**2. What is ozone therapy?**

Ozone therapy is a general termination of a medical therapy that medical ozone gas is used as drug by several methods. Some of these methods are systemic where many others are local applications. Ozonated autohaemotherapy (O3-AHT) widely known by people firstly descri‐ bed by Wehrli and Steinbart and since 1954 it has been used in millions of patients in different pathologies with apparent clinical benefit. AHT might be applied in two forms, Major AHT simply driving 100-150 ml of venous blood into a sterile bottle made of neutral glass or other ozone resistant material where blood and medical ozone is mixed in therapeutic doses and then reinfused back to the donor without side effects. 3.13 % Natrium Citrate solution is used as an anticoagulant during the procedure with short lasting effect. In some patients Heparin might be used instead of Natrium Citrate depending on the patient's case.

© 2014 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

**Figure 1.** Schematic drawing of the components necessary to perform major autohematherapy

Minor AHT is very similar to major AHT method with a few differences, where 5-10 ml of blood is mixed with precise dose of medical ozone in a syringe and reinjected by intramuscular route to the donor that no anticoagulant is used. Rectal insufflation (RI) of medical ozone gas is another method of systemic ozone therapy that is applied on some cases if others methods cannot be done or this method is preferred over others due to diseases.

Ozone is normally present as gas made of three atoms of oxygen with a cyclic structure. The medical generator of ozone produces it from pure oxygen passing through a high voltage gradient (5-13 mV) according to the reaction.

$$\text{2SO}\_2 + \text{68}, \text{400cal} \rightarrow \text{2O}\_3 \tag{1}$$

**b.** On the other hand, at variance with oxygen, ozone reacts immediately as soon as it is

Where atomic oxygen behaves as a very reactive atom. Contrary to the incorrect belief that ozone penetrates through the skin and mucosae or enters into the cells, it is emphasized that, after the mentioned reaction, ozone does not exist any longer. In order of preference, ozone reacts with polyunsaturated fatty acids (PUFA), antioxidants such as ascorbic and uric acids, thiol compounds with-SH groups such as cysteine, reduced glutathione (GSH) and albumin. Depending upon the ozone dose, carbohydrates, enzymes, DNA and RNA can also be affected.

shows the simultaneous formation of one mole of hydrogen peroxide (included among reactive

The fundamental ROS molecule is hydrogen peroxide, which is a non-radical oxidant able to act as an ozone Messenger responsible for eliciting several biological and therapeutic effects [13,14]. The concept that ROS are always harmful has been widely revised because, in physiological amounts, they act as regulators of signal transduction and represent important mediators of host defense and immune responses. Presence of traces of Fe++should be avoided because, in the presence of hydrogen peroxide, via the Fenton's reaction, they will catalyze the

It is determined [15] that the formation of nitrogen monoxide (NO,) in human endothelial cells exposed to ozonated serum. Attention should be paid to the fact that an excess of ROS can lead to the formation of other toxic compounds such as peroxynitrite (O=NOO-) and hypochlorite

Although ROS have a lifetime of less than a second, they can damage crucial cell components and, therefore, their generation must be precisely calibrated to achieve a biological effect without any damage. This can be achieved by regulating the ozone dose (ozone concentration as mg/mL of gas per mL of blood in 1:1 ratio) against the antioxidant capacity of blood that can be measured and, if necessary, strengthened by oral administration of antioxidants before

**d.** LOPs production follows peroxidation of PUFA present in the plasma: they are hetero‐ geneous and can be classified as lipoperoxides (LOO,), alkoxyl radicals (LO,), lipohydro‐ peroxides (LOOH), isoprostanes and alkenals, among which are 4-hydroxy-2,3 transnonenal (HNE) and malonyldialdehyde (MDA). Radicals and aldehydes are intrinsically toxic and must be generated in very low concentrations. They are in vitro far

oxygen species, ROS) and of two moles of lipid oxidation products (LOPs) [12].


