Urine Myoglobin is Associated with Different Severities and Depths for Burn Patients

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However, the identification of burn depth based upon clinical assessment was not accurate. It still needs more biomarkers to rapidly distinguish different depths for burn specialists. The potential value of urine myoglobin (UM) is still unknown in burn patients. Patients and methods The urine of 30 burn patients and 10 Healthy volunteers (Discover set) were collected from January 2017 to December 2020. Urine proteomics of all samples was identified by Label-free mass spectrometry and functional analysis for differential expression proteins (DEPs) was also analyzed. The Receiver Operating Characteristic curve to evaluate the value of UM from another 67 burn patients (Validation set). Results A total of 8253 peptides and 1465 proteins were identified in four groups. For severe burn patients, 402 DEPs (279 up-regulated and 123 down-regulated) were identified and functional analysis demonstrated that neutrophil-mediated immunity, response to stimulus, and complement and coagulation cascades pathway were the most enrichment function. UM had significant differences in burn patients with different severities and depths (Discover and Validation set; P <0.05). UM as a novel biomarker could rapidly distinguish different depths for burn patients (Discover set, AUC=0.8203, 95%CI: 0.6448~0.9957; Validation set, AUC=0.7393, 95%CI: 0.618~0.8606). Conclusion The urine DEPs were mainly identified in the severe group. UM might as a novel biomarker help burn specialists to rapidly determine different depths for burn patients. Burns Urine Proteomics Burn Depth Severity Label-free Mass Spectrometry Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Introduction Approximately 800,000 people were burned each year in China, and about half of patients need to be hospitalized[ 1 ]. There were 3275 burn patients who eventually died in the USA in 2016 and also face serious challenges to decrease the rate of mortality[ 2 ]. The depths of burn were commonly be divided into three degrees, including superficial (I degree), partial-thickness (II degrees), and full-thickness (III degrees). The depth of the burn has a tremendous impact on the course of treatment and prognosis. Patients with different depths have been received various treatments, such as observation, common treatment, and skin grafting[ 3 ]. When it comes to the diagnostics of burn depth, the main challenge is the ability to distinguish between superficial partial-thickness and deep partial-thickness burns, which impact the dermis to varying degrees[ 4 , 5 ]. The significance of distinguishing partial-thickness burns and full-thickness burns was influenced the critical clinical decision that whether a patient should be allowed to heal on their own or if the wound should be surgically excised and replaced with a graft[ 6 ]. Burn depth analysis has traditionally been estimated via physical examination by the burn specialist. The surgeon would assess pain and tenderness, formation of blisters, the appearance of the dermis, color, and if the skin blanched when pressure was applied. However, this method has been shown to be only 60 to 75% accurate in correctly identifying the depth[ 7 ]. Many technologies have been emerged to distinguish the depth of burn, including radioactive phosphorus, vital dyes, and thermography[ 8 – 10 ]. The most recent wave of depth analysis techniques includes laser Doppler imaging and indocyanine green video angiography [ 11 ]. However, based on clinical assessment and various technologies are still not accurately assessing burn depth. Therefore, it still needs more biomarkers to easily distinguish different depths for burn patients and make an early clinical decision. Mass spectrometry-based proteomic analysis has been being a powerful screening tool for the early detection of renal function and cancer-related biomarkers. This analysis has been successfully utilized to identify protein biomarkers from body fluids [ 12 – 14 ]. Unlike blood samples, urine could be collected non-invasively and timely, moreover, urine could better reflect the changes in the human body. Besides, the protein compositions of urine samples are relatively simple, stable, and easier to analysis [ 15 ]. Based on these, Label-free mass spectrometry (MS) was used to analyze the changes in urine proteins between burn patients with different severity and healthy volunteers. bioinformatics analysis was also carried out for different expression of proteins (DEPs). Our study is to find specific urine proteins for different severities and depths and further evaluate the potential clinical value, which may lay a foundation for potential biomarkers for burn patients. Patients And Methods Patients Thirty burn patients (10 mild, 10 moderate, and 10 severe), who were hospitalized for treatment within 24 hours after burned, were selected before they underwent treatment at Beijing Shijitan Hospital, Capital Medical University from April 2017 to December 2020. Exclusion criteria: patients whose age was less than 18 or more than 65 years old were excluded. Patients who have chronic diseases such as chronic kidney disease, diabetes, hyperlipidemia, rheumatism, hepatitis, history of cancers, and blood system diseases were also excluded. Besides, Patients with the usage history of medicine 2 weeks prior to the collection of the urine sample were also excluded. The detailed information for our study was shown in Fig. 1 . According to the classification proposed by the burn conference, the mild group refers to Ⅱ degree burn patients who had < 9% total body surface area (TBSA) burn; moderate group refers to Ⅱ degree burn patients who had 10 ~ 29% TBSA burn; severe group refers to 30%~49% TBSA burn (Ⅱ degree) or 10%~19% TBSA burn (Ⅲ degree), or the total burn area is less than 30%, but one of the following conditions exists: patients with severe general condition or shock; patients with multiple injuries or combined injury; patients with moderate or severe inhalation injury; especially severe burn refers to total area ≥ 50% or Ⅲ degree burn area ≥ 20%. The detailed clinical features were shown in Table 1 . Table 1 Demographics and clinical characteristics of burn patients and HC Characteristics Discovery cohort Validation cohort HC (N = 10) Mild (N = 10) Moderate (N = 10) Severe (N = 10) P value Mild and Moderate (N = 33) Severe (N = 34) P value Age, range (median) 23 ~ 46 (33.5) 28 ~ 54 (36.0) 24 ~ 48 (35.0) 24 ~ 56 (34.0) 0.519 23 ~ 67 (46) 21 ~ 66 (40) 0.214 Gender, male, n (%) 5 (50%) 4 (40) 5 (50) 5 (50) 1.0 26 (78.78) 28 (82.35) 0.765 Total protein (g/L) 57.8 ~ 75.9 (66.15) 62.8 ~ 79.5 (73.4) 52.5 ~ 78.0(63.55) 32.4 ~ 53.1(49.55) 0.001 53.3 ~ 75.9 (62.7) 37.8 ~ 71.8(56.35) 0.001 ALB (g/L) 31.7 ~ 47.4 (38.05) 36.2 ~ 50.4 (44.1) 33.1 ~ 44.9 (41.3) 15.5 ~ 29 (26.0) 0.001 26.2 ~ 47.4 (41.3) 18 ~ 44.5(30.35) 0.001 BUN 3.52 ~ 9.12 (5.09) 3.9 ~ 7.12 (5.775) 1.8 ~ 6.88 (5.22) 4.1 ~ 9.5 (6.4) 0.089 3.2 ~ 9.12 (4.99) 2.7 ~ 16.5(5.7) 0.233 Serum creatinine 47 ~ 78 (59.7) 38 ~ 81 (54) 42.5 ~ 70 (50.6) 36.9 ~ 178.1(73.95) 0.496 41 ~ 97 (61) 40.3 ~ 116.8(60.2) 0.792 Note : a: P value was compared between severe and HC group. TP: total protein; ALB: albumin. To ensure the accuracy and comprehensiveness of reporting in this case-control biomarker study. ten healthy volunteers were selected as the healthy control group (HC) and the detailed clinical features were also shown in Table 1 . Evaluation of burn depth Burn depth evaluation has traditionally been based on the histological assessment of dermal microvascular occlusion in burn biopsies in our hospital. The analysis of UM In the present study, each patient’s midstream urine was obtained before treatment and the value of urine MB was tested by Beckman ACCESS2, USA. The test of urine MB was carried out under the guidance of the instructions. Urine samples collection Midstream urine samples were collected in the morning into dry and clean containers from all volunteers. Immediately after collection, urine samples were centrifuged at 4000r/min for 5 minutes to remove cell debris and casts. Then we divided the supernatants into aliquots and froze them at -80°C until use. Protein extraction and digestion To found more urine proteins, ten samples in each group were mixed into one sample and was repeatedly tested three times. The detailed information for test as followed: The frozen urine sample was reconstituted at 4°C, centrifuged at 2000g for 10min, and the protein concentration was determined using Bradford method, rest was frozen to -80℃. Take 10µg of each sample for SDS-PAGE electrophoresis, stain with Coomassie blue for 30 minutes, and decolor until the background is clear. The gel was then washed by destained buffer and treated with ACN as above. When the enzyme solution is completely absorbed by colloidal particles, then made up to 100 µL with 25 mM ABC, proteins were digested overnight at 37℃. Next day, after centrifugation in low speed, the supernatant was collected, 200 µL of ACN was added, put under vortex and mixed. the supernatant was extracted in 100 µL of 0.1% formic acid (FA). Combined the supernatant and centrifuged at 12000 g for 5 min at room temperature and then lyophilized. The powder was dissolved and mixed in 0.1% of formic acid. The supernatant was slowly loaded to the C18 desalting column, washed with washing solution (0.1% FA, 3% ACN) 3 times, then eluted twice by some elution buffer (0.1% FA, 70% ACN). The eluents were collected and lyophilized. Peptide identification, quantitative profiling and statistics by LC–MS/MS Mobile phase A (100% water, 