L-Glutathione

Glutathione is a tripeptide. Its full name is gamma-L-glutamyl-L-cysteinyl-glycine. Its reduced form is written GSH. Nearly all eukaryotic cells contain it, as do many prokaryotes. In both, it occurs at millimolar concentrations. In animals, it is made in the cytosol (Oestreicher and Morgan, 2019). Glutathione is the low molecular weight thiol that cells make in the largest amount. It protects cells against oxidants and reactive electrophiles (Forman et al., 2009).

Its first bond is unusual. Glutamate joins cysteine through its side-chain (gamma) carboxyl group. Ordinary peptide bonds use the alpha carboxyl group. A dedicated cell-surface enzyme, gamma-glutamyl transpeptidase, cleaves this gamma bond (Hanigan, 2014).

Amounts and storage also come from these pages. The sequence comes from PubChem.

The glutathione literature is very large. The studies below cover six areas. Each summary states the model used.

Synthesis and breakdown

Cells make glutathione in the cytosol in two steps. The first uses glutamate-cysteine ligase. This is the rate-limiting enzyme, earlier called gamma-glutamylcysteine synthetase. The second uses glutathione synthetase. The main controls are the supply of cysteine and how active the ligase is. The genes respond to transcription factors, including Nrf2 (Lu, 2013).

In work with the purified enzyme, glutathione inhibited its own synthesis. It acted through non-allosteric feedback on gamma-glutamylcysteine synthetase (Richman and Meister, 1975). Breakdown starts outside the cell. Gamma-glutamyl transpeptidase releases glutamate. Cell-surface dipeptidases cleave the cysteinyl-glycine that is left (Hanigan, 2026). Models: purified enzymes, cell and animal biochemistry, reviews.

The GSH/GSSG redox couple

The GSSG/2GSH couple is the redox couple found in the largest amount in a cell. The Nernst equation was used to estimate its half-cell reduction potential. This value was tied to the state of the cell. It was about -240 mV in proliferating cells.

It was -200 mV in differentiating cells and -170 mV in apoptotic cells (Schafer and Buettner, 2001). Compartments differ. A peptide probe was trapped in the secretory pathway. The GSH/GSSG ratio there was 1:1 to 3:1. The whole-cell ratio was 30:1 to 100:1 (Hwang et al., 1992). Models: redox theory and cultured cells.

Enzymes that use glutathione

Mammals have eight enzymes called glutathione peroxidases, GPx1 to GPx8. These enzymes remove hydroperoxides. Studies report a role for GPx1 in insulin signalling. They also report roles for GPx4 in apoptosis and male fertility (Brigelius-Flohé and Maiorino, 2013). Glutathione transferases attach glutathione to electrophilic xenobiotics. They also attach it to oxidative stress products such as 4-hydroxynonenal.

Tests with disrupted genes in mice showed that the cytosolic enzymes broadly protect cells. They are cytoprotective (Hayes et al., 2005). Glutathione can also attach to protein cysteine residues. This is a change made after translation, a post-translational modification. It is called S-glutathionylation (Dalle-Donne et al., 2009). Models: enzymology, knockout mice, reviews.

Depletion as an experimental tool

Much of the evidence on glutathione's function comes from removing it. Buthionine sulfoximine was about 20 times more effective than prothionine sulfoximine at inhibiting gamma-glutamylcysteine synthetase. It lowered kidney glutathione in mice to under 20% of control (Griffith and Meister, 1979). In cancer cells, erastin blocks cystine uptake through the cystine/glutamate antiporter, system xc-.

It triggers iron-dependent, non-apoptotic cell death. The authors named this ferroptosis (Dixon et al., 2012). Follow-up work showed that glutathione depletion stops glutathione peroxidases from working. Another class of compounds inhibits GPX4 directly. This work also profiled how sensitive 177 cancer cell lines were (Yang et al., 2014). Models: purified enzyme, mice, cancer cell lines, xenograft mice.

Measuring GSH and GSSG

The common enzymatic recycling assay uses DTNB to read GSH at 412 nm. Glutathione reductase and NADPH recycle GSSG back to GSH (Rahman et al., 2006). Sample handling is the main source of error. In human blood, oxidation during acid deproteinization made GSSG values seem too high. Oxidation after neutral to alkaline pH was restored did the same.

The authors concluded that artifacts may affect most published GSSG data (Rossi et al., 2002). Derivatizing GSH with N-ethylmaleimide before deproteinization prevents artifactual oxidation. Without this step, artifactual oxidation of 5 to 15% of the GSH in a sample can occur during acid deproteinization (Giustarini et al., 2013). Models: analytical methods, human blood.

Oral glutathione in healthy volunteers

Human studies of oral glutathione do not agree. In 7 healthy volunteers, a single oral amount did not cause a significant rise in plasma glutathione over 270 minutes. The authors attributed this finding to hydrolysis by gamma-glutamyltransferase in the intestine and liver (Witschi et al., 1992). A 4-week trial studied 40 healthy adults. The trial was randomized and placebo-controlled.

It found no change in oxidative stress biomarkers or glutathione status (Allen and Bradley, 2011). A 6-month trial studied 54 non-smoking adults. The trial was randomized and placebo-controlled. It reported higher glutathione in blood, erythrocytes and plasma. Glutathione was also higher in lymphocytes and buccal cells. Levels returned to baseline after a 1-month washout (Richie et al., 2015). Model: healthy human volunteers.