Amino acid and protein turnover in human skeletal muscle

Sammanfattning: Critically ill patients are characterised by a severe net protein catabolism. The rate of muscle protein loss is in the magnitude of 10% per week. A consequence of muscle wasting is increased weakness, which is associated with high rates of mortality and morbidity. Protein wasting is a result of either a decrease of protein synthesis or an increase of protein degradation or a combination of both. To understand the underlying mechanisms determinations of both protein synthesis and degradation on the tissue level are necessary, since the regulation of protein turnover is different between tissues. Protein synthesis on the tissue level can be measured quantitatively, however protein degradation is more difficult to measure quantitatively and the methods to study muscle protein breakdown in humans are not fully validated. One aim of this thesis was to descriptively study the amino acid and protein metabolism of skeletal muscle longitudinally in intensive care unit (ICU) patients. In a volunteer study endotoxin was used to obtain a human model of the initial phase of sepsis. To assess protein turnover in skeletal muscle, techniques to quantify synthesis as well as degradation in muscle tissue are required. Hence developing of new and accurate quantitative techniques to measure muscle protein breakdown are necessary. Another aim of the thesis was to develop and validate new and existing techniques to measure muscle protein breakdown rates which were validated in volunteers in the basal state and following endotoxin administration. The temporal pattern of amino acid net balances across the leg and plasma concentrations, in particular glutamine and glutamate, in long-staying ICU patients were investigated. Neither glutamine concentration nor net release from the leg changed significantly during the initial two weeks of ICU stay, despite a net release of phenylalanine indicating a progressive net loss of skeletal muscle proteins. In addition, the net uptake of glutamate across the leg muscle was not altered during this period. Studying of amino acid metabolism in the initial phase of illnesses in ICU patients is not possible. Therefore endotoxin administration was used as a human model of the early phase of sepsis to obtain information of the initial pattern of amino acid metabolism in skeletal muscle. Concentrations of all amino acids in plasma and skeletal muscle decreased after endotoxin administration and in addition the efflux of most amino acid from the leg increased. For studies of protein turnover at the tissue level quantitatively accurate methods to measure protein kinetics are necessary. 3-Methylhistidine (3-MH) can be used as a marker of contractile protein degradation. Arterio-venous differences of 3-MH concentrations across limbs have been used to assess skeletal muscle protein degradation, but the methods to measure 3-MH net balances by HPLC techniques are not precise enough to measuring of the small arteriovenous concentration differences. The use of tracer techniques, employing 2H3-3-MH and 2 2H5-phenylalanine, enabled calculation of the rates of appearance for 3-MH and phenylalanine respectively. In the basal state these values were shown to be different from zero during the isotopic steady state. The effects of endotoxin on muscle protein metabolism were studied by three different models using tracer techniques. Whole body phenylalanine rate of appearance increased indicating an increase in whole body protein degradation. From the leg there was an increased efflux of phenylalanine indicating a net protein loss, but no alteration in 3-MH net balance or rate of appearance. A 3-compartment model for phenylalanine turnover was used, which showed a decreased muscle protein synthesis but an unchanged degradation. So endotoxin administration cause altered muscle protein synthesis but does not influence protein degradation, although whole body protein degradation increases. In summary alterations in amino acid metabolism in skeletal muscle are established early during the illnesses. During the initial two weeks of ICU stay no temporal changes in amino acid concentrations and net balances were seen, despite continuous protein loss from skeletal muscle. An endotoxin challenge decreased plasma and muscle amino acid concentrations and in addition the efflux of amino acids from the leg increased. Measurement of 3-MH rate of appearance from leg muscle using isotopically labelled 3-MH resulted in consistent and accurate values of contractile protein degradation rates. Endotoxin administration decreased human skeletal muscle synthesis rate while protein breakdown was not affected.