WO2020039259A1 - Methods and reagents for measuring glutathione - Google Patents

Abstract

Disclosed herein are methods and kits of reagents for measuring glutathione.

Description

METHODS AND REAGENTS FOR MEASURING GLUTATHIONE

CROSS-REFERENCE

[001] This application claims the benefit of U.S. Provisional Application No. 62/720,553, filed August 21, 2018, which is incorporated herein by reference in its entirety.

INCORPORATION BY REFERENCE

[002] All publications, patents, and patent applications disclosed herein are incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. In the event of a conflict between a term disclosed herein and a term in an incorporated reference, the term herein controls.

BRIEF SUMMARY

[003] Disclosed herein is a kit for measuring a glutathione concentration or glutathionylation in a biological sample, wherein the kit comprises glutaredoxin, nicotinamide adenine dinucleotide phosphate (NADPH), glutathione reductase (GR), or a fluorescent substrate comprising an eosin- labeled glutathione disulfide, or any combination thereof. In some instances, the eosin-labeled glutathione disulfide has a strongly quenched fluorescence. In some instances, the kit further comprises a stabilizing agent in a form a modified serum albumin. In some instances, the modified serum albumin is a alkylated bovine serum albumin. In some instances, the alkylated bovine serum albumin blocks a flee sulfhydryl group to a concentration of about 50 pg/ml. In some instances, the glutaredoxin is present in about 0.1 mM to about 0.5 mM, or about 0.5 mM to about 1 mM, about 0.1 mM to about 1 mM, or about 1 mM to about 2.6 mM. In some instances, the glutaredoxin is present in about 0.52 mM. In some instances, the glutaredoxin is human glutaredoxin 1 (hGrx-1). In some instances, the NADPH is present in about 0.1 mg/ml to about 0.4 mg/ml, or about 0.4 mg/ml to about 1 mg/ml. In some instances, the NADPH is present in about 0.2 mg/ml. In some instances, the glutathione reductase is present in about 0.05 mM to about 0.2 mM, about 0.2 mM to about 0.5 mM, or about 0.5 mM to about 5 mM. In some instances, the glutathione reductase is present in about 0.1 mM. In some instances, the fluorescent substrate is di-eosin-glutathione disulfide (Di-E-GSSG). In some instances, tire fluorescent substrate is present in about 1 mM to about 10 mM, about 5 mM to about 15 mM, about 10 mM to about 25 mM, or about 10 mM to about 40 mM. In some instances, the fluorescent substrate is present in about 10 mM. In some instances, the kit comprises a buffer. In some instances, the buffer comprises a potassium phosphate. In some instances, the potassium phosphate is present in about 0.1 M to about 0.2 M, or about 0.2 M to about 0.9 M. In some instances, the buffer comprises a chelating agent. In some instances, the chelating agent is EDTA. In some instances, the chelating agent is present in about 1 mM to about 5mM, or about 5mM to about 20 mM. In some instances, the buffer is at a pH value of about 7 to about 7.5, or about 7.5 to about 8. In some instances, the kit further comprises sulfosalicyclic acid, polyethylene glycol p-tert-octylphenyl ether (TRITON-X-IOO), a vinylpyridine, triethanolamine, or any combination thereof. In some instances, the biological sample is a plasma, a serum, a tissue, or a cell lysate, for example of an animal such as a human. In some instances, the biological sample has about 1 pg to about 5 pg of protein. In some instances, the biological sample is less than 1 ml. In some instances, the biological sample is about 5 pi to about 20 pi, about 10 pi to about 30 pi, or about 30 pi to about 100 pi. In some instances, the sample is frozen. In some instances, the sample is snap frozen.

[004] Disclosed herein is a method for measuring a glutathione concentration or

glutathionylation in a biological sample, comprising 1) incubating the biological sample for a period of time to reduce glutathione disulfide in the biological sample to generate a free sulfhydryl group (thiol), and 2) detecting the free sulfhydryl group by fluorescence. In some instances, the period of time is about 5 minutes to about 30 minutes. In some instances, the period of time is about 5 minutes to about 10 minutes, or about 10 minutes to about 20 minutes. In some instances, the incubation is carried out in a solution that comprises glutaredoxin, nicotinamide adenine dinucleotide phosphate (NADPH), or glutathione reductase (GR), or any combination thereof. In some instances, the method further comprises contacting the biological sample with a fluorescent substrate. In some instances, the fluorescent substrate complies an eosin-labeled glutathione disulfide. In some instances, the fluorescent substrate is di-eosin- glutathione disulfide (Di-E-GSSG). In some instances, the method further comprises shaking the biological sample for at least: about 10 seconds to about 30 seconds. In some instances, the detecting by fluorescence is at about 540 nm to about 550 nm (for example about 545 nm) after exciting at about 515 nm to about 525 nm (for example about 520 nm) for at least: about 10 minutes to about 20 minutes, or about 20 minutes to about 60 minutes. In some instances, the method further comprises contacting the biological sample with sulfosalicyclic acid, polyethylene glycol p-tert-octylphenyl ether (TRITON-X-100), or a combination thereof, before the measuring to be able to measure glutathione disulfide (GSSG). In some instances, the method further comprises contacting the biological sample with an alkylating agent, before the measuring. In some instances, the alkylating agent is a vinylpyridine or N-ethyl-maleimide. In some instances, the vinylpyridine is 2-vinylpyridine or 4-vinylpyridine. In some instances, the vinylpyridine is present in a concentration of about 0.1% to about 1% w/w or v/v, or about 1% to about 5% w/w or v/v. In some instances, the method further comprises contacting the biological sample with triethanolamine, before the measuring. In some instances, the triethanolamine is present in a concentration of about 0.1% to about 1% w/w or v/v, or about 1% to about 5% w/w or v/v. In some instances, the detecting comprises using a standard fluorescent plate reader. In some instances, the biological sample is a plasma, a serum, a tissue, or a cell lysate, for example of an animal such as a human. In some instances, the biological sample has about 1 pg to about 5 pg of protein. In some instances, the biological sample is less than 1 ml. In some instances, the biological sample is about 5 pi to about 20 pi, about 10 pi to about 30 pi, or about 30 pi to about 100 pi. In some instances, the sample is frozen. In some instances, the sample is snap frozen.

BRIEF DESCRIPTION OF THE DRAWINGS

[005] Figures la and lb illustrate standard curves for GSH-GSSG-CSSC-CSSG. 50pl of master mix of alkylated-BSA 0.1 mg/ml, NADPH 0.4 mg/ml, GR 0. lpM, hGrxl lpM were added to lOpl of different standard solution at different concentration. After addition of fluorescent substrate, fluorescence at 545 nm was recorded after excitation at 520nm. Slope of the linear part of the curve (fluorescence, time) were plotted against the GSH or disulfide concentration (n for “a”>8; n for“b” =4 result are average +/- STD).

[006] Figure 2 illustrates a total-GSH measurement. Total-GSH and GSSG concentration was measured in different cell line. The same sample was aliquoted in two and analysed in parallel with the DTNB-established method and the present fluorescent method. The value obtained were normalised for the total protein content. No significant differences were observed comparing the two methods. (n=2 mean+/-STD).

