Glutathione (GSH) Part I

Today’s post is where I am going to get seriously scientific.  There is another Epiphany at the end, but you will read about it in Part II.  Part I is the primer.  To fully understand Part II and see why it really does lead to another epiphany moment, you should read it.  If you plan on actually implementing this at home, I suggest you read Part I, Part II and go and see your paediatrician.  My research should not be seen as medical advice.  Doctor always knows best and I am not a doctor.

Introduction

I mentioned earlier, that in January 2013,when I decided to launch my ANA project, the plan was as follows; start work first with my own observations and look for a hypothesis that I could develop entirely myself.  Having found a treasure trove of existing scientific research, I decided that I would also develop a Plan B.  Plan B is very simple, to just read the research and apply my own little grey cells.

Plan A went very fast and with a few days I had developed my first hypothesis.  I have not given it a name yet, but the letters TRH will feature prominantly.  Having developed a hypothesis you then have to figure out what to do with it.  In the case of my second hypothesis, concerning Hypokalemic Sensory Overload, it was really easy to test it.  For me, it is proven, although maybe one day I will do a double-blind, randomized, placebo-controlled study, to prove it to the rest of the world.

One weekend in early February, I had an evening off at home with no kids nor any obligations.  So I thought this would be a good time to tinker away on Google Scholar.  This is a special kind of search that only lists serious scientific research.

If it was not for Google Scholar, I would have a lot more free time and you would be doing something much more fun and read my ramblings.

I started reading some research into the pseudo-science of autism.  Having travelled through hyperbaric oxygen  therapy, I arrived at methyl B12 treatment.  It turns out that in the US, parents are injecting their autistic kids with vitamin B12 in their rears.  There are whole discussions on various websites as to how best to do this.  Apparently, the best way is to wait till the kid is asleep, apply lidocaine cream to numb the skin and then jab in the needle.  This is not something I plan doing to Monty, nor I hope him to me.  Then I found some research dedicated to see if methyl B12 treatment actually works.

Well, the study concluded that “methyl B12 is ineffective in treating behavioral symptoms of autism”.  But then the author a caveat “However, detailed data analysis suggests that methyl B12 may alleviate symptoms of autism in a subgroup of children, possibly by reducing oxidative stress”

I was aware that I was in the dreaded territory of  “DAN Doctors” and the paper was published in a something called The Journal of Alternative and Complimentary Medicine, so big red warning lights were flashing.  I could buy the full paper for $51 or live with the abstract.  I choose the latter and moved on.

Now after 20 munites of "Google Scholaring" I had something juicy to investigate.  What is oxidative stress? what is glutathione redox status (GSH/GSSG)? and what was the relevance of the subgroup that had increased plasma concentrations of GSH?

My new book on Human Physiology has yet to arrive, but I have pretty much figured it out anyway.  I do love Amazon and I guess they must love me, by now.

So what is Glutathione (GSH)? Well, if you live in the world of  pseudo-science, it is very easy;  it’s an antioxidant “period”.

I’d be wasting my time and yours if I left it at that.

 

First a bit of chemistry

A thiol is a type of compound that contains  the following bond   R–SH, where R is a carbon containing group of atoms. (Hopefully, from schooldays you will recall that S is sulphur and H is hydrogen).

Thiols tend to smell terrible, like rotten eggs or garlic and thiols are readily oxidized

Thiols play a very important role in human biology.  I took a quick look at a list thiols, to see if any bells starting ring between my ears. They did.

You have guessed that Glutathione is a thiol, make a mental note of another important one, cysteine.

 

Selected Thiols

 

Glutathione  C₁₀H₁₇N₃O₆S

Cysteine   C₃H₇NO₂S

Thioctic acid  C₈H₁₄O₂S₂

  

I included the third thiol for a reason;  I used to buy vials of the stuff on business trips to Romania.  It was not cheap, maybe EUR 300 for a whole box, I do not remember.  I do remember that it is used as a therapy for peripheral diabetic neuropathy.  It is known to be a powerful antioxidant.

