Tracer Methods Defining Amino Acid Kinetics

Isotopically labeled tracers are used to follow flows of endogenous metabolites in the body. The labeled tracers are identical to the endogenous metabolites in terms of chemical structure with substitution of one or more atoms with isotopes different from those usually present. The isotopes are substituted to make the tracers distinguishable (measurable) from the normal metabolites. We usually think first of the radioactive isotopes (e.g., 3H for hydrogen and 14C for carbon) as tracers that can be measured by the particles they emit when they decay, but there are also non-radioactive, stable isotopes that can be used. Because isotopes of the same atom only differ in the number of neutrons that are contained, they can be distinguished in a compound by mass spectrometry, which determines the abundance of compounds by mass. Most of the lighter elements have one abundant stable isotope and one or two isotopes of higher mass of minor abundance. The major and minor isotopes are 1H and 2H for hydrogen, 14N and 15N for nitrogen, 12C and 13C for carbon, and 16O, 17O, and 18O for oxygen. Except for some isotope effects, which can be significant for both the radioactive (3H) and nonradioactive (2H) hydrogen isotopes, a compound that is isotopically labeled is essentially indistinguishable from the corresponding unlabeled endogenous compound in the body. Because they do not exist in nature and so little of the radioactive material is administered, radioisotopes are considered "weightless" tracers that do not add material to the system. Radioactive tracer data are expressed as counts or disintegrations per minute per unit compound. Because the stable isotopes are naturally occurring (e.g., ยป1% of all carbon in the body is 13C), the stable isotope tracers are administered and measured as the "excess above the naturally occurring abundance" of the isotope in the body as either the mole ratio of the amount of tracer isotope divided by the amount of unlabeled material or the mole fraction (usually expressed as a percentage: mole % excess or atom % excess, the latter being an older, less appropriate term in the literature) (64).

The basis of most tracer measurements to determine amino acid kinetics is the simple concept of tracer dilution. This concept is illustrated in Fig.yie,,2.;.9 for the determination of the flow of water in a stream. If you infuse a dye of known concentration (enrichment) into the stream, go downstream after the dye has mixed well with the stream water, and take a sample of the dye, then you can calculate from the measured dilution of the dye the rate at which water must be flowing in the stream to make that dilution. The necessary information required is infusion rate of dye (tracer infusion rate) and measured concentration of the dye (enrichment or specific activity of the tracer). The calculated value is the flow of water through the stream (flux of unlabeled metabolite) causing the dilution. This simple dye-dilution analogy is the basis for almost all kinetic calculations in a wide range of formats for a wide range of applications. A few of the more important approaches are discussed below.

Figure 2.9. Basic principal of the "dye-dilution" method of determining tracer kinetics.

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