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Effects of ozone on blood components

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Title:
Effects of ozone on blood components
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English
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Sloan, Daniela
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University of South Florida
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Subjects / Keywords:
Erythrocyte
Glutathione
C-reactive protein
Autohemoadministration
Dissertations, Academic -- Environmental & Occupational Health -- Doctoral -- USF   ( lcsh )
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non-fiction   ( marcgt )

Notes

Abstract:
ABSTRACT: Previous studies on the medical use of ozone therapies show a very diverse array of results, from ozone reducing the amount of HIV virus in the blood, to no effect, to causing the death of several patients due to pulmonary embolism and infections. However, ozone therapies are widely used in Europe and considered medically safe. In the U.S., doctors in 28 states use ozone therapies. The objectives of this study were to investigate the effects of medical grade ozone at varying concentrations used in ozone therapies. These were achieved by evaluating the C-reactive protein, erythrocyte sedimentation rate, total reduced and oxidized glutathione content of erythrocytes which were all markers used to determine ozone injury/inflammation. Despite the fact that ozone is a very strong oxidant, previous research indicates that depending on the dose and the health status of the biological system, sometimes ozone can act as an antioxidant. The medical exposure range for ozone is between 20 -80 mg/ml with an average of 50 mg/ml. The concentrations used in this study were 20, 40, 80 and 160 mg/ml. Ozone was generated in the "Breath Lab" at USF from medical grade oxygen obtained through electrical corona arc discharge using an OL80C ozone generator. De-identified blood samples of 10 ml blood/sample containing EDTA as anticoagulant were obtained from the James A. Haley VA Hospital patients. Equal volumes of blood and ozone gas mixture were allowed to mix in ozone-resistant syringes prior to dividing each sample into three parts, one for each corresponding parameter to be studied. The C-reactive protein was analyzed through ELISA using the colorimetric method available from Helica Biosystems; erythrocyte sedimentation rate was measured in graduated sedimentation tubes; the total reduced glutathione (GSH) and oxidized glutathione (GSSG) content of erythrocytes was determined according to the colorimetric method developed by the Oxford Biomedical Research. Overall, the concentrations of ozone used did not have a statistically significant effect on the parameters investigated. However, a small percentage of the blood samples showed an improvement in the parameters studied, especially at the highest ozone concentration.
Thesis:
Dissertation (Ph.D.)--University of South Florida, 2010.
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Includes bibliographical references.
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by Daniela Sloan.
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Includes vita.

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Effects Of Ozone On Blood Components by Daniela Sloan A dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy Department of Environmental and Occupational Health College of Public Health University of South Florida Major Professor: Yehia Hammad, Ph.D. Azliyati Azizan, Ph.D. Skai Schwartz, Ph.D. Thomas Truncale, D.O. Date of Approval: April 7, 2010 Keywords: erythrocyte, glutathione, c-reactive prot ein, autohemoadministration Copyright 2010 Daniela Schiopu

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Dedication To my husband and parents for all their love and su pport

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Acknowledgments A heartfelt thank you for Dr. Yehia Hammad, without whom this research would not have been possible and Dr. Thomas Truncale, Dr. Azliyati Azizan and Dr. Skai Schwartz for all the help with the study design and execution. We are very grateful for all the help provided by the Phlebotomy Lab and the Res earch & Development Committee staff from the James A. Haley Veterans Affairs Hosp ital in collecting the blood samples and guiding us through the IRB approval process. A special thank you goes to all VA Hospital patients who agreed to take part in our st udy.

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i Table of Contents List of Tables..................................... ................................................... .............................iii List of Figures.................................... ................................................... .............................iv Abstract........................................... ................................................... ..................................v List of abbreviations.............................. ................................................... ........................vii Introduction....................................... ................................................... ................................1 Background........................................ ................................................... ...................1 Significance of the research...................... ................................................... ..........16 Methods............................................ ................................................... ...............................17 Blood sample collection and preparation........... ................................................... .17 Erythrocyte sedimentation rate.................... ................................................... .......21 C-reactive protein................................ ................................................... ................23 Glutathione....................................... ................................................... ...................25 Statistical analysis.............................. ................................................... .................28 Results ........................................... ................................................... ..................................29 Erythrocyte sedimentation rate.................... ................................................... .......29 C-reactive protein................................ ................................................... ................37 Glutathione....................................... ................................................... ...................40 Combined results.................................. ................................................... ..............42

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ii Discussion......................................... ................................................... ..............................45 References......................................... ................................................... ..............................49 Appendix A: Calibration Data for the Rotameters and Needle Valve...............................55 Appendix B: Calibration Curve for C-Reactive Protei n.................................................. ..62 Appendix C: Calibration Curves for Glutathione..... ................................................... ......64 Appendix D: Erythrocyte Sedimentation Rates........ ................................................... ......67 Appendix E: C-Reactive Protein Results............. ................................................... ...........70 Appendix F: Glutathione Absorbancies for Ozone Trea tments.........................................73 About the Author................................... ................................................... .............End Page

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iii List of Tables Table 1 Summary of Previous Studies on Ozone...... ...............................................7 Table 2 Summary of Previous Studies on Blood and Oz one..................................12 Table 3 Calibration Data for the Rotameters and Nee dle Valve.............................55 Table 4 Normal Erythrocyte Sedimentation Rates..... ............................................21 Table 5 C-Reactive Protein Standards............... ................................................... ..24 Table 6 Glutathione Standards...................... ................................................... .......26 Table 7 Erythrocyte Sedimentation Rate Results.... ...............................................30 Table 8 C-Reactive Protein Results................ ................................................... .....37 Table 9 Glutathione Ratio Results................. ................................................... ......40 Table 10 Patient Response to all Three Tests....... ................................................... .43

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iv List of Figures Figure 1 Calibration Data for the Rotameters and N eedle Valve.............................60 Figure 2 Wintrobe Tubes for Erythrocyte Sedimentat ion Rate................................22 Figure 3 Calibration Curve for C-Reactive Protein.. ................................................62 Figure 4 Calibration Curves for Glutathione........ ................................................... .64 Figure 5 Erythrocyte Sedimentation Rate............ ................................................... .67 Figure 6 Erythrocyte Sedimentation Rate for Control s............................................32 Figure 7 Erythrocyte Sedimentation Rate for Ozone Treatments............................33 Figure 8 Combined Erythrocyte Sedimentation Rates.. ...........................................33 Figure 9 Difference in Erythrocyte Sedimentation Ra te Between the Highest Ozone Treatment and Baseline............................ ..................................................3 5 Figure 10 Difference in Erythrocyte Sedimentation R ate between the Lowest Ozone Treatment and Baseline............................ ................................................. 3 6 Figure 11 C-Reactive Protein Results............... ................................................... ......70 Figure 12 Combined C-Reactive Protein Results...... .................................................39 Figure 13 Glutathione Absorbancies for Ozone Treatm ents......................................73

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vii Acronyms CC16 protein – Clara cell serum protein CHD – coronary heart disease CRP – C-reactive protein hsCRP – high sensitivity C-reactive protein DTNB 5,5'-Dithio-Bis 2-nitrobenzoic acid ESR – erythrocyte sedimentation rate GSH –glutathione (reduced) GSHPx – glutathione peroxidase GSSH – glutathione (oxidized) IgG Immunoglobulin MPA – metaphosphoric acid NADPH nicotinamide adenine dinucleotide phosphate NOS2 – nitric oxide synthase PAHs – polyaromatic hydrocarbons SOD – superoxide dismutase TMB 3,3’, 5,5’-tetramethylbenzidine VOCs – volatile organic compounds

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i Effects of Ozone on Blood Components Daniela Sloan ABSTRACT Previous studies on the medical use of ozone thera pies show a very diverse array of results, from ozone reducing the amount of HIV v irus in the blood, to no effect, to causing the death of several patients due to pulmon ary embolism and infections. However, ozone therapies are widely used in Europe and considered medically safe. In the U.S., doctors in 28 states use ozone therapies. The objectives of this study were to investigate th e effects of medical grade ozone at varying concentrations used in ozone therapies. These were achieved by evaluating the C-reactive protein, erythrocyte sedimentation rate, total reduced and oxidized glutathione content of erythrocytes which were all markers used to determine ozone injury/inflammation. Despite the fact that ozone is a very strong oxidan t, previous research indicates that depending on the dose and the health status of the biological system, sometimes ozone can act as an antioxidant. The medical exposure range for ozone is between 20 -80 m g/ml with an average of 50 m g/ml. The concentrations used in this study were 20 40, 80 and 160 m g/ml.

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ii Ozone was generated in the “Breath Lab” at USF from medical grade oxygen obtained through electrical corona arc discharge using an OL 80C ozone generator. De-identified blood samples of 10 ml blood/sample containing EDTA as anticoagulant were obtained from the James A. Haley VA Hospital patients. Equal volumes of blood and ozone gas mixture were allowed to mix in ozone-resistant syri nges prior to dividing each sample into three parts, one for each corresponding parame ter to be studied. The C-reactive protein was analyzed through ELISA using the colori metric method available from Helica Biosystems; erythrocyte sedimentation rate w as measured in graduated sedimentation tubes; the total reduced glutathione (GSH) and oxidized glutathione (GSSG) content of erythrocytes was determined accor ding to the colorimetric method developed by the Oxford Biomedical Research. Overall, the concentrations of ozone used did not have a statistically significant effect on the parameters investigated. However, a s mall percentage of the blood samples showed an improvement in the parameters studied, es pecially at the highest ozone concentration.

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1 Introduction Background Ozone is one of the five major air pollutants along with carbon monoxide, sulfur dioxide, nitrogen dioxide and particulates. The dif ference between ozone and these four pollutants is the fact that it is not emitted direc tly into the air from industrial facilities, power plants or automobiles. Instead, ozone is a ph otochemical pollutant, a major component of photochemical smog, created when the s unlight mediates chemical reactions with other pollutants (Breslin, 1995). Du e to this effect, human exposures to atmospheric ozone are of interest and thus have bee n studied for a long time. Most commonly the effects of ozone are lung injury, resp iratory infections and inflammation caused by ozone concentrations of 0.1 ppm in adults and as little as 0.085 ppm in children. These findings prompted the USEPA to revi se its 0.12 ppm standard and set a limit of 0.08 ppm ozone/8 hours, based on the decis ion that this level protects the public health (Moore 1999). However, studies on southern C alifornians showed that the pulmonary function changes at 0.5 ppm are less seve re during a high-ozone season compared to a low-ozone season, suggesting an incre ased tolerance to ozone but not less cellular damage (Munzer et al ., 1995). On the other hand, ozone has been widely use d in the medical field for ozone therapies, the most com mon being ozone autohemotherapy or ozonated autohemoadministration (Hiromichi et al. 2 006,

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2 en.wikipedia.org/wiki/Ozone_therapy). Autohemothera py is an alternative medical technique practiced for about 50 years in Europe, w hich involves withdrawing up to 200 ml of venous blood, then immediately mixing it with therapeutic concentrations of ozone gas and a minimal amount of anticoagulant, usually heparin, then re-injecting it into the basilic vein at the elbow. Other techniques are ozo ne bagging, when all parts of the body except for the head are placed in a bag full of ozo ne at a concentration of 100 m g of ozone /ml air mixture for up to 2 hours; ozone rectal ins ufflation, where an average of about 1 1/2 liters of 27 m g/ml O3 gas are introduced into the colon; ozone vaginal i nsufflation, where the vagina is insufflated for about 5 minutes ; ozone ear insufflation, where O3 is introduced in the ear cavity for an average of 5 mi nutes; ozone air purification, where low levels of ozone sterilize and rejuvenate the room a ir and lastly ozone charged drinking water, where O3 is bubbled into water which must be imbibed immedi ately while the O3 is still in the glass (oxygenmedicine.com). Experim ental evidence suggests that these therapies may boost the immune system, reduce the n umber of viruses in the blood and a reduction in lung, breast and uterine tumors (Sweet et al. 1980). A study by Wells et al. (1991) demonstrated that ozone was able to inactiva te HIV-1 virions in a dosedependent manner. Ozone concentrations of 1200 ppm achieved g reater than 11-log virus inactivation within 2 hours from ozone administrati on (2-log means 99% inactivation). The authors developed and used a T cell line – HUT 78/HIV-1AAV stably infected with HIV-1. The ozone was delivered into the cell medium through a closed hollow fiber system as a stream of ozone/oxygen, using nitrogen as the carrier. Ozone therapy is used legally in 16 countries, mostly in Europe. In the U.S., recently passed Alternative Therapy Legislation has made ozo ne therapy an option for patients in

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3 13 states. In Alaska, Arizona, Colorado, Georgia, M innesota, New York, New Jersey, North Carolina, Ohio, Oklahoma, Oregon, South Carol ina, and Washington, physicians can legally use ozone treatments in their practice without fear of prosecution. (wikipedia.org). Scientific papers that described studies on the ef fects of ozone and/or ozone therapies in relation to blood provided contradicti ng results. First, there are the studies that found no effect of ozone on blood and blood co nstituents. Biedunkiewicz et al. (2006) found no evidence that ozone affects blood c oagulation and fibrinolysis. It would have been expected that ozone reduces blood viscosi ty and inhibits coagulation, which are important side effects for patients undergoing hemodialysis. Autohemoadministration lead to no statistical differences between C-reacti ve protein at baseline and after ozone treatment in patients undergoing hemodialysis (Tyli cki et al. 2004). This result proved that autohemoadministration is safe for the patient s. Zimran et al. (1999) showed that ozone does not affect red blood cell enzymes and in termediates or red blood cell integrity. Furthermore, ozone neither damage erythr ocytes, nor induced oxidation of intracellular hemoglobin in the case of heparin-tre ated blood (wikipedia.org). A study by Travagli et al. (2006) did not yield significant he molysis or methemoglobin when whole blood was treated with a therapeutic concentration of ozone. In contrast, there are studies that showed either a negative or a positive effect of ozone. Bocci et al. (1999) showed that during ozonated autohemotherapy, ozone induced formation of platelet aggregation (blood clots) in heparin (anticoagulant)-treated bl ood (Bocci et al. 1996). Larini and Bocci (2004) showed that cytokine production was de pressed at ozone concentrations above 40 m g/ml. Bocci (2006) advocated the use of ozone thera pies and ending the

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4 labeling of ozone therapies as a dangerous or toxic while recognizing that atmospheric ozone can be responsible for respiratory system dam age. In this case, ozone therapies were shown to improve blood circulation and oxygen delivery to ischemic tissues and induce a mild activation of the immune system (Bocc i 2006). The same author, Bocci (2007) later demonstrated that ozone can activate b iochemical pathways in leukocytes, erythrocytes and platelets without acute or chronic toxicity, and decreases blood plasma antioxidant capacity for about 20 minutes (Bocci, 2 007). Patients under maintenance hemodialysis who were given autohemoadministration showed a decrease in blood access recirculation, which is the return of the dialyzed blood into the extracorporeal circuit through the arterial needle, rather than returning to the systemic circulation. This is a positive effect, helping to maintain the effectiven ess of hemodialysis, even though these results were not statistically significant (Tylicki et al. 2004). Blomberg et al. (2003) showed that exposure to atmo spheric ozone impairs lung function, induces airway inflammation and alters ep ithelial permeability, as shown by analysis of CC16 protein from peripheral blood (Blo mberg et al. 2003). Animal studies showed that ozone exposure results in local bronchi al inflammation and also affects the nervous system and thymocyte proliferation, and pla ces mice under oxidative stress (Feng et al. 2006). In rats, ozone exposed animals had an increased lyzozyme activity and a decreased total protein, both being an indicator of liver disease (Jakubowski et al. 2004). Another study on mice by Kenyon et al. (2006 ) demonstrated that ozone induced acute lung injury but the NOS2 enzyme present in so me mice had a protective effect against lung injury. Experiments on male rats resul ted in a positive linear relationship between ozone concentration and the concentrations of serum total lipoprotein free

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5 cholesterol (FCh) and high-density lipoprotein tota l cholesterol (HDL-Ch) (Mole et al. 1985). A study reported by Hiromochi et al. (2006) on the effects of ozone autohemoadministration in cows, showed significant changes in leukocyte populations following ozone blood stimulation (Hiromichi et al. 2006). Other studies showed were that exposure to ozone increases sensitivity to the toxicity of other chemicals like 1nitronaphtalene (Schmelzer et al. 2006); external o zone exposure combined with internal exposure to PAHs and VOCs resulted in low level of DNA damage in teenagers but it is not clear if ozone alone can be responsible for the mutations (Koppen et al. 2007). Air pollutants, including ozone, were shown to cause pu lmonary inflammation in both human and animals under experimental conditions; this cau ses an increase in the liver inflammatory markers, fibrinogen and C-reactive pro tein. However, when air pollutant exposure of 40 healthy volunteers was studied over the course of a 1-year period, there was no relationship between air pollutants and the amount of fibrinogen and CRP (Rudez et al. 2009). Lab experiments on healthy volunteers showed that exposure to an ambient air ozone concentration of 0.5 ppm induced a signif icant decrease in vital capacity and total lung capacity, expiratory flow rates and an i ncrease of respiratory frequency on exercise (Hazucha et al. 1989).This concurs with a study by Bowler and Crapo (2002) showing that ozone exposure decreases the forced ex piratory volume FEV1 and children playing in areas with high concentrations of enviro nmental ozone have a higher incidence of asthma. Despite these results, a review of 24 st udies concluded that a threshold concentration below which no effects on pulmonary f unction are elicited cannot be defined (Hazucha, 1987).

