There now seems to be intense interest in EBO2 techniques. Copious volumes of research have provided evidence that Ozone’s dynamic resonance structures facilitate physiological interactions useful in treating a myriad of pathologies. The comment of Ozone opponents is that ozone therapy looks like a panacea for all diseases. Indeed, it seems so, but in reality, this is due to the multitude of compounds originated at first from the reaction of ozone with body fluids, and eventually able to display pleiotropic effects delivered by different organs.
Ozone can be considered a “PRODRUG”. What exactly is a Prodrug? A prodrug is a medication or compound that, after administration, is metabolized into a pharmacologically active drug. Instead of administering a drug directly. The following illustration shows how a prodrug works:
One of the issues raised by the scientific community is: how does Ozone really acts on Humans? Ozone is quite different from a drug and its action is not a consequence of a binding reaction between one molecule (drug) and one receptor (cellular membrane protein). For this reason, we cannot look at Ozone in the classical terms of pharmacology.
Ozone like other agents, and unlike the common drugs that act on a specific receptor, induces small stress to the whole cell when used at adequate doses. This, in turn, triggers a series of intracellular metabolic processes and promotes a myriad of intracellular activities. Because of these reactions, the defense mechanisms of the cell are alarmed and pushed to improve cell activity, explaining in part the surprising therapeutic actions of Ozone. By the same token Ozone is not truly a prodrug since it can have direct effects on phospholipids, lipoproteins, the cell membrane envelopes of bacteria and viruses. These bioactivities can eradicate bacteria and viruses.
In order to fully understand the biochemical basis underlying the pharmacological effects of ozone, it is important to illustrate its effects on various coenzymes. These coenzymes are responsible for ozone cell metabolism regulation. These effects on metabolism have profound effects on many aspects in the body. One of the significant effects of ozone is the acceleration of glycolysis. Glycolysis results in breaking glucose into pyruvate and getting a very valuable H+ hydrogen ion. The free energy released in this process is used to form the high-energy molecules ATP (adenosine triphosphate) and NADH (reduced nicotinamide adenine dinucleotide). ATP is cellular energy that all cells depend upon. Ozone will allow the ratio of NAD/NADH to be about 700/1. A fundamental condition for guaranteeing the continuity of this process is the reoxidation of NADH as it occurs following ozone exposure. The NADH is converted to NAD+.
The above diagram shows some important relationships. The ratio of NAD/NADH should be about 700/1. Ozone will stimulate the NQO1 gene which has profound effects on this ratio. NQO1 also stimulates the Sirtuin genes which are very significant in anti-aging and general well-being.
As far as protein metabolism, ozone intervenes mainly due to its remarkable affinity towards sulfhydryl groups. A sulfhydryl is a functional group consisting of a sulfur bonded to a hydrogen atom.
The sulfhydryl group is one of the most reactive and ubiquitous binding molecules in biological systems. It is found in most proteins and also in a few low molecular-weight substances such as glutathione, CoA (Coenzyme A, notable for its role in the synthesis and oxidation of fatty acids), lipoate, thioglycolate, and free cysteine. It is the most studied of chemical groups, particularly in relation to its role in enzymatic activity and properties of proteins.
Similarly, ozone reacts with essential amino acids such as methionine, tryptophan, and other amino acids containing sulfur (i.e., cysteine). In this case, the amino acids are protected from ozone inactivation by two reactions that prevent their degradation: first the oxidation of glutathione and then the oxidation of the coenzymes NADH and NADPH, which are key reactions in the biochemical mechanism of ozone. Nicotinamide adenine dinucleotide, or NAD, is in all living cells, where it functions as a coenzyme. It exists in either an oxidized form, NAD+, which can accept a hydrogen atom (i.e., a proton), or a reduced form, NADH, which can donate a hydrogen atom. Note that "donate a proton" and "accept a pair of electrons" translates to the same thing in biochemistry. Nicotinamide adenine dinucleotide phosphate, or NADP+, is a similar molecule with a similar function, differing from NAD+ in that it contains an additional phosphate group. The oxidized form is NADP+, while the reduced form is NADPH.
Finally, ozone reacts directly with unsaturated fatty acids, which have a double carbon bond and are therefore available for an oxidative reaction, leading to the formation of peroxides following cleavage of the lipid chains. In addition to the direct contributions to cellular metabolism described above, both NADH and NADPH may take part in other important physiological processes, including mitochondrial functions, calcium regulation, antioxidation and its counterpart (the generation of oxidative stress), gene expression, immune functions, the aging process and cell death. As a result, some biochemistry researchers have proposed that further investigation
Ozone dissolved in the plasma reacts immediately with a number of biomolecules producing two compounds. There are two compounds Reactive Oxygen Species (ROS) and Lipid Oxidative Products (LOPS). They represent the “ozone messengers” and are responsible for many of the biological and therapeutic effects attributed to ozone. ROS are produced immediately in the early phase (mainly Hydrogen peroxide or H2O2) and are responsible for the early biological effects on blood (erythrocytes, leucocytes, platelets). Hydrogen peroxide, now universally recognized as one of the main intracellular signaling molecules, acts on the different blood cells. Hydrogen peroxide is one of the most significant cytokine inducers in white blood cells. The mass of erythrocytes mops up the bulk of hydrogen peroxide. H2O2 diffuses easily from the plasma into the cells and its sudden appearance in the cytoplasm represents a triggering stimulus. This stimulus depends upon the cell types. Different biochemical pathways can be concurrently activated in erythrocytes, leukocytes and platelets resulting in numerous biological effects.
