N-acetylcysteine (NAC)

 

 

NAC is a thiol-containing amino acid; it is readily absorbed and is non-toxic. It has many beneficial effects; perhaps the most surprising is the likely ability of this molecule to destroy chlamydial elementary bodies. It is also a powerful antioxidant, and replenishes intracellular glutathione. It may also be effective in inactivating fungal gliotoxins. These properties are reviewed below.

 

 

Destroying the elementary bodies: one of the many possible benefits of N-acetylcysteine


Chlamydiae are apparently unique amongst Gram negative organisms in possessing cysteine-rich proteins on the surface membrane of the spore-like EB. Cysteine is an amino acid containing a sulfhydryl ( —S-H ) group; a disulphide bond ( —S-S— ) can form between two cysteine molecules. Such cross-linking bonds are common within and between the EB surface proteins. They are thought to preserve the integrity and shape of the EB. Furthermore, these disulphide bonds are accessible for cleavage. Why do chlamydial EBs have this surface structure? Structure, in the realm of the prokaryote, implies function: there is no room for ornament or unnecessary redundancy. Evolution works with swift efficiency at this scale. Reduction of these bonds disrupts the integrity of the EB's surface, and may be the mechanism by which the EB rapidly opens within the endosome. Rapidity is important, because the chlamydial body, now truly inside the host cell, must avoid fusion with the host lysosome or it will be killed. Raulston and co-workers have shown that the disulphide bonds between the chlamydial surface proteins must be opened up just prior to or during attachment to the host cell.
[Raulston JE et al., Surface accessibility of the 70-kilodalton Chlamydia trachomatis heat shock protein following reduction of outer membrane protein disulfide bonds. Infect Immun. 2002; 70(2): 535-43.] The mechanisms of chlamydial attachment to the host cell membrane; its entry into the cell; its ability to rapidly open and to subvert host cellular activities; its ability to evade host-defences and to track actively across the host cytoplasm taking what it needs from various host organelles — as if it were in a department store — are fascinating to consider. The parasite probably uses an intricate system of microscopic syringes and needles (the type III secretion system) to inject subversional proteins into host cytoplasm to induce endosomal formation. (Other bacteria, easier to study - for instance, Shigella flexneri - do this by injecting proteins which cause the host-cell surface to ruffle, overarch and then seal behind the parasite.) Once the EB is in the endosome, time is of the essence; it has only a limited amount of preformed proteins and it must get to work metabolizing as fast as possible.

It is reasonable to think that, were the EB coat to be opened up before achieving attachment to a nutrient-rich host cell, the unprotected EB would perish through starvation.

Destruction of EBs may be very important, as there is evidence that they may accumulate in extracellular spaces awaiting their chance to enter host cells - this may be analogous to the 'acellular (or extracellular) load' seen in HIV infections
[Chuck Stratton, personal communication.]

Penicillamine (dimethyl cysteine), which reduces disulphide bonds, inactivates EBs in vitro and prevents the initiation of infection in vivo. Amoxycillin, an inexpensive
b-lactam antibiotic, is also effective in inactivating EBs in vivo; penicillamine is one of its major metabolites. [Chuck Stratton, personal communication.]

N-acetyl cysteine, which, being a thiol antioxidant, is a good candidate for the reduction of disulphide bonds in EB coating proteins. It is readily available as a health supplement without the need for prescription.


The NAC Test

One indirect indicator of chronic infection with this organism is the N-acetyl cysteine test. This relies on the ability of NAC to rupture the extracellular Elementary Body by opening up surface disulphide bonds in the organism’s geodesic coat, as described above. The EB opens and perishes. The release of naked bacterial components causes local inflammatory symptoms. Because EBs are more numerous in primary respiratory infections, the acellular load of EBs is likely to be highest around respiratory structures. In a positive NAC test the daily administration of 2.4 G of NAC will cause, after a few days, sinusitis-like symptoms, with watery mucous; also a cough productive of a clear, moderately viscous sputum. Systemic symptoms — 'NAC flu' — may also occur. If symptoms are severe, the dose of NAC may be cut down to 600mg and slowly built up as may be tolerated. Symptoms wane, sometimes quickly, after a few days if the chlamydial load is small; if the load is large they may continue for a month or more as the EBs are destroyed and their remains removed by the immune system. As far as I am aware, NAC is unlikely to produce die-off reactions with any other genus.


