Insights into Ozone-Induced Alterations of Serum Albumin in Relapsing Remitting Multiple Sclerosis (MS) Patients and a Non-MS Individual

Insights into Ozone-Induced Alterations of Serum Albumin in Relapsing Remitting Multiple Sclerosis (MS) Patients and a Non-MS Individual | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Insights into Ozone-Induced Alterations of Serum Albumin in Relapsing Remitting Multiple Sclerosis (MS) Patients and a Non-MS Individual Zahra Hassani-Nejad Pirkouhi, Ramin Naderi Beni, Mehrnaz Hosseini Jafari, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6564545/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Multiple sclerosis (MS) is a chronic autoimmune disorder that affects the central nervous system and is characterized by neurological impairments. At present, there is no cure for MS and existing treatments merely modulate the course of the disease or alleviate symptoms. Autohemotherapy, the most common form of ozone therapy, is gaining attention for treating neurological ailments, and a number of studies suggest that it may have potential therapeutic benefits in the management of MS, given its ability to regulate the immune system responses and reduce inflammation. In the present study, the effects of different concentrations of medical ozone (40, 60, and 80 µg/ml) on human serum albumin (HSA) of four relapsing-remitting MS (RRMS) patients, the most common form of MS, and a non-MS individual was investigated. The ischemia modified albumin (IMA) levels of each participant were measured before and after ozonation using albumin-cobalt binding assay. Additionally, the HSA protein of each subject was analyzed pre- and post-ozone treatment using a set of spectroscopic techniques. Altogether, the results showed that the medical ozone concentrations used in this study led to alterations in HSA by increasing IMA levels and inducing aggregation without causing major changes in the protein’s overall secondary structure. Moreover, the extent to which ozone affected HSA varied among each individual, highlighting the importance of prior testing and using innocuous and personalized concentrations of ozone in autohemotherapy of RRMS patients. Multiple sclerosis Autohemotherapy Human serum albumin Personalized therapy Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 1. Introduction Multiple sclerosis (MS) is a prevalent autoimmune disease that affects the brain and the spinal cord (central nervous system). Its symptoms are initiated when the myelin sheath that insulates nerve cells is attacked and destructed, potentially causing permanent nerve damage to varying extents [ 1 ]. Clinical manifestations of MS are unpredictable and differ among patients depending on the location of lesions. Symptoms may include numbness, vision impairment, bladder and bowel incontinence and other physical or cognitive dysfunctions [ 2 ]. While the underlying cause of MS remains unclear, both genetic susceptibility and environmental factors can contribute. Environmental factors include vitamin deficiency, particularly vitamins D and B12, viral infections such as mononucleosis [ 3 ], smoking, diet, exposure to UV radiation and so forth [ 4 ]. Although MS is not hereditary, having a family history of the disease has been shown to increase the likelihood of developing the disease [ 5 , 6 ]. MS is known to initiate with disturbances in the blood brain barrier (BBB), alterations in the immune system, T cell infiltration into the CNS, and the production of pro-inflammatory cytokines [7]. These processes activate monocytes and macrophages by producing cytokines and contributing to the destruction of the myelin sheath. The CD4 + T-helper cells (Th), particularly Th1 and Th17, along with CD8 + T cells, migrate across the BBB. Once inside, antigen-presenting cells reactivate Th1 and Th17 cells, driving the secretion of pro-inflammatory cytokines [8]. Together, these neuroinflammatory responses exacerbate demyelination and neurodegeneration. Mast cells can also play a role by promoting demyelination, presenting myelin antigens to T cells, further disrupting the BBB [9]. Regulatory T (Tregs) cells, on the other hand, can suppress autoreactive immune responses from T and B cells [10]. After crossing the BBB, Tregs exert suppressive effects within the CNS and have been reported to exhibit regenerative properties, aiding remyelination in the CNS. Impaired maturation of Tregs increases the risk of MS, highlighting their role as potential therapeutic targets for alleviating MS progression [11,12]. MS manifests in several clinical subtypes, including relapsing-remitting MS (RRMS), primary progressive MS (PPMS), secondary progressive MS (SPMS), and progressive-relapsing MS (PRMS) [13]. RRMS, the most prevalent subtype, is marked by periods of symptom exacerbation (relapses) followed by remission. PPMS, affecting about 10% of MS patients, is characterized by a steady progression of symptoms without relapses. SPMS arises in some individuals with an initial relapsing-remitting disease course and is defined by ongoing relapses and progressive neurological decline. PRMS, the rarest form occurring in roughly 5% of cases, involves continuous disease progression with no remissions [14]. Current treatments for MS cannot completely halt the progression of neurodegeneration or reverse the damage but can only ameliorate disease severity and regulate its progression by decreasing inflammation. Autohemotherapy, regarded as the most common form of ozone therapy, has been receiving increasing attention in treating various neurological disorders, including MS [15]. This therapy involves treating patient’s blood with ozone ex- vivo and reinfusing it into the bloodstream [16,17]. In essence, ozone gas introduces mild and controlled oxidative stress, ultimately enhancing the expression of antioxidant enzymes [18–20]. Although its precise mechanism remains unclear, ozone is believed to act indirectly since it lacks cellular receptors. Upon entering the bloodstream, ozone interacts with polyunsaturated fatty acids in cell membranes, forming secondary messengers known as ozone peroxides. These peroxides activate casein kinase 2, which stimulates nuclear factor-erythroid 2-related factor 2, ultimately driving the transcription of antioxidant response elements [18]. Ozone therapy can help modulate the immune system responses, which are typically overreactive in MS [18]. Thus far, a number of studies have investigated the efficacy of ozone therapy in treating MS patients. As mentioned previously, the proinflammatory cytokines produced by Th17 cells are known to exacerbate inflammation and demyelination, contributing to the progression of MS [21]. However, one study has shown that receiving ozone therapy twice per week for 6 months could lower Th17 levels and its associated inflammatory factors in peripheral blood of MS patients [22]. In another similar report, it was demonstrated that undergoing ozone therapy decreases inflammation and elevates Treg cell responses and their associated factors in MS patients [23]. Furthermore, two separate studies have also indicated that ozone therapy alleviates disease severity in MS patients by reducing tumor necrosis factor-alpha, interleukin (IL)-33, and IL-22 cytokines which are typically elevated in MS patients [18,24]. Despite ongoing efforts towards understanding the effectiveness of ozone therapy in reducing MS symptoms, the overall effect of ozone therapy on the blood proteins of MS patients has not yet been investigated. Human serum albumin (HSA) is well-known for its antioxidant activity and is likely to be the primary target of oxidation and nitration reactions due to its high abundance in plasma, where it constitutes approximately 55–60% of the total protein content [25]. It is a globular heart-shaped protein (~ 66.5 kDa) consisting of 585 amino acid residues. Structurally, HSA has three homologous domains (I-III), each consisting of two subdomains, A and B [25,26]. As an allosteric protein, HSA can undergo conformational changes in response to ligand binding. Its structural flexibility enables it to bind a variety of ligands and maintain stability under diverse conditions [27]. HSA is recognized for its broad range of functions, including protective roles. It is crucial for maintaining blood oncotic pressure, binding and transporting atty acids, metabolites, hormones, and drugs, and regulating blood pH by binding to hydrogen and other ions [28]. Previously, the effect of different concentrations of ozone on the hemoglobin of four type II diabetic patients and a healthy individual has been investigated by our group [29,30]. Since HSA is the most abundant protein in the mammalian plasma and its oxidation by ozone is likely to cause protein conformational and structural changes, in the present study, the effect of three different concentrations of medical ozone (40, 60, and 80 µg/ml) was investigated on HSA of four RRMS patients and one non-MS individual, to gain insights into the effect of ozonation on blood HSA. 2. Methods 2.1 Sample collection and blood ozonation Blood samples from four RRMS patients and one healthy individual were obtained with informed consent under ethical approval from the University of Tehran’s Medical Sciences Department (IR.UT.SCIENCE.REC.1401.006). The demographic information including gender, age, body mass index, as well as disease duration of each participant is shows in Table 1 . Samples were collected at the Saghdoosh Wound Centre Clinic in Tehran, Iran. Each sample was divided into four portions: three were treated with ozone at concentrations of 40, 60, and 80 µg/ml for 5 minutes at a 1:1 volumetric ratio in a syringe, similar to the protocol developed by Mehraban et al. [31], while the fourth served as a non-ozonated control. Ozonated samples appeared lighter in color compared to the control (Fig. S1 ). Table 1 The demographic information of the 4 RRMS patients and the healthy individual (control). BMI: Body mass index. Samples Gender Age BMI (kg/m 2 ) Disease duration Non-MS (a) Female 25 21.5 N/A RRMS patient (b) Male 33 25.1 2 years RRMS patient (c) Female 41 23.8 5 years RRMS patient (d) Male 31 24.9 2 years RRMS patient (e) Male 37 24.4 3 years 2.2 HSA Purification Purification of HSA through salt precipitation