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

Abstract

Small blood volumes commonly obtained from small mammals during field studies are only sufficient for a single biochemical assay. In this study, we used blood collected from a population of wild eastern chipmunks (Tamias striatus) and developed modified methods to improve analytical selectivity and sensitivity required for measuring markers of oxidative stress using small blood volumes.Specifically, we proposed a modified malondialdehyde (MDA) analysis protocol by high performance liquid chromatography (HPLC) and also optimized both the uric acid independent ferric reducing antioxidant power (FRAP) and hypochlorous acid shock capacity (HASC) assays. We present methods in which a total volume of less than 60 μl of plasma is required to obtain a comprehensive portrait of an individual's oxidative profile.

Inflammatory and Oxidative Stress Markers—Mirror Tools in Rheumatoid Arthritis

“…Previous studies showed that the ROS act as a trigger that switch on the NLRP3 inflammasome and contribute to disease progression in a vicious cycle [54]. In accord with previous studies, we found that MLR increased in RA patients compared to the healthy-matched group [29]. Our study revealed that MLR has an increased accuracy compared to NLR, to an AUC of 0.93, and 95% CI between 0.82 and 1.03, as a result of ROC analysis.…”

“…Blood cells were removed from the plasma immediately following centrifugation. Plasma aliquots were properly stored (at −80 • C, with avoiding repeated freezing/refreezing cycle) for further analysis of oxidative stress markers, these are in accord with other studies that showed the sample stability for oxidative stress markers in biological fluids (for 1 year at −80 • C or two days at 4 • C) [29,30]. For inflammatory markers, venous blood samples were collected on a vacutainer from Becton Dickinson (BD) and the blood was allowed to clot for 20 min at room temperature (RT).…”

“…The lipid peroxidation analysis was established by quantifying TBARS using a spectrophotometric method. The TBARS assay is the most common used method to measure the level of malondialdehyde (MDA), the major lipid oxidation product in biological fluids, cell extract, and tissue sample, and correlated with other oxidative stress markers can offer a valuable window on the complex phenomenon of lipid peroxidation [29,[32][33][34]. The TBARS testing principle is based on the properties of thiobarbituric acid to react with the lipids hydroperoxide (Sigma Aldrich, Germany) and generate malondialdehyde that are generated from the lipid peroxidation reaction.…”