Determination of intracellular glutathione in human skeletal muscle by reversed-phase high-performance liquid chromatography |
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| Affiliation: | 1. Anesthesiological Metabolism Unit, Clinical Research Centre, Department of Anaesthesiology and Intensive Care, Huddinge University Hospital, Karolinska Institute, S-141 86 Huddinge, Stockholm, Sweden;2. Department of Surgery, St. Göran''s Hospital, Stockholm, Sweden;3. Division of Toxicology, Institute of Environmental Medicine, Karolinska Institute, Stockholm, Sweden;4. Department of Medical Laboratory Technology, Stockholm University, College of Health Sciences, Stockholm, Sweden;1. Department of Biophysics, School of Pharmacy with the Division of Laboratory Medicine in Sosnowiec, Medical University of Silesia, Katowice, Poland, Jedności 8, 41-200 Sosnowiec, Poland;2. Department of Microbiology, University of Bialystok, Ciolkowskiego 1J, 15-245 Bialystok, Poland;3. Laboratory of Applied Microbiology, University of Bialystok, Ciolkowskiego 1J, 15-245 Bialystok, Poland;1. Center of Studies in Health and Environment, School of Science and Technology, National University of San Martín, San Martín, Buenos Aires, Argentina;2. Department of Pharmaceutical Science, Hampton University, VA, USA;3. Department of Histology and Embryology, School of Dentistry, University of Buenos Aires, Buenos Aires, Argentina;1. CNRS/UPPA, Institut of Analytical and Physical Chemistry for the Environment and Materials (IPREM), UMR5254, Hélioparc, F-64053, Pau, France;2. Institut de Génomique Fonctionnelle de Lyon, IGFL, CNRS/ENS UMR5242, 69007 Lyon, France;3. Centre International de Recherche en Infectiologie, CIRI, 69007 Lyon, France;4. INSERM U1111, 69007 Lyon, France;5. CNRS/ENS/UCBL1 UMR5308, 69007 Lyon, France;1. Institute of Physiology, Czech Academy of Sciences, Prague, Czech Republic;2. Department of Analytical Chemistry, Institute of Chemical Technology, Prague, Czech Republic;3. Laboratory of Molecular Structure Characterization, Institute of Microbiology, v.v.i., Czech Academy of Sciences, Prague, Czech Republic |
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| Abstract: | A chromatographic method for the specific determination of cellular low molecular mass thiols has been applied to human muscle tissue. The method is based on the derivatisation of thiols using monobromobimane, which is a specific reagent for the sulphydryl group. The glutathione and cysteine bimane adducts were separated by reversed-phase HPLC, whilst quantitation of the cysteine and glutathione adducts was achieved by fluorescence spectroscopy. The method was found to yield a quantitative recovery of glutathione (ca. 96%), to be sensitive (down to 20 pmol glutathione/per injection) and reveal a low intra-individual coefficient of variation (C.V. < 5%) of the glutathione concentrations in human skeletal muscle. The concentrations of reduced and total glutathione were 1320 ± 37 μmol/kg wet weight (mean ± S.E.M.) and 1525 ± 66 μmol/kg wet weight, respectively. The method was also applied to tissues from nine healthy volunteers to determine if fluctuations in glutathione level occurred over a 24-h period. No diurnal variation of glutathione level in human skeletal muscle was observed. |
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