Extracorporeal blood oxygenation and ozonation: clinical and biological implications of ozone therapy

Abstract

Some lines of evidence have suggested that the challenge to antioxidants and biomolecules provoked by pro-oxidants such as ozone may be used to generate a controlled stress response of possible therapeutic relevance in some immune dysfunctions and chronic, degenerative conditions. Immune and endothelial cells have been proposed to be elective targets of the positive molecular effects of ozone and its derived species formed during blood ozonation. On the bases of these underlying principles and against often prejudicial scepticism and concerns about its toxicity, ozone has been used in autohemotherapy (AHT) for four decades with encouraging results. However, clinical application and validation of AHT have been so far largely insufficient. Latterly, a new and more effective therapeutic approach to ozone therapy has been established, namely extracorporeal blood oxygenation and ozonation (EBOO). This technique, first tested in vitro and then in vivo in sheep and humans (more than 1200 treatments performed in 82 patients), is performed with a high-efficiency apparatus that makes it possible to treat with a mixture of oxygen–ozone (0.5–1 μg/ml oxygen) in 1 h of extracorporeal circulation up to 4800 ml of heparinized blood without technical or clinical problems, whereas only 250 ml of blood can be treated with ozone by AHT. The EBOO technique can be easily adapted for use in hemodialysis also. The standard therapeutic cycle lasts for 7 weeks in which 14 treatment sessions of 1 h are performed. After a session of EBOO, the interaction of ozone with blood components results in 4–5-fold increased levels of thiobarbituric acid reactants and a proportional decrease in plasma protein thiols without any appreciable erythrocyte haemolysis. On the basis of preliminary in vitro evidence, these simple laboratory parameters may represent a useful complement in the routine monitoring of biological compliance to the treatment. The clinical experience gained so far confirms the great therapeutic potential of EBOO in patients with severe peripheral arterial disease, coronary disease, cholesterol embolism, severe dyslipidemia, Madelung disease, and sudden deafness of vascular origin. Extensive investigation on oxidative stress biomarkers and clinical trials are under way to validate this new technique further.     

A realistic evaluation of the action of ozone on whole human blood

We have clarified the role of the ozone concentration in relation to the resistance of human erythrocytes in whole human blood or in blood diluted either in saline or in distilled water.

Spectrophotometric data related to haemoglobin were evaluated by exposing samples of fresh human blood directly to ozone doses (ratio 1:1 volume), within the therapeutic range (0.21–1.68 mM) and to one toxic dose (3.36 mM). Furthermore, the same determinations have been carried out after previous dilution of the same blood with either pure water or physiological saline (1 ml blood + 29 ml diluent) followed by ozonation with the above reported ozone doses. Addition of either saline or water implies a dilution of plasma antioxidants and also total haemolysis after water dilution. Particularly the latter case represents a most unphysiological situation because the osmotic shock causes the solubilization of the erythrocytic content. While it is possible to demonstrate that after haemolysis there is an ozone-concentration dependent transformation of some oxyhaemoglobin to methaemoglobin, no such process occurs after ozonation of whole blood.

The results of this study fully confirm our previous data that judicious ozone doses neither damage erythrocytes, nor induce oxidation of intracellular haemoglobin. We hope that our conclusions will definitively clarify the absence of toxicity of ozonetherapy.     

Vascular access for extracorporeal therapy in the rabbit

A technique allowing short-term blood access for extracorporeal therapy in rabbits is described. The technique utilizes silicone rubber cannulae and teflon vessel tips to construct an externalized carotid-jugular arteriovenous shunt. The insertion procedure takes 1 h and extracorporeal blood flows in excess of 10 ml/min are obtainable for up to 7 days. Blood flow may still be obtained following shunt clotting by insertion of smaller diameter catheters through the arterial cannula. This technique has been successfully used for extracorporeal therapy in rabbit disease models.     

