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'''Understanding the Effects of Different Chlorine Dioxide Preparations on Human Blood, Characteristics''' 

'''Fundación de medicina electromolecular'''

Lead scientist: Dr. h.c. Andreas Ludwig Kalcker Assistants: 

Scarlet

Gabriela Castillo

…

'''Abstract'''

This study investigates the effects of chlorine dioxide on live blood samples, comparing the NaClO2 + acid mixture (acidified sodium chlorite) with pure ClO2 gas bubbled through water (CDS). Live blood samples from human volunteers were observed under a Nikon phase contrast microscope following exposure to the experimental substances. The NaClO2 + acid mixture exhibited limited positive effects, alongside potential side effects and instability, while the pure ClO2 gas demonstrated intermediate oxygenation and improved blood flow. 

Contrary to common beliefs, oral ingestion of CDS did not result in methemoglobinemia, highlighting its safety profile. Immediate hemostatic effects observed with concentrated CDS suggest a potential application in managing bleeding conditions. The study findings underline the importance of the form of chlorine dioxide used and its physiological implications. CDS shows promise as a safer alternative with therapeutic potential, warranting further investigation into its role as an oxygen carrier and metabolic acidity compensator. This research provides valuable insights for future studies and potential applications of chlorine dioxide in physiological enhancement without adverse effects.

'''Introduction'''

Chlorine dioxide has been a subject of controversy and confusion in various contexts due to its different forms and the associated effects it may have. Nevertheless there are studies in humans with very positive results for different applications (Lubbers et al., 1982). In this clinical study, we aim to delve into the intricate world of chlorine dioxide by investigating and comparing the effects of two distinct forms on live blood samples. The first form under scrutiny is a combination of NaClO2 and an acid, commonly referred to as acidified sodium chlorite (also known as MMS). This particular formulation has garnered attention for its perceived instability and potential side effects, raising concerns within the scientific community and beyond.

Contrastingly, the second form involves a process of bubbling pure ClO2 gas through water to create an aqueous solution, also known as CDS. Notably, this form does not contain Chlorite, and research suggests that it exhibits stability over the long term, presenting an intriguing alternative for potential applications (Aparicio-Alonso et al., 2021).By conducting a comparative analysis of these two forms of chlorine dioxide on live blood samples, we aim to contribute valuable insights to the ongoing discourse surrounding this compound and its diverse manifestations. This study holds promise for shedding light on the effects of chlorine dioxide in a controlled setting, paving the way for a deeper understanding of its implications in biological systems. 

'''Hypothesis'''

''Alternative Hypothesis''

There will be significant differences in the observed effects between acidified sodium chlorite and chlorine dioxide solution on blood samples during the 12-minute period.

''Null Hypothesis  ''

There will be no significant differences in the observed effects between acidified sodium chlorite and chlorine dioxide solution on blood samples during the 12-minute period.

'''Objectives''' 

''General objective''

To compare the effects of acidified sodium chlorite and chlorine dioxide solution on live blood samples over a period of 12 minutes

''Specific objectives''

# To demonstrate the side effects and reactions generated by acidified sodium chlorite in group 1 of live blood samples.
# Analyze the reactions in group 2 of chlorine dioxide in blood samples and assess hemostatic response.
# Examine the interaction with Calcium (Ca) and contrast the absence of methemoglobinemia when applying chlorine dioxide to blood samples.

'''Study Design'''

In this study, live blood samples were meticulously collected from Swiss human human volunteers of both 30-40 years of age and prepared for observation under a high-resolution Nikon phase contrast microscope to assess the effects of chlorine dioxide. The experimental groups were divided into two categories: Group 1 received exposure to the NaClO2 + acid mixture, commonly known as acidified sodium chlorite , while Group 2 was subjected to the pure ClO2 gas by electrolytic method without acid bubbled through water, creating an aqueous solution known as CDS. The samples were carefully monitored for stability, ongoing reactions, and immediate effects following exposure to the respective chlorine dioxide forms. Observations were made on changes in blood cell morphology, oxygen levels, and overall condition under the microscope. Additionally, the response to a concentrated saline solution and the immediate effects on bleeding control with concentrated CDS were evaluated to determine the impact of chlorine dioxide on live blood samples. Quantitative data were meticulously collected and statistically analyzed to compare the effects of acidified sodium chlorite  and CDS on live blood samples, providing valuable insights into the potential benefits and risks associated with these chlorine dioxide forms.

