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A Simple HPLC-UV Method for the Determination of Glutathione in PC-12 Cells.

Appala RN, Chigurupati S, Appala RV, Krishnan Selvarajan K, Islam Mohammad J - Scientifica (Cairo) (2016)

Bottom Line: Due to its own sulfhydryl (SH) group, GSH readily reacts with Ellman's reagent to form a stable dimer which allows for quantitative estimation of GSH in biological systems by UV detection.The separation was achieved using a C8 column with a mobile phase consisting of phosphate buffer adjusted to pH 2.5 (mobile phase A) and acetonitrile (mobile phase B), running in a segmented gradient manner at a flow rate of 0.8 mL/min, and UV detection was performed at 280 nm.Limit of detection (LOD) and limit of quantification (LOQ) were 0.05 and 0.1 μg/mL, respectively.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmaceutical Chemistry, Sultan Ul Uloom College of Pharmacy, Telangana, Hyderabad 500 034, India.

ABSTRACT
A highly sensitive and simple HPLC-UV method was developed and validated for the assay of glutathione (GSH) in PC-12 cells. Glutathione is a major intracellular antioxidant having multiple biological effects, best known for its cytoprotective effects against cell damage from reactive oxygen species and toxic reactive metabolites and regulating the cellular redox homeostasis. Due to its own sulfhydryl (SH) group, GSH readily reacts with Ellman's reagent to form a stable dimer which allows for quantitative estimation of GSH in biological systems by UV detection. The separation was achieved using a C8 column with a mobile phase consisting of phosphate buffer adjusted to pH 2.5 (mobile phase A) and acetonitrile (mobile phase B), running in a segmented gradient manner at a flow rate of 0.8 mL/min, and UV detection was performed at 280 nm. The developed HPLC-UV method was validated with respect to precision, accuracy, robustness, and linearity within a range of 1-20 μg/mL. Limit of detection (LOD) and limit of quantification (LOQ) were 0.05 and 0.1 μg/mL, respectively. Furthermore, the method shows the applicability for monitoring the oxidative stress in PC-12 cells.

No MeSH data available.


Related in: MedlinePlus

Representative chromatogram from 6 replicates is shown. (a) 2-Nitro-5-mercapto-benzoic (NMB) acid, Peak 1 with RT 7.43 min; glutathione dimer (GSH Dimer), Peak 2 with RT 10.93 min, and Ellman's reagent (DTNB), Peak 3 RT 14.12 min in untreated PC-12 cells (control). (b) NMB, Peak 1 with RT 7.44 min, GSH Dimer, Peak 2 with RT 10.88 min, and DTNB, Peak 3 retention time 14.10 min in PC-12 cells treated with methylglyoxal. (c) NMB, Peak 1 with RT 7.59 min, glutathione dimer, Peak 2 with RT 10.92 min, and DTNB, Peak 3 RT 14.44 min in PC-12 cells treated with CoCl2.
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fig3: Representative chromatogram from 6 replicates is shown. (a) 2-Nitro-5-mercapto-benzoic (NMB) acid, Peak 1 with RT 7.43 min; glutathione dimer (GSH Dimer), Peak 2 with RT 10.93 min, and Ellman's reagent (DTNB), Peak 3 RT 14.12 min in untreated PC-12 cells (control). (b) NMB, Peak 1 with RT 7.44 min, GSH Dimer, Peak 2 with RT 10.88 min, and DTNB, Peak 3 retention time 14.10 min in PC-12 cells treated with methylglyoxal. (c) NMB, Peak 1 with RT 7.59 min, glutathione dimer, Peak 2 with RT 10.92 min, and DTNB, Peak 3 RT 14.44 min in PC-12 cells treated with CoCl2.

Mentions: Glutathione is required for the detoxification of methylglyoxal, a toxic metabolite of liver. It is evident from earlier reports that methylglyoxal accumulated in cells due to GSH depletion is the major cause for cellular dysfunction and oxidative stress [17]. Such stress is also observed in conditions such as inflammation, and the cell copes with the stress with intracellular antioxidants. GSH is a major antioxidant present in cells and it exists in reduced as well as oxidized form, depending upon the oxidative state of the cell. An accurate measurement of intracellular GSH provides a means of determining the oxidative stress caused by an agent and the cellular response to it. Both methylglyoxal (Figure 3(b)) and CoCl2 (Figure 3(c)) treatment significantly altered the level of intracellular GSH compared to control (Figure 3(a)). It is possible that CoCl2 also activates GSH synthesizing enzymes, resulting in a higher level of GSH. Further studies will need to be done to test this possibility. There is a previous report that methylglyoxal interferes with GSH synthesis and secretion [18], which could be why methylglyoxal treated cells show a lower level of GSH, compared to control and CoCl2 treated cells. The present study was performed twice with the same sample procedure and HPLC chromatographic conditions. When compared with the control, in both the studies authors found a significant decrease in GSH upon methylglyoxal treatment. Further experimentation is needed to study the more accurate and precise results.


