Follow on from The Master Regulator of Aging? Redox, Glutatione and Cysteine, Part 1

An increase in levels of intra-cellular reduced glutathione, and a shift to a more reduced redox state, happens by two primary means.

  1. De novo synthesis of glutathione from cysteine, glycine and glutamine.
  2. Recycling of oxidised glutathione (GSSG) back to its reduced form.
  3. Exporting (efflux of) oxidised glutathione out of the cell (at the cost of depleting the total glutathione pool)
The first two paths required particular enzymes and energy sources (ATP or NADPH). Also some of the direct functions of glutathione require additional enzymes. All these enzymes are produced by genetic transcription from the ARE/EpRE (Antioxidant Response Element, also known as the Electrophile Response Element) which is initiated by translocation of Nrf2 into the nucleus.

Here we will look at a number of factors that can affect cysteine supply, GSH synthesis (transcription of the ARE via Nrf2, levels and efficiency of the enzymes), recycling, function and oxidization/depletion of GSH (all which in turn affects the redox state).


Dietary Cysteine Supply

Cysteine is the least abundant of the 3 amino acids glutathione is synthesized from, and also the primary functional amino with it’s sulfhydryl group. Hence its supply is a critical factor in the de novo synthesis of glutathione. Due to the reactive nature of the sulfhydryl group, means to effectively deliver cysteine into the cell via various forms such as N-Acetyl-Cysteine, Ribose-Cysteine and others pro-drugs is a topic in itself. A number of studies have shown benefits from supplementing with cysteine.

From Oxidative stress and ageing: is ageing a cysteine deficiency syndrome? Philos Trans R Soc Lond B Biol Sci. 2005 December “In several clinical trials, cysteine supplementation improved skeletal muscle functions, decreased the body fat/lean body mass ratio, decreased plasma levels of the inflammatory cytokine tumour necrosis factor a (TNF-a), improved immune functions, and increased plasma albumin levels. As all these parameters degenerate with age, these findings suggest: (i) that loss of youth, health and quality of life may be partly explained by a deficit in cysteine and (ii) that the dietary consumption of cysteine is generally suboptimal and everybody is likely to have a cysteine deficiency sooner or later.”

From Glutathione synthesis is diminished in patients with uncontrolled diabetes and restored by dietary supplementation with cysteine and glycine. Diabetes Care. 2011 Jan “Patients with uncontrolled type 2 diabetes have severely deficient synthesis of glutathione attributed to limited precursor availability. Dietary supplementation with GSH precursor amino acids can restore GSH synthesis and lower oxidative stress and oxidant damage in the face of persistent hyperglycemia.”

From Deficient synthesis of glutathione underlies oxidative stress in aging and can be corrected by dietary cysteine and glycine supplementation Am J Clin Nutr. 2011 September Glutathione deficiency in elderly humans occurs because of a marked reduction in synthesis. Dietary supplementation with the glutathione precursors cysteine and glycine fully restores glutathione synthesis and concentrations and lowers levels of oxidative stress and oxidant damages.

Practical intervention: As mentioned in the articles above, dietary supplementation with GSH precursor amino acids. A detailed look at various cysteine options will be discussed in a further post.


Trans-sulfuration pathway

Of the 22 standard amino acids cysteine is considered one of the non-essential amino acids, despite its critical functions, as the body can synthesize it from methionine via the trans-sulfuration pathway. The intermediate molecule in this conversion is homocysteine Other than supplementing directly, this is the other primary source of cysteine for the body.

From Redox regulation of homocysteine-dependent glutathione synthesis. Redox Rep. 2003 “studies in our laboratory have shown that approximately 50% of the cysteine in glutathione is derived from homocysteine in human liver cells… These studies provide the first evidence for the reciprocal sensitivity of the trans-sulfuration pathway to pro- and antioxidants, and demonstrate that the upstream half of the glutathione biosynthetic pathway (i.e. leading to cysteine biosynthesis) is redox sensitive

From Age-associated perturbations in glutathione synthesis in mouse liver. Biochem J. 2007 Aug “The amount of the toxic trans-sulfuration/glutathione biosynthetic pathway intermediate, homocysteine, was 154% higher (P<0.005) in the liver of old mice compared with young mice. The conversion of homocysteine into cystathionine, a rate-limiting step in trans-sulfuration catalysed by cystathionine -synthase, was comparatively less efficient in the old mice, as indicated by cystathionine/homocysteine ratios. Incubation of tissue homogenates with physiological concentrations of homocysteine caused an up to 4.4-fold increase in the apparent Km of GCL for its glutamate substrate, but had no effect on Vmax. The results suggest that perturbation of the catalytic efficiency of GCL and accumulation of homocysteine from the trans-sulfuration pathway may adversely affect de novo GSH synthesis during aging.”

