Quantification of Carbon Monoxide (CO) in Postmortem Human Brain Tissues After CO Poisoning

Quantification of Carbon Monoxide (CO) in Postmortem Human Brain Tissues After CO Poisoning | 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 Article Quantification of Carbon Monoxide (CO) in Postmortem Human Brain Tissues After CO Poisoning Kazuya Mori, Jun Yoshida, Fumiya Morioka, Qiyue Mao, Munehiro Katagi, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6293118/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 11 Sep, 2025 Read the published version in Scientific Reports → Version 1 posted 9 You are reading this latest preprint version Abstract In this study, we aimed to quantify carbon monoxide (CO) in human brain tissue to better understand the toxic mechanism of CO poisoning. Currently, conventional CO measurement methods are limited; however, the hemoCD assay has proven to be a simple and rapid method for quantifying CO in human tissues. Using this method, CO concentrations were measured in various brain regions, revealing significantly higher CO concentrations in the CO-exposed group (approximately 30-50 pmol/mg) compared to the non-exposed group (approximately 20-30 pmol/mg). However, the absence of elevated CO concentrations in specific brain regions suggests that CO inhalation is not selectively associated with areas that have a high affinity for CO or those that typically show abnormal signals during CO intoxication, as previously confirmed by MRI. The observed difference of 10-20 pmol/mg between the CO-exposed and non-exposed groups suggests that an additional 10-20 pmol/mg of external CO could reach lethal levels, potentially causing death. The results of this study are expected to contribute to the elucidation of the pathogenesis of CO poisoning and ultimately aid in the development of effective treatment strategies. Biological sciences/Biological techniques Health sciences/Health care Health sciences/Medical research Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Introduction Carbon monoxide (CO) poisoning is one of the most common forms of poisoning in modern society. According to a report by the National Research Institute of Police Science, CO poisoning accounts for approximately 70% of all poisoning-related deaths in Japan, with several thousand fatalities reported annually (1). Most cases of CO poisoning result from fires or suicides involving charcoal briquette combustion. In forensic practice, it is often necessary to assess the presence and severity of CO poisoning symptoms, its effects on bodily functions, and its role in the cause of death. CO also has physiological effects, such as vasodilation, neurotransmitter release, and inhibition of inflammatory responses (2-5). Furthermore, it has been suggested that a feedback mechanism for CO production exists, wherein endogenous CO is synthesized to levels comparable to those prior to its removal, even after endogenous CO has been eliminated (6). However, since CO is a lethal substance, it is essential to elucidate its toxicity mechanisms for future drug development and treatment strategies. Although CO toxicity has primarily been explained by theories such as hypoxic injury and cellular damage, many aspects remain unclear (7). In general, to clarify the toxicity mechanism of a drug or poison, it is necessary to determine the absolute amount and concentration of the substance that accumulates in tissues. However, only few studies have addressed this in relation to CO. Clinically, brain dysfunction is the primary consequence of acute CO poisoning, and delayed neurological damage can also occur (8). MRI imaging of CO poisoning cases in the acute phase has revealed various abnormal findings, with numerous reports describing typical signal abnormalities in specific brain regions (9-11). The prognosis for patients with late-onset encephalopathy varies widely, ranging from complete recovery to prolonged loss of consciousness or death. The reasons for these differences are not yet clear (8), but they may be related to differences in CO concentrations across different brain regions. Therefore, quantifying CO levels in various regions of the brain is crucial for understanding these variations. In forensic practice, one method of evaluating CO toxicity involves measuring CO intoxication levels in the body, including CO-hemoglobin (Hb) saturation in the blood, which serves as key evidence in determining whether a death resulted from CO poisoning (12). The relationship between CO-Hb saturation and clinical symptoms has been established, and a lethal CO-Hb saturation level is generally considered to be at least 50% (13). However, this method does not allow for the measurement of absolute CO amounts or concentrations in tissues. On the other hand, Gas chromatography (GC) has been reported as a method for quantifying CO (12,14,15). However, it is not a quick or simple method and lacks versatility due to the complexity of sample pretreatment, detection sensitivity issues, and the need for CO-specific analytical columns and detectors. Furthermore, while high-sensitivity detection methods such as hollow-core antiresonant fiber (HC-ARF) and fluorescent probes for CO have been explored, they remain impractical due complex experimental procedures and detection sensitivity limitations (16,17). In this study, we investigated a rapid and simple method for CO quantification using hemoCD, as originally reported by Kitagishi et al (18,19) (Fig.1). Among these methods, hemoCD-P has an affinity for CO approximately 100 times greater than that of normal Hb, a property that may have potential therapeutic applications for CO poisoning (20). The hemoCD assay, which enables CO quantification using hemoCD-P, does not require specialized equipment. This method allows for rapid analysis using a common and inexpensive spectrophotometer, involves simple pretreatment, and eliminates the need for complex calibration curves. Mao et al. have already used this method to measure ultra-trace amounts of endogenous CO in various organ tissues in rat experiments, and have proposed the mechanism that external CO once accumulated in tissues then transferred to Hb in blood due to its high CO affinity, and the storage capacity for CO should be different depending on the amount of heme contained in the tissues/organs as a CO binding site (19). If the hemoCD assay can be successfully applied to human organs, it will facilitate the rapid and simple quantification of CO in human tissues and contribute to the elucidation of CO poisoning mechanisms. Therefore, in this study, we applied the hemoCD assay to postmortem human brain tissue to quantify CO levels in different brain regions and compare them in cases suspected to have died from CO poisoning. Results Investigation of the application of the hemoCD assay to postmortem human tissues Using the hemoCD assay, we investigated the feasibility of quantifying endogenous CO in postmortem human tissues, specifically their brain tissue. The amount of sample used for analysis was 10 mg per analysis, following the hemoCD assay protocol described by Mao et al. ( 19 ). A portion of the cadaveric brain tissue was homogenized in phosphate-buffered saline (PBS) and hemoCD-P was added. Additionally, the homogenized liquid was further sonicated (Fig. 2 ). After sonication, samples were centrifuged for 15 minutes to obtain a clear supernatant, which was then filtered through a 0.45 µm filter. Following filtration, 4.5 mg of sodium hydrosulfite (Na 2 S 2 O 4 ) was added to the sample, and the analysis was performed using a spectrophotometer. As a result, absorption spectra with two characteristic absorption maxima were observed for deoxy-hemoCD-P (434 nm) and CO-hemoCD-P (422 nm) (Fig.