In this study, live blood samples were meticulously prepared for observation under a Nikon phase contrast microscope to assess the effects of chlorine dioxide. The experimental groups were divided into two categories: Group 1, exposed to the NaClO2 + acid mixture (acidified sodium chlorite ), and Group 2, subjected to pure ClO2 gas (3000 ppm) bubbled through water (CDS). Detailed observations were made to monitor the ongoing reactions and stability of each form of chlorine dioxide within the live blood samples. The effects of both forms on live blood morphology, oxygen levels, and overall condition were carefully examined under the microscope to capture any observable changes. Furthermore, the response of the blood samples to a concentrated saline solution  was evaluated to understand their reactivity. 

A parallel test was conducted simultaneously to evaluate the immediate effects on bleeding control using a concentrated CDS solution (over 1000 ppm). This test aimed to gain a deeper understanding of the hemostatic properties of chlorine dioxide by observing its impact on live blood samples. The comprehensive observations sought to elucidate the physiological responses to various forms of chlorine dioxide and their potential implications for health and well-being.

'''Methods'''

''Sample Preparation''

Live blood samples were collected from human volunteers and prepared for observation under a Nikon phase contrast microscope. The samples were handled with care to maintain their integrity and prevent contamination.

In the experimental groups of the study, Group 1 was exposed to the NaClO2 + acid mixture, commonly referred to as acidified sodium chlorite . Live blood samples were carefully subjected to this mixture to investigate its immediate effects and any ongoing reactions within the sample. Observations were meticulously recorded to capture the dynamic responses of the live blood samples to the NaClO2 + acid mixture. In contrast, Group 2 received exposure to pure ClO2 gas bubbled through water, resulting in the creation of an aqueous solution known as CDS. The live blood samples in this group were observed to assess the intermediate oxygenation effects and stability induced by the pure ClO2 gas. Detailed observations were conducted to monitor the changes in blood oxygenation and overall stability of the samples under the influence of the ClO2 gas bubbling process, providing valuable insights into the physiological effects of this form of chlorine dioxide on live blood samples.

Throughout the study, various key observations were carefully conducted to evaluate the effects of chlorine dioxide on live blood samples. The stability and ongoing reactions of the NaClO2 + acid mixture (acidified sodium chlorite ) were closely monitored over time to understand its dynamic behavior. Similarly, the stability of the ClO2 gas in the CDS solution was rigorously assessed within a sealed glass enclosure to determine its long-term stability. Microscopic analysis played a crucial role in the study, with live blood samples from both experimental groups meticulously observed under a Nikon phase contrast microscope. Changes in blood cells, oxygen levels, and overall condition were meticulously recorded and analyzed to capture the detailed physiological responses induced by the different forms of chlorine dioxide. Following exposure to the experimental substances, a concentrated saline solution of 3000 ppm was applied to the live blood samples to observe any discernible changes in their reactivity. 

Moreover, the immediate effects of a concentrated CDS solution (over 1000 ppm) on stopping bleeding in wounds were assessed, shedding light on the potential hemostatic properties of chlorine dioxide in live blood samples. 

''Data Collection and Analysis''

Throughout the comprehensive study, a series of meticulous observations were systematically executed to explore the impact of chlorine dioxide on live blood samples. The dynamic behavior and stability of the NaClO2 + acid mixture (acidified sodium chlorite ) were meticulously monitored over an extended period to elucidate its reactivity profile. Similarly, an in-depth assessment of the stability of ClO2 gas within the CDS solution was conducted, utilizing a sealed glass enclosure to ensure a controlled environment for long-term stability evaluations. Central to the study, microscopic analysis under a high-resolution Nikon phase contrast microscope meticulously scrutinized live blood samples from both experimental groups. Detailed examination of blood cell morphology, oxygen levels, and overall physiological conditions provided a comprehensive understanding of the intricate responses elicited by the diverse forms of chlorine dioxide. Subsequent exposure to a concentrated saline solution of 3000 ppm enabled the observation of any notable alterations in reactivity within the live blood samples post-treatment. 

