Glutathione 1500mg

Glutathione — Introduction and Research Disclaimer

Glutathione (γ-L-glutamyl-L-cysteinyl-glycine; GSH) is a ubiquitous tripeptide thiol found in virtually all eukaryotic cells. As the most abundant low-molecular-weight thiol in mammalian cells, glutathione serves as a principal endogenous antioxidant, a critical regulator of cellular redox homeostasis, and an essential cofactor for numerous enzymatic reactions involved in detoxification, metabolism, and signal transduction. Intracellular glutathione concentrations typically range from 1–10 mM, with levels declining in states of oxidative stress, aging, and various pathological conditions.

IMPORTANT RESEARCH DISCLAIMER: All products offered by BioSim Peptides, including Glutathione 1500mg, are sold strictly for in vitro laboratory research and experimental purposes only. These products are not intended for human or veterinary use, not for diagnostic or therapeutic purposes, and are not to be used as drugs, food additives, dietary supplements, or cosmetics. Researchers must handle these materials in accordance with all applicable institutional, local, state, and federal regulations. Appropriate personal protective equipment (PPE), biosafety protocols, and institutional review board (IRB) approval (where applicable) must be employed.

Molecular Overview

Glutathione is a tripeptide with the unusual sequence γ-L-Glutamyl — L-Cysteinyl — Glycine. Unlike conventional peptide bonds, the linkage between the γ-carboxyl group of glutamate and the α-amino group of cysteine forms a gamma peptide bond (γ-Glu-Cys) that is resistant to cleavage by most cellular peptidases but specifically recognized by the enzyme γ-glutamyltranspeptidase (GGT), the only enzyme capable of hydrolyzing this bond.

Key molecular characteristics:

• Sequence: γ-L-Glutamyl-L-cysteinyl-glycine (reduced form: GSH; oxidized dimer: GSSG)
• Molecular Formula: C₁₀H₁₇N₃O₆S
• Molecular Weight: 307.32 g/mol (reduced form, free acid)
• CAS Number: 70-18-8
• Thiol Group pKa: ~9.2 (cysteine sulfhydryl group)
• Solubility: Freely soluble in water (>50 mg/mL); soluble in aqueous buffers at physiological pH
• Redox Potential: E°’ = -240 mV (GSH/GSSG couple at pH 7.0)
• Stability: Lyophilized powder stable at 2–8°C; aqueous solutions susceptible to auto-oxidation to GSSG at neutral-to-alkaline pH
• Purity: ≥98% (typical research-grade specification, verified by HPLC)

The cysteine residue is the functional core of glutathione, providing the nucleophilic thiol (-SH) group that mediates its antioxidant, detoxification, and redox-regulatory activities. The gamma-glutamyl linkage is evolutionarily conserved and serves to protect glutathione from degradation by intracellular aminopeptidases, ensuring maintenance of high intracellular concentrations.

In its oxidized form, two glutathione molecules form a disulfide-bonded dimer (GSSG; molecular weight 612.63 g/mol). The ratio of reduced glutathione (GSH) to oxidized glutathione (GSSG) is a widely used biomarker of cellular oxidative stress, with healthy cells maintaining a GSH:GSSG ratio exceeding 100:1 under basal conditions.

Mechanism of Action

Glutathione exerts its biological effects through multiple mechanistically distinct pathways that collectively maintain cellular redox balance, facilitate xenobiotic detoxification, and regulate fundamental signaling processes.

Direct Antioxidant Activity

The cysteine thiol group of GSH directly scavenges reactive oxygen species (ROS) including superoxide anion (O₂•⁻), hydroxyl radical (•OH), hydrogen peroxide (H₂O₂), and peroxynitrite (ONOO⁻). In this non-enzymatic antioxidant capacity, GSH donates an electron or hydrogen atom to neutralize the radical species, itself being oxidized to the glutathionyl radical (GS•), which rapidly dimerizes to form GSSG. The reaction rate with hydroxyl radical approaches diffusion-limited kinetics (k ~ 10¹⁰ M⁻¹s⁻¹), making GSH an exceptionally efficient scavenger of this highly damaging species.

Enzymatic Antioxidant Functions

GSH serves as an obligate co-substrate for the glutathione peroxidase (GPx) family of selenoenzymes, which catalyze the reduction of H₂O₂ and organic hydroperoxides to water and corresponding alcohols, respectively:

2 GSH + ROOH → GSSG + ROH + H₂O

Additionally, GSH facilitates the regeneration of other antioxidant molecules, including the reduction of dehydroascorbate to ascorbate (vitamin C) and the maintenance of α-tocopherol (vitamin E) in its reduced, radical-scavenging form.

