Protein S

Dietary intake was assessed at baseline using validated food frequency questionnaires (FFQ). A slightly different approach was applied to the first two cohorts of the Rotterdam Study (RS-I and RS-II) than to the third cohort (RS-III). For the first two cohorts, an FFQ was applied in a two-stage approach. In the first stage, participants indicated which foods they consumed at least twice a month in the preceding year using a self-administered checklist of 170 food items. In a second stage, a trained dietician used this list to identify how often and in which amounts the foods were consumed. This FFQ was validated against fifteen 24 h food records and four 24 h urinary urea excretion samples in a subsample of the Rotterdam Study (n = 80), which demonstrated that it was able to adequately rank participants according to their intake: Pearson’s correlation for nutrient intakes with the food records ranged between 0.44 and 0.85 and Spearman’s correlation for protein intake against urinary urea was 0.67 [4 (link)]. For the third cohort, a self-administered semi quantitative FFQ was used to assess dietary intake. This FFQ was based on 389 items and was previously validated in two other Dutch populations using a 9-day dietary record [5 (link)] and a 4 week dietary history [6 (link)], which showed Pearson’s correlations for intakes of different nutrients varying from 0.40 to 0.86. For each food item, the frequency of consumption (in times per month or per week), the number of servings per day (expressed in standardized household measures) as well as the preparation methods were included. Information on portion size, type of food item, and preparation method were collected. Nutrient data were calculated from the Dutch Food Composition Table, using 1993’s version for RS-I, the 2001’s version for RS-II, and 2011’s update for RS-III to account for the changes in nutritional composition of foods. We excluded participants who had an unreliable dietary intake according to the trained nutritionist who performed the interview or because their estimated daily energy intake was implausible, for which cut-offs were set at <500 or >5000 kcal/day.

Cells were divided into the following five groups for western blotting: control group (untreated RAW 264.7 cells), model group (RAW 264.7 cells treated with ox-LDL), and three treatment groups (RAW 264.7 cells treated with ox-LDL and 0.2, 0.6, and 1.8 g/L of GXHP, respectively). Cells were collected, and a protein extraction kit (Gene pool, Beijing, China) was used to extract proteins according to the manufacturer’s protocol. The protein concentration was determined using a BCA protein assay (Multi Sciences, Hangzhou, China). Protein separation using 12% SDS-PAGE gel was then performed, and the protein samples were transferred to a PVDF membrane. After blocking, the PVDF membrane was incubated with specific primary antibodies (Cell Signaling Technology, Danvers, MA, USA), such as PI3 kinase p85 antibody (dilution ratio was 1:500), phospho-PI3 kinase p85 antibody (dilution ratio was 1:1,000), AKT1 antibody (dilution ratio was 1:1500), and phospho-AKT1 antibody (dilution ratio was 1:500), overnight at 4°C. This was followed by 1 hour incubation at room temperature with horseradish peroxidase conjugated goat anti-rabbit IgG (Abcam, Cambridge, MA, USA)(dilution ratio was 1:5000).Antigen–antibody binding was detected using enhanced chemiluminescence reagents (ThermoFisher Scientific, Waltham, MA, USA). Quantification of each protein was determined using Quantity One v.4.6.2.

Venous blood was collected in the early morning after an overnight fast. Plasma was separated and stored at −80°C until processing. Measurements of glucose, total and high-density lipoprotein cholesterol, triglycerides was performed with chemical methods in authomated devices, as previously reported (39 (link)). Low-density lipoprotein level was calculated by the Friedewald formula. Glomerular filtration rate was assessed by duplicate measurements of 24-h creatinine clearance. Coagulation parameters were measured in plasma as previously described (40 (link)). In brief, fibrinogen was assayed in an automatic coagulometer by a functional test, D-dimer was assayed immunoenzymatically, prothrombin fragment 1 + 2 (F1 + 2) and tissue-plasminogen activator (t-PA) by an enzyme-linked immunosorbent assay, plasminogen activator inhibitor-1 (PAI-1) by immunoassay, antithrombin III (ATIII), protein C, protein S, and von Willebrand factor (vWF) by functional chromogenic assays.