Having got the suspicious items through customs they finally ended up going to the military hospital, along with my father in law, the final recipient. It is administered intravenously.  As you will see from the study, only when give IV was there an effect, the oral version had no beneficial therapeutic effect.  This is a very common problem, crossing the BBB (blood brain barrier) and the same you will notice later, will apply to GSH.  In US and  UK, this treatment for peripheral diabetic neuropathy is not used and is merely experimental.  In some east European countries, it has been a standard therapy for decades.

I told you that this particular thiol is called Thioctic acid, but just confuse the lay person, it has a further three names - Thiotacid, Lipoic acid and Alpha Lipoic Acid (ALA).

Now did I choose to add ALA to my list of three thiols to talk about, because I already knew something else about it?  It often seems to be the case, in my 5 weeks reading about human physiology, that it's a very small world, full of coincidences.

ALA has another quite unrelated use, in heavy metal chelation.  I read that "Lipoic acid administration can significantly enhance biliary excretion of inorganic mercury in rat experiments".  It is the agent of choice of some DAN Doctors for their young patients with autism.

Do not confuse alpha lipoic acid with alpha-Linolenic acid, which is an omega 3 fatty acid.

By the way, we are actually doing some research currently into omega 3 oil.  Please note that there is no such thing as omega 3 oil as such, it is the name to a big group of quite different individual polyunsaturated fatty acids. It is believed that three are important in human physiology, those being alpha-linolenic acid (ALA), eicosapentaenoic acid (EPA), and docosahexaenoic acid (DHA). More of this and what to make of them, will be in a later post.

Summary so far

• So we know that GSH is a thiol, thiols incorporate a sulphur-hydrogen bond. Thiols are great antioxidants and thiols tend to stink.

• GSH’s formula is  C₁₀H₁₇N₃O₆S and it looks like:-

• In its oxidized form GSH becomes GSSG
• GSSG’s formula is C₂₀H₃₄N₆O₁₃S₂

 

As you would expect GSSG goes by numerous aliases, namely :-

Glutathione Hydrate Oxidized, Glutathione Oxidized GSSG Hydrate;GSSG Hydrate Oxidize; and even Glutathione Disulphide

If you compare the molecular formulas of GSH and GSSG,  you will notice that one molecule of GSSG = 2 molocules of GSH plus  O²⁻

Redox (reduction- oxidation)

You may have learnt about his at school.  The process often involves oxygen, hence oxidation, but it does not have to.  The strict definitions are :

Oxidation = all processes that involve loss of electrons

Reduction = all processes that involve the gain of electrons

In redox processes, the reductant or reducing agent loses electrons and is oxidized, and the oxidant or oxidizing agent gains electrons and is reduced. The pair of  oxidizing agent and reducing agent is called a redox pair. A redox couple is a reducing species and its corresponding oxidized form, e.g., Fe²⁺/Fe³⁺.

 So when a piece of your caste iron fence rusts, you get:

 

4 Fe + 3 O₂ → 2 Fe₂O₃

 

(the next part is taken from Colorado State University’s fantastic web resource covering Pathophysiology and more)  I just added maybe 5% and corrected the spelling.

 

A radical (aka free radical)  is an atom or group of atoms that have one or more unpaired electrons. Radicals can have positive, negative or neutral charge. They are formed as necessary intermediates in a variety of normal biochemical reactions, but when generated in excess or not appropriately controlled, radicals can wreak havoc on a broad range of macromolecules. A prominent feature of radicals is that they have extremely high chemical reactivity, which explains not only their normal biological activities, but how they inflict damage on cells.  Their chief danger comes from the damage they can do when they react with important cellular components such as DNA, or the cell membrane. Cells may function poorly or die if this occurs. To prevent free radical damage the body has a defence system of antioxidants.

Oxygen Radicals

There are many types of radicals, but those of most concern in biological systems are derived from oxygen, and known collectively as reactive oxygen species. Oxygen has two unpaired electrons in separate orbitals in its outer shell. This electronic structure makes oxygen especially susceptible to radical formation.

