Development and Validation of a Sensitive UHPLC-MS/MS–Based Method for the Analysis of Folylpolyglutamate Synthetase Enzymatic Activity in Peripheral Blood Mononuclear Cells: Application in Rheumatoid Arthritis and Leukemia Patients

Abstract

Background: Folylpolyglutamate synthetase (FPGS) is a crucial enzyme in both cellular folate homeostasis and the intracellular retention of folate analogue drugs such as methotrexate (MTX), which is commonly used for the treatment of (pediatric) leukemia and the anchor drug in rheumatoid arthritis (RA) treatment. To date, assessment of FPGS catalytic activity relies on assays using radioactive substrates that are labor-intensive and require relatively large numbers of cells. Here, we describe a nonradioactive, ultra–high-performance liquid chromatography–tandem mass spectrometer (UHPLC-MS/MS)–based method allowing for sensitive and accurate measurements of FPGS activity in low cell numbers (ie, 1–2 × 106) of biological specimens, including leukemic blast cells of acute lymphoblastic leukemia patients and peripheral blood mononuclear cells of patients with RA. Methods: The UHPLC-MS/MS assay was validated with 2 CCRF-CEM human leukemia cells, one proficient and one deficient in FPGS activity. Linearity of time and protein input were tested by measuring FPGS activity at 30–180 minutes of incubation time and 10–300 mcg protein extract. In addition, FPGS enzyme kinetic parameters were assessed. Results: The FPGS enzymatic assay showed a linear relation between FPGS activity and protein input (R2 ≥ 0.989) as well as incubation time (R2 ≥ 0.996). Moreover, the UHPLC-MS/MS method also allowed for evaluation of FPGS enzyme kinetic parameters revealing Km values for the substrates MTX and L-glutamic acid of 64 µmol/L and 2.2 mmol/L, respectively. The mean FPGS activity of acute lymphoblastic leukemia blast cells (n = 4) was 3-fold higher than that of CCRF-CEM cells and 44-fold and 88-fold higher than that of peripheral blood mononuclear cells from MTX-naive (n = 9) and MTX-treated RA patients (n = 6), respectively. Conclusions: Collectively, given its sensitivity with low cell numbers and avoidance of radioactive substrates, UHPLC-MS/MS–based analysis of FPGS activity may be eligible for routine therapeutic drug monitoring of MTX in RA and leukemia for therapy (non)response evaluations.

Pharmacokinetics of oral and subcutaneous methotrexate in red and white blood cells in patients with early rheumatoid arthritis: the methotrexate monitoring trial

“…Furthermore, along with MTX dosing and plasma pharmacokinetics in the RA setting, it has to be considered that RFC and FPGS operate far below their maximal capacities (Michaelis Menten constant (Km) of 5 µmol/L and 60–100 µmol/L, respectively,30 while intracellular total MTX-PG concentrations in RA PBMCs (figure 1) are around 0.3–0.5 µmol/L (calculated from MCVs of 200 fl for lymphocytes and 400 fl for monocytes) 37. Given the low intracellular MTX-PG concentrations, unfavourable FPGS enzyme kinetics, low FPGS enzyme activity in PBMCs and a shorter lifespan,30 these combinations will hamper to convert MTX-PG 1 to higher MTX-PG species.…”

“…For this reason, a formal sample size calculation was not necessary. However, a sample size of 40 patients was reasoned to be sufficient, since we anticipated that levels of MTX-PGs in PBMCs with a regulated folate/MTX metabolism would be higher than in RBCs 29 30…”

“…As enucleated and non-proliferating cells, mature RBCs have no regulated folate metabolism and other than some residual activity of the folate/MTX transporter RFC and ABCC1/4/5 efflux transporter activity,25 RBCs lack activities of key enzymes (including FPGS) in MTX metabolism 26 27. Therefore, it can be hypothesised that white blood cells, specifically peripheral blood mononuclear cells (PBMCs), might serve as better candidates for TDM, as FPGS is tightly regulated in these cells 28–30. Preliminary approaches to measure MTX-PGs in PBMCs have been reported, but came with analytical challenges and were not followed up 31 32…”

Patient-derived oral mucosa organoids as an in vitro model for methotrexate induced toxicity in pediatric acute lymphoblastic leukemia

“…FPGS catalytic activity analysis in organoids and leukemic cell lines was performed essentially as described by Muller et al [56]. In short, FPGS protein was isolated from organoids and cell lines by sonication (Sonoplus Mini 20; Bandelin, Berlin, Germany) on ice for 2 � 10 seconds (10 second intervals with 90% amplitude and 30-second intervals between samples) in FPGS extraction buffer (…”

Are long-chain methotrexate polyglutamate levels the reason for LD-MTX related adverse events in inflammatory arthritis?

“…MTX is actively transported inside blood cells with the help of solute carrier family 19 member 1 (SLC19A1, also known as reduced folate carrier 1) and forms its metabolites by the addition of glutamic acid residue with the help of folylpolyglutamate synthase (FPGS) enzyme [2]. These sequential additions of glutamate residue create methotrexate polyglutamates (MTXPG 2-n ).…”