༓), which was quite similar to the previous study for rat tissues ( 19 ). These findings indicate that the assay is adaptable for use in human tissues. Determination of endogenous CO in various fractions of the postmortem human brain in the non-CO-exposed group Next, we attempted to quantify CO in various regions of the postmortem human brain in cases where no CO exposure had occurred (control group: Ctrl group) (Fig. 4 ). As shown in Fig. 5 A, CO concentrations in each brain fraction were as follows: 21.3 ± 4.0 pmol/mg in the cortex of the frontal lobe, 24.5 ± 5.3 pmol/mg in the medulla of the frontal lobe, 25.1 ± 7.2 pmol/mg in the putamen, 20.8 ± 4.4 pmol/mg in the globus pallidus, 21.0 ± 5.4 pmol/mg in the internal capsule, 25.1 ± 7.2 pmol/mg in the caudate nucleus, 24.0 ± 4.3 pmol/mg in the cortex of the temporal lobe, and 23.6 ± 6.9 pmol/mg in the medulla of the temporal lobe. There were no significant differences in CO concentrations among these brain fractions. Furthermore, no specific accumulation of CO was detected in any individual samples. Determination of CO in various fractions of the postmortem human brain in the CO-exposed group We then quantified CO levels in different brain regions of individuals exposed to CO (CO group). As shown in Fig. 5 B, CO concentrations in each brain fraction were as follows: 34.6 ± 9.3 pmol/mg in the cortex of the frontal lobe, 35.5 ± 5.6 pmol/mg in the medulla of the frontal lobe, 44.4 ± 7.9 pmol/mg in the putamen, 37.0 ± 9.8 pmol/mg in the globus pallidus, 35.2 ± 10.0 pmol/mg in the internal capsule, 40.0 ± 7.9 pmol/mg in the caudate nucleus, 36.5 ± 8.0 pmol/mg in the cortex of the temporal lobe, and 40.2 ± 5.8 pmol/mg in the medulla of the temporal lobe. Compared to the Ctrl group, CO concentrations were significantly higher in all brain fractions (Fig. 6). However, there were no significant differences in CO concentrations among individual brain fractions. Furthermore, CO concentration in the brain did not increase proportionally with blood CO-Hb saturation. No correlation between brain CO concentrations and blood CO-Hb saturation was observed in this study. Discussion Previously, the hemoCD assay was shown to quantify CO in various tissues, including the rat brain. This study demonstrates for the first time that the method can be applied to human brain tissue without additional complex processing. Mao et al. used the hemoCD assay to analyze whole rat brains that had not been exposed to CO and found that 26.4 ± 6.2 pmol/mg of endogenous CO had accumulated ( 19 ). Our results also indicate that CO concentrations in all examined brain regions of the Ctrl group ranged from 20 to 30 pmol/mg. Interestingly, the amounts of endogenous CO were quite comparable between rat and human brains, which revealed in this study for the first time. As reported, endogenous CO is continuously generated at an average rate of approximately 0.4 mL/h in human adults ( 21 ). It is also estimated that 70% of CO production in living organisms is associated with the breakdown of heme, with 80–90% of this heme being derived from Hb. Hb levels in rats are comparable to those in humans, and most endogenous CO production results from heme breakdown ( 22 , 23 ). Therefore, the similar CO concentrations observed in rats and humans are not contradictory. In each brain fraction, CO was present at approximately 20–30 pmol/mg in all samples from the Ctrl group. In other words, the presence of similar CO concentrations in all fractionated tissues suggest that these levels are not toxic to the human body. At the very least, the findings suggest that CO concentrations at the levels observed in this study play an essential role in all parts of the brain. According to the literature, heme oxygenase (HO)-2 expression is highly abundant in mammalian brains, where the brain produces CO constantly via the heme degradation catalyzed by HO-2 ( 24 , 25 ). Unlike cortisol, which is secreted urgently in response to stress, CO appears to exert physiological effects as part of daily biological activities ( 26 ). Mao et al. compared CO levels in various organs, including the brain, between groups with and without CO exposure, and showed that the CO-exposed group had significantly higher CO accumulated than the non-exposed group ( 19 ). Our results also showed CO concentrations of approximately 30–50 pmol/mg in the CO-exposed group, with significant differences observed across all brain regions compared to the Ctrl group. The concentration values were also similar to those reported by Vreman et al.; however, it is unclear which part of the human brain they analyzed ( 27 ). Consequently, direct comparisons between our results and those of other studies are challenging due to the lack of site-specific measurements. Most importantly, these findings suggests that CO inhalation does not result in selective uptake of CO within specific regions of the brain. MRI and other imaging studies have reported that abnormal signals are typically found in the bilateral globus pallidus in acute cases, with additional abnormalities observed in the putamen, caudate nucleus, thalamus, cerebral cortex, hippocampus, cerebellum, etc. in some cases ( 9 – 11 ). Histological studies of postmortem brains from patients who survived long after CO exposure have shown heterogeneous destruction of basic structures in the cerebral cortex and basal ganglia, as well as glial cell damage in the globus pallidus and cerebral medulla ( 28 ). These findings suggest the existence of site-specific CO localization. However, our results did not show elevated CO concentrations corresponding to the sites of abnormal signal development identified by MRI. This strongly suggest that there is no selective uptake of CO by inhalation. The brain contains several heme proteins, such as cytochrome c oxidase, neuroglobin, cystathionine β-synthase. CO may inhibit their functions by coordinating directly to the heme of these enzymes ( 25 , 29 , 30 ). The cell types composing each brain regions analyzed in this study vary, meaning that different areas of the brain may have differing susceptibilities to CO. Our results support this possibility and may contribute to a better understanding of CO toxicity mechanisms. Furthermore, these results could help explain the individual differences in the onset of sequelae often observed in survivors of CO poisoning. Furthermore, CO concentrations in the CO-exposed group, with blood CO-Hb saturation of 50% or higher, remained at approximately 30–50 pmol/mg, regardless of variations in blood CO-Hb saturation. This suggests that these CO concentrations represent the maximum amount of CO that can accumulate in brain tissue, that is, the saturation threshold. Alternatively, the CO concentration in the brain may have remained at 30–50 pmol/mg due to fatal organ damage, even though the potential accumulation capacity might exceed this range. Therefore, the difference in CO concentrations observed between the CO-exposed and Ctrl groups may reflect an additive effect of endogenous CO, potentially leading to fatal outcomes ( 29 ). In other words, these levels may be lethal and provide critical insights into the toxicity of CO. It has been pointed out that the reported relationship between blood CO-Hb saturation and clinical symptoms is based on observations made during the process