NMR-based metabolomic analysis of biological fluids to monitor relevant unsolved diseases

2015 - 2016Metabolomics and metabonomics encompass the comprehensive profiling of multiple metabolite concentrations and their cellular and systemic fluctuations in response to drugs, diet, lifestyle, environment, stimuli and genetic modulations, in order to characterize the beneficial and adverse effects of such interactions. In the context of biomedical applications, metabolomics will have a preferential role with respect to the other "Omics" sciences for its ability to detect in real time the response of the organisms to pathological stressors. The application of the NMR technique for the metabolomics analysis was applied to bio-fluids deriving from populations of patients respectively affected by salivary gland tumor, antiphospholipid autoimmune syndrome and altered lipid profile. This NMR metabolomic screening was aimed i) at the definition of a metabolomic profile that may be patognomonic of the disease under scrutiny and ii) at the identification of biomarkers to be used with diagnostic and prognostic scope. In the present work, we present a NMR-based metabolomic study of saliva of patients suffering of salivary gland tumors. Our data show that individuals suffering parotid tumor have a characteristic metabolomic profile with abnormalities associated to the metabolism of acetate, alanine, lactate, methanol, phenylalanine, propionate, succinate. We have identified for the first time the metabolomic fingerprint characterizing parotid tumor patients disease having potential application to improve timely diagnosis and appropriate therapeutic approaches. Salivary gland tumor, as many other cancers, is a complex disease, resulting from an interdependent series of biochemical alterations, rather than a single disruptive event. In this case our approach aimed at the identification of a panel of metabolite markers rather than a single biomarker, will improve the sensitivity and specificity for detection. Integrating the protocols of tumor grading and histological classification. Our NMR-based metabolomic study revealed different metabolomic profiles in saliva of male patients affected by salivary gland tumors compared with the profiles of age, gender, and sampling-date matched control individuals. Our approach provide preliminary data for the identification of metabolites that can be used as metabolomics fingerprint of salivary gland tumor. Determination of metabolomics fingerprint, rather than single metabolic biomarker, may fully reflect the multifactorial nature of oncogenesis and the heterogeneity of oncogenic pathways, providing precious elements to integrate diagnostic laboratory and clinical tests. Antiphospholipid syndrome (APS) is a rheumatic inflammatory chronic autoimmune disease inducing hypercoagulable state associated with vascular thrombosis and pregnancy loss in women. Cardiac, cerebral and vascular strokes in these patients are responsible for reduction in life expectancy. Timely diagnosis and accurate monitoring of disease is decisive to improve the accuracy of therapy. In the present work, we present a NMR-based metabolomic study of blood sera of APS patients. Our data show that individuals suffering APS have a characteristic metabolomic profile with abnormalities associated to the metabolism of methyl group donors, ketone bodies and amino acids. We have identified for the first time the metabolomic fingerprint characterizing APS disease having potential application to improve APS timely diagnosis and appropriate therapeutic approaches. The first stratification of APS patients according to the gender offers preliminary indications for the management of the disease according to the gender oriented medicinal approach. Human serum includes a large number of components which derive from endogenous metabolism and nutritional intake. Serum components vary in response to diet. Serum lipid composition is probably the most important benchmark in assessing cardiovascular risk and disease progression. Serum components, also derived from nutritional intake, can affect general metabolism and, more specifically, affect molecular mechanisms and pathways linking nutritional intake and chronic disease risk. To identify the effect exerted by altered lipid composition on the genome expression pattern, response of gene expression to serum samples from hypercholesterolemic and normocholesterolemic male subjects was previously studied. In the present part of my PhD thesis, using a NMR metabolomics approach I studied the metabolomics profile of the aforementioned hypercholesterolemic and normocholesterolemic sera to correlate the previously identified trascriptomic signature of human hepatoma cells to the relative metabolomics profile. Hypercholesterolemic sera previously proved to increase in human hepatoma cells, the mRNA expression of HMGCS2, an enzyme involved in the pathway of keton bodies. Our NMR based metabolomics analysis evidences abnormal concentrations of metabolites involved in the keton bodies pathway. This indicates a correlation between the trascriptomic profile of hepatoma cells treated with hypercholesterolemic sera, and the