1. The 'flooding dose' technique for measuring the rate of protein synthesis in tissues in vivo involves the injection of a large amount of unlabelled amino acid together with the tracer to minimize differences in isotopic enrichment of the free amino acid in plasma and tissue compartments. This approach has been investigated in human muscle by taking biopsies from postabsorptive male volunteers given [1-13C]leucine. 2. Intravenous injection of 4 g of unlabelled leucine resulted in a rapid rise in free leucine concentration of seven- to eleven-fold in plasma and five-fold in muscle. Values were still elevated by two-fold after 2 h. 3. Five minutes after injection of [1-13C]leucine (0.05 g/kg) the isotopic enrichment of plasma leucine was 82% that of the injected material, falling to 44% at 120 min. The enrichment of free leucine in sequential muscle biopsies was close to that in plasma and almost identical to that for plasma alpha-ketoisocaproate. 4. The rate of protein synthesis was determined from the increase in leucine enrichment in protein of muscle biopsies taken before and 90 min after injection of [1-13C]leucine (0.05 g/kg; 19 or 39 atom% excess) and the average plasma alpha-ketoisocaproate enrichment over this period (taken to represent muscle free leucine). The mean rate of muscle protein synthesis in 10 subjects was 1.95 (SEM 0.12) %/day. Rates of protein synthesis calculated from plasma leucine as precursor enrichment were only 5% lower than those calculated from plasma alpha-ketoisocaproate.(ABSTRACT TRUNCATED AT 250 WORDS)
The individual tissues responded differently to trauma, and showed a wide range of values. The responses were not significantly correlated with each other and no pattern of tissue protein synthesis characteristic of critical illness was observed. However, both muscle protein and albumin synthesis rates correlated with metabolic status and clinical indices of the severity of illness.
The extension of the flooding method for measuring the rate of protein synthesis, from animal to human tissues, has led to criticism. This is based on the observation that in human muscle, unlike animal tissues, the rate of synthesis in the fasting state measured with constant infusion is lower than that obtained with the flooding technique. Moreover, incorporation of infused tracer can be enhanced with a simultaneous flood, although an inhibition of incorporation has also been reported. Explanations for these observed discrepancies are explored. Evidence from studies in human muscle both with flooding and with a nonisotopic technique have given no indication of a stimulation of protein synthesis during flooding. It is therefore concluded that the most likely explanation for the discrepancy between methods is that changes in the isotopic enrichment of the precursor amino acid, which are minimized by the flooding procedure, are not adequately accounted for with the constant infusion method.
The role of insulin in the regulation of muscle protein synthesis in adult humans has been investigated with intravenous infusion of insulin at levels comparable with those observed after normal feeding. Glucose was also infused to maintain euglycemia. Muscle protein synthesis was measured in six healthy subjects before and during insulin and glucose infusion from the incorporation of L-[2H5]phenylalanine into the protein of vastus lateralis sampled by percutaneous biopsy. L-[2H5]phenylalanine was given as a single injection of a flooding amount (45 mg/kg). The relatively low levels of enrichment of phenylalanine in protein (0.005 atom%) were measured by modified gas chromatography-mass spectrometry and verified by comparison with incorporation of L-[2,6-3H]phenylalanine. Similarity of enrichment in tissue-free and plasma pools (flooding) and linear incorporation over the period of measurement were also verified. The fractional rate of muscle protein synthesis in the group of postabsorptive subjects was 1.65 +/- 0.11% (SE)/day. The rate was unaltered by insulin and glucose infusion, 1.66 +/- 0.16%/day.
1. The rate of protein synthesis in quadriceps muscle of healthy subjects estimated from the incorporation of l-[1-13C]leucine given by continuous infusion was 1.1%/day. The estimate of protein synthesis from the incorporation of a flooding amount of labelled leucine was 1.8%/day (sd 0.65). The possibility that the higher rate obtained with the flooding technique arose from stimulation of protein synthesis by the large amount of leucine is unlikely.
2. The same rate of protein synthesis (1.7%/day, sd 0.3) was obtained with a flooding amount (0.05 g/kg) of a different amino acid, l-[1-13C]phenylalanine, as was obtained with leucine.
3. Incorporation of l-[1-13C]phenylalanine was not affected by simultaneous injection of leucine (1.7%/day, sd 0.7) or valine (1.6%/day, sd 0.4).
4. Protein synthesis, assessed in a completely different way from the proportion of polyribosomes isolated from the skeletal muscle, was unaltered by the injection of 0.05 g of l-leucine/kg (44.6%, sd 8.5 versus 43.8%, sd 7.7).
5. Good agreement in estimates of protein synthesis was observed in subjects in whom both legs were measured with both l-[1-13C]leucine (mean difference 0.16%/day) and l-[1-13C]phenylalanine (mean difference 0.2%/day).
Treatment with GH for 5 days in patients with multiple organ failure stimulated muscle protein synthesis, increased muscle free glutamine, and increased intracellular muscle water.
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