Validation of the Calcineurin Phosphatase Assay

• Published in: Clinical chemistry (Baltimore, Md.) Md.), 2004-12, Vol.50 (12), p.2331-2337
• Main Authors: Koefoed-Nielsen, Pernille B, Karamperis, Nikolaos, Jorgensen, Kaj Anker
• Format: Article
• Language: English
• Subjects: Amino acids; Analytical, structural and metabolic biochemistry; Biological and medical sciences; Calmodulin - pharmacology; Drug dosages; Enzyme kinetics; Female; Fundamental and applied biological sciences. Psychology; Humans; Immunosuppressive agents; Immunosuppressive Agents - administration & dosage; Immunosuppressive Agents - pharmacology; Immunosuppressive Agents - therapeutic use; Inhibitors; Investigative techniques, diagnostic techniques (general aspects); Kidney Transplantation; Kidney transplants; Kinases; Kinetics; Male; Medical sciences; Middle Aged; Peptides; Pharmacodynamics; Pharmacokinetics; Phosphatase; Phosphoric Monoester Hydrolases - antagonists & inhibitors; Phosphoric Monoester Hydrolases - blood; Phosphorylation; Proteins; Radioactivity; Sensitivity and Specificity
• ISSN: 0009-9147; 1530-8561
• DOI: 10.1373/clinchem.2004.034066

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. 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. 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. 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.