood substrates in a practically
quantitative fashion. During the 5 minute mixing, we know all the biochemical reactions going on
to activate blood cells so that, by the time of the reinfusion, O3 has disappeared. The beauty of the
system is that, by using O3 within the well-determined therapeutic window, not only toxicity is
avoided but one can control every step and fashion the ozonation process on the patient's disease.
Since 1994, I was interested in finding a blood substitute and eventually I also landed in trying
ozonating saline. As you know, it was demonstrated that ozonation of medical physiological saline
(0.9% NaCl) with various O3 concentrations (50-70-100 μg/mL) induced at the same time formation
of hydrogen peroxide and chemiluminescent effects indicating the generation of free radicals (Bocci
et al., 1998). The production of H2O2 was progressive and by using an O3 concentration of 100
μg/mL reached the value of about 20 μM after 60 min of O3 insufflation. Without further bubbling,
the infusion of 250 mL of this solution in healthy volunteers caused considerable pain along the
venous path of the infused arm after about 24 hours. This indicated that the solution has irritated the
endothelium with the risk of a phlebitis and we were concerned that, besides H2O2, a transitory
formation of HOCl or perchlorate may be the noxious agent. Although chloride could be oxidized
by O3 to perchlorate (Truong et al., 2004; Rao et al., 2010), the saline solution containing traces of
Fe2+ allows to the Fenton's reactions with formation of oxydryl ralicals Hypochlorous acid
constitutes an inflammatory agent of the endothelium during an infusion, even at a trace
concentration. Moreover, it may activate platelets and induce a microcoagulation. Although it is
well known that ClO- is physiologically produced by phagocytic cells and it is an efficacious
bactericidal compound, it remains either confined in phagosomes or released in plasma near
endothelial cells (Goldmann et al., 2009). However, ClO- is one of the most noxious reactive
oxygen species (ROS) during a chronic inflammation. It is unfortunate that the practice of using
ozonated saline has become common in Russia and is widely used because it is inexpensive and less
time-consuming than major AHT and simultaneously applicable to many patients. As it could be
foreseen, physicians have started to use it also in Italy, Spain, Greece and Turkey. Ikonomidis et al.
(2005) in Greece, have reported that they maintain the saline solution under a constant flow of O3
during transfusion but they warned that the maximum amount of O3 daily administered is usually 4-
5 mg and should never exceed 8-10 mg. In their publication they also stated "if we exceed these
rates, the over coagulation syndrome starts" and they strongly recommended to perform coagulation
tests before starting therapy. These precautions reinforce our preliminary objection to this approach.
Moreover, Foksinski et al. (1999) have measured 8-oxodeoxyguanosine, a typical oxidative DNA
damage in lymphocytes of atherosclerotic patients after the IV infusion of ozonated saline, that is a
worrisome result never detected after O3-AHT.
Fortunately to the best of our knowledge, Russian physicians ozonize the saline with very low O3
concentrations (2-3 μg/mL) and this precaution certainly reduces toxicity but it leaves open the
aspect of therapeutic efficacy.
During the last couple of years I had been lucky to work together on this topic with two researchers
of the Department of Pharmaceutical and Applied Chemistry at the University of Siena. We have
ascertained that the procedure of ozonation of absolutely pure water is a far simpler procedure
because this is the only case when the unstable O3 obeys Henry's law. However if the water
contains NaCl, the extremely high reactivity of O3 induces a complex series of reactions with the
possible progressive formation of H2O2, unstable OCl- , NaClO4, ·OH, 1O2 and some unstable O3.
Razumovski, Ershov et al (2008); Bocci et al, (2009) have evaluated the complexity of O3 reactions
and rapidity of its decomposition. Here we enclose our diagram.