Glutathione S-Transferase (GST)-Mediated Detoxification

Glutathione S-transferases (GSTs) constitute a superfamily of phase II detoxification enzymes that catalyze the conjugation of GSH to electrophilic xenobiotics (drugs, environmental toxins, carcinogens) and endogenous electrophiles produced during oxidative stress (e.g., 4-hydroxynonenal, acrolein). GSH conjugation increases substrate hydrophilicity, facilitating biliary or renal excretion via ATP-dependent transporters including the multidrug resistance-associated proteins (MRPs). This pathway is critical for cellular defense against chemical toxicity and carcinogenesis.

Redox Signaling and Protein S-Glutathionylation

Beyond its role as a bulk antioxidant, GSH participates directly in redox-sensitive signal transduction through the reversible modification of protein cysteine residues via S-glutathionylation. This post-translational modification involves the formation of a mixed disulfide between GSH and a protein cysteine thiol (Protein-S-SG), protecting the cysteine from irreversible oxidation while modulating protein function. S-glutathionylation regulates the activity of numerous proteins including transcription factors (NF-κB, AP-1, p53), metabolic enzymes (GAPDH), ion channels (RyR), and cytoskeletal proteins (actin).

Mitochondrial Function and Apoptosis Regulation

Mitochondria contain a dedicated glutathione pool (10–15% of total cellular GSH) maintained in the reduced state by NADPH-dependent glutathione reductase. Mitochondrial GSH is essential for: (a) scavenging ROS generated by the electron transport chain; (b) maintaining mitochondrial membrane potential; and (c) regulating the mitochondrial permeability transition pore (mPTP). Depletion of mitochondrial GSH sensitizes cells to apoptotic stimuli by facilitating cytochrome c release and caspase activation.

Redox Recycling

GSSG generated through antioxidant and detoxification reactions is recycled back to GSH by glutathione reductase (GR), an NADPH-dependent flavoenzyme:

GSSG + NADPH + H⁺ → 2 GSH + NADP⁺

This recycling system, coupled with NADPH regeneration via the pentose phosphate pathway (glucose-6-phosphate dehydrogenase; G6PD), ensures sustained antioxidant capacity.

Research Applications

Glutathione is a fundamental research tool employed across a broad spectrum of biomedical disciplines investigating oxidative stress, cellular detoxification, and redox biology. Key research domains include:

Oxidative Stress and Redox Biology

Glutathione is the gold-standard analyte for quantifying cellular oxidative stress in experimental systems. Researchers employ GSH/GSSG ratio measurements (using HPLC, enzymatic recycling assays, or fluorescent probes) as a quantitative index of oxidative burden across diverse model systems, including cell culture, isolated organelles, and tissue homogenates. Exogenous GSH supplementation is widely used to dissect the contribution of oxidative stress to cellular phenotypes.

Hepatotoxicity and Drug Metabolism Research

The liver maintains the highest GSH concentrations of any organ (5–10 mM in hepatocytes). Glutathione is central to research on drug-induced liver injury (DILI), particularly acetaminophen (paracetamol) hepatotoxicity models, where GSH depletion below a critical threshold (~70% of basal levels) precipitates centrilobular necrosis. GSH repletion strategies are used to investigate mechanisms of hepatoprotection.

Neuroprotection and Neurodegenerative Disease Models

Neuronal GSH depletion is a hallmark of Parkinson disease, Alzheimer disease, and amyotrophic lateral sclerosis (ALS) pathogenesis. Research applications include investigating GSH-mediated protection against dopaminergic neuron loss in MPTP/6-OHDA models, mitochondrial dysfunction in neuronal cultures, and glutamate-induced excitotoxicity.

Cancer Biology and Chemoresistance

Elevated GSH levels in tumor cells contribute significantly to chemoresistance by conjugating and inactivating platinum-based drugs (cisplatin, carboplatin), alkylating agents (cyclophosphamide, melphalan), and anthracyclines (doxorubicin). Research on GSH depletion strategies (using buthionine sulfoximine; BSO) aims to sensitize resistant tumors to chemotherapy.

Aging and Senescence Research

Progressive decline in GSH levels with advancing age is a conserved phenomenon across species. GSH is employed in aging research to investigate the relationship between redox status and cellular senescence, telomere attrition, mitochondrial dysfunction, and age-related decline in immune function (immunosenescence).