Blood proteome of acute intracranial hemorrhage in infant victims of abusive head trauma

Abusive head trauma (AHT) is a leading cause of mortality and morbidity in infants. While the reported incidence is close to 40 cases per 100′000 births/year, misdiagnoses are commonly observed in cases with atypical, subacute, or chronic presentation. Currently, standard clinical evaluation of inflicted intracranial hemorrhagic injury (ICH) in infants urgently requires a screening test able to identify infants who need additional investigations. Blood biomarkers characteristic of AHT may assist in detecting these infants, improving prognosis through early medical care. To date, the application of innovative omics technologies in retrospective studies of AHT in infants is rare, due also to the blood serum and cerebrospinal fluid of AHT cases being scarce and not systematically accessible. Here, we explored the circulating blood proteomes of infants with severe AHT and their atraumatic controls. We discovered 165 circulating serum proteins that display differential changes in AHT cases compared with atraumatic controls. The peripheral blood proteomes of pediatric AHT commonly reflect: (i) potentially secreted proteome from injured brain, and (ii) proteome dysregulated in the system's circulation by successive biological events following acute ICH. This study opens up a novel opportunity for research efforts in clinical screening of AHT cases.     

d-Alanine as a biomarker and a therapeutic option for severe influenza virus infection and COVID-19

Since the outbreak of coronavirus disease 2019 (COVID-19), biomarkers for evaluating severity, as well as supportive care to improve clinical course, remain insufficient. We explored the potential of d-amino acids, rare enantiomers of amino acids, as biomarkers for assessing disease severity and as protective nutrients against severe viral infections. In mice infected with influenza A virus (IAV) and in patients with severe COVID-19 requiring artificial ventilation or extracorporeal membrane oxygenation, blood levels of d-amino acids, including d-alanine, were reduced significantly compared with those of uninfected mice or healthy controls. In mice models of IAV infection or COVID-19, supplementation with d-alanine alleviated severity of clinical course, and mice with sustained blood levels of d-alanine showed favorable prognoses. In severe viral infections, blood levels of d-amino acids, including d-alanine, decrease, and supplementation with d-alanine improves prognosis. d-Alanine has great potentials as a biomarker and a therapeutic option for severe viral infections.     

Functional group analysis during ozonation of sunflower oil methyl esters by FT-IR and NMR

Ozonation of neat sunflower oil (SFO) methyl esters was monitored by FT-IR and 1H and 13C NMR spectroscopy. During the early stage of ozonation, ozone absorption was essentially quantitative. This was accompanied by the formation of 1,2,4-trioxolane. IR and NMR spectra of ozonated samples showed that scission of ozonide to give aldehyde were minimal. 1H NMR analysis revealed that the amount of ozonide relative to aldehyde was more than 90% regardless of the extent of ozonation. Complete ozonation was attained after supplying around 0.20 g O3/ml methyl ester after which ozone absorption suddenly dropped to around 25%. At the latter part of ozonation, ozonide and aldehyde reacted with excess ozone to give carboxylic acid. Reaction products were identified according to Criegee mechanism.     

Water disinfection using the novel approach of ozone and a liquid whistle reactor

A novel approach of ozone treatment assisted by a liquid whistle reactor (LWR), which generates hydrodynamic cavitation, has been explored for water disinfection using a simulated effluent containing Escherichia coli (E. coli), one of the dominant markers in faecal coliforms. A suspension having an E. coli concentration of approximately 10⁸ to 10⁹ CFU mL⁻¹ was introduced into the LWR to examine the effect of hydrodynamic cavitation alone and in combination with ozone. Operating conditions of inlet pressure and ozone doses as well as time of ozonation for individual operation along with the combined operation have been varied with the aim of maximizing the extent of disinfection and arriving at an optimum strategy for treatment. It has been observed that nearly 75% disinfection can be achieved in about 3 h of treatment time using an optimized combination of hydrodynamic cavitation and ozonation. This combination has been found to be a cost-effective technique for achieving maximum disinfection compared to the individual operation of hydrodynamic cavitation (lower extent of disinfection) and ozonation (higher costs of treatment usually due to higher cost of ozone generation).     

