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vista
pañola de
zonoterapia
evista
spañola de
zonoterapia
junio 2010
aceoot informa
3
curso de experto universitario
en ozonoterapia - 2ª Edición 2010-2011
PLAZAS LIMITADAS
Introducción.
El presente curso nace de la colaboración entre la Universidad de Sevilla y ACEOOT con el objetivo de dar la
ozonoterapia una formación universitaria, con profesorado de reconocido prestigio. Aportara al alumno los conocimientos
necesarios teóricos y prácticos para aplicar esta técnica.
Datos básicos del curso:
Nombre: Ozonoterapia
Tipo de curso: Experto Universitario
Número de créditos: 25.80 LRU
Número de Horas: 258 horas
Unidad Organizadora:
Universidad: Sevilla
Departamento: Cirugia
Director de estudios: Prof. Andrés Carranza Bencano (Catedrático de Traumatología y Cirugia Ortopédica)
Requisitos:
Requisitos para admisión: Licenciatura en Medicina
Requisitos para obtención del titulo: Superar cuestionarios de cada modulo y trabajo final de curso.
Preinscripción:
Desde 1 al 30 de Septiembre 2010
Datos de Matriculación:
Fecha de inicio: 01/10/10
Fecha de fin: 20/10/10
Precio: 1297.40 € (tasas incluidas)
Impartición:
Fecha inicio: 19/11/2010
Fecha fin: 22/10/2011
Modalidad: Semipresencial
Lugar de Impartición: Facultad de Medicina Universidad de Sevilla
Más información:
El numero de plazas es limitada. Se puede hacer una reserva de plaza enviando un email a: infogeneral@aceoot.org
PUBLICIDAD
evista
spañola de
zonoterapia
junio 2010
4
aceoot informa
I premio científico a la investigación
en ozonoterapia "velio bocci"
La Asociación Científica Española de Aplicación de Oxigeno-Ozonoterapia, con el fin
de fomentar el interés investigador y estimular el desarrollo de trabajos relacionados
con la ozonoterapia, convoca el I Premio Científico a la Investigación en ozonoterapia
“Velio Bocci”, con arreglo a las siguientes:
BASES
1º- Premio
Un premio de 600 € y diploma al mejor trabajo de investigación.
2º- Requisitos
- Podrán optar a este premio todos los medicos o investigadores nacionales o extranjeros.
- Los trabajos tendrán como tema la ozonoterapia.
- Los trabajos pueden ser de investigación sobre los efectos de la ozonoterapia o
revisiones de casos clínicos.
3º- Lugar, plazo y forma de presentación de los trabajos.
- Quienes opten a este premio deberán enviar la documentación requerida a la sede
oficial ( Colegio Oficial de Medicos de Valladolid c/ Pasión 13 Valladolid ) o por correo
electrónico al Secretario Técnico (secretaria.ozono@hotmail.es)
- El plazo de presentación de trabajos finalizara a las 14 horas del día 30 de Septiembre
de 2010.
- Presentación:
* Los trabajos se presentaran completos y redactados en castellano o ingles con el
siguiente formato: introducción, material y métodos, resultados, conclusiones y en PDF o
WORD.
* Se entregaran 2 copias, una con los datos del autor del trabajo y otra donde no figure
ningún dato identificativo.
*Si el envío es por correo postal los trabajos irán en 2 sobres cerrados, indicando
“ORIGINAL” en la copia que incluye los datos del autor.
*Si el envío es por e-mail se enviaran 2 copias, indicando en el nombre del archivo la
palabra “ORIGINAL” en la copia con los datos del autor.
SOLICITAR BASES COMPLETAS A : infogeneral@aceoot.org
5
revista
española
ozonotera
junio 2010
eventos
congresos, symposiums, jornadas,....
IX JORNADAS INTERNACIONALES DE OZONOTERAPIA
Granada, 12 y 13 de Noviembre de 2010.
Organizado por:
Asociación Científica Española de OxígenoOzonoterapia Médica.
Más información: www.aceoot.org
6º SYMPOSION INTERNACIONAL DE APLICACIONES
DEL OZONO
II CONGRESO INTERNACIONAL DE OZONOTERAPIA
DE LA FIOOT.
Palacio de Convenciones. Ciudad de la Habana (CUBA)
Del 28 de Junio al 1 de Julio de 2010
Más información: www.congresoniccuba.com
PUBLICIDAD
Empresa biotecnológica dedicada a ofrecer, tanto productos como servicios, aplicados
a la regeneración tisular y a la medicina regenerativa.
www.proteal.com
(+34) 902 501 609
(+34) 93 163 02 27
proteal@proteal.com
evista
spañola de
zonoterapia
junio 2010
temas a debate
6
uso de bolsas de plástico en
autohemoterapias. utilidad del
suero salino ozonificado.
En esta sección propondremos un tema a debatir sobre cuestiones en las que tenemos discrepancias y
cada uno puede mandar su opinión al correo infogeneral@aceoot.org indicando en la referencia el tema
sobre el que opina. La opiniones recibidas serán publicadas en el siguiente número de la revista
THE OPEN LETTER TO PROFESSOR V. BOCCI
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.
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.
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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 twenty-four 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.
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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 sub-groups. 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.
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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.
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ANSWER ON LETTER TO PROFESSOR E.I.NAZAROV (FROM VELIO BOCCI)
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.
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 inflow-outflow 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.
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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 m 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% st polypropylene that
you have presented at the recent Istabul Congress (1st International th th 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.
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 (50-70100 mg/mL) induced at the same time formationof 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 mg/mL reached the value of about 20 mM 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 2+Fe 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 timeconsuming 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
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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 O3AHT.
Fortunately to the best of our knowledge, Russian physicians ozonize the saline with very low O3 concentrations (23 mg/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, O2 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.
Figure 1. The diagram shows the rapid increase and decrease of O3 bubbled at 70 mg/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 mg/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 mg/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.
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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 agerelated 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
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% heatinactivated fetal calf serum (FCS), 2 mM L-glutamine, 100 U/mL penicillin and 100 mg/mL streptomycin (all from
Life Technologies, Gaithersburg, MD) at the final concentration of 1 x 10 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 mg/mL (Sigma Chemical Co.). After
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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 mm 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 mg/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 O2O3 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 PVCDEHP 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.
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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 sterilefiltered O2-O3 via an antibacterial (0.2 mm), hydrophobic ozone-resistant 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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tema del mes
Velio Bocci, Emma Borrelli,
Valter Travagli and Iacopo
Zanardi
15
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.
Key words: oxidative stress; antioxidants; oxidative preconditioning; ozone; ozonated autohemotherapy
1. INTRODUCTION
A. A Brief Historical Review
Christian Friedrich Schönbein, 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. In nature ozone is continuously produced in the stratosphere
(at 25–30 km from the Earth surface) by UV radiation (< 183nm) 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.
It is also produced during the electric discharge of lightning, which catalyzes the formation
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
(49mL in 100 mL, 0.02 M, at 01C), even though an ample variability is present in the literature.
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
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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 (NO2) will be very toxic.4 The most recent medical ozone generator
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 concentration
can be only 5%.
2. BEHAVIOR OF OZONE
A. Ozone as an Oxidant
Ozone has a cyclical structure assessed by the absorption at 253.7nm with a distance among
oxygen atoms of 1.26A
and exists in several mesomeric states in dynamic equilibrium (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 mitoc hondrial respiration. Phagocytes
reacting with pathogens6–8 produce anion superoxide (O_2 ), H2O2, and hypoc hlorous
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, H2O2 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.
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 320nm) and C (from 100 to 280 nm), whic h are mutagenic and can enhance skin
carcinogenesis. Unfortunately, during the last decades, short-sighted human activities, by
releasing chlorofluorocarbons in the atmosphere, have led to a decreased ozone concentration,
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/m3 (0.001 ppm),
ten times lower than our odor perception threshold for ozone about 20 mg/m3 (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/m3. Moreover, additional anthropogenic
emissions of NO, NO2, 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/m3 as an 8 hr mean concentration to
protect the health of workers. Evaluation of recent Studies allows establishing an average
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environmental ozone concentration of 90710 mg/m3. However, ozone concentration in urban air
can exceed 0.8ppm 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
0.70–0.77mg 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/m3,
reducing pulmonary functions and markedly enhancing the risk of cardiovascular deaths.
Ozone levels of 500 mg/m3 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 ozonecontaminated air opposes
only the ELF’s volume of about 20–40mL,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, the generated ROS, LOP, and nitrating species damage the epithelial lining. The phosphorylation
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, and TGFb1.
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, tachykinins, proteases, alkenals,
and F2-isoprostanes able to selfmaintain 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 70m2) of the bronchial–alveolar space. Recent
studies have detected 4-hydroxynonenal (4-HNE), isoprostanes, H2O2, and
malondialdehyde (MDA) in the bronchoalveolar lavage fluid. The interesting
study by Last et al 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 though the half-life of these compounds is brief, 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. 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–CO2 at pH 7.3 but with a pO2 of 160mmHg, i.e. more
than double of cells in vivo. Even more important is the fact that culture media have a significantly
lower level of antioxidants than plasma, particularly of albumin. 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.
Moreover, antioxidant components are not dinamically replenished in vitro while cells remain exposed to a constant
ozone concentration.
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 after intravenous infusion in patients with agerelated 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 contributed
to consider ozone as a deleterious gas.
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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
3O2 + 68,400 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 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) 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’’. In 1936, a Frenc h 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 O2–O3 gas
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 (O3-AHT) by insufflating ex vivo the gas into the
blood contained in a dispensable ozoneresistant 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.).
This negative concept may only be changed by valid scientific and clinical data. It is worthwhile to mention what
Timbrell55 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.’
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.
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. 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. Moreover, the presence of proteins such as transferrin and ceruloplasmin
quenches oxidizing reactions by chelating transition metals (mainly Fe2+ and Cu+). 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.
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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 (1–5mM) but also thioredoxin and several lipophilic compounds such as atocopherol, 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 enzymes (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 components
will be briefly reviewed.47,63 After having performed thousands of treatments, the
standard procedure is to add 200mL of a gas mixture composed of medical oxygen (495%)
with ozone (o5%) to 180mL of blood after the previous addition of 20mL 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 1.5mL of O2 and 2.4mL of
O3 dissolve in the blood water but their fate is quite different. Oxygen physically diffuses
into erythrocytes and fully saturates hemoglobin (Hb4O8) but in spite of the pO2 as high
as 450mmHg, the therapeutic value of oxygenation is irrelevant because the successive infusion
of oxygenated–ozonated blood (about 15 mL/min) hardly modify the pO2 (_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 8mg for 100mL 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 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. Some ozone reacts with PUFA as
follows
leading to the simultaneous formation of 1 mol of H2O2 (included among ROS) and 2 mol of
LOP.
The fundamental ROS molecule is H2O2, whic h is not ionized but is an oxidant able to
act as an ozone messenger responsible for eliciting several biological and therapeutic
effects.
As it was mentioned, the old concept that H2O2 is always harmful has been widely
revised because, in physiological amounts, it acts as a regulator of signal transduction and
represents a cr ucial mediator of host defense and imm une responses. While exposure
to oxygen is ineffective, ozone causes the generation of H2O2 and of the chemiluminescent
reaction in both physiological saline and plasma. However, while in saline there is a
consistent and prolonged increase in H2O2, in the ozonated plasma both c hemiluminescence
and H2O2 increase immediately but decay very rapidly with a half-life of less than 2 min
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suggesting that both antioxidants and traces of enzymes rapidly reduce H2O2 to water. In
ozonated blood the reduction of H2O2 is so fast that it has been experimentally impossible to
measure it. H2O2 is able to easily pass through the cell membrane, but the intracellular
concentration increases only 1/10 of the extracellular one. Its relative stability allows
measuring it in plasma; in normotensive subjects its concentration is of 2.5 mM. In this case the intracellular
concentration of H2O2 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. Depending upon its local concentration and cell-type, H2O2
can either induce proliferation or cell death. 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.