0 50 100 150 200 250
0,0
0,2
0,4
0,6
0,8
1,0
1,2
O3 bubbling in ultrapure water
O3 disappearance in ultrapure water
O3 bubbling in saline (0.9% NaCl) solution
O3 disappearance in saline (0.9% NaCl) solution
Absorbance (260 nm)
Time (min)
Figure 1. The diagram shows the rapid increase and decrease of O3 bubbled at 70 μg/mL concentration (gas
inflow 1.5 L/min) in either ultrapure water (400 mL) or physiological saline (400 mL). Ozone bubbling was
stopped after 25 min and absorbance was measured every 2 min at 260 nm. At O3 concentration of 10 μg/mL the
curves are very similar, but absorbance is considerably lower than shown in the diagram. (Unpublished data).
It is therefore necessary to enumerate and discuss the problems occurring during the preparation of
ozonated saline:
1) For human use it would be unwise to use O3 concentration over 4 μg/mL (4 mg/L).
Moreover it is essential to establish the volume per minute of the gas mixture O2-O3. The
problem is that different ozone generators have variable gas output: if it is 1 L per minute,
the O3 delivered to 200 mL of saline would be 4 mg/L but, if the output per minute is
equivalent to 3 litres of gas, then the actual dose of O3 delivered will be 12 mg/L! As a
consequence one must properly instruct the ozonetherapist in relation to the owned ozone
generator as otherwise one risk to poison the patient.
2) The period of ozonation time also ought to be well defined in relation to the volume of
saline because in the case of saline solution an ozonation time of 20 min appears enough to
reach a plateau. Obviously a shorter or longer ozonation period will differently modifies the
concentration of hydrogen peroxide, O3 and other radicals.
3) Another aspect to be clearly defined if gas bubbling will continue or not during the IV
infusion period. This is because, as soon as the gas bubbling is stopped, the concentration of
H2O2 remains fairly stable but the O3 concentration will halve during the next 30 min and
this affects the therapeutic result. As a trivial example, I doubt that in a large clinic all the
saline infusions are all under a continuous O3 bubbling and it is likely that saline bottles will
be ozonated and then distributed implying a more or less long delay before the infusion.
After one hour delay, O3 is not longer present.
4) As a preliminary conclusion, one must ponder on the validity of using the infusion of
ozonated saline. It is certainly less dangerous than the direct IV infusion of the gas mixture
that some quacks, without a medical qualification, still dare to perform with the serious risk
of causing an oxygen embolism. However it does not represent a good improvement because
the variable presence of H2O2, O3, etc does not insure neither a good reproducibility, nor a
consistent therapeutic effect. Moreover the blood flow in the cubital vein varies
considerably in different patients and in women and this implies that a fairly constant
infusion of ozonated saline versus a variable blood flow and content of antioxidants implies
an uncertain blood/H2O2- O3 relationship with possibly a too low or too high bio-oxidation.
By comparison, a fundamental pillar of the classical O3-AHT is that we can maintain
precisely the blood/ O3 ratio within the known therapeutic range.
5) I regret to say that this approach contains too many uncertain parameters and, in any case, it
needs to be carefully standardized to avoid risking to perform a placebo infusion or an
excessive and risky treatment. Nonetheless if, on the basis of the critical need to treat too
many patient, it is allowed by Russian Health Authorities it will be never accepted by
neither the FDA, USA or but the EC Authorities.
6) Owing to the fact that H2O2 is one of the most important ROS generated by O3, since 2005
(Bocci et al. 1998), in women with very difficult venous access, by using a G27 needle, we
have intravenously infused the solution of pure H2O2 in glucose(5 %) or saline solutions at
the concentrations ranging from 0.03-0.06 % (8-16mM). The bio-oxidative therapy with
H2O2 was first described by Dr. I.N. Love in 1888 (Love, 1888) and then promoted by Dr.
C.H. Farr in 1993 (Farr, 1993). We showed a modest but consistent activity in women with
age-related macular degeneration. In contrast to ozonated saline, this compromise, very
simple to prepare, does not contain other dangerous ROS and one does not need an ozone
generator. Obviously, the glucose solution should not be used in diabetic patients.
Finally, I am also very interested in treating chronic heart disease (CHD) and I would like to call
your attention to the disastrous result published in the Lancet (Torre-Amione et al., 2008), by using
a bad copy of minor AHT after an extremely high oxidation and heat stress on 10 mL of blood. This
is a dreadful example of an irrational ozonation procedure that has severely compromised the future
of ozonetherapy.
