Pull Your Blood and Mix It With Saline and Ozone and Have It Go Through Your Body Again

J Transl Med. 2011; 9: 66.

Ozone acting on homo claret yields a hormetic dose-response relationship

Velio A Bocci

¹Dipartimento di Fisiologia, Università degli Studi di Siena, Viale Aldo Moro, 2, 53100, Siena, Italy

Iacopo Zanardi

ⁱⁱDipartimento Farmaco Chimico Tecnologico and European Research Center for Drug Discovery and Evolution, Università degli Studi di Siena, Viale Aldo Moro, ii, 53100, Siena, Italy

Valter Travagli

ⁱⁱDipartimento Farmaco Chimico Tecnologico and European Research Centre for Drug Discovery and Development, Università degli Studi di Siena, Viale Aldo Moro, ii, 53100, Siena, Italy

Received 2010 Nov 29; Accepted 2011 May 17.

Abstruse

The aim of this paper is to analyze why ozone can be medically useful when information technology dissolves in blood or in other biological fluids. In reviewing a number of clinical studies performed in Peripheral Arterial Diseases (PAD) during the last decades, it has been possible to confirm the long-held view that the inverted U-shaped bend, typical of the hormesis concept, is suitable to represent the therapeutic activeness exerted by the and so-called ozonated autohemotherapy. The quantitative and qualitative aspects of human blood ozonation have been also critically reviewed in regard to the biological, therapeutic and prophylactic of ozone. Information technology is hoped that this gas, although toxic for the pulmonary arrangement during prolonged inhalation, will be presently recognized every bit a useful amanuensis in oxidative-stress related diseases, joining other medical gases recently thought to be of therapeutic importance. Finally, the elucidation of the mechanisms of action of ozone besides as the obtained results in PAD may encourage clinical scientists to evaluate ozone therapy in vascular diseases in comparison to the current therapies.

Introduction

Ozone is a double-faceted gas. Information technology has a crucial protective relevance in partially blocking mutagenic and carcinogenic UV radiations emitted by the sunday (wavelengths of 100-280 nm) in the stratosphere [1], while its increasing concentration in the troposphere causes severe pulmonary damage and increased mortality [2,iii]. In spite of this drawback, there are growing experimental and clinical evidences most the medical use of ozone [4-11]. Since Xvi Century, Paracelsus had ingeniously guessed that "all things are poison and zippo is without poison and merely the correct dose differentiates a poison from a remedy". In 2005, Timbrell reiterated the concept in his volume: "The poison paradox; chemicals as friends and foes" [12]. During the Earth evolution, harnessing oxygen by metazoans has allowed a fantastic biodiversity and growth simply it has too created a irksome interim "poison". It is reasonable to believe that the antioxidant arrangement slowly evolved and specialized during the concluding two billion years for counteracting the daily formation (3-v one thousand in humans) of anion superoxide in the mitochondria and the release of H_iiO_ii past ubiquitous NADPH oxidases. However, there is a general consensus that the physiological production of H₂O_two is essential for life. Olivieri et al. [13] and Wolff [xiv] were the showtime to draw the result of either depression concentrations of radioactive thymidine or of a very low dose of radiation inducing an adaptive response in human cells in comparing to a loftier dose. Goldman [xv] introduced the term "hormesis" to mean "the benign issue of a low level exposure to an agent that is harmful at loftier levels". It goes to the merit of Calabrese [sixteen-nineteen] to have experimentally controlled this concept and to have presented a number of examples of stimulatory responses following stimuli below the toxicological threshold. Until 2002 ozone therapy was pharmacologically conceived as a therapy where depression ozone doses were stimulatory, while loftier doses were inhibitory. This conception, reflecting the classical idea that a low antigen dose is stimulatory, where an antigen overdose is inhibitory, was vague and unsuitable because ozone acts in a complex way and a loftier dose can nevertheless be effective but accompanied past side-furnishings. Indeed, ane of united states of america in 2002 amply delineated the sequence of biochemical reactions elicited ex vivo later the addition of a certain book of O₂-O₃ gas mixture to an equal book of human blood [twenty]. Outset of all, mixing blood with an oxidant implies a calculated and precise oxidative stress, i.e. a homeostatic change with production of highly reactive messengers. The oxidative stress, like many others, induces a biological response leading to an adaptive phenomenon. The teleological significance of this response is universal, from leaner to plants and Mammals, and small repetitive stresses induce an extremely useful adaption response represented past the revival of disquisitional defense force mechanisms [20-22]. At the same fourth dimension, Calabrese and Baldwin described the "overcompensation stimulation hormesis" (OCSH) as the result of a compensatory biological procedure following an initial disruption in homeostasis [17]. Afterward a reviewer's information also Re later on had expressed this possibility [23]. Ozone presents some subtle differences that volition be explained by clarifying the biochemical reactions occurring between the organic compounds of plasma and this gas.