A very enlightening finding was achieved by evaluating the variation of the total antioxidant
status (TAS) as measured by the Rice-Evans and Miller’s method85 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
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level progressively decreases at first and then remain stable after 20 min. 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 intraerythrocytic
GSH has been found oxidized to GSSG within 1 min after ozonation, but promptly
reduced to normal after 20 min. 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 electron
donations.
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), F2-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, O2, or either 40 or 80 mg/mL ozone concentrations.
Plasma TBARS in vitro are far more stable than ROS, 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 impor tant. It is an amphipathic
molecule and reacts with a variety of compounds such as albumin, enzymes, GSH, carnosine,
and phospholipids. 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 molecules
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, 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 transferred 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 mechanisms.
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.
Needless to say that a constant increase in peroxidation as it happens
after ischemia-reperfusion, CCl4 intoxication,
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ADP-iron overload, and c hronic 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 systems37,95–99
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 sc hematically indicated in Figure 5 clarifies why 4-HNE is
not a risk:
(1) Dilution: The highest concentration of 4-HNE measured after exposing 180mL of human blood to the highest
ozone amount (16 mg) is less than 1mM 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. Several authors96,101,102
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 4.2+-1.7 min. 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.
(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.
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 F2-isoprostanes, may act as a signaling event
of an ongoing acute oxidative stress (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, H2O2 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.
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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 approac h and experimental results exclude this possibility because when
blood is gently mixed ex vivo with O2–O3, 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 K1 and lactate dehydrogenase, or a change of osmotic fragility, or of electrophoretic
mobility, or increased methemoglobin. Levels (mg/dL) of fibrinogen, cholesterol, triglycerids, HDL, and LDL in plasma
are not modified even using the excessive ozone concentration of 160 mg/mL per mL of blood. 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 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. 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. Our thinking is well supported by other data 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. Interestingly, heparinized samples were far more susceptible to ozone most likely because of
the remaining physiological Ca21 level: in fact, a further addition of 2.5–5mM Ca enhanced
the hemolysis up to 40%. Second, how ozone messengers activate blood cells? Initially, the sudden formation of
an H2O2 gradient between the ozonated plasma and the intracellular fluid causes the rapid
passage of about 10% H2O2 into the blood cell cytoplasms and represents the triggering
stimulus: depending upon the cell type, different bioc hemical pathways can be concurrently
activated in erythrocytes, leukocytes, and platelets resulting in numerous biological effects.
The rapid reduction of H2O2 to water is operated by the high concentration of intracellular
GSH, CAT, and GSPase but, nonetheless, H2O2 must be above the threshold concentration
for activating several biochemical pathways as follows.
The mass of erythrocytes mops up the bulk of H2O2: 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
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pathway, of whic h glucose-6-phosphate dehydrogenase (G6PDH) is the key enzyme. In
patients with ARMD, after 13 O3-AHT, a small increase in ATP formation has been determined
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 autohemotherapeutic 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 O3-AHT, an increase in some antioxidant enzymes
has been determined (Fig. 8). This result has been reported also by other authors 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 O3-AHT
treatments (in which ca. 3.8L of blood were ozonated within 7 weeks), isopycnic centrifugation of
blood separated old (heavy) and young (light) erythrocytes (RBC), whic h 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
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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, respectively.
In the so-called old erythrocytes, whic h practically include the bulk of cells (20–120 days
old), G6PDH obviously increased only from 3107127 up to 435787 nmol/hr/mgHb. It is necessary
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 isc hemia (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 shown124,125 that
ozone cannot inactivate bacteria, viruses, and fungi in vivo because, paradoxically, the pathogens
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. Once again the crucial messenger is H2O2 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-inflammatory cytokines. This process, c hecked by a phosphatase or inhibited by cytoplasmic
antioxidants, is very transitory. The release of several cytokines from ozonated blood upon in
vitro incubation has been measured since 1990. 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, 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 obser ving the variable individual production of IL-8 by
blood donors in 13 blood ozonated samples. 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.
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During ozonation of blood, particularly if it is anticoagulated with heparin, an ozonedosedependent increase in activation of platelets has been noted with a consequent release of typical growth factors,
which will enhance the ulcers in ischemic patients (Fig. 10). Whenever possible, albeit with caution, the use of heparin
as an anticoagulant is preferable to sodium citrate because, by not chelating plasmatic Ca21, 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. Figure 11 shows the in vitro production
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 O3, while it was inhibited in the presence of the NO_ inhibitor N-onitroL-arginine-methyl ester (L-NAME). While NO has a half-life of less than 1 sec, proteinbound 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 beneficial,
137 while it is well established that micromolar levels can be toxic. 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 ‘‘therapeutic
shock’’ able to restore homeostasis.138 These aspects are critical and imply two drawbacks:
first, if the ozone generator is not well calibrated or periodically c hecked, 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 approac h.
Other aspects regarding the future of ozone therapy will be evaluated in Section 9.
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D5. 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. Any change of theexternal 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 c hronic infections, whic h 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 isc hemic insult was pioneered by Murry et al. ‘‘Oxidative preconditioning’’
has been also well demonstrated. 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.’’
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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,” it has been postulated that LOP, by acting as
long-distance messengers, can transmit to all organs the information of an acute oxidative
stress.The bone marrow is par ticular ly 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 c hronic oxidative
stress has been now demonstrated experimentally. The increased synthesis of enzymes
suc h as SOD, GSPase, GSH-Rd, and CAT has been repeatedly determined in experimental
animals and in patients (reviewed in 57). Iles and Liu have demonstrated the 4-HNE, by
inducing the expression of g-glutamate cysteine ligase, causes an intracellular increase in
GSH, whic h plays a key role in antioxidant defence. Furthermore LOP induce oxidative
stress proteins, one of whic h 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. Fe21 is promptly chelated by the upregulated synthesis of ferritin. The induction of HO1 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. 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 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 therapy.
That is also the reason why we always start using low ozone concentrations just above
the threshold level to better ac hieve 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 approac h has been the O3-AHT because, on the basis of the patient’s body
weight, a predetermined volume of blood (200–250 mL) to whic h 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 (O2–O3) in a stoichiometric fashion, with the ozone concentration precisely
determined by using an ozone-resistant, disposable 500mL 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, have been extended: they include the quasi-total body exposure
to O2–O3 and the extracorporeal blood circulation against O2–O3.The latterprocedure is
rather invasive because blood collected from a vein circulates through an ozone-resistant
gas exchanger180,181 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 5mL of blood to be
immediately and vigorously mixed for 1 min with an equal volume of O2–O3 at an ozone
concentration ranging between 80 and 100 mg/mL of gas per mL of blood already extensively
described. 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.
The slight hemolysis (_2%) is purposefully required because the heme released in the
gluteal muscle will stimulate the synthesis of HO-1.
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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 isc hemic 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 antioxidant
capacity; (f) it does not procure acute or late side effects; (g) it procures a surprising 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
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 isc hemia and any minor trauma facilitate the formation of an ulcer, whic h 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 O3-AHT. According to FontaineLeric he classification, patient at either stage II (inter mittent claudication and transitory
pain), or stage III (continuous pain, cyanosis, and possibly initial ulcers) ac hieve the best
results. Stage IV includes incipient necrosis of toes and unbearable pain leads to surgical
amputation that can be avoided with O3-AHT in about 50% of cases.183–185 In comparison
to pentoxyfilline and prostanoids (the gold standard of orthodox treatment), O3-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. All patients continued conventional treatment with statins, antihyper tensive,
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.Previous studies have shown the validity of O3-AHT in this complex pathology, but it
is a mistake to stop therapy too early in these patients because O3-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 O3-AHT
(225 mL blood plus 25mL 3.8% sodium citrate solution), given weekly for at least 4 months.
Topical therapy perfor med with ozonated olive oil is extremely useful when initial dry
gangrene or ulcers are present. The frequency of O3-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 O3-AHT works it appears evident by the fact that the nocturnal
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excruciating pain disappears after the first two to three treatments, indicating the improvement
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 Yor k Hear t Association (NYHA) functional
classes II–IV c hronic hear t failure (CHF). Beside standard medication, the experimental
group during a period of some 24 weeks, underwent about 25 intragluteal injections eac h
patient receiving 10mL of its own blood heavily oxidized with ozone associated with UV
irradiation and heating at 42.51C. It is unbelivable that 10mL of blood were oxidized with as
many as 75 mg of ozone, a dose that kills all cells and denature plasma proteins. This
procedure, whic h is a sort of minor O3-AHT, 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 difference in the composite endpoint of death for cardiovascular
reasons between the control and the experimental group were noted. A few researchers have criticized the
approach that had been also a failure in the previous Simpadico trial in patients with chronic limb
isc hemia. Actually this trial was stopped because of the risk of inducing neoplasia. This
approac h has been discussed here because, being based on an irrational concept, may
undermine the progress of the real O3-AHT that utilizes the minimal amount of ozone just
sufficient for triggering useful biological activities.
Millions of people suffer from c hronic limb, brain, and heart isc hemia, whic h represent
the major cause of death worldwide. This has a huge socio-economic impact, particularly in
the developing wor ld. If only or thodox medicine will accept O3-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 O3-AHT, but all over the world there are about 30 million
people searching for a therapy. Nonetheless, ophthalmologists can only prescribe antioxidants
and zinc, which are minimally effective. Since 1995, almost 1,000 patients with
the dry form of ARMD have been treated with O3-AHT at our polyclinic and three-quarters
have shown an improvement of one to two lines on the visual acuity chart.
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 O3-AHT is the only treatment able to dramatically improve the patient’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 whic h is c hronic hypoxia. Although O3-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 O3-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). The exudative form,
c haracterized by an aberrant c horoidal 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 O3-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 protozoa,
when free in water, are readily oxidized. 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. 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
O3-AHT as an adjuvant in c hronic viral infections (e.g., HIV, HCV, HSV), in combination
with highly active anti-retroviral therapy (HAART), pegylated interferon-a plus either lamivudine or ribavirin and
the acyclovir.
On the other hand, bacterial septicaemia must be treated with the most suitable antibiotics
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); or (ii) as ozonated water; or (iii) ozonated oils (where ozone is firmly stabilized
as a triozonide) for the treatment of bacterial, viral, and fungal infections, aphthous
ulcers, burns, abscesses, and osteomyelitis. Topical therapy is most effective when combined
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with O3-AHT owing to the improved oxygenation of hypoxic tissues. Radiodermatitis215 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 publichealth
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 lowerextremity
amputation. Although the constant use of rectal–colon insufflation of O2–O3 is not the optimal approach, it seems
to improve the prognosis of diabetes by combining topical therapy with ozonated oil and O3-AHT. 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. 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, but so
far official medicine has not yet discovered this excellent preparation far more effective than ointments containing
often ineffective antibiotics and corticosteroids, which delays healing. With the current increase in health-care
costs, O3-AHT and ozonated oils deser ve attention because they reduce hospital assistance
and are inexpensive.