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6 A positive effect of ambient ozone was noticed whe n cancer cells extracted from lung, breast and uterine tumors were exposed to ozo ne concentrations between 0.3 to 0.8 ppm in ambient air. Concentrations between 0.3 to 0 .5 ppm inhibited cancer growth between 40 and 60 percent. A concentration of 0.8 p pm inhibited cancer cell growth more than 90 percent. This shows that human cancer cells have an impaired defense mechanism against ozone, compared to normal cells ( Sweet et al. 1980). A summary of some of the previous studies on ozone therapies and their effects, positive, negative or no effect is given below in T able 1.

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7 Table 1. Summary of Previous Studies on Ozone. Previous studies Sample size Positive effects Negative effects No effect Guven et al. (2009) rats Reduced intestinal damage oxidative stress Rodriguez et al. (2009) rats Increase in antioxidant enzymes, decrease myeloperoxidase (damage marker) in lungs Labuschagne et al (2009) baboons Up-regulated antioxidant capacity Schultz et al. (2008) rabbits Remission of squamous cell carcinoma Jiao & Peng (2008) 42 Improvement in liver function for hep. B Mustafaev et al. (2007) 20 Prevention of pyoinflammatory complications following transurethral resection of prostatic adenoma Ohtsuka et al. (2006) cows Increased CD4+/CD8+ ratio Jakubowski et al. (2004) rats Increased lysozyme activity, decreased total protei n level Clavo et al. (2004) 18 Improved oxygenation in hypo xic tumors Simonetti et al. (2003) 600 Additive effect for lum bar disk herniation Al-Dalain et al (2001) rats Improvement in glycemic control Sweet et al. (1980) cells Inhibited cancer cell gro wth

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8 Table 1 (Continued) Blomberg et al. (2003) 22 Increased CC16 serum (marker for ozone-induced lung damage) Hazucha et al. (1989) 14 Inhibited inspiration, reduced total lung capacity and vital capacity Forsberg et al. 3430 Increased blood fibrinogen Gornicki & Gutse (2000) cells Lead to changes in erythrocyte membranes, cytoskeletal proteins Schmelzer et al. (2006) rats On cytokine production Biedunkiewicz et al. (2006) 11 On blood coagulation parameters Tylicki et al. (2004) 12 On inflammation response in hemodialyzed patients

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9 The main difference between the studies that did n ot find any effects of ozone on blood and the studies that found a wide diversity o f effects, can be attributed to the different methodology used. In general, the finding s of the studies with no effects were based on a single concentration of ozone to work w ith; whereas the studies that were able to prove that ozone had either a positive or n egative effect looked at several ozone concentrations. However, the latter analyzed a spec ific problem such as heparinized blood from male donors (wikipedia.org), heparinized or citric acid treated blood from volunteers between 23 and 27 years old, plasma (Boc ci et al. 1999), release of cytokines from mononuclear cells (Larini & Bocci 2005), total cholesterol in male rats (Mole et al. 1985) or methemoglobin (Bocci & Aldinucci 2006). An other possible explanation for the in vitro damaging effect of ozone on blood componen ts is that the ozone toxicity is exerted when cells are incubated in antioxidant-f ree culture media and therefore do not benefit of the antioxidant capacity of the blood (L arini & Bocci, 2005). According to Hernandez (2007), one of the reasons why ozone in medicine has not been approved as a common practice is its use witho ut an appropriate control. The main ozone therapy mechanism of action is based on an ex tremely transitory and regulated oxidative stress imposed ex vivo (Bocci, 2002). At the same time, ozone therapy acts as an efficient oxidative stress regulator stimulating the antioxidant system of the cell. As reactive oxygen species attack a variety of organic substrates, oxidative stress can be evaluated by measuring reaction products of oxidati ve damage. Because of this, it would be necessary to assess the patient's redox status b efore and during application of ozone therapy in order to control the safety doses of ozo ne to be applied in each application. Previous reports remarked on the lack of studies o n the effects of ozone on the

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10 immune responses, on peripheral blood leukocytes, t he mechanism of ozone therapies and that there are controversies on whether the blo od should be diluted or not. Furthermore, while the fact that ozone damages the membrane of erythrocytes is well known, the ozone therapies are considered by many a s safe but others claim that ozone concentrations within the medical range cause degra dation of the proteins in erythrocyte membranes (Fischbach, 2000). Common markers of inflammatory responses to infect ions and chemical agents are an increase in Creactive protein and a short-term increase in GSH (glutathione) levels. Glutathione is an antioxidant that protects cells f rom toxins such as free radicals generated by the powerful oxidative properties of o zone. The C-reactive protein is usually absent in the blood of healthy persons and appears rapidly in blood and body fluids as a response to injurious stimuli (Fischbac h, 2000). However, another study by Ridker et al. (2000) showed that high levels of the high sensitivity C-reactive protein were found in the blood of healthy postmenopausal w omen. Later on, these women developed various forms of cardiovascular disease a nd hsCRP was the significant predictor of cardiovascular risk out of 12 plasma v ariables. This result confirmed a previous study (Kuller et al., 1996) which was the first to show a direct correlation between CRP and coronary heart disease (CHD) mortal ity in healthy but high risk men. The correlation between CRP and CHD mortality is st rengthened when other risk factors like smoking are present. However, the results of t his study were not able to show a correlation between CRP and nonfatal myocardial inf arctions, only a correlation between CRP and CHD deaths. Ridker et al. (2005) pursued th is topic and for high sensitivity assays of CRP, their cut-offs were less than 1 mg/L for low risk, 1 to 3 mg/L for

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11 moderate risk, and greater than 3 mg/L for high ris k. The drawback is that the continuum extends beyond that. The patients with the very hig hest levels of hsCRP —5 to 10, 10 to 20, or even greater than 20 mg/L—are at the very hi ghest risk. These were not false positives and they helped to explain why people wit h periodontal disease, arthritis, and other systemic inflammatory disorders had higher va scular risk. A plausible explanation is that inflammation from any cause has an adverse effect on the vascular endothelium. Antioxidant enzymes like copper/zinc superoxide di smutase (SOD), catalase and gluthatione peroxidase (GSHPx) are part of the intr acellular protection mechanism important in overcoming oxidative stress and are k nown to be activated in vascular diseases and acute stroke (Zimmermann et al. 2004). In this study, nearly two thirds of the patients with a stroke in the past showed decre ased GSH levels and the authors speculate it was possibly associated with increased oxidative stress and arteriosclerosis. The GSH levels and antioxidant capacity are also de creased following an organ transplant, which may indicate the need for glutath ione supplementation to improve antioxidant status (Wierzbicka et al. 2007). Glutat hione (GSH) is an important tripeptide thiol ( g -glutamyl cysteinyl glycine) antioxidant and its in tracellular concentration is indicative of oxidative stress. The oxidative stres s is a common marker of many diseases such as chronic lung diseases, neurodegenerative di seases rheumatoid arthritis, amyotrophic lateral sclerosis and most recently AID S (Rahman et al. 2005, Halliwell 1996). Within the cell glutathione is found in two forms: GSH, the reduced sulfhydryl form and GSSG, the oxidized disulfide form (Rahman et al. 2005). A summary of some of the previous studies on ozone therapies and thei r effect on blood is given below in Table 2.

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12 Table 2. Summary of Previous Studies on Blood and O zone EFFECTS OF OZONE ON FINDINGS FROM PREVIOUS STUDIES SAMPLE SIZE ESR CRP GSH OTHER Bocci et al. 2006 1 n/a n/a n/a Total antioxidant status decreased temporarily Travagli et al. 2007 3 No effect Decrease in GSH enzymes but not significant Decrease in total antioxidant status, no effect on fibrinogen, cholesterol Bocci et al. 1999 5 No effect Decrease in total antioxidant status, reversible platelet aggregation Biedunkiewicz et al. 2006 11 No effect on blood coagulation, fibrinolysis Tylicki et al. 2004 12 No effect Gornicki & Gutsz, 2000 21? Effect on erythrocyte membrane fluidity is dose dependent Clavo et al. 2004 18 Ozone increased tumor oxyge nation Goran et al. 2009 40 No effect Ozone increased platelet aggregation, thrombin generation Mustafaev et al. 2007 20 Decrease Increase in leu kocytes, phagocytes Haddad et al. 2009 horses Decrease in gamma glutamyltransferase, increase in fibrinogen Ohtsuka et al.2005 cows Increase in plasma protein, serum protein, and globulin, CD4+T cells

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13 Table 2 (Continued) Lambuschagne et al. 2009 baboons Decrease in tota l GSH Rodriguez et al. 2009 -O2/O3 mix insufflated in to lower abdomen, not blood rats Increase in GSH enzyme activity Guven et al. 2009 rats Increase in GSH enzyme activity Al-Dalain et al. 2001 rats Ozone prevented oxidative stress damage INHALED OZONE Forsberg et al -poster 3430 Ozone increases the amount of fibrinogen Blomberg et al. 2003 22 Ozone increased serum Clara cell protein Jakubowski et al. 2004 rats Decreased levels Rudez et al. 2009 40 No effect

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14 During the past couple of decades, ozone has been w ildly used in the medical field as ozone therapies, the most common being ozo ne autohemotherapy or ozonated autohemoadministration. However, its effects are st ill controversial. Advocates of these techniques sustain that ozone is beneficial for tre ating a large array of diseases including inflammatory and degenerative conditions of the bon es and joints, cardiovascular diseases, reducing viral load in HIV infections and stopping cancer proliferation. At the other end of the spectrum, there are ozone treatmen ts that resulted in the death of the patients or infections. The early techniques of inj ecting ozone gas into the patients veins, lead to pulmonary embolism and death of the patient s. Most recently, the only fatality was caused by septicemia as a result of using conta minated needles and a more frequent effect was infection with hepatitis virus; however, these are not a consequence of ozone exposure, they are a result of improper administrat ion of medical techniques. It is common knowledge that ozone is a very strong oxidant, with a solubility 10times higher than oxygen. Therefore, it would be ex pected that ozone would cause cell membrane damage, oxidative stress and inflammation. The reasons why it is so hard to assign ozone therapies to a definite class of effec ts can be explained by the “poison paradox: chemicals can behave as friends or foes de pending on the dose and the biological system”. Taking it a step further, it is known that “most drugs produce many effects, all drugs produce at least two effects (Wa lsh, 2005). In conclusion, even though ozone is a strong oxidant, sometimes it can act as an antioxidant. The objectives of this study are to improve the stu dy design, used previously by other researchers, to determine optimal sample size and o zone concentration interval, to evaluate a

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15 combination of inflammation and oxidative stress ma rkers and to provide an answer to the controversy over the effects of ozone therapies. The primary hypotheses tested were: 1. The concentration of C-reactive protein in the bloo d does not increase with increasing concentrations of ozone in blood. 2. Erythrocyte sedimentation rate is not affected by i ncreasing concentrations of ozone in blood. 3. The ratio of reduced/oxidized glutathione does not change with increasing concentrations of ozone in blood. The secondary hypotheses tested were: 1a. Ozone concentrations above 100 m g of ozone/ml of blood will increase the concentration of Creactive protein in blood. 2a. Erythrocyte sedimentation rate increases with increasing concentrations of ozone in blood. 3a. The amount of oxidized glutathione incre ases with increasing concentrations of ozone in blood.

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16 Significance of the research This study does not approve or disapprove of the us e of ozone therapies, but it does intend to shed some light onto the controversy that surrounds the health effects of ozone. The purpose of the study is to look at the effects of varying ozone concentrations that are within medical range (50-100 g ozone/ml blood) compared to untreated blood, and the effects of ozone concentrations up to 3 times highe r than the most common blood ozone therapy concentration, 50 g ozone/ml blood and detect the concentration where ozone starts to have a deleterious effect on blood compon ents that are primary markers of injury and/or inflammation. Previous studies analyzed diff erent parameter combinations than the ones chosen in this study, and generally used o nly one ozone concentration and had a very small sample size consisting of patients with little variation in their health status. In general, autohemotherapy uses a concentration of 50 g ozone /ml blood. Bagging techniques use higher concentrations, usually 100 g ozone /ml air mixture.

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17 Methods Blood sample collection and preparation The experiments were conducted on de-identified blo od samples collected from the James A. Haley VA Hospital patients who were sc heduled to have blood drawn by the Phlebotomy Lab staff. The sample size was set t o include 20 patients and the blood from each patient was split into five subsamples, o ne to serve as a control and the other four to be treated with various ozone concentration s. The tests chosen to assess ozone damage were erythrocyte sedimentation rate (ESR), C -reactive protein (CRP) and glutathione ratio (GSH/GSSG). According to the stud y protocols used, the amounts of blood needed for each subsample were 1 ml blood for ESR, 150 l blood for GSH/GSSG and 850 l blood for CRP for a total of 2 ml/subsam ple and a total of 10 ml for a full set of experiments for each patient. Initially, the stu dy design required each patient to donate 30 ml of blood, aside from the blood needed for the VA Hospital tests, in order to provide three replicates for each test. Following consultat ions with the VA Hospital and the IRB committee, it was decided that each patient will do nate 10 ml of blood for this study. All patients who agreed to take part in this study were given information about the study, asked to acknowledge if they meet the exclusion cri teria and then asked to sign a consent form. Because the parameters studied can be affecte d by certain inflammatory conditions, the exclusion criteria for the study were patients with HIV, hepatitis, rheumatoid arthritis,

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18 pneumococcal meningitis, chronic lung disease, cong estive heart failure, sickle cell anemia, polycythemia, inflammatory bowel disease an d post-transplant patients. The blood was collected in tubes coated with EDTA to pr event blood coagulation. Due to a high concern for the privacy of the patien ts it was not possible to persuade the IRB committee that a label of random s equences of numbers and letters would be de-identified enough to protect the privac y of the patients. As a result, in order to have to tubes labeled with random and unrelated codes, astronomical data for sun rising and setting as provided by the U.S. Navy Oce anography portal for different cities in the U.S. was used. None of these cities or data were associated with Tampa, FL or the James A. Haley VA Hospital. Once the blood was coll ected, the tubes were placed on ice and transported to the “Breath Lab” in the College of Public Health where the experiments were conducted. The ozone was produced by an ozone generator, using medical-grade oxygen. The ozone concentrations used in the study were 20, 40, 80 and 160 g/ml of blood. Prior to exposing the blood to ozone, the OL80 ozone generator (from Ozone Services and Ozone Lab, Burton, BC, Can ada) and the needle valve on the oxygen cylinder were calibrated for the concentrati ons used with a low flow rotameter and a high flow rotameter (see Appendix A, Table 3a b and c). Appropriate settings were developed from the calibr ation data to conduct the experiment. When using the low flow rotameter, the steel ball was chosen over the glass ball because it provided a better fit for the data, R2= 0.975 compared to an R2= 0.94. Similarly, when using the high flow rotameter, the steel ball was chosen over the glass ball because it provided a better fit for the data, R2= 0.886 compared to an R2= 0.865 (see Appendix A, Figure 1a and 1b).