0.1% formic acid) and B solution (100% acetonitrile, 0.1% formic acid) were prepared. Peptides were separated in a analytical column, using a linear gradient elution. The separated peptides were analyzed by Orbitrap Fusion mass spectrometer (Thermo Fisher) and Full scan range from m/z 350 to 1550 with resolution of 120000 (at m/z 200), an automatic gain control (AGC) target value was 2×10 5 and a maximum ion injection time was 50 ms. The resulting spectra from each fraction were searched separately against human database by the search engines MaxQuant. The MS/MS spectra results were queried against the SwissProt human database within Uniprot ( www.uniprot.org ) using the Proteome Discoverer software suite (v2.1, Thermo Fisher Scientific). Searches were performed using a peptide tolerance of 20 ppm and a product ion tolerance of 0.05 Da. At the protein level, a 1% FDR was used as a filter, and each protein contained at least 1 unique peptide. After filtering the results as described above, the peptide abundances in the different reporter ion channels of the MS/MS scan were normalized. The protein abundance ratios were based on unique peptide results and proteins with a fold change ≥ 1.5 were considered significantly altered. Functional evaluation and pathway analysis Online resources such as Database for Annotation, Visualization, and Integrated Discovery (Metascape: https://metascape.org ) and Kyoto Encyclopedia of Genes and Genomes (KEGG: https://www.genome.jp),an d Gene ontology: http://geneontology.org ) were applied to investigate gene ontology and signaling pathways of candidate biomarkers. STRING protein-protein interactions network analysis ( https://string-db.org ) was used to determine the relationships and net-works between identified proteins. Statistical analysis Statistical analyses were performed R 4.0.1 version and visualized by GraphPad Prism 8.0 (GraphPad Software, San Diego, California, USA). Data were expressed as median (range) and were tested by ANOVA test and Student-Newman-Keuls (SNK) test. The results were considered to be statistically significant at P < 0.05. Results Demographic characteristics of burn patients and HC There were no significant differences in age and gender in each group. In discover set, compared with HC, patients with mild or moderate only had lower level of TP and other features were not found. However, burn patients with severe had lower level of TP and ALB ( P < 0.05, Table 1 ) than HC. In validation set, there were also significant difference in TP and ALB between severe and non-severe group ( P < 0.05, Table 1 ) and other clinical features were not found. Overview of urinary proteomics for burn patients Our study compared the urine protein expression between burn patients with mild, moderate, severe degree and HC through the approach of Label-free mass spectrometry and tandem mass spectrometry as described in Methods. A total of 8253 peptides and 1465 proteins with a protein FDR of < 1% and with at least two unique peptides were identified in four groups of urine sample (Fig. 2 A, B, and C ). Heatmap and Venn diagram were performed based on the logarithm of peak intensity in Label-Free Quantification to visualize proteins with differential expression in burn patients. A total of 402 DEPs were identified in severe group (log 2 FC > 1.5; P < 0.05; FDR < 1%; Fig. 2 F), of which 279 proteins were up-regulated, and 123 proteins were down-regulated. However, there was only one DEP between the mild and HC groups (Fig. 2 D), whereas 11 proteins (7 up-regulated and 2 down-regulated; Fig. 2 E) were found between the moderate and HC groups. In summary, our study found that mild and moderate groups were not identified obviously urine DEPs. Bioinformatics analysis of proteomes for severe group To further understand the function of DEPs, GO analysis showed that neutrophil-mediated immunity for biological process (BP), collagen-containing for cellular component, and enzyme inhibitor for molecular function were the most enrichment function (Fig. 3 A). Complement and coagulation cascades pathway was most enrichment function through KEGG pathway analysis ( P adjust < 0.05; Fig. 3 B). Furthermore, the metascape analysis further proved that the top three levels of BP were negative regulation of the biological process, immune system process, and response to stimulus, which might further support that urine proteomics could reflect the dynamic change of immunity functions for burn patients (Fig. 3 C). The STRING analysis of MB As one of top up-regulated urine protein, the results of STRING analysis demonstrated that the function of MB was most related to the creatine kinase activity and oxygen carrier activity ( P < 0.05, Fig. 4 ). Besides, Hp , APOA1 , TNNI2 , and CYCS were detected in the data of discover set and related information was shown in Table 2 . Table 2 The related urine protein for UM in discover set Protein ID Protein name Gene Log 2 FC P value FDR P00738 Haptoglobin HP 3.124 0.000006 0.00036 P02647 Apolipoprotein A-I APOA1 2.703 0.000218 0.00201 A0A087WXS0 Troponin I, fast skeletal muscle TNNI2 3.128 0.000422 0.00291 C9JFR7 Cytochrome c CYCS 2.972 0.001749 0.00661 The difference of UM in patients with different severities and depths In discover set, there was a significant difference in UM among mild, moderate, and severe groups ( P = 0.0058, Fig. 5 A) and also in III degrees depth and non-III degrees depth groups ( P = 0.0437, Fig. 5 B). In validation set, the same differences in burns patients were also founded ( P < 0.05, Fig. 5 C and 5 D). The value of UM to distinguish different depths for burn patients To further evaluate the potential clinical value of UM, UM might could as a biomarker to rapidly distinguish patients with III degrees depth and non-III degrees depth through the ROC curve (Discover set, AUC = 0.8203, 95% CI: 0.6448 ~ 0.9957, Fig. 6 A; Validation set, AUC = 0.7393, 95% CI: 0.618 ~ 0.8606, Fig. 6 B). Discussion Blood and urine are common frequently liquids used to the discovery of biomarkers of human diseases as both can be collected frequently and non-invasively. It is a challenge to obtain peripheral venous blood and will increase the incidence of severe infection by the usage of central venous catheter for severe patients. Therefore, Urine as a body fluid has several advantages over blood: easily obtained in large volumes, less complex, and lower dynamic range allowing low abundance but functionally important proteins[ 16 , 17 ]. In the present study, only little significant DEPs were found in mild group, which could be interpreted that the lower TBSA could not or slightly activate the response to a stimulus or the immune response. For the moderate group, only 9 DEPs were identified and 5 proteins were also found in the severe group (data was not shown). However, there were 402 DEPs in severe group, moreover, GO and KEGG analysis for DEPs demonstrated that the response to stimulus and complement and coagulation cascades pathway were the most enrichment. Burns that affect ≥ 20% of the TBSA trigger a major inflammatory response and lead to capillary leakage, which mainly caused hypoalbuminemia [ 18 ]. Acute kidney injury, Severe infections, and Hypoproteinemia are the most adverse complications for burn patients who were admitted to hospital. Furthermore, the prognosis of those patients seems not well[ 19 – 21 ]. The severe burn patients had lower albumin compared healthy volunteers and further support the results of our study. It is important to make reasonable clinical decision based on precise burn depth for burn specialists and found more reliable biomarker to distinguish the depth of burn. MB as one of the up-regulated protein was founded in severe group and our study demonstrated that there were significant differences in different severities and depths. Interestingly, our study revealed that UM as a novel biomarker could distinguish different depths in burn patients, which might assist burn specialist to quickly assess the depth of burn. Our study showed that UM play an important role in predicting the depth of burn. We further consider the reasons why UM plays a key role in burn patients. Mb is a in the cytoplasm of muscle and cardiac tissue, iron-containing porphyrin and hemoglobin Homologous intracellular chromoprotein containing a 153 amino Polypeptide chain composed of acid residues and a heme prosthetic group, moreover, Mb could promote the oxygen supply in tissues and has functions such as oxygen transporter, intracellular catalyst and oxygen storage[ 22 ]. As we all know, Trauma, infection, drugs, immune diseases, endocrine and metabolic disorders and other factors can cause damage to skeletal muscle and cardiac muscle, followed by dissolution, changes in cell membrane integrity, and the contents of muscle cells enter the extracellular fluid and blood circulation system[ 23 , 24 ]. Resulting in the increase of serum Mb content, this process involves many pathophysiological mechanisms such as activation of phospholipase A2, mitochondrial dysfunction, continuous contraction of muscle cells, and oxidative stress caused by free radicals. UM was mainly used to predicting the acute Kendy injury for burn patients[ 25 ]. However, Gosling P and colleagues found that UM was not increased in burn patients[ 26 ]. In our study, UM was greatly increased patients with three degrees depth. We further hypothesized that the onset of burn caused the broken of red blood cell. Mb was released into the blood and further excretion in the urine. To the best of our knowledge, our study is the first to report on the identification of urine proteins as a urinary biomarker for burn patients. DEPs