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371757 GSH/GSSG Ratio Assay Kit

GSH/GSSG Ratio Assay Kit MSDS (material safety data sheet) or SDS, CoA and CoQ, dossiers, brochures and other available documents.
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      Overview

      Replacement Information

      Key Spec Table

      Detection Methods
      Colorimetric
      Description
      Overview

      This product has been discontinued.



      A spectrophotometric assay kit used for the measurement of reduced to oxidized glutathione ratio (GSH/GSSG) in biological samples.
      Catalogue Number371757
      Brand Family Calbiochem®
      Features and benefits• Highly Sensitive and Specific: Lower limit of detection in sample mixture is 9 nM GSSG. Over 13 different compounds were tested to assess any interference and exhibited no interference.
      • Easy to Use: A simplified colorimetric assay. the GSH/GSSG assay uses the thiol-scavenging reagent that rapidly scavenges GSH, but does not interfere with glutathione reductase assay. Each kit can be used for up to 100 tests for determining total glutathione/GSSG ratios or 200 tests for either of the two.
      Assay time: About 1.5 h
      • Versatile: Can be used with whole blood, cell lysates, and tissue extracts.
      Application Data
      A four point calibration curve A412/min VS. µ GSH used to determine the concentration of GSSG.

      Stability of GSSG, from a rested subject, in the intact red blood cell at 4°C. (GSH-159).

      Stability of GSSG in the red blood cell lysate at 4°C. (GSH-161)
      Materials Required but Not Delivered• Spectrophotometer
      • Centrifuge 1000 x g or greater
      • Spectrophotometric cuvettes with a 1 cm optical path length (glass or polystyrene).
      • Disposable tubes and stoppers (glass or polypropylene).
      • Pipettes, preferably adjustable capable of accurately pipetting 10, 50, 100, 200, 700 and 3000 µl.
      • Balance
      • Metaphosphoric acid
      References
      ReferencesAnderson, M. 1996. Glutathione in Free Radicals, A Practical Approach, ed. N.A. Punchard and F.J. Kelly Oxford University Press, New York, p 213.
      Richie, J.P. Jr., et al. 1996. Clinical Chemistry 42, 64.
      Griffith, O.W. 1980. Analytical Biochemistry 106, 207.
      Tietze, F. 1969. Analytical Chemistry 27, 502.
      Guntherberg, H., and Rost, J. 1966. Analytical Biochemistry 15, 205.
      Product Information
      Detection methodColorimetric
      Form200 Tests
      FormatCuvette
      Kit containsChromogen DTNB, Glutathione Reductase Enzyme, NADPH, Scavenger, Assay Buffer, GSSG Buffer, Standard, and a user protocol. Each kit can be used to perform 100 tests representing the ratio of total Glutathione to the oxidized form or 200 tests of either.
      Quality LevelMQ100
      Applications
      Biological Information
      Assay time1.5 h
      Sample Typecell lysates, tissue extracts, blood
      Physicochemical Information
      Sensitivity9 nM GSSG
      Dimensions
      Materials Information
      Toxicological Information
      Safety Information according to GHS
      Safety Information
      Product Usage Statements
      Storage and Shipping Information
      Ship Code Blue Ice Only
      Toxicity Standard Handling
      Storage +2°C to +8°C
      Storage ConditionsWhen not in use, place the bottles at 4°C. Do not allow the capped reagent bottles to sit at room temperature for long periods of time.
      Protect from Light Protect from light
      Do not freeze Ok to freeze
      Packaging Information
      Transport Information
      Supplemental Information
      Kit containsChromogen DTNB, Glutathione Reductase Enzyme, NADPH, Scavenger, Assay Buffer, GSSG Buffer, Standard, and a user protocol. Each kit can be used to perform 100 tests representing the ratio of total Glutathione to the oxidized form or 200 tests of either.
      Specifications
      Global Trade Item Number
      Catalogue Number GTIN
      371757 0

      Documentation

      GSH/GSSG Ratio Assay Kit Certificates of Analysis

      TitleLot Number
      371757

      References

      Reference overview
      Anderson, M. 1996. Glutathione in Free Radicals, A Practical Approach, ed. N.A. Punchard and F.J. Kelly Oxford University Press, New York, p 213.
      Richie, J.P. Jr., et al. 1996. Clinical Chemistry 42, 64.
      Griffith, O.W. 1980. Analytical Biochemistry 106, 207.
      Tietze, F. 1969. Analytical Chemistry 27, 502.
      Guntherberg, H., and Rost, J. 1966. Analytical Biochemistry 15, 205.
      User Protocol

