The diagnostic ability of serum Fe concentrations as a marker of inflammation in horses

The diagnostic ability of serum Fe concentrations as a marker of inflammation in horses | 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 The diagnostic ability of serum Fe concentrations as a marker of inflammation in horses Marina OTSUKA, Yoshiki MURAKAMI, Haruyuki HIRATA, Minoru OKAMOTO, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7656132/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 The present study investigated whether serum Fe concentrations may be used as an inexpensive and simple inflammatory marker in clinical practice as an alternative to existing acute phase proteins (APPs). The control group included 42 healthy adult horses and 20 healthy yearlings. Forty-two horses were enrolled in the inflammatory disease group. Plasma serum amyloid A (SAA) concentrations were significantly higher in the inflammatory disease group than in the control group. When the cut-off value for plasma SAA concentrations was set at > 99.45 µg/dL by a ROC analysis, AUC was 0.960 and sensitivity and specificity were 83.8 and 98.4%, respectively, reconfirming that SAA is an inflammatory marker that diagnoses inflammatory disease with a high probability. In addition, serum Fe concentrations were significantly lower in the inflammatory disease group than in the control group, with AUC = 0.842, indicating a high diagnostic ability. These results suggest that the diagnostic ability of serum Fe concentrations is similar to that of existing APPs and, thus, may be widely applied as an inexpensive inflammatory marker that is easy to measure. acute phase protein horse inflammation iron serum amyloid A Figures Figure 1 Figure 2 Figure 3 INTRODUCTION Inflammatory diseases have a significant impact on the productivity and prognosis of horses. Therefore, veterinarians need to understand the inflammatory state of horses and control it appropriately. To achieve this, it is desirable to incorporate indicators of the inflammatory state, i.e., inflammatory markers, into daily medical practice and use them in treatment. Inflammatory markers are already being used in human and companion animal medicine, and many are classified as acute phase proteins (APPs) [ 1 – 3 ]. APPs are produced in vivo as a result of the systemic response of the innate immune system during various inflammatory processes [ 4 , 5 ]. Representative APPs include alpha-1 acid glycoprotein, C-reactive protein, haptoglobin (HPT), and serum amyloid A (SAA) [ 3 , 4 , 6 ]. The blood concentration of SAA in horses was previously shown to be more than 1,000-fold higher under inflammatory conditions than under physiological conditions and, thus, it has attracted attention as an inflammatory marker [ 2 , 7 ]. SAA is highly inflammatory and reaches its maximum blood concentration within approximately 6 hours after the onset of inflammation; however, it has a short half-life and decreases within 12 hours after inflammation has resolved [ 24 ]. In other words, SAA reflects the real-time inflammatory state in horses. One characteristic of SAA is that its reactivity differs depending on whether the disease is infectious [ 7 ]. Since infectious diseases are inflammatory diseases that are commonly encountered in equine medicine, the differentiation of infectious and non-infectious diseases based on the expression pattern of SAA highlights the significance of its measurement [ 8 ]. Collectively, these findings indicate the importance of measuring APPs in equine clinical medicine. However, APPs are less frequently used as inflammatory markers in equine medicine than in human and companion animal medicine because of the economic constraints of livestock medicine and the limitations of the measurement environment [ 9 , 10 ]. Therefore, for veterinarians to understand the inflammatory state of living organisms in actual clinical cases and use inflammatory markers to select treatment plans, an indicator that is inexpensive, easy to measure, and an alternative to inflammatory markers is needed. It is important to consider not only the proteins induced in the liver by IL-6, but also non-protein substances activated by inflammation as indicators of inflammation, such as trace elements in blood. In the bodies of animals with inflammation, a number of enzymes are responsible for the antioxidant response to reactive oxygen species generated by inflammatory reactions [ 11 – 14 ]. Trace elements called antioxidant elements, such as iron (Fe), zinc (Zn), copper, selenium, and manganese, are located at the center of the structures of these enzymes. Therefore, trace element concentrations may be selected as alternative candidates for inflammation markers. Based on these findings, our research team conducted a screening survey of serum trace element concentrations in cows with peracute mastitis using the Particle-Induced X-ray Emission method [ 15 ], and found that serum Fe and Zn concentrations were significantly lower in cows with peracute mastitis than in healthy cows. The use of Zn concentrations as an indicator represents a better approach to evaluating the state of inflammation; however, current biochemical testing technology requires large testing equipment to measure Zn concentrations in serum. Since few general testing companies are willing to undertake Zn measurements, it does not necessarily meet the requirements of simplicity and low cost as an alternative to inflammation markers. A number of methods are used to measure serum Fe concentrations, all of which are classical and simple. For example, the 2-nitroso-5-(N-propyl-N-sulfopropylamino) phenol (Nitroso-PSAP) method, which was developed for water quality testing, is widely used in the field of clinical biochemistry. A commercially available kit is employed for this measurement, which takes approximately 15 minutes. By implementing it on an automatic