The reaction of ozone with so many moleculesimplies two fundamental processes:
I call the first “THE OZONE INITIAL REACTION” because some of the ozone dose is unavoidably consumed during oxidation of ascorbic and uric acids, sulphydryl (SH)-groups of proteins and glycoproteins. Although albumin, ascorbic and uric acids tame the harsh reactivity of ozone (Halliwell, 1996), they allow this first reaction that is important because it generates reactive oxygen species (ROS), which triggers several biochemical pathways in blood ex vivo (ie, in the glass bottle). ROS are neutralized within 0.5-1 minute by the antioxidant system.
The second, well characterized reaction is known as “LIPID PEROXIDATION” (Pryor et al., 1995). In the hydrophilic plasma environment, one mole of an olefin (particularly arachidonic acid present in plasma triglycerides and chylomicrons) and one mole of ozone give rise to two moles of aldehydes and one mole of hydrogen peroxide (H2O2). These two reactions, completed within seconds, use up the total dose of ozone that generates hydrogen peroxide, an oxidant but not a radical molecule (usually included in the ROS family) and a variety of aldehydes known as LIPID OXIDATION PRODUCTS (LOPs).
FROM NOW ON, NOT OZONE, BUT ONLY ROS (MOSTLY HYDROGEN PEROXIDE) AND LOPs ARE RESPONSIBLE FOR THE SUCCESSIVE AND MULTIPLE BIOCHEMICAL REACTIONS HAPPENING IN DIFFERENT CELLS ALL OVER THE BODY.
Therefore it should be clear that a good deal of ozone is consumed by the antioxidants present in plasma and only the second reaction is responsible for the late biological and therapeutic effects . This should clarify why a very low ozone dose can be ineffective or equivalent to a placebo. ROS include several radicals as anion superoxide (O2.-), nitrogen monoxide (NO.), peroxynitrite (O=NOO-), the already mentioned hydroxyl radical and other oxidant compounds such as hydrogen peroxide and hypoclorous acid (HClO). All of these compounds are potentially cytotoxic (Fridovich, 1995; Pullar et al, 2000; Hooper et al., 2000), luckily have a very short half-life (normally a fraction of a second) and both the plasma and cells have antioxidants able to neutralize them, if their concentrations do not overwhelm the antioxidant capacity.
LOPs generated after peroxidation of a great variety of PUFAs are heterogenous and briefly are represented by peroxyl radicals (ROO.), a variety of hydroperoxides (R-OOH) and a complex mixture of low molecular weight aldehydic end products, namely malonyldialdeyde (MDA), and alkenals, among which 4-hydroxy-2,3 transnonenal (4-HNE), is one of the most cytotoxic. The chemistry and biochemistry of these compounds has been masterfully described by Esterbauer’s group (1991). If one thinks about the wealth and chemical heterogeneity of lipids, glycolipids and phospholipids present in plasma, it becomes difficult to imagine how many potent, potentially noxious, compounds can be generated by the lipids reacting with ozone. During one of my several disputes with American referees, a distinguished scientist wrote: “It is grotesque to think that any Western World Drug Regulating Agency would condone infusing the hodgepodge of ozonized products to treat diseases, although it is probable that the products would initiate and/or modulate a wide spectrum of inflammatory-immune processes to varying degrees”.
In my opinion, this referee missed what I believe is the formidable strength of ozonetherapy: provided that we can control (by using precise ozone concentrations exactly related to the blood volume and antioxidant capacity) the amount of LOPs, we can achieve a multitude of biological effects unthinkable with a single drug(Figure 1).
The scheme ought to fix in the reader’s mind this crucial point and the sequence of events eventually leading to the therapeutic results: ROS are produced only during the short time that ozone is present in the glass bottle, ex vivo, and they yield EARLY biological effects on blood, whereas LOPs, which are simultaneously produced, have a far longer half-life and, during the reinfusion of ozonated blood in the donor, they reach the vascular system and practically all the organs where they trigger LATE effects (Figure 2).
We have come to a critical point: how can we reconcile the production of toxic compounds with the idea that these compounds exert important biological and therapeutic effects?