were mainly founded burn in severe burn patients. Interestingly, our study revealed that UM could be served as a biomarker to rapidly distinguish different burn depths. However, our study also has some limitations: The sample was not large and collected from a single center. The UM still has certain diagnostic limitations and larger samples need to be validated in the future. Conclusions This is the first comprehensive study to demonstrate the landscape of urine proteomics for burn patients. UM could serve as a novel and non-invasive biomarker for distinguishing burn depth. No matter what, our study provides a new insight for burn patients and looking forward to seeing these results translated into clinical practice after validation based on a large sample. Declarations Acknowledgement None Authors’ contributions TL data analysis and draft the manuscript; QM Z, JT W, and TC L sample and data collection; M Z designed the study and revised the manuscript. All authors reviewed the manuscript. Funding This work was supported by the foundation of Beijing Key Clinical Specialty Program (2020ZDZK2). Availability of data and materials The data sets generated and analysed during the current study are available from the corresponding author upon a reasonable request. Ethics approval and consent to participate For the human urine samples, all methods were carried out in accordance with relevant guidelines and regulations. The study was approved by the Ethics Committee of Beijing Shijitan Hospital, and all burn patients and healthy volunteers were provided written consent and assent. All experiments conformed to the Declaration of Helsinki. Consent for publication Not applicable. Competing interests The authors declare that they have no competing interests. References Xie B, Xiao SC, Peng XD, Zhu SH, Lv KY, Li HY, Xia ZF: Epidemiology and outcome analysis of severe extensive burns: a 12-year summary of 103 cases in a burn center in China. Journal of burn care & research: official publication of the American Burn Association 2012, 33(3):e127-132. doi: 10.1097/BCR.0b013e3182335a5d . Burn Wound Healing and Scarring Pathophysiology: Total Scar Management; 2020. Watts AM, Tyler MP, Perry ME, Roberts AH, McGrouther DA: Burn depth and its histological measurement. Burns: journal of the International Society for Burn Injuries 2001, 27(2):154–160. doi: 10.1016/s0305-4179(00)00079-6 . Papp A, Kiraly K, Härmä M, Lahtinen T, Uusaro A, Alhava E: The progression of burn depth in experimental burns: a histological and methodological study. Burns: journal of the International Society for Burn Injuries 2004, 30(7):684–690. doi: 10.1016/j.burns.2004.03.021 . Chen J, Yan H, Luo G, Luo Q, Li X, Zhang J, Yuan Z, Peng D, Peng Y, Hu J et al : Characteristics of burn deaths from 2003 to 2009 in a burn center: A retrospective study. Burns & trauma 2013, 1(2):80–86. doi: 10.4103/2321-3868.118933 McUmber H, Dabek RJ, Bojovic B, Driscoll DN: Burn Depth Analysis Using Indocyanine Green Fluorescence: A Review. Journal of burn care & research: official publication of the American Burn Association 2019, 40(4):513–516. doi: 10.1093/jbcr/irz054 Monstrey S, Hoeksema H, Verbelen J, Pirayesh A, Blondeel P: Assessment of burn depth and burn wound healing potential. Burns: journal of the International Society for Burn Injuries 2008, 34(6):761–769. doi: 10.1016/j.burns.2008.01.009 Bennett JE, Dingman RO: Evaluation of burn depth by the use of radioactive isotopes; an experimental study. Plastic and reconstructive surgery (1946) 1957, 20(4):261–272. doi: 10.1097/00006534-195710000-00001 Green HA, Bua D, Anderson RR, Nishioka NS: Burn depth estimation using indocyanine green fluorescence. Archives of dermatology 1992, 128(1):43–49. Miccio J, Parikh S, Marinaro X, Prasad A, McClain S, Singer AJ, Clark RA: Forward-looking infrared imaging predicts ultimate burn depth in a porcine vertical injury progression model. Burns: journal of the International Society for Burn Injuries 2016, 42(2):397–404. doi: 10.1016/j.burns.2015.07.006 Stanga PE, Lim JI, Hamilton P: Indocyanine green angiography in chorioretinal diseases: indications and interpretation: an evidence-based update. Ophthalmology 2003, 110(1):15–21; quiz 22 – 13. doi: 10.1016/s0161-6420(02)01563-4 Frantzi M, Metzger J, Banks RE, Husi H, Klein J, Dakna M, Mullen W, Cartledge JJ, Schanstra JP, Brand K et al : Discovery and validation of urinary biomarkers for detection of renal cell carcinoma. Journal of proteomics 2014, 98:44–58. doi: 10.1016/j.jprot.2013.12.010 Zhang C, Leng W, Sun C, Lu T, Chen Z, Men X, Wang Y, Wang G, Zhen B, Qin J: Urine Proteome Profiling Predicts Lung Cancer from Control Cases and Other Tumors. EBioMedicine 2018, 30:120–128. doi: 10.1016/j.ebiom.2018.03.009 Li Y, Long J, Chen J, Zhang J, Qin Y, Zhong Y, Liu F, Peng Z: Analysis of Spatiotemporal Urine Protein Dynamics to Identify New Biomarkers for Sepsis-Induced Acute Kidney Injury. Frontiers in physiology 2020, 11:139. doi: 10.3389/fphys.2020.00139 Li M, Zhao M, Gao Y: Changes of proteins induced by anticoagulants can be more sensitively detected in urine than in plasma. Science China Life sciences 2014, 57(7):649–656. doi: 10.1007/s11427-014-4661-y Barratt J, Topham P: Urine proteomics: the present and future of measuring urinary protein components in disease. CMAJ: Canadian Medical Association journal = journal de l'Association medicale canadienne 2007, 177(4):361–368. doi: 10.1503/cmaj.061590 Jakobsen KR, Paulsen BS, Bæk R, Varming K, Sorensen BS, Jørgensen MM: Exosomal proteins as potential diagnostic markers in advanced non-small cell lung carcinoma. Journal of extracellular vesicles 2015, 4:26659. doi: 10.3402/jev.v4.26659 Fishel RS, Are C, Barbul A: Vessel injury and capillary leak. Critical care medicine 2003, 31(8 Suppl):S502-511. doi: 10.1097/01.CCM.0000081431.50015.46 Folkestad T, Brurberg KG, Nordhuus KM, Tveiten CK, Guttormsen AB, Os I, Beitland S: Acute kidney injury in burn patients admitted to the intensive care unit: a systematic review and meta-analysis. Critical care 2020, 24(1):2. doi: 10.1186/s13054-019-2710-4 Zavlin D, Chegireddy V, Boukovalas S, Nia AM, Branski LK, Friedman JD, Echo A: Multi-institutional analysis of independent predictors for burn mortality in the United States. Burns & trauma 2018, 6:24. doi: 10.1186/s41038-018-0127-y Zhou J, Tan J, Gong Y, Li N, Luo G: Candidemia in major burn patients and its possible risk factors: A 6-year period retrospective study at a burn ICU. Burns: journal of the International Society for Burn Injuries 2019, 45(5):1164–1171. doi: 10.1016/j.burns.2019.01.005 Meyer RA: Aerobic performance and the function of myoglobin in human skeletal muscle. American journal of physiology Regulatory, integrative and comparative physiology 2004, 287(6):R1304-1305. doi: 10.1152/ajpregu.00541.2004 Knapik JJ, O'Connor FG: Exertional Rhabdomyolysis: Epidemiology, Diagnosis, Treatment, and Prevention. Journal of special operations medicine: a peer reviewed journal for SOF medical professionals 2016, 16(3):65–71. Mair J, Artner-Dworzak E, Dienstl A, Lechleitner P, Morass B, Smidt J, Wagner I, Wettach C, Puschendorf B: Early detection of acute myocardial infarction by measurement of mass concentration of creatine kinase-MB. The American journal of cardiology 1991, 68(17):1545–1550. doi: 10.1016/0002-9149(91)90307-7 Bhavsar P, Rathod K, Rathod D, Chamania C: Utility of Serum Creatinine, Creatine Kinase and Urinary Myoglobin in Detecting Acute Renal Failure due to Rhabdomyolysis in Trauma and Electrical Burns Patients. The Indian journal of surgery 2013, 75(1):17–21. doi: 10.1007/s12262-012-0451-6 Gosling P, Sutcliffe A, Cooper M, Jones A: Burn and trauma associated proteinuria: the role of lipid peroxidation, renin and myoglobin. Annals of clinical biochemistry 1988:53–59. doi: 10.1177/000456328802500107 Additional Declarations No competing interests reported. Cite Share Download PDF Status: Posted Version 2 posted You are reading this latest preprint version Show more versions Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-1311947","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":87495174,"identity":"f490d41d-a20f-4b7c-9546-cd0758c36359","order_by":0,"name":"Tao Li","email":"","orcid":"","institution":"Capital Medical University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Tao","middleName":"","lastName":"Li","suffix":""},{"id":87495175,"identity":"93cd475f-c155-42dd-953c-fbf9ff441d3a","order_by":1,"name":"Qiaomei Zhao","email":"","orcid":"","institution":"the First Medical Center of Chinese PLA General Hospital","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Qiaomei","middleName":"","lastName":"Zhao","suffix":""},{"id":87495176,"identity":"6ef3dc48-3b29-4f6d-b75f-6a65fc4abb38","order_by":2,"name":"Jitu Wang","email":"","orcid":"","institution":"Capital Medical University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Jitu","middleName":"","lastName":"Wang","suffix":""},{"id":87495177,"identity":"1e5bf647-3ce0-4288-b639-ebc56efa9b32","order_by":3,"name":"Tianci Liu","email":"","orcid":"","institution":"Capital Medical University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Tianci","middleName":"","lastName":"Liu","suffix":""},{"id":87495178,"identity":"5c514b4f-01e8-4e3e-ad1a-4bf765053dac","order_by":4,"name":"Man Zhang","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA3klEQVRIiWNgGAWjYDACCSjNL8GQAOQcIEGL5AyQlgRStBjcAJHEaJGf3fzs4dccuzzj2w2PP1j+uMPAP7sBvxbGOcfMjWW3JReb3TmQJiGR8IxB4g4Bm5glEsykJbcxJ267kZAG9MthBgOJBPxa2CTSvwG11CdunpGQ/IEoLTwSOWaSH7cdTtwgkZAgQZQWCYmcMmnGbccTZ4D9knaYR+IGAS3yM9K3Sf7cVp3YP7sn+bOEzWE5/hkEtIAAMw/EjQnMwEjiIaweCBh/gCn2A4wfiFI/CkbBKBgFIw0AAEcrRZE1o/2CAAAAAElFTkSuQmCC","orcid":"","institution":"Capital Medical University","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"Man","middleName":"","lastName":"Zhang","suffix":""}],"badges":[],"createdAt":"2022-01-30 13:59:05","currentVersionCode":2,"declarations":"","doi":"10.21203/rs.3.rs-1311947/v2","doiUrl":"https://doi.org/10.21203/rs.3.rs-1311947/v2","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":18840622,"identity":"83cd5a50-0f59-49fa-8531-03243459b7db","added_by":"auto","created_at":"2022-03-03 22:03:41","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":299999,"visible":true,"origin":"","legend":"\u003cp\u003eThe workflow of this study.