Hk315 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.

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).

Surrogate measures of gut permeability consisted of circulating antibodies to Escherichia coli anti-core LPS by enzyme immunoassay (EIA) and plasma concentrations of LBP (HK315-02; Hycult Biotech). LBP was performed per the manufacturer’s instructions, with plasma diluted to 1:1000. Measurement of LPS antibody was performed as published,²⁶ (link) with plasmas diluted to 1:1000 in 0.5% bovine serum albumin (BSA) in phosphate-buffered saline with 0.05% Tween-20, and repeated with greater or lesser dilutions for samples in which the EIA values were outside the bounds of the standard curve. All assays were performed in duplicate. The EIA uses as antigen the core LPS purified from E coli O157:H7 strain 86-24^nalR ΔrfbE that does not express the O157 side chain, so antibodies detected reflect anti-E coli and not anti-E coli O157 LPS. The secondary antibody for these EIAs was goat anti-human IgG/F(ab)2-horseradish peroxidase (Millipore Sigma; diluted 1:10 000 in 0.5% BSA in phosphate-buffered saline with 0.05% Tween-20). Results for the anti-LPS antibodies are expressed as EIA units, normalized to a positive control as we have done previously.²⁶ (link)