Dissolved ozone in biological fluid monitored by optical device operating in the red-infrared region

ORIGINAL ARTICLE

Dissolved ozone in biological fluid monitored by optical device operating in the red-infrared region

Henrique Cunha CarvalhoI, ^*; Milene da Silva Melo^I; Carlos José de Lima^{I, II}; Renato Amaro Zângaro^{I, II}

^IInstituto de Engenharia Biomédica, Universidade Camilo Castelo Branco - UNICASTELO, Parque Tecnológico de São José dos Campos, Estrada Doutor Altino Bondesan, 500, Eugênio de Melo, CEP 12247-016, São José dos Campos, SP, Brasil

^IIAssociação Cidade da Ciência Tecnologia e Educação - CITÉ, São José dos Campos, SP, Brasil

ABSTRACT

INTRODUCTION: When a gas is used for therapy, often the kinetic behavior and their distribution in biological systems is not known, leading to unsatisfactory results for clinical application. The use of ozone in living organisms has been scientifically released worldwide under the name of ozone therapy. The efficacy of this technique is determined primarily by the diffusion of gas within the tissues or fluids and which determines their action in the entire target region. We propose the development of technique to monitoring the O₃ dissolved in the biological fluid using an optical device operating in the red-infrared region.

METHODS: The recombination of O₃ in O₂ enables the monitoring of the latter by the measurement of SpO₂, and, based on this phenomenon, we propose to use an optical device operating in the red-infrared region to monitoring indirectly the diffusion of O₃ in fluids. The system was based on optomechanical arrangement using a capsule containing fluid that was ozonated or oxygenated during the process. A pulse oximeter is a noninvasive device used for continuously measure of SpO₂ resulting from the recombination of ozone.

RESULTS: The measurements of SpO₂ when subjected to ozone and oxygen, showed an increased rate of SpO₂ function of time for both cases reaching its peak in 80s and 160s, respectively. The experimental data concerning the SpO₂ saturation as a function of time can be fitted by the theoretical model, showing a good correlation between them.

CONCLUSION: A technique was developed using an optical device operating in the red-infrared region to monitoring ozone dissolved in biological fluid, showing a simple and effective way to indirectly monitoring the presence of ozone in fluids.

Keywords: Ozone, Biological fluid, Diffusion, Recombination, Optical device, Red-infrared region.

Introduction

The use of ozone in living organisms has been widely publicized primarily on its action on bacteria, fungi, protozoa and viruses (Bocci et al., 2011a; Loeb, 2011). Numerous other biological and medical applications has been scientifically tried and reported around the world under the name of ozone therapy.

Ozone involved in redox balance, being a powerful oxidant that reacts immediately when in contact with biological fluids, inducing molecules directly on the balance of reactive oxygen species (ROS), which influence many biochemical events in the cell metabolism, providing antimicrobial effects benefits in addition to repair and balance of the target organism (Bocci et al., 2011a).

Thus, satisfactory results have been obtained in the treatment of diabetic foot (Wainstein et al., 2011), hepatitis (Zaky et al., 2011), cancer (Schulz et al., 2008), coronary heart disease (Martínez-Sánchez et al., 2012), atherosclerosis (Delgado-Roche et al., 2013), as a disinfecting agent in microorganisms causing nosocomial infections (Doan et al., 2012; Zoutman et al., 2011), and others.

When a gas is used for therapeutic purposes, often their kinetic behavior and their distribution in biological systems is not known, leading to unsatisfactory results when clinical application. The application of ozone therapy efficacy is determined primarily by the diffusion of gas within the tissues or fluids and which determines their action in the entire target region. The techniques commonly used to monitoring the diffusion of ozone fluids are invasive (Buchan et al., 2005; Loeb, 2011), which prevents monitoring systems such as sterile blood. Noninvasive optical techniques may be used in the UV region at 254 nm (Gao et al., 2012; Kalnajs and Avallone, 2010), but this spectral region cannot be applied in biological fluids due to high radiation absorption UV blood.

The recombination of O₃ in O₂, allows the monitoring of the latter by the measurement of SpO₂ and based on this phenomenon, we propose a new approach to monitoring the O₃ dissolved in the biological fluid using a simple and ingenious optomechanical device without the need to use O₃ meter.