Alterations in the transsulfuration pathway can have two negative effects on GSH synthesis.

  1. A decrease in conversion of methionine to cysteine available for GSH synthesis (a pathway which itself is redox sensitive).
  2. An increase in homocysteine which reduces the efficiency of the enzymes for GSH synthesis (further detailed in the section below titled “Decline in Catalytic Efficiency of GCL”)
The high level of enzymes in the transsulfuration pathway is believed to contribute to the extended longevity of the Ames dwarf mouse. From Methionine flux to transsulfuration is enhanced in the long living Ames dwarf mouse. Mech Ageing Dev. 2006 May “This, along with data from previous studies support the hypothesis that altered methionine metabolism plays a significant role in the oxidative defense of the dwarf mouse and that the mechanism for the enhanced oxidative defense may be through altered GSH metabolism as a result of the distinctive methionine metabolism.”

From Homocysteine and Familial Longevity: The Leiden Longevity Study PLoS One. 2011 “Increased concentrations of homocysteine have consistently been associated with ischemic cardiac events, stroke, venous thrombosis, Alzheimer’s disease, osteoporosis and depression. In line with these associations, higher levels of homocysteine have shown to independently predict all cause mortality in large population cohorts as well as in clinical populations”, however “The results suggest that homocysteine metabolism is not likely to predict familial longevity.” Also “Other interesting metabolites in the methionine cycle include S-adenosyl methionine (SAM) and S-adenosylhomocysteine (SAH), as their ratio is a representation of methylation status, and both markers have been associated with cardiovascular abnormalities more strongly than homocysteine itself”

The trans-sulfuration pathway is a whole topic in itself. One gender specific factor is men have higher plasma levels of total homocysteine than do women. It has been shown that testosterone downregulates cystathionine beta-synthase, which catalyzes the committing step in the transsulfuration pathway (ref).

Practical Interventions: DHA, CLA, Vitamin B6 and B12. Homocysteine can either be recycled back into methionine which requires B12, or converted into cysteine which requires B6. The polyunsaturated fats DHA (an Omega-3) and CLA (conjugated linoleic acid) have been shown to reduce homocysteine via changes in enzymes gene expression(ref).


Decline in Catalytic Efficiency of GCL

A number of studies have shown than that efficiency of the enzyme in the rate-limiting step in glutathione synthesis declines with age. In particular this has shown to be caused by the age-related increase in homocysteine as previously disccused in the trans-sulfuration pathway section.

From Pro-oxidant shift in glutathione redox state during aging. Adv Drug Deliv Rev. 2008 Experimental studies suggest that age-related accumulation of homocysteine, an intermediate in the trans-sulfuration pathway, may be responsible for causing the loss of affinity between GCL and its substrates.

From Age-associated perturbations in glutathione synthesis in mouse liver. Biochem J. 2007 August The results suggest that perturbation of the catalytic efficiency of GCL and accumulation of homocysteine from the trans-sulfuration pathway may adversely affect de novo GSH synthesis during aging… Results of the present study are the first to suggest that the relatively high levels of homocysteine that accumulate during aging can interfere with de novo GSH synthesis. Although the precise nature of the underlying mechanism is at present unclear, our results demonstrate that physiological concentrations of free homocysteine can inhibit the efficiency of cysteine utilization by GCL in a competitive manner. Homocysteine has been shown to bind GCLc at the active site in vitro as well as in vivo, forming ?-glutamylhomocysteine, which is then rapidly degraded enzymatically. An additional mechanism by which an age-related increase in free homocysteine can inhibit the catalytic efficiency of GCL in a competitive manner is described in [35]. It is possible that an aging-related increase in the competition between cysteine and homocysteine for the cysteine binding site of GCL might lead to a decrease in GC synthesis.