D. Pulmonary Diseases
Lung diseases, suc h as c hronic obstructive pulmonary disease (COPD), will soon become the
fourth most common cause of death, whic h, with emphysema and asthma, make significant
incapacity. Using corticosteroids, long-acting b2-agonists, and antibiotics, orthodox medicine
has certainly proved helpful, 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 (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 O3-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), or indirectly, via intramuscular administration into the paravertebral
muscles. This latter type of administration has been assimilated to a ‘‘chemical acupuncture.’’ 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) 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).
Finally, ozone has proved very useful in dentistry for eliminating infections and blocking
primary root carious lesions. The interested reader will appreciate the notable book.
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) 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. Fisch Could not receive a more enthusiastic
appreciation by Prof. Lynch.
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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 muc h better
known hyperbaric oxygen therapy (HOT) 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.
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 orthodox
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 c hronic 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 appropriate
ozone dose reacts with biomolecules it yields a number of compounds that in spite of their intrinsic toxicity, thanks
for their pharmacodynamic, stimulate important biochemical 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 multiplicity 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 reactivity
and the stage of the disease.
Since the discovery of NO as a physiological messenger, other gaseous molecules suc h as
CO, H2S, and H2, in spite of being known as potentially toxic molecules, if used 668 K BOCCI ETAL.
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 hypoglycemic
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 pO2 of about 99mmHg) 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 c hange this
severe outlook? Today we have a comprehensive framework for understanding the biochemical
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 pharmaceutical
industry, and negligence of health authorities. As ozone therapy is a very cheap treatment, especially if it will be
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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.
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artículos nacionales
ozonoterapia sistémica. fundamento
médico, utilidad en el tratamiento del
dolor. (1ª parte) en el siguiente número va la segunda parte.
Fco. Javier Hidalgo Tallón (Instituto de
Neurociencias, Universidad de Granada.)
Nelly Albesa Caro (Unidad del Dolor.
Policlínico Ruber)
1.- INTRODUCCIÓN.
2.- ESTRÉS OXIDATIVO.
3.- ESTRÉS OXIDATIVO, HIPEREXCITABILIDAD NEURONAL
Y DOLOR.
4.- APLICACIONES SISTÉMICAS DE OXÍGENO-OZONO.
GENERALIDADES.
5.- ISQUEMIA/REPERFUSIÓN Y ESTRÉS OXIDATIVO.
6.- NEUROPATÍA DIABÉTICA Y E.O.
7.- FIBROMIALGIA Y E.O.
8.- CONSIDERACIÓN FINAL.
1.- INTRODUCCIÓN.
La ozonoterapia consiste en la aplicación de una
mezcla de oxígeno médico con ozono a muy baja
concentración. El ozono ha de ser producido “in situ” para
cada aplicación, y es una molécula formada por tres átomos
de oxígeno (O3) en lugar de los dos (O2) de los que se
compone la molécula de oxígeno. La idea no es nueva, y
las aplicaciones de ozono médico se remontan a principios
del siglo pasado. El Dr. Kellogg, en su libro sobre difteria,
ya mencionaba el ozono como desinfectante allá por el
1881, y en 1898 los doctores Thauerkauf y Luth fundaron
en Berlín el “Instituto para oxígenoterapia”, llevando a cabo
los primeros ensayos con animales. En el año 1911, salió a
la luz el libro "A Working Manual of High Frequency
Currents", publicado por el Dr. Noble Eberhart, jefe del
siglo pasado. El Dr. Kellogg, en su libro sobre difteria, ya
mencionaba el ozono como desinfectante allá por el 1881,
y en 1898 los doctores Thauerkauf y Luth fundaron en Berlín
el “Instituto para oxígenoterapia”, llevando a cabo los
primeros ensayos con animales. En el año 1911, salió a la
luz el libro "A Working Manual of High Frequency Currents",
publicado por el Dr. Noble Eberhart, jefe del departamento
de terapeutica fisiológica de la Universidad de Loyola,
donde se hablaba del uso del ozono medico en el tratamiento
de enfermedades como la tuberculosis, la anemia, el asma,
la bronquitis, la fiebre del heno, la diabetes, etc. Pero a
pesar de los éxitos obtenidos, las máquinas generadoras
de ozono no han sido muy fiables, y es la tecnología actual
la que nos permite obtener con toda fiabilidad la mezcla
idónea de ambos gases. (1) Al aplicar este tipo de terapia,
siendo el ozono altamente oxidante, realmente estamos
induciendo una “microoxidación” controlada e inocua, cuya
respuesta orgánica será tipo “efecto vacuna”, y activará
a nuestro favor el sistema antioxidante celular. Se ha
demostrado en clínica y en laboratorio que este estímulo
redunda en la producción de unos “súper glóbulos rojos”,
capaces de transportar y de ceder mayores cantidades de
oxígeno a todas las células. (2)
2.- EL ESTRÉS OXIDATIVO.
Nuestras células funcionan con oxígeno; éste es un
gas fundamental para que tenga lugar la degradación de
glucosa con el fin de obtener la energía necesaria para la
subsistencia. Pero a la misma vez que necesario, el oxígeno
es un tóxico, ya que de su degradación resultan las llamadas
especies reactivas del oxigeno (anión superóxido, peróxido
de hidrógeno y radical hidroxilo); éstas son altamente
reactivas, y su acumulo es capaz de bloquear e incluso
destruir las funciones celulares.
PUBLICIDAD
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El ambiente altamente oxidativo obligó a las células
a desarrollar sistemas de defensa (los denominados sistemas
antioxidantes) cruciales para el bienestar celular; el
incremento excesivo de especies reactivas del oxígeno está
implicado, tanto en el envejecimiento fisiológico, como en
procesos morbosos tan dispares como la aterosclerosis, la
fibrosis quística, el Alzhéimer o incluso el dolor crónico. (3)
El estrés oxidativo es inevitable cuando la vida se
desarrolla en un medio rico en oxígeno, y cuando los procesos
antioxidantes no son suficientes o se ven desbordados la
célula puede entrar en una situación crítica que le lleve a
la muerte programada, o apoptosis. De hecho, se sabe
que muchos agentes inductores de apoptosis son oxidantes
o dan lugar a procesos de estimulación del metabolismo
oxidativo celular, al igual que muchos inhibidores de la
apoptosis tienen carácter antioxidante. (4) La célula, para
conservar y mantener la salud, necesita mantener un estado
saludable en su equilibrio oxidante-antioxidante, de lo
contrario puede caer enferma, arrastrando consigo al
sistema orgánico que la sustenta.
Lo deseable para cualquier átomo o molécula es tener
estabilidad, lo que supone presentar en su orbital más
externo dos electrones apareados que giren en sentidos
opuestos. Un radical libre es una especie química (átomo
o molécula) inestable por presentar un electrón desapareado
en su última capa; el tener un número impar de electrones
hace que el radical esté deseoso de completarse y que
tenga avidez por “soltar” el electrón sobrante o “aceptar”
un electrón desde otro elemento. Los radicales libres, en
busca de su estabilidad, inician reacciones en cadena que
se propagan con el intercambio electrónico de una molécula
a otra, formándose así una cadena de nuevos radicales
hasta que terminan por unirse entre sí dos compuestos de
naturaleza radical, que compartiendo sus electrones de
última capa generan un nuevo compuesto estable no radical.
En su avidez por estabilizarse, los radicales,
altamente reactivos, se adhieren a moléculas presentes en
el medio celular e interfieren la dinámica de las reacciones
químicas, lo que dará lugar a disfunciones celulares y
enfermedades. La vinculación del estrés oxidativo con
enfermedades en las que el sistema inmune resulta deficiente
ha sido sobradamente contrastada, y la función distorsionada
de los infiltrados de linfocitos, macrófagos y neutrófilos da
lugar a una sobreexpresión del estallido inflamatorio, con
exceso de especies reactivas del oxígeno, hiperproducción
de proteasas y daño orgánico. Además, la hiperactividad
inflamatoria cursará con niveles excesivos de sustancias
algogénicas como la sustancia P, el péptido relacionado
con el gen de la calcitonina, la bradiquinina, las
prostaglandinas o los leucotrienos. (5) Partiendo de que se
trata de enfermedades relacionadas con el estrés oxidativo
que a menudo cursan con dolor, del papel del ozono como
estabilizador del sistema inmune (6)(7) y de la inofensividad
de la técnica, algunos autores han difundido series de casos
con resultados no poco interesantes (8)(9)(10)
De todas maneras el propio organismo produce
radicales libres y otras especies reactivas del oxígeno; como
ejemplos tenemos la síntesis de anión superóxido (O.-) en
la vacuola neutrofílica durante el estallido inflamatorio,
fundamental para la acción germicida, e incluso la síntesis
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de ozono en éste mismo proceso ha sido sugerida. (11) Los
radicales libres son necesarios para la vida, y un nivel
adecuado de estrés oxidativo es beneficioso por ser un
estímulo propicio para que las células mantengan un sistema
antioxidante adecuado.
Como se ha dicho, la implicación de las especies
reactivas del oxígeno en multitud de enfermedades es una
realidad ampliamente aceptada; el ozono, actuando como
un activador biológico, ha demostrado ser capaz de activar
el sistema antioxidante celular e invertir el estrés oxidativo,
fruto del desequilibrio entre los factores prooxidantes y
antioxidantes. (12)
3.- GLUTÁMICO, HIPEREXCITABILIDAD NEURONAL, ESTRÉS
OXIDATIVO Y NOCICEPCIÓN.
La génesis de un impulso es de carácter puramente
eléctrico y la comunicación entre neuronas es, en la mayoría
de los casos, de tipo químico. La señal que surge en los
receptores periféricos (terminaciones libres para el dolor)
se propagan a lo largo del axón en el seno de la misma
neurona, que comienza a sintetizar una serie de sustancias
de tipo excitatorio tales como el glutámico, la sustancia P
ó el PRGC (péptido relacionado con el gen de la calcitonina).
El glutámico, neurotransmisor excitatorio universal,
se encuentra almacenado en la terminal presináptica de la
neurona, en el seno de unas vesículas, y está en espera de
que se desencadene un potencial de acción que lo libere
al espacio sináptico. La llegada del potencial despolarizador
provoca la entrada de iones Ca++, condición indispensable
en el proceso. Bloqueando experimentalmente los canales
del Ca++ se consigue que el neurotransmisor no se libere,
aunque el potencial de acción esté presente.
Una vez tenemos al glutámico en el espacio sináptico,
éste puede ser recaptado de nuevo, tanto hacia la misma
neurona que lo liberó como hacia una célula glial vecina.
En ambos casos se necesita una bomba de transporte que
trabaja con un aporte energético supletorio. Cuanto más
glutámico haya en la sinapsis, más cantidad de
neurotransmisor se acoplará a los receptores postsinápticos.
En concreto para el glutámico se definen varios
receptores (AMPA, KAINATO, NMDA...), y la respuesta al
producirse el acoplamiento neurotransmisor–receptor dará
lugar a la excitación de la neurona postsináptica, que
interpretará una orden determinada. Los NMDA son
receptores ligados a canales iónicos para el Ca++, cuya
apertura facilita la entrada del ión con resultados excitatorios.
El glutámico se acopla en el borde extracelular de los
receptores y actúa abriendo el canal, lo que produce una
corriente de iones Ca++ fluyendo hacia el interior celular
(excitación). (Figura 1) Puede ocurrir que la respuesta
neuronal sea tal que la entrada de iones Ca++ sea excesiva,
ó se perpetúe en el tiempo, en cuyo caso la respuesta al
estímulo puede resultar exagerada. Estos fenómenos son
cruciales en los mecanismos de excitabilidad, y el incremento
espaciotemporal de la actividad celular resulta en un
exceso de radicales libres que pueden no ser adecuadamente
tamponados, favoreciendo la apoptosis o el desarrollo de
fenómenos de facilitación central y neuropáticos, lo que en
clínica explicaría los procesos de hiperespuesta al estímulo
(alodinia e hipersensibilidad).