+ ®+O biomolecules O O, 3 2 (2)

General Aspects of Ozone Therapy http://dx.doi.org/10.5772/57470 161

dissolved in biological water (physiological saline, plasma, lymph, urine)

All of these compounds act as electron donor and undergo oxidation.

formation of the most reactive OH, (hydroxyl radical).

**c.** The main reaction:

anion (ClO-).

and throughout ozone therapy.

Ozone is 1.6 fold denser and 10-fold more soluble in water (49.0 mL in 100 mL water at 0\_C) than oxygen. Although ozone is not a radical molecule, it is the third most potent oxidant (E\_5 12.076 V) after fluorine and persulfate. Ozone is an unstable gas that cannot be stored and should be used at once because it has a half life of 40 min at 20\_C

#### **2.1. What is the behavior and fate of ozone after coming in contact with body fluids?**

The essential concepts to bear in mind are the following;

**a.** As any other gas, ozone dissolves physically in pure water according to Henry's Law in relation to temperature, pressure and ozone concentration. Only in this situation ozone does not react and in a tightly closed glass bottle, the ozonated water is useful as a disinfectant that remains active for a couple of days

**b.** On the other hand, at variance with oxygen, ozone reacts immediately as soon as it is dissolved in biological water (physiological saline, plasma, lymph, urine)

$$\text{O}\_3 + \text{biomlecules} \rightarrow \text{O}\_2 + \text{O}\_2 \tag{2}$$

Where atomic oxygen behaves as a very reactive atom. Contrary to the incorrect belief that ozone penetrates through the skin and mucosae or enters into the cells, it is emphasized that, after the mentioned reaction, ozone does not exist any longer. In order of preference, ozone reacts with polyunsaturated fatty acids (PUFA), antioxidants such as ascorbic and uric acids, thiol compounds with-SH groups such as cysteine, reduced glutathione (GSH) and albumin. Depending upon the ozone dose, carbohydrates, enzymes, DNA and RNA can also be affected. All of these compounds act as electron donor and undergo oxidation.

**c.** The main reaction:

**Figure 1.** Schematic drawing of the components necessary to perform major autohematherapy

cannot be done or this method is preferred over others due to diseases.

should be used at once because it has a half life of 40 min at 20\_C

The essential concepts to bear in mind are the following;

disinfectant that remains active for a couple of days

gradient (5-13 mV) according to the reaction.

160 Pharmacology and Nutritional Intervention in the Treatment of Disease

Minor AHT is very similar to major AHT method with a few differences, where 5-10 ml of blood is mixed with precise dose of medical ozone in a syringe and reinjected by intramuscular route to the donor that no anticoagulant is used. Rectal insufflation (RI) of medical ozone gas is another method of systemic ozone therapy that is applied on some cases if others methods

Ozone is normally present as gas made of three atoms of oxygen with a cyclic structure. The medical generator of ozone produces it from pure oxygen passing through a high voltage

Ozone is 1.6 fold denser and 10-fold more soluble in water (49.0 mL in 100 mL water at 0\_C) than oxygen. Although ozone is not a radical molecule, it is the third most potent oxidant (E\_5 12.076 V) after fluorine and persulfate. Ozone is an unstable gas that cannot be stored and

**2.1. What is the behavior and fate of ozone after coming in contact with body fluids?**

**a.** As any other gas, ozone dissolves physically in pure water according to Henry's Law in relation to temperature, pressure and ozone concentration. Only in this situation ozone does not react and in a tightly closed glass bottle, the ozonated water is useful as a

3O 68,400cal 2O 2 3+ ® (1)

$$\text{R}-\text{CH}=\text{CH}-\text{R} \rightarrow \text{O}\_3 \rightarrow \text{H}\_2\text{O} \rightarrow \text{R}-\text{CH}=\text{O} + \text{R}-\text{CH}=\text{O} + \text{H}\_2\text{O}\_2\tag{3}$$

shows the simultaneous formation of one mole of hydrogen peroxide (included among reactive oxygen species, ROS) and of two moles of lipid oxidation products (LOPs) [12].

