Studying apolipoprotein turnover with stable isotope tracers: correct analysis is by modeling enrichments - PubMed (original) (raw)
Review
Studying apolipoprotein turnover with stable isotope tracers: correct analysis is by modeling enrichments
Rajasekhar Ramakrishnan. J Lipid Res. 2006 Dec.
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.
Figures
Fig. 1
Adapted from Ref. to show the essential part of their Fig. 1. Leucine pools and synthesis of a protein of interest [e.g., VLDL apolipoprotein B (apoB)]. The larger pool is for the tracee, and the smaller pool is for the tracer. Under the assumption of tracee steady state, tracee incorporation into VLDL apoB is unaltered during the study, but tracer mass and incorporation change with time, indicated by (t); the tracer incorporation rate constant is the same as for the tracee. Other pathways for leucine and for VLDL apoB are not shown. The dashed rectangles denote total leucine and VLDL apoB.
Fig. 2
Schemata of three amino acid precursors, and their rates of incorporation into a specific apolipoprotein in a hypothetical study with a primed constant infusion of a tracer of leucine and a simultaneous bolus injection of a tracer of glycine. Under the assumption of tracee steady state, A shows the precursor pools, with the small pools representing tracers. If the tracee (unlabeled) masses Uleu, Ugly, and Uala are constant, the rates of incorporation vary with time, as given by the formulas under the arrows and as shown in B. The numbers along the y axis are in arbitrary units. The curves begin at the leucine, glycine, and alanine contents of apolipoprotein B before tracer infusion. Soon after 0 h, leucine is at 14 instead of 12.5, and glycine is at 9 instead of 4.5, resulting in a changing stoichiometry of the apoB product, an impossibility. C shows the tracer-to-tracee ratio (TTR) data from Fig. 4 of Parhofer et al. (17), presented here, under the assumption of tracee steady state, as the change in total mass of VLDL apoB. If tracee apoB were constant, total apoB increased by nearly 4% to a new steady level, according to the leucine TTR data, whereas the glycine TTR data would suggest that total apoB increased quickly by nearly 4% and then declined. The dashed horizontal line indicates that an untraced amino acid should be interpreted as no change in VLDL apoB. Thus, the assumption of a tracee steady state is contradicted by the data of Parhofer et al. (17). AA, amino acid.
Fig. 3
The tracer and tracee precursor pools for an amino acid whose tracer is introduced. Both pool sizes can vary with time, indicated by (t), but the total (tracer plus tracee) incorporation rate into an apolipoprotein is unchanged. The separate incorporation rates of tracer and tracee are proportional to their respective masses, as given by the formulas. The fluxes on other pathways, such as oxidation, clearance, or other storage pools, may bear different relationships to the masses, as indicated by question marks to mean “unknown.”
Fig. 4
Schematic bar graphs showing the changes with time in precursor amino acid (P), incorporation rate into apolipoprotein (S), and amino acid (AA) in apolipoprotein (M) for two types of tracer studies. The full height of each bar represents the total of tracer and tracee, whereas the hatched portion is for the tracer. The subscript T stands for total (tracer plus tracee), L stands for tracer (label), C stands for constant infusion, B stands for bolus, and 1, 8, and 15 indicate 1, 8, and 15 h. The left panels are for a primed constant infusion. The upper left panel shows that the tracer in the precursor increases and stays at a constant fraction from 1 to 15 h. The middle left panel shows that apolipoprotein incorporation is unchanged, with the tracer contributing that same fraction from 1 to 15 h. The bottom left panel shows that the apolipoprotein mass remains the same while the amount of label increases from 0 to 1 to 8 h, approaching a plateau at 15 h. The right panels are for a bolus study. The upper right panel shows that the tracer in the precursor increases and then declines from 1 to 15 h. The middle right panel shows that the apolipoprotein incorporation is unchanged, with the tracer contributing a fraction equal to its fraction in the precursor. The bottom right panel shows that the apolipoprotein mass remains the same while the amount of label increases from 0 to 1 h and then decreases to 15 h.
Fig. 5
Scheme of the amino acid and apolipoprotein systems. When a tracer is introduced, that amino acid is in an unsteady state, but the apolipoprotein remains in a steady state. The dashed line separates the unsteady amino acid system from the steady apolipoprotein system. The number of pools is for illustrative purposes. The unaltered synthesis paths are shown by double arrows. Total masses are denoted by M, and fluxes are denoted by R. Tracer masses are denoted by m; labeled fluxes, which equal the corresponding total fluxes multiplied by the source pool enrichments, are given in terms of total fluxes and tracer-to-total mass ratios or enrichments. For instance, the total flux from protein pool 1 to pool 2 is R21, and the corresponding tracer flux is R21m1(t)/M1, or R21 multiplied by the tracer enrichment in pool 1. Tracer quantities are shown in italics, below or to the right of the corresponding total quantities.
Fig. 6
In a bolus study, precursor TTR is higher and sharper than precursor tracer enrichment (E). A: Theoretical situation with a single precursor pool whose enrichment declines monoexponentially. B: TTR data taken from Parhofer et al. (17) and enrichments calculated from their TTR data, along with fitted curves. The area under the TTR curve is 39% higher in A and 35% higher in B than the corresponding area under the enrichment curve. This overestimation of the forcing function leads to an underestimate of fractional synthetic rates with TTR modeling.
References
- Phair RD, Hammond MG, Bowden JA, Fried M, Fisher WR, Berman M. Preliminary model for human lipoprotein metabolism in hyperlipoproteinemia. Fed Proc. 1975;34:2263–2270. - PubMed
- Kissebah AH, Alfarsi S, Adams PW, Wynn V. The metabolic fate of plasma lipoproteins in normal subjects and in patients with insulin resistance and endogenous hypertriglyceridaemia. Diabetologia. 1976;12:501–509. - PubMed
- Packard CJ, Third JL, Shepherd J, Lorimer AR, Morgan HG, Lawrie TD. Low density lipoprotein metabolism in a family of familial hypercholesterolemic patients. Metabolism. 1976;25:995–1006. - PubMed
- Kekki M, Miettinen TA, Wahlstrom B. Measurement of cholesterol synthesis in kinetically defined pools using fecal steroid analysis and double labeling technique in man. J Lipid Res. 1977;18:99–114. - PubMed
Publication types
MeSH terms
Substances
LinkOut - more resources
Full Text Sources
Research Materials
Miscellaneous