blood biochemistry analyzer, it is simple and provides a result within 30 minutes, including pretreatment [ 16 ]. Therefore, many testing companies perform contract testing of serum Fe concentrations at a cost of about 500 yen per sample [ 17 ]. Fe is regulated in vivo by hepcidin in the liver [ 18 – 20 ] Hepcidin is an antimicrobial peptide in humans [ 21 , 22 ]. Similar to other APPs, it is secreted from hepatocytes in response to inflammatory IL-6 [ 19 , 23 , 24 ]. It binds to the Fe-excreting factor ferroportin [ 24 – 26 ] and reduces blood Fe levels. It has been shown to inhibit the transfer of Fe from ferroportin-rich cells, such as intestinal cells, macrophages, hepatocytes, and placental cells [ 27 ], to plasma [ 28 ]. Based on this mechanism, Tsukano et al. [ 29 ] investigated the time course of changes in serum Fe concentrations and hepcidin gene expression in the liver using experimental endotoxin-shocked cattle, and reported that the hepatic mRNA expression levels of IL-6 and hepcidin were significantly higher 2 to 8 hours after an endotoxin challenge than their pre-challenge levels. However, it currently remains unclear whether serum Fe concentrations may replace APPs, which are representative of existing inflammation markers. In other words, APPs must be used as the golden standard to evaluate the responsiveness of serum Fe concentrations to inflammation. Therefore, in the present study, we set existing APPs as the gold standard and clarified the extent to which serum Fe concentrations in horses are similar to the diagnostic results of existing APPs in an evaluation of inflammation in animals with and without an inflammatory condition. We also investigated whether inflammation in horses may be evaluated using serum Fe concentrations. By clarifying whether serum Fe concentrations are useful as an inflammation marker in clinical settings, the present results will contribute to the routine evaluation of inflammation in horses, which is expensive, complicated, and difficult, and will enable the simple diagnosis of and treatment planning for inflammatory diseases, thereby improving production efficiency in production sites. MATERIALS AND METHODS The present study was conducted with the approval of the Rakuno Gakuen University Experimental Animal Committee (approval number: VH18C10). The control group included 42 healthy adult horses (34 Thoroughbreds, 5 Japanese ponies, 1 Welsh Mountain pony, 1 crossbreed, and 1 unknown) and 20 healthy yearlings (Thoroughbreds) kept at farms under the jurisdiction of the Hidaka Branch of the Hokkaido Agricultural Mutual Aid Association, the JRA Hidaka Breeding Farm, and farms near Rakuno Gakuen University. Forty-two horses (21 yearlings and 21 adult horses, all Thoroughbreds) referred to the Hokkaido Agricultural Mutual Aid Association Livestock Advanced Medical Center were enrolled as the inflammatory disease group. Ten-milliliter blood samples were collected from the jugular vein of all study horses and immediately stored in serum separation vacuum blood collection tubes (Venoject II vacuum blood collection tubes; Terumo, Tokyo, Japan) and heparinized vacuum blood collection tubes (Venoject II vacuum blood collection tubes; Terumo, Tokyo, Japan). Blood samples were centrifuged at 1,500 × g at room temperature for 15 minutes to obtain serum and heparinized plasma, respectively, on the same day. Serum and plasma samples were stored at -80°C for later analyses. Plasma SAA concentrations were assessed using an automatic chemical analyzer with an SAA measurement reagent (VET-SAA test 'Eiken' SAA; Eiken Chemical Co., Ltd., Tokyo, Japan). Aspartate transaminase, alanine transaminase (ALT), alkaline phosphatase, γ-glutamyltransferase (γ-GTP), total bilirubin (T-Bil), blood urea nitrogen, and creatinine (Cre) were also measured using an automatic chemical analyzer (Hitachi 7170S; Hitachi, Ltd., Tokyo, Japan). Furthermore, serum Fe concentrations were evaluated by the Nitroso-PSAP method using a commercially available kit (N-assay L Fe-H Nittobo; Nittobo Medical Co., Ltd., Tokyo, Japan). All statistical analyses were performed using IBM SPSS Statistics software v.27 (IBM Co., Somers, NY, USA). Blood test results are shown as the mean ± standard deviation for a normal distribution and medians [minimum-maximum] for a non-normal distribution. The two-sample t- test was used for comparisons between groups if the items were normally distributed, and the Mann-Whitney U-test when items were non-normally distributed. An ROC analysis was used to assess diagnostic performance, and the area under the curve (AUC), the ideal cut-off value, sensitivity, and specificity were calculated. The effect of each item on the difference between the control and inflammatory disease groups was analyzed using a binomial logistic regression analysis. The ideal cut-off value was calculated using the Youden index ( J ), and a value of p < 0.05 was considered to indicate a significant difference. Results Biochemical test results are shown in Table 1. Although significant differences in ALT, γ-GTP, and T-Bil concentrations were observed between the control and inflammatory disease groups ( p < 0.001, p < 0.001, and p = 0.009, respectively), the respective values did not significantly deviate from the reference values [ 30 ], and, thus, data from all study horses were used in statistical analyses. Plasma SAA concentrations were 2.80 [1.00-145] mg/L in the control group and 1782.75 [2.00-6576] mg/L in the inflammatory disease group, which was significantly higher ( p < 0.001). Serum Fe concentrations were 155 [93–441] µg/dL in the control group and 69 [30.0-317.00] µg/dL in the inflammatory disease group, which was significantly lower ( p < 0.001, Fig. 1 ). To examine the relationships between the presence or absence of inflammatory disease and the test items, a binomial logistic regression analysis was performed on all