Tracers to investigate protein and amino acid metabolism in human subjects

Abstract

Abbreviations: FSR, fractional synthesis rate; IRMS, isotope-ratio mass spectrometry; α-KIC, α-ketoisocaproic acid; MS, mass spectrometry; nb, net balance of tracer; NB, net balance of tracee; Ra, rate of appearance; Rd, rate of disappearance. The contribution of whole-body methods to the future gain of knowledge is expected to be limited due to the fact that most physiological disturbances have been investigated extensively, and due to the lack of information on the relative contribution of various tissues and proteins to whole-body changes.Tracer amino acid-incorporation methods are most suited to investigate these latter aspects of protein metabolism. These methods have shown that some tissues (liver and gut) have much higher turnover rates and deposit much more protein than others (muscle). Massive differences also exist between the fractional synthesis rates of individual proteins. The incorporation methods have been properly validated, although minor disagreements remain on the identity of the true precursor pool (the enrichment of which should be used in the calculations). Arterio-venous organ balance studies have shown that little protein is deposited in skeletal muscle following a proteincontaining meal, while much more protein is deposited in liver and gut. The amount deposited in the feeding period in each of these tissues is released again during overnight fasting. The addition of tracers to organ balance studies allows the simultaneous estimation of protein synthesis and protein breakdown, and provides information on whether changes in net protein balance are caused primarily by a change in protein synthesis or in protein breakdown. In the case of a small arterio-venous difference in a tissue with a high blood flow, estimates of protein synthesis and breakdown become very uncertain, limiting the value of using the tracer. An additional measurement of the intracellular free amino acid pool enrichment allows a correction for amino acid recycling and quantification of the inward and outward transmembrane transport. However, in order to obtain reliable estimates of the intramuscular amino acid enrichment and, therefore, of muscle protein synthesis and breakdown in this so-called three-pool model, the muscle should be freeze-dried and the resulting fibres should be freed from connective tissue and small blood clots under a dissection microscope. Even when optimal precautions are taken, the calculations in these tracer balance methods use multiple variables and, therefore, are bound to lead to more variability in estimates of protein synthesis than the tracer amino acid incorporation methods. In the future, most studies should focus on the measurement of protein synthesis and breakdown in specific proteins in order to understand the mechanisms behind tissue adaptation in response to various stimuli (feeding, fasting, exercise, trauma, sepsis, disuse and disease). The tracer laboratories, therefore, should improve the methodology to allow the measurement of low tracer amino acid enrichments in small amount...

Postexercise protein ingestion increases whole body net protein balance in healthy children

“…In addition, the noninvasive study design precluded our ability to measure other potential biological effectors of protein metabolism such as circulating insulin, which can suppress whole body protein breakdown (Arslanian) and, provided there was a differential response between conditions, may have also contributed to the changes in WBPB observed herein. Nevertheless, other stable isotope methodologies are available to study whole body protein metabolism such as the primed constant [ 13 C]leucine infusion (45), which has been utilized successfully in research in children (13) and is arguably the current gold standard methodology (45). Despite the disadvantage of requiring repeated breath and blood samples that is not always feasible in children, the advantages of [ 13 C]leucine infusion include its preferential metabolism within skeletal muscle, the ability to measure changes in circulating amino acid and insulin concentrations, and the greater time resolution over which changes in protein turnover can be measured.…”

Action of GH on skeletal muscle function: molecular and metabolic mechanisms

“…Amino acids released from protein breakdown are either reutilised in protein synthesis or irreversibly lost via oxidation. Over the last two decades, isotope tracer methods such as leucine turnover technique have made it possible to accurately measure these components of whole-body protein metabolism by tracking the metabolic fate of a labelled amino acid (Wagenmakers 1999).…”

“…To address the direct effects of GH on skeletal muscle protein turnover, investigators have measured the arterio-venous difference of labelled and unlabelled amino acids across the forearm or leg (Wagenmakers 1999). Using this technique, Fryburg et al (1991) and Fryburg & Barrett (1993) reported that GH induced an increase in protein synthesis without affecting the rate of protein breakdown in forearm muscles.…”

Stable isotopes in obesity research

“…GC-MS and IRMS are thus complementary techniques. Sometimes both are used in different parts of a tracer study, e.g., in total-body protein turnover determinations (Wagenmakers, 1999).…”