Glutathione is a tripeptide of glycine, glutamic acid and cysteine. In the red blood cell, the reduced form of glutathione is vital in maintaining hemoglobin in a reduced state and hence protecting the cells from oxidative damage. Glutathione is involved in detoxification of hydrogen peroxide through glutathione oxidase. Low levels of glutathione are found in deficiencies of key enzymes involved in glutathione metabolism, such as glucose-6-phosphate dehydrogenase, glutathione synthase and glutathione reductase.

Simple, direct and automation-ready procedures for measuring reduced glutathione are becoming popular in Research and Drug Discovery. BioAssay Systems' QuantiChromTM Glutathione Assay Kit is designed to accurately measure reduced glutathione in biological samples. The improved 5,5'-dithiobis(2-nitrobenzoic acid (DTNB) method combines deproteination and detection (Reagent A) into one reagent. DTNB reacts with reduced glutathione to form a yellow product. The optical density, measured at 412 nm, is directly proportional to glutathione concentration in the sample. The optimized formulation has a long shelf life and is completely free of interference due to sample turbidity.

Simple and convenient. The procedure involves mixing the DTNB Reagent with sample, removing protein precipitates for proteinaceous samples, adding a second Reagent and reading the optical density.

Low interference. Amino acids and common buffers do not interfere.

Drug Discovery/Pharmacology: effects of drugs on glutathione metabolism.

Precautions: reagents are for research use only. Normal precautions for laboratory reagents should be exercised while using the reagents. Please refer to Material Safety Data Sheet for detailed information.

Samples: whole blood samples should be diluted 20-fold with water prior to the assay (n = 20). Cell lysate can be prepared as follows: collect 2 x 106 cells by centrifugation at 1,000g for 10 min at 4°C. Wash cells in cold PBS. Lyse cells by homogenization or sonication in 1-2 mL of cold buffer containing 50 mM MES or phosphate (pH 6-7) and 1 mM EDTA. Centrifuge at 10,000g for 15 min at 4°C. Use supernatant for assay.

Note: b-mercaptoethanol, dithiothreitol and cysteine are known to interfere in this assay. Avoid using these compounds during sample preparation. Amino acids do not interfere.

Mix 120 μL sample with 120 μL Reagent A in 1.5-mL centrifuge tubes. Vortex to mix well. If turbidity occurs, pellet 5 min at 14,000 rpm in a table centrifuge. If the mixture remains clear, no centrifugation is necessary.

3. Transfer 200 μL sample/Reagent A mixture into wells of the 96- well plate. Add 100 μL Reagent B. Tap plate lightly to mix.

4. Incubate 25 min at room temperature. Read OD412nm.

Mix 400 μL sample with 400 μL Reagent A, centrifuge sample tubes if precipitation occurs. Transfer 600 μL supernatant and mix with 400 μL Reagent B. Incubate 25 min at room temperature. Measure OD412nm against water. Transfer 400 μL Calibrator and 800 μL Water into a clean cuvet and measure OD412nm against water.

Subtract blank OD (water) from the Calibrator and Sample OD values. The glutathione concentration of Sample is calculated as

ODSAMPLE, ODSTD and ODBLANK are optical density values of the sample, Calibrator and sample “Blank” (water or buffer in which the sample was dissolved). n is the dilution factor (20 for blood samples).

Conversions: 1 mg/dL glutathione equals 32.5 μM, 0.001% or 10 ppm.

Pipeting devices, centrifuge tube and table centrifuge.

Clear bottom 96-well plates (e.g. Corning Costar) and plate reader.

Spectrophotometer and cuvets for measuring OD at 412 nm.

EXAMPLE. 20 μL fresh mouse blood was mixed quickly with 380 μL water. Assays in 96-well plate gave blood glutathione concentration of 1124 ± 8 μM (n = 2)

Standard Curve with Freshly Prepared Glutathione in 96-well plate assay

1. Lindenmaier, H. et al (2005). Interaction of progestins with the human multidrug resistance-associated protein 2 (MRP2). Drug Metab Dispos. 33(11):1576-9.

2. Blenn, C. et al (2006). Poly(ADP-ribose) glycohydrolase silencing protects against H2O2-induced cell death. Biochem J. 396(3):419- 29.

GLUTATHIONE PEROXIDASE (GPX, EC 1.11.1.9) represents an enzyme family with peroxidase activity whose main biological role is to protect the organism from oxidative damage. It helps prevent lipid peroxidation of cellular membranes by removing free peroxide in the cell. GPX catalyzes the following reaction with glutathione reductase (GR),

2 GSH + H2O2 GS-SG + 2 H2O,

GS-SG + NADPH 2 GSH + NADP+

Simple, direct and high-throughput assays for GPX activity find wide applications. BioAssay Systems' improved assay directly measures NADPH consumption in the enzyme coupled reactions. The measured decrease in optical density at 340nm is directly proportional to the enzyme activity in the sample.

Drug Discovery/Pharmacology: effects of drugs on GPX activity.

NADPH: 40 μL H2O2 Solution: 100 μL 3% H2O2

Positive Control: 9 μL Glutathione Peroxidase (GPX)

Storage conditions. The kit is shipped on ice. Store all components at -20 °C. Shelf life of three months after receipt.

Precautions: reagents are for research use only. Normal precautions for laboratory reagents should be exercised while using the reagents. Please refer to Material Safety Data Sheet for detailed information.

Transfer 10 μL sample and 10 μL reconstituted GPX Positive Control into separate wells of the 96-well plate. In addition, for each assay run, include a background control that only contains 10 μL Assay Buffer.