Sequential reduction of molecular oxygen (equivalent to sequential addition of electrons) leads to formation of a group of reactive oxygen species:

• superoxide anion
• peroxide (hydrogen peroxide)
• hydroxyl radical

The structure of these radicals is shown in the figure below, along with the notation used to denote them. Note the difference between hydroxyl radical and hydroxyl ion, which is not a radical.

Another radical derived from oxygen is singlet oxygen, designated as ¹O₂. This is an excited form of oxygen in which one of the electrons jumps to a superior orbital following absorption of energy.

Formation of Reactive Oxygen Species

Oxygen-derived radicals are generated constantly as part of normal aerobic life. They are formed in mitochondria as oxygen is reduced along the electron transport chain. Reactive oxygen species are also formed as necessary intermediates in a variety of enzyme reactions. Examples of situations in which oxygen radicals are overproduced in cells include:

• White blood cells such as neutrophils specialize in producing oxygen radicals, which are used in host defence to kill invading pathogens.
• Cells exposed to abnormal environments such as hypoxia or hyperoxia generate abundant and often damaging reactive oxygen species. A number of drugs have oxidizing effects on cells and lead to production of oxygen radicals.
• Ionizing radiation is well known to generate oxygen radicals within biological systems. Interestingly, the damaging effects of radiation are higher in well oxygenated tissues than in tissues deficient in oxygen.

Biological Effects of Reactive Oxygen

It is best not to think of oxygen radicals as "bad". They are generated in a number of reactions essential to life and, as mentioned above, phagocytic cells generate radicals to kill invading pathogens. There is also a large body evidence indicating that oxygen radicals are involved in intercellular and intracellular signaling. For example, addition of superoxide or hydrogen peroxide to a variety of cultured cells leads to an increased rate of DNA replication and cell proliferation - in other words, these radicals function as mitogens.

Despite their beneficial activities, reactive oxygen species clearly can be toxic to cells. By definition, radicals possess an unpaired electron, which makes them highly reactive and thereby able to damage all macromolecules, including lipids, proteins and nucleic acids.

One of the best known toxic effects of oxygen radicals is damage to cellular membranes (plasma, mitochondrial and endomembrane systems), which is initiated by a process known as lipid peroxidation. A common target for peroxidation is unsaturated fatty acids present in membrane phospholipids.

Reactions involving radicals occur in chain reactions. Note that a hydrogen is abstracted from the fatty acid by hydroxyl radical, leaving a carbon-centered radical as part of the fatty acid. That radical then reacts with oxygen to yield the peroxy radical, which can then react with other fatty acids or proteins.

Peroxidation of membrane lipids can have numerous effects, including:

• increased membrane rigidity
• decreased activity of membrane-bound enzymes
• altered activity of membrane receptors.
• altered permeability

In addition to effects on phospholipids, radicals can also directly attack membrane proteins and induce lipid-lipid, lipid-protein and protein-protein crosslinking, all of which obviously have effects on membrane function.

Mechanisms for Protection Against Radicals

Life on Earth evolved in the presence of oxygen, and necessarily adapted by evolution of a large battery of antioxidant systems. Some of these antioxidant molecules are present in all life forms examined, from bacteria to mammals, indicating their appearance early in the history of life.

Many antioxidants work by transiently becoming radicals themselves. These molecules are usually part of a larger network of cooperating antioxidants that end up regenerating the original antioxidant. For example,vitamin E becomes a radical, but is regenerated through the activity of the antioxidants vitamin C and glutathione.

Enzymatic Antioxidants

Three groups of enzymes play significant roles in protecting cells from oxidant stress:

Superoxide dismutases (SOD) are enzymes that catalyze the conversion of two superoxides into hydrogen peroxide and oxygen. The benefit here is that hydrogen peroxide is substantially less toxic that superoxide. SOD accelerates this detoxifying reaction roughly 10,000-fold over the non-catalyzed reaction.