Ozone is a Stiff Oxidant Gas

The iii oxygen atoms in gas-stage ozone class an isosceles triangle with a altitude amid the equal sides of 1.26 Å, and exist in several mesomeric states in dynamic equilibrium [24]. In terms of oxidation potential (E°), ozone (ii.07 Five) is the third subsequently fluorine (3.06 V) and hydroxyl radical (two.80 V). Other pertinent oxidants are: hydrogen peroxide (one.77 V), hypochlorous acrid (1.49 Five) and chlorine (ane.36 V). Ozone has a paired number of electrons in the external orbit and, although information technology is non a radical molecule, information technology is far more reactive than oxygen and readily generates some of the ROS produced by oxygen. Ozone is very unstable and at xx °C, with a one-half-life of about 40 min, it decomposes according to the exothermic reaction:

Such an attribute has generated the idea that ozone will donate its free energy to the organism by reacting with specific trunk compartments [twenty]. However, afterwards having ascertained the complexity of the mechanism of action, the conclusion is that ozone dissolved in the water of plasma acts as a pro-drug, generating chemical messengers which volition accelerate transfer of electrons and the overall metabolism. Information technology goes to the merit of Hans Wolff (1927-1980), a German dr., to have developed the O₃-AHT by insufflating ex vivo a gas mixture composed of medical oxygen (95%) and ozone (5%) into the claret independent in a disposable ozone-resistant and sterile glass bottle [25].

Which are the Blood Components Reacting with Ozone?

For most three decades ozone therapy was used simply in Germany by practitioners who, by using empirical procedures, elicited skepticism and prejudice in academic clinical scientists. Just during the last fifteen years, past using mod ozone generators able to photometrically (253.7 nm) measure the ozone concentration in a specified gas volume, in real time, and in a precise mode (hence the precise ozone dose per ml of claret), it has been possible to accurately written report the reactions of ozone with human being blood. Information technology has been clarified that ozone toxicity depends upon its dose and, more than important, that judicious ozone dosages can be neutralized by biological defenses [4,twenty-22,26]. Claret contains some 55% of plasma and almost 45% of cells, the majority of which is represented by erythrocytes. The composition of plasma is complex simply, merely said, information technology contains: well-nigh 92% of water; dissolved ions such as HCO_three ^- and PO_iv ^3- regulate the pH within the range of 7.3-seven.4; both hydrophilic (glucose, uric acid, ascorbic acrid, cysteine and other amino acids) and lipophilic (bilirubin, vitamin E, carotenoids, lycopene) molecules; near 5 mg lipids (triglycerides, cholesterol, phospholipids and lipoproteins); proteins, among which albumin (four.5 g/dl), fibrinogen as well as globulins, amidst which either transferrin or ceruloplasmin binds either Fe²⁺ or Cu⁺, respectively, coagulation factors and hormones. Amongst the plasma main functions, one is the antioxidant activity performed by a diverseness of molecules such equally uric acrid (4.0-7.0 mg/dl, 400 μM), ascorbic acrid (Aa) (0.iv - ane.5 mg/dl, 22,7-85 µ;M), GSH (0.5-1.0 μM), the mentioned lipophilic compounds as well as albumin. In detail, erythrocytes have a great reservoir of GSH (about 1 mmol/l), thioredoxin with 2 available cysteine, and stiff antioxidant enzymes (catalase, GSH-Rd, GSH-Px, GSH-Tr, and SOD). They tin apace wipe out nifty amounts of oxidants such as ^·OH, H₂O_ii, OCl^-, ONOO^- and, at the same time, recycle protons back to oxidised compounds past using protons donated by NADPH continuously regenerated by the activity of G6PD via the pentose phosphate pathway. It must be noted that nearly of these antioxidants work in concert accelerating the reduction of noxious oxidants (Effigy ​ 1). Albumin on its own is the nearly important because it holds nucleophilic residues, such as one gratuitous Cys34 besides as multiple Lys199 and His146 [27,28].

Cellular responses to oxidant exposure. ROOH and ROO• bespeak lipohydroperoxide and its oxygen centered organic radicals formed by radical reactions with cellular components, respectively. GSH and GSSG represent the sulfhydryl/disulfide pair of glutathione species. Nicotinamide adenine dinucleotide phosphate, NADP(H), is the chief electron source, regenerated by the cellular reduction systems.

