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

Uric Acid and Cognition: What Is the Connection?

Cognitive decline is one of the most frequent and disabling non motor features in ageing. The relationship between uric acid (UA) levels and cognitive impairment varies with dementia subtype. In this context, dysmetabolism, oxidative stress and neuroinflammation play a role in cognitive impairment and are all affected by UA. The mechanisms of neuroprotection against oxidative stresses include both the antioxidant activity against reactive oxygen species (ROS) and the induction of the gene expression of the antioxidant enzymes. On the other hand, hyperuricemia induces the production of ROS and inflammatory cytokines, leading to neuroinflammation and neurodegeneration. The increased brain gliosis could link neuroinflammation and dysmetabolism. Also the gender-related differences in the clinical features of cognitive impairment could be partly related to UA and should be taken into account for the `normal` range set for UA concentration. In conclusion, UA affects cognitive function, by interacting with neural, immune, cardiometabolic and, probably, adipose pathways and could be a target of the neuro-immune-adipose interactions

Relationship between the peroxidation of leukocytes index ratio and the improvement of postprandial metabolic stress by a functional food

For the first time, we investigated the relationship between postprandial dysmetabolism and the Peroxidation of Leukocytes Index Ratio (PLIR), a test that measures the resistance of leukocytes to exogenous oxidative stress and their functional capacity of oxidative burst upon activation. Following a blind, placebo controlled, randomized, crossover design, ten healthy subjects ingested, in two different occasions, a high fat and high carbohydrates meal with Snello cookie (HFHCM-S) or with control cookies (HFHCM-C). Snello cookie, 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) and reduced the postprandial increase of uric acid. HFHCM-S improved PLIR of lymphocytes, but not of monocytes and granulocytes. Both meals increased granulocytes' count and reduced the lipoperoxidation induced by both exogenous free radicals and reactive oxygen species (ROS) produced by oxidative burst. Our results suggest that the healthy status of the subjects could be a limitation of this pilot study for PLIR evaluation on cells that produce ROS by oxidative burst. In conclusion, the relationship between PLIR and postprandial dysmetabolism requires further investigation

The peroxidation of leukocytes index ratio reveals the prooxidant effect of green tea extract

Despite tea increased plasma nonenzymatic antioxidant capacity, the European Food Safety Administration (EFSA) denied claims related to tea and its protection from oxidative damage. Furthermore, the Supplement Information Expert Committee (DSI EC) expressed some doubts on the safety of green tea extract (GTE). We performed a pilot study in order to evaluate the effect of a single dose of two capsules of a GTE supplement (200mg x 2) on the peroxidation of leukocytes index ratio (PLIR) in relation to uric acid (UA) and ferric reducing antioxidant potential (FRAP), as well as the sample size to reach statistical significance. GTE induced a prooxidant effect on leukocytes, whereas FRAP did not change, in agreement with the EFSA and the DSI EC conclusions. Besides, our results confirm the primary role of UA in the antioxidant defences. The ratio based calculation of the PLIR reduced the sample size to reach statistical significance, compared to the resistance to an exogenous oxidative stress and to the functional capacity of oxidative burst. Therefore, PLIR could be a sensitive marker of redox status

Nutraceutical-based integrative medicine: adopting a mediterranean diet pyramid for attaining healthy ageing in veterans with disabilities

Veterans with disability represent a big burden worldwide and often require long-term rehabilitation. Unhealthy dietary and lifestyle habits, including smoke and alcohol abuse, are common in veterans. In the context of integrative medicine approaches, the "complementary and alternative medicine" has been suggested for the management of chronic diseases. However, the potential risk of interaction between herbal products, dietary supplements and drugs must be considered in veterans. The Mediterranean diet has been suggested as a natural, non-pharmacological nutraceutical for healthy ageing. Although there is a broad consensus on the positive effect of plant foods consumption, the presence of glucosinolates, flavonoids and furanocoumarins in some plant foods and beverages must be taken into consideration owing to their potential interfering with drugs metabolism and bioavailability. Albeit seasonality could ensure the maintenance of the single dose of phytochemical below that at which adverse effects in some individuals genetically predisposed or unpleasant drug interactions in diseased subjects can occur, a personalized nutrition is recommended in veterans who are in treatment for comorbidities. Furthermore, sports practice can lead veterans with motor disabilities and mental impairments to excel in some disciplines, giving rise to the phenomenon of the Paralympics and the development of "recreational therapy". Moreover, outdoor lifestyle, through vitamin D synthesis, and conviviality, improving socialization, could account for the Mediterranean lifestyle health benefits. In this work we propose for veterans a Mediterranean Pyramid, which could be the basis for integrative medicine for veterans with disabilities, patient-centered approaches and interprofessional (including physical medicine and rehabilitation clinicians, pharmacists and nutritionists) interventions

In vitro evaluation of antimicrobial and antioxidant activities of algal extracts

The aim of this study was to evaluate the antioxidant capacity, the antimicrobial proprieties of algae Ascophyllum nodosum and Schizochytrium spp. against one of major swine enteric pathogen Escherichia coli O138 by broth macro-dilution method in Luria-Bertani (LB) medium. The antimicrobial effect of the algal extracts at supplementation of 0.12%, 0.06% and 0.03% (v/v) on E. coli O138, genetically characterised by PCR, was evaluated by following the bacterial growth. The antioxidant activity was determined by the ABTS Radical Cation Decolorisation Assay. In particular, the log(10) E. coli used as control resulted significantly higher than 0.12% at 3 hours (8.82 +/- 0.07 and 8.18 +/- 0.07 log(10) cells/mL, respectively; p<.01) suggesting an inhibitory activity related to the dose. No effect activity was observed with Schizochytrium spp. against E. coli growth. A. nodosum and Schizochytrium spp. exhibited antioxidant capacity (p<.05). The combination of them (1:1) exhibited antioxidant activity suggesting a synergistic effect (p<.05). The different proprieties of algal species that can modulate the O138 E. coli growth, one of the major pathogen of swine species, together with the antioxidant capacity, make them a promising functional feed additive to improve the gut health, therefore further studies are needed to confirm these activities in vivo

Association of antioxidant nutraceuticals and acetaminophen (paracetamol): Friend or foe?