is infrequently reported in the literature. In this study, we used a modified approach of ammonium sulfate precipitation following a protocol similar to the one developed by Cioloboc et al. [32]. The method is simple and has the advantage of obtaining highly purified HSA without the use of a column. First, the control sample and the ozonated blood samples were centrifuged at 3000 rpm for 20 min at room temperature to obtain clean serum. Serum proteins were then sequentially precipitated by stepwise addition of 35%, 45%, 55%, and 65% solid ammonium sulfate with continuous stirring followed by 15 min centrifugation at 9000 rpm after the addition of 35% and 45% ammonium sulphate and 20 min centrifugation at 12000 rpm after precipitation with 55% and 65% ammonium sulfate as reported in Table 2 . The purity of HSA was then checked using 15% SDS-PAGE (Fig S2). Table 2 Percentage of ammonium sulfate with stirring and centrifugation time for HSA purification. Ammonium sulphate (%) Stirring duration (min) Centrifugation speed (RPM) and duration (min) Temperature (°C) 35 15 15 min 9000 RPM 5 45 20 15 min 9000 RPM 5 55 20 20 min 12000 RPM 5 65 30 20 min 12000 RPM 5 2.3 Albumin-cobalt binding assay Ischemia Modified Albumin (IMA) was measured using albumin-cobalt binding assay according to the rapid colorimetric albumin cobalt binding assay developed by Bar-Or et al. [33]. In this assay, 200 \(\:\mu\:\) L of ozonated and non-ozonated serum was mixed with 50 \(\:\mu\:\) L of 0.1% cobalt chloride and incubated for 10 min to ensure adequate binding of cobalt to albumin. Following this, 50 \(\:\mu\:\) L of a 1.5 mg/ml dithiothreitol (DTT) solution was added as a colorizing reagent. After a 2-min incubation, 1000 \(\:\mu\:\) L of 0.9% NaCl was added to quench the reaction. A blank was prepared in the same manner but without the addition of DTT. Finally, absorbance was measured at 470 nm in absorbance units (ABSU). All results were expressed as the average of three measurements for each ozone concentration. 2.4 UV-Vis Absorption spectroscopy The concentration of the HSA samples were determined by measuring absorbance at 280 nm, using a molar extinction coefficient (ε) of 35,700 M⁻¹ cm⁻¹. For each sample, the average of three absorbance measurements was used. The UV-Vis absorption spectra of the HSA samples were recorded using a UV-Visible spectrophotometer (Varian, Cary 100 Bio, Australia) over a wavelength range of 205 to 305 nm. A 200 mM phosphate buffer at pH 7.4 was used as the blank. 2.5 Circular dichroism spectroscopy To examine the effect of ozonation on the secondary structure of HSA, circular dichroism (CD) measurements were performed in the far-UV region (190–260 nm) using an AVIV 215 spectropolarimeter (Aviv Associates, Lake-wood, NJ, USA) and a quartz cell with a path length of 0.1 cm. The blank used for CD measurements was 200 mM phosphate buffer at pH 7.4. The resulting data were plotted as ellipticity (in millidegrees. cm 2 dmol -1 ) against wavelength (in nanometers). 2.6 Intrinsic fluorescence measurements Intrinsic fluorescence measurements of non-ozonated and ozonated samples were done using a spectrofluorometer (Varian, Carry eclipse, Australia). The emission spectra were collected at an excitation wavelength of 295 nm ( \(\:{\lambda\:}_{ex}\) = 295 nm), corresponding to tryptophan’s (Trp) fluorescence. 2.7 Dynamic light scattering (DLS) Dynamic light scattering (DLS) was used to detect the diameter changes in HSA following ozonation. The hydrodynamic sizes were measured using a nanoparticle size analyzer DLS instrument (SZ-100-Horiba, Japan). Samples were loaded into disposable cuvettes with a 10 mm optical path. The refractive index was set to 1.59, and the absorption was set to 0.01, with the dispersion medium viscosity of 0.893 mPa·s and a refractive index of 1.33. 3. Results 3.1 Albumin cobalt binding assay IMA is a sensitive marker for ischemic heart disease and an important biochemical indicator for assessing oxidative stress levels. Under normal conditions, HSA can bind to transition metal ions including cobalt, copper, and nickel at its N terminal region (Asp-Ala-His-Lys). However, elevated oxidative stress impairs HSA’s ability to bind these metals. The colorimetric cobalt binding assay is used for measuring the IMA levels by introducing a specific amount of cobalt ions to the serum sample. These cobalt ions can only bind to unmodified HSA but not to IMA. With the addition of DTT as the coloring agent, the unbound cobalt ions react with DTT and lead to the formation of a colored complex that can be quantified spectrophotometrically. Figure 1 illustrates the IMA levels for the non-MS participant (a) and the four RRMS patients (b-e). For participant a, increasing the ozone concentration caused little to no change in overall IMA levels in the ozonated samples. In contrast, IMA levels in RRMS patient b increased noticeably in samples ozonated with 60 µg/ml ozone and rose even further with 80 µg/ml ozone, compared to the non-ozonated control. In patient c, IMA levels rose moderately when the ozone concentration reached 40 µg/ml and continued to increase with 60 µg/ml, showing the highest elevation at 80 µg/ml. For patient d, IMA levels slightly declined with 40 µg/ml ozone, then increased modestly with 60 µg/ml, and showed a marked rise at 80 µg/ml compared to the non-ozonated sample. Among the RRMS patients, patient e exhibited the least change in IMA levels, with no substantial variations observed across different ozone concentrations. Altogether, these results suggest that HSA in RRMS patients is more susceptible to ozone-induced modifications compared to non-MS individual. 3.2 UV-Vis analysis UV-Vis absorption spectroscopy in the range of 200–300 nm was employed to analyze the structural changes of ozonated and non-ozonated HSA, focusing on peak intensities at 222 nm (indicative of peptide bonds) and 278 nm (characteristic of aromatic amino acids). Figure 2 demonstrated that the UV-Vis spectra of HSA samples ozonated with 40, 60, and 80 µg/ml ozone in both the healthy individual (a) and MS patients (b–e) showed no significant deviations compared to the non-ozonated control. This indicates that the peptide bonds of the protein, corresponding to the 222 nm region, remained largely unaffected, likely due to the protective effects of endogenous blood antioxidants against ozone-induced damage. Additionally, no substantial alterations were observed in the signals corresponding to aromatic amino acids—such as tyrosine (Tyr), Trp, and phenylalanine—at 278 nm. Minor variations were detected only in patient c, where increasing ozone concentrations resulted in a reduction in peak intensity at 278 nm. Similarly, in patient d, ozone concentration of 40, 60 and 80 µg/ml led to a minor decrease in peak intensity at 278 nm. 3.3 Far-UV CD analysis CD is an absorption spectroscopy technique that measures the differential absorption of left- and right-circularly polarized light [34]. In this study Far-UV CD was used to evaluate protein destabilization and reductions in alpha-helix content. About 70% of HSA is composed of alpha helices with turns and extended loops. The CD spectrum of HSA in the far-UV region showed two negative absorption bands (minima) at 208 nm and 222 nm, and a strong maximum band at 191–193 nm characteristic of the alpha-helix structure (Fig. 3 ). The percent change in the alpha helical content of each sample were calculated using BeStSel webserver [35]. In the non-MS individual (a), the alpha-helical content of the ozonated samples remained comparable to that of the non-ozonated control sample, which is also shown by the perfectly superimposable CD spectra curves. In patient b, however, with an increase in ozone concentration to 40 µg/ml, there is a 5% decrease in the alpha-helical percentage. When ozone concentration increases to 60 and 80 µg/ml, the alpha-helical percentage decreases by 9% compared to the non-ozonated HSA. In patient c, a similar trend is observed: in the HSA sample ozonated with 40 µg/ml of ozone, there is a 4% decrease compared to the control sample. In the sample ozonated with 60 µg/ml, the alpha-helical content decreases by 11%, and in the 80 µg/ml ozonated sample, by 12%, compared to the non-ozonated HSA. In patient d, however, the alpha-helical content of HSA did not significantly change in the 40 µg/ml ozonated sample, showing only a 0.7% decrease compared to the non-ozonated HSA. The HSA sample ozonated with 60 µg/ml exhibited the lowest alpha-helical percentage, with a 7% decrease while the 80 µg/ml ozonated sample showed a 5% decrease compared to the control non-ozonated sample. In patient e, upon ozonation, as indicated by the overlapping CD spectra curves, the alpha-helical percentage changes were negligible, ranging from a 1–2% decrease in the ozonated samples compared to non-ozonated control. Furthermore, the overall shape and peak position of the CD spectra curves across all five cases remained unaltered. 3.4 Intrinsic fluorescence analysis In order to gain further insights on conformational changes of HSA due to ozonation, we analyzed the intrinsic fluorescence spectra of the samples. Intrinsic fluorescence is commonly used to investigate protein folding and conformational changes. Figure 4 depicts the intrinsic fluorescence emission spectra at \(\:{\lambda\:}_{ex}\) = 295 nm for the non-MS individual (a) and four RRMS patients (b-e). In all cases, the changes in intrinsic fluorescence intensity are minimal. For the HSA samples from the healthy individual (a) and patient b, increasing ozone concentration did not significantly alter fluorescence intensity, with only a slight decrease in the intensity observed. However, in patient b, a shift to shorter wavelengths was detected, which may indicate albumin oxidation or changes in the local environment of the tryptophan fluorophore at position 214 (Trp214), which is situated in a hydrophobic pocket within domain II. In the case of patient c, increasing ozone concentrations to 60 and 80 µg/ml, resulted in a noticeable decrease in peak intensity. In patient e, a similar trend is observed where increasing ozone concentration has led to a decreased peak intensity. In patient e, similar to the non-MS individual and patient b, the changes in peak intensity were minimal. 