NMR-Based Metabolomics to Evaluate Individual Response to Treatments.

The aim of this chapter is to highlight the various aspects of metabolomics in relation to health and diseases, starting from the definition of metabolic space and of how individuals tend to maintain their own position in this space. Physio-pathological stimuli may cause individuals to lose their position and then regain it, or move irreversibly to other positions. By way of examples, mostly selected from our own work using 1H NMR on biological fluids, we describe the effects on the individual metabolomic fingerprint of mild external interventions, such as diet or probiotic administration. Then we move to pathologies (such as celiac disease, various types of cancer, viral infections, and other diseases), each characterized by a well-defined metabolomic fingerprint. We describe the effects of drugs on the disease fingerprint and on its reversal to a healthy metabolomic status. Drug toxicity can be also monitored by metabolomics. We also show how the individual metabolomic fingerprint at the onset of a disease may discriminate responders from non-responders to a given drug, or how it may be prognostic of e.g., cancer recurrence after many years. In parallel with fingerprinting, profiling (i.e., the identification and quantification of many metabolites and, in the case of selected biofluids, of the lipoprotein components that contribute to the 1H NMR spectral features) can provide hints on the metabolic pathways that are altered by a disease and assess their restoration after treatment.

Antcin A, a phytosterol regulates SARS-CoV-2 spike protein-mediated metabolic alteration in THP-1 cells explored by the 1 H-NMR-based metabolomics approach.

The mechanism of SARS-CoV-2 spike protein-mediated perturbations of metabolic pathways and modulation of antcin A, a steroid-like compound isolated from Taiwanofungus camphoratus, are not studied. Here, we investigated the metabolic alteration by SARS-CoV-2 spike protein and the regulatory effect of antcin A on SARS-CoV-2 spike protein-induced metabolic changes in the Phorbol 12-myristate 13-acetate (PMA)-induced human monocytes (THP-1) using proton nuclear magnetic resonance (1 H-NMR) and MetaboAnalyst 5.0 software. The cytotoxic potential of SARS-CoV-2 spike protein, antcin A, and dexamethasone was assessed by MTT assay. The metabolomic perturbations and their relation to human coronaviruses' receptors were evaluated by qPCR. This study indicated that the altered metabolites mediated by SARS-CoV-2 protein, such as methionine, phosphoenolpyruvic acid, canadine, glutamine, ethanolamine, and phenylalanine, were significantly reversed by antcin A. In addition, antcin A significantly inhibited SARS-CoV-2 spike protein-mediated up-regulation of TLR-4 and ACE2 receptors, while GRP78 inhibition was not statistically significant. This is the first study to use 1 H-NMR to investigate SARS-CoV-2 spike protein-induced metabolomic changes in PMA-induced THP-1 cells. Antcin A significantly reversed metabolomic alters while dexamethasone failed to fix them. Therefore, we believe that antcin A could be a potential candidate for therapeutic agents for viral infections related to a metabolic abnormality.

Quantitative 1HNMR Spectroscopy: Analysis of Zinc Gluconate in UtozincR tablets, a Mixture of Zinc Gluconate and Vitamin C.

BACKGROUND: Zinc is an essential metal for humans and plays key roles in several biological events such as immunity, allergy, growth, and inflammation. The deficiency in zinc causes an increased infection rate with pathogens. Organo-zincates such as zinc gluconate are known for better absorption compared with their inorganic zinc salts. Its role in enhancing the immune system has driven a huge demand for organo-zinc supplements and in the treatment protocol of coronavirus disease, the causative agent of the COVID-19 pandemic. OBJECTIVE: Herein, we report on a quantitative analysis of zinc gluconate in the authentic form in presence of vitamin C and the method was applied to their dosage form (UtozincR tablets). The method is simple, accurate, and validated according to ICH guidelines. METHODS: Quantification of zinc gluconate formulated with vitamin C (UtozincR tablets) using Q-1HNMR. Maleic acid and deuterium oxide were used as internal standards and solvents, respectively. RESULTS: The linearity range, the limit of detection and quantification, stability, precision, and accuracy, were validated. The validation of the method within five concentration levels (from 10 to 50 mg/0.5 mL D2O) afforded a limit of detection of 4.58 mg/mL, a quantification limit of 15.27 mg/mL, and excellent linearity. CONCLUSION: The method proposed in the present study is simple, fast, non-destructive, and accurate. Zinc gluconate quantification values obtained by the Q-1HNMR method were found to show an acceptable correlation with those obtained by the thin-layer chromatographic technique. HIGHLIGHTS: The method was successfully applied on UtozincR tablets, and the results were compared with the reported reference pharmacopeial method. The salt exchange between maleic acid (IS) and zinc gluconate was tested by noticing the change in the chemical shift of IS and zinc gluconate.