The fundamental ROS molecule is hydrogen peroxide, which is a non-radical oxidant able to act as an ozone Messenger responsible for eliciting several biological and therapeutic effects [13,14]. The concept that ROS are always harmful has been widely revised because, in physiological amounts, they act as regulators of signal transduction and represent important mediators of host defense and immune responses. Presence of traces of Fe++should be avoided because, in the presence of hydrogen peroxide, via the Fenton's reaction, they will catalyze the formation of the most reactive OH, (hydroxyl radical).

It is determined [15] that the formation of nitrogen monoxide (NO,) in human endothelial cells exposed to ozonated serum. Attention should be paid to the fact that an excess of ROS can lead to the formation of other toxic compounds such as peroxynitrite (O=NOO-) and hypochlorite anion (ClO-).

Although ROS have a lifetime of less than a second, they can damage crucial cell components and, therefore, their generation must be precisely calibrated to achieve a biological effect without any damage. This can be achieved by regulating the ozone dose (ozone concentration as mg/mL of gas per mL of blood in 1:1 ratio) against the antioxidant capacity of blood that can be measured and, if necessary, strengthened by oral administration of antioxidants before and throughout ozone therapy.

**d.** LOPs production follows peroxidation of PUFA present in the plasma: they are hetero‐ geneous and can be classified as lipoperoxides (LOO,), alkoxyl radicals (LO,), lipohydro‐ peroxides (LOOH), isoprostanes and alkenals, among which are 4-hydroxy-2,3 transnonenal (HNE) and malonyldialdehyde (MDA). Radicals and aldehydes are intrinsically toxic and must be generated in very low concentrations. They are in vitro far more stable (6) than ROS but fortunately, upon blood reinfusion, they undergo a marked dilution in body fluids, excretion (via urine and bile), and metabolism by GSH-transferase (GSH-Tr) and aldehyde dehydrogenases. Thus, only submicromolar concentrations can reach all organs, particularly bone marrow, liver, central nervous system (CNS), endocrine glands, etc., where they act as signaling molecules of an ongoing acute oxidative stress [16].

**2.2. Which are the biological effects elicited by ROS and LOPs?**

late and long-lasting messengers.

factors released from platelets and endothelial cells.

pathways.

The ozonation process is therefore characterized by the formation of ROS and LOPs acting in two phases. This process happens either ex vivo (as a typical example in the blood collected in a glass bottle) or in vivo (after an intramuscular injection of ozone) but while ROS are acting immediately and disappear (early and short-acting messengers), LOPs, via the circulation, distribute throughout the tissues and eventually only a few molecules bind to cell receptors. Their pharmacodynamics allow minimizing their potential toxicity and allows them to become

General Aspects of Ozone Therapy http://dx.doi.org/10.5772/57470 163

Formation of ROS in the plasma is extremely rapid and is accompanied by a transitory and small ozone dosedependent decrease (ranging from 5 to 25%) of the antioxidant capacity. Importantly, this return to normal within 15–20 min owes to the efficient recycling of oxidized compounds such as dehydroascorbate to ascorbic acid [20]. H2O2 diffuses easily from the plasma into the cells and its sudden appearance in the cytoplasm represents the triggering stimulus: depending upon the cell type, different biochemical pathways can be concurrently activated in erythrocytes, leukocytes and platelets resulting in numerous biological effects. It must be noted that between the plasma and the cytoplasm compartments there is a gradient and the intracellular H2O2 concentration is only about 1/10 of the plasmatic one [21]. The rapid reduction to water is operated by the high concentration of GSH, CAT and GSH-Px; nonethe‐ less, H2O2 must be above the threshold concentration for activating several biochemical

