A Guide to “Chronic Fatigue Syndrome A Biological Approach” Chapter 7 (Edited by Patrick Englebienne Ph.D., Kenny DeMeirleir M.D, Ph.D., CRC Press. Washington D.C. 2002)
Chapter Seven: RNase L, Symptoms, Biochemistry of Fatigue and Pain and Co-Morbid Disease by Neil R. McGregor, Pascale De Becker, and Kenny De Meirleir
The RNase L system is mainly involved in antiviral defense and ‘controlled cellular degradation’ or cell suicide. Cell suicide is used to rid the body of infected or otherwise damaged cells. IIt does this by targeting and destroying mRNA involved in cell suicide. The authors believe that 37-kDa RNase L fragment destroys enough mRNA that the synthesis of proteins, the main workers of the cell, is effected and this imperils the ability of the cell to maintain its ‘homeostatic mechanisms’.
This reduced protein synthesis could lead to inadequate levels of receptors, membrane pumps, and many other intracellular proteins that are essential in controlling cellular homeostasis. The increased presence of viruses, or bacterial pathogens that are often found in CFS only further exacerbate the biochemical irregularities present and may impair brain, immune and other functions.
Given the wide variety of effects RNase L dysregulation may have, it was important to demonstrate that RNase L expression in CFS was, in fact linked to symptom expression. In HIV, another complex disease, alterations in basic immune factors (CD4 or T helper cells) not only cause immune and neural system problems, but also leave the patient vulnerable to attack by opportunistic infections and altered central nervous system activity. In CFS these researchers believe a disrupted 2-5A synthetase/RNase L system similarly leads to a very complex disease process.
SYMPTOM CLUSTERS IN CFS
The multiple effects RNase L dysregulation may have suggests that understanding the influence of the RNase L system upon patient biochemistry requires differentiating symptom groupings in CFS patients. To this end a factor analysis of symptoms found in CFS and fatigued but non-CFS patients was done.
Four symptom groupings were revealed. One grouping most clearly differentiated CFS patients from the fatigued but non-CFS patients. This group (see Table 7.1) called ‘general CFS symptoms’, included many of the symptoms found in the CDC case definition (fever, sore throat, flu-like symptoms), as well as gastrointestinal disturbances, symptoms associated with viral reactivation (ulcerations, shingles, cold sores), and a group of general symptoms (urinary frequency, dry eyes, non-refreshed sleep, allergies, new sensitivities to foods, drugs, etc.) Interestingly enough, the neurocognitive (memory problems, difficulty with words, attention deficit, etc.) and muscoskeletal symptoms (myalgia, athralgia, numbness and tingling, chest pain, etc.) which are part of the CFS case definition were separated from the major defining symptoms.
(I believe this means that while all the CFS patients were differentiated from the controls by the general symptoms, only a subset of CFS patients had high neurocognitive and muscoskeletal scores). The authors suggest that neurocognitive and muscoskeletal symptoms may be due to ‘separate influences’ or ‘host responses’. (This will be clearer later but has to do with the varied co-morbid diseases or disruptions generally found in conjunction with CFS).
That mood changes and psychiatric factors did not differentiate CFS patients from controls suggests they are not core symptoms of CFS. Patients who’d had CFS the longest had the highest symptom scores. CFS patients with a sudden onset of the disease had higher general CFS scores and reduced mood/psychiatric scores relative to CFS patients with a delayed onset.
Interestingly, exercise capacity (as measured by VO2 max) was strongly negatively correlated with muscoskeletal scores (i.e. the CFS patents who experienced the most athralgia, myalgia, muscle twitching, numbness, weakness, chest pain, had the hardest time exercising). None of the other groupings were strongly associated with reductions in the ability to exercise.
RNase L PROTEINS , sIL-2r, IL-6, CFS and SYMPTOMS
Our understanding of RNase L dysfunction in CFS has increased rapidly since Suhadolnik et. al. first demonstrated that CFS patients exhibited significantly increased RNase L activity and reduced cellular protein levels in 1994. By 2000 De MeirLeir et. al. reported that CFS patients exhibited increased levels of the 37-kDa and 40-kDa compared to the native 83-kDa enzyme, as well as increased quantities of all three enzymes. The 37/83-kDa ratio was found to be a good predictor of CFS.