of CO inhalation and gradual increase in blood CO-Hb levels. Therefore, in clinical practice, it is also said that the severity of acute CO poisoning does not necessarily correlate with blood CO-Hb saturation at the time of presentation ( 31 , 32 ). In other words, this saturation value alone does not provide a comprehensive understanding of the CO toxicity mechanism. The findings from the present study therefore are important for elucidating the toxicological mechanisms of CO. Methods Cardiac blood CO-Hb saturation measurement CO-Hb saturation was measured using cardiac blood collected from the cadavers. The sample was diluted approximately 1:200 with 0.1% sodium carbonate solution. Subsequently, 4.5 mg of sodium hydrosulfite was added to the solution and mixed. After adding 0.2 mL of 1N sodium hydroxide solution and allowing it to stand, absorbance at (a) 530 nm and (b) 558 nm was measured using a spectrophotometer. CO-Hb saturation was calculated using the formula (2.21 - b/a) × 79, as previously described. The CO-Hb saturation levels for each cadaver are shown in Table 1 . This study was conducted in accordance with the principles of the Declaration of Helsinki and was approved by the Clinical Research Review Committee of Osaka Medical and Pharmaceutical University (2023-078-1). While we made every effort to obtain consent from the deceased’s family, in cases where it was not feasible, an opt-out approach was employed. Information about the study was made publicly available through the Department of Forensic Medicine, Osaka Medical and Pharmaceutical University Website (URL: https://www.ompu.ac.jp/u-deps/leg/contact/index.html ), allowing families to decline participation if desired. Table 1 Background information of samples used in this study. No. Age Sex Cause of death CO-Hb saturation (%) Time since death Situation 1 68 M acute myocardial infarction 0.6 3days found in supine position at home 2 35 F cardiac sudden death 0.7 2days found in supine position at work 3 82 M lethal arrhythmia 0.7 2days found in lateral decubitus position at home. 4 78 M hypothermia 2.0 2days found in supine position at home 5 19 F exsanguination 5.0 2days murder case 6 25 M carbon monoxide poisoning 51.6 3days suicide by charcoal briquettes 7 76 M fatal thermal injuries 52.2 19h fire at home 8 74 M fatal thermal injuries 55.5 13h fire at home 9 51 M carbon monoxide poisoning 66.7 3days suicide by charcoal briquettes 10 75 M fatal thermal injuries 72.5 3days suicide by charcoal briquettes 11 25 F carbon monoxide poisoning 72.5 2days suicide by charcoal briquettes 12 26 M carbon monoxide poisoning 72.7 3days suicide by charcoal briquettes Samples A total of 12 cadavers were available for analysis. Details of the cadavers are presented in Table 1 . These cadavers were classified into two groups based on blood CO-Hb saturation levels and the results of the environmental investigation by the investigative agency. Cadavers with CO-Hb saturation below 10% were classified as the Ctrl group (n = 5), indicating no CO exposure. Cadavers with CO-Hb saturation above 50% were assigned to the CO exposure (CO) group (n = 7). The brain was removed from each cadaver for analysis. However, previous reports indicate that CO can be produced in decomposed drowned bodies ( 33 , 34 ). Therefore, cadavers suspected of decomposition-related CO production, based on external findings at autopsy or autopsy results, were excluded from sample collection. Sample Preparation The brain was sectioned into 2-cm-thick slices in the coronal plane, and the following regions were excised, as shown in Fig. 4 : cortex of the frontal lobe, medulla of the frontal lobe, putamen, globus pallidus, internal capsule, caudate nucleus, cortex of the temporal lobe, cortex of the temporal lobe, and medulla of the temporal lobe. The excised tissues were placed in 2mL tubes, rapidly frozen, and stored at -80°C. CO determination in brain tissue by hemoCD assay Sample (10mg) thawed at room temperature was weighed and homogenized using an Automill (Tokken Inc. Japan) in PBS (0.5ml). After homogenization, hemoCD-P (3 µL) with Na 2 S 2 O 4 (4 mg) in PBS was added, followed by sonication on ice (10s×2, amplitude: 30; QSO-NICA). Samples were then centrifuged (14,000×g, 15min), and the supernatants were filtered through a 0.45㎛ pore filter (TecholabSC;LTD. Japan). The filtrates were treated with Na 2 S 2 O 4 (4 mg) before measurement using a UV-Vis-NIR Spectrometer (V-730, Japan Spectroscopy Co., Ltd. Japan). All analyses were processed using JASCO Spectrum Manager Ver. 2 (Japan Spectroscopy Co., Ltd. Japan). Absorbance was measured at 434 nm for deoxy-hemoCD-P and 422 nm CO-hemoCD-P. CO concentrations (pmol/mg) were calculated using the equation reported by Mao et al. ( 19 ). Statistics and Reproducibility Statistical analyses were performed using GraphPad Prism, version 9.5.1 (GraphPad Software). All data are presented as means ± standard error from at least three independent experiments and were analyzed by one-way analysis of variance (ANOVA) and Student’s t-test. Differences with P values less than 0.05 were considered statistically significant. Declarations Acknowledgements This work was supported by JSPS KAKENHI (24K13561 and 24K01640), AMED (24ym0126808j), and JST (JPMJSF2305). We sincerely appreciate the professional English language editing provided by Dr. Maiko Kusano (KMD). Author contribution K.M and M.K. conceived the study. Material preparation conducted by F.M and T.S. Data collection and analysis were performed by K.M. and J.Y. The first draft of the manuscript was written by K.M. hemoCD was provided by H.K. All authors commented on previous versions of the manuscript. All authors reviewed the results and approved the final version of the manuscript. Competing interests The authors declare no competing interests. Data Availability All relevant data are in the manuscript. References Tsujikawa, K. Fatal poisoning situation in Japan in 2007 to 2018 based on Annual case reports of drug and toxic poisoning in Japan issued by National Research Institute of Police Science. Jpn J. Clin. Toxico . 36 , 361–368 (2023). Marks, G. S., Brien, J. F., Nakatsu, K. & McLaughlin, B. E. Does carbon monoxide have a physiological function? Trends Pharmacol. 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Cite Share Download PDF Status: Published Journal Publication published 11 Sep, 2025 Read the published version in Scientific Reports → Version 1 posted Editorial decision: Revision requested 29 Jul, 2025 Reviewers agreed at journal 27 May, 2025 Reviews received at journal 18 Apr, 2025 Reviewers agreed at journal 17 Apr, 2025 Reviewers invited by journal 15 Apr, 2025 Editor assigned by journal 11 Apr, 2025 Editor invited by journal 04 Apr, 2025 Submission checks completed at journal 30 Mar, 2025 First submitted to journal 30 Mar, 2025 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. