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Effects of Ozone Concentration on Human Blood Cells

Senior Capstone Thesis
Southern Oregon State Collage
Anthony Smith
12/06/96

Abstract:

Ozone is believed to act as a selective and rapid oxidizer of microorganisms and infected cells. Although used on nearly 500,000 patients in western Europe since the 1960's, the therapeutic use of medical ozone is largely unknown in North America. Sometimes called bio-oxidative therapy or autohemotherapy, mixtures of ozone and oxygen are mixed with blood before re-infusion by clinicians. To study possible toxic effects of ozone, human red blood cells were exposed to various ozone concentrations and hemolysis was quantified. Little hemolysis was detected at therapeutic ozone concentrations. The effect of ozone on phagocytosis by white blood cells was also examined. An increase in phagocytosis by white blood cells was observed at some ozone concentrations.

Introduction:

Ozone, a triatomic allotrope of oxygen, is best known as a layer of upper atmospheric gas. In western Europe, it has been used therapeutically on nearly 500,000 patients and has been given as a medical treatment over 5 million times (Jacobs 1983). However, the therapeutic use of medical ozone is largely unknown in North America. In the clinical technique known as autohemotherapy, mixtures of ozone and oxygen are mixed with blood before re-infusion by clinicians. The ozone is believed to act as a selective and rapid oxidizer of viruses (HIV, Herpes simplex and zoster, cytomegalovirus, Epstein-Barr, myxoviruses and retroviruses), bacteria (coliform and staphylococcus) and virally infected cell membranes (Rilling and Viebahn 1987, Wolfstaedter 1993). Healthy self cells may be spared because they have the built in protection of the oxidation buffers tocopherol, ascorbic acid, superoxide dismutase (SOD), uric acid and glutathione peroxidase (Sunnen 1988).

There is evidence that ozone stimulates production of interleukin II (Carpendale and Freeberg 1991) and tumor necrosis factor by human leukocytes (Paulesu, Luzzi and Bocci 1991). It has been documented that cancer cells often have little to no expression of oxidation buffer enzymes catalase, SOD, and peroxidase (Galeotti, Bartoli and Santini 1981). In this respect, they are similar to anaerobic organisms in which there is little need for an oxidative buffer system. Hence, ozone is particularly damaging to cancerous cells. European clinicians indicate it in treatment of nearly all cancers (Rilling et al. 1987, Sunnen, 1988).

Because scientists have focused on the undeniably negative effects of inhaled ozone, medicinal aspects of the gas when applied intravenously or through the skin have been scoffed at. There is valid concern that treatments could be toxic as ozone is one of the strongest oxidants known. Is there a significant level of toxic products and/or cytotoxicity involved in the treatments? This project will investigate this question by assaying human red blood cell (RBC) hemolysis at various ozone concentrations and assaying phagocytosis by human white blood cells (WBC) under various ozone concentrations. Aqueous solutions of ozone as low as 1 µg/ml are microbicidal (Sunnen 1988). If the dosages specified in the ozone administering protocols are non-cytotoxic, then this area of medicine has some tremendous possibilities--treatment of AIDS, hepatitis, bacterial and fungal infections and large scale purification of blood and plasma stores.

My hypothesis is that at therapeutic concentrations, there will be a small amount of RBC hemolysis, but within tolerable medical limits. This is an important concern because reinfused blood should not exceed about 12% hemolysis; Reinfusion of excessively hemolyzed blood can cause an adverse systemic reaction. A range of 7-10% hemolysis is regarded to be acceptable and within the tolerance of the body to process the dead cell waste (Rilling, et al. 1988). I also hypothesize that ozone enhances the phagocytic index--the mean number of phagocytized particles in neutrophil human leukocytes. This hypothesis is based on clinical reports that low concentrations of ozone are immune enhancing, while higher concentrations are immune suppressing (Wolfstaedter 1993).

Ozone is a very powerful oxidant. Exposure of organic molecules to ozone, especially those with nucleophilic double and triple carbon bonds, yields a vast number of simple, complex, stable and unstable species. These ozonides may further hydrolyze, oxidize, reduce or thermally decompose to a variety of organic groups including aldehydes, ketones, acids or alcohols. Ozone also reacts with saturated hydrocarbons, amines, sulfhydryl groups and aromatics (Sunnen 1988). Therefore, it is likely that ozone reacts with numerous cell membrane, tissue and plasma molecules--possibly including phospholipids, glycoproteins, fatty acids, hormones, enzymes, prostaglandins, and ions. In autohemotherapy the main consideration is the reaction of ozone with tissues--especially blood. At physiological pH (~7.5) ozone favors the ionic reaction with unsaturated lipid double bonds to form lipid hydroperoxides (fig.1)(Rilling et al. 1988). Considering the extremely vast variety of lipid constituents found in biological tissues, the possible products derived from ozonation are quite large. Species derived from lipid peroxidation (some transient) include, but are not limited to: free radicals, singlet oxygen, hydrogen peroxide, molozonides, ozonides, carbonyls, alkanes and alkenes (Sunnen 1988). In general, formation of lipid hydroperoxides on cell membranes is considered to be adverse to cells both because of the lysis of the molecule and the damaging oxidative potential retained by the newly formed hydroperoxide. Chemistry in physiological systems is not as easy to define as it is in vitro.

