Markers of Oxidative Stress in Inflammatory Bowel Diseases: Risk Factors and Implications for a Dietetic Approach (OxIBDiet)

Markers of Oxidative Stress in Inflammatory Bowel Diseases: Risk Factors and Implications for a Dietetic Approach

Markers of Oxidative Stress in Inflammatory Bowel Disease in Children and Adults: Risk Factors and Implications for a Dietetic Approach

Inflammatory bowel disease (IBD), including Crohn's disease (CD), Ulcerative Colitis (UC) and IBD-unclassified (IBD-U) is a chronic inflammatory intestinal disorders that affect both children and adults. Patients with IBD can present with severe gastrointestinal symptoms, require frequent hospitalizations, expensive medical treatments and can develop invalidating complications requiring surgery. The incidence of IBD is increasing worldwide. The pathogenesis is multifactorial with immunological, environmental and genetic factors contributing to the disease. There is evidence that oxidative stress (OS) imbalance is involved in IBD onset and evolution, although the exact contribution to the pathogenes is unclear. An antioxidant dietetic approach is promising as an adjunctive treatment of IBD. The main aims of this project are to characterize the OS imbalance in IBD in relation to disease's features and to genetic factors and to evaluate the efficacy of an antioxidant dietetic treatment

IBD is a complex disorder that is thought to be the result of an aberrant immune response to commensal bacteria in a genetically susceptible host. The chronic inflammation along the gastrointestinal tract that characterizes IBD results from an imbalance of effector lymphocytes and pro-inflammatory cytokines. Some of the cytokines, as well as the triggered leukocytes and activated macrophages, can produce large amounts of reactive oxygen species (ROS) thus predisposing to oxidative stress disturbances. Many of the clinical and pathophysiological features of IBD, particularly tissue injury (mucosal erosions) and fibrosis have been associated to redox imbalance due to continuous ROS production and a net decrease of antioxidant molecules. Although uncontrolled oxidative stress is destructive in inflammatory conditions, the body's antioxidant defenses can counteract the effects caused by excess of ROS. Antioxidants are protective molecules/compounds toward pro-oxidant molecules. They can be endogenous or/and come from the diet. Endogenous compounds include intracellular enzymatic antioxidants such as superoxide dismutases (SODs), glutathione peroxidase (GPX), and catalase (CAT), intracellular nonenzymatic antioxidant such as glutathione (GSH) and extracellular antioxidants such as vitamins (Vit. A-C-E-B group). GSH is considered the major non-protein low molecular weight defender against oxidative (or redox) stress and the most important cellular thiol buffer. Moreover it acts as cofactor for the antioxidant enzymes GPxs and GST. GSH has been used as a biomarker for inflammation and several studies showed reduced levels of GSH in inflammatory conditions. Experimental colitis models showed decreased GSH levels that can be restored to a normal level by antioxidants supplementation. Also antioxidant enzymes as SODs, CAT and GPxs were found dysregulated in IBD condition. The differences in the regulation of expression of SOD, CAT and GPxs may not only reflect their importance in physiology, but may be also insufficient in removal of ROS under inflammatory conditions such as IBD. Recently an association between SOD1, CAT and GSHPX1 polymorphisms and the risk of inflammatory bowel disease in the Polish population has been described. Kosaka et al. found a correlation between age of onset and severity of IBD with polymorphisms in SOD2 manganese superoxide dismutase and NAD(P)H quinone oxidoreductase. In this respect, IBD disease can be regarded as multifactorial disease. There are several lines of evidence to suggest that diet is a key player in the onset, perpetuation and management of IBD. The most important evidence linking diet to IBD comes from exclusive enteral nutrition (EEN) that is the primary induction treatment of active paediatric Crohn Disease (CD). Epidemiological evidence associates certain dietary nutrients and components to the increased risk of IBD. There is emerging evidence that some diets, including the Specific Carbohydrate Diet (SCD) and the CD Exclusion Diet (CDED) could treat or prevent subsequent disease flare. Data previously presented induce to the hypothesis that an antioxidant dietetic approach, could have a role in the treatment of IBD. Dietary antioxidants may include ascorbic acid, vitamin E, glutathione, methionine, carotenoids, polyphenolic compounds, selenium and vitamin A. Clinical experience evaluating antioxidant dietetic approach in IBD patients is limited to few studies, mostly investigating the effects of single antioxidants in small number of patients. So far pediatric data regarding the oxidative status in children with IBD have rarely been reported. Collecting data in IBD children and comparing these with adults data, particularly in subjects at diagnosis, would give the unique opportunity to evaluate the role of oxidative stress in IBD pathogenesis. OxIBDiet working hypothesis is that oxidative stress imbalance is a key feature of IBD and the persistence of such imbalance is likely to contribute to the development of complications and more broadly to the evolution of the disease. A comparison between oxidative stress imbalance in children and adults with IBD and controls will address the question whether the stress imbalance is a consequence or a primary event in the inflammatory burden of IBD. Addressing these pathways and targeting the oxidative damage can have potential implications in IBD monitoring and treatment.

