Spectrophotometric assay for calcineurin activity in leukocytes isolated from human blood

The calcineurin inhibitors, cyclosporine and tacrolimus, still constitute the cornerstone of immunosuppressive regimen after organ transplantation. An efficient and feasible way to measure calcineurin activity and inhibition by these drugs may improve therapeutic monitoring of these drugs in transpl...

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Bibliographic Details
Published in:Analytical biochemistry 2006-11, Vol.358 (1), p.104-110
Main Authors: Sellar, Kathryn J., van Rossum, Huub H., Romijn, Fred P.H.T.M., Smit, Nico P.M., de Fijter, Johan W., van Pelt, Johannes
Format: Article
Language:English
Subjects:
Calcineurin
Calcineurin - blood
Calcineurin Inhibitors
Cyclosporine
Cyclosporine - pharmacology
Cyclosporine - therapeutic use
Female
Humans
Kidney Transplantation
Leukocytes - chemistry
Male
Middle Aged
Pharmacodynamic monitoring
Spectrophotometry - methods
Tacrolimus
Tacrolimus - pharmacology
Tacrolimus - therapeutic use
Transplantation
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Summary:The calcineurin inhibitors, cyclosporine and tacrolimus, still constitute the cornerstone of immunosuppressive regimen after organ transplantation. An efficient and feasible way to measure calcineurin activity and inhibition by these drugs may improve therapeutic monitoring of these drugs in transplant recipients. Calcineurin activity was measured in leukocyte lysates isolated from human blood using spectrophotometric phosphate quantification. The dephosphorylation of a 19-amino acid peptide substrate of calcineurin was determined using the Malachite green phosphate reagent in the presence of okadaic acid and with and without the calcium chelator EGTA. Sample storage and lysis buffer components were among the variables optimized, and the inhibitory effect of calcineurin inhibitors was investigated. Observed loss of calcineurin activity during sample storage was eliminated by adding ascorbic acid to lysis buffer. The final inter- and intraassay variation coefficients were 10 and 4.5%, respectively, and the detection limit was 15 pmol min −1 × 10 6 WBC −1, where WBC is white blood cells (leukocytes). In vitro IC 50 values were 212 and 34 μg/L for cyclosporine and tacrolimus, respectively. In vivo calcineurin inhibition was observed when calcineurin activity was measured in transplant recipients on maintenance therapy with cyclosporine and tacrolimus.
ISSN:0003-2697
1096-0309
DOI:10.1016/j.ab.2006.08.013