Hk406

We analyzed different domains of markers: hematology (e.g., leukocytes), muscle physiology (e.g., bicarbonate, creatinine), metabolic activity (e.g., glucose, insulin) and intestinal function (e.g., zonulin, intestinal fatty acid binding protein). A list of the parameters in the heatmap (Figure 3), including abbreviations, is provided as Supplementary Material (Supplement 2). Hematologic markers were analyzed using Advia 1200 (Siemens). Other markers were measured using the Cobas 6000 (Roche) (Star-shl, Etten-Leur, Netherlands), according to standard procedures. Zonulin concentrations in serum were determined using an ELISA kit (Immundiagnostik AG, Bensheim, Germany) and measured with an ELISA plate reader at 450 nm against 620 nm as reference (Wegh et al., 2019 (link)). Serum intestinal fatty acid binding protein (iFABP) levels were measured using a commercial human ELISA Test Kit (HK406, Hycult Biotech, Uden, Netherlands) according to the manufacturer’s instructions, and analyzed with a multi-detector microplate reader VICTOR^TM X3 (PerkinElmer) using Workout v2.5 software.

Urine was collected for 6 h by placing a cotton ball near the urethral meatus within the diaper to avoid stool contamination. Once the cotton ball was saturated, urine was obtained by squeezing it into a sterile syringe and collected in a urine tube. The samples were centrifuged, and the supernatants were frozen at −80 °C until evaluation [13 (link)]. The urinary concentration of I-FABP was quantified in duplicate samples using a commercially available enzyme-linked immunosorbent assay (HK406; Hycult Biotech, Plymouth Meeting, PA, USA). The urinary creatinine concentration (creatinine_u) was determined in duplicate samples using a colorimetric assay (Cayman Chemical Co., Ann Arbor, MI, USA). Urine was analyzed for creatinine content to calculate the I-FABP/creatinine_u ratio.

Heparin plasma samples were analyzed for plasma levels of I‐FABP using an enzyme‐linked immunosorbent assay (ELISA) kit (HK406, Hycult Biotech, the Netherlands) according to the manufacturer's instructions.