2.3. Laboratory Measurements
Inflammation, TNF-α, HT
BLOOD, HEART, INFLAMMATION, SEPARATION
Body weight, waist circumference, body fat, and blood pressure were measured by clinicians at the Nutrition Research Center at Loma Linda University during three out of the four clinic visits for each phase of the study. Measurements were performed under a normal setting with participants wearing light loose clothing. Blood pressure was measured after at least 5 min rest using an ambulatory blood pressure monitor and appropriately sized cuffs according to the guidelines of the American Heart Association as previously explained [After an overnight fast (≥10 h), blood samples were collected at baseline and the end of each 12-week period of the trial. After separation by centrifugation, serum, and plasma were aliquoted and stored immediately at −80 °C until the project was completed. Blood lipids were analyzed at the Analytical Core Laboratory, John Mayer USDA Human Research Center at Tufts University (Medford, NJ, USA) on an automated AU480 Clinical Chemistry Analyzer (Beckman Coulter, Inc., Brea, CA, USA), as specified in the manufacturer’s procedural documentation, and high sensitivity C-reactive protein (hsCRP) was measured by solid-phase, two-site chemiluminescent immunometric assays using the IMMULITE 2000, (Siemens Healthcare Diagnostics, Los Angeles, CA, USA). Tests per subject were conducted in the same analytical run to reduce systematic error and inter-assay variability. Inflammation and oxidation markers were assayed at the LLU nutrition laboratory using commercial ELISA and colorimetric kits. Tests for plasma human (IL-1), interleukin-1 receptor inhibitor (IL-1Ra), TNF-α, haptoglobin, and thiobarbituric acid reactive substances (TBARS) were assayed using ELISA kits obtained from R&D Systems (Minneapolis, MN, USA). The human IL-6 was from Abcam (Cambridge, MA, USA) and the colorimetric kit used to assay total antioxidant status (TAS) was from Millipore Sigma (Burlington, MA, USA). All samples were assayed in duplicate according to the manufacturer’s directions and absorbance was measured on an automatic microplate reader (Bio Tek Synergy HT, Winooski, VT, USA). Intra- and inter-assay CV were <10% on all assays.