Acetaminophen (paracetamol or APAP) is an analgesic and antipyretic drug that can induce oxidative stress-mediated hepatotoxicity at high doses. Several studies reported that antioxidant nutraceuticals, in particular phenolic phytochemicals from dietary food, spices, herbs and algae have hepatoprotective effects. Others, however, suggested that they may negatively impact the metabolism, efficacy and toxicity of APAP. The aim of this review is to discuss the pros and cons of the association of antioxidant nutraceuticals and APAP by reviewing the in vivo evidence, with particular reference to APAP pharmacokinetics and hepatotoxicity. Results from the murine models of APAP-induced hepatotoxicity showed amelioration of liver damage with nutraceuticals coadministration, as well as reductions in tissue markers of oxidative stress, and serum levels of hepatic enzymes, bilirubin, cholesterol, triglycerides and inflammatory cytokines. On the other hand, both increased and decreased APAP plasma levels have been reported, depending on the nutraceutical type and route of administration. For example, studies showed that repeated administration of flavonoids causes down-regulation of cytochrome P450 enzymes and up-regulation of uridine diphosphate glucuronosyltransferases (UGT). Moreover, nutraceuticals can alter the levels of APAP metabolites, such as mercapturate glucuronide, sulfate and cysteine conjugates. Overall, the reviewed in vivo studies indicate that interactions between APAP and nutraceuticals or plant foods exist. However, the majority of data come from animal models with doses of phytochemicals far from dietary ones. Human studies should investigate gene-diet interactions, as well as ethnic variability in order to clarify the pros and cons of co-administering antioxidant nutraceuticals and APAP. Keywords: Acetaminophen, Antioxidants, Food-drug interaction, Nutraceuticals, Paracetamo

Evaluation of antigens stability of tobacco seeds as edible vaccine against VTEC strains

Plants have represent a promising alternative for biopharmaceutical proteins (Ma et al., 2003; Rossi et al., 2014). Many plant based edible vaccines have been shown to be effective in inducing local immune responses (Rossi et al., 2013). Edible vaccines can activate both mucosal and systemic immunity, as they come in contact with the digestive tract lining. This dual effect would provide first-line defense against pathogens invading through the mucosa. The antigens are released in the intestines are taken up by M cells that are present over the Payer’s patches (in the ileum) and the gut associated lymphoid tissue (GALT). Edible vaccines represent an important worldwide goal for the prevention of the enteric diseases, also in livestock. In particular, the enteric infections are a significant clinical problem in pigs. Verocytotoxic Escherichia (E.) coli strains are responsible for serious enterotoxaemia that causes important economic losses in the pig industry. The production of a vaccine for oral administration of transgenic seeds could be a practical and efficient system to prevent the infection and to reduce the antibiotic use. This study was focused on tobacco plants, previously transformed by agroinfection for the seed-specific expression of antigenic proteins (F18 adhesive fimbriae and the B subunit of the Vt2e toxin) as model of edible vaccines against verocytotoxic E. coli strains. The dietary administration of transgenic tobacco seeds promotes a significant increase in the number of mucosal IgA-producing cells of the tunica propria in both small and large intestine in mice (Rossi et al., 2013). A protective effect of oral administration of transgenic tobacco seeds was also observed against verocytotoxic Escherichia coli infection in piglets (Rossi et al., 2014). The aim of this study was to assess the seed-expression stability, that is a important requirement in the vaccine production, of F 18 and Vt2e-B heterologous genes into the progeny of transformed tobacco plants

Proteomic/peptidomic profile and Escherichia coli growth inhibitory effect of in vitro digested soya protein

Plant proteins contain bioactive peptides with functional properties and physiological activities. In the present work, the bioactive peptides produced during in vitro gastrointestinal digestion of soya protein isolate were investigated. Protein samples were subjected to simulated gastrointestinal digestion with a generation of permeate (3 kDa) fractions. The permeate was analysed by nano-liquid chromatography electrospray ionisation tandem mass spectrometry (LC-nano ESI MS/MS) using a shotgun peptidomic approach, and the retentate was further digested with trypsin and analysed using a shotgun proteomic approach. Based on protein profile observed, the retentate was further tested for its potential antimicrobial activity by evaluating the inhibitory effect on E. coli growth. In the present study the peptidomic/ proteomic characterisation of permeate and retentate fractions revealed the presence of bioactive peptides and proteins associated with antioxidant, ACE-inhibitory, anti-hypertensive and antimicrobial activities. The presence of potentially antimicrobial proteins in the retentate fraction is supported by a marked E. coli F18þ growth inhibitory activity of the same fraction. In particular, the growth inhibitory effect was significant from one until six hours of incubation with 0.65–2.6 mg/ml of in vitro digested soya. The obtained data confirmed that soya-based supplements may have potential beneficial effects after human consumption, and they may be recycled for animal nutrition in line with the circular economy concept