blood test items, and only the plasma concentration of SAA (OR = 1.014 [1.005–1.022]) was associated with inflammatory disease. The diagnostic abilities of plasma SAA concentrations and serum Fe concentrations were evaluated using an ROC analysis. The results obtained showed that the AUC for plasma SAA concentrations was 0.960 and the ideal cut-off value was > 99.45 mg/L. Sensitivity and specificity were 83.8 and 98.4%, respectively. The AUC for serum Fe concentrations was 0.842, and the ideal cut-off value for inflammatory disease was < 103.00 µg/dL. Sensitivity and specificity were 91.9 and 75.7%, respectively (Fig. 2 ). Furthermore, the diagnostic ability, sensitivity, specificity, positive predictive value, and negative predictive value of serum Fe concentrations were evaluated based on the diagnostic results of plasma SAA concentrations in the present study. Serum Fe concentrations had a high diagnostic ability for diagnosing equine inflammatory disease associated with elevated SAA levels, with an AUC of 0.820 and ideal cut-off value of < 95.5 µg/dL. Sensitivity and specificity at this time were 92.5 and 71.9%, respectively, with a positive predictive value of 64.5% and a negative predictive value of 94.5% (Fig. 3 ). DISCUSSION The purpose of the present study was to clarify whether serum Fe concentrations, which are inexpensive and easy to measure, may be used as an alternative indicator to APPs, existing inflammation markers in horses. Since HPT and/or SAA have high diagnostic abilities in horses, similar to human and companion animal medicine, it is considered desirable to use these APPs as a method to objectively evaluate inflammation in the field of veterinary medicine for production animals. However, measurements are expensive and labor-intensive, making it difficult to use APPs as indicators on a daily basis in veterinary medicine for production animals. On the other hand, the concentrations of many trace elements are affected by inflammation, such as antioxidant reactions. A previous study reported that Fe and Zn significantly decreased in an experimental inflammation model in cattle [ 31 ]. Fe plays many roles in enzyme activity and is an essential element for both hosts and pathogens [ 32 ], exists in two reversible redox states: reduced Fe 2+ and oxidized Fe 3+ , it may take different states depending on the environment. Due to these properties, Fe has played many roles as a biocatalyst and the center of redox reactions during the evolution of early life [ 33 ]. Fe is an essential nutrient for pathogens, and pathogens that invade the body attempt to use Fe complexes, such as transferrin and hemoglobin, as nutrients [ 33 ] causes a nutrient shortage for bacteria in the body, leading to a decrease in enzyme activity and the inhibition of growth; therefore, it functions as a bacteriostatic immune system [ 16 ]. A general method for measuring serum Fe concentrations has been established, and even when requested to do so by a testing institution, it may be measured for approximately 500 yen per sample [ 31 ]. Therefore, in veterinary medicine for livestock, serum Fe concentrations are useful as an inflammation marker because they are inexpensive to measure. Furthermore, a decrease in serum Fe concentrations associated with inflammatory diseases has been observed in clinical cases of veterinary medicine other than cattle. For example, serum Fe concentrations significantly decreased not only during systemic inflammation, but also during local inflammation in horses [ 34 – 36 ]. Furthermore, a reduction was observed in serum Fe concentrations 24 hours after surgery [ 37 ], while a significant decrease was detected within 24 hours after experimentally inducing inflammation [ 17 , 38 , 39 ]. Therefore, serum Fe concentrations decrease during inflammation regardless of the species, indicating their potential as an inflammation marker that may be applied clinically. If serum Fe concentrations replace APPs as an inexpensive and easy inflammatory marker, they may be used in veterinary medicine for production animals, which is often limited by financial, labor, and time constraints, making it possible to easily diagnose inflammatory diseases and develop treatment plans. Plasma SAA concentrations were significantly higher in the inflammatory disease group than in the control group. In addition, when the cut-off value for plasma SAA concentrations was set at > 99.45 µg/dL by the ROC analysis, the AUC was 0.960, and sensitivity and specificity were 83.8 and 98.4%, respectively, reconfirming that SAA is an inflammatory marker that diagnoses inflammatory disease with a high probability. In horses, SAA shows up to 1,000-fold increases within 6 hours after an inflammatory stimulation and has a very short half-life of 30 to 120 minutes [ 40 – 42 ] making it an excellent inflammatory marker with a concentration that decreases within 12 hours after the inflammatory stimulation has disappeared. In addition, the results of the binomial logistic regression analysis showed a very strong correlation with inflammatory diseases. Serum Fe concentrations were significantly lower in the inflammatory disease group than in the control group, with a high diagnostic ability of AUC = 0.842; however, a relationship with inflammatory disease was not observed in the binomial logistic regression analysis. Although median serum Fe concentrations differed between the inflammatory disease and control groups, the concentration distributions of each group overlapped, making it difficult to clearly distinguish between these groups at a specific Fe concentration. Therefore, we recommend using Fe as a screening tool rather than as a marker of inflammation. The present study investigated the potential of Fe as an alternative to existing inflammatory markers, particularly SAA, for the diagnosis of inflammation in horses. The results obtained suggest that Fe is a