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27 Oct '17

Science behind ozone therapy part 2

Posted by Marin Crangaci in immunity, ozone, ozone therapy, research, science

This article is a second part of the series of articles with the generic "Science behind ozone therapy".  In this part we want to  describe some of the fundamentals of ozone and its pharmacodynamics as well as the impact ozone has on the immune system. The characteristics of ozone as a potent oxidant is fundamental in its effect on physiology and the immune system. These topics are discussed in some detail as it pertains to ozone as a medical treatment using various modalities.

To understand about we speak for first we need to define some terms:

Pharmacodynamics: Molecular Mechanisms of Drug Action

Pharmacodynamics is the study of the biochemical and physiological effects of drugs and their mechanisms of action. Understanding pharmacodynamics can provide the basis for the rational therapeutic use of a drug and the design of new and superior therapeutic agents.
Simply stated, pharmacodynamics refers to the effects of a drug on the body. In contrast, the effects of the body on the actions of a drug are pharmacokinetic processes, and include absorption, distribution, metabolism, and excretion of drugs (often referred to collectively as ADME). Many adverse effects of drugs and drug toxicities can be anticipated by understanding a drug’s mechanism(s) of action, its
pharmacokinetics, and its interactions with other drugs. Thus, both the pharmacodynamic properties of a drug and its pharmacokinetics contribute to safe and successful therapy. The effects of many drugs, both salutory and deleterious, may differ widely from patient to patient due to genetic differences that alter the pharmacokinetics and the pharmacodynamics of a given drug. This aspect of pharmacology is termed pharmacogenetics. 

Reactive Oxygen Species

Reactive oxygen species (ROS) include molecules such as hydrogen peroxide (H2O2), neutral free radicals such as the hydroxyl radical (.OH), and anionic radicals such as the superoxide anion radical (). Free radicals have an unpaired electron in the outer orbital.

These free radicals are extremely unstable because they react with a target molecule to capture an electron, so that they become a stable molecule with only paired electrons in the outer shell. However, the target molecule left behind becomes a free radical, which initiates a chain reaction that continues until two free radicals meet to create a product with a covalent bond. ROS—particularly .OH, which is the most reactive of them all—have the potential to damage important biological molecules, such as proteins, lipids, and DNA. However, ROS also play important physiological roles in the oxidation of iodide anions by thyroid peroxidase in the formation of thyroid hormone, as well as in the destruction of certain bacteria by reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase and myeloperoxidase in phagocytic cells. Finally, the highly reactive signaling molecule nitric oxide is a free radical.

Quantitatively, the most important source of ROS is the mitochondrial electron transport chain . Complex I and complex III of the electron transport chain generate  as byproducts . The enzyme superoxide dismutase (SOD) converts  to hydrogen peroxide, which in turn can yield the highly reactive .OH.

Only a small fraction of the oxygen used in aerobic metabolism (<1%) generates ROS. However, even that amount would be lethal in the absence of protective mechanisms. Fortunately, organisms have two potent antioxidant defenses. The major defense is enzymatic, specifically SODs, catalase, and glutathione peroxidase . In addition, low-molecular-weight antioxidants, such as vitamins C and E, play a minor role in the defense against the metabolically produced radicals.

Because these antioxidant defense mechanisms are not fully protective, the dominant concept of the oxidative stress theory is that an imbalance between the production and removal of ROS by antioxidant defenses is the major cause of aging. Nevertheless, recent studies using genetically engineered mice—with either deficient or overexpressed antioxidant enzymes—do not support this theory.

Since ozone is an unstable gas, it is imperative in its medical use to be aware of the concentration of ozone being administered to patients in order to avoid toxicity. A key in avoiding toxicity is knowledge of the effects of ozone to certain organs; while ozone contact with blood is acceptable, contact with the lungs and eyes should be strictly avoided.

Ozone dissolved in pure water can be used as a disinfectant, as long as it is maintained in a tightly closed glass bottle. Under these circumstances ozone does not react with its environment including the container. Ozone reacts immediately and is broken down after coming in contact with fluids such as physiological plasma, saline, urine, and lymph .