Let us now examine how hydrogen peroxide, now universally recognized as one of the main intracellular signalling molecules [13], acts on the different blood cells. The mass of erythro‐ cytes mops up the bulk of hydrogen peroxide: GSH is promptly oxidized to GSSG and the cell, extremely sensitive to the reduction of the GSH/GSSG ratio, immediately corrects the unbal‐ ance by either extruding GSSG or reducing it with GSH-Rd at the expense of ascorbate or of the reduced nicotinamide adenine dinucleotide phosphate (NADPH), which serves as a crucial electron donor. Next, the oxidized NADP is reduced after the activation of the pentose phosphate pathway, of which glucose-6-phosphate dehydrogenase (G-6PD) is the key enzyme. It is determined that a small but significant increase of ATP formation [10,11], but whether this is due to the activation of the pentose cycle or to phosphofructokinase or to both remains to be clarified. Moreover, for a brief period the reinfused erythrocytes enhance the delivery of oxygen into ischemic tissues because of a shift to the right of the oxygen–hemoglobin disso‐ ciation curve, due either to a slight decrease of intracellular pH (Bohr effect) or/and an increase of 2,3-diphosphoglycerate (2,3-DPG) levels. Obviously, one AHT treatment has a minimal effect and we need to ozonate at least 2.5–4 L of blood within a period of 30–60 days. During this period, LOPs act as repeated stressors on the bone marrow and these frequent stimuli cause the adaptation to the ozone stress during erythrogenesis with upregulation of antioxi‐ dant enzymes. As a consequence, a patient with chronic limb ischemia undergoing ozone therapy can have a clinical improvement due to the formation of successive cohorts of erythrocytes progressively more capable of delivering oxygen to his/her ischemic tissues. However, the final improvement is also due to the localized release of NO, CO and growth

If the stage of the disease is not too far advanced, these molecules can elicit the upregulation of antioxidant enzymes such as superoxide dismutase (SOD), GSH-peroxidases (GSH-Px), GSH-reductase (GSH-Rd) and catalase (CAT). Interestingly, Iles and Liu [17] have just demonstrated that HNE, by inducing the expression of glutamate cysteine ligase, causes an intracellular increase of GSH, which plays a key role in antioxidant defense. Furthermore, LOPs induce oxidative stress proteins, one of which is heme-oxygenase I (HO-1 or HSP-32) which, after breaking down the heme molecule, delivers very useful compounds such as CO and bilirubin [18]. Bilirubin is a significant lipophilic antioxidant and a trace of CO cooperates with NO in regulating vasodilation by activating cyclic GMP. Fe++is promptly chelated by upregulated ferritin. The induction of HO-1 after an oxidative stress has been described in hundreds of papers as one of the most important antioxidant defense and protective enzyme. Moreover, LOPs exert a neuroimmunomodulatory effect highlighted by a feeling of well being reported by patients during ozone therapy.

Although it remains hypothetical, it is possible that LOP, throughout the treatments, acting as acute oxidative stressors in the bone marrow microenvironments activate the release of metalloproteinases, of which MP-9 particularly may favor the detachment of staminal cells [11]. These cells, once in the blood circulation, may be attracted and home at sites where a previous injury (a trauma or an ischemic-degenerative event) has taken place. The potential relevance of such an event would have a huge practical importance and will avoid the unnatural, costly and scarcely effective practice of the bone marrow collection with the need of the successive and uncertain reinfusion [19].

It is emphasized that submicromolar LOPs levels can be stimulatory and beneficial, whereas high levels can be toxic. This conclusion, based on many experimental data [16], 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. In conclusion, it must be clear that the ozonation process either happening in blood, or intradiscal or in an intramuscular site represents an acute oxidative stress. However, provided that it is precisely calculated according to a judicious ozone dosage, it is not delete‐ rious but is actually capable of eliciting a multitude of useful biological responses and, possibly, can reverse a chronic oxidative stress due to aging, chronic infections, diabetes, atherosclerosis, degenerative processes and cancer. Indeed, the ozonotherapeutic act is interpreted as an atoxic but real ''therapeutic shock'' able to restore homeostasis.