Let us first examine the behaviour and pharmacodynamic of hydrogen peroxide, which in practical terms is the most important ROS. As soon as ozone dissolves in the plasmatic water and reacts with PUFAs, the concentration of hydrogen peroxide starts to increase but, just as rapidly, decreases because this unionized molecule diffuses quickly into erythrocytes, leukocytes and platelets, where it triggers several biochemical pathways.
Does the increased intracellular concentration of hydrogen peroxide become toxic for the cell? Absolutely no! Because, at the same time, it undergoes reduction to water in both plasma and intracellular water, thanks to the presence of powerful antioxidant enzymes such as catalase, glutathione-peroxidase (GSH-Px) and free reduced glutathione (GSH). Perhaps for one second, the plasma-intracellular concentration has been estimated to range from 1 to 10 micromolars, which avoids any toxicity (Stone and Collins, 2002). The transitory presence of hydrogen peroxide in the cytoplasm means that it acts as one of the ozone chemical messengers and that its level is critical: it must be above acertain threshold to be effective but not too high to become noxious. In ourstudies, performed with human blood exposed to ozone concentrations ranging from 20 to 80 mcg/ml per ml of blood, the process of hydrogen peroxide generation, diffusion and reduction wasfound always extremely transitory (Bocci et al.,1993a;b; 1998a;b).
Figure 2. The multivaried biological response of the organism to ozonized blood can be envisaged by considering that ozonized blood cells and the generated LOPs interact with a number of organs. Some of these represent real targets (liver in chronic hepatitis, vascular system for vasculopathies), while other organs are probably involved in restoring normal homeostasis.
ER: erythrocytes, PLAT: platelets, BMC: blood mononuclear cells, GRAN: granulocytes, CNS: central nervous system, GIT: gastrointestinal tract, MALT: mucosal associated lymphoid tissue.
Hydrogen peroxide is now widely recognised as an intracellular signaling molecule able to activate a tyrosine kinase, which phosphorylates a transcription factor (Nuclear Factor KB, NFKB), which allows the synthesis of a number of different proteins (Baeuerle and Henkel, 1994; Barnes and Karin., 1997). Basically hydrogen peroxide functions by oxidizing cysteines (Rhee et al., 2000), and we and Others have found that it acts on blood mononuclear cells (Bocci and Paulesu, 1990; Bocci et al., 1993b; 1998a; Reth, 2002), on platelets (Bocci et al., 1999a), on endothelial cells (Valacchi and Bocci, 2000) and on erythrocytes (Bocci, 2002).
ROS entering into the erythrocytes are almost immediately reduced (hydrogen peroxide to water and lipoperoxides to hydroperoxides) at the expense of GSH. The enormous mass of erythrocytes can easily mop up hydrogen peroxide and, within 10-15 minutes, marvellously recycle back oxidized antioxidants in reduced form (Mendiratta et al., 1998a, b). While glutathione reductase (GSH-Rd) utilises the reduced nicotinamide adenine dinucleotide phosphate (NADPH, this coenzyme serves as an electron donor for various biochemical reactions) to recycle oxidized glutathione(GSSG) to the original level of GSH, the oxidized NADP is reduced after the activation of the pentose phosphate pathway, of which glucose-6-phosphate dehydrogenase (G-6PD) is the key enzyme . Thus, glycolysis is accelerated with a consequent increase of ATP levels. Moreover the reinfused erythrocytes, for a brief period, enhance the delivery of oxygen into ischemic tissues because of a shift to the right of the oxygen-haemoglobin dissociation curve due either to a slight decrease of intracellular pH (Bohr effect) or/and an increase of 2,3-diphosphoglycerate (2,3-DPG) levels.
Figure 3. A summary of the biological effects elicited during exposure of human blood to oxygen-ozone, ex vivo and during its reinfusion in the donor.
