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