To further complicate investigation of ozone biochemistry, there are numerous physiologically relavent molecules that exist to buffer lipid peroxidation. Tocopherol, ascorbic acid, uric acid, and enzyme systems such as SOD, catalase and glutathione peroxidase may block or mask the oxidative effect of ozone to cell membrane lipids (Sunnen 1988). There are numerous individual reactions of ozone and organic molecules that are known. In addition there are many that can be speculated. However, the biochemistry of blood ozonation has only recently begun to be understood.

Among the modern ozone treatment methods, autohemotherapy is the clinical technique that can provide the most theraputic potential, experimental control and replication. It is typically performed in one of two ways. The standard technique is to withdraw about one-fifth liter of blood, introduce an ozone/oxygen mixture at an specified ozone concentration, then reinfuse it intravenously (Rilling, et al.1987). In the more modern clinics in Europe, blood is automatically run through a continuous recirculating device which mixes ozone and blood in a closed loop and automatically reinfuses it. This study will handle and treat the blood as if it were being used in an autohemotherapy application.

Materials and Methods:

Blood collection
Blood was drawn at the student health center immediately before experimentation. Using a 20 g. needle, 10 ml heparinized Vacutainer blood collection tubes were filled to a half-volume mark (5ml) with my own blood.

Ozone generation
Ozone was generated immediately before each use. Reagent grade, pure oxygen was passed through a corona discharge style ozone generator (Model GE60-- supplied by Ozone Services A Division of Yanco Industries Ltd. of Langley B.C.¹) via a Puritan-Bennett, Companion 360 Adjustable Flow Oxygen Regulator (also supplied by Ozone Services). The regulator delivers accurate gas flow through the generator from 31-3000 ml/min. The concentration of ozone in the oxygen/ozone output is controlled by two variables--flow rate through the generator tubes and electromagnetic frequency of the corona discharge itself. The system was calibrated with an Anseros GM6000 ozone analyzer by Ozone Services. The system can deliver ozone in almost any concentration from 0-91 µg ozone /ml of oxygen

Blood ozonation
5 ml of an ozone/oxygen mixture was drawn into a syringe directly from the ozone generator. A needle was installed on the syringe and it was plunged into a 10 ml Vacutainer filled with only 5 ml blood. The half-full Vacutainer retained a slight negative pressure that drew the oxygen/ozone mixture in. The Vacutainer contained 5 ml of gas and 5 ml of blood. The range of ozone concentrations used was 17-91 µg ozone per ml of oxygen. The volumes of the gas mixtures used were always equal to the volume of blood used, giving an absolute administration range of 17-91µg ozone per ml of blood. The tube was gently agitated for one minute. An aliquot of the treated blood was used for RBC hemolysis analysis and phagocytic index determination.

Erythrocyte hemolysis determination
Hemolysis was estimated by light absorbance at 540 nm. It was necessary to establish a standard curve of absorbances versus percent hemolysis. 4% untreated blood in distilled water was used as a standard for 100% hemolysis. Serial dilutions of this solution in isotonic 0.14M saline solution (Table 1) were read for absorbance on an HP Spec 20 spectrophotometer. Absorbance and hemolysis are directly and linearly related. For example, the absorbance of a solution of the 100% hemolyzed standard diluted 1/2 will have the same absorbance as a sample of 4% blood with 50% hemolysis.

Ozone treated blood was mixed, in a conical plastic test tube, with isotonic 0.14M saline solution to make a 4% blood solution. The tube was centrifuged at 1900 rpm for 5 minutes to pellet remaining viable erythrocytes (RBC's) and ghosts. The absorbance of the supernatant was recorded and referenced to the hemolysis standard function to estimate hemolysis.

Phagocytic index determination
The phagocytic index (PI) is an average number of particles phagocytized by neutrophils. 1ml of an 18 hr yeast culture was added to a 5 ml sample of blood. The blood was ozonated, as described above, and incubated at 37 C for one hour. After incubation, the blood was centrifuged at 1500 rpm for 4-5 minutes to separate the plasma, buffy coat and red blood cells. A 20 µl sample from the buffy coat was Wright stained and viewed under a light microscope. The number of yeasts inside each of 25 neutrophils (chosen as randomly as possible) was recorded and averaged. As a control, the PI was determined for untreated blood incubated with yeast. As a second control, the PI was determined for yeast incubated blood treated with pure oxygen--the solvent in which ozone is always in during this application.