There is an ample literature regarding the cytotoxicity of LOPs. These compounds, when tested either in tissue culture, or examined in the context of the delicate respiratory system, are toxic even at a concentration of 1 micromolar. Surprisingly, submicromolar concentrations (0.01-0.5 microM) tested in several cell types can stimulate proliferation and useful biochemical activities. These findings lead to believe that toxicity of ozonated lipid products depends upon their final concentrations and tissue-localization, so that they can act either as injurious or useful signals (Dianzani, 1998; Parola et al., 1999; Bosch-Morell et al., 1999; Larini et al., 2004). Blood, in comparison to the lungs, is a much more ozone-resistant “tissue” and we have never observed any damage. However, when we reinfuseozonated blood, what is the fate of LOPs? We have often measured the kinetic of their disappearance from blood and their half-life in six patients with age-related macular degeneration (ARMD) was equivalent to 4.2±1.7 min. On the other hand, if the same ozonated blood samples were incubated in vitro, levels of LOPs hardly declined during the next two hours, a result clarifying their toxicity in static cell cultures. As far as cholesteryl ester hydroperoxide is concerned, Yamamoto (2000) has emphasized the role of the enzymatic degradation and hepatic uptake. Thus LOPs toxicity in vivo is most likely irrelevant for the following reasons:
DILUTION (up to 150-200 folds) of these compounds in blood and body fluids rapidly lowers their initial concentration to pharmacological, but not toxic levels. Obviously the ozone dose must be within the therapeutic range.
NEUTRALISATION of LOPs due to the antioxidant capacity in body fluids and cells.
DETOXIFICATION of LOPs (scarcely observable in vitro) due to the interaction with billions of cells endowed with detoxifying enzymes such as aldehyde- and alcohol-dehydrogenases, aldose reductase and GSH-transferases (GSH-T).
EXCRETION of LOPs into the urine and bile after hepatic detoxification and renal excretion.
BIOACTIVITY without toxicity. As already mentioned, submicromolar concentrations of LOPs can act as physiological messengers able to reactivate a biological system gone awry.
From a pharmacokinetic point of view, trace amounts of LOPs, can reach all organs and particularly the bone marrow and the Central Nervous System (Figure 2). LOPs are extremely important in informing specific cell receptors of a minimal and calculated oxidative stress eliciting the adaptive response. In regard to erythrocytes, LOPs can influence the erythroblastic lineage, allowing the generation of cells with improved biochemical characteristics. These “supergifted erythrocytes” as I called them, due to a higher content of 2,3-DPG and antioxidant enzymes, during the following four months, are able to deliver more oxygen into ischemic tissues. The consequence of repeated treatments, obviously depending upon the volume of ozonated blood, the ozone concentration and the schedule is that, after a few initial treatments, a cohort (about 0.8 % of the pool) of “supergifted erythrocytes” will enter daily into the circulation and, relentlessly, will substitute old erythrocytes generated before the therapy. This means that, during prolonged ozonetherapy, the erythrocyte population will include not only cells with different ages but, most importantly, erythrocytes with different biochemical and functional capabilities. In the course of ozone therapy, we have already measured a marked increase of G-6PD and other antioxidant enzymes in young erythrocytes. (Bocci, 2004). The process of cell activation is very dynamic and don’t last for ever because blood cells have a definite life-time and a limited biochemical memory; therefore, the therapeutic advantage MUST BE MAINTAINED WITH LESS FREQUENT TREATMENTS.
zone toxicity to blood, biological fluids and internal organs can be totally avoided when the ozone dose reduces only in part and transitorily the multiform and potent antioxidant capacity. The antioxidant system has evolved during the last two billions years as an essential defence against oxygen: it is made up of scavengers components, namely albumin, vitamins C and E, uric acid, bilirubin, cysteine, ubiquinol, alpha-lipoic acid and of intracellular antioxidants, such as GSH, thioredoxin and enzymes (superoxide dismutase, SOD; GSH-Px, GSH-Rd, GSH-T, catalase, etc.,) and proteins such as transferrin and caeruloplasmin, able to chelate free iron and copper that, otherwise, can favour the formation of hydroxyl radicals. The wealth and the variety of extracellular and intracellular antioxidants, thoroughly described by Chow and Kaneko (1979), Halliwell (1994; 1999a, b; 2001), Frei (1999), Holmgren, (1989), Di Mascio et al., (1989), Jang et al., (1997), Packer et al., (1997), Bustamante et al., (1998) and Chae et al., (1999), are able to explain how bland amounts of ozone can be tamed with the results of stimulating several biological systems without deleterious effects. Until this key point is understood, the dogma of ozone toxicity will continue to linger.