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Una neurona puede llegar a excitarse tanto que
acabe destruida por la toxicidad de los radicales libres
generados (excitoxicidad). Supongamos una activación de
los NMDA con entrada masiva de Ca++ en la célula; la
neurona responde y la ingeniería nuclear se pone manos
a la obra, por ejemplo, codificando la síntesis de más
receptores NMDA. Si estos NMDA se activan la excitación
celular aumentará y la maquinaria celular se verá más
forzada, con una mayor producción de “desechos” tóxicos
para la célula (especies reactivas de oxígeno) cuyo acumulo
puede ser fatal. (Fig. 2)
De acuerdo con lo expuesto la literatura es limitada, pero
recientemente se han publicado trabajos relacionando el
estrés oxidativo a nivel neuronal con los procesos de dolor
neuropático. En el modelo de la diabetes mellitus tipo II,
donde la efectividad de la ozonoterapia sistémica ha sido
constatada (tanto a nivel preclínico como clínico) (13) Hayden
y Tyagi (2004) exponen cómo los fenómenos de toxicidad
metabólica dan lugar a la formación de múltiples especies
reactivas de oxígeno. (14) Se admite que en el desarrollo
de la polineuropatía diabética toman parte diversos factores:
metabólico, vascular, autoinmune, alteración del factor de
crecimiento neuronal y remodelación de la matrix extracelular
neuronal; en el trabajo que mencionamos se relaciona cada
uno de estos fenómenos con el estrés oxidativo. Más
recientemente, en un modelo de neuropatía inducida por
ligazón en el nervio ciático de rata, Di Cesare y
colaboradores (2007) constatan la existencia de fenómenos
apoptóticos que se revierten con la administración de acetilL-carnitina, de efecto neuroprotector y regulador de la
muerte celular en el nervio dañado. (15)
Por definición, el envejecimiento celular es el acumulo
de especies reactivas del oxígeno relacionado con el
deterioro en las capacidades antioxidantes. El fenómeno
de envejecimiento se ha relacionado con el estrés oxidativo
en el sistema opioide en estudios preclínicos. Se ha observado
una correlación negativa entre la antinocicepción por morfina
y la oxidación proteica en las células del cortex, núcleo
estriado e hipocampo, y los mismos autores demuestran
cómo la capacidad analgésica inducida por fentanilo
decrece conforme se registran marcadores elevados de
estrés oxidativo. (16)
4.- APLICACIONES SISTÉMICAS DE OXÍGENO-OZONO.
GENERALIDADES.
Aparte de las aplicaciones tópicas o la infiltración
de la mezcla de gases (frecuentemente empleada en dolores
musculares y osteoarticulares), el objeto de este capítulo es
la ozonoterapia sistémica, cuyos efectos se proyectan de
forma simultánea sobre la totalidad del organismo para
conseguir estimular las defensas antioxidantes. La
administración de la mezcla de gases se puede llevar a
cabo fundamentalmente mediante dos vías: la
autohemoterapia y la insuflación rectal. La autohemoterapia
puede ser “mayor” o “menor”; la autohemoterapia mayor
consiste en la extracción de una cantidad determinada de
sangre, que sin salir de un circuito cerrado es puesta en
contacto con la mezcla de gases, con los que reaccionará
hasta la dilución de los mismos; tras unos minutos la sangre
se reinfunde. En la llamada “autohemoterapia menor” la
mezcla tiene lugar en una jeringa, y la sangre ozonizada
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se inyecta por vía intramuscular. (Foto Autohemoterapia)
Mediante la insuflación rectal la mezcla de gases
es amplia y rápidamente absorbida a través de la mucosa
intestinal; esta técnica, fácil de aplicar, muy segura y
cómoda, es de elección, tanto en los trabajos preclínicos
sobre ratas como en numerosos estudios clínicos en humanos.
(Foto Insuflación rectal)
Es una realidad el hecho de que las aplicaciones
sucesivas de ozono médico a nivel general (sistémicas)
redundan en un aumento significativo del aporte de oxígeno
en todas las células del organismo, lo que supone, a grandes
rasgos una mejora en la capacidad celular para auto
repararse (17)
La acción reparadora del ozono ha demostrado
ser capaz de recuperar la pared interna de los pequeños
vasos sanguíneos, y una constatación de esta realidad son
los excelentes resultados publicados recientemente, en un
ensayo clínico aleatorizado, en una prestigiosa revista
europea de farmacología, donde las recuperaciones de
úlceras en pacientes diabéticos son altamente significativas.
(18) También se ha podido demostrar el efecto beneficioso
de este gas sobre otro, el óxido nítrico, crucial en mantener
a niveles óptimos la dilatación de las arterias, y por lo tanto
el flujo de sangre a nivel de todo el organismo. (19)
Hoy podemos asegurar que mediante esta terapia,
exenta de todo riesgo, decrece sustancialmente el daño
celular por falta de oxigenación, independientemente de
la enfermedad subyacente. Además los productos fruto de
la descomposición del ozono se comportan como activadores
biológicos, lo que mejora el nivel de energía y la capacidad
de nuestro sistema defensivo, en beneficio de enfermedades
de tipo alérgico-autoinmune como pueden ser la psoriasis,
el asma o la artritis reumatoide. (20)(21)
Se ha demostrado científicamente que las
aplicaciones controladas de ozono médico mejoran la
maquinaria antioxidante celular al haberse medido en el
interior de las células cantidades superiores de agentes
antioxidantes, tales como el glutatión reducido o la superóxido
dismutasa. (22) Como consecuencia directa el ozono actúa
como un verdadero “basurero” celular, limpiando los radicales
libres. De acuerdo con este concepto, la ozonoterapia
preventiva tendría un “efecto antienvejecimiento celular”.
Según los estudios en hígado y riñón de rata, cuando
un organismo se preacondiciona con ozono rectal, el daño
después de sufrir un accidente tóxico o isquémico será
bastante menor, y esto se avala por seguimientos clínicos
en pacientes con accidentes isquémicos cerebrales. (23)(24)
Lo importante de este concepto es que avalaría, tanto las
aplicaciones preventivas en condiciones de riesgo
cerebrovascular (25)(26) como los tratamientos tempranos
del dolor isquémico.
Trabajos de excelente rigor y calidad pueden leerse
actualmente en revistas tan prestigiosas como Nature,
Transplant Internacional, Shock, Free Radicals, Mediators
of Inflamation, Internacional Journal of Pharmacology,
Pharmacological Research, Liver International, etc. Incluso
en la revista de la Sociedad Española del Dolor se dedicó
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recientemente un monográfico completo sobre las
capacidades del ozono médico y su utilidad en el tratamiento
del dolor.
FIGURA 2
Aunque es deseable un mayor nivel de evidencia, sí se
conoce que la ozonoterapia debidamente aplicada no tiene
ningún riesgo y es una realidad médica cuyo uso puede
justificarse como tratamiento paliativo en medicina del dolor,
aunque siempre complementando a la medicina clásica, con
la cual no interfiere. Aunque escasos, los artículos científicos
sobre ozonoterapia en tumorales no deben ser desdeñados.
(27)(28)(29)(30)(31)(32)
FIGURA 1
AUTOHEMOTERAPIA
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37
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artículos nacionales
hipertrofia benigna de próstata (HBP)
tratada con ozonoterapia
Dr. José Faus Victoria
Gandía - Valencia (SPAIN)
email: drfaus@ono.com
SUMARIO:
Este articulo refleja mi experiencia sobre la infiltracion
de ozono en la glándula prostática. Los resultados
son alentadores en esta afeccion que aflige a gran
parte de la poblacion masculina entre 45-50 años
de edad.
La técnica usada viene de la Terapia neural
descubierta y promulgada por los hermanaos Huneke.
Segun la teoria de la Terapia neural, todas las
glandulas organicas inflamadas pueden constituir
un “campo de interferencia” que se manifestará a
nivel local o a nivel general. La infiltracion de la
misma próstata, hecha ya sea con Procaina u otra
sustancia repolariza las membranas celulares
haciendo desaparecer dicho campo de interferencia.
Invito a todos los medicos ozonoterapéuticos a que
la practiquen sin miedo pues el riesgo es minimo y
es muy simple de aplicar.
La próstata crece con la edad del hombre. En la pubertad
duplica su tamaño y va creciendo progresivamente a partir
de los 25 años. El peso de una próstata adulta está entre
20-25 gr. Raramente provoca problemas antes de los 40
años, pero a los 60, la mitad de los hombres sufren las
consecuencias de su exceso de tamaño (puede llegar a 100
gr o mas), y a los 70 casi el 90 %, por no decir que todos
los hombres tienen problemas de próstata.
Durante su crecimiento, los tejidos circundantes impiden su
crecimiento por lo que comprimen la uretra impidiendo el
flujo normal desde la vejiga. Ante esta estenosis, la vejiga
debe de contraerse con más vigor y al final se debilita.
Los síntomas que provoca la hipertrofia prostática los
resumimos
1. Dificultad del chorro con goteo en la última fase de la
micción, con sensación de insatisfacción (disuria). Interrupción
del chorro urinario
2. Urgencia en ir al baño y con mas frecuencia (polaquiuria)
3. Posibles infecciones orina por vaciamiento incompleto de
la vejiga (cistitis-prostatitis)
4. Problemas en la eyaculación e incluso en la erección
(impotencia)
5. Posibilidad de retención completa de orina con el
consiguiente globo vesical (estadios finales)
Paradójicamente, algunos hombres con grandes crecimientos
prostáticos presentan menos síntomas comparados con otros,
con menores crecimientos. Con ello manifestamos que no
necesariamente a mayor crecimiento hay más obstrucción
En EEUU hubo 4,5 millones de visitas por HBP (hipertrofia
benigna de próstata) en el año 2000. Posiblemente estas
cifras irán en aumento, dada el aumento de expectativa
de vida alcanzada.
La dificultad en la micción constituye de por sí un factor
importantísimo en calidad de vida de los hombres por
arriba de los 60 años. El buen funcionamiento vesicoprostático implica no solo la micción sino que además la
calidad en el contexto sexual y existencial (Peters 2001)
Welch-Weinger-Barry (2002) en un estudio efectuado sobre
8.406 hombres destacaron que hombres con sintomatología
moderada-severa en ésta zona, tienen un status de salud
mas deteriorado que aquellos pacientes afectados de gota,
hipertensión arterial, angina pectoris o diabetes mellitas.
Al respecto Rosen (2003) en el estudio denominado
Multinational Survey of the Aging
Male MSAM-7 publicó un excelente estudio donde menciona
que más del 50% deM hombres de 50 o mas años presentan
disfunción sexual como consecuencia de presentar síntomas
urinarios bajos
¿Por qué se produce la HPB?
