Mass Spectrometry and Metabolomics

Research Activity

Metabolomics and Lipidomics: A New Frontier for Research in Pediatrics

Metabolomics is the most recent of the “omic” sciences. Metabolomics and Lipidomics can be defined as the quantitative analysis of all the metabolites (small molecules) of a biological sample aiming to the investigation of the multiparametric metabolic response of a living system to pathophysiological stimuli or genetic modifications. A metabolic profile consists of the set of metabolites reflecting enzyme expression and activity, and includes the building blocks and breakdown products of the DNA, RNA, proteins, and cellular components. Also, it is affected by several factors unrelated to the genome, such as interactions with commensal microorganisms, nutritional factors, environmental agents, and any exposure to drugs or toxic substances resulting in discordance between genotype and phenotype. In many fields of medicine, there is a growing interest in characterizing diseases at molecular level with a view to developing an individually tailored therapeutic approach. Metabolomics and Lipidomics is a novel area that promises to contribute significantly to the characterization of various disease phenotypes and to the identification of personal metabolic features that can predict response to therapies. Based on analytical platforms such as mass spectrometry or NMR spectroscopy, the metabolomics approach enables a comprehensive overview of the metabolites, leading to the characterization of the metabolic fingerprint of a given sample. These metabolic fingerprints can then be used to distinguish between different disease phenotypes and to predict a drug’s effectiveness and/or toxicity. Several studies published in the last few years applied the metabolomic approach in the field of pediatric medicine. Being a highly informative technique that can be used on samples collected non-invasively (e.g. urine or exhaled breath condensate), metabolomics has appeal for the study of pediatric diseases.

Summary of ongoing research and main recent results achieved

When Time is Brain: A Multi-Omic Approach for Rapid Diagnosis and Prognostic Characterization of Hypoxic-Ischemic Encephalopathy
Hypoxic-ischemic encephalopathy (HIE) is characterized by inadequate oxygen supply to vital organs and peripheral tissues in newborns, resulting in tissue hypoxia and injury. HIE may lead to adverse early outcomes like neonatal death as well as long-term consequences, including cerebral palsy, epilepsy, behavioral disorders, cognitive impairment, and neurodevelopmental disability. Currently, therapeutic hypothermia stands as the sole approved neuroprotective treatment for HIE. Our research on this topic began in 2015, involving untargeted metabolomic analysis of urine samples from 53 asphyctic newborns, 21 exhibiting HIE signs at brain MRI and 32 without. Key metabolites—L-alanine, L-lysine, creatine, and L-3-methylhistidine—differentiated the HIE group from the non-HIE group. Further analysis of samples from HIE newborns and healthy controls revealed three impacted metabolic pathways during HIE: steroidogenesis, lysine degradation, and carnitine synthesis. To explore the predictive potential of the urinary metabolome at birth, we conducted a longitudinal follow-up of our cohort up to 7 years, revealing correlations between birth levels of pipecolic acid, L-pyroglutamic acid, gamma-aminobutyric acid, and L-3-methylhistidine, and long-term neurological outcomes. Our future work until 2026 is encapsulated in the “MultiOmicAsphyxia” study, integrating diverse -omic techniques and miRNA analysis for a comprehensive understanding of HIE. The objectives of this project include validating prior findings through targeted metabolomics, advancing comprehension of HIE pathophysiology with multi-modal -omic techniques and miRNA analysis, analyzing data for early biomarkers and metabolic targets, and developing a bedside device using low-field NMR for early metabolome characterization in each HIE newborn, to provide tailored care and assess specific outcomes efficiently. (Ongoing research project)

Urinary metabotypes of newborns with perinatal asphyxia undergoing therapeutic hypothermia

