Validation of the Calcineurin Phosphatase Assay

Abstract

AbstractBackground: The calcineurin inhibitors cyclosporine and tacrolimus are used as primary immunosuppressive drugs in transplant patients. Measuring calcineurin phosphatase (CaN) activity is a proposed pharmacodynamic approach to optimize dosing of these drugs.Methods: Whole blood samples were obtained from 10 patients treated with calcineurin inhibitors and 20 healthy volunteers and frozen at −80 °C. CaN activity was measured by its ability to dephosphorylate a 19-amino acid peptide previously phosphorylated with [γ-32P]ATP. Radioactivity was quantified by liquid scintillation, and results were converted from cpm to U of CaN. Validation of the assay included enzyme kinetics, linearity, precision (at low and normal CaN activities), analytical recovery, and limit of detection.Results: The enzyme followed simple Michaelis-Menten-type kinetics: Vmax was estimated as 240 nmol 32P · L−1 · min−1 and Km as 70 μmol/L. The assay was linear within the concentration range examined. Analytical recovery varied from 68% to 72%. The total analytical SD was 0.059 and 0.053 U of CaN for high and low CaN activity, respectively. The within-day SD for high and low activity was 0.032 and 0.039 U of CaN, respectively. The limit of detection was 0.04 U of CaN, which is far below the values measured in patients treated with CaN inhibitors.Conclusions: In addition to the pharmacokinetic monitoring applied today, the CaN assay can be used to monitor patients treated with calcineurin inhibitors, hopefully leading to prolonged graft survival.