[007] Figures 3a to 3f illustrate levels of total GSH and GSSG in different mice tissues. Total GSH (Fig. 3a), GSSG (Fig. 3b) and PSSG (Fig. 3c) were measured in snap frozen tissues. Each sample was divided in two and analysed in parallel following the different methods. No significant differences were obtained comparing the DTNB-based assay and the present fluorescent-based method (n=4). Total GSH (Fig. 3d), GSSG (Fig. 3e) and PSSG (Fig. 3f) were measured in fresh collected tissues. The results shows that for glutathione measurement, snap frozen tissue stored at -80°C must be handle with extra careful due to artefact oxidations. The DTNB assay was not sensitive enough to measure the level of PSSG in flesh collected tissues.

[008] Figures 4a to 4c illustrate levels of total GSH and GSSG in mice liver. Total GSH (Fig. 4a), GSSG (Fig. 4b) and PSSG (Fig. 4c) were measured in snap frozen liver. Each sample was divided in two and analysed in parallel following the different methods. No significant differences were obtained comparing the DTNB-based assay and the present fluorescent-based method when the samples were in relatively high concentration (total GSH and GSSG) but at very low concentration (PSSG) the DTNB-method is not sensitive enough to give a reliable result ( n=4). [009] Figures 5a to 5b illustrate levels of total GSH in human plasma. Total-GSH was measured in human plasma from different donors (Fig. 5a). The 5 samples with higher total GSH level were analysed in parallel following the different methods. Significant differences were obtained comparing the DTNB based assay and the present fluorescent-based method (Fig. 5b). The DTNB method is not sensitive enough to measure the glutathione in plasma.

[0010] Figure 6 illustrates atypical glutathione standard curve, recorded at 545 nm emission after excitation at 520 nm. Slope (AFluorescence/minute) between 5 and 50 minutes plotted against GSH content for the corresponding well.

DETAILED DESCRIPTION

[0011] Disclosed herein are unique methods and kits for directly measuring glutathione and protein glutathionylation with ultrahigh sensitivity. The methods and kits of reagents disclosed herein are suitable for determing glutathione and glutaihionylated proteins in a wide range of biolgical samples, including complex samples such as cell lysates, tissues, plasma, and serum. The methods and kits disclosed herein can use a fluorescent substrate that comprises an eosin- labeled glutathione disulfide, for example di-eosin-glutathione disulfide (Di-E-GSSG). The methods and kits disclosed herein can further use an assay system that comprises nicotinamide adenine dinucleotide phosphate (NADPH), glutathione reductase, and glutaredoxin. The methods and kids disclosed herein can be used directly with frozen samples, to solve the issue that freezing often leads to oxidation of glutathione and formation of mixed disulfides. The methods and kit disclosed herein can be used in the study of redox signalling, and also can have high applicability in clinical studies when the samples are in small amounts and the concentration of glutathione is very low.

[0012] In some instances, the methods and kits disclosed herein can be dramatically more sensitive than the conventional method such as the DTNB assay and can easily be used with plasma and serum samples to determine total glutathione. For example, the present methods and kits can detect a glutathione concentration of 0.1 mM or lower, while known methods can only detect a glutathione concentration at a much higher value, such as 1 raM or more. Conventional methods would have to use DTT (dithiothreitol) which breaks disulfides and precipitates proteins and requires lots of controls. The colorimetric assay using DTNB is also an enzymatic method but much less sensitive for plasma samples (e.g., up to 1 ml has to be concentrated). The presence of thioredoxin reductase in the DTNB assay sample may give false positives since DTNB is a substrate for mammalian thioredoxin reductase (e.g., can be present in plasma and serum). The sensitivity limit of the method/kit here is lower than a conventional colorimetric method, allowing the measurement of GSH from plasma and from glutathionylated proteins in complex samples.

[0013] In some instances, a kit or method disclosed herein can be combined with a kit or method for measuring glutaredoxin and thioredoxin in the same serum or plasma samples, which can lead to a measurement of the redox sate of the thioredoxin and glutaredoxin systems in a subject such as a person, and the levels of oxidative stress and inflammation.

[0014] In some instances, the methods and kits herein are used in vitro. In some instances, the methods may be addpated for applicability in vivo.

[0015] In some aspects, disclosed herein is an assay system used for a kit or method for measuring a glutathione concentration or glutathionylation in a biological sample, wherein the assay system comprises glutaredoxin (e.g., 0.1 mM), nicotinamide adenine dinucleotide phosphate (NADPH, e.g., at 0.2 mg/ml), glutathione reductase (GR, e.g., hGrx-1 at 0.52 mM), and a fluorescent substrate comprising an eosin-labeled glutathione disulfide (e.g., Di-E-GSSG at 10 mM), and optionally a stablizing agent (e.g., alkylated BSA at 0.05 mg/ml). In some instances, the assay system comprises a buffer (e.g, 0.18 M potassium phosphate pH 7.5 with EDTA).

[0016] In some instances, the kits and methods herein comprise a reduced glutathione, which can be used to construct a standard curve to calculate the amount of glutathione the sample (for example serum or plasma) contains.

[0017] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of the ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the compositions or unit doses herein, some methods and materials are now described. Unless mentioned otherwise, the techniques employed or contemplated herein are standard methodologies. The materials, methods and examples are illustrative only and not limiting.

[0018] The details of one or more inventive embodiments are set forth in the accompanying drawings, the claims, and the description herein. Other features, objects, and advantages of the inventive embodiments disclosed and contemplated herein can be combined with any other embodiment unless explicitly excluded.

[0019] Unless otherwise indicated, open terms for example“contain,”“containing,”“include,” “including,” and the like mean comprising. [0020] The singular forms“a”,“an”, and“the” are used herein to include plural references unless the context clearly dictates otherwise. Accordingly, unless the contrary is indicated, the numerical parameters set forth in this application are approximations that may vary depending upon the desired properties sought to be obtained by the present invention.

[0021] Unless otherwise indicated, some embodiments herein contemplate numerical ranges. When a numerical range is provided, unless otherwise indicated, the range includes the range endpoints. Unless otherwise indicated, numerical ranges include all values and subranges therein as if explicitly written out. Unless otherwise indicated, any numerical ranges and/or values herein can be at 90-110% of the numerical ranges and/or values. Throughout, unless otherwise noted,“about” means within ±10% of a value.

Glutathione

[0022] Glutathione is a tripeptide (g-Glu-Cys-Gly) present at very high concentrations throughout living organisms, for example from 1 to 10 mM in cells, and at a 1000-fold lower concentration in plasma or serum. Glutathione is an abundant low-molecular-weight thiol, up to lOmM in cells, and exists in three major forms: reduced sulphydryl (a.k.a. thiol-reduced form, GSH), glutathione disulfide (GSSG) or glutathione bound to Cys residues in proteins (PSSG). The ratio GSH/GSSG has been used as an indicator of the redox level in cells but this parameter can also be estimated by the quantification of PSSG. In fact, PSSG has the advantage of being more stable than GSSG.

