The Master Regulator of Aging? – Redox, Glutatione and Cysteine, Part 1

More important than a cure for cancer.

A bold claim, however a recently published study showed that with major advances in cancer treatment or heart disease, a 51-year-old can expect to live about one more year.  A modest improvement in delaying aging would double this to two additional years — and those years are much more likely to be spent in good health.

“Even a marginal success in slowing aging is going to have a huge impact on health and quality of life.” said co-author S. Jay Olshansky of the School of Public Health at the University of Illinois-Chicago.

Finding a way to slow the biological processes of aging will do more to extend the period of healthy life in humans than attacking individual diseases alone, according to some of the nation’s top gerontologists writing in the latest issue of Public Policy & Aging Report.

These sentiments are echoed by Calico, the new Google funded start-up, on it’s ambitious quest to reverse the ageing process and extend human life.  Google CEO Larry Page said “One of the things I thought was amazing is that if you solve cancer, you’d add about three years to people’s average life expectancy.  We think of solving cancer as this huge thing that’ll totally change the world. But when you really take a step back and look at it, yeah, there are many, many tragic cases of cancer, and it’s very, very sad, but in the aggregate, it’s not as big an advance as you might think.”

But perhaps Dr Herbert Nagasawa has beaten them to the punch with the technology he’s lead the development of over his accomplished 40+ year long career in medicinal chemistry.  More on that in another post.

Vincent Giuliano, and more recently James (Jim) Watson, have been assembling a wealth of knowledge connecting the latest publish research into the aging process at www.anti-agingfirewalls.com, which has been an invaluable source of information and ideas.

Early this year Jim proposed to Vince an intellectual wager to determine the most important “signaling mechanism” for longevity and the most important cellular adaptation mechanism.  The abridged version is:

  1. Wager #1 - What is the most important signal?  ROS, nutritional substrates, or hypoxia?  [i.e.ROS signaling (via Nrf2) vs Nutritional & Hypoxic Signaling (via HIF-1a and SIRTs)] (I, Vince, think this refers to anti-aging interventions)

Proposal:   I propose that we make an “intellectual wager” for the lump sum of one dollar.  To allow time for the debate, the winner will be “paid up” at end of 2013.  The “wager” is on which is the most important “signaling mechanism” for longevity – “ROS signaling” [Vince's bet] or “Nutrient/Oxygen signaling” [Jims bet].

  1. Wager #2 - What is the most important cellular adaptation mechanism? (i.e. hormetic response).  (i.e. anti-oxidant response element upregulation, unfolded protein response, mitochondrial biogenesis, DNA repair mechansims, autophagy, etc.)

Proposal:   I propose that we make an “intellectual wager” for the lump sum of one dollar.  The question is what is the most important cellular adaptation mechanism? (i.e. hormetic response) that promotes health and longevity.  (i.e. anti-oxidant response element upregulation, unfolded protein response, mitochondrial biogenesis, DNA repair mechanisms, autophagy, etc.)  You say it is upregulating the AREs [Anti-oxidant Response Element] and I say it is upregulating autophagy

To throw my hat into the ring I’ve finally put together my own particular research on the subject.  If I had to summarize it to the most concise form possible I would say maintenance of cellular redox homeostasis is the most important longevity factor.  To reword it for wager #1 my premise is that redox signalling is the most important. My answer for wager #2 would then have to be the response to maintain redox homeostasis, with ARE/EpRE upregulation being one primary factor.

This ties tightly into ROS signaling via Nrf2, they are pieces of the same puzzle and I’ll attempt to tease apart some the subtle differences.  I see redox state, implying GSH levels (and it’s direct functions), to be the primary factor,  with increasing Nrf2 expression as one of two ways to achieve it. (1. Increasing the enzymes, 2. Increasing the substrates).  I’ll cover the subtleties I’ve found of Nrf2 expression and GSH synthesis in Part 2.

One of the first main clues which set me down the redox path a while ago when investigating cysteine supply for GSH synthesis was this conclusion from Oxidative stress and ageing: is ageing a cysteine deficiency syndrome?

“In humans, an age-related oxidative shift in the ratio of reduced to oxidized glutathione, i.e. the glutathione redox status, has been demonstrated in whole blood, and peripheral blood mononuclear cells. … Responses of signalling cascades to changes in redox status (see §1b) are, therefore, not merely experimental artefacts. As the thiol/disulfide redox status shifts to more oxidative conditions in old age, there is inevitably a shift in the set points of physiological signals.

On further investigation redox state appeared to be the most far reaching factor, the one with a finger in every pie, and of growing interest in the research arena.  Two quick indicative examples are this article’s title, Changing paradigms in thiology from antioxidant defense toward redox regulation. (Methods Enzymol. 2010), and the following from Basic Principles and Emerging Concepts in the Redox Control of Transcription Factors (Antioxid Redox Signal. 2011 October)

“Activation of gene transcription has for long been considered to be primarily, if not exclusively, regulated by cascades of protein phosphorylation and de-phosphorylation… During the nineties, though, a second area was recognized to be intimately related to transcriptional regulation, the ubiquitin/proteasome system…

The multiple ways of redox regulations that became obvious over the last two decades lead us to presume that most, if not all, of the classical routes to transcriptional activation are modulated by redox processes or even critically depend on oxidant signals”

and Glutathione and apoptosis Free Radic Res. 2008 August

“Increasingly, we have witnessed a growing appreciation of the role of GSH in redox signalling beyond its traditionally recognized role as the main cellular antioxidant against oxidative challenge. The heightened interest in GSH in post-translational control of cellular processes has brought to the fore the versatility of this ubiquitious molecule that is present in millimolar concentrations in most cells and whose homeostasis is rigorously controlled by GSH redox enzymes and glutamate-cysteine ligase-driven GSH synthesis”

 

A definition of Redox

“Redox state is the energetic force for electron transfer, much as pH is a measure of the strength of proton transfer. I.e., redox state measures the ability of a compound to donate or receive electrons, just as pH is a measure of the ability of a compound to donate or receive protons. Technically, redox state, E, is the electromotive force in mV relative to the standard state of hydrogen as follows. An example is also shown for the most abundant intracellular reductant, glutathione:

gsh-redox

where Eo is the standard reduction state at pH 7 (−264 mV for glutathione, R is the gas constant, T is temperature (oK), n is the number of transferred electrons and F is Faraday’s constant.” (ref)

 

Redox Glutathione?

I’ll be taking some liberty of alternating between referring to redox state and glutathione levels, as GSH/GSSG is the major intra-cellular redox couple.  Partly because my initial research initially focused on glutathione before looking more closely at redox and also because GSH has it’s own direct functions such as detoxification via the GST enzymes. Plus some of the authors of the articles referenced may not have been aware of the significance of GSH levels and redox.  Being the major player the I’ll focus on many factors of GSH synthesis and function, and the practical interventions, in Part 2.

“Cellular redox state, or the balance between oxidation/reduction reactions, is collectively determined by the reduction potentials and reducing capacities of the redox couples, such as GSH/GSSG, NADPH/NADP+, NADH/NAD+, cysteine/cystine, thioredoxin (reduced)/thioredooxin (oxidized) and glutaredoxin (reduced)/glutaredoxin (oxidized). Nonetheless, the GSH/ GSSG couple is regarded as the primary arbiter of the tissue redox state because it is comparatively 2 to 4 orders of magnitude higher in abundance than the other redox couples and it is also metabolically linked to the less abundant redox couples via direct or indirect donations of reducing equivalents for the reduction of their oxidized forms” (ref)

 

GSH, More Powerful than ATP

Having always thought of ATP as the energy currency of the cell, interestingly the prior quote follows on to say:

“The mitochondrial electron transport chain efficiently oxidizes NADH with molecular oxygen over numerous smaller steps to siphon about 98% of the energy as a proton and electrical gradient across the mitochondrial inner membrane.   We commonly think of ATP as a high energy molecular currency, but hydrolysis of the phosphate bond produces only −7.3 kcal/mol, compared to almost −60 kcal/mol of oxidized NADH. Since the reducing energies of NADH, NADPH and GSH are so large, they can provide the power for a large number of reduction reactions in the cell including glycolysis, ATP generation, disulfide bond formation in numerous enzymes, transporters, signaling molecules and transcription factors. The driving force for these reactions depends on the relative concentrations of the oxidized and reduced forms of each redox couple.” (ref)

(image source Copywright Exp Gerontol. 2010 March; 45(3): 173–179.)

