General description
A spectrophotometric assay kit that is specific for reduced glutathione (GSH) or total mercaptans. Absorbance is read at ~400 nm for GSH and ~356 nm for total mercaptens.
Note: 1 T = 1 test.
Oxidative stress occurs in most, if not all, human diseases. Our understanding of the role played by reactive oxygen species in the progression of pathological changes is still evolving. Convenient, accurate and reproducible methodologies to measure oxidative stress parameters have been lacking. Glutathione (γ-glutamylcysteinylglycine or GSH) is a naturally occuring tripeptide whose nucleophilic and reducing properties play a central role in metabolic pathways, as well as in the antioxidant system of most aerobic cells. GSH plays a critical role as a coenzyme with a variety of enzymes including, glutathione peroxidase, glutathione S-transferase, and thiol transferase. GSH also plays major roles in drug metabolism, calcium metabolism, the γ-glutamyl cycle, blood platelet, and membrane functions. In addition, GSH is crucial to a variety of life processes, including the detoxification of xenobiotics, maintenance of the -SH level of proteins, thiol-disulfide exchange, removal of hydroperoxides and free radicals, and amino acid transport across membranes. Physiological values of intracellular GSH generally range from 1 to 10 mM. Although many methods have been described for the assay of GSH, the reliable ones are labor intensive and not easy to use.
Biochem/physiol Actions
The Calbiochem® Glutathione Assay Kit makes it possible to assay for glutathione with only one sampling and one colorimetric measurement. A modification of this method makes it possible to assay other mercaptans. This alternate protocol is based on the measurement of substitution products that absorb maximally at 356 nm in the absence of reagent R2. Due to its ease of use, this method is well adapted for the quantification of glutathione and total mercaptans in a large number of biological samples. The main advantage of the method is that it does not require any enzyme as a reagent.
Features and Benefits
Assay Time: 1.5 h
Preparation Note
Erythrocyte Lysate
1. Centrifuge a minimum of 500 l of whole blood at 2500 X g at 4°C for 5 min.
2. Discard plasma supernatant. If not assayed immediately, store erythrocyte pellet at -70°C (erythrocyte pellet can be stored at -70°C for 15 days).
3. Resuspend erythrocyte pellet in 4 volumes of MPA working solution, 0-4°C.
4. Thoroughly mix and centrifuge at 3000g at 4°C for 10 min.
5. Collect the upper clear aqueous layer and keep at 0-4°C for the assay (within 1 h).
Liver Homogenates
1. Wash tissue in 0.9% NaCl solution.
2. Blot tissue on paper and weigh.
3. Mince tissue in ice-cold MPA working solution.
4. Homogenize minced tissue.
5. Centrifuge homogenate at 3000 x g, 4°C for 10 min.
6. Collect the upper clear aqueous layer* and keep at 0-4°C for the assay (within 1 h).
*Cloudy supernatant should be filtered through 0.2 m filters.
Hepatocyte Lysates
1. Resuspend hepatocyte* pellet, from rats or mice, in 500 l of ice-cold MPA working solution.
2. Homogenize cell suspension.
3. Centrifuge homogenate at 3000 x g, 4°C for 10 min.
4. Collect the upper clear aqeous layer and keep at 0-4°C for the assay (within 1 h).
*Approximately 2.5-3.5 x 10⁶ cells are used (5-8 mg of total protein).
Upon arrival store the entire kit contents at 4°C.
• All solutions of reagents and buffer have been tightly sealed and are stable if properly stored between 4°C. Do Not Freeze. Protect from light.
• After removing the required amount of each reagent for immediate use, all bottles should be tightly closed and stored between 2-4°C. Do not leave the reagent bottles open or at room temperature.
Analysis Note
Measurement of Total Mercaptans (RSH): Assay at 356 nm
The Calbiochem Glutathione Assay Kit can be used for the measurement of other mercaptans (RSH), which include GSH. The assay is carried out in the absence of reagent R2 at 356 nm. The absorbance at 356 nm is a linear function of [RSH] concentration in the sample, but it is not GSH-specific. If the sample essentially contains GSH and a single other mercaptan i.e., N-acetylcysteine, these two mercaptans can be assayed by using the same single sample for two measurements. The first measurement is made at 356 nm before the addition of reagent R2 as described below, and the second measurement is made, after the addition of R2, at 400 nm.
Adjust the spectrophotometer absorbance to zero at 356 nm with buffer only (solution 3). The reaction mixture is prepared as in Section 3, with the omission of step 4. The absorbance (A) is measured at 356 nm.
A standard curve must be prepared with the corresponding mercaptan in Table 2, to calculate the concentration. Figure 2 gives two examples of standard curves obtained with glutathione and Nacetylcysteine at 356 nm.
GSH Concentration
The calculation is based on the following equation:
[GSH] = {(A-A₀)/(E x l)} x D
where:
[GSH] is the initial glutathione concentration in the sample, expressed as molar concentration.
A and A₀ are the absorbances measured in the presence and in the absence of sample, respectively.
E is the apparent molar extinction coefficient of the product measured at 400 nm.
l is the optical path (cm).
D is the dilution factor of the sample.
Total Mercaptan Concentration
The calculation is based on the modified version of the equation used for GSH:
[total RSH] = { (A - A₀) / (E x I) } x D
where :
[total RSH] is the concentration of total mercaptan in the sample.
A and A₀ are the absorbances measured in the presence and in the absence of sample, respectively.
E is the apparent molar extinction coefficient of the product measured at 356 nm.
I is the optical path (cm).
D is the dilution factor of the sample.
Note:
Do not add reagents R1 and R2 in reverse order.
The temperature, (25 ± 3°C), should be kept constant throughout the experiment.
The final reaction volume (1 ml) should not vary from one measurement to another.
The absorbance at 400 nm is proportional to glutathione concentration. It is stable for min, provided that the reaction mixture is kept in the dark.
The sensitivity of the assay for mercaptans at 356 nm is of the same order of magnitude as that of glutathione at 400 nm as shown in Table 2.
Sensitivity: From 30 repeated measurements performed on the control ([RSH] = 0) on the same day, under the same experimental conditions, the detection limit of the assay was 5 µmol/l in the final reaction mixture (spectrophotometric cuvette). For example, using the maximum volume of 300 µl of a sample would therefore give a detection limit for glutathione of about 17 µmol/l.
Other Notes
A Chromogen, 30% NaOH, Buffer, and a user protocol.
Due to the nature of the Hazardous Materials in this shipment, additional shipping charges may be applied to your order. Certain sizes may be exempt from the additional hazardous materials shipping charges. Please contact your local sales office for more information regarding these charges.
Ji, L.L., and Fu, R. 1992. J. Appl. Physiol.72, 549.
Anderson M.E. 1989. Enzymatic and chemical methods for the determination of
Glutathione; In: Glutathione: chemical, biochemical and medical aspects, Vol. A, Dolphin D., Poulson R. and Avramovic O. Eds., John Wiley and Sons, pp. 339-365.
Dolphin D., et al. 1989. Glutathione: Chemical, Biochemical and Medical Aspects, Vols A & B, J. Wiley and Sons.
Legal Information
CALBIOCHEM is a registered trademark of Merck KGaA, Darmstadt, Germany
Disclaimer
Toxicity: Multiple Toxicity Values, refer to MSDS (O)