NAC's other valuable properties


NAC's ability to replenish depleted glutathione in an animal model with defective transporter neuronal proteins is dramatic.
[Aoyama K, Suh SW, Hamby AM, Liu J, Chan WY, Chen Y, Swanson RA. Neuronal glutathione deficiency and age-dependent neurodegeneration in the EAAC1 deficient mouse. Nat Neurosci. 2005 Nov 27; {Epub ahead of print}] The mice with this gene-defect aged prematurely and lost brain substance. They had depleted glutathione levels in their neurones. The authors comment in an interview: For several days, a group of gene-deficient mice were fed N-acetylcysteine, an oral form of cysteine that is readily taken up by neurons. When their neuron slices were compared with slices from untreated gene-deficient mice, it was found that N-acetylcysteine "had completely corrected the biochemical defect" in their neurons, recounts Swanson. "Their glutathione levels were normal, their ability to withstand hydrogen peroxide toxicity was normal, and the oxidants we saw in the neurons in response to oxidative challenges were normal." This is remarkable. Major oxidative stress is a cause of cell-death in MS. NAC's antioxidant benefits may not be limited to the replenishment of depleted glutathione. [Gosset P, Wallaert B, Tonnel AB, Fourneau C. Thiol regulation of the production of TNF-alpha, IL-6 and IL-8 by human alveolar macrophages. Eur Respir J. 1999 Jul;14(1):98-105.]


NAC may prove useful for other reasons, having been credited with many beneficial effects:

replenishing glutathione, a major intracellular antioxidant; see above.

preventing further new infections with C. pneumoniae and thus averting the risk of MS relapse which this can produce.

ameliorating intracellular infections (such as influenza A) which are known to precede MS relapse;

modulating the immune response, making cells more resistant to the effects of proinflammatory cytokines such as TNF-
a.

is itself a major antioxidant. It is non-toxic in standard supplemental doses.

chelates heavy metals, which are known to accentuate oxidative stress.

may moderate the effect of endotoxins, which are made by chlamydiae.

NAC may be of particular importance in demyelinating conditions. Not only may it help moderate lipid peroxidation as an antioxidant, it may also moderate the induction of ceramide production by TNF alpha and consequent cell-death.[Singh I, Pahan K, Khan M, Singh AK. Cytokine-mediated induction of ceramide production is redox-sensitive. Implications to proinflammatory cytokine-mediated apoptosis in demyelinating diseases. J Biol Chem. 1998 Aug 7;273(32):20354-62.] These authors also found that thiol depletion of itself could induce ceramide production independently of TNFalpha. Furthermore, they comment: 'NAC, which has been used to block the cytokine-induced ceramide production in this study and to inhibit cytokine-mediated induction of inducible nitric oxide synthase in a previous study, is a nontoxic pharmaceutical drug that enters the cell readily and serves both as a scavenger of ROS and a precursor of GSH, the major intracellular thiol. Therefore, the use of reductants such as NAC or other thiol compounds may be beneficial in restoring cellular redox and in inhibition of cytokine-mediated induction of inducible nitric oxide synthase and breakdown of sphingomyelin thus reducing NO-mediated cytotoxicity as well as ceramide-mediated apoptosis in neuroinflammatory diseases.'

Persons with a high EB load may experience a variety of symptoms as the EBs are ruptured and endotoxin and other bacterial material are released. Common symptoms include pain round the eyes and over the sinuses; wheezing and sputum production. This is not surprising, as the organism will have been in the respiratory tract - producing EBs- for a long time before it was forced to become a persistent intracellular form. Other symptoms depend on the location of the EBs, but joint pain, pain in soft tissues and abdominal discomfort have been experienced. Symptoms tend to last a month or so.

(NAC is used medically to liquefy the sputum of patients with lung diseases characterized by overproduction of mucus; this makes expectoration easier. This liquefying action is carried out by reduction of the disulphide bonds between protein molecules in the sputum; the more of these bonds the more viscid the sputum.)




N-acetyl cysteine and Gliotoxin, a poisonous metabolite secreted by Candida sp.


N-acetyl cysteine may also be helpful in preventing the establishment of fungal infections such as candidiasis. Candida (and fungi in other and often distant genera) produce a toxin called gliotoxin [Shah DT, Larsen B. Clinical isolates of yeast produce a gliotoxin-like substance. Mycopathologia. 1991 Dec;116(3):203-8.]