 

Not a one-size-fits-all

Redox state and glutathione levels are maintained at different levels in various parts of the cells and body.

“GSH concentration in brain cells is about 400-times higher than in blood. … The mitochondrial GSH accounts for approximately 15% of cellular glutathione. In general the mitochondrial GSH:GSSG ratio is greater than that of the cytosol, resulting in a more reducing environment. One study of the GSH and GSSG levels in rat mitochondria reported that liver GSH concentration was 8.4 um and GSSG was 0.02 um, corresponding to approximately 250:1 GSH:GSSG ratio. However, in brain mitochondria the GSH was reported to be 5.5 um and GSSG 0.09 um, giving an approximately 50:1 GSH:GSSG ratio. … Unlike most of the cell (which maintains a 100:1 GSH:GSSG ratio), the endoplasmic reticulum has an unusually oxidative environment estimated at a 5:1 GSH:GSSG ratio” (ref)

Tissue levels of GSH vary widely between different tissues, with the highest concentrations in the eye lens (∼10 mM) and the lowest concentrations in fast-twitch skeletal muscles (∼0.5 mM). (ref)

“Although synthesized exclusively in the cytosol, GSH is distributed to different cellular compartments where it maintains distinct redox environments uniquely suited to the function of the organelle, be it protein folding in the endoplasmic reticulum or gene transcription in the nucleus.” (ref)

And from a further reference below “stem cells reside in redox niches with low ROS levels”

 

Dysregulation of redox state is more important and independent to (preceedes) ROS damage

The “redox stress hypothesis” proposes aging-associated functional losses are primarily caused by a progressive pro-oxidizing shift in the redox state of the cells, which leads to the over-oxidation of redox-sensitive protein thiols and the consequent disruption of the redox-regulated signaling mechanisms.  Another article on the topic, Epigenetic oxidative redox shift (EORS) theory of aging unifies the free radical and insulin signaling theories (Exp Gerontol. 2010 March) says:

“In order to begin to resolve these paradoxes, I propose that an oxidized redox state is upstream of the commonly observed ROS damage. Certainly, ROS damage is affected by the balance of oxyradical generation and anti-oxidant defenses. Numerous sources of oxyradical generation have been documented, but less appreciation exists for the essential role that ROS or redox signaling plays in metabolism. The common impression that the mitochondrial electron transport chain is the major source of oxyradical generation often overlooks other sources in the cytoplasm and plasma membrane and an upstream oxidized redox state.”

Experimental evidence in  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. finds:

“To determine whether glutathione (GSH) loss or increased reactive oxygen species (ROS) are more important to neuron loss, aging, and Alzheimer’s disease (AD), we stressed or boosted GSH levels in neurons isolated from aging 3xTg-AD neurons compared with those from age-matched nontransgenic (non-Tg) neurons. … Remarkably, the rate of neuron loss with ROS did not increase in old age and was the same for both genotypes, which indicates that cognitive deficits in the AD model were not caused by ROS. … These stress tests and neuroprotective treatments suggest that the redox environment is more important for neuron survival than ROS”

Testing of the structural damage-based oxidative stress hypothesis (ref):

“In summary, the presently accessible information suggests that although the steady-state amounts of macromolecular oxidative damage tend to increase with age, the molar ratios of oxidized:unoxidized macromolecules are very low. Furthermore, the oxidized macromolecules, rather than being stored, are generally rapidly eliminated and replaced via nascent biosynthesis. Arguably, the age-related functional losses would be expected to depend more upon the pool size of the parent unoxidized macromolecules rather than the amounts of the oxidized macromolecules, unless it could be demonstrated that the very presence of oxidized macromolecules was itself deleterious, analogous to dominant negative mutations. Because the physiological losses in the latter part of life are often quite severe and mortality increases exponentially, whereas the accrued amounts of macromolecular oxidative damage are relatively minuscule, the case for a possible causal association remains tenuous. Nonetheless, this ambiguity does not imply that ROS or oxidative stress do not play an important role in the aging process. Rather, the steady-state amounts of oxidative structural damage are not synonymous with oxidative stress nor are they a reliable indicator of functional losses.”

 

Under normal conditions hydrogen peroxide function as signalling molecule and its levels are maintained by  catalase and the peroxidase enzymes.  Dysregulation of redox leading to an over oxidized state can cause an increase in the generation of the highly reactive and more more damaging hydroxyl free radical (OH·) via the iron-catalyzed, Haber-Weiss- and Fenton-type reactions, and an increase in reactions causing irreversible bonds on redox-sensitive protein thiols.  A more detailed description is here.  Furthermore another article below finds there is an “irreversible consequence of nuclear GSH depletion” even after GSH levels are restored.

 

Supporting Evidence

Vincent Guiliano has detailed his 14 Theories of Aging here, I’ll use these to group together some the of the research showing the extensive role redox and glutathione play in cellular processes and health.

1. Oxidative Damage

Glutathione is often referred to as the master anti-oxidant in the published literature.  A fairly obvious link that a reduced redox state and high levels of glutathione will be able to reduce oxidative damage.

2. Cell DNA Damage (and cell proliferation regulation)

Nuclear glutathione. Biochim Biophys Acta. 2013 May

“The sequestration of GSH in the nucleus of proliferating animal and plant cells suggests that common redox mechanisms exist for DNA regulation in G1 and mitosis in all eukaryotes. We propose that glutathione acts as “redox sensor” at the onset of DNA synthesis with roles in maintaining the nuclear architecture by providing the appropriate redox environment for the DNA replication and safeguarding DNA integrity. In addition, nuclear GSH may be involved in epigenetic phenomena and in the control of nuclear protein degradation by nuclear proteasome. Moreover, by increasing the nuclear GSH pool and reducing disulfide bonds on nuclear proteins at the onset of cell proliferation, an appropriate redox environment is generated for the stimulation of chromatin decompaction.”

The primary role of glutathione against nuclear DNA damage of striatum induced by permethrin in rats. Neuroscience. 2010 Jun

“On the contrary, GSH played a crucial role on striatum since it was able to protect the cells against nuclear DNA damage induced by PERM. In conclusion our outcomes suggested that nuclear DNA damage of striatum cells was directly related to GSH depletion due to PERM insecticide.”

The Depletion of Nuclear Glutathione Impairs Cell Proliferation in 3t3 Fibroblasts

“We show here and in a previous report that nuclear glutathiolation changes during the cell cycle and that the depletion of nuclear GSH changes the pattern of nuclear glutathionylated proteins. The suggestion that reduced nuclear environment could protect oxidant sensitive proteins from oxidation could be confirmed by our results: there was less glutathionilated and more oxidised proteins when the nuclear GSH was depleted by DEM. However, after nuclear GSH increased (72 h) the glutathionylation of nuclear protiens in DEM treated cells reached the values of control, while the level of protein oxidation remained high. This reflects the irreversible consequence of nuclear GSH depletion early in the culture

So, the presence of the high glutathione level in the nucleus appears to be a prerequisite for the start of the cell proliferation. Our findings are in line with several other studies aimed to elucidate the fine redox regulatory mechanisms that lie behind the correct cell cycle progression. Conour et al., suggested that the reduction of the intracellular environment as cells progress from G1 to G2/M phase, as shown in our study, may protect genomic DNA from oxidative damage upon brake down of the nuclear envelope. Indeed, one of the assertions in support of this premise derives from the study of oxidative stress related to genotoxicity, recently published by Green; oxidative DNA modifications displayed a negative linear correlation with nuclear GSH. This is of special importance considering the report of Menon et al on the necessity of the oxidative event in early G1 phase to allow G1-S transition. Even more, it has recently been postulated that the restriction of DNA synthesis to the reductive phase of the cycle in yeast may be an evolutionarily important mechanism for reducing oxidative damage to DNA during replication, which implies the common mechanism of the DNA protection during S phase in all eukaryotes.”

ii. Glutathione S-transferase and DNA damage

Glutathione S-transferase are a family of enzymes which catalyze the conjugation of the reduced form of glutathione to xenobiotic substrates, which can damage DNA, for the purpose of detoxification. While these studies, and more, look at GST polymorphisms causing reduced GST levels, the ability to detoxify DNA damaging compounds will be determined by both the concentrations of the enzyme and substrate, i.e. GST and GSH levels.