Practical interventions: First the interventions above for reducing homocysteine levels. Secondly, increasing the levels of cysteine and GCL via supplementing with Nrf2 activators and cysteine pro-drugs to compensate.


Intra-cellular cysteine supply

Neural/CNS cells have some particular intricacies in regards to cysteine supply and GSH synthesis. The antiporter system x©(-) imports cystine (CySS), the oxidized form of cysteine (Cys), into the cells with a 1:1 counter-transport of glutamate. Glutamate is a major neurotransmitter in the central nervous system, which can cause issues when in excess. High extra-cellular neural glutamate caused by concussive brain injuries, excitotoxicity (which can occur in autism, Alzheimers etc), and neuro-inflammatory conditions can reduce intra-cellular cysteine supply by competing with the transporter.

From Glutathionea review on its role and significance in Parkinsons disease The FASEB Journal October 2009 Although both neurons and glial cells can synthesize GSH, glial cells, specifically astrocytes, also have important roles to play in supplying GSH substrates to neurons. Astrocytes synthesize and export GSH, which can then undergo transpeptidation to cysteinylglycine and ?-glutamyl amino acid by the ecto-enzyme ?-glutamyl transpeptidase (?-GT). The cysteinylglycine generated can then be utilized by neurons to manufacture GSH, probably undergoing dipeptide cleavage to its constituent amino acids first. This mechanism of substrate supply minimizes the neurotoxic effects of large amounts of extracellular cysteine, which can activate glutamate receptors. A full discussion of the functions of GSH and its maintenance in neuronal cells is beyond the scope of this review, and the reader is referred to Zeevalk et al. and Dringen for further information.

From Immunoexcitotoxicity as a central mechanism in chronic traumatic encephalopathy-A unifying hypothesis Surg Neurol Int 2011 The cystine/glutamate X c antiporter is an exchange system where intracellular glutamate is exchanged for extracellular cystine, so as to supply cysteine for glutathione (GSH) generation. [147] Excess extracellular glutamate prevents exchange and lowers astrocytic GSH. The astrocyte is the major source of neuronal GSH. Under such conditions, the neuron becomes highly vulnerable to conditions of oxidative stress, as seen with concussive brain injuries and immunoexcitotoxicity.

Practical Intervention: Do your best to avoid head concussions which can spike glutamate levels to 20x the normal levels, and 4x the toxic levels! Nicotinamide has been shown to protect against glutamate excitotoxicity (ref). Alpha-lipoic acid can reduce cystine to cysteine, which can then use a different transporter into the cell.


Mitochondrial glutathione transport

From Mitochondrial glutathione transport is a key determinant of neuronal susceptibility to oxidative and nitrosative stress. J Biol Chem. 2013 Feb The mitochondrial glutathione (GSH) pool is a critical antioxidant reserve that is derived entirely from the larger cytosolic pool via facilitated transport. The mechanism of mitochondrial GSH transport has not been extensively studied in the brain. However, the dicarboxylate (DIC) and 2-oxoglutarate (OGC) carriers localized to the inner mitochondrial membrane have been established as GSH transporters in liver and kidney. .. These findings demonstrate that maintenance of the mitochondrial GSH pool via sustained mitochondrial GSH transport is essential to protect neurons from oxidative and nitrosative stress.

From Mitochondrial glutathione transport: physiological, pathological and toxicological implications. Chem Biol Interact. 2006 Oct Overexpression of the cDNA for the DIC and OGC in a renal proximal tubule-derived cell line, NRK-52E cells, showed that enhanced carrier expression and activity protects against oxidative stress and chemically induced apoptosis. This has implications for development of novel therapeutic approaches for treatment of human diseases and pathological states. Several conditions, such as alcoholic liver disease, cirrhosis or other chronic biliary obstructive diseases, and diabetic nephropathy, are associated with depletion or oxidation of the mitochondrial GSH pool in liver or kidney.

