Hk320 02

LGMs were measured in the serum or EDTA plasma samples of the 500 individuals selected from the NQplus study [22 (link)]. These samples were collected in the fasting state (after an overnight fasting period) and were stored at −80 °C at the Division of Human Nutrition of Wageningen University. Enzyme-linked immunosorbent assays (ELISA) were used to measure zonulin (K5601, Immundiagnostik AG, Bensheim, Germany), LBP (HK315-02, Hycult Biotech, Uden, The Netherlands), and sCD14 (HK320-02, Hycult Biotech), according to the manufacturers’ protocol. After measurement of LGMs, CRP, a marker for inflammation, was measured in EDTA plasma (CRP range: 0.25–16 ng/mL) (EC1001-1, Assaypro LLC, St. Charles, MO, USA). All samples were analyzed in duplicates.

Soluble CD14 (sCD14), high-mobility group box-1 (HMGB-1), lipopolysaccharide (LPS), and d-lactate were measured from peripheral blood samples. Peripheral blood samples were collected on a day of the annual follow-up from all participants in the present translational study (median 9 years after treatment, range: 4-32 years). FACT-Cog questionnaires were completed during the same follow up visit. Peripheral blood samples were collected into BD Vacutainer^® K2EDTA Tubes (BD Biosciences, Franklin Lakes, NJ, USA) containing ethylenediaminetetraacetic acid in the morning of the annual follow-up visit (n=142). Blood samples (1 ml) were initially centrifuged at 2,500 × g for 10 min and subsequently at 3,500 × g for 10 min to separate the plasma from the blood cells. Plasma aliquots were stored at −80°C until further analysis.
HMGB1 (LS-F4038; LSBio, Seattle, WA, USA), LPS (abx150357; Abbexa, Cambridge, United Kingdom) and sCD14 (HK320-02; Hycult Biotech, Uden, Holland) were all measured using commercially available ELISA kits and the corresponding calibration standards. D-lactate was assessed using an enzymatic colorimetric assay (MAK058; Sigma-Aldrich, St. Louis, MO, USA). Intra-assay and inter-assay coefficients of variation for all assays were below 5% and 10%, respectively.

In the fasting blood sample taken prior to consumption of the MS mix solution, zonulin, LBP, and sCD14 were evaluated. Enzyme-linked immunosorbent assay (ELISA) was used to measure zonulin (K5601, Immundiagnostik AG, Bensheim, Germany) in serum and LBP (HK315-02, Hycult Biotech, Uden, The Netherlands) and sCD14 (HK320-02, Hycult Biotech) in plasma, according to the manufacturer’s protocols. Additionally, to gain a more accurate insight into the metabolic health of the subjects, the body mass index (BMI) was calculated from the heights and weights of the subjects, and the metabolic health markers glucose, LDL cholesterol, HDL cholesterol, total cholesterol, total triglycerides, ALT, GGT, and the inflammatory marker C-reactive protein (CRP) were measured in the fasting blood sample. These metabolic health markers in the blood were assessed at the hospital Gelderse Vallei (Ede, The Netherlands) using standardized clinical procedures. All blood biomarkers were assessed twice (once with and once without an acetylsalicylic acid challenge prior to the gut permeability test).