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.
Type de document :
Article dans une revue
Clinical Chemistry, American Association for Clinical Chemistry, 2013, epub ahead of print. 〈10.1373/clinchem.2013.213264〉
Liste complète des métadonnées

http://www.hal.inserm.fr/inserm-00925869
Contributeur : Pierre Marquet <>
Soumis le : mercredi 8 janvier 2014 - 16:54:27
Dernière modification le : vendredi 16 février 2018 - 15:25:02

Lien texte intégral

Identifiants

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〉

Partager

Métriques

Consultations de la notice

99