Peroxidation of leukocytes index ratio and a functional mathematical index: their potential application in screening of non-communicable diseases

This PhD thesis has been conducted in the context of a project aimed to evaluate the possible use of the PLIR, a test that measures both the resistance of leukocytes to an exogenous oxidative stress and the leukocytes functional capacity of oxidative burst upon activation, as redox marker in humans. The clinical relevance of a biomarker must be critically evaluated before the use in large trials. For the above reason we have planned pilot studies. In order to evaluate the redox status in particular conditions (smoking habit, disease states), ex vivo free-radical production and oxidative stress in body fluids are measured. These methods are used also in human intervention studies to associate the levels of ingested antioxidants (by foods or supplements) with improvement of the body antioxidant status. Despite the fact that it has been suggested that nutraceuticals are capable of improving health, significant methodological bias must be taken into account in the interpretation of data from the measurement of reactive species in leukocytes and platelets by flow cytometry, from the evaluation of markers based on ROS-induced modifications, from the assay of the enzymatic players of redox status, and from the measurement of the total antioxidant capacity of human body fluids. It has been suggested that the bias of each method could be overcome by the evaluation of oxidative stress by using more than one criterion and indexes of redox status have been proposed [Marrocco et al. 2017]. The OXY-SCORE was computed by subtracting the protection score (GSH, alpha- and gamma-tocopherol levels, and antioxidant capacity) from the damage score (plasma free and total malondialdehyde, GSSG/GSH ratio, and urine F2-IsoPs). In some diseases, the choice of the markers that must be considered in the global index should dictate the clinical relevance in the subjects selected. In patients with chronic venous insufficiency (CVI) was used for OXyVen index calculation the normalized and standardized plasma parameters which showed a significant statistical difference between CVI patients and controls (SH, MDA-bound protein, protein carbonyls, and CAT activity). However, the major bias of these index is the use of markers that do not have at the moment normal values. The oxidative-INDEX was calculated by subtracting the OXY (the antioxidant capacity measured with the OXY adsorbent test) standardized variable from the ROM (the reactive oxygen metabolites measured with the d-ROM) standardized variable. Although normal values have been proposed for these variables, the OXY adsorbent test quantifies the ability of the plasma nonenzymatic antioxidant compounds to cope with the in vitro oxidant action of hypochlorous acid (HOCl; an oxidant endogenously produced). This type of approach does not consider the important role of free radicals in the innate response and in the resistance to infection, that declines in some conditions, such as overtraining. Within the total antioxidant capacity assays the FRAP, exploits the same principle of biological antioxidant potential (BAP) (i. e. the reduction of ferric to ferrous ions), matches the antioxidant capacity to the reducing ability. It is well known that reduced iron is critical in the onset of oxidative stress due to the Fenton reaction, that generates the hydroxyl radical initiator of lipid peroxidation. Furthermore, the antioxidant capacity is strongly influenced by UA. The latter is a well known pathogenic factor when at high concentrations. In a previous postprandial study we observed that a functional food covered by dark chocolate and containing glucomannan, inulin, fructooligosaccharides, and Bacillus coagulans strain GanedenBC30 significantly improved postprandial metabolic stress (insulin, glucose, and triglycerides), reduced the postprandial increase of UA, and improved PLIR of lymphocytes, but not of monocytes and granulocytes. We suggested that, although PLIR is a functional index that is independent of baseline levels of oxidation, measuring the ratio between the resistance to exogenous and the resistance to endogenous ROS injury, this ratio calculation could mask the effect of foods that inhibit both the exogenous ROS injury and the oxidative burst. From that the aims of this thesis were: 1. Evaluate the relationships between PLIR and FRAP, its major endogenous determinant UA and FRAP-UA, by using a GTE due to its reported UA-lowering and potential pro-oxidant effects. 2. Study the relationships between PLIR and a mathematical index that considers health-related habits and UA plasma levels.