Sample selection
Serum samples were obtained from an experimental group of 25 patients with OTLF and a control group of 23 healthy volunteers. All samples were acquired from Peking Union Medical College Hospital. Written informed consent was obtained from all patients and the study was approved by the hospital institutional review board (JS-981). The inclusion criterion of the experimental group was a confirmed radiological diagnosis of OTLF. All patients had a slowly progressive history of neurological symptoms and signs which is suspected to be caused by thoracic stenosis (TSS). The axial plain CT scan of the thoracic vertebrae and sagittal reconstruction provides analysis of calcified ligaments. MRI can show signal changes and deformation of the spinal cord. The combination of these two imaging examination methods can differentially diagnose OTLF from other causes of TSS (such as OPLL). The exclusion criteria include (1) participants had any history of spinal deformity, other spinal disease or trauma were removed; (2) participants had any systemic metabolic diseases (such as skeletal fluorosis) were removed; (3) participants who were not willing to sign the written consent form were removed; (4) the cases were removed where clinical information was lacking or missing and therefore statistical analysis could not be performed. The healthy controls were recruited from among healthy subjects during an annual health check at Peking Union Medical College Hospital. Standard meals was provided three days before sampling.
Sample treatment
Morning fasting blood samples were taken from a peripheral vein and collected into ethylenediaminetetraacetic acid (EDTA) tubes. Samples were stored at 4 °C for three hours before centrifugating at 3000 r/min at room temperature for 20 minutes. The supernatants were separated and stored at -80 °C until further analyses.
Before further analysis, the samples were thawed at room temperature. 100 μL serum was extracted and mixed with 400 μL methanol. The mixture was vortexed for 5 minutes and then centrifuged at 13000 r/min for 15 minutes. 400 μL of supernatants were then taken for UPLC-MS analysis.
UPLC-MS analysis
UPLC-MS analysis was performed using a Waters ACQUITY system (Waters Corporation, Milford, USA) combined with a Thermo Scientific high-resolution mass spectrometer (MS) system (Thermo Fisher Scientific Inc, San Jose, USA) . C18 column (Acquity UPLC BEH C18-2.1x100 mm, 1.7 µm) was used to achieve the separation purpose. 0.2% formic acid solution was used for mobile phase A, pure acetonitrile was used for mobile phase B. Linear gradient elution methods was used for separation. Each sample was injected three times with 5 μL on each occasion. The temperature of the column oven was held at 40 °C and the flow rate was controlled at 0.4 mL/min. Both positive and negative ion mode was applied for data collection of MS.
Metabolites and Transcriptomic Data analysis
MarkerLynx (Waters, USA) was used for peak finding, filtering and alignment in the original spectrums. SIMCA-P+12.0 (Umetrics, Umea, Sweden) was used to perform statistical analyses between two groups such as principal component analysis (PCA) and orthogonal partial least squares (OPLS). MedCalc® Version 11.4.2.0 software was used to plot the receiver operating characteristic curve (ROC) analysis. The differential metabolites statistic analyses were carried out by SPSS 16.0. Metabolites were mapped to the KEGG (Kyoto Encyclopedia of Genes and Genomes Database)[28] and HMDB (The Human Metabolome Database)[29]. Transcriptomic data GSE69787 was acquired from GEO (Gene Expression Omnibus) platform[30]. R project[31] was used to ascertain the differentially expressed metabolites and genes. Volcano plot and heatmaps were also carried out through R project.