The study by Dr A.V. Artiomenko (Odessa, 2004) performed with ozonated saline seems to have
given a "significant" (statistic is missing!) improvement of 63 CHD's patients. However, what is
missing in this study is a direct comparison with a similar group of patients properly treated with
O3-AHT. As it is, results not published in an international peer reviewed journals are lost for the
scientific community.
Summary We would like to thank you for your letter and for compelling us to express our
objective judgements. Our aim is to try to demonstrate the validity and reliability of ozonetherapy
and all our efforts should be directed to allow the acceptance of ozonetherapy as an effective
approach within orthodox medicine. The Western world is either against or it has prejudice towards
this approach and, in spite on lack of sponsors and funds, we must strive hard to succeed.
V. Bocci, MD, Emeritus Professor of Physiology, Department of Physiology,
Prof. V. Travagli, and Dr. I. Zanardi, PhD, Department of Pharmaceutical and Applied Chemistry
at the University of Siena, Italy
16/04/2010
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Appendix 1
1. MATERIALS AND METHODS
Particles were measured by an automatic counter (Royco) by Dr. G. Gavioli and collaborators at
Braun Carex, Mirandola (Modena, Italy) while several chemical compounds among which phthalates
were detected by HPLC by a specialized Institute (Istituto di Ricerche Agroindustria, Director: Dr.
G.C. Angeli, Modena, Italy).
The proliferation index (PI) of blood mononuclear cells (BMC) has been assessed after isolation of
BMC from human blood of normal donors. PBMC were isolated by Ficoll-Hypaque (Sigma
Chemical Co., St. Louis, MO) gradient centrifugation, washed twice in RPMI-1640 medium
supplemented with 20 mM HEPES buffer, spun down at low speed to remove platelets, and
resuspended in RPMI-1640 medium supplemented with 2 mM HEPES, 10% heat-inactivated fetal
calf serum (FCS), 2 mM L-glutamine, 100 U/mL penicillin and 100 μg/mL streptomycin (all from
Life Technologies, Gaithersburg, MD) at the final concentration of 1 x 106 viable cells/mL. Cell
viability was assayed by the trypan blue exclusion technique and light microscope observation.
Aliquots (0.1 mL) of BMC suspension were added per well in triplicate wells to 96-well flat
bottomed tissue culture plates (Costar, Cambridge, MA). BMC were cultured without stimulation or
stimulated with PHA at a final concentration of 5 μg/mL (Sigma Chemical Co.). After 12 hours
incubation, either control saline, or ozonized saline in a glass syringe, or in blood bags was added to
the culture medium in a 1:4 proportion. Thereafter incubation continued for 40 and 64 hours. Cell
proliferation was evaluated by a colorimetric immunoassay (Boehringer Mannheim, Mannheim,
Germany) based on BrdU incorporation. Briefly, after either 40 and 64 hours of incubation at 37°
with 5% CO2 in air and 100 % humidity, the cells were labelled with BrdU for 6 hr (10 IU/well).
The cells were then fixed, anti-BrdU-POD antibody added and the immune complexes detected by
the subsequent substrate reaction. The proliferative index (PI) was obtained, calculating the ratio
between PHA-stimulated cells and unstimulated ones, after subtraction of the corresponding blanks.
It is emphasized that all tests were carried out with the same procedure and timing used during a
conventional autohaemotherapy.
All tests were performed in double blind fashion by two external firms specialized in the pertinent
assays. PI and all other analyses were assessed in the Institute of General Physiology, University of
Siena. Results were expressed as mean ± SD.
