Glutathione HPLC Assay kit:
For Research Use Only
Size: 100 Tests
Incubation Time: 40 minutes
Sample Type: EDTA Blood
Sample Size: 100 µL
Product Developed and Manufactured in Germany.
Product Support in the USA.
Reference Values (Normal)
GSH total: 783 – 1346 µmol/l
GSH reduced: 639 – 1146 µmol/l
GSH reduced/total: > 80 %
It is suggested that each laboratory establish its own normal ranges
The Eagle Biosciences Glutathione HPLC Assay Kit is intended for the quantitative determination of glutathione in EDTA blood. The Glutathione HPLC Assay Kit is for research use only and should not be used for diagnostic procedures.
For the determination of glutathione the sample is divided in two aliquots. One is reduced and the total amount of glutathione is measured. The other aliquot is treated without reduction solution, which determines only the reduced glutathione. During the derivatisation reaction glutathione is converted into a fluorescent probe. The following precipitation step removes high molecular substances. After centrifugation the fluorescent probe is cooled (2-8°C) and injected into the HPLC system. The isocratic separation via HPLC at 30°C uses a reversed phase column in two runs. One run lasts 4 minutes. The chromatograms are recorded by a fluorescence detector. The quantification is performed with the delivered EDTA-blood calibrator; the concentration is calculated by the internal standard method. The amount of oxidized glutathione is calculated by subtraction of:
Glutathione (total) – Glutathione (reduced)
Please take in mind that the difference must be divided by two because oxidized glutathione (GSSG) consists of two reduced GSH molecules.
1. Pipette into 1.5 ml reaction tubes: 100 µl sample, CAL or CTRL + 200 µl SOL
2. Vortex briefly. The sample is then divided in two portions:
Add 50 µl diluted sample
100 µl IS
20 µl RED
100 µl DERIVAT
Add 50 µl diluted sample
100 µl REAC
100 µl DERIVAT
3. Incubate for 20 minutes at 60 °C.
4. Add 100 µl PREC
5. Incubate for 10 minutes at 2 -8 °C and centrifuge for 10 min at 10.000 g.
6. Add 100 µl supernatant to 200 µl REAC in autosampler-vials
7. Inject 20 µl in the HPLC-system.
Glutathione (GSH) an intracellular tripeptide consisting of glycine, cysteine and glutamic acid. It is common in all tissues and protects the cells against oxidative stress. It is important for the activation or inhibition of enzymes and transport proteins. The transport of amino acids is also controlled by glutathione. Glutathione is very important for the stabilization of protein and non-protein sulfhydryl-groups to maintain a reducing intracellular environment.
Most of the intracellular glutathione is reduced (approx. 90 % in EDTA-blood), only 10 % is oxidized (GSSG). The NADPH-dependent glutathione reductase maintains this steady state. Alterations in the glutathione status are involved in the pathogenesis of several diseases. In discussion are reperfusion damage, liver injury, cancer, diabetes mellitus, cataract, inflammatory diseases, chronic lymphatic edema and radiation damages. Altered glutathione concentrations might also be due to pollution, cigarette smoke, side effects of drugs and aging. In case of oxidative stress the amount of reduced glutathione is diminished. The relation of reduced to oxidized glutathione gives information about the redox and detoxification status of cells and tissue.
The Eagle Biosciences Glutathione HPLC Assay Kit makes it possible to determine glutathione in an easy, fast and precise way. The Glutathione HPLC Assay Kit includes all reagents in ready to use form for preparation and separation of the samples with exception of the columns (IC1800rp) and the controls (IC1800ko). Both can be supplied by Eagle Biosciences. Beside the complete test kit, it is possible to order all components separately. Please request our single component price list.
Limitations of the Method
Serum or plasma should not be used because the content of glutathione is much low. It is not possible to distinguish between oxidized and reduced glutathione. Don´t use lipemic samples.