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19 The OLC80 ozone generator needed two different oxyg en flows to generate the four ozone concentrations needed, 31 ml/min and 125 ml/m in. Based on the two figures mentioned above it was determined that we needed to use the low flow rotameter on a setting of 79 for a flow rate of 31 ml/min and the high flow rotameter on a setting of 23 for a flow rate of 125 ml/min. The next step was to calibrate the needle valve on the oxygen cylinder based on the rotameter settings. For the low flow rotameter the steel ball and a polynomial function provided a better fit for the data. For the high f low rotameter the steel ball and a polynomial function provided a better fit for the d ata. (see Appendix A, Figure 1c and 1d). Based on the above mentioned two figures, in o rder to obtain the flow rates needed by the ozone generator, the needle valve had to be positioned on a setting of 8 when using the low flow rotameter and a setting of 3.5 when us ing the high flow rotameter. Using ozone-resistant syringes, 2 ml of blood were extracted from the 10 ml blood sample into each syringe, resulting in five s yringes/blood sample or five syringes/patient. The five syringes corresponded to one control and four ozone treatments. The ozone generator and needle valve we re set for the first ozone concentration used and the generator was allowed to run for five minutes to ensure that the concentration goal was reached. Then, a 2 ml vo lume of gas mixture at the desired concentration was extracted into the corresponding syringe, previously filled with blood, resulting in a 1:1 blood:gas mixture by volume rati o. The syringe was then placed on a platform mixer and allowed to mix 20 minutes at low speed to prevent foaming. This procedure was repeated for the other ozone concentr ations. The control samples received 2 ml air and were then placed on the platform mixer

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20 Five blood samples were collected at one time for three out of the four collection days. During one of the collection days, one of the patients did not have enough blood to donate more than 5 ml of blood, therefore the sampl e had to be discarded and an additional patient enrolled in the study. The total sample size was twenty patients (n=20), each donating 10 ml of blood which was further divi ded into 2 ml subsamples. After the blood was divided into amounts specific for the thr ee tests, the empty test tubes were disposed of appropriately, no later than 8 hours af ter blood collection.

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21 Erythrocyte sedimentation rate (ESR) After mixing, 1 ml of blood was taken from each syr inge and inserted into the Wintrobe reservoir of its corresponding graduated t ube for ESR. The graduated tube was inserted into the reservoir, adjusting its depth so the blood level reaches the “0” mm mark on the tube. The tubes had to be placed in a vertic al position to prevent any bias in determining the sedimentation rate (see figure 2). One hour later, the difference between the blood level and the initial “0” mm level was de termined. The difference, expressed as mm of blood/hour represents the erythrocyte sedimen tation rate. The erythrocyte sedimentation rates are affected by age and gender. Table 4 below shows the expected ESR values. Table 4. Normal Erythrocyte Sedimentation Rates nnr r n rn All the tubes were discarded following ESR determi nation. The total number of tubes was 100, with five tubes for each of the twen ty patients.

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22 F igure 2. Wintrobe Tubes for Erythrocyte Sedimentati on Rate.

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23 C-reactive protein (CRP) The C-reactive protein was assessed according to th e Helica Biosystems research ELISA (enzyme-linked immunosorbent assay) protocol from blood serum. The serum was extracted from the 850 l of whole blood left a vailable from the 2 ml blood subsample. The blood was placed into microcentrifug e tubes and centrifuged to separate the serum. The serum was pipetted into fresh centri fuge tubes and frozen at -70C for one month, until CRP was determined. The total number o f CRP samples evaluated was 100, which was five samples for each one of the twenty p atients. The reagents and the five standards were prepared a ccording to the Helica protocol. The serum samples underwent a two-step di lution with wash buffer, the first at a 1:1,000 ratio and the second at a 1:4 ratio for a 1:4,000 total dilution. One hundred l from each of the diluted serum samples was added in each of the corresponding microplate wells. The wells were coated with an aff inity purified rabbit antihuman CRPIgG. This was the antibody for human serum CRP (ant igen). The microplates were then incubated at room temperature for 30 minutes to all ow the samples to react with the antibody coating of the microplate wells. After the incubation, the microplates were washed four to five times with buffer (phosphate-bu ffered saline with Tween 20) and placed on paper towels to dry. Each well received 1 00 l of conjugate, (a horseradish peroxidase (HRP)-labeled rabbit anti-human CRP-IgG with stabilizers and a preservative) followed by incubation and buffer was hing as above. The purpose of the conjugate is to react with and tag the antigen-anti body complexes. After these steps, 100 l of TMB (3,3’, 5,5’-tetramethylbenzidine) were ad ded in each well and allowed to

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24 incubate for 10 minutes. If a blue color developed, that was an indication of a positive sample. Next, 100 l of Stop solution (phosphoric a cid) were added to each well. The stop solution causes the color to turn yellow, maki ng it possible to be read in the microplate reader at 450 nm. Each microplate was ru n with a set of standard solutions at predetermined concentrations. A standard concentrat ion curve was constructed using the absorbancy readings for each of the standards used (Table 5, Figure 3 in Appendix B). Table 5. C-Reactive Protein Standards Concentration (ng/ml) Absorbance 3.33 1.292 3.33 1.355 1.11 0.561 1.11 0.517 0.37 0.234 0.37 0.234 0.12 0.122 0.12 0.151 The standard concentration curve was used to conve rt the absorbancy readings of the serum samples into C-reactive protein concentra tions, multiplying by four to get the actual C-reactive protein serum concentration in g /ml. The normal C-reactive protein levels are those between 0 and 5 g/ml. However, so me researchers consider 10 g/ml to be the upper limit for normal CRP values. In this s tudy, 5 g/ml was the cut-off value used for C-reactive protein because a high risk of heart disease is associated with CRP values as low as of 3 g/ml. Higher levels of C-reactive protein are found durin g late pregnancy, mild inflammation and viral infections (10–40 mg/L), act ive inflammation, bacterial infection (40–200 mg/L) and severe bacterial infections and b urns (>200 mg/L) (wikipedia.com).

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25 Glutathione The remaining 150 l of blood from the original 2 m l blood sample treated with ozone were used to prepare the reduced (GSH) and ox idized (GSSG) glutathione samples. There were a total of 100 GSG and 100 GSSG samples, which were ten samples for each of the twenty patients. Reduced glutathione is a tripeptide that contains a free thiol group. In a glutathione peroxidase catalized reaction, two mole cules of GSH are bound together to form one molecule of GSSH. For the accurate measurement of GSSG and the GSH/GS SG ratio, a glutathione assay needs to prevent the oxidation of GSH in the sample. In this case, a pyridine derivative was used as a thiol-scavenging reagent, which reacts quickly with GSH but does not interfere with the activity of the glutath ione reductase enzyme. For a GSSG sample, thirty l of thiol scavenger were added to a microcentrifuge tube then 100 l of blood were added to the centrifuge tube and mixed g ently. The purpose of the scavenger was to keep the glutathione in its oxidized form. F or a GSG sample, 50 l of blood were added into an empty centrifuge tube. All the sample s were frozen at -70C until they were used for glutathione determination. Prior to glutathione determination, the samples wer e thawed and prepared according to Oxford Biomedical GT-35 protocol as fo llows. Into the GSSG sample centrifuge tube were added 270 l ice-cold 5% MPA ( metaphosphoric acid), making a dilution factor x 4 and then the tube was vortexed briefly. The sample was then centrifuged at 1000 x g and 4C for 10 minutes. After centrifuging, 50 l of the

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26 supernatant was collected with a pipette and added to 700 L assay buffer in a new microcentrifuge tube. This step added to a dilution factor x 15 therefore making the total dilution factor x 60. The GSH sample centrifuge tub e received 350 L ice-cold 5% MPA, a dilution factor x 8 and was vortexed briefly. The sample was then centrifuged at 1000 x g and 4C for 10 minutes. After centrifuging, 25 l of the supernatant was collected with a pipette and added to 1.5 ml assay buffer in a new microcentrifuge tube. This step added to a dilution factor x 61 therefore making the tota l dilution factor x 488. In parallel, all the reagents from the assay kit we re reconstituted from received stock and assay buffer. The seven standard solution s used to make the calibration curve for GSG and GSSG were prepared (Table 6). Next, 200 l of the blank solution (assay buffer) was added to a cuvette along with 200 l DTNB (5,5'-Dithio-Bis 2-Nitrobenzoic Acid) solution and 200 l reductase solution. The solutions in the cuvette were mixed and were incubated at room temperature for five minutes After incubation 200 l NADPH (nicotinamide adenine dinucleotide phosphate) solut ion added to the cuvette, causing the solution to turn yellow. The change of absorbance a t 412 nm was recorded by taking readings every minute for 10 minutes. This procedur e was repeated for all the standards and the GSH and GSSG samples. Table 6. Glutathione Standards n B0 0.000 0.000 0 0 S1 0.100 0.050 0.0234 0.0234 S2 0.250 0.125 0.0293 0.0059 S3 0.500 0.250 0.0374 0.0315 S4 1.000 0.500 0.0588 0.0273 S5 1.500 0.750 0.068 0.0407 S6 2.000 1.000 0.0708 0.0301 S7 3.000 1.500 0.1018 0.0717

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27 Because the concentration of GSSG is much lower in the reaction mixture compared to GSHt the protocol recommends that selec ted data ranges from the calibration curve should be plotted separately. For GSHt, linear regression was done on a three-point curve using the 0, 0.50, 0.75, 1.0, and 1.50 M GSSG (0, 1.0, 1.5, 2.0, and 3.0 M GSH) data points. In the case of GSSG, the 0, 0.0 5, 0.125, and 0.25 M GSSG data points were used (see Appendix C, Figure 4). After all the samples were read, an 11-point graph was generated for all samples; the slope of the line wass equal to sample rate. Th e calibration is described by the regression equation: Net Rate = Slope x GSH + Intercept In order to calculate the total GSH (GSHt) or GSSG concentration from the GSH calibration curve: GSH =((Net Rate – Intercept)/slope) x Dilution Fact or The GSH/GSSG Ratio was calculated using the formula : Ratio =(GSHt-2xG SSG)/GSSG This assay measured the reduction of GSH to GSSG. T he rate of the reaction was proportional to the GSH and GSSG concentration. The smaller the GSH/GSSG ratio, the higher the oxidative stress, as it would indicate a high amount of oxidized glutathione.

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28 Statistical analysis The data was tested for normality using leaf plots, box plots and normal probability plots. All three data sets were skewed to the left, indicating smaller numbers (smaller erythrocyte sedimentation rates, smaller c oncentrations of C-reactive protein, smaller glutathione ratios) were predominant. SAS statistical package and GLM procedure (General Linear Models) were used to detect differences among the ozone treatmen ts and among the samples from the patients. The independent variables were the ozone treatments and the patients (samples). In the case of ESR, models using additional indepen dent variables –age, gender, age nested within gender, interaction between age and g ender were also used but only age was statistically significant. Least square means w ere computed for the independent variables, with p-values for differences in LS mean s. Any difference with a probability p< 0.05 was consi dered statistically significant.

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29 Results Erythrocyte sedimentation rate An indication of a positive effect of ozone therapi es is a decrease in erythrocyte sedimentation rate. An increase in the erythrocyte sedimentation rate is a marker of inflammatory damage. The erythrocyte sedimentation results observed in t his study follow the pattern shown in previous studies of some positive effects, some negative effects, and some no effect. This is further complicated by the fact tha t some patients may exhibit both positive and adverse effects, depending on the ozone concent ration used (see results in Table 7 below).

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30 Table 7. Erythrocyte Sedimentation Rate Results Erythrocyte Sedimentation Rate (mm/hr) Concentration 0 1 2 3 4 Patient A 6 5 7.3 6.5 5 B 4 9 9 9 7 C 7 14 12.5 10 7.5 D 14 14 14 10 15 E 45 48 42 49 44 F 39 24 20 40 21 G 60 18 34 75 57 H 34 36 32 27 34 I 4 3 4 4 3 J 7 8 4 8 8 K 15 20 12 10 15 L 29 30 24 25 25 M 1 16 6 13 52 N 21 18 25 26 28 O 10 11 7 7 6 P 39 40 28 37 27 Q 48 32 39 29 18 R 7 8 9 8 9 S 40 24 18 16 40 T 1 4 7 4 4 *Where the ozone concentrations are: 0 =0 g/ml, 1 =20 g/ml, 2=40 g/ml, 3= 80 g/ml, 4= 160 g/ml

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31 A decrease in erythrocyte sedimentation rate, a pos itive effect, was noticed in six out of the 20 patients. For the graphical represent ation see Appendix D, Figures 5c, 5l, 5o, 5p, 5q and 5s for patients C, L, O, P, Q, and S The ages of these patients ranged between 31 and 73 years old. Out of these six patie nts, four of them showed a steadily decreasing trend in erythrocyte sedimentation rate from the control to the highest ozone concentration (Figure 5l, 5o, 5p and 5q). One of th e six patients showed a decrease in erythrocyte sedimentation rate only for the lowest three ozone concentrations, 20 80 g/ml (Figure 5s). The last of the six patients sho wed a decreasing trend in erythrocyte sedimentation rate from the lowest ozone concentrat ion, 20 g/ml to the highest ozone concentration 160 g/ml but all these rates were hi gher than the baseline, 0 g ozone/ml blood (Figure 5c). An increase in erythrocyte sedimentation rate, whic h is an adverse effect as it indicates an inflammatory condition was noticed in four out of the 20 patients. These results are shown in Appendix B, Figures 5g, 5m, 5n and 5s for patients G, M, N, and S. The ages of these patients ranged from 29 to 63 yea rs old. Two of these four patients showed an increasing trend from the control to the highest ozone concentration (Figure 5m and 5n). One of the four patients showed an incr ease in the erythrocyte sedimentation rate from the lowest ozone concentration to the hig hest but the results for the two lowest ozone concentrations were still better than the pat ient’s baseline (Figure 5g). The last of the four patients showed an increase in erythrocyte sedimentation rate only for the highest ozone concentration (Figure 5s). A total of eleven patients showed no effect on the erythrocyte sedimentation rate when comparing the control and the four ozone conce ntrations (Figure 5a, 5b, 5d, 5e, 5f,