#### **2.2. Which are the biological effects elicited by ROS and LOPs?**

more stable (6) than ROS but fortunately, upon blood reinfusion, they undergo a marked dilution in body fluids, excretion (via urine and bile), and metabolism by GSH-transferase (GSH-Tr) and aldehyde dehydrogenases. Thus, only submicromolar concentrations can reach all organs, particularly bone marrow, liver, central nervous system (CNS), endocrine glands, etc., where they act as signaling molecules of an ongoing acute oxidative stress

If the stage of the disease is not too far advanced, these molecules can elicit the upregulation of antioxidant enzymes such as superoxide dismutase (SOD), GSH-peroxidases (GSH-Px), GSH-reductase (GSH-Rd) and catalase (CAT). Interestingly, Iles and Liu [17] have just demonstrated that HNE, by inducing the expression of glutamate cysteine ligase, causes an intracellular increase of GSH, which plays a key role in antioxidant defense. Furthermore, LOPs induce oxidative stress proteins, one of which is heme-oxygenase I (HO-1 or HSP-32) which, after breaking down the heme molecule, delivers very useful compounds such as CO and bilirubin [18]. Bilirubin is a significant lipophilic antioxidant and a trace of CO cooperates with NO in regulating vasodilation by activating cyclic GMP. Fe++is promptly chelated by upregulated ferritin. The induction of HO-1 after an oxidative stress has been described in hundreds of papers as one of the most important antioxidant defense and protective enzyme. Moreover, LOPs exert a neuroimmunomodulatory effect highlighted by a feeling of well being

Although it remains hypothetical, it is possible that LOP, throughout the treatments, acting as acute oxidative stressors in the bone marrow microenvironments activate the release of metalloproteinases, of which MP-9 particularly may favor the detachment of staminal cells [11]. These cells, once in the blood circulation, may be attracted and home at sites where a previous injury (a trauma or an ischemic-degenerative event) has taken place. The potential relevance of such an event would have a huge practical importance and will avoid the unnatural, costly and scarcely effective practice of the bone marrow collection with the need

It is emphasized that submicromolar LOPs levels can be stimulatory and beneficial, whereas high levels can be toxic. This conclusion, based on many experimental data [16], 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. In conclusion, it must be clear that the ozonation process either happening in blood, or intradiscal or in an intramuscular site represents an acute oxidative stress. However, provided that it is precisely calculated according to a judicious ozone dosage, it is not delete‐ rious but is actually capable of eliciting a multitude of useful biological responses and, possibly, can reverse a chronic oxidative stress due to aging, chronic infections, diabetes, atherosclerosis, degenerative processes and cancer. Indeed, the ozonotherapeutic act is interpreted as an atoxic

[16].

reported by patients during ozone therapy.

162 Pharmacology and Nutritional Intervention in the Treatment of Disease

of the successive and uncertain reinfusion [19].

but real ''therapeutic shock'' able to restore homeostasis.

The ozonation process is therefore characterized by the formation of ROS and LOPs acting in two phases. This process happens either ex vivo (as a typical example in the blood collected in a glass bottle) or in vivo (after an intramuscular injection of ozone) but while ROS are acting immediately and disappear (early and short-acting messengers), LOPs, via the circulation, distribute throughout the tissues and eventually only a few molecules bind to cell receptors. Their pharmacodynamics allow minimizing their potential toxicity and allows them to become late and long-lasting messengers.

Formation of ROS in the plasma is extremely rapid and is accompanied by a transitory and small ozone dosedependent decrease (ranging from 5 to 25%) of the antioxidant capacity. Importantly, this return to normal within 15–20 min owes to the efficient recycling of oxidized compounds such as dehydroascorbate to ascorbic acid [20]. H2O2 diffuses easily from the plasma into the cells and its sudden appearance in the cytoplasm represents the triggering stimulus: depending upon the cell type, different biochemical pathways can be concurrently activated in erythrocytes, leukocytes and platelets resulting in numerous biological effects. It must be noted that between the plasma and the cytoplasm compartments there is a gradient and the intracellular H2O2 concentration is only about 1/10 of the plasmatic one [21]. The rapid reduction to water is operated by the high concentration of GSH, CAT and GSH-Px; nonethe‐ less, H2O2 must be above the threshold concentration for activating several biochemical pathways.