[0023] Glutathione plays several important roles in cellular physiology', such as the maintenance of redox status and signalling through protein glutathionylation. Glutathione is present at high concentrations in the intracellular environment and changes in the GSH/GSSG ratio may be considered an indirect measure of intracellular redox status. Moreover tire focus on redox regulation via protein glutathionylation has increased during the past decade both in physiological and pathological conditions such as diabetes, neurodegenerative diseases, and cancer. Glutathione plays a key role in heavy metals and xenobiotics detoxification, cell redox homeostasis, gene expression, cell proliferation and apoptosis mainly binding Cys residues in protein, known as protein glutathionylation. Glutathione also acts coupled to different enzymes like Glutathione Peroxidase and Glutaredoxin (Grx). In some instances for Grx it represents the reducing power for the enzyme and indirectly can regulate the activity of other important enzymes like in the case of Ribonucleotide Reductase (RNR) that catalyzes the rate limiting step of the DNA synthesis. In some instances, the RNR activity with Grx is very much dependent on the concentrations of glutathione. In some instances, glutathione functions in the iron-sulfur cluster structure of Grx mitochondrial isoforms. [0024] Within mammalian cells, glutathione exists mainly (>98%) in the thiol-reduced form (GSH) in feet the oxidized form. In some instances, GSSG can be toxic in cytosol, and it can be either reduced by Glutathione Reductase (GR) in a reaction requiring NADPH as electron donor, or exported from cells when its cytosolic concentration increases.

[0025] In some aspects, disclosed herein is a new protocol to detect GSH, GSSG and PSSG in cell lines, tissues, serum, and plasma. The fluorescent substrate, Di-E-GSSG, can be cleaved by Grx. This generates two molecules of E-GSH increasing the fluorescence emission coupled to oxidation of the active site in Grx (reaction 1-3).

[0026] Oxidized Grx is reduced by one molecule of GSH and this mixed disulfide is subsequently reduced by a second GSH molecule releasing GSSG that is in turn reduced by GR in the presence of NADPH. GSH represents the limiting factor of the Grx cycle meaning that the increase in fluorescence depends on the GSH concentration.

[0027] In some aspects, the method and kit herein relates to an enzymatic method for quantitative determination of glutathione and protein S-glutathionylation using fluorescent eosin- glutathione. In some instances, the method and kit herein utilizes the enzymatic reaction of glutaredoxin (Grx) keeping the GSH from the samples as limiting factor for the kinetic, using as substrate, the fluorescent compound Di-E-GSSG. The conjugation of two molecules of eosin to glutathione disulfide (Di-E-GSSG) shows very low fluorescence that increases, up to 20 times, after its reduction to E-GSH.

[0028] In some aspects, disclosed herein is a highly sensitive fluorescent-based method for detection of low concentrations of glutathione and PSSG in complex samples such as cell lysate, tissues and plasma. The whole procedure was optimised to measure the fluorescence increase of the di-eosin-glutathione disulfide (Di-E-GSSG) reduced by glutaredoxin in the presence of glutathione reductase and NADPH, keeping glutathione as limiting factor to drive the reaction. [0029] In some instances for example in samples with high GSH concentration, a method disclosed herein is reliable as the conventional DTNB assay or more reliable than the DTNB assay, and additionally it has higher sensitivity demonstrated by the ability to detect GSH in plasma and PSSG in tissues under physiological conditions. In some instances, an assay method disclosed herein is easy to perform and it can be undertaken in 96-well plates using a standard fluorescent plate reader.

Kits

[0030] Disclosed herein is a kit for measuring a glutathione concentration or glutathionylation (e.g., protein S-glutathionylation) in a biological sample, wherein the kit comprises glutaredoxin, nicotinamide adenine dinucleotide phosphate (NADPH), glutathione reductase (GR), or a fluorescent substrate comprising an eosin-labeled glutathione disulfide, or any combination thereof. In some instances, the eosin-labeled glutathione disulfide has a strongly quenched fluorescence. In some instances, the kit further comprises a stabilizing agent in a form a modified serum albumin. In some instances, the modified serum albumin is a alkylated bovine serum albumin. In some instances, the alkylated bovine serum albumin blocks a flee sulfhydryl group to a concentration of about 50 pg/rnl. In some instances, the glutaredoxin is present in about 0.1 mM to about 0.5 mM, or about 0.5 mM to about 1 mM, about 0.1 mM to about 1 mM, or about 1 mM to about 2.6 mM. In some instances, the glutaredoxin is present in about 0.52 mM. In some instances, the glutaredoxin is human glutaredoxin 1 (hGrx-1). In some instances, the NADPH is present in about 0.1 mg/ml to about 0.4 mg/ml, or about 0.4 mg/ml to about 1 mg/ml. In some instances, the NADPH is present in about 0.2 mg/ml. In some instances, the glutathione reductase is present in about 0.05 mM to about 0.2 mM, about 0.2 mM to about 0.5 mM, or about 0.5 mM to about 5 mM. In some instances, the glutathione reductase is present in about 0.1 mM.

In some instances, the fluorescent substrate is di-eosin-glutathione disulfide (Di-E-GSSG). In some instances, the fluorescent substrate is present in about 1 mM to about 10 mM, about 5 mM to about 15 mM, about 10 mM to about 25 mM, or about 10 mM to about 40 mM. In some instances, the fluorescent substrate is present in about 10 mM. In some instances, the kit comprises a buffer. In some instances, the buffer comprises a potassium phosphate. In some instances, the potassium phosphate is present in about 0.1 M to about 0.2 M, or about 0.2 M to about 0.9 M. In some instances, the buffer comprises a chelating agent. In some instances, the chelating agent is EDTA. In some instances, the chelating agent is present in about 1 mM to about 5mM, or about 5mM to about 20 mM. In some instances, the buffer is at a pH value of about 7 to about 7.5, or about 7.5 to about 8. In some instances, the kit further comprises sulfosalicyclic acid, polyethylene glycol p-tert-octylphenyl ether (TRITON-X-100), a vinylpyridine, triethanolamine, or any combination thereof. In some instances, the biological sample is a plasma, a serum, a tissue, or a cell lysate, for example of an animal such as a human. In some instances, the biological sample has about 1 pg to about 5 pg of protein. In some instances, the biological sample is less than 1 ml. In some instances, the biological sample is about 5 pi to about 20 pi, about 10 pi to about 30 pi, or about 30 pi to about 100 pi. In some instances, the sample is frozen. In some instances, the sample is snap frozen.

