Non-enzymatic antioxidant capacity and oxidative damage
OXY Adsorbent test- OXY was measured in the plasma (concentration unit: mmol l−1 of HClO neutralized; Diacron International, Grosseto, Italy). OXY directly measures the ability of a plasma sample to quench the oxidant hypochlorous acid. In addition to directly reacting with biological molecules, hypochlorous acid can form more deleterious hydroxyl radicals (Candeias et al. 1993). OXY provides an index of non-enzymatic antioxidant capacity (e.g. dietary and non-dietary antioxidants), without being complicated by inclusion of uric acid (Alan and McWilliams, 2013; Cooper-Mullin et al. 2019; Costantini, 2011; Skrip and McWilliams, 2016).
Oxygen radical absorbance capacity- In preparation for the measurement of ORAC and LPO, approximately 250 mg of liver or pectoralis was homogenized on ice in 9 volumes of 0.1 M phosphate buffer, pH 7 with 3 x 10 sec pulses of a high-speed stainless-steel homogenizer (Tissue Master 125, Omni International, Kennesaw GA USA). Homogenate was centrifuged at 10,000 g for 10 minutes at 4°C (Beckman Coulter Allegra 21R), and the supernatant was aliquoted to 2 separate tubes to conduct the two separate assays (ORAC, LPO). Supernatant was immediately frozen at -80°C until the time of the assays (5-8 months after homogenization). We estimated antioxidant capacity against two of the more damaging forms of ROS that readily react with lipids (Halliwell and Gutteridge 2007), peroxyl and hydroxyl radicals (concentration unit: arbitrary units per gram of tissue, a.u.g-1), by using a microplate-based version of the competitive ORAC assay (Cao and Prior 1999) following (Jimenez et al. 2020). When in vitro production of the radicals exceeds the antioxidant capacity of the tissue, these ROS modify the algal pigment phycoerythrin (545 nm/575 nm) and decrease its fluorescence. Peroxyl radicals were generated by 320 mmol l−12,2′-azobis (2-amidinopropane) dihydrochloride, and hydroxyl radicals were generated in separate plates by adding 0.25 μl per well of 10 mmol l−1 CuSO4 and 0.667 mol l−1ascorbate mixture. ORAC values for peroxyl and hydroxyl radicals were determined by integrating the area under the fluorescence decay curve.
Uric acid -Uric acid is a by-product of protein catabolism that acts as an antioxidant (Halliwell and Gutteridge 2007). Methods and results for this metabolite were reported previously (citation redacted for initial review). Briefly, we assayed uric acid concentration (concentration unit: mmol/L) using an absorbance endpoint assay adapted for small volumes (TECO Diagnostics, Anaheim, CA) on 96-well plates in duplicate.
Lipid oxidative damage d-ROMs- Oxidative damage in the plasma was measured using the d-ROMs test (concentration unit: mmol l−1 H2O2 equivalents; Diacron International). This test works by first decreasing the pH of the plasma to release metal ions from proteins to cleave circulating ROMs through incubation with a solution of 0.01 mol l−1 acetic acid/sodium acetate buffer. The subsequent products react with a chromogen (N,N-diethyl-p-phenylenediamine) which has a color intensity that is proportional to the concentration of reactive oxygen metabolites (ROMs) in the plasma and was measured at 505 nm (Costantini et al. 2007, Costantini 2016, Cooper-Mullin et al. 2019). ROMs measured in this test are primarily hydroperoxides, and in plasma are primarily produced when reactive species interact with lipids (Davies 2016, Ito et al. 2017).
Lipid hydroperoxides- Oxidative damage in the liver and pectoralis was measured using the LPO test (concentration unit: hydroperoxide concentration µM; Cayman Chemical, Ann Arbor, MI). This assay provides a general measure of lipid peroxidation by directly measuring hydroperoxides compared to assays that measure the byproducts of specific fatty acid peroxidation (e.g. MDA, 4-HNE). Hydroperoxides in the sample were extracted using a Chloroform and methanol method. Hydroperoxides in the extraction react with ferrous ions detected by thiocyanate ion chromogen and was read at 500 nm in a glass 96-well plate.
Plasma or tissue from each individual was measured in triplicate for LPO and in duplicate for all other assays (OXY, peroxyl and hydroxyl radical absorbance capacity, d-ROMs); all coefficient of variations were under 10%, and replicates were averaged prior to statistical analyses.