Methods

The present study aims to determine the diffusion of ozone gas in fluids by measuring SpO₂. The model was proposed in order to be able to perform real-time monitoring, noninvasively and nondestructively, using such a pulse oximeter operating in the region red - infrared. A system was developed based on the use of an optical oximeter (MD-300C - Moriya JG), which operates at two emission wavelengths, λ₁ and λ₂, the first of which presents high absorption by oxyhemoglobin and second low absorption. The SpO₂ absorption corresponds to the difference between these two variables. As a biological fluid, was used 0.8 mL of whole bovine milk, type C, with 10 samples, five submitted to the flow of oxygen and five others submitted to the flow of ozone. This fluid was used due to optical scattering characteristics, which facilitates the detection of the SpO₂, and the model can be easily implemented in blood.

Pulse oximetry is based on light transmittance of hemoglobin. The light transmission coefficient of a substance is determined by the Beer-Lambert Law, which states that the concentration of an unknown solute in a solvent may be determined by absorption/scattering of light. In this case, solutes are oxyhemoglobin and reduced hemoglobin, and blood as the solvent.

During systole, the blood system is filled, increasing blood volume and absorption/scattering of light. During diastole, blood volume and light absorption/scattering reach their lowest point. The pulse oximeter operates synchronously with the cardiac pulse and SpO₂ is obtained from the difference between the maximum and minimum absorption/scattering intensities during systole and diastole. In this case, two wavelengths are used, λ₁ (660 nm) that is highly absorbed by oxyhemoglobin and λ₂ (910 nm) which is absorbed by all the others components present in the tissue (Figure 1). The difference of intensity between the two signals expressed SpO₂. In this work, the solvent is milk and solutes are ozone and oxygen.

The system of SpO₂ was developed according to the diagram shown in Figure 2, using an optical oximeter having in its interior a capsule containing milk that received the gas flow. In this case, the finger is a capsule and the biological tissue is the milk inside of the capsule.

To observe the ability of diffusion in the milk a preliminary experiment used pure oxygen, and the results were used as reference of oxygen saturation. A second experiment was then conducted in order to determine the recombination of O₃ in O₂, and in this case, ozone was infused into the milk and SpO₂ were monitored by the oximeter.

For their timing, the oximeter requires mimicking the human physiological system, which is based on the systole/diastole. In this case, this condition was obtained by moving the milk contained in the capsule. The movement is developed with the aid of an eccentric device, powered by a stepper motor operating at 60 rpm. The diffusion in the milk was provided by two gas lines, O₂ and O₃, both operating at a flow rate of ¹/₃₂ L/min, at different times, and the ozone generator used (OzonLife - Medical Systems) was able to provide 40 mg/L.

Several models have been proposed to describe the transfer of ozone from the gas phase to the liquid phase (Bin, 2006). Generally, these models postulate that the concentration in both phases is homogeneous, except in the gas-liquid interface area. To determine the mass transfer coefficient (K_La) is necessary to calculate the mass balance of the limiting phase (liquid), which is given by Equation 1 (Kunz et al., 1999):

where, K_La: volumetric mass transfer coefficient [min ^{- 1}]; C_L^sat: saturation concentration of gas in solution [mg.L ^{- 1}]; C_L: concentration of ozone in solution [mg.L ^{- 1}]; K_d: kinetic constant of ozone decomposition [min ^{- 1}]; C_L: t: time of ozonation [min].

Results

The experiment was conducted to determine the SpO₂ sample of 0.8 mL of milk, when this was ozonated or oxygenated. The results showed that in both cases, saturation occurs at relatively short times, of the order of a few minutes, which can be seen in Figures 3 and 4.

Because the specifics of the experiment, the oximeter response starts from 75%, and therefore the Figures 3 and 4, has its origins located in this region. The measurements of SpO₂ when subjected to ozone and oxygen, showed an increased rate of SpO₂ function of time for both cases reaching its peak in 80 s and 160 s, respectively. The experimental data concerning the SpO₂ saturation as a function of time can be fitted by the theoretical curves obtained from Equation 2, showing a good correlation between them.

where, C_O3: ozone concentration [mg.L ^{- 1}]; a, b and c: parameters; t: time [min].