Furthermore, the immediate hemostatic effects of a concentrated CDS solution exceeding 1000 ppm were methodically evaluated, shedding light on the potential therapeutic properties of chlorine dioxide in managing bleeding conditions within live blood samples. Overall, these meticulous and extensive observations yielded valuable insights into the nuanced physiological responses and effects of chlorine dioxide on live blood samples across varied experimental settings, enriching our understanding of its potential implications in biological systems. 


'''Results'''

The results of the study revealed intriguing findings regarding the effects of chlorine dioxide on live blood samples. In the group exposed to the NaClO2 + acid mixture (acidified sodium chlorite), limited positive effects were observed, accompanied by known potential side effects in oral intake such as diarrhea and vomiting at high doses (Loh & Shafi, 2014). The instability of this mixture due to ongoing reactions was evident during the observations. 

In contrast, the group exposed to pure ClO2 gas bubbled through water (CDS) exhibited an immediate increase of the Z potential of the RCB and  oxygenation of oxygen-depleted blood and improved blood flow. 

Interestingly, challenging common belief and highlighting its potential safety in this context. 

The immediate cessation of bleeding with high concentrated CDS (3000 ppm) indicated a possible interaction with the calcium (CA+) in the smooth muscles of veins, leading to vascular spasm. These results shed light on the contrasting effects of acidified sodium chlorite  and CDS forms of chlorine dioxide on live blood samples and provide valuable insights for further research and application.

# '''NaClO2 + Acid Mixture (acidified sodium chlorite )''':
#* Limited positive effects observed
#* Potential side effects like diarrhea and vomiting in high doses
#* Unstable nature due to ongoing reactions
# '''Pure ClO2 Gas (CDS)''':
#* Intermediate oxygenation of oxygen-depleted blood
#* Improved blood flow and oxygen levels
#* No observed methemoglobinemia with oral ingestion

In the images captured during the study, intricate details of live blood samples exposed to different forms of chlorine dioxide are visually depicted. The microscopic images reveal the cellular reactions and structural changes within the blood samples following exposure to the NaClO2 + acid mixture (acidified sodium chlorite) and pure ClO2 gas bubbled through water (CDS). Distinct differences in blood cell morphology, oxygen levels, and overall condition can be observed between the experimental groups. The images provide a visual representation of the ongoing reactions and stability of each form of chlorine dioxide within the live blood samples, offering insights into their respective effects on cellular dynamics. Additionally, the visual data showcase the immediate effects of concentrated saline solution (3000 ppm) on the blood samples and the hemostatic responses triggered by concentrated CDS solution (over 1000 ppm). These images serve as valuable visual aids, complementing the microscopic observations and enhancing the understanding of the physiological responses of live blood samples to different forms of chlorine dioxide in the study.

''Photomicrograph taken with a bright-field microscope stained with acidic staining, blood. Infiltration of acidified chlorite in RBC'' 

''Photomicrograph taken with a bright-field microscope stained with acidic staining, blood. No direct positive effect observed with acidified chlorite''

''Photomicrograph taken with a bright-field microscope stained with acidic staining, blood. Inicial RBC with echinocytes in a bad state and infiltration in blood thrombus''

''Photomicrograph taken with a bright-field microscope stained with acidic staining, blood. Oxygenation observed immediately''

''Photomicrograph taken with a bright-field microscope stained with acidic staining, blood. After 12 minutes fully recovered Z potential and oxygenated blood''

'''Discussion'''

The discussion of the study results highlights the contrasting effects of the NaClO2 + acid mixture (acidified sodium chlorite) and pure ClO2 gas bubbled through water (CDS) on live blood samples. The observed limited positive effects and potential side effects of acidified sodium chlorite , such as diarrhea and vomiting at high doses, raise concerns about its stability and safety for use as stated in a report of the FDA before U.S. FOOD & DRUG Administration (FDA) (2019).  In contrast, the intermediate oxygenation and improved blood flow seen with CDS suggest its potential as a safer alternative with fewer adverse effects. The absence of methemoglobinemia with oral ingestion of CDS challenges previous beliefs and opens up new possibilities for its application. 

