Il 18

Serum malondialdehyde (MDA) was measured with a thiobarbituric acid reactive substances kit using fluorometric procedures (TBARS Assay Kit, Cayman Chemical Company, Ann Arbor, MI). Lipopolysaccharide-binding protein (LBP) was measured in lithium heparinized plasma diluted 1:200 with the provided assay buffer using an enzyme-linked immunosorbent assay kit (Hycult Biotech, Plymouth Meeting, PA). Plasma total antioxidant capacity (TAC) was measured using a colorimetric assay (Antioxidant Assay Kit, Cayman Chemical Company, Ann Harbor, MI). Immediately after centrifugation, 100 μL of fresh plasma was diluted 1:50 with the provided assay buffer. TAC of plasma was defined as all antioxidants within a sample that inhibited the oxidation of 2,2’-Azino-di-3-ethylbenzthiazoline sulphonate by metmyoglobin relative to a water-soluble tocopherol analog and quantified as millimolar Trolox equivalents as described by Miller et al. (1993) (link). Serum cytokines were measured using a 13-plex Immunoassay targeting IFNy, IL-10, IL-12, IL-18, IL-1α, IL-1β, IL-4, IL-8, TNF-α, GM-CSF, IL-1ra, and IL-2 (Eve Technologies, Calgary, AB). A CV threshold of less than 5% was used for all biomarker analyses.

One day prior to sacrifice, animals underwent a dual x-ray absorptiometry (DXA) scan (Hologic ODR 4500; Hologic Inc., Marlborough, MA, USA) under light anesthetic (isoflurane). Lean mass (g), fat mass (g), body fat %, bone mineral content (g), and bone mineral density (BMD) (g/cm²) were assessed using Hologic QDR software for small animals. Following 12 h of feed deprivation, rats were anesthetized with isoflurane and blood collected from the portal vein. From this sample, a Milliplex Rat Cytokine Array/Chemokine Array (Millipore, St. Charles, MO, USA) was used to measure serum TNFα, IL-1α, IL-1β, IL-5, IL-10, IL-18, and leptin (Eve Technologies, Calgary, AB, Canada). Rats were subsequently killed by overanesthetization and decapitation. The cecum, colon, and jejunum were excised, cleaned, weighed, snap-frozen, and stored at −80 °C.