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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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-6293118","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":446173232,"identity":"2092aa76-741f-4e93-b519-fc18042593fa","order_by":0,"name":"Kazuya Mori","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA70lEQVRIiWNgGAWjYBACCQSTsfEBNmG8WpoNSNXCwIZPIQJIth9/Js1TwxDNL324reJjzp3EBvbDDxgsd+DWIs2TkCbNc4whd2ZfYtvNmdueJTbwpBkwSJ7BrUWOIeGYNA8bQ+6GM4xtt3m3HU5sYMgBWt6GRwv/wzZpnn8QLcVgLfxv8GuRlkhmk+Ztg2hhBmuRIGCL5IxnzJZz+yRyZ/YwNksC/WLcJvHM4AA+v0icT3944803m9x+HvaHHz5uuyPbz5/88LEknhADASYeRPQcYGADkoclG/BrYfyBYB+AiHwkoGUUjIJRMApGFAAAHvFPKacHdrUAAAAASUVORK5CYII=","orcid":"","institution":"Osaka Medical and Pharmaceutical University","correspondingAuthor":true,"prefix":"","firstName":"Kazuya","middleName":"","lastName":"Mori","suffix":""},{"id":446173233,"identity":"04220da6-f64c-427c-bbae-e39bb05c9922","order_by":1,"name":"Jun Yoshida","email":"","orcid":"","institution":"Osaka Medical and Pharmaceutical University","correspondingAuthor":false,"prefix":"","firstName":"Jun","middleName":"","lastName":"Yoshida","suffix":""},{"id":446173234,"identity":"2a66580d-993e-42cd-9c5c-67b446fabd2f","order_by":2,"name":"Fumiya Morioka","email":"","orcid":"","institution":"Osaka Medical and Pharmaceutical University","correspondingAuthor":false,"prefix":"","firstName":"Fumiya","middleName":"","lastName":"Morioka","suffix":""},{"id":446173235,"identity":"616552f1-47f8-4dc1-9b3d-bf2d69c207df","order_by":3,"name":"Qiyue Mao","email":"","orcid":"","institution":"Doshisha University","correspondingAuthor":false,"prefix":"","firstName":"Qiyue","middleName":"","lastName":"Mao","suffix":""},{"id":446173236,"identity":"90002059-6a49-42e9-9930-b7e5e720a3e3","order_by":4,"name":"Munehiro Katagi","email":"","orcid":"","institution":"Osaka Medical and Pharmaceutical University","correspondingAuthor":false,"prefix":"","firstName":"Munehiro","middleName":"","lastName":"Katagi","suffix":""},{"id":446173237,"identity":"9ba0bf0b-19b7-49dd-812c-76fc293f2161","order_by":5,"name":"Hiroaki Kitagishi","email":"","orcid":"","institution":"Doshisha University","correspondingAuthor":false,"prefix":"","firstName":"Hiroaki","middleName":"","lastName":"Kitagishi","suffix":""},{"id":446173238,"identity":"4da760d7-9f3b-4d70-b118-c83b2dfb1b56","order_by":6,"name":"Takako Sato","email":"","orcid":"","institution":"Osaka Medical and Pharmaceutical University","correspondingAuthor":false,"prefix":"","firstName":"Takako","middleName":"","lastName":"Sato","suffix":""}],"badges":[],"createdAt":"2025-03-24 08:23:28","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6293118/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6293118/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1038/s41598-025-15661-x","type":"published","date":"2025-09-11T15:57:17+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":82119790,"identity":"472cb77a-f054-4574-81b3-ad9362779220","added_by":"auto","created_at":"2025-05-07 03:11:07","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":141018,"visible":true,"origin":"","legend":"\u003cp\u003eStructures of deoxy-hemoCD1 and CO-hemoCD1\u003c/p\u003e\n\u003cp\u003eStructures of deoxy-hemoCD1 and CO-hemoCD1 complexes are shown. hemoCD1 is composed of 5,10,15,20-tetrakis(4-sulfonatophenyl)porphinatoiron (Ⅱ)(Fe\u003csup\u003eⅡ\u003c/sup\u003eTPPS) and aper-O-methyl-β-cyclodextrin dimer having a pyridine linker(Py3CD). Adapted from refs 19.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-6293118/v1/debff7e79ca43c7c0ec6be25.png"},{"id":82119789,"identity":"16177b51-639d-44c8-be03-38992b1b6836","added_by":"auto","created_at":"2025-05-07 03:11:07","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":41332,"visible":true,"origin":"","legend":"\u003cp\u003eProtocol for the hemoCD1 assay in human brain tissue\u003c/p\u003e\n\u003cp\u003eExperimental procedure describing the various steps of the hemoCD1 assay for measuring CO in human brain tissue.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-6293118/v1/bfcf126169624264644e4efb.png"},{"id":82117799,"identity":"1240dc01-09d1-4a42-a4aa-7f93aaa8b636","added_by":"auto","created_at":"2025-05-07 03:03:07","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":17058,"visible":true,"origin":"","legend":"\u003cp\u003eAbsorbance of hemoCD assay in human brain tissue\u003c/p\u003e\n\u003cp\u003eAbsorbance spectrum（422nm:deoxy-hemoCD1, 434nm:CO-hemoCD1）observed in human brain tissue.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-6293118/v1/63fddd07c8a7756d8223322e.png"},{"id":82117804,"identity":"acc9f190-f47d-4ba1-8464-e7c56a21e6d0","added_by":"auto","created_at":"2025-05-07 03:03:07","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":586259,"visible":true,"origin":"","legend":"\u003cp\u003eBrain regions used for analysis\u003c/p\u003e\n\u003cp\u003eThe brain regions shown are as follows:\u003c/p\u003e\n\u003cp\u003e1: cortex of the frontal lobe, 2: medulla of the frontal lobe, 3: putamen, 4: globus pallidus,\u003c/p\u003e\n\u003cp\u003e5: internal capsule, 6: caudate nucleus, 7: cortex of the temporal lobe, 8: medulla of the temporal lobe\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-6293118/v1/0996414681363980a8f14adb.png"},{"id":82117803,"identity":"17a8958c-c0f1-42d2-b8ea-f94dd44cd5f6","added_by":"auto","created_at":"2025-05-07 03:03:07","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":396944,"visible":true,"origin":"","legend":"\u003cp\u003eComparison of carbon monoxide (CO) concentration in different brain regions between\u003c/p\u003e\n\u003cp\u003ethe control group (A) and the CO-exposed group (B)\u003c/p\u003e\n\u003cp\u003eBox plots showing carbon monoxide (CO) concentrations in different brain regions for the control group (A) and the CO-exposed group (B). Asterisks indicate mean values. One-way ANOVA was conducted for each group, but no significant difference was found. A significance level of p \u0026lt; 0.05 was considered statistically significant.\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-6293118/v1/8add99efda2829d295c5bae1.png"},{"id":82122177,"identity":"dfba1cb0-dc38-488a-aaed-74da88201d2f","added_by":"auto","created_at":"2025-05-07 03:27:07","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":258741,"visible":true,"origin":"","legend":"\u003cp\u003eComparison of CO concentrations in various brain regions between Ctrl and CO-exposed groups.\u003c/p\u003e\n\u003cp\u003eIndependent t-test was performed to compare the Ctrl group and the CO-exposed group. Data are presented as mean ± standard deviation (SD). A significance level of p \u0026lt; 0.05 was considered statistically significant.\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-6293118/v1/fa6a11f4bb205e1e84996e27.png"},{"id":91359061,"identity":"0f0ca1fd-2419-40dc-8c18-bcb5ef1e1e43","added_by":"auto","created_at":"2025-09-15 16:04:42","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2355848,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6293118/v1/f3869bfa-8c69-46a2-b489-b0f9a9188722.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Quantification of Carbon Monoxide (CO) in Postmortem Human Brain Tissues After CO Poisoning","fulltext":[{"header":"Introduction","content":"\u003cp\u003eCarbon monoxide (CO) poisoning is one of the most common forms of poisoning in modern society. According to a report by the National Research Institute of Police Science, CO poisoning accounts for approximately 70% of all poisoning-related deaths in Japan, with several thousand fatalities reported annually (1). Most cases of CO poisoning result from fires or suicides involving charcoal briquette combustion. In forensic practice, it is often necessary to assess the presence and severity of CO poisoning symptoms, its effects on bodily functions, and its role in the cause of death. CO also has physiological effects, such as vasodilation, neurotransmitter release, and inhibition of inflammatory responses (2-5). Furthermore, it has been suggested that a feedback mechanism for CO production exists, wherein endogenous CO is synthesized to levels comparable to those prior to its removal, even after endogenous CO has been eliminated (6). However, since CO is a lethal substance, it is essential to elucidate its toxicity mechanisms for future drug development and treatment strategies.