Are glutathione S transferases involved in DNA damage signalling? Interactions with DNA damage and repair revealed from molecular epidemiology studies. Mutat Res. 2012 Aug

“Our results show that GST polymorphisms and GST activity can apparently influence DNA stability and repair of oxidised bases, suggesting a potential new role for these proteins in DNA damage processing via DNA damage signalling.”

Protection against oxidative DNA damage and stress in human prostate by glutathione S-transferase P1. Mol Carcinog. 2012 Jul

“Loss of GSTP1 expression via promoter hypermethylation is the most common epigenetic alteration observed in human prostate cancer. Silencing of GSTP1 can increase generation of reactive oxygen species (ROS) and DNA damage in cells… These results suggest that loss of GSTP1 expression in human prostate cells, a process that increases their susceptibility to oxidative stress-induced DNA damage”

 

3. Mitochondrial Damage

Unearthing the secrets of mitochondrial ROS and glutathione in bioenergetics Trends in Biochemical Sciences, October 2013

“In excess, ROS can be detrimental; however, at low concentrations oxyradicals are essential signaling molecules. Mitochondria thus use a battery of systems to finely control types and levels of ROS, including antioxidants. Several antioxidant systems depend on glutathione. Here, we review mitochondrial ROS homeostatic systems, including emerging knowledge about roles of glutathione in redox balance and the control of protein function by post-translational modification.”

Mitochondrial glutathione, a key survival antioxidant. Antioxid Redox Signal. 2009 Nov

“Among the arsenal of antioxidants and detoxifying enzymes existing in mitochondria, mitochondrial glutathione (mGSH) emerges as the main line of defense for the maintenance of the appropriate mitochondrial redox environment to avoid or repair oxidative modifications leading to mitochondrial dysfunction and cell death. mGSH importance is based not only on its abundance, but also on its versatility to counteract hydrogen peroxide, lipid hydroperoxides, or xenobiotics, mainly as a cofactor of enzymes such as glutathione peroxidase or glutathione-S-transferase (GST). Many death-inducing stimuli interact with mitochondria, causing oxidative stress; in addition, numerous pathologies are characterized by a consistent decrease in mGSH levels, which may sensitize to additional insults. From the evaluation of mGSH influence on different pathologic settings such as hypoxia, ischemia/reperfusion injury, aging, liver diseases, and neurologic disorders, it is becoming evident that it has an important role in the pathophysiology and biomedical strategies aimed to boost mGSH levels.”

 

4. Tissue Glycation

Functional Consequences of Age-Dependent Changes in Glutathione Status in the Brain. Antioxid Redox Signal. 2013 Feb

“Changes in redox homeostasis can also potentiate the accumulation of advanced glycation endproducts, resulting in defects in protein processing and function as well as a further increase in inflammation.”

Glutathione reverses early effects of glycation on myosin function

“It is concluded that glucose modifies myosin function in a dose-dependent manner and that glutathione reverses the effect of glucose on myosin function.

The present results demonstrate that GSH reverses the formation of early glycation products. The restoration of motility by GSH after incubation with a reducing sugar implies a reversal of Schiff base formation.

The present results suggest that GSH, in addition to its antioxidant function, could play an important role in preventing the progress of glycation of intracellular proteins.”

 

5.  Lipofuscin Accumulation

Influence of intracellular glutathione concentration of lipofuscin accumulation in cultured neonatal rat cardiac myocytes.

“The authors show that reduced [decreased] GSH level leads to a simultaneous increase in accumulation of lipofuscin in cardiac myocytes, possibly by increasing the level of cytosolic hydrogen peroxide.”

Nothing specific in the follow but a good recent review of lipofuscin  published coincidentally in Redox Biology

Lipofuscin: formation, effects and role of macroautophagy Redox Biol. 2013

 

6. Chronic or Excess Inflammation

Searching PubMed for redox, or glutathione, and inflammation returns a multitude of articles.  In particular Nuclear factor-kappaB is a redox sensitive transcription factor critical to immune and inflammatory response.

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3166203/figure/f15/

NF-κB and Nrf2, the Yin and Yang of the inflammatory response.

 

7. Immune System Compromise

Glutathione is important for immune cells protect to protect themselves when they attack pathogens with ROS blasts.  Also many studies have shown, a number listed below, that high levels of glutathione inhibit entry and/or replication for many types of commonly known viruses.

Glutathione: A key player in autoimmunity Autoimmun Rev. 2009 Jul

“Altered glutathione concentrations may play an important role in many autoimmune pathological conditions prevalently elicited, detrimed and maintained by inflammatory/immune response mediated by oxidative stress reactions.

Inhibition of influenza infection by glutathione

“Accumulating evidence suggests that cellular redox status plays an important role in regulating viral replication and infectivity…  Together, the data suggest that the thiol antioxidant GSH has an anti-influenza activity in vitro and in vivo.”

Glutathione Inhibits HIV Replication by Acting at Late Stages of the Virus Life Cycle

” Overall data suggest that GSH can interfere with late stages of virus replication.  This would be in agreement with data obtained in cells exposed to herpesvirus type 1 (a DNA virus) or to Sendai (an RNA virus), showing that the suppression of virus replication by GSH is related to the selective inhibition of envelope glycoproteins.”

Evidence for antiviral activity of glutathione: in vitro inhibition of herpes simplex virus type 1 replication

“Data suggest that exogenous GSH inhibits the replication of HSV-1 by interfering with very late stages of the virus life cycle, without affecting cellular metabolism.”

Natural Killer Cells, Glutathione, Cytokines, and Innate Immunity Against Mycobacterium tuberculosis

“Furthermore, NK cell functions are dependent on adequate levels of glutathione.  Our results strongly indicate that glutathione in combination with IL-2+IL-12 augments NK cell functions, leading to control M. tuberculosis infection.”

Glutathione and infection

“In this review we describe how GSH works to modulate the behavior of many cells including the cells of the immune system, augmenting the innate and the adaptive immunity as well as conferring protection against microbial, viral and parasitic infections. This article unveils the direct antimicrobial effects of GSH in controlling Mycobacterium tuberculosis (M. tb) infection within macrophages. In addition, we summarize the effects of GSH in enhancing the functional activity of various immune cells such as natural killer (NK) cells and T cells resulting in inhibition in the growth of M. tb inside monocytes and macrophages. Most importantly we correlate the decreased GSH levels previously observed in individuals with pulmonary tuberculosis (TB) with an increase in the levels of pro-inflammatory cytokines which aid in the growth of M. tb.”

Inhibitory Effect of Glutathione on Oxidative Liver Injury Induced by Dengue Virus Serotype 2 Infections in Mice

“in vitro treatment of HepG2 cells with antioxidants such as GSH inhibited viral entry as well as the production of reactive oxygen species in HepG2 cells.”

 

8  Neurological Degeneration

High levels of GSH in neurons and white matter, suggests astrocytes rather than neurons may be particularly vulnerable to oxidative stress (ref).  Also astrocytes appear to be susceptible high extra cellular glutamate from immunoexcitotoxicity etc, which reduces their ability to uptake cysteine for GSH synthesis, however I will cover this in Part 2.

Dysregulation of Glutathione Homeostasis in Neurodegenerative Diseases  Nutrients. 2012 October

“Over the past several decades the role of intracellular GSH status in neurodegenerative diseases has been studied intensively. Such research continues to provide mechanistic insights pertaining to the cellular dysfunctions of the neurodegenerative diseases, including Parkinson’s disease, Alzheimer’s disease, Huntington’s disease, amyotrophic lateral sclerosis, and Friedreich’s ataxia. Disruption in GSH homeostasis and modification of the enzymes that are dependent on GSH as a substrate have been linked to initiation and progression of the neurodegenerative diseases. The dysregulation of GSH and GSH-dependent enzymes induces a variety of cellular problems that can lead to mitochondrial dysfunction, accumulation of ROS/RNS damage, disruption of signaling pathways, protein aggregation, and ultimately cell death.”

Functional Consequences of Age-Dependent Changes in Glutathione Status in the Brain.  Antioxid Redox Signal. 2013 Feb

“Decreases in GSH are also associated with microglial activation and endothelial dysfunction, both of which can contribute to impairments in brain function. Changes in redox homeostasis can also potentiate the accumulation of advanced glycation endproducts, resulting in defects in protein processing and function as well as a further increase in inflammation.”

