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Validation of the Calcineurin Phosphatase Assay

¹ Department of Renal Medicine C, Research Laboratory, Aarhus University Hospital, Skejby Sygehus, Brendstrupgaardsvej, DK-8200 Aarhus N, Denmark.

^aAuthor for correspondence. Fax 45-89496003; e-mail bundgaardpernille{at}dadlnet.dk.

Background: 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 [-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: V_max was estimated as 240 nmol ³²P · L^–1 · min^–1 and K_m 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.

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