The reader can appreciate the complexity of this system in Table 2
Superoxide dismutases (SOD)
Gluthatione redox system
Reducing equivalents via NADPH and NADH
Glucose, Cysteine, Cysteamine, taurine, Tryptophane, Hystidine, Methionine
The interaction among antioxidants, enzymes and the metabolic system is very important as it allows their rapid regeneration and the maintenance of a normal antioxidant status. The following scheme, drawn by Prof. L. Packer, beautifully illustrates the cooperation among various antioxidant system in order to neutralize a lipoperoxide radical ROO. (shown on the left hand side) to a less reactive hydroperoxide, ROOH. The reducing activity is continuously generated by cellular metabolism via the continuous reduction of NAD(P)+ to NAD(P)H.
It suffices here to say that, during the transient exposure of blood to appropriate concentrations of ozone, the antioxidant reservoir decreases between 2-25 % in relation to ozone doses between 10-80 mcg/ml of gas per ml of blood. It is important to add that this partial depletion is corrected in less than 20 min thanks to the recycling of dehydroascorbic acid, GSSG, alpha-tocopheryl radical to the reduced compounds.
CONCLUSIONS:What happens when human blood is exposed to a therapeutic dose of oxygen-ozone?
Both gases dissolve in the water of plasma depending upon their solubility, partial pressure and temperature. While oxygen readily equilibrates between the gas and the blood phases, the ten-fold more soluble ozone cannot equilibrate because IT REACTS with biomolecules (PUFA, antioxidants) present in the plasma. The reaction yields hydrogen peroxide (among other possible ROS) and lipid oxidation products (LOPs). The sudden rise in plasma of the concentration of hydrogen peroxide generates a gradient, which causes its rapid transfer into blood cells where, in a few seconds, it activates several biochemical processes and simultaneously undergoes reduction to water by the efficient intracellular antioxidant system (GSH, catalase, GSH-Px).This critical step corresponds to a controlled, acute and transient oxidative stress necessary for biological activation, without concomitant toxicity, provided that the ozone dose is compatible with the blood antioxidant capacity.
While ROS are responsible for immediate biological effects (Figure 1), LOPs are important as late effectors, when the blood, ozonated ex vivo, returns into the circulation upon reinfusion ( Figures 2 and 3).
LOPs can reach any organ, particularly the bone marrow where, after binding to receptors in submicromolar concentrations, elicit the adaptation to the repeated acute oxidative stress, which is the hallmark of ozonated autohemotherapy. Upon prolonged therapy, LOPs activity will culminate in the upregulation of antioxidant enzymes, appearance of oxidative stress proteins (haeme-oxygenase I as a typical marker) and probable release of stem cells, which represent crucial factors explaining some of the extraordinary effects of ozonetherapy (Chapter 8).
It must be emphasized that BLOOD EXPOSED TO OZONE UNDERGOES A TRANSITORY OXIDATIVE STRESS necessary to activate biological functions without detrimental effects. The stress must be adequate (not subliminal) to activate physiological mechanisms, BUT NOT EXCESSIVE to overwhelm the intracellular antioxidant system and cause damage. Thus, an excessive ozone dose or incompetence in manipulating this gas can be deleterious. On the other hand, very low ozone doses (below the threshold), are fully neutralised by the wealth of plasma antioxidants and can produce only a placebo effect.
The concept that ozonetherapy is endowed with an acute oxidative stress bothers the opponents of this approach because they consider it as a damage inflicted to the patients, possibly already under a chronic oxidative stress. THEY DO NOT BELIEVE THAT OZONETHERAPY INDUCES A MULTIVARIED THERAPEUTIC RESPONSE ALREADY WELL DOCUMENTED IN SOME DISEASES. Moreover THEY DO NOT DISTINGUISH THE CHRONIC OXIDATIVE STRESS (COS) DUE TO AN ENDOGENOUS AND UNCONTROLLED HYPEROXIDATION WITH THE SMALL AND TRANSIENT OXIDATIVE STRESSES that we can precisely perform EX VIVO with the ozone dose .
The THERAPEUTIC RESPONSE achieved after these repeated oxidative stresses can be envisaged as a PRECONDITIONING EFFECT eventually able to reequilibrate the redox system altered by pathogenetic stimuli.