3.5 DLS analysis DLS was used to monitor the changes in the size and aggregation state of HSA following ozone treatment. The DLS results for the non-MS and the four RRMS patients are shown in number mode (Fig. 5 ). The average hydrodynamic diameter of the HSA monomer typically ranges from ~ 5 to ~ 7 nm [36–38]. In the healthy individual (a), the size of the sample increased from 7.5 nm to 9.4 nm when treated with 80 µg/ml ozone. For patient b, the sample size increased notably in the 60 µg/ml ozonated sample, with the sizes of the 40 and 80 µg/ml ozonated samples being similar. In patient c, the size of the HSA monomeric form increased in an ozone concentration-dependent manner. Similar to the results obtained from the albumin cobalt binding assay, CD and fluorescence spectroscopy, patient c’s HSA exhibited the largest change upon ozonation. Patient d exhibited a similar trend with the HSA size increasing from 7.8 nm in the non-ozonated sample to 11.3 nm in the 80 µg/ml ozonated sample. In patient e, the HSA sample size also increased with ozone treatment, with the 40 µg/ml ozonated sample showing similar results to the 60 µg/ml ozonated sample. Overall, the DLS changes due to ozonation were dynamic, indicating that the HSA size did not consistently increase in every sample with increasing ozone concentration. This variability suggests that DLS is more sensitive to polydispersity and protein aggregation than to small conformational alterations induced by ozone. Additionally, unlike ozonated Hb samples reported in previous studies [30,39], aggregate formation in HSA was observed to a lesser extent upon ozonation with 40, 60, and 80 µg/ml of ozone. 4. Discussion In this study, we investigated the effects of three different concentrations of medical grade ozone (40, 60 and 80 µg/ml) on HSA from four RRMS patients and one non-MS individual using different spectroscopic techniques and albumin cobalt binding assay. Due to its high abundance in the blood plasma, HSA serves as a key protein and an ideal target for evaluating the effect of ozonation on blood proteins. Therefore, we analyzed IMA levels and spectroscopic properties of both ozonated and non-ozonated HSA from all subjects to understand how ozonation affects protein structure and conformation in a personalized matter. Free radicals, especially hydroxyl radicals, lead to the formation of IMA. Consequently, IMA levels serve as a biomarker to assess whether oxidative stress induced by the specified ozone concentrations affects HSA. The N-terminal region of HSA is highly sensitive to biochemical changes caused by oxidative stress and its ability to bind metal ions is altered under high oxidative stress conditions [40]. In our study, IMA levels in both the healthy individual and RRMS patients remained within the normal range (typically below ~ 0.40 ABSU) [33] after whole blood ozonation. Having said that, IMA levels in patients b, c and d showed a slight increase with higher ozone concentrations. Additionally, RRMS patients exhibited higher IMA levels compared to the non-MS individual, which is consistent with previous reports indicating elevated IMA concentrations in RRMS patients compared to non-MS controls [41]. This increase is likely attributed to greater oxidative stress and reduced total antioxidant capacity in RRMS patients [42]. Another reaction induced by ozone is the potential alteration of the secondary and tertiary structures of soluble proteins. Ozone causes oxidation and ozonolysis of amino acids such as Trp, cysteine (Cys), and Tyr; as a result, proteins may undergo changes in folding, potentially losing their ligand-binding capacity following ozonation. It is important to note that the amide bonds of proteins are resistant to high concentrations of ozone, and the protein backbone remains intact upon ozonation[43,44]. Using UV-Vis absorption spectroscopy in the 200–300 nm range, information related to the 222 nm peak (indicative of peptide bonds) and the 278 nm peak (associated with aromatic amino acids) was analyzed for both ozonated and non-ozonated albumin samples. The UV-Vis spectra of HSA from whole blood ozonated with concentrations of 40, 60, and 80 µg/ml in RRMS patients and the healthy individual showed no significant changes compared to the non-ozonated control sample. This suggests that the peptide bonds of HSA remained largely unaffected. This stability may be due to the presence of blood antioxidants or the ineffectiveness of the applied ozone concentrations in altering peptide bonds, which is consistent with previous studies [29,30,45]. The secondary structure of HSA is primarily composed of alpha helices. The far-UV CD spectra showed that ozonation did not significantly alter the alpha helical content of HSA, with notable exceptions observed in patients b and c. The overall shape and the peak position of the spectra remained unchanged, consistent with previous where ozonation did not affect the overall secondary structure of hemoglobin compared to non-ozonated samples [29–31]. This is because only at very high concentrations, ozone reacts with the double bonds present in the backbone of the protein, leading to the formation of carbonyl groups (-C = O) and potential disruption of protein’s secondary structure [43]. Intrinsic fluorescence spectroscopy offers valuable insights into biochemical environment and changes in the tertiary structure of proteins. The intrinsic fluorescence property of proteins primarily arises from aromatic residues, particularly Trp and Tyr. In the native protein, the emission by Tyr is often quenched, while Trp fluorescence is highly sensitive to changes in the protein’s environment. HSA contains a single Trp residue (Trp214), which is very sensitive to oxidation and is excited at 295 nm. Intrinsic fluorescence spectra of HSA at \(\:{\lambda\:}_{ex}\) = 295 nm, showed no significant changes between non-ozonated and ozonated samples in the non-MS individual a and RRMS patient e. However, in patient b, there was a slight shift to shorter wavelengths in the ozonated samples. In patient c and d, increasing ozone concentration led to a decrease in intrinsic fluorescence peak intensity. This decrease may result from oxidation-induced chemical changes in tryptophan, leading to the formation of kynurenine (Kyn214), a metabolite of Trp. Such modifications can contribute to changes in the absorption and fluorescence spectra of HSA [46]. DLS was used to further examine the changes in HSA post-ozonation and to assess the size distribution of the samples. The DLS results indicated that ozonation, particularly at higher concentrations, led to the formation of HSA aggregates, as evidenced by increased diameters of the protein particles. This finding aligns with previous studies reporting that ozonation promotes protein aggregation [29–31,47] .The aggregate formation could be due to cross-linking events, such as di-Tyr cross-links, di-sulfide bonds, or other covalent modifications[48]. Furthermore, ozone-induced chemical alterations typically involve reaction with certain amino acid residues, especially aromatic and sulfur-containing residues[43], potentially resulting in protein aggregation or fragmentation, depending on the extent and location of these modifications. Ozone-induced oxidation can also disrupt the forces that stabilize protein’s native structure by reducing side-chain hydrophobicity, enhancing hydrogen bonding capacity, and altering electrostatic and Van der Waals interactions. In addition, the amino acids most vulnerable to oxidation in HSA are the reduced Cys34 and six methionine (Met) residues (Met87, Met123, Met298, Met329, Met446 and Met548) [49]. Specifically, the oxidation of Met and Cys34 residues is thought to act as a defense mechanism against reactive oxygen species, shielding the protein from structural damage. Once the aforementioned residues are fully oxidized, the chemical properties of HSA is altered and its propensity for aggregation increases [50–52]. 5. Conclusions In summary, the reported results highlight the importance of personalized ozone concentrations in the autohemotherapy of RRMS patients. While the IMA levels of all studied subjects remained within a normal range, the HSA metal binding capacity, as indicated by the albumin cobalt binding assay, was slightly altered in the ozonated samples compared to the non-ozonated samples in three of the four patients. Ozone also induced HSA aggregation in a dose-dependent manner without significantly affecting the secondary structure of the protein. Additionally, the extent to which ozone affected HSA varied among individuals. Lastly, the stability of ozonated HSA was found to be higher than that of ozonated Hb reported in previous studies. Furthermore, it is worth noting that larger clinical trials are needed to verify the safety of autohemotherapy and its effect on blood proteins. Declarations Acknowledgements Many thanks to Mr. Vahid Mirzaaghaei, the founder of Gardina Corporation and manufacturer of ozone therapy medical devices in Iran, for providing the Gardina ozone generator. Ethical Approval This study has been approved by the University of Tehran’s Medical Sciences Department; Ethical code: IR.UT.SCIENCE.REC.1401.006. Authors Contributions Z.H.P. was responsible for methodology development, sample collection, data curation, data analysis, and drafting the initial version of the manuscript. R.N.B. contributed to methodology, sample collection, and assisted with data analysis. M.H.J. and F.M. contributed to methodology development. M.F. assisted with sample collection and ozonation. A.S. was responsible for conceptualization, data analysis, validation, supervision, methodology development, acquiring funding, and review and editing. Funding The Research Council of the University of Tehran has provided financial support for conducting this research. This funding is a routine funding available to graduate students for conducting research. Competing Interests The authors declare no competing interests. Consent to Participate Informed consent was obtained from all five participants included in this study. Consent to Publish Not applicable Competing Interests The authors have no competing interests to disclose. Availability of Data and Materials The data that support the findings of this study are available on request. References Koriem KMM. Multiple sclerosis: New insights and trends. Asian Pac J Trop Biomed [Internet]. 