Variations of photosynthetic activity and growth of freshwater algae according to ozone contact time in ozone treatment

The activity of freshwater algae in drinking water supply at various ozone contact times was investigated to evaluate ozonation processes used for sterilization of algae. O production rates of algae just after ozonation were severely retarded even by short ozone contact. The algal growth rate was also declined with increase of ozone contact time and completely inhibited by ozonation for 60 min. These results implied that the usual treatment time applied in conventional ozonation processes is not sufficient but at least 60 min of ozonation is required for a complete sterilization of freshwater algae in drinking water source.     

The effect of ozone on the biodegradation of 17α-ethinylestradiol and sulfamethoxazole by mixed bacterial cultures

The potential development of antibacterial resistance and endocrine disruption has led to increased research investigating the removal of contaminants from wastewater (WW) such as sulfamethoxazole (SMX) and 17α-ethinylestradiol (EE2). These compounds react quickly with ozone (O₃), thus ozonation during WW treatment may result in their complete removal. Also, O₃ has demonstrated the ability to increase the biodegradability of WW and certain pharmaceuticals, suggesting its potential as a pretreatment to activated sludge (AS, biological treatment). The objective of this study was to determine whether ozonation, conducted at doses lower than commonly applied to treated WW, would lead to an increased biodegradability of SMX and EE2. The results show that after ozonation performed at lab-scale the bacterial mixtures removed 5 % to 40 % more SMX; however, 2 % to 23 % less EE2 was removed, which was attributed to the observed preferential degradation of a by-product of EE2 ozonation. These results suggest that although ozonation, used as a pretreatment, was shown in literature to increase the overall biodegradability of AS as well as some specific antibiotic compounds and a blood lipid regulator, the potential for increased removal of pharmaceuticals seems to be compound-dependent and cannot yet be extrapolated to this entire class of compounds.     

Mechanism of Ozone Inactivation of Bacteriophage f2

The inactivation kinetics of bacteriophage f2 were studied by using ozone under controlled laboratory conditions. The phage were rapidly inactivated during the first 5 s of the reaction by 5 and 7 logs at ozone concentrations of 0.09 and 0.8 mg/liter, respectively. During the next 10 min, the phage were further inactivated at a slower rate in both treatments. The [³H]uridine-labeled f2 phage and its ribonucleic acid (RNA) were examined to elucidate the mechanism of ozone inactivation, utilizing adsorption to host bacteria, sucrose density gradient analysis, and electron microscopy. The specific adsorption of the phage was reduced by ozonation in the same pattern as plaque-forming unit reduction. RNA was released from the phage particles during ozonation, although it had reduced infectivity for spheroplasts. Electron microscopic examination showed that the phage coat was broken by ozonation into many protein subunit pieces and that the specific adsorption of the phage to host pili was inversely related to the extent of phage breakage. The RNA enclosed in the phage coat was inactivated less by ozonation than were whole phage, but inactivated more than naked RNA. These findings suggest that ozone breaks the protein capsid into subunits, liberating RNA and disrupting adsorption to the host pili, and that the RNA may be secondarily sheared by a reduction with and/or without the coat protein molecules, which have been modified by ozonation.     

pH-Sensitive mutagenic activity of ozone-treated 1,2-dimethylhydrazine in the Salmonella/microsome assay

Treatment with ozone inactivates the mutagenicity of many carcinogens in aqueous solution. The colon carcinogen, 1,2-dimethylhydrazine (DMH) has been reported an exception; ozone treatment convenrts dimethylhydrazine from a non-mutagen into a mutagen. In the Salmonella/microsone assay, the mutagenicity of ozone-treated dimethylhydrazine was dependent on pH. The ozonation product was a strong mutagen in acidic but was not mutagenic in basic solution. The mutagenicity of the acidic ozonation product was inactivated by raising the pH of the solution. Unlike untreated dimethylhydrazine, its ozonation product in basic solution was not converted to a mutagen in this ozone-low pH system.     