From The ketogenic diet increases mitochondrial glutathione levels. J Neurochem. 2008 Aug KD-fed rats showed a twofold increase in hippocampal mitochondrial GSH and GSH/GSSG ratios compared with control diet-fed rats. As GSH is a major mitochondrial antioxidant that protects mitochondrial DNA (mtDNA) against oxidative damage, we measured mitochondrial H2O2 production and H2O2-induced mtDNA damage. Isolated hippocampal mitochondria from KD-fed rats showed functional consequences consistent with the improvement of mitochondrial redox status i.e. decreased H2O2 production and mtDNA damage. Together, the results demonstrate that the KD up-regulates GSH biosynthesis, enhances mitochondrial antioxidant status, and protects mtDNA from oxidant-induced damage.

From Mitochondrial glutathione depletion in alcoholic liver disease. Alcohol. 1993 Nov-Dec GSH in mitochondria originates from cytosol by a transport system which translocates GSH into the matrix. This transport system is impaired in chronic ethanol-fed rats, which translates in a selective and significant depletion of the mitochondrial GSH content

Practical Interventions: A sufficient level of GSH in the cytosol by other means discussed here should be enough to maintain mitochondrial levels. Avoid alcohol as it has been shown to selectively deplete mitochondrial glutathione by interfering with the transporter. The improvement from a ketogenic diet may be to a general intra-cellular improvement, and not mitochondrial specific.


GSH Depletion Greatly Diminishes the Expression Levels of Antioxidant Genes

From Genetic dissection of the Nrf2-dependent redox signaling-regulated transcriptional programs of cell proliferation and cytoprotection Physiolgenomics September 2007 To determine whether depletion of GSH would have an effect on antioxidant gene expression, Nrf2 cells were treated with BSO for 6 and 24 h, RNA was isolated, and quantitative and semiquantitative PCR analyses were used to analyze the expression levels of several antioxidant genes. The mRNA expression levels of several antioxidant enzymes, such as gclc, gclm, gsta3, gsta4, gsta1, and gsta2, were markedly lower in Nrf2+/+ cells treated with BSO as early as 6 h and remained low at 24 h compared with vehicle-treated control group.

The sub-heading above was taken from the article, however the enzymes with lowered expression were not just the antioxidant enzymes, but also include those for the synthesis of GSH (gclc and gclm)

Practical Intervention: This appears to be somewhat of a vicious cycle, low glutathione levels lowers the levels of the synthesis enzymes further. Supplementing with GSH/cysteine precursors and Nrf2 activators would help return to normal levels.


Epigenetic Silencing

Epigenetic silencing of Nrf2 and the glutathione enzymes can cause a reduction in glutathione synthesis and function.

From Nrf2 Expression Is Regulated by Epigenetic Mechanisms in Prostate Cancer of TRAMP Mice PLoS One. 2010 These results indicate that the expression of Nrf2 is suppressed epigenetically by promoter methylation associated with MBD2 and histone modifications in the prostate tumor of TRAMP mice.

From DNA hypermethylation regulates the expression of members of the Mu-class glutathione S-transferases and glutathione peroxidases in Barrett’s adenocarcinoma. Gut. 2009 Jan CONCLUSION: Epigenetic inactivation of members of the glutathione pathway can be an important mechanism in Barrett’s tumourigenesis. From Glutathione peroxidase 7 has potential tumour suppressor functions that are silenced by location-specific methylation in oesophageal adenocarcinoma. Gut. 2013 Apr Our data suggest that GPX7 possesses tumour suppressor functions in OAC and is silenced by location-specific promoter DNA methylation.

Practical interventions: Interestingly two of our favourite Nrf2 activators also appear to work via epigenetic modifications. Many other factors such as exercise, diet, emotional state can also induce positive epigenetic changes, however I’m unsure if there any specific effects on the Nrf2/ARE promoter region.

From Sulforaphane enhances Nrf2 expression in prostate cancer TRAMP C1 cells through epigenetic regulation. Biochem Pharmacol. 2013 May Taken together, our current study shows that SFN regulates Nrf2’s CpGs demethylation and reactivation in TRAMP C1 cells, suggesting SFN may exert its chemopreventive effect in part via epigenetic modifications of Nrf2 gene with subsequent induction of its downstream anti-oxidative stress pathway.

From Pharmacodynamics of curcumin as DNA hypomethylation agent in restoring the expression of Nrf2 via promoter CpGs demethylation. Biochem Pharmacol. 2011 Nov Taken together, our current study suggests that CUR can elicit its prostate cancer chemopreventive effect, potentially at least in part, through epigenetic modification of the Nrf2 gene with its subsequent induction of the Nrf2-mediated anti-oxidative stress cellular defense pathway.