SODs are metal-containing enzymes that depend on a bound manganese, copper or zinc for their antioxidant activity. In mammals, the manganese-containing enzyme is most abundant in mitochondria, while the zinc or copper forms predominant in cytoplasm. Interestingly, SODs are inducible enzymes - exposure of bacteria or vertebrate cells to higher concentrations of oxygen results in rapid increases in the concentration of SOD.

Catalase is found in peroxisomes in eucaryotic cells. It degrades hydrogen peroxide to water and oxygen, and hence finishes the detoxification reaction started by SOD.

Glutathione peroxidase is a group of enzymes, the most abundant of which contain selenium. These enyzmes, like catalase, degrade hydrogen peroxide. They also reduce organic peroxides to alcohols, providing another route for eliminating toxic oxidants.

In addition to these enzymes, glutathione transferase, ceruloplasmin, hemoxygenase and possibly several other enzymes may participate in enzymatic control of oxygen radicals and their products.

Non-enzymatic Antioxidants

Three non-enzymatic antioxidants of particular importance are:

Vitamin E is the major lipid-soluble antioxidant, and plays a vital role in protecting membranes from oxidative damage. Its primary activity is to trap peroxy radicals in cellular membranes.

Vitamin C or ascorbic acid is a water-soluble antioxidant that can reduce radicals from a variety of sources. It also appears to participate in recycling vitamin E radicals. Interestingly, vitamin C also functions as a pro-oxidant under certain circumstances.

Glutathione may well be the most important intracellular defense against damage by reactive oxygen species. It is a tripeptide (glutamyl-cysteinyl-glycine). The cysteine provides an exposed free sulphydryl group (SH) that is very reactive, providing an abundant target for radical attack. Reaction with radicals oxidizes glutathione, but the reduced form is regenerated in a redox cycle involving glutathione reductase and the electron acceptor NADPH.

 

 ** Now we have left Colorado ** 
 
(Colorado State University is located in Fort Collins, Colorado, in case you were wondering) 
You kept that one quiet Colin, I thought an Englishman's home was supposed to be a castle, and preferably in North London, not over there where Eric Cartman and Stan Marsh come from)

An antioxidant is a molecule inhibits the oxidation of other molecules

Oxidation reactions can produce free radicals. In turn, these radicals can start chain reactions. When the chain reaction occurs in the cell, it can cause damage or death to the cell. Antioxidants terminate these chain reactions by removing free radical intermediates, and inhibit other oxidation reactions. They do this by being oxidized themselves, so antioxidants are often reducing agents such as thiols.

Oxidative Stress

I found a great definition:-

An imbalance between oxidants and antioxidants, in favour of the oxidants, potentially leading to damage is called oxidative stress.

Wikipedia itself has gone for a dumbed down version:-

Oxidative stress reflects an imbalance between the systemic manifestation of reactive oxygen species and a biological system's ability to readily detoxify the reactive intermediates or to repair the resulting damage

 

I prefer the former definition. Every time I hear “detoxify, toxins or detox”  I assume I am talking to somebody who does not know which end of a screwdriver to hold.

Pro-oxidant

A pro-oxidant helps induce oxidative stress.

Let’s sum up again:-

• Glutathione (GSH) and Glutathione (GSSG) are dating, they are a redox couple.
• GSH is a reducing agent and antioxidant, during redox it loses electrons and is itself oxidized to form GSSG.
• GSH is the most important of the 3 most important antioxidants in your body.
• Free radicals are not always bad. They have both a positive/necessary role plus a negative/redundant role.
• Oxidative stress is always bad. The oxidants have got the whip hand.

 

Coming up in Part II

 

• Brain region-specific glutathione redox imbalance in autism

 

• Regulation of cellular glutathione

 

• Clinical trial of glutathione supplementation in autism spectrum disorders

 

• Glutathione precursors to raise GSH levels in plasma (N-acetylcysteine, whey protein)

 

• N-acetylcysteine in psychiatry

 

• And finally, having understood the science behind it, what you have all been waiting for, and what I was shocked find had already been tested:-  A Randomized Controlled Pilot Trial of Oral N-Acetylcysteine in Children with Autism

Glutathione (GSH) Part II

Glutathione (GSH) Part III