Isotope Incorporation

Metabolic pathways are studied at DPMSR through the relative quantitation of isotope incorporation after cell culture or tissue exposure to isotopically labeled compounds.  In general, isotope incorporation experiments allow for the resolution of differences between many interconnected metabolic pathways1.  Metabolic isotope incorporation has primarily been performed on glycolysis and the TCA cycle.  However, it is also possible to study the pentose phosphate pathway, acetyl-CoA, fatty acids, purines and pyrimidines along with redox metabolomics with isotope incorporation.  These metabolic pathways are crucial for cardiovascular function2, development of Alzheimer’s disease, and inborn errors of metabolism3.  

The DPMSR uses a specialized capillary electrophoresis system called a ZipChipTM from 908 Devices coupled to a high resolution orbitrap MS/MS to analyze isotope incorporation metabolomics samples.  The ZipChip platform optimizes separation of amino acids within a short method analysis time, and when combined with a high resolution orbitrap, co-migrating pairs can be identified and distinguished from interference as well based on differences in m/z.  The figure below shows some of the details of isotope incorporation analysis with a ZipChip. 

ZipChip Metabolomics Isotope Flux

The sample types accepted for isotope incorporation analysis include cell cultures, tissues, plasma, and dried blood spots on paper.  The figure below shows a partial summary of glutamine metabolism4. The molecules measured in the assay include the compounds in the figure below with red annotations, which denote the number of carbons measured as incorporated into each compound.  An example of the data generated from this platform details the quantitative isotopic incorporation of arginine +10 into the plasma of pediatric malaria patients5. 

If you would like to submit samples for isotope incorporation metabolomic analysis, please contact either Kendra Adams or Will Thompson to discuss the details of your experiment and note that samples cannot be accepted without prior consultation.

Isotope Incorporation Glutamine Citrulline Arginine Ornithine TCA Cycle

References

1. Integration of Flux Measurements and Pharmacological Controls to Optimize Stable Isotope-Resolved Metabolomics Workflows and Interpretation.  Lorkiewicz, P. K., Gibb, A. A., Rood, B. R., et alNature Sci. Reports 913705 (2019).  doi:10.1038/s41598-019-50183-3.

2. Metabolomics and Isotope Tracing.   Jang C, Chen L, Rabinowitz JD.  Cell 173822 (2018).  doi:10.1016/j.cell.2018.03.055

3. Cardiovascular Metabolomics.  McGarrah, R. W., Crown, S. B., Zhang, G.-F., et al.   Circulation Res. 1229 (2018).  doi:10.1161/CIRCRESAHA.117.311002.

4. Metabolic Fate and Function of Dietary Glutamate in the Gut.  Burrin, D. G., and Stoll, B.  The American Journal of Clinical Nutrition903 (2009). doi:10.3945/ajcn.2009.27462Y.

5. Kinetic and Cross-Sectional Studies on the Genesis of Hypoargininemia in Severe Pediatric Plasmodium falciparum Malaria.  Rubach, M. P., Zhang, H., Florence, S. M., et al.  Infection and Immunity 874, (2019). doi.org/10.1128/IAI.00655-18.