While on the epigenetic topic, curiously some cancers increase their resistance to oxidative stress by promoter methylation silencing of Keap1 expression, which results in more free Nrf2.



The enzyme Glutathione Reductase requires NADPH as an electron donor to recycle two oxidised glutathione molecules (GSSG) back to reduced GSH. At the cellular level, replenishment of the NADPH is dependent on the metabolism of glucose (e.g., though the action of glucose-6-phosphate dehydrogenase). (ref) The pentose phosphate pathway regulates the GSH/GSSG ratio by providing nicotinamide-adenine dinucleotide phosphate (NADPH), which is required for the reduction of GSSG to GSH by glutathione reductase. (ref). The Glutathione Reductase enzyme is synthesised via the Nrf2/ARE pathway, so the factors affecting Nrf2 will be also be applicable here too.

From Nrf2-regulated glutathione recycling independent of biosynthesis is critical for cell survival during oxidative stress. Free Radic Biol Med. 2009 Feb Overall, Nrf2 is critical for maintaining the GSH redox state via transcriptional regulation of GSR [glutathione reductase] and protecting cells against oxidative stress.

In the following research article we can see that providing a NADH precursor increased redox state an neuron survival independent of Nrf2 activation.

From Dual-energy precursor and nuclear erythroid-related factor 2 activator treatment additively improve redox glutathione levels and neuron survival in aging and Alzheimer mouse neurons upstream of reactive oxygen species. Neurobiol Aging. 2014 Jan By combining the Nrf2 activator together with the NADH precursor, nicotinamide, we increased neuron survival against amyloid beta stress in an additive manner.

Nicotinamide is the amide of nicotinic acid (vitamin B3 / niacin).

From The Redox Stress Hypothesis of Aging Free Radic Biol Med. 2012 February Transgenic studies have shown that augmentation of reducing power, provided by NADPH and GSH, is the most effective currently known experimental manipulation for the prolongation of lifespan.

From Time line of redox events in aging postmitotic cells

Here, we report the discovery that chronologically aging yeast cells undergo a sudden redox collapse, which affects over 80% of identified thiol-containing proteins. We present evidence that this redox collapse is not triggered by an increase in endogenous oxidants as would have been postulated by the free radical theory of aging. Instead it appears to be instigated by a substantial drop in cellular NADPH, which normally provides the electron source for maintaining cellular redox homeostasis.

These findings raised the intriguing possibility that these processes are directly connected (Figure 7) and that loss of NADPH might be the trigger for the observed redox collapse. Consistent with this idea, analysis of the cellular GSH/GSSG ratio in chronologically aging yeast cells, which is dependent on the NADPH-dependent glutathione reductase, revealed a pro-oxidizing shift in the GSH redox potential that coincided with the decrease in cellular NADPH levels.

Further reading: A Biophysically-based Mathematical Model for the Catalytic Mechanism of Glutathione Reductase. Free Radic Biol Med. 2013 Oct

Practical interventions: The NAD+/NADH precursor nicotinamide (the amide of vitamin B3 / niacin), or possibly Nicotinamide Riboside. Activating the AMPK pathway through exercise, caloric restriction, ketogenic diet or supplements such alpha-lipoic acid and resveratrol result in more NAPDH being produced. See my post here for more details.


The Parkinsons Paradox

From Expression of Nrf2 in Neurodegenerative Diseases J Neuropathol Exp Neurol. 2007 January “In Parkinson’s Diseases, nuclear localization of Nrf2 is strongly induced, but this response may be insufficient to protect neurons from degeneration.”

From the previous factors covered and the two examples below we can see a number possible reasons why this could be the case, such as low cysteine supply, excess extra-cellular glutamate, epigenetic silencing, high levels of homocysteine and insufficient NADPH for recycling by glutathione reductase.

From Glutathionea review on its role and significance in Parkinsons disease The FASEB Journal October 2009 This study suggests that cysteine supply to neurons could be altered in PD; this finding is supported by increased activity and levels of ?-GT both in dopaminergic cells and in PD patients as an attempt to generate neuronal GSH… This increase in glutathione reductase levels again suggests that the cells of the SN are attempting to maintain GSH levels.

