Abstract 1196: Correct Analysis of Mass Isotope Data to Study Lipoprotein Kinetics is by Modeling Enrichments, not Tracer-to-Tracee Ratios
Fractional synthetic rates (FSR) and other kinetic parameters of lipoprotein metabolism in humans are calculated from mass spectrometry data obtained after administering stable isotopes of amino acids (AA), which label proteins endogenously. The method used in nearly all publications is to express the isotopic content of AA and of proteins as tracer-to-tracee ratios (TTR), and to fit a simple or multicompartmental model to the TTR data to estimate the kinetic parameters. The use of TTR has been justified on the grounds that AA infusion alters the masses of body pools. A theoretical study into the validity of modeling TTR was undertaken.
Results: AA masses are indeed altered by isotope infusion; however, since AA levels are seldom rate-limiting in transcription/translation, protein synthesis is not affected. Tracer enrichment of a precursor AA incorporates tracer in newly synthesized proteins, but with a corresponding decrease in incorporation of unlabeled AA. Since protein synthesis is unaltered, total protein masses, fluxes and kinetic parameters are not affected - the protein system is in a steady state. Therefore, enrichment (E, tracer mass divided by tracer+tracee) and not TTR is the appropriate quantity to be analyzed. TTR is numerically larger than E by a factor equal to (1+TTR), thus overstating the driving force for tracer incorporation in protein synthesis. Use of TTR leads to a consistent underestimate of FSR. Integral calculus was used to derive the error with TTR in two experimental designs used in most publications. In pulsed constant infusion studies, if FSR is estimated by dividing the slope of tracer enrichment in a protein by the plateau enrichment (PE), use of TTR underestimates FSR by a factor equal to 3(1-PE)[ln(1/(1-g)-g-g^2/2]/g^3, where g is the highest protein enrichment in the linear phase; the error roughly equals PE, generally 5–10%. In bolus studies, TTR use underestimates the FSR of a slowly turning over protein by a factor equal to the ratio of the areas under precursor E and TTR curves, which can be 0.75 or even lower.
Conclusion: To avoid errors introduced by using TTR, researchers should model E in studying lipoprotein kinetics.