Ozone’s paired number of electrons are found in the external orbit and it is not considered a radical molecule despite the formation of radicals when undergoing mitochondrial respiration. Within plasma, ozone reacts with a number of biological molecules. Hierarchically, ozone reacts first with polyunsaturated fatty acids (PUFA), antioxidants such as ascorbic acid, compounds with thiol groups, reduced glutathione (GSH) and albumin . Each of these molecules is oxidized similarly by ozone to generate hydrogen peroxide (H2O2) and two moles of lipid oxidation products (LOPs) . Similarly, the production of anion superoxide, hydrogen peroxide, and hypochlorous acid are a result of phagocytes reacting with pathogens .

Ozone is commonly mistaken as always being cytotoxic since sensitivity of normal and neoplastic cells in culture exposed to ozone at low concentrations has been documented . These results are misleading since cultured cells have significantly less antioxidants than found in blood plasma and is present in typically five to ten fold less albumin in cellular culture as compared to blood plasma . The range of therapeutic dosage of ozone in blood is between 10 μg/ml gas and 80 μg/ml gas per mL of anticoagulated blood . Under these circumstances, the entirety of ozone was consumed in under 5 min . There are no known side effects of autohemotherapy under these conditions. There is risk of blood clot if blood collected is not properly handled. However, proper handling, the use of heprin, and filtration can eliminate the opportunity to produce or introduce blood clots in the body.

The reaction between ozone and PUFA generates two aldehyde constituents in the addition to the formation of hydrogen peroxide. Since the production of aldehydes are particularly deleterious, only therapeutic dosages of ozone should be administered to reduce their formation. The latter product, hydrogen peroxide, has several biological and therapeutic effects and is considered to be a fundamental reactive oxygenated species (ROS) . The production of hydrogen peroxide itself is controversial but like ozone it is important to understand that the concentration of hydrogen peroxide is key. Hydrogen peroxide is a crucial mediator in host defense and immune response in addition to a regulator in signal transduction in physiological amounts . The concentration of hydrogen peroxide dictates whether cells will undergo proliferation or cell death .

The known effects of ozone on the metabolomics of human blood samples indicate a significant increase in the presence of certain metabolites including formate, allantoine, acetate, and acetoacetate. Of those metabolites listed, formate, aceteate, and acetoacetate indicate a linear increase in concentration as determined by their corresponding NMR peaks . Allantoine expressed a hyperbolic curve suggesting a potential saturation point beyond an ozone dose of 800 μg/ml of ozone gas per blood sample.

Pyruvate’s relationship was expressed as a biphasic sigmoidal monotonically decreasing function in response to increasing ozone concentration in tested blood samples . Although these results do give us a glimpse of the shift in blood sample metabolomics, they do not explain how some patients who undergo extracorpeal blood oxygenation and ozonation (EBOO) treatment experience an increase of energy. The decrease in pyruvate might even suggest the contrary since pyruvate is a critical metabolite in glycolysis since it is the end product of glycolysis. However, metabolomics research on rats exposed to ozone gas offers a more thorough understanding of the metabolic shifts and physiological changes after exposure. 313 named metabolites were analyzed primarily after immediate exposure to ozone gas and showed 81 metabolites were significantly increased while 48 were decreased. Of these 313 named metabolites there is the curious case of pyruvate which was shown to increase after the rats were exposed to ozone. As for the account of extra energy after exposure of ozone, the study also shows a marked increase in epinephrine in the rats exposed to ozone. However, it is unclear whether the increase in epinephrine is due to psychological distress from the rats who are exposed to ozone gas or if the ozone physiologically induced the increase of epinephrine. Further studies on humans are warranted to determine the causality of the increase in epinephrine after exposure to ozone.

Cellular physiology can be impacted by ozone in other ways. Oxidative stress impairs oxygen delivery and induce aging in red blood cells. Superoxide, a metabolite formed in some chemical reactions following ozone being reduced by antioxidants, inhibits platelet-derived growth factor receptors  but hydrogen peroxide, another metabolite formed in a reaction with ozone and antioxidants is required for platelet derived growth factor signal transduction. The effect of ozone on human umbilical vein endothelial cells (HUVECs) in vitro shows a marked increase in nitic oxide (NO) and interleukin-8 (IL-8).

