Myalgic Encephalomyelitis/Chronic Fatigue Syndrome as a

NO/ONOO- Cycle Disease

Martin L. Pall, Professor Emeritus of Biochemistry and Basic Medical Sciences, Washington State University and Research Director of the Tenth Paradigm Research Group

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Additional NO/ONOO- cycle diseases – Chronic Inflammatory Diseases

The Five Principles

Chronic fatigue syndrome (CFS), sometimes called myalgic encephalomyelitis or myalgic encephalopathy (ME) was the first of these multisystem illnesses to be proposed to be caused by a vicious cycle (1-11) that has recently been named the NO/ONOO- (no, oh no!) cycle (8) and is still one where an increasingly strong case for this etiology can be made. While I think that there is also an increasingly strong case for calling this illness ME, I will refer to it here as CFS/ME because both of these terms are being used in the scientific literature.

CFS/ME appears to have the largest group of initiating short-term stressors described in the literature of all the multisystem illnesses, stressors where each may be expected to act to increase nitric oxide levels. Most of these are documented in a my book and recent review (8,9) and some of these are also documented elsewhere (1,2,14). The apparent initiating stressors implicated in CFS/ME cases are as follows:

1. Viral infections

2. Bacterial infections

3. A protozoan infection, toxoplasmosis

4. Carbon monoxide exposure

5. Physical trauma

6. Organophosphorus poisoning

7. Severe psychological stress

8. Ciguatoxin poisoning

9. Ionizing radiation exposure

While the first two of these are implicated most commonly in the initiation of CFS/ME cases, we need explanations for the apparent roles of all nine. The fact that all nine can initiate a sequence of events that leads to increased nitric oxide synthesis (8,9,11) must be considered to be a striking coincidence that provides a key clue to CFS/ME etiology. Indeed the genetic evidence implicating corticosteroid-binding globulin gene (15,16) and the serotonin transporter gene (17) in determining susceptibility to CFS/ME, as discussed in Chapter 5, ref.8, also provides support for a nitric oxide role in CFS/ME initiation. Both of these genes can act to determine cortisol function and cortisol is known to lower the induction of the inducible nitric oxide synthase (iNOS) and therefore partially determine levels of nitric oxide (8,9).

Of the nine stressors listed above, the first three act primarily by inducing iNOS, as does the ionizing radiation (stressor 9) (1,2,8,11). The others are thought to act, at least in part, by increasing NMDA activity which acts, in turn, through the other two nitric oxide synthases, nNOS and possibly eNOS (2,8,12). Thus the common feature of these initiating stressors is their common increase in nitric oxide, not the specific form or forms of nitric oxide synthase involved.

The connection between each of the first six initiating stressors and nitric oxide is very clear. I’d like to discuss the last three. Number 7, severe psychological stress has been mainly linked to nitric oxide synthesis through animal models of posttraumatic stress disorder, where it has been shown to increase NMDA activity and consequently levels of nitric oxide (7,8).

Ciguatoxin exposure is known to greatly delay closing of certain sodium channels and this is known, in turn to be able to stimulate NMDA activity (Chapter 5, ref. 8). The role of ciguatoxin in increasing nitric oxide levels is inferred, therefore, from the known role of NMDA receptors activity in increasing nitric oxide and its oxidant product peroxynitrite.

Ionizing radiation initiates cases of post-radiation syndrome, a CFS/ME-like illness (11). Ionizing radiation is known to act to stimulate the transcription factor NF- kB, leading in turn to increased iNOS activity and consequent nitric oxide. It so acts at doses of radiation reported to have initiated cases of post-radiation syndrome following the Chernobyl disaster (11).

It can be seen, from the above, that the pattern of evidence implicating elevated nitric oxide synthesis activity in the initiation of CFS/ME cases is quite striking. How can nitric oxide act to initiate these illnesses? By acting mainly through its oxidant product peroxynitrite to initiate the vicious cycle mechanism (1), the NO/ONOO- cycle, that is responsible for chronic illness. The NO/ONOO- cycle mechanism, given previously on my main web page, book (8) and elsewhere (9), is presented again below in Fig. 1.

