Abstract

BACKGROUND: Folylpolyglutamate synthetase (FPGS) is a crucial enzyme in both cellular folate homeostasis and the intracellular retention of folate analogue drugs like 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 utilizing radioactive substrates which are labor-intensive and require relatively large numbers of cells. Here, we describe a non-radioactive, UHPLC-MS/MS-based method allowing sensitive and accurate measurements of FPGS activity in low cell numbers (i.e. 1 - 2 x 10) of biological specimens, including leukemic blast cells of acute lymphocytic leukemia (ALL) patients and peripheral blood mononuclear cells (PBMCs) of RA patients.

METHODS: The UHPLC-MS/MS assay was validated with two 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 incubation time and 10 - 300 µg 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 (R ≥ 0.989) as well as incubation time (R ≥ 0.996). Moreover, the UHPLC-MS/MS method also allowed 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. Mean FPGS activity of ALL blast cells (n=4) was 3-fold higher than CCRF-CEM cells and 44-fold and 88-fold higher than PBMCs 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.

Original languageEnglish
JournalTherapeutic Drug Monitoring
DOIs
Publication statusE-pub ahead of print - 16 Apr 2019

Cite this

@article{671f3bbde000489ab8fa04741f17a4bd,
title = "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 like 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 utilizing radioactive substrates which are labor-intensive and require relatively large numbers of cells. Here, we describe a non-radioactive, UHPLC-MS/MS-based method allowing sensitive and accurate measurements of FPGS activity in low cell numbers (i.e. 1 - 2 x 10) of biological specimens, including leukemic blast cells of acute lymphocytic leukemia (ALL) patients and peripheral blood mononuclear cells (PBMCs) of RA patients.METHODS: The UHPLC-MS/MS assay was validated with two 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 incubation time and 10 - 300 µg 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 (R ≥ 0.989) as well as incubation time (R ≥ 0.996). Moreover, the UHPLC-MS/MS method also allowed 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. Mean FPGS activity of ALL blast cells (n=4) was 3-fold higher than CCRF-CEM cells and 44-fold and 88-fold higher than PBMCs 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.",
author = "Muller, {Ittai B} and Marry Lin and Struys, {Eduard A} and Paniz Heydari and Hebing, {Renske C F} and Nurmohamed, {Mike T} and {van der Laken}, Conny and Lems, {Willem F} and Jacqueline Cloos and Gerrit Jansen and {de Jonge}, Robert",
year = "2019",
month = "4",
day = "16",
doi = "10.1097/FTD.0000000000000638",
language = "English",
journal = "Therapeutic Drug Monitoring",
issn = "0163-4356",
publisher = "Lippincott Williams and Wilkins",

}

TY - JOUR

T1 - Development and validation of a sensitive UHPLC-MS/MS-based method for the analysis of folylpolyglutamate synthetase enzymatic activity in peripheral blood mononuclear cells

T2 - application in rheumatoid arthritis and leukemia patients

AU - Muller, Ittai B

AU - Lin, Marry

AU - Struys, Eduard A

AU - Heydari, Paniz

AU - Hebing, Renske C F

AU - Nurmohamed, Mike T

AU - van der Laken, Conny

AU - Lems, Willem F

AU - Cloos, Jacqueline

AU - Jansen, Gerrit

AU - de Jonge, Robert

PY - 2019/4/16

Y1 - 2019/4/16

N2 - BACKGROUND: Folylpolyglutamate synthetase (FPGS) is a crucial enzyme in both cellular folate homeostasis and the intracellular retention of folate analogue drugs like 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 utilizing radioactive substrates which are labor-intensive and require relatively large numbers of cells. Here, we describe a non-radioactive, UHPLC-MS/MS-based method allowing sensitive and accurate measurements of FPGS activity in low cell numbers (i.e. 1 - 2 x 10) of biological specimens, including leukemic blast cells of acute lymphocytic leukemia (ALL) patients and peripheral blood mononuclear cells (PBMCs) of RA patients.METHODS: The UHPLC-MS/MS assay was validated with two 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 incubation time and 10 - 300 µg 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 (R ≥ 0.989) as well as incubation time (R ≥ 0.996). Moreover, the UHPLC-MS/MS method also allowed 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. Mean FPGS activity of ALL blast cells (n=4) was 3-fold higher than CCRF-CEM cells and 44-fold and 88-fold higher than PBMCs 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.

AB - BACKGROUND: Folylpolyglutamate synthetase (FPGS) is a crucial enzyme in both cellular folate homeostasis and the intracellular retention of folate analogue drugs like 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 utilizing radioactive substrates which are labor-intensive and require relatively large numbers of cells. Here, we describe a non-radioactive, UHPLC-MS/MS-based method allowing sensitive and accurate measurements of FPGS activity in low cell numbers (i.e. 1 - 2 x 10) of biological specimens, including leukemic blast cells of acute lymphocytic leukemia (ALL) patients and peripheral blood mononuclear cells (PBMCs) of RA patients.METHODS: The UHPLC-MS/MS assay was validated with two 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 incubation time and 10 - 300 µg 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 (R ≥ 0.989) as well as incubation time (R ≥ 0.996). Moreover, the UHPLC-MS/MS method also allowed 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. Mean FPGS activity of ALL blast cells (n=4) was 3-fold higher than CCRF-CEM cells and 44-fold and 88-fold higher than PBMCs 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.

U2 - 10.1097/FTD.0000000000000638

DO - 10.1097/FTD.0000000000000638

M3 - Article

JO - Therapeutic Drug Monitoring

JF - Therapeutic Drug Monitoring

SN - 0163-4356

ER -