A vascular spasm is a sudden, involuntary constriction of a blood vessel, leading to a reduction in blood flow. This physiological response can occur in response to various stimuli, such as trauma, cold temperatures, or electromolecular charge increase of oxidation reduction potential (Gomes et al., 2012). In the context of chlorine dioxide of concentrated gas exposure, vascular spasms may be induced by direct contact with the compound due to increased ORP levels in the lungs, particularly in the smooth muscles of veins. However, it is important to note that vascular spasms associated with chlorine dioxide are primarily linked to inhalation or direct contact scenarios rather than oral intake. When chlorine dioxide is ingested orally, it undergoes specific metabolic processes within the gastrointestinal tract and bloodstream, which differ from the mechanisms involved in triggering vascular spasms. Therefore, the risk of vascular spasm formation is not typically associated with oral intake of chlorine dioxide due to the distinct pathways and interactions involved in systemic absorption and distribution within the body (Young, 2016). Methemoglobinemia, a condition characterized by elevated levels of methemoglobin in the blood, has been a subject of contradictory results in common publications regarding the effects of chlorine dioxide exposure. The formation of methemoglobin can inhibit the normal oxygen-carrying capacity of red blood cells, leading to tissue hypoxia. Vascular spasm effects on veins induced by chlorine dioxide may contribute to the development of methemoglobinemia, particularly in cases of inhalation or direct exposure. The danger of breathing chlorine dioxide lies in the potential for vascular spasms in lung tissue, which can impede the flow of red blood cells to the alveoli, disrupting oxygen uptake. However, oral intake of chlorine dioxide presents a different scenario, as it is absorbed as a gas without reactivity in the stomach. Upon reaching the bloodstream, chlorine dioxide dissociates with intermediate steps into sodium chloride (NaCl) and oxygen (O2), potentially playing a role in compensating for metabolic acidity within the body. This distinct pathway of absorption and dissociation highlights the differential effects of chlorine dioxide based on the route of exposure and underscores the importance of considering these mechanisms in assessing its physiological impact.

These findings prompt further research to explore the therapeutic potential and safety profile of CDS as an oxygen carrier and metabolic acidity compensator in various health conditions.

'''Conclusion'''

In conclusion, this study elucidates the contrasting effects observed between the NaClO2 + acid mixture (acidified sodium chlorite ) and pure ClO2 gas bubbled through water (CDS) on live blood samples, underscoring critical implications for physiological responses. While acidified sodium chlorite  demonstrated limited positive effects coupled with potential side effects and instability, CDS showcased significant advantages including intermediate oxygenation, improved blood flow, and safety in oral ingestion without inducing methemoglobinemia. The immediate hemostatic properties of concentrated CDS suggest a promising application in managing bleeding conditions, emphasizing its potential therapeutic utility. Moreover, the distinct pathways of absorption and dissociation of CDS, contributing to its role as an oxygen carrier and metabolic acidity compensator, highlight its favorable safety profile and therapeutic potential. These findings emphasize the necessity of considering the specific form of chlorine dioxide utilized and its consequential effects on physiological responses. Moving forward, further research investigating the diverse applications of CDS in various health conditions and metabolic processes is warranted to fully harness its benefits. This study offers valuable insights into the contrasting effects of acidified sodium chlorite  and CDS forms of chlorine dioxide on live blood samples, providing a comprehensive understanding of their potential benefits and risks, thereby paving the way for future advancements in utilizing chlorine dioxide to enhance physiological functions without adverse effects.

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