Studying apolipoprotein turnover with stable isotope tracers: correct analysis is by modeling enrichments

Abstract

Lipoprotein kinetic parameters are determined from mass spectrometry data after administering mass isotopes of amino acids, which label proteins endogenously. The standard procedure is to model the isotopic content of the labeled precursor amino acid and of proteins of interest as tracer-to-tracee ratio (TTR). It is shown here that even though the administered tracer alters amino acid mass and turnover, apolipoprotein synthesis is unaltered and hence the apolipoprotein system is in a steady state, with the total (labeled plus unlabeled) masses and fluxes remaining constant. The correct model formulation for apolipoprotein kinetics is shown to be in terms of tracer enrichment, not of TTR. The needed mathematical equations are derived. A theoretical error analysis is carried out to calculate the magnitude of error in published results using TTR modeling. It is shown that TTR modeling leads to a consistent underestimation of the fractional synthetic rate. In constant-infusion studies, the bias error percent is shown to equal approximately the plateau enrichment, generally ,10%. It is shown that, in bolus studies, the underestimation error can be larger. Thus, for mass isotope studies with endogenous tracers, apolipoproteins are in a steady state and the data should be fitted by modeling enrichments.-Ramakrishnan, R. Studying apolipoprotein turnover with stable isotope tracers: correct analysis is by modeling enrichments. Beginning in the 1970s, apolipoprotein kinetics were routinely studied with exogenous tracers, for instance by isolating VLDL or LDL from a subject, radioiodinating it, and injecting it back into the subject (1-4). Endogenous labeling, with a labeled precursor of the metabolite of interest, has the virtue of labeling the synthetic pathways and not altering tracer metabolic properties, as might happen with exogenous labeling. Whole body cholesterol metabolism was studied with tritiated water (5), and tritiated leucine has been used to study lipoprotein kinetics (6-8). Highly sensitive gas chromatography-mass spectrometry and refinements thereof, and the availability of synthetic amino acids and other molecules that are multiply labeled with mass isotopes, have altered the field to the point that endogenous labeling with mass isotopes is now the norm in human turnover studies (9).An important aspect of mass isotopes is that the amount of tracer introduced is not negligible in relation to the amount in plasma of the tracee. Cobelli, Toffolo, and Foster (10) and Foster et al. (11) considered this problem and advocated the use of tracer-to-tracee ratio (TTR) in place of the previously standard use of tracer enrichment in atoms percent excess or moles percent excess (12-15). Since then, nearly all investigators have used TTR in analyzing mass isotope data to calculate lipoprotein turnover parameters. In what follows, we revisit this issue and derive the mathematical relationships needed for the analysis of tracer data from endogenous labeling. In particular, we show that the apolipoprotein syste...

Alternative equations for whole-body protein synthesis and for fractional synthetic rates of proteins

“…Such a change in protein composition is clearly impossible. Elsewhere, we [16] have presented data from the work of Parhofer et al [33] with two tracers to explain this contradiction in detail. There is no such contradiction under the PSS assumption.…”

“…The concept of PSS was first advanced by Garlick et al [9]. The development here follows one we used in a recent article [16] concerning apolipoprotein kinetics.…”

“…apolipoprotein studies. Barrett et al [17] have reviewed modeling TTR; this author [16] has provided the framework for modeling enrichment. We confine the analysis here to single-pool models (ignoring any tracer recycling due to protein breakdown) used commonly to calculate protein FSRs.…”

“…Elsewhere [16], we have analyzed single-pool models for apolipoprotein turnover where tracer loss is not ignored. The extent of FSR underestimation with TTR is similar to the result above.…”

Proteome Kinetics: Coupling the Administration of Stable Isotopes with Mass Spectrometry-Based Analyses

“…Foster et al (Foster et al 1993) www.intechopen.com have elegantly outlined the rationale behind different mathematical treatments of a given data set, they demonstrate the impact of various assumptions in regards to the modeling of data on the apparent FSR. It is also important to note the differences when modeling data that are expressed as a tracer-to-tracee ratio vs isotopic enrichment, the former is commonly reported but the latter may be preferred in many instances (Cobelli, Toffolo, and Foster 1992;Ramakrishnan 2006;. A second major factor to consider regarding the logic that is applied in kinetic studies centers on heterogeneity in the product pool (note that there are concerns regarding heterogeneity in labeling of the precursor pool, those will be considered in more detail in Section 3) .…”