The Biochemical Reactions of Ozone with Claret

During the virtually precise and safe methodological ex vivo O₃-AHT approach, oxygen-ozone mixture dissolves into the water of plasma. Oxygen has a low solubility, but the pO_ii slowly raises upwards to about 400 mmHg [29]. Hemoglobin get fully oxygenated (Hb₄O₈) but this is hardly relevant because, during the infusion menstruation, it mixes with venous claret which has a pO₂ of about 40 mmHg. On the other hand, ozone behaves quite differently because, past immediately reacting with ions and biomolecules, it does non follow the classical Henry's law in terms of linear solubility variation with pressure level. Beginning of all ozone is about tenfold more soluble than oxygen and, every bit ozone dissolves in the plasmatic water, it instantaneously reacts with hydrophilic antioxidants: by using an ozone concentration of twoscore μg/ml, respective to 0.84 µ;mol/ml per ml of blood, within five min an average of 78% of Aa has been oxidized to dehydroascorbate and almost twenty% of uric acid has been oxidized to allantoin [30]. But virtually 10% of alpha tocopherol has formed an alpha tocopheryl radical. At the aforementioned time the remaining ozone performs the peroxidation of available unsaturated fatty acids, which stand for an elective substrate and are mostly albumin-bound. Peroxidation of n-6 PUFA leads to the formation of H_iiO₂ and 4-hydroxy-2E-nonenal (4-HNE) [31], while due north-iii PUFA leads to the formation of 4-hydroxy-2E-hexenal (four-HHE) [32,33]:

As all of these reactions happen in a few seconds, ozone, until nowadays in the gas phase, continues to dissolve in the plasmatic water and instantly reacts. Within the canonical 5 min, ozone is fully extinct with both a rather small-scale depletion of hydrosoluble antioxidants and the simultaneous plasmatic increase of ROS and LOP. The ozonated blood is and so infused into the donor patient.

What is the Significance and Fate of These Ozone Messengers?

Showtime of all the brief life-span of H₂O_ii will be discussed. During the 5 min of mixing blood with the gas ex vivo, H₂O₂ will dynamically increase its concentration: rapid at start and progressively slowing down as ozone is being depleted. With the therapeutically loftier ozone concentration of 80 μg/ml per ml blood, the H₂O₂ concentration measured in plasma after 2.5 min is at most 40 μM considering the rate of synthesis is equilibrated by multiple degradation routes. Some H_twoO₂ is reduced by gratuitous soluble antioxidants including traces of catalase and GSH-Px. As the hemolysis is negligible (<0.5%), free Fe²⁺ or Cu⁺ are non nowadays and information technology is unlikely that hydroxyl ions are ever formed by either the Fenton-Jackson or the Haber-Weiss reactions. Equally H₂O₂ is unionized, it freely diffuse into all claret cells although the majority is mopped upward by erythrocytes. The establishment of a dynamic, all the same transitory, H_twoO₂ gradient between the plasma and the cytoplasmatic water of claret cells makes this oxidant a very early effector. Its final intracellular concentration may exist not higher than 10%, hence iii-4 μmoles, as it has been demonstrated in other studies [34-39]. The smartness of this system is that the H₂O_ii concentration, though minor, is enough to trigger several crucial biochemical reactions without toxicity considering the internal cell environment contains a wealth of GSH, thioredoxin, catalase and GSH-Px, which practice not allow a dangerous increase. In spite of a threshold of only a few micromoles, it has a critical relevance and ways that an ozone amount below 0.42 μmol for each ml volume of the gas mixture (medical grade O₂ ≥95% and O₃ ≤5%) reacting in a i:1 ratio with autologous blood may be ineffective, resulting in a therapeutic failure of O₃-AHT. It is also necessary to remind that the ozonation process greatly differs whether it occurs either in plasma or in claret. In plasma, TAS levels was, as expected, ozone-dose dependent and decreased between 46 and 63% in relation to ozone concentrations of either 0.84 μmol/ml or 1.68 μmol/ml per ml of plasma, respectively. On the other hand, in blood taken from the same donors, subsequently being treated with the same ozone concentrations, TAS only decreased from 11 to 33% in the first minute afterwards ozonation, respectively. Moreover, it was surprising to determine that they both recovered and returned to the original value inside xx min, indicating the capacity of blood cells to apace regenerate dehydroascorbate and GSH disulfide [34]. It has been also brilliantly demonstrated that, thanks to erythrocytes, dehydroascorbate was recycled dorsum to Aa within 3 min [xl]. On the same way, only nearly xx% of the intraerythrocytic GSH had been oxidized to GSSG within one min later on ozonation and promptly reduced to normal after 20 min [41]. Aa, blastoff-tocopherol, GSH and lipoic acid undergo an orderly reduction by a cooperative sequence of electron donation continuously supplied by NADPH-reducing equivalents to GSH-Rd and thioredoxin reductase [42] (Figure ​ 1). These data, by showing that the therapeutic ozonation simply temporarily and reversibly modifies the cellular redox homeostasis were reassuring regarding the safety of ozone every bit a medical drug. In summary, the initial disruption of homeostasis due to ozone oxidation is followed by the rapid reestablishment of homeostasis with two main advantages: the first being the value of triggering several biochemical reactions in claret cells and the second mediated by LOP compounds, the consecration of an adaptive process due to the up-regulation of the antioxidant enzymes. This is in line with the temporal sequence of the OCSH dose-response relationship.

