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WO2002103036A1 - Methode enzymatique de quantification rapide de la glutathione oxydee et reduite - Google Patents

Methode enzymatique de quantification rapide de la glutathione oxydee et reduite Download PDF

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Publication number
WO2002103036A1
WO2002103036A1 PCT/KR2001/001053 KR0101053W WO02103036A1 WO 2002103036 A1 WO2002103036 A1 WO 2002103036A1 KR 0101053 W KR0101053 W KR 0101053W WO 02103036 A1 WO02103036 A1 WO 02103036A1
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Prior art keywords
glutathione
oxidized
reduced glutathione
gsh
sample
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Inventor
Young-Mee Park
Eun-Mi Choi
Jin-Hee Choi
Sung-Hoon Kim
Sun-Hee Baek
Sang-Seop Lee
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Priority to KR1020000005954A priority Critical patent/KR20010078585A/ko
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Priority to PCT/KR2001/001053 priority patent/WO2002103036A1/fr
Priority to JP2002553796A priority patent/JP2004518426A/ja
Priority to CN01802073A priority patent/CN1392901A/zh
Publication of WO2002103036A1 publication Critical patent/WO2002103036A1/fr
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/26—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving oxidoreductase
    • E—FIXED CONSTRUCTIONS
    • E02—HYDRAULIC ENGINEERING; FOUNDATIONS; SOIL SHIFTING
    • E02B—HYDRAULIC ENGINEERING
    • E02B15/00—Cleaning or keeping clear the surface of open water; Apparatus therefor
    • E02B15/04—Devices for cleaning or keeping clear the surface of open water from oil or like floating materials by separating or removing these materials
    • E02B15/10—Devices for removing the material from the surface
    • E—FIXED CONSTRUCTIONS
    • E02—HYDRAULIC ENGINEERING; FOUNDATIONS; SOIL SHIFTING
    • E02B—HYDRAULIC ENGINEERING
    • E02B15/00—Cleaning or keeping clear the surface of open water; Apparatus therefor
    • E02B15/04—Devices for cleaning or keeping clear the surface of open water from oil or like floating materials by separating or removing these materials
    • E02B15/08—Devices for reducing the polluted area with or without additional devices for removing the material
    • E02B15/0814—Devices for reducing the polluted area with or without additional devices for removing the material with underwater curtains

