Hs crp

Highly sensitive CRP, leptin, IL-6, TNF-α and adiponectin were assayed by an in-house ELISA technique (hs-CRP, Abcam; remainder, R&D Systems). Insulin levels were measured using a radiolabelling technique as described previously [9 (link)]. The homeostasis model assessment (HOMA) [10 (link)] was used to estimate beta cell function (HOMA-β) and peripheral insulin sensitivity (HOMA-S).

Plasma, isolated from EDTA-treated peripheral blood, and serum samples were stored at −80 °C until analyses. Peripheral blood mononuclear cells (PBMCs) were isolated from EDTA-treated blood using Hypaque-Ficoll (GE Healthcare) density gradient centrifugation, counted with Nucleocounter® and then cryopreserved at −150 °C in fetal bovine serum (Sigma-Aldrich) containing 10% DMSO (Sigma-Aldrich), at a concentration of 10⁶ cells/ml of cryopreservation media. Soluble markers of inflammation and microbial translocation, and metabolites of tryptophan catabolism pathway were analyzed in plasma by ELISA (hs-CRP (Abcam, UK), sCD14 (R&D, Minnesota, USA), IL-6 (R&D), LBP (Hycult Biotech, The Netherlands)) or HPLC (http://bevital.no), respectively, according to manufacturer’s instructions.

Resting blood samples were collected at baseline and after the 9-month intervention using Serum Separating Tubes (BD Vacutainers). The serum samples were stored at −80 until analysis via commercially available ELISAs to characterize changes in kappa and lambda FLCs (Seralite; Abingdon Health, Oxford, United Kingdom) and hs-CRP (Abcam Cambridge, MS). Using published guidelines for hs-CRP (Haffner, 2006 (link)) and circulating FLCs (Heaney et al., 2020 (link)), 51.8% of participants had abnormal hs-CRP at baseline (greater than 3 mg∙L^-1), and 41.5% of participants had abnormally high levels of kappa FLC (8.72–23.0 mg∙L^-1). Creatinine and Cystatin c were measured to control for kidney function (Abcam, Cambridge, MS) according to the CKD-EPI equation (Inker et al., 2021 (link)).