Role of a Redox-Based Methylation Switch in mRNA Life Cycle (Pre- and Post-Transcriptional Maturation) and Protein Turnover: Implications in Neurological Disorders Front Neurosci. 2012

“A “holonarchy” for synaptic plasticity can be imagined, beginning at mRNA synthesis, transcription, translation, protein turnover, methylation reactions, and at the highest level redox status serves as the central regulatory switch.”

(See a definition for holonarchy here)

Glutathione and Parkinson’s disease: Is this the elephant in the room? Biomed Pharmacother. 2008 Apr-May

“At least 2 decades have past since the demonstration of a 40% deficit in total glutathione (GSH) levels in the substantia nigra in patients with Parkinson’s disease (PD). The similar loss of GSH in the nigra in Incidental Lewy body disease, thought to be an early form of PD, indicates that this is one of the earliest derangements to occur in the pre-symptomatic stages of PD”

Impaired Glutathione Synthesis in Neurodegeneration Int. J. Mol. Sci. 2013

 

11        Susceptibility to Cardiovascular Disease

In Vince’s 14 Theories of Aging, this section says:

The age-related remodeling appears to involve an imbalance between omega-3 and omega-6 fatty acids in these membranes as well as dysfunctional Ca2+ metabolism. (ref)

A articles few which look at the Calcium aspect.

Redox regulation of cardiac calcium channels and transporters Cardiovascular Research 71 (2006)

“Changes in the intracellular redox environment can affect many cellular processes, including the gating properties of ion channels and the activity of ion transporters. Because cardiac contraction is highly dependent on intracellular Ca2+ levels ([Ca2+]I ) and [Ca2+]I regulation, redox modification of Ca2+ channels and transporters has a profound effect on cardiac function.”

Because levels of ROS and RNS can increase significantly after stimulation of specific signal transduction pathways or during certain pathological conditions of the heart (e.g. ischemia– reperfusion) the redox defense system is essential for the maintenance of cellular homeostasis.

In many mammalian cells, including cardiomyocytes, glutathione is considered the major cytosolic redox buffer.”

Redox regulation of calcium release in skeletal and cardiac muscle Biol Res. 2002;35(2):183-93.

“In skeletal and cardiac muscle cells, specific isoforms of the Ryanodine receptor channels mediate Ca2+ release from the sarcoplasmic reticulum. These channels are highly susceptible to redox modifications, which regulate channel activity.”

Crosstalk between calcium and redox signaling: from molecular mechanisms to health implications. Antioxid Redox Signal. 2008 Jul;

“Studies done many years ago established unequivocally the key role of calcium as a universal second messenger… Furthermore, it is becoming increasingly apparent that there are significant interactions between calcium and redox species, and that these interactions modify a variety of proteins that participate in signaling transduction pathways and in other fundamental cellular functions that determine cell life or death.”

 

12        Telomere Shortening and Damage

Chronic oxidative stress compromises telomere integrity and accelerates the onset of senescence in human endothelial cells.

“These findings demonstrate a key role for glutathione-dependent redox homeostasis in the preservation of telomere function in endothelial cells”

Glutathione regulates telomerase activity in 3T3 fibroblasts.

“Telomerase activity is maximal under reduced conditions i.e. when the reduced/oxidized glutathione ratio is high. Consequently glutathione concentration parallels telomerase activity.”

 

14. Stem Cell Supply Chain Breakdown

Redox homeostasis: the linchpin in stem cell self-renewal and differentiation January 2013

“Recently, a growing body of literature has shown that stem cells reside in redox niches with low ROS levels. The balance of Redox homeostasis facilitates stem cell self-renewal by an intricate network. Thus, to fully decipher the underlying molecular mechanisms involved in the maintenance of stem cell self-renewal, it is critical to address the important role of redox homeostasis in the regulation of self-renewal and differentiation of stem cells.”

 

Furthermore I’ll look at two additional topics redox and GSH weave their way into.

Epigenetics

Redox regulation of the epigenetic landscape in cancer: a role for metabolic reprogramming in remodeling the epigenome. Free Radic Biol Med. 2012 Dec

“We further speculate that redox biology can change epigenetic events through oxidation of enzymes and alterations in metabolic cofactors that affect epigenetic events such as DNA methylation. Combined, these metabolic and redox changes serve as the foundation for altering the epigenotype of normal cells and creating the epigenetic progenitor of cancer.”

The redox basis of epigenetic modifications: from mechanisms to functional consequences. Antioxid Redox Signal. 2011 Jul

“Recent research is revealing that redox metabolism is an increasingly important determinant of epigenetic control that may have significant ramifications in both human health and disease. Numerous characterized epigenetic marks, including histone methylation, acetylation, and ADP-ribosylation, as well as DNA methylation, have direct linkages to central metabolism through critical redox intermediates such as NAD(+), S-adenosyl methionine, and 2-oxoglutarate.”

Blood glutathione redox status and global methylation of peripheral blood mononuclear cell DNA in Bangladeshi adults. Epigenetics. 2013 Jul

“Our findings support the hypothesis that a more oxidized blood GSH redox status is associated with decreased global methylation of peripheral blood mononuclear cell DNA”

 

Cell Cycle/Death - Apoptosis and Post-mitotic

Apoptosis and glutathione: beyond an antioxidant Cell Death and Differentiation (2009)

“GSH depletion was initially ascribed to its oxidation by RS generated during oxidative stress. However, it is now recognized that under more physiological stimulation of apoptosis, such as activation of death receptors, GSH depletion occurs as an active process involving its extrusion across the plasma membrane. This phenomenon has also been shown to precede oxidative stress generated by the accumulation of RS and to be necessary for the progression of apoptosis. Indeed, GSH depletion has been shown to induce or potentiate apoptosis, and excessive oxidative stress. Although the exact mechanisms involved in the regulation of apoptosis by GSH remain elusive, recent reports show that oxidative post-translational modifications in proteins regulated by GSH content such as glutathionylation (protein-SSG) and nitrosylation (protein-SNO) are important regulators of apoptosis.”

Glutathione and apoptosis Free Radic Res. 2008 August

“With regards to apoptosis, the mitochondrial GSH redox status is emerging to be a central player. Our studies have provided new perspectives on the role of mitochondrial GSH in mitochondrial DNA integrity and cell survival and the availability of genetic approaches targeting mitochondrial GSH transporters offers new strategies for studying the importance of this redox compartment in apoptosis. The current understanding of protein S-glutathiolation in conjunction with protein S-nitrosation as important post-translational regulatory mechanisms has contributed to recent advances in apoptosis research.”

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. [For recycling oxidized GSSG to reduced GSH]”

And follow up comments to this article here: A new answer to old questions

Atmospheric oxygen accelerates the induction of a post-mitotic phenotype in human dermal fibroblasts: the key protective role of glutathione

“Increasing oxidative stress by addition of hydrogen peroxide or depletion of glutathione also induced a switch from a mitotic to a post-mitotic phenotype in these cells, whereas addition of the anti-oxidant N-acetylcysteine under atmospheric (20%) oxygen tension potently inhibited this process. In addition, a statistically significant correlation was observed between the magnitude of intracellular glutathione depletion and the reduction in the population of mitotic cells in this model. We propose that the switch from a mitotic to a post-mitotic phenotype represents a process of cellular ageing and that standard atmospheric oxygen tension imposes a substantial oxidative stress on dermal fibroblasts which accelerates this process in culture. The data also suggest that intracellular glutathione levels strongly influence the induction of a post-mitotic phenotype and that, by implication, depletion of glutathione may play a significant role in the progression of cellular ageing in human skin.”

 

Further Proof Required?

As mentioned previously redox regulation is seeing a greater interest in the research community.  Still further research will be required to elucidate the roles and mechanisms redox plays.  In this article, published in Front Neurosci. 2012, the authors suggests a number of study designs which could provide further evidence, saying:

“These and other such studies would allow researchers to test the underlying major hypothesis that redox state is the ultimate source of regulatory control over mRNA methylation, mRNA processing, protein synthesis, and protein turnover.”

 

While searching for further supporting evidence, I very recently came across this article which would be the most detailed research article supporting the redox stress argument, taking into account transgenic species, organism fitness and more, and aided in putting together some of the previous points..  So take a moment to pause here and read it in its entirety.