Definitions

  • the present invention relates to a method for rapid
  • oxidized and reduced glutathione ⁇ glutamylcysteinylglycine
  • GSSG • GSH oxidized and reduced glutathione
  • Glutathione a low-molecular weight thiol coumpound, exists in cells mostly in free form, but partly forms mixed-disulfide bond with cysteine or coenzyme A.
  • the glutathione exists in forms of reduced glutathione (GSH) and oxidized glutathione (or .glutathione disulfide, GSSG) depending on oxidized/reduced state of the cell.
  • GSH reduced glutathione
  • GSSG oxidized glutathione
  • Most of the intracellular free glutathione normally exist as reduced form (GSH) , meanwhile, the level of oxidized form is extremely low.
  • GSH in eucaryotic cells is known to account for over 80% of total cytoplasmic low molecular weight thiol compounds, and 10 to 15% of mitochodrial low molecular weight thiol compounds.
  • ROS reactive oxygen species
  • Methods for quantitative measurement of glutathione are classified into chromatographic, enzymatic and chemical methods, that is, a method using HPLC(high performance liquid chromatography) , a method using spectrophotometer for assaying enzyme activity and a method using o- phthalaldehyde (OPT) which is known to react with glutathione specifically (see : Sies, H., Assay of glutathione, glutathione disulfide, and glutathione mixed disulfides in biological samples, Methods Enzymol . r 77:373- 383, 1981; Tietze, Enzymatic method for quantitative determination of nanogram amounts of total and oxidized glutathione: applications to mammalian blood and other tissues, Anal .
  • OPT o- phthalaldehyde
  • a chromatographic method using HPLC a method for quantitative measurement by treating a glutathione- contained sample with light-absorbing material or fluorescent material followed by measuring absorbance or fluorescence of the materials separated by HPLC, can detect the minor level of 10 ⁇ 9 mol glutathione, however, has disadvantages that it takes over 30min for one cycle of HPLC analysis, and two cycles of HPLC analysis have to run for the measurement of GSH and GSSG separately in one sample, thus, it is very time consuming and inappropriate for analysis of a large number of samples.
  • Enzymatic methods include a method using an oxidizing agent such as DTNB (5, 5 '-dithiobis- (2-nitrbenzoic acid)) and glutathione reductase, a glyoxalase method which measures lactoylglutathione produced by glyoxalase I-catalyzed reaction between GSH and methylglyoxal, and a method using GSH transferase which produces a mixture of chlorodinitrobenzene and GSH.
  • the enzymatic methods have an advantage of speediness of sample analysis (in a few minutes) .
  • Another, a relatively simple method for quantitation of glutathione is a flourometric method, which uses the property of OPT forming fluorescence complex with GSH or GSSG in the absence of other sulfur compounds.
  • Flouorometric method using OPT is simple and useful for analysing a large number of samples, however, has revealed the low specificity caused by inhibition of OPT reaction with GSH or GSSG by other intracellular sulfur compounds and a ino acids, as well as, is not appropriate for tissue samples because measurement of GSSG in NEM(N- ethylmaleimide) -treated samples using OPT is carried out under a condition of high pH.
  • a recent method which apply aforementioned method to the multiwell plate was neither appropriate.
  • the present inventors have made an effort to develop a simple and accurate method for the quantitative measurement of GSH and GSSG in cell, and discovered that GSH and GSSG can be quantitated in a simple and accurate manner by introducing a step of treating a sample with a GSH trapping agent to the conventional enzymatic assay method of glutathione which employs oxidizing agent and glutathione reductase.
  • a primary object of present invention is, therefore, to provide a method for the simultaneous quantitative measurement of oxidized and reduced glutathione by employing oxidizing agent, glutathione reductase and GSH trapping agent.
  • Figure la is a HPLC chromatogram of oxidized form of glutathione (GSSG) and reduced form of glutathione (GSH) from a sample without NEM treatment.
  • Figure lb is a HPLC chromatogram of GSSG and GSH from a sample treated with NEM.
  • Figure 2 is a graph showing the measurement of absorbance of TNB produced in a sample treated with DTNB and glutathione reductase over a certain period of time.
  • Figure 3a is a standard curve of protein established by Bradford method.
  • Figure 3b is a GSH standard curve showing the changes in slopes of absorbance plot.
  • Figure 3c is a GSSG standard curve showing the changes in slopes of absorbance plot.
  • Figure 4a is a graph showing the changes in slopes of absorbance plot which represent the amounts of total glutathione measured with increasing amount of tissue samples.
  • Figure 4b is a graph showing the changes in slopes of absorbance plot which represent the amounts of GSSG measured with increasing amount of tissue samples.
  • the method for rapid quantitation of oxidized and reduced glutathione comprises the steps of: obtaining a tissue sample and then preparing a homogenate of the tissue whose metabolism is blocked; preparing equal volume of the homogenate fractions with and without a GSH trapping agent, and adding phosphate buffer, distilled water and NADPH solution to each of the fractions; adding glutathione reductase and oxidizing agent to each of the fractions to initiate reaction; and, measuring absorbances of the reaction mixture containing the fractions with and without a GSH trapping agent to quantitate oxidized glutathione (GSSG) and total glutathione, respectively, then, subtracting the amount of GSSG from that of total glutathione to determine the amount of reduced glutathione (GSH) .
