Nuclear magnetic resonance as an attractive resource for monitoring surveillance candidates of acute and chronic lung disorders

Metabolomics is the comprehensive study of metabolites, i.e. substrates and end-products of cell metabolism. These are low-molecular weight molecules which include amino, nucleic and organic acids, peptides, carbohydrates, vitamins, polyphenols, alkaloids and inorganic species. Being metabolite concentration influenced by both genetic and environmental factors, their amount directly reflects the underlying biochemical activity and state of cells, tissues or organisms. Profiling the metabolome could thus represent the molecular phenotype better than other approaches such as genomics and proteomics. Among the available procedures (Gas Chromatography-/Liquid Chromatography-Mass Spectrometry), high-resolution nuclear magnetic resonance spectroscopy (HR-NMR) is currently one of the leading analytical tools for metabolomic research due to its peculiarities. The distinctive advantage of NMR over other methods is the possibility to perform an inherent quantitative and untargeted analysis, also with respect to the chemical nature of metabolites. In addition, NMR shows a good reproducibility, a rapid acquisition time of spectra, and it is not destructive with regard to the sample for which little or no preparation is required. Taken together, these features have promoted NMR-assisted metabolomics to the rank of a valuable method for an efficient investigation of a variety of lung diseases. s S sAim of this chapter is to provide an overview of the applications of metabolomics to the study of acute and chronic lung disorders. Why focus on pulmonary disorders? First, by involving tens of million people, lung diseases are some of the most common medical conditions in the world. Second, the depth of analysis ultimately reached by current metabolomic procedures has provided a new and larger context for future studies on the biology of these conditions. This has allowed for the generation of metabolite profiles that could be useful for exploring pathological mechanisms and/or discovering new potential therapeutic targets for a variety of pulmonary disorders

(1)H NMR To Explore the Metabolome of Exhaled Breath Condensate in α(1)-Antitrypsin Deficient Patients: A Pilot Study.

The metabolomic analysis of exhaled breath condensate (EBC) may provide insights on both the pathology of pulmonary disorders and the response to therapy. This pilot study describes the ability of nuclear magnetic resonance (NMR)-based metabolomics to discriminate α1-antitrypsin deficient (AATD)-patients, who were diagnosed with moderate to severe emphysema, from healthy individuals. Comparative analysis of samples from these two homogeneous cohorts of individuals resulted in the generation of NMR profiles that were different from both a qualitative and a quantitative point-of-view. Among the identified metabolites that separated patients from controls, acetoin, propionate, acetate, and propane-1,2 diol were those presenting the biggest difference. Unambiguous confirmation that the two groups could be completely differentiated on the basis of their metabolite content came from the application of univariate and multivariate statistical analysis (principal component analysis, partial least squares discriminant analysis (PLS-DA), and orthogonal PLS-DA). MetaboAnalyst 3.0 platform, used to define a relationship among metabolites, allowed us to observe that pyruvate metabolism is the most-involved pathway, most of metabolites being originated from pyruvate. These preliminary data suggest that NMR, with its ability to differentiate the metabolic fingerprint of EBC of AATD patients from that of healthy controls, has a potential "clinical applicability" in this area

¹H NMR To Explore the Metabolome of Exhaled Breath Condensate in α₁‑Antitrypsin Deficient Patients: A Pilot Study

The metabolomic analysis of exhaled breath condensate (EBC) may provide insights on both the pathology of pulmonary disorders and the response to therapy. This pilot study describes the ability of nuclear magnetic resonance (NMR)-based metabolomics to discriminate α1-antitrypsin deficient (AATD)-patients, who were diagnosed with moderate to severe emphysema, from healthy individuals. Comparative analysis of samples from these two homogeneous cohorts of individuals resulted in the generation of NMR profiles that were different from both a qualitative and a quantitative point-of-view. Among the identified metabolites that separated patients from controls, acetoin, propionate, acetate, and propane-1,2 diol were those presenting the biggest difference. Unambiguous confirmation that the two groups could be completely differentiated on the basis of their metabolite content came from the application of univariate and multivariate statistical analysis (principal component analysis, partial least squares discriminant analysis (PLS-DA), and orthogonal PLS-DA). MetaboAnalyst 3.0 platform, used to define a relationship among metabolites, allowed us to observe that pyruvate metabolism is the most-involved pathway, most of metabolites being originated from pyruvate. These preliminary data suggest that NMR, with its ability to differentiate the metabolic fingerprint of EBC of AATD patients from that of healthy controls, has a potential “clinical applicability” in this area

