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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.
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.
Sensitivity for detection of MAVP by UPLC-MS/MS was improved by derivatization with butanol–HCl (LLOQ, 5.0fmol) and the method was linear from 0.5 to 250μmol/L (R2>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%.
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.
•In MKD severity of disease correlates with residual enzyme activity.•Assessment of MK activity requires high accuracy measurement methods.•Isotope dilution UPLC-MS/MS allowed precise quantification of mevalonate-5-phosphate.•Determination of MK activity was highly accurate (±2.7%) and precise (CV ≤11%).•The novel method may improve diagnosis and enable screening for drug candidates.