2016;6:429–40. Available from: https://www.sciencedirect.com/science/article/pii/S2221169116302453 Gelfand JM. 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Molecular insights into the effect of ozone on human hemoglobin in autohemotherapy: Highlighting the importance of the presence of blood antioxidants during ozonation. Int J Biol Macromol. 2018;119:1276–85. Cioloboc D, Arkosi M-K, Silaghi-Dumitrescu R. A new protocol for purifying human serum albumin. Stud Ubb Chem. 2013;3:27–32. Bar-Or D, Lau E, V. Winkler J. A novel assay for cobalt-albumin binding and its potential as a marker for myocardial ischemiaI—a preliminary report. J Emerg Med. 2000;19:311–5. Daniel HAC a, Carlos HIR. The use of circular dichroism spectroscopy to study protein folding, form and function. African J Biochem Res. 2009;3:164–73. Micsonai A, Wien F, Bulyáki É, Kun J, Moussong É, Lee Y-H, et al. BeStSel: a web server for accurate protein secondary structure prediction and fold recognition from the circular dichroism spectra. Nucleic Acids Res. 2018;46:W315–22. Velichko E, Makarov S, Nepomnyashchaya E, Dong G. Molecular Aggregation in immune system activation studied by dynamic light scattering. Biology (Basel). 2020;9:123. Sun X, Ferguson HN, Hagerman AE. Conformation and aggregation of human serum albumin in the presence of green tea polyphenol (EGCg) and/or palmitic acid. Biomolecules. 2019;9:705. Zhou C, Qi W, Neil Lewis E, Carpenter JF. Concomitant Raman spectroscopy and dynamic light scattering for characterization of therapeutic proteins at high concentrations. Anal Biochem [Internet]. 2015;472:7–20. Available from: https://www.sciencedirect.com/science/article/pii/S0003269714005454 Mahlooji M, Naderi Beni R, Mehraban F, Seyedarabi A. The molecular effects of ozone on human hemoglobin oligomerisation pre- and post-COVID-19 infection accompanied by favoured antioxidant roles of cinnamaldehyde and phenyl ethyl alcohol. J Mol Struct [Internet]. 2025;1321:140131. Available from: https://www.sciencedirect.com/science/article/pii/S0022286024026401 Shevtsova A, Gordiienko I, Tkachenko V, Ushakova G. Ischemia-modified albumin: origins and clinical implications. Dis Markers. 2021;2021:1–18. Aydin O, Ellidag HY, Eren E, Kurtulus F, Yaman A, Yılmaz N. Ischemia modified albumin is an indicator of oxidative stress in multiple sclerosis. Biochem Medica. 2014;24:383–9. Hadžović-Džuvo A, Lepara O, Valjevac A, Avdagić N, Hasić S, Kiseljaković E, et al. Serum total antioxidant capacity in patients with multiple sclerosis. Bosn J Basic Med Sci. 2011;11:33. Cataldo F. On the action of ozone on proteins. Polym Degrad Stab. 2003;82:105–14. Cataldo F. Ozone Degradation of Biological Macromolecules: Proteins, Hemoglobin, RNA, and DNA. Ozone Sci Eng. 2006;28:317–28. Mehraban F, Seyedarabi A. Molecular effects of ozone on amino acids and proteins, especially human hemoglobin and albumin, and the need to personalize ozone concentration in major ozone autohemotherapy. Crit Rev Clin Lab Sci [Internet]. 2023;1–16. Available from: https://doi.org/10.1080/10408363.2023.2185765 Goswami N, Makhal A, Pal SK. Toward an alternative intrinsic probe for spectroscopic characterization of a protein. J Phys Chem B. 2010;114:15236–43. Rosenfeld MA, Leonova VB, Konstantinova ML, Razumovskii SD. Self-assembly of fibrin monomers and fibrinogen aggregation during ozone oxidation. Biochem. 2009;74:41–6. Taguchi K, Chuang VTG, Maruyama T, Otagiri M. Pharmaceutical aspects of the recombinant human serum albumin dimer: structural characteristics, biological properties, and medical applications. J Pharm Sci. 2012;101:3033–46. Roche M, Rondeau P, Singh NR, Tarnus E, Bourdon E. The antioxidant properties of serum albumin. FEBS Lett [Internet]. 2008;582:1783–7. Available from: https://doi.org/10.1016/j.febslet.2008.04.057 Levine RL, Berlett BS, Moskovitz J, Mosoni L, Stadtman ER. Methionine residues may protect proteins from critical oxidative damage. Mech Ageing Dev. 1999;107:323–32. Bourdon E, Loreau N, Lagrost L, Blache D. Differential effects of cysteine and methionine residues in the antioxidant activity of human serum albumin. Free Radic Res. 2005;39:15–20. Levine RL, Mosoni L, Berlett BS, Stadtman ER. Methionine residues as endogenous antioxidants in proteins. Proc Natl Acad Sci. 1996;93:15036–40. Supplementary Files SupplementaryABB.docx GraphicalAbstract.tiff Fig1SSDS.tiff Fig2SBloodSample.tiff 2S Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6564545","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":463994637,"identity":"ced64457-4fea-409b-824e-5e4bdf95957a","order_by":0,"name":"Zahra Hassani-Nejad Pirkouhi","email":"","orcid":"","institution":"University of Tehran Institute of Biochemistry and Biophysics","correspondingAuthor":false,"prefix":"","firstName":"Zahra","middleName":"Hassani-Nejad","lastName":"Pirkouhi","suffix":""},{"id":463994638,"identity":"db087558-d6b2-45de-a701-30a23f3878b9","order_by":1,"name":"Ramin Naderi Beni","email":"","orcid":"","institution":"University of Tehran Institute of Biochemistry and Biophysics","correspondingAuthor":false,"prefix":"","firstName":"Ramin","middleName":"Naderi","lastName":"Beni","suffix":""},{"id":463994639,"identity":"9dfeca41-976c-4b8a-82c2-a6856d47856c","order_by":2,"name":"Mehrnaz Hosseini Jafari","email":"","orcid":"","institution":"University of Tehran Institute of Biochemistry and Biophysics","correspondingAuthor":false,"prefix":"","firstName":"Mehrnaz","middleName":"Hosseini","lastName":"Jafari","suffix":""},{"id":463994640,"identity":"74a2ca61-62af-4322-9707-e2ec78a79a79","order_by":3,"name":"Fouad Mehraban","email":"","orcid":"","institution":"University of Tehran Institute of Biochemistry and Biophysics","correspondingAuthor":false,"prefix":"","firstName":"Fouad","middleName":"","lastName":"Mehraban","suffix":""},{"id":463994641,"identity":"134f92fe-5fa3-4240-abcf-2920787e03a0","order_by":4,"name":"Maryam Fahimifar","email":"","orcid":"","institution":"University of Tehran","correspondingAuthor":false,"prefix":"","firstName":"Maryam","middleName":"","lastName":"Fahimifar","suffix":""},{"id":463994642,"identity":"cc144ea1-5566-45c0-b5d6-eb33c1a6cbc2","order_by":5,"name":"Arefeh Seyedarabi","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA7ElEQVRIiWNgGAWjYPACZiDmYTjwAco9QLSWgzMYDEjUwswD1YIX8DewP3zMU2Etb95+9uBh27Y/DPztBxgPV+DRInGAx9iY50y64ZwzeQmHc9sMGCTOJDAcPIPPmgM8bNK8bYcZZzDkGIC1MNxgYDjYgEeH/AH25795/x22n8H/xuCwJVCLPCEtBgcYzJh5Gw4nzpAA2sII1GJASIvhYR5jyTnH0pNnSLxLONhzzpjH8ExiA14tcsfbH354U2NtO4M/9/CHH2VycnLHDx/+iE8LKEaYeJD4QDYjXg1gwPiDoJJRMApGwSgY0QAAi5lNN9RebCAAAAAASUVORK5CYII=","orcid":"https://orcid.org/0000-0003-4234-9799","institution":"University of Tehran Institute of Biochemistry and Biophysics","correspondingAuthor":true,"prefix":"","firstName":"Arefeh","middleName":"","lastName":"Seyedarabi","suffix":""}],"badges":[],"createdAt":"2025-04-30 11:29:43","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6564545/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6564545/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":83899281,"identity":"1a526132-f16b-432c-b2aa-da4817ac1dc0","added_by":"auto","created_at":"2025-06-04 09:14:55","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":130281,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eIMA differences between non-ozonated and ozonated samples of the non-MS individual (a) and four RRMS patients (b-e). \u003c/strong\u003eStatistical significance was assessed using Student’s t-test. P values are denoted by *, **, and ***, corresponding to significance levels of \u0026lt;0.05, \u0026lt;0.01, and \u0026lt;0.001 respectively.\u003c/p\u003e","description":"","filename":"image2.png","url":"https://assets-eu.researchsquare.com/files/rs-6564545/v1/58ff90b806e810d812a56a9f.png"},{"id":83897979,"identity":"d5fbc647-3f49-4aa3-9ef0-4fe65becb17c","added_by":"auto","created_at":"2025-06-04 09:06:55","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":198906,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eUV-VIS absorbance spectra of HSA ozonated at different concentrations for the healthy individual (a) and the four RRMS patients (b-e).\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"image3.png","url":"https://assets-eu.researchsquare.com/files/rs-6564545/v1/6af0da911c4806d32e431e48.png"},{"id":83897986,"identity":"977bbc65-77ec-4c8b-b45a-159fba3161a5","added_by":"auto","created_at":"2025-06-04 09:06:55","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":207769,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eNon-ozonated and ozonated HSA CD spectral changes of the healthy individual (a) and the four RRMS patients (b-e) in the far-UV region.\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"image4.png","url":"https://assets-eu.researchsquare.com/files/rs-6564545/v1/fbaae873963ca15fa2f6b340.png"},{"id":83899283,"identity":"eca1ceb9-a1ec-47c1-8096-c9d914b8bbb5","added_by":"auto","created_at":"2025-06-04 09:14:55","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":199376,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe intrinsic fluorescence spectra of the healthy individual (a) and the four RRMS patients (b-e). The samples were excited at λ\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003eex \u003c/strong\u003e\u003c/sub\u003e\u003cstrong\u003e= 295 nm and fluorescence was recorded over 320-420 nm.\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"image5.png","url":"https://assets-eu.researchsquare.com/files/rs-6564545/v1/4bdab63c28b719421f3a37bc.png"},{"id":83897981,"identity":"f300c3a4-fc5a-4af9-bb43-eabaa9391f26","added_by":"auto","created_at":"2025-06-04 09:06:55","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":136581,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eDLS profiles of non-ozonated and ozonated HSA samples for the non-MS individual (a) and the four RRMS patients (b-e). The non-ozonated sample is shown in black, 40 µg/ml ozonated samples in gray, 60 µg/ml samples in blue, and 80 µg/ml samples are in red. The peak diameter values are shown in number mode in each graph.