metabolomics profile of the same sera. [edited by author]La metabolomica e la metabonomica comprendono il profilo completo di numerosi metaboliti con riferimento alle varie concentrazioni e fluttuazioni sia cellulari che sistemiche in risposta a farmaci, dieta, stile di vita, influenza dell'ambiente, stimoli e modulazioni genetiche, al fine di caratterizzare gli effetti benefici e negativi di tali interazioni. Nel contesto delle applicazioni biomediche, la metabolomica avrà in futuro un ruolo preferenziale rispetto alle altre scienze 'omiche' per la possibilità di rilevare in tempo reale la risposta degli organismi agli stress patologici. L' applicazione della tecnica NMR è stata utilizzata per l' analisi metabolomica di bio-fluidi derivanti da popolazioni di pazienti affetti rispettivamente da tumore delle ghiandole salivari; da sindrome da antifosfolipidi; pazineti con profilo lipidico alterato. Questo screening metabolomico NMR è mirato i) alla definizione di un profilo metabolomico che potrebbe essere patognomonico delle malatte monitorate e ii) l'identificazione di biomarcatori da utilizzare in ambito diagnostico e prognostico. In questo studio metabolomico basato su analisi NMR della saliva di pazienti affetti dai tumori delle ghiandole salivari i nostri dati mostrano caratteristiche anomalie nel profilo metabolomico connesse con il metabolismo di acetato , alanina, lattato, metanolo, fenilalanina, propionato, succinato. Abbiamo identificato per la prima volta l'impronta digitale metabolomica che caratterizza pazienti con tumori della parotide con una potenziale applicazione per migliorare la diagnosi tempestiva ed un approccio terapeutico adeguato. I tumori alle ghiandole salivari, come molti altri tipi di cancro, sono patologie complesse, risultanti da una serie interdipendente di alterazioni biochimiche, piuttosto che un singolo evento dirompente. In questo caso, con un approccio rivolto all'identificazione di un panel di metaboliti marcatori, piuttosto che ad un singolo biomarcatore, miglioreranno ed aumenteranno la sensibilità e la specificità per il rilevament, integrando i protocolli diagnostici classici e la classificazione istologica. Il nostro studio metabolomico NMR-based ha rivelato diversi profili nella saliva di pazienti affetti da tumori delle ghiandole salivari, confrontati in base all' età e al sesso, abbinati con i controlli. Il “finger print”, piuttosto che i singoli biomarkers, può riflettere in pieno la natura multifattoriale ed etrogenea della oncogenesi , fornendo preziosi elementi per integrare i test diagnostici clinici e di laboratorio. La sindrome antifosfolipidi (APS) è una malattia autoimmune, reumatica, infiammatoria cronica associata ad uno stato di ipercoagulabilità: inducendo trombosi vascolari ed aborti spontaeni nelle donne. Ictus cerebrali e vascolari in questi pazienti sono responsabili della riduzione della aspettativa di vita: una diagnosi tempestiva ed un accurato monitoraggio della malattia è determinante per migliorare la precisione della terapia. Nel presente lavoro, vi presentiamo uno studio di metabolomica NMR su siero di pazienti affetti da APS. I nostri dati mostrano che gli individui che soffrono di APS hanno un profilo metabolomico caratteristico con anomalie del metabolismo associate ai donatori di gruppi metilici, di aminoacidi e corpi chetonici. Abbiamo identificato per la prima volta il “finger print” della sindrome da APS con la potenziale applicazione di migliorare la diagnosi tempestiva e favorire un approccio terapeutico adeguato. La prima stratificazione di pazienti APS pazienti in base al sesso offre indicazioni per la gestione della malattia secondo un approccio medico gender oriented. Il siero umano comprende un gran numero di componenti derivanti sia dal metabolismo endogeno sia dall' apporto nutrizionale i quali variano in risposta alla dieta. La composizione lipidica del siero è probabilmente il punto di riferimento più importante nella valutazione del rischio cardiovascolare e della progressione della malattia. Inoltre la composizione lipidica può influenzare il metabolismo e più in particolare, i percorsi molecolari che collegano l' apporto nutrizionale ed rischio di malattia cronica. L'effetto esercitato dalla composizione lipidica modificata sul pattern genomico in risposta all' espressione su campioni di siero da sogetti maschi ipercolesterolemici, confrontati con normocholesterolemici è stato oggetto di un precedente studio. Nell' ultimaparte di questa tesi di dottorato, utilizzando l'approccio metabolomico NMR ho studiato il profilo dei supramenzionati ipercolesterolemici e normocholesterolemici per correlare il profilo trascrittomico ottenuto dalle cellule epatiche umane con il profilo metabolomico del siero umano utilizzato per la cultura, mostrando una aumentata espressione di mRNA di HMGCS2, un enzima coinvolto nel percorso di corpi chetonici. Dall' analisi NMR sono emerse concentrazioni alterate di metaboliti coinvolti della via biosintetica dei corpi chetonici. Questo indica una correlazione tra il profilo trascriptomico di cellule epatiche trattate con sieri ipercolesterolemici, e il profilo metabolomica dei sieri stessi. [a cura dell'autore]XV n.s. (XXIX

