Determining the Viability of Biomarkers for Oxidative Stress Detection in Clinically Afflicted Cohorts

Intro: In the US, there are more than 5 million Americans living with Alzheimer’s disease (AD) and approximately 500,000 more are suffering from Parkinson’s disease (PD). Oxidative stress (OS) and its deregulation of reactive oxygen species (ROS) have been implicated as a component of neurodegenerative diseases. Accumulation of ROS is accountable for increasing mitochondrial dysfunction that leads to neuronal apoptosis. In addition to ROS accumulation, OS can result in a number of cellular by-products such as Malondialdehyde (MDA), 8-Isoprostane, and Advanced Oxidation Protein Products (AOPP). Recent studies also suggest that high levels of testosterone can depress cellular resistance to oxidative stress. Purpose: The objective of this research is to determine the viability of laboratory assays as a detection tool for the evaluation of neurodegenerative diseases in aging males. Methods: 352 clinical plasma samples from the Texas Alzheimer’s Research and Care Consortium were used in assay evaluation. The samples were collected from either Caucasian or Hispanic males and evaluated based on 6 previously determined disease statuses. The AOPP Assay was used to detect chlorinated oxidation of plasma proteins. For AOPP, plasma samples were performed with a 1:7 dilution factor. Following preparation, the samples were then plated in duplicates and read at an absorbance of 340nm. To correlate with a previous testosterone study, a Testosterone ELISA was conducted with the use of Rabbit Anti-Free T-Antibody without sample dilution. Once plated, the samples were incubated for 60 minutes, washed, and read at an absorbance of 450nm. MDA can be accounted for in OS-resultant lipid peroxidation (LP). MDA levels in the clinical samples were examined using a Colorimetric TBARS Assay. The assay subjected the samples to boiling, centrifuging, and incubation in an ice bath. The supernatant was then removed, plated, and read at an absorbance of 532nm. Results: The absorbance reading from the AOPP assay and Colorimetric TBARS assay, yielded similar results across all six-disease statuses. Despite the differences in disease status, samples were determined to have triple the amount of MDA concentration compared the control. Conclusion: The similarity in values and discrepancy from the control indicates that AOPP and TBARS cannot accurately determine OS in banked plasma samples. T-ELISA is currently undergoing further scrutiny with the use of mass spectrometry.