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eAlthough CO toxicity has primarily been explained by theories such as hypoxic injury and cellular damage, many aspects remain unclear (7). In general, to clarify the toxicity mechanism of a drug or poison, it is necessary to determine the absolute amount and concentration of the substance that accumulates in tissues. However, only few studies have addressed this in relation to CO. Clinically, brain dysfunction is the primary consequence of acute CO poisoning, and delayed neurological damage can also occur (8). MRI imaging of CO poisoning cases in the acute phase has revealed various abnormal findings, with numerous reports describing typical signal abnormalities in specific brain regions (9-11). The prognosis for patients with late-onset encephalopathy varies widely, ranging from complete recovery to prolonged loss of consciousness or death. The reasons for these differences are not yet clear (8), but they may be related to differences in CO concentrations across different brain regions. Therefore, quantifying CO levels in various regions of the brain is crucial for understanding these variations.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eIn forensic practice, one method of evaluating CO toxicity involves measuring CO intoxication levels in the body, including CO-hemoglobin (Hb) saturation in the blood, which serves as key evidence in determining whether a death resulted from CO poisoning (12). The relationship between CO-Hb saturation and clinical symptoms has been established, and a lethal CO-Hb saturation level is generally considered to be at least 50% (13). However, this method does not allow for the measurement of absolute CO amounts or concentrations in tissues. On the other hand, Gas chromatography (GC) has been reported as a method for quantifying CO (12,14,15). However, it is not a quick or simple method and lacks versatility due to the complexity of sample pretreatment, detection sensitivity issues, and the need for CO-specific analytical columns and detectors. Furthermore, while high-sensitivity detection methods such as hollow-core antiresonant fiber (HC-ARF) and fluorescent probes for CO have been explored, they remain impractical due complex experimental procedures and detection sensitivity limitations (16,17).\u003c/p\u003e\n\u003cp\u003eIn this study, we investigated a rapid and simple method for CO quantification using hemoCD, as originally reported by Kitagishi et al (18,19) (Fig.1). Among these methods, hemoCD-P has an affinity for CO approximately 100 times greater than that of normal Hb, a property that may have potential therapeutic applications for CO poisoning (20). The hemoCD assay, which enables CO quantification using hemoCD-P, does not require specialized equipment. This method allows for rapid analysis using a common and inexpensive spectrophotometer, involves simple pretreatment, and eliminates the need for complex calibration curves. Mao et al. have already used this method to measure ultra-trace amounts of endogenous CO in various organ tissues in rat experiments, and have proposed the mechanism that external CO once accumulated in tissues then transferred to Hb in blood due to its high CO affinity, and the storage capacity for CO should be different depending on the amount of heme contained in the tissues/organs as a CO binding site (19). If the hemoCD assay can be successfully applied to human organs, it will facilitate the rapid and simple quantification of CO in human tissues and contribute to the elucidation of CO poisoning mechanisms. Therefore, in this study, we applied the hemoCD assay to postmortem human brain tissue to quantify CO levels in different brain regions and compare them in cases suspected to have died from CO poisoning.\u003c/p\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec2\" class=\"Section2\"\u003e \u003ch2\u003eInvestigation of the application of the hemoCD assay to postmortem human tissues\u003c/h2\u003e \u003cp\u003eUsing the hemoCD assay, we investigated the feasibility of quantifying endogenous CO in postmortem human tissues, specifically their brain tissue. The amount of sample used for analysis was 10 mg per analysis, following the hemoCD assay protocol described by Mao et al. (\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e). A portion of the cadaveric brain tissue was homogenized in phosphate-buffered saline (PBS) and hemoCD-P was added. Additionally, the homogenized liquid was further sonicated (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e2\u003c/span\u003e). After sonication, samples were centrifuged for 15 minutes to obtain a clear supernatant, which was then filtered through a 0.45 \u0026micro;m filter. Following filtration, 4.5 mg of sodium hydrosulfite (Na\u003csub\u003e2\u003c/sub\u003eS\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e) was added to the sample, and the analysis was performed using a spectrophotometer. As a result, absorption spectra with two characteristic absorption maxima were observed for deoxy-hemoCD-P (434 nm) and CO-hemoCD-P (422 nm) (Fig.༓), which was quite similar to the previous study for rat tissues (\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e). These findings indicate that the assay is adaptable for use in human tissues.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eDetermination of endogenous CO in various fractions of the postmortem human brain in the non-CO-exposed group\u003c/b\u003e \u003c/p\u003e \u003cp\u003eNext, we attempted to quantify CO in various regions of the postmortem human brain in cases where no CO exposure had occurred (control group: Ctrl group) (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e4\u003c/span\u003e). As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e5\u003c/span\u003eA, CO concentrations in each brain fraction were as follows: 21.3\u0026thinsp;\u0026plusmn;\u0026thinsp;4.0 pmol/mg in the cortex of the frontal lobe, 24.5\u0026thinsp;\u0026plusmn;\u0026thinsp;5.3 pmol/mg in the medulla of the frontal lobe, 25.1\u0026thinsp;\u0026plusmn;\u0026thinsp;7.2 pmol/mg in the putamen, 20.8\u0026thinsp;\u0026plusmn;\u0026thinsp;4.4 pmol/mg in the globus pallidus, 21.0\u0026thinsp;\u0026plusmn;\u0026thinsp;5.4 pmol/mg in the internal capsule, 25.1\u0026thinsp;\u0026plusmn;\u0026thinsp;7.2 pmol/mg in the caudate nucleus, 24.0\u0026thinsp;\u0026plusmn;\u0026thinsp;4.3 pmol/mg in the cortex of the temporal lobe, and 23.6\u0026thinsp;\u0026plusmn;\u0026thinsp;6.9 pmol/mg in the medulla of the temporal lobe. There were no significant differences in CO concentrations among these brain fractions. Furthermore, no specific accumulation of CO was detected in any individual samples.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eDetermination of CO in various fractions of the postmortem human brain in the CO-exposed group\u003c/h2\u003e \u003cp\u003eWe then quantified CO levels in different brain regions of individuals exposed to CO (CO group). As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e5\u003c/span\u003eB, CO concentrations in each brain fraction were as follows: 34.6\u0026thinsp;\u0026plusmn;\u0026thinsp;9.3 pmol/mg in the cortex of the frontal lobe, 35.5\u0026thinsp;\u0026plusmn;\u0026thinsp;5.6 pmol/mg in the medulla of the frontal lobe, 44.4\u0026thinsp;\u0026plusmn;\u0026thinsp;7.9 pmol/mg in the putamen, 37.0\u0026thinsp;\u0026plusmn;\u0026thinsp;9.8 pmol/mg in the globus pallidus, 35.2\u0026thinsp;\u0026plusmn;\u0026thinsp;10.0 pmol/mg in the internal capsule, 40.0\u0026thinsp;\u0026plusmn;\u0026thinsp;7.9 pmol/mg in the caudate nucleus, 36.5\u0026thinsp;\u0026plusmn;\u0026thinsp;8.0 pmol/mg in the cortex of the temporal lobe, and 40.2\u0026thinsp;\u0026plusmn;\u0026thinsp;5.8 pmol/mg in the medulla of the temporal lobe. Compared to the Ctrl group, CO concentrations were significantly higher in all brain fractions (Fig.