What is the Action of Ozone in the Blood Cells?

- Erythrocytes

Probably the activation of phosphofructokinase accelerates glycolysis with a demonstrated increase of ATP and 2,three-DPG [4,20]. Functionally, the oxyhemoglobin sigmoid curve shifts to the right attributable to the Bohr upshot, i.due east. a small pH reduction (nigh 7.25) and a slight increase of 2,3-DPG. This metabolite increases only in patients who take a very low level only it remains to be clarified how the phosphoglyceromutase is activated. The shift to the correct is advantageous for improving tissue oxygenation every bit the chemical bonding of oxygen to hemoglobin is attenuated, facilitating oxygen extraction from ischemic tissues. Rokitansky et al., had previously shown that the pO_ii was lowered to twenty-25 mm Hg in the femoral vein of PAD's patient throughout O₃-AHT sessions [43]. Information technology seems obvious that erythrocytes ozonated ex vivo may be modified only for a brief period. Only repeated therapeutic sessions may permit to LOP compounds to reach the bone-marrow and actuate a subtle development at the erythropoietic level, favouring the formation of new erythrocytes with improved biochemical characteristics, which provisionally were named "supergifted erythrocytes" [20]. If this hypothesis is right, every day, during prolonged ozonetherapy, the bone marrow may release a cohort (about 0.9% of the pool) of new erythrocytes with improved biochemical characteristics. In fact, the therapeutic reward does non abruptly stop with the abeyance of the therapy only rather persists for 2-iii months, probably in relation to the life-bridge of the circulating supergifted erythrocytes [26]. It is interesting that during prolonged ozonetherapy, by isolating through a sedimentation slope the small portion of very immature erythrocytes, it has been demonstrated that they have a significant college content of G6PD [44]. Such a issue strengthens the postulation that just a cycle of more than than 15 treatments (not less than 3 liters of ozonated blood) could amend an ischemic pathology.

- Leukocytes

Homo neutrophils are able to generate an ozone-similar molecule [45] and volatile compounds [46] as a office of their phagocyte activity. Neutrophil phagocytic activity has been constitute enhanced during ozonetherapy [47]. Moreover, H₂O₂ activates a tyrosin-kinase with subsequent phosphorylation of IkB, one of the trimeric components at rest of the ubiquitous transcription factor denominated NF-kB [48,49]. The phosphorylated IkB detaches from the trimer and information technology is broken down in the proteasome. The remaining eterodimer p50-p65 is transferred into the nucleus, where information technology tin can actuate about 100 genes up-regulating the synthesis of astute-phase proteins, several proinflammatory cytokines (IFN-γ, TNF-α, IL-8) and fifty-fifty HIV proteins [l]. At that place is no doubt that H₂O_ii is the trigger as the activation is related to a cysteine oxidation that can exist prevented by an backlog of thiol. Although ozone is a very modest inducer of some cytokines [fifty], the consequent immunomodulatory effect may be useful in immune-depressed patients after chemotherapy, or in chronic infectious diseases. It must be clear that ozone in itself cannot exist in the apportionment and moreover, due to the strong antioxidant capacity of plasma, information technology is unable to kill any pathogens in vivo whereas an activated immune system may be helpful [51].

- Platelets

During O_iii-AHT, the detection of PDGF-B, TGF-β_i, IL-8 and EGF released in heparinized plasma in ozone- dose dependent quantities was not surprising because platelets are exquisitely sensitive to a progressive acute oxidative stress [20,52]. The increased level of these growth factors in the circulation may have the benign effect of enhancing the healing of foot-related problems from diabetes or PAD.