Perinatal asphyxia (PA) still occurs in about three to five per 1,000 deliveries in developed countries; 20% of these infants show hypoxic-ischemic encephalopathy (HIE) on brain magnetic resonance imaging (MRI). The aim of our study was to apply metabolomic analysis to newborns undergoing therapeutic hypothermia (TH) after PA to identify a distinct metabotype associated with the development of HIE on brain MRI. We enrolled 53 infants born at >35 weeks of gestation with PA: 21 of them showed HIE on brain MRI (the “HIE” group), and 32 did not (the “no HIE” group). Urine samples were collected at 24, 48 and 72 hours of TH. Metabolomic data were acquired using high-resolution mass spectrometry and analyzed with univariate and multivariate methods. Considering the first urines collected during TH, untargeted analysis found 111 relevant predictors capable of discriminating between the two groups. Of 35 metabolites showing independent discriminatory power, four have been well characterized: L-alanine, Creatine, L-3-methylhistidine, and L-lysine. The first three relate to cellular energy metabolism; their involvement suggests a multimodal derangement of cellular energy metabolism during PA/HIE. In addition, seven other metabolites with a lower annotation level (proline betaine, L-prolyl-L-phenylalanine, 2-methyl-dodecanedioic acid, S-(2-methylpropionyl)-dihydrolipoamide-E, 2,6 dimethylheptanoyl carnitine, Octanoylglucuronide, 19-hydroxyandrost-4-ene-3,17-dione) showed biological consistency with the clinical picture of PA. Moreover, 4 annotated metabolites (L-lysine, L-3-methylhistidine, 2-methyl-dodecanedioic acid, S-(2-methylpropionyl)-dihydrolipoamide-E) retained a significant difference between the “HIE” and “no HIE” groups during all the TH treatment. Our analysis identified a distinct urinary metabotype associated with pathological findings on MRI, and discovered 2 putative markers (L-lysine, L-3-methylhistidine) which may be useful for identifying neonates at risk of developing HIE after PA. (PLoS One 2016 Oct 18;11(10):e0164211)

Unravelling the role of lipids in ZIKA virus Infection and traversing of the Placenta

ZIKA virus (ZIKV) is a member of the Flaviviridae family. Infection of pregnant women might cause variable combinations of neurological and developmental disorders, known as the congenital ZIKA syndrome (CZS). Little is known about ZIKA’s ability to infect and cross the placenta. Likewise, we ignore the direct and indirect pathological effects associated with placental infection. This gap in knowledge seriously hampers our ability to diagnose and treat infected patients. Only recently, lipidomics has offered invaluable insights into the field of flavivirology, describing a fascinating interplay, in which viruses lead to a profound rearrangement of the host lipid metabolism. Interestingly, for some flaviviruses, severity and incidence of the disease is worsened in overweight populations, suggesting a role of nutrition in the regulation of infection. Our project aims to investigate: 1) how ZIKA infections affect the lipid metabolism of the placenta and the associated fetal sequelae. 2) How lipid composition of the viral envelope defines placental tropism. 3) How nutrition can determine susceptibility to infection and vertical transmission (VT). Liquid chromatography and mass spectrometry will be applied for the lipidomic analyses. Generated data will: 1) improve our understanding of vertical transmission, indicating potential markers of placental infection; 2) create the basis for the development of antivirals selectively targeting the placenta; 3) inform cohort retrospective studies to verify the role of nutrition as risk factor for CZS, during pregnancy. (submitted)

Inborn errors of bile acid synthesis (BASD) are rare genetic disorders of liver metabolism that cause chronic liver diseases

Inborn errors of bile acid synthesis are rare genetic disorders of liver metabolism that cause chronic liver diseases, fat malabsorption, and fat-soluble vitamin deficiency during child-hood. These defects, due to a defective functioning of enzymes, are characterized by a failure to produce normal bile acids (BAs) and an accumulation of unusual BAs and BAs intermediates. BAs are potent digestive surfactants that promote the absorption of cholesterol, lipids and fat-soluble vitamins acting as emulsifiers. They provide the primary driving force for the promotion and secretion of bile and are essential for the development of the biliary excretory route for the elimination of endogenous and exogenous toxic substances, including bilirubin, xenobiotics, and drug metabolites. Inborn errors in the BA biosynthetic pathways include cerebrotendinous xanthomatosis (CTX, sterol 27-hydroxylase deficiency), 3β-hydroxysteroid- Δ5-C27-steroid dehydrogenase deficiency (HSD3B7), D4-3-oxosteroid-5β-reductase deficiency, oxysterol 7α-hydroxylase deficiency, cholesterol 7α-hydroxylase deficiency, α-methylacyl-CoA racemase (AMACR), peroxisomal β-oxidation, and amino acid N-acyltransferase. The most useful screening test is the analysis of urinary bile acids and bile alcohols by flow injection analysis (FIA) electrospray ionization-tandem mass spectrometry (ESI–MS/MS). The screening procedures indicate that inborn errors of BA metabolism probably account for 1% to 2% of the cases of liver disease in infants, children, and adolescents, making this an important and specific category of metabolic liver disease. Early diagnosis of inborn errors of BAs synthesis is important because if the disorder remains untreated, progressive liver disease, together with neurologic disease, may develop and lead to death or require liver transplantation. The treatment of these defects is based on the oral administration of primary bile acids as cholic acid and chenodeoxycholic acid. However, their use was associated with an increased risk of serious possible adverse events, and treatment needs to be accurately monitored. (Chem Phys Lipids. 2017 Apr; 204:43-56. doi: 10.1016/j.chemphyslip.2017.03.004) (Ongoing research project)