Raw concentration data - Serum metabolomic features of diabetic retinopathy in glucose well-controlled diabetic patients
Description
To investigate systematic metabolic biomarkers for early diagnosis of glucose-well-controlled DM patients who still developed diabetic retinopathy (GW-DR) and explore potential biomarker-based precise treatment strategies for DR, a prospective hospital-based diabetes cohort was established at the Department of endocrinology and metabolism at Shanghai General Hospital. a total of 2126 diabetic patients have been recruited to receive diabetes management including glycemia control and regular follow-up care. A total of 42 glucose well-controlled diabetic participants, including 21 GW-DR subjects and 21 control subjects (GW-C) with matched HbA1c, fasting blood glucose (FBG), homeostasis model assessment for insulin resistance (HOMA-IR), homeostasis model assessment-b (HOMA-b), age, body mass index (BMI), and other clinical information both at baseline and follow-up using propensity score matching (PSM) were enrolled for serum metabolites analysis. To reveal the metabolic features of GW-DR patients, we used liquid chromatography-mass spectrometry to profile the metabolomic patterns in the serum of GW-DR and GW-C subjects.
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Sample collection Fasting blood samples were collected at both baseline and every follow-up. After at least 8-hour fasting, 10 mL of venous blood was collected under complete aseptic precautions from all the subjects and stored at 4℃. The serum was centrifuged at 2000 rpm for 10 min within 30 min. 5mL serum for clinical glucose and lipid profile evaluation and the remaining 5mL serum was then transferred into a 1.5 mL sterile tube and stored at −80℃ immediately for metabolomics analysis. After all participants were enrolled, serum samples were shipped in dry ice to the Zhejiang Provincial Key Laboratory for Cancer Molecular Cell Biology for metabolomics analysis. Metabolite extraction Each serum sample was extracted as previously described in our laboratory. In brief, 50μL of serum sample from each subject was used for analysis. The serum was added to 200 μL of methanol and the mixture was mixed in 96-well plates. After vortexing, the extract precipitated in the 96-well plates and the protein was removed by centrifugation (4 °C, 10 min, 500 X g). Then, the extract was lyophilized and stored at -80 °C. Before analysis, each sample was reconstituted with water/methanol (80:20, v/v). After vortexing and centrifugation, the 96-well plates were transferred into a liquid chromatography–mass spectrometry (LC-MS) system for analysis. Pooled QC samples were pretreated following the same procedure as described above. The QC samples were processed and detected after every ten actual serum samples. Metabolite quantitation The target metabolites were analyzed by LC-MS/MS with a triple quadrupole mass spectrometer (the QTRAP 6500 + System, ABSCIEX) using previously established methods. The separation was performed on 150 × 2.1mm, 5µm SeQuant ZIC-pHILIC HPLC Column (Merk), using a high-performance UHPLC system (Exion LC AD system) coupled to a triple quadrupole mass spectrometer (QTRAP 6500 + System, AB SCIEX). For targeted metabolomics, we performed liquid chromatography on the pHILIC column at a flow rate of 0.15 mL/min for 34 minutes, with 20 mM ammonium carbonate and 0.1% (v/v) ammonium hydroxide as Solvent A and acetonitrile as Solvent B. The gradient program was as follows: 0-1 min, 20% B; 1-3 min, 20%-60% B; 3-13 min, 60%-98% B; 13-13.1 min, 98%-20% B; 13.1-16 min, 20% B. Column temperature was 45°C and autosampler was charged to 4°C. Metabolites were detected by MRM transitions in positive and negative modes. Sample analysis was performed in positive/negative switching mode. The detection conditions of MRM-MS/MS are defined as follows: curtain gas (CUR) 45 psi, ion spray voltages (IS) 5500 V (positive) and -4500 V (negative), temperature (TEM) 350°C, ion source gas 1 (GS1): 45 psi, ion source gas 2 (GS2): 40 psi. Data were normalized and analyzed by MetaboAnalyst.