The “Glutathione Reductase Assay Kit” provides an indirect and highly reproducible method of quantifying the activity of total cellular glutathione reductase. The activity of the enzyme is an important measure of the antioxidant status of the cell.

Oxidative stress has been implicated in aging and in the pathogenesis of a number of disorders. The extent of injury is generally related to an increase, or decrease of one or more free radical scavenging enzymes ⁽²⁾ . High levels of glutathione reductase have been found in erythrocytes from patients with rheumatoid arthritis ⁽⁶⁾ .

* The Glutathione Reductase Assay Kit is for Research Purposes Only.

The oxidation of NADPH to NADP + is accompanied by a decrease in absorbance at 340nm (A ₃₄₀ ), thus providing a spectrophotometric means of detection which is directly proportional to the GR activity in the sample. The reaction is thus measured by the decrease in absorbance at 340 nm using the extinction coefficient 6220 M ^-1 cm ^-1 for NADPH. One unit of NADPH causes the oxidation of 1.0 mmole NADPH at 25°C at pH 7.0. The Zeptometrix Glutathione Reductase Kit can be used to measure GR activity in plasma, erythrocyte lysates, tissue homogenates, and yeast cell lysates. Preferred sample is diluted RBC hemolysate.

The Kit provides reagents sufficient for 100 (1ml) manual tests. If using an automated system such as the Cobas Mira or Fara, considerably more tests can be run.

• Please read all instructions carefully prior to performing the assay.
• To avoid cross contamination, use separate pipet tips for each sample.
• Universal safety precautions while working with bio-hazardous materials should be adopted.
• Wear gloves, lab coats and safety glasses at all times.
• All contaminated materials and biohazardous materials should be properly disposed and work surfaces appropriately decontaminated.
• The guidelines provided in this insert are intended to assist the researcher
  with performing the assay.
   

• GR-Assay Buffer: contains potassium phosphate and EDTA.
• GPR-Reagent 1 (4 vials/kit): contains oxidized glutathione.
• GPR Reagent 2 (4 vials/kit): contains ß-NADPH.
• Gpx-Reagent 3 (25 ml/kit): contains Tris/HCI.
• QC Material (1 vial/kit): contains Human source material. Use Universal Precautions.

Handling and Storage

Store the assay buffer at 2-4°C. NADPH and oxidized glutathione should be stored at -70°C. The components of the Kit are stable for 1 year when stored properly.

Materials/Equipment/Procedures Required But Not Supplied

• UV/Vis spectrophotometer with a kinetic program. Should preferably be
equipped with temperature controlled cuvette chamber.
• Quartz cuvettes with a 1 cm path length.
• Adjustable pipettors with disposable pipette tips.
• Serological pipets.
• Beakers or flask to make the working solution.
• Deionized distilled water (sterile).
• Hemoglobin measurement for red blood cell hemolysate.
• Protein measurements for clarified homogenates from tissues.

1. Add 2 ml deionized distilled water to one vial of GR Reagent 1 (GSSG). Mix well for complete recovery.

2. Add 4 ml GR Reagent 3 to one vial of GR Reagent 2 (NADPH). Mix well for complete recovery.

3. Working Solution: Aseptically transfer 25 ml GR Assay Buffer to a beaker. Add 2.0 ml of Reagent 1 and 2.0 ml Reagent 2 to the GR assay buffer beaker and mix well. Rinse the vial of Reagent 1 with the working solution to be assured of complete recovery of GSSG. This volume is sufficient for 25 manual tests.

4. QC Material: Add 0.5 ml of deionized water to the vial. Allow to sit for 5 min, then vortex gently until completely resuspended. Keep on ice.

5. If samples are frozen, remove them from the freezer and allow to thaw to room temperature (RT). Vortex samples once they are at RT. Fresh samples may be kept at 4ºC 4-6 hrs.

6. Turn on spectrophotometer and allow to warm up for at least 15 minutes. Set kinetic parameters as follows: wavelength 340 nm, lag time 40 sec, rate time 60 sec, total measurement time 100 sec, and read intervals every 15-30 sec. The assay is to be run at 25ºC.

Notes:
• Samples should be run in duplicate.

1. Collect blood using EDTA, heparin or citrate as the anticoagulant.
2. Centrifuge at 3000 rpm for 10 min at 4ºC.
3. Remove plasma from the cells by drawing it off from the top.
4. Freeze at -70º C for up to 6 months if not used immediately.
5. Thaw out samples before analysis. Vortex well to mix.