Measurement methods

[0031] Disclosed herein is a method for measuring a glutathione concentration or

glutathionylation (e.g., protein S-glutathionylation) in a biological sample, comprising 1) incubating the biological sample for a period of time to reduce glutathione disulfide in the biological sample to generate a free sulfhydryl group (thiol), and 2) detecting the free sulfhydryl group by fluorescence. In some instances, the period of time is about 5 minutes to about 30 minutes. In some instances, the period of time is about 5 minutes to about 10 minutes, or about 10 minutes to about 20 minutes. In some instances, the incubation is carried out in a solution that comprises glutaredoxin, nicotinamide adenine dinucleotide phosphate (NADPH), or glutathione reductase (GR), or any combination thereof. In some instances, the method further comprises contacting the biological sample with a fluorescent substrate. In some instances, the fluorescent substrate complies an eosin-labeled glutathione disulfide. In some instances, the fluorescent substrate is di-eosin-glutathione disulfide (Di-E-GSSG). In some instances, the method further comprises shaking the biological sample for at least: about 10 seconds to about 30 seconds. In some instances, the detecting by fluorescence is at about 540 nm to about 550 nm (for example about 545 nm) after exciting at about 515 nm to about 525 nm (for example about 520 nm) for at least: about 10 minutes to about 20 minutes, or about 20 minutes to about 60 minutes. In some instances, the method further comprises contacting the biological sample with sulfosalicyclic acid, polyethylene glycol p-tert-octylphenyl ether (TRITON-X-100), or a combination thereof, before the measuring to be able to measure glutathione disulfide (GSSG). In some instances, the method further comprises contacting the biological sample with an alkylating agent, before the measuring. In some instances, the alkylating agent is a vinylpyridine or N-ethyl-maleimide. In some instances, the vinylpyridine is 2-vinylpyridine or 4-vinylpyridine. In some instances, the vinylpyridine is present in a concentration of about 0.1% to about 1% w/w or v/v, or about 1% to about 5% w/w or v/v. In some instances, the method further comprises contacting the biological sample with triethanolamine, before the measuring. In some instances, the triethanolamine is present in a concentration of about 0.1% to about 1% w/w or v/v, or about 1% to about 5% w/w or v/v. In some instances, the detecting comprises using a standard fluorescent plate reader. In some instances, the biological sample is a plasma, a serum, a tissue, or a cell lysate, for example of an animal such as a human. In some instances, the biological sample has about 1 pg to about 5 pg of protein. In some instances, the biological sample is less than 1 ml. In some instances, the biological sample is about 5 mΐ to about 20 mΐ, about 10 mΐ to about 30 mΐ, or about 30 mΐ to about 100 mΐ. In some instances, the sample is frozen. In some instances, the sample is snap frozen.

Example 1.

Summary

[0032] The efficacy and sensitivity of the present method and kit against the standard DTNB- based assay were compared. Three different samples were used: cell lysate, mice tissue and human plasma. The low molecular weight thiols and then the PSSG were measured.

[0033] In cell lines, where the concentration of GSH is in mM range, total-GSH showed no significant differences across methods. This means that the fluorescent-method is comparable with the DTNB assay. Moreover, different cell lines and their GSH content were used. GSSG measurements accounted for 10-20% of total-GSH in samples using both assays.

[0034] After checking in cell lines that the DTNB and fluorescent-methods were equivalent, the glutathionylation profile of tissue samples was measured. Different conditions were tested to check if the variations observed across both methods and laboratories can also be due to the sample storage and manipulation.

[0035] All the organs used presented comparable amount of total-GSH, except for the liver, which showed a higher concentration. This difference is possiblly because in the liver GSH is utilized for detoxification and it is produced for exportation to other parts of the body such as bile and blood. No statistically significant differences were observed between methods using tissues samples where the amount of GSH and GSSG was abundant confirming the cell results about the comparability of the two assays for total-GSH and GSSG measurement.

[0036] Furthermore PSSG concentration was analysed and in snap frozen samples (Fig. 3c, Fig. 4c) the value obtained were similar comparing the DTNB and fluorescent assay results. However comparing the data with fresh tissues, the previous values may possiblly be artefacts due to sample manipulation. The effect is particularly visible in brain tissue.

[0037] It was possible to measure with small variation the PSSG content in fresh collected samples only using the fluorescent-method (Fig. 3f). [0038] Liver analysis using the present fluorescent assay shows low levels of PSSG in physiological condition and an increasing concentration, as expected, after diamide treatment. It was not possible to obtain the same results using the DTNB method.

[0039] Therefore, data comparing snap frozen and fresh samples underlined first, the importance of a careful handle of samples in order to avoid artefact oxidation during storage and manipulation; and second the high sensitivity' of the fluorescent assay to measure PSSG in physiological conditions.

[0040] GSH content in plasma can pose a problem when it comes to detection because its range is low (usually between 1-10 mM) and the presence of mixed disulfide could represent a problem of underestimation depending of the approach used. Here, with the present fluorescent-method, it was possible to measure total-GSH in plasma with a concentration in the expected range (Figs. 5a, 5b) opening the possibility to use the present method/kit in clinical studies. Moreover another advantage is the possibility to store the plasma and be able to measure the total GSH content considering also the different disulfides forms that are rapidly formed during the sample preparation and storage.

[0041] As seen in figures 2, 3 and 4, the measurements were comparable between methods when the samples had high concentration of glutathione, vice versa for plasma samples with low concentration, it was able to detect total-GSH including all form of GSH oxidation

(GSSG,CSSG and PSSG), proving the method high sensitivity. The values obtain are in line with the literature where more sophisticated techniques were used.

[0042] The major strategies utilized to measure protein glutathionylation consist in three main approaches: (i) labelling GSH; (ii) Immunoblotting using antibodies against GSH; (iii) break the disulfide bond with GSH and label these now free SH groups.

Materials and Methods

Reagents

[0043] Except when indicated, all general reagents were of molecular biology or higher grade and purchased from Sigma-Aldrich. L-cysteine-glutathione disulfide (CSSG) from Cayman chemical. Di-E-GSSG was prepared separately. Spectrapor dialysis membranes were purchased from Spectrum Laboratories and Amicon ultra centrifuge filter tubes with 14000 MW cut off were from Merk Millipore.

Preparation of alkylated BSA [0044] BSA (0.3 mM) was incubated with Iodoacetamide (IAM - 1 mM) in 0.1 M potassium phosphate buffer, pH 7.5 for 60 min at 37 °C. Alkylated-BSA was separated from the non- reacted IAM by dialysis against 0.1 M potassium phosphate buffer pH 7.5 using dialysis membranes with 14000 MW cut off. The concentration of alkylated-BSA was determined both by the method of Bradford [21], calibrated with BSA, and measuring the absorbance at 280-310 nm using the molar absorption extinction coefficient of 44000 M^cm^"1 in 0.1 M potassium phosphate buffer pH 7.5.

Fluorescence measurements

[0045] The reduction of Di-E-GSSG and further release of E-GSH was measured by- fluorescence in black 96-well microtiter plates in 100 pL final volume, recording fluorescence emission at 545 nm after excitation at 520 nm using an Enspire 2300 Multilable Reader, from PerkinElmer. The standard curves for GSH, GSSG , CSSC, CSSG were made using 10 mΐ of each different standard concentration, 30 mΐ of 0.1 M phosphate buffer - EDTA 5 mM pH 7.5 (KPE), 50 mΐ of master mix (alkylated-BSA 0.1 mg/ml, NADPH 0.4 mg/ml, GR 0.1 mM, hGrxl 1 mM (IMCO, Sweden) in KPE) and 10 mΐ of fluorescent substrate. Fluorescence was read at 545 nm after excitation at 520 nm for 30 min every 30 seconds.

Cell culture

[0046] Cells (HEK293, MCF7, PC3 and PANC-1) were cultivated in appropriate medium supplemented with 10% (v/v) of fetal bovine serum (FBS) and maintained at an atmosphere of 37°C and a 5% (v/v) CO2.