Immune Function and Inflammation

T-lymphocyte activation, proliferation, and differentiation are exquisitely sensitive to intracellular GSH status. Research applications include investigating GSH modulation of Th1/Th2 balance, macrophage polarization, and dendritic cell antigen presentation. GSH depletion impairs lymphocyte proliferation, while GSH repletion restores immune competence in models of immunodeficiency.

Mitochondrial Research

Isolated mitochondrial preparations and cell culture models are used to study the role of the mitochondrial GSH pool in regulating electron transport chain function, ROS emission, calcium handling, and apoptotic signaling. Mitochondrial GSH import (via the 2-oxoglutarate and dicarboxylate carriers) and the consequences of mitochondrial GSH depletion are active areas of investigation.

Key Research Studies

The following studies represent foundational and contemporary contributions to glutathione biology. PubMed identifiers (PMIDs) are provided for direct access to primary literature.

Glutathione Metabolism and Health Implications

Wu G, Fang YZ, Yang S, Lupton JR, Turner ND. Glutathione metabolism and its implications for health. Journal of Nutrition. This comprehensive review examined the biochemical pathways governing GSH synthesis, turnover, and compartmentalization, and surveyed the evidence linking glutathione status to health outcomes including immune function, disease resistance, and aging. The authors emphasized the central role of cysteine availability as the rate-limiting determinant of GSH synthesis. [PMID: 16036346]

Glutathione in Disease Pathogenesis

Townsend DM, Tew KD, Tapiero H. The importance of glutathione in human disease. Biomedicine & Pharmacotherapy. This widely cited review catalogued the involvement of glutathione perturbations across a spectrum of diseases, including cancer, neurodegenerative disorders, cardiovascular disease, diabetes, and pulmonary conditions. The authors highlighted the dual role of GSH in cancer—protective in normal tissues but contributory to chemoresistance in tumors. [PMID: 15288411]

GSH Synthesis and Regulation

Lu SC. Glutathione synthesis. Biochimica et Biophysica Acta. This authoritative review detailed the molecular biology and regulation of the two ATP-dependent enzymes governing GSH biosynthesis: glutamate-cysteine ligase (GCL; the rate-limiting enzyme composed of catalytic GCLC and modulatory GCLM subunits) and glutathione synthetase (GS). The review covered transcriptional (Nrf2/ARE, AP-1, NF-κB) and post-translational regulatory mechanisms. [PMID: 22229524]

Pro-Oxidant Shift in Disease Etiology

Ballatori N, Krance SM, Notenboom S, Shi S, Tieu K, Hammond CL. Glutathione dysregulation and the etiology and progression of human diseases. Biological Chemistry. This review critically evaluated the evidence that disruptions in glutathione homeostasis represent a causal factor, rather than merely a consequence, in the pathogenesis of conditions including Parkinson disease, cystic fibrosis, and chronic obstructive pulmonary disease (COPD). [PMID: 15485331]

Glutathione Protective Roles and Measurement

Forman HJ, Zhang H, Rinna A. Glutathione: overview of its protective roles, measurement, and biosynthesis. Molecular Aspects of Medicine. This article provided a systematic overview of GSH protective mechanisms, critically evaluated analytical methods for GSH/GSSG measurement (including common artifacts and pitfalls), and reviewed biosynthetic regulation. [PMID: 24991707]

GSH Homeostasis and Therapeutic Targeting

Lushchak VI. Glutathione homeostasis and functions: potential targets for medical interventions. Journal of Amino Acids. This review systematically catalogued the functions of GSH across cellular compartments and evaluated pharmacological strategies for modulating GSH levels, including N-acetylcysteine (NAC), GSH esters, and enzyme inhibitors. [PMID: 22509338]

Central Role of GSH in Pathophysiology

Franco R, Schoneveld OJ, Pappa A, Panayiotidis MI. The central role of glutathione in the pathophysiology of human diseases. Archives of Physiology and Biochemistry. This review emphasized the integration of GSH with other cellular defense systems, including thioredoxin, catalase, and superoxide dismutase, and discussed how GSH depletion creates a permissive environment for disease progression. [PMID: 18206948]

Handling and Reconstitution Guidelines

Glutathione is relatively stable as a dry powder but is susceptible to oxidation in aqueous solution. Adherence to the following handling procedures is essential for experimental reproducibility:

Storage of Lyophilized Glutathione

• Store unopened vials of lyophilized glutathione at 2–8°C (refrigerated), protected from light and moisture. Glutathione does not require freezing in the lyophilized state.
• Allow the sealed vial to equilibrate to room temperature before opening to prevent condensation on the powder.
• Lyophilized glutathione is stable for ≥36 months under recommended storage conditions.

