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 of which GPx is one. ⁽⁸⁾ Various diseases show different levels of the universally present GPx in all tissues. A reduction in enzyme level is associated with Parkinson's disease ⁽⁹⁾ and in Chronic Glomerulonephritis patients. ⁽¹⁰⁾ In patients with end-stage renal disease (ESRD), GPx activity in adult patients was comparable to that in the control groups (children and adults); the GPx in children with ESRD was almost twice as high than in the other groups. ⁽¹¹⁾ GPx activity decreased significantly in liver and increased in kidney in three-week-old diabetic rats that showed a reversal with a change in diet. ⁽¹²⁾

The OXItek “Total Glutathione Peroxidase Assay Kit“ provides a method of quantifying the activity of total glutathione peroxidase.

*The Total Glutathione Peroxidase Assay Kit is for Research Purposes Only.

In this assay, cumene hydroperoxide is used as the peroxide substrate (ROOH), and glutathione reductase (GSSG-R) and NADPH (ß-Nicotinamide Adenine Dinucleotide Phosphate, Reduced) are included in the reaction mixture. The formation of GSSG (glutathione) catalyzed by GPx is coupled to the recycling of GSSG back to GSH using GSSG-R. NADPH is oxidized to NADP ⁺ . The change in A ₃₄₀ due to NADPH oxidation is monitored and is indicative of GPx activity. Since all other reagents are provided in excess, the amount of GPx in the test sample is the rate-limiting factor. The over-all 2-step reaction is: ⁽¹³⁾

The oxidation of NADPH to NADP ⁺ is monitored spectrophotometrically by a decrease in absorbance at 340 nm (A ₃₄₀ ). Under conditions in which the GPx activity is rate limiting, the rate of decrease in the A ₃₄₀ is directly proportional to the GPx activity in the sample . ⁽¹³⁾

GPx activity both in plasma and in Red Blood Cells (hemolysate) can be determined with this kit. The kit can also be adapted to GPx activity determination in cells from culture and tissue homogenates.

The Kit provides reagents sufficient for 100 (~1ml) tests.

• Please read all instructions carefully prior to performing assay.
Recommendations in the insert are to be used as guidelines.
• To avoid cross contamination, use separate pipette tips for each sample.
• Universal safety precautions while working with bio-hazardous materials should
be adopted. ⁽¹⁴⁾
• Wear gloves, lab coats and safety glasses at all times.
• All contaminated materials and biohazardous material should be properly
disposed and work surfaces appropriately decontaminated.
• The source for the QC Material is Human. All tests should be conducted using
precautions recommended for blood borne pathogens, as defined by OSHA
regulations.

Materials Supplied:

• GPx-Assay Buffer: contains Potassium Phosphate and EDTA.
• GPx-Reagent 1: contains reduced Glutathione (GSH).
• GPx Reagent 2: contains NADPH.
• Gpx-Reagent 3: contains Cumene Hydroperoxide, Sodium Bicarbonate.
• GPx-Reagent 4: contains Potassium Phosphate and GSSG-R (Glutathione Reductase).
• QC Material: contains Human source material. Use Universal Precautions.

Handling and Storage

Store GPx-Reagent 1, GPx-Reagent 2 and GPx-Reagent 4 at -70ºC. GPx-Reagent 3 and the GPx-Assay Buffer should be stored at 4ºC . The components of the Kit are stable for 1 year when stored properly. The QC material when stored at -70ºC is stable for 6 months unreconstituted.

Materials/Equipment/Procedures Required But Not Supplied

• UV/Vis spectrophotometer with a kinetic program. Should preferably be
equipped with temperature controlled cuvette chamber.
• Spectrophotometric cuvettes.
• Adjustable pipettors with disposable pipette tips.
• Serological pipettes.
• Saline.
• Beakers/flasks to make reagents and working solutions.
• Deionized distilled water (DI).
• Hemoglobin measurement for red blood cell hemolysate.
• Protein measurements for clarified homogenates from tissues.