El crecimiento prostático dependerá del desbalance entre
la proliferación celular y muerte celular (apoptosis) tanto
en el compartimiento estroma como del epitelio. Esto se
manifiesta más a nivel periuretral y de transición. Se habla
de una proporción de crecimiento estroma/epitelio de 2,7
+- 0,1, en un hombre asintomáticos mientras que en los que
presentan síntomas obstructivos alcanza a ser 4,6 +-0,3
(Shapiro, 1992)
A lo largo de la historia se ha asociado la HBP al
envejecimiento del hombre. Este fenómeno no ocurría en
aquéllos a quienes les extirparon los testículos antes de la
pubertad. Por esta razón, algunos investigadores creen que
los factores relacionados al envejecimiento y los testículos
pueden ser los detonantes en el desarrollo del crecimiento
prostáticos. Con el envejecimiento, la cantidad de testosterona
activa en la sangre disminuye, dando paso a una mayor
proporción de estrógenos. Estudios realizados en animales
sugieren que la HPB puede ocurrir debido a que los altos
niveles de estrógeno dentro de la glándula aumentan la
actividad de sustancias que promueven el crecimiento celular.
Otra teoría enfoca hacia la dihidrotestosterona (DHT), una
sustancia derivada de la testosterona en la próstata, que
puede ayudar a su crecimiento. La mayoría de animales
pierden su capacidad de producir DHT con la edad. Sin
embargo, algunas investigaciones indican que aún con una
caída en los niveles sanguíneos de testosterona, los hombres
ancianos continúan produciendo y acumulando altos niveles
de DHT en la próstata. Esta acumulación de DHT puede
promover el crecimiento celular. Los científicos han notado
también que los hombres que no producen DHT no desarrollan
HPB.
En algunos casos un hombre puede no saber que tiene
obstrucción urinaria hasta que súbitamente presenta dificultad
total para orinar. Esta situación, llamada retención aguda
de orina, es provocada tras la toma de medicamentos
para la gripe o alergia (pseudoefedrina o fenilefrina
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simpaticomimético bloquea la relajación del cuello de la
vejiga y evitar el vaciamiento urinario.
Con respecto a la actividad sexual se ha especulado que
la abundancia favorece, pero tambien la escasez. La
actividad sexual NO TIENE ninguna relación con el desarrollo
de esta enfermedad. Tampoco se ha podido demostrar
consistentemente, que algún alimento favorezca este
problema. Sin embargo si puede agravar la BPH con la
ingesta de alcohol, temperaturas frías o largos periodos
de inmovilidad, en especial sentado.
Es importante hacer el diagnostico diferencial entre cáncer
e hipertrofia benigna de próstata. El cálculo de PSA (proteín
specific antigen) nos sacará de dudas.
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TRATAMIENTO FARMACOLÓGICO
Medicamentos bloqueantes de los receptores al faadrenérgicos (tamsulozina, alfuzosina, doxazosina y
terazosina )
Inhiben la excesiva actividad alfa-adrenérgica existente
por el aumento del tono simpático. Producen pues, una
relajación del músculo liso contenido en el estroma prostático.
El músculo liso representala prostática hipertrofiada y
benigna y el 45% de su constitución cuando se trata de
glándulas prostáticas sanas.
Medicamentos bloqueantes de la enzima alfa- reductasa
(finasteride,dutasteride)
Son potentes inhibidores reversibles de la 5-alfa reductasa.
Al inhibir la 5-alfa reductasa se bloquea la conversión de
testosterona a dihidrotestosterona (DHT) el andrógeno
intraprostrotatico en el hombre responsable no sólo del
crecimiento prostático benigno sino también de los proceso
neoproliferativos del cáncer prostático
Relajantes muscarinicos por un componente de hiperactividad
vesical (Oxibutinina, Tolterodine)
Diagnóstico
Además de la clínica sugestiva de obstrucción prostática se
ha practicar el tacto rectal. También se debe de evaluar
la función renal con pruebas de sangre, así como la medición
de la orina residual por medio de los ultrasonidos. También
es importante la detección precoz de un posible cáncer de
próstata porque este es asintomático y puede asociarse
con la BPH.
Tratamientos clásicos
La cirugía prostática abierta ha dado paso a la
prostatectomía transuretral. No obstante como dato curioso
Kaplan 1995 demostró que la mayoría de hombres con
prostatismo prefieren alternativas terapéuticas no quirúrgicas
en lugar de una resección prostática transuretral y luego
de 1 año de evolución estaban satisfechos con su elección
terapéutica.
Debo de hacer mención del tratamiento por el Láser verde
desarrollado por la Clínica Mayo (Rochester-MinnesotaUSA) desde 1998. Dicho Laser emite una luz verde con una
longitud de onda de 532 nm y una potencia de 120 vatios
que permite vaporizar el tejido prostático que comprime
el conducto de la orina (uretra). El paciente experimenta
una inmediata mejoría del flujo urinario.. Al retirar la sonda
vesical a las 12 horas, el paciente orina con un excelente
flujo miccional y puede marcharse a su casa. Los resultados
se mantienen a largo plazo y paciente ha requerido una
nueva intervención por hiperplasia benigna de la próstata.
Son Medicamentos que relajan el detrusor en hiperactividad.
Los agentes anticolinérgicos logran que se retarde la
sensación inicial de
urgencia urinaria, inhibiendo las contracciones involuntarias
de la vejiga
que necesiten terapias a largo plazo. Los usuales efectos
colaterales de los anticolinergicos son: sequedad de las
mucosas, particularmente de los ojos, sensación de boca
seca, cefaleas, estreñimiento, taquicardia, confusión …..etc
hacen bastante difícil su prescripción .
Fármacos bloqueadores de impulsos periféricos
a través de los receptores purinergios,
opiáceos.
Tendrán su lugar asegurado en la terapéutica de los estados
hiperactivos vesicales ya que bloquean los impulsos aferentes
1-Toxina botulínica:
Inhibe la acetilcolina disminuyendo la contractilidad vesical
y produciendo atrofia muscular en el sitio de la inyección.
El proceso es reversible y los axones se regeneran en tres
a seis meses. La molécula de toxina botulínica no atraviesa
la barrera hematoencefálica y por ende no tendrá efecto
sobre el sistema nervioso central. Interés en discinergia
vesico-esfinteriana, esclerosis múltiple…etc
Chuang (2005) revela que la toxina botulínica inyectada
dentro de la
Glándula prostática tiene visos de convertirse en un
tratamiento promisorio en pacientes con crecimiento prostático
benigno sintomático.
Gonadotrofina coriónica humana
Puede actuar directa o indirectamente sobre la glándula
prostática y tejidos asociados a través de mecanismos
independientes de el bloqueo alfa-1- adrenérgico o
independiente del mecanismo de inhibición de la 5-alfa
reductasa
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Fitoterapia (Medicina Complementaria o
Natural)
La creciente utilización de estos productos alternativos en
los últimos años en todas partes del mundo ha sido motivada
por el sentimiento creciente de rechazo
hacia otras formas de terapia, bien sea médicas o
mínimamente invasivas o propiamente quirúrgicas así como
a los resultados no adecuados obtenidos con éstos tipos de
tratamientos. Pero otras de las razones de su amplio uso es
que no se necesita prescripción médica para su compra así
como al hecho de que estén etiquetados para promocionar
la salud prostática así como rodeados por un aureola de
seguridad por el hecho de ser productos naturales y quizás
el factor mas importante de su uso masivo es que se vende
en lugares no acostumbrados de venta de productos para
la salud como por ejemplo automercados tiendas de
suplementos nutricionales y parafarmacias
El Ginseng asiático
Utilizado para la astenia psicofísica con el objetivo de
mejorar la resistencia física y mental podrá ocasionar
hipertensión arterial y alterar los tiempos de sangrado.
Nunca se deberán administrar con corticosteroides o
warfarina sódica o estrógenos ni con vitamina E.
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los leucotrienos y otros metabolitos de la 5-Lipooxigenasa
.Dentro de los principios activos que conforman el Pygeum
africanum tenemos los esteroles, los terpenoides y los
alcoholes vegetales que mejorarán los síntomas urinarios
porque inhibirán los factores de crecimiento betafibroblástico (TGFb) y los factores de crecimiento epidermico
EGF que inducirán la proliferación fibroblástica disminuyendo
la inflamación y el edema. Efecto antiestrogénico, debido
a que inhibe la Aromaterasa que convierte la Testosterona
a la a la androsten y a estradiol.
Isoflavonas de soja
Las isoflavonas de la soya comprenden tres grupos principales
y sus formas
Glicosiladas: genisteina, daidzeina y la gliciteina. Los
Isoflavonas o Fitoestrogenos son compuestos no esteroideos
derivados de las plantas que poseen actividad biologica
como los estrógenos. Actúan compitiendo a nivel del receptor
estrogénico pero su actividad estrogénica es bajo. Inhiben
de la 5-alfa reductasa Estados Unidos de Norteamerica
fallecen 4 a 5 veces más hombres por cancer de
prostata que en Japon debido a su alimentación rico en
soja.
Kava®
Utilizado para el stress la ansiedad y el insomnio podrá
ocasionar alteración de los reflejos motores de la capacidad
cognoscitiva y podrá aumentar los efectos producidos por
el consumo alcohlico y los barbitúricos. También podrá
causar dermatosis ictericia e incoordinación motora.
Urtica dioica
Tiene efecto de inhibir los factores de crecimiento o inclusive
la interacción de dichos factores de crecimiento y por último
se le asignan también efectos de inhibición
del metabolismo de la celula prostáticas así como su
crecimiento por acciones sobre
dicha membrana a nivel del sodio, potasio y la ATPasa
Serenoa repens (Saw Palmetto)
Metodología para la punción e infiltración de ozono
Es la más utilizada y la más conocida. Puede inducir apoptosis
o muerte celular programada así como inhibición de la
proliferación celular lo cual lleva a una reducción en el
volumen prostático y a una mejoría sintomática y de la
uroflujometra en el estudio urodinamico (Vacherot 1999).
La Serenoa repens lo podemos conseguir individualmente
o unido a formulas prostáticas combinado con bioflavonoides,
licopeno daidzeina genisteina selenio, vitamina E, entre
algunos de los principios herbarios y vitamínicos mas
conocidos.. etc.
Como efectos colaterales están las molestias gástricas
El paciente en decúbito supino y con las rodillas dobladas
se rebate con las manos los testículos hacia el abdomen. El
médico enfrente con el dedo índice de la mano izquierda
localiza la próstata a través del ano (como si de una
exploración rutinaria se tratara). Con al otra mano pincha
a través del rafe del periné buscando imaginariamente la
próstata localizada.
Pygeum Africanum
Dada la sencillez de su práctica y la mejora rápida de los
síntomas que ocasiona, recomendamos a todo profesional
médico que se enfrente a este tipo de patología, en especial
los urólogos.
Fitofarmaco utilizado en Europa debido a sus propiedades
de reducir la inflamación en el área prostática y de mejorar
sustancialmente los síntomas asociados al crecimiento
prostatico benigno Andro (1995), Pansadoro (1993). Entre
sus propiedades se cuentan la inhibición de los factores de
crecimiento fibroblástico tiene efectos antiestrogenicos inhibe
Dosificación: La dosis oscilará entre 30 -40 mcgr/ml,
dependiendo del nivel de stress oxidativo que presente el
paciente.
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PREMISAS HIPOTÉTICAS
1. El ozono promueve una activación en la síntesis de las
enzimas involucradas en la eliminación de los radicales
libres (glutatión peroxidasa, catalasa y súper oxidodismutasa.
2. El ozono tiene afinidad especial por los dobles enlaces
de los fosfolípidos de las membranas provocando la escisión
de los ácidos grasos (ozonolisis) (Criegge, 1973; Srisankar
y Patterson, 1979). Dichos ácidos grasos insaturados quedan
“partidos” en forma de peróxidos, con menos átomos de
Carbono, convirtiéndose en hidrófilos (“amigos del agua”),
y con esto se facilita su eliminación».
