Glutathione metabolism of human skeletal muscle in surgical trauma

Abstract: Glutathione Metabolism of Human Skeletal Muscle in Surgical Trauma Jia-Li Luo Dissertation from the Department of Anesthesiology and Intensive Care, Huddinge University Hospital, KARO Institution, Karolinska Institutet. Stockholm, Sweden In man, muscle tissue serves as a protein reservoir which is mobilized to meet the specific metabolic needs associated with various catabolic conditions, such as surgical trauma and critical illness. Glutathione (GSH) is one of the most abundant short-chain peptides and a major source of nonprotein thiol in the body, and tissue GSH concentration is related to its oxidative capacity. Skeletal muscle is relative specific in a variety of metabolic properties, such as oxidative potential, patterns of amino acid utilization, and antioxidant enzyme activity. Therefore, the levels of muscle GSH and glutathione disulfide, as well as other thiol containing amino acids, are expected to reflect these metabolic characteristics in surgical trauma. A HPLC technique based on the use of monobromobimane was adapted and developed in order to measure the concentrations of glutathione, cysteine and their respective disulfides. Quantitation of the bimane adducts of glutathione and cysteine is achieved by reversed-phase HPLC. Additionally, the method is coupled with the prederivatization procedures of dithiothreitol (DTT) and N-ethylmaleimide (NEM) for measuring the thiol disulfides. Full validation results for quantitative measurements are demonstrated in acid extracts of muscle tissue with high analytic recoveries of 97% for GSH and >92% for GSSG, as well as low variations of <3% in within-run and <10% in between-run. The tissue free GSH in various tissue homogenates of healthy human subjects were determined. The highest concentration was found in duodenal mucosa (4.47±0.41 mmol/kg wet weight), followed by liver (3.69±0.78 mmolrkg ww), gastric mucosa (2.49±0.33 mmol/kg ww), and skeletal muscle (1.34±0.16 mmol/kg ww). The GSH concentration in muscle tissue was not influenced by food intake, neither by food deprivation. Moreover, there was no diumal variation on muscle GSH levels. The effect of elective abdominal surgery on the concentration of GSH in skeletal muscle were evaluated before operation and 6, 24 and 48 hours after operation in one group of patient and at 24 and 72 hours after surgery in another group of patients. In skeletal muscle, the concentration of GSH decreased by 40% 24 hours postoperatively and remained low 48 and even 72 hours postoperatively. However, a sign of recovery of the GSH concentration was seen at 72 hours as compared to their 24 hours' values. There was a concomitant decrease in the concentration of total GSH in skeletal muscle, whereas the concentration of GSSG remained unaltered after surgery. During critical illness, the concentration of GSH and total GSH in skeletal muscle were 43 and 38% lower than the matched controls. Moreover, the redox status of glutathione was changed in critically ill patients, indicating an increasing exposure for oxidative stress. A statistical correlation was found between muscle free glutamine and muscle total GSH. The metabolism of muscle GSH was studied by measuring the GSH synthetic enzyme activity after surgical trauma. The activities of y-glutamylcysteine synthetase did not change significantly after surgery. In contrast, GSH synthetase activity decreased postoperatively. In addition, decreases in the GSH peroxidase activity were seen at both 24 and 72 hours postoperatively. The decrease of muscle GSH is mainly due to a diminished GSH synthetic capacity as reflected by the decrease on the GSH synthetase activity. A statistical correlation between GSH concentration and GSH synthetase activity existed postoperatively. In summary, methodology for a selective measurement of GSH and GSSG in various human tissues biopsy specimens is developed, which enables extensive studies of glutathione metabolism. Key words- glutathione, cysteine, skeletal muscle, surgical trauma, critical illness, human. ISBN 91-628-2395-7 Stockholm, 1997