CLASSICAL AND INNOVATIVE APPLICATIONS OF NMR SPECTROSCOPY IN THE METABOLOMIC PROFILING OF BIOLOGIAL SAMPLES

METABOLOMICS IS AN OMIC SCIENCE WITH RAPIDLY EXPANDING APPLICATIONS, INCLUDING DRUG DISCOVERY. WITHIN THIS FIELD, PHARMACOMETABOLOMICS HAS DEVELOPED AS A BRANCH DEDICATED TO STUDYING DRUG-INDUCED METABOLIC EFFECTS. IT PLAYS A CRUCIAL ROLE IN HYPOTHESIS GENERATION, AS DEMONSTRATED BY ITS ABILITY TO UNCOVER PREVIOUSLY UNRECOGNIZED PATHWAYS INVOLVED IN DRUG ACTION. A MAJOR STRENGTH OF PHARMACOMETABOLOMICS LIES IN ITS USE TO EXPLORE CELLULAR MECHANISMS OF ACTION (MOA), OFFERING AN UNBIASED ALTERNATIVE TO TRADITIONAL HYPOTHESIS-DRIVEN APPROACHES THAT TYPICALLY FOCUS ON SPECIFIC RECEPTORS OR ISOLATED PATHWAYS. IN CONTRAST, UNTARGETED METABOLOMICS PROVIDES A COMPREHENSIVE VIEW OF TREATMENT-INDUCED METABOLIC CHANGES AND CAN REVEAL UNEXPECTED OR OFF-TARGET EFFECTS. AS PART OF MY PHD RESEARCH, I INVESTIGATED THE IN VITRO BIOLOGICAL MOA OF NICOTINE AND NERVE GROWTH FACTOR (NGF) USING THE POTENTIAL OF UNTARGETED METABOLOMICS. EMPLOYING SH-SY5Y NEUROBLASTOMA CELLS, I ANALYZED BOTH THE EXOMETABOLOME (CULTURE MEDIUM) AND ENDOMETABOLOME (CELLULAR EXTRACTS) FOLLOWING TREATMENT WITH THESE MOLECULES, WHICH ARE OF POTENTIAL INTEREST FOR ALZHEIMER’S DISEASE (AD) THERAPY. AS A PRELIMINARY STEP, WE CONFIRMED THAT EXPOSURE TO RECOMBINANT AMYLOID Β PEPTIDE AΒ(1–42) REPRODUCED IN OUR MODEL THE KEY METABOLIC DYSREGULATIONS CHARACTERISTIC OF AD. WE THEN EVALUATED WHETHER NICOTINE AND NGF COULD COUNTERACT THESE ALTERATIONS. TO CAPTURE THE DYNAMIC EXCHANGE OF METABOLITES BETWEEN COMPARTMENTS  ESSENTIAL FOR INTERPRETING CELLULAR FUNCTION  WE BUILT FOR THE FIRST TIME A STATISTICAL MODEL THAT COMBINES THE QUANTIFICATION MATRICES OF INTRA- AND EXTRACELLULAR METABOLITES. OUR FINDINGS DEMONSTRATE THAT NICOTINE EXERTS NEUROPROTECTIVE EFFECTS BY RESTORING METABOLIC IMBALANCES ASSOCIATED WITH AD PATHOLOGY. IN PARTICULAR, NICOTINE REVERSED THE MAJORITY OF AΒ(1–42)-INDUCED ALTERATIONS, INCLUDING DISRUPTIONS IN NEUROTRANSMISSION-RELATED PATHWAYS, ENERGY METABOLISM, AND MEMBRANE PHOSPHOLIPID TURNOVER. NGF, BY CONTRAST, EXHIBITED NEUROPROTECTIVE ACTIVITY PRIMARILY BY REDUCING GLUTAMATE AND GLYCINE CONCENTRATIONS AND FUNCTIONING AS AN EARLY MODULATOR OF KEY BIOCHEMICAL PATHWAYS, THEREBY PREVENTING REDOX DISEQUILIBRIUM AND ALTERATIONS IN ENERGY METABOLISM. DURING MY INTERNATIONAL RESEARCH PERIOD, I FURTHER SPECIALIZED IN NMR SPECTROSCOPY WITH FOCUSING ON NON-HYDROGENATIVE PARAHYDROGEN-INDUCED HYPERPOLARIZATION (NHPHIP). AT THE MAGNETIC RESONANCE RESEARCH CENTER OF RADBOUD UNIVERSITY, UNDER THE SUPERVISION OF DR. MARCO TESSARI, I CONDUCTED THE FIRST TARGETED AND CHIRAL METABOLOMIC ANALYSIS OF AMINO ACID CONTENT IN ANIMAL TISSUES USING THIS APPROACH. THIS TECHNIQUE ENABLED THE DISCRIMINATION BETWEEN D- AND L-AMINO ACID ENANTIOMERS AND REVEALED AMINO ACID IMBALANCES ASSOCIATED WITH ALTERED D-ASPARTATE LEVELS. NHPHIP PROVED PARTICULARLY VALUABLE FOR THIS STUDY, AS SEVERAL AMINO ACIDS ARE DIRECTLY OR INDIRECTLY INVOLVED IN NEUROTRANSMISSION. TAKEN TOGETHER, THE APPLICATION OF NHPHIP IN THIS CONTEXT HIGHLIGHTS THE POTENTIAL OF NMR HYPERPOLARIZATION TECHNIQUES FOR TARGETED METABOLOMICS INVESTIGATIONS OF AMINO ACID DYSREGULATION.