From Redox homeostasis and cellular stress response in aging and neurodegeneration. Methods Mol Biol. 2010 Recent findings emphasize a relationship between elevated homocysteine (Hcy) levels and neurodegeneration, which can be observed in Alzheimer’s and Parkinson’s diseases. As discussed in the previous section increased homocysteine reduces the catalytic efficiency of GCL for glutathione synthesis.



As to be expected genetic polymorphisms/defects can affect the function of all these contributing factors: Transporters, Nrf2, glutathione enzymes, transsulfuration pathway etc.

From Genomic Structure and Variation of Nuclear Factor (Erythroid-Derived 2)-Like 2 Oxid Med Cell Longev. 2013 Compilation of publically available SNPs and other genetic mutations shows that human NRF2 is highly polymorphic with a mutagenic frequency of 1 per every 72 bp. Functional at-risk alleles and haplotypes have been demonstrated in various human disorders.”

From Dysregulation of Glutathione Homeostasis in Neurodegenerative Diseases Nutrients. 2012 October “Analogous to patients with Parkinson’s Disease, mutations in GSH-dependent enzymes are reported to confer increased susceptibility to Alzheimer’s disease. These data are consistent with a report that indicates that serum samples from AD patients have decreased GPx activity compared to healthy age-matched controls. Additionally, polymorphisms in GST genes have been linked to early onset as well as faster cognitive decline in AD patients”

Patients with genetic defects in the transsulfuration pathway are characterized by high levels of homocysteine, low levels of GSH, and increased incidence of age-related pathologies. (ref)

Cystic Fibrosis is caused by a mutation in the gene for the protein cystic fibrosis transmembrane conductance regulator (CFTR). The functions of this protein includes glutathione transport, which is believed to a major factor contributing to the pathology of the condition.

From Systemic deficiency of glutathione in cystic fibrosis. J Appl Physiol. 1993 Dec The glutathione deficiency observed in [respiratory] epithelial lining fluid in CF patients is not limited to the site of the inflammation but is systemic.

From A new model of cystic fibrosis pathology: lack of transport of glutathione and its thiocyanate conjugates. Med Hypotheses. 2007 “The authors describe how this disruption of the redox state caused by excess cellular GSH, will naturally prevent the delivery of zinc as a cofactor for various enzymatic processes, and how these disruptions in normal redox may cause alterations in both humoral and cell-mediated immunity. “

Autism has numerous studies showing decreased glutathione levels and function, adversely affecting the ability to detoxify, possibly due to genetic polymorphisims.

From Mercury and autism: Accelerating Evidence? Neuro Endocrinol Lett. 2005 Oct The process of cysteine and glutathione synthesis, which are crucial for natural mercury detoxification, are reduced in autistic children, possibly due to genetic polymorphisms. Therefore, autistics have 20% lower plasma levels of cysteine and 54% lower levels of glutathione, which, among others, adversely affect their ability to detoxify and excrete metals like mercury


Increased mitochondrial H202 production

From The Redox Stress Hypothesis of Aging Free Radic Biol Med. 2012 “One of the most commonly observed correlates of aging is the increase in the mitochondrial production of H2O2, which implies that this particular alteration should be considered as a possible triggering factor in the initiation of the senescent decline. As described above, increased production of H2O2 would quite likely decrease the GSH/GSSG redox potential and elevate the levels of protein glutathionylation, mixed disulfide bonding and over-oxidation of protein cysteine thiolate-based redox switches. This mechanism is, in general, compatible with the existing data”

Sedentary lifestyle

The Epigenetic oxidative shift theory hypothesises that low demands for energy switches energy metabolism more towards glycolysis, which causes an oxidative shift due to the requirement for more NAD+.

From Epigenetic oxidative redox shift (EORS) theory of aging unifies the free radical and insulin signaling theories Exp Gerontol. 2010 March “Aging is often associated with a sedentary life style. If there are no demands for the extra energy that can be produced by aerobic oxidative phosphorylation, then cells and organs may down-regulate the electron transport chain components and survive adequately on glycolysis. Increased consumption of sugar in beverages may also enforce reliance on glycolysis. An oxidative shift is proposed to ensure ample supplies of the requisite NAD+”

Practical Interventions: Exercise! The decreased demands for energy, i.e. excessive caloric intake, may tie in with the effects seen from caloric restriction. Exercise has also been shown to cause positive changes in epigenetics and cellular signalling.