2. RESULTS AND DISCUSSION
These can be summarized as follows:
Table 1 reports the number of plastic particles ranging in size among 2, 5, 10, 20 and 25 μm in
either the control saline (test no. 10), or in saline withdrawn from blood bags with no exposure to
O2-O3 (test no. 1) or in saline as before but exposed to O2-O3 (70 μg/mL per mL saline, ratio 1:1)
for 10 min (test no. 2), or in saline from other PVC bags, control (test no. 15) or in saline exposed to
O2-O3 for 10 min (test no. 16). It appears very clear that the number of plastic particles released
from different PVC blood bags far exceed the number of control samples. According to the
European Pharmacopea, values of particles released after ozonation exceed the maximal tolerated
value of 3.3-10.7 fold. All the saline samples collected from the plastic bags after ozonation showed
by HPLC examination, several compounds as phthalates, caprolactamate and linear chain
hydrocarbons not readily identifiable.
Interestingly, the same examination of tubing in PVC-DEHP normally used for collecting blood and
insufflating O2-O3 do not show an abnormal release of plastic particles (Table 2) even though the
O3 exposure was prolonged for 30 min. This is not surprising because tubings have far less
additives than bags. Thus as the time of contact with O2-O3 is very transient, these tubings could
still be used although we have preferred to substitute them with a new brand made up of more
resistant material (PVC additioned with tris(2-ethylhexyl) trimellitate, TEHT, C33H54O6) known as
Staflex TOTM. Material released from this type of tubing is less than 100 fold than from tubing
PVC-DEHP so that this new type is absolutely safe.
Besides the potential risk propounded by plastic particles and chemical compounds during the
reinfusion of ozonated blood, we thought important to investigate whether BMC withdrawn from
the bags show any modification of the PI. A series of analyses carried out after two different periods
of incubation (40 and 64 hours) clearly show a consistent depression of the PI that can be as high as
27.2% (Table 3). Taking into account the small volume of ozonated saline added to the culture
medium this value is possibly underestimated and therefore is worrisome. This negative effect is not
directly due to O3 but rather to unknown compounds released into the saline during ozonation of the
blood bags. It is obvious that we do not want the same phenomenon occurring in vivo and
moreover, owing to the variety of compounds released from the plastic material, we don't know
which is (are) the compound(s) responsible for the inhibition.
Table 1. Numbers of plastic particles (size of 2, 5, 10, 20 and 25 μm) counted for each mL of physiological saline
after the indicated tests.
Table 2. Numbers of plastic particles (size of 2, 5, 10, 20 and 25 μm) counted for each mL of physiological saline
after tests in PVC tubings in current use.
Tabella 3. Evaluation of the proliferation index (PI) of human isolated blood mononuclear cells after 40 and 64
hours of incubation in culture medium after addition (see Methods) of physiological saline (PS) collected from
control or from ozonized saline (80 μg/mL) in glass syringes (GS) or from saline previously ozonized in a blood
bag for either 10 min (A) or for 12 hours (B).
For all of these reasons, the use of a new device is now strongly recommended. This is composed of
a) a neutral 500 mL glass bottle (sterile and under vacuum), b) a new atoxic tubing for collecting
blood and insufflating sterile-filtered O2-O3 via an antibacterial (0.2 μm), hydrophobic ozoneresistant
filter and c) an appropriate tubing with filter that is used, firstly for infusing saline, and secondly for returning the ozonated blood to the donor.
It is important that the exposure of blood to O2-O3 lasts only the necessary 5 min because mixing of
blood must be gentle to avoid foaming. Because blood is very viscous, it takes about 5 min to achieve
a complete and homogenous equilibrium. It can be noted that the pO2 slowly reaches
supraphysiological values (up to 400 mmHg) and then it remains constant. On the other hand, O3
rapidly dissolves in the water of plasma and then reacts instantaneously so that all of the O3 dose is
exhausted within 5 min.
The ozonetherapists must follow this procedure for avoiding either negative effects on the
patients, or being found guilty of medical malpractice.
The worrisome problem is the widespread use of plastic autotransfusion bags that, while suitable for
storing blood, release various plastic compounds into the blood, especially lipids, even during a
short time.
The data were sent to The Italian National Research Health Institute in Rome. The answer was that
plastic bags allowed for storing blood CANNOT BE USED WITH O2-O3 IN ANY CIRCUMSTANCE. The prohibition became and remains effective since 2000: Today CE neutral glass bottles fitted with ozone-resistant plastic cork and idoneous tubings are currently used.



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