Inactivation of Viruses and Bacteria by Ozone, With and Without Sonication

Selected organisms with public health significance were placed in a reaction chamber for treatment by ozonation, by ozonation and sonication, by sonication, or by sonication during oxygenation. Vesicular stomatitis virus, encephalomyocarditis virus, GDVII virus, Staphylococcus aureus, Pseudomonas fluorescens, Salmonella typhimurium, enteropathogenic Escherichia coli, Vibrio cholerae, and Shigella flexneri were inactivated by treatment with ozone. When microorganisms were suspended in phosphate-buffered saline, they were inactivated rapidly by treatment with ozone. However, microorganisms suspended in secondary effluent from a wastewater treatment plant required longer contact times with ozone for complete inactivation. Simultaneous treatments by ozonation and sonication reduced the contact time for complete inactivation of microorganisms in secondary effluent. Treatment by sonication alone or sonication and oxygenation did not inactivate microorganisms. Therefore, the simultaneous treatment of microorganisms in secondary effluent with ozone and sonication resulted in a synergistic effect.     

Delignification of straw with ozone to enhance biodegradability

Summary Wheat straw was treated with ozone to remove the lignin and increase its biodegradability. The attack of ozone on straw is not selective. Lignin and carbohydrates are oxidized concurrently though the rate of reaction with the latter is slower. A 50% reduction of the original lignin content is optimal for enzymatic hydrolysis. After treatment, 75% of the cellulose in straw is degraded within 24 h as compared to 20% in untreated straw. During ozonation lignin is converted to soluble products which to a great extent are biodegradable and thus yield a useful byproduct. At the moment, ozonation ranks among the more expensive methods of treatment. However, the economics may be improved by reducing the cost of ozone production; this is likely to take place in the near future due to technological improvements, and by reducing the ozone consumption by optimizing the process of ozonation.     

Selectins and monocyte chemotactic peptide as the markers of atherosclerosis activity

The role of adhesive selectin molecules in the process of atherogenesis is an open question. These molecules are known as markers of atherosclerosis activity, however, only some biological mechanisms are known up to now. In this study we examined the levels of soluble forms of E-, P-selectin and monocyte chemotactic protein (MCP-1) in the process of extracorporeal cholesterol elimination by LDL-apheresis. We measured the levels of sE-, sP-selectin and MCP-1 in the plasma before and after LDL-apheresis and in the washout solution from immunoabsorption columns Lipopak. Eighty measurements were performed repeatedly in 6 patients with severe familial hypercholesterolemia (FH) on long-term LDL-apheresis treatment. Before the procedure P-selectin levels were 204+/-179 ng/ml, E-selectin 32.1+/-33.7 ng/ml, MCP-1 323.8+/-121 pg/l, whereas after the procedure we found P-selectin levels 131.6+/-34 ng/ml, E-selectin 33.1+/-51 ng/ml, and MCP-1 200.4+/-15 pg/l. Levels of P-selectin were increased in the blood of patients with FH in spite of long-term intensive extracorporeal LDL-elimination, documenting thus the activity of atherosclerosis. The levels of P-selectin and MCP-1 decreased significantly after the hypolidemic procedure and could be used as another marker showing the effectivity of the extracorporeal LDL-cholesterol elimination (immediately after the procedure), and, after further verification, may serve as a marker for controlling the therapy efficacy.     

