Development of small blood volume assays for the measurement of oxidative stress markers in mammals

Abstract

Measuring oxidative stress has become increasingly valuable in ecological studies, especially when different markers are measured on the same individual. However, many of the current methods lack sensitivity for analysis of low blood volume samples, which represent a challenge for longitudinal field studies of small organisms. Small blood volumes can usually only be analysed by using a single assay, therefore providing limited information on individual’s oxidative profile. In this study, we used blood collected from a population of wild eastern chipmunks (Tamias striatus) and modified methods presented in the literature to improve analytical selectivity and sensitivity required for small blood volumes. Specifically, we proposed a modified malondialdehyde (MDA) analysis protocol by HPLC and also optimized both the uric acid independent ferric reducing antioxidant power (FRAP) and hypochlorous acid shock capacity (HASC) assays. Development of the three modified methods was achieved with a sensitivity and repeatability that meets standards of field ecology while allowing measurement of all three assays in duplicate using less than 60 μL of plasma. Availability of these tests using small blood volumes will provide ecologists with a more comprehensive portrait of an individual’s oxidative profile and a better understanding of its determinants and interactions with the environment.

Optimized Oxidative Stress Protocols for Low-microliter Volumes of Mammalian Plasma

“…researchers to perform comprehensive analyses of oxidative stress profile of small wild animals. We present in-house assays optimized to yield the most selective analysis for each marker using the smallest blood volumes possible (Langille et al, 2018).…”

Spatio-temporal variation in oxidative status regulation in a small mammal

“…Plasma oxidative damage and antioxidant levels were assayed following Langille et al.’s (2018) methods, which are optimised for small blood volumes. We thus only provide a brief overview of these assays.…”

“…Total non-enzymatic antioxidant power was assessed with two markers: the ferric reducing/antioxidant power (FRAP) assay and the hypochlorous acid shock capacity (HASC) assay. These markers assay antioxidant power with contrasting mechanisms (see Langille et al, 2018 for details) and comparative studies have found no correlation or a weak positive correlation between them (Costantini, 2011; Vassalle et al, 2004). They are, therefore likely to depict different and complementary pictures of the antioxidant status in sampled individuals (Langille et al, 2018; Vassalle et al, 2004).…”

“…This assay has been criticized for being highly sensitive to uric acid levels (Benzie & Strain, 1996; Cohen, Mcgraw & Robinson, 2009; Costantini, 2011), so here we used a uric acid independent FRAP assay. A 5 µL aliquot of plasma or standard (FeSO 4 , see Langille et al., 2018 for details) was mixed 1:1 with ultrapure water-containing uricase (1.00 U/mL) and incubated 5 min at room temperature on a 2D shaker. Then, 200 µL of FRAP reagent was added and absorbance was read at 593 nm using an iMark Filter absorbance microplate reader (Bio-Rad Laboratories Ltd., Mississauga, ON, Canada) after a 30 min incubation with shaking at room temperature.…”

“…It yields the total non-enzymatic antioxidant power through the amount of HOCl (mmol) required to fully oxidize a plasma sample. A 1 µL aliquot of plasma and 100 µL of oxidant solution (hypochlorous acid, see Langille et al., 2018 for details) were added to 100 µL of ultrapure water, followed by a 10 minutes incubation at room temperature on a 2D shaker. After that, 20 µL of N, N-dimethyl-p-phenylenediamine (83 µL in 50 mL anhydrous ethanol) was quickly added and absorbance was read at 515 nm after a 60 s incubation with shaking using the iMark Filter absorbance microplate reader.…”