The Redox Stress Hypothesis of Aging Free Radic Biol Med. 2012 February 1; 52(3): 539–555.

“Presently, the balance of evidence seems to favor the view that the role of structural damage is relatively minor compared to that emanating from the disruptions of the redox-based molecular switches. We have termed this phenomenon “redox stress”, as there are indications of a disturbance in the thiol redox state in the post-reproductive phase of life. Furthermore, 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.”

 

The Redox Shift with Age

So now we know how important redox is, here is a quick look at how it changes with age.  Also health conditions, lifestyle choices and environmental conditions can accelerate the loss of GSH and oxidative shift in the redox state, for example: “We conclude that subjects with type 2 diabetes have decreased oxidant capacity, evidenced by reduced synthesis of glutathione, and they are under increased oxidative stress” (ref)

Epigenetic oxidative redox shift (EORS) theory of aging unifies the free radical and insulin signaling theories

Dean Jones at Emory University was the first to show that human plasma GSH/GSSG is controlled at a relatively constant redox state of −137 mV in 740 healthy adults through age 50. However, an oxidative shift of about 7 mV/decade occurs over the next two decades, followed by a further decline to −110 mV in the 70 to 85-year-old group.

Oxidative stress and ageing: is ageing a cysteine deficiency syndrome?

“In humans, an age-related oxidative shift in the ratio of reduced to oxidized glutathione, i.e. the glutathione redox status, has been demonstrated in whole blood, and peripheral blood mononuclear cells. The mean plasma cysteine/cystine redox status of human subjects shows a significant oxidative shift between the third and the ninth decade of life. This age-related oxidative shift is accompanied by a decrease in the plasma glutathione level and a decrease in the ratio of reduced versus oxidized forms of plasma albumin and other thiol/disulfide redox couples.”

Extracellular Redox State: Refining the Definition of Oxidative Stress in Aging

“Plasma GSH/GSSG redox in humans becomes oxidized with age, in response to chemotherapy, as a consequence of cigarette smoking, and in association with common age-related diseases (e.g., type 2 diabetes, cardiovascular disease). However, the GSH/GSSG redox is not equilibrated with the larger plasma cysteine/cystine (Cys/CySS) pool, and the Cys/CySS redox varies with age in a pattern that is distinct from that of GSH/GSSG redox.”

 

Cysteine’s other roles

While cysteine is the critical functional amino, and least abundant, of the 3 amino acids required for glutathione synthesis, it also plays key roles in other enzymes and proteins, including glutathione peroxidase 1 (GPX1)

Cysteine-Based Redox Switches in Enzymes Antioxid Redox Signal. 2011 March
“While cysteine residues often play critical roles in enzyme catalysis, they also act as redox switches in many enzymes, allowing for communication between the global or local cellular redox properties and enzymatic function.”

Cysteine is also important for forming the proper 3D structure of proteins from the disulfide bonds between cysteine groups.

Metal and redox modulation of cysteine protein function. Chem Biol. 2003 Aug

“In biological systems, the amino acid cysteine combines catalytic activity with an extensive redox chemistry and unique metal binding properties. The interdependency of these three aspects of the thiol group permits the redox regulation of proteins and metal binding, metal control of redox activity, and ligand control of metal-based enzyme catalysis. Cysteine proteins are therefore able to act as “redox switches,” to sense concentrations of oxidative stressors and unbound zinc ions in the cytosol, to provide a “storage facility” for excess metal ions, to control the activity of metalloproteins, and to take part in important regulatory and signaling pathways.”

 

The Future?

While there are simple nutritional strategies for maintaining glutathione levels and redox homeostasis which I will discuss in Part 2,  the increasing interest and research in redox biology is already showing promising therapeutic potential for chronic conditions and drug development.

Site-Specific Antioxidative Therapy for Prevention of Atherosclerosis and Cardiovascular Disease

“In addition to the improvement of lifestyle, recently emerging drugs that are effective in treating CVD have a property to eliminate ROS with a site-specific manner without interrupting favorable redox signaling, thereby ameliorating oxidative stress to endothelial cells.”

Redox-directed cancer therapeutics: molecular mechanisms and opportunities. Antioxid Redox Signal. 2009 Dec

“Redox dysregulation originating from metabolic alterations and dependence on mitogenic and survival signaling through reactive oxygen species represents a specific vulnerability of malignant cells that can be selectively targeted by redox chemotherapeutics.

The impressive number of ongoing clinical trials that examine therapeutic performance of novel redox drugs in cancer patients demonstrates that redox chemotherapy has made the crucial transition from bench to bedside.”

Discovery of a statistically significant and interpretable relationship between redox reactivity and lethality of drugs. Curr Drug Metab. 2013 Sep

“A statistically significant and interpretable relationship between electrophilicity as a redox reactivity indicator and LD50 as a lethality indicator of drugs was discovered, and this relationship could be interpreted by the action of the cytochrome P450. The drugs chosen in this study were Topoisomerase II inhibitor anticancer drugs, and the electrophilicity of drugs was obtained by quantum chemical calculation. Since the P450 detoxification mechanism is the catalytic oxidation of drug molecules, it may infer that the drug molecules being easily oxidized (low electrophilicity) will be weak in lethality in general. In addition, this relationship revealed two structural scaffolds for the anthracycline-based topoisomerase II inhibitors, and their lethality mechanisms are not totally the same. Such relationship can assist in designing new drugs that candidates possessing low electrophilicity are recommended for lowering of lethality, and moieties providing a large inductive effect can reduce the electrophilicity of the anthracycline-based topoisomerase II inhibitors.”

 

Further Reading:

Teaching the basics of redox biology to medical and graduate students: Oxidants, antioxidants and disease mechanisms Redox Biol. 2013; 1(1): 244–257.

Reactive oxygen species (ROS) homeostasis and redox regulation in cellular signaling Cell Signal. 2012 May; 24(5): 981–990.

 

Part 2

Factors Affecting Redox, GSH Levels and Function – Redox, Glutathione and Cysteine, Part 2

 

Part 3 coming soon detailing the 6 available types of GSH supplementation (2 Cysteine pro-drugs, 1 GSH pro-drug and 2 GSH delivery mechanisms and optimized dietary sources i.e. whey protein) along with other supporting nutrients.

Found this informative? Share the knowledge with a Facebook Like, Tweet, posting the link etc. or leave a comment below!

 

Reviewing Glutathione Supplement Options – Redox, Glutathione and Cysteine, Part 3

 WORK IN PROGRESS – Aiming to be essentially complete in the next month

 

Glutathione is a tri-peptide, made from the three amino acids cysteine, glutamine and glycine. In the stomach glutathione is rapidly broken down into the three separate amino acids.  The key amino, cysteine, is reactive due to its sulfhydryl group, and rapidly reacts with other molecules or form bonds together to create cystine (note the subtle spelling difference).

 

Oral Reduced GSH

The reduced (active) form of glutathione is cheaply available as a supplement. On hearing how glutathione is the body’s master antioxidant and detoxifier (and the primary determinate of the intra-cellular redox status) a bottle of reduced L-Glutathione seems like a bargain, but unfortunately it is very poor at increasing cellular glutathione levels.

It’s long known taking glutathione orally has minimal benefits. Witschi et al. showed that patients given 3000 mg of oral GSH led to no significant change in blood GSH or cysteine

So what to do?

Effective glutathione supplementation can be broken down in the 3 main area’s/methods.

The first two are the pro-drug approach.  A pro-drug is a compound which is metabolised into the active drug in the body.  This is done by attaching additional molecules to the compound, which are then.  For glutathione supplementation this is done in two ways,

  1. Glutathione pro-drugs, such as S-Acetyl-Glutathione, and
  2. Cysteine pro-drugs, such as N-Acetyl-Cysteine and Ribose-Cysteine.

The third method is delivery mechanisms for reduced glutathione, such as in a liposomal form or intravenous.

The 2010 article tk published in Molecules, looks at the research of a number of cysteine and GSH pro-drugs in the laboratory.  However I will only look closely at the different practical (i.e. commercially available) options to choose from.

 

 

ALA and extracellular GSH

 

 

Intravenous (IV)

To bypass the breakdown of GSH in the digestive tract another method of delivery which has been used for a while is intravenous injection. More common uses are for treatment of conditions such as Parkinson’s, which has a tell tale loss of glutathione in the substantia nigra, and toxicity from heavy metals etc.