\u003c/p\u003e","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-1311947/v2/61ab8b6321110c0b2650c8de.png"},{"id":18840893,"identity":"39cba6f9-ac78-4d19-b775-cbee5587eb8d","added_by":"auto","created_at":"2022-03-03 22:06:41","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1487198,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eOverview of urinary proteomics for burn patients \u003c/strong\u003e\u003c/p\u003e\u003cp\u003eA: QC control for our study; B: The heatmap of HC and burn patients with differential degrees; C: Venn diagram of different burn groups; D, E, and F: volcano plot of among HC, Mild, Moderate, and Severe groups.\u0026nbsp;\u003c/p\u003e","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-1311947/v2/0760a1ec1eedcc2e49d71fd5.png"},{"id":18840894,"identity":"c15d54ab-a8f1-4811-b035-724efa74a7d4","added_by":"auto","created_at":"2022-03-03 22:06:41","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":497391,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eBioinformatics analysis of proteomics in severe group\u003c/strong\u003e\u003c/p\u003e\u003cp\u003eA, B and C: GO, KEGG pathway, and metascape analysis for severe group\u003c/p\u003e","description":"","filename":"Figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-1311947/v2/04b56975cec0f8c9f3083dcc.png"},{"id":18840626,"identity":"08f2faab-6c64-4788-b971-a767e3c6d00e","added_by":"auto","created_at":"2022-03-03 22:03:41","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":345865,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe STRING analysis of MB\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"Figure4.png","url":"https://assets-eu.researchsquare.com/files/rs-1311947/v2/e811c9d472b63281e2777493.png"},{"id":18840624,"identity":"3abded5a-710e-4885-9759-5654890691f6","added_by":"auto","created_at":"2022-03-03 22:03:41","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":518533,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe difference of UM in patients with different severities and depths \u003c/strong\u003e\u003c/p\u003e\u003cp\u003eA and B: the difference of UM between different severities and depths in discover set. \u003c/p\u003e\u003cp\u003eC and D: the difference of UM between different severities and depths in validation set.\u0026nbsp;\u003c/p\u003e","description":"","filename":"Figure5.png","url":"https://assets-eu.researchsquare.com/files/rs-1311947/v2/d481e002bc8be5befbbf43b8.png"},{"id":18840627,"identity":"c228bbf9-1b2d-4d29-b85b-8a2ecff621ea","added_by":"auto","created_at":"2022-03-03 22:03:41","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":453078,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe ROC curve in discover set and validation set \u003c/strong\u003e\u003c/p\u003e\u003cp\u003eA: The ROC curve in discover set (AUC=0.8203, 95%CI: 0.6448~0.9957).\u003c/p\u003e\u003cp\u003eB: The ROC curve in Validation set (AUC=0.7393, 95%CI: 0.618~0.8606).\u003c/p\u003e","description":"","filename":"Figure6.png","url":"https://assets-eu.researchsquare.com/files/rs-1311947/v2/295f2710e289ec22cb57026e.png"},{"id":20903138,"identity":"89b8419c-fb23-4d1f-9078-98ce3fdce264","added_by":"auto","created_at":"2022-04-29 03:59:22","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1790680,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-1311947/v2/9d40a059-e072-4d48-b386-2519db57e083.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Urine Myoglobin is Associated with Different Severities and Depths for Burn Patients","fulltext":[{"header":"Introduction","content":"\u003cp\u003eApproximately 800,000 people were burned each year in China, and about half of patients need to be hospitalized[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. There were 3275 burn patients who eventually died in the USA in 2016 and also face serious challenges to decrease the rate of mortality[\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. The depths of burn were commonly be divided into three degrees, including superficial (I degree), partial-thickness (II degrees), and full-thickness (III degrees). The depth of the burn has a tremendous impact on the course of treatment and prognosis. Patients with different depths have been received various treatments, such as observation, common treatment, and skin grafting[\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. When it comes to the diagnostics of burn depth, the main challenge is the ability to distinguish between superficial partial-thickness and deep partial-thickness burns, which impact the dermis to varying degrees[\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. The significance of distinguishing partial-thickness burns and full-thickness burns was influenced the critical clinical decision that whether a patient should be allowed to heal on their own or if the wound should be surgically excised and replaced with a graft[\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eBurn depth analysis has traditionally been estimated via physical examination by the burn specialist. The surgeon would assess pain and tenderness, formation of blisters, the appearance of the dermis, color, and if the skin blanched when pressure was applied. However, this method has been shown to be only 60 to 75% accurate in correctly identifying the depth[\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Many technologies have been emerged to distinguish the depth of burn, including radioactive phosphorus, vital dyes, and thermography[\u003cspan additionalcitationids=\"CR9\" citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. The most recent wave of depth analysis techniques includes laser Doppler imaging and indocyanine green video angiography [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. However, based on clinical assessment and various technologies are still not accurately assessing burn depth. Therefore, it still needs more biomarkers to easily distinguish different depths for burn patients and make an early clinical decision.\u003c/p\u003e \u003cp\u003eMass spectrometry-based proteomic analysis has been being a powerful screening tool for the early detection of renal function and cancer-related biomarkers. This analysis has been successfully utilized to identify protein biomarkers from body fluids [\u003cspan additionalcitationids=\"CR13\" citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. Unlike blood samples, urine could be collected non-invasively and timely, moreover, urine could better reflect the changes in the human body. Besides, the protein compositions of urine samples are relatively simple, stable, and easier to analysis [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eBased on these, Label-free mass spectrometry (MS) was used to analyze the changes in urine proteins between burn patients with different severity and healthy volunteers. bioinformatics analysis was also carried out for different expression of proteins (DEPs). Our study is to find specific urine proteins for different severities and depths and further evaluate the potential clinical value, which may lay a foundation for potential biomarkers for burn patients.\u003c/p\u003e"},{"header":"Patients And Methods","content":"\u003cdiv class=\"Section2\" id=\"Sec7\"\u003e\n \u003ch2\u003ePatients\u003c/h2\u003e\n \u003cp\u003eThirty burn patients (10 mild, 10 moderate, and 10 severe), who were hospitalized for treatment within 24 hours after burned, were selected before they underwent treatment at Beijing Shijitan Hospital, Capital Medical University from April 2017 to December 2020. Exclusion criteria: patients whose age was less than 18 or more than 65 years old were excluded. Patients who have chronic diseases such as chronic kidney disease, diabetes, hyperlipidemia, rheumatism, hepatitis, history of cancers, and blood system diseases were also excluded. Besides, Patients with the usage history of medicine 2 weeks prior to the collection of the urine sample were also excluded. The detailed information for our study was shown in Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e.\u003c/p\u003e\n \u003cp\u003eAccording to the classification proposed by the burn conference, the mild group refers to Ⅱ degree burn patients who had\u0026thinsp;\u0026lt;\u0026thinsp;9% total body surface area (TBSA) burn; moderate group refers to Ⅱ degree burn patients who had 10\u0026thinsp;~\u0026thinsp;29% TBSA burn; severe group refers to 30%~49% TBSA burn (Ⅱ degree) or 10%~19% TBSA burn (Ⅲ degree), or the total burn area is less than 30%, but one of the following conditions exists: patients with severe general condition or shock; patients with multiple injuries or combined injury; patients with moderate or severe inhalation injury; especially severe burn refers to total area\u0026thinsp;\u0026ge;\u0026thinsp;50% or Ⅲ degree burn area\u0026thinsp;\u0026ge;\u0026thinsp;20%. The detailed clinical features were shown in Table \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e.