      Revision30-May-2017 JSW
      Form200 Tests
      FormatCuvette
      Detection methodColorimetric
      StorageWhen not in use, place the bottles at 4°C. Do not allow the capped reagent bottles to sit at room temperature for long periods of time.
      BackgroundReduced glutathione (GSH), a tripeptide (γ-glutamylcysteinylglycine) with a free thiol group, is a major antioxidant in human tissues that provides reducing equivalents for the glutathione peroxidase (GPx) catalyzed reduction of hydrogen peroxide and lipid hydroperoxides to water and the respective alcohol. During this process GSH becomes oxidized glutathione (GSSG). The GSSG is then recycled into GSH by gutathione reductase (GR) and β-nicotinamide adenine dinucleotide phosphate (NADPH). When mammalian cells are exposed to increased oxidative stress, the ratio of GSH/GSSG will decrease as a consequence of GSSG accumulation. Measurement of the GSSG level or determination of the GSH/GSSG ratio, is a useful indicator of oxidative stress and can be used to monitor the effectiveness of antioxidant intervention strategies.

      Figure 1: Formulation
      Principles of the assayThe accurate measurement of GSSG levels has proved very difficult due to the low amount of GSSG in tissues and because of the absence of effective methods to prevent oxidation of GSH to GSSG during sample preparation. To measure GSSG in tissues, Guntherberg and Rost first introduced N ethylmaleimide (NEM) to eliminate the GSH. Although NEM can react with GSH to form a stable complex and prevent the participation of the reduced form in the enzymatic assay, NEM also inhibits GR. For this reason, Griffith (3) first introduced 2 vinylpyridine (2-VP), which does not inhibit GR significantly, to derivatize GSH. However, the 2-VP reaction is relatively slow and the reagent has little solubility in an aqueous medium.

      The GSH/GSSG assay uses the thiol-scavenging reagent, 1-methyl-2-vinylpyridinium trifluoromethane-sulfonate¹ (M2VP) at a level that rapidly scavenges GSH but does not interfere with the GR assay.

      Figure 2: Principal of the Assay

      Reaction of M2VP with a thiol to form 1-Methyl-2-(2-thioethyl)-pyridinium salt. 2-VP at 10 mM, usually takes 60 min to remove 70% of the GSH in the sample during which time oxidation of GSH may occur, resulting in significant overestimation of the GSSG concentration. Using M2VP, complete scavenging of GSH is accomplished in less than one min.


      In 1969, Tietze first introduced an enzymatic method for quantitative determination of amounts of total (reduced and oxidized or GSHt) glutathione. The method employs Ellman's reagent (5,5'-dithiobis-2-nitrobenzoic acid or DTNB), which reacts with GSH to form a spectrophotometrically detectable product at 412 nm. GSSG can be determined by the reduction of GSSG to GSH, which is then determined by the reaction with Ellman's reagent. In brief, the Tietze method utilizes the change in color development during the reaction, and the reaction rate is proportional to the GSH and GSSG concentrations.

      Figure 3: Principal of the Assay
      Materials provided• Assay Buffer (Kit Component No. KP19601): 1 vial, lyophilized NaPO₄ with EDTA
      • GSSG Buffer (Kit Component No. KP19602): 1 bottle, 150 ml, NaPO₄ with EDTA
      • Enzyme (Kit Component No. KP19603): 1 bottle, 40 ml, Glutathione reductase (GR) in NaPO₄ with EDTA
      • NADPH (Kit Component No. KP19604): 6 vials, lyophilized β-Nicotinamide adenine dinucleotide phosphate with Tris base and mannitol.
      • Scavenger (Kit Component No. KP19605): 1 vial, 2 ml, 1-Methyl-2-vinyl-pyridium trifluoromethane sulfonate (M2VP) in HCl
      • Chromogen (Kit Component No. KP19606): 2 bottles, 20 ml, 5,5'-Dithiobis-(2-nitrobenzoic acid) (DTNB) in NaPO₄ with EDTA, with ethanol
      • Standard 0.0 µM GSH (Kit Component No. KP19607): 1 vial, 2 ml, 0.0 µM GSH
      • Standard 0.1 µM GSH (Kit Component No. KP19608): 1 vial, 2 ml, 0.1 µM GSH
      • Standard 0.25 µM GSH (Kit Component No. KP19609): 1 vial, 2 ml, 0.25 µM GSH
      • Standard 0.5 µM GSH (Kit Component No. KP19610): 1 vial, 2 ml, 0.5 µM GSH
      • Standard 1.5 µM GSH (Kit Component KP19611): 1 vial, 2 ml, 1.5 µM GSH
      • Standard 3.0 µM GSH (Kit Component No. KP19612): 1 vial, 2 ml, 3.0 µM GSH
      Note: Standard GSSG in KPO₄ buffer with EDTA, 2 ml each. Each GSSG molecule is equivalent to two GSH molecules: therefore, the values are expressed as µM GSH:

