Glutathione metabolism of human skeletal muscle in surgical trauma
Author: Luo, Jia-Li
Date: 1997-04-04
Location: Hörsalen plan 4, Novum, Huddinge sjukhus
Time: 9.00
Department: Centrum för kirurgisk vetenskap CFSS / Center for Surgical Sciences CFSS
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
Issue date: 1997-03-14
Publication year: 1997
ISBN: 91-628-2395-7
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