IBD subjects (40 children and 40 adults, 50% with CD and 50% with UC) will be enrolled in different conditions: at diagnosis, in remission, at relapse. For each IBD subject an age and gender matched control subject will be enrolled (40 children and 40 adults). In a subgroup of subjects (20 children and 20 adults, 50% with CD and 50% with UC) in clinical remission/mild disease a dietetic approach will be proposed. Patients on steroids and patients with strictures will not be enrolled for this part of the study. Patients will be randomized in 2 groups: Group Antioxidant diet will receive 8 weeks of antioxidant dietary treatment and group Normal diet will continue a normal dietetic scheme (corresponding to a isocaloric, normolipidic diet for age and sex).

Group Normal diet will continue a normal dietetic scheme (corresponding to a isocaloric, normolipidic diet for age and sex).

The "antioxidant diet" will include the principal antioxidant molecules/nutrients previously shown to be beneficial in IBD: Flavonoids, particularly resveratrol and curcumin; Olive oil; Glutathione, Vitamins A, C, E; Carotenoids; Selenium and Omega 3 Fatty Acids (PUFAs). The daily amount of these substances will be calculated following the published evidence, whether these amounts will not be achievable by a standard diet, a supplementary formulation will be proposed.

Blood levels of ROS will be determined by cytofluorimetry, blood levels of glutathione by spectrofluorimetry

Total antioxidant capacity (Trolox equivalent and ferrous equivalent) in patients with IBD and controls

Trolox equivalent will be assessed by antioxidant assay Kit, ferrous equivalent instead wille require a manual assessment.

Antioxidant enzymes pattern (GST, SOD, GPxs, GR enzyme levels, activity and transcripts) in patients with IBD and controls

Spectrophotometer will be used to assess enzyme levels and activity, cDNA kit to determine enzyme transcripts.

Plasma hydroperoxides will be detected by LPO ELISA kit, thiobarbituric acid reactive species by TBARS assay kit.

Polymorphisms of genes implicated in oxidative stress defense (SOD1, SOD2, SOD3; GPX1, GPX2, GPX3; GPX4; GPX5; GSTA1; GSTA2; GSTM1; GSTM2; GSTM3; GSTP1; GSTO1; NQ01 NQ02; NOX1; NOX3; NOX4 and NOX5 genes) in patients with IBD and controls

Blood levels of ROS and glutathione in patients with IBD after a specific antioxidant dietary treatment

Blood levels of ROS will be determined by cytofluorimetry, blood levels of glutathione by spectrofluorimetry at baseline and after 12 weeks of dietary treatment.

Total antioxidant capacity (Trolox equivalent and ferrous equivalent) in patients with IBD after a specific antioxidant dietary treatment

Trolox equivalent will be assessed by antioxidant assay Kit, ferrous equivalent instead wille require a manual assessment at baseline and after 12 weeks of dietary treatment.

Antioxidant enzymes pattern (GST, SOD, GPxs, GR enzyme levels, activity and transcripts) in patients with IBD after a specific antioxidant dietary treatment

Spectrophotometer will be used to assess enzyme levels and activity, cDNA kit to determine enzyme transcripts at baseline and after 12 weeks of dietary treatment

Plasma hydroperoxides and thiobarbituric acid reactive species in patients with IBD after a specific antioxidant dietary treatment

Plasma hydroperoxides will be detected by LPO ELISA kit, thiobarbituric acid reactive species by TBARS assay kit at baseline and after 12 weeks of dietary treatment.

Urine oxidized guanines (8-OHdG, 8-OHG e guanosine) levels in patients with IBD after a specific antioxidant dietary treatment

DNA/RNA oxydate damage kit will be used to study urine oxidized guanines levels at baseline and after 12 weeks of dietary treatment

Phisician Global Assessment (PGA) score in patients with IBD after a specific antioxidant dietary treatment

Calprotectin levels will be analyzed in stool samples collected at baseline and after 12 weeks of dietary treatment

Inclusion Criteria: - Diagnosis of IBD Exclusion Criteria: permanent stoma cancer cardiovascular disease ischemic disease Alzheimer's disease type 2 diabetes

SOD Clinica di Gastroenterologia, Epatologia ed Endoscopia Digestiva, Azienda Ospedaliero Universitaria Ospedali Riuniti di Ancona

Individual participant data that underlie the results reported in this article after deidentification will be shared

Proposals may be submitted up to 36 months following article publication. After 36 months the data will be available in our University's data warehouse but without investigator support other than deposited metadata.

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