Guaranteed reagent

TN, TP, and TDP were analyzed following the National Standard method (HJ636-2012, GB11893-89) with UV–visible spectrophotometer (DR6000, HACH Co. Ltd., Loveland, CO, United States). The settled water samples were digested by heating with alkaline potassium persulfate and added hydrochloric acid. Subsequently, we performed the concentration measure of TN in the processed water samples by comparing calibration curves (R² = 0.9992). The settled water samples were digested by heating with potassium persulfate and adding 10% ascorbic acid solution and molybdate solution to coloration. Next, we performed the concentration measurement of TP in the processed water samples by comparing calibration curves (R² = 0.9991). TDP was determined the same way (Liu et al., 2020 (link); Bai et al., 2022 (link); de Anda et al., 2022 (link); Zhong et al., 2022 (link); Zhen-Zhen et al., 2023 (link)). All chemical reagents in this study were applied with guaranteed reagent (Merck Co. Ltd., Darmstadt Germany).

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. Sixty-two 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 (0.1 g) were placed in 50 ml PP centrifuge tube. After adding 0.5 ml HNO₃ solution (Guaranteed reagent, Merck, Germany) and 0.5 ml H₂O₂ solution (Guaranteed reagent, LookChem, China), the mixtures were put in a graphite digester for digestion at 120 °C for 2 h. 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 0.003 μg/g and 3.2 μg/g, respectively. The detection of Se level was conducted by professional researchers from Shanghai Jiao Tong University School of Agriculture and Biology.

Biological samples were placed in a freeze dryer (Scientz-100F) for vacuum freeze-drying. The freeze-dried fruit was crushed using a mixer mill (MM 400, Retsch, Dusseldorf, Germany) with zirconia beads for 1.5 min at 30 Hz. A 100-mg sample of powder was weighted and extracted overnight at 4 °C with 0.6 mL 70% aqueous methanol (GR: Guaranteed reagent, Merck, Darmstadt, Germany). Following centrifugation at 10,000× g for 10 min, the extracts were absorbed (CNWBOND Carbon-GCB SPE Cartridge, 250 mg, 3 mL; ANPEL, Shanghai, China, www.anpel.com.cn/cnw, accessed on January 2020) and filtrated (SCAA-104, 0.22-μm pore size; ANPEL, Shanghai, China, http://www.anpel.com.cn/, accessed on January 2020). The samples were stored in an injection flask for UPLC-MS/MS analysis. Quality control (QC) samples were prepared using mixed L. maackii fruit sample extracts to monitor the reproducibility of the samples under the same treatment method. One QC sample was inserted in each of the 10 detected samples during stability evaluation of the instrumental analysis conditions.