These findings suggested that RNase L dysfunction is the central event in CFS pathogenesis. Further findings documented increased RNase L levels and reductions in its natural inhibitor RLI are present in CFS, and illustrated that both viral and chemical triggers can, via mediation of the IFN-b pathway, activate the dysfunctional RNase L pathway in CFS patients. (Does this mean that toxins can trigger the initial dysregulation or do they just exacerbate after it is present?)
The effects that increased RNase L levels and fragmentation have on symptom expression have been addressed in two recent studies. (Tying symptom expression to laboratory is a very big deal. Several seemingly promising research efforts on CFS have foundered when it was found that changes in the factor under examination had no effect on symptom expression. If laboratory markers are not correlated with symptomology then they are most likely not central to the disease.) The most basic expression of RNase L dysfunction in CFS – increased RNase L activity – was, consistent with earlier studies, strongly correlated with the general CFS and muscoskeletal symptoms (p<.0004, p<.0005) as well as the neurocognitive symptoms (p<.006). It was not correlated with the mood change/psychiatric symptoms.
Interestingly enough, the 37-kDa/80-kDa ratio was strongly associated only with the core CFS symptoms. Because most of the core CFS symptoms are associated with infectious activity, it appears that increased RNase L fragmentation reflects increased infections and/or viral reactivations. That increases in two components (IL-6, C-reactive protein) of the acute phase reaction are similarly elevated further suggests that infection/viral reactivation in CFS is a central component of increased RNase L activity and fragmentation. (The ‘acute phase reaction’ takes place in the early stages of the inflammation response.) While total RNase L activity is most closely associated with general and muscoskeletal symptoms, RNase L fragmentation is at its height when ‘pathogen associated events’ appear to be occurring. Based on these findings the authors suggest that the muscoskeletal/neurocognitive and mood change/psychiatric symptoms experienced by CFS patients may be the consequences of pathogen associated activity.
A statistical analysis indicated that increases in different cytokines (IL-2r, Il- 6) resulted in different symptoms. The authors suggest that the interaction between RNase L and the different cytokines may be the basis for the significant heterogeneity in symptom expression found in CFS patients. This study, then, underscores and begins to elucidate the complex interactions found between RNase L disruption, symptoms, the host response and environmental influences (It begins, in effect, to disentangle the maze of sometimes conflicting results that have bedeviled researchers in the last 15 years. Basic cellular processes are disrupted in CFS. Symptoms will vary – as they do in AIDS – depending on variety of factors.).
THE COMPLEX DISEASE PROCESS
The examination of symptom expression between CFS patients and fatigued but non-CFS patients indicated that the general CFS symptoms – which are mostly indicative of the infectious process – most broadly differentiated the two groups. An examination of symptom expression in a different study found in CFS patients and healthy controls indicated that muscoskeletal symptoms (p<0.00001(!)) followed by general CFS and neurocognitive symptoms most broadly differentiated the two groups. (Why did the core CFS symptoms not best differentiate the two groups? Perhaps because the most discriminatory factors will be the most unusual ones. While few people experience at any given time muscoskeletal symptoms (muscle numbness, fasiculations, chest pain, etc.), some people in any group will experience general CFS symptoms (sore throat, allergies, rashes, cough, etc. ?). Once again mood change/psychiatric symptom scores did not differentiate the two groups. The authors developed a disease model based on this data and on clinical observations.
The data suggest that from 25-50% of CFS patients at any given time experience symptoms similar to those occurring after viral reactivation or other infectious events. While in healthy controls these events are simply associated with increased RNase L activity, in CFS patients they are associated with increased RNase L activity and fragmentation. CFS patients also exhibit increases in immune (C-reactive protein) and oxidative markers (methaemoglobin, 2-3 bpg, malondialdehyde) which suggest an increase in the acute phase process.