  • the method for quantitative measurement of intracellular glutathione is further illustrated by the following steps .
  • Step 1 Preparation of samples
  • a tissue sample is prepared to give a homogenate of the tissue whose metabolism is blocked: the tissue sample is obtained from animal or plant source, freeze-dried at a temperature of -70 °C and treated with 5% PCA (perchloric acid) solution to give a homogenate of the tissue whose metabolism is blocked.
  • PCA perchloric acid
  • N-ethylmaleimide is preferably used as the GSH trapping agent, which is preferably added to the fractions to reach a final concentration of 20 to lOOmM, and 2 to 3 volumes of phosphate buffer is preferably added to the fraction to reach a final concentration of 50 to 150mM, 6 to 7 volumes of distilled water is preferably added to the fractions, and 20 to 30% (v/v) of the NADPH solution is preferably added to the fractions to reach a concentration of 0.1 to 0.3mM.
  • NAM N-ethylmaleimide
  • the reaction is initiated by adding glutathione reductase and oxidizing agent to each of the fractions: 10 to 15% (v/v) of the glutathione reductase is preferably added to the fractions to reach a final concentration of 0.05 to 0.2U/ml, and 5, 5 ' -dithiobis- (2-nitrobenzoic acid) (DTNB) is preferably used as an oxidizing agent, of which 10 to 15% (v/v) is preferably added to the fractions to reach a final concentration of 0.02 to O.lmM.
  • Step 4 Measurement of absorbance
  • Oxidized glutathione (GSSG) and total glutathione are quantitated, respectively, by measuring absorbances of the assay mixtures containing the fractions with and without a GSH trapping agent, then the amount of reduced glutathione (GSH) is determined by subtracting the amount of GSSG from that of total glutathione: the absorbance is preferably measured at 405nm.
  • the glutathione in the tissue or cell is susceptible to the intracellular glutathione metabolism- related enzymes in the course of extracting glutathione, it has to be bewared, in conducting the quantitation of oxidized and reduced glutathione using the method of the invention, that the analysis results may not reflect the status of the tissue sample per se.
  • Another problem reside in the method of the invention is that oxygen and metal ions present in the sample would react with sulfhydryl group of reduced glutathione (GSH) to yield oxidized glutathione (GSSG) during the step of treating samples to measure the amount of glutathione.
  • the present inventors have employed a strategy to block changes in the glutathione pool of a tissue sample obtained from an animal to measure the amount of GSH and GSSG in a tissue sample correctly.
  • red/ox ratio of glutathione in the tissue sample is changed by the metabolism-related enzymes such as glutathione reductase and NADPH after the tissue sample is obtained from an animal
  • the metabolism-related enzymes such as glutathione reductase and NADPH
  • changes in red/ox ratio of glutathione by metabolism was blocked by immediate freeze-clamping of the removed tissue sample.
  • the tissue samples were fixed and frozen with liquid nitrogen and stored at -70 °C until use.
  • another strategy of blocking metabolism of the removed tissue sample is that metabolism-related enzymes are denatured by homogenizing the frozen tissue sample in the presence of protein denaturing agent, 5% (v/v) PCA solution.
  • the method for quantitative measurement of GSSG requires the pre-removal of GSH in a sample. Also, to quantitate intracellular oxidized glutathione accurately, artificial oxidation of GSH into GSSG after the tissue sample has been obtained from an animal should be prevented, and the prevention of oxidation of GSH can be attained by masking the sulfhydryl group of GSH.
  • the present inventors have employed the strategy of masking GSH in the sample by using a GSH trapping agent such as NEM which inhibits the disulfide linkage formation between sulfhydryl groups by formation of a covalent bond with sulfhydryl group of glutathione.
  • the reaction rate of NEM at neutral pH is as fast as less than lmin, making its use very effective. Since NEM also inhibits the activity of glutathione reductase, residual NEM should be removed after complete masking of GSH, thus, in accordance with the invention, for using in GSSG assay, the reaction mixture containing NEM was extracted with the equal volume of ether to remove residual NEM.
  • Amount of GSH is determined by continuous reduction rate of DTNB to TNB resulting from the reaction between GSH and DTNB, and GSSG contained in the sample or produced by the reaction between GSH and DTNB is reduced to GSH by the reaction of glutathione reductase and NADPH.
  • amount of total glutathione, sum of amount of GSH and GSSG is expressed by the sum of reduction rate to TNB.
  • the reduction rate to TNB is accurately proportional to the amount of total glutathione and is determined by measuring absorbance at 405nm.
  • total amount of glutathione is determined by measuring the slope of absobance plot over the range of linear increase, which represent amount of GSH to which GSH and GSSG in the sample are converted as described above, and, from the NEM-treated sample, amount of oxidized glutathione (GSSG) is determined by measuring absorbance.
  • standard curves for oxidized and reduced glutathione are established by measuring absorbances of certain amounts of oxidized and reduced glutathione respectively, and then, amount of GSSG is determined by comparing absorbances of total glutathione and GSSG to the absorbances in standard curves, and converted into nmol glutathione/ g of protein after analyzing protein content in the sample.
