2.1. Study subjects
This study is a community-based cross-sectional investigation at Hangxin Hospital, Beijing, China, from July to November 2010. A total of 4000 community subjects underwent the health screening program. According to the diagnosis and classification criteria proposed by the American Diabetes Association (ADA) in 2010 19, diabetes was defined as someone with (1) a fasting plasma glucose (FPG) level of 7.0 mmol/L or higher, or (2) a 2h oral glucose tolerance test (2h OGTT) level of 11.1 mmol/L or higher, or (3) a glycosylated hemoglobin A1c (HbA1c) concentration of 6.5% or more. If the subject met one or more of these criteria, he/she was categorized as diabetes. Prediabetes was defined as someone with (1) FPG ≥ 5.6 mmol/L and < 7.0 mmol/L, or (2) 2h OGTT level of ≥ 7.8 mmol/L and < 11.1 mmol/L, respectively, or (3) a HbA1c concentration of ≥ 5.7% and < 6.5%, subjects without a prior diagnosis of diabetes. If the subject met one or more of these criteria, he/she was categorized as prediabetes. The inclusion criteria included age level of > 18 and < 75 years, local residents in Beijing, complete data measurements and informed consents. Subjects with cardiovascular and cerebrovascular diseases, mental disorders, gastrointestinal disease, nephropathy, metabolic syndrome, malignant tumors, pregnancy or incomplete recorded information were excluded from this project based on their medical records.
After investigation, a total of 105 subjects (69 males and 36 females) including 35 subjects for diabetes group, 35 subjects for prediabetes group and 35 subjects for healthy control group with complete data were finally enrolled in this study. The study was performed according to the guidelines of the Helsinki Declaration. A standard protocol was designed by Institute of Basic Research in Clinical Medicine and was approved by the Ethics Committee of China Academy of Chinese Medical Sciences. Written informed consent was obtained from all subjects.
2.2. Data measurements and blood sampling
All subjects were asked to fill out a questionnaire focusing on demographic characteristics (age, gender, education, etc.), anthropometrics (height, weight and waist circumference (WC)), medical history, and health-related behavior under the guidance of physicians. Fasting blood samples and 2h OGTT blood samples were drawn via venipuncture from the study subjects by clinical nurses. After storage for 2 h at 4°C, the blood samples were centrifuged at 3000 rpm for 10 min. The obtained serum was divided into two parts: one part was used for the measurement of FPG, HbA1c, 2h OGTT, total cholesterol (TC), triglyceride (TG), high- and low-density lipoprotein cholesterol (HDL-C, LDL-C), and uric acid (UC) concentrations according to the manufacturers’ instructions for the respective commercial test kits. The remaining 100 µL serum was added to 320 µL of methanol, and the mixture was vortexed for 60 s. After centrifugation at 15000 rpm for 10 min at 4°C, the supernatant was stored at -80°C for GC-MS analysis.
Blood sample (6mL) was randomly divided into six parts and extracted identically. These six samples were injected continuously to verify the repeatability of the sample preparation method. 20 µL are extracted from each blood sample to produce a mixed quality control (QC) sample and 100µL aliquot is extracted from this mixed sample by the same method. The mixed sample is used to provide a representative "mean" sample containing all the analytes encountered during the analysis and to verify the stability of the GC-MS system.
2.3. GC-MS analysis
GC-MS analysis was performed using GCMS-QP2010 Plus (Shimadzu, Kyoto) and capillary column (Rxi-50, 30 m×0.25 mm, 0.25 ft m).With helium as carrier gas, the rate was 1.0 mL/min. The oven temperature varied from 60 ~ 80℃ at 5℃/min, then from 80 ~ 90℃ at 2℃/min (keep for 3 min), from 90 ~ 150℃ at 10℃/min (keep for 1 min), from 150 ~ 220℃ at 1℃/min, and from 220 ~ 290℃ at 10℃/min. The injector and interface temperature were maintained at 250℃.The mass spectrum in electron impact mode was generated at 70 eV. The ion source temperature was maintained at 250℃.One sample of 1μL was injected with a split mode injection (split ratio，60:1).Based on the linear retention index (RI) and the comparison of MS data with reference compounds, these components were preliminarily identified. \The linear retention indices of all components were determined by homologous n-alkanes (C10–C40).These components were identified by comparing with the mass spectra of NIST05 and NIST05S.20
2.4. Data processing and statistical analysis
The number of components in different samples was selected according to the retention time of the common peaks. The retention time and peak areas of GC-MS were obtained in one table. The table is then used as input data for multivariate statistical analysis. A multivariate statistical analysis, including unsupervised principal component analysis (PCA) and partial least-squares-discriminant analysis (PLS-DA), was used for metabolic profiles using SIMCA-P 11.0 statistical package (Umetrics AB, Umeå, Sweden).SAS 9.1.3 Statistical package (order No.195557) for statistical analysis. Chi-square test was used in attribute data analysis. The measured data were normally distributed. Analysis of variance was used for the comparison between groups. P values < 0.05 were set as significant for all of the statistical tests.
2.5. Pathway analysis
The analysis of bio-functions and canonical pathways for the candidate metabolites were conducted by using the Ingenuity Pathway Analysis system (IPA, Ingenuity® Systems, http://www.ingenuity.com), to gain insight into the typical metabolic alterations associated with the biomarkers and the mechanisms related to the transition from prediabetes to diabetes.