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32 5h, 5i, 5j, 5k, 5r, and 5t for patients A, B, D, E, F, H, I, J, K, R, and T). The ages of these patients cover the entire spectrum from 22 to 74 ye ars old. When plotting the results for controls only (Figure 6 below), age does not appear to have an influence on erythrocyte sedimentation r ate. An equal number of people below and over 50 years old have normal erythrocyte sedim entation rates. Figure 6. Erythrocyte Sedimentation Rates for Contr ols The patients are denoted by letters from A to T, arranged based on their age an d the number 0 next to the patient identification letter represents the baseline, 0 g ozone/ml blood. Similarly, when plotting the results for the treatm ents only (Figure 7 below), people below and over 50 years old had equal number s of erythrocyte sedimentation rates above the normal limit. Age Females Males Controls only 0 10 20 30 40 50 60 70 F0T0N0S0O0P0J0B0R0H0D0K0L0I0Q0M0E0G0C0A0 Patient ESR (mm/hr) 0 10 20 30 40 50 60 70 80 Age (yrs)

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33 Figure 7. Erythrocyte Sedimentation Rates for Ozone Treatments. The patients are denoted by letters from A to T, arranged based on t heir age with the four bars/patient representing each ozone concentration in ascending order. A closer look at the combined effects of ozone and age reveals that people below 40 years old generally showed a decrease in erythro cyte sedimentation rate with increasing ozone concentrations. This is a positive result. In contrast, people above 45 years old had an increase in erythrocyte sedimentat ion rate under the four ozone concentrations compared to the control. This is an adverse result, indicating an inflammatory response (Figure 8a and b). Fig. 8a. comparison between normal and above normal values Above normal Normal values Age Females Males Treatments only 0 10 20 30 40 50 60 70 80 FTNSOPJBRHDKLIQMEGCA Patient ESR (mm/hr) 0 10 20 30 40 50 60 70 80 Age (yrs) 0 10 20 30 40 50 60 70 80 2225293137394143434553606061616263647374 Age (years)ESR (mm/hr)

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34 Fig. 8b. normal values for men and women Figure 8. Combined Erythrocyte Sedimentation Rates. Next, the results for the erythrocyte sedimentation rate obtained at the highest ozone concentration, C4=160 g/ml, were compared to the baseline (C0) erythrocyte sedimentation rate (Figure 9a). A negative differen ce between the erythrocyte sedimentation rate at the highest ozone concentrati on and the one at baseline indicates a decrease in the erythrocyte sedimentation rate due to ozone treatment, indicating a positive result (shown in Figure 9b). Most of the p atients who showed a negative C4-C0 difference were over 60 years old, indicating that they are the category that responds best to high ozone concentrations. men women 0 10 20 30 40 50 60 70 80 2225293137394143434553606061616263647374 Age (years)ESR (mm/hr)

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35 a. Values above show a positive (C4-C0) difference, va lues below show a negative difference b. Nine of the 20 patients showed a negative C4-C0 di fference Figure 9. Difference in Erythrocyte Sedimentation R ate between the Highest Ozone Treatment and Baseline When the same comparison was done between the lowe st ozone concentration C1=20 g/ml and the baseline, C0 a similar number o f people under and over 60 years ESR -40 -20 0 20 40 602562436174414373375360296045223931636164Age (yrs) C4-C0 (mm/hr) 0 10 20 30 40 50 60 70 C0 (baseline) in mm/hr C4-C0 C0 ESR -35 -30 -25 -20 -15 -10 -5 0 20304050607080 Age (yrs) C4-C0 (mm/hr)

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36 old had negative C1-C0 differences indicating a dec rease in erythrocyte sedimentation rate as a result of ozone treatment (Figure 10). Figure 10. Difference in Erythrocyte Sedimentation Rate Between the Lowest Ozone Treatment and Baseline However, the statistical models show that there are no statistically significant differences among the ozone concentrations, p=0.56. The age of the patients and the differences between patients are statistically sign ificant, both with p=0.0001 but for the ozone concentrations used the statistical model cou ld not detect a difference among ozone treatments. ESR -50 -40 -30 -20 -10 0 20304050607080 Age (yrs) C1-C0 (mm/hr)

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37 C-reactive protein Similar to the erythrocyte sedimentation rate, an i ndication of a positive effect of ozone therapies is also a decrease in the concentra tion of C-reactive protein. An increase in C-reactive protein is a marker of inflammatory d amage. Again, the C-reactive protein results observed in t his study follow the pattern shown in previous studies of some positive effects, some negative effects, and some showing no effect. This is further complicated by t he fact that some patients may exhibit both positive and adverse effects, depending on the ozone concentration (see results in Table 8). Table 8. C-Reactive Protein Results C-reactive protein (mg/l) Concentration 0 1 2 3 4 Patient A 0.87 0.51 0.59 0.64 0.66 B 0.73 0.92 2.23 1.69 3.72 C 3.14 2.94 4.36 2.16 4.91 D 0.79 0.48 0.55 0.61 1.34 E 3.99 1.62 0.98 2.94 0.92 F 0.56 0.61 0.42 1.25 0.68 G 2.21 1.72 0.59 1.02 0.66 H 0.71 1.90 0.79 1.09 0.56 I 3.86 1.83 0.69 1.71 0.6 J 0.87 2.64 0.63 1.15 0.83 K 0.92 0.92 0.66 0.82 0.98 L 0.77 1.44 1.2 1.11 0.68 M 12.35 0.93 0.93 4.27 11.45 N 7.71 6.83 5.03 0.77 0.9 O 1.26 0.42 0.57 0.44 0.47 P 0.61 0.72 0.45 0.5 0.49 Q 1.73 0.56 0.54 0.5 0.56 R 0.42 0.44 0.41 0.45 0.56 S 0.99 0.78 0.48 0.58 0.48 T 0.43 0.44 0.49 0.58 1.09 Where the ozone concentrations are: 0 =0 g/ml, 1 = 20 g/ml, 2=40 g/ml, 3= 80 g/ml, 4= 160 g/ml

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38 A decrease in C-reactive protein, which is a positi ve effect, was noticed in five out of the 20 patients. For the graphical represent ation see Appendix E, Figures 11e, 11g, 11i, 11m, and 10n for patients E, G, I, M, and N. W ith the exception of patient N the age of these five patients ranged between 61 and 64 yea rs old. Out of these five patients, four of them showed a steadily decreasing trend in C-rea ctive protein from the control to the highest ozone concentration (Figure 11e, 11g, 11i, and 11n). One of the five patients, patient M showed a decrease in C-reactive protein o nly for the lowest two ozone concentrations, 20, respectively 40g/ml (Figure 11 m). An increase in C-reactive protein was observed in t wo out of the 20 patients (Figure 11b and previous 11m for patients B and M). The cut-off value used, between normal and abnormal C-reactive protein concentratio ns was of 5 mg/ml. Patient M showed previously a decrease in C-reactive protein for the lowest two ozone concentrations, with the C-reactive protein increas ing for the highest two ozone concentrations, 80, respectively 160 g/ml. This wo uld be interpreted as a negative result, however, even with the increase in C-reacti ve protein these levels were lower than the baseline. Overall, fourteen out of the 20 patients showed no change in the C-reactive protein concentration as a results of ozone treatme nts. When the results for the 20 patients are combined, it becomes obvious that most of the patients had normal C-reactive protein value s, except for 3 patients, two of which had elevated values only for the highest ozone conc entration treatment. The other patient had elevated values for the lowest two ozone concen trations (Figure 12).

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39 Figure 12. Combined C-Reactive Protein Results The statistical models showed that there are no sta tistically significant differences among the ozone concentrations, p=0.177. Only the d ifferences between patients are statistically significant, with p=0.0001. 0.00 2.00 4.00 6.00 8.00 10.00 12.00 14.00 ABCDEFGHIJKLMNOPQRST PatientCRP (mg/l) Conc 1 Conc 2 Conc 3 Conc 4 Combined CRP results

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40 Glutathione An indication of a positive effect of ozone therapi es is an increase in the reduced to oxidized glutathione ratio. A decrease in the re duced to oxidized glutathione ratio is a marker of oxidative stress. Similar to erythrocyte sedimentation rate and C-rea ctive protein, the glutathione ratio results observed in this study follow the pat tern shown in previous studies of a combination of positive, negative effects, and no e ffects (see Table 9 below). This is further complicated by the fact that some patients may exhibit both positive and adverse effects, depending on the ozone concentration. Table 9. Glutathione Ratio Results GSH/GSSG ratio Concentration 0 1 2 3 4 Patient A 1.95 1.92 2.47 1.87 2.92 B 1.97 2.47 1.87 2.09 1.97 C 2 2.05 2.01 1.99 2.01 D 2.04 1.96 1.99 1.97 1.95 E 2.17 1.98 1.3 2.37 1.98 F 2.04 1.68 1.64 1.7 2.28 G 2.04 1.68 1.64 1.7 2.28 H 0.16 1.75 1.97 1.9 4.6 I 0.99 1.65 1.88 1.86 1.89 J 1.73 2.05 2.1 2.34 2.75 K 1.85 1.44 2.4 2.21 3.77 L 2.01 2.59 1.48 2.21 3.27 M 1.28 1.6 1.86 1.87 2.13 N 3.13 2.42 1.72 1.68 1.91 O 1.37 1.68 1.98 1.21 0.96 P 1.86 1.82 1.85 1.84 2.08 Q 1.93 1.34 0.63 1.55 0.81 R 2.16 1.45 3.51 3.32 2.65 S 2.5 2.08 2.49 2.22 2.66 T 2.38 2.68 2.14 2.82 2.83 Where the ozone concentrations are: 0 =0 g/ml, 1 = 20 g/ml, 2=40 g/ml, 3= 80 g/ml, 4= 160 g/ml

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41 An increase in the glutathione ratio, a positive ef fect, was noticed in six out of the 20 patients. For the graphical representation see A ppendix F, Figures 13a, 13f, 13h, 13k, 13l, and 13r for patients A, F, H, K, L, and R. The se patients ranged in age from 22 to 74 years old. One of the six patients, F, experienced an increase in the glutathione ratio only for the two highest ozone concentrations, the two l owest ozone concentrations having an adverse effect (Figure 13f). A decrease in the glutathione ratio, which is an ad verse effect, was noticed in four out of the 20 patients (Figure 13b, 13f, 13o, and 1 3q for patients B, F, O, and Q. with ages ranging from 22 to 61 years old). Out of these four patients, one showed a decrease in the glutathione ratio only for the highest two o zone concentrations (Figure 13o) while another showed a decrease in the glutathione ratio only for the lowest two ozone concentrations (Figure 13f). A total of ten patients showed no effect on the glu tathione ratio when comparing the control and the four ozone concentrations (Figu re 13d, 13e, 13g, 13i, 13j, 13m, 13n, 13p, 13s, and 3t for patients D, E, G, I, J, M, N, P, S, and T). The ages of these patients cover the entire spectrum from 25 to 64 years old. The statistical models showed that there are no sta tistically significant differences among the ozone concentrations, p=0.2379 and among the patients, p=0.2495.

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42 Combined results The response of the patients to the three tests was combined in Table 10 below. The responses were coded with numbers, 0 meaning no effect, positive numbers meaning an increase in the parameter tested and negative nu mbers a decrease in the parameter tested. A decrease erythrocyte sedimentation rate or C-rea ctive protein as a result of ozone treatment is a desired positive effect while for glutathione, an increase in the GSH/GSSH ratio as a result of ozone treatment is a positive effect. Based on their score for the three tests combined, the patients were assigned into categories from I to V with I indicating the highes t overall change and V meaning no change (see legend below). Legend: -2 decreasing I highest change for all 3 tests -1 slightly decreasing II high change across all 3 tests 0 unchanged III moderate change across all 3 tests +1 slightly increasing IV least change across all 3 tests +2 increasing V no change

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43 Table 10. Patient Response to all Three Tests TEST PATIENT Gender Age ESR CRP GSH/GSSG ratio Overall change A M 74 0 0 +1 IV B M 43 0 +2 -1 II C M 73 0 +1 n/a IV D F 53 0 0 0 V E M 63 0 -2 0 III F M 22 0 0 0 V G M 64 +1 -1 0 III H M 45 0 0 +1 IV I M 61 0 -2 0 III J M 41 0 0 0 V K M 60 0 0 0 V L M 60 0 0 +2 III M M 62 +2 +2* 0 I N F 29 +1 -2 0 II O M 37 0 0 0 V P M 39 -1 0 0 IV Q M 61 -2 0 -1 II R M 43 0 0 +1 IV S F 31 -1 0 0 IV T M 25 0 0 +1 IV *INCREASE AMONG OZONE CONCENTRATIONS, CONTROL EXCLUD ED Most patients (12 patients) experienced little to n o change to this three tests. Four of the patients showed moderate change across all t hree tests. Three patients showed high change across all three tests and only one was extr emely responsive to the three tests. The patient with the highest responsiveness had the hig hest increase in erythrocyte sedimentation rate compared to the control and C-re active protein concentration

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44 compared to the lowest two ozone concentrations. Th e patients with high change mostly had negative changes as a result of ozone treatment while the patients with moderate changes showed positive improvements.

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45 Discussion The main purpose of this study was to determine if ozone therapies are harmful for the samples and should not be offered as altern ative treatment techniques in hospitals. Based on the parameters studied, ozone therapies wo uld have a harmful effect if the samples experienced a statistically significant increase in erythrocyte sedimentation rate and C-reactive protein concentration and a sta tistically significant decrease in the glutathione ratio. Ozone therapies typically use an ozone concentration of 50 g of ozone/ml blood. Ozone bagging techniques utilize co ncentrations up to 100 g of ozone/ml air mixture. The range of ozone concentrat ions used in this study were chosen to cover the therapeutical spectrum and ranged from 20 g of ozone/ml blood to160 g/ml. Our results were not able to reject the primary hyp otheses and infer that the ozone therapies are harmful. While most of the samples di d not have any reaction to the ozone treatments, about forty percent of the samples had either a positive or negative response. Overall, 30 % of the samples experienced a decrease in erythrocyte sedimentation rate for the four ozone concentrations, which is a positive effect of ozone treatments. If the ozone concentrations were studied individually, the most efficient ozone concentration was 40 g/ml, with 55% of the samples showing a decrease i n erythrocyte sedimentation rate. When the blood was treated with a concentration of 160 g/ml, 45% of the samples showed a decrease in erythrocyte sedimentation rate A concentration of 80 g/ml caused

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46 an erythrocyte sedimentation rate decrease in 40 % of the samples while a concentration of 20 g/ml was least efficient, with 35 % of the s amples showing a decrease in erythrocyte sedimentation rate. If the samples are evaluated individually, 20 % of them had the highest decrease in the erythrocyte sedimen tation rate at the 80 g/ml ozone concentration, 15 % at 40 g/ml and 160 g/ml and 1 0 % at 20 g/ml. A negative response to the four ozone treatments wa s found in 20 % of the samples. Sixty percent of the samples had a decreas e in erythrocyte sedimentation rate at 20 g/ml, 55 % of the samples at 80 g/ml, 40 % at 160 g/ml and 35 % at 40 g/ml. An overall decrease in the C-reactive protein for e ach of the four ozone concentrations was noticed in 25 % of the samples ( changes less than 0.05 mg/l were ignored). However, individual ozone concentrations had a bigger impact than their combined effect. Most samples showed a positive eff ect at a particular ozone concentration only, while the other concentrations had no effect. The most efficient ozone concentration was again 40 g/ml, with 75% of the samples showing a decrease in C-reactive protein. Concentrations of 160 g/ml and 80 g/ml caused a decrease in Creactive protein in 65 % of the samples. The lowest ozone concentration, 20 g/ml determined a decrease in C-reactive protein in 55 % of the samples. When the samples are evaluated individually, 35 % of them had the hi ghest decrease in C-reactive protein at the 40 g/ml ozone concentration, 20 % at 20 g/ml and 160 g/ml and 10 % of the samples at 80 g/ml. A negative C-reactive protein response to the four ozone treatments was noticed in 10 % of the samples. Each of the two highest ozo ne concentrations, 80 and 160 g/ml, and the lowest ozone concentration, 20 g/ml caused a negative response in 30 % of the

