Reagent diluent

Tissues were fixed in 1% paraformaldehyde (PFA) overnight at 4°C, dehydrated in 30% sucrose and frozen in optimum cutting temperature (OCT) compound (VWR). Frozen sections were stained with antibodies (1:100) in buffer containing 0.1 M TRIS, 1% bovine serum albumin (Reagent diluent, R&D), 1% mouse serum and 0.3% Triton X-100 (Sigma) overnight at 4°C. Images were acquired using a Leica TCS SP8 microscope and 40X oil immersion objective.

Circulating levels of human total PON-1 were measured in Lithium-heparin plasma by an enzyme-linked immunosorbent assay (ELISA) purchased from R&D Systems (catalog No. DYC5816-5) and performed according to the manufacturer’s recommendations. Samples for ELISA were prepared at 100× dilution in sample diluent purchased from R&D Systems (catalog No. DYC001). The ELISA assay kit contained human total PON-1 capture antibody, detection antibody, PON-1 standard and streptavidin HRP. Additional reagents such as reagent diluent (catalog No. DY995), substrate solution (catalogue No. DY999), and stop solution (catalog No. DY994) were also purchased from R&D systems. The minimum and maximum amount of detectable PON-1 were 0.15 ng/mL and 10 ng/mL, respectively. Western blot was performed independently using a monoclonal antibody to PON-1 [31 (link)] to validate the ability of the ELISA to detect the presence of circulating PON-1 protein in plasma (Figure S4).
Circulating lactonase activity of PON was measured in the patient serum samples with a commercially available fluorometric assay (BioVision Incorporated, catalog # K999-100). Serum PON lactonase activity was calculated as the hydrolytic activity toward a fluorogenic benzopyran-2-one substrate of PON in the presence and absence of a specific PON inhibitor (2-hydroxyquinoline) according to the manufacturer’s protocol.

HUVEC or HCAEC were seeded at 80 000 cells/well in fibronectin coated 24‐well plates. Cells were treated for 30 min with peak metabolite profiles identified previously at 1, 6, 24 h post consumption (Table 1) or 0.01% DMSO (vehicle control) prior to the addition of 10 ng/mL (HUVEC) or 0.1 ng/mL (HCAEC) TNF‐α (determined as providing maximal induction while maintaining physiologically relevant concentrations following time‐ and concentration‐response experiments), and incubated for 24 h at 37°C, 5% CO₂, in a humidified atmosphere. Supernatants were collected on ice, centrifuged at 2000 ×g for 10 min at 4°C, and stored at ‐80°C prior to ELISA. Samples were thawed at room temperature and vortexed for 3 × 5 s immediately prior to analysis. Supernatants were diluted 1:1 in Reagent Diluent (R&D Systems) and protein expression of soluble VCAM‐1 (sVCAM‐1; hereby referred to as VCAM‐1) and IL‐6 were determined by commercially available DuoSet enzyme‐linked immunosorbent assay (ELISA) (R&D Systems), according to the manufacturer's instructions. Absorbance values for all ELISA plates were recorded using an OMEGA plate reader from BMG LABTECH (Bucks, UK).