Abstract
The earliest studies of human protein metabolism involved determination of nitrogen balance (N balance). By determining the amount of N ingested, and subtracting N lost in urine, feces, sweat, and minor miscellaneous losses, one can calculate the N balance. Because almost all of the N in the human body is contained in proteins, a positive N balance generally indicates that the protein mass of the body has increased, and a negative N balance that it has decreased. However, the nonprotein N content of the body can vary enough to invalidate short-term N balance as a measure of protein balance. Each gram of N represents about 6.25 g of protein (Forbes 1987a). Because, in theory, N balance could be measured with a precision of a few tenths of a gram each day, and a 70 kg man contains about 1800 g of N (Forbes 1987a), this method could detect changes in total protein mass on the order of 0.1%.
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Notes
If a radioactive isotope is used, the ratio of radioactive to nonradioactive amino acid is termed the specific activityof the amino acid. This formula is accurate for both stable and radioactive isotopes. However, when radioactive isotopes are used their mass is negligible, so that the infusion rate of the tracer typically is not subtracted from first term. The specific activity is measured as disintegrations per minute (dpm) of the tracer divided by concentration of the amino acid, and the infusion rate is expressed as dpm infused per minute rather than as the mass of tracer infused per minute. Equations will be given for stable isotope tracers, because they are applicable to both stable and radioactive tracers. Investigators using radioactive tracers can disregard terms in the equation that are designed to remove the influence of tracer mass.
The literature on the use of the 15N end product method usually employs the term flux, or N flux, rather than Ra, because the Ra of the amino acid is not being measured, only the dilution of its N in the free N pool.
Some investigators advocate using the tracer to tracee ratio rather than the enrichment (i.e., ratio of tracer to tracee + tracer) of the protein and aminoacyl-tRNA index when using a stable isotopic tracer. However, this method assumes that there is an increase in protein synthesis that is proportional to the increase in the mass of the free amino acid pool caused by the stable tracer. In my opinion, there is not adequate theoretical or experimental evidence to support such an assumption, and I prefer to use the enrichment values. The two methods will deviate significantly only when the tracer enrichment is high, such as during flooding dose experiments.
The solution for FRSin this equation is FRS =(-1/t)ln[(Eaa-tRNA-EPt)/Eaa-tRNA] This solution has limited usefulness because it applies to a situation in which there is only a single sample of enriched protein. Ideally, if nonlinear tracer incorporation is expected, multiple samples should be taken over time to ensure a monoexponential increase in tracer enrichment. If this equation is used to estimate FRSbetween two time points at which the protein is enriched with tracer relative to the pre-tracer baseline, trefers to the time difference between these points, EPt refers to the difference in enrichment, and Eaa-tRNA refers to the difference between Eaa-tRNA and EPat the first time point.
The natural 13C /12C ratio is 1.1%, so a compound with 15 carbons has a 15% chance of having one or more 13C atoms. Moreover, many reagents used for derivatizing compounds for GC contain silicon, and the 29Si/28Si ratio is about 5%. It is not unusual for the background “enrichment ” to be 20% or more, so that detecting a difference of only 0.5% or less is difficult. With IRMS, the background enrichment is only 1.1% for CO2 and 0.75% for N2.
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© 1999 Springer Science+Business Media New York
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Welle, S. (1999). Methods for Studying Protein Metabolism in Humans. In: Human Protein Metabolism. Springer, New York, NY. https://doi.org/10.1007/978-1-4612-1458-8_3
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DOI: https://doi.org/10.1007/978-1-4612-1458-8_3
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