\u0026nbsp;6). However, there were no significant differences in CO concentrations among individual brain fractions. Furthermore, CO concentration in the brain did not increase proportionally with blood CO-Hb saturation. No correlation between brain CO concentrations and blood CO-Hb saturation was observed in this study.\u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003ePreviously, the hemoCD assay was shown to quantify CO in various tissues, including the rat brain. This study demonstrates for the first time that the method can be applied to human brain tissue without additional complex processing.\u003c/p\u003e \u003cp\u003eMao et al. used the hemoCD assay to analyze whole rat brains that had not been exposed to CO and found that 26.4\u0026thinsp;\u0026plusmn;\u0026thinsp;6.2 pmol/mg of endogenous CO had accumulated (\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e). Our results also indicate that CO concentrations in all examined brain regions of the Ctrl group ranged from 20 to 30 pmol/mg. Interestingly, the amounts of endogenous CO were quite comparable between rat and human brains, which revealed in this study for the first time. As reported, endogenous CO is continuously generated at an average rate of approximately 0.4 mL/h in human adults (\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e). It is also estimated that 70% of CO production in living organisms is associated with the breakdown of heme, with 80\u0026ndash;90% of this heme being derived from Hb. Hb levels in rats are comparable to those in humans, and most endogenous CO production results from heme breakdown (\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e). Therefore, the similar CO concentrations observed in rats and humans are not contradictory.\u003c/p\u003e \u003cp\u003eIn each brain fraction, CO was present at approximately 20\u0026ndash;30 pmol/mg in all samples from the Ctrl group. In other words, the presence of similar CO concentrations in all fractionated tissues suggest that these levels are not toxic to the human body. At the very least, the findings suggest that CO concentrations at the levels observed in this study play an essential role in all parts of the brain. According to the literature, heme oxygenase (HO)-2 expression is highly abundant in mammalian brains, where the brain produces CO constantly via the heme degradation catalyzed by HO-2 (\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e). Unlike cortisol, which is secreted urgently in response to stress, CO appears to exert physiological effects as part of daily biological activities (\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e). Mao et al. compared CO levels in various organs, including the brain, between groups with and without CO exposure, and showed that the CO-exposed group had significantly higher CO accumulated than the non-exposed group (\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e). Our results also showed CO concentrations of approximately 30\u0026ndash;50 pmol/mg in the CO-exposed group, with significant differences observed across all brain regions compared to the Ctrl group. The concentration values were also similar to those reported by Vreman et al.; however, it is unclear which part of the human brain they analyzed (\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e). Consequently, direct comparisons between our results and those of other studies are challenging due to the lack of site-specific measurements.\u003c/p\u003e \u003cp\u003eMost importantly, these findings suggests that CO inhalation does not result in selective uptake of CO within specific regions of the brain. MRI and other imaging studies have reported that abnormal signals are typically found in the bilateral globus pallidus in acute cases, with additional abnormalities observed in the putamen, caudate nucleus, thalamus, cerebral cortex, hippocampus, cerebellum, etc. in some cases (\u003cspan additionalcitationids=\"CR10\" citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e). Histological studies of postmortem brains from patients who survived long after CO exposure have shown heterogeneous destruction of basic structures in the cerebral cortex and basal ganglia, as well as glial cell damage in the globus pallidus and cerebral medulla (\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e). These findings suggest the existence of site-specific CO localization. However, our results did not show elevated CO concentrations corresponding to the sites of abnormal signal development identified by MRI. This strongly suggest that there is no selective uptake of CO by inhalation. The brain contains several heme proteins, such as cytochrome c oxidase, neuroglobin, cystathionine β-synthase. CO may inhibit their functions by coordinating directly to the heme of these enzymes (\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e). The cell types composing each brain regions analyzed in this study vary, meaning that different areas of the brain may have differing susceptibilities to CO. Our results support this possibility and may contribute to a better understanding of CO toxicity mechanisms. Furthermore, these results could help explain the individual differences in the onset of sequelae often observed in survivors of CO poisoning.\u003c/p\u003e \u003cp\u003eFurthermore, CO concentrations in the CO-exposed group, with blood CO-Hb saturation of 50% or higher, remained at approximately 30\u0026ndash;50 pmol/mg, regardless of variations in blood CO-Hb saturation. This suggests that these CO concentrations represent the maximum amount of CO that can accumulate in brain tissue, that is, the saturation threshold. Alternatively, the CO concentration in the brain may have remained at 30\u0026ndash;50 pmol/mg due to fatal organ damage, even though the potential accumulation capacity might exceed this range. Therefore, the difference in CO concentrations observed between the CO-exposed and Ctrl groups may reflect an additive effect of endogenous CO, potentially leading to fatal outcomes (\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e). In other words, these levels may be lethal and provide critical insights into the toxicity of CO.\u003c/p\u003e \u003cp\u003eIt has been pointed out that the reported relationship between blood CO-Hb saturation and clinical symptoms is based on observations made during the process of CO inhalation and gradual increase in blood CO-Hb levels. Therefore, in clinical practice, it is also said that the severity of acute CO poisoning does not necessarily correlate with blood CO-Hb saturation at the time of presentation (\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e, \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e). In other words, this saturation value alone does not provide a comprehensive understanding of the CO toxicity mechanism. The findings from the present study therefore are important for elucidating the toxicological mechanisms of CO.