Validation of the calcineurin phosphatase assay.
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 degrees C. CaN activity was measured by its ability to dephosphorylate a 19-amino acid peptide previously phosphorylated with [gamma-(32)P]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 (32)P x L(-1) x min(-1) and K(m) as 70 micromol/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.
Koefoed-Nielsen
Pernille B
PB
Department of Renal Medicine C, Research Laboratory, Aarhus University Hospital, Skejby Sygehus, Brendstrupgaardsvej, DK-8200 Aarhus N, Denmark. bundgaardpernille@dadlnet.dk
Karamperis
Nikolaos
N
Jørgensen
Kaj Anker
KA
D002147:Calmodulin / Q000494:pharmacology; D005260:Female; D006801:Humans; D007166:Immunosuppressive Agents / Q000008:administration & dosage / Q000494:pharmacology / Q000627:therapeutic use; D016030:Kidney Transplantation; D007700:Kinetics; D008297:Male; D008875:Middle Aged; D010744:Phosphoric Monoester Hydrolases / Q000037:antagonists & inhibitors / Q000097:blood*; D012680:Sensitivity and Specificity
D002147:Calmodulin; D007166:Immunosuppressive Agents; C120817:calcineurin phosphatase; D010744:Phosphoric Monoester Hydrolases