High‐throughput screening of oxidative stress biomarkers – significance, precision and cost‐effectiveness

Abstract

Since Reactive Oxygen (ROS) and Nitrogen Species (NOS) provoke a wealth of molecular, biochemical and immunological modifications, it is important to develop biomarkers to estimate the oxidative stress (OS) burden. In recent decades, several oxidative stress biomarkers have been developed to determine both antioxidative defense mechanisms as well as indicators for the pro-oxidative radical attack. Due to the interlocked network of antioxidants, free radicals, immunological reactions and lipid peroxidation products, a battery of biomarkers is needed to provide an overall impression with respect to the pro- and anti-oxidative balance in humans. This is of great importance to prevent carelessness administration of highly dosed antioxidant supplements as well as to contribute to diagnostics in health and disease. These aspects were the basis and challenge in designing distinct biomarkers at predetermined junctions, i.e. the measurement of antioxidants in the first line - with special attention to the kinetics of diverse antioxidants including TAC® (Total Antioxidant Capacity)1, PPm®(Polyphenols microtitre)] and the endogenous antioxidant system, e.g. peroxidase activity (EPA®)2. Total peroxides [TOC®] indicate short-term as well as long-term oxidative stress on lipids and proteins and are diagnostically as conclusive as isoprostanes3. Last but not least, antibodies against oxidized LDL are essential in assessing the overall health of individuals. Accordingly, different ELISA methods are available for IgM [MDA-LDL IgM®] and IgG antibodies [oLAb®] for both malondialdehyde (MDA)-modified LDL as well as copper-oxidized LDL4. As a remarkable feature we equipped the IgM MDA-LDL antibody ELISA with standards of a human monoclonal antibody emerging from the fusion of a female B-cell with a mouse myeloma cell line. These biomarkers were designed as high-throughput methods to determine a multitude of samples within a short space of time with high sensitivity, thereby minimizing the cost of a single determination. We have developed an assortment of assays to determine the interplay of pro- and antioxidative variables at distinct key events in the course of lipid peroxidation. Such tools are suitable for both scientific purposes and routine diagnosis to supply clinicians as well as scientists with additional information on the OS status. In conclusion, the main advantages of these biomarkers can be listed as follows: high capacity, low cost, suitability for every body fluid, high precision and sensitivity, specificity and short time of analysis. These methods have proved to be reliable and sensitive5 in such varied settings as epidemiologic studies on OS as a function of increasing body-mass index6 or the coincidence of deteriorating performance and increasing OS in top athletes7.