3. El ozono desarrolla todo su poder a través de sus
metabolitos: los peróxidos. ( Dr. Frank Shallemberger)
4. Teniendo como sustrato el Acido Araquidónico presente
en las membranas, la cicloxigensa o prostaglandinsintetasa
y la lipoxigenasa son enzimas microsomales que catalizan
la inserción de O2 en varias posiciones en el ácido
araquidónico libre, promoviendo la formación de
Prostaglandinas, tanto las pro como las anti-inflamatorias
5.
Las prostaglandinas son mediadoras de la
transmisión del mensaje que las hormonas troficas como la
LH, la TSH y la ACTH producen sobre las células efectoras.
Estas a su vez probablemente, actuando sobre unos receptores
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CONCLUSIONES
Teniendo en cuenta que el aumento de tamaño “benigno”
de la próstata (hipertrofia) se debe a un acúmulo de
metabolitos o radicales libres en el estroma que mantienen
un estado inflamatorio “larvado” tanto del estroma como
del epitelio celular, la acción del ozono “licuando” dichos
metabolitos sería beneficioso. Asimismo el ozono por el
mecanismo de la ozonolisis a nivel de las membranas
aumenta la síntesis de PGs a partir del acido Araquidónico
del tipo 2 por mediación de la cicloxigenasas facilitaria
la acción mediadora del influjo de las hormonas en dichos
tejidos. Así como la liberación de citokinas promoverían la
activación del sistema inmunitario.
Todo ello, en conjunto, facilitaría su reducción y evitaría la
obstrucción a la hora de lo micción
BIBLIOGRAFIA
Dr Julio Cesar Potenziani Bigel l i *Especial ista Uro logo Hospital Privado
Centro Médico de Caracas
Dr. Rafael F. Velazquez Macias (Vias urinarias y sexualidad)
h t t p : / / w w w. u ro c i r u g i a . c o m / U ro n o ve d a d e s / u ro n _ h p o. h t m
Ziada A. Rosemblum M, Crawford E, Benign prostatic hyperplasia: An
overview. Urology .Mar 1999
Luis W. Lu, MD, FACS.Director, Elk County Eye Clinic
S e n i o r S t a f f M e m b e r, Pe n n s y l va n i a E ye C o n s u l t a n t s
. untyeyeclinic.com
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artículos nacionales
ozone therapy for tumor oxygenation:
a pilot study
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artículos nacionales
ozone therapy in the functional recovery
from diseases involving damage to
central nervous system cells.
PhD. GOMEZ MORALEDA, M.A.
(MEDOZONO S.L. SPAIN)
Paper presented at the 12th World
Congress of the International Ozone
Association. MAY/95, LILLE, FRANCE
Abstract
On the basis of personal experience during several years
in preclinical and clinical trials, sets of results are presented,
concerning the Ozone Therapy treatment of different groups
of subjects suffering from various diseases which involve
degenerative or ischemic damage of central nervous system
cells (Senile Dementia, Cerebrovascular Ischemia, Hipoacusia,
Optic Nerve Dysfunctions, Sequels of Glaucoma and Retinitis
Pigmentosa).
Regarding previously mentioned results, some interrelated
hypothesis are formulated to explain the different effects
achieved in functional recovery of patients with the Ozone
Therapy. These are based in some of the already known
effects of ozone on living cells, so as in the cells physiology
of those subsystems of the central nervous system involved
in each disease.
INTRODUCTION
Ozone has been widely applied in many diseases caused
by peripheral circulatory disturbances, infections, alterations
of the immune system, aesthetics, etc., but less attention has
been paid to research about nervous cells damage produced
by degenerative and ischemic diseases affecting subsystems
of the central nervous system (CNS), so as by other causes
as traumatic or compressive events, etc.
Several studies were performed in Cuba, under the scientific
direction of the author in the National Centre for Scientific
Research, Ozone Research Centre and in various Hospitals
and Health Institutes, concerning different diseases with their
common basis on Central Nervous System dysfunctions. I
would like to emphasize special recognition to all valuable
professionals participating in the individual clinical trials
mentioned in present paper.
Present paper shows a brief summary of these diseases,
some of their main characteristics, and the most important
positive results achieved after the ozone therapy treatments
of groups of patients suffering from them. Ozone was
administered to patients by systemic way, mainly by Major
Autohemo Therapy, in dosis over 5.000 µg/session. A set
of hypothesis is also proposed to explain such results.
MAIN TEXT
DISEASES:
• Retinitis Pigmentosa (RP) . RP is a
degeneration of the retina which primarily affects
photoreceptors and pigmented epithelium, being the main
symptoms: night blindness, followed by a progressive reduction
of the visual field (Daugman, 1986, DukeElder, 1984). It is
hereditary in nature, and can be transmitted under different
autosomal dominant, autosomal recessive, xlinked and
sporadic. Visual alterations generally start in the medium
periphery, provoking an annular scotoma, which evolves
towards tubular vision and finally complete blindness
(Márquez, 1962, Wirt, 1984).
The histopathology of this disease (Marquez 1962, Daugman,
1986 and Scuderi & Moreno, 1986) show vascular alterations
due to retinal vessels attenuation. Also choroidal sclerosis
was reported, which contributes to tissue anoxia and leads
to atrophy of retina layers. Nevertheless, drugs with
oxygenating action on the CNS tissues and the retina, used
so far, did not give satisfactory response to treatments.
Other theories (Wirt, 1984, DukeElder, 1984 and Vodovozov,
1986) invoked encephalon disturbs, immunological alterations,
lack of vitamins, etc. Nevertheless, therapeutics directed to
these causes did not give the expected results as well.
• Optic Nerve Dysfunction (OND). One
of the most frequent causes of blindness around the world
is OND, or optic atrophy (OA), which can be the result of
different disturbances affecting the visual pathway, like
ischemic, toxic, metabolic, hereditary and degenerative
phenomena, injuries or compression, among others, producing
this type of irreversible sequel.
Today, neuroplasticity is recognized as a nervous tissue
property in spite of neurons incapacity for multiplication
(BachRita, 1980, Estrada, 1988 Boisson, 1990). Peripheral
nervous system possibilities of regeneration are well known
and recent research emphasized this property, though in
lower degree, in the CNS (Varon et al.,1986, Aguayo et
al.,1986, Aguayo et al.,1985).
Optic nerve (ON) as a part of the CNS is deprived of myelin
sheath and has been the target of some experimental work
on neuroplasticity. Aguayo et al., in 1986 showed the ON
axons possibilities of regeneration in rats when they are
able to make appropriate contact with the coliculli cells,
through peripheral nerve segment grafts.
Neurotransplantology is gaining ground at present, but
investigations on the ON injuries are still in animal
experimental stage (1989, Carmignoto et al., 1989, Sievers
& Hausmann, 1989) and no conclusive results have been
reported in man. In the process of the OND not all nerve
fibers are fully degenerated and this depends on the intensity
of the injury, time of evolution, glial reactions, etc. Such
criteria are taken into consideration to try to develop
different therapeutic methods, including hyperbaric
oxygenation and photic and electric stimulation, with poor
effect up to date.
• Primary Open Angle Glaucoma (POAG).
It is, according to etiology, a multi factorial chronic disease,
suffered by approximately 2% of population over 40 years
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(Speath & Jandra,1989, Tielsch et al., 1991). It is associated
to diseases with large vascular component (Kashintseva,
1986).
The characteristic pathologic rise in intra ocular tension,
joined to the circulatory deficiency and the metabolic changes
in retina and O.N. deteriorate the nervous function, leading
to the glaucomatous optic atrophy. Changes in circulation
cause the variation of the blood rheological properties,
hypoxia and modifications in the oxygen metabolism, which
in sum deprive the metabolism and produce alterations in
ocular tissue trophism (Kashintseva & Kribonski, 1979, Mukha,
1990).
Experimental models of glaucoma show destructive damage
in ocular vessels and tissue, mainly in the draining system,
provoking not only ocular tension alterations, but also altered
metabolic processes (Conde & Gurtobaya, 1977, Nesterov,
1982).
Some years ago, in addition to the local hypo tensors,
pharmacologic complexes were attempted to improve
haemodinamics and restore the normal oxidation processes
in ocular tissue, with the aim of to stimulate the deteriorated
nervous function (Conde, 1977). They exhibited poor evidence
of improvement and no long lasting effects.
• Senile Dementia (SD). SD is probably one
of the most fearful central nervous system diseases for the
aging and mostly the worse managed from the clinical point
of view, still aggravating its prognosis, not only for the
patients but also for families and society, in which they live.
Classification scale of senile dementias recognized as
"incurable", published by Hachinsky and in agreement with
other authors (Smith & Kiloh, 1981) was applied, where
Alzheimer dementia constituted approximately 60 % of
cases, those of vascular nature represented 20 % and mixed
types (vascular - degenerative), the remaining 20 %. It
allowed studying "incurable" dementias, excluding all those
cases potentially reversible and different in nature, which
in elderly patients can be expressed as pseudo demential
state.
In geriatrics, the concept of health is defined in terms of
function, a healthy elderly is not that one, who does not
suffer from any pathology, but one who is autonomous. The
fundamental objective for incurable SD treatments (of
vascular or degenerative etiology) is to increase the autonomy
of elderly, especially in regards to mental condition, self
medication capacity and daily life activities performance,
giving special attention to multidimensional evaluation and
validity.
Lot of drugs have been tested throughout the time for
therapeutics in this disease, from psycho pharmacs to neural
metabolic activators, including, of course, cerebral
vasodilators. Many authors (Pfeiffer, 1980, Greenblatt et
al., 1975, Jenike, 1987, Kane & Smith, 1982, Jenike, 1988)
accept that adverse reactions produced with the use of
these medications must be constantly considered, especially
because no really positive effects have been found.
• Ischemic Cerebrovascular Disease
(ICVD). The majority of these brain processes are in great
extent dependent from, and consequence of different
diseases, as arteriosclerosis, arterial hypertension and
dislipidemies (Gimenes, 1988). Aging is a physiological
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condition, associated with increase in the incidence of the
before mentioned processes, producing an augmentation of
cerebra vascular disease in the third age (Otsfeld, 1980).
In ischemic cerebrovascular disease, reduction in the
oxygenation pattern of brain takes place. Consequently,
normal nutritional cycle of neurons is reversed and anaerobic
glycolisis prevails, producing drop in available ATP for the
energetic cycle of neurons (Heiss, 1983, Heiss et al., 1983).
Several treatments apply for this disease. Some of them try
to avoid the appearance of phenomenon (Devesa, 1992),
while others try to diminish mortality and sequels in those
who already present symptoms. In both groups, some progress
has been achieved, especially in the first group, by means
of appropriate handling of preventive aspects. Results in
the second group are not so promising, specially concerning
sequels.
• Cochleo-Vestibular Syndrome (CVS).
Regarding biochemical and metabolic aspects of some
pathologies of the internal ear, it has been recognized that
one third of patients suffering from neurosensorial bilateral
Hipoacusia present abnormal glucose tolerance curves
(Paparella, 1982). Same author reports audition dysfunctions
in patients under hypoglycemic conditions and diabetes. It
is presumable the important role of glucose in normal function
of cochlear organ, being the perilinfa's glucose concentration
similar to that of serum and knowing that ciliated cells, as
all other somatic cells, involve aerobic metabolism with high
energy yield, stored as ATP.
