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update 69: NAC, glutathione, oxidative stress, thrombosis

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glutathione – far more than just an antioxidant

So we start with a closer look at glutathione, and its backbone amino acid chain, including the amino acid cysteine. Cysteine has the formula HO2CCH(NH2)CH2SH. The thiol sub-chain (SH) is important because it can bind to another form of the molecule, with S binding to S (oxidised form) rather than binding to H (reduced form) as here. So, as Dr Seheult explains, if you have two glutathiones, in this reduced form (2GSH), oxidised via hydrogen peroxide (H2O2), you will create a bond (GS-SG) between the two oxidised glutathiones, together with water. This happens in the oxidisation processes in our cells.

Seheult next mentions ADAMTS13, which is also known as von Willibrand factor-cleaving protease, so it’s a zinc-containing enzyme. VWF polymerises via disulphide bonds, and ADAMTS13 can help in disrupting that process, I think. Seheult diverts us by mentioning the disulphide bonds that connect the spiral strands of keratin in hair. A ‘perm’ reduces the molecular structure, breaking the disulphide bonds, so that the individual strands can be straightened, or made more curly, after which ‘you neutralise the perm agent’?? via H2O2, allowing disulphide bonds to re-form keeping the new hair structure in place. That was almost interesting.

So what can we do to assist these glutathione-based processes in relieving oxidative stress? This is apparently where N-Acetylcysteine (NAC) comes in. This molecule, which is ‘the N-acetyl derivative of the natural amino acid L-cysteine’, is ‘an antioxidant and disulphide breaking agent’, according to a 2018 review article in the Journal of Free Radical Research (not a political journal). So NAC is a reducing agent, which, like cysteine, has an SH bond. It breaks disulphide bonds and adds hydrogen, reducing viscosity. NAC has been used as a mucolytic inhalant, and as an agent against tylenol (paracetamol) overdose. How this last effect works is complex and I’ll try to comprehend it.

As Seheult explains it, NAC would act on the metabolite of paracetamol in situations of overdose. In such cases the liver metabolises paracetamol via an alternative pathway, by means of the toxic metabolite NAPQI, which depletes the liver’s glutathione. NAC replenishes the glutathione, but I won’t try to analyse the mechanism here. The main point is that NAC’s glutathione-boosting effects may have potential in dealing with Covid-19 symptoms. According to the above-mentioned review article, glutathione depletion is related to oxidative stress associated with a wide range of illnesses and pathologies, as well as in general ageing. So, a 1997 study in Italy looked at H1N1 flu and NAC treatment in a randomised, double-blind trial of 262 individuals of both sexes, most of them suffering from non-respiratory chronic degenerative diseases. They were divided into a placebo group and a NAC tablet group for a period of six months. No difference was found in both groups contracting the virus, but the majority of the placebo group (79%) came down with symptomatic forms, compared to only 25% of the treatment group, a significant difference. The study concluded that NAC treatment ‘appears to provide a significant attenuation of influenza and influenza-like episodes, especially in elderly high-risk individuals.’

So, recognising that this update is 2-3 months old now, I went online to see if NAC treatment is being used, or more comprehensive trials are being undertaken, as I note that, though case-rates are still disturbingly high, especially in the USA, death-rates are somewhat reduced.

An article from NCBI (the National Center for Biotechnology Information), which post-dates update 69 by a couple of weeks, presents only a hypothesis:

that NAC could act as a potential therapeutic agent in the treatment of COVID-19 through a variety of potential mechanisms, including increasing glutathione, improving T cell response, and modulating inflammation.

However, it didn’t seem as if any effective clinical trials focusing specifically on Covid-19 had been completed at the time of the article. A much more recent article (July 14) in Future Medicine (not such a promising name, given the urgency), presents more biochemical detail of NAC’s action, along with the anticoagulant heparin, and mentions ongoing clinical trials, but not specific results. It also mentions NAC treatment as a preventive for frontline ICU workers and general healthcare workers. It may be that such treatment is already being applied.

So, returning to update 69, Seheult cites another article from 2010 in Biochemical Pharmacology which showed that NAC inhibited viral replication (here the virus was H5N1) and reduced inflammatory cytokines, and again they suggested it as a potential treatment in the case of future influenza pandemics. Another small trial suggested some limited efficacy for NAC in the treatment of acute respiratory distress syndrome (ARDS).