1. Working Solution:

• Use aseptic handling to aliquot 25 ml of GPx-Assay Buffer to a glass
beaker. Add entire contents of 1 vial of GPx-Reagent 1. Wash vial with
buffer to ensure complete recovery.
• Add 200 µl GPx-Reagent 4 to the above solution. Bring to room
temperature. Working Solution is good for 25 (1 ml) assays.
• This solution can be kept at ambient temperature (24-25ºC) for a period of
8-10 hours with no significant effect on assay performance. Do not freeze
solution.

2. Start Solution:

• Add 2.14 ml GPx-Reagent 3 to 1 vial of GPx-Reagent 2. Bring to room temperature.
• The start solution can be kept at ambient temperature (24-25ºC) for a period of 8-10 hours with no significant effect on assay performance. Do not freeze solution.

3. QC Material:

• Add .5 ml of DI water to the QC Material vial. Allow to sit 5 min, then vortex
gently until completely resuspended. Keep on ice.

4. Turn on spectrophotometer, set to measure absorbance at 340 nm in the kinetic mode. Lag time is 40 sec, rate time 60 sec, read intervals 15-30 sec. Assay temperature should be set at 24-25ºC.

Notes:

• Samples should be run in duplicate.
• Reactions are very sensitive to temperature changes.
• Blanks (DI water replaces sample) should be run.
• Sodium azide inhibits the reaction.
• Thawed and resuspended QC Material cannot be refrozen.
Recommend each lab have plasma they can run each time.

1. Collect blood using EDTA, heparin or citrate as the anticoagulant.
2. Spin down the RBC by centrifugation at 3000 rpm for 10 min at 4 º C
3. Remove plasma from the cells by drawing it off from the top.
4. If not used directly, quick freeze samples in either liquid nitrogen or ethanol-dry ice bath and store at -70º C freezer until analysis.
5. Thaw out samples before analysis. Vortex well to mix.

RED BLOOD CELLS

1. Collect and process blood as above till step 3.
2. Remove the buffy coat (the white interface between the pelleted RBCs and the plasma) and discard.
3. Wash RBC pellet with saline at 4 º C. Centrifuge at 3000 rpm for 10 min at 4 º C. Discard clear saline from top. Repeat once.
4. To an aliquot of RBC add an equal volume of DI water, vortex well for complete lysis of the cells.
5. Perform a hemoglobin measurement of this 1:2 diluted RBC hemolysate. Convert from g/dl to g/L.
6. If not used directly, quick freeze hemolysate sample aliquots in either liquid nitrogen or ethanol-dry ice bath and store at -70ºC freezer until analysis.
7. Thaw out frozen samples before analysis. Vortex well.
8. Dilute thawed out red blood cell lysate in DI water to 6-7 g/L of hemoglobin.

   • Mix well.
   • Leave diluted samples at RT or on ice before analysis.
   •Diluted samples are stable for 8 hours at RT or 48 hours at 0-5ºC.

TISSUES

1. Homogenize tissue samples in 4-6 volumes (per wet weight of tissues) of cold GPx Assay Buffer and 1
   mM ß-mercaptoethanol.
2. Centrifuge mixture for 10-15 min at 8000 rpm at 4ºC.
3. Remove supernatant from the top of the tube for the assay.
4. Determine protein concentration of the supernatant.
5. If not used directly, quick freeze samples in either liquid nitrogen or ethanol-dry ice bath and store at -70º C freezer until analysis.
6. Thaw out samples before use. Vortex well.

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 until analysis.
8. Thaw out samples before use. Vortex well.

Step 2: Zero spectrophotometer at 340 nm with DI water.

Step 3: Pipette the following reagents into the cuvette:

• 870 µl Working Solution A
• 60 µl Start Solution

Step 4: Start the reaction by adding 30 µl sample or DI water for blank determinations. Pipette up and down to mix thoroughly. Avoid bubbling.