This is one member of a large family of mycotoxins which cause damage to the immune system, provoking caspase-mediated apoptosis in monocytes and macrophages. Gliotoxin molecules contain a highly active surface disulphide bond which, on being reduced, damages host proteins by altering their structure. Gliotoxin may also deplete host glutathione, removing host antioxidant potential and increasing free radical damage; the molecule has been shown to oscillate between oxidized and reduced forms with the production of unstable peroxides. From the yeast's point of view the production of gliotoxin may be fundamental to the establishment of chronic colonization; locally high levels are found, for instance, in the genital tract of women with severe vaginal candidiasis
[Shah DT, Glover DD, Larsen B. In situ mycotoxin production by Candida albicans in women with vaginitis. Gynecol Obstet Invest. 1995; 39(1): 67-9.] Gliotoxin is a potent neurotoxin, and may alter gut motility. It impairs the efficiency of host polymorphonuclear neutrophils [Shah DT, Jackman S, Engle J, Larsen B. Effect of gliotoxin on human polymorphonuclear neutrophils. Infect Dis Obstet Gynecol. 1998; 6(4): 168-75.] There is some evidence that intestinal gliotoxin may cause dysfunction of the gut barrier by damaging enterocytes [Upperman JS, Potoka DA et al., Mechanism of intestinal-derived fungal sepsis by gliotoxin, a fungal metabolite. J Pediatr Surg. 2003 Jun; 38(6): 966-70.] Gliotoxin has been shown to be a virulence factor in other fungal infections such as invasive aspergillosis.

N-acetyl cysteine, on theoretical grounds, may be expected to neutralize gliotoxin by opening the disulphide bond, and indeed has been shown to be protective in vitro.
[Zhou X, Zhao A, Goping G, Hirszel P. Gliotoxin-induced cytotoxicity proceeds via apoptosis and is mediated by caspases and reactive oxygen species in LLC-PK1 cells. Toxicol Sci. 2000 Mar; 54(1): 194-202.] It probably acts by opening the gliotoxin disulphide bond. Upperman and co-workers [reference above] found that dithiothreitol, which also opens available disulphide bonds, protected gut cells from gliotoxin-mediated apoptosis. Not all orally taken N-acetyl cysteine is absorbed; that which remains in the gut may be useful in counteracting gliotoxin produced by intestinal candida. It should be noted that N-acetyl cysteine does not possess intrinsic activity against the yeast itself; Candida albicans is not inhibited even by high concentrations (> 100 mg/L) of NAC [Wheldon D: unpublished data.]

Gardiner and co-authors provide a very good overview of gliotoxin and related mycotoxins [Gardiner DM, Waring P, Howlett BJ. The epipolythiodioxopiperazine (ETP) class of fungal toxins: distribution, mode of action, functions and biosynthesis. Microbiology. 2005 Apr;151 (Pt 4): 1021-32.] the full text of which is available as a pdf file.

Caveat There is confusion over the term gliotoxin. The term was first used in the 1930s to describe a toxic metabolite of the fungus Gliocladium fimbriatum; the name is derived from the name of the organism. This toxin is the small molecule with the heterocyclic nucleus spanned by a disulphide bond as described above. Unfortunately the same name — gliotoxin — has also been applied to a protein, toxic to the glial (gliocyte) classes astrocytes and oligodendrocytes, which has been found in the CSF of persons with MS [Menard A, Amouri R, Dobransky T, et al., A gliotoxic factor and multiple sclerosis. J Neurol Sci. 1998 Feb 5; 154 (2) : 209-21.] These two toxins have categorically different structures and have nothing in common except their name. Despite this, Internet articles have appeared which assume a common identity and which forward the assertion that mycotoxins have been demonstrated in the CSF of persons with MS.

Other sulphur-containing organic molecules have anti-candidal activity; the best known being allicin, which is found in garlic
[Yamada Y, Azuma K. Evaluation of the in vitro antifungal activity of allicin. Antimicrob Agents Chemother. 1977 Apr;11(4):743-9.]

(On a personal note I have to say I love garlic. So, fortunately, does Sarah. However, it has got me into trouble. A friend of mine and I - both aged 11 - were hauled in front of the headmaster one day. Our crime? Introducing wild garlic into our dinner to remedy the dull, repetitive school-food blandness. Our accuser, the housekeeper, bristled with indignation. I can see her face now as she held out the pungent evidence. Forty-five years have passed, and I'm pleased to say that garlic is much more acceptable in the anglophone world.)

 

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page revised 15th January 2008