Sample preparation: cells and plasma

[0047] Sample preparation was performed with the purpose to compare directly the results obtained by DTNB-based method versus the fluorescent-based method. Cells were scraped from 100 mm dish plates, washed twice in ice-cold PBS and lysed in 0.1% Triton-X and 0.6% sulfosalicylic acid (SSA) in KPE. The acidified cell lysate supernatants were divided in two and used directly for total-GSH or derivatized for GSSG assay'. For GSSG measurement the samples were first added of vinylpyridine (4m1 of 1: 10 diluted vinylpyridine in KPE for IOOmI of supernatant). Standards of different concentration of GSSG were prepared and treated in parallel. All samples were vortex 15 sec and incubate for 2h at room temperature in a fume hood. To inactivate the non-reacted vinylpyridine, 6m1 of triethanolamine (1:6 diluted in KPE) were added each sample, incubated for lOmin and divided in two aliquots for the DTNB method and fluorescent method analyses. Human plasma was obtained from healthy blood donors at the Karolinska Hospital, Stockholm, Sweden. Plasma was aliquoted and stored at -20 °C until usage in further studies. Standard curves of GSH and GSSG were run in parallel to the samples. The linear part of the kinetic curve was used to calculate the slope for each curve.

Sample preparation: tissues

[0048] A novel protocol was used here to avoid any GSH or GSSG contamination in the protein pellet and oxidation due to sample manipulation. Snap-frozen or fresh heart, kidney, brain, spleen and liver samples were homogenized and sonicated in an ice-cold solution containing 5% phosphoric acid and 0.6% SSA mixture and clarified by centrifugation. The supernatants were immediately processed for GSH and GSSG measurement, as described below, while the pellets were resuspended in 500 mΐ of 6 M urea buffer (0.1 M phosphate and 10 mM N-ethylmaleimide (NEM), pH 7.4) and incubated at room temperature for 30 min, 10 mΐ of each sample were aliquoted for protein quantification.

[0049] Proteins were precipitated with acetonitrile and centrifuged at 15000 xg for 5 min. The supernatants were discarded, whereas pellets were rinsed three times with acetonitrile to remove excess of NEM. Pellets were then resuspended in 500 mΐ of 6 M urea/phosphate buffer (0.2 M, pH 7.4) with sonication and then precipitated again with 5% SSA solution and centrifuged three times to remove any residual GSH and GSSG. The final pellet was resuspended in 500 mΐ of 6 M urea/borate buffer (25 mM, pH 9.0) to release the GSH from protein and 10 mΐ/sample were collected for protein concentration measurement. SSA 5% was added and after centrifugation, the supernatants were collected for measurements.

[0050] The respective final supernatants far GSH, GSSG and PSSG preparation were divided in two aliquots and processed as follows, for PSSG a standard curve of GSH was used as a reference.

DTNB-based method

[0051] DTNB (5,5-dithio-bis-(2-nitrobenzoic acid) - Ellman’s reagent) 1.5mg/ml mixed with 1.65 U/ml of GR solution was added to the samples and after 30 second, allowing time for GRto reduced GSSG, 0.7 mg/ml of NADPH were added and the reaction was immediately followed by reading Abs at 412 nm for 5 min every 20 sec.

Fluorescent-based method

[0052] A master mix (alkylated-BSA 0.1 mg/ml, NADPH 0.4 mg/ml, GR 0.1 mM, hGrxl 1 mM (IMCO, Sweden) in KPE) was added to the samples. After 60 sec at room temperature the fluorescent substrate was added and fluorescence was read at 545 nm after excitation at 520 nm for 30 min every 30 sec. Statistics

[0053] Data are presented as the mean ± SD and n > 3. Statistical significance was determined by paired Student's / test. P < 0.05 was considered statistically significant.

Results

Standard curves

[0054] To use this method as a GSH-content assay, the conditions to measure GSH

concentration using Grx system were optimized to keep GSH as limiting factor. A broad range of GSH dilutions was tested, and the slope of the linear part of the curve“fluorescence vs time” shows a strong positive correlation with increases in GSH concentration. The results (Fig. la) demonstrated that using this method, it is possible to detect GSH concentration as low as 0.1 mM. Moreover, it was checked if GSSG and mixed glutathione disulfide can be detected using this method and Figure lb shows that only the disulfide with glutathione gives an increase in fluorescence over time proportional to the increase in concentration while CSSC does not lead to any increase in fluorescence.

Comparison between DTNB-method and fluorescent-method in different cell lines

[0055] The results from the present fluorescent method and the conventional DTNB -method on the total-GSH and GSSG content were in the same range and did not show any statistical difference between the methods (Fig. 2). The total-GSH measurements were compared to those obtained elsewhere with established methods in complex samples (DTNB, HPLC and biotinylated-GSH).

Comparison between DTNB-method and fluorescent-method in different organs and conditions

[0056] The analysis of total thiol-disulphide state of glutathione is challenging not only for the low concentration of specific forms (PSSG) or samples (plasma) but also for the risk to artefact formation during sample collection, storage and manipulation. For this consideration, comparison was done between the measurements of fresh samples and snap frozen tissues stored in -80°C for a month.

[0057] The glutathione content in tissues is usually higher than the limit detectable by the DTNB-method. The conventional DTNB-method was compared with the results from the present fluorescent method in measuring total-GSH and GSSG in different samples.

[0058] Mice tissues were homogenized and the obtained supernatants (see materials and methods) were analyzed. There are no differences between the concentration values for total- GSH and GSSG comparing the two methods (Figs. 3a, 3b). The values obtained for total-GSH are according to the data already reported in literature.

[0059] Moreover, it is interesting to notice that the levels of PSSG in brain (Fig. 3c) are quite high compared to the other samples.

[0060] Figures 3d, 3e, and 3f show the results obtained on the flesh collected tissues and despite the total-GSH (3d) value are the same using both methods and in both conditions, the GSSG concentrations (3e) are lower in the flesh collected tissues in particular in the brain. PSSG (3f) had a dramatic decrease in concentration in flesh samples and the level were lower than the detection limit of DTNB assay in two out of three cases.

[0061] The majority of studies on GSH transport and metabolism have been done in liver because its high concentration, result of equilibrium between its synthesis and its efflux into bile and blood plasma, and function, in feet in liver glutathione plays a key role in detoxification reactions.

[0062] Analysis of GSH was performed in liver as for the other tissues samples, the

concentration obtained with the two method were comparable and despite the not statistical significance it is possible to see a slightly difference between the flesh samples and the snap frozen ones, in both assays (Fig. 4a).

[0063] GSSG instead (Fig. 4b) showed a decrease of about 3 times comparing the fresh samples with the snap frozen. It was possible to measure the concentration changes with DTNB and fluorescent-based method with high accuracy thanks to the high concentration of total glutathione in liver.

[0064] Detection of PSSG in fresh liver (Fig. 4c) was not possible using the DTNB assay however there was a lower difference between flesh and snap frozen values of PSSG respect to the values of the other tissues (Figs. 3a to 3f).

[0065] An aliquot of the fresh homogenized liver was treated with lOmM diamide for 20 minutes and only using the fluorescent-method, it was able to measure the expected increase of PSSG.

Total-GSH measurement in human plasma

[0066] Measurement of GSH in plasma samples can be used for clinical purposes when samples usually present very low amounts of GSH. The substrate specificity and sensitivity of the present method were tested by measuring total-GSH in plasma from healthy blood donors. The fluorescence increase over time was plotted and the concentration of GSH was calculated from the standard curve run at the same time as the samples.