On the other hand, Lipid Oxidative Products (LOPS), which are simultaneously produced at the same time as ROS have a far longer half-life. They reach the vascular system and interact with several organs, where they trigger late effects. Some of these real targets are liver, vascular system, while other organs are probably involved in restoring normal homeostasis including the central nervous system, gastrointestinal tract, mucosal associated lymphoid tissue. The LOPS molecules can elicit the upregulation of antioxidant enzymes such as superoxide dismutase (SOD), GSH-peroxidases (GSH-Px), GSH-reductase (GSH-Rd) and catalase (CAT). Moreover, LOPS exert a neuroimmunomodulatory effect highlighted by a feeling of well-being reported by patients during ozone therapy.
OZONE THE WONDER DRUG
Ozone is a wonder drug because it can produce four extraordinary phenomena: 1) the induction of Oxidative Shock Proteins (OSP) 2) the upregulation of antioxidant enzymes (catalase, superoxide dismutase, glutathione peroxidase) 3) the reduction and/or normalization of oxidative stress; and 4) the release of bone marrow stem cells.
Ozone mobilizes bone marrow stem cells (BMSC) and produces LOPS, which induce Nitric Oxide synthase (NOs). This produces inhibition of platelet-leukocyte aggregation.NO is responsible for producing neovascularization and neoangiogenisis. Also, NO activates MMP-9 (matrix metalloproteinase 9) which is indispensable for stem cell mobilization. MMP-9 actually releases the bond that holds stem cells in the bone marrow and releases them to the circulation. The production of Nitric Oxide is one of the main mechanisms by which hyperbaric oxygen will increase stem cell output from the bone marrow. These numbers will include a wide variety of the stem cells that are released from the marrow including Hematopoietic and Mesenchymal Stem cells. Ozone forms lipid oxidative products which in turn produce nitric oxide which makes proteases which release stem cells from the bone marrow into the circulation. The release of stem cells from the marrow is a multi-step process dependent on many different factors.
We can see from the following diagram the very process of how Ozone affects stem cell release:
The above diagram shows release of stem cells (HSC) from the bone marrow to the circulation. This is very dependent upon the production of MMP-9. This stimulation releases the stem cells from the bone marrow and allows them to go into circulation.
The fact that Ozone releases stem cells from the bone marrow is very important. It should be mentioned that this concept is similar to the mechanism of hyperbaric oxygen. In the above diagram it is demonstrates that LOPS, throughout the treatments, act as acute oxidative stressors in the bone marrow microenvironments activate the release of metalloproteinases, of which MP-9 particularly may favor the detachment of stem cells. 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 re-infusion.
OZONE AND OXIDATIVE STRESS
One of the most important aspects of Ozone therapy is activation the Nuclear Factor Erythroid 2 Related Factor 2 commonly known as NrF2. NrF2 is like the body’s “missile defense system”. It helps keep oxidative stress under control. Oxidative stress arises from free radicals. Free radicals are compounds which have free electrons. These rogue molecules can cause havoc by “stealing” electrons from other molecules. Some free radicals are capable of damaging the DNA or crippling the proteins and lipids that make up various tissues. As a result, these crucial molecules become damaged, weak, dysfunctional, or otherwise incapable of fulfilling their roles. Produced deep within cells as a byproduct of ordinary energy-producing processes, free radicals are thought to be responsible for aging and disease, including even cancer. How does NrF2 protect us from free radicals?
NrF2 is basically like a thermostat. But instead of regulating temperature, it regulates stress levels known as oxidative stress. It responds by binding with the DNA, signaling the cells to make thousands of molecules to shield the cells. They will later activate a new response to form a new barrier. This “barrier” will protect the cells from future stress. Furthermore, Nrf2 will remove the toxins that cause cell damage and boost the normal function. Both of these functions will return the stress levels to normal, minimizing the negative effects.
We must realize that we do not want to constantly have NrF2 activated. Ozone is a selective NrF2 activator. How does this work? Ozone produces Antioxidant Responsive Elements (ARE). Antioxidant responsive elements (AREs) mediate the transcriptional induction of a battery of genes which comprise much of this chemoprotective response system and ultimately the NrF2 pathway.
