Quantification of mevalonate-5-phosphate using UPLC-MS/MS for determination of mevalonate kinase activity

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 0.99) 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.