\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"image6.png","url":"https://assets-eu.researchsquare.com/files/rs-6564545/v1/3d0322fcdfe0828bbd3ec141.png"},{"id":83901212,"identity":"cf29428d-dcaf-4043-8686-ea6f7dcad540","added_by":"auto","created_at":"2025-06-04 09:30:58","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1816743,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6564545/v1/e50f945c-82ad-4171-b392-ab5727f9f339.pdf"},{"id":83897980,"identity":"8476b8c2-89a3-4c37-988e-8adfd6127e04","added_by":"auto","created_at":"2025-06-04 09:06:55","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":1209865,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryABB.docx","url":"https://assets-eu.researchsquare.com/files/rs-6564545/v1/e17088134cbd8c2d22a0fb18.docx"},{"id":83897987,"identity":"3e64ee39-9581-4d06-a263-2f5acccf9bc3","added_by":"auto","created_at":"2025-06-04 09:06:55","extension":"tiff","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":4835254,"visible":true,"origin":"","legend":"","description":"","filename":"GraphicalAbstract.tiff","url":"https://assets-eu.researchsquare.com/files/rs-6564545/v1/884559894f91579c797ffcb3.tiff"},{"id":83899282,"identity":"c6494859-95fe-48d1-ab0d-f1adc48c6fb8","added_by":"auto","created_at":"2025-06-04 09:14:55","extension":"tiff","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":1020324,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cbr\u003e\u003c/p\u003e","description":"","filename":"Fig1SSDS.tiff","url":"https://assets-eu.researchsquare.com/files/rs-6564545/v1/42b663714b8d0fcfa9d8b9b8.tiff"},{"id":83897988,"identity":"52dcae2c-081e-4831-b451-305627282aa1","added_by":"auto","created_at":"2025-06-04 09:06:55","extension":"tiff","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":748340,"visible":true,"origin":"","legend":"\u003cp\u003e2S\u003c/p\u003e","description":"","filename":"Fig2SBloodSample.tiff","url":"https://assets-eu.researchsquare.com/files/rs-6564545/v1/5f1b39446306c5b4131a7cd6.tiff"}],"financialInterests":"","formattedTitle":"Insights into Ozone-Induced Alterations of Serum Albumin in Relapsing Remitting Multiple Sclerosis (MS) Patients and a Non-MS Individual","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eMultiple sclerosis (MS) is a prevalent autoimmune disease that affects the brain and the spinal cord (central nervous system). Its symptoms are initiated when the myelin sheath that insulates nerve cells is attacked and destructed, potentially causing permanent nerve damage to varying extents [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Clinical manifestations of MS are unpredictable and differ among patients depending on the location of lesions. Symptoms may include numbness, vision impairment, bladder and bowel incontinence and other physical or cognitive dysfunctions [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. While the underlying cause of MS remains unclear, both genetic susceptibility and environmental factors can contribute. Environmental factors include vitamin deficiency, particularly vitamins D and B12, viral infections such as mononucleosis [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e], smoking, diet, exposure to UV radiation and so forth [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. Although MS is not hereditary, having a family history of the disease has been shown to increase the likelihood of developing the disease [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eMS is known to initiate with disturbances in the blood brain barrier (BBB), alterations in the immune system, T cell infiltration into the CNS, and the production of pro-inflammatory cytokines [7]. These processes activate monocytes and macrophages by producing cytokines and contributing to the destruction of the myelin sheath. The CD4\u003csup\u003e+\u003c/sup\u003e T-helper cells (Th), particularly Th1 and Th17, along with CD8\u003csup\u003e+\u003c/sup\u003e T cells, migrate across the BBB. Once inside, antigen-presenting cells reactivate Th1 and Th17 cells, driving the secretion of pro-inflammatory cytokines [8]. Together, these neuroinflammatory responses exacerbate demyelination and neurodegeneration.\u003c/p\u003e \u003cp\u003eMast cells can also play a role by promoting demyelination, presenting myelin antigens to T cells, further disrupting the BBB [9]. Regulatory T (Tregs) cells, on the other hand, can suppress autoreactive immune responses from T and B cells [10]. After crossing the BBB, Tregs exert suppressive effects within the CNS and have been reported to exhibit regenerative properties, aiding remyelination in the CNS. Impaired maturation of Tregs increases the risk of MS, highlighting their role as potential therapeutic targets for alleviating MS progression [11,12].\u003c/p\u003e \u003cp\u003eMS manifests in several clinical subtypes, including relapsing-remitting MS (RRMS), primary progressive MS (PPMS), secondary progressive MS (SPMS), and progressive-relapsing MS (PRMS) [13]. RRMS, the most prevalent subtype, is marked by periods of symptom exacerbation (relapses) followed by remission. PPMS, affecting about 10% of MS patients, is characterized by a steady progression of symptoms without relapses. SPMS arises in some individuals with an initial relapsing-remitting disease course and is defined by ongoing relapses and progressive neurological decline. PRMS, the rarest form occurring in roughly 5% of cases, involves continuous disease progression with no remissions [14].\u003c/p\u003e \u003cp\u003eCurrent treatments for MS cannot completely halt the progression of neurodegeneration or reverse the damage but can only ameliorate disease severity and regulate its progression by decreasing inflammation. Autohemotherapy, regarded as the most common form of ozone therapy, has been receiving increasing attention in treating various neurological disorders, including MS [15]. This therapy involves treating patient\u0026rsquo;s blood with ozone ex-\u003cem\u003evivo\u003c/em\u003e and reinfusing it into the bloodstream [16,17]. In essence, ozone gas introduces mild and controlled oxidative stress, ultimately enhancing the expression of antioxidant enzymes [18\u0026ndash;20]. Although its precise mechanism remains\u003c/p\u003e \u003cp\u003eunclear, ozone is believed to act indirectly since it lacks cellular receptors. Upon entering the bloodstream, ozone interacts with polyunsaturated fatty acids in cell membranes, forming secondary messengers known as ozone peroxides. These peroxides activate casein kinase 2, which stimulates nuclear factor-erythroid 2-related factor 2, ultimately driving the transcription of antioxidant response elements [18].\u003c/p\u003e \u003cp\u003eOzone therapy can help modulate the immune system responses, which are typically overreactive in MS [18]. Thus far, a number of studies have investigated the efficacy of ozone therapy in treating MS patients. As mentioned previously, the proinflammatory cytokines produced by Th17 cells are known to exacerbate inflammation and demyelination, contributing to the progression of MS [21]. However, one study has shown that receiving ozone therapy twice per week for 6 months could lower Th17 levels and its associated inflammatory factors in peripheral blood of MS patients [22]. In another similar report, it was demonstrated that undergoing ozone therapy decreases inflammation and elevates Treg cell responses and their associated factors in MS patients [23]. Furthermore, two separate studies have also indicated that ozone therapy alleviates disease severity in MS patients by reducing tumor necrosis factor-alpha, interleukin (IL)-33, and IL-22 cytokines which are typically elevated in MS patients [18,24].\u003c/p\u003e \u003cp\u003eDespite ongoing efforts towards understanding the effectiveness of ozone therapy in reducing MS symptoms, the overall effect of ozone therapy on the blood proteins of MS patients has not yet been investigated. Human serum albumin (HSA) is well-known for its antioxidant activity and is likely to be the primary target of oxidation and nitration reactions due to its high abundance in plasma, where it constitutes approximately 55\u0026ndash;60% of the total protein content [25]. It is a globular heart-shaped protein (~\u0026thinsp;66.5 kDa) consisting of 585 amino acid residues. Structurally, HSA has three homologous domains (I-III), each consisting of two subdomains, A and B [25,26]. As an allosteric protein, HSA can undergo conformational changes in response to ligand binding. Its structural flexibility enables it to bind a variety of ligands and maintain stability under diverse conditions [27]. HSA is recognized for its broad range of functions, including protective roles. It is crucial for maintaining blood oncotic pressure, binding and transporting atty acids, metabolites, hormones, and drugs, and regulating blood pH by binding to hydrogen and other ions [28].\u003c/p\u003e \u003cp\u003ePreviously, the effect of different concentrations of ozone on the hemoglobin of four type II diabetic patients and a healthy individual has been investigated by our group [29,30]. Since HSA is the most abundant protein in the mammalian plasma and its oxidation by ozone is likely to cause protein conformational and structural changes, in the present study, the effect of three different concentrations of medical ozone (40, 60, and 80 \u0026micro;g/ml) was investigated on HSA of four RRMS patients and one non-MS individual, to gain insights into the effect of ozonation on blood HSA.\u003c/p\u003e"},{"header":"2. Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1 Sample collection and blood ozonation\u003c/h2\u003e \u003cp\u003eBlood samples from four RRMS patients and one healthy individual were obtained with informed consent under ethical approval from the University of Tehran\u0026rsquo;s Medical Sciences Department (IR.UT.SCIENCE.REC.1401.006). The demographic information including gender, age, body mass index, as well as disease duration of each participant is shows in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. Samples were collected at the Saghdoosh Wound Centre Clinic in Tehran, Iran. Each sample was divided into four portions: three were treated with ozone at concentrations of 40, 60, and 80 \u0026micro;g/ml for 5 minutes at a 1:1 volumetric ratio in a syringe, similar to the protocol developed by Mehraban et al. [31], while the fourth served as a non-ozonated control. Ozonated samples appeared lighter in color compared to the control (Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003e\u003cb\u003eThe demographic information of the 4 RRMS patients and the healthy individual (control).