Under normal conditions, Nrf2 is expressed at very low levels, and is mainly sequestered in the cytoplasm by its specific inhibitor called Kelch-like ECH associated protein-1 (Keap-1) that also promotes its rapid degradation. The effectiveness of this mechanism allows a rapid turnover of Nrf2, which displays a half-life of a few minutes. Under specific stimuli, Nrf2 dissociates from Keap1 and translocates into the nucleus and transactivates the ARE-driven genes. These genes encode for proteins involved in a multitude of vital biological functions which include protein homeostasis, oxidative stress response, detoxication, DNA repair, proliferation, autophagy (body's way of cleaning out damaged cells), mitochondrial biogenesis and function, inflammation, and the metabolism of lipids, carbohydrates and amino acids. When you stimulate NrF2 you have a healthier patient.
The following illustration shows these concepts:
NrF2 goes on to control a wide variety of processes in the body including proper formation of proteins, control of inflammation, healthy mitochondrial function, and healthy adipose tissue.
WHAT ARE SOME OF THE OTHER EFFECTS OF OZONE THERAPY
The oxidation chemistry of ozone is known to produce Hydrogen Peroxide (H2O2) that enters cells where it has various effects. In red blood cells (RBCs), it shifts the hemoglobin dissociation curve to the right and facilitates release of oxygen, while in leukocytes (WBCs) and endothelial cells it can stimulate the production of interleukins, interferons, growth factors and nitric oxide production. In platelets it favors release of growth factors. As a further aspect of the therapeutic action of ozone, there is the capacity to regulate the cell antioxidant network positively. This aspect is of key relevance in all those conditions in which an imbalance between production and neutralization of ROS (Reactive Oxygen Species) may develop, resulting in oxidative stress. Again, this relates to the implications of the NrF2 pathway and its activation or inactivation. These events may turn into a self-feeding cycle in which oxidative stress is sustained by micro- and macro-inflammatory reactions that lead to cell and tissue degeneration and necrosis. This scenario features the pathogenetic role of oxidative stress in several chronic, degenerative disease states such as chronic viral infections, atherosclerosis, tumor growth, neurodegenerative diseases and accelerated aging. Ozone can help stop these problems in their tracks.
HEAT SHOCK PROTEINS
Another aspect of Ozone therapy is the production of HEAT SHOCK PROTEINS (HSPS). Shock proteins are a family of proteins that are produced by cells in response to exposure to stressful conditions. They were first described in relation to actual heat shock, but are now known to also be expressed during other stresses including exposure to cold, UV light (this is one of the reasons we are utilizing a UVA light to stress the blood cells) and during wound healing or tissue remodeling. We can also stimulate Heat Shock Proteins by using a sauna or by subjecting our body to very cold temperatures.
Many members of the Heat Shock Protein group perform chaperone functions by stabilizing new proteins to ensure correct folding or by helping to refold proteins that were damaged by the cell stress. This increase in expression is genetically regulated. In the packed, busy confines of a living cell, hundreds of chaperone proteins vigilantly monitor and control protein folding. From the moment proteins are generated in and then exit the ribosome until their demise by degradation, chaperones act like helicopter parents, jumping in at the first signs of bad behavior to nip misfolding in the bud or to sequester problematically folded proteins before their aggregation causes disease. People often mistakenly think that proteins are free to live out their lives in a cell. As it becomes increasingly clear that folding is not a once-in-a-lifetime event for proteins but instead a part of day-to-day life in the cell. Scientists are discovering that problems in this sophisticated system of protein folding are implicated in diseases as diverse as cancer, diabetes, and Alzheimer’s. As time goes on we are finding more and more diseases associated with misfolded proteins. This will open up a new realm in medicine in which Ozone therapy and Photo modulation may be integral players on taking the science of Heat Shock Proteins to the next level. Not only is Heat Shock Proteins important in disease control but it seems to have a place in improving athletic performance.
The dramatic upregulation of the heat shock proteins is a key part of the heat shock response and is induced primarily by heat shock factor (HSF). The following illustrations demonstrate the very important work of the Heat Shock proteins:
HSPs are found in virtually all living organisms, from bacteria to humans. HSPs are known to modulate the effects of inflammation cascades leading to the generation of ROS and intrinsic apoptosis through inhibition of pro-inflammatory factors, thereby playing crucial roles in the pathogenesis of human inflammatory diseases and cancer. There is a scientific push for studying the HSPs for the treatment of various inflammatory diseases and cancer. Also, it appears that a safe and effective method of stimulating HSPs is by the use of Ozone in blood. Heat shock proteins may play a critical role in reducing recovery time and boosting muscle development.
The following illustration shows the mechanism of HSPs:
We can see that the HSPs can have profound implications for a person’s well-being. This is a field where more research will certainly be needed. We can see that when we have an altered expression of Heat Shock proteins many bad things happen. It appears that many autoimmune diseases are related to altered Heat Shock proteins. The following diagram is a good illustration of this.
Chronic inflammatory conditions are accompanied by a partial or sometimes large resetting of the immune system to a pro-inflammatory and pro-oxidant state. This response has gross implications also in the integrity of vascular components and may represent a sensitive therapeutic target of chronic, degenerative conditions. On the basis of these theoretical foundations, reported therapeutic applications for ozone therapy were the activation of the immune system in infectious diseases and cancer, and an improved oxygen utilization and release of growth factors that can reduce the extent of ischemic lesions in vascular diseases.
- Dr. P