Skip to Main content Skip to Navigation
Journal articles

Calcineurin Activity Assay Measurement by Liquid Chromatography-Tandem Mass Spectrometry in the Multiple Reaction Monitoring Mode.

Abstract : BACKGROUND: Blood concentrations of the calcineurin inhibitors (CNIs) cyclosporine and tacrolimus are currently measured to monitor immunosuppression in transplant patients. The measurement of calcineurin (CN) phosphatase activity has been proposed as a complementary pharmacodynamic approach. However, determining CN activity with current methods is not practical. We developed a new method amenable to routine use.METHODS: Using liquid chromatography-multiple reaction monitoring mass spectrometry (LC-MRM-MS), we quantified CN activity by measuring the dephosphorylation of a synthetic phosphopeptide substrate. A stable isotope analog of the product peptide served as internal standard, and a novel inhibitor cocktail minimized dephosphorylation by other major serine/threonine phosphatases. The assay was used to determine CN activity in peripheral blood mononuclear cells (PBMCs) isolated from 20 CNI-treated kidney transplant patients and 9 healthy volunteers.RESULTS: Linearity was observed from 0.16 to 2.5 μ mol/L of product peptide, with accuracy in the 15% tolerance range. Intraassay and interassay recoveries were 100.6 (9.6) and 100 (7.5), respectively. Michaelis-Menten kinetics for purified CN were Km = 10.7 (1.6) μ mol/L, Vmax = 2.8 (0.3) μ mol/min/mg, and for Jurkat lysate, Km = 182.2 (118.0) μ mol/L, Vmax = 0.013 (0.006) μ mol/min/mg. PBMC CN activity was successfully measured in a single tube with an inhibitor cocktail.CONCLUSIONS: Because LC-MRM-MS is commonly used in routine clinical dosage of drugs, this CN activity assay could be applied, with parallel blood drug concentration monitoring, to a large panel of patients to reevaluate the validity of PBMC CN activity monitoring.
Document type :
Journal articles
Complete list of metadatas

https://www.hal.inserm.fr/inserm-00925869
Contributor : Pierre Marquet <>
Submitted on : Wednesday, January 8, 2014 - 4:54:27 PM
Last modification on : Friday, February 16, 2018 - 3:25:02 PM

Links full text

Identifiers

Collections

Citation

Lynn Carr, Anne-Laure Gagez, Marie Essig, François-Ludovic Sauvage, Pierre Marquet, et al.. Calcineurin Activity Assay Measurement by Liquid Chromatography-Tandem Mass Spectrometry in the Multiple Reaction Monitoring Mode.. Clinical Chemistry, American Association for Clinical Chemistry, 2013, epub ahead of print. ⟨10.1373/clinchem.2013.213264⟩. ⟨inserm-00925869⟩

Share

Metrics

Record views

183