[0067] The levels of total-GSH measured ranged from 1 mM to 7 mM (Fig. 5a) with values. The values with higher GSH content could be due to partial haemolysis but it may be difficult to exclude individual variations among the subjects.

[0068] The samples were pooled 3x3 with high GSH level to have 5 new plasma samples, each of which was divided in two sub-aliquots. As it can be seen in Fig. 5b using the same amount of plasma (20m1), it was not possible to determine a reliable result with the DTNB-method. This would also be explained by the feet that the total-GSH measured in the fluorescent assay includes both the GSSG and the mixed disulfide form with Cys (CSSG). Exploiting the catalytically features of Grx were able to measure all the disulfide formed by GSH (CSSG, GSSG, PSSG) without interference of CSSC (Fig. lb).

Example 2.

FLUORESCENT GLUTATHIONE ASSAY KIT

Materials

[0069] Fluorescence micro plate reader with 520 nm excitation / 545 nm emission like Perkin Elmer Enspire.

[0070] 96 black micro titer plates.

[0071] Sulfosalicylic acid, Triton-X-100, 2-vinylpyridine, triethanol amine (for sample preparation).

[0072] Distilled water.

[0073] Supplied components:

[0074] Assay buffer (KE buffer) x2 - white cap

Potassium phosphate, pH 7.5 and EDTA.

Preparation procedure:

Dissolve the content of each tube with 0.8 ml distilled water, yielding 2.5 M potassium phosphate pH 7.5

and 10 mM EDTA (2.5 M KE buffer).

Use one tube to prepare the MASTER MIX. Dilute the other tube 2.5 times with distilled water in a larger container, yielding 0.1 M potassium phosphate pH 7.5 and 4 mM EDTA (0.1M KE buffer). Use this to dilute samples when needed.

Store at -20°C.

[0075] Reduced glutathione (GSH) - yellow' cap

Lyophilized GSH.

Preparation procedure :

Dissolve the content with 50 mΐ distilled water yielding 0.1 M GSH.

Store at -20°C.

Make a 10 mM GSH aliquot from the 0.1 M GSH stock using distilled water.

Not stabile in low concentrations, prepare fresh before each assay

[0076] Assay stabilizing reagent - transparent cap

Lyophilized stabilizing reagent.

Preparation procedure :

Dissolve the content with 100 mΐ distilled water to make 10 mg/ml

Store at -20°C.

[007h b-NADPH - purple cap

Lyophilized reduced b-NADPH containing stabilizing reagents.

Preparation procedure:

Dissolve the content with 50 mΐ distilled water, yielding 40mg/ml reduced b-NADPH. Store at -20°C. Not stabile for long durations once diluted.

[0078] Baker yeast glutathione reductase (GR) - browm cap

20 mΐ of 50 mM glutathione reductase in 50 percent glycerol, 75 mM Tris-Cl, 1 mM EDTA, pH 7.5.

Preparation procedure:

Add 30 mΐ of 0.1 M KE buffer to make 50 mΐ of 20 mM glutathione reductase.

Cap and shake the tube. For long term use make aliquots before dilution.

Store at -20°C.

[0079] Human glutaredoxin 1 (hGrx-1) - blue cap

156 pg recombinant hGrx-1 lyophilized from 10 mΐ of 50 mM Tris-HCl, 1 mM EDTA, pH 7.5 (TE).

Preparation procedure:

Dissolve the content with 100 mΐ distilled w'ater yielding 1.56 mg/ml (130 mM) of hGrx-

1. Store at -20°C.

For long term use make aliquots.

[0080] Fluorescent substrate - green cap

Lyophilized fluorescent substrate.

Preparation procedure:

Dissolve the content in 1.0 ml distilled water yielding 200mM of fluorescent substrate. Dilute to 40mM before use in assay.

Store at -20°C.

SUGGESTED PREPARATION OF SAMPLES

[0081] Plasma/Serum

To obtain plasma, collect blood using anticoagulant, centrifuge lOOOxg for 10 min at 4°C and take the supernatant.

To obtain serum, collect blood without anticoagulant, leave the sample at room temperature for 30 min to allow the blood to clot, centrifuge 2000xg 15 min at 4°C and take the supernatant.

Plasma and serum can be used directly; it may be desired using 5-30 mΐ plasma in the assay and adjust the volume to fit the sample to the standard curve.

The assay can be optimized for 384 well plates using 3-5m1 of plasma or serum sample.

[0082] Tissues and Cells

Cell preparation:

1. Collect cells (1-10 x 10⁶) and centrifuge at lOOOxg for 10 min. Scrape the cells if adherent rather than use trypsin.

2. Remove supernatant; wash the cell pellet with 1 ml PBS save 10m1 to perform protein determination and centrifuge at lOOOxg for 5 min.

3. Remove the supernatant. From now on the samples should be kept in ice. Dissolve the pellet in 0.3ml 0.1% Triton-X-100 and 2% sulfosalicylic acid (flesh prepared) in 0.1 M KE pH 7.5 to lyse the cells and sonicate with 10 burst for 10 sec.

4. Centrifuge lOOOOxg for 10 min at 4°C and collect the supernatant.

5. The acidified cell lysate supernatants can be divided in two and used directly for total -GSH or derivatized for GSSG assay following the Rahman et al sample preparation. For GSSG measurement the samples must be fast mixed with vinylpyridine (4m1 of 1 : 10 diluted vinylpyridine in 0.1 KE for IOOmI of supernatant). After vortexing for 15s, samples are incubated for 2h at RT in a fume hood. To inactivate non-ieacted vinylpyridine add 6 mΐ of triethanol amine (1:6 diluted in KE buffer) and incubate 10 min.

Tissue preparation:

1. Homogenize the tissue sample in 0.1% Triton-X in 0.1 M KE buffer pH 7.5 ice cold.

2. Centrifuge at lOOOxg for 5 min in 4°C to remove the debris. Collect supernatant and save 10m1 to perform protein determination.

3. Add 2% sulfosalicylic acid (fresh prepared) and centrifuge at lOOOxg for 10 min in 4°C. Collect the supernatant.

4. The acidified supernatants can be divided in two and used directly for total-GSH or

derivatized for GSSG assay following the Rahman et al sample preparation. For GSSG measurement the samples must be fast mixed with vinylpyridine (4m1 of 1 : 10 diluted vinylpyridine in 0.1 KE for IOOmI of supernatant). After vortexing for 15s, samples are incubated for 2h at RT in a fume hood. To inactivate non-reacted vinylpyridine add 6 mΐ of triethanol amine (1:6 diluted in KE buffer) and incubate 10 min.

5. It may be desired measuring the protein concentrations of all non-plasma samples and make diluted aliquots of supernatant corresponding to 2pg/pl protein to use in the assay. This makes it easier to fit the samples in the standard curve and to calculate [GSHJ / [protein].

[0083] GLUTATHIONE ASSAY

[0084] General information:

The final volume of the assay is 100 mΐ per well and performed at room temperature.

The final assay should be carried out in triplicate to increase accuracy but it is the user’s choice.