Discussion

The ozone decomposes in water spontaneously through complex mechanisms involving free radical generation. Due to the high water concentration in the milk, can be considered in this case that the mechanism of decomposition of ozone in milk is similar to that of water.

During the recombination of O₃ in O₂, two reaction mechanisms are understood in the liquid, the direct oxidation of compounds by the ozone and indirect oxidation of compounds by hydroxyl free radicals produced during the decomposition of O₃. In this case, the free radicals present, determine the rate of reaction, and also the regeneration of the superoxide radical O₂-ion or proton form HO₂, derived from the hydroxyl radical (OH), which means the consumption of 1 mol of ozone (Guerrero, 2005). As a result, all species capable of consuming hydroxyl radicals without regenerating the superoxide ion will produce a stabilizing effect on the ozone molecule in the sample. This effect associated with those produced by-products, provides greater quantity of molecular oxygen, resulting in a smaller time interval for increased saturation SpO₂.

The recombination of O₃ in O₂ depends mainly on the type of flow, gas solubility and temperature. The type of flow is defined by the Reynolds number (Re) according to Equation 3, and values of "Re" greater than 2400, as set turbulent flow. This experiment was carried out under atmospheric pressure and the calculated Reynolds number was equal to 16405, characterizing it as turbulent flow. In order to reduce the influence of temperature on the process, the experience was performed at a constant temperature equal to 21 °C.

Where, Re: Reynolds number [dimensionless]; D: diameter of capsule = 0.08 [m]; v: velocity of flow = 6.217 [m.s ^{- 1}]; ρ: density of milk at 21 °C = 1029 [Kg/m³] and ∝: viscosity of milk at 21 °C = 2.1 [cP].

The concentration of ozone versus time expressed by Equation 2 is dependent on the solubility of the gas, which in turn directly affects the mass transfer. The data concerning the solubility of gases in liquids have great theoretical and practical interest, where the processes involved in solubility/mass transfer between gas and liquid are crucial to monitoring the diffusion of gas in the interface processes.

As shown in Figures 3 and 4, to reach the oxygen saturation (SpO₂) at 95% produced by ozonized fluid requires half time if compared when the fluid is oxygenated, which is supported by the calculations obtained from Equation 2. This analysis sparked numerous questions about which mechanisms are involved in this phenomenon. Analyzing the variables involved in the process, different studies have reported a significant difference in solubility of ozone and oxygen (Bin, 2006; Bocci et al., 2011a) when dissolved in water at normal temperature and pressure, and this difference according to our analysis the most plausible answer to explain this result. Bin (2006) also presented several relationships between different expressions of the solubility of ozone in liquids. Several authors have also published data on solubility of ozone in water (Rischbieter et al., 2000; Kuosa et al., 2004; Levanov et al., 2008), indicating that it is about ten times more soluble than molecular oxygen (Bocci et al., 2011b), supporting this hypothesis. Another aspect to be considered when the milk is ozonized, that is, the ozone decomposition constant (K_d) increases exponentially as a function of the presence in milk of different molecules, increasing the rate of recombination O₃, with consequent reduction in the time to reach saturation. The relative high pH of the sample, in this case close to 7.0, also contributes to this phenomenon. In this situation the value of K_d can be as high enough to compete with the value of K_La, making the recombination-O₃ O₂ occurs immediately after diffusion of ozone in liquid medium, explaining why the SpO₂ induced by the action of ozone is faster than that induced by oxygen.

The results allow us to conclude that the technique, developed using an optical device operating in the red-infrared region to monitoring ozone dissolved in biological fluid, provides a simple and effective way to indirectly monitor the presence of ozone in fluids.

Acknowledgments

CAPES (Coordination for the Improvement of Higher Education Personnel) and FAPESP (São Paulo Research Foundation) for the financial support.

Received: 21 July 2013

Accepted: 27 January 2014

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Publication Dates

• Publication in this collection
  14 July 2014
• Date of issue
  June 2014

History

• Accepted
  27 Jan 2014
• Received
  21 July 2013