Ozone Therapy Review
Original Source The International Journal of Artificial Organs
Ozone in Nature
Ozone, a gas discovered in the mid-nineteenth century, is a molecule consisting of three atoms of oxygen in a dynamically unstable structure due to the presence of mesomeric states (1). The gas is colourless, acrid in odour and explosive in liquid or solid form. It has a half-life of 40 min at 20°C and about 140 min at 0°C (2). In nature it is abundant only in the stratosphere (20,000- 30,000 m) where its concentrations reach 16-20 mg/m3. In this layer, it is produced by the action of ultraviolet solar radiation and in turn, protects the earth from ultraviolet solar radiation. Ozone occurs at less than 20 µg/m3 at theEarth’s surface, concentrations that are perfectlycompatible with life (2).
In recent decades, photochemical pollution of the lower atmosphere, caused by degradation of petroleum gas and volatile combustion products of oil, coal and a great variety of other compounds, ranging from gaseous mixtures prepared in chemical laboratories to forest fires, has led to much higher ozone levels, especially in cities. In the stratosphere, chlorofluorocarbons in liquid refrigerants and spray cans have destroyed part of the protective layer, causing a "hole" at the south pole. These events, widely reported in the mass media, have created considerable apprehension among the public and doctors, who see ozone as a dangerous toxic substance and have difficulty accepting that it can have therapeutic effects (2).
Ozone is toxic for animals and humans, affecting the lungs and eyes. It irritates the eyes, and its effects on the lungs depend on concentration, temperature, humidity and exposure time. Inhalation of low concentrations of ozone may cause coughing and irritation of the throat (3, 4). Higher concentrations damage the bronchial mucosa and pneumocytes, and may lead to pulmonary edema (5). It has been calculated that breathing pure ozone at a concentration
of 0.02 µg/mL leads to death in 4 h. No other toxic effects have been demonstrated. It should be recalled that oxygen, nitrogen and carbon dioxide, the main gases in the air we breathe, are also toxic and lethal if breathed in abnormal concentrations (2, 6).
Mechanisms of Therapeutic Action of Ozone
In about 1940, Kleinmann (7) demonstrated bactericidal properties of ozone which is used today to sterilise water. Fish (8) observed that ozone has topical therapeutic activity in various skin diseases. In 1974, Wolff (9) described a method in which a certain quantity of blood was exposed to ozone in closed glass recipients and then reinfused into the patient, with interesting therapeutic responses. Since then, apart from sterilisation of water, ozone has been used in therapy in an empirical way, albeit with encouraging results (1, 10-13).
Only recently has the medical literature begun to show serious interest in the topic, despite the fact that thousands of doctors throughout the world have been using ozone in various applications with positive and often surprising results. This use has occurred in the absence of codified procedures, specific rationale, scientific rigour or practical knowledge. The main therapeutic use of ozone is that already recorded and described by Wolff, known today as ozone autohemotherapy (OAHT) (9). Recent studies to clarify the mechanism of action have shown that contact between ozone and blood gives rise to effects that can be exploited in medicine.
Exposure of human blood to a mixture of oxygen and ozone is not toxic for blood, providing exposure times and concentrations are appropriate (14- 17). Indeed, unlike the respiratory system, human blood, the components of which are in a highly dynamic state, is able to neutralise the oxidising power of ozone by a potent defence system. Like other gases (O2, CO2, ..), ozone must be dissolved in water in order to act at the biochemical level. On contact with blood, it dissolves in plasma and instantly decomposes in a cascade of reactive oxygen species (ROS), for example hydrogen peroxide (H2O2), superoxide anion (O2 •¯) and hydroxyl radical (OH•) (18). These compound are highly reactive and have a short half-life. Moreover, during peroxidation of plasma lipids, there occurs formation of late effectors denominated Lipid Oxidation Products (LOPS). ROS are also produced by the body during cell respiration by mitochondria and during bacterial phagocytosis by leucocytes. Normally it is by virtue of production of hydrogen peroxide and hypochlorite that animals and humans defend themselves from continuous invasion by pathogenic agents (19, 20). ROS have their own toxicity, however, and aerobic organisms have in turn developed an antioxidant system, consisting of substances in the plasma, such as uric acid, ascorbic acid, albumin, vitamin E and bilirubin, and of intracellular enzymes such as superoxide dismutase (SOD), catalase (T), glutathione peroxidase (GSH-Px), glutathione reductase (GSH R), glutathione transferase (GSH T) and the redox system of glutathione (GSHGSSG), kept at optimal level by enzymes and the pentose cycle (via NADPH) (21, 22). Most of the dose of ozone that comes into contact with blood is partly reduced by hydrosoluble antioxidants and partly transformed into ROS and LOPS, which are also checked by the antioxidant system of the body before they can damage blood cells.
A first pharmacological effect of ozone is due to the slight excess of ROS acting as chemical messengers for membrane receptors and various biological functions (23, 24), while LOPS act on practically all cells after blood reinfusion. The oxidising action of ozone leads to the formation of hydrogen peroxide that enters cells with various effects: in red blood cells it shifts the hemoglobin dissociation curve to the right and facilitates release of oxygen (25, 26); in leucocytes and endothelial cells it induces production of interleukins, interferon, TGF, nitrogen oxide and antacoids 27, 28); in platelets it favours release of growth factors 29); in all cells (30, 31) it stimulates long term efficiency of antioxidant systems in adaptation to its oxidant action. Another likely effect, not yet demonstrated, is activation of endogenous stem cells. On contact with blood, ozone therefore causes a very transitory imbalance between oxidants and antioxidants, as an acute, exogenous oxidative stress.
