NMR-based metabolomics applications: from food to human biofluids

Metabolomics is the scientific discipline that identifies and quantifies endogenous and exogenous metabolites in different biological samples. Metabolites are crucial components of a biological system and they are highly informative about its functional state, due to their closeness to the organism’s phenotype. This approach finds an increasing number of applications in many areas including medical, pharmaceutical, food and environmental sciences. The combined use of NMR spectroscopy and chemometrics techniques, is able to provide the metabolic “fingerprint” of the various samples. This PhD project focused on the analysis of various samples covering a wide range of fields, namely, food and nutraceutical sciences, cell metabolomics and medicine using a metabolomics approach. Indeed, the first part of the thesis describes two exploratory studies performed on Algerian extra virgin olive oil and apple juice from ancient Danish apple cultivars. Both studies revealed variety-related peculiarities that would have been difficult to detect by means of traditional analysis. The second part of the project includes four metabolomics studies performed on samples of biological origin. In particular, the first study is related to a recent emerging field: cell metabolomics. Indeed, tumour cells (HTC116) were treated with novel anticancer drugs in order to understand their in vitro action. The aim of this study was also the development of a reliable experimental protocol for an efficient harvesting, quenching and extraction of cellular metabolites of human adherent cancer cell lines. The second and the third studies concern the evaluation of the effects of functional food ingredients, namely β-glucans and phytosterols, on in vivo animal models. In particular, the hypocholesterolemic action of β-glucans was investigated by analysing rat plasma and faecal samples. This study confirmed the role of barley β-glucans in increasing faecal bile acids excretion in hypercholesterolemic rats and showed, for the first time, a modulation of the primary and secondary bile acid excretion, depending on the molecular weight of the β-glucan employed. In the other study, the effects of phytosterols on a murine colitis model, was investigated. NMR measurements on the liver metabolome revealed the role of these plant sterols in restoring the homeostatic equilibrium of the living system. Thus, in both cases, the results suggest the appropriate use of these nutraceutical products. The last study explores the differences in the follicular fluid metabolome of hyper- and normoinsulinemic women affected with Polycystic Ovary Syndrome (PCOS). The study provides preliminary but interesting relationships between serum hormones and metabolites in follicular fluids

Development of an optimized protocol for NMR metabolomics studies of human colon cancer cell lines and first insight from testing of the protocol using DNA G-quadruplex ligands as novel anti-cancer drugs

The study of cell lines by nuclear magnetic resonance (NMR) spectroscopy metabolomics represents a powerful tool to understand how the local metabolism and biochemical pathways are influenced by external or internal stimuli. In particular, the use of adherent mammalian cells is emerging in the metabolomics field in order to understand the molecular mechanism of disease progression or, for example, the cellular response to drug treatments. Hereto metabolomics investigations for this kind of cells have generally been limited to mass spectrometry studies. This study proposes an optimized protocol for the analysis of the endo-metabolome of human colon cancer cells (HCT116) by NMR. The protocol includes experimental conditions such as washing, quenching and extraction. In order to test the proposed protocol, it was applied to an exploratory study of cancer cells with and without treatment by anti-cancer drugs, such as DNA G-quadruplex binders and Adriamycin (a traditional anti-cancer drug). The exploratory NMR metabolomics analysis resulted in NMR assignment of all endo-metabolites that could be detected and provided preliminary insights about the biological behavior of the drugs tested

Comparison of the biological effects of gadodiamide (Omniscan) and gadoteridol ({ProHance}) by means of multi-organ and plasma metabolomics

Gadolinium-based contrast agents (GBCAs) are massively employed in radiology to increase the diagnostic power of MRI. However, investigations aiming at detecting possible metabolic perturbations or adverse health effects due to gadolinium deposition are still lacking. In this work, aqueous organs extract and plasma samples were analyzed by GC-MS and H-1-NMR, respectively, to investigate the effects of multiple administrations of one linear (Omniscan) and one macrocyclic (ProHance) GBCA, on the main metabolic pathways in healthy mice. Multivariate analysis revealed that plasma metabolome was not differently perturbed by the two GBCAs, while, the multiorgan analysis displayed a clear separation of the Omniscan-treated from the control and the ProHance-treated groups. Interestingly, the most affected organs were the brain, cerebellum and liver. Thus, this work paves the way to both the safest use of the commercially available GBCAs and the development of new GBCAs characterized by lower general toxicity

Metabolic Alkalosis resulting from a Congenital Duodenal Diaphragm

Duodenal diaphragm is an unusual cause of upper intestinal obstruction. We present here a neonate with duodenal diaphragm who presented with features of metabolic alkalosis. Further, an algorithm of management of metabolic alkalosis in a newborn is suggested

Models for preclinical studies in aging-related disorders: one is not for all

Preclinical studies are essentially based on animal models of a particular disease. The primary purpose of preclinical efficacy studies is to support generalization of treatment–effect relationships to human subjects. Researchers aim to demonstrate a causal relationship between an investigational agent and a disease-related phenotype in such models. Numerous factors can muddle reliable inferences about such causeeffect relationships, including biased outcome assessment due to experimenter expectations. For instance, responses in a particular inbred mouse might be specific to the strain, limiting generalizability. Selecting well-justified and widely acknowledged model systems represents the best start in designing preclinical studies, especially to overcome any potential bias related to the model itself. This is particularly true in the research that focuses on aging, which carries unique challenges, mainly attributable to the fact that our already long lifespan makes designing experiments that use people as subjectsextremely difficult and largely impractical

Screening of DNA G-quadruplex stabilizing ligands by nano differential scanning fluorimetry

G-quadruplex (G4) nucleic acid structures are involved in a number of different diseases and their drug-induced stabilization is deemed to be a promising therapeutic approach. Herein is reported a proof of principle study on the use of nano differential scanning fluorimetry for a rapid and accurate analysis of G4-stabilizing ligands, exploiting the fluorescence properties of a 2-aminopurine modified G4-forming oligonucleotide

Novel Therapeutic Targets in Metabolic Disorders: From the Bench to the Bedside

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Effect of saline irrigation and plant-based biostimulant application on fiber hemp (Cannabis sativa L.) growth and phytocannabinoid composition

Phytocannabinoids represent the hallmark of the secondary metabolism of Cannabis sativa. The content of major phytocannabinoids is closely related to genetic variation as well as abiotic elicitors such as temperature, drought, and saline stress. The present study aims to evaluate hemp response to saline irrigation supplied as NaCl solutions with an electrical conductivity (EC) of 2.0, 4.0, and 6.0 dS m-1 (S1, S2, and S3, respectively) compared to a tap water control (S0). In addition, the potential beneficial effect of a plant-based biostimulant (a legume protein hydrolysate) in mitigating the detrimental effects of saline irrigation on crop growth and phytocannabinoid composition was investigated. Sodium chloride saline irrigation significantly reduced biomass production only with S2 and S3 treatments, in accordance with an induced nutrient imbalance, as evidenced by the mineral profile of leaves. Multivariate analysis revealed that the phytocannabinoid composition, both in inflorescences and leaves, was affected by the salinity level of the irrigation water. Interestingly, higher salinity levels (S2-S3) resulted in the predominance of cannabidiol (CBD), compared to lower salinity ones (S0-S1). Plant growth and nitrogen uptake were significantly increased by the biostimulant application, with significant mitigation of the detrimental effect of saline irrigations