A Simple HPLC-UV Method for the Determination of Glutathione in PC-12 Cells.

Appala RN, Chigurupati S, Appala RV, Krishnan Selvarajan K, Islam Mohammad J - Scientifica (Cairo) (2016)

Representative chromatogram from 6 replicates is shown. (a) 2-Nitro-5-mercapto-benzoic (NMB) acid, Peak 1 with RT 7.43 min; glutathione dimer (GSH Dimer), Peak 2 with RT 10.93 min, and Ellman's reagent (DTNB), Peak 3 RT 14.12 min in untreated PC-12 cells (control). (b) NMB, Peak 1 with RT 7.44 min, GSH Dimer, Peak 2 with RT 10.88 min, and DTNB, Peak 3 retention time 14.10 min in PC-12 cells treated with methylglyoxal. (c) NMB, Peak 1 with RT 7.59 min, glutathione dimer, Peak 2 with RT 10.92 min, and DTNB, Peak 3 RT 14.44 min in PC-12 cells treated with CoCl2.
© Copyright Policy - open-access
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4834400&req=5

fig3: Representative chromatogram from 6 replicates is shown. (a) 2-Nitro-5-mercapto-benzoic (NMB) acid, Peak 1 with RT 7.43 min; glutathione dimer (GSH Dimer), Peak 2 with RT 10.93 min, and Ellman's reagent (DTNB), Peak 3 RT 14.12 min in untreated PC-12 cells (control). (b) NMB, Peak 1 with RT 7.44 min, GSH Dimer, Peak 2 with RT 10.88 min, and DTNB, Peak 3 retention time 14.10 min in PC-12 cells treated with methylglyoxal. (c) NMB, Peak 1 with RT 7.59 min, glutathione dimer, Peak 2 with RT 10.92 min, and DTNB, Peak 3 RT 14.44 min in PC-12 cells treated with CoCl2.
Mentions: Glutathione is required for the detoxification of methylglyoxal, a toxic metabolite of liver. It is evident from earlier reports that methylglyoxal accumulated in cells due to GSH depletion is the major cause for cellular dysfunction and oxidative stress [17]. Such stress is also observed in conditions such as inflammation, and the cell copes with the stress with intracellular antioxidants. GSH is a major antioxidant present in cells and it exists in reduced as well as oxidized form, depending upon the oxidative state of the cell. An accurate measurement of intracellular GSH provides a means of determining the oxidative stress caused by an agent and the cellular response to it. Both methylglyoxal (Figure 3(b)) and CoCl2 (Figure 3(c)) treatment significantly altered the level of intracellular GSH compared to control (Figure 3(a)). It is possible that CoCl2 also activates GSH synthesizing enzymes, resulting in a higher level of GSH. Further studies will need to be done to test this possibility. There is a previous report that methylglyoxal interferes with GSH synthesis and secretion [18], which could be why methylglyoxal treated cells show a lower level of GSH, compared to control and CoCl2 treated cells. The present study was performed twice with the same sample procedure and HPLC chromatographic conditions. When compared with the control, in both the studies authors found a significant decrease in GSH upon methylglyoxal treatment. Further experimentation is needed to study the more accurate and precise results.

Bottom Line: Due to its own sulfhydryl (SH) group, GSH readily reacts with Ellman's reagent to form a stable dimer which allows for quantitative estimation of GSH in biological systems by UV detection.The separation was achieved using a C8 column with a mobile phase consisting of phosphate buffer adjusted to pH 2.5 (mobile phase A) and acetonitrile (mobile phase B), running in a segmented gradient manner at a flow rate of 0.8 mL/min, and UV detection was performed at 280 nm.Limit of detection (LOD) and limit of quantification (LOQ) were 0.05 and 0.1 μg/mL, respectively.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmaceutical Chemistry, Sultan Ul Uloom College of Pharmacy, Telangana, Hyderabad 500 034, India.

ABSTRACT
A highly sensitive and simple HPLC-UV method was developed and validated for the assay of glutathione (GSH) in PC-12 cells. Glutathione is a major intracellular antioxidant having multiple biological effects, best known for its cytoprotective effects against cell damage from reactive oxygen species and toxic reactive metabolites and regulating the cellular redox homeostasis. Due to its own sulfhydryl (SH) group, GSH readily reacts with Ellman's reagent to form a stable dimer which allows for quantitative estimation of GSH in biological systems by UV detection. The separation was achieved using a C8 column with a mobile phase consisting of phosphate buffer adjusted to pH 2.5 (mobile phase A) and acetonitrile (mobile phase B), running in a segmented gradient manner at a flow rate of 0.8 mL/min, and UV detection was performed at 280 nm. The developed HPLC-UV method was validated with respect to precision, accuracy, robustness, and linearity within a range of 1-20 μg/mL. Limit of detection (LOD) and limit of quantification (LOQ) were 0.05 and 0.1 μg/mL, respectively. Furthermore, the method shows the applicability for monitoring the oxidative stress in PC-12 cells.

No MeSH data available.


Related in: MedlinePlus