Let us now examine how hydrogen peroxide, now universally recognized as one of the main intracellular signalling molecules [13], acts on the different blood cells. The mass of erythro‐ cytes mops up the bulk of hydrogen peroxide: GSH is promptly oxidized to GSSG and the cell, extremely sensitive to the reduction of the GSH/GSSG ratio, immediately corrects the unbal‐ ance by either extruding GSSG or reducing it with GSH-Rd at the expense of ascorbate or of the reduced nicotinamide adenine dinucleotide phosphate (NADPH), which serves as a crucial electron donor. Next, the oxidized NADP is reduced after the activation of the pentose phosphate pathway, of which glucose-6-phosphate dehydrogenase (G-6PD) is the key enzyme. It is determined that a small but significant increase of ATP formation [10,11], but whether this is due to the activation of the pentose cycle or to phosphofructokinase or to both remains to be clarified. Moreover, for a brief period the reinfused erythrocytes enhance the delivery of oxygen into ischemic tissues because of a shift to the right of the oxygen–hemoglobin disso‐ ciation curve, due either to a slight decrease of intracellular pH (Bohr effect) or/and an increase of 2,3-diphosphoglycerate (2,3-DPG) levels. Obviously, one AHT treatment has a minimal effect and we need to ozonate at least 2.5–4 L of blood within a period of 30–60 days. During this period, LOPs act as repeated stressors on the bone marrow and these frequent stimuli cause the adaptation to the ozone stress during erythrogenesis with upregulation of antioxi‐ dant enzymes. As a consequence, a patient with chronic limb ischemia undergoing ozone therapy can have a clinical improvement due to the formation of successive cohorts of erythrocytes progressively more capable of delivering oxygen to his/her ischemic tissues. However, the final improvement is also due to the localized release of NO, CO and growth factors released from platelets and endothelial cells.

Although ozone is one of the most potent disinfectants, it 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, as it was proposed a long time ago [22,23], ozone acts as a mild enhancer of the immune system by activating neutrophils and stimulating the synthesis of some cytokines (2,5–7). Once again, the crucial messenger is hydrogen peroxide, which after entering into the cytoplasm of blood mononuclear cells (BMC) by oxidizing selected cysteines, activates a tyrosine kinase, which then phosphorylates the transcription factor nuclear factor kB [24], allowing the release of a heterodimer (p50+p65).

represents the critical factor for overcoming chronic viral infections, ischemia and celldegen‐

General Aspects of Ozone Therapy http://dx.doi.org/10.5772/57470 165

**3. A concise summary of biological effects observed after ozone therapy**

These cells respond with an activation of glycolysis due to activation of the pentose hosphate pathway. It is found that increased adenosine triphosphate levels (from 13899/260 to 19689/232 mM) in patients with age-related macular degeneration (ARMD) (atrophic form) after a therapeutic cycle (14 sessions) of O3-AHT.26 Moreover, Viebahn [27] reported the same effect in athletes and elderly patients after rectal insufflation of O2\_/O3. Ozonation implies a small but consistent oxidation of GSH to glutathione disulfide, and GSH reductase utilizes the reduced form of the coenzyme nicotinamide adenine dinucleotide phosphate supplied by G6PDH to reduce glutathione disulfide to GSH, which indeed returns rapidly to the original level. [28] The increase of 2,3-diphosphoglycerate varies depending on the basic level in ARMD patients and only those who had a low level showed a marked increase with therapy. Viebahn, [27] after a longer cycle of therapy in elderly people, observed a significant increase. An increase of 2,3-diphosphoglycerate level in oxyhemoglobin shifts to the right (p50 value increases); its dissociation curve implies an increased delivery of O2 into the hypoxic tissues. The life-span of ozonated 99Tc-labeled erythrocytes and their uptake by liver and spleen are