In next part we will discuss in more detail how ozone interact and change effect of ROS and its benefic effects.

22 Oct '17

Science behind ozone therapy

Posted by Marin Crangaci in immunity, ozone therapy, research, science

Ozone therapy has been marred by conventional science for years due to many flawed experimental designs or small sample sizes of the population in which it intends to study. For this reason, many physicians have dismissed ozone therapy and limited funds have been delegated to furthering the knowledge of its therapeutic effects. However, there is evidence that suggests that ozone does have various therapeutic effects that range from disinfection of pathogens, anticancer properties, and treatment of back ailments.

In general meaning, ozone therapy results in the short-term production of reactive oxygen species such as hydrogen peroxide, and the long-term production of lipid oxidation products (LOPs) that induce repeated oxidative stress. By these mechanisms, ozone therapy increases the production of antioxidant enzymes from the body’s cells and tissues while providing protection against malignant cells and cellular lesions of diverse etiology (hypoxia and ischemia, toxins, infectious agents, immunologic reactions, physical agents and aging).

The beneficial physiological effects of ozone therapy have been thoroughly investigated in some instances and a wealth of published research exists on the subject. The relative simplicity of this therapy, coupled with the low potential for severe side effects or adverse events, makes ozone an attractive adjuvant therapy for a number of health conditions.

To understand the characteristics of ozone as a potent oxidant and its effect on physiology and the immune system is necessary to speak about physiochemical properties of ozone.

PHYSICO-CHEMICAL PROPERTIES OF OZONE

The word ozone derives from the Greek ozein (ὄζειν), whieh means "to give off a smell". It is an unstable gas of a soft sky-blue colour, with a pungent, acrid smell already perceptible at a concentration of 0.005-0.01 ppmv. The molecule is composed of three oxygen atoms (03) and has a molecular weight of 48.00. It has a cyclical structure assessed by the spectrum absorption in the infrared region, with a distanee among oxygen atoms of 1.26 A.

 

 Mesomeric states ot ozone

In the liquid state, ozone has a dark blue colour and has a boiling point quite different from oxygen (O2 boiling point (at 760 mmHg) is -182 .96°C and of O3 is -111.9 °C). In the liquid and solid state, ozone is highly explosive. Among oxidant agents, it is the third strongest, after fluorine and persulphate. It violently reacts with oxidizable organic compounds such as benzol, dienes and alkanes. Ozone is soluble in methanol and CFC in equal volumes and, of interest from the medieal point of view , is far more soluble in water than is oxygen (At 25 °C, ozone solubility is 109 mg/l. The solubility of oxygen is 8 mg/l. Ozone is 13 times more soluble than oxygen.)

Ozone is formed from oxygen via an endothermic process:

For the production of medical ozone, it is indispensable to use pure oxygen for medicaI use. As shown above, the reaction is reversible and the dissociation velocity from ozone to oxygen depends on the temperature. This means that ozone is a metastable gas with a temperature-dependent half-life and it is hardly storable.

Therefore, for the biomedical application of ozone, it must be prepared ex tempore and used at once for treating the patient. This means that the ozone generator must be situated near the patient, in a suitably aerated room so that even traces of ozone in the air are quickly removed.

For medical purposes, it is not possible to transport ozone directly, but we can to “put” ozone in solvent (ex. water or oil) to increase its lifetime.

Chronic torpid ulcers, exposed dirty traumatic lesions, infected wounds, burns, insect stings, herpetic skin lesions, fungal infections, etc. are advantageously treated with ozonized water or oil rather than gas because it is easy to apply a compress soaked with ozonized water or oil to any part of the body. Moreover, there is no risk of breathing ozone, particularly with a generator not equipped with a suction pump connected to an ozone destructor.

Solution of ozone in water takes place according to the law defined by Henry in 1803: under ideal thermodynamic conditions, the saturation concentration of a gas in water is proportional to its concentration. However, this is correct only if the water is absolutely pure (bidistilled) and the temperature and ozone pressure remain constant.  