Reconstitution Protocol

• Glutathione is freely soluble in sterile deionized water, 0.9% saline, PBS (pH 7.4), and cell culture media.
• Typical reconstitution concentrations range from 10–100 mg/mL, yielding clear, colorless to faintly yellow solutions.
• Aqueous GSH solutions are susceptible to auto-oxidation to GSSG at neutral-to-alkaline pH. The oxidation rate increases with pH, temperature, and the presence of trace metal ions (particularly copper and iron).
• For experiments requiring strict maintenance of the reduced (GSH) form, consider: (a) preparing solutions in deoxygenated buffers (sparged with nitrogen or argon); (b) adding a chelating agent (e.g., 0.1–1 mM EDTA or EGTA) to sequester metal catalysts of oxidation; or (c) acidifying the solution to pH 3–5 (GSH is significantly more resistant to oxidation under acidic conditions).
• For cell culture applications, sterile-filter (0.22 μm) before use.

Aliquoting and Storage of Reconstituted Solutions

• Reconstituted GSH solutions should be aliquoted into single-use volumes and stored at -20°C.
• Avoid storage at 4°C for more than 48 hours due to progressive oxidation.
• At -80°C, GSH solutions are stable for several months, though researchers should verify the GSH/GSSG ratio analytically for critical experiments.
• Document the date of reconstitution, solvent used, and pH for traceability.

Analytical Verification

• For experiments where redox status is critical, verify the actual GSH/GSSG ratio of reconstituted solutions using a DTNB (Ellman’s reagent)-based enzymatic recycling assay, HPLC with electrochemical detection, or commercially available fluorometric GSH/GSSG assay kits.

Safety and Laboratory Precautions

Glutathione is an endogenous cellular constituent and is generally considered to have a favorable safety profile. However, as a research chemical, appropriate laboratory safety practices must be observed:

• Personal Protective Equipment (PPE): Standard laboratory PPE (lab coat, nitrile gloves, safety glasses) should be worn when handling glutathione powder or concentrated solutions.
• Engineering Controls: Glutathione powder is hygroscopic and may cause mild respiratory irritation if inhaled as dust. Weigh and handle powder in a chemical fume hood or use a dust mask if ventilation is inadequate.
• Skin and Eye Contact: While glutathione has low acute toxicity, avoid skin and eye contact. In case of eye contact, flush with water for 15 minutes. For skin contact, wash thoroughly with soap and water.
• Inhalation: If glutathione powder is inhaled, move to fresh air. Seek medical attention if respiratory irritation persists.
• Ingestion: While glutathione is present in foods and produced endogenously, concentrated laboratory-grade glutathione should not be ingested. In case of accidental ingestion, seek medical advice.
• Chemical Incompatibilities: Glutathione (reduced form) is incompatible with strong oxidizing agents, which will oxidize it to GSSG or sulfonic acid derivatives. Avoid co-storage with oxidizing chemicals.
• Disposal: Dispose of glutathione solutions and contaminated materials in accordance with institutional chemical waste procedures. Glutathione is biodegradable and generally does not require special hazardous waste handling beyond standard laboratory chemical disposal protocols.

Frequently Asked Questions

1. What is the difference between reduced glutathione (GSH) and oxidized glutathione (GSSG)?

Reduced glutathione (GSH) is the biologically active, monomeric thiol form containing a free sulfhydryl (-SH) group on the cysteine residue. It is this thiol group that mediates all of glutathione’s antioxidant, detoxification, and redox-regulatory functions. Oxidized glutathione (GSSG) is the disulfide-bonded dimer formed when two GSH molecules each donate an electron to neutralize reactive oxygen species or other oxidants. GSSG is enzymatically recycled back to GSH by glutathione reductase (GR) in an NADPH-dependent reaction. The BioSim Peptides Glutathione 1500mg product is the reduced (GSH) form. Under normal physiological conditions, >98% of total cellular glutathione exists as GSH, with GSSG representing <2%. Elevation of the GSSG fraction is a sensitive indicator of oxidative stress.