Fig. 1 legend. Vicious (NO/ONOO-) cycle diagram. Each arrow represents one or more mechanisms by which the variable at the foot of the arrow can stimulate the level of the variable at the head of the arrow. It can be seen that these arrows form a series of loops that can potentially continue to stimulate each other. An example of this would be that nitric oxide can increase peroxynitrite which can stimulate oxidative stress which can stimulate NF-kappaB which can increase the production of iNOS which can, in turn increase nitric oxide. This loop alone constitutes a potential vicious cycle and there are a number of other loops, diagrammed in the figure that can collectively make up a much larger vicious cycle. The challenge, according to this view, in these illnesses is to lower this whole pattern of elevations to get back into a normal range. You will note that the cycle not only includes the compounds nitric oxide, superoxide and peroxynitrite (abbreviated PRN) but a series of other elements, including the transcription factor NF-kappaB, oxidative stress, five inflammatory cytokines (in box, upper right), all three different forms of nitric oxide synthases (iNOS, nNOS and eNOS), and neurological receptors including some of the TRP receptors and the NMDA receptor. Mitochondrial dysfunction leads to ATP depletion. Tetrahydrobiopterin (BH4) depletion is shown to be an important aspect of the cycle.

Are NO/ONOO- Cycle Elements Elevated in the Chronic Phase of CFS/ME?

The mechanisms involved in the NO/ONOO- cycle are discussed in many of my papers (1,2,7-12) and summarized most completely in my book (8). One very important question is in determining whether any specific chronic disease/illness is a NO/ONOO- cycle disease is whether elements of the cycle are elevated in the chronic phase of illness. The answer, in CFS/ME as is also true of the other multisystem illnesses, MCS, FM and PTSD: Where they have been looked at, they are elevated!

Some of the specific CFS/ME evidence is as follows (8):

1. Oxidative stress has been reported in 13 earlier studies of CFS/ME, published by seven research groups on four continents (reviewed in 5,8,13) and two additional studies have been reported recently (22,23). Such oxidative stress is also supported by reports of glutathione depletion and cyst(e)ine depletion in CFS/ME (20,21) and by reported depletion of essential fatty acids in CFS/ME (24,25). Oxidative stress is expected to lead to both glutathione/cysteine depletion and essential fatty acid depletion as I proposed earlier (1). Thus oxidative stress is probably the best documented change in CFS/ME. Having said that, it is not a specific response to CFS/ME! Many inflammatory diseases will lead to elevation of markers of oxidative stress and in addition, many cases of CFS/ME (presumably among the more modestly effected) fall into the “normal” range of these markers. The same pattern occurs with many other changes that are reported in CFS/ME, where on average there are statistically significant changes but many individual CFS/ME cases fall well within the normal range.

2. Mitochondrial/energy metabolism dysfunction is part of the NO/ONOO- cycle mechanism because peroxynitrite attacks a number of components of mitochondria, and nitric oxide and superoxide also inhibit certain mitochondrial functions (8,9). 18 different studies provide evidence for mitochondrial and/or energy metabolism dysfunction in CFS/ME (reviewed in 1,8). This again provides extensive evidence supporting NO/ONOO- cycle biochemistry in CFS/ME. Among these are studies showing that agents predicted to improve mitochondrial function such as carnitine/acetyl carnitine, coenzyme Q10 and lipids designed to help regenerate the mitochondrial inner membrane are all helpful in the treatment of CFS/ME. These provide evidence that not only is there mitochondrial dysfunction but that it contributes to the CFS/ME pathophysiology.