Ages (Ages, 1989) reports certain metabolic alterations to
occur in internal ear as cochleo-vestibular manifestations of
hypothyroidism, so as in the vertigo syndrome of cervical
origin, exhibiting positive response to cinarizine (Bartual,
1988). Noise induced auditive dysfunction causes lowering
of oxygen tension, as in the Manasse otosclerosis, with
consequent hypoxia in the membranous labyrinth and
accumulation of toxic metabolic detritus, damaging it.
Presbiacusia involves depletion of cellular population, due
to extra cellular deposition of material and accumulation
of toxic substances in the cells.
EXPERIMENTAL RESULTS
Most important positive results achieved by ozone therapy
in the clinical trials performed (Menendez et. al., 1990,
Menendez et. al., 1992, Santiesteban et. al., 1993, Ferrer
et. al., 1992, Devesa et. al., 1993, Rodriguez et. al., 1993,
Basabe et. al., 1992), are as follows:
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DISCUSSION
Ozone presents important properties which make it very
useful in medical field, related to its stimulating effect on
oxygen metabolic processes and blood circulation, where
it modifies the rheological properties of the blood, preventing
erythrocytes from aggregation and increasing their
deformability and permeability. To understand the ozone
action mechanism it is important to consider not only its
possible direct effect as an oxygenating agent, but also a
complex of biochemical processes. It is known that ozone
selectively reacts with the unsaturated fatty acids of the
phospholipidic layer of cell membranes, producing a series
of short chain peroxides "ozone metabolites" with important
functions in the organism.
These include increasing of the oxygen absorption capacity
of erythrocytes as well as its transference to tissue, stimulation
of oxygen metabolization through the reactivation of several
biochemical cycles, activation of enzymatic redox systems
which protect against degenerative processes, and modulating
effect on biological and immune responses. At a first glance,
the mechanisms which could explain the satisfactory
improvements in the majority of patients under the different
clinical trials presented, all of them involving damage to
CNS cells, are neuroplasticity and CNS cells revitalization.
It is already demonstrated in previous reports the ability of
ozone to enhance blood flow and oxygen supply to tissue.
This fact could be part of the effects on the diseases
discussed, but not enough to explain the observed degree
of stimulation of those CNS cells processes mentioned above.
This is supported by the evidence that other drugs with
similar oxygenating effects were applied to the same
diseases with no such positive results.
In the past few years, it has been recognized that in some
diseases affecting CNS cells, at least part of the damaged
and dysfunctional ones could be still alive in some kind of
sub functional state during some time. This opens the possibility
to find a way to reactivate them and restore their functions.
Also neuroplasticity, makes possible the substitution of the
already died cells functions by other neighboring cells, which
in turn should improve their capabilities.
Stimulation of glycolisis by ozone metabolites could be one
of the keys to understand the functional recovery of those
cells severely damaged but still not completely died. These
cells, as a result of hypoxic conditions due to different causes
(ischemia, compression, toxicity, trauma, edema, expression
of inherited dysfunctions, degenerative processes, etc.), could
have been forced to change their metabolism from the
normal aerobic, to anaerobic. It is very well known that
anaerobic glycolisis yield much less energy than the aerobic,
and probably the cells forced to that condition, under lack
of ATP, would slowly die. In those cells not having the ability
to spontaneously change their metabolism to aerobic again,
subsequent increases of oxygen availability would not be
enough to restore normal metabolic pathways, specially
aerobic glycolisis, and the cells, would continue suffering
deterioration, not having energy enough for their vital
functions.
Ozone metabolites, being able to stimulate aerobic glycolisis
in the cells by means of activation of the enzymes involved,
and improving the availability of energy as ATP, would be
the trigger for both: revitalization with functional recovery
of those deprived, but still alive cells, and also neuroplasticity,
it means assumption (partially at least) of the functions of
those already died by adjacent healthy and activated
neighboring cells. These hypotheses agree with the observed
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fact that ozone therapy was more effective in such
diseases, the earlier the treatment was applied, being this
parameter quantitatively related to the survivance of
dysfunctional cells. Not less important is that similar success
was achieved in diseases of different etiology, as is the
case for Alzheimer disease and vascular dementia, O.N.
dysfunctions (except for Optic Leber Atrophy, in which no
improvement was observed) and CVS.
Lack or poor improvement in those patients who were
completely blind or deaf, or presented signs of deep or
complete atrophy, could suggest that good results in the
application of the ozone therapy could be expected when
some degree of the functions remain and atrophy is not
complete. In other words, it seems probable that structural
substratum and or functional reserves are necessary, so that
the neuroplasticity or functional recovery mechanisms could
be activated. This idea would be supported also by the fact
that recovery was faster and more evident in the less severe
or recent cases.
The mechanism of ozone actions in some of these entities
could also be related to its ability to significantly stimulate
other enzymatic processes in cells, some of them vital for
cell protection against degenerative processes, such as
metabolization of aggressive oxygen species and
detoxification. Examples could be degenerative processes
in which detritus of cells are accumulated, so aggravating
the damage to other cells. In such cases, some specialized
cells of the immune systems should also perform their specific
cleaning function, such as macrophages. Some reports about
ozone stimulation or modulation of macrophages, lymphocytes,
etc point out the dose dependent effect of ozone on the
immune system, and establish the marked stimulation which
can be achieved with the appropriate dose of ozone in the
phagocitosis of macrophages.
Regarding RP, for example, it was reported that the cells
of the pigmented epithelium, adjacent to the photoreceptors
layer, have the responsibility for phagocitosis of the segments
of photoreceptors that are periodically rejected from them
when already exhausted. One of the mechanisms proposed
to explain the degeneration of photoreceptors is that the
pigmented epithelium cells in certain extent loss their
phagocityc capacity and rejected segments are accumulated
and decomposed, damaging the photoreceptors themselves
by toxemia. In such process, one of the beneficial effects of
ozone could be the stimulation of the phagocityc capacity
of pigmented epithelium cells to eliminate toxemia and
recover the functionality of photoreceptors. Similar mechanisms
could also take place in the cochlear organ, were similar
damage due to toxemia was already mentioned in the
introduction.
CONCLUSIONS
In our opinion, the ozone treatment, at least, favors certain
reversion of unfavorable conditions of vascular or metabolic
nature in those neurons which are not completely damaged
and/or in some specific adjacent cells which must protect
them and so they could potentially recover their functions,
at least partially.
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artículos internacionales
ozone treatment reduces markers of
oxidtive and endothelial damage in an
experimental diabetes model in rats
SAID MOHAMMED AL DALAIN,
GREGORIO MARTÍNEZ, EDUARDO
CANDELARIO-JALIL, SILVIA MENÉNDEZ,
LAMBERTO RE, ATTILIA GIULIANI and
OLGA SONIA LEÓN
Ozone has been used as a therapeutical agent and beneficial effects have been observed. However
so far only a few biochemical and pharmacodynamic mechanisms have been elucidated. We demonstrate that controlled
ozone administration may promote an oxidative preconditioning or adaptation to oxidative stress, preventing the damage
induced by reactive oxygen species (ROS). Taking into account that diabetes is a disorder associated with oxidative stress,
we postulate that ozone treatment in our experimental conditions might protect antioxidant systems and maintain, at a
physiological level, other markers of endothelial cell damage associated with diabetic complications. Five groups of rats
were classified as follows: (1) control group treated only with physiological saline solution; (2) positive control group using
streptozotocin (STZ) as a diabetes inductor; (3) ozone group, receiving 10 treatments (1.1 mg kg_1), one per day after
STZ-induced diabetes; (4) oxygen group (26 mg kg_1), one per day, as in group 3 but using oxygen only; (5) control
ozone group, as group 3, but without STZ. The ozone treatment improved glycemic control and prevented oxidative stress,
the increase of aldose reductase, fructolysine content and advanced oxidation protein products. Nitrite and nitrate levels
were maintained without changes with regard to non-diabetic control. The results of this study show that repeated
administration of ozone in non-toxic doses might play a role in the control of diabetes and its complications.
INTRODUCTION
Diabetes produces a large number of changes in vessels
that affect the reactivity of smooth muscle and endothelium,
the production of vasoactive substances by endothelium,
vessel wall permeability to macromolecules, susceptibility
to atherosclerosis and activity of the thrombolytic system
[1–3]. These events are related to the chronic vascular
complications of this disorder. The vascular lesion in
diabetes consists of microangiopathy, distinguished by
thickening of capillary basement membranes resulting
in increased vascular permeability. These changes are
clinically manifested as diabetic retinopathy and/or
microangiopathy, which consists of atheromatous involvement
of large blood vessels. Macroangiopathy is morphologically
similar to non-diabetic atheroma, but tending to occur earlier
and be more extensive [4].
Vascular endothelium appears to be a vulnerable target
for hyperglycemia-induced metabolic changes. High glucose
concentrations promote endothelial cell damage by different
mechanisms, probably through mutual facilitatory interactions
between them [5]. Activation of polyol pathway, nonenzymatic glycosylation of proteins and the increase of
reactive oxygen species (ROS) play an important role in
diabetic complications.
Ozone, administered by rectal insufflation in a Lumber of
controlled treatments, has shown protective effects
against the damage induced by carbon tetrachloride and
hepatic and renal isc hemia-reperfusion through
a probable mechanism of oxidative preconditioning which
confers protection by stimulation of antioxidant
endogenous systems, accumulation of adenosine and by
blocking the xanthine/xanthine oxidase pathway for
ROS generation [6–9]. In addition, a decrease of blood
cholesterol and stimulation of antioxidative response
in cardiopathy patients treated with intravenous ozone
therapy has been demonstrated [10].
Taking the view that diabetes promotes an oxidative damage
and ozone protects the cells in oxidative stress
situations, we investigated the actions of ozone on
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mechanisms, probably through mutual facilitatory interactions
between them [5]. Activation of polyol pathway, nonenzymatic glycosylation of proteins and the increase of
reactive oxygen species (ROS) play an important role in
diabetic complications.
Ozone, administered by rectal insufflation in a Lumber of
controlled treatments, has shown protective effects
against the damage induced by carbon tetrachloride and
hepatic and renal isc hemia-reperfusion through
a probable mechanism of oxidative preconditioning which
confers protection by stimulation of antioxidant
endogenous systems, accumulation of adenosine and by
blocking the xanthine/xanthine oxidase pathway for
ROS generation [6–9]. In addition, a decrease of blood
cholesterol and stimulation of antioxidative response
in cardiopathy patients treated with intravenous ozone
therapy has been demonstrated [10].
Taking the view that diabetes promotes an oxidative damage
and ozone protects the cells in oxidative stress
situations, we investigated the actions of ozone on
streptozotocin-induced diabetes, characterizing the
redox balance and its relation with markers of polyol
pathway, non-enzymatic glycosylation of proteins and
the levels of nitrates and nitrites, as a measure of nitric
oxide (NO) production.
MATERIALS AND METHODS
Animals
Male Sprague–Dawley rats weighing 250–278 g
were obtained from CENPALAB (Bejucal, Havana,
Cuba). Animals were housed in temperature- and
lightcontrolled rooms and allowed free access to normal
diet pellets and tap water. All procedures were performed
as approved by the Institutional Animal Care Committees
(ARCA No. 012) and in accordance with the European
Union Guidelines for animal experimentation.
Induction of experimental hyperglycemia
Experimental diabetes was induced by a single
intraperitoneal (i.p.) injection of 45 mg kg_1
streptozotocin (STZ) (Sigma, St Louis, MO, USA)
to overnight fasted rats [11]. STZ was dissolved in citrate
buffer solution (0.1 M, pH 4.5) and freshly prepared
immediately before injection. Animals were considered
hyperglycemic when non-fasting serum glucose levels
were higher than 20 mM after 48 h of STZ injection [12].