STRUCTURAL FEATURES OF PEPTIDES DERIVED FROM FELINE IMMUNODEFICIENCY VIRUS TM-GLYCOPROTEIN

Feline immunodeficiency virus (FIV) is a pathogen that causes an AIDS-like syndrome in domestic cats and is extensively used as a model system by which criteria for anti-lentiviral vaccines and drugs development can be tested. Despite little homology sequence, the surface and TM gp of FIV and HIV-1 exhibit a common structural framework and appear to play similar roles in Initiation of cell infection mediating the final event of membrane fusion and virus entry. Recently a 20-mer synthetic peptide spanning amino acids 767L-G786 of the membrane-proximal ectodomain of FIV (TM) gp - gp41(767-786)- was found to be endowed with potent antiviral activity. Testing deleted or substituted peptides, the 8-mer gp41(770-777), designated C8, including in the sequence three Trp residues was identified as the minimal sequence needed for full antiviral activity. NMR conformational analysis of gp41(770-777) evidenced the presence of a b-turn conformation centered on the residues 773-776. Here we report the conformational analysis of the 20-mer TM-767-786 by means of NMR spectroscopy. A comparison between structural properties of gp41(767-786) and of gp41(770-777) was carried out in order to define the role played by the sequence length and by the Trp side chains on the active fragments stability

Intermittent fasting for health care, a review

The incidence of obesity and overweight in the world has been increasing in recent years due to poor diet and lack of physical activity; people suffering obesity and overweight, related with malnutrition due to excess, often resort to calorie restriction diets that are usually not very effective. In this context, intermittent fasting (IF) has become popular due to the possibilities for weight loss that it offers. This diet consists of alternating periods of fasting with unrestricted eating; however, its effectiveness and consequences are unknown to most users. This narrative review analyzes whether intermittent fasting contributes to the improvement of body and metabolic composition. The purpose of the review was to examine the available data on the contribution of intermittent fasting to the improvement of body and metabolic composition, in order to provide information and to define the parameters that condition safe achievement of its benefits. IF dieting triggers adaptive cell responses that cause a decrease in lipid oxidative stress markers in individuals with obesity and prediabetes. Metabolic alterations have been found to go hand in hand with the alteration of circadian rhythms; if IF contributes to this effect, it may assist in treating and preventing obesity and associated diseases. However, there are also disadvantages, such as the loss of lean muscle mass by wasting, and increased hypoglycemia

El ayuno intermitente, ¿un éxito o una desventaja para el cuidado de la salud?

The incidence of obesity and overweight in the world has been increasing in recent years due to poor diet and lack of physical activity; people suffering obesity and overweight, related with malnutrition due to excess, often resort to calorie restriction diets that are usually not very effective. In this context, intermittent fasting (IF) has become popular due to the possibilities for weight loss that it offers. This diet consists of alternating periods of fasting with unrestricted eating; however, its effectiveness and consequences are unknown to most users. This narrative review analyzes whether intermittent fasting contributes to the improvement of body and metabolic composition. The purpose of the review was to examine the available data on the contribution of intermittent fasting to the improvement of body and metabolic composition, in order to provide information and to define the parameters that condition safe achievement of its benefits. IF dieting triggers adaptive cell responses that cause a decrease in lipid oxidative stress markers in individuals with obesity and prediabetes. Metabolic alterations have been found to go hand in hand with the alteration of circadian rhythms; if IF contributes to this effect, it may assist in treating and preventing obesity and associated diseases. However, there are also disadvantages, such as the loss of lean muscle mass by wasting, and increased hypoglycemia.La incidencia de obesidad y sobrepeso en el mundo ha ido en aumento en los últimos años debido a la mala alimentación y la falta de actividad física; Las personas que padecen obesidad y sobrepeso, relacionadas con la desnutrición por exceso, suelen recurrir a dietas de restricción calórica que suelen ser poco efectivas. En este contexto, el ayuno intermitente (AI) se ha popularizado debido a las posibilidades de pérdida de peso que ofrece. Esta dieta consiste en alternar períodos de ayuno con alimentación sin restricciones; sin embargo, su eficacia y consecuencias son desconocidas para la mayoría de los usuarios. Esta revisión narrativa analiza si el ayuno intermitente contribuye a la mejora de la composición corporal y metabólica. El objetivo de la revisión fue examinar los datos disponibles sobre la contribución del ayuno intermitente a la mejora de la composición corporal y metabólica, con el fin de aportar información y definir los parámetros que condicionan la consecución segura de sus beneficios. Se ha encontrado que las alteraciones metabólicas van de la mano con la alteración de los ritmos circadianos; si AI contribuye a este efecto, puede ayudar a tratar y prevenir la obesidad y las enfermedades asociadas. Sin embargo, también existen desventajas, como la pérdida de masa muscular magra por atrofia y el aumento de la hipoglucemia