RED BLOOD CELLS

1. Collect blood using EDTA, heparin or citrate as the anticoagulant.
2. Collect the RBCs by centrifugation at 3000 rpm for 10 min at 4ºC.
3. Discard the plasma and buffy coat (the white interface between the pelleted RBCs and the
plasma) by removing from the top. Optional: aliquot and save the plasma.
4. Wash RBC pellet once with cold saline at 4ºC filled to the top of the tube. Invert several times, then centrifuge at 3000 rpm for 10 min at 4ºC. Discard clear saline and any remaining buffy coat from the top. Repeat once.
5. Lyse the RBCs by adding cold deionized water (1:1) to the packed cells. Allow to mix 10 min.
6. Perform a hemoglobin measurement of this 1:2 diluted RBC hemolysate. Commercial standards and Drabkins reagent (Sigma or Randox) provide a simple spectrophotometric method of measuring hemoglobin in g/dl. Convert units to g/L.
7. Aliquot and freeze at -70ºC for up to 6 months if not used immediately.
8. Thaw out frozen samples before analysis. Vortex well.
9. Dilute a small aliquot of the red blood cell hemolysate to 7 g/L with deionized water.

TISSUES

1. Homogenize or sonicate tissue samples that have been flash-frozen in liquid nitrogen in 4-6 volumes (per wet weight of tissues) of cold Glutathione Reductase Assay Buffer and 1 mM ß-mercaptoethanol.
2. Centrifuge mixture for 10 -15 min at 8000 rpm at 4ºC.
3. Draw off supernatant from the top of the tube for the assay.
4. Freeze samples at -70ºC for up to 6 months if not used directly.
5. Determine protein concentration of the supernatant.

YEAST CELLS (Maximum sample: 10⁹ cells)

1. Pellet yeast cells (10⁹ cells) by centrifugation at 2500 rpm for 10 min in 13 X 100 mm glass test tubes.
2. Resuspend cell pellet in 1.25 ml cold extraction buffer (20 mM Tris pH 8.0).
3. Add 0.48 g glass beads (0.22 mm).
4. Vortex samples for 5 min.
5. Centrifuge samples at 2500 rpm for 10 min.
6. Aliquot supernatants to Eppendorf tubes.
7. If not used directly, quick freeze samples in either liquid nitrogen or ethanol-dry ice bath and store at -70ºC freezer for up to 6 months until analysis.
8. Thaw out samples before use. Vortex well.

2. Zero at 340 nm with deionized water.

3. Pipette the following into the sample cuvette:

a.935 ml Working Solution
b. 35 ml sample

4. Place parafilm on top and gently invert the cuvette several times to mix. Avoid bubbling.

5. Place cuvette in the correct position in the spectrophotometer.

6. Record the change in A 340 for 100 sec at 15-30 sec intervals. Most spectrophotometers allowing kinetic parameters will show the reaction as it proceeds. For manual plotting of absorbance points, user should have at least 3 points. For automated runs, such as with the Cobas Mira or Fara, the user may view all raw data and their plots.
 

2. The net A ₃₄₀ /min for the test sample can be converted to NADPH consumed using the following relationship:

1 unit of Glutathione Reductase will cause the formation of 1 mmol NADP + from NADPH per min at pH 7.0 at 25º C .

Extinction coefficient for NADPH is 0.00622 mM^-1 cm^-1 at 340 nm.

3. Activity of GR can be expressed as International Unit/Liter (U/L) of the sample or in terms of the protein or hemoglobin content.

4. A theoretical unique factor is determined to convert change in absorbance per minute ( Δ/min) to the corresponding units of enzyme activity. This factor is calculated using the following equation:

U/L = ΔA/minute X F; where F= factor

F = (TV/SV) X 10³ / 6.22 where

TV = Total Volume in ml
SV = Sample Volume in ml
10³ = converts ml to L
6.22 = millimolar absorbance coefficient

For this assay, with a 1 cm light path, the factor calculates to be 4455. This factor can be programmed into the spectrophotometer and the machine directly converts the change in absorbance at 340 nm (ΔA/min) to activity in U/L, or alternately, results can be calculated manually.