Blood glucose was measured using a diagnostic kit
obtained from Sigma 315–100 (Sigma, St Louis, MO,
USA) based on a colorimetric reaction.
Animals and treatment
The protocol consisted of five experimental groups
(n D 10 each). (1) Control group treated only with
physiological saline solution; (2) positive control group
using STZ as a diabetes inductor; (3) ozone group,
receiving 10 treatments (1.1 mg kg_1, a dose of ozone
in which the phenomenon of oxidative preconditioning is
achieved without appreciable toxicity [6–9]) one per day
after STZ-induced diabetes; (4) oxygen group, vehicle of
O3 (26 mg kg_1, dose equivalent to the O2 concentration
present in one O3 dose) one per day, as in group 3 but
using oxygen only; (5) control ozone group, as group 3,
but without STZ. The ozone concentration in the O3/O2
mixture was 50 _g ml_1.
Ozone was generated by OZOMED equipment
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manufactured by the Ozone Research Center (Cuba)
and was administered by rectal insufflation. Ozone
was obtained from medical grade oxygen, was used
immediately upon generation and represented only
about 3% of the gas .O2 C O3/ mixture. The ozone
concentration is measured by using a built-in UV
spectrophotometer set at 254 nm (accuracy, 0.002 Å at
1 Å, repeatability 0.001 Å and calibrated with internal
standard). The ozone dose is the product of the ozone
concentration (expressed as mg l_1 by the gas .O2 C O3/
volume (l)). By knowing the body weight of the rat the
ozone dose is calculated as mg kg_1 as in our previous
papers [6–9]. After 11 days of diabetic induction, blood
glucose was measured, the body weight of the animals
was monitored and then they were killed by diethyl
ether anesthesia. Afterwards the pancreas was promptly
removed for biochemical studies. Pancreas homogenates
were obtained using a tissue homogenizer Edmund
Bühler at 4 _C. The homogenates were prepared using
a 50 mM KCl/histidine buffer pH 7.4, 1 : 10 (w/v) and
were spun down with a Sigma Centrifuge 2K15, at 4 _C
and 8500 g for 20 min. Supernatants were taken for
biochemical determinations.
Biochemical determinations
The biochemical parameters were evaluated in the
supernatants of pancreas homogenates 11 days after
STZinduced diabetes and 24 h after the last treatment with
ozone or oxygen, respectively. The different parameters
were determined by spectrophotometric methods using an
Ultrospect Plus Spectrophotometer from Pharmacia LKB.
Catalase activity was measured by following the
decomposition of hydrogen peroxide at 240 nm at 10 s
intervals for 1 min [13]. Superoxide dismutase (SOD) and
glutathione peroxidase (GSH-Px) were measured using kits
supplied by Randox Laboratories Ltd., Ireland (Cat. No.
SD125 and No. RS505). Concentrations of malondialdehyde
(MDA) were analyzed using the LPO-586 kit obtained
from Calbiochem (La Jolla, CA,USA). In the assay,
the production of a stable chromophore after 40 min
of incubation at 45 _C was measured at a wavelength
of 586 nm. For standards, freshly prepared solutions of
malondialdehyde bis [dimethyl acetal] (Sigma St Louis,
MO, USA) were employed and assayed under identical
conditions [14].
Quantification of total hydroperoxides was measured
by Bioxytech H2O2-560 kit (Oxis International Inc.,
Portland, OR, USA) using xylenol orange to form a
stable colored complex, which can be measured at 560
nm. Total protein concentration was determined by
the method of Bradford with bovine serum albumin as
standard [15]. After precipitation of thiol proteins using
TCA 10%, the reduced glutathione (GSH) was measured
according to the method of Sedlak and Lindsay [16] with
Ellman’s reagent (5,50 dithiobis (2-nitrobenzoic acid)
10_2 M (Sigma St Louis, MO, USA)), the absorbance was
measured at 412 nm . Nitrite/nitrate levels
were
determined by the Griess reaction
by first converting nitrates to nitrites
using nitrate reductase
(Boehringer Mannheim Italy SpA,
Milan, Italy). Then the Griess
reagent (1% sulphanilamide,
0.1% N-(1-naphthyl)ethylenediamine dihydrochloride
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acid) was added [17]. Samples were incubated at room
temperature for 10 min and absorbance was measured
at 540 nm using a microplate reader. The advanced
oxidation protein products (AOPP) were measured
through the oxidation of iodide anion to diatomic iodine
by AOPP [18]. Relative fructolysine content (Amadori’s
product of glycated serum protein) was measured by
reduction of the redox indicator nitrobluetetrazolium
(NBT) at 530 nm [19]. Aldose reductase activity was
determined using a conventional procedure [20].
Statistical analysis.
The OUTLIERS preliminary test for detection of
error values was initially applied for statistical analysis.
Afterward, the ANOVA method (single way) was used
followed by the homogeneity variance test (BartlettBox). In addition, a multiple comparison test was
used (Duncan test). Data were expressed as the mean
_ standard deviation of 10 animals. The level of
statistical significance employed was at least P < 0:05
for all experiments..
RESULTS
Body weights and blood analysis Rats treated with
streptozotocin (STZ) and STZ C O2 were hyperglycemic and
lost weight over the experimental period (Table I). Ozone
treatment reduced hyperglycemia by 40% in comparison
with STZ-treated rats. Body weight of the rats was increased
in a similar way as for the non-diabetic control.
Antioxidant–prooxidant balance
The O3 C STZ treatment increased glutathione
(GSH) concentrations with regard to the remaining
groups [Fig. 1(a)]. The enzymes superoxide dismutase
(SOD) and catalase (CAT) showed a similar trend
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57
[Fig. 1(b, c)]. Neither GSH nor SOD were different
in the remaining groups (non-diabetic, STZ-induced
diabetes, O2-treated diabetic or O3-treated rats).
Treatment with ozone caused a reduction in glutathione
peroxidase with regard to STZ (43%) and STZ C O2
(36%) groups; however, concentrations in ozone-treated
diabetic rats were still raised above those seen in nondiabetic
control rats [Fig. 1(d)]. Total peroxides were
reduced in the ozone-treated group with regard to all
treatments, including the control non-diabetic [Fig. 2(a)],
whereas malondialdehyde (MDA) concentrations were
maintained at the level of the control in the animals
treated with O3 or in the group treated with O3 C STZ
(P < 0:05) and a significant increase was noted in the
treatments with STZ and O2 C STZ (P < 0:05) with
respect to control group.Biomarkers of the polyol pathway,
non-enzymatic glycosylation, protein oxidation and nitric
oxide The results obtained for these parameters are shown
in Table II. Aldose reductase activity which catalyzes
the reduction of glucose to sorbitol and the relative
fructolysine content, precursor of Advanced Glycation
Endproducts (AGEs) was significantly (P < 0:05)
increased in STZ and O2-STZ diabetic rats. On the
other hand, there was no significant differences when
comparing the diabetic rats treated with ozone and
the control non-diabetic. The ozone group did not
significantly (P < 0:05) modify the aldose reductase
activity with regard to normal control rats. A close
relation was found (r D 0:78, P < 0:05) between
relative fructolysine content and AOPP concentrations.
The levels of NO2/NO3, in the ozone-treated group, did
not differ from the control group. Both groups showed
significantly higher concentrations with regard to STZ
and STZ C O2.
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DISCUSSION
Most previous studies have focused on immediate or
concurrent factors, which contribute to the phenomenon
of diabetes-induced endothelial dysfunction. In the
present study we have integrated some of the most
important metabolic events associated with the
diabetic endotheliopathy process and its control by
ozone treatment.
It is of critical importance to maintain the antioxidant
potential of the pancreatic cell in order to ensure both
its survival and insulin secretory capacity during times
of increased oxidative stress. On the other hand, the
pancreas is the main target of STZ.
The antioxidant–prooxidant balance, associated with
the control of oxidative stress was favored by ozone
treatment, while the group treated with oxygen (vehicle of
ozone) did not differ from the STZ-induced diabetic rats.
Ozone reduced STZ-induced hyperglycemia and it
increased the antioxidant defenses (GSH, SOD and CAT
levels) of the pancreas [Fig. 1(a, b, c)]. The capacity
of ozone to enhance antioxidant endogenous systems, in
front of oxidative stress by oxidative preconditioning or
adaptative mechanisms, has been demonstrated [6].
There is evidence that hyperglycemia can lower both
the activity of a number of enzymes including SOD [21]
and GSH synthesis, presumably by glycation [22]. It
is not possible at this knowledge state to define how
ozone treatment decreases hyperglycemia. However
the observation that diabetic patients have lowered
antioxidant defenses, both enzymatic (SOD, CAT,
GSH-Px) and non-enzymatic (vitamin C, E or A,
free radical scavengers or ‘total radical-trapping
antioxidant capacity’) is almost as well established as
the observation of increased oxidative damage [21].
Therefore, these results suggest that ozone protective
effects on antioxidant endogenous defenses improve
glucose metabolism.
In line with the increase in antioxidant systems there
was a reduction of total peroxides and the concentrations
of MDA were at the level of the control group (Fig. 2).
MDA and peroxides have been associated with diabetes
and its complications. An approximately three-fold increase
in ROS production accompanied by a similar elevation
of MDA, an index of lipid peroxidation, was seen
in rat aorta after 1 month of diabetes [23]. In addition,
a role for H2O2 has been demonstrated in protein crosslinking
junio 2010
58
in diabetes [24].
No differences were observed in GSH and SOD
among non-diabetic, STZ-induced diabetes and
oxygentreated diabetic groups. This behavior may be due
to compensating mechanisms similar to the one which was
found for (mRNA) SOD in STZ-treated rats [24].
When analyzed, the treatment with ozone maintained
the necessary antioxidant–prooxidant balance. Nevertheless,
endothelium integrity and function depend not only
on the ROS control but also on possible modes of action
and some potential interactions between the polyol pathway,
ROS production, advanced glycation endproducts
and NO generation [5, 25, 26].
The concentrations of the mediators derived from the
increased flux of glucose through the polyol pathway
(aldose reductase and fructolysine) were reduced by
ozone treatment while AOPP were not increased in the
ozone treatment group. Corresponding with these results,
a close relation between fructolysine contents and AOPP
concentrations was found (r D 0:78, P < 0:05).
The regulative effects of ozone on aldose reductase activity
represent another interesting action of this complementary
medical approach since aldose reductase is
a key enzyme of the polyol pathway and its inhibitors
have been used as therapeutical drugs linked to improving
NO production or release [27]. This is brought about
through NADPH-sparing activity that helps to replenish
antioxidant reserves, thus having an indirect antioxidant
action in mild diabetic neuropathy or in preventing
periphereal and autonomic neuropathy in unaffected diabetic
patients [28].
Substantial evidence exists that diabetes results in
impaired endothelial dysfunction suggesting diminished
nitric oxide production from diabetic endothelium [29].
Ozone treatment prevented depletion of NO2/NO3
(Table II). This result indicates that NO production
has not been affected by STZ-induced diabetes. Thus,
ozone may protect against the imbalance in NO–ROS
interactions, improve NO-mediated relaxation and
decrease microvessel reactivity, in this experimental
model of diabetes.
In summary, ozone treatment improved glycemic
control and prevented oxidative stress, the increase of
aldose reductase, fructolysine content and advanced
oxidation protein products. NO2/NO3 levels were
maintained without changes with regard to non-diabetic
control. These events are closely related with endothelial