Enfermedad inflamatoria intestinalherramientas analíticas y metabolómicas para su diagnóstico y valoración del estado nutricional

Abstract

Inflammatory bowel disease (IBD) is a chronic, multifactorial immune disorder characterised primarily by inflammation of the intestine, with periods of activity and remission that diminish patients' quality of life. In general, IBD encompasses two different pathologies: ulcerative colitis (UC) and Crohn's disease (CD). Clinically, both diseases share similar symptoms, such as bloody diarrhoea, abdominal pain, weakness and weight loss; while they often differ in complications and prevalence, as well as in the location and depth of inflammation. Diagnosis of IBD and its different entities is currently not a simple task, and involves the observation of different clinical, histological, endoscopic and analytical factors and is a costly, invasive and time-consuming process. In this regard, recent advances in analytical instrumentation and metabolomics strategies have played an important role in the study of IBD. In this PhD thesis, the volatile organic compounds (VOCs) profile of IBD patients has been studied using non-targeted metabolomics approaches with the aim of developing chemometric models for the diagnosis and monitoring of the disease. The methods will be applied to urine and blood serum analysis. Urine has advantages such as easy collection, being one of the main routes of excretion of water-soluble metabolites and xenobiotics, while blood serum provides an overview of metabolism, although with a lower level of specificity compared to urine. In Chapter 1, a gas chromatography-mass spectrometry (GC-MS)-based methodology for monitoring the VOC profile in serum is developed. In addition, a headspace analysis (HS) is proposed as sample treatment and introduction system, avoiding the more complex procedures described to date. The chemometric treatment consists of an orthogonal partial least squares discriminant analysis (OPLS-DA) that allows the generation of prediction models useful for the differentiation between IBD patients and healthy individuals without IBD. In Chapter 2, a method based on GC coupled to ion mobility spectrometry (IMS) is optimised and validated using also headspace injection for the determination of VOCs in both serum and urine samples. IMS is an emerging analytical technique whose applications in the clinical field are becoming more and more frequent due to its many advantages over other analytical techniques. IMS performs a two-dimensional separation as analytes are separated according to their interaction with the GC column and their drift behaviour under the influence of an electric field, increasing the resolving power and enabling the motorisation of a larger number of VOCs. The VOCs determined by GC-IMS are used to differentiate between IBD patients and healthy individuals, as well as the different stages of the disease (active disease or in remission) by creating OPLS-DA-based models. In addition, a targeted strategy is simultaneously developed that allows the quantification and identification of volatiles in biological samples of interest to determine which compounds contribute most to which classes of IBD. The methods developed in both chapters could be used as a complementary tool in the diagnosis of IBD, avoiding high-cost invasive methods such as endoscopy. On the other hand, this PhD Thesis also studies and compares malnutrition and malabsorption in patients with CD or UC with active disease or in remission. Both syndromes directly affect IBD patients causing alterations in iron metabolism, lipid profile and nutritional status. Specifically, in Chapter 3, iron metabolism is assessed through serum iron, ferritin, ferritin saturation index, haemoglobin and mean corpuscular volume. Ferritin levels may increase in case of inflammation causing a confounding factor in the results obtained, so in this study C-reactive protein (CRP) levels were also assessed together. To evaluate the lipid profile, the parameters of cholesterol, triglycerides, LDL and HDL are investigated, as well as the atherogenic risk associated with the disease using the Castelli index. Finally, a study of nutritional status is performed by microscopic observation of faeces and CONUT screening correlating it with faecal calprotectin (FC) levels.