Caloric restriction

From Caloric restriction and redox state: does this diet increase or decrease oxidant production? Redox Rep. 2011 Recently, some authors have suggested that CR acts through hormesis, enhancing the production of reactive oxygen species (ROS), activating stress response pathways, and increasing lifespan. Here, we review the literature on the effects of CR and redox state. We find that there is no evidence in rodent models of CR that an increase in ROS production occurs… Overall, the largest body of work indicates that CR improves redox state, although it seems improbable that a global improvement in redox state is the mechanism through which CR enhances lifespan.

Countering the authors conclusion, from the theory proposed in Part 1 a global improvement in redox state would be a factor in enhancing lifespan from CR.

From Modulation of glutathione and thioredoxin systems by calorie restriction during the aging process. Exp Gerontol. 2003 May “ The results of our study showed that GSH and GSH-related enzyme activities decreased with age in ad libitum (AL)-fed rats, while CR rats consistently showed resistance to decreases in these activities… Our conclusion is that a redox imbalance occurs during aging and that redox changes are minimized through the anti-oxidative action of CR.”

From Effects of caloric restriction on glutathione redox state in mouse Adv Drug Deliv Rev. 2008 A decrease in the amount of food consumption, relative to the ad libitum (AL) fed level, has been shown to extend life span of certain laboratory strains of mice and rats. There is also some evidence that caloric restriction (CR) retards the onset and progression of some age-associated changes linked to oxidative stress. A comparison between 22-month-old AL mice and those fed a diet containing 40% fewer calories than the AL group since the age of 4 months, indicated that CR had no effect on GSH content of tissue homogenates, whereas GSSG concentration was significantly lowered in the brain (42 %) and testis (37 %). In mitochondria, CR had no effect on GSH amount except in the heart and eye, where the increases were 24 % and 9 %, respectively. The mitochondrial glutathione redox state was significantly more proreducing (-5 to -10 mV) in the heart, kidney, eye and testis of CR mice, compared to the AL mice. This proreducing shift was primarily due to a decrease in GSSG content. CR had no effect on glutathione redox potential in the brain or liver. In most tissues examined, CR caused a decrease in the amount of protein-SSG. In general, these studies indicated that CR attenuates the age-related oxidizing shift in the glutathione redox state.



Many natural and man-made environmental toxins are neutralised by conjugation with glutathione via the glutathione transferase enzymes. These conjugates are then excreted from the body which depletes glutathione and the key amino acid cysteine. Additionally these toxins may induce ROS production as they metabolised before they are conjugated and excreted from the body.

The popular pain killer paracetamol/acetaminophen (Panadol/Tylenol) is primarily metabolized in the liver by glucuronidation and sulfation (sulfate conjugation). However the 10-15% metabolised by glutathione conjugation is the cause of liver damage and death from overdose by glutathione depletion.

From Advances in metal-induced oxidative stress and human disease. Toxicology. 2011 May Cadmium, arsenic and lead show their toxic effects via bonding to sulphydryl groups of proteins and depletion of glutathione.

From Mitochondrial glutathione depletion in alcoholic liver disease. Alcohol. 1993 The profound and selective mitochondrial GSH depletion precedes the onset of alcoholic liver disease, mitochondrial lipid peroxidation, and progression of liver damage.


Too Much Of A Good Thing?

There is a less known flip side to oxidative stress. Reductive stress can also become an issue when the redox state becomes too reduced. While unlikely an issue for the average person, especially as we age, as the feedback mechanisms should restore the redox state. It is something to consider when taking high dose supplements.

Glutathione-dependent reductive stress triggers mitochondrial oxidation and cytotoxicity. FASEB J. 2012 Apr

Reductive stress in young healthy individuals at risk of Alzheimer disease. Free Radic Biol Med. 2013 Oct

Reductive stress linked to small HSPs, G6PD, and Nrf2 pathways in heart disease. Antioxid Redox Signal. 2013 Mar

Nrf2 deficiency prevents reductive stress-induced hypertrophic cardiomyopathy Cardiovasc Res. 2013 Oct


Next - Part 3, Reviewing Glutathione Supplementation Options