3. Two studies report increased nitric oxide levels (3,26). In addition, studies of neopterin levels, a marker of high level iNOS induction reported statistically significant elevation in three of five studies of CFS/ME, suggesting that iNOS induction contributes to the nitric oxide elevation (1). The hydroxocobalamin form of vitamin B 12, a potent nitric oxide scavenger (4) was reported to produce statistically significant improvements of CFS/ME-like patients in a placebo-controlled trial (27). Hydroxocobalamin has been used clinically to treat CFS/ME-like illnesses for over 60 years, being used in at least 9 countries on three continents, mainly by IM injection. Patients report rapid improvement of their entire spectrum of symptoms in response of hydroxocobalamin injection. The pattern of apparent efficacy suggests that not only is nitric oxide elevated in CFS/ME, but that it contributes in a major way to its etiology.

4. There have not been any published studies on peroxynitrite, but Dr. Tapan Audhya has shown me his data on this. He finds that many CFS/ME patients have elevated levels of 3-nitrotyrosine, a marker of peroxynitrite elevation.

4. There are 10 studies that report that one or more of the inflammatory cytokines in the right, upper corner of Fig. 1 are elevated in CFS/ME (8). These elevations are relatively modest suggesting that they contribute to but do not dominate the CFS/ME etiology.

5. Two physicians and one research group report clinical observations supporting an elevation of NMDA activity in CFS/ME, again supporting the NO/ONOO- cycle etiology (8).

6. Two recent studies have reported elevated NF-kappaB activity in CFS/ME, while mistakenly calling it NF-kappa beta [28,29].

While these are all important observations supporting a NO/ONOO- cycle etiology, additional such observations from clinical trials are discussed on my therapy web page. However, there are also some additional clinical observations from therapies that may also suggest elevation of cycle components. For example, some physicians have used agents known to lower NF-kappaB activity as part of their CFS/ME treatment protocols and others have used the drug guaifenesin, a drug reported to lower capsaicin responses and therefore vanilloid receptor action (8). The drug thiacetarsamide was reported by Tarello to produce great improvement animal models of CFS/ME and has been found by me to scavenge both nitric oxide and peroxynitrite (reviewed in 8). These studies suggest that elevation of NO/ONOO- cycle elements and also suggest that because lowering those elements may produce the improvements reported, the elements may contribute substantially, to the etiology of CFS/ME.

Genetic Studies

We have already discussed the genetic studies that provide evidence for a role of nitric oxide in CFS/ME. There are several additional genetic studies that should be mentioned.

Vladutiu and Natelson (30) reported a role for a polymorphism in the angiotensin concerting enzyme (ACE) in determining susceptibility to CFS/ME. This gene regulates the levels of angiotensin II, which determines, in turn the levels of tetrahydrobiopterin (BH4), an important cycle element. BH4 depletion produces in turn increased superoxide, so that this genetic study implicates two NO/ONOO- cycle elements in CFS/ME, BH4 depletion and increased superoxide.

Two studies (31,32) have implicated inflammatory cytokines in determining CFS/ME, susceptibility, arguing in turn for another set of NO/ONOO- cycle elements.

Boles and coworkers (33,34) have reported that people with mutations in their mitochondrial DNA have CFS/ME-like symptoms, along with a variety of other symptoms, implicating mitochondrial dysfunction and consequent ATP depletion, other NO/ONOO- cycle elements.

All of these genetic studies are discussed on somewhat more detail in ref 9. These genetic studies, taken together, implicate the following NO/ONOO- cycle elements in CFS/ME: Nitric oxide, BH4 depletion, superoxide, inflammatory cytokines and mitochondrial dysfunction. Together, they provide substantial support, therefore, for a NO/ONOO- cycle etiology for CFS/ME.

Gene Expression Studies and NO/ONOO- Cycle Elements

There are a number of gene expression studies that implicate various elements of the NO/ONOO- cycle in CFS/ME, studies that are reviewed in Ref. 9 and will not be cited separately here. Specifically, gene expression studies implicate oxidative stress, mitochondrial dysfunction, chronic inflammatory responses and excitotoxicity including possible excessive NMDA activity. Consequently, a number of NO/ONOO- cycle elements are implicated in CFS/ME, from gene expressions studies alone.

