Reversed-Phase Chromatographic Method for Specific Determination of Glutathione in Cultured Malignant Cells

Details

• Personal Author:
  Ahmad F ; Ahmad H ; Awasthi S ; Sharma R
• Description:
  A chromatographic method for the specific determination of glutathione in malignant cell lines is described. The method is based on the ability of glutathione-S-transferase to specifically and quantitatively conjugate glutathione to 1-chloro-2,4-dinitrobenzene and chromatographic quantitation of the resultant conjugate, dinitrophenyl-S-glutathione, by reversed-phase liquid chromatography. The assay can be performed on 20 000 g supernatants of cell homogenates without acid extraction. 2-Mercaptoethanol, a sulfhydryl compound often used as a thiol-protective agent to preserve enzymatic activities of a number of enzymes, did not interfere with glutathione determination by this method. The dinitrophenyl-S-glutathione isolated from either standard glutathione samples or from cell homogenates was shown to be identical to authentic dinitrophenyl-S-glutathione using mass spectrometry. Recovery of glutathione in standard samples by the current method was identical to that determined using 5,5'-dithiobis(2-nitrobenzoic acid). Exogenous glutathione added to supernatants of cell homogenate in the presence or absence of 2-mercaptoethanol was also completely recovered. [Description provided by NIOSH]
• Subjects:
  Chromatography Enzymes Neoplasms Occupational Health Safety
• Keywords:
  Cell Function Chromatographic Analysis Enzyme Activity Malignancy
• ISSN:
  0021-9673
• Document Type:
  Text
• Funding:
  Grant
• Genre:
  Journal Article
• Place as Subject:
  OSHA Region 6 ; Texas
• CIO:
  National Institute for Occupational Safety and Health (NIOSH)
• Topic:
  Workplace Safety & Health
• Location:
  North America
• Pages in Document:
  167-173
• Volume:
  584
• Issue:
  2
• NIOSHTIC Number:
  nn:20056728
• Citation:
  J Chromatogr 1992 Dec; 584(2):167-173
• Contact Point Address:
  H. Ahmad, Department of Chemistry, PAN-American, 1201 W. University Drive, Edinburg, TX 78539, USA
• CAS Registry Number:
  Glutathione (CAS RN 70-18-8)
• Federal Fiscal Year:
  1993
• Performing Organization:
  University of Texas Medical Branch, Galveston, Texas
• Peer Reviewed:
  True
• Start Date:
  19860101
• Source Full Name:
  Journal of Chromatography
• End Date:
  19960630
• Collection(s):
  National Institute for Occupational Safety and Health
• Main Document Checksum:
  urn:sha-512:e796a0026988fba0056c678c92476e4a1994c0243283377627e1e280e762bfbe512bde1950fee18485ab104e34959041d14b56f23cea8b9034456dd314121a4c
• Download URL:
  https://stacks.cdc.gov/view/cdc/214279/cdc_214279_DS1.pdf
• File Type:
  [PDF - 489.24 KB ]