Glutathione in Whole Blood - HPLC

Order No.: 66000, for 100 tests
Parameters:
Free Glutathione (GSH), Glutathionedisulfide (GSSG)

Detection of reduced and oxidized form
10 µl sample volume
High reliability by inclusion of an internal standard

Free Glutathione (GSH)

Glutathionedisulfide (GSSG)

Clinical relevance

Glutathione is a tripeptide, which is present in nearly all cells at high concentrations and is one of the most important antioxidant substances in the body. Glutathione exists in a reduced (GSH) and an oxidized form (GSSG) and the ratio of both is a measure for the redox status of the cell. Glutathione is especially important for the erythrocytes. Here it is involved in reducing methaemoglobin to haemoglobin, which can then bind oxygen molecules once again. Glutathione is also required for many reductive reactions in the body cells, thereby regulating the concentration of free radicals. Glutathione forms conjugates in the liver that eliminate toxic contaminants. There it also inactivates reactive intermediate products resulting from the metabolisation of certain drugs. Therefore, a high concentration of GSSG can indicate a liver necrosis or dystrophy.

 

Product advantages

  • Detection of reduced and oxidised form
  • Sample volume of only 10 µl is sufficient
  • High reliability by using an internal standard

 

GSH as well as GSSG can be reliably quantified with this kit by using only 10 µl of whole blood. The sample preparation is based on protein precipitation and derivatisation followed by HPLC determination with fluorescence detection. After precipitation, the sample is split into two halves. One aliquot is derivatised immediately for the determination of reduced glutathione; the second aliquot is chemically reduced before derivatisation which leads to the detection of the sum of both, oxidised and reduced glutathione. The use of an optimised internal standard minimises any analytical variations that could be crucial when calculating the GSSG/GSH ratio.

More Information
Number of Tests 100
Limit of quantification 5 µmol/l
Linearity up to 15000 µmol/l
Recovery 99 %
Intraassay

CV ≤ 4.6 %

Interassay CV ≤ 4 %
Analysis Time 5 min
Sample Preparation

1) Preparation of whole blood samples:

  • Place 150 μl Internal Standard into a light-protected reaction vial.
  • Add 10 μl whole blood and mix by rinsing the tip.
  •  Mix briefly (vortex).
  • Add 400 μl Precipitation Reagent and mix 30 s (vortex).
  • Centrifuge 7 min at 9000 x g.

2) Determination of free glutathione:

  • Place 50 μl of the supernatant from step 1) into a new, light-protected reaction vial.
  • Add 100 μl Derivatisation Mix and mix well.
  • Incubate 10 min at +50 to +55 °C in a water bath, cool down immediately.
  • Inject 20 μl into the HPLC system.

3) Determination of the sum of oxidised and free glutathione:

  • Place 50 μl of the supernatant from 1) in a new, light-protected reaction vial.
  • Add 10 μl Reduction Mix and mix well (vortex).
  • Incubate 5 min at room temperature.
  • Add 100 μl Derivatisation Mix and mix well.
  • Incubate 10 min at +50 to +55 °C in a water bath, cool down immediately.
  • Inject 20 μl into the HPLC system.
Sample Stability The prepared samples are stable for 2 days at room temperature, at +2 to +8 °C for 5 days. The non-derivatised or reduced supernatant is stable up to 3 days at +2 to +8 °C.
Specimen Whole Blood
Pre-analytic Treatment Information on the collection and storage of patient samples can be gathered from the specific literature.
Injection Volume 20 µl
Flow rate 1.3 ml/min
Column temperature ambient (~ 25 °C)
Wavelength EX 385 nm, EM 515 nm
Gradient Isocratic
Please note The freely available information on this website, in particular on the sample preparation, are not sufficient to work with our products. Please read instructions and warning notices on products and/or instruction manuals.
Method of Analysis HPLC
Parameter Free Glutathione (GSH), Glutathionedisulfide (GSSG)
Additional Info Any isocratic HPLC system with fluorescence detector is suitable.

  1. 66001 HPLC mobile phase glutathione whole blood
    Mobile Phase
    Order no.: 66001
    Glutathione in Whole Blood - HPLC
    Details
  2. 66001 HPLC mobile phase glutathione whole blood
    Mobile Phase, 10 x 1000 ml
    Order no.: 66002
    Glutathione in Whole Blood - HPLC
    Details
  3. 66004 HPLC internal standard glutathione whole blood
    Internal Standard
    Order no.: 66004
    Glutathione in Whole Blood - HPLC
    Details
  4. 66005 HPLC precipitation reagent glutathione whole blood
    Precipitation Reagent
    Order no.: 66005
    Glutathione in Whole Blood - HPLC
    Details
  5. 66006 HPLC derivatisation reagent 1 glutathione whole blood
    Derivatisation Reagent 1 (lyoph.)
    Order no.: 66006
    Glutathione in Whole Blood - HPLC
    Details
  6. 66007 HPLC derivatisation reagent 2 glutathione whole blood
    Derivatisation Reagent 2
    Order no.: 66007
    Glutathione in Whole Blood - HPLC
    Details
  7. 66008 HPLC reduction reagent glutathione whole blood
    Reduction Reagent
    Order no.: 66008
    Glutathione in Whole Blood - HPLC
    Details
  8. Reaction Vials, 1.5 ml
    Reaction Vials, 1.5 ml
    Order no.: 33005
    Amber colour, light protected
    Details
  9. 66003 HPLC glutathione whole blood calibration standard
    Glutathione Whole Blood Calibration Standard
    Order no.: 66003
    Glutathione in Whole Blood - HPLC
    Details
  1. 66003 HPLC glutathione whole blood calibration standard
    Glutathione Whole Blood Calibration Standard
    Order no.: 66003
    Glutathione in Whole Blood - HPLC
    Details
  1. 0077 0078 0079 HPLC glutathione whole blood controls
    Glutathione Whole Blood Controls
    Order no.: 0077/0078/0079
    Details
  1. PEEK Prefilter Housing
    PEEK Prefilter Housing
    Order no.: 15010
    Details
  2. 66100 HPLC column glutathione whole blood
    HPLC column
    Order no.: 66100
    Glutathione in Whole Blood - HPLC
    Details
  3. PEEK-encased Prefilter, 5 µm
    Details
  4. Precolumn cartridge 4/10
    Precolumn cartridge 4/10
    Order no.: 18066
    Glutathione in Whole Blood - HPLC
    Details
  5. Precolumn Cartridge Holder 4/10
    Details
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