The pleiotropic LOP activities

As shown in Figure ​ 2, LOP product follows peroxidation of PUFA present in the plasma: they are heterogeneous and tin exist classified as lipoperoxide radicals, alkoxyl radicals, lipohydroperoxides, F₂-isoprostanes, as well as aldehydes like acrolein, MDA and terminal hydroxyl alkenals, amongst which iv-HNE and 4-HHE. As free radicals and aldehydes are intrinsically deleterious, only precise and appropriate ozone doses must be used in order to generate them in very low concentrations. Amid the aldehydes, 4-HNE is quantitatively the virtually important. It is an amphipathic molecule and it has a brief-half-life in saline solution. On the other hand it reacts with a multifariousness of compounds such equally albumin, enzymes, GSH, carnosine, and phospholipids [31,53]. There is no receptor for 4-HNE but information technology has been reported that, in concentration above 1 μM in vitro, afterward binding to more seventy biochemical targets, it exerts some deleterious activeness [31]. On the other manus, during the rapid reaction of ozone with blood, the generated hydroxy-alkenals, will form adducts both with GSH or with the abundant albumin molecules. This possibility is supported by findings which have shown that homo albumin, rich in accessible nucleophilic residues, tin can quench upwards to nine 4-HNE molecules, the first being Cys34, followed by Lys199 and His146 [27,28]. Interestingly, when samples of ozonated human plasma were incubated at 37 °C for 9 hours, 4-HNE, nigh probable bound to albumin, remained stable [54]. These data clarify why a judicious ex vivo ozonation of blood does not harm the vascular system during the infusion into the donor. Aerobic organisms, in order to tolerate the continuous generation of aldehydic compounds accept adult detoxifying systems every bit follows: the first is the dilution of these products in both the plasma and the extracellular fluid involving a volume of about 11 L in humans. The second is the detoxification operated past aldehyde dehydrogenase, aldose reductase and GSH-Tr [55,56] and the 3rd is the excretion via bile and urine excretion [57-59]. The relevance of these catabolic pathways was appreciated when the one-half-life of infused alkenals present in ozonated blood in a patient was less than five min [60]. The interesting aspect is that albumin can transport iv-HNE in all trunk tissues, from liver to endocrine glands and the CNS. 4-HNE-Cys adducts, released at many sites, inform a variety of cells of a transient, acute oxidative stress and represent an of import biochemical trigger. At submicromolar or picomolar levels, 4-HNE can human action every bit a signaling molecule capable of activating the synthesis of γ-glutamate cysteine ligase, γ -glutamyl transferase, γ -glutamyl transpeptidase, HSP-70, HO-i, and antioxidant enzymes such as SOD, GSH-Px, catalase and last just not least, G6PDH, a critical electron-donor enzyme during erythropoiesis in the bone marrow. There is a wide consensus on the relevance of the induction of protective molecules during small simply repeated oxidative stress [20,61-65]. In other words, the concept that a precisely controlled oxidative stress tin strengthen the antioxidant defenses is well accustomed today. Once once more, the low level of stress past enhancing the fettle of the defense force organization, is consequent with the hormetic concept. Moreover at the time of ozonated claret infusion, 4-HNE-Cys adduct tin can as well act on the vast expanse of endothelial cells and enhance the production of NO [35]. Such a crucial mediator on its own or as a nitrosothiol, with a trace of CO released with bilirubin via HO-1 activity, allows vasodilation, thus improving tissue oxygenation in ischemic tissues [66]. H₂Due south is another potentially toxic molecule that, when released in trace amounts, it becomes an important physiological vasodilator similar NO and CO [67,68]. Moreover, every bit it happens for the mentioned physiological traces of other gases, the pocket-sized corporeality of ozone necessary to trigger useful biological effects is in line with the concept of the hormesis theory [69].

Generic scheme of polyunsaturated fatty acids peroxidation. Arachidonic acid reactions have been detailed, but similar pathways are applicable to other polyenoic fatty acids. MDA: malondialdehyde. HHE: iv-hydroxy-2E-hexenal. HNE: 4-hydroxy-2E-nonenal.