After reaching the plateau phase, the ozonized, bidistilled water can be used or stored in a glass bottle tightly closed with a teflon cap, possibly in a refrigerator. Ozone decomposition depends largely on the temperature. If it is kept at +5 "C, the theoretical half-life (t/2) is about 110 hours, i.e. after this period the initial ozone concentration of 20.8 ug/ml (26% of 80 ug/ml) would decrease to about 10 ug/ml. This is of practical importance because ozonized water, if maintained properly, can be used for at least 48 hours at the patient's horne for domiciliary treatment.

Therefore, the instability of ozone is due not only to its metastable nature but mainly to its high reactivity with ions and an array of organic molecules such as those present in biological fluids, namely PUFAs, compounds with sulphydryl groups (-SH), several amino acids and carbohydrates. In such cases, ozone solubility no longer follows Henry's law because as soon as ozone dissolves in water, it encounters a molecule and reacts immediately with it. As a corollary, if ozone is still present in the gas phase, it will dissolve in the water phase but again it will react instantaneously with other molecules and disappear from the water.

In the last decade, there has been a growing interest in the application of ozonized oils. In Cuba, probably because of the lack of other pomades, ozonized oil has been widely employed in torpid ulcers, bacterial, viral, fungal and parasitic infections. As a natural preparation, ozonized oil is now available in several countries. In 2001 ozonized sunflower oil (Oleozon) from Cuba was tested by Sechi et al. (2001) and it was found to have valuable antimicrobial activity against all the micro-organisms tested. Pure olive oil (sunflower oil is employed in Cuba) is ozonized by bubbling O2-03 gas through it for two days until it solidifies. Olive oil contains about 80% oleic acid (18:1 n-6) and, according to Miura (who works at the College of Medical Technology, Hokkaido University, Sapporo, Japan), 1.0 g of oil can absorb up to 160 mg of ozone. After ozonization, characteristic modifications were assessed by observing a 13C-nuclear magnetic resonance (NMR) spectrum: new carbon signals could be related to Criegee's ozonides peaking at around 105 ppm, with complete disappearance of olefinic carbon signals at about 130 ppm. The absence of carbonyl compounds and carboxylic acids, in conjunction with data obtained by NMR spectrum, elemental analysis and high-performance liquid chromatography (HPLC), led to the conclusion that prolonged ozonization results in exclusive formation of triozonides of triolein.

Remarkably, no spectrum changes were noted in ozonized olive oil stored in the refrigerator for two years. There is no real need to have a solid oil preparation, except for commercial purposes and long stability. Indeed, the pathological situations are so variable they require great flexibility; thus one can use fairly liquid or very viscous ozonized olive oiI after keeping it in the cold.

How ozonized olive oil acts is open to speculation. However, it seems likely that when the triozonide comes into contact with the wound, the body temperature and the presenee of serum favour its decomposition to reactive ozone, which readily dissolves in water, generating H202 and a variety of oxidized compounds. This may explain the strong and prolonged disinfectant activity, which however must be tempered so as not to damage the living tissue. This reasoning implies that we should have titrated preparations with either high, medium or low triozonide concentrations to be used during either the inflammatory pus-rich phase I, regenerating phase II or remodelling phase III, respectively.

In the Department of Surgery, Chiba-Tokushukai Hospital, Japan, Matsumoto et al. (2001) tested the efficacy ofthe oil prepared by Miura et al. in intractable fistula and wounds after surgical operations (acute appendicitis with peritonitis, intrapelvie, abdominal and perianal abseesses, ete.). In aseries of 28 patients, the ozonized oil proved to be fully effcetive in 27 cases, without adverse side effeets. Finally, it’s good to be mention that there are several pharmaceutieal vehicles for ozonized oil, such as capsules, pessaries, suppositories and collyriums, to be used in various infections. The smell of ozonized oil is similar to rancid fat (hence unpleasant) and capsules ingested by mouth, although distasteful, are tolerable.

Therefore, in the near future, we will be posting a series of articles here to provide you with the information you require in order to ensure that you achieve the goals you have in mind for your ozonated oil. We will be posting articles that obtain their information from documented studies and professional information. The internet is full of contradictory information regarding ozonated oil - we aim to provide you with simple, clear, and precise information. Stay Tuned.