Why increased oxidative markers in CFS? Possibly because every inflammatory event is accompanied by increased free radical production. Phagocytosis – the ingestion of pathogens – in particular generates high amounts of free radicals. We should note that the only cells thus far found with RNase L dysfunction in CFS, monocytes/macrophages, are phagocytes. As phagocytes flood the injured areas they use free radicals in order to kill invaders or to break down damaged cells. An intense ‘respiratory burst’ that monocytes use to awaken at warp speed from a rather somnolent existence (powered exclusively by anaerobic respiration) produces a host of free radicals as well. During this process free radicals are released outside the infected cell in order to degrade the cells membranes as it is being engulfed. The inflammatory process is exacerbated when free radicals induce the release of prostaglandins from damaged membranes. A weak free radical, nitric oxide (NO) is released when the blood vessels dilate in order to speed immune products to the infected or wounded site. NO can combine with superoxide to form an extremely reactive free radical peroxynitrite (OONO-). Peroxynitrite is able to deform many of the molecules or proteins it comes into contact with.
The increased free radical formation occurring during an inflammatory episode does not present a severe problem in people with adequately functioning antioxidant systems. Malondialdehyde is a decomposition product formed during lipid peroxidation. Because polyunsaturated fatty acids (PUFA’s) such as linoleic and arachidonic acids, which are susceptible to peroxidation, are found in great abundance in cellular membranes, membranes are among the first components to suffer when free radical levels are high. Peroxides are formed when hydroxyl (OH-), an extremely reactive free radical, is produced when hydrogen peroxide and superoxide interact. The body has no real defenses against OH-; its strategy is to avoid hydroxyl formation by completely degrading H202 and O2- before they have a chance to interact. If the levels of the two enzymes that degrade these substances, superoxide dismutase or catalase, are low, however, then hydroxyl formation and the oxidation that accompanies it, are inevitable. Nitric oxide, which the authors of this text and Martin Pall believe is upregulated, inhibits hydrogen peroxide degradation by disrupting catalase.
A positive correlation of oxidative markers with symptom factor scores indicated that increased oxidative radicals in CFS exacerbate CFS symptoms. Increased free radical levels can cause cyt-c release in the mitochondria and thus initiate the apoptotic process.
An examination of the four symptom factor groupings in light of the total urinary and amino and organic acid excretions and serum free fatty acids revealed that reduced organic and amino acid levels were associated with rises in all four symptom scores. (Reduced amino acid secretions may reflect a dysregulated protein turnover process that results in increased proteolysis (protein degradation) relative to protein synthesis. Enhanced proteolysis occurs in order to increase amino acid levels during trauma, infection or highly stressful situations. It appears that the reduced amino acid secretions seen in CFS patients occur simply because the body is using all the amino acids available to build proteins. RNase L and PKR upregulation, RNase L fragmentation and reduced growth hormone could all result in decreased protein synthesis.) Conversely, free fatty acid levels were positively correlated with muscoskeletal symptom scores and unrelated to the other symptom groups.
In order to investigate possible patterns in symptom expression over time, the scores for the four symptom groups for CFS patients and controls were plotted over eight sequential periods. In contrast to the scores for the controls, which were lower and independent of each other, the scores for CFS patients were higher and the general CFS and muscoskeletal symptoms appeared to rise and fall together. The authors suggest that this pattern reflects periods of increased cytokine and RNase L activity and RNase L fragmentation caused by viral reactivations and/or infections.
CHANGES IN BIOCHEMISTRY ASSOCIATED WITH MUSCLE PAIN AND FATIGUE
Increased excretions in tyrosine and 3-methylhistidine amino acids also differentiated CFS patients from controls. Increased tyrosine was associated with declining leucine levels and increased pain. It reflects the degradation of non-fibrillar proteins in the cell. Increased 3-methylhistidine, because it is found only in cytoskeletal or fibrillar actin, comes from the increased degradation of contractile or fibrillar proteins.
Increased tyrosine levels indicate that ‘non-fibrillar’ protein stores in the cells cytoplasm are being degraded to provide amino acids. Increased urinary 3-methyl-histidine levels indicate a mobilization of fibrillar muscle proteins has occurred in response to an exhaustion of cytoplasmic protein stores. It indicates a much higher level of stress. The very low levels in CFS patients of a by-product of protein degradation (leucine) that regulates this process suggests an ongoing proteolytic process. Disruption in leucine homeostasis has, in fact, been suggested as the cause of the chronic proteolysis believed to occur in CFS.