\u003c/p\u003e"},{"header":"Methods","content":"\u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eCardiac blood CO-Hb saturation measurement\u003c/h2\u003e \u003cp\u003eCO-Hb saturation was measured using cardiac blood collected from the cadavers. The sample was diluted approximately 1:200 with 0.1% sodium carbonate solution. Subsequently, 4.5 mg of sodium hydrosulfite was added to the solution and mixed. After adding 0.2 mL of 1N sodium hydroxide solution and allowing it to stand, absorbance at (a) 530 nm and (b) 558 nm was measured using a spectrophotometer. CO-Hb saturation was calculated using the formula (2.21 - b/a) \u0026times; 79, as previously described. The CO-Hb saturation levels for each cadaver are shown in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. This study was conducted in accordance with the principles of the Declaration of Helsinki and was approved by the Clinical Research Review Committee of Osaka Medical and Pharmaceutical University (2023-078-1). While we made every effort to obtain consent from the deceased\u0026rsquo;s family, in cases where it was not feasible, an opt-out approach was employed. Information about the study was made publicly available through the Department of Forensic Medicine, Osaka Medical and Pharmaceutical University Website (URL: \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.ompu.ac.jp/u-deps/leg/contact/index.html\u003c/span\u003e\u003cspan address=\"https://www.ompu.ac.jp/u-deps/leg/contact/index.html\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e), allowing families to decline participation if desired.\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\u003eBackground information of samples used in this study.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"7\"\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=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNo.\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eAge\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eSex\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eCause of death\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eCO-Hb saturation (%)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eTime since death\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003eSituation\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e68\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eM\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eacute myocardial infarction\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e3days\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003efound in supine position at home\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e35\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eF\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003ecardiac sudden death\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e2days\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003efound in supine position at work\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e82\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eM\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003elethal arrhythmia\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e2days\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003efound in lateral decubitus position at home.\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e78\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eM\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003ehypothermia\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e2.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e2days\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003efound in supine position at home\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e19\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eF\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eexsanguination\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e5.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e2days\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003emurder case\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e25\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eM\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003ecarbon monoxide poisoning\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e51.6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e3days\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003esuicide by charcoal briquettes\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e76\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eM\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003efatal thermal injuries\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e52.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e19h\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003efire at home\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e74\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eM\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003efatal thermal injuries\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e55.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e13h\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003efire at home\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e51\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eM\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003ecarbon monoxide poisoning\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e66.7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e3days\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003esuicide by charcoal briquettes\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e75\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eM\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003efatal thermal injuries\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e72.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e3days\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003esuicide by charcoal briquettes\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e11\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e25\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eF\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003ecarbon monoxide poisoning\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e72.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e2days\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003esuicide by charcoal briquettes\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e26\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eM\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003ecarbon monoxide poisoning\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e72.7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e3days\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003esuicide by charcoal briquettes\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\n\u003ch3\u003eSamples\u003c/h3\u003e\n\u003cp\u003eA total of 12 cadavers were available for analysis. Details of the cadavers are presented in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. These cadavers were classified into two groups based on blood CO-Hb saturation levels and the results of the environmental investigation by the investigative agency. Cadavers with CO-Hb saturation below 10% were classified as the Ctrl group (n\u0026thinsp;=\u0026thinsp;5), indicating no CO exposure. Cadavers with CO-Hb saturation above 50% were assigned to the CO exposure (CO) group (n\u0026thinsp;=\u0026thinsp;7). The brain was removed from each cadaver for analysis. However, previous reports indicate that CO can be produced in decomposed drowned bodies (\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e, \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e). Therefore, cadavers suspected of decomposition-related CO production, based on external findings at autopsy or autopsy results, were excluded from sample collection.