title: Calcineurin activity assay measurement by liquid chromatography-tandem mass spectrometry in the multiple reaction monitoring mode.
abstract: BACKGROUND
  Blood concentrations of the calcineurin inhibitors (CNIs) cyclosporine and tacrolimus are currently measured to monitor immunosuppression in transplant patients. The measurement of calcineurin (CN) phosphatase activity has been proposed as a complementary pharmacodynamic approach. However, determining CN activity with current methods is not practical. We developed a new method amenable to routine use.
  
  
  METHODS
  Using liquid chromatography-multiple reaction monitoring mass spectrometry (LC-MRM-MS), we quantified CN activity by measuring the dephosphorylation of a synthetic phosphopeptide substrate. A stable isotope analog of the product peptide served as internal standard, and a novel inhibitor cocktail minimized dephosphorylation by other major serine/threonine phosphatases. The assay was used to determine CN activity in peripheral blood mononuclear cells (PBMCs) isolated from 20 CNI-treated kidney transplant patients and 9 healthy volunteers.
  
  
  RESULTS
  Linearity was observed from 0.16 to 2.5 μmol/L of product peptide, with accuracy in the 15% tolerance range. Intraassay and interassay recoveries were 100.6 (9.6) and 100 (7.5), respectively. Michaelis-Menten kinetics for purified CN were Km = 10.7 (1.6) μmol/L, Vmax = 2.8 (0.3) μmol/min · mg, and for Jurkat lysate, Km = 182.2 (118.0) μmol/L, Vmax = 0.013 (0.006) μmol/min · mg. PBMC CN activity was successfully measured in a single tube with an inhibitor cocktail.
  
  
  CONCLUSIONS
  Because LC-MRM-MS is commonly used in routine clinical dosage of drugs, this CN activity assay could be applied, with parallel blood drug concentration monitoring, to a large panel of patients to reevaluate the validity of PBMC CN activity monitoring.
authors.affiliation: INSERM U850, Limoges, France;
authors.forename: Lynn
authors.initials: L
authors.lastname: Carr
authors.forename: Anne-Laure
authors.initials: AL
authors.lastname: Gagez
authors.forename: Marie
authors.initials: M
authors.lastname: Essig
authors.forename: François-Ludovic
authors.initials: FL
authors.lastname: Sauvage
authors.forename: Pierre
authors.initials: P
authors.lastname: Marquet
authors.forename: Louis Noel
authors.initials: LN
authors.lastname: Gastinel
mesh_terms: D000328:Adult; D019703:Calcineurin / Q000097:blood*; D065095:Calcineurin Inhibitors; D002138:Calibration; D002851:Chromatography, High Pressure Liquid / Q000295:instrumentation / Q000379:methods*; D016572:Cyclosporine / Q000097:blood / Q000494:pharmacology / Q000627:therapeutic use; D005260:Female; D006801:Humans; D007165:Immunosuppression Therapy / Q000379:methods; D007166:Immunosuppressive Agents / Q000097:blood / Q000494:pharmacology / Q000627:therapeutic use; D019169:Jurkat Cells; D016030:Kidney Transplantation; D007963:Leukocytes, Mononuclear / Q000201:enzymology; D008297:Male; D010748:Phosphopeptides / Q000737:chemistry*; D012015:Reference Standards; D015203:Reproducibility of Results; D013379:Substrate Specificity; D016559:Tacrolimus / Q000097:blood / Q000494:pharmacology / Q000627:therapeutic use; D053719:Tandem Mass Spectrometry / Q000295:instrumentation / Q000379:methods*
chemical_list: D065095:Calcineurin Inhibitors; D007166:Immunosuppressive Agents; D010748:Phosphopeptides; D016572:Cyclosporine; D019703:Calcineurin; D016559:Tacrolimus