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

Abstract

BackgroundMethotrexate (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-PG2–6). Loss of FPGS activity is associated with reduced MTX activity and although red blood cell (RBC) MTX-PGn levels correlate with disease activity in RA patients,1 it is anticipated to be more relevant to measure MTX-PGn 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-PGn in PBMCs.2 ObjectivesTo validate a rapid, sensitive and non-radioactive assay to measure FPGS activity and MTX-PGn in PBMCs based on LC-MS/MS technology.MethodsProtein 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-PG2 formation was analysed with AB Sciex 4000 Q Trap tandem mass spectrometer coupled to an Acquity Ultra Performance LC system. Measurement of PBMC-MTX-PGn (n=5) was performed by extraction of MTX-PGn from PBMCs by perchloric acid precipitation. Quantification was performed with 13C515N-labelled MTX-PG1–5 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.ResultsIn 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.3 FPGS activity in CEM/R30dm was <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-PGn in RA patient PBMCs were 22,1% (range: 8.2%–36.2%) for MTX-PG2, 32.8% (27.1%–43.6%) for MTX-PG3, 34.4% (30.4%–41.3%) for MTX-PG4 and 10.6% (0.0%–28.4%) for MTX-PG5. Average total MTX-PGn 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.ConclusionsA sensitive LC-MS/MS based method was developed for the measurement of FPGS activity and MTX-PGn levels in PBMCs of RA patients. This method holds promise to guide future MTX-therapy response evaluations.References[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.Disclosure of InterestNone declared

AB0235 Polymorphisms of hla-drb1 and tnf-308 g/a are associated with radiographic joint destruction in patients with very early rheumatoid arthritis

“…Substrate affinity parameters (Km) for MTX (65 mM) and l-glutamic acid (2.2 mM) were consistent with earlier reports. 3 FPGS activity in CEM/R30dm was <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.…”

Personalized medicine in rheumatoid arthritis: methotrexate polyglutamylation revisited

“…Thus, conceivably analysis of MTX-PG n levels in peripheral blood mononuclear cells (PBMCs) of RA patients during MTX treatment might be of greater clinical relevance, but on the other hand more analytically challenging. Building on the expertise with MTX-PG n analysis and quantification in RBCs by stable isotope dilution liquid chromatography/mass spectrometry (LC-MS/MS) with 13 C 5 15 N-labeled MTX-PG 1-7 internal standards [17,21], we were able to modify this method for the analysis of MTX-PG n in low amounts (<5 x 10 6 ) of PBMCs of RA patients [22]. The availability of a sensitive and quantitative LC-MS/MS method to determine MTX-PG n levels in PBMCs allows implementation in future MTX-TDM studies and may help to define whether MTX-PG n analysis in PBMCs is of added value for predicting MTX response.…”

“…Indeed FPGS activity in bone-marrow cells is very low and even using a sensitive radio-active assay was not measurable in human PBMCs [25]. To meet these shortcomings, we adapted the above-mentioned LC-MS/MS method for MTX-PG n analyses in PBMCs to quantify the FPGS activity reaction product MTX-Glu2 in extracts of 5 × 10 6 RA patient's PBMCs [22]. The feasibility of FPGS analysis in PBMCs of RA patients combined with molecular analysis for FPGS pre-mRNA splicing aberrations could then provide insight into the contributing role of FPGS in variations of MTX-PG n accumulation and MTX response in RA.…”