The use of stable isotope tracers as metabolic probes of whole-body and limb metabolism.

We have been drawn to the use of stable isotopes rather than radioisotope tracers (Halliday & Rennie, 1982) as probes of human metabolism for a number of reasons. The most important of these are the lack of radioactive hazard, the higher sensitivity in some techniques such as measurement of tissue protein synthesis, and the ease of measurement of fractional enrichment in one instrument leading to much greater precision than can be obtained by separate measurements of radioactivity and concentration. We have for the most part used very simple models for our studies following on in the footsteps of Professor John Waterlow (Waterlow et al. 1978), and we routinely use a primed constant-infusion method (Matthews et al. 1980; Rennie et al. 1982a,b) (Fig. I ) . Since a major aim of our studies is to investigate the nature of protein metabolism, we use an essential amino acid, leucine, as our probe. The aim of the infusion is to achieve plateau labelling of leucine in the free amino acid pool: this facilitates the calculation of the metabolic flux of leucine through that pool and ensures that processes of protein synthesis or amino acid oxidation draw on a precursor pool which is in a steady-state with regard to labelling. By sampling blood we can get some estimate of the labelling of the free pool, although this is inevitably an overestimate because of the problems of partition of amino acids within tissues. However, for accessible tissues this is not a major problem and the same techniques (gas chromatography-mass spectrometry) can be applied to both plasma and intracellular water to measure leucine enrichment. Leucine oxidation may be measured in the whole body by means of collection of expired carbon dioxide with subsequent cryogenic purification and measurement of 13C0, enrichment by isotope-ratio mass spectrometry (Halliday & Read, 1981). Protein synthesis in tissues is measured by the removal of tissue samples either by needle biopsy under local anaesthetic or by open biopsy during surgical procedures, followed by the chromatographic purification of amino acid from hydrolysed protein and the measurement of enrichment of protein-bound amino acid. Although we originally used ion-exchange methods (Rennie et al. 1982a,b) to purify the amino acid, a preparative gasliquid chromatographic method used to fractionate N-propyl amino acid esters has superseded this (W. W. Read, M. J. Rennie and D. Halliday, unpublished results). How confident can we be of the answers we obtain by these kinds of methods? We have examined the effects of biological variability and the precision and accuracy of the methods. In addition, since there are a number of assumptions