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47 samples. The 40 g/ml concentration caused a negati ve response in 15 % of the samples. Individual results showed 30 % of the samples had t he highest negative response at the highest ozone concentration, 160 g/ml, 20 % of the samples at the lowest ozone concentration, 0.2 g/ml and 10 % at the 80 g/ml c oncentration. For the glutathione ratio, a positive effect was re presented by an increase in the ratio as a result of exposure to ozone (changes les s than 0.1 were ignored). Thirty % of the samples had a positive response for all ozone c oncentrations. Some samples responded to only one particular ozone concentratio n. The highest ozone concentration, 160 g/ml proved to be the most effective with 65 % of the samples experiencing an increase in the glutathione ratio. This was followe d by the 80 g/ml concentration with 50 % of the samples and the lowest ozone concentrat ions each with 40 % of the samples. When the samples are evaluated individually, 60 % o f them had the highest increase in the glutathione ratio at the 160 g/ml ozone concen tration, 10 % at 20 g/ml and 40 g/ml and 5 % of the samples at 80 g/ml. A negative glutathione ratio response to the four o zone treatments was noticed in 20 % of the samples. Each of the two lowest ozone c oncentrations, 20 and 40 g/ml, caused a negative response in 40 % of the samples, followed by the 80 g/ml concentration with 30 % of the samples and 160 g/m l with 20 % of the samples. When the samples are evaluated individually, 10 % of the m had the highest increase in the glutathione ratio at the 160 g/ml ozone concentrat ion, respectively 80 g/ml, 25 % at 20 g/ml and 35 % of the samples at 40 g/ml. Most of the samples showed none to little response to ozone treatments for the three tests studied, as it was reinforced by the la ck of statistical significance when the

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48 differences between treatments were evaluated. The statistical models as a whole, which included the samples as the independent variable an d their age and gender (for erythrocyte sedimentation rate) were statistically significant. However the results for the three tests were not able to determine a difference between the ozone concentrations used. This means that the study could not prove tha t ozone therapies are harmful, even though some of the concentrations used were signifi cantly higher than the therapeutical concentrations. However, most of the samples (12) showed an improve ment in one of the tests. This suggests that ozone therapies can be used to i mprove the general health status of a patient. Even though the ozone therapies may not ha ve the potency to cure cancer or AIDS, they could be a very useful tool if they are used as an adjuvant for established medical procedures if their effect is additive. The results obtained in this study demonstrate the difficulty that previous studies ha d in pinpointing an effect of ozone therapies. It appears clear that the ozone therapie s have to be tailored for the individual as there is no general formula for their effectiveness It is also possible that the blood may not be an appropriate media for testing the effect of ozone therapies as the blood antioxidant capacity may be able to counteract some of these effects. For future research, it would be very useful to dev elop a better test, using parameters that correlate with ozone therapy concen trations, with a greater sensitivity and lower variability for the data.

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49 References cited 1. Al-Dalain, S. M. et al. Ozone treatment reduce s markers of oxidative and endothelial damage in an experimental diabetes model in r ats. Pharmacol Res 2001, 44(5): 391396 2. Andreula, C. F., et al. Minimally invasive oxygen-o zone therapy for lumbar disk herniation. Am J Neuroradiol 2003, 24(5): 784-787 3. Ashfaq, S. et al. The relationship between plasma l evels of oxidized and reduced thiols and early atherosclerosis in healthy adults. J Am Coll Cardiol 2006; 47(5): 1005-1011 4. Biedunkiewicz, B. et al. Brief Report: Blood Coagul ation Unaffected by Ozonated Autohemotherapy in Patients on Maintenance Hemodial ysis. Arch MedRes 2006; 37, 1034-1037 5. Blomberg, A. et al. Clara cell protein as a biomark er for ozone-induced lung injury in humans. Eur Respir J 2003; 22: 883–888 6. Bocci, V. A. Brief Report: Tropospheric Ozone Toxic ity vs. Usefulness of Ozone Therapy. Arch Med Res 2007; 38, 265-267 7. Bocci, V. Is it true that ozone is always toxic? Th e end of a dogma. Toxicol Appl Pharmacol 2006; 216, 493 – 504 8. Bocci, V. and C. Aldinucci. Biochemical Modificatio ns Induced in Human Blood by Oxygenation-Ozonation. J Biochem Molec Toxicol 2006; 20 (3), 133-138 9. Bocci, V. 2002. Oxygen-Ozone Therapy. A Critical Ev aluation, Kluwer Academic Publishers, Dordrecht 10. Bocci, V. et al. Studies on the biological effects of ozone: 9. Effects of ozone on human platelets. Platelets 1999; 10, 110-116 11. Bowler, R. P. and J. D. Crapo. Oxidative stress in allergic respiratory diseases. J Allergy Clin Immunol 2002;110(3):349-56. 12. Breslin, K. The impact of ozone. Env Health Perspect 1995; 103(7/8): 660-664

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50 13. Clavo, B. et al. Ozone therapy for tumor oxygenatio n: a pilot study. eCAM 2004; 1(1): 93-98 14. Durand, P. et al. Acute methionine load-induced hyp erhomocysteinemia enhances platelet aggregation, thromboxane biosynthesis, and macrophage-derived tissue factor activity in rats. FASEBJ. 1997; 11, 1157-1168 15. Eto, K. et al. Platelet Aggregability Under Shear i s Enhanced in Patients With Unstable Angina Pectoris Who Developed Acute Myocar dial Infarction Jpn Circ J 2001; 65, 279–282 16. Feng, R. et al. Ozone exposure impairs antigen-spec ific immunity but activates IL7-induced proliferation of CD4-CD8thymocytes in B ALB/c mice. J Toxicol Environ Health A 2006; 69 (16), 1511-26 17. Fishbach, F. 2000. A Manual of Laboratory & Diagnos tic Tests, 6th ed. Lippincott Williams & Wilkins, Philadelphia, PA 1910 18. Grnicki, A. and A. Gutsze. In vitro effects of ozo ne on human erythrocyte membranes: An EPR study. Acta Biochim Polon 2000; 47 (4), 963–971 19. Guven, A, et al. Medical ozone therapy reduces oxid ative stress and intestinal damage in an experimental model of necrotizing ente rocolitis in neonatal rats. J Ped Surg 2009; 44, 1730-1735 20. Hazucha, M. J. Relationship between ozone exposure and pulmonary function changes. J Apply Physiol 1987; 62(4): 1671-1680 21. Hazucha, M. J. et al. Mechanism of action of ozone on the human lung. J Appl Physiol, 1989; 67(4): 1535-1541 22. Halliwell, B. Antioxidants in human health and dise ase. Annu Rev Nutr 1996; 16: 33-50 23. Hernandez, F. A. To What Extent Does Ozone Therapy Need a Real Biochemical Control System? Assessment and Importance of Oxidat ive Stress. Arch Med Res 2007; 38: 571-78 24. Herrmann, W., H. Schorr. Total Homocysteine, Vitami n B12, and Total Antioxidant Status in Vegetarians. Clin Chem, 2001; 47(6): 1094 –1101 25. Hiromichi, O. et al. Changes in leukocyte populatio n after ozonated autohemoadministration in cows with inflammatory di seases. J Vet Med Sci, 2006; 68 (2): 175-178

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51 26. Jakubowski, K. et al. The level of some acute phase proteins, total protein, gammaglobulins and activity of lysozyme in blood plasma of rats supplemented with vitamin E and exposed to ozone. Pol J Vet Sci 2004; 7(4): 283-287 27. Jiao, X. J. and X. Peng. Clinial study of medical o zone therapy in chronic hepatitis B of 20 patients. Zhonhua Shi Yan He Lin Chuang Bing Du Xue Za Zhi 2008; 22(6): 484-485 28. Kelly, F. J. et al. The free radical basis of air p ollution:focus on ozone. Resp Med 1995; 89, 647-656 29. Kenyon, N. J. et al. Differentiation of the roles o f NO from airway epithelium and inflammatory cells in ozone-induced lung inflammati on. Toxicol Appl Pharmacol 2006; 215, 250-259 30. Koppen, G. et al. A battery of DNA effect biomarker s to evaluate environmental exposure of Flemish adolescents. J Appl Toxicol 2007; 27(3): 238-46. 31. Kotake, Y. et al. Platelet Dysfunction during Cardi opulmonary Bypass Assessed by a novel Whole Blood Aggregometer. J Cardiothor Vasc Anesthesia 2006; 20(4): 536-540 32. Kuller, L. H., R. P. Tracy et al. Relation of C-Re active Protein and Coronary Heart Disease in the MRFIT Nested Case-Control Study. Am J Epid 1996; 144(6): 537547 33. Labuschagne, C. F. et al. Ozone concentration depen dent autohaemotherapy effects on baboon antioxidant capacity and DNA integrity an d repair capacity of lymphocytes. Afr J Biotech 2009; 8(5): 715-720 34. Larini, A. and V. Bocci. Effects of ozone on isola ted peripheral blood mononuclear cells. Toxicol. in Vitro 2005; 19 (1), 55-61 35. Lin, Y.C., and S.C. Wu. Effects of ozone exposure o n inactivation of intraand extracellular enterovirus 71. Antiviral Res 2006; 70 (3), 147-153 36. Macy, E. M., T. E. Hayes, and R. P. Tracy. Variabil ity in the measurement of Creactive protein in healthy subjects: implications for reference intervals and epidemiological applications. Clin Chem 1997; 43(1): 52–58 37. Mole Jr, M. L. et al. Effect of ozone on serum lipi ds and lipoproteins in the rat. Toxicol Appl Pharmacol 1985; 80 (3), 367-376 38. Moore, G. 1999. Living with the Earth; Concepts in Environmental Science. CRC Press LLC, Boca Raton, FL 33431

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52 39. Mustafaev, E. M. et al. The role of ozone therapy i n prevention of pyoinflammatory complications after transurethral resection of pros tatic adenoma. Urol 2007; 1,18-23 40. Nakamura, T. Synergistic effect of cilostazol and d ipyridamole mediated by adenosine on shear-induced platelet aggregation. Thrombosis Res 2007; 119, 511 — 516 41. Ohtsuka, H. et al. Changes in leukocyte population after ozonated autohemoadministration in cows with inflammatory di seases. J Vet Med Sci 2006; 68(2): 175-178 42. Pastore, A., G. Federici et al. Analysis of glutath ione: implication in redox and detoxification, Review. Clin Chim Acta, 2003; 333, 19 – 39 43. Rahman, I. et al. Assay for quantitative determinat ion of glutathione and glutathione disulfide levels using enzymatic recycl ing method. Nature Protocols 2005; 1(6): 3159-65 44. Rebrin, I. et al. Effects of age and caloric restri ction on glutathione redox state in mice. Free Radic Biol Med 2003;35(6): 626-635 45. Ridker, P. M. C-Reactive Protein, Inflammation, and Cardiovascular Disease, Clinical Update. Tex Heart Inst J 2005; 32(3): 384-386 46. Ridker, P. M., C. H. Hennekens et al. C-Reactive Pr otein and Other Markers of Inflammation in the Prediction of Cardiovascular Di sease in Women. N Engl J Med 2000; 343:512 47. Richards, N. P. et al. Can the rapid semiquantitati ve estimation of serum C reactive protein be adapted for the management of bacterial infection? J Clin Pathol 1985; 38, 464-467 48. Rodrigo, R., S. Trujillo et al. Changes in (Na + K) -Adenosine Triphosphatase Activity and Ultrastructure of Lung and Kidney Asso ciated With Oxidative Stress Induced by Acute Ethanol Intoxication. Chest 2002; 121: 589-596 49. Rodriguez, Z. Z. et al. Preconditioning with ozone/ oxygen mixture induces reversion of some indicators of oxidative stress an d prevents organic damage in rats with fecal peritonitis. Inflamm Res 2009; 58, 371-375 50. Rossi, R., A. Milzani et al. Blood Glutathione Disu lfide: In Vivo Factor or in Vitro Artifact? Clin Chem 2002; 48(5): 742–753 51. Rudez, G. et al. Effects of ambient air pollution o n hemostasis and inflammation. Env Health Perspect 2009; 117(6): 995-1001

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53 52. Schalla,W, O., R. J. Arko, And S. E. Thompson. Eval uation of a C-Reactive Protein Latex Agglutination Detection Test with Sera from P atients with Sexually Transmitted Diseases. J Clin Microbiol 1984; 20(6): 1171-1173 53. Schmelzer, K. R. et al. The Role of Inflammatory Me diators in the Synergistic Toxicity of Ozone and 1-Nitronaphthalene in Rat Air ways. Environ Health Perspect 2006; 114,1354–1360. 54. Schultz, S. et al. Treatment with ozone/oxygen-pneu moperitoneum results in complete remission of rabbit squamos cell carcinoma s. Int J Cancer 2008; 122, 2360-2367. 55. Sweet, F. et al. Ozone selectively inhibits growth of human cancer cells. Science 1980; 209(4459): 931-933 56. Thomson, J. M. (ed). 1980. Blood Coagulation and Ha emostasis, a practical guide. Churchill Livingstone, New York, NY 10036 57. Travagli, V., I. Zanardi, V. Bocci. Short communica tion: A realistic evaluation of the action of ozone on whole human blood. Internat J Biol Macromolec 2006; 39, 317–320 58. Troxler, M. et al. Platelet function and antiplatel et therapy. Brit J Surg 2007; 94: 674 – 682 59. Turgeon, M.L. 1999. Clinical Hematology theory and procedures, 3rd ed. Lippincott Williams & Wilkins, Philadelphia, PA 19106 60. Tylicki, L. et al. No effects of ozonated autohemot herapy on inflammation response in hemodialyzed patients. Mediat Inflamm 2004, 13(5/6): 513-517 61. Tylicki, L. et al. Fistual function and dialysis ad equacy during ozonotherapy in chronically hemodialyzed patients. Artif Organs 2004, 28(5): 377-380 62. Valabhji, J. et al. Total Antioxidant Status and Co ronary Artery Calcification in Type 1 Diabetes. Diabetes Care 2001; 24(9) 63. Valiance, H., and G. Lockitch. Rapid, Semi-Quantita tive Assay of C-Reactive Protein Evaluated. Clin Chem 1991; 37(11): 1981-1982 64. Vincent Corbett, J. 2000. Laboratory Tests and Diag nostic Procedures with nursing diagnoses, 5th ed. Prentice Hall Health, Upper Saddle River, NJ 0 7458 65. Walsh, C. T. and R. D. Schwartz-Bloom, 2005. Levine 's pharmacology: drug actions and reactions, 7th ed. Taylor & Francis Group, 2 Park Square, Milton Park, Abingdon, Oxon, OX14 4RN

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54 66. Wells, K. H. et al. Inactivation of Human Immunodef iciency Virus Type 1 by Ozone In Vitro. Blood, 1991; 78(7):1882-1890 67. Wierzbicka, A., J. Pawnowska et al. Lipid, Carbohyd rate Metabolism, and Antioxidant Status in Children After Liver Transpla ntation. Transplantation Proceedings 2007; 39, 1523–1525 68. Zimmermann, C., K.Winnefeld S.Streck M.Roskos R.L.H aberl. Antioxidant Status in Acute Stroke Patients and Patients at Stroke Ris k. Eur Neurol 2004; 51:157–161 69. Zimran, A. et al. Effect of Ozone on Red Blood Cell Enzymes and Intermediates. Acta Haematologica 1999;102 (3),148-151 70. http://en.wikipedia.org/wiki/Ozone_therapy 71. http://www.oxygenmedicine.com/ozonetherapyed.html

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55 Appendix A Calibration Data for the Rotameters and Needle Valv e