Another interesting aspect observed in almost 2/iii of patients is a sense of wellness and physical energy throughout the ozonetherapy [lxx]. It is not all the same known whether these feelings are due to the power of the generated ozone messengers which tin alter or improve the hormonal secretion. On the other hand, the feeling of euphoria may be due to improved oxygenation or/and enhanced secretion of growth hormone, ACTH-cortisol and dehydroepiandrosterone [26,71]. Furthermore, when LOP reach the hypothalamic area they may better the release of serotonin and endorphins, as it was observed afterward intense dynamic do [72]. Feel acquired after thousands O_three-AHT has clarified that there is neither objective nor subjective toxicity, or to use Calabrese's acronyms, in that location is no observable agin effects (NOAEL). Moreover, neither structural nor enzymatic amercement have been observed in blood components afterward ozonation of blood within the therapeutic window [73,74]. On the other hand, patients with more than advanced disease during the initial session especially if performed with a high ozone dosage, oftentimes study to feel very tired and sleepy. This is the lowest observed adverse effect level (LOAEL) that has been observed in about ten% of PAD'due south patients with stage Iii and IV of the Leriche-Fontaine's classification. Such a knowledge compels to begin always with low ozone dosage and advisedly discover the patient'southward response.

Which is the Almost Suitable Term for Describing the Dose-Response Human relationship Between Ozone and Claret?

Ozone is a toxic gas and it cannot be compared to either any usual immunological stimulus or to stable chemic compounds: firstly, nobody has ever described a cell receptor for ozone, and secondly the biochemical reactions with blood components generate diverse messengers with quite different half-lives, finalities and fate. Moreover, non but biological but also clinical responses take to exist taken into account when using ozonetherapy in quite different pathologies such as cardiovascular, or autoimmune or orthopedic diseases. The hormetic dose response appears to be useful for describing the dual pharmacological response elicited by ozone, basically acting as a pro-drug. The most common class of the hormetic dose response bend, depicting depression dose stimulatory and high dose inhibitory and toxic responses is the ß- or inverted U-shaped curve shown in Figure ​ 3, panel a. All the same, the graphic analogy of the hormetic dose-response relationship between ozone and claret needs an explanation considering it slightly differs from graphs presented on the upshot of other stressors (Figure ​ 3, panel b) [26,75-78]. It has been establish that an ozone dose of just x µ;g/ml (0.21 μmol/ml) per ml of claret is fully neutralized by both uric acrid and Aa, particularly when the TAS of individual blood is between one.5-1.9 mM [79]. Information technology follows that the minimal reaction, if any, with PUFA will not generate plenty messengers every bit ROS and LOP to trigger biological effects. In this case the small ozone dose is totally consumed by available gratuitous antioxidants and the ozonated claret will not display therapeutic activity. Gaseous ozone doses between twenty and fourscore µ;g/ml (0.42-one.68 μmol/ml) per ml of claret are well calibrated against claret'due south TAS and both biological and therapeutic effects will ensue. A recent metabonomic written report has shown that the blood antioxidant chapters is almost exhausted when the ozone dose has been raised to 160 µ;g/ml per ml of blood [74]. In simple words, too lilliputian ozone, unable to modify the homeostatic equilibrium, is unable to elicit the hormetic response. On the basis of the last observation, it would be near interesting to clarify the response in normal volunteers.

The hypothetical inverted U-shaped curve describing an platonic dose-response human relationship (panel A). The inverted U-shaped bend drawn on the basis of the therapeutic effect in PAD's patients by using an ozone concentration range between 15 and 80 μg/ml of gas per ml of blood. During a course of 15-twenty sessions, the initial ozone concentration of 10 μg/ml has been slowly upgraded to the concentration of 80 μg/ml (panel B). The stop-points that have been considered to decide the therapeutic effects are: claudication; ankle-brachial alphabetize; disappearance of pain; healing of skin ulcers.

