Fruit, vegetables and some other foods are rich in flavonoids, carotenoids and vitamins that provide antioxidant protection. The body also has mechanisms to produce its own ‘endogenous’ antioxidants including glutathione, coenzyme Q10, alpha lipoic acid, L-carnitine and melatonin.
In this and next week’s blog we will be reviewing two of these endogenous antioxidant – glutathione and coenzyme Q10. Today we start with glutathione – the most abundant free radical scavenger synthesised endogenously in humans. There is increasing mechanistic, clinical, and epidemiological evidence which demonstrates that glutathione status is significant in acute and chronic diseases.
Biosynthesis of glutathione
Glutathione (GSH) is produced in the cytoplasm of all human cells but especially in the liver, spleen, kidney, lens and both white and red blood cells and synthesis is upregulated in response to oxidative stress. The liver is a major exporter of glutathione to plasma and also to bile (some other organs also export to plasma in lesser amounts).
Glutathione is a tripeptide of L-glutamine, L-cysteine and glycine. Cells make glutathione in two ATP (adenosine triphosphate) dependent steps:
- In the first, rate-limiting, step the enzyme gamma-glutamylcysteine synthetase catalyses the synthesis of gamma-glutamylcysteine from L-glutamate and L-cysteine. Both the availability of the substrate cysteine (from protein), and/or certain genetic polymorphisms (ie mutations) that reduce enzyme activity, can rate-limit this step
- Second, glycine is added to the C-terminal of gamma-glutamylcysteine via the enzyme glutathione synthetase
Glutathione synthesis is directly connected to the methylation cycle through homocysteine and cysteine. Approximately 50% of homocysteine leaves the folate-dependent methylation cycle and is metabolised via the trans-sulfuration pathway to cystathionine and cysteine. Thus, glutathione synthesis is linked to adequate availability of folate, other co-factors, as well as methionine and S-adenosylmethionine.
Cysteine contains a sulfhydryl (-SH) group or thiol group. Thiol groups are strong reducing agents – thus glutathione serves as an electron donor and in the process is converted to its oxidised form (GSSG). Once oxidised, glutathione can be recycled (ie reduced back) by the enzyme glutathione reductase using NADPH as an electron donor.
The ratio of reduced glutathione (GSH) to oxidised ie ‘spent’ glutathione (GSSG) within cells is used as a measure of cellular oxidative stress. In healthy cells and tissue, more than 90% of the total glutathione pool is in the reduced form (GSH) and less than 10% exists in the oxidised form (GSSG).
In general, the mitochondrial GSH:GSSG ratio is greater than that of the cytosol, resulting in a more ‘reducing environment’ in the mitochondria (reflecting the high degree to which reactive oxygen species are produced within mitochondria).
Functions of glutathione
The enzymes glutathione peroxidase (GPx), glutaredoxin (Grx), glutathione reductase (GR), and glutathione-S-transferase (GST) use glutathione (GSH) as a substrate in many cellular reactions. It can also participate in reactions directly ie without enzymatic reaction.
Antioxidant action: Glutathione can effectively scavenge free radicals both directly and through enzymatic reaction (eg hydroxyl radical, lipid peroxyl radical, peroxynitrite, and hydrogen peroxide). It can also maintain exogenous antioxidants such as vitamins C and E in their reduced (ie active) forms.
For example, as a co-factor for glutathione peroxidase (a selenium-containing enzyme) it catalyses the glutathione-dependent reduction of hydrogen peroxide, and other peroxides, to their respective alcohols and water. The reduction of hydrogen peroxide results in the formation of oxidised glutathione (GSSG) which needs to be recycled back to the reduced form (GSH) as described above.
Detoxification: Glutathione is a substrate in reactions which are catalysed by glutathione-S-transferase enzymes in cytosol, microsomes and mitochondria. Glutathione-S-stransferase are a family of enzymes which catalyse the specific addition of glutathione to substrates to allow them to be eliminated. Conjugation using glutathione is an important Phase II detoxification pathway in the liver – glutathione is a hydrophilic (ie water soluble) molecule that is conjugated to various lipophilic (ie fat soluble) physiological metabolites (eg oestrogens) and xenobiotics, biotransforming them so they can be excreted in the bile (and then eliminated in the stool).