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56 Appendix A (Continued) Table 3. Calibration Data for the Rotameters and Ne edle Valve a. Low Flow (0-0.05 l/min) Rotameter CALIBRATION OF LOW FLOW ROTAMETERGLASS BALL SETTING VOLUME TIME TIME FLOW RATE SETTING FLOW RATE CC SECONDS MINUTES CC/MIN ML/MIN 51 5 57 0.95 5.3 46 5.3 46 5 59 0.98 5.1 51 5.3 101 5 24 0.40 12.5 94 12.5 98 5 25 0.42 12.0 96 12.0 96 5 27 0.45 11.1 98 11.1 94 5 27 0.45 11.1 101 11.1 117 5 19 0.32 15.8 111 15.8 115 5 21 0.35 14.3 113 14.3 113 5 20 0.33 15.0 115 15.0 111 5 21 0.35 14.3 117 14.3 140 5 14 0.23 21.4 138 21.4 139 5 15 0.25 20.0 139 20.0 138 5 15 0.25 20.0 140 20.0 CALIBRATION OF LOW FLOW ROTAMETERSTAINLESS STEEL BALL SETTING VOLUME TIME TIME FLOW RATE SETTING FLOW RATE CC SECONDS MINUTES CC/MIN ML/MIN 14 5 57 0.95 5.3 12 5.26 12 5 59 0.98 5.1 14 5.08 38 5 24 0.40 12.5 34 12.50 36 5 25 0.42 12.0 35 12.00 35 5 27 0.45 11.1 36 11.11 34 5 27 0.45 11.1 38 11.11 47 5 19 0.32 15.8 44 15.79 46 5 21 0.35 14.3 45 14.29 45 5 20 0.33 15.0 46 15.00 44 5 21 0.35 14.3 47 14.29 68 5 14 0.23 21.4 62 21.43 66 5 15 0.25 20.0 66 20.00 62 5 14 0.23 21.4 68 21.43 110 5 7 0.12 42.9 105 42.86 109 5 8 0.13 37.5 108 37.50 108 5 7 0.12 42.9 109 42.86 105 5 7 0.12 42.9 110 42.86 136 5 5 0.08 60.0 135 60.00 136 5 5 0.08 60.0 136 60.00 136 5 5 0.08 60.0 136 60.00 135 5 5 0.08 60.0 136 60.00

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57 Appendix A (Continued) Table 3 (Continued) b. High Flow (0.05-0.8 l/min) Rotameter CALIBRATION OF HIGH FLOW ROTAMETER GLASS BALL SETTING VOLUME TIME TIME FLOW RATE SETTING FLOW RATE CC SECONDS MINUTES CC/MIN ML/MIN 27 5 6 0.10 50.0 27 50.00 27 5 6 0.10 50.0 27 50.00 27 5 6 0.10 50.0 27 50.00 27 5 7 0.12 42.9 27 42.86 27 5 7 0.12 42.9 27 42.86 27 5 7 0.12 42.9 27 42.86 40 5 7 0.12 42.9 39 42.86 40 5 4 0.07 75.0 39 75.00 40 5 5 0.08 60.0 39 60.00 39 5 5 0.08 60.0 39 60.00 39 5 5 0.08 60.0 40 60.00 39 5 4 0.07 75.0 40 75.00 39 5 5 0.08 60.0 40 60.00 60 5 5 0.08 60.0 60 60.00 60 5 3 0.05 100.0 60 100.00 60 5 3 0.05 100.0 60 100.00 60 5 3 0.05 100.0 60 100.00 60 5 3 0.05 100.0 60 100.00 60 5 3 0.05 100.0 60 100.00 97 5 3 0.05 100.0 97 100.00 97 5 2 0.03 150.0 97 150.00 97 5 2 0.03 150.0 97 150.00 97 40 11 0.18 218.2 97 218.18 97 40 11 0.18 218.2 97 218.18 146 30 5 0.08 360.0 146 360.00 146 40 6 0.10 400.0 146 400.00

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58 Appendix A (Continued) Table 3b. ( Continued) CALIBRATION OF HIGH FLOW ROTAMETER STAINLESS STEEL BALL SETTING VOLUME TIME TIME FLOW RATE SETTING FLOW RATE CC SECONDS MINUTES CC/MIN ML/MIN 2 5 6 0.10 50 2 50.00 2 5 6 0.10 50 2 50.00 2 5 6 0.10 50 2 50.00 2 5 7 0.12 43 2 42.86 2 5 7 0.12 43 2 42.86 2 5 7 0.12 43 2 42.86 2 5 7 0.12 43 2 42.86 10 5 5 0.08 60 10 60.00 10 5 5 0.08 60 10 60.00 10 5 4 0.07 75 10 75.00 10 5 5 0.08 60 10 60.00 10 5 4 0.07 75 10 75.00 10 5 5 0.08 60 10 60.00 10 5 5 0.08 60 10 60.00 24 5 3 0.05 100 24 100.00 24 5 3 0.05 100 24 100.00 24 5 3 0.05 100 24 100.00 24 5 3 0.05 100 24 100.00 24 5 3 0.05 100 24 100.00 24 5 3 0.05 100 24 100.00 44 5 2 0.03 150 44 150.00 44 5 2 0.03 150 44 150.00 44 5 1 0.02 300 44 300.00 44 40 11 0.18 218 44 218.18 44 40 11 0.18 218 44 218.18 69 30 5 0.08 360 69 360.00 69 40 6 0.10 400 69 400.00

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59 Appendix A (Continued) Table 3 (Continued) c. Calibration of the Needle Valve for the Low Flow an d High Flow Rotameters Low flow rotameter High flow rotameter Setting Setting Needle valve Glass Steel Needle valve Glass Steel setting ball ball setting ball ball 6.5 0.0 5 2 0 8 6.5 0.0 6 2 0 11 6.5 0.0 6 2 0 11 6.5 0.0 6 4 0 25 7 0.0 9 4 0 25 7 0.0 10 4 0 26 7 0.0 10 6 11 41 7 0.0 10 6 11 41 7.5 0.0 11 6 11 41 7.5 0.0 11 8 29 69 7.5 0.0 12 8 29 69 7.5 0.0 12 8 29 69 8 18.0 73 10 33 84 8 30.0 74 10 37 86 8 21.0 80 10 36 86 8 24.0 85 8.5 71.0 140 8.5 73.0 143 8.5 74.0 143 8.5 66.0 150

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60 Appendix A (Continued) a. Calibration of the low flow (0-0.05 l/min) rotam eter using the steel ball. b. Calibration of the high flow (0.05-0.8 l/min) ro tameter using the steel ball. High flow -steel ball y = 4.5774x + 17.979 R2 = 0.8861 0.00 50.00 100.00 150.00 200.00 250.00 300.00 350.00 400.00 450.00 020406080 setting flow (ml/min) Low flow -steel ball y = 0.4556x 5.1495 R2 = 0.9752 0.00 10.00 20.00 30.00 40.00 50.00 60.00 70.00 050100150 setting flow (ml/min)

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61 Appendix A (Continued) c. Calibration of the needle valve for the low flow rotameter. d. Calibration of the needle valve for the high flo w rotameter. Figure 1. Calibration Data for the Rotameters and N eedle Valve Low flow rotameter y = 53.929x2 739.98x + 2539.2 R2 = 0.9804 0.0 10.0 20.0 30.0 40.0 50.0 60.0 70.0 80.0 90.0 100.0 110.0 120.0 130.0 140.0 150.0 160.0 5678910 needle valve rotameter setting High flow rotameter y = 9.7167x 12.167 R2 = 0.9868 0 10 20 30 40 50 60 70 80 90 100 024681012 needle valve rotameter setting

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62 Appendix B Figure 3. Calibration Curve for C-Reactive Protein

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63 Appendix B (Continued) CRPy = 0.3684x + 0.1042 R2 = 0.9970 0.5 1 1.5 00.511.522.533.5 conc (ng/ml)absorbance Figure 3. Calibration Curve for C-Reactive Protein

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64 Appendix C Figure 4. Calibration Curves for Glutathione

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65 Appendix C (Continued) Standard 1 y = 0.0234x 0.004 R2 = 0.9999 -0.05 0 0.05 0.1 0.15 0.2 0.25 024681012 time (min) absorbancy Standard 2 y = 0.0293x 0.0012 R2 = 0.999 -0.05 0 0.05 0.1 0.15 0.2 0.25 0.3 0.35 024681012 time (min)absorbancy Standard 3 y = 0.0374x 0.0099 R2 = 0.999 -0.1 0 0.1 0.2 0.3 0.4 024681012 time (min) absorbancy Standard 4 y = 0.0588x + 0.0047 R2 = 0.9986 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 024681012 time (min)absorbancy

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66 Appendix C (Continued) Standard 5 y = 0.068x + 0.0123 R2 = 0.9976 0 0.2 0.4 0.6 0.8 024681012 time (min) absorbancy Standard 6 y = 0.0708x + 0.0041 R2 = 0.9982 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 024681012 time (min) absorbancy Standard 7 y = 0.1018x + 0.021 R2 = 0.9974 0 0.2 0.4 0.6 0.8 1 1.2 024681012 time (min) absorbancy

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67 Appendix D Figure 5. Erythrocyte Sedimentation Rates Note: In the following figures, the capital letter denotes the patient (from A to T), the 0-4 numbers represent the ozone concentration (4 being the highest ozone concentration), the small letter ‘s’ denotes a GSH sample and the doubl e letter ‘ss’ denotes a GSSG sample. The green line indicates a positive effect and the red line an adverse effect.

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68 Appendix D (Continued) Fig. 5e. Fig.5a. Fig. 5c. Fig. 5b. Fig. 5d. Fig. 5f Fig. 5g Fig. 5h Fig. 5j Fig. 5i 0 5 10 J0J1J2J3J4 JESR (mm/hr) 0 2 4 6 I0I1I2I3I4 IESR (mm/hr) 0 20 40 H0H1H2H3H4 HESR (mm/hr) 0 50 100 G0G1G2G3G4 GESR (mm/hr) 0 20 40 60 F0F1F2F3F4 FESR (mm/hr) 35 40 45 50 E0E1E2E3E4 EESR (mm/hr) 0 10 20 D0D1D2D3D4 DESR (mm/hr) 0 5 10 15 C0C1C2C3C4 CESR (mm/hr) 0 5 10 B0B1B2B3B4 BESR (mm/hr) 0 5 10 A0A1A2A3A4 A ESR (mm/hr)

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69 Appendix D (Continued) Fig. 5m Fig. 5k Fig. 5n Fig. 5o Fig. 5l Fig. 5q Fig. 5r Fig. 5p Fig. 5t Fig. 5s 0 20 40 L0L1L2L3L4 LESR (mm/hr) 0 10 20 30 K0K1K2K3K4 KESR (mm/hr) 0 20 40 60 M0M1M2M3M4 MESR (mm/hr) 0 10 20 30 N0N1N2N3N4 NESR (mm/hr) 0 5 10 15 O0O1O2O3O4 OESR (mm/hr) 0 20 40 60 P0P1P2P3P4 PESR (mm/hr) 0 5 10 R0R1R2R3R4 RESR (mm/hr) 0 20 40 60 Q0Q1Q2Q3Q4 QESR (mm/hr) 0 20 40 60 S0S1S2S3S4 SESR (mm/hr) 0 5 10 T0T1T2T3T4 TESR (mm/hr)

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70 Appendix E Figure 11. C-Reactive Protein Results Note: In the following figures, the capital letter denotes the patient (from A to T), the 0-4 numbers represent the ozone concentration (4 being the highest ozone concentration). Green lines represent a positive effect, red lines an adverse effect. For example: B2 means sample from patient B, 2nd highest ozone concentration treatment

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71 Appendix E (Continued) 0.00 2.00 4.00 6.00 8.00 10.00 12.00 14.00 A0A1A2A3A4 treatments Conc (mg/l) 0.00 2.00 4.00 6.00 8.00 10.00 12.00 14.00 B0B1B2B3B4 treatments Conc (mg/l) 0.00 2.00 4.00 6.00 8.00 10.00 12.00 14.00 C0C1C2C3C4 treatments Conc (mg/l) 0.00 2.00 4.00 6.00 8.00 10.00 12.00 14.00 D0D1D2D3D4 treatments Conc (mg/l) 0.00 2.00 4.00 6.00 8.00 10.00 12.00 14.00 E0E1E2E3E4 treatments Conc (mg/l) 0.00 2.00 4.00 6.00 8.00 10.00 12.00 14.00 F0F1F2F3F4 treatments Conc (mg/l) 0.00 2.00 4.00 6.00 8.00 10.00 12.00 14.00 G0G1G2G3G4 treatments Conc (mg/l) 0.00 2.00 4.00 6.00 8.00 10.00 12.00 14.00 H0H1H2H3H4 treatments Conc (mg/l) 0.00 2.00 4.00 6.00 8.00 10.00 12.00 14.00 I0I1I2I3I4 treatments Conc (mg/l) 0.00 2.00 4.00 6.00 8.00 10.00 12.00 14.00 J0J1J2J3J4 treatments Conc (mg/l) Fig. 11a Fig. 11b Fig. 11c Fig. 11d Fig.11e Fig. 11f Fig. 11g Fig. 11h Fig. 11i Fig. 11j

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72 Appendix E (Continued) 0.00 2.00 4.00 6.00 8.00 10.00 12.00 14.00 K0K1K2K3K4 treatments Conc (mg/l) 0.00 2.00 4.00 6.00 8.00 10.00 12.00 14.00 L0L1L2L3L4 treatments Conc (mg/l) 0.00 2.00 4.00 6.00 8.00 10.00 12.00 14.00 M0M1M2M3M4 treatments Conc (mg/l) 0.00 2.00 4.00 6.00 8.00 10.00 12.00 14.00 N0N1N2N3N4 treatments Conc (mg/l) 0.00 2.00 4.00 6.00 8.00 10.00 12.00 14.00 O0O1O2O3O4 treatments Conc (mg/l) 0.00 2.00 4.00 6.00 8.00 10.00 12.00 14.00 P0P1P2P3P4 treatments Conc (mg/l) 0.00 2.00 4.00 6.00 8.00 10.00 12.00 14.00 Q0Q1Q2Q3Q4 treatments Conc (mg/l) 0.00 2.00 4.00 6.00 8.00 10.00 12.00 14.00 R0R1R2R3R4 treatments Conc (mg/l) 0.00 2.00 4.00 6.00 8.00 10.00 12.00 14.00 S0S1S2S3S4 treatments Conc (mg/l) 0.00 2.00 4.00 6.00 8.00 10.00 12.00 14.00 T0T1T2T3T4 treatments Conc (mg/l) Fig. 11t Fig. 11r Fig. 11q Fig. 11o Fig. 11p Fig. 11n Fig. 11m Fig. 11k Fig. 11l Fig. 11s

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73 Appendix F Figure 13. Glutathione Absorbancies for Ozone Treat ments Note: In the following figures, the capital letter denotes the patient (from A to T), the 0-4 numbers represent the ozone concentration (4 being the highest ozone concentration), the small letter ‘s’ denotes a GSH sample and the doubl e letter ‘ss’ denotes a GSSG sample. For example: A2s means patient A, 2nd highest ozone concentration treatment, GSH sample.