A problem still under study regards the generation of biochemically improved erythrocytes during prolonged ozone therapy. While ROS have an extremely short life, LOPs, during the reinfusion of ozonated blood, return into the donor's circulation. While they are fairly stable in vitro, they rapidly disappear from blood in vivo owing to considerable dilution into body fluids, degradation by aldehyde dehydrogenases, excretion into bile and urine, and uptake in various organs including bone marrow cells. This process is crucial for explaining the mech‐ anism of ozone tolerance: during erythrogenesis, submicromolar LOP concentrations can upregulate the synthesis of antioxidant enzymes and indeed, after appropriate density gradient separation, it is found that young (lighter) erythrocytes contain more G6PDH than older (heavier) cells generated before the therapy. [25] This result suggests that ozone therapy enhances the generation of erythrocytes with improved metabolic characteristics, a sort of

These were the cells that were examined first as it is hypothesized that ozone could act as an IFN-g inducer.[29] Since then it is shown [30,31] that ozone behaves as a weak (compared with mitogens) cytokine (such as tumor necrosis factor-a, interleukin-2, interleukin-6, interleukin-8, transforming growth factor-b [TGF-b]) inducer. Several studies [32 - 35] have confirmed that ozone can stimulate bronchoalveolar cells to release proinflammatory cytokines and eicosa‐ noids. Thanks to parallel progress in understanding the role of antioxidants and redox

'supergifted erythrocytes' able to correct hypoxia in vascular diseases.

eration.

**3.1. Erythrocytes**

**3.2. Leukocytes**

comparable with oxygenated erythrocytes.(3)

This complex moves on to the nucleus and switches on some hundred genes eventually responsible for causing the synthesis of several proteins, among which are the acute-phase reactants and numerous interleukins. In the past, it was measured the release of several cytokines from ozonated blood upon in vitro incubation (2–7). Once the ozonated leukocytes return to 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 [25]. This process, described as the physiological cytokine response, is part of the innate immune system and helps us to survive in a hostile environment.

During ozonation of blood, particularly if it is anticoagulated with heparin, we have noted an ozone dose-dependent increase of activation of platelets [8, 26] with a consequent release of typical growth factors, which will enhance the healing of chronic ulcers in ischemic patients. Whenever possible, the use of heparin as an anticoagulant is preferable to sodium citrate because, by not chelating plasmatic Ca++, it reinforces biochemical and electric events.

During reinfusion of the ozonated blood into the donor, the vast expanse of the endothelial cells will be activated by LOPs, resulting in an increased production of NO, plasma Snitrosothiols and S-nitrosohemoglobin [15, 27]. Whereas NO has a half-life of less than 1 sec, protein-bound-NO can exert vasodilation also at distant ischemic vascular sites with relevant therapeutic effect.

Moreover, on the basis of the phenomenon of ozone tolerance that says the exposure of an organism to a low level of an agent, harmful at high levels, induces an adaptive and beneficial response [28,29], it is postulated that LOPs, by acting as long-distance messengers, can transmit to all organs the information of an acute oxidative stres [10, 11]. The bone marrow is particu‐ larly relevant because it can upregulate antioxidant enzymes during erythrogenesis and allows the release of staminal cells for possibly regenerating infarcted organs. Moreover, the stimu‐ lation of the endocrine and central nervous systems may help to understand why most 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 neurotransmitter release.

The paradoxical concept that ozone eventually induces an antioxidant response capable of reversing a chronic oxidative stress is common in the animal and vegetal kingdom and there is good experimental evidence [30–34] that this phenomenon is present in the animal and vegetal kingdom. Moreover, it is already supported by findings of an increased level of antioxidant enzymes and HO-1 during ozone therapy [10,11]. It also suggests that a judicious use of ozone, in spite of acting as an oxidant, enhances the antioxidant capacity, which represents the critical factor for overcoming chronic viral infections, ischemia and celldegen‐ eration.