Combined Biological Treatment/Ozonation of Wastewaters for the Mineralisation of Non‐Biodegradable Naphthalene‐1,5‐Disulphonic Acid

This paper deals with the ozonation and biological treatment for achieving the mineralisation of the non‐biodegradable compound naphthalene‐1,5‐disulphonic acid (NADSA; C₁₀H₈O₆S₂), a basic chemical for dyes and building materials. This combination of ozonation and biological treatment is expected to minimise the ozone consumption compared to ozonation alone. To examine the influence of ozone on the biodegradability of NADSA, a lab‐scale plant was run semi‐continuously and batch tests were conducted for comparison. For total ozonation, 1.8 mol O₃/mol DOC have to be used. For optimum biodegradation, only 0.8 mol O₃/mol DOC are needed. The results show that by using a combination of ozonation and biological treatment the ozone consumption can be reduced by more than 50% compared to ozonation alone. Some of the intermediate products are isolated and suggestions for their chemical structures are made. A proposal for the first steps of ozonation is also presented to illustrate a possible pathway of ozonation. All the major identifiable compounds are non‐biodegradable. From these results, it is quite clear that further intermediate products are formed, which are biodegradable.     

The ozone tolerance: I) Enhancement of antioxidant enzymes is ozone dose-dependent in Jurkat cells

We have begun to examine the biological and toxic effects of ozone on Jurkat T cells incubated thereafter for 24, 48 and 72 h. Tissue culture medium was strengthened by adding 20% fetal calf serum with an albumin content of about 6 mg/ml. Ozonization was performed by exposing for 10 min a volume of cell suspension (4 x 10(5)/ml) to an equal volume of a gas mixture composed of oxygen-ozone with precise ozone concentrations ranging from 1.5 up to 72 microg/ml (31.5-1512 microM). The proliferation index declined progressively and was ozone dose-dependent. The response of enzymatic activities varied depending upon the enzyme and ozone concentrations: glucose-6-phosphate dehydrogenase begins to increase at an ozone dose of 6 microg/ml (126 microM), reached a peak at 12 microg/ml (252 microM) and rapidly declined thereafter. On the other hand activities of superoxide dismutase, glutathione peroxidase and glutathione reductase increased progressively from the ozone concentration of 12 microg/ml. Thus, as we have observed in blood, the biological response is linked to the ozone dose that must reach a threshold to be effective.     

Ozone treatment of process water from a dry-mill ethanol plant

Fuel ethanol production in corn dry milling plants is a rapidly expanding industrial sector. Whole stillage, the residue from the distillation of the fermented corn, is centrifuged and the concentrate, thin stillage, is found to have a chemical oxygen demand (COD) of approximately 75,000 mg/L. This thin stillage is partly recycled, but much of it needs to be evaporated to concentrate the solubles for addition to the animal feed coproduct from corn dry milling. This research is an exploration into lowering COD from thin stillage using ozonation as a simple single-step unit process to facilitate a larger reusable fraction. The ozonation would usually be a pretreatment before additional flocculation or biological treatment. Also, COD removal by ozonation with and without a catalyst has been studied. Three different application rates of ozone O(3,1)=7 mg/min, O(3,2)=21 mg/min, and O(3,3)=33 mg/min were used for 8h into samples of 2L each of three dilutions 20x, 30x and 40x. COD removal of 85% was observed with an ozone dosage of 4000 mg/L into a 40x-diluted sample. This would correspond to about 0.5 mg COD removed per mg ozone dosed. However, at lower dosages and smaller dilutions, more than 1mg COD removal was achieved per mg ozone dosed. Two different catalysts, Fe(II) and Fe(III), were used and the samples were ozonated for 4 h. Five different dosages of each of the two catalysts were used and better COD removal was observed compared to ozonation alone with all 5 dosages. COD removal rate was increased from 45% to 74% with Fe(III) and was increased to 77% with Fe(II). Up to 10mg/L COD was removed per mg O(3) dosed well below the maximum dosage. Both the catalysts resulted in almost the same improved COD removal rates when compared to ozonation alone.     

