Study participant enrollment
To determine the differences between MetS and non-MetS subjects, regarding of changes in lipids and glucose levels before and after meal intake, the associations by MetS of LA remodeling, and the correlation of VLDL-χ with LA remodeling, this prospective cohort study was conducted at a single Medical Center. Consecutively, 167 participants were enrolled (age range, 28–74 years old). The exclusion criteria included significant coronary heart disease, myocardial infarction, congenital heart diseases, heart failure, significant heart valve diseases, cerebrovascular diseases, cancers, insulin therapy, women with pregnancy or breastfeeding. The inclusion of MetS participants shall meet requirement of criteria with any 3 of the following components: (1) central obesity (waist ≥ 80 cm for women and ≥ 90 cm for men); (2) raised blood pressure (BP) (systolic BP ≥130mmHg or diastolic BP ≥85 mmHg or treatment of previously diagnosed HTN); (2) raised fasting glucose (≥100 mg/dL or diagnosed type 2 DM); (3) raised triglyceride (≥150mg/dL or on triglyceride-lowering treatment); and (4) reduced HDL-C (< 50 mg/dL for women and < 40 mg/dL for men). Among these participants, 87 and 80 had MetS and did not have MetS, respectively. All study participants with MetS were seen by cardiologists or endocrinologists at Kaohsiung Medical University Hospital (Kaohsiung, Taiwan). The study protocol was approved by the Kaohsiung Medical University Hospital Institutional Review Board (IRB) (KMUHIRB-E(I)-20170256). An informed consent form was signed and returned by all participants before joining the study and undergoing plasma collection. This study adhered to the principles of the Declaration of Helsinki.
Sample and demographic data collection
All study participants were instructed to fast before beginning at midnight and came to the hospital at 8 AM. Each participant underwent two venous blood draws (20 mL for each sampling, in BD VACUETTE® EDTA Blood Tubes (Becton, Dickinson and Company, Franklin Lakes, NJ, USA)); one blood draw was performed in a fasting state, and the other was after finishing the combo meal of 620 calories including a ham-egg sandwich, hashed browns, and a sweetened tea. To abide by the IRB approved protocol, in which the amount of blood drawing shall not exceed 40 mL over a week, a postprandial time was randomly selected for each participant from one of 0.5 h, 1 h, 2 h, and 4 h. In addition to blood sample collection, each participant underwent measurements of height, body weight, abdominal and hip circumferences, blood pressure and heart rate measurements. The medical record, if available, was reviewed, data related to medical history (hypertension and type 2 DM) and medication use were recorded.
Laboratory testing for biochemical indicators and the quantification of negatively charged low-density lipoprotein (LDL-χ) and negatively charged VLDL (VLDL-χ)
The analysis of biochemical parameters was performed in the Department of Laboratory Medicine at Kaohsiung Medical University Hospital according to the standard operating procedures. Technicians who performed tests were blinded to participants identity and data. Randomly duplicate samples were used to determine the analytical accuracy and measurement precision. As described previously, pairs of plasma samples (in fasting and postprandial states) were obtained from 167 participants. Plasma samples were immediately supplemented with the following after collection to prevent bacterial contamination and oxidation: protease inhibitor cocktail (Roche Diagnostics, Indianapolis, IN), 1% penicillin/streptomycin/neomycin mixture (Invitrogen, Carlsbad, CA), and 0.5 mM EDTA. Plasma LDL and VLDL were isolated by using sequential potassium bromide density-gradient ultracentrifugation between a density range of 1.006 and 1.063 g/mL. Fractions of LDL and VLDL samples that were isolated by density were resolved into subfractions with most negatively charged lipoproteins, i.e., LDL-χ and VLDL-χ, respectively, by increasing the negative charge on UnoQ12 columns (BioRad, Hercules, CA) in the ion-exchange fast-protein liquid chromatography system (FPLC, GE Healthcare, Chicago, IL), as described previously [26, 31]. In short, the columns were first equilibrated with buffer A (0.02 M Tris–HCl, pH 8.0; 0.5 mM EDTA). With a multistep linear gradient of buffer B (1 M NaCl in buffer A) at a flow rate of 2 mL/min under observation at 280 nm, the LDL-χ and VLDL-χ subfractions were eluted, separately concentrated by using Centriprep filters (YM-30; EMD Millipore Corp., Billerica, MA) and sterilized by being passed through 0.22-μm filters. The protein concentration of the LDL and VLDL samples was measured by using the Lowry method [31, 32].
Electrocardiographic (ECG) parameters
Twelve-lead ECG was performed by experienced medical technicians. The parameters that were measured and recorded by one experienced technician who was blinded to the other data and clinical information included P wave durations, PR intervals, QRS width, QTc intervals, and the terminal force of P waves in lead V1 [33]. Cardiac rhythms were interpreted, and any rhythm other than regular sinus rhythm (such as AF and/or flutter, pacemaker rhythm, ventricular tachycardia, supraventricular tachycardia, and second- or third-degree atrial-ventricular block) was discarded before further analyses.
Echocardiographic assessment
Echocardiography was performed by one experienced cardiologist using a transthoracic cardiac probe (Vivid 7; General Electric Medical Systems, Horten, Norway), with the participant in the left decubitus position. Two-dimensional and two-dimensional-guided M-mode images were obtained, and LA diameter, left ventricle (LV) size, and LV function were assessed according to the standards of the American Society of Echocardiography [34]. LV ejection fraction (LVEF) was derived by the modified Simpson’s method [34]. The raw data were measured and recorded while the researchers were blinded to the other data.
Statistical analysis
All continuous variables are presented as the mean ± standard deviation. For all parameters examined in this study, the Shapiro-Wilk normality test was used to determine whether a random sample of values followed a normal distribution. To compare differences between the non-MetS and MetS groups, Student’s t test was used for continuous data, and a chi-square test or Fisher’s exact test was used for binary data. The associations of LA diameter and ECG parameters with body mass index (BMI), waist circumference, hip circumference, systemic blood pressure, pulse rate, fasting and postprandial plasma glucose, triglycerides, HDL-C, LDL-χ and VLDL-χ were evaluated by using the Pearson or Spearman (nonparametric) correlation. To evaluate the determining and confounding factors for LA diameter, simple linear regression and hierarchical multivariable linear regression with the stepwise method in the MetS group, non-MetS group, and total group were used to analyze all the variables, including age, hypertension, DM, BMI, waist circumference, hip circumference, heart rate, systolic blood pressure, diastolic blood pressure, VLDL-χ, triglycerides, high-density lipoprotein cholesterol, left ventricular size, LVEF, E/E’ med, and E/E’ lat. To validate robustness of main findings, several procedures were also carried out. First, a matching procedure by random selection of subjects from the non-MetS group by age within ± 2 years was conducted in order to reduce imbalance of age distribution between two groups. Second, multivariable analyses were re-performed by MetS for the overall and the age-matched 47 pairs subjects. Results were considered statistically significant based on a P value < 0.05. Statistical analyses were performed by using the statistical package in GraphPad Prism (version 8; GraphPad Software, Inc., San Diego, CA, USA) software system and SPSS statistical software (version 22, IBM Corp., Armonk, NY, USA) and SAS 9.4 software (SAS Institute Inc., Cary, NC, USA).