  • a multiwell plate may be employed for analysis of a large number of samples for oxidized and reduced glutathione. That is, the quantitation of oxidized and reduced glutathione in a large number of samples can be conducted in parallel by steps of: dispensing phosphate buffer, distilled water and NADPH solution in each well of a multiwell plate; adding equal volumes of NEM-treated or untreated sample into each well followed by adding glutathione reductase and DTNB to each well to initiate reaction; and, measuring absorbance of the reaction mixture containing the NEM-treated sample to quantitate oxidized glutathione (GSSG) and the NEM-untreated sample to quantitate total glutathione, then, subtracting the amount of GSSG from that of total glutathione to determine the amount of reduced glutathione (GSH) : herein, the condition of each step is identical to that of method for quantitative measurement of glutathione described above.
  • Example 1 Preparation of tissue sample and NEM treatment for quantitative measurement of glutathione
  • NEM was used as a GSH trapping agent in the invention.
  • NEM solution very unstable at room temperature, was prepared just prior to use and kept on ice bath, while, relatively stable under a condition of low pH, was prepared to a concentration of 250mM in 5% (v/v) PCA.
  • PCA homogenate of tissue prepared above was divided into 60 fd and 400 id, and then, 100 fd of 250mM NEM was added to 400 fd aliquot of PCA homogenate to yield a final concentration of NEM of 250mM, which was immediately followed by neutralization by addition of
  • NEM-treated and NEM-untreated fractions prepared above were centrifuged respectively to obtain a supernatant, and then, the supernatant of NEM-untreated samples were diluted 5 to 10 folds with lOO M phosphate buffer (pH 7.0) containing ImM EDTA to prepare samples for quantitative measurement of total glutathione, and the supernatant of NEM-treated samples were subjected to pretreatment for removal- of unreacted residual NEM, which includes 10 times of extraction with an equal volume of ether and subsequent removal of ether by drying with a vacuum pump to prepare samples for quantitative measurement of GSSG.
  • lOO M phosphate buffer pH 7.0
  • the protein precipitates obtained by centrifugation of NEM- untreated homogenate of tissue were dissolved in 100 fd of IN NaOH, diluted 10 fold, and then subjected to measurement of protein by Bradford assay method.
  • a protein standard curve was established by using bovine serum albumin (BSA) at concentrations shown in Table 1 by Bradford method (see: Figure 3a) .
  • Figure 3a is a standard curve of protein established by Bradford method.
  • Example 2 Quantitative measurement of glutathione using a multiwell plate
  • the amount of glutathione in tissues was measured by using a multiwell plate for assaying a large number of samples as follows: first, a mixture of 50 fd of 5x phosphate buffer (500mM phosphate buffer containing 5mM EDTA, pH 7.0), 125 fd of distilled water and 5 fd of lOmM NADPH was distributed into the each well.
  • 5x phosphate buffer 500mM phosphate buffer containing 5mM EDTA, pH 7.0
  • 5 fd of lOmM NADPH was distributed into the each well.
  • a glutathione standard curve was established by measuring the absorbance of glutathione standard solution prepared in. Example 2 (see: Tables 2 and 3, Figures 3b and 3c) .
  • Tables 2 and 3 above show concentrations of GSH and GSSG, and corresponding slopes of standard curves, respectively.
  • Figures 3a and 3b show a GSH standard curve and a GSSG standard curve, respectively.
  • the slope of absorbance plot over the range of linear increase was determined to be 3.1871
  • the slope was determined to be 5.1794.
  • Example 3 Total glutathione and GSSG concentration (nmole/ml sample) measured in Example 3 were converted into the amount of glutathione (nmole) per mg of protein.
  • the amount of protein was measured by Bradford method and standard curve in a similar manner as in Example 2 where the amount pelleted protein was measured following centrifugation of NEM-untreated homogenate. The protein concentration was determined to be 13.88 mg/ml.
  • the values of total glutathione concentration (467.45nmol/ml) and GSSG concentration (9.63nmol/ml) were converted into 33.68nmol/mg and 0.69nmol/mg, respectively. Therefore, specific amount of GSH in the tissue sample was turned out to be 32.99nmol/mg.
  • the present invention provides a method for the simultaneous quantitation of oxidized and reduced glutathione by introducing a step of treating a sample with a GSH trapping agent to the conventional enzymatic assay method of glutathione employing oxidizing agent and glutathione reductase.
  • the invented method for rapid quantitation of oxidized and reduced glutathione comprises the steps of: obtaining a tissue sample and then preparing a homogenate of the tissue whose metabolism is blocked; preparing equal volume of the homogenate fractions with and without a GSH trapping agent, and adding phosphate buffer, distilled water and NADPH solution to each of the fractions; adding glutathione reductase and oxidizing agent to each of the fractions to initiate reaction; and, measuring absorbances of the reaction mixtures containing the fractions with and without a GSH trapping agent to quantitate oxidized glutathione (GSSG) and total glutathione, respectively, then, subtracting the amount of GSSG from that of total glutathione to determine the amount of reduced glutathione (GSH) .
  • the amount of oxidized and reduced glutathione can be simultaneously measured in a simple and accurate manner, which makes possible its practical application in the diagnosis and treatment of diseases caused by the abnormality of oxidized/reduce

