Population
Vegetarians were recruited from Shanghai, China through vegetarian restaurants, vegetarian social activities, and online advertisements between the period of March 2016 and May 2016. The inclusion criteria were as follows: the participants had to 1) be at least 18 years of age; 2) reside in Shanghai at least for more than a period of 6 months; 3) follow a vegetarian diet for at least a year; 4) understand the contents of questionnaires. Exclusion criteria were based on 1) history of severe nutritional malabsorption or systemic diseases; 2) history of pregnancy or breastfeeding within the preceding 12 months. Omnivores were recruited within friends and relatives of the vegetarians and were matched respectively for the same sex and age (±1 year) to that of vegetarian participants. This study was approved by the Institutional Review Board of the Shanghai Jiao Tong University School of Medicine, and all participants provided written informed consent.
Anthropometric indexes, clinical and dietary assessment
All subjects were questioned about their demographics and personal behavior information, including age, sex, income, alcohol consumption, smoking, physical activity, sedentary time,sleep duration, vegetarian pattern (vegan or lacto-ovo-vegetarian), and vegetarian duration, with a questionnaire. Vegetarians were defined as people who followed a vegetarian diet at all daily meals for at least 1 year; otherwise, they were defined as omnivores. Vegans were defined as those who did not consume any sort of foods of animal origin, while those who consumed meat, eggs, dairy products, or fish were defined as “lacto-ovo-vegetarians”.
Participants' height, body weight, waist circumference, and hip circumference were obtained from the physical examination by trained dietitians following the standardized protocol. Body mass index (BMI) was calculated as weight (kilograms) divided by height (meters) squared. Waist-to-hip ratio was calculated as the measured waist circumference divided by hip circumference.
A 24-hour diet recall questionnaire was used to assess their daily consumption of different nutrients. Daily nutrient intakes were calculated from the 24-hour dietary recall data using Nutrition Calculator v2.5 software, which was developed by the Institute for Nutrition and Food Safety of the Chinese Centre for Disease Control and Prevention and Beijing B-win Technology Co. Ltd.
Assessment of toenail Se exposure 282 vegetarians and 282 matched omnivores were recruited in the study. Participants were asked to provide toenail clippings from all ten toes and return them to the researchers. 62 subjects did not provide their toenail samples. In this study, toenail samples were obtained from 220 vegetarians and 220 matched omnivores who met the criteria for inclusion. All toenail samples were pretreated by graphite digestion. The concentrations of toenail Se were measured according to inductively coupled plasma-mass spectrometry from the National Standard for Food Safety Determination of Multi Elements in Food (GB 5009.268-2016). Briefly, toenail samples (0.1g) were placed in 50ml PP centrifuge tube. After adding 0.5ml HNO3 solution (Guaranteed reagent, Merck, Germany) and 0.5ml H2O2 solution (Guaranteed reagent, LookChem, China), the mixtures were put in a graphite digester for digestion at 120 ° C for 2 hours. After digestion, the mixtures were taken out and cooled to room temperature. An inductively coupled plasma mass spectrometer (ICP-MS) ThermoFisher i CAP Q (ThermoFisher, USA) was used to analyze the levels of toenail Se. The lower detection limit and upper detection limit of Se in nails by ICP-MS, were 0.003μg/g and 3.2μg/g, respectively. The detection of Se level was conducted by professional researchers from Shanghai Jiao Tong University School of Agriculture and Biology.
Assessment of insulin resistance
Peripheral venous blood samples were collected after at least 12 hours of fasting. Fasting blood glucose (FG) and fasting insulin (FI) concentrations were tested by the Clinical Laboratory Center of Shanghai Xinhua Hospital. Insulin resistance and b-cell function were evaluated by the homeostasis model assessment (HOMA) method (18), in which FI (mU/L) and FG (mmol/L) were used
HOMA-IR= FI (mU/L)×FG (mmol/L) / 22.5 (16, 19)
Insulin resistance is defined as HOMA-IR > 2.60 (18, 20).
HOMA-B(%) = 20×FI (mU/L) / [ FG (mmol/L)- 3.5 ] (16, 19)
Statistical analysis
All statistical analyses were performed with the use of Stata software version 14.0 (StataCorp, College Station, TX, USA), and two-sided P values <0.05 were considered as statistically significant. The continuous variables were shown as mean ± standard deviation (SD), while the categorical variables were expressed as number and percentage (%). To compare the differences between vegetarian group and omnivore group, the paired Student’s t-test study for continuous variables and paired chi-square test for categorical variables were used. Covariance analysis was used to test the differences in glucose metabolism and insulin resistance between the vegan group and the lacto-ovo-vegetarian group after controlling the covariates, including age, sex, BMI, alcohol consumption, income, and daily dietary intakes (energy, protein, fat, carbohydrate, and fiber). Then multi-linear regression was used to investigate the association between nail concentrations of Se and insulin resistance after adjusting for all major confounders. Interaction and stratified analyses were conducted according to sex (male and female) and vegetarian type (Vegan and Lacto-ovo-vegetarian).