Animal Models and the NO/ONOO- Cycle

There are two animal (mouse) models of CFS/ME, on each of which there are multiple published studies. In one of these animal models (reviewed in 8,9), cases are initiated by a stressor that increases nitric oxide and produces elevated levels of inflammatory cytokines and also mitochondrial dysfunction. In the other (also reviewed in 8,9), there is substantial evidence for oxidative stress. Both of these animal models seem to be consistent with a NO/ONOO- cycle etiology and they collectively provide evidence for roles of several cycle elements.

Local Nature

The fourth principle underlying the NO/ONOO- cycle is that the basic mechanism is primarily local. This primarily local nature shows up in CFS/ME in two distinct ways. One is that there is stunning variation among different CFS/ME cases which are difficult to explain without such a local mechanism (discussed in Chapter 4, ref. 8). This variation is what has made it very difficult to come up with a simple case definition.

A second way in which this primarily local nature shows up is in brain scan studies where one can directly visualize the tissue variation, both quantitative and qualitative variation. Perhaps the most convincing such study for CFS/ME are the two MRI studies published by the Natelson group (35,36), where the variation is obvious from the published scans. Other relevant brain scan studies were discussed in Chapter 4, Ref. 8.

Chronic Fatigue Syndrome—Where Should We Search for a Specific Biomarker?

Searching for specific biomarkers for specific multisystem illnesses should focus, according to the NO/ONOO- cycle model, on the effects of the impact of that cycle on whatever tissue impact is most characteristic of a particular illness. In the case of MCS, FM and PTSD, the issue of specificity is readily apparent because the most characteristic symptoms or signs of illness are readily apparent. In the case of CFS/ME, they are not. However the recent deliberations on CFS/ME have focused on the symptom of post-exertional malaise as the most characteristic symptom (37) and Jason and coworkers have also focused on post-exertional malaise as perhaps THE critical symptom of CFS/ME (38,39). It is there that we should focus our search for a specific biomarker, in my view.

Post-exertional malaise is a phenomenon where exercise in CFS/ME patients produces an exacerbation of their entire spectrum of symptoms, an exacerbation that the NO/ONOO- cycle model predicts is likely to be due to up-regulation of NO/ONOO- cycle biochemistry. The view that excessive exercise in CFS/ME may up-regulate the basic causal mechanism is supported by some of the pioneering observations of Dr. Melvin Ramsay. Ramsay observed that CFS/ME sufferers who continued to work longest before collapse had a much poorer prognosis than those who were diagnosed early and underwent prolonged bed rest (40).

How might one use the phenomenon of post-exertional malaise to develop a specific biomarker for MCS? When I discussed this issue with Dr. Paul Cheney, suggesting that this is where we should focus our efforts to develop a specific biomarker for CFS/ME, he told me that his CFS/ME patients show a characteristic difference from normal controls—whereas normal controls show an increase in corticol levels after exercise, his CFS/ME patients do not. The notion that changes in cortisol response may cause post-exertional malaise in an attractive one. Cortisol (like other glucocorticoids) is known to lower the induction of the inducible nitric oxide synthase (iNOS) and may have a substantial role, therefore, in controlling nitric oxide levels. A deficient cortisol response to exercise may lead, therefore, to increased nitric oxide levels after exercise in CFS/ME patients vs. controls, leading, in turn, to up-regulation of the NO/ONOO- cycle.

Is there other evidence supporting a specific deficiency in cortisol control in CFS/ME? After all, the HPA axis control controls cortisol production and HPA axis dysfunction is known to occur in all of the multisystem illnesses (8), and so such dysfunction is not specific to CFS/ME. Two reviews suggest that HPA axis control in CFS/ME is distinct from that in FM (41,42). Consistent with such specificity, Ottenweller et al (43) reported changes in cortisol control in response to exercise in CFS/ME patients, similar to those found by Cheney. Dinan and coworkers reported (44) that the adrenal glands of CFS/ME patients were substantially smaller than those of controls, consistent with an aberration of HPA axis control. Certain other studies also provide support (Chapter 5, ref.8).

