Study design and participants
The data were extracted from electronic medical record of Hunan Provincial People's Hospital according to inclusion criteria and exclusion criteria in 2019. This study's protocol was established according to the ethical guidelines of the Helsinki Declaration and was approved by the Human Ethics Committee of the First Affiliated Hospital of Hunan Normal University (approval NO.2018-20). The inclusion criteria for patients with coronary heart disease were (1) The diagnosis of coronary heart disease was in accordance with WHO diagnostic standard in 1999, and was confirmed as coronary heart disease by electrocardiogram, color doppler echocardiography and coronary angiography; (2) There was no anti-anxiety and depression drugs taking before; (3) The patients have normal reading and cognitive ability, and can cooperate to fill out the Hamilton Anxiety Scale (HAMA); (4) Hamilton Anxiety Scale-17 (HAMD-17) score ≤14; (5) Age ranges from ≥ 18 to ≤ 80 years old. (6) Resting blood pressure value ≤180/120 mmHg. The exclusion criteria were (1) Cardiac function grading of New York Heart Association(NYHA) grade ≥ Ⅲ grade; (2) Comorbidity with severe arrhythmia or severe cardiac dilatation; (3) Resting blood pressure value above 180/120 mmHg; (4) Comorbidity with diabetes or blood sugar still has not been improved after treatment; (5) Complicated with chronic infectious diseases, serious liver, brain, kidney and lung related diseases; (6) History of depression or anxiety, and used psychotropic drugs, alternative drugs or psychotherapy in the first four weeks or included in the electric shock treatment (ECT) eight weeks ago, substance abuse or dependence in the first three months. (7) Severe anxiety or depression, cognitive impairment, nervous system disease, or other mental illness. (8) In recent 3 months patients have had or will have traumatic heart surgery. (9) Tumor patients. (10) Abnormal thyroid function.
In addition to the above diagnostic criteria for CHD, patients with CHD comorbid anxiety also met the criteria diagnostic criteria of Chinese classification and diagnostic criteria for mental disorders (3rd Edition), and the international HAMD-17 score≥14.
The data of patients contained detailed information including age, gender, diagnosis, body mass index(BMI), chronic diseases including hypertension (no/yes), or hyperlipidemia (no/yes), medical insurance (with/without) and employment status (employment/retirement), left ventricular ejection fraction(LVEF), N terminal pro B type natriuretic peptide(NT-proBNP), fasting blood glucose(FBS), triglyceride(TG), cholesterol(TC), low density cholesterol(LDL-C), routine blood test, hepatic and renal functions. These above data were extracted as baseline demographic characteristics, and were divided into two groups: CHD and CHD comorbidity anxiety group (HAMD-17 score≥14). The informed consent was obtained from all patients.
Collection, treatment and analysis of blood samples
Blood was collected from veins, kept in heparinized tubes, and centrifuged at 3,000 × g at 4◦C for 5min to obtain plasma. Plasma neuropeptide Y(NPY) levels were measured by enzymelinked immunosorbent assay (CUSABIO, USA) according to instruction.
LC/MS-grade acetonitrile and HPLC-grade methanol were purchased from Merck (Darmstadt, Germany). Methanoic acid was purchased from CNW Company (Germany). All other chemicals were of analytical grade and were purchased from Sigma (St. Louis, MO, USA). Watson’s distilled water was used.
Metabolome Analysis Using UPLC-QTOF-MS
Blood was collected as above, and the supernatant plasma was stored at −80◦C. All samples were then thawed for 15min and vortexed for 5s prior to analysis. A total of 200 µL of plasma sample was mixed with 600 µL of methanol, vortexed for 40s, and left undisturbed for 20min. After centrifugation at 12,000 × g for 15min at 4◦C, 600 μL supernatant dried in vacuum at room temperature, then the residue dissolved in 200 μL 50% acetonitrile, vortexed for 40 s, and centrifuged at low temperature and high speed (4 ℃, 12000 RPM / min) for 10 min. 100 µL supernatant was extracted for ultra performance liquid chromatography coupled with quadrupole time-of-flight tandem mass spetrometry (UPLC-QTOF-MS) assay.
UPLC-QTOF-MS Analysis (Impact II, Bruker) were used to analyze the samples. The chromatographic separation conditions and quality control (QC) procedures referred to the experimental method of Ren Yu et al33. The details were as follows:each sample (10µL) was injected into a Acclaim TM 120 C18 column (Themo Fisher, USA, 100 × 2.1mm, 2.2 µm) at 4◦C, and the following mobile phases were used at a flow-rate of 0.2mL/min at 30◦C: H2O with 0.1% methane acid (A) and acetonitrile with 0.1% methane acid (B). Mass spectrometry analysis was performed in V flight tube detection mode with nitrogen as the atomization cone gas in both positive and negative ion modes. The source temperature was set at 200◦C, the extraction cone was at 4v, and a cone gas flow of 8.0 L/min was used in both modes. Capillary voltage was set at 4.0 and 3.5 kV, the sampling cone was 35 and 50 kV, the desolvation temperature was 350◦C and 300◦C, respectively, and the desolvation gas ﬂow was set at 600 and 700 L/h, respectively. Mass spectrometric data was collected in centroid mode from 20 to 1,000 m/z.
The peak height for the internal standard was continuously monitored during analysis to ensure signal stability. Quality control (QC) procedures were used to validate the methods and to ensure stability. 50 µL QC samples prepared by pooling identical volumes of individual plasma sample. To ensure that the system was suitable for use, 6 pooled QC samples were run prior to analysis in each ion mode. Six ions (min_m/z) were selected to evaluate the relative standard deviation (RSD) of retention time, m/z, and peak area.
The scatter plot of the first principal component is shown in Figure 5. The results show that all QC samples are distributed within the scope of 2SD , indicating that the consistency of experimental operation and the stability of the instrument system are within the controllable range.
Identiﬁcation of Metabolites
The UPLC-QTOF-MS data were imported into the Metaboscape version 3.0 analysis software (Bruker company), and the positive and negative Brooke matrix tables were established respectively to detect and align the peaks of all samples. The parameters were set as follows: the chromatographic peaks with retention time of 0-30min were intercepted, the peak intensity threshold was 1000, the minimum peak length was 5 spectra, the peaks were screened according to the 80% rule, and the mass spectrum data were corrected with sodium formate. After being identified and aligned, we normalized the strength of each ion with the total strength of the total ions in each chromatogram. We used the standard database, HMDB database and online search database of Bruker company to identify the secondary mass spectra and get the corresponding compounds. Finally, the three-dimensional matrix information includes retention time(RT), mass charge ratio (M/Z), ionic strength information (variables) and compound name. In addition, the Kyoto encyclopedia of genes and genomes (KEGG, http://www.genome.jp/kegg/) biochemical database was used to interpret possible pathways involving the identiﬁed metabolites.
The continuous variables were expressed as means ± standard errors of the mean, compared by Student’s t-test before the Levene test to ensure the equality of variances, otherwise using wilcoxon rank-sunm test. Categorical variables were expressed as numbers and percentages, compared by Chi-square test. All statistical analysis was performed with SPSS 24.0 software; significant diﬀerences were indicated by p ＜0.05.
Mass Profile software was used for peak extraction, retention time (RT) alignment, peak alignment, and deconvolution analysis. Finally, data were imported into SIMCA-P software (v14.0, Umetric, Umea, Sweden) for principal component analysis(PCA) and orthogonal partial least squares discriminant analysis(OPLS-DA). Variable Importance in Projection (VIP )> 1 and p ＜ 0.05 were considered statistically significant.