A serum nuclear magnetic resonance-based metabolomic signature of antiphospholipid syndrome

Antiphospholipid syndrome (APS) is a rheumatic inflammatory chronic autoimmune disease inducing hypercoagulable state associated with vascular thrombosis and pregnancy loss in women. Cardiac, cerebral and vascular strokes in these patients are responsible for reduction in life expectancy. Timely diagnosis and accurate monitoring of disease are decisive to improve the accuracy of therapy. In the present work, we present a NMR-based metabolomic study of blood sera of APS patients. Our data show that individuals suffering APS have a characteristic metabolomic profile with abnormalities associated to the metabolism of methyl group donors, ketone bodies and amino acids. We have identified for the first time the metabolomic fingerprint characterizing APS disease having potential application to improve APS timely diagnosis and appropriate therapeutic approaches

NMR-based metabolomic profile of hypercholesterolemic human sera: Relationship with in vitro gene expression?

Hypercholesterolaemia is considered an important cause of atherosclerotic cardiovascular disease. In a previous investigation, we demonstrated that cultured hepatoma cells treated with hypercholesterolaemic sera compared with cells treated with normocholesterolaemic sera show overexpression of mRNAs related to mitochondrial 3-hydroxy-3-methylglutaryl-coenzyme A synthase (HMGCS2). In the present work, using an NMR metabolomic analysis, we demonstrate that the hypercholesterolaemic blood sera previously used to treat cultured hepatoma cells are characterized by a metabolomic profile that is significantly different from the normocholesterolaemic sera. Acetate, acetone, 2-hydroxybutyrate, cysteine, valine, and glutamine are the metabolites distinguishing the two groups. Abnormalities in the concentrations of these metabolites reflect alterations in energy-related pathways, such as pantothenate and CoA biosynthesis, pyruvate, glycolysis/gluconeogenesis, the citrate cycle, and ketone bodies. Regarding ketone bodies, the pathway is regulated by HMGCS2; therefore, serum samples previously found to be able to increase HMGCS2 mRNA levels in cultured cells also contain higher amounts of the metabolites of its encoded enzyme protein product

Saliva of patients affected by salivary gland tumour: An NMR metabolomics analysis

Cancers affecting the salivary glands have been an increasing incidence. Salivary gland cancer is not detected until it reaches an advanced stage, which would generally result in a poor prognosis and survival rate. Therefore, early detection as well as the screening of high risk populations with precancerous lesions remains an unmet medical need. In the present work, we present a NMR-based metabolomic study of the saliva of patients suffering from salivary gland tumours. Analysis of data was done using a combined approach based on PRICONA quantitative analysis and statistical multivariate analysis. Interestingly, both the analytical methods indicate that individuals affected by parotid tumour have a characteristic metabolomic profile characterized by abnormalities in the concentration of several aminoacids. Among these the most significant are those relative to Alanine and Leucine suggestive of an alteration in the metabolic pathways of glycogenic aminoacids and ketone bodies. Our data, describing the preliminary metabolomics fingerprint of parotid tumour, are consistent with the recent view that oncogenic signalling corresponds to alteration in the metabolism of nutrient pull (Vander Heiden et al., 2009), rather than to a single metabolite

NMR applications in food analysis. Part B

Applications of low-field NMR relaxometry and NMR-imaging in the analysis of food samples are described using examples of different food matrices and different problems related to food processing, maturation and ageing, authenticity, shelf-life, perishability etc

NMR methodologies in food analysis

Nuclear Magnetic Resonance (NMR) methodologies offer a comprehensive characterization of foodstuffs owing to the possibility to study a sample from different points of view including structural, compositional, functional, morphological etc. aspects. High resolution NMR spectroscopy applied to semi-solid food samples or to extracts in solution is used to determine the foodstuff composition. Here, some features of high resolution NMR methodologies related to food analysis such as quantitative analysis, chemometrics, and use of databases are included. Other NMR methodologies such as relaxometry and imaging described in this chapter give precious information regarding morphology and texture of intact food samples

NMR Applications in Food Analysis: Part B

Applications of low-field NMR relaxometry and NMR-imaging in the analysis of food samples are described using examples of different food matrices and different problems related to food processing, maturation and ageing, authenticity, shelf-life, perishability, etc