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 2×105 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),and 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.