\u003c/b\u003e BMI: Body mass index.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"5\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSamples\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eGender\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eAge\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eBMI (kg/m\u003csup\u003e2\u003c/sup\u003e)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eDisease duration\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNon-MS (a)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eFemale\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e25\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e21.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eN/A\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eRRMS patient (b)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMale\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e33\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e25.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e2 years\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eRRMS patient (c)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eFemale\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e41\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e23.8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e5 years\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eRRMS patient (d)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMale\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e31\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e24.9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e2 years\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eRRMS patient (e)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMale\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e37\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e24.4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e3 years\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2 HSA Purification\u003c/h2\u003e \u003cp\u003ePurification of HSA through salt precipitation is infrequently reported in the literature. In this study, we used a modified approach of ammonium sulfate precipitation following a protocol similar to the one developed by Cioloboc et al. [32]. The method is simple and has the advantage of obtaining highly purified HSA without the use of a column. First, the control sample and the ozonated blood samples were centrifuged at 3000 rpm for 20 min at room temperature to obtain clean serum. Serum proteins were then sequentially precipitated by stepwise addition of 35%, 45%, 55%, and 65% solid ammonium sulfate with continuous stirring followed by 15 min centrifugation at 9000 rpm after the addition of 35% and 45% ammonium sulphate and 20 min centrifugation at 12000 rpm after precipitation with 55% and 65% ammonium sulfate as reported in Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e. The purity of HSA was then checked using 15% SDS-PAGE (Fig S2).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003ePercentage of ammonium sulfate with stirring and centrifugation time for HSA purification.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"4\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAmmonium sulphate (%)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eStirring duration (min)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eCentrifugation speed (RPM) and duration (min)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eTemperature (\u0026deg;C)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e35\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e15\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e15 min 9000 RPM\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e45\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e20\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e15 min 9000 RPM\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e55\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e20\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e20 min 12000 RPM\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e65\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e30\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e20 min 12000 RPM\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3 Albumin-cobalt binding assay\u003c/h2\u003e \u003cp\u003eIschemia Modified Albumin (IMA) was measured using albumin-cobalt binding assay according to the rapid colorimetric albumin cobalt binding assay developed by Bar-Or et al. [33]. In this assay, 200 \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\mu\\:\\)\u003c/span\u003e\u003c/span\u003eL of ozonated and non-ozonated serum was mixed with 50 \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\mu\\:\\)\u003c/span\u003e\u003c/span\u003eL of 0.1% cobalt chloride and incubated for 10 min to ensure adequate binding of cobalt to albumin. Following this, 50 \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\mu\\:\\)\u003c/span\u003e\u003c/span\u003eL of a 1.5 mg/ml dithiothreitol (DTT) solution was added as a colorizing reagent. After a 2-min incubation, 1000 \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\mu\\:\\)\u003c/span\u003e\u003c/span\u003eL of 0.9% NaCl was added to quench the reaction. A blank was prepared in the same manner but without the addition of DTT. Finally, absorbance was measured at 470 nm in absorbance units (ABSU). All results were expressed as the average of three measurements for each ozone concentration.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4 UV-Vis Absorption spectroscopy\u003c/h2\u003e \u003cp\u003eThe concentration of the HSA samples were determined by measuring absorbance at 280 nm, using a molar extinction coefficient (ε) of 35,700 M⁻\u0026sup1; cm⁻\u0026sup1;. For each sample, the average of three absorbance measurements was used. The UV-Vis absorption spectra of the HSA samples were recorded using a UV-Visible spectrophotometer (Varian, Cary 100 Bio, Australia) over a wavelength range of 205 to 305 nm. A 200 mM phosphate buffer at pH 7.4 was used as the blank.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.5 Circular dichroism spectroscopy\u003c/h2\u003e \u003cp\u003eTo examine the effect of ozonation on the secondary structure of HSA, circular dichroism (CD) measurements were performed in the far-UV region (190\u0026ndash;260 nm) using an AVIV 215 spectropolarimeter (Aviv Associates, Lake-wood, NJ, USA) and a quartz cell with a path length of 0.1 cm. The blank used for CD measurements was 200 mM phosphate buffer at pH 7.4. The resulting data were plotted as ellipticity (in millidegrees. cm\u003csup\u003e2\u003c/sup\u003e dmol\u003csup\u003e-1\u003c/sup\u003e ) against wavelength (in nanometers).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e2.6 Intrinsic fluorescence measurements\u003c/h2\u003e \u003cp\u003eIntrinsic fluorescence measurements of non-ozonated and ozonated samples were done using a spectrofluorometer (Varian, Carry eclipse, Australia). The emission spectra were collected at an excitation wavelength of 295 nm (\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:{\\lambda\\:}_{ex}\\)\u003c/span\u003e\u003c/span\u003e= 295 nm), corresponding to tryptophan\u0026rsquo;s (Trp) fluorescence.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e2.7 Dynamic light scattering (DLS)\u003c/h2\u003e \u003cp\u003eDynamic light scattering (DLS) was used to detect the diameter changes in HSA following ozonation. The hydrodynamic sizes were measured using a nanoparticle size analyzer DLS instrument (SZ-100-Horiba, Japan). Samples were loaded into disposable cuvettes with a 10 mm optical path. The refractive index was set to 1.59, and the absorption was set to 0.01, with the dispersion medium viscosity of 0.893 mPa\u0026middot;s and a refractive index of 1.33.\u003c/p\u003e \u003c/div\u003e"},{"header":"3. Results","content":"\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003e3.1 Albumin cobalt binding assay\u003c/h2\u003e \u003cp\u003eIMA is a sensitive marker for ischemic heart disease and an important biochemical indicator for assessing oxidative stress levels. Under normal conditions, HSA can bind to transition metal ions including cobalt, copper, and nickel at its N terminal region (Asp-Ala-His-Lys). However, elevated oxidative stress impairs HSA\u0026rsquo;s ability to bind these metals. The colorimetric cobalt binding assay is used for measuring the IMA levels by introducing a specific amount of cobalt ions to the serum sample. These cobalt ions can only bind to unmodified HSA but not to IMA. With the addition of DTT as the coloring agent, the unbound cobalt ions react with DTT and lead to the formation of a colored complex that can be quantified spectrophotometrically.\u003c/p\u003e \u003cp\u003eFigure\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e illustrates the IMA levels for the non-MS participant (a) and the four RRMS patients (b-e). For participant a, increasing the ozone concentration caused little to no change in overall IMA levels in the ozonated samples. In contrast, IMA levels in RRMS patient b increased noticeably in samples ozonated with 60 \u0026micro;g/ml ozone and rose even further with 80 \u0026micro;g/ml ozone, compared to the non-ozonated control. In patient c, IMA levels rose moderately when the ozone concentration reached 40 \u0026micro;g/ml and continued to increase with 60 \u0026micro;g/ml, showing the highest elevation at 80 \u0026micro;g/ml. For patient d, IMA levels slightly declined with 40 \u0026micro;g/ml ozone, then increased modestly with 60 \u0026micro;g/ml, and showed a marked rise at 80 \u0026micro;g/ml compared to the non-ozonated sample. Among the RRMS patients, patient e exhibited the least change in IMA levels, with no substantial variations observed across different ozone concentrations. Altogether, these results suggest that HSA in RRMS patients is more susceptible to ozone-induced modifications compared to non-MS individual.