Application of 13C flux analysis for diagnosis of metabolic disorders

Introduction

Significant cost reduction of DNA sequencing results in discovery of multiple new gene variants. However, their pathogenicity and molecular phenotypes needs to be uncovered and verified by functional tests. Labeling with stable isotopes and targeted metabolomics can be used to measure metabolic fluxes within central carbon and sugar metabolism providing a unified functional assay capable of diagnosing multiple genetic defects.

Technological and methodological innovation 

Targeted LC-MS/MS methods were optimized for detection of incorporation of ¹³C labels into intermediates of sugar and central carbon metabolism. In silico MS/MS fragmentation was performed to predict massed of fragment ions for selective detection of isotopomers. Patient-derived fibroblasts were cultured until near confluency. After a shift to a medium supplemented with ¹³C glucose the incorporation of isotopic label was followed for up to 48 hours.

Results and impact

The method allowed detection of ¹³C incorporation into TCA cycle intermediates on positions consistent with action of pyruvate dehydrogenase and citric acid synthase. Significantly lower PDH-dependent flux into TCA cycle was observed for cells derived from patient with prior diagnosis of PDH deficiency.

The results show potential to use MS/MS to distinguish positional isotopomers of metabolites of central carbon metabolism and deconvolute fluxes through alternative pathways.

References

 [1] Anke P Willems et al. 2019. Biochimica et Biophysica Acta (BBA) - General Subjects. Vol 1863, 1471-1479