marker with high sensitivity and a high negative predictive value for diagnosing inflammatory diseases, whereas its specificity is slightly lower than that of SAA. These results suggest that serum Fe concentrations are useful as a screening test item for diseases with a low prevalence in horses. Serum Fe concentrations may be affected by [ 43 ]. The consistently low specificity shown in this study may be due to the large number of confounding factors. The high sensitivity and negative predictive value of Fe make it less likely to produce false negatives and, thus, it is regarded as a very useful test item, particularly in the diagnosis of diseases with a low prevalence, where negative results are often obtained. In addition, as described above, the measurement of plasma Fe concentrations has the characteristics of being easier and less expensive than existing APPs, which suggests that it may be performed with lower costs and less labor than existing APPs when the number of tests is high and the probability of disease is low, such as the screening of all cattle and blood tests on animals introduced to the farm. Declarations CONFLICT OF INTERSTING The authors have no conflicts of interest directly relevant to the content of this article. Funding: The authors declare that no funds, grants, or other support were received during the preparation of this manuscript. Author Contributions: All authors contributed to the study conception and design. Material preparation, data collection and analysis were performed by Marina Otsuka, Yoshiki Murakami and Kazuyuki Suzuki. The first draft of the manuscript was written by Marina Otsuka and all authors commented on previous versions of the manuscript. All authors read and approved the final manuscript. Ethics Approval: All procedures were reviewed and approved by the Guide for the Care and Use of Laboratory Animals of the School of Veterinary Medicine at Rakuno Gakuen University (Approval#: VH18C10). Competing Interests: The authors declare no competing interests. References Baumann H, Gauldie J (1994) The acute phase response. Immunol Today 15:74-80. https://doi.10.1016/0167-5699(94)90137-6 Cray C (2012) Acute phase proteins in animals. Prog Mol Biol Transl Sci 105:113-150. https://doi.10.1016/B978-0-12-394596-9.00005-6 Eckersall PD, Conner JG (1988) Bovine and canine acute phase proteins. 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Supplementary Files OtsukaTable1.xlsx Table 1 Results of biochemical tests in the control and inflammatory disease groups 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. 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. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. 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-7656132","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":526640979,"identity":"cc45f943-c5ef-47e7-b713-4cb822285302","order_by":0,"name":"Marina OTSUKA","email":"","orcid":"","institution":"Rakuno Gakuen University","correspondingAuthor":false,"prefix":"","firstName":"Marina","middleName":"","lastName":"OTSUKA","suffix":""},{"id":526640980,"identity":"bd900c4d-e11e-4ed4-aa81-4ee1ab641809","order_by":1,"name":"Yoshiki MURAKAMI","email":"","orcid":"","institution":"Rakuno Gakuen 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1","display":"","copyAsset":false,"role":"figure","size":206328,"visible":true,"origin":"","legend":"\u003cp\u003eComparison of plasma SAA and serum Fe concentrations between control and inflammatory disease groups\u003c/p\u003e","description":"","filename":"OtsukaFig1.png","url":"https://assets-eu.researchsquare.com/files/rs-7656132/v1/2111d7172369bd07c5b6fc33.png"},{"id":93140782,"identity":"2602f0cc-2193-49ef-82d2-5ed633dd2369","added_by":"auto","created_at":"2025-10-09 13:01:42","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":230914,"visible":true,"origin":"","legend":"\u003cp\u003eROC analysis of plasma SAA and serum Fe concentrations in the inflammatory disease group\u003c/p\u003e","description":"","filename":"OtsukaFig2.png","url":"https://assets-eu.researchsquare.com/files/rs-7656132/v1/465cd530b2d5663b415f6842.png"},{"id":93138956,"identity":"c1c1c6fc-57db-459d-b31c-98d24990f66f","added_by":"auto","created_at":"2025-10-09 12:45:42","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":286280,"visible":true,"origin":"","legend":"\u003cp\u003eROC analysis of serum Fe concentrations in horses that tested positive at the cut-off value for plasma SAA concentrations\u003c/p\u003e","description":"","filename":"OtsukaFig3.png","url":"https://assets-eu.researchsquare.com/files/rs-7656132/v1/7ae0fec176c85818dbae3dab.png"},{"id":99314304,"identity":"186b2627-2e10-4305-9f99-3dcb229ff652","added_by":"auto","created_at":"2025-12-31 16:21:08","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1021325,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7656132/v1/92b42ec3-05a0-4739-9944-4d3ebe9f9dc4.pdf"},{"id":93140119,"identity":"7861520d-0231-48aa-9875-8f71c13558cf","added_by":"auto","created_at":"2025-10-09 12:53:42","extension":"xlsx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":10727,"visible":true,"origin":"","legend":"\u003cp\u003eTable 1 Results of biochemical tests in the control and inflammatory disease groups\u003c/p\u003e","description":"","filename":"OtsukaTable1.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-7656132/v1/422a008f4e13bb775756b5a2.xlsx"}],"financialInterests":"No competing interests reported.","formattedTitle":"The diagnostic ability of serum Fe concentrations as a marker of inflammation in horses","fulltext":[{"header":"INTRODUCTION","content":"\u003cp\u003eInflammatory diseases have a significant impact on the productivity and prognosis of horses. Therefore, veterinarians need to understand the inflammatory state of horses and control it appropriately. To achieve this, it is desirable to incorporate indicators of the inflammatory state, i.e., inflammatory markers, into daily medical practice and use them in treatment.