\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eSample Preparation\u003c/h2\u003e \u003cp\u003eThe brain was sectioned into 2-cm-thick slices in the coronal plane, and the following regions were excised, as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e4\u003c/span\u003e: cortex of the frontal lobe, medulla of the frontal lobe, putamen, globus pallidus, internal capsule, caudate nucleus, cortex of the temporal lobe, cortex of the temporal lobe, and medulla of the temporal lobe. The excised tissues were placed in 2mL tubes, rapidly frozen, and stored at -80\u0026deg;C.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eCO determination in brain tissue by hemoCD assay\u003c/h3\u003e\n\u003cp\u003eSample (10mg) thawed at room temperature was weighed and homogenized using an Automill (Tokken Inc. Japan) in PBS (0.5ml). After homogenization, hemoCD-P (3 \u0026micro;L) with Na\u003csub\u003e2\u003c/sub\u003eS\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e (4 mg) in PBS was added, followed by sonication on ice (10s\u0026times;2, amplitude: 30; QSO-NICA). Samples were then centrifuged (14,000\u0026times;g, 15min), and the supernatants were filtered through a 0.45㎛ pore filter (TecholabSC;LTD. Japan). The filtrates were treated with Na\u003csub\u003e2\u003c/sub\u003eS\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e (4 mg) before measurement using a UV-Vis-NIR Spectrometer (V-730, Japan Spectroscopy Co., Ltd. Japan). All analyses were processed using JASCO Spectrum Manager Ver. 2 (Japan Spectroscopy Co., Ltd. Japan). Absorbance was measured at 434 nm for deoxy-hemoCD-P and 422 nm CO-hemoCD-P. CO concentrations (pmol/mg) were calculated using the equation reported by Mao et al. (\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e).\u003c/p\u003e\n\u003ch3\u003eStatistics and Reproducibility\u003c/h3\u003e\n\u003cp\u003eStatistical analyses were performed using GraphPad Prism, version 9.5.1 (GraphPad Software). All data are presented as means\u0026thinsp;\u0026plusmn;\u0026thinsp;standard error from at least three independent experiments and were analyzed by one-way analysis of variance (ANOVA) and Student\u0026rsquo;s t-test. Differences with P values less than 0.05 were considered statistically significant.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by JSPS KAKENHI (24K13561 and 24K01640), AMED (24ym0126808j), and JST (JPMJSF2305). We sincerely appreciate the professional English language editing provided by Dr. Maiko Kusano (KMD).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contribution\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eK.M and M.K. conceived the study. Material preparation conducted by F.M and T.S. Data collection and analysis were performed by K.M. and J.Y. The first draft of the manuscript was written by K.M. hemoCD was provided by H.K. All authors commented on previous versions of the manuscript. All authors reviewed the results and approved the final version of the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e\u003cp\u003eAll relevant data are in the manuscript.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eTsujikawa, K. Fatal poisoning situation in Japan in 2007 to 2018 based on Annual case reports of drug and toxic poisoning in Japan issued by National Research Institute of Police Science. \u003cem\u003eJpn J. Clin. Toxico\u003c/em\u003e. \u003cb\u003e36\u003c/b\u003e, 361\u0026ndash;368 (2023).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMarks, G. S., Brien, J. F., Nakatsu, K. \u0026amp; McLaughlin, B. E. Does carbon monoxide have a physiological function? \u003cem\u003eTrends Pharmacol. 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Int.\u003c/em\u003e \u003cb\u003e22\u003c/b\u003e, 131\u0026ndash;135 (1983).\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-6293118/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6293118/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"In this study, we aimed to quantify carbon monoxide (CO) in human brain tissue to better understand the toxic mechanism of CO poisoning. Currently, conventional CO measurement methods are limited; however, the hemoCD assay has proven to be a simple and rapid method for quantifying CO in human tissues. Using this method, CO concentrations were measured in various brain regions, revealing significantly higher CO concentrations in the CO-exposed group (approximately 30-50 pmol/mg) compared to the non-exposed group (approximately 20-30 pmol/mg). However, the absence of elevated CO concentrations in specific brain regions suggests that CO inhalation is not selectively associated with areas that have a high affinity for CO or those that typically show abnormal signals during CO intoxication, as previously confirmed by MRI. The observed difference of 10-20 pmol/mg between the CO-exposed and non-exposed groups suggests that an additional 10-20 pmol/mg of external CO could reach lethal levels, potentially causing death. The results of this study are expected to contribute to the elucidation of the pathogenesis of CO poisoning and ultimately aid in the development of effective treatment strategies.","manuscriptTitle":"Quantification of Carbon Monoxide (CO) in Postmortem Human Brain Tissues After CO Poisoning","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-05-07 03:03:02","doi":"10.21203/rs.3.rs-6293118/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-07-29T07:14:46+00:00","index":"","fulltext":""},{"type":"reviewerAgreed","content":"7277727099640708377002981693439837631","date":"2025-05-27T10:39:11+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-04-18T06:48:50+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"195773513443992713534831896346700703430","date":"2025-04-17T06:13:50+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-04-15T06:05:29+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-04-11T06:36:44+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2025-04-04T06:37:48+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-03-30T12:23:07+00:00","index":"","fulltext":""},{"type":"submitted","content":"Scientific Reports","date":"2025-03-30T12:21:58+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"476311f0-f201-4014-9867-f8af4274f37b","owner":[],"postedDate":"May 7th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":47489889,"name":"Biological sciences/Biological techniques"},{"id":47489890,"name":"Health sciences/Health care"},{"id":47489891,"name":"Health sciences/Medical research"}],"tags":[],"updatedAt":"2025-09-15T16:01:28+00:00","versionOfRecord":{"articleIdentity":"rs-6293118","link":"https://doi.org/10.1038/s41598-025-15661-x","journal":{"identity":"scientific-reports","isVorOnly":false,"title":"Scientific Reports"},"publishedOn":"2025-09-11 15:57:17","publishedOnDateReadable":"September 11th, 2025"},"versionCreatedAt":"2025-05-07 03:03:02","video":"","vorDoi":"10.1038/s41598-025-15661-x","vorDoiUrl":"https://doi.org/10.1038/s41598-025-15661-x","workflowStages":[]},"version":"v1","identity":"rs-6293118","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6293118","identity":"rs-6293118","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}