Record the emission at 545 nm after excitation at 520 run for 20-60 minutes

[0085] It may be desired as first step to check that samples fit within the standard curve.

a) For cell samples, about 1-5 pg of total protein.

b) For plasma samples, about 10-30 mΐ pure plasma.

If a sample is not within the standard curve, dilute the samples accordingly.

Assay procedure

[0086] Steps:

1. Incubate for 10 min to ensure all GSSG is reduced to GSH.

2. Add 25 mΐ of 40 mM fluorescent substrate to each well.

3. Make sure to shake the plate properly (for ca 30 seconds) - If the substrate is not fully mixed it will make the first 5-10 minutes unusable.

4. Record the emission at 545 nm after excitation at 520 nm for maximum 60 minutes.

[008h DATA TREATMENT AND CALCULATION OF RESULTS

1. Select the linear range of the standard curve (for example 10-30 minutes) and the same range for the samples to determine rate of reaction (Afluorescence per minute).

2. Calculate the rate of reaction as AFluorescence/ATime, meaning the slope of the curve.

3. Subtract all samples’ slopes with their corresponding background’s slopes

4. If you have duplicates or triplicates, calculate the mean values.

5. Using the standard curve equation extrapolate the amount of GSH in samples.

6. Normalize for amount of protein considering every dilution done to the sample. [0088] Additional Considerations:

Plasma and semm samples can have a very different background activity and can be influenced by the healthy condition of the blood donor and other variables. To obtain maximum accuracy when calculating the concentration of samples, it may be suitable to measure the same sample with three different concentrations, like using 5-10-20 mΐ of plasma to get a linear correlation (if all of them are within the standard curve) that you can use when you are calculating results. Plasma and serum samples with visible sign of hemolysis should be discarded and are unreliable.

To calculate the concentration of GSH:

- Run 5-10-20m1 sample

- Plot sample slope against the volume used for each sample and calculate the intercept of the trend line. The three different measures should be aligned.

- GSH concentration in well = (Sample slope -intercept) /Standard curve slope

Normalize for amount of protein and consider every dilution done in the process.

Example 3

1.0 General kit information

[0089] This exemplary SOP standard operating procedure is suitable for the preparation of 10 GSH kit™. Each of the kits’ components is described thoroughly in the upcoming sections of this document. The GSH kit™ is composed of:

[0090] Except for Bakers yeast glutathione reductase and Recombinant hGrxl all the components are lyophilized once prepared. The kit is to be stored at -20 °C.

2.0 Assay stabilizing reagent (alkylated BSA) [0091] General information: Each vial or tube contains 50 pL 10 mg/mL alkylated BSA.

1. Dissolve 10 mg 99 % (protease flee, essentially g-globulin) bovine serum albumin (BSA) in 0.5 rtiL 0.1 M potassium phosphate buffer pH 7.5 yielding 20 mg/mL or 0.29 mM.

2. Add iodoacetamide to 2 mM final concentration; e.g. add 6 pL 200 mM iodoacetamide to 500 pL BSA solution.

3. Incubate 60 minutes at 37 °C.

4. During the incubation, prepare 4 L 0.1 M potassium phosphate buffer pH 7.5.

5. After incubation, transfer the mixture to a semi-permeable membrane with 12000-15000 cut-off.

6. Put the membrane with the content in 2 L 0.1 M potassium phosphate buffer pH 7.5 and dialyze for at least 2-3 hours with stirring at 4 °C.

7. Transfer the membrane to the remaining 2 L 0.1 M potassium phosphate buffer pH 7.5 and dialyze for at least 2-3 hours at 4 °C. Comment: this dialysis can be left over night.

8. Carefully collect the content from the membrane and transfer it to a tube.

9. Desalting of the content is recommended using sephadex column for effective removal of any alkylating agents.

10. To calculate the final concentration, dilute 50 and 100 times with phosphate buffer pH 7.5 in a cuvette, measure the absorbance at A260, Azbo, A310 and calculate as follow:

11. This step is optional, but recommended. To make sure BSA is completely alkylated, calculate the flee thiols by following the scheme:

12. Dilute the alkylated BSA to 10 mg/mL alkylated BSA in 0.5 mL 0.1 M potassium phosphate buffer pH 7.5 containing 2mM EDTA.

13. Aliquot 50 pL per tube in the micro tubes and lyophilize.

14. Once lyophilized cap the tubes with TRANSPARENT caps.

15. Store at -20 °C.

3.0 Assay buffer (2 tubes/kit)

[0092] General information: Each vial or tube contains 0.8 mL lyophilized assay buffer.

1. Prepare 20 mL 1 M potassium phosphate pH 7.5 and 10 mM EDTA.

2. Aliquot 1 mL per tube in the micro tubes and lyophilize.

3. Once lyophilized cap the tubes with WHITE caps.

4. Store at -20 °C.

4.0 Reduced glutathione (GSH)

[0093] General information: Each vial or tube contains 50 pL O.l M GSH.

1. Dissolve 15 mg reduced glutathione (GSH) in 0.5 mL 0.1 M potassium phosphate pH 7.5 and 1 mM EDTA yielding 0.1 M GSH.

2. Aliquot 50 pL per tube in the micro tubes and lyophilize.

3. Once lyophilized cap the tubes with YELLOW caps.

4. Store at -20 °C.

5.0 b-NADPH

[0094] General information: Each vial or tube contains 50 pL 40 mg/ml or 50 mM b-NAΌRH

1. Dissolve 20 mg b-NADPH in 0.5 mL buffer containing 10 mM Tris-Cl pH 9.2 and 200 mM NaCl, yielding 40 mg/mL or 50 mM.

2. Aliquot 50 pL per tube in the micro tubes and lyophilize.

3. Once lyophilized cap the tubes with PURPLE caps.

4. Store at -20 °C. 6.0 Human glutaredoxin 1 (hGrx-l)

[0095] General information: Each vial or tube contains 156 pg recombinant hGrx-l lyophilized from 10 mΐ of 50 mM Tris-HCl, 1 mM EDTA, pH 7.5 (GE).

1. Aliquot 10 pL per tube in the micro tubes of 15.6 mg/ml and cap the tubes with BLUE caps.

2. Store at -20 °C.

7.0 Bakers yeast glutathione reductase

[0096] General information: Each vial or tube contains 20 pL 50 pM Baker yeast glutathione reductase in 50 percent glycerol-75 mM Tris-Cl-1 ntM EDTA, pH 7.5 solution.

1. Take one vial 100 UN glutathione reductase from bakers yeast (Sigma) and transfer the content to an Eppendorf tube.

2. Wash the vial with additional 200 pL 85 % (NH*)2SO* pH 7 solution and transfer to the same tube.

3. Spin the content at 10000 rpm for 10 minutes.

4. Carefully remove the supernatant and wash the pellet with 200 pL (NHOzSCU pH 7 solution.

5. Spin the content at 10000 rpm for 10 minutes and carefully discard the pellet.

6. Dissolve the pellet with 100 pL 50 percent glycerol-75 mM Tris-Cl-1 mM EDTA, pH 7.5 solution.

7. To calculate the final concentration, dilute 125 and 250 times in TE buffer in a cuvette, measure the absorbance at A₄63 and calculate as follow:

AJM x dilution factor

= [glutathione reductase] M

11 300 M-ⁱ cm-ⁱ

8. Prepare 0.1 mL 50 pM glutathione reductase with 50 percent glycerol-75 mM Tris-Cl-1 mM EDTA, pH 7.5 solution (dilute if needed).