IV GSH has also quietly been used in anti-aging regimes where the high cost doesn’t deter those who can afford it, including a number of well-known celebrities (ref).

IV delivers GSH straight into the blood stream, however we still run into another roadblock into raising intra-cellular levels.  Most cells are unable to transport extra-cellular GSH inside the cell. (some epithelial cells are a main exception ref ). Glutathione first has to be broken down into the constituent amino acids, which then can be transported into the cell.

Cysteine (Cys) is rapidly converted to Cystine (CySS), with Cys/CySS being the major extra-cellular redox couple. Cystine is the major source of cysteine for the cells, and is transported in by the X-c transporter. However this transporter is weakly expressed in lymphocytes and inhibited by glutamate.

Also Cysteine is readily transported into cells for GSH synthesis by the efficient ASC transport system for neutral amino acids (ref).  Cysteine is in much lower concentrations, but another mechanism by which extracellular GSH may increase intracellular GSH levels in cultured cells is by reducing cystine to cysteine, which is then rapidly transported and used as a substrate for intracellular GSH synthesis. (ref)

The other downsides to intravenous glutathione therapy include the costs and inconvenience of regular injections. To maintain increased glutathione levels required injections about every 3 to 4 days.

 

Lipsomal Glutathione

Another form of glutathione which has become more popular which has the advantages of intravenous delivery is the liposomal form.  Liposomes are small bubbles made out of the same material as a cell membrane, phospholipids. Liposomes can be filled with drugs/compounds to enhance their delivery.

Can liposomal glutathione deliver it into the cell?

“Blocking γ-glutamylcysteine synthetase with buthionine sulfoxamine prevented replenishment with liposomal-GSH demonstrating the requirement for catabolism and resynthesis.”

Liposomal-glutathione provides maintenance of intracellular glutathione and neuroprotection in mesencephalic neuronal cells. Neurochem Res. 2010 Oct

“Because they are made of the same type of material as our cell membranes, liposomes penetrate mucosal tissues allowing for rapid release into the blood stream.  Nutrients that are not in liposomes have to pass through the stomach to reach the liver where they are metabolized and released into the bloodstream.  Some nutrients are destroyed or compromised by stomach acids.  Liposomes avoid the digestive system by penetrating the mucosal tissue.” – http://www.lipoglut.com/

Never the less the published studies on liposomal glutathione have shown potential benefits for pulmonary heart neuronal maintenance atherosclerosis

Many liposomal glutathione products can be found on Amazon.

Dave Asprey’s Bulletproof liposomal glutathione which claims to be stronger than other liposomal glutathione due to the innovative and pharmaceutical grade delivery system.

 

 

Undenatured Whey

Before moving onto the next section, I’ll quickly cover high quality undenatured whey protein is also used/sold as a glutathione booster.  One product in this space is Immunocal.  The human clinical showed a 36% increase in lymphocyte glutathione (compared to 276% for a NAC formulation). While not as effective as dedicated glutathione options, quality whey has additional health benefits from the other valuable aminos and compounds it contains.

 

N-Acetyl-Cysteine (NAC)

The most commonly used form of glutathione supplementation in a clinical setting and by consumers would be the cysteine pro-drug N-Acetyl-Cysteine.

Many published studies have shown benefits of NAC over a wide range of disease conditions.

There are some downsides to NAC which preclude it from being a highly effective means to raise intra-cellular GSH levels.  Its use has been limited by several drawbacks, including low membrane penetration and low systemic bioavailability (ref). Studies have shown while NAC is good at preventing liver damage from acetaminophen (e.g. Tylenol, Panadol) it is not so effective in protecting the kidneys (ref).

NAC is rapidly de-acetylated in the blood tk

 

“Administration of NAC [over 5 days] significantly increased cysteine levels in human plasma and rat bronchoalveolar lavage, but the levels in human neutrophils and rat alveolar macrophages after NAC did not differ from control levels. GSH levels were not altered significantly by NAC.” (ref)

The other downside is that it has its own activity separate to being a GSH precursor and can have pro-oxidant effects.

“Because of a potential prooxidative action indicated by lower reduced glutathione and higher oxidised glutathione plasma levels in healthy subjects at a dose of only 1.2 g NAC (Kleinveld et al., 1992), Kelly (1998) concluded, that the chronic daily supplementation with NAC by healthy individuals, who are not subject to excessive oxidative stress, may be ill advised” (ref)

One study of a 3-week protocol in which mice received high-dose NAC in vivo found the NAC-treated mice developed pulmonary arterial hypertension (PAH) that mimicked the effects of chronic hypoxia due to in vivo conversion of NAC to S-nitroso-N-acetylcysteine (ref). Uowever it’s not unlikely to be a concern with modern moderate dose NAC formulations, as many studies have shown NAC has positive pulmonary effects.

NAC is also used intravenously for ac

There is a large number of studies on PubMed showing many benefits to NAC supplementation. It is widely accepted now that NAC should be taken in a cocktail with other anti-oxidants such as Vitamin C and Alpha-Lipoic Acid to enhance its absorption, utilization and recycling.

Which was refined over a decade of use in a clinical practice by the late Dr Robert Keller, an accomplished doctor and researcher

A daily serve contains 750mg NAC, 500mg Vitamin C and 150mg Alpha-Lipoic acid.

Dr Robert Keller’s NAC formulation was originally launched as MaxGXL in 2007 and can also be found slightly cheaper as the Original Glutathione Formula at the Rob Keller MD website

DIY

 

 

S-Acetyl-Glutathione (SAG)

SAG is a more recently commercially available glutathione pro-drug which in unique in being able to deliver GSH into the cell.  tk?? However the published studies have only been in vitro, so it is not clear if SAG would be deacetylated to normal GSH in the digestive tract and blood stream before it can be absorbed into the cells.  If it is de-aceteylated in the digestive tract and blood stream, as NAC is, then it would be similar to taking a liposomal form for GSH.

My last look showed 4 published articles looking at glutathione levels in regards to herpesfibroblastscancer and HIV (If you know of any more please leave a comment)

As noted in one article for people with a reduced ability to synthesise glutathione (e.g. deficiency in glutathione synthetase) then SAG is likely the best option as it can deliver GSH directly into the cell.

There are a handful of S-Acetyl Glutathione products on Amazon

Another interesting option is SynergiaGSH which contains SAG along with 23 other ingredients for a convenient all-in-one anti-aging supplement.

Nrf2 activators

A number of products exist on the market which aim the increase glutathione levels solely via compounds which activate the Nrf2 pathway (eg ProVantage, Nuley).

They are capable of increasing glutathione levels in the short term by increasing the activity of the enzymes which synthesize glutathione. However the supply of cysteine will eventually be the limiting substrate in glutathione synthesis.  Glutathione, and the crucial cysteine amino, is lost from the body when it conjugated to toxins and removed by the kidneys.

Furthermore some of the compounds which activate Nrf2 such as tk are metabolised by conjugation with glutathione.

 

Nrf2 activators have shown promise in conditions such as tk. Pharmaceutical companies spending big to develop their own patentable version, such as tk.

The Nrf2 pathway creates more than just the various glutathione enzymes, and its benefits tk.

 

Ribose-Cysteine (RibCys, RiboCeine)

One of the oldest studied cysteine pro-drugs is also one of the newest commercially available glutathione enhancers, D-Ribose-L-Cysteine.  The first published study on it was in 1982, many years before glutathione had any mainstream recognition. Since then there is a total of 22 published studies on RibCys.  RibCys was originally developed by a research team led by Dr Herbert Nagasawa at the Veterans Administration medical research center. The aim was to protect the livers of alcoholic war veterans by effectively and safely delivering cysteine inside the cell for glutathione production.

RiboCeine studies

RibCys is able to be absorbed through the digestive tract and intra-cellularly. Blocking the enzyme for glutathione synthesis inhibits the action of RibCys indicating it works solely by supply cysteine for glutathione synthesis (ref).

Inside the cell RibCys is cleaved by a non-enzymatic hydrolisis reaction, which releases ribose and cysteine.

Ribose is sugar which is used as substrate for synthesis of nucleotides (eg ATP, NAD+), and it is part of the building blocks that form DNA and RNA molecules. Under hypoxic conditions ribose synthesis can be a limiting factor in ATP synthesis.