\u003c/p\u003e\n \u003cdiv class=\"gridtable\"\u003e\u003ctable border=\"1\" id=\"Tab1\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003eDemographics and clinical characteristics of burn patients and HC\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003ccolgroup cols=\"9\"\u003e\u003c/colgroup\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eCharacteristics\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\" colspan=\"4\"\u003e\n \u003cp\u003eDiscovery cohort\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\" colspan=\"4\"\u003e\n \u003cp\u003eValidation cohort\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eHC\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e(N\u0026thinsp;=\u0026thinsp;10)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eMild\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e(N\u0026thinsp;=\u0026thinsp;10)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eModerate\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e(N\u0026thinsp;=\u0026thinsp;10)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eSevere\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e(N\u0026thinsp;=\u0026thinsp;10)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cspan class=\"BoldItalic\" name=\"Emphasis\" type=\"BoldItalic\"\u003eP\u003c/span\u003e \u003cstrong\u003evalue\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eMild and Moderate\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e(N\u0026thinsp;=\u0026thinsp;33)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eSevere\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e(N\u0026thinsp;=\u0026thinsp;34)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cspan class=\"BoldItalic\" name=\"Emphasis\" type=\"BoldItalic\"\u003eP\u003c/span\u003e \u003cstrong\u003evalue\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eAge, range (median)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e23\u0026thinsp;~\u0026thinsp;46 (33.5)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e28\u0026thinsp;~\u0026thinsp;54 (36.0)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e24\u0026thinsp;~\u0026thinsp;48 (35.0)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e24\u0026thinsp;~\u0026thinsp;56 (34.0)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.519\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e23\u0026thinsp;~\u0026thinsp;67 (46)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e21\u0026thinsp;~\u0026thinsp;66 (40)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.214\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eGender, male, n (%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e5 (50%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e4 (40)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e5 (50)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e5 (50)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e26 (78.78)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e28 (82.35)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.765\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eTotal protein (g/L)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e57.8\u0026thinsp;~\u0026thinsp;75.9 (66.15)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e62.8\u0026thinsp;~\u0026thinsp;79.5 (73.4)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e52.5\u0026thinsp;~\u0026thinsp;78.0(63.55)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e32.4\u0026thinsp;~\u0026thinsp;53.1(49.55)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.001\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e53.3\u0026thinsp;~\u0026thinsp;75.9 (62.7)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e37.8\u0026thinsp;~\u0026thinsp;71.8(56.35)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.001\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eALB (g/L)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e31.7\u0026thinsp;~\u0026thinsp;47.4 (38.05)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e36.2\u0026thinsp;~\u0026thinsp;50.4 (44.1)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e33.1\u0026thinsp;~\u0026thinsp;44.9 (41.3)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e15.5\u0026thinsp;~\u0026thinsp;29 (26.0)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.001\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e26.2\u0026thinsp;~\u0026thinsp;47.4 (41.3)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e18\u0026thinsp;~\u0026thinsp;44.5(30.35)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.001\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eBUN\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3.52\u0026thinsp;~\u0026thinsp;9.12 (5.09)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3.9\u0026thinsp;~\u0026thinsp;7.12 (5.775)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.8\u0026thinsp;~\u0026thinsp;6.88 (5.22)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e4.1\u0026thinsp;~\u0026thinsp;9.5 (6.4)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.089\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3.2\u0026thinsp;~\u0026thinsp;9.12 (4.99)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2.7\u0026thinsp;~\u0026thinsp;16.5(5.7)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.233\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eSerum creatinine\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e47\u0026thinsp;~\u0026thinsp;78 (59.7)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e38\u0026thinsp;~\u0026thinsp;81 (54)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e42.5\u0026thinsp;~\u0026thinsp;70 (50.6)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e36.9\u0026thinsp;~\u0026thinsp;178.1(73.95)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.496\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e41\u0026thinsp;~\u0026thinsp;97 (61)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e40.3\u0026thinsp;~\u0026thinsp;116.8(60.2)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.792\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" colspan=\"9\"\u003e\n \u003cp\u003e\u003cstrong\u003eNote\u003c/strong\u003e: a: P value was compared between severe and HC group. TP: total protein; ALB: albumin.\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n \u003c/div\u003e\n \u003cp\u003eTo ensure the accuracy and comprehensiveness of reporting in this case-control biomarker study. ten healthy volunteers were selected as the healthy control group (HC) and the detailed clinical features were also shown in Table \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e.\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003eEvaluation of burn depth\u003c/strong\u003e\u003c/p\u003e\n\u003c/div\u003e\n\u003cp\u003eBurn depth evaluation has traditionally been based on the histological assessment of dermal microvascular occlusion in burn biopsies in our hospital.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eThe analysis of UM\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIn the present study, each patient\u0026rsquo;s midstream urine was obtained before treatment and the value of urine MB was tested by Beckman ACCESS2, USA. The test of urine MB was carried out under the guidance of the instructions.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eUrine samples collection\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMidstream urine samples were collected in the morning into dry and clean containers from all volunteers. Immediately after collection, urine samples were centrifuged at 4000r/min for 5 minutes to remove cell debris and casts. Then we divided the supernatants into aliquots and froze them at -80\u0026deg;C until use.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eProtein extraction and digestion\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo found more urine proteins, ten samples in each group were mixed into one sample and was repeatedly tested three times. The detailed information for test as followed: The frozen urine sample was reconstituted at 4\u0026deg;C, centrifuged at 2000g for 10min, and the protein concentration was determined using Bradford method, rest was frozen to -80℃.\u003c/p\u003e\n\u003cp\u003eTake 10\u0026micro;g of each sample for SDS-PAGE electrophoresis, stain with Coomassie blue for 30 minutes, and decolor until the background is clear. The gel was then washed by destained buffer and treated with ACN as above. When the enzyme solution is completely absorbed by colloidal particles, then made up to 100 \u0026micro;L with 25 mM ABC, proteins were digested overnight at 37℃.\u003c/p\u003e\n\u003cp\u003eNext day, after centrifugation in low speed, the supernatant was collected, 200 \u0026micro;L of ACN was added, put under vortex and mixed. the supernatant was extracted in 100 \u0026micro;L of 0.1% formic acid (FA). Combined the supernatant and centrifuged at 12000 g for 5 min at room temperature and then lyophilized. The powder was dissolved and mixed in 0.1% of formic acid. The supernatant was slowly loaded to the C18 desalting column, washed with washing solution (0.1% FA, 3% ACN) 3 times, then eluted twice by some elution buffer (0.1% FA, 70% ACN). The eluents were collected and lyophilized.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePeptide identification, quantitative profiling and statistics by LC\u0026ndash;MS/MS\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMobile phase A (100% water, 0.1% formic acid) and B solution (100% acetonitrile, 0.1% formic acid) were prepared. Peptides were separated in a analytical column, using a linear gradient elution. The separated peptides were analyzed by Orbitrap Fusion mass spectrometer (Thermo Fisher) and Full scan range from m/z 350 to 1550 with resolution of 120000 (at m/z 200), an automatic gain control (AGC) target value was 2\u0026times;10\u003csup\u003e5\u003c/sup\u003e and a maximum ion injection time was 50 ms.