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003e3.2 UV-Vis analysis\u003c/h2\u003e \u003cp\u003eUV-Vis absorption spectroscopy in the range of 200\u0026ndash;300 nm was employed to analyze the structural changes of ozonated and non-ozonated HSA, focusing on peak intensities at 222 nm (indicative of peptide bonds) and 278 nm (characteristic of aromatic amino acids). Figure\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e demonstrated that the UV-Vis spectra of HSA samples ozonated with 40, 60, and 80 \u0026micro;g/ml ozone in both the healthy individual (a) and MS patients (b\u0026ndash;e) showed no significant deviations compared to the non-ozonated control. This indicates that the peptide bonds of the protein, corresponding to the 222 nm region, remained largely unaffected, likely due to the protective effects of endogenous blood antioxidants against ozone-induced damage. Additionally, no substantial alterations were observed in the signals corresponding to aromatic amino acids\u0026mdash;such as tyrosine (Tyr), Trp, and phenylalanine\u0026mdash;at 278 nm. Minor variations were detected only in patient c, where increasing ozone concentrations resulted in a reduction in peak intensity at 278 nm. Similarly, in patient d, ozone concentration of 40, 60 and 80 \u0026micro;g/ml led to a minor decrease in peak intensity at 278 nm.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003e3.3 Far-UV CD analysis\u003c/h2\u003e \u003cp\u003eCD is an absorption spectroscopy technique that measures the differential absorption of left- and right-circularly polarized light [34]. In this study Far-UV CD was used to evaluate protein destabilization and reductions in alpha-helix content. About 70% of HSA is composed of alpha helices with turns and extended loops. The CD spectrum of HSA in the far-UV region showed two negative absorption bands (minima) at 208 nm and 222 nm, and a strong maximum band at 191\u0026ndash;193 nm characteristic of the alpha-helix structure (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). The percent change in the alpha helical content of each sample were calculated using BeStSel webserver [35]. In the non-MS individual (a), the alpha-helical content of the ozonated samples remained comparable to that of the non-ozonated control sample, which is also shown by the perfectly superimposable CD spectra curves. In patient b, however, with an increase in ozone concentration to 40 \u0026micro;g/ml, there is a 5% decrease in the alpha-helical percentage. When ozone concentration increases to 60 and 80 \u0026micro;g/ml, the alpha-helical percentage decreases by 9% compared to the non-ozonated HSA. In patient c, a similar trend is observed: in the HSA sample ozonated with 40 \u0026micro;g/ml of ozone, there is a 4% decrease compared to the control sample. In the sample ozonated with 60 \u0026micro;g/ml, the alpha-helical content decreases by 11%, and in the 80 \u0026micro;g/ml ozonated sample, by 12%, compared to the non-ozonated HSA. In patient d, however, the alpha-helical content of HSA did not significantly change in the 40 \u0026micro;g/ml ozonated sample, showing only a 0.7% decrease compared to the non-ozonated HSA. The HSA sample ozonated with 60 \u0026micro;g/ml exhibited the lowest alpha-helical percentage, with a 7% decrease while the 80 \u0026micro;g/ml ozonated sample showed a 5% decrease compared to the control non-ozonated sample. In patient e, upon ozonation, as indicated by the overlapping CD spectra curves, the alpha-helical percentage changes were negligible, ranging from a 1\u0026ndash;2% decrease in the ozonated samples compared to non-ozonated control. Furthermore, the overall shape and peak position of the CD spectra curves across all five cases remained unaltered.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003e3.4 Intrinsic fluorescence analysis\u003c/h2\u003e \u003cp\u003eIn order to gain further insights on conformational changes of HSA due to ozonation, we analyzed the intrinsic fluorescence spectra of the samples. Intrinsic fluorescence is commonly used to investigate protein folding and conformational changes. Figure\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e depicts the intrinsic fluorescence emission spectra at \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:{\\lambda\\:}_{ex}\\)\u003c/span\u003e\u003c/span\u003e= 295 nm for the non-MS individual (a) and four RRMS patients (b-e). In all cases, the changes in intrinsic fluorescence intensity are minimal. For the HSA samples from the healthy individual (a) and patient b, increasing ozone concentration did not significantly alter fluorescence intensity, with only a slight decrease in the intensity observed. However, in patient b, a shift to shorter wavelengths was detected, which may indicate albumin oxidation or changes in the local environment of the tryptophan fluorophore at position 214 (Trp214), which is situated in a hydrophobic pocket within domain II. In the case of patient c, increasing ozone concentrations to 60 and 80 \u0026micro;g/ml, resulted in a noticeable decrease in peak intensity. In patient e, a similar trend is observed where increasing ozone concentration has led to a decreased peak intensity. In patient e, similar to the non-MS individual and patient b, the changes in peak intensity were minimal.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003e3.5 DLS analysis\u003c/h2\u003e \u003cp\u003eDLS was used to monitor the changes in the size and aggregation state of HSA following ozone treatment. The DLS results for the non-MS and the four RRMS patients are shown in number mode (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). The average hydrodynamic diameter of the HSA monomer typically ranges from ~\u0026thinsp;5 to ~\u0026thinsp;7 nm [36\u0026ndash;38]. In the healthy individual (a), the size of the sample increased from 7.5 nm to 9.4 nm when treated with 80 \u0026micro;g/ml ozone. For patient b, the sample size increased notably in the 60 \u0026micro;g/ml ozonated sample, with the sizes of the 40 and 80 \u0026micro;g/ml ozonated samples being similar. In patient c, the size of the HSA monomeric form increased in an ozone concentration-dependent manner. Similar to the results obtained from the albumin cobalt binding assay, CD and fluorescence spectroscopy, patient c\u0026rsquo;s HSA exhibited the largest change upon ozonation. Patient d exhibited a similar trend with the HSA size increasing from 7.8 nm in the non-ozonated sample to 11.3 nm in the 80 \u0026micro;g/ml ozonated sample. In patient e, the HSA sample size also increased with ozone treatment, with the 40 \u0026micro;g/ml ozonated sample showing similar results to the 60 \u0026micro;g/ml ozonated sample.\u003c/p\u003e \u003cp\u003eOverall, the DLS changes due to ozonation were dynamic, indicating that the HSA size did not consistently increase in every sample with increasing ozone concentration. This variability suggests that DLS is more sensitive to polydispersity and protein aggregation than to small conformational alterations induced by ozone. Additionally, unlike ozonated Hb samples reported in previous studies [30,39], aggregate formation in HSA was observed to a lesser extent upon ozonation with 40, 60, and 80 \u0026micro;g/ml of ozone.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eIn this study, we investigated the effects of three different concentrations of medical grade ozone (40, 60 and 80 \u0026micro;g/ml) on HSA from four RRMS patients and one non-MS individual using different spectroscopic techniques and albumin cobalt binding assay. Due to its high abundance in the blood plasma, HSA serves as a key protein and an ideal target for evaluating the effect of ozonation on blood proteins. Therefore, we analyzed IMA levels and spectroscopic properties of both ozonated and non-ozonated HSA from all subjects to understand how ozonation affects protein structure and conformation in a personalized matter.\u003c/p\u003e \u003cp\u003eFree radicals, especially hydroxyl radicals, lead to the formation of IMA. Consequently, IMA levels serve as a biomarker to assess whether oxidative stress induced by the specified ozone concentrations affects HSA. The N-terminal region of HSA is highly sensitive to biochemical changes caused by oxidative stress and its ability to bind metal ions is altered under high oxidative stress conditions [40]. In our study, IMA levels in both the healthy individual and RRMS patients remained within the normal range (typically below ~\u0026thinsp;0.40 ABSU) [33] after whole blood ozonation. Having said that, IMA levels in patients b, c and d showed a slight increase with higher ozone concentrations. Additionally, RRMS patients exhibited higher IMA levels compared to the non-MS individual, which is consistent with previous reports indicating elevated IMA concentrations in RRMS patients compared to non-MS controls [41]. This increase is likely attributed to greater oxidative stress and reduced total antioxidant capacity in RRMS patients [42].\u003c/p\u003e \u003cp\u003eAnother reaction induced by ozone is the potential alteration of the secondary and tertiary structures of soluble proteins. Ozone causes oxidation and ozonolysis of amino acids such as Trp, cysteine (Cys), and Tyr; as a result, proteins may undergo changes in folding, potentially losing their ligand-binding capacity following ozonation. It is important to note that the amide bonds of proteins are resistant to high concentrations of ozone, and the protein backbone remains intact upon ozonation[43,44]. Using UV-Vis absorption spectroscopy in the 200\u0026ndash;300 nm range, information related to the 222 nm peak (indicative of peptide bonds) and the 278 nm peak (associated with aromatic amino acids) was analyzed for both ozonated and non-ozonated albumin samples. The UV-Vis spectra of HSA from whole blood ozonated with concentrations of 40, 60, and 80 \u0026micro;g/ml in RRMS patients and the healthy individual showed no significant changes compared to the non-ozonated control sample. This suggests that the peptide bonds of HSA remained largely unaffected. This stability may be due to the presence of blood antioxidants or the ineffectiveness of the applied ozone concentrations in altering peptide bonds, which is consistent with previous studies [29,30,45].