title: Quantification of mevalonate-5-phosphate using UPLC-MS/MS for determination of mevalonate kinase activity.
abstract: OBJECTIVE
  Mevalonate kinase deficiency, a rare autosomal recessive autoinflammatory disease, is caused by mutations in the MVK gene encoding mevalonate kinase (MK). MK catalyzes the phosphorylation of mevalonic acid to mevalonate-5-phosphate (MVAP) in the pathway of isoprenoid and sterol synthesis. The disease phenotype correlates with residual activity ranging from <0.5% for mevalonic aciduria to 1-7% for the milder hyperimmunoglobulinemia D and periodic fever syndrome (HIDS). Hence, assessment of loss-of-function requires high accuracy measurements. We describe a method using isotope dilution UPLC-MS/MS for precise and sensitive determination of MK activity.
  
  
  METHODS
  Wild-type MK and the variant V261A, which is associated with HIDS, were recombinantly expressed in Escherichia coli. Enzyme activity was determined by formation of MVAP over time quantified by isotope dilution UPLC-MS/MS. The method was validated according to the FDA Guidance for Bioanalytical Method Validation.
  
  
  RESULTS
  Sensitivity for detection of MAVP by UPLC-MS/MS was improved by derivatization with butanol-HCl (LLOQ, 5.0 fmol) and the method was linear from 0.5 to 250 μmol/L (R(2) > 0.99) with a precision of ≥ 89% and an accuracy of ± 2.7%. The imprecision of the activity assay, including the enzymatic reaction and the UPLC-MS/MS quantification, was 8.3%. The variant V261A showed a significantly decreased activity of 53.1%.
  
  
  CONCLUSIONS
  Accurate determination of MK activity was enabled by sensitive and reproducible detection of MVAP using UPLC-MS/MS. The novel method may improve molecular characterization of MVK mutations, provide robust genotype-phenotype correlations, and accelerate compound screening for drug candidates restoring variant MK activity.
authors.affiliation: Department of Molecular Pediatrics, Dr. von Hauner Children's Hospital, Ludwig-Maximilians-University, Munich 80337, Germany.
authors.forename: Lukas
authors.initials: L
authors.lastname: Reitzle
authors.affiliation: Institute of Laboratory Medicine, Ludwig-Maximilians-University, Munich 81377, Germany.
authors.forename: Barbara
authors.initials: B
authors.lastname: Maier
authors.affiliation: Department of Molecular Pediatrics, Dr. von Hauner Children's Hospital, Ludwig-Maximilians-University, Munich 80337, Germany; Department of Child and Adolescent Psychiatry, Psychotherapy and Psychosomatics, University Medical Center Hamburg-Eppendorf, Hamburg 20246, Germany.
authors.forename: Silvia
authors.initials: S
authors.lastname: Stojanov
authors.affiliation: Institute of Laboratory Medicine, Ludwig-Maximilians-University, Munich 81377, Germany.
authors.forename: Daniel
authors.initials: D
authors.lastname: Teupser
authors.affiliation: University Children's Hospital, University Medical Center Hamburg-Eppendorf, Hamburg 20246, Germany.
authors.forename: Ania C
authors.initials: AC
authors.lastname: Muntau
authors.affiliation: Institute of Laboratory Medicine, Ludwig-Maximilians-University, Munich 81377, Germany.
authors.forename: Michael
authors.initials: M
authors.lastname: Vogeser
authors.affiliation: Department of Molecular Pediatrics, Dr. von Hauner Children's Hospital, Ludwig-Maximilians-University, Munich 80337, Germany. Electronic address: soeren.gersting@med.uni-muenchen.de.
authors.forename: Søren W
authors.initials: SW
authors.lastname: Gersting
mesh_terms: D002853:Chromatography, Liquid / Q000379:methods; D006801:Humans; D054078:Mevalonate Kinase Deficiency / Q000201:enzymology; D008798:Mevalonic Acid / Q000031:analogs & derivatives* / Q000737:chemistry / Q000378:metabolism; D008958:Models, Molecular; D010766:Phosphorylation; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000032:analysis / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D017433:Protein Structure, Secondary; D053719:Tandem Mass Spectrometry / Q000379:methods
chemical_list: C045038:phosphomevalonic acid; D017853:Phosphotransferases (Alcohol Group Acceptor); C021796:mevalonate kinase; D008798:Mevalonic Acid
keywords: Activity assay; HIDS; Hyperimmunoglobulinemia D and periodic fever syndrome; MA; MKD; Mevalonate kinase; Mevalonate kinase deficiency; Mevalonate-5-phosphate; UPLC-MS/MS; mevalonic aciduria