Step 5: Cover cuvette with parafilm and invert gently 2-3 times.

Step 6: Place cuvette in the correct position in the spectrophotometer.

Step 7: Record the change in A ₃₄₀ for 1 min, following a 40 sec lag time.

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

1 unit of Glutathione Peroxidase will cause the formation of 1 µmol NADP + from NADPH per min at pH 8.0 at 25º C .

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

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

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

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

This factor can be programmed into the spectrophotometer and the machine directly converts the change in absorbance at 340 nm ( /min) to activity in U/L. For this assay, with the proper volumes, the factor calculates to be 5144.7.

Determination of the Reaction rate:

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.

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

0.808-0.674/100 sec-40 sec = 0.134/min

Sample Rate, ₃₄₀/min = 0.134
Blank Rate, ₃₄₀ /min = 0.015
Net Rate ₃₄₀ /min = 0.12

Calculation of activity

GPx Activity U /L = 1 µmol/min/L = ( ₃₄₀ /min)/0.00622 X d X (TV/SV in µl)

For a 1 cm cuvette path length (d) = 0.12/0.00622 X 960/30 = 19.29 X 32 = 617.28 U/L
Unit definition: 1 unit of glutathione peroxidase will form 1.0 µmol NADP+ from NADPH per min at pH 8.0 at 25 º C.

Precision and Reproducibility of Method

Linearity : Shown below is activity in U/L for the QC material spiked with increasing volume of purified bovine erythrocyte Glutathione Peroxidase enzyme (10,000 U/L stock solution; Sigma). The assay is linear within a change in absorbance range of 0.130 to 0.275 A₃₄₀ /minute which for our QC material spiked with bovine erythrocyte GPx, corresponds to 510 U/L to 1324 U/L enzyme activity respectively. Further experiments have established an accurate measurement of change in absorbance to 0.05 A₃₄₀ /minute corresponding to activity value as low as 261 U/L (results not shown). Accordingly, change in absorbance values below or above the lower and upper limits (0.05 and 0.275 A₃₄₀ /min) indicate the need for use of a more concentrated or more diluted sample.

Figure 2

Figure 3 shows the range of dilutions that the Total Glutathione Peroxidase Assay kit can accurately measure GPx activity in human RBC hemolysate. Dilutions of 1:4, 1:8, 1:10,1:20, 1:30, 1:40, 1:50 and 1:100 of a 160 g/L hemoglobin in a RBC hemolysate were tested. Activity is expressed in U/g Hemoglobin. The Kit is effectively accurate over a range of 2.5 fold dilution from 1:20 to 1:50 of the hemolysate that corresponds to 8.05 g/L and 3.22 g/L hemoglobin respectively.

Figure 3

Experiment:

The Total Glutathione Peroxidase Assay Kit was used to calculate total GPx activity from 15 random samples of blood that were obtained commercially. The plasma and red blood cells were separated and appropriate dilutions of the RBC hemolysate were made. The GPx activity for plasma and the RBC hemolysate were calculated both manually and by automation using the Cobas Fara II Chemistry System. * Correlation between the two methods used (manual vs automated) was 0.97 for the plasma and 0.969 for the WRBC.

* Parameters for the automated system are available upon request.

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TURN ON SPECTROPHOTOMETER SET AT 340 nm

SET ASSAY TEMPERATURE AT 25 º C

PREPARE REAGENTS

PREPARE SAMPLES

SET ZERO at 340 nm WITH DI WATER

PIPETTE FOLLOWING REAGENTS INTO A CUVETTE

870 µl REACTION BUFFER
60 µl START SOLUTION
30 µl SAMPLE

PIPETTE GENTLY UP AND DOWN

COVER WITH PARAFILM AND TURN GENTLY 2-3 TIMES

PLACE CUVETTE IN CORRECT POSITION IN SPECTROPHOTOMETER

RECORD THE CHANGE IN A ₃₄₀ FOR 1 MIN

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