Figure 1 shows the slope (rate) of the linear portion of the curve when the absorbance
(A₃₄₀ ) values are plotted as a function of time (in seconds) in the sample above. Figure 1

Select the highest and lowest points of the linear curve and determine the change in absorbance at 340 nm during the time interval. In this example it is: A₃₄₀ (Time 2)-A₃₄₀ (Time 1)/T2-T1
 

Sample Rate ΔA₃₄₀ /min = 0.0098
 

Blank Rate ΔA₃₄₀ /min = 0.0004
 

Net Rate ΔA₃₄₀ /min = 0.0094

 

Calculation of activity

GR Activity U/L = 1mmol/min/L = ( ΔA₃₄₀/min)/0.00622 x d x (TV/SV in µl)

For a 1 cm cuvette path length (d) = (0.0094/0.0062) X (935/35) = 1.52 X 26.7 = 40.58 IU/L = 5.8 U/g hemoglobin.

Unit definition: 1 unit of glutathione reductase will form 1.0 mmol NADP + from NADPH per min at pH 7.0 at 25ºC

Note: Enzyme activity can be decreased by negative feedback from excess substrate or from damage by oxidative modification ⁽⁷⁾ . For very high or very low results, check the plots of absorbance points over time. Lines should be linear. A curve followed by a plateau indicates substrate depletion and will require diluting samples.

Linearity:

Shown below is the activity in U/L for a RBC hemolysate spiked with increasing volume of purified Glutathione Reductase (Sigma) enzyme (250 U/L stock solution). The assay is linear within a change of absorbance range (ΔA/min) of approximately 0.0061 to beyond 0.02 which in our experiment corresponds to 27 U/L and 200 U/L enzyme activity respectively. Accordingly, change in absorbance values below or above the lower and upper limits ( 0.003 and 0.025 A₃₄₀ /min) indicate the need for the use of a more concentrated or diluted sample.

Figure 3 shows the range of dilutions that the Glutathione Reductase Kit can accurately and consistently measure GR activity in human RBC hemolysate. Dilutions of 1:10,1:20,1:30,1:40, 1:50 and 1:100 of a 166.56 g/L hemoglobin in a RBC hemolysate was tested. Activity is expressed in U/g hemoglobin. The Kit is effectively accurate over a range of 2.0 fold dilution from 1:20 to 1:40 of the hemolysate that corresponds to 8.3 g/L and 4.1 g/L hemoglobin respectively.

Experiment:

The Glutathione Reductase Assay Kit was used to measure GR activity in 15 random samples of blood that were obtained commercially. The plasma and red blood cells were separated and appropriate dilutions of RBC hemolysates were made. The GR activity was measured both manually and by automation. Correlation between the 2 methods was 0.93.

*Parameters for the automated system are available upon request.

2.  Armstrong D., (1998) Free Radical and Antioxidant Protocols Edited by Donald Armstrong, Humana Press Totowa, NJ, pp 299-313

3.  Bompart GJ, Prevot DS, and Bascands JL (1990) Rapid automated analysis of glutathione reductase, peroxidase, and s-transferase activity. Clin Biochem 23 , 501-504.

4.  Gastani GF, Kirkman, HN, Mangerini, R, and Ferraris, AM (1994) Importance of catalase in the disposal of hydrogen peroxide within human erythrocytes. Blood 84 , 325-330.

5.  Harmening D (ed) (1992 ) Clinical Hematology and Fundamentals of Hemostasis (2^nd ed.) F.A Davis, Co., Philadelphia, PA, pp 251, 540.

6.  Mulherin DM, Thurnham DI, Situnayake RD (1996) Glutathione reductase activity, riboflavin status, and disease activity in rheumatoid arthritis. Ann Rheum Dis 55 , 837-40.

7.  Tabatabaie T and Floyd RA (1994) Susceptibility of glutathione peroxidase and glutathione reductase to oxidative damage and the protective effect of spin trapping agents. Arch Biochem Biophys 314 , 112-119.

TURN ON SPECTROPHOTOMETER AND SET AT KINETIC
PARAMETERS

SET ASSAY TEMPERATURE AT 25°C

PREPARE REAGENTS

PREPARE SAMPLES

AUTO ZERO at 340 nm WITH DEIONIZED WATER

PIPETTE FOLLOWING REAGENTS INTO A CUVETTE

935 ml WORKING SOLUTION
35 ml SAMPLE
MIX BY INVERSION

PLACE CUVETTE IN CORRECT POSITION IN SPECTROPHOTOMETER

RECORD THE CHANGE IN A₃₄₀ FOR 1 MIN

ZeptoMetrix Corporation
872 Main Street
Buffalo, New York 14202

Office Phone: 716-882-0920
Fax: 716-882-0959