Body protein pool

Oxidative losses, O, and N excretion li

Figure 2 Single pool model for study of amino acid flux and protein turnover by tracer-labeled amino acid infusion. Movement of tracer is shown as dotted lines.

body, and the isotopic enrichment of tRNA-bound amino acids is lower than the measurable extracellular amino acid pool. Because it is difficult to measure labeling of amino acyl tRNA, a variety of indirect approaches have been used in an attempt to circumvent the problem. Equilibrium labeling of apolipoprotein B-100 has also been used to measure isotopic enrichment of hepatic amino acids. Thus, because apo B-100 turns over with a half-life of less than 1 h, during an infusion of several hours the protein labeling will reach a plateau representative of the hepatic precursor pool, and this has indicated a complex relationship between plasma and precursor enrichments for phenylalanine and leucine.

Flux rate measurements define whole body rates of protein synthesis and proteolysis, and each measurement is subject to error associated with the precursor assumption. Alternative methods have attempted to measure protein synthesis and proteo-lysis separately. In animal studies, a ''flooding large dose'' measurement of protein synthesis has been developed that enables all free pools to become equally labeled. Protein synthesis rates are calculated from measurement of isotope uptake into protein and free amino acid labeling during short periods after the dose. The method has been adapted for human use with a stable isotope, but there is evidence that the large quantities of the single amino acid stimulate protein synthesis.

Quantification of rates of proteolysis is especially problematic. In animal studies, proteolysis can be estimated from rates of synthesis and growth of tissue protein, and with careful design proteolysis rates can be measured over relatively short periods (e.g., 6h following the administration of an endotoxin). Urinary excretion rates of 3-methyl histidine, a post-translationally modified amino acid not metabolized in the organism and excreted quantitatively in the urine, were proposed as a measure of myofi-brillar protein degradation. Although the substantial contribution of small, rapidly turning over pools in microfilaments invalidates this approach for whole body studies, its release from incubated or perfused muscle can be used to determine myofibrillar protein degradation.

Simultaneous determination of protein synthesis and degradation can be made, in principle at least, from organ tracer balance studies (i.e., measurements of concentrations and isotopic enrichments of tracer amino acids across tissues such as the leg or forearm combined with measurements of 3-methyl histidine release). Such studies have identified a selective inhibitory effect of insulin on nonmyofibrillar protein degradation and stimulatory influences of amino acids on muscle protein synthesis.

All of these methods allow study of turnover of individual amino acids in protein and measurement of their nonprotein metabolic fate (e.g., oxidation). A different approach is to use 15N glycine to study overall amino nitrogen turnover. Because of nitrogen exchange between amino acids by transamination, this label acts as a tracer for total free amino nitrogen rather than for any individual amino acid. The whole body nitrogen flux is estimated from the relative proportion of administered tracer excreted in the end product. This is then resolved into protein synthesis and proteolysis from measurements of N intake and excretion. The application of the method can be made simple by giving the 15N label orally as a single dose. Although simple in concept, this approach is metabolically complicated with two urinary end products of nitrogen metabolism, urea and ammonia, each deriving from different pathways and each giving different flux values.

The choice of method must depend on the questions asked and circumstances of the subjects under study. 13C carbon labeling is more suited to short-term (e.g., 3 or 4 h to 24 h) clinical measurements for which frequent blood and breath sampling is possible. Thus, the efficiency and mechanisms of postprandial protein utilization during meal feeding can be measured by means of 13C leucine balance and turnover measurements. 15N methods are more suitable for free-living subjects and patients, when urine sampling is possible but regular blood and breath sampling is inconvenient. The most famous example is the use of this method in an unassisted Antarctic crossing. Both methods involve many assumptions, but in practice the two approaches have been shown to give similar results.

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