\u003c/p\u003e\u003cp\u003eInflammatory markers are already being used in human and companion animal medicine, and many are classified as acute phase proteins (APPs) [\u003cspan additionalcitationids=\"CR2\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. APPs are produced \u003cem\u003ein vivo\u003c/em\u003e as a result of the systemic response of the innate immune system during various inflammatory processes [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Representative APPs include alpha-1 acid glycoprotein, C-reactive protein, haptoglobin (HPT), and serum amyloid A (SAA) [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. The blood concentration of SAA in horses was previously shown to be more than 1,000-fold higher under inflammatory conditions than under physiological conditions and, thus, it has attracted attention as an inflammatory marker [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. SAA is highly inflammatory and reaches its maximum blood concentration within approximately 6 hours after the onset of inflammation; however, it has a short half-life and decreases within 12 hours after inflammation has resolved [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. In other words, SAA reflects the real-time inflammatory state in horses.\u003c/p\u003e\u003cp\u003eOne characteristic of SAA is that its reactivity differs depending on whether the disease is infectious [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Since infectious diseases are inflammatory diseases that are commonly encountered in equine medicine, the differentiation of infectious and non-infectious diseases based on the expression pattern of SAA highlights the significance of its measurement [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eCollectively, these findings indicate the importance of measuring APPs in equine clinical medicine. However, APPs are less frequently used as inflammatory markers in equine medicine than in human and companion animal medicine because of the economic constraints of livestock medicine and the limitations of the measurement environment [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. Therefore, for veterinarians to understand the inflammatory state of living organisms in actual clinical cases and use inflammatory markers to select treatment plans, an indicator that is inexpensive, easy to measure, and an alternative to inflammatory markers is needed.\u003c/p\u003e\u003cp\u003eIt is important to consider not only the proteins induced in the liver by IL-6, but also non-protein substances activated by inflammation as indicators of inflammation, such as trace elements in blood. In the bodies of animals with inflammation, a number of enzymes are responsible for the antioxidant response to reactive oxygen species generated by inflammatory reactions [\u003cspan additionalcitationids=\"CR12 CR13\" citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. Trace elements called antioxidant elements, such as iron (Fe), zinc (Zn), copper, selenium, and manganese, are located at the center of the structures of these enzymes. Therefore, trace element concentrations may be selected as alternative candidates for inflammation markers. Based on these findings, our research team conducted a screening survey of serum trace element concentrations in cows with peracute mastitis using the Particle-Induced X-ray Emission method [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e], and found that serum Fe and Zn concentrations were significantly lower in cows with peracute mastitis than in healthy cows. The use of Zn concentrations as an indicator represents a better approach to evaluating the state of inflammation; however, current biochemical testing technology requires large testing equipment to measure Zn concentrations in serum. Since few general testing companies are willing to undertake Zn measurements, it does not necessarily meet the requirements of simplicity and low cost as an alternative to inflammation markers.\u003c/p\u003e\u003cp\u003eA number of methods are used to measure serum Fe concentrations, all of which are classical and simple. For example, the 2-nitroso-5-(N-propyl-N-sulfopropylamino) phenol (Nitroso-PSAP) method, which was developed for water quality testing, is widely used in the field of clinical biochemistry. A commercially available kit is employed for this measurement, which takes approximately 15 minutes. By implementing it on an automatic blood biochemistry analyzer, it is simple and provides a result within 30 minutes, including pretreatment [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. Therefore, many testing companies perform contract testing of serum Fe concentrations at a cost of about 500 yen per sample [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eFe is regulated \u003cem\u003ein vivo\u003c/em\u003e by hepcidin in the liver [\u003cspan additionalcitationids=\"CR19\" citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e] Hepcidin is an antimicrobial peptide in humans [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. Similar to other APPs, it is secreted from hepatocytes in response to inflammatory IL-6 [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. It binds to the Fe-excreting factor ferroportin [\u003cspan additionalcitationids=\"CR25\" citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e] and reduces blood Fe levels. It has been shown to inhibit the transfer of Fe from ferroportin-rich cells, such as intestinal cells, macrophages, hepatocytes, and placental cells [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e], to plasma [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. Based on this mechanism, Tsukano et al. [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e] investigated the time course of changes in serum Fe concentrations and hepcidin gene expression in the liver using experimental endotoxin-shocked cattle, and reported that the hepatic mRNA expression levels of IL-6 and hepcidin were significantly higher 2 to 8 hours after an endotoxin challenge than their pre-challenge levels.