AB0236 Development and validation of a sensitive lc-ms/ms-based method for analysis of enzymatic activity of folylpolyglutamate synthetase and methotrexate polyglutamates in peripheral blood mononuclear cells of rheumatoid arthritis patients
Authors: I.B. Muller, P. Heydari, M. Lin, E.A. Struys, R. Hebing, C.J. van der Laken, D. van Schaardenburg, M.T. Nurmohamed, W.F. Lems, J. Cloos, G. Jansen, R. de Jonge
Journal: Annals of the Rheumatic Diseases (2018)

<h3>Background</h3> Methotrexate (MTX) is a widely applied anti-rheumatic and anti-leukemic drug. For its intracellular retention and pharmacologic activity, MTX relies on the enzymatic activity of folylpolyglutamate synthetase (FPGS) to convert MTX into its polyglutamate forms (MTX-PG<sub>2–6</sub>). Loss of FPGS activity is associated with reduced MTX activity and although red blood cell (RBC) MTX-PG<sub>n</sub> levels correlate with disease activity in RA patients,<sup>1</sup> it is anticipated to be more relevant to measure MTX-PG<sub>n</sub> in peripheral blood mononuclear cells (PBMCs). Thus, the aim of our study was to develop a LC-MS/MS method to 1) measure FPGS activity replacing laborious radioactive assays, and 2) to measure MTX-PG<sub>n</sub> in PBMCs.<sup>2</sup> <h3>Objectives</h3> To validate a rapid, sensitive and non-radioactive assay to measure FPGS activity and MTX-PG<sub>n</sub> in PBMCs based on LC-MS/MS technology. <h3>Methods</h3> Protein extracts (n=5) of PBMCs of MTX-treated RA patients were incubated for 2 hours at 37°C in FPGS assay buffer (pH8.8) containing 250 µM MTX and 4 mM l-glutamic acid as substrates. Next, MTX-PG<sub>2</sub> formation was analysed with AB Sciex 4000 Q Trap tandem mass spectrometer coupled to an Acquity Ultra Performance LC system. Measurement of PBMC-MTX-PG<sub>n</sub> (n=5) was performed by extraction of MTX-PG<sub>n</sub> from PBMCs by perchloric acid precipitation. Quantification was performed with <sup>13</sup>C<sub>5</sub><sup>15</sup>N-labelled MTX-PG<sub>1–5</sub> internal standards. In FPGS activity and MTX-PG validation studies, human CCRF-CEM leukaemia cells, CEM/R30dm (a FPGS-deficient, MTX-resistant subline of CCRF-CEM), and human acute lymphoblastic leukemic (ALL) cells served as reference. <h3>Results</h3> In CCRF-CEM, the FPGS enzymatic assay showed linearity with protein input (10–250 µg) and incubation time (0.5–3 hours). Substrate affinity parameters (Km) for MTX (65 µM) and l-glutamic acid (2.2 mM) were consistent with earlier reports.<sup>3</sup> FPGS activity in CEM/R30dm was &lt;1% of CCRF-CEM. FPGS activity in ALL blasts was similar to CCRF-CEM while FPGS activity in RA patient PBMCs was 1%–5% of CCRF-CEM, and was non-detectable in RBCs. Average individual fractions of total MTX-PG<sub>n</sub> in RA patient PBMCs were 22,1% (range: 8.2%–36.2%) for MTX-PG<sub>2</sub>, 32.8% (27.1%–43.6%) for MTX-PG<sub>3</sub>, 34.4% (30.4%–41.3%) for MTX-PG<sub>4</sub> and 10.6% (0.0%–28.4%) for MTX-PG<sub>5</sub>. Average total MTX-PG<sub>n</sub> levels per number of RA patient PBMCs were 30–50 fold higher than matched numbers of erythrocytes, and 6–9 fold lower than ALL blasts incubated for 24 hours with 1 µM MTX. <h3>Conclusions</h3> A sensitive LC-MS/MS based method was developed for the measurement of FPGS activity and MTX-PG<sub>n</sub> levels in PBMCs of RA patients. This method holds promise to guide future MTX-therapy response evaluations. <h3>References</h3> [1] de Rotte MC, et al. Ann Rheum Dis2015;74:408–414. [2] Jansen G, et al. Oncology Res1992;4:299–305. [3] Liani E, et al. Int. J. Cancer2003;103:587–599. <h3>Disclosure of Interest</h3> None declared