\u003c/p\u003e\n\u003cp\u003eThe resulting spectra from each fraction were searched separately against human database by the search engines MaxQuant. The MS/MS spectra results were queried against the SwissProt human database within Uniprot (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e\u003ca href=\"http://www.uniprot.org\" target=\"_blank\"\u003ewww.uniprot.org\u003c/a\u003e\u003c/span\u003e\u003c/span\u003e) using the Proteome Discoverer software suite (v2.1, Thermo Fisher Scientific). Searches were performed using a peptide tolerance of 20 ppm and a product ion tolerance of 0.05 Da. At the protein level, a 1% FDR was used as a filter, and each protein contained at least 1 unique peptide. After filtering the results as described above, the peptide abundances in the different reporter ion channels of the MS/MS scan were normalized. The protein abundance ratios were based on unique peptide results and proteins with a fold change\u0026thinsp;\u0026ge;\u0026thinsp;1.5 were considered significantly altered.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunctional evaluation and pathway analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eOnline resources such as Database for Annotation, Visualization, and Integrated Discovery (Metascape: \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://metascape.org\u003c/span\u003e\u003c/span\u003e) and Kyoto Encyclopedia of Genes and Genomes (KEGG:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.genome.jp),an\u003c/span\u003e\u003c/span\u003ed Gene ontology: \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://geneontology.org\u003c/span\u003e\u003c/span\u003e) were applied to investigate gene ontology and signaling pathways of candidate biomarkers. STRING protein-protein interactions network analysis (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://string-db.org\u003c/span\u003e\u003c/span\u003e) was used to determine the relationships and net-works between identified proteins.\u003c/p\u003e\n\u003cdiv class=\"Section2\" id=\"Sec14\"\u003e\n \u003ch2\u003eStatistical analysis\u003c/h2\u003e\n \u003cp\u003eStatistical analyses were performed R 4.0.1 version and visualized by GraphPad Prism 8.0 (GraphPad Software, San Diego, California, USA). Data were expressed as median (range) and were tested by ANOVA test and Student-Newman-Keuls (SNK) test. The results were considered to be statistically significant at \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05.\u003c/p\u003e\n\u003c/div\u003e"},{"header":"Results","content":"\u003cdiv class=\"Section2\" id=\"Sec16\"\u003e\n \u003cp\u003e\u003cstrong\u003eDemographic characteristics of burn patients and HC\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003eThere were no significant differences in age and gender in each group. In discover set, compared with HC, patients with mild or moderate only had lower level of TP and other features were not found. However, burn patients with severe had lower level of TP and ALB (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05, Table \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e) than HC. In validation set, there were also significant difference in TP and ALB between severe and non-severe group (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05, Table \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e) and other clinical features were not found.\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003eOverview of urinary proteomics for burn patients\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003c/div\u003e\n\u003cp\u003eOur study compared the urine protein expression between burn patients with mild, moderate, severe degree and HC through the approach of Label-free mass spectrometry and tandem mass spectrometry as described in Methods. A total of 8253 peptides and 1465 proteins with a protein FDR of \u0026lt;\u0026thinsp;1% and with at least two unique peptides were identified in four groups of urine sample (Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eA, B, \u003cstrong\u003eand C\u003c/strong\u003e).\u003c/p\u003e\n\u003cp\u003eHeatmap and Venn diagram were performed based on the logarithm of peak intensity in Label-Free Quantification to visualize proteins with differential expression in burn patients. A total of 402 DEPs were identified in severe group (log\u003csub\u003e2\u003c/sub\u003eFC\u0026thinsp;\u0026gt;\u0026thinsp;1.5; \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05; FDR\u0026thinsp;\u0026lt;\u0026thinsp;1%; Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eF), of which 279 proteins were up-regulated, and 123 proteins were down-regulated.\u003c/p\u003e\n\u003cp\u003eHowever, there was only one DEP between the mild and HC groups (Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eD), whereas 11 proteins (7 up-regulated and 2 down-regulated; Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eE) were found between the moderate and HC groups. In summary, our study found that mild and moderate groups were not identified obviously urine DEPs.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eBioinformatics analysis of proteomes for severe group\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo further understand the function of DEPs, GO analysis showed that neutrophil-mediated immunity for biological process (BP), collagen-containing for cellular component, and enzyme inhibitor for molecular function were the most enrichment function (Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eA). Complement and coagulation cascades pathway was most enrichment function through KEGG pathway analysis (\u003cem\u003eP\u003c/em\u003e adjust\u0026thinsp;\u0026lt;\u0026thinsp;0.05; Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eB).\u003c/p\u003e\n\u003cp\u003eFurthermore, the metascape analysis further proved that the top three levels of BP were negative regulation of the biological process, immune system process, and response to stimulus, which might further support that urine proteomics could reflect the dynamic change of immunity functions for burn patients (Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eC).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eThe STRING analysis of MB\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAs one of top up-regulated urine protein, the results of STRING analysis demonstrated that the function of MB was most related to the creatine kinase activity and oxygen carrier activity (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05, Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003e). Besides, \u003cem\u003eHp\u003c/em\u003e, \u003cem\u003eAPOA1\u003c/em\u003e, \u003cem\u003eTNNI2\u003c/em\u003e, and CYCS were detected in the data of discover set and related information was shown in Table \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e.\u003c/p\u003e\n\u003cdiv class=\"gridtable\"\u003e\u003ctable border=\"1\" id=\"Tab2\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003eThe related urine protein for UM in discover set\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003ccolgroup cols=\"6\"\u003e\u003c/colgroup\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eProtein ID\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eProtein name\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eGene\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eLog\u003csub\u003e2\u003c/sub\u003eFC\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eP value\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eFDR\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eP00738\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eHaptoglobin\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eHP\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3.124\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.000006\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.00036\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eP02647\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eApolipoprotein A-I\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eAPOA1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2.703\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.000218\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.00201\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eA0A087WXS0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eTroponin I, fast skeletal muscle\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eTNNI2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3.128\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.000422\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.00291\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eC9JFR7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eCytochrome c\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eCYCS\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2.972\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.001749\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.00661\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n\u003c/div\u003e\n\u003cp\u003e\u003cstrong\u003eThe difference of UM in patients with different severities and depths\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIn discover set, there was a significant difference in UM among mild, moderate, and severe groups (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.0058, Fig. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eA) and also in III degrees depth and non-III degrees depth groups (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.0437, Fig. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eB).\u003c/p\u003e\n\u003cp\u003eIn validation set, the same differences in burns patients were also founded (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05, Fig. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eC and \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eD).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eThe value of UM to distinguish different depths for burn patients\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo further evaluate the potential clinical value of UM, UM might could as a biomarker to rapidly distinguish patients with III degrees depth and non-III degrees depth through the ROC curve (Discover set, AUC\u0026thinsp;=\u0026thinsp;0.8203, 95% CI: 0.6448\u0026thinsp;~\u0026thinsp;0.9957, Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003eA; Validation set, AUC\u0026thinsp;=\u0026thinsp;0.7393, 95% CI: 0.618\u0026thinsp;~\u0026thinsp;0.8606, Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003eB).\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eBlood and urine are common frequently liquids used to the discovery of biomarkers of human diseases as both can be collected frequently and non-invasively. It is a challenge to obtain peripheral venous blood and will increase the incidence of severe infection by the usage of central venous catheter for severe patients. Therefore, Urine as a body fluid has several advantages over blood: easily obtained in large volumes, less complex, and lower dynamic range allowing low abundance but functionally important proteins[\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. In the present study, only little significant DEPs were found in mild group, which could be interpreted that the lower TBSA could not or slightly activate the response to a stimulus or the immune response. For the moderate group, only 9 DEPs were identified and 5 proteins were also found in the severe group (data was not shown). However, there were 402 DEPs in severe group, moreover, GO and KEGG analysis for DEPs demonstrated that the response to stimulus and complement and coagulation cascades pathway were the most enrichment. Burns that affect\u0026thinsp;\u0026ge;\u0026thinsp;20% of the TBSA trigger a major inflammatory response and lead to capillary leakage, which mainly caused hypoalbuminemia [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. Acute kidney injury, Severe infections, and Hypoproteinemia are the most adverse complications for burn patients who were admitted to hospital. Furthermore, the prognosis of those patients seems not well[\u003cspan additionalcitationids=\"CR20\" citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. The severe burn patients had lower albumin compared healthy volunteers and further support the results of our study. It is important to make reasonable clinical decision based on precise burn depth for burn specialists and found more reliable biomarker to distinguish the depth of burn.\u003c/p\u003e \u003cp\u003eMB as one of the up-regulated protein was founded in severe group and our study demonstrated that there were significant differences in different severities and depths. Interestingly, our study revealed that UM as a novel biomarker could distinguish different depths in burn patients, which might assist burn specialist to quickly assess the depth of burn. Our study showed that UM play an important role in predicting the depth of burn. We further consider the reasons why UM plays a key role in burn patients. Mb is a in the cytoplasm of muscle and cardiac tissue, iron-containing porphyrin and hemoglobin Homologous intracellular chromoprotein containing a 153 amino Polypeptide chain composed of acid residues and a heme prosthetic group, moreover, Mb could promote the oxygen supply in tissues and has functions such as oxygen transporter, intracellular catalyst and oxygen storage[\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. As we all know, Trauma, infection, drugs, immune diseases, endocrine and metabolic disorders and other factors can cause damage to skeletal muscle and cardiac muscle, followed by dissolution, changes in cell membrane integrity, and the contents of muscle cells enter the extracellular fluid and blood circulation system[\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. Resulting in the increase of serum Mb content, this process involves many pathophysiological mechanisms such as activation of phospholipase A2, mitochondrial dysfunction, continuous contraction of muscle cells, and oxidative stress caused by free radicals. UM was mainly used to predicting the acute Kendy injury for burn patients[\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. However, Gosling P and colleagues found that UM was not increased in burn patients[\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. In our study, UM was greatly increased patients with three degrees depth. We further hypothesized that the onset of burn caused the broken of red blood cell. Mb was released into the blood and further excretion in the urine.\u003c/p\u003e \u003cp\u003eTo the best of our knowledge, our study is the first to report on the identification of urine proteins as a urinary biomarker for burn patients. DEPs were mainly founded burn in severe burn patients. Interestingly, our study revealed that UM could be served as a biomarker to rapidly distinguish different burn depths. However, our study also has some limitations: The sample was not large and collected from a single center. The UM still has certain diagnostic limitations and larger samples need to be validated in the future.\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eThis is the first comprehensive study to demonstrate the landscape of urine proteomics for burn patients. UM could serve as a novel and non-invasive biomarker for distinguishing burn depth. No matter what, our study provides a new insight for burn patients and looking forward to seeing these results translated into clinical practice after validation based on a large sample.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNone\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026rsquo; contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTL data analysis and draft the manuscript; QM Z, JT W, and TC L sample and data collection; M Z designed the study and revised the manuscript. All authors reviewed the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by the foundation of Beijing Key Clinical Specialty Program (2020ZDZK2).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe data sets generated and analysed during the current study are available from the corresponding author upon a reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFor the human urine samples, all methods were carried out in accordance with relevant guidelines and regulations. The study was approved by the Ethics Committee of Beijing Shijitan Hospital, and all burn patients and healthy volunteers were provided written consent and assent. All experiments conformed to the Declaration of Helsinki.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no competing interests.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eXie B, Xiao SC, Peng XD, Zhu SH, Lv KY, Li HY, Xia ZF: Epidemiology and outcome analysis of severe extensive burns: a 12-year summary of 103 cases in a burn center in China. Journal of burn care \u0026amp; research: official publication of the American Burn Association 2012, 33(3):e127-132. doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1097/BCR.0b013e3182335a5d\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBurn Wound Healing and Scarring Pathophysiology: Total Scar Management; 2020.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWatts AM, Tyler MP, Perry ME, Roberts AH, McGrouther DA: Burn depth and its histological measurement. Burns: journal of the International Society for Burn Injuries 2001, 27(2):154\u0026ndash;160. doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/s0305-4179(00)00079-6\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePapp A, Kiraly K, H\u0026auml;rm\u0026auml; M, Lahtinen T, Uusaro A, Alhava E: The progression of burn depth in experimental burns: a histological and methodological study. Burns: journal of the International Society for Burn Injuries 2004, 30(7):684\u0026ndash;690. doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/j.burns.2004.03.021\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eChen J, Yan H, Luo G, Luo Q, Li X, Zhang J, Yuan Z, Peng D, Peng Y, Hu J \u003cem\u003eet al\u003c/em\u003e: Characteristics of burn deaths from 2003 to 2009 in a burn center: A retrospective study. Burns \u0026amp; trauma 2013, 1(2):80\u0026ndash;86. doi: \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.4103/2321-3868.118933\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMcUmber H, Dabek RJ, Bojovic B, Driscoll DN: Burn Depth Analysis Using Indocyanine Green Fluorescence: A Review. Journal of burn care \u0026amp; research: official publication of the American Burn Association 2019, 40(4):513\u0026ndash;516. doi: \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1093/jbcr/irz054\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMonstrey S, Hoeksema H, Verbelen J, Pirayesh A, Blondeel P: Assessment of burn depth and burn wound healing potential. Burns: journal of the International Society for Burn Injuries 2008, 34(6):761\u0026ndash;769. doi: \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/j.burns.2008.01.009\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBennett JE, Dingman RO: Evaluation of burn depth by the use of radioactive isotopes; an experimental study. Plastic and reconstructive surgery (1946) 1957, 20(4):261\u0026ndash;272. doi: \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1097/00006534-195710000-00001\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGreen HA, Bua D, Anderson RR, Nishioka NS: Burn depth estimation using indocyanine green fluorescence. Archives of dermatology 1992, 128(1):43\u0026ndash;49.