In other cells, glutathione is similarly conjugated and exported to plasma for elimination via the urine. Thus in these reactions glutathione is ‘lost’ from the system and not recycled. Glutathione is also capable of participating in non-enzymatic conjugation with some chemicals.
The reducing power of glutathione is also important in DNA synthesis, cell signalling and neuromodulation. Glutathione also has a role in protein synthesis, prostaglandin and leukotriene synthesis, amino acid transport and iron metabolism. Thus, every system in the body can be affected by the state of the glutathione system, especially the immune system, the nervous system, the gastrointestinal system, the lungs and the eyes.
Glutathione is an essential antidote to paracetamol toxicity which occurs when glutathione is depleted by an overdose of acetaminophen; at this point a reactive cytochrome P450 metabolite reacts with cellular proteins, killing cells in the process.
The genes encoding the glutathione enzymes are known to be highly polymorphic and genetic variations can change an individual’s susceptibility to carcinogens and toxins as well as affect the toxicity of certain drugs.
For example, mutations in glutathione peroxidase enzymes have been linked to Alzheimer’s; and mutations in glutathione-s-transferase enzymes to Parkinson’s.
Glutathione, Health and Disease
Low glutathione is commonly observed in wasting and negative nitrogen balance, as seen in cancer, HIV/AIDS, sepsis, trauma, burns, starvation and athletic overtraining. Levels also decline with age. Glutathione depletion is linked to a number of disease states including pulmonary, liver, circulatory diseases and diabetes. Here we look in a little more detail at links between glutathione and neurodegenerative disease, autism, oral and eye health.
Neurodegenerative diseases – Dysregulation of glutathione homeostasis and alterations in glutathione-dependent enzyme activities are increasingly implicated in the development and progression of neurodegenerative diseases, including Alzheimer’s, Parkinson’s and Huntington’s diseases, amyotrophic lateral sclerosis, and Friedreich’s ataxia.
Apoptosis is key to neurodegenerative disease progression, and there is evidence that mitochondrial glutathione levels may play a key role in regulating apoptosis. The brain is particularly susceptible to oxidative stress for a number of reasons. Firstly it uses about 20% of the body’s oxygen supply for energy production (and energy production is a significant source of ROS); secondly, relative to other organs, the brain has lower levels of ROS scavenging enzymes and thirdly the brain is 60% fat – a high proportion of which is unsaturated fatty acids that are highly susceptible to lipid peroxidation.
ROS generation in the brain comes from multiple sources but leakage of electrons to molecular oxygen at various points in the mitochondrial electron transport chain is the main source of ROS. Monoamine oxidase (MAO) reactions are another source – MAO enzymes catalyse the oxidation of neurotransmitters, thereby terminating their activity.
For example, dopamine is oxidised by MAO-B, and hydrogen peroxide is produced as a by-product. Dopamine oxidation via MAO occurs primarily in the substantia nigra where oxidative stress is associated with loss of neurons, the pathological basis for Parkinson’s disease. ROS-mediated damage has been cited as a probable contributing cause for all of the common neurodegenerative diseases, suggesting that the natural antioxidant defence mechanisms are overwhelmed in these cases.
The brain also has an abundant supply of nitric oxide (NO) which can generate the toxic oxidant peroxynitrite (ONOO−). Like ROS, ONOO− leads to the oxidation of proteins, lipids, and DNA.
Another mechanism whereby glutathione is important in neurodegenerative disease is in assisting regulation of protein degradation pathways involved in regulation of cell survival. These pathways remove misfolded, damaged or otherwise non-functional proteins. When misfolded proteins are not adequately removed from cells, protein aggregation may occur and these are hallmarks of some neurodegenerative diseases eg Alzheimer’s (beta amyloid plaques), Parkinson’s (alpha synuclein-containing Lewy bodies) and Huntingdon’s (huntingtin protein aggregates).
Autism – Increased oxidative stress and abnormalities in glutathione metabolism have been shown in several studies of children with autism. Decreased glutathione(GSH) concentration and GSH/GSSG ratio, and increased oxidised glutathione (GSSG) concentration have been reported in mitochondria, plasma, brain and other tissues of individuals with autism.
