Method for the Improvement of Enrichment Estimation in Stable Isotope Metabolic Studies
Authors: Malagaris Ioannis , Porter Craig , Herndon David N. , Yu Yong‐Ming 
Published: June 2021

Background

Stable isotope methods are currently the most accurate in vivo approach to estimate protein kinetics. Tracers are molecules where atoms on their structure have been exchanged with heavier isotopes. The determination of substrate kinetics is possible through the administration of a tracer molecule and subsequent measurement of enrichment (ratio of tracer‐to‐tracee, TTR) over time. The attachment of heavier atoms on molecules results in a shift in the tracer's mass distribution therefore, mass spectrometry readings should be processed to obtain the true TTR. Usually, the correction is done by applying the “skew” correction factor. While improvement is achieved, differences remain between theoretical estimations and actual experimental results. We hypothesize that accounting for tracer's isotopic purity would further improve TTR estimation.

Methods

The L‐[ring‐13C6] Phenylalanine (Cambridge Isotope Laboratories, Andover, MA, USA) with nominal isotopic purity of 99% was used. The TBDMS derivative was prepared and signal intensities at mass spectra 340, 341, 342, 343 (M4, M5, M6, M7) (corresponding to the fragment C17H30N1Si2) were measured using GC‐MS (model 5973, Hewlett‐Packard Co., Palo Alto, CA). Using the binomial and multinomial distributions the most likely value of isotopic purity of the tracer was estimated. Subsequently, three mixtures of unlabeled phenylalanine with varying amounts of tracer were prepared yielding samples with TTR of 0.5%, 4% and 20% respectively. The samples were processed as described above and intensities at mass spectra 336 (M0), 341 (M5) and 342 (M6) were measured. Having established the tracer's isotopic purity, intensities at M0, M5 and M6 were modeled as linear combination of tracer and tracee masses using least squares method and TTR was estimated as the ratio of the two regression coefficients. TTR was also estimated with the traditional method by applying the “skew” correction factor to the signal intensities ratio M6/M0.

Results

The tracer's isotopic purity was estimated at 99.1%, corresponding to 71% and 4.7% of relative mass at mass numbers 342 (M6) and 341 (M5) respectively. The estimated TTR of the samples with known TTR of 0.5%, 4% and 20% using the traditional method were 0.46%, 3.75% and 19.1% (error of 8%, 6.25% and 4.5%) respectively. With the new method estimated TTR were 0.52%, 3.97%, 20.2% (errors of 4%, 0.7%, 1%) respectively.

Conclusions

Our preliminary data suggest mild improvement in TTR estimation. We expect the new method to significantly improve TTR estimates in samples containing multiple tracers with overlapping spectra and more accurate determination of muscle protein kinetics calculated through complicated formulas and rely on exponential decay modeling.

Support or Funding Information

This work was supported by grants from NIH (P50 GM060338, R01 GM056687, T32 GM008256, R01 HD049471, K01 HL070451), NIDILRR (90DP00430100), Shriners of North America (SHC 84080, SHC 84090)

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.