Ozone Therapy in Oxidative-Stress Related Diseases

The metabolic syndrome is recognized as one of the most serious affliction in Western countries acquired past a number of metabolic alterations such as type-ii diabetes, hypercholesterolaemia, atherosclerosis and renal dysfunction with the mutual denominator represented by a chronic oxidative stress. Diabetic patients, particularly those with pes ulcers, are critical and today they still have a gloomy prognosis. This is because they need a multiform therapy aiming to eliminate the peripheral ischemia, the neuropathy and the infected skin lesions. The range of ozone concentrations between 15 and 35-l µ;g/ml is prophylactic also in individuals with a low TAS level and it appears to be particularly effective in PAD [43,80-85]. Several clinical studies performed in different hospitals seem to establish the validity of the inverted U-shaped curve in this frequent pathology (Figure ​ 3, panel B). In line with "the concept of a benign consequence inside the context of a dose-response study is hard to determine due to considerable biological complication and the fact that beneficial effects are often seen with reference to a specific and relative setting" [17], a word of caution is necessary. This is particularly truthful when ozone therapy is performed in unlike patients within the variety of 3 PAD's Two, Iii and Four stages, according to the Leriche-Fontaine classification [86]. First of all it is necessary to trust the precision of ozone's dosages used by unlike clinicians and secondly, ozone activity cannot be compared with that expressed by a single chemical compound (see, eg, Arsenic [76], and homocysteine [77]) in cultured cells. As it has been antiseptic, the real ozone messengers are H₂O₂ every bit a ROS and a variety of alkenals as LOP. These messengers act on different cells, have a quite different lifetime and alkenals are intrinsically toxic. Furthermore, each patient has his ain medical history and his own psycho-physical reactivity. Consequently, ozone dosages between 0.42-0.84 µ;mol/ml generate less alkenals than dosages in the range 0.84-1.68 µ;mol/ml, and therefore patients with a low antioxidant capacity become more than susceptible to a side effect like deep fatigue afterwards the therapy session. Attention must be besides paid to the type of pharmacological response accomplished in different pathologies as either muscular-orthopedic or autoimmune diseases. Then far, in the latter information technology remains unknown the ozone dosage, if whatever, able to increment the T-cell regulatory levels and activity. Consequently, at this phase the U-shaped curve remains meaningful only for PAD and only time to come trials will be able to ascertain the ozone behavior in either stroke or chronic heart disease. Martinez-Sanchez et al. take also reported that the theoretical U-shaped bend fits the ozone therapy results [87]. Claret ozonation, even if performed within the therapeutic range and for a few minutes, represents always a calibrated acute oxidative stress. In order to never damage the patient, the strategy: "start low-become slow" is a gilt dominion to induce a valid adaptation to the far more than unsafe chronic oxidative stress, typical of inflammatory and degenerative diseases [88]. Such an aspect implies that the terminal therapeutic effect is due to an average of progressively increasing ozone dosages.

The gas mixture medical course oxygen-ozone can be proficiently used for the ozonation of blood because this incomparable liquid tissue contains an imposing assortment of antioxidants, which are able to tame not only its oxidant power but also its messengers (ROS and LOP) generated by the reactions with blood components. Therefore, if ozone is judiciously used within the established therapeutic window (0.42-1.68 μmol/ml per ml of autologous blood) in PAD, it tin exert better therapeutic furnishings than the current therapy by prostacyclin analogue. Moreover, regarding the accompanying foot-related problems, both some ozone derivatives similar ozonated water and different gradation of standardized ozonated vegetable oils will be used until complete healing [89,90]. As stroke, heart infarction and PAD are cumulatively the offset crusade of expiry and inability, if it will go possible to employ ozone therapy in the public hospitals of the developed Countries, it may be possible to enter a phase where ozone volition become an extensive remedy. Moreover, there is no doubt that either infective or autoimmune glomerulo-nephritis as well as end stages of renal failure associated with hemodialysis are characterized, to a different extent, by an imbalance between pro- and antioxidative mechanisms [91]. Moreover the kidney does not take the regenerative ability of liver and this is one of the reasons for explaining why too often "nephropaties lack a specific treatment and progress relentlessly to end-stage renal affliction" [92]. Another important reason is that till today a valid strategy to reduce oxidative stress in renal diseases is not available. Ozone therapy, non merely may correct a chronic oxidative stress, but it may also stimulate untapped resources able to afford some improvement [ix,93]. Information technology appears therefore reasonable to suggest the combination of conventional treatments with mild O₃-AHT in any initial nephropathy for preventing the risk of progression towards a chronic disease.

In several Countries, among others Republic of cuba, Russia, and Ukraine, treatments by ozone are already a reality, although unlike assistants modalities, such as the infusion of ozonated saline and of the rectal insufflations of ozone, are in current use because inexpensive and applicative to thousands of patients every day [94]. Nevertheless, it is hoped that adequate ozone-based therapeutic treatments for patients affected by oxidative-stress related diseases could be implemented in every public hospital.

Conclusions

During the last two decades the paradoxical behaviour of ozone has been clarified: when information technology is chronically inhaled, it is highly toxic for the pulmonary system because the enormous alveolar surface, unprotected by sufficient antioxidants, is exposed to the cumulative ozone dose, which causes a chronic inflammation. This is non surprising considering even for oxygen [95], equally well as for glucose and uric acid levels a modification of the physiological concentrations is deleterious.