      Table 1: Standard GSSG/GSH
      Materials Required but not provided• Spectrophotometer
      • Centrifuge 1000 x g or greater
      • Spectrophotometric cuvettes with a 1 cm optical path length (glass or polystyrene).
      • Disposable tubes and stoppers (glass or polypropylene).
      • Pipettes, preferably adjustable capable of accurately pipetting 10, 50, 100, 200, 700 and 3000 µl.
      • Balance
      • Metaphosphoric acid
      Preparation• Whole Blood: The sample preparation for whole blood is described below. This assay has only been validated for whole blood containing EDTA as the anticoagulant. Whole blood is difficult to pipette with precision. Using positive displacement pipetting techniques will improve the precision of the GSH and GSSG assays with whole blood. The freezing step serves to lyse the red blood cell and maximize the concentration of GSSG in the sample. Blood samples that have been frozen without prior treatment with the Scavenging Reagent are not suitable for the GSSG assay. Note: GSH Linearity: Because GSH is at high concentrations in whole blood, approximately 1 mM of the whole clood sample should be diluted at least 244 times in order to maintain linearity of the reaction rate. 1. Add 10 µl M2VP to a microcentrifuge tube (recommended). 2. Carefully add 100 µl whole blood (or 100 ml PBS for the GSSG Blank) to the bottom of the centrifuge tube. 3. Mix gently. 4. Freeze the sample at -70°C. (Sample is stable for at least 30 days at -70°C). 5. Thaw the sample and immediately mix, incubate at room temperature for 2-10 min. 6. Add 290 µl cold 5% MPA to the tube (1/4 dilution of original sample). 7. Vortex the sample for 15-20 s. 8. Centrifuge at 1000 x g or greater for 10 min. 9. Add 50 µl MPA extract to 700 µl GSSG buffer (1/15 dilution of the acid extract). 10. Place the diluted extract on ice until use (Final sample dilution is 1/60). • GSSG Blank 1. Add 50 µl MPA to 700 µl GSSG buffer (1/15 dilution of the acid extract). 2. Place the diluted MPA on ice until use (final sample dilution is 1/60) • GSH Sample 1. Carefully add 50 µl of whole blood to the bottom of a microcentrifuge tube (recommended). 2. Freeze the sample at -70°C. (Sample is stable for at least 30 days at -70°C). 3. Thaw the sample and immediately mix. 4. Add 350 µl cold 5% MPA to the tube (1/8 dilution of original sample). 5. Vortex the sample for 15-20 s. 6. Centrifuge at 1000 x g or greater for 10 min. 7. Add 50 µl MPA extract to 3 ml Assay Buffer (1/61 dilution of the acid extract). 8. Place diluted extract on ice until use (Final sample dilution is 1/488). • Tissues Tissues have not been tested with the GSH/GSSG Assay. The following issues need to be considered when preparing tissue samples for analysis: • GSH oxidation in vitro likely occurs rapidly in disrupted tissues. The M2VP reagent should be added as rapidly as possible. • GSSG diffusion from the cell may result in underestimating the GSSG. This is likely to be only an issue with extensively washed cells. • γ-Glutamyltranspeptidase will metabolize GSH. This membrane bound enzyme is especially high in kidney, pancreas, ciliary body, choroids plexus, intestinal epithelia, bile duct cells, lymphoid cells, and many tumor cells. • Normal Plasma GSSG in normal resting plasma is at or below the lowest level of detection for the assay. • Urine GSSG is not detectable in urine.
      Reagent preparation• NADPH: Just prior to use, reconstitute the lyophilized NADPH Reagent with 7.5 ml of Assay Buffer. The reconstituted NADPH Reagent is stable for 6 h at room temperature. • Assay Buffer: Reconstitute the lyophilized powder with 650 ml of deionized water. The reconstituted reagent is stable at 4°C. • 5% Metaphosphoric Acid: Prepare fresh daily. Weigh 1 g MPA and dissolve in 20 ml deionized water. Note: MPA and NADPH are intended for same day use following reconstitution. • Preparing GSSG Standard: Standards are ready to use.
      Detailed protocol1. Add 200 µl of standards, blank or samples to the cuvettes.
      2. Add 200 µl of Chromogen to each cuvette.
      3. Add 200 µl of Enzyme to each cuvette.
      4. Mix and incubate at room temperature for 5 min.
      5. Add 200 µl of NADPH to each cuvette.
      6. Record the change of absorbance at 412 nm for 3 min.
      CalculationsThe calculation of the GSH and GSSG concentrations and the GSH/GSSG ratio requires four steps: 1. Reaction Rate Determination: Note: The rate curves must be linear. If they are not, this generally means the GSH concentration is too high. Dilute the sample and re-run the assay. The change in absorbance at 412 nm is a linear function of the GSH concentration in the reaction mixture, is described by the following equation of a line:
                                              A412 = slope x Min + intercept
      where the slope of the regression equation is equal to the rate. Note that the intercepts for these rate curves are ignored because they are dependent on the DTNB background and the time interval between the addition of the NADPH (reaction start) and the first recorded A412 measurement. In the examples below, linear regression gave the following equation of the line for the GSHt and the GSSG samples:

      Figure 4: Reaction Rate

      Reaction rate for an untreated sample. The rate is proportional to the concentration of GSHt (Left). Reaction rate for a M2VP treated sample (•) and the GSSG Blank (o). The rate is proportional to the concentration of GSSG (Right).

