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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

Reaction of Ellman's reagent with glutathione.
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fig1: Reaction of Ellman's reagent with glutathione.

Mentions: Glutathione (GSH) is chemically known as (2S)-2-amino-4-[[(1R)-[(carboxymethyl) carbamoyl]-2-sulfanylethyl] carbamoyl] butanoic acid. GSH is a tripeptide (Figure 1), often considered as the mother of all antioxidants and is present in almost every cell. Because GSH exists within the cells, it is in a prime position to neutralize free radicals. The strong antioxidant effect of GSH helps keep cells running smoothly and also helps the liver to remove chemicals that are foreign to the body such as drugs/pollutants [1, 2]. In addition, GSH has the potential to fight almost any disease; particularly those associated with ageing, since free radical damage is the cause of many of the diseases of old age. GSH is nucleophilic at the sulfur and attacks poisonous electrophilic conjugate acceptors. Thiol groups are kept in a reduced state at a concentration of approximately ~5 mM in animal cells. In effect, GSH reduces any disulfide bond formed within cytoplasmic proteins to cysteines by acting as an electron donor. In the process, GSH is converted to its oxidized form glutathione disulfide (GSSG). Glutathione is found almost exclusively in its reduced form, since the enzyme that reverts it from its oxidized form, GSSG, is constitutively active and inducible upon oxidative stress. In fact, the ratio of GSH to GSSG within cells is often used scientifically as a measure of cellular toxicity [3–5].


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)

Reaction of Ellman's reagent with glutathione.
© Copyright Policy - open-access
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC4834400&req=5

fig1: Reaction of Ellman's reagent with glutathione.
Mentions: Glutathione (GSH) is chemically known as (2S)-2-amino-4-[[(1R)-[(carboxymethyl) carbamoyl]-2-sulfanylethyl] carbamoyl] butanoic acid. GSH is a tripeptide (Figure 1), often considered as the mother of all antioxidants and is present in almost every cell. Because GSH exists within the cells, it is in a prime position to neutralize free radicals. The strong antioxidant effect of GSH helps keep cells running smoothly and also helps the liver to remove chemicals that are foreign to the body such as drugs/pollutants [1, 2]. In addition, GSH has the potential to fight almost any disease; particularly those associated with ageing, since free radical damage is the cause of many of the diseases of old age. GSH is nucleophilic at the sulfur and attacks poisonous electrophilic conjugate acceptors. Thiol groups are kept in a reduced state at a concentration of approximately ~5 mM in animal cells. In effect, GSH reduces any disulfide bond formed within cytoplasmic proteins to cysteines by acting as an electron donor. In the process, GSH is converted to its oxidized form glutathione disulfide (GSSG). Glutathione is found almost exclusively in its reduced form, since the enzyme that reverts it from its oxidized form, GSSG, is constitutively active and inducible upon oxidative stress. In fact, the ratio of GSH to GSSG within cells is often used scientifically as a measure of cellular toxicity [3–5].

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