So on it goes. A 2018 article found that ‘[NAC] improves oxidative stress and inflammatory response in patients with community acquired pneumonia [CAP]’. This oxidative stress reduction may be more important for Covid-19 cases because of the possibility of thrombosis due to the effect on VWF. A 2013 study found a significant decrease in a number of coagulation factors with NAC treatment. Of course, with this blood-thinning facility, NAC should not be used for patients with increased bleeding risk during or resulting from surgery. In any case I note that NAC is on the WHO list of most safe drugs or treatments.

And there are more studies. Another 2018 study found that NAC could reverse cerebral injury from strokes exacerbated by diabetes. The study concludes that ‘the diabetic blood and brain become more susceptible to platelet activation and thrombosis’, and that NAC appears to offer protection against the risk of stroke. The study’s explanation of the process here gives me an opportunity for further revision:

[NAC protects against stroke] by altering both systemic and vascular prothrombotic responses via enhancing platelet GSH, and GSH-dependent MG elimination, as well as correcting levels of antioxidants such as SOD1 and GPx-1.

So that’s platelet glutathione, and glutathione-dependent methylglyoxal, and the antioxidants mentioned are superoxide dismutase 1 and glutathione peroxidase 1. The ScienceDirect website does an amazing job of informing us about every known aspect of molecular biochemistry, just saying. Its material on glutathione and its catalysis is exhaustive and exhausting. And it looks as though the silver lining to the tragedy of Covid-19 may be a spike in further research into this and other essential elements of the molecular basis of immune systems.

Dr Seheult goes on to cite one more study, which found that ‘NAC administration promotes lysis of arterial thrombi that are resistant to conventional approaches…’, principally by acting on VWF, and that it is even more effective in combination with ‘a nonpeptidic GpIIa/IIIb (glycoprotein) inhibitor’, with no observed worsening of symptoms or outcome vis-a-vis normal haemostasis.

So I’ll end this piece wondering how things are going with NAC and other applications to reduce both respiratory and thrombotic symptoms in regions where the virus continues to be spread through a mixture of government, business and personal irresponsibility and stupidity. The battle to keep people alive and relatively healthy will, I think, ultimately win over the stupidity of some, but at a terrible and tragic cost. Vaccines are in the offing, but fear, indifference and ignorance will probably have the most adverse influence on their effectiveness.

References

Coronavirus Pandemic Update 69: “NAC” Supplementation and COVID-19 (N-Acetylcysteine)

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7261085/

https://www.futuremedicine.com/doi/10.2217/fmb-2020-0074

https://www.sciencedirect.com/science/article/pii/S0304416512002735

https://www.sciencedirect.com/topics/biochemistry-genetics-and-molecular-biology/methylglyoxal

Written by stewart henderson

August 2, 2020 at 12:46 pm

stuff about Covid-19: cytokine problems

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got all that?

Canto: So what are cytokines? They’re ‘signalling proteins, usually less than 80kDa in size’ – that means kilodaltons, and it refers to molecular mass. Proteins have a huge variety of shapes and sizes, the largest being titin, with a mass of 3,816,188.13 Da. I don’t know why they don’t keep to kilodaltons. I presume the daltons measurement is in memory of the pioneering British chemist John Dalton, a truly inspiring character. Cytokines are quite small proteins, I think. Or peptides, which are described on other science sites as not being proteins, or not quite, which is confusing.

Jacinta: We’re looking at a ScienceDirect website which is pretty technical, but it says cytokines regulate many biological functions including those related to innate and acquired immunity. Here’s its ‘operational definition’:

Cytokines can be defined operationally as polypeptides secreted by leukocytes and other cells that act principally on hematopoietic cells, the effects of which include modulation of immune and inflammatory responses.

So peptides are short strings of amino acids, and proteins are longer strings of amino acids, so polypeptides are apparently more than just peptides but not quite proteins. Very weird. Leukocytes are white blood cells, of which there are three main types (I think): monocytes, lymphocytes (T cells and B cells) and granulocytes (neutrophils, eosinophils and basophils). Leukocytes are made in our bone marrow and are found in our blood and lymph. I’d love to learn about lymph one day.

Canto: So leukocytes are part of our immune system, as are the cytokines they secrete. Hematopoietic cells – always worth breaking things down: hema, or haema, always refers to blood, and poiesis, from ancient Greek, essentially means production or bringing into being. Presumably, then, these hematopoietic cells exist in the bone marrow, where they produce leukocytes. And yet… that all seems to mean that cytokines are secreted (and presumably produced) by leukocytes to act on hematopoietic cells that produce leukocytes… It seems a bit circular to me.