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMiccio J, Parikh S, Marinaro X, Prasad A, McClain S, Singer AJ, Clark RA: Forward-looking infrared imaging predicts ultimate burn depth in a porcine vertical injury progression model. Burns: journal of the International Society for Burn Injuries 2016, 42(2):397\u0026ndash;404. doi: \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/j.burns.2015.07.006\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eStanga PE, Lim JI, Hamilton P: Indocyanine green angiography in chorioretinal diseases: indications and interpretation: an evidence-based update. Ophthalmology 2003, 110(1):15\u0026ndash;21; quiz 22 \u0026ndash; 13. doi: \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/s0161-6420(02)01563-4\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFrantzi M, Metzger J, Banks RE, Husi H, Klein J, Dakna M, Mullen W, Cartledge JJ, Schanstra JP, Brand K \u003cem\u003eet al\u003c/em\u003e: Discovery and validation of urinary biomarkers for detection of renal cell carcinoma. Journal of proteomics 2014, 98:44\u0026ndash;58. doi: \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/j.jprot.2013.12.010\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhang C, Leng W, Sun C, Lu T, Chen Z, Men X, Wang Y, Wang G, Zhen B, Qin J: Urine Proteome Profiling Predicts Lung Cancer from Control Cases and Other Tumors. \u003cem\u003eEBioMedicine\u003c/em\u003e 2018, 30:120\u0026ndash;128. doi: \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/j.ebiom.2018.03.009\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLi Y, Long J, Chen J, Zhang J, Qin Y, Zhong Y, Liu F, Peng Z: Analysis of Spatiotemporal Urine Protein Dynamics to Identify New Biomarkers for Sepsis-Induced Acute Kidney Injury. Frontiers in physiology 2020, 11:139. doi: \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.3389/fphys.2020.00139\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLi M, Zhao M, Gao Y: Changes of proteins induced by anticoagulants can be more sensitively detected in urine than in plasma. Science China Life sciences 2014, 57(7):649\u0026ndash;656. doi: \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1007/s11427-014-4661-y\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBarratt J, Topham P: Urine proteomics: the present and future of measuring urinary protein components in disease. CMAJ: Canadian Medical Association journal = journal de l'Association medicale canadienne 2007, 177(4):361\u0026ndash;368. doi: \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1503/cmaj.061590\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eJakobsen KR, Paulsen BS, B\u0026aelig;k R, Varming K, Sorensen BS, J\u0026oslash;rgensen MM: Exosomal proteins as potential diagnostic markers in advanced non-small cell lung carcinoma. Journal of extracellular vesicles 2015, 4:26659. doi: \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.3402/jev.v4.26659\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFishel RS, Are C, Barbul A: Vessel injury and capillary leak. Critical care medicine 2003, 31(8 Suppl):S502-511. doi: \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1097/01.CCM.0000081431.50015.46\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFolkestad T, Brurberg KG, Nordhuus KM, Tveiten CK, Guttormsen AB, Os I, Beitland S: Acute kidney injury in burn patients admitted to the intensive care unit: a systematic review and meta-analysis. Critical care 2020, 24(1):2. doi: \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1186/s13054-019-2710-4\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZavlin D, Chegireddy V, Boukovalas S, Nia AM, Branski LK, Friedman JD, Echo A: Multi-institutional analysis of independent predictors for burn mortality in the United States. Burns \u0026amp; trauma 2018, 6:24. doi: \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1186/s41038-018-0127-y\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhou J, Tan J, Gong Y, Li N, Luo G: Candidemia in major burn patients and its possible risk factors: A 6-year period retrospective study at a burn ICU. Burns: journal of the International Society for Burn Injuries 2019, 45(5):1164\u0026ndash;1171. doi: \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/j.burns.2019.01.005\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMeyer RA: Aerobic performance and the function of myoglobin in human skeletal muscle. American journal of physiology Regulatory, integrative and comparative physiology 2004, 287(6):R1304-1305. doi: \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1152/ajpregu.00541.2004\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKnapik JJ, O'Connor FG: Exertional Rhabdomyolysis: Epidemiology, Diagnosis, Treatment, and Prevention. Journal of special operations medicine: a peer reviewed journal for SOF medical professionals 2016, 16(3):65\u0026ndash;71.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMair J, Artner-Dworzak E, Dienstl A, Lechleitner P, Morass B, Smidt J, Wagner I, Wettach C, Puschendorf B: Early detection of acute myocardial infarction by measurement of mass concentration of creatine kinase-MB. The American journal of cardiology 1991, 68(17):1545\u0026ndash;1550. doi: \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/0002-9149(91)90307-7\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBhavsar P, Rathod K, Rathod D, Chamania C: Utility of Serum Creatinine, Creatine Kinase and Urinary Myoglobin in Detecting Acute Renal Failure due to Rhabdomyolysis in Trauma and Electrical Burns Patients. The Indian journal of surgery 2013, 75(1):17\u0026ndash;21. doi: \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1007/s12262-012-0451-6\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGosling P, Sutcliffe A, Cooper M, Jones A: Burn and trauma associated proteinuria: the role of lipid peroxidation, renin and myoglobin. Annals of clinical biochemistry 1988:53\u0026ndash;59. doi: \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1177/000456328802500107\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Burns, Urine Proteomics, Burn Depth, Severity, Label-free Mass Spectrometry","lastPublishedDoi":"10.21203/rs.3.rs-1311947/v2","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-1311947/v2","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eBackgrounds\u003c/strong\u003e\u003c/p\u003e\u003cp\u003eIt is important to make reasonable clinical decision based on precise burn depth for burn specialists. However, the identification of burn depth based upon clinical assessment was not accurate. It still needs more biomarkers to rapidly distinguish different depths for burn specialists. The potential value of urine myoglobin (UM) is still unknown in burn patients. \u003c/p\u003e\u003cp\u003e\u003cstrong\u003ePatients and methods\u003c/strong\u003e\u003c/p\u003e\u003cp\u003eThe urine of 30 burn patients and 10 Healthy volunteers (Discover set) were collected from January 2017 to December 2020. Urine proteomics of all samples was identified by Label-free mass spectrometry and functional analysis for differential expression proteins (DEPs) was also analyzed. The Receiver Operating Characteristic curve to evaluate the value of UM from another 67 burn patients (Validation set). \u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eResults\u003c/strong\u003e\u003c/p\u003e\u003cp\u003eA total of 8253 peptides and 1465 proteins were identified in four groups. For severe burn patients, 402 DEPs (279 up-regulated and 123 down-regulated) were identified and functional analysis demonstrated that neutrophil-mediated immunity, response to stimulus, and complement and coagulation cascades pathway were the most enrichment function. UM had significant differences in burn patients with different severities and depths (Discover and Validation set; \u003cem\u003eP\u003c/em\u003e\u0026lt;0.05). UM as a novel biomarker could rapidly distinguish different depths for burn patients (Discover set, AUC=0.8203, 95%CI: 0.6448~0.9957; Validation set, AUC=0.7393, 95%CI: 0.618~0.8606).\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eConclusion\u003c/strong\u003e\u003c/p\u003e\u003cp\u003eThe urine DEPs were mainly identified in the severe group. UM might as a novel biomarker help burn specialists to rapidly determine different depths for burn patients.\u0026nbsp;\u003c/p\u003e","manuscriptTitle":"Urine Myoglobin is Associated with Different Severities and Depths for Burn Patients","msid":"","msnumber":"","nonDraftVersions":[{"code":2,"date":"2022-03-03 22:03:39","doi":"10.21203/rs.3.rs-1311947/v2","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}},{"code":1,"date":"2022-02-04 19:52:24","doi":"10.21203/rs.3.rs-1311947/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":"12034018-c025-4ea4-8296-61cf149ee5db","owner":[],"postedDate":"March 3rd, 2022","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2022-04-29T03:59:14+00:00","versionOfRecord":[],"versionCreatedAt":"2022-03-03 22:03:39","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v2","identity":"rs-1311947","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-1311947","identity":"rs-1311947","version":["v2"]},"buildId":"WrCJVZZCHTDjtuVLN7oU0","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}