Study design and participants
The present study included 1145 patients diagnosed with T2D, aged from 18 to 75 years, from March 2018 to January 2020 at the outpatient department of the Endocrinology of Ningbo First Hospital, Zhejiang province, China and the National Standardized Metabolic Disease Management Center (MMC). T2D was diagnosed based on the definition proposed by the American Diabetes Association [12]. Patients were excluded from this study according to the exclusion criteria of T2D patients: (1) age > 75 years and age < 18 years; (2) diagnosis with any kind of cancer; (3) positive islet autoantibodies; (4) glomerular filtration rate (eGFR) < 30 mL/min; (5) severe liver dysfunction; (6) acute infectious diseases; (7) pregnancy or lactation; (8) incompletion of standard questionnaires.
The research project was approved by the Ethics Committee of Ningbo First Hospital, China (No. 2019-R057) and followed the Declaration of Helsinki. Written informed consents were obtained from all participants.
Dietary assessment
Dietary information was collected through a standard food frequent questionnaire (FFQ), following the guidelines proposed by Ningbo first hospital. All the dietary information was collected by well-trained nurses. Quantitative dietary information was collected on how often usual foods (vegetables, fruits, soya and soya products) was consumed per day, how often meats (red meat, poultry, fish and shrimp) were consumed per week, and how many times seasoning (salt and sugar) was consumed per day. Dietary salt was categories into 4 groups: <4g/d, 4-6g/d, 6-8g/d and ≥8g/d. The missing reports were asked to fill in and the misreports were evaluated and corrected based on daily reasonable consumption.
Demography and lifestyle
All patients, who were willing to participate in the study, were invited to complete the MMC standard questionnaires on their demography [education (low education: lower than colleagues/universities and high education: colleagues/universities or above)], lifestyle [smoking status (no current smoking, sometimes and every day); drinking alcohol status (no current drinking, sometimes and every day); physical activity], medical history and medication records.
Anthropometric measurements and blood pressure
Anthropometric measurements including body weight, height and waist circumference (WC) were measured with light clothing by well-trained endocrinology nurses. Body weight was measured using an electronic scale to the nearest 0.1kg and height was measured using a metal column height meter to the nearest 0.1cm. WC was measured at the midpoint between the inferior costal margin and the iliac crest in the midaxillary line. BMI, defining a general obesity, was calculated as weight (kg) / height (m2). Patients were classified into four BMI categories according to China Obesity Task Force as follows: underweight (<18.5 kg/m2), normal weight (18.5-23.9 kg/m2), overweight (24.0-27.9 kg/m2), and obesity (≥ 28.0 kg/m2). Abdominal obesity was defined according to WC values: WC > 90 cm in men or > 85cm in women [13].
Blood pressure was measured using an electronic sphygmomanometer on the right or left arm after a 10-minute rest.
Biomarker measurements
After a 10-12h-overnight fasting, blood samples were obtained in the early morning to measure the levels of blood profiles including fasting plasma glucose (FPG), fasting insulin (FINS), glycated hemoglobin A1c (HbA1c), total cholesterol (TC), triglyceride (TG), high-density lipoprotein cholesterol (HDL-C) and low-density lipoprotein cholesterol (LDL-C). Then the 100g carbohydrate (steamed bread meal) test was performed in all subjects to assess the 2 hour postprandial plasma glucose (2hPG) and 2 hour postprandial insulin (2hINS) concentrations.
FPG and 2hPG were assessed by the glucose oxidase method and chemiluminescence immunoassay, respectively. FINS was measured by radioimmunoassay. Lipid profiles were analyzed by enzymatic procedures using an autoanalyzer (Hitachi 747; Hitachi, Tokyo, Japan). HbA1c was determined by high-pressure liquid chromatography.
Statistical analysis
The percentage of patients, mean and median values with standard deviation (SD) among the categories of salt intakes were presented as descriptive analysis. ANOVA with Bonferroni correction/ Games-Howell, and Mann-Whitney U test and Kruskal-Wallis were used to examine mean and median values of biomarkers within and between the categories of salt intakes.
Generalized linear models (GLM) were used to assess associations of salt intakes with biomarkers (FPG, 2hPG, FINS, 2hINS and HbA1c) after adjusting for confounding factors (gender, age and education levels), lifestyle factors (physical activity, smoking status and drinking status), BMI and interactions. Interactions between salt intake and confounding factors were only remained in the model.
Results were considered statistically significant at a two-tailed level of 0.05. Statistical analyses were conducted using IBM SPSS Statistics version 26.0.