Implementation of a fast method for the measurement of carnitine palmitoyltransferase 2 activity in lymphocytes by tandem mass spectrometry as confirmation for newborn screening

Carnitine palmitoyltransferase II (CPT2) is a rare autosomal recessive inherited disorder affecting mitochondrial β-oxidation. Confirmation diagnostics are mostly based on molecular sequencing of the CPT2 gene, especially to distinguish CPT2 and carnitine:aclycarnitine translocase deficiencies, which present with identical acylcarnitine profiles on newborn screening (NBS). In the past, different enzyme tests in muscle biopsies have been developed in order to study the functional effect in one of the main target organs. In this study, we implemented a method for measurement of CPT2 enzyme activity in human lymphocytes with detection of the reaction products via liquid chromatography mass spectrometry to enable the simultaneous evaluation of the functional impairment and the clear diagnosis of the disease. CPT2 activity was measured in samples collected from CPT2 patients (n = 11), heterozygous carriers (n = 6), and healthy individuals (n = 52). Seven patients out of 11 were homozygous for the common mutation c.338T>C and showed a residual activity with median values of 19.2 ± 3.7% of healthy controls. Heterozygous carriers showed a residual activity in the range of 42% to 75%. Four individuals carrying the heterozygous mutation c.338T>C showed a 2-fold higher residual activity as compared to homozygous individuals. Our optimized method for the measurement of CPT2 activity is able to clearly discriminate between patients and healthy individuals and offers the possibility to determine CPT2 activity in human lymphocytes avoiding the need of an invasive muscle biopsy. This method can be successfully used for confirmation diagnosis in case of positive NBS and would markedly reduce the time to define diagnosis.