DEVELOPMENT OF ANALYTICAL METHODS IN METABOLOMICS FOR THE STUDY OF HEREDITARY AND ACQUIRED GENETIC DISEASE

METABOLOMICS AND MASS SPECTROMETRY The research project take place in the branch of metabolomics, which involves the systematic study of the metabolites present in a cell and in this area MS, thanks to its potential to carry out controlled experiments of fragmentation, plays a role as a key methodology for identification of various metabolites. The work of thesis project is focused on the analytical methods development for the diagnosis of metabolic diseases and is divided as follows: ? Newborn screening of inborn error of metabolism; ? Therapeutic drug monitoring in CML patients. NEWBORN SCREENING OF INBORN ERROR OF METABOLISM Newborn screening is the process by which infants are screened shortly after birth for a list of disorders that are treatable, but difficult or impossible to detect clinically. The history of neonatal screening of inherited metabolic disease has seen a continuous and positive change limits screening to duplicate the examinative confirmation, through the introduction of new analytical technologies, such as tandem mass spectrometry (MS/MS), which allow testing not only qualitative but also quantitative. It has allowed to move from single test to multiple screening tests for other diseases such as metabolic aminoacidopathies a lower incidence, organic acidurias and defects of fatty acid oxidation. Disorders detected by most MS/MS newborn screening programs can be divided in three major categories: ? Amino acid disorders including urea cycle defects ? Organic acid disorders ? Fatty acid oxidation defects Fatty acid oxidation disorders are a group of inherited metabolic conditions that lead to an accumulation of fatty acids, and a decrease in cell energy metabolism. Each fatty acid oxidation disorder is associated with a specific enzyme defect in the fatty acid metabolic pathway and affects utilization of dietary and stored fat. Newborn screening includes testing for a panel of acylcarnitines. In some cases, an elevated level of a particular acylcarnitine may indicate the possibility of one of several different fatty acid oxidation disorders; the specific disorder cannot be determined without diagnostic further testing. It has been demonstrated that the following fatty acid oxidation disorders may be detected in newborn dried blood spot samples using a testing panel. Fatty acids with carbon chain lengths of primarily 18 carbons or less are metabolized in the mitochondria by a process known as ?-oxidation. Several disorders in mitochondrial ?-oxidation have been characterized and include very long-chain acyl-CoA dehydrogenase (VLCAD) deficiency, medium-chain acyl-CoA dehydrogenase (MCADD) deficiency, short-chain acyl-CoA dehydrogenase (SCAD) deficiency, multiple acyl-CoA dehydrogenase deficiency (MADD) etc. A pilot expanded newborn screening programme to detect inherited metabolic disorders, by means of liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS), began in the Campania region, southern Italy, in 2007. By October 2011, >23.753 dried blood samples on filter paper from 14 hospitals in the region had been screened in our laboratory. Within this programme, we detected two cases of Medium Chain acyl CoA Dehydrogenase (MCADD) deficiency, one case of ?-ketothiolase Deficiency, one case of Short Branched Chain Amino acid Deficiency, two cases of maternal vitamin B12 deficiency, one case of Propionic aciduria, one case of Isovaleric aciduria, one case of Methymlonic aciduria and one case of Isobutirryl CoA Dehydrogenase Deficiency. In the elaborate, it is reported a clinical case describing the situation of a newborn to whom is diagnosed a Medium-Chain Acyl CoA Dehydrogenase Deficiency at birth. Immediately after the diagnosis, the patient underwent emergency therapy with glucose, after which the carnitine concentration decreased significantly as described in the Figure 9 where is shown the profile of acylcarnitine during therapy. The results of therapy are evident: LC-MS/MS analysis shows that C0 decreases from 0.62 µmol/L to 0.2 µmol/L , the C8 decreases from 6.14 µmol/L to 0.98 µmol/L , the C10 decreases from 0.60 µmol/L to 0,11 µmol/L and C10: 1 decreases from 0.51 µmol/L to 0.25 µmol/L . The biochemical and molecular analysis are extended to family members, and in this way, the early detection allows not only to discover that the relatives of affected child were healthy carriers of the disease, heterozygous for the mutation, but also to discover that her brother, one year and three months old, was affected with the same pathology, thereby allowing to save the baby treating him with proper medical care. THERAPEUTIC DRUG MONITORING IN CML PATIENTS The second line of research has focused on the development of analytical methods for qualitative and quantitative assessments of drugs in biological fluids, especially on the assay of imatinib, a drug used to treat patients with chronic myeloid leukemia. Chronic Myeloid Leukemia (CML) is a malignant neoplasia caused by an acquired alteration of the totipotent stem cell of the bone marrow. The genetic abnormality of CML is the translocation between chromosomes 9 and 22, which causes the shaping of a shorter chromosome 22 (Philadelphia chromosome) and creates an hybrid gene, the BCR/ABL gene, which encodes an oncoprotein (Bcr/Abl) with tyrosine-kinase activity deregulated. In 1998, when imatinib was distributed in the marked, a great improvement in the treatment of CML was made. Imatinib is the first of a new category of chemotherapeutic agents that act by inhibiting enzymes with tyrosine-kinase activity rather than inhibiting the rapid cells division; imatinib acts as an allosteric inhibitor: it inhibits the substrate phosphorylation by ATP, blocking the enzymatic protein in a conformation that prevents ATP access to the active site. Imatinib plasma monitoring is important for treatment because patients being treated can acquire resistance to the drug after 4-5 years of treatment; in case of failure of the therapy and occurrence of severe adverse events it is important to intervene promptly by adjusting the dose of imatinib or making other therapeutic choices based on the administration of II generation drugs such as nilotinib and dasatinib. The doctorate was conducted in a laboratory included in the project EUTOS imatinib BLT (Blood Level Testing) as reference center in central and southern Italy and islands to evaluate plasma imatinib concentrations. To evaluate plasma imatinib concentrations, an analytical procedure was developed that uses LC-MS/MS. The spectrometer on which the method has been developed is a triple quadrupole mass spectrometer, it consists of three quadrupoles in series, in which the first and third, Q1 and Q3, work as mass filters while the second acts as a collision cell. The possibility to set individually the quadrupoles on specifics mass range allows a highly selective and specific analysis. We make scans of the first mass analyzer to obtain the maximum sensitivity on the mass/charge ratio of the precursor ions through the optimization of a series of instrumental parameters. Once the precursor ion was optimized in terms of sensitivity and selectivity, we proceed to its fragmentation in the collision cell (Q2), in order to establish its characteristic product ions. The choice of product ions is very important because, under certain conditions, a molecule fragment in the same way and they will be always the same fragments, so the product ions selected at this stage will be those that will characterize the molecule and will allow the identification in very complex matrices. Once optimized the parameters for the characteristics fragmentation, chromatographic conditions are optimized too. Once developed and optimized the method, imatinib plasma concentrations for patients enrolled in the EUTOS project were dosed. Imatinib blood-level testing data are processed through a digital platform, the "Labnet" that acts as a direct connection between hematology centers associated and reference centers for imatinib dose monitoring. A further objective of the project was to evaluate the correlation of two methods for the assay of imatinib plasma concentration: LC-MS/MS e HPLC-UV/VIS.