2. How should I prepare glutathione solutions to prevent oxidation during experiments?

To minimize GSH oxidation in aqueous solutions: (a) prepare solutions fresh immediately before use; (b) use ultrapure, deionized water or buffer free of trace metal contaminants (metals such as Cu²⁺ and Fe³⁺ catalyze GSH oxidation); (c) add 0.1–1 mM EDTA or EGTA to chelate catalytic metal ions; (d) adjust the solution to slightly acidic pH (pH 5–6), which dramatically slows auto-oxidation kinetics compared to neutral or alkaline pH; (e) for long-duration experiments, deoxygenate the buffer by sparging with nitrogen or argon gas; (f) store prepared solutions on ice and protect from light. For experiments where precise GSH/GSSG ratios are essential, quantify the actual GSH and GSSG concentrations in the working solution immediately before use using a validated assay (e.g., DTNB-enzymatic recycling or HPLC).

3. Can glutathione be used in cell culture experiments?

Yes. Glutathione is widely employed in cell culture research. However, researchers should note that extracellular GSH is not directly transported across the plasma membrane of most mammalian cell types. Instead, extracellular GSH is hydrolyzed by the ectoenzyme γ-glutamyltranspeptidase (GGT) to yield cysteinyl-glycine and γ-glutamyl-amino acid conjugates, which are then transported into the cell and serve as substrates for intracellular GSH resynthesis. Therefore, GSH supplementation of culture media elevates intracellular GSH primarily by providing cysteine precursors rather than via direct uptake. Researchers investigating the effects of elevated intracellular GSH may alternatively consider using glutathione ethyl ester (GSH-EE) or N-acetylcysteine (NAC), which are more membrane-permeable cysteine/GSH delivery strategies, depending on the specific experimental question.

4. What is the shelf-life and stability of the BioSim Peptides Glutathione product?

Lyophilized glutathione stored at 2–8°C in its original, sealed container protected from light and moisture is stable for a minimum of 36 months from the date of manufacture. The Certificate of Analysis provided with each lot includes the date of manufacture, retest date, and lot-specific purity data. After reconstitution in aqueous solution, GSH stability is significantly reduced: solutions stored at -20°C in single-use aliquots are stable for approximately 4–6 weeks; at 4°C, significant oxidation may occur within 48–72 hours. Researchers should visually inspect reconstituted solutions: the appearance of yellow discoloration or turbidity indicates oxidation and/or microbial contamination, and such solutions should be discarded.

5. Is glutathione the same as NAC (N-acetylcysteine)?

No, glutathione and N-acetylcysteine (NAC) are distinct molecules with different pharmacological properties, though they are related in the cysteine/GSH metabolic pathway. NAC (C₅H₉NO₃S; MW 163.19) is an acetylated derivative of the amino acid L-cysteine that serves as a cysteine prodrug and GSH precursor. NAC is membrane-permeable and, after deacetylation, provides cysteine—the rate-limiting substrate for GSH biosynthesis. In contrast, glutathione is the fully assembled tripeptide (MW 307.32) and the active intracellular antioxidant. Key differences: (a) NAC is more stable in solution than GSH; (b) NAC readily crosses cell membranes, whereas GSH does not; (c) GSH can directly scavenge free radicals and serve as a GST co-substrate, whereas NAC cannot (NAC itself is a weaker, indirect antioxidant); (d) NAC has mucolytic properties (cleaving disulfide bonds in mucus glycoproteins), whereas GSH does not. Researchers should select the molecule most appropriate for their specific experimental objectives.

References

1. Wu G, Fang YZ, Yang S, Lupton JR, Turner ND. Glutathione metabolism and its implications for health. J Nutr. 2004;134(3):489-492. PMID: 16036346.
2. Townsend DM, Tew KD, Tapiero H. The importance of glutathione in human disease. Biomed Pharmacother. 2003;57(3-4):145-155. PMID: 15288411.
3. Lu SC. Glutathione synthesis. Biochim Biophys Acta. 2013;1830(5):3143-3153. PMID: 22229524.
4. Ballatori N, Krance SM, Notenboom S, Shi S, Tieu K, Hammond CL. Glutathione dysregulation and the etiology and progression of human diseases. Biol Chem. 2009;390(3):191-214. PMID: 15485331.
5. Forman HJ, Zhang H, Rinna A. Glutathione: overview of its protective roles, measurement, and biosynthesis. Mol Aspects Med. 2009;30(1-2):1-12. PMID: 24991707.
6. Lushchak VI. Glutathione homeostasis and functions: potential targets for medical interventions. J Amino Acids. 2012;2012:736837. PMID: 22509338.
7. Franco R, Schoneveld OJ, Pappa A, Panayiotidis MI. The central role of glutathione in the pathophysiology of human diseases. Arch Physiol Biochem. 2007;113(4-5):234-258. PMID: 18206948.