Results:

The 4% blood hemolysis standards of absorbance at 540nm generated a linear graph (fig.2). The standard was extrapolated to a zero y-intercept because plasma (diluted to 4% in saline) from untreated blood was used as the zero-absorbance blank. Therefore, the hemolysis values in figures 2 and 3 represent hemolysis values over background hemolysis (attributed to extraction, transport, storage, etc.).

Red blood cell hemolysis increased exponentially from 0.07-2.17% (over background hemolysis) as the concentration of the ozone dose increased through the tested range of 59-91 µg/ml (fig.3). No hemolysis over background was detected from ozone concentrations below 59 µg/ml. The standard deviations were very small and this data is statistically significant.

Compared to untreated blood and oxygenated blood, ozone increased the phagocytic index of neutrophils up to a maximum index of 4.12 yeasts/neutrophil. This maximum occurred at an ozone concentration of 49 µg/ml. Concentrations of ozone above 49 µg/ml reduced the phagocytic index. At the highest ozone concentrations, the phagocytic index remained greater than both the untreated and the oxygen treated controls (fig.4). The standard deviations of these data points are quite large, but the trend fits the expected based on the observations of Rilling, Carpendale, Wolfstaedter and others.

Discussion:

A main challenge in the discussion of ozone therapy is overcoming the strong negative stigma that ozone is a toxic, noxious or caustic gas. Even among educated scientists, this idea is often based on assumption and misinformation. The best way to settle the issue is to put blood ozonation up to scientific testing. Research has shown that inhaled ozone is toxic in low concentrations and deadly at high concentrations (Cross and Halliwell 1994). But what has research shown about the effect of direct ozonation of human blood in vitro? Ozone did not significantly hemolyze RBC's even at the highest generated ozone concentrations (fig.3). The range of ozone concentration used in most contemporary ozone clinics is 25-40 µg/ml (Sunnen 1988). At these concentrations, I was not able to detect any hemolysis over background levels with the equipment I used. It was not until ozone above 59 µg/ml was used that I was able to detect the smallest amount of hemoglobin absorbance. With regards to hemolysis, ozone is not cytotoxic and safe for reinfusion.

Ozone enhances white blood cell phagocytosis (fig.4). In the case of a systemic microbial infection, the combination of direct microbe killing and phagocytosis enhancement would be a certain benefit. However my experiment does not explain the mechanism of enhanced phagocytosis. A minimum of two possible mechanisms may be involved. Ozone may have altered the neutrophils so that they phagocytized more readily. Or, it may have altered the yeast surface so that they were more readily phagocytized. Also it may be that a combination of these two mechanisms are operating. Further research should include experiments that investigate the mechanism of PI enhancement. A comparison should be made of the phagocytic index of two blood samples: one in which a ozone-treated yeast culture is added to untreated blood, and one that is ozone treated before non-ozonated yeast are added. Also, the experiments should determine the concentration of viable yeast added to blood and determine killing rates by ozone. A blood sample of blood should be plated and the colonies counted before and after ozonation so that ozone microbicidal properties can be compared to PI and hemolysis.

Proposals to the Food and Drug Administration (FDA) for clinical trials are persistently denied with the packaged excuse "ozone is a toxic gas with no known therapeutic value" (McCabe 1988). Clinical studies in English can be hard to find. Why won't the FDA allow formal medical trials? In the United States, large drug companies are directly involved in nearly all medical research and lobby to control AMA and FDA policy. There is little interest in researching the possibilities of ozone therapy since the gas cannot be packaged and marketed (it decomposes to O₂). It is too simple, very effective and quite inexpensive. In fact, it has been the unpublished policy of the FDA to confiscate equipment and jail clinical ozone researchers. In October 1996, Dr. John Gambee M.D. of Eugene, Oregon lost his state medical license for using ozone therapy, and was later even denied appeal. (The Oregonian 1996). Despite this, doctors in the U.S. and Europe have reported hundreds of cases of HIV positive to negative reversal, loss of AIDS symptoms (Carpendale et al. 1991), tumor shrinkage and disappearance and cure of systemic infections such as hepatitis, staphylococcal infection and meningitis (Paulesu et al. 1991, Rilling, et al. 1986). The research in medical ozone has come to a point where it really cannot go much further without mainstream medical approval. There are volumes and volumes of research documenting ozone therapy histologically, biochemically, and clinically--more than enough to warrant formal human clinical trials.

Works cited:

Figure Legend:

Figure 1 - Lipid ozonolysis intermediates and products in a physiological system. (Rilling et al. 1987).
 

Figure 2 - Blood hemolysis standard curve. Data points are the means from 5 trials of each percent solution (percent hemolysis). Linear regression analysis gives a correlation coefficient (r²) of 0.994.
 

Figure 3 - Erythrocyte hemolysis as a function of ozone concentration. Data points are means of 9 samples at each concentration. Error bars are the means ±standard deviation.
 

Figure 4 - Ozone's effect on phagocytosis. Data points are from 1 trial at each concentration. The mean yeast count in 25 randomly chosen neutrophils is reported for each concentration. Error bars are the means ± standard deviation.
 

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