Lp pla2

A subgroup of paired human AbdSc and omental adipose tissue biopsies from participants who were lean (age 43.6 ± 6.2 years; BMI 22.5 ± 2.2 kg/m²; n = 9), overweight (age 47.5 ± 11.5 years; BMI 27.4 ± 1.5 kg/m²; n = 10) or obese (age 48.1 ± 8.5 years; BMI 34.0 ± 2.9 kg/m²; n = 5) was used for protein analysis. The adipose tissue was homogenised in Phosphosafe extraction buffer (Novagen, Merck, Darmstadt, Germany) and cultured adipocytes were harvested in RIPA buffer (Cell Signaling, Denver, MA, USA) with a cocktail of protease inhibitors, to extract total protein. Protein concentrations were measured using the Bio-Rad Detergent Compatible protein assay kit (Bio-Rad, San Diego, CA, USA) [16 (link)]. Western blotting was performed as described elsewhere [17 (link)], and protein levels of cPLA2 (1:100, Cell Signaling), iPLA2 (1:500, Sigma, Poole, UK) and Lp-PLA2 (1:200, R&D Systems) were assessed with rabbit and goat monoclonal antibodies.

Participants’ fasting, cryopreserved plasma samples were shipped to Temple University (Philadelphia, PA) for centralized quantification of levels of immune/inflammatory biomarkers using enzyme-linked immunosorbent assay kits: soluble CD14 (sCD14; R&D), soluble CD163 (sCD163; IQ Products), MCP-1 (R&D), high-sensitivity IL-6 (R&D), Lp-PLA2 (R&D), and oxLDL (Mercodia) [7 (link)]. D-dimer was measured using the HemosIL D-dimer HS 500 kit on the ACL TOP (Werfen). Fasting, cryopreserved serum samples were shipped to Quest Diagnostics for quantification of high-sensitivity C-reactive protein (hs-CRP) by commercially available assays.