Like many, if not most, elements and compounds in the body there is a delicate balancing act.  Like other sugars ribose can react in a process called ribosylation (as a glucose reaction is glycation).  However many studies have shown significantly higher doses of ribose to have beneficial effects (ref)

One acetaminophen (APAP) toxicity study showed “RibCys is nontoxic to CD-1 mice at the doses employed (up to 3g or 11.86 mmol/kg over 3 hours) and thus may be a candidate for use as an alternative antidote for treatment of APAP poisoning. The apparently large dose of RibCys used in these studies is not very different from the large amount of NAC that is routinely employed for treatment of APAP overdose in humans.” (ref)

Release-On-Demand

A property of RibCys (referred to as ‘Release On Demand’) is being able to be absorbed into the cell and utilized as required. On the low stress end of the scale a mouse study showed an increase in glutathione levels 16 hours after a single dose.  Other toxicology studies shows it is able to rapidly provide cysteine for glutathione synthesis under stressful conditions such as acetaminophen poisoning.

- Ability for slow release under low stress conditions as to not adversely affect redox state.

- Indescriminate anti-oxidant use has shown to be detrimental by interfering with ROS signalling.  Transient oxidative stress allows Nrf2 activation which produces the other enzymes which are required for glutathione to perform its detoxification and anti-oxidant functions , autophagy,

Why RibCys?

So what are my reasons for continuing to take RiboCeine over other front runners (liposomal and SAG)

  • More studies (including in vivo) over longer period detailing bio-distribution, efficacy and safety.
  • Doesn’t have a direct effect interfering with ROS signalling/redox, works with normal synthesis pathways and allows maximum Nrf2 activation and transient oxidative stress to induce autophagy and other beneficial stress responses.
  • Provides Ribose as a bonus.
  • Herb Nagasawa credentials are unmatached in the glutathione space, researching glutathione technology for over 25 years, and a world renowned medicinal chemist.  If he’s gone to this effort to make RibCys available then that’s a sign.

 

Cellgevity is available to order online here on the Max International website.

 

A New Year, New Insights. Interplay between Redox, AMPK, SirTs, NAD+, NADPH

How quickly a year flys by. I was hoping to have this post done before the first month of a year has passed! 2013 was a noteworth year with the publishing of my first big post on the topic of Redox and GSH ( and part 2) which was also accepted onto http://www.anti-agingfirewalls.com here.

My fellow authors there recently posted up a recap of their new insights over the last year at:

http://www.anti-agingfirewalls.com/2014/01/21/jim-watsons-top-12-list-of-things-i-learned-about-aging-in-2013/

http://www.anti-agingfirewalls.com/2014/01/09/editorial-13-personal-health-and-longevity-science-headlines-for-2013/

Having gained a decent understanding of the GSH piece of the puzzle, I’ve started to look further into redox and the interactions with other the couples (NAD+/NADH, NADP/NADPH) and how they interact with other key areas of interest, ATP, Caloric Restriction(CR), AMPK and SIRTs.

One thing which was confusing originally is that NAD/NADH ratio is predominately NAD+ as an energy acceptor/oxidiser and maintained ‘opposite’ to the GSH/GSSG and NADP/NADPH couples which provide reducing power.  This required brushing up on some basic biochem to make sense of it.  The following exceprt from Molecular Biology of the Cell. 4th edition. was enlightening.

Like ATP, NADPH is an activated carrier that participates in many important biosynthetic reactions that would otherwise be energetically unfavorable.

The difference of a single phosphate group has no effect on the electron-transfer properties of NADPH compared with NADH, but it is crucial for their distinctive roles. The extra phosphate group on NADPH is far from the region involved in electron transfer and is of no importance to the transfer reaction. It does, however, give a molecule of NADPH a slightly different shape from that of NADH, and so NADPH and NADH bind as substrates to different sets of enzymes. Thus the two types of carriers are used to transfer electrons (or hydride ions) between different sets of molecules.

Why should there be this division of labor? The answer lies in the need to regulate two sets of electron-transfer reactions independently. NADPH operates chiefly with enzymes that catalyze anabolic reactions, supplying the high-energy electrons needed to synthesize energy-rich biological molecules. NADH, by contrast, has a special role as an intermediate in the catabolic system of reactions that generate ATP through the oxidation of food molecules, as we will discuss shortly. The genesis of NADH from NAD+ and that of NADPH from NADP+ occur by different pathways and are independently regulated, so that the cell can independently adjust the supply of electrons for these two contrasting purposes. Inside the cell the ratio of NAD+ to NADH is kept high, whereas the ratio of NADP+ to NADPH is kept low. This provides plenty of NAD+ to act as an oxidizing agent and plenty of NADPH to act as a reducing agent—as required for their special roles in catabolism and anabolism, respectively.

A big story in late 2013 was David Sinclair and colleagues from both Harvard and Australia publication showing that supplementation with an NAD+ precursor could reverse this mitochondrial dysfunction and “Warburg-like” metabolic state in mice in just one week.

Keeping that in mind, one of the key references (The Redox Stress Hypothesis of Aging Free Radic Biol Med. 2012) in my first post stated:

 Furthermore, 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.

Given Sinclair’s results I would be be interested to see the results of supplementing both with a GSH precursor and NAD+ precursor(s), ensuring sufficient levels of both oxidising and reducing activated carrier molecules. 2013 also marked the introduction of a new commercial Nicotinamide riboside product called Niagen which also acts as a NAD precursor.  This could be an interesting one to stack with the newer GSH/Cysteine pro-drugs available such as Ribose-Cysteine.

Looking back at an article I had previously referenced indicates this to be true.  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[/NAD+] precursor, nicotinamide, we increased neuron survival against amyloid beta stress in an additive manner.

The alcohol dehydrogenase and aldehyde dehydrogenase enzymes both require NAD+ for the two catabolic steps to break down ethanol.  Perhaps some Nicotinamide Riboside wouldn’t go astray after enjoying a few alcoholic beverages.  I’ll be personally testing this hypothesis this year.

NAPDH 

While NAD+ has been in the limelight again recently, I’ve been piecing together some articles looking at NAPDH, which is critical for maintaining redox homoeostasis as it is required to recycle GSSG back to 2GSH.

This following article was particularly interesting. While it is in regards to tumor cell survial, the energy stress (caloric restriction?) situation seems applicable for normal cells too.

AMPK regulates NADPH homeostasis to promote tumour cell survival during energy stress (Nature. 2012 May)

The underlying mechanisms of cell death and survival under metabolic stress are not well understood. A key signalling pathway involved in metabolic adaptation is the liver kinase B1 (LKB1)–AMP-activated protein kinase (AMPK) pathway2,3. Energy stress conditions that decrease intracellular ATP levels below a certain level promote AMPK activation by LKB1. Previous studies showed that LKB1-deficient or AMPK-deficient cells are resistant to oncogenic transformation and tumorigenesis4–6, possibly because of the function of AMPK in metabolic adaptation. However, the mechanisms by which AMPK promotes metabolic adaptation in tumour cells are not fully understood. Here we show that AMPK activation, during energy stress, prolongs cell survival by redox regulation. Under these conditions, NADPH generation by the pentose phosphate pathway is impaired, but AMPK induces alternative routes to maintain NADPH and inhibit cell death. The inhibition of the acetyl-CoA carboxylases ACC1 and ACC2 by AMPK maintains NADPH levels by decreasing NADPH consumption in fatty-acid synthesis and increasing NADPH generation by means of fatty-acid oxidation. Knockdown of either ACC1 or ACC2 compensates for AMPK activation and facilitates anchorage-independent growth and solid tumour formation in vivo, whereas the activation of ACC1 or ACC2 attenuates these processes. Thus AMPK, in addition to its function in ATP homeostasis, has a key function in NADPH maintenance, which is critical for cancer cell survival under energy stress conditions, such as glucose limitations, anchorage-independent growth and solid tumour formation in vivo.

Briefly back to NAD+, AMPK also increase the NAD+/NADH ratio, a nice double whammy, by increasing mitochondrial β-oxidation. From AMPK regulates energy expenditure by modulating NAD+ metabolism and SIRT1 activity Nature 2009.