¹H NMR To Evaluate the Metabolome of Bronchoalveolar Lavage Fluid (BALf) in Bronchiolitis Obliterans Syndrome (BOS): Toward the Development of a New Approach for Biomarker Identification

This report describes the application of NMR spectroscopy to the profiling of metabolites in bronchoalveolar lavage fluid (BALf) of lung transplant recipients without bronchiolitis obliterans syndrome (BOS) (stable, S, <i>n</i> = 10), and with BOS at different degrees of severity (BOS 0p, <i>n</i> = 10; BOS I, <i>n</i> = 10). Through the fine-tuning of a number of parameters concerning both sample preparation/processing and variations of spectra acquisition modes, an efficient and reproducible protocol was designed for the screening of metabolites in a pulmonary fluid that should reflect the status of airway inflammation/injury. Exploiting the combination of mono- and bidimensional NMR experiments, 38 polar metabolites, including amino acids, Krebs cycle intermediates, mono- and disaccharides, nucleotides, and phospholipid precursors, were unequivocally identified. To determine which signature could be correlated with the onset of BOS, the metabolites’ content of the above recipients was analyzed by multivariate (PCA and OPLS-DA) statistical methods. PCA analysis (almost) totally differentiated S from BOS I, and this discrimination was significantly improved by the application of OPLS-DA, whose model was characterized by excellent fit and prediction values (<i>R</i>² = 0.99 and <i>Q</i>² = 0.88). The analysis of <i>S</i> vs BOS 0p and of BOS 0p vs BOS I samples showed a clear discrimination of considered cohorts, although with a poorer efficiency compared to those measured for <i>S</i> vs BOS I patients. The data shown in this work assess the suitability of the NMR approach in monitoring different pathological lung conditions

Structure-activity relationship (SAR) in monosaccharide-based Toll-like receptor 4 (TLR4) antagonists

The structure-activity relationship was investigated in a series of synthetic TLR4 antagonists formed by a glucosamine core linked to two phosphate esters and two linear carbon chains. Molecular modeling showed that the compounds with 10, 12 and 14 carbons chains are associated to higher stabilization of the MD-2/TLR4 antagonist conformation than in the case of the C16 variant. Binding experiments with human MD-2 showed that the C12 and C14 variants have higher affinity than C10, while the C16 variant did not interact with the protein. The molecules, with the exception of the C16 variant, inhibited the LPS-stimulated TLR4 signal in human and murine cells and the antagonist potency mirrored the MD-2 affinity calculated from in vitro binding experiments. FT-IR, NMR, and SAXS measurements suggested that the aggregation state in aqueous solution depends on fatty acid chains lengths and that this property can influence TLR4 activity in this series of compounds

Structure–Activity Relationship in Monosaccharide-Based Toll-Like Receptor 4 (TLR4) Antagonists

The structure–activity relationship was investigated in a series of synthetic TLR4 antagonists formed by a glucosamine core linked to two phosphate esters and two linear carbon chains. Molecular modeling showed that the compounds with 10, 12, and 14 carbons chains are associated with higher stabilization of the MD-2/TLR4 antagonist conformation than in the case of the C16 variant. Binding experiments with human MD-2 showed that the C12 and C14 variants have higher affinity than C10, while the C16 variant did not interact with the protein. The molecules, with the exception of the C16 variant, inhibited the LPS-stimulated TLR4 signal in human and murine cells, and the antagonist potency mirrored the MD-2 affinity calculated from <i>in vitro</i> binding experiments. Fourier-transform infrared, nuclear magnetic resonance, and small angle X-ray scattering measurements suggested that the aggregation state in aqueous solution depends on fatty acid chain lengths and that this property can influence TLR4 activity in this series of compounds

Structure–Activity Relationship in Monosaccharide-Based Toll-Like Receptor 4 (TLR4) Antagonists

The structure–activity relationship was investigated in a series of synthetic TLR4 antagonists formed by a glucosamine core linked to two phosphate esters and two linear carbon chains. Molecular modeling showed that the compounds with 10, 12, and 14 carbons chains are associated with higher stabilization of the MD-2/TLR4 antagonist conformation than in the case of the C16 variant. Binding experiments with human MD-2 showed that the C12 and C14 variants have higher affinity than C10, while the C16 variant did not interact with the protein. The molecules, with the exception of the C16 variant, inhibited the LPS-stimulated TLR4 signal in human and murine cells, and the antagonist potency mirrored the MD-2 affinity calculated from <i>in vitro</i> binding experiments. Fourier-transform infrared, nuclear magnetic resonance, and small angle X-ray scattering measurements suggested that the aggregation state in aqueous solution depends on fatty acid chain lengths and that this property can influence TLR4 activity in this series of compounds