9. Aliquot 20 pL per tube in the micro tubes and lyophilize.

10. Once lyophilized cap the tubes with BROWN caps.

11. Store at -20 °C. 8.0 Fluorescent substrate (f-GSSG)

1. 1ml of 200mM f-GSSG

2. Lyophilize

9.0 Quality control of the GSH kit™

9.1 Reconstitution of the kit contents

[0097] Reconstitute the each of the kit components with distilled water as follow:

[0098] Prepare a MASTER MIX (for 20 wells) as follow:

[0099] Dilute the F-GSSG 200mM to 40mM in distilled water.

[00100] To execute the GSH assay, follow the protocol suggested in the table above and below.

9.3 Data treatment

[00101] The protocol is as follows:

1. Once the readings are finished, select a linear range from the results in order to determine rate of reaction (Afluorescence per minute).

2. Calculate the rate of reaction as follow:

Afluorescence fluorescence aim i) - fluorescence o¾ne n

minute Time 2 (mm.) - Time 1 (min)

3. Plot the rate of reaction against its’ corresponding concentration.

4. Include a copy of this result in each kit.

[00102] The examples and embodiments described herein are for illustrative purposes only and various modifications or changes suggested to persons skilled in the art are to be included within the spirit and purview of this application and scope of the appended claims.

Claims

CLAIMS WHAT IS CLAIMED IS:

1. A kit for measuring a glutathione concentration or glutathionylation in a biological sample, wherein the kit comprises glutaredoxin, nicotinamide adenine dinucleotide phosphate (NADPH), glutathione reductase (GR), and a fluorescent substrate comprising an eosin-labeled glutathione disulfide.

2. The kit of claim 1, wherein the eosin-labeled glutathione disulfide has a strongly

quenched fluorescence.

3. The kit of claim 1 or 2, wherein the kit further comprises a stabilizing agent in a form of a modified serum albumin.

4. The kit of claim 3, wherein the modified serum albumin is an alkylated bovine serum albumin.

5. The kit of claim 4, wherein the alkylated bovine serum albumin blocks a free sulfhydryl group to a concentration of about 50 pg/ml.

6. The kit of any preceding claim, wherein the glutaredoxin is present in about 0.1 mM to about 0.5 mM, or about 0.5 mM to about 1 mM.

7. The kit of any preceding claim, wherein the glutaredoxin is present in about 0.52 mM.

8. The kit of any preceding claim, wherein the glutaredoxin is human glutaredoxin 1 (hGrx-

1).

9. The kit of any preceding claim, wherein the NADPH is present in about 0.1 mg/ml to about 0.4 mg/ml.

10. The kit of any preceding claim, wherein the NADPH is present in about 0.2 mg/ml.

11. The kit of any preceding claim, wherein the glutathione reductase is present in about 0.05 mM to about 0.2 mM.

12. The kit of any preceding claim, wherein the glutathione reductase is present in about 0.1 mM.

13. The kit of any preceding claim, wherein the fluorescent substrate is di-eosin-glutathione disulfide (Di-E-GSSG).

14. The kit of any preceding claim, wherein the fluorescent substrate is present in about 1 mM to about 10 mM, or about 10 mM to about 40 mM.

15. The kit of any preceding claim, wherein the fluorescent substrate is present in about 10 mM.

16. The kit of any preceding claim, wherein the kit comprises a buffer.

17. The kit of claim 16, wherein the buffer comprises a potassium phosphate.

18. The kit of claim 17, wherein the potassium phosphate is present in about 0.1 M to about 0.2 M.

19. The kit of any one of claims 16 to 18, wherein the buffer comprises a chelating agent.

20. The kit of claim 19, wherein the chelating agent is EDTA.

21. The kit of claim 19 or 20, wherein the chelating agent is present in about 1 mM to about

5mM.

22. The kit of any one of claims 16 to 21, wherein the buffer is at a pH value of about 7 to about 7.5, or about 7.5 to about 8.

23. The kit of any preceding claim, wherein the kit further comprises sulfosalicyclic acid, polyethylene glycol p-tert-octylphenyl ether (TRITON-X-IOO), a vinylpyridine, triethanolamine, or any combination thereof.

24. A method for measuring a glutathione concentration or glutathionylation in a biological sample, comprising 1) incubating the biological sample for a period of time to reduce glutathione disulfide in the biological sample to generate a free sulfhydryl group (thiol), and 2) detecting the flee sulfhydryl group by fluorescence.

25. The method of claim 24, wherein the period of time is about 5 minutes to about 30 minutes.

26. The method of claim 25, wherein the period of time is about 5 minutes to about 10 minutes, or about 10 minutes to about 20 minutes.

27. The method of any preceding claim, wherein the incubation is carried out in a solution that comprises glutaredoxin, nicotinamide adenine dinucleotide phosphate (NADPH), and glutathione reductase (GR).

28. The method of any preceding claim, further comprising contacting the biological sample with a fluorescent substrate.

29. The method of claim 28, wherein the fluorescent substrate compries an eosin-labeled glutathione disulfide.

30. The method of claim 29, wherein the fluorescent substrate is di-eosin-glutathione

disulfide (Di-E-GSSG).

31. The method of any preceding claim, further comprising shaking the biological sample for at least: about 10 seconds to about 30 seconds.

32. The method of any preceding claim, wherein the detecting by fluorescence is at about 545 nm after exciting at about 520 nm for at least about 20 minutes to about 60 minutes.

33. The method of any preceding claim, further comprising contacting the biological sample with sulfosalicyclic acid, polyethylene glycol p-tert-octylphenyl ether (TRITON-X-100), or a combination thereof, before the measuring to be able to measure glutathione disulfide (GSSG).

34. The method of any preceding claim, further comprising contacting the biological sample with an alkylating agent, before the measuring.

35. The method of claim 34, wherein the alkylating agent is a vinylpyridine or N-ethyl- maleimide.

36. The method of claim 35, wherein the vinylpyridine is 2-vinylpyridine or 4-vinylpyridine.

37. The method of claim 34 or 35, wherein the vinylpyridine is present in a concentration of about 0.1% to about 1% w/w or v/v.

38. The method of any one of claims 34 to 37, further comprising contacting the biological sample with triethanolamine, before the measuring.

39. The method of claim 38, wherein the triethanolamine is present in a concentration of about 0.1% to about 1% w/w or v/v.

40. The method of any preceding claim, wherein the detecting comprises using a standard fluorescent plate reader.

41. The kit or method of any preceding claim, wherein the biological sample is a plasma, a serum, or a cell lysate.

42. The kit or method of any preceding claim, wherein the biological sample has about 1 pg to about 5 pg of protein.

43. The kit or method of any preceding claim, wherein the biological sample is less than 1 ml.

44. The kit or method of any preceding claim, wherein the biological sample is about 5 mΐ to about 20 mΐ, about 10 mΐ to about 30 mΐ, or about 30 mΐ to about 100 mΐ.

45. The kit or method of any preceding claim, wherein the sample is frozen.

46. The kit or method of any preceding claim, wherein the sample is snap frozen.