\u003c/p\u003e\u003cp\u003eHowever, it currently remains unclear whether serum Fe concentrations may replace APPs, which are representative of existing inflammation markers. In other words, APPs must be used as the golden standard to evaluate the responsiveness of serum Fe concentrations to inflammation. Therefore, in the present study, we set existing APPs as the gold standard and clarified the extent to which serum Fe concentrations in horses are similar to the diagnostic results of existing APPs in an evaluation of inflammation in animals with and without an inflammatory condition. We also investigated whether inflammation in horses may be evaluated using serum Fe concentrations. By clarifying whether serum Fe concentrations are useful as an inflammation marker in clinical settings, the present results will contribute to the routine evaluation of inflammation in horses, which is expensive, complicated, and difficult, and will enable the simple diagnosis of and treatment planning for inflammatory diseases, thereby improving production efficiency in production sites.\u003c/p\u003e"},{"header":"MATERIALS AND METHODS","content":"\u003cp\u003e The present study was conducted with the approval of the Rakuno Gakuen University Experimental Animal Committee (approval number: VH18C10). The control group included 42 healthy adult horses (34 Thoroughbreds, 5 Japanese ponies, 1 Welsh Mountain pony, 1 crossbreed, and 1 unknown) and 20 healthy yearlings (Thoroughbreds) kept at farms under the jurisdiction of the Hidaka Branch of the Hokkaido Agricultural Mutual Aid Association, the JRA Hidaka Breeding Farm, and farms near Rakuno Gakuen University. Forty-two horses (21 yearlings and 21 adult horses, all Thoroughbreds) referred to the Hokkaido Agricultural Mutual Aid Association Livestock Advanced Medical Center were enrolled as the inflammatory disease group. Ten-milliliter blood samples were collected from the jugular vein of all study horses and immediately stored in serum separation vacuum blood collection tubes (Venoject II vacuum blood collection tubes; Terumo, Tokyo, Japan) and heparinized vacuum blood collection tubes (Venoject II vacuum blood collection tubes; Terumo, Tokyo, Japan). Blood samples were centrifuged at 1,500 \u0026times; \u003cem\u003eg\u003c/em\u003e at room temperature for 15 minutes to obtain serum and heparinized plasma, respectively, on the same day. Serum and plasma samples were stored at -80\u0026deg;C for later analyses. Plasma SAA concentrations were assessed using an automatic chemical analyzer with an SAA measurement reagent (VET-SAA test 'Eiken' SAA; Eiken Chemical Co., Ltd., Tokyo, Japan). Aspartate transaminase, alanine transaminase (ALT), alkaline phosphatase, γ-glutamyltransferase (γ-GTP), total bilirubin (T-Bil), blood urea nitrogen, and creatinine (Cre) were also measured using an automatic chemical analyzer (Hitachi 7170S; Hitachi, Ltd., Tokyo, Japan). Furthermore, serum Fe concentrations were evaluated by the Nitroso-PSAP method using a commercially available kit (N-assay L Fe-H Nittobo; Nittobo Medical Co., Ltd., Tokyo, Japan).\u003c/p\u003e\u003cp\u003eAll statistical analyses were performed using IBM SPSS Statistics software v.27 (IBM Co., Somers, NY, USA). Blood test results are shown as the mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation for a normal distribution and medians [minimum-maximum] for a non-normal distribution. The two-sample \u003cem\u003et-\u003c/em\u003etest was used for comparisons between groups if the items were normally distributed, and the Mann-Whitney U-test when items were non-normally distributed. An ROC analysis was used to assess diagnostic performance, and the area under the curve (AUC), the ideal cut-off value, sensitivity, and specificity were calculated. The effect of each item on the difference between the control and inflammatory disease groups was analyzed using a binomial logistic regression analysis. The ideal cut-off value was calculated using the Youden index (\u003cem\u003eJ\u003c/em\u003e), and a value of \u003cem\u003ep\u0026thinsp;\u0026lt;\u003c/em\u003e\u0026thinsp;0.05 was considered to indicate a significant difference.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003eBiochemical test results are shown in Table\u0026nbsp;1. Although significant differences in ALT, γ-GTP, and T-Bil concentrations were observed between the control and inflammatory disease groups (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001, and \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.009, respectively), the respective values did not significantly deviate from the reference values [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e], and, thus, data from all study horses were used in statistical analyses.\u003c/p\u003e\u003cp\u003ePlasma SAA concentrations were 2.80 [1.00-145] mg/L in the control group and 1782.75 [2.00-6576] mg/L in the inflammatory disease group, which was significantly higher (\u003cem\u003ep\u0026thinsp;\u0026lt;\u003c/em\u003e\u0026thinsp;0.001). Serum Fe concentrations were 155 [93\u0026ndash;441] \u0026micro;g/dL in the control group and 69 [30.0-317.00] \u0026micro;g/dL in the inflammatory disease group, which was significantly lower (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001, Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). To examine the relationships between the presence or absence of inflammatory disease and the test items, a binomial logistic regression analysis was performed on all blood test items, and only the plasma concentration of SAA (OR\u0026thinsp;=\u0026thinsp;1.014 [1.005\u0026ndash;1.022]) was associated with inflammatory disease.