\u003c/p\u003e \u003cp\u003eThe secondary structure of HSA is primarily composed of alpha helices. The far-UV CD spectra showed that ozonation did not significantly alter the alpha helical content of HSA, with notable exceptions observed in patients b and c. The overall shape and the peak position of the spectra remained unchanged, consistent with previous where ozonation did not affect the overall secondary structure of hemoglobin compared to non-ozonated samples [29\u0026ndash;31]. This is because only at very high concentrations, ozone reacts with the double bonds present in the backbone of the protein, leading to the formation of carbonyl groups (-C\u0026thinsp;=\u0026thinsp;O) and potential disruption of protein\u0026rsquo;s secondary structure [43].\u003c/p\u003e \u003cp\u003eIntrinsic fluorescence spectroscopy offers valuable insights into biochemical environment and changes in the tertiary structure of proteins. The intrinsic fluorescence property of proteins primarily arises from aromatic residues, particularly Trp and Tyr. In the native protein, the emission by Tyr is often quenched, while Trp fluorescence is highly sensitive to changes in the protein\u0026rsquo;s environment. HSA contains a single Trp residue (Trp214), which is very sensitive to oxidation and is excited at 295 nm. Intrinsic fluorescence spectra of HSA at \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:{\\lambda\\:}_{ex}\\)\u003c/span\u003e\u003c/span\u003e= 295 nm, showed no significant changes between non-ozonated and ozonated samples in the non-MS individual a and RRMS patient e. However, in patient b, there was a slight shift to shorter wavelengths in the ozonated samples. In patient c and d, increasing ozone concentration led to a decrease in intrinsic fluorescence peak intensity. This decrease may result from oxidation-induced chemical changes in tryptophan, leading to the formation of kynurenine (Kyn214), a metabolite of Trp. Such modifications can contribute to changes in the absorption and fluorescence spectra of HSA [46].\u003c/p\u003e \u003cp\u003eDLS was used to further examine the changes in HSA post-ozonation and to assess the size distribution of the samples. The DLS results indicated that ozonation, particularly at higher concentrations, led to the formation of HSA aggregates, as evidenced by increased diameters of the protein particles. This finding aligns with previous studies reporting that ozonation promotes protein aggregation [29\u0026ndash;31,47] .The aggregate formation could be due to cross-linking events, such as di-Tyr cross-links, di-sulfide bonds, or other covalent modifications[48]. Furthermore, ozone-induced chemical alterations typically involve reaction with certain amino acid residues, especially aromatic and sulfur-containing residues[43], potentially resulting in protein aggregation or fragmentation, depending on the extent and location of these modifications. Ozone-induced oxidation can also disrupt the forces that stabilize protein\u0026rsquo;s native structure by reducing side-chain hydrophobicity, enhancing hydrogen bonding capacity, and altering electrostatic and Van der Waals interactions. In addition, the amino acids most vulnerable to oxidation in HSA are the reduced Cys34 and six methionine (Met) residues (Met87, Met123, Met298, Met329, Met446 and Met548) [49]. Specifically, the oxidation of Met and Cys34 residues is thought to act as a defense mechanism against reactive oxygen species, shielding the protein from structural damage. Once the aforementioned residues are fully oxidized, the chemical properties of HSA is altered and its propensity for aggregation increases [50\u0026ndash;52].\u003c/p\u003e"},{"header":"5. Conclusions","content":"\u003cp\u003eIn summary, the reported results highlight the importance of personalized ozone concentrations in the autohemotherapy of RRMS patients. While the IMA levels of all studied subjects remained within a normal range, the HSA metal binding capacity, as indicated by the albumin cobalt binding assay, was slightly altered in the ozonated samples compared to the non-ozonated samples in three of the four patients. Ozone also induced HSA aggregation in a dose-dependent manner without significantly affecting the secondary structure of the protein. Additionally, the extent to which ozone affected HSA varied among individuals. Lastly, the stability of ozonated HSA was found to be higher than that of ozonated Hb reported in previous studies. Furthermore, it is worth noting that larger clinical trials are needed to verify the safety of autohemotherapy and its effect on blood proteins.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMany thanks to Mr. Vahid Mirzaaghaei, the founder of Gardina Corporation and manufacturer of ozone therapy medical devices in Iran, for providing the Gardina ozone generator.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthical Approval\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study has been approved by the University of Tehran\u0026rsquo;s Medical Sciences Department; Ethical code: IR.UT.SCIENCE.REC.1401.006.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors Contributions\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eZ.H.P. was responsible for methodology development, sample collection, data curation, data analysis, and drafting the initial version of the manuscript. R.N.B. contributed to methodology, sample collection, and assisted with data analysis. M.H.J. and F.M. contributed to methodology development. M.F. assisted with sample collection and ozonation. A.S. was responsible for conceptualization, data analysis, validation, supervision, methodology development, acquiring funding, and review and editing.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe Research Council of the University of Tehran has provided financial support for conducting this research. This funding is a routine funding available to graduate students for conducting research.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting Interests\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interests.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent to Participate\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eInformed consent was obtained from all five participants included in this study.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent to Publish\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting Interests\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors have no competing interests to disclose.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of Data and Materials\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe data that support the findings of this study are available on request.\u0026nbsp;\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eKoriem KMM. 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J Pharm Sci. 2012;101:3033\u0026ndash;46. \u003c/li\u003e\n\u003cli\u003eRoche M, Rondeau P, Singh NR, Tarnus E, Bourdon E. The antioxidant properties of serum albumin. FEBS Lett [Internet]. 2008;582:1783\u0026ndash;7. Available from: https://doi.org/10.1016/j.febslet.2008.04.057\u003c/li\u003e\n\u003cli\u003eLevine RL, Berlett BS, Moskovitz J, Mosoni L, Stadtman ER. Methionine residues may protect proteins from critical oxidative damage. Mech Ageing Dev. 1999;107:323\u0026ndash;32. \u003c/li\u003e\n\u003cli\u003eBourdon E, Loreau N, Lagrost L, Blache D. Differential effects of cysteine and methionine residues in the antioxidant activity of human serum albumin. Free Radic Res. 2005;39:15\u0026ndash;20. \u003c/li\u003e\n\u003cli\u003eLevine RL, Mosoni L, Berlett BS, Stadtman ER. Methionine residues as endogenous antioxidants in proteins. Proc Natl Acad Sci. 1996;93:15036\u0026ndash;40. \u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Multiple sclerosis, Autohemotherapy, Human serum albumin, Personalized therapy","lastPublishedDoi":"10.21203/rs.3.rs-6564545/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6564545/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"Multiple sclerosis (MS) is a chronic autoimmune disorder that affects the central nervous system and is characterized by neurological impairments. At present, there is no cure for MS and existing treatments merely modulate the course of the disease or alleviate symptoms. Autohemotherapy, the most common form of ozone therapy, is gaining attention for treating neurological ailments, and a number of studies suggest that it may have potential therapeutic benefits in the management of MS, given its ability to regulate the immune system responses and reduce inflammation. In the present study, the effects of different concentrations of medical ozone (40, 60, and 80 µg/ml) on human serum albumin (HSA) of four relapsing-remitting MS (RRMS) patients, the most common form of MS, and a non-MS individual was investigated. The ischemia modified albumin (IMA) levels of each participant were measured before and after ozonation using albumin-cobalt binding assay. Additionally, the HSA protein of each subject was analyzed pre- and post-ozone treatment using a set of spectroscopic techniques. Altogether, the results showed that the medical ozone concentrations used in this study led to alterations in HSA by increasing IMA levels and inducing aggregation without causing major changes in the protein’s overall secondary structure. Moreover, the extent to which ozone affected HSA varied among each individual, highlighting the importance of prior testing and using innocuous and personalized concentrations of ozone in autohemotherapy of RRMS patients.","manuscriptTitle":"Insights into Ozone-Induced Alterations of Serum Albumin in Relapsing Remitting Multiple Sclerosis (MS) Patients and a Non-MS Individual","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-06-04 09:06:50","doi":"10.21203/rs.3.rs-6564545/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"829a3d56-f794-43bb-83fe-fb470d8b1af8","owner":[],"postedDate":"June 4th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-06-04T09:06:52+00:00","versionOfRecord":[],"versionCreatedAt":"2025-06-04 09:06:50","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6564545","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6564545","identity":"rs-6564545","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}