With appropriate exposure time and ozone dose, the oxidative stress may be exactly calculated and transient with respect to endogenous toxicity of ROS produced over a lifetime. This calculated imbalance activates messengers that trigger biological effects, without exceeding the capacity of the antioxidant system (32). Ozone, therefore, acts like a drug with a precise therapeutic window: it is not toxic if administered within the therapeutic range, but it may be ineffective if the dose is too low (1) because totally quenched by antioxidants. A further aspect of its action could be important and is currently being researched. It regards the capacity to positively regulate the antioxidant system (33). The body is besieged by continuous production of ROS. For example, production of ROS is high during respiration, in the metabolic cycle of fatty acids, in cytochrome P450 reactions to xenobiotics, in the presence of phagocytosis and in many pathological situations (34). There are situations in the course of a lifetime in which a vicious circle of imbalance between production and neutralisation of ROS develops: the former continue to increase while the antioxidant system becomes weaker. This happens during chronic viral infections, atherosclerosis, tumour growth, neurodegenerative diseases and aging (34).
Excessive production of ROS and/or antioxidant deficit may become chronic and irreversible at certain times, leading to death. Administration of exogenous antioxidants could, at best, slow down the process, but if the latter is not too advanced, prolonged ozone therapy with therapeutic and progressively increasing doses, may restore the balance between ROS produced and neutralised, inducing a potentiation of the intracellular antioxidant system, with adaptation to chronic oxidative stress (35). Indeed, we know that cells may react to oxidative stress in two ways: if the stress is excessive and continuous, the cell dies; if the stress is modest and transient, the cell has time to react and become resistant, activating expression of silent or rarely expressed genes and producing shock proteins, such as heat shock protein (HSP), glucose-regulated protein (GRP) and oxidative shock protein (OSP). Production of all these proteins is stimulated during ozone therapy (1, 36).
Monitoring of Ozone Therapy
It is technically impossible to measure ozone directly in the blood or assay ROS in ozonated plasma because of their very brief half-life (fractions of a second) (1). However, there are indirect methods of monitoring the oxidising action of ozone in the body through terminal products or biochemical modifications of the plasma antioxidant system. Indeed, it is possible to measure lipid peroxidation, antioxidant capacity, markers typical of oxidative status and enzyme activities in plasma. Many of these parameters are cumbersome to measure (for example, assay of isoprostanes and 8-hydroxyguanosine as markers of oxidative status) (37) or time-consuming (enzyme activities) or without commercially available kits (2-3 diphosphoglyceric acid) (38). Our group has been using two parameters of lipid peroxidation that are relatively easy and give reproducible results: 1)Assay of thiobarbituric acid reactive substances (TBARS) (1).
Ozone in plasma reacts with unsaturated fatty acids to produce a vast range of aldehydes, including malonyldialdehyde (MDA). Determination of MDA gives an indication of the degree of peroxidation. The method, described by Buege & Aust (39), is a colorimetric determination based on reaction with thiobarbituric acid (TBA). This determination is useful in clinical practice, providing an indication of the degree of peroxidation of treated blood. The greater the peroxidation, the greater the concentration of TBARS. 2) Assay of protein thiol groups (PTG) (40). Plasma protein sulphydryl groups are the first line of defence against oxidants. PTG are released in the reaction and can be detected by the Ellman reagent which produces a coloured compound, measured by spectrophotometry. Ozone causes a decrease in PTG in plasma. The patterns of TBARS and PTG provide sufficient indication of peroxidation status induced by ozone in clinical practice (1, 2).
OAHT is practised today in all countries of Europe, being first proposed, as we have seen, by Wolff in 1974 9). Minor O3 autohemotherapy and major O3 autohemotherapy have been described; the former uses 5-10 mL and the latter 200-250 mL of blood. The technique is simple: blood is collected in a glass recipient containing either heparin or sodium citrate, placed in contact with an oxygen/ozone mixture at concentrations ranging between 15-80 µg/mL for 5-10 min and then reinfused into the patient. This is usually done twice a week for 7-8 weeks. Both methods are indicated for the following disorders: peripheral vasculopathy (11, 41, 42) Burger disease, atherosclerotic vasculopathy, diabetic vasculopathy) chronic ischemic cardiopathies (43, 44) not susceptible to surgical treatment, acute cerebral ischemi chronic virus infections (1, 45): hepatitis, herpes I and II, herpes Zoster chronic bacterial and fungal infections (46, 47), refractory to conventional therapy degenerative eye diseases such as retinal maculopathy of the elderly, diabetic ischemic retinitis, pigmented retinitis (with which Bocci et al have extensive experience: (1) orthopedic pathology (48) osteoarthritis (1, 2) various pain syndromes (1, 2). To these major pathologies affecting a large number of patients we could also add the vast branch of aesthetic medicine. Here, however, we shall only consider clinical application for severe pathologies. Although many papers have been published all over the world, there have been few studies with experimental animals confirming ozone efficacy. Controlled clinical studies have only just begun to appear in the literature (11, 41, 42, 49). OAHT is associated with induction of production of interferon alpha, beta and gamma, TNF alpha, interleukin (1, 2, 50, 51) granulopoietin (GM-CSF) and transforming growth factor beta (TGF beta), and it seems likely that many other proteins are also stimulated (1). An increase in intraerythrocyte SOD activity has also been observed, suggesting an increase in antioxidant defences. These modifications can be observed for hours and days after OAHT, suggesting that once leucocytes are activated by ozone, they migrate into lymphoid environments where cytokine release triggers other immune cells (52, 53).
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