Others have expressed views similar to those I write here. Torpy (45) described CFS/ME patients with “altered dynamic responses to stress, especially cortisol to stimuli.” Neeck and Crofford (46) reported “abnormalities of central components of the HPA axis” in CFS/ME.

The prediction, then, is that exercise, acting in part or in whole through aberrent cortisol control, will act in CFS/ME patients to up-regulate NO/ONOO- cycle biochemistry in a response that will not be seen in normal controls . Is there any evidence for this? Jammes et al (47) reported large increases in markers of oxidative stress in CFS/ME patients after exercise, whereas only small increases were seen in controls. LaManca et al (48) reported much larger cognitive deficits after exercise in CFS/ME patients compared with controls, consistent with such cognitive deficits being caused by NO/ONOO- cycle biochemistry.

Even the major changes in cardiac function found in CFS/ME patients by Peckerman et al (49,50) and by Cheney (51 and personal communication) may be caused by lowered cortisol levels because cardiac dysfunction in humans and animals can be caused by lowered cortisol levels (reviewed in ref 8, chapter 5).

The notion that dysfunctional cortisol control in response to exercise is behind the phenomenon of post-exertional malaise in CFS/ME should allow one to use exercise control of almost any easily measurable NO/ONOO- cycle element to develop a specific biomarker for CFS/ME. In addition, almost any easily measureable symptom or sign of CFS/ME might also be used. My own prejudice is that we should use exercise control of markers of nitric oxide production before and after exercise or perhaps cortisol levels themselves, but no doubt others may have other parameters they may prefer to measure.

CFS/ME and the NO/ONOO- Cycle

Important relevant evidence supporting a NO/ONOO- cycle etiology for CFS/ME is provided on my main web page in my therapy web page and in my book (8), as well as above. The evidence for a presumptive nitric oxide mechanism for initiation is strong for CFS/ME and indeed stronger than it is for the other multisystem illnesses.

The importance of the NO/ONOO- cycle model of CFS/ME is not just that it is supported by a diversity of experimental observations. That is important, obviously, but that is just the beginning of what is needed. The need here is for an explanatory model of great breadth, one that explains not just one or two of the puzzling features of this illness, but each of its important features and the features, both similar and distinctive of this whole group of multisystem illnesses. It has been the perceived lack of such a model that has led CFS/ME and this group of illnesses to be repeatedly described as unexplained. Now we can argue that the whole group of illnesses is explained. Some of these explanation that have particular relevance to CFS/ME are as follows:

We have the list of nine distinct short-term stressors that can all apparently initiate cases of CFS/ME, all of which can act to start a sequence, leading to increased nitric oxide levels. We have, for the first time an explanation for how this diversity of stressors can lead to a common response – initiation of cases of CFS/ME.

CFS/ME is chronic because of the action of the NO/ONOO- cycle mechanism. We can explain the generation of both non-specific and specific symptoms and signs of CFS/ME, the latter discussed here and the former in my book (8). We can explain its comorbidity with other multisystem illnesses and of other well-accepted diseases such as migraine and asthma as being due to each of these illnesses having similar causal mechanisms.

We can explain the action of certain agents and entire treatment protocols which appear to be effective in the treatment of CFS/ME, some discussed above and others discussed on my main web page and in my book (8). Indeed it is the great promise of this mechanism as a predictor of therapeutic approaches that is its most important feature for the many who suffer from CFS/ME and related illnesses.

We can explain the stunning variation, both quantitative and qualitative in the symptoms and signs of illness among CFS/ME patients and, indeed, among patients of the whole group of multisystem illnesses.

We can explain the role of a series of genes reported to influence CFS/ME susceptibility, roles that to my knowledge have not been previously explained.

It has been the many previously unexplained features of CFS/ME and these multisystem illnesses that has led others to argue we needed a new paradigm of human of disease in order to explain them. That is exactly what we have.

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51. http://phoenix-cfs.org/Cardio%20IVa%20Superoxide.htm