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74 Appendix F (Continued) nr r rn r rn r r n r r A2s y = 0.0594x + 0.0836 R2 = 0.973 0 0.2 0.4 0.6 0.8 024681012 time (min) absorbancy A2ss y = -0.0021x + 0.72 R2 = 0.1993 0.68 0.69 0.7 0.71 0.72 0.73 0.74 024681012 time (min) absorbancy A3s y = 0.0255x + 0.7104 R2 = 0.8874 0 0.5 1 1.5 024681012 time (min) absorbancy A3ss y = -0.0034x + 1.0101 R2 = 0.1702 0.94 0.96 0.98 1 1.02 1.04 024681012 time (min) absorbancy A A4s y = -0.1365x + 0.2417 R2 = 0.483 -1.5 -1 -0.5 0 0.5 1 024681012 time (min) absorbancy A4ss y = -0.0033x + 0.7411 R2 = 0.2017 0.68 0.7 0.72 0.74 0.76 0.78 0.8 024681012 time (min) absorbancy a. Glutathione absorbencies for patient A for the f ive treatments

PAGE 85

75 Appendix F (Continued) B0s y = 0.0751x 0.2039 R2 = 0.9958 -0.4 -0.2 0 0.2 0.4 0.6 0.8 051015 time (min) absorbancy B0ss y = 0.0528x + 0.6061 R2 = 0.8745 0 0.5 1 1.5 051015 time (min) absorbancy B1s y = 0.0594x + 0.0836 R2 = 0.973 0 0.2 0.4 0.6 0.8 024681012 time (min) absorbancy B1ss y = -0.0021x + 0.72 R2 = 0.1993 0.68 0.69 0.7 0.71 0.72 0.73 0.74 024681012 time (min) absorbancy B2s y = 0.0255x + 0.7104 R2 = 0.8874 0 0.5 1 1.5 024681012 time (min) absorbancy B2ss y = -0.0034x + 1.0101 R2 = 0.1702 0.94 0.96 0.98 1 1.02 1.04 024681012 time (min) absorbancy B3s y = -0.0033x + 0.7411 R2 = 0.2017 0.68 0.7 0.72 0.74 0.76 0.78 0.8 024681012 time (min) absorbancy B3ss y = 0.0003x + 0.106 R2 = 0.2215 0.102 0.104 0.106 0.108 0.11 0.112 024681012 time (min) absorbancy B4s y = 0.0121x + 0.7918 R2 = 0.9135 0.75 0.8 0.85 0.9 0.95 024681012 time (min) absorbancy B4ss y = 0.0053x + 0.1399 R2 = 0.9264 0 0.05 0.1 0.15 0.2 0.25 024681012 time (min) absorbancy b. Glutathione absorbencies for patient B for the f ive treatments

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76 Appendix F (Continued) D0s y = 0.0009x 0.0295 R2 = 0.7343 -0.04 -0.03 -0.02 -0.01 0 024681012 time (min) absorbancy D1s y = 0.0004x + 0.0177 R2 = 0.8299 0 0.005 0.01 0.015 0.02 0.025 024681012 time (min) absorbancy D1ss y = 0.0017x 0.0259 R2 = 0.7101 -0.04 -0.03 -0.02 -0.01 0 024681012 time (min) absorbancy D2s y = 0.0006x 0.0216 R2 = 0.8124 -0.025 -0.02 -0.015 -0.01 -0.005 0 024681012 time (min) absorbancy D2ss y = 0.0013x + 0.06 R2 = 0.8993 0 0.02 0.04 0.06 0.08 024681012 time (min) absorbancy D3s y = 0.0006x 0.018 R2 = 0.8961 -0.02 -0.015 -0.01 -0.005 0 024681012 time (min) absorbancy D3ss y = 0.0008x + 0.0663 R2 = 0.7783 0.06 0.065 0.07 0.075 0.08 024681012 time (min) absorbancy D4s y = 0.0007x 0.0175 R2 = 0.8405 -0.02 -0.015 -0.01 -0.005 0 024681012 time (min) absorbancy D4ss y = 0.0012x + 0.0251 R2 = 0.635 0 0.01 0.02 0.03 0.04 024681012 time (min) absorbancy D0ss y = 0.0012x + 0.0258 R2 = 0.896 0 0.01 0.02 0.03 0.04 024681012 time (min)absorbancy d. Glutathione absorbencies for patient D for the f ive treatments

PAGE 87

77 Appendix F (Continued) E0s y = 0.0005x 0.0114 R2 = 0.8746 -0.015 -0.01 -0.005 0 024681012 time (min)absorbancy E0ss y = 0.0016x + 0.3552 R2 = 0.6403 0.35 0.355 0.36 0.365 0.37 0.375 024681012 time (min)absorbancy E1s y = 0.0005x 0.0074 R2 = 0.6828 -0.01 -0.008 -0.006 -0.004 -0.002 0 024681012 time (min)absorbancy E1ss y = 0.0003x + 0.2583 R2 = 0.2227 0.252 0.254 0.256 0.258 0.26 0.262 0.264 024681012 time (min)absorbancy E2s y = 0.0003x 0.01 R2 = 0.3932 -0.015 -0.01 -0.005 0 024681012 time (min)absorbancy E2ss y = 0.0006x + 0.1509 R2 = 0.1832 0.14 0.145 0.15 0.155 0.16 024681012 time (min)absorbancy E3s y = 0.0003x 0.0111 R2 = 0.5476 -0.015 -0.01 -0.005 0 024681012 time (min)absorbancy E3ss y = 0.0003x + 0.2404 R2 = 0.2473 0.236 0.238 0.24 0.242 0.244 0.246 024681012 time (min)absorbancy E4s y = 0.0004x 0.0086 R2 = 0.569 -0.015 -0.01 -0.005 0 024681012 time (min)absorbancy E4ss y = -0.0001x + 0.172 R2 = 0.0109 0.155 0.16 0.165 0.17 0.175 0.18 024681012 time (min)absorbancy e. Glutathione absorbencies for patient E for the f ive treatments

PAGE 88

78 Appendix F (Continued) F0s y = 0.0004x 0.007 R2 = 0.5114 -0.01 -0.008 -0.006 -0.004 -0.002 0 024681012 time (min) absorbancy F0ss y = 0.0034x + 0.2549 R2 = 0.8334 0.25 0.26 0.27 0.28 0.29 0.3 024681012 time (min) absorbancy F1s y = 0.0004x 0.0053 R2 = 0.6245 -0.008 -0.006 -0.004 -0.002 0 024681012 time (min) absorbancy F1ss y = 0.0005x + 0.0649 R2 = 0.4408 0.06 0.062 0.064 0.066 0.068 0.07 0.072 024681012 time (min)absorbancy F2s y = 0.0005x 0.004 R2 = 0.7634 -0.006 -0.004 -0.002 0 0.002 024681012 time (min) absorbancy F2ss y = 0.0528x + 0.0568 R2 = 0.9987 0 0.2 0.4 0.6 0.8 024681012 time (min)absorbancy F3s y = 0.0578x + 0.022 R2 = 0.9982 0 0.2 0.4 0.6 0.8 024681012 time (min) absorbancy F3ss y = 0.0215x + 0.0718 R2 = 0.9995 0 0.1 0.2 0.3 0.4 024681012 time (min)absorbancy F4s y = 0.0622x 0.0214 R2 = 0.998 -0.2 0 0.2 0.4 0.6 0.8 024681012 time (min) absorbancy F4ss y = 0.0428x + 0.1325 R2 = 0.9973 0 0.2 0.4 0.6 024681012 time (min) absorbancy f. Glutathione absorbencies for patient F for the f ive treatments

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79 Appendix F (Continued) G0s y = 0.0619x 0.1111 R2 = 0.9949 -0.2 0 0.2 0.4 0.6 024681012 time (min)absorbancy G0ss y = 0.0381x + 0.2952 R2 = 0.9926 0 0.2 0.4 0.6 0.8 024681012 time (min)absorbancy G1s y = 0.0569x 0.0711 R2 = 0.9825 -0.2 0 0.2 0.4 0.6 024681012 time (min)absorbancy G1ss y = -0.0559x + 0.5617 R2 = 0.6743 0 0.2 0.4 0.6 0.8 024681012 time (min)absorbancy G2s y = -0.0414x + 0.6645 R2 = 0.6167 0 0.2 0.4 0.6 0.8 024681012 time (min)absorbancy G2ss y = -0.0693x + 1.0429 R2 = 0.8481 0 0.5 1 1.5 024681012 time (min) absorbancy G3s y = -0.0494x + 0.7714 R2 = 0.7569 0 0.2 0.4 0.6 0.8 1 024681012 time (min)absorbancy G3ss y = -0.0707x + 0.9106 R2 = 0.7965 0 0.2 0.4 0.6 0.8 1 024681012 time (min)absorbancy G4s y = -0.0366x + 0.7093 R2 = 0.6564 0 0.2 0.4 0.6 0.8 024681012 time (min)absorbancy G4ss y = -0.0081x + 0.2552 R2 = 0.0526 0 0.1 0.2 0.3 0.4 0.5 024681012 time (min)absorbancy g. Glutathione absorbencies for patient G for the f ive treatments

PAGE 90

80 Appendix F (Continued) H0s y = 0.012x + 0.3055 R2 = 0.2014 0 0.1 0.2 0.3 0.4 0.5 024681012 time (min)absorbancy H0ss y = -0.0277x + 0.4519 R2 = 0.2872 0 0.2 0.4 0.6 0.8 024681012 time (min)absorbancy H1s y = -0.0372x + 0.6713 R2 = 0.5172 0 0.2 0.4 0.6 0.8 024681012 time (min)absorbancy H1ss y = -0.0485x + 0.6111 R2 = 0.6922 0 0.2 0.4 0.6 0.8 024681012 time (min)absorbancy H2s y = -0.0963x + 0.7372 R2 = 0.7975 -0.5 0 0.5 1 024681012 time (min) absorbancy H2ss y = -0.0681x + 0.8095 R2 = 0.7031 0 0.2 0.4 0.6 0.8 1 024681012 time (min)absorbancy H3s y = -0.0773x + 0.5594 R2 = 0.7151 -0.4 -0.2 0 0.2 0.4 0.6 0.8 024681012 time (min) absorbancy H3ss y = -0.0648x + 0.5194 R2 = 0.655 -0.2 0 0.2 0.4 0.6 0.8 024681012 time (min) absorbancy H4s y = -0.0475x + 0.3309 R2 = 0.5684 -0.2 0 0.2 0.4 0.6 024681012 time (min) absorbancy H4ss y = 0.0086x + 0.0082 R2 = 0.7848 -0.05 0 0.05 0.1 024681012 time (min) absorbancy h. Glutathione absorbencies for patient H for the f ive treatments

PAGE 91

81 Appendix F (Continued) I0s y = 0.0065x 0.1165 R2 = 0.6806 -0.2 -0.15 -0.1 -0.05 0 024681012 time (min) absorbancy I0ss y = -0.0239x + 0.3829 R2 = 0.4689 0 0.2 0.4 0.6 024681012 time (min) absorbancy I1s y = -0.0432x + 0.3165 R2 = 0.4634 -0.2 0 0.2 0.4 0.6 024681012 time (min) absorbancy I1ss y = -0.0709x + 0.8398 R2 = 0.6169 0 0.5 1 024681012 time (min) absorbancy I2s y = -0.0854x + 0.7101 R2 = 0.6035 -0.5 0 0.5 1 024681012 time (min) absorbancy I2ss y = -0.0752x + 0.9215 R2 = 0.582 0 0.5 1 1.5 024681012 time (min) absorbancy I3s y = -0.072x + 0.561 R2 = 0.5188 -0.4 -0.2 0 0.2 0.4 0.6 0.8 024681012 time (min)absorbancy I3ss y = -0.0671x + 0.7969 R2 = 0.5627 0 0.2 0.4 0.6 0.8 1 024681012 time (min)absorbancy I4s y = -0.0673x + 0.5058 R2 = 0.5633 -0.4 -0.2 0 0.2 0.4 0.6 0.8 024681012 time (min)absorbancy I4ss y = -0.058x + 0.7008 R2 = 0.5826 0 0.2 0.4 0.6 0.8 1 024681012 time (min) absorbancy i. Glutathione absorbencies for patient I for the f ive treatments

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82 Appendix F (Continued) J0s y = -0.0669x + 0.4575 R2 = 0.6285 -0.4 -0.2 0 0.2 0.4 0.6 0.8 024681012 time (min)absorbancy J0ss y = -0.0861x + 0.6979 R2 = 0.7306 -0.5 0 0.5 1 024681012 time (min)absorbancy J1s y = -0.0738x + 0.5155 R2 = 0.6908 -0.4 -0.2 0 0.2 0.4 0.6 0.8 024681012 time (min) absorbancy J1ss y = -0.0419x + 0.4069 R2 = 0.4616 -0.2 0 0.2 0.4 0.6 0.8 024681012 time (min)absorbancy J2s y = -0.0904x + 0.6829 R2 = 0.839 -0.4 -0.2 0 0.2 0.4 0.6 0.8 024681012 time (min) absorbancy J2ss y = -0.0451x + 0.635 R2 = 0.9158 0 0.2 0.4 0.6 0.8 024681012 time (min)absorbancy J3s y = -0.0287x + 0.1987 R2 = 0.8385 -0.2 -0.1 0 0.1 0.2 0.3 024681012 time (min) absorbancy J3ss y = -0.0037x + 0.158 R2 = 0.2592 0 0.05 0.1 0.15 0.2 0.25 024681012 time (min)absorbancy J4s y = -0.0005x 0.0408 R2 = 0.0117 -0.08 -0.06 -0.04 -0.02 0 024681012 time (min)absorbancy J4ss y = 0.0072x + 0.0177 R2 = 0.789 0 0.02 0.04 0.06 0.08 0.1 024681012 time (min)absorbancy j. Glutathione absorbencies for patient J for the f ive treatments

PAGE 93

83 Appendix F (Continued) K0s y = 0.001x + 0.0194 R2 = 0.6333 0 0.01 0.02 0.03 0.04 024681012 time (min)absorbancy K0ss y = -0.0016x + 0.3459 R2 = 0.5154 0.325 0.33 0.335 0.34 0.345 0.35 0.355 024681012 time (min)absorbancy K1s y = -0.0076x + 0.091 R2 = 0.2816 0 0.05 0.1 0.15 0.2 024681012 time (min)absorbancy K1ss y = -0.036x + 0.5472 R2 = 0.503 0 0.2 0.4 0.6 0.8 024681012 time (min)absorbancy K2s y = -0.0581x + 0.4889 R2 = 0.6442 -0.2 0 0.2 0.4 0.6 0.8 024681012 time (min)absorbancy K2ss y = -0.0098x + 0.3317 R2 = 0.0996 0 0.2 0.4 0.6 024681012 time (min)absorbancy K3s y = 0.0326x + 0.209 R2 = 0.4993 0 0.2 0.4 0.6 0.8 024681012 time (min) absorbancy K3ss y = -0.0095x + 0.4906 R2 = 0.1156 0 0.2 0.4 0.6 0.8 024681012 time (min)absorbancy K4s y = 0.0143x + 0.1101 R2 = 0.3417 0 0.1 0.2 0.3 0.4 024681012 time (min)absorbancy K4ss y = 0.0084x + 0.2201 R2 = 0.9588 0 0.1 0.2 0.3 0.4 024681012 time (min) absorbancy k. Glutathione absorbencies for patient K for the f ive treatments