      GSHt: A412 = 0.2209 x Min + 0.2363 with an r2 value of 1.0000. Therefore, the rate for the GSHt sample is 0.2209 A412/min. GSSG: A412 = 0.05938 x Min + 0.1651 with an r2 value of 0.9999. Therefore, the rate for the GSSG sample is 0.05938 A412/min. GSSG BLANK: A412 = 0.04238 x Min + 0.1454 with an r2 value of 0.9999. Therefore, the rate for the GSSG Blank is 0.04238 A412/min. 2. Construction of Calibration Curves: Note: Calibration curves must be linear. The GSH/GSSG-412 assay uses a six-point standard curve for both GSHt and GSSG determinations. The Net Rate is the difference between the rate at each concentration of GSH and the Blank rate.

      Table 2: A Typical 6 Point Calibration of the GSH/GSSG Assay

      Because the concentration of GSSG is much lower in the reaction mixture compared to GSHt, it is recommended that selected data ranges from the calibration curve be plotted separately. For GSHt, perform linear regression on a three-point curve using the 0, 1.50, and 3.00 µM GSH data points. In the case of GSSG, use the 0, 0.10, 0.25, and 0.50 µM GSH data points.

      Figure 5: GSH Calibration Curve

      A three point calibration curve A412/min VS. µ GSH used to determine the concentration of GSH.

      Figure 6: GSSG Calibration Curve

      A four point calibration curve A412/min VS. µ GSH used to determine the concentration of GSSG.

      3. Calculation of Analyte Concentrations (GSHt and GSSG) and 4. GSH/GSSG Ratio The general form of the regression equation describing the calibration curve is:
                                              Net Rate = slope x GSH + intercept

      Figure 7: Calculations
      Sensitivity9 nM GSSG
      Sensitivity NotesThe enzymatic assay is for total GSH, including both GSH and GSSG. The Lower Limit of Detection (LLD) is a measure of the lowest GSSG concentration based on the differences of blank rates, which will be defined as 3.29 standard deviations above zero.

      Table 3: Sensitivity
      PrecisionThe precision of the GSH/GSSG assay was determined using a modification of the NCCLS EP5-T2 guideline. A sample containing a high and low concentration of GSSG was assayed twice per day for 13 days. At the same time, the GSHt and GSSG were determined from whole blood samples stored at -70°C. Each precision parameter was expressed as the mean, standard deviation, and coefficient of variation of the net rate were observed in each sample set.

      Table 4: Precision
      RecoveryGSHt and GSSG were measured in a whole blood (WB) sample, with known concentrations of GSH or GSSG added to determine the addition recovery for the assay. The recovery of the GSH assay was 98% and the GSSG assay was 96%.

      Table 5: Recovery
      LinearityGSSG was diluted in Assay Buffer and tested using the GSH/GSSG assay. the results show that the assay is linear from 0 to 5 µM GSH in the reaction mixture which was equivalent to 0-2.5 µM GSH in the orginal sample.

      Figure 8: Linearity on Dilution
      SpecificityThe GSH analogues were tested in buffer solution and the interference was calculated by comparing with the slope of the GSSG standard. The values are expressed as a percent of the GSSG standard. All tested analogues have no interfering effect on the GSH/GSSG assay.

      Table 6: Specificity


      Sample Stability. Glutathione and oxidized glutathione are relatively stable in intact "resting" cells for up to 24 h at 4°C, Figure 6. The stability of "elevated" GSSG in the intact red blood cell has not been determined. It is recommended that blood samples be treated with M2VP as soon as possible and frozen immediately.

      Figure 9: GSSG Stability in Intact Red Blood Cells at 4°C

      Stability of GSSG, from a rested subject, in the intact red blood cell at 4°C. (GSH-159).


      Upon disruption of the cell, GSH is rapidly oxidized. To minimize the in vitro oxidation of GSH to GSSG, the Scavenger Reagent should be added to the sample prior to lysis or homogenization.

      Figure 10: Stability of GSSG in Red Blood Cell Lysate

      Stability of GSSG in the red blood cell lysate at 4°C. (GSH-161)
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