PLASMA AND LEUKOCYTE METABOLOMICS AND METABOLISATION STUDY OF PATIENTS WITH CHRONIC MYELOID LEUKAEMIA

Background
Metabolomics refers to the interpretation of information-rich data with the aim to understand metabolic processes and to find changes in metabolite levels as a potential biomarkers of the disease. Due to drug metabolizing enzymes several bioactive or toxic metabolites of tyrosine kinase inhibitors are produced. Therefore determination of drug metabolites may have clinical relevance and could be helpful for treatment adjustment. 

Aims
The first aim of this work was to examine the influence of tyrosine kinase inhibitors and hydroxyurea on the leukocyte and plasma metabolite profiles from patients with CML compared with healthy controls. The second aim of this work was profiling of imatinib metabolites in plasma from patients with chronic myeloid leukaemia.

Methods

Leukocytes and plasma samples from healthy controls (n=10), patients before treatment (n=9), treated with hydroxyurea (n=5), imatinib (n=24), dasatinib (n=8) and nilotinib (n=10) were prepared from whole blood and metabolites were extracted by the chloroform/methanol/water mixture. Analysis of 400 metabolites was performed using high performance liquid chromatography (Ultimate 3000, Dionex) coupled with tandem mass spectrometry (QTRAP 5500, AB Sciex). Metabolites were quantified using MultiQuant 3.0 software and statistically evaluated in R programme language with statistics packages. For metabolisation study plasma samples were taken from patients on treatment by imatinib (24h after dose of 400mg). Separation of imatinib metabolites was performed on Phenomenex Kinetex C18 column using UltiMate 3000 RS (Thermo Scientific) liquid chromatography. For detection in full scan mode and MS² fragmentation experiments Orbitrap Elite mass spectrometer (Thermo Scientific) based on exact mass measurement was used. Data were evaluated using MetWorks 1.3 SP3 and Mass Frontier 7.0 software.

Results

Total 170 metabolites were identified on the basis of its transition and retention time in all leukocyte and plasma samples. Significant changes in metabolic pathways were found. Both unsupervised and supervised statistic methods (PCA, PLS-DA, OPLS-DA, cluster analysis) separated all the studied groups. In plasma samples more than 90 potential metabolites in concentration range of 4 orders of magnitude were found. All metabolites were identified by exact m/z values and confirmed by MS2 and MS3 exact m/z fragmentation experiments. Changes in profiles of several metabolisations between patients were followed.

Summary
In this study metabolome of human leukocytes and plasma was defined and changes of metabolite levels were observed in all groups of patients compared to healthy controls. High Resolution Mass spectrometry using Orbitrap principle is able to detect and confirm high as well as low intensive metabolites and its fragments. Tyrosine kinase inhibitor – Imatinib offers complex metabolite profile. It has diagnostic potential for individualized treatment of patients with adverse effects or resistance on treatment.

The work was supported by grant of IGA Ministry of Health, Czech Republic NT12218-4/2011. 

Keyword(s): Leukocytes, Plasma, Tyrosine kinase inhibitor

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