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.