PAGE 94

84 Appendix F (Continued) L0s y = 0.0523x + 0.119 R2 = 0.7837 0 0.2 0.4 0.6 0.8 024681012 time (min)absorbancy L0ss y = -0.018x + 0.5086 R2 = 0.2123 0 0.2 0.4 0.6 0.8 024681012 time (min)absorbancy L1s y = 0.0541x + 0.2491 R2 = 0.6779 0 0.2 0.4 0.6 0.8 1 024681012 time (min)absorbancy L1ss y = -0.0026x + 0.5697 R2 = 0.0069 0 0.2 0.4 0.6 0.8 024681012 time (min)absorbancy L2s y = 0.024x + 0.3189 R2 = 0.2392 0 0.2 0.4 0.6 0.8 024681012 time (min) absorbancy L2ss y = -0.0189x + 0.5968 R2 = 0.2413 0 0.2 0.4 0.6 0.8 1 024681012 time (min)absorbancy L3s y = 0.0214x + 0.2131 R2 = 0.3018 0 0.2 0.4 0.6 024681012 time (min)absorbancy L3ss y = 0.0124x + 0.4503 R2 = 0.1379 0 0.2 0.4 0.6 0.8 024681012 time (min)absorbancy L4s y = 0.0856x + 0.106 R2 = 0.9015 0 0.5 1 1.5 024681012 time (min) absorbancy L4ss y = 0.0061x + 0.3164 R2 = 0.2056 0 0.1 0.2 0.3 0.4 0.5 024681012 time (min) absorbancy l. Glutathione absorbencies for patient L for the f ive treatments

PAGE 95

85 Appendix F (Continued) M0s y = 0.006x + 0.2728 R2 = 0.4532 0 0.1 0.2 0.3 0.4 024681012 time (min)absorbancy M0ss y = -0.0113x + 0.4346 R2 = 0.8851 0 0.1 0.2 0.3 0.4 0.5 024681012 time (min)absorbancy M1s y = -0.0019x + 0.2341 R2 = 0.0196 0 0.1 0.2 0.3 0.4 024681012 time (min)absorbancy M1ss y = -0.014x + 0.4033 R2 = 0.3032 0 0.2 0.4 0.6 0.8 024681012 time (min)absorbancy M2s y = -0.013x + 0.2884 R2 = 0.3274 0 0.1 0.2 0.3 0.4 0.5 024681012 time (min)absorbancy M2ss y = -0.0138x + 0.4065 R2 = 0.2924 0 0.2 0.4 0.6 0.8 024681012 time (min)absorbancy M3s y = -0.0044x + 0.2652 R2 = 0.1893 0 0.1 0.2 0.3 0.4 024681012 time (min)absorbancy M3ss y = -0.0058x + 0.3235 R2 = 0.0984 0 0.1 0.2 0.3 0.4 0.5 024681012 time (min) absorbancy M4s y = -0.0003x + 0.2274 R2 = 0.001 0 0.1 0.2 0.3 0.4 024681012 time (min)absorbancy M4ss y = 0.0025x + 0.1514 R2 = 0.1696 0 0.05 0.1 0.15 0.2 0.25 024681012 time (min)absorbancy m. Glutathione absorbencies for patient M for the f ive treatments

PAGE 96

86 Appendix F (Continued) N0s y = 0.0062x + 0.1609 R2 = 0.1973 0 0.1 0.2 0.3 024681012 time (min)absorbancy N0ss y = 0.009x + 0.1962 R2 = 0.9268 0 0.1 0.2 0.3 0.4 024681012 time (min)absorbancy N1s y = 0.0039x + 0.4181 R2 = 0.6125 0.38 0.4 0.42 0.44 0.46 024681012 time (min)absorbancy N1ss y = 0.0066x + 0.1434 R2 = 0.8183 0 0.05 0.1 0.15 0.2 0.25 024681012 time (min)absorbancy N2s y = 0.0032x + 0.2943 R2 = 0.2602 0 0.1 0.2 0.3 0.4 024681012 time (min)absorbancy N2ss y = -0.0025x + 0.344 R2 = 0.0108 0 0.2 0.4 0.6 024681012 time (min) absorbancy N3s y = -0.0008x + 0.329 R2 = 0.0057 0 0.1 0.2 0.3 0.4 0.5 024681012 time (min) absorbancy N3ss y = -0.0087x + 0.1785 R2 = 0.1855 0 0.1 0.2 0.3 0.4 024681012 time (min) absorbancy N4s y = 0.0034x + 0.2181 R2 = 0.3772 0 0.1 0.2 0.3 024681012 time (min) absorbancy N4ss y = 0.0016x + 0.1805 R2 = 0.0451 0 0.1 0.2 0.3 024681012 time (min) absorbancy n. Glutathione absorbencies for patient N for the f ive treatments

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87 Appendix F (Continued) O0s y = 0.0282x 0.6567 R2 = 0.9998 -0.8 -0.6 -0.4 -0.2 0 024681012 time (min)absorbancy O0ss y = -0.0403x + 0.2123 R2 = 0.3823 -0.4 -0.2 0 0.2 0.4 024681012 time (min) absorbancy O1s y = -0.0533x + 0.1602 R2 = 0.3503 -0.6 -0.4 -0.2 0 0.2 0.4 024681012 time (min) absorbancy O1ss y = -0.0793x + 0.2052 R2 = 0.6258 -0.8 -0.6 -0.4 -0.2 0 0.2 0.4 024681012 time (min) absorbancy O2s y = -0.0351x 0.0933 R2 = 0.2468 -0.6 -0.4 -0.2 0 0.2 024681012 time (min) absorbancy O2ss y = -0.0238x + 0.0628 R2 = 0.3731 -0.2 -0.1 0 0.1 0.2 0.3 024681012 time (min) absorbancy O3s y = 0.006x 0.3919 R2 = 0.0583 -0.6 -0.4 -0.2 0 024681012 time (min) absorbancy O3ss y = -0.0145x + 0.0013 R2 = 0.1118 -0.4 -0.2 0 0.2 0.4 024681012 time (min) absorbancy O4s y = 0.0036x 0.3376 R2 = 0.0261 -0.5 -0.4 -0.3 -0.2 -0.1 0 024681012 time (min) absorbancy O4ss y = -0.0439x + 0.1358 R2 = 0.5334 -0.4 -0.2 0 0.2 0.4 024681012 time (min) absorbancy o. Glutathione absorbencies for patient O for the f ive treatments

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88 Appendix F (Continued) P0s y = -0.0232x 0.264 R2 = 0.3314 -0.6 -0.4 -0.2 0 0.2 024681012 time (min)absorbancy P0ss y = -0.023x 0.1916 R2 = 0.3887 -0.5 -0.4 -0.3 -0.2 -0.1 0 024681012 time (min) absorbancy P1s y = -0.0211x 0.3045 R2 = 0.4407 -0.6 -0.4 -0.2 0 024681012 time (min) absorbancy P1ss y = -0.0243x 0.1669 R2 = 0.3348 -0.5 -0.4 -0.3 -0.2 -0.1 0 0.1 024681012 time (min) absorbancy P2s y = -0.0284x + 0.0592 R2 = 0.403 -0.4 -0.2 0 0.2 0.4 024681012 time (min) absorbancy P2ss y = -0.0289x + 0.1439 R2 = 0.3556 -0.2 0 0.2 0.4 024681012 time (min) absorbancy P3s y = -0.0164x 0.2974 R2 = 0.2081 -0.6 -0.4 -0.2 0 024681012 time (min) absorbancy P3ss y = -0.0184x 0.1264 R2 = 0.3699 -0.4 -0.3 -0.2 -0.1 0 0.1 024681012 time (min) absorbancy P4s y = -0.0137x 0.3796 R2 = 0.2994 -0.6 -0.4 -0.2 0 024681012 time (min)absorbancy P4ss y = 0.0232x + 0.5425 R2 = 0.2541 0 0.2 0.4 0.6 0.8 1 024681012 time (min)absorbancy p. Glutathione absorbencies for patient P for the f ive treatments

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89 Appendix F (Continued) Q0ss y = 0.0037x + 0.2672 R2 = 0.9577 0.26 0.27 0.28 0.29 0.3 0.31 024681012 time (min)absorbancy Q0s y = 0.0186x + 0.1679 R2 = 0.8357 0 0.1 0.2 0.3 0.4 024681012 time (min) absorbancy Q1s y = 0.0049x + 0.27 R2 = 0.3061 0 0.1 0.2 0.3 0.4 024681012 time (min)absorbancy Q1ss y = -0.0121x + 0.3435 R2 = 0.252 0 0.2 0.4 0.6 024681012 time (min) absorbancy Q2s y = 0.0127x + 0.2565 R2 = 0.7447 0 0.1 0.2 0.3 0.4 0.5 024681012 time (min)absorbancy Q2ss y = -0.0029x + 0.2082 R2 = 0.1058 0 0.1 0.2 0.3 024681012 time (min) absorbancy Q3s y = 0.0055x + 0.1929 R2 = 0.4514 0 0.1 0.2 0.3 024681012 time (min)absorbancy Q3ss y = -0.0033x + 0.2585 R2 = 0.1061 0 0.1 0.2 0.3 0.4 024681012 time (min)absorbancy Q4s y = 0.0095x + 0.2095 R2 = 0.6492 0 0.1 0.2 0.3 0.4 024681012 time (min)absorbancy Q4ss y = -0.0132x + 0.6351 R2 = 0.31 0 0.2 0.4 0.6 0.8 1 024681012 time (min)absorbancy q. Glutathione absorbencies for patient Q for th e five treatments

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90 Appendix F (Continued) R0s y = 0.0689x + 0.0712 R2 = 0.9073 0 0.2 0.4 0.6 0.8 1 024681012 time (min)absorbancy R0ss y = -0.0177x + 0.6423 R2 = 0.1923 0 0.2 0.4 0.6 0.8 1 024681012 time (min)absorbancy R1s y = 0.0263x + 0.5215 R2 = 0.1715 0 0.2 0.4 0.6 0.8 1 024681012 time (min)absorbancy R1ss y = -0.0268x + 0.8435 R2 = 0.4107 0 0.5 1 1.5 024681012 time (min)absorbancy R2s y = 0.1581x 0.0242 R2 = 0.9967 -0.5 0 0.5 1 1.5 2 024681012 time (min) absorbancy R2ss y = 0.0117x + 0.4643 R2 = 0.5518 0 0.2 0.4 0.6 0.8 024681012 time (min) absorbancy R3s y = 0.2322x + 0.1411 R2 = 0.9562 0 1 2 3 024681012 time (min)absorbancy R3ss y = 0.0016x + 0.4413 R2 = 0.0107 0 0.2 0.4 0.6 024681012 time (min) absorbancy R4s y = 0.1149x + 0.0611 R2 = 0.9508 0 0.5 1 1.5 024681012 time (min) absorbancy R4ss y = 0.0239x + 0.247 R2 = 0.993 0 0.2 0.4 0.6 024681012 time (min)absorbancy r. Glutathione absorbenci es for patient R for the five treatments

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91 Appendix F (Continued) S0s y = 0.0704x + 0.0377 R2 = 1 0 0.2 0.4 0.6 0.8 024681012 time (min)absorbancy S0ss y = 0.0209x + 0.2443 R2 = 0.9978 0 0.1 0.2 0.3 0.4 0.5 024681012 time (min)absorbancy S1s y = 0.0352x + 0.086 R2 = 0.7958 0 0.2 0.4 0.6 024681012 time (min)absorbancy S1ss y = -0.0043x + 0.4117 R2 = 0.033 0 0.2 0.4 0.6 024681012 time (min)absorbancy S2s y = 0.1088x + 0.1983 R2 = 0.8927 0 0.5 1 1.5 024681012 time (min)absorbancy S2ss y = -0.0124x + 0.7893 R2 = 0.1508 0 0.5 1 1.5 024681012 time (min)absorbancy S3s y = 0.1238x + 0.107 R2 = 0.948 0 0.5 1 1.5 2 024681012 time (min) absorbancy S3ss y = 0.0533x + 0.4036 R2 = 0.9768 0 0.5 1 1.5 024681012 time (min) absorbancy S4s y = 0.1449x 0.0369 R2 = 0.9953 -0.5 0 0.5 1 1.5 2 024681012 time (min) absorbancy S4ss y = 0.0278x + 0.3489 R2 = 0.9956 0 0.2 0.4 0.6 0.8 024681012 time (min)absorbancy s. Glutathione absorbencies for patient S for the f ive treatments

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92 Appendix F (Continued) T0ss y = 0.0323x + 0.3273 R2 = 0.9992 0 0.2 0.4 0.6 0.8 024681012 time (min)absorbancy T0s y = 0.0978x 0.0412 R2 = 0.9938 -0.5 0 0.5 1 1.5 024681012 time (min)absorbancy T1s y = 0.0833x + 0.1967 R2 = 0.843 0 0.5 1 1.5 024681012 time (min)absorbancy T1ss y = -0.0012x + 0.4576 R2 = 0.003 0 0.2 0.4 0.6 0.8 024681012 time (min)absorbancy T2s y = 0.0827x + 0.1942 R2 = 0.8072 0 0.5 1 1.5 024681012 time (min)absorbancy T2ss y = -0.0259x + 0.7408 R2 = 0.2781 0 0.2 0.4 0.6 0.8 1 024681012 time (min)absorbancy T3s y = 0.0798x + 0.1533 R2 = 0.8456 0 0.5 1 1.5 024681012 time (min)absorbancy T3ss y = 0.0018x + 0.3908 R2 = 0.0152 0 0.2 0.4 0.6 024681012 time (min)absorbancy T4s y = 0.1087x + 0.004 R2 = 0.973 0 0.5 1 1.5 024681012 time (min)absorbancy T4ss y = 0.0182x + 0.2414 R2 = 0.9732 0 0.1 0.2 0.3 0.4 0.5 024681012 time (min)absorbancy t. Glutathione absorbencies for patient T for the f ive treatments

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About the Author Daniela Sloan received a Bachelor’s Degree in Ecol ogy in 1996 followed by a Master’s degree in Environmental Protection in 1997 from University of Bucharest. She received a second Master’s degree in Marine Biology in 2003 from Georgia Southern University. She is first author on three publicatio ns on Echinoderms.


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Sloan, Daniela.
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Effects of ozone on blood components
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by Daniela Sloan.
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[Tampa, Fla] :
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2010.
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Dissertation (Ph.D.)--University of South Florida, 2010.
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ABSTRACT: Previous studies on the medical use of ozone therapies show a very diverse array of results, from ozone reducing the amount of HIV virus in the blood, to no effect, to causing the death of several patients due to pulmonary embolism and infections. However, ozone therapies are widely used in Europe and considered medically safe. In the U.S., doctors in 28 states use ozone therapies. The objectives of this study were to investigate the effects of medical grade ozone at varying concentrations used in ozone therapies. These were achieved by evaluating the C-reactive protein, erythrocyte sedimentation rate, total reduced and oxidized glutathione content of erythrocytes which were all markers used to determine ozone injury/inflammation. Despite the fact that ozone is a very strong oxidant, previous research indicates that depending on the dose and the health status of the biological system, sometimes ozone can act as an antioxidant. The medical exposure range for ozone is between 20 -80 mg/ml with an average of 50 mg/ml. The concentrations used in this study were 20, 40, 80 and 160 mg/ml. Ozone was generated in the "Breath Lab" at USF from medical grade oxygen obtained through electrical corona arc discharge using an OL80C ozone generator. De-identified blood samples of 10 ml blood/sample containing EDTA as anticoagulant were obtained from the James A. Haley VA Hospital patients. Equal volumes of blood and ozone gas mixture were allowed to mix in ozone-resistant syringes prior to dividing each sample into three parts, one for each corresponding parameter to be studied. The C-reactive protein was analyzed through ELISA using the colorimetric method available from Helica Biosystems; erythrocyte sedimentation rate was measured in graduated sedimentation tubes; the total reduced glutathione (GSH) and oxidized glutathione (GSSG) content of erythrocytes was determined according to the colorimetric method developed by the Oxford Biomedical Research. Overall, the concentrations of ozone used did not have a statistically significant effect on the parameters investigated. However, a small percentage of the blood samples showed an improvement in the parameters studied, especially at the highest ozone concentration.
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Advisor: Yehia Hammad, Sc.D.
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C-reactive protein
Autohemoadministration
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