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eThe diagnostic abilities of plasma SAA concentrations and serum Fe concentrations were evaluated using an ROC analysis. The results obtained showed that the AUC for plasma SAA concentrations was 0.960 and the ideal cut-off value was \u0026gt;\u0026thinsp;99.45 mg/L. Sensitivity and specificity were 83.8 and 98.4%, respectively. The AUC for serum Fe concentrations was 0.842, and the ideal cut-off value for inflammatory disease was \u0026lt;\u0026thinsp;103.00 \u0026micro;g/dL. Sensitivity and specificity were 91.9 and 75.7%, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Furthermore, the diagnostic ability, sensitivity, specificity, positive predictive value, and negative predictive value of serum Fe concentrations were evaluated based on the diagnostic results of plasma SAA concentrations in the present study. Serum Fe concentrations had a high diagnostic ability for diagnosing equine inflammatory disease associated with elevated SAA levels, with an AUC of 0.820 and ideal cut-off value of \u0026lt;\u0026thinsp;95.5 \u0026micro;g/dL. Sensitivity and specificity at this time were 92.5 and 71.9%, respectively, with a positive predictive value of 64.5% and a negative predictive value of 94.5% (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e"},{"header":"DISCUSSION","content":"\u003cp\u003eThe purpose of the present study was to clarify whether serum Fe concentrations, which are inexpensive and easy to measure, may be used as an alternative indicator to APPs, existing inflammation markers in horses. Since HPT and/or SAA have high diagnostic abilities in horses, similar to human and companion animal medicine, it is considered desirable to use these APPs as a method to objectively evaluate inflammation in the field of veterinary medicine for production animals. However, measurements are expensive and labor-intensive, making it difficult to use APPs as indicators on a daily basis in veterinary medicine for production animals.\u003c/p\u003e\u003cp\u003eOn the other hand, the concentrations of many trace elements are affected by inflammation, such as antioxidant reactions. A previous study reported that Fe and Zn significantly decreased in an experimental inflammation model in cattle [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. Fe plays many roles in enzyme activity and is an essential element for both hosts and pathogens [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e], exists in two reversible redox states: reduced Fe\u003csup\u003e2+\u003c/sup\u003e and oxidized Fe\u003csup\u003e3+\u003c/sup\u003e, it may take different states depending on the environment. Due to these properties, Fe has played many roles as a biocatalyst and the center of redox reactions during the evolution of early life [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. Fe is an essential nutrient for pathogens, and pathogens that invade the body attempt to use Fe complexes, such as transferrin and hemoglobin, as nutrients [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e] causes a nutrient shortage for bacteria in the body, leading to a decrease in enzyme activity and the inhibition of growth; therefore, it functions as a bacteriostatic immune system [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. A general method for measuring serum Fe concentrations has been established, and even when requested to do so by a testing institution, it may be measured for approximately 500 yen per sample [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. Therefore, in veterinary medicine for livestock, serum Fe concentrations are useful as an inflammation marker because they are inexpensive to measure. Furthermore, a decrease in serum Fe concentrations associated with inflammatory diseases has been observed in clinical cases of veterinary medicine other than cattle. For example, serum Fe concentrations significantly decreased not only during systemic inflammation, but also during local inflammation in horses [\u003cspan additionalcitationids=\"CR35\" citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. Furthermore, a reduction was observed in serum Fe concentrations 24 hours after surgery [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e], while a significant decrease was detected within 24 hours after experimentally inducing inflammation [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e, \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. Therefore, serum Fe concentrations decrease during inflammation regardless of the species, indicating their potential as an inflammation marker that may be applied clinically. If serum Fe concentrations replace APPs as an inexpensive and easy inflammatory marker, they may be used in veterinary medicine for production animals, which is often limited by financial, labor, and time constraints, making it possible to easily diagnose inflammatory diseases and develop treatment plans.\u003c/p\u003e\u003cp\u003ePlasma SAA concentrations were significantly higher in the inflammatory disease group than in the control group. In addition, when the cut-off value for plasma SAA concentrations was set at \u0026gt;\u0026thinsp;99.45 \u0026micro;g/dL by the ROC analysis, the AUC was 0.960, and sensitivity and specificity were 83.8 and 98.4%, respectively, reconfirming that SAA is an inflammatory marker that diagnoses inflammatory disease with a high probability. In horses, SAA shows up to 1,000-fold increases within 6 hours after an inflammatory stimulation and has a very short half-life of 30 to 120 minutes [\u003cspan additionalcitationids=\"CR41\" citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e] making it an excellent inflammatory marker with a concentration that decreases within 12 hours after the inflammatory stimulation has disappeared. In addition, the results of the binomial logistic regression analysis showed a very strong correlation with inflammatory diseases.