High-Resolution Magic-Angle Spinning NMR Spectroscopy for Evaluation of Cell Shielding by Virucidal Composites Based on Biogenic Silver Nanoparticles, Flexible Cellulose Nanofibers and Graphene Oxide.

Antiviral and non-toxic effects of silver nanoparticles onto in vitro cells infected with coronavirus were evaluated in this study using High-Resolution Magic-Angle Spinning Nuclear Magnetic Resonance (HR-MAS NMR) spectroscopy. Silver nanoparticles were designed and synthesized using an orange flavonoid-hesperetin (HST)-for reduction of silver(I) and stabilization of as obtained nanoparticles. The bio-inspired process is a simple, clean, and sustainable way to synthesize biogenic silver nanoparticles (AgNP@HST) with diameters of ∼20 nm and low zeta potential (-40 mV), with great colloidal stability monitored for 2 years. The nanoparticles were used for the fabrication of two types of antiviral materials: colloids (AgNP@HST spray) and 3D flexible nanostructured composites. The composites, decorated with AgNP@HST (0.05 mmol L-1), were made using cellulose nanofibers (CNF) obtained from orange peel and graphene oxide (GO), being denominated CNF@GO@AgNP@HST. Both materials showed high virucidal activity against coronaviruses in cell infection in vitro models and successfully inhibited the viral activity in cells. HR-MAS 1H-NMR technique was used for determining nanomaterials' effects on living cells and their influences on metabolic pathways, as well as to study viral effects on cells. It was proven that none of the manufactured materials showed toxicity towards the intact cells used. Furthermore, viral infection was reverted when cells, infected with the coronavirus, were treated using the as-fabricated nanomaterials. These significant results open possibilities for antiviral application of 3D flexible nanostructured composite such as packaging papers and filters for facial masks, while the colloidal AgNP@HST spray can be used for disinfecting surfaces, as well as a nasal, mouth, and eye spray.

Extra Virgin Olive Oil Secoiridoids Modulate the Metabolic Activity of Dacarbazine Pre-Treated and Treatment-Naive Melanoma Cells.

Nowadays, many individuals, whether healthy or diagnosed with disease, tend to expose themselves to various easily accessible natural products in hopes of benefiting their health and well-being. Mediterranean populations have traditionally used olive oil not only in nutrition but also in cosmetics, including skincare. In this study, the phenolic profile-composed of twelve compounds altogether, including the secoiridoids oleocanthal (OCAL) and oleacein (OCEIN)-of extra virgin olive oil (EVOO) from autochthonous cultivars from Croatia was determined using 1H qNMR spectroscopy and HPLC-DAD analysis, and its biological activity was investigated in melanoma cell lines. The EVOO with the highest OCEIN content had the strongest anti-cancer activity in A375 melanoma cells and the least toxic effect on the non-cancerous keratocyte cell line (HaCaT). On the other hand, pure OCAL was shown to be more effective and safer than pure OCEIN. Post-treatment with any of the EVOO phenolic extracts (EVOO-PEs) enhanced the anti-cancer effect of the anti-cancerous drug dacarbazine (DTIC) applied in pre-treatment, while they did not compromise the viability of non-cancerous cells. The metastatic melanoma A375M cell line was almost unresponsive to the EVOO-PEs themselves, as well as to pure OCEIN and OCAL. Our results demonstrate that olive oils and/or their compounds may have a potentially beneficial effect on melanoma treatment. However, their usage can be detrimental or futile, especially in healthy cells, due to inadequately applied concentrations/combinations or the presence of resistant cells.

SARS-CoV-2 infection paralyzes cytotoxic and metabolic functions of the immune cells.

The SARS-CoV-2 virus is the causative agent of the global COVID-19 infectious disease outbreak, which can lead to acute respiratory distress syndrome (ARDS). However, it is still unclear how the virus interferes with immune cell and metabolic functions in the human body. In this study, we investigated the immune response in acute or convalescent COVID-19 patients. We characterized the peripheral blood mononuclear cells (PBMCs) using flow cytometry and found that CD8+ T cells were significantly subsided in moderate COVID-19 and convalescent patients. Furthermore, characterization of CD8+ T cells suggested that convalescent patients have significantly diminished expression of both perforin and granzyme A. Using 1H-NMR spectroscopy, we characterized the metabolic status of their autologous PBMCs. We found that fructose, lactate and taurine levels were elevated in infected (mild and moderate) patients compared with control and convalescent patients. Glucose, glutamate, formate and acetate levels were attenuated in COVID-19 (mild and moderate) patients. In summary, our report suggests that SARS-CoV-2 infection leads to disrupted CD8+ T cytotoxic functions and changes the overall metabolic functions of immune cells.