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Abstract

L'invention concerne une méthode de mesure quantitative simultanée de la glutathione réduite et de la glutathione oxydée, par rapport à un dosage de glutathione classique, dans laquelle un agent oxydatif et une glutathione réductase sont utilisés, et qui consiste à traiter l'agent de piégeage de la glutathione réduite (GSH). Une méthode enzymatique de quantification rapide de la glutathione oxydée/réduite consiste à : (I) prélever un tissu et préparer un homogénat à partir du tissu dont le métabolisme est bloqué ; (ii) préparer un volume égal de fractions d'homogénat avec ou sans neutralisant de GSH ; et ajouter une solution saline dans un tampon phosphate, de l'eau distillée et une solution de NADPH à chaque fraction ; (iii) ajouter la glutathione réductase et l'agent oxydatif à chaque fraction, de sorte qu'une réaction soit provoquée ; (iv) mesurer l'absorbance des fractions avec ou sans agent de piégeage de GSH, de sorte que la teneur en glutathione oxydée (GSSG) et en glutathione totale soit quantifiée, et soustraire la quantité de GSSG de la quantité totale de glutathione, de manière que la glutathione réduite (GSH) soit quantifiée. Selon la méthode de l'invention, la mesure quantitative de la glutathione oxydée et réduite peut s'effectuer de manière simultanée avec une grande précision, ce qui permet son utilisation dans le diagnostic et le traitement de maladies induites par des taux anormaux de GSH/GSSG dans les cellules.
PCT/KR2001/001053 2001-06-20 2001-06-20 Methode enzymatique de quantification rapide de la glutathione oxydee et reduite Ceased WO2002103036A1 (fr)

Priority Applications (4)