\u003c/p\u003e\u003cp\u003eSerum Fe concentrations were significantly lower in the inflammatory disease group than in the control group, with a high diagnostic ability of AUC\u0026thinsp;=\u0026thinsp;0.842; however, a relationship with inflammatory disease was not observed in the binomial logistic regression analysis. Although median serum Fe concentrations differed between the inflammatory disease and control groups, the concentration distributions of each group overlapped, making it difficult to clearly distinguish between these groups at a specific Fe concentration. Therefore, we recommend using Fe as a screening tool rather than as a marker of inflammation.\u003c/p\u003e\u003cp\u003eThe present study investigated the potential of Fe as an alternative to existing inflammatory markers, particularly SAA, for the diagnosis of inflammation in horses. The results obtained suggest that Fe is a marker with high sensitivity and a high negative predictive value for diagnosing inflammatory diseases, whereas its specificity is slightly lower than that of SAA. These results suggest that serum Fe concentrations are useful as a screening test item for diseases with a low prevalence in horses.\u003c/p\u003e\u003cp\u003eSerum Fe concentrations may be affected by [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]. The consistently low specificity shown in this study may be due to the large number of confounding factors. The high sensitivity and negative predictive value of Fe make it less likely to produce false negatives and, thus, it is regarded as a very useful test item, particularly in the diagnosis of diseases with a low prevalence, where negative results are often obtained. In addition, as described above, the measurement of plasma Fe concentrations has the characteristics of being easier and less expensive than existing APPs, which suggests that it may be performed with lower costs and less labor than existing APPs when the number of tests is high and the probability of disease is low, such as the screening of all cattle and blood tests on animals introduced to the farm.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eCONFLICT OF INTERSTING\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors have no conflicts of interest directly relevant to the content of this article.\u003c/p\u003e\u003cp\u003eFunding: The authors declare that no funds, grants, or other support were received during the preparation of this manuscript.\u003c/p\u003e\n\u003cp\u003eAuthor Contributions: All authors contributed to the study conception and design. Material preparation, data collection and analysis were performed by Marina Otsuka, Yoshiki Murakami and Kazuyuki Suzuki. The first draft of the manuscript was written by Marina Otsuka and all authors commented on previous versions of the manuscript. All authors read and approved the final manuscript.\u003c/p\u003e\n\u003cp\u003eEthics Approval: All procedures were reviewed and approved by the Guide for the Care and Use of Laboratory Animals of the School of Veterinary Medicine at Rakuno Gakuen University (Approval#: VH18C10).\u003c/p\u003e\n\u003cp\u003eCompeting Interests: The authors declare no competing interests.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eBaumann H, Gauldie J (1994) The acute phase response. Immunol Today 15:74-80. https://doi.10.1016/0167-5699(94)90137-6\u003c/li\u003e\n\u003cli\u003eCray C (2012) Acute phase proteins in animals. 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Academic Press\u003c/li\u003e\n\u003c/ol\u003e"},{"header":"Table 1","content":"\u003cp\u003eTable 1 is available in the Supplementary Files section.\u003c/p\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":"acute phase protein, horse, inflammation, iron, serum amyloid A","lastPublishedDoi":"10.21203/rs.3.rs-7656132/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7656132/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThe present study investigated whether serum Fe concentrations may be used as an inexpensive and simple inflammatory marker in clinical practice as an alternative to existing acute phase proteins (APPs). The control group included 42 healthy adult horses and 20 healthy yearlings. Forty-two horses were enrolled in the inflammatory disease group. Plasma serum amyloid A (SAA) concentrations were significantly higher in the inflammatory disease group than in the control group. When the cut-off value for plasma SAA concentrations was set at \u0026gt;\u0026thinsp;99.45 \u0026micro;g/dL by a ROC analysis, AUC was 0.960 and sensitivity and specificity were 83.8 and 98.4%, respectively, reconfirming that SAA is an inflammatory marker that diagnoses inflammatory disease with a high probability. In addition, serum Fe concentrations were significantly lower in the inflammatory disease group than in the control group, with AUC\u0026thinsp;=\u0026thinsp;0.842, indicating a high diagnostic ability. These results suggest that the diagnostic ability of serum Fe concentrations is similar to that of existing APPs and, thus, may be widely applied as an inexpensive inflammatory marker that is easy to measure.\u003c/p\u003e","manuscriptTitle":"The diagnostic ability of serum Fe concentrations as a marker of inflammation in horses","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-10-09 12:45:37","doi":"10.21203/rs.3.rs-7656132/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":"3ceae004-4423-43b6-9a31-72b87cd1656e","owner":[],"postedDate":"October 9th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-12-27T04:08:43+00:00","versionOfRecord":[],"versionCreatedAt":"2025-10-09 12:45:37","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7656132","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7656132","identity":"rs-7656132","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}