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Calcineurin Activity Assay Measurement by Liquid Chromatography-Tandem Mass Spectrometry in the Multiple Reaction Monitoring Mode.
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Edité par CCSD ; American Association for Clinical Chemistry -
International audience. 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.
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