To determine how AMPK acutely increases the NAD+/NADH ratio, we pharmacologically targeted different possible sources of cellular NAD+ production. Inhibition of the glycolytic enzyme lactate dehydrogenase with oxamic acid did not affect the ability of AICAR to increase NAD+ levels and the NAD+/NADH ratio. In contrast, inhibition of mitochondrial fatty acid oxidation with etomoxir was enough to hamper the increase in NAD+/NADH induced by AMPK, indicating that an increase in mitochondrial β-oxidation is required for AMPK to increase the NAD+/NADH ratio.

 

From Sirt3 Mediates Reduction of Oxidative Damage and Prevention of Age-related Hearing Loss under Caloric Restriction Cell. 2010 November

Collectively, these results provide evidence that during CR, Sirt3 induces the deacetylation and activation of Idh2 [isocitrate dehydrogenase], leading to increased levels of NADPH in mitochondria of multiple tissues.

 

This recent thinking took me back to an article I referenced which stood out in my mind when doing my previous redox/GSH research.

Time line of redox events in aging postmitotic cells eLife. 2013;

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. This decrease in NADPH levels occurs very early during lifespan and sets into motion a cascade that is predicted to down-regulate most cellular processes. Caloric restriction, a near-universal lifespan extending measure, increases NADPH levels and delays each facet of the cascade. Our studies reveal a time line of events leading up to the system-wide oxidation of the proteome days before cell death.

Thioredoxin reductase: an early oxidation target in yeast

Oxidation of at least 28 proteins significantly preceded the general oxidation of proteins under standard or caloric restriction conditions (Figure 3, clusters D and E and Table 1). Of these early-oxidized proteins, 20 had oxidation states of more than 45% at day 2 of cultivation, which was 1.5- to 3.8-fold higher than their oxidation status during exponential growth. One of these early oxidation targets is the highly conserved enzyme thioredoxin reductase, the key component of the thioredoxin system. Although we cannot exclude that oxidation of any one of the other early oxidation targets directly or indirectly affects or even controls S. cerevisiae lifespan, we decided to focus our subsequent studies on thioredoxin reductase, as this enzyme is the central player in maintaining cellular redox homeostasis. Loss of thioredoxin reductase activity has been shown to cause widespread protein oxidation.

This is one example where redox stress disables the enzymes required to maintain redox homoeostasis, further increasing the stress. Is there hope?

It was intriguing to observe that early protein oxidation is, at least in its initial stage, a fully reversible event in yeast. Moreover, we found that more than 80% of viable cells were recovered from day 3- and day 4-old cultures despite an almost fully oxidized thiol proteome.

 

More on Redox Stress

One article published at the start of this year was on SirT1 mutants which replaced oxidation-sensitive cysteine with serine to  stop inactivation by redox stress.

This appears to be another one of many pathways in the viscous cycle of aging, as increased redox stress inactives SIRT1 (possibly by  S-glutathiolation of a SirT1 cysteine residue decreasing the binding affinity of NAD+).  In the second two quoted sections also note that Glutaredoxin-1 (Glrx) uses glutathione as a co-factor.

A redox-resistant sirtuin-1 mutant protects against hepatic metabolic and oxidant stress J Biol Chem. 2014 Jan.

We show in SirT1 overexpressing HepG2 cells that oxidants (nitrosocysteine or hydrogen peroxide) or metabolic stress (high palmitate and high glucose) inactivate SirT1 by reversible oxidative post-translational modifications (OPTM) on cysteines.

To prove that OPTMs of SirT1 are glutathione (GSH) adducts, glutaredoxin-1 (Glrx) was overexpressed to remove this modification. Glrx overexpression maintains endogenous SirT1 activity and prevents proapoptotic signaling in metabolically stressed HepG2 cells.

By demonstrating that SIRT1 can be regulated by S-glutathiolation of specific Cys residues, our results have uncovered the potential for SIRT1 to be directly regulated by oxidative modifications that may link its activity to GSH redox and production of cellular reactive oxygen and nitrogen species. These novel findings also indicate that the GSH redox state may affect the response of SIRT1 to polyphenols. These results imply that antioxidants might significantly potentiate the response to small molecule activators of SIRT1.

 

Further reading AMPK and SIRT1: a long-standing partnership? Am J Physiol Endocrinol Metab. 2010 April;

 

Markers of Aging

<Work in progress>

Glycans

Glycans Are a Novel Biomarker of Chronological and Biological Ages J Gerontol A Biol Sci Med Sci (2013)

Fine structural details of glycans attached to the conserved N-glycosylation site significantly not only affect function of individual immunoglobulin G (IgG) molecules but also mediate inflammation at the systemic level. By analyzing IgG glycosylation in 5,117 individuals from four European populations, we have revealed very complex patterns of changes in IgG glycosylation with age. Several IgG glycans (including FA2B, FA2G2, and FA2BG2) changed considerably with age and the combination of these three glycans can explain up to 58% of variance in chronological age, significantly more than other markers of biological age like telomere lengths. The remaining variance in these glycans strongly correlated with physiological parameters associated with biological age. Thus, IgG glycosylation appears to be closely linked with both chronological and biological ages. Considering the important role of IgG glycans in inflammation, and because the observed changes with age promote inflammation, changes in IgG glycosylation also seem to represent a factor contributing to aging.

 

Methylation

http://www.sciencedaily.com/releases/2013/10/131020203006.htm

 

Telomere length

 

 

Redox state

“Dean Jones at Emory University was the first to show that human plasma GSH/GSSG is controlled at a relatively constant redox state of −137 mV in 740 healthy adults through age 50. However, an oxidative shift of about 7 mV/decade occurs over the next two decades, followed by a further decline to −110 mV in the 70 to 85-year-old group.” (ref)

“In humans, an age-related oxidative shift in the ratio of reduced to oxidized glutathione, i.e. the glutathione redox status, has been demonstrated in whole blood, and peripheral blood mononuclear cells. The mean plasma cysteine/cystine redox status of human subjects shows a significant oxidative shift between the third and the ninth decade of life. This age-related oxidative shift is accompanied by a decrease in the plasma glutathione level and a decrease in the ratio of reduced versus oxidized forms of plasma albumin and other thiol/disulfide redox couples.” (ref)

Factors Affecting Redox, GSH Levels and Function – Redox, Glutathione and Cysteine, Part 2

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  it’s 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 β-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 γ-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(c)(-) 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, Alzheimer’s etc), and neuro-inflammatory conditions can reduce intra-cellular cysteine supply by competing with the transporter.

From Glutathione—a review on its role and significance in Parkinson’s 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 γ-glutamyl amino acid by the ecto-enzyme γ-glutamyl transpeptidase (γ-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.

 

Recycling

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 Parkinson’s 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 Glutathione—a review on its role and significance in Parkinson’s 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 γ-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.

 

Genetics

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 author’s 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.”

 

Environmental

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

SIRT1

SIRT1 activation by curcumin pretreatment attenuates mitochondrial oxidative damage induced by myocardial ischemia reperfusion injury.

 

http://www.sciencedaily.com/releases/2013/09/130903123041.htm

“We found that only the mice that overexpressed Sirt1 in the brain (called BRASTO) had significant lifespan extension and delay in aging, just like normal mice reared under dietary restriction regimens,”. The BRASTO mice demonstrated significant life span extension without undergoing dietary restriction.

Having narrowed control of aging to the brain, Imai’s team then traced the control center of aging regulation to two areas of the hypothalamus called the dorsomedial and lateral hypothalamic nuclei. They then were able to identify specific genes within those areas that partner with Sirt1 to kick off the neural signals that elicit the physical and behavioral responses observed.

“We found that overexpression of Sirt1 in the brain leads to an increase in the cellular response of a receptor called orexin type 2 receptor in the two areas of the hypothalamus,” said first author Akiko Satoh, PhD, a postdoctoral staff scientist in Imai’s lab.

 

Antidepressant-like activity of resveratrol treatment in the forced swim test and tail suspension test in mice: The HPA axis, BDNF expression and phosphorylation of ERK. Pharmacol Biochem Behav. 2013 Oct 11

A novel pathway regulates memory and plasticity via SIRT1 and miR-134 Nature 466, 1105–1109 

SIRT1-mediated deacetylation of MeCP2 contributes to BDNF expression Epigenetics. 2012 July 1; 7(7): 695–700.

Sirt1 mediates neuroprotection from mutant huntingtin by activation of TORC1 and CREB transcriptional pathway Nat Med. 2011 December 18; 18(1): 159–165.