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

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.