臭氧自血疗法及其在糖尿病治疗中的应用

目前,糖尿病及其并发症已经成为我国一个重大的公共卫生问题。大量研究证实,氧化应激与糖尿病大血管和微血管并发症的发生及发展密切相关。臭氧自血疗法的应用已有半个世纪,低强度、精确计算且可重复的臭氧自血治疗可诱发标准化的急性氧化应激,使机体的抗氧化防御系统得到锻炼和增强,而在此过程中产生的一定数量的信使,可以与血液和实质细胞产生相互作用,改善缺血组织的血液循环和氧供,使免疫系统轻度活化,增加生长因子的释放,激活神经保护系统,改善患者的健康状态。虽然有证据显示臭氧治疗在改善血糖和血脂控制方面的贡献,且安全性良好,但迄今为止,臭氧自血疗法的临床试验数据还相当欠缺,有必要开展设计良好的临床试验,进行长期观察,以获得其应用和推广所需的更为确凿的证据。本综述旨在探讨臭氧自血疗法的作用机制及应用前景。     

Rapid Removal of Tetrabromobisphenol A by Ozonation in Water: Oxidation Products,Reaction Pathways and Toxicity Assessment

Tetrabromobisphenol A (TBBPA) is one of the most widely used brominated flame retardants and has attracted more and more attention. In this work, the parent TBBPA with an initial concentration of 100 mg/L was completely removed after 6 min of ozonation at pH 8.0, and alkaline conditions favored a more rapid removal than acidic and neutral conditions. The presence of typical anions and humic acid did not significantly affect the degradation of TBBPA. The quenching test using isopropanol indicated that direct ozone oxidation played a dominant role during this process. Seventeen reaction intermediates and products were identified using an electrospray time-of-flight mass spectrometer. Notably, the generation of 2,4,6-tribromophenol was first observed in the degradation process of TBBPA. The evolution of reaction products showed that ozonation is an efficient treatment for removal of both TBBPA and intermediates. Sequential transformation of organic bromine to bromide and bromate was confirmed by ion chromatography analysis. Two primary reaction pathways that involve cleavage of central carbon atom and benzene ring cleavage concomitant with debromination were thus proposed and further justified by calculations of frontier electron densities. Furthermore, the total organic carbon data suggested a low mineralization rate, even after the complete removal of TBBPA. Meanwhile, the acute aqueous toxicity of reaction solutions to Photobacterium Phosphoreum and Daphnia magna was rapidly decreased during ozonation. In addition, no obvious difference in the attenuation of TBBPA was found by ozone oxidation using different water matrices, and the effectiveness in natural waters further demonstrates that ozonation can be adopted as a promising technique to treat TBBPA-contaminated waters.     

Basic examinations on chemical pre-oxidation by ozone for enhancing bioremediation of phenanthrene contaminated soils

Biological treatment of polycyclic aromatic hydrocarbons (PAH) has been demonstrated to be a feasible and common remediation technology which has been successfully applied to the clean-up of contaminated soils. Because bioavailability of the contaminants is of great importance for a successful bioremediation, a chemical pre-oxidation step by ozone was tested to enhance the subsequent biodegradation steps. Oxidation of PAH by ozone should result in reaction products that have a better solubility in water and thus a better bioavailability. A major part of this work was done by examinations of the model substance phenanthrene as a typical compound of PAH. After initial ozonation of phenanthrene, analysis by GC-MS showed at least seven identified conversion-products of phenanthrene. In comparison with phenanthrene these conversion products were more efficiently biodegraded by Sphingomonas yanoikuyae or mixed cultures when the ozonation process resulted in monoaromatic compounds. Primary ozonation products with biphenylic structures were found not to be biodegradable. Investigations into the toxicity of contaminated and ozonated soils were carried out by well-established toxicity assays using Bacillus subtilis and garden cress. The ozonated soils surprisingly showed higher toxic or inhibitory effects towards different organisms than the phenanthrene or PAH itself. The microbial degradation of phenanthrene in slurry reactors by S. yanoikuyae was not enhanced significantly by preozonation of the contaminated soil.