One study examined the activity of several glutathione pathway enzymes in brain tissues from ten individuals with autism and ten age-matched controls. The enzymes examined included glutathione-s-transferase, glutathione peroxidase, glutamate cysteine ligase (the rate-limiting enzyme for glutathione synthesis) and glutathione reductase (recycles glutathione).
The activity of the first three, but not glutathione reductase were found to be reduced in brain tissue of ASD compared with control individuals. This is alongside other metabolic abnormalities such as folate and methylation abnormalities and zinc/copper ratio. Studies have demonstrated that interventions that address metabolic abnormalities in the glutathione, methylation and folate pathways may improve both metabolic biomarkers and core symptoms of autism spectrum disorder.
The reason for abnormalities in glutathione enzyme activity in autism is not clear but genetic polymorphisms in glutathione pathway enzymes are thought to be a contributory factor, along with nutritional factors.
Eye health – Glutathione is found at high concentrations in the lens especially in the epithelial layer where it has many important functions including helping to maintain lens transparency by preventing the formation of high molecular weight crystalline aggregates, removal of xenobiotics and protection against oxidative damage.
A study of human lenses ranging from birth to 92 years of age showed that over the years glutathione levels reduce up to 73%, and oxidised glutathione levels (ie ‘spent’ GSSH) increase from 2% to 18%.
Glaucoma, the leading cause of irreversible blindness worldwide, is characterised by progressive damage or degeneration to the optic nerve. Since the retina and optic nerve are part of the central nervous system, glaucoma has been suggested to share similarities with other neurodegenerative diseases. The retina is one of the most vascularised tissues in the body and is particularly vulnerable to oxidative stress due to its high consumption of oxygen and exposure to light.
Glutathione deficiency is manifested largely through an increased susceptibility to oxidative stress, and the resulting damage is thought to be a key step in the onset and progression of glaucoma.
Oral health – Periodontal disease is a chronic condition associated with increased inflammation in response to plaque biofilm (produced by bacteria) and decreased antioxidant activity in saliva, resulting in loss of periodontal and bone support. Certain bacteria species eg Fusobacterium spp and others, are capable of degrading glutathione to form toxic hydrogen sulfide within the periodontal pocket. Cigarette smoking can also deplete glutathione within periodontal tissues.
In addition, certain glutathione-s-transferase polymorphisms significantly increase the risk of periodonitis. Studies have shown reduced levels of glutathione in saliva, gingival crevicular fluid (fluid in gum crevices) and blood serum in periodonitis patients. Increasing glutathione levels through supplementation with eg N-acetyl cysteine has been shown to block activation of inflammatory cytokines (NF-kB etc), limit tissue damage and support wound healing.
Adequate protein intake is important for endogenous glutathione synthesis. In addition:
- Calcitriol, the active metabolite of vitamin D3, increases glutathione levels in the brain and appears to be a catalyst for glutathione production
- S-adenosylmethionine (SAMe), a co-substrate involved in methyl group transfer, has been shown to increase cellular glutathione content. SAM production requires adequate B vitamins – folate, B12, B6 – and zinc in particular. B6 is also a cofactor for the CBS enzyme in the trans-sulfuration pathway which converts homocysteine to cysteine
- Supplementation with N-acetyl cysteine – which provides the cysteine substrate has been shown to increase levels
- Up regulation of the transcription factor Nrf2 will increase enzymes needed for glutathione synthesis. Nrf2 can be upregulated by alpha lipoic acid and cruciferous vegetables
Until now, oral supplementation with glutathione has been difficult. Because glutathione is a tripeptide, it is broken down in the gastrointestinal tract by proteases to its constituent amino acids. In addition, if it does survive intact then there is the problem of transport across the cell membrane. Supplements therefore need to be in in ‘liposomal’ form – the liposome provides protection from digestion and enables transport across cell membranes. Liposomal glutathione has also been shown to cross the blood-brain barrier.
Glutathione is a key antioxidant produced in the body and involved in multiple metabolic processes. When synthesis cannot keep pace with requirements or where enzyme systems are not functioning well, then sub-optimal glutathione activity may be a key factor in ageing and the progression of many diseases.
If you have any questions regarding the topics that have been raised, or any other health matters please do contact me (Clare) by phone or email at any time.
firstname.lastname@example.org, 01684 310099
The Cytoplan editorial team: Clare Daley & Joseph Forsyth
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