Application Number Priority Date Filing Date Title
KR1020000005954A KR20010078585A (ko) 2001-06-20 2000-02-09 효소역학을 이용한 산화형·환원형 글루타치온의 정량적고속측정방법
PCT/KR2001/001053 WO2002103036A1 (fr) 2001-06-20 2001-06-20 Methode enzymatique de quantification rapide de la glutathione oxydee et reduite
JP2002553796A JP2004518426A (ja) 2001-06-20 2001-06-20 酸化型及び還元型グルタチオンの迅速定量のための酵素学的方法
CN01802073A CN1392901A (zh) 2001-06-20 2001-06-20 酶法快速定量测定氧化型和还原型谷胱甘肽

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RU2436101C1 (ru) * 2010-06-25 2011-12-10 Государственное Образовательное Учреждение Высшего Профессионального Образования "Кубанский государственный медицинский университет" (ГОУ ВПО КГМУ) Способ диагностики нарушений метаболизма в организме в условиях окислительного стресса
WO2012030960A1 (fr) * 2010-09-01 2012-03-08 Promega Corporation Dosage de glutathion oxydé
CN112362646A (zh) * 2020-10-27 2021-02-12 华南理工大学 一种基于纳米酶的谷胱甘肽传感器及其制备方法与应用
CN114113064A (zh) * 2021-12-27 2022-03-01 郑州大学 基于苯并双噻唑的光响应类氧化物酶及其制备方法和在比色检测食品中谷胱甘肽的应用

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KR20010078585A (ko) * 2001-06-20 2001-08-21 복성해 효소역학을 이용한 산화형·환원형 글루타치온의 정량적고속측정방법
CN102978276B (zh) * 2012-12-24 2015-11-04 丁兆明 一种快速检测细胞中谷胱甘肽的方法
CN105004689B (zh) * 2015-07-31 2017-09-12 中国烟草总公司郑州烟草研究院 卷烟烟气总粒相物诱导细胞氧化应激gsh/gssg的测定方法
CN106338476A (zh) * 2016-08-16 2017-01-18 南京林业大学 一种谷胱甘肽还原酶活性单位换算方法
EP3719500B1 (fr) * 2017-11-28 2023-01-04 Cell2in, Inc. Procédé d'amélioration de la qualité d'une cellule thérapeutique par la mesure du glutathion en temps réel
CN109884031A (zh) * 2019-03-14 2019-06-14 中国人民解放军军事科学院军事医学研究院 检测还原型谷胱甘肽和/或氧化型谷胱甘肽的方法
WO2020262297A1 (fr) * 2019-06-28 2020-12-30 株式会社島津製作所 Procédé de mesure de distribution de glutathion réduit sur un tissu biologique, et procédé d'acquisition de données de diagnostic
CN110656155A (zh) * 2019-09-29 2020-01-07 江西乐成生物医疗有限公司 谷胱甘肽还原酶测定试剂质控品及制备方法
CN117292748B (zh) * 2023-09-25 2024-06-07 程静为 一种用于酶法生产谷胱甘肽的酶活性优化方法

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Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
RU2436101C1 (ru) * 2010-06-25 2011-12-10 Государственное Образовательное Учреждение Высшего Профессионального Образования "Кубанский государственный медицинский университет" (ГОУ ВПО КГМУ) Способ диагностики нарушений метаболизма в организме в условиях окислительного стресса
WO2012030960A1 (fr) * 2010-09-01 2012-03-08 Promega Corporation Dosage de glutathion oxydé
US9816127B2 (en) 2010-09-01 2017-11-14 Promega Corporation Oxidized glutathione assay
US10889849B2 (en) 2010-09-01 2021-01-12 Promega Corporation Oxidized glutathione assay
CN112362646A (zh) * 2020-10-27 2021-02-12 华南理工大学 一种基于纳米酶的谷胱甘肽传感器及其制备方法与应用
CN114113064A (zh) * 2021-12-27 2022-03-01 郑州大学 基于苯并双噻唑的光响应类氧化物酶及其制备方法和在比色检测食品中谷胱甘肽的应用
CN114113064B (zh) * 2021-12-27 2023-09-08 郑州大学 基于苯并双噻唑的光响应类氧化物酶及其制备方法和在比色检测食品中谷胱甘肽的应用

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