66000 HPLC kit glutathione whole blood

Glutathione in Whole Blood - HPLC

Order no.: 66000
100 Tests
  • Glutathione
  • Glutathionedisulfide (GSSG)

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

Description

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.

Chromatogram

Technical Data

Method of Analysis

HPLC

Number of Tests

100

Parameter

Glutathione , Glutathionedisulfide (GSSG)

Analysis Time

< 5 min

Limit of quantification

30 µmol/l

Linearity

up to 15000 µmol/l

Recovery

99 %

Intraassay

CV < 6 %

Interassay

CV 2.6-10.8 %

Gradient

Isocratic

Information

For the Chromsystems HPLC analysis of glutathione in whole blood any isocratic HPLC system with fluorescence detector is suitable.

Column temperature

ambient (~25 °C)

Flow rate

1.3 ml/min

Specimen

Whole Blood

Sample Preparation

A) Precipitation

  • Place 150 μl Internal Standard into an amber coloured reaction vial.
  • Add 10 μl whole blood and mix by aspirating the pipette tip.
  •  Mix briefly (vortex).
  • Add 400 μl Precipitation Reagent and mix 30 s (vortex).
  • Centrifuge 7 min at 9000 x g.


B) Determination of reduced glutathione

  • Place 50 μl of the supernatant from A) into a new, amber coloured reaction vial.
  • Add 100 μl Derivatisation Mix and mix well.
  • Incubate 10 min at +50 to +55 °C in a water bath.
  • Inject 20 μl into the HPLC system.

C) Determination of the sum of oxidised and reduced glutathione

  • Place 50 μl of the supernatant from A) in a new, amber coloured 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.
  • Inject 20 μl into the HPLC system.
  • The concentration of oxidised glutathione is calculated by subtracting the concentration of reduced glutathione from the sum of both (oxidised + reduced).

Sample Stability

The stability of patient samples depends on the conditions during storage as well as on the use of different anticoagulants. More information can be gathered from specific literature.

Injection Volume

20 µl

Flow

1.3 ml/min

Additional Info

For the Chromsystems HPLC analysis of glutathione in whole blood any isocratic HPLC system with fluorescence detector is suitable.

Wavelength

EX 385 nm, EM 515 nm

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.

Kit Components

Components available separately

Calibrators

Calibrators available separately

Controls

Controls available separately

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