On the ground of the mechanisms of activity, ozone therapy appears to be a safety, economical, effective treatment for patients with cardiovascular disorders based on the post-obit biological responses [26]:

a) information technology improves claret circulation and oxygen delivery to ischemic tissue owing to the concerted event of NO and CO and an increment of intraerythrocytic two,3-DPG level;

b) by improving oxygen delivery, it enhances the general metabolism;

c) it upregulates the cellular antioxidant enzymes and induces HO-ane and HSP-70;

d) it induces a mild activation of the immune arrangement and enhances the release of growth factors from platelets;

e) information technology procures a surprising wellness in about of the patients, probably past stimulating the neuro-endocrine system. However, ozone dosages must exist calibrated confronting the antioxidant chapters of the patient'south plasma, or otherwise the "commencement low-go slow" strategy must be used evaluating the subjective feeling of the patient later on each session.

It remains to be clarified whether some messengers nowadays in the ozonated blood are able to stimulate the release of staminal cells in the patient's bone marrow.

The evaluation of results obtained in several clinical trials performed in PAD has allowed to establish that the dose-response relationship in PAD tin can be depicted every bit an inverted U-shaped hormetic model with a brief, initial lack of effect due to the potency of claret antioxidants. A mild acute oxidative stress induced by ozone in blood ex vivo, as several other mild stresses due to either heat or cold exposure, a transient ischemia, other chemicals and physical exercise are able to induce a sort of "preconditioning response" oft leading to both a repair and an increased defense chapters well within the "overcompensation stimulation hormesis". This new achievement, added to an increasing broad consensus in advisedly using gases as NO, CO, H₂South, N₂O and H₂ equally existent medical drugs [68], suggests that as well ozone may exist soon included into this category. I of the basic functions of ozone, subsequently dissolving in the water of plasma is to accelerate the substitution of protons and electrons or, in simple words, to reactivate the metabolism all over the body. In this manner, crucial biological functions gone off-target tin recover indicating that ozone operated as both a biological response modifier and an antioxidant inducer.

Information technology is hoped that this paper will elicit the interest of clinical scientists in evaluating ozone therapy in vascular, renal and diabetic diseases, thus translating the laboratory results to the patient'south bed.

Abbreviations

2,three-DPG: 2,3-diphosphoglycerate; 4-HHE: iv-hydroxy-2E-hexenal; 4-HNE: 4-hydroxy-2E-nonenal; Aa: ascorbic acid; ACTH: adrenocorticotropic hormone; ATP: adenosine triphosphate; CNS: central nervous organization; EGF: epidermal growth factor; G6PD: glucose-half-dozen-phosphate dehydrogenase; GSH: glutathione; GSH-Rd: glutathione reductase; GSH-Px: glutathione peroxidase; GSH-Tr: glutathione transferase; GSSG: oxidized glutathione; HIV: homo immunodeficiency virus; HO-1: heme-oxygenase-I; HSP-seventy: oestrus shock proteins (70 kDa); IFN-γ: interferon γ; IkB: inhibitor of NF-kB; LOAEL: lowest observed adverse effect level; LOP: lipid oxidation products; IL-eight: interleukin 8; MDA: malondialdehyde; NADPH: nicotinamide adenine dinucleotide phosphate; NF-kB: nuclear factor kappa-calorie-free-chain-enhancer of activated B cells; NOAEL: no observable adverse event level; OCSH: overcompensation stimulation hormesis; PaO₂: partial pressure of arterial oxygen; PO_ii: fractional force per unit area of oxygen; O₃-AHT: ozonated autohemotherapy; PAD: peripheral arterial diseases; PDGF-B: platelet-derived growth factor, subunit B; PUFA: polyunsaturated fat acids; ROS: reactive oxygen species; SOD: superoxide dismutase; TAS: total antioxidant status; TGF-β₁: transforming growth factor β₁; TNF-α: tumor necrosis factor.

Competing interests

The authors declare that they have no competing interests.

Authors' contributions

VAB and VT conceived, outlined the direction of, provided data to shape the manuscript content and discussion, gathered references, and drafted the manuscript. IZ refined the search for information, gathered references, and generated the figures. All authors have read and approved the final manuscript.

Writer Details

VAB, Grand.D., Emeritus professor of Physiology, Department of Physiology, University of Siena, Viale Aldo Moro, ii, 53100, Siena, Italian republic

IZ, in charge equally mail-doc position at the Section of Pharmaceutical Chemistry and Technology, Viale Aldo Moro, ii, 53100, Siena, Italy

VT, Associate professor in Pharmaceutical Technology and Chief of the Postal service-Graduate School of Hospital Chemist's shop, University of Siena, Viale Aldo Moro, two, 53100, Siena, Italy

Acknowledgements

This paper is defended to Mrs Helen Carter Bocci who for decades has generously linguistically corrected our papers.

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Source: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3125221/

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