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 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)x10(6) WBC(-1), where WBC is white blood cells (leukocytes). In vitro IC50 values were 212 and 34 microg/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.
Sellar
Kathryn J
KJ
Department of Clinical Chemistry, Leiden University Medical Center, Leiden 2333 ZA, The Netherlands.
van Rossum
Huub H
HH
Romijn
Fred P H T M
FP
Smit
Nico P M
NP
de Fijter
Johan W
JW
van Pelt
Johannes
J
D019703:Calcineurin / Q000097:blood*; D065095:Calcineurin Inhibitors; D016572:Cyclosporine / Q000494:pharmacology / Q000627:therapeutic use; D005260:Female; D006801:Humans; D016030:Kidney Transplantation; D007962:Leukocytes / Q000737:chemistry*; D008297:Male; D008875:Middle Aged; D013053:Spectrophotometry / Q000379:methods; D016559:Tacrolimus / Q000494:pharmacology / Q000627:therapeutic use
D065095:Calcineurin Inhibitors; D016572:Cyclosporine; D019703:Calcineurin; D016559:Tacrolimus