Jacinta: Certainly complex. Let’s barge on. The ScienceDirect site has it that cytokines are secreted by many cell types, often at high concentrations, and are mostly involved in cell-to-cell interactions with neighbouring cells. This is called paracrine signalling, as opposed to other forms of signalling (endocrine, juxtacrine and autocrine). However, cytokines can sometimes use those other forms. There are many different groups of cytokines, usually named for their most significant effects, as we see them, but they’re actually pleiotropic, meaning they have each a variety of functions, and those functions can be mediated by other cytokine groups. So, certainly complex, but in terms of their function in response to airways diseases…

Canto: But now I’m hearing that Covid-19 isn’t necessarily an airways disease, or only an airways disease. It may affect the brain and the nervous system, the kidneys, the heart, the blood…

Jacinta: Hmmm, so much more to explore, before we all die. But knowledge is power, the more we know, the more we can defend ourselves. Let’s all be Popperian optimists and rise to the challenge. Here’s an overview, from a 2009 article on cytokines as related to asthma and COPD:

The major classes of cytokines include: pro- and anti-inflammatory cytokines, cytokines of neutrophil and eosinophil recruitment and activation, cytokines derived from T-helper (Th) and T-regulatory (Tregs) cells, and cytokines of T-cell recruitment and growth factors.

The cells mentioned are all leukocytes. But the storm of cytokines may well be causative of those other symptoms found in Covid-19 sufferers, such as blood clots. A very recent article in the Lancet has this to say in reference to what we’re seeing:

the overproduction of early response proinflammatory cytokines (tumour necrosis factor [TNF], IL-6, and IL-1β) results in what has been described as a cytokine storm, leading to an increased risk of vascular hyperpermeability, multiorgan failure, and eventually death when the high cytokine concentrations are unabated over time. Therefore, therapeutic strategies under investigation are targeting the overactive cytokine response with anticytokine therapies or immunomodulators, but this must be balanced with maintaining an adequate inflammatory response for pathogen clearance.

Canto: Wow, I suppose one thing we’ll be learning fast from this pandemic will be a lot more about cytokine production and how it can be abated without risk to the immune system. I wonder if there are any ‘anticytokine therapies’ at present?

Jacinta: Well I’ve read this Lancet article and I can’t pretend to comprehend all that’s in it, but of course it tries to address all we’re concerned about here so I’m going to try to explain it in my way. Hospitalised Covid-19 patients are presenting with pneumonia, ARDS and other respiratory conditions, and sepsis. Sepsis is a broad term, referring to an unbalanced blood immune response which, at its worst, can lead to multiple organ failure. Vascular hyperpermeability, mentioned above, is defined as ‘the excessive leakage of fluid and proteins from blood vessels to the interstitial space‘, being the fluid-filled space around tissue cells. The protease thrombin, which is apparently a coagulant (among other things) and not itself a cytokine, is in normal circumstances tightly regulated in the body by multiple factors, all of which can be impaired by hyperinflammatory conditions. The procoagulant-anticoagulant balance is disrupted, which can lead to microthrombosis and ‘disseminated intravascular coagulation’. Which I think is self-explanatory, and not good. The article refers to ‘raised d-dimer concentrations’ which has to do with fibrin, a fibrous protein involved in blood-clotting. The difficulty is that treatment with ‘endogenous anticoagulants’ has its dangers, shown in previous negative trials. There’s this important factor in respiratory physiology called the ventilation/perfusion (V/Q) ratio, with V being the measure of air getting to the alveoli, and Q the measure of blood getting to the alveoli. A mismatch there can affect the possibility of venous thromboembolism – blood clots, to oversimplify. What this forbiddingly technical Lancet article is suggesting, finally, is that studies conducted on murine [rat/mouse] models of PAR-1 antagonists (PAR-1 being protease-activated receptor, the main thrombin receptor mediating platelet aggregation) have shown some promise, and need to be further investigated tout de suite. Here are the authors’ final words:

Targeting thrombin, coagulation factor Xa or PAR-1, might therefore be an attractive approach to reduce SARS-CoV-2 microthrombosis, lung injury, and associated poor outcomes.

References

https://en.wikipedia.org/wiki/John_Dalton

https://www.sciencedirect.com/topics/neuroscience/cytokines

https://www.medicalnewstoday.com/articles/326701

https://www.cancer.gov/publications/dictionaries/cancer-terms/def/leukocyte

https://www.thelancet.com/journals/lanres/article/PIIS2213-2600(20)30216-2/fulltext

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3049450/

Written by stewart henderson

May 2, 2020 at 4:51 pm