Tyrosine is a building block for epinephrine and norepinephrine, the two main stress related hormones. It is believed to be an adaptogen that helps the body cope with physical and psychological stress. Tyrosine aids adrenal, thyroid and pituitary functioning.
Cytokine upregulation and the myalgia and lethargy that often accompanies it, are often seen in CFS patients. One common outcome of cytokine upregulation is increased nitric oxide (NO) activity. High NO levels alter the redox potential (NADPH/NADP+) and effect the regulation of the citric acid cycle found in the mitochondria.
The citric acid cycle, in contrast to the glycolytic pathway, the other energy producing pathway, functions aerobically. It is the main generator the fuel the body runs on, ATP. NO, a close structural analogue to O2, competes with O2 for its binding sites on cytochrome oxidase which sits at the end of the electron transport chain in the mitochondria.
As electrons are passed down the chain they give off energy. This energy is used to produce the hydrogen gradient that drives the transformation of ADP to ATP. O2 accepts the electrons after they have discharged their energy. It’s a garbage collector! O2 is valuable because (a) it is a good electron acceptor, and (more importantly) because (b) after accepting electrons it can easily be degraded to an innocuous substance, water, that does not harm the body. 90% of the 02 in the body is consumed in this process! NO on the other hand is not a good electron acceptor. It stops the process in its tracks and leaves the electron transport chain in a reduced (i.e. electron rich) state which results in the production of more free radicals. When O2 levels are low or when NO levels are high NO wins the battle. This is a serious problem.
While O2 is easily degraded to water by a variety of enzymes, as it is collects electrons (is oxidized), a free radical 02-, is formed. Most of 02- spontaneously reverts to hydrogen peroxide (H202) which catalase then converts to H20. The rest of 02- is easily converted by glutathione. But what if, as appears to be the case in CFS, glutathione levels are low and 02- builds up, while, as often happens in CFS, an inflammatory process is co-occurring? Because NO and 02-, as noted earlier, produce the very destructive free radical peroxynitrite (OONO-) you perhaps have a recipe for cellular disaster. If this situation goes on long enough, the citric acid cycle, probably because of free radical damage, can be damaged irreversibly. If that happens ATP production drops and cellular processes are inhibited or the apoptotic program is invoked and the cell dies.
NO is not, however, the only potential inhibitor of the citric acid cycle in CFS. IFN-y and TNF increase anerobic energy production (glycolysis) and inhibit aerobic energy production as well. Short term upregulation of NO by IFN-y and bacterial lipopolysaccharides (LPS) results in inhibited cytochrome–c oxidase and (eventually) succinate-cytochrome-c reductase. Mitochondrial functioning, especially in regard to malate and succinate associated respiration is depressed when IFN-y and LPS are present, and ATP levels are lowered.
As noted earlier reducing the oxygen available for oxidative phosphorylation, high NO levels can reduce ATP output significantly. In an attempt to offset this deficit the anaerobic component of the energy production cycle is upregulated. Because it is so much less efficient than the citric acid cycle, however, energy production is still significantly reduced. One negative outcome of increased glycolytic activity is increased accumulations of lactic acid that can reduce pH levels in the blood and cause ‘metabolic acidosis’.) Cells operating on energy produced by glycolysis are less hardy than those operating aerobically. Increased glycolytic activity in cells exposed to IFN-y and LPS, results in decreased cell survival rates. After prolonged exposure to IFN-y and LPS (as could happen in someone with a chronic bacterial infection) even the administration of glycolytic inhibitors will not prevent cell death. Mitochondrial functioning, especially with regard to malate and succinate associated respiration, is depressed when IFN-y and LPS are present.
So CFS patients appear to be presented with two problems here; high IFN-y or LPS levels stress the cell at the precise moment the cell is struggling because its operating largely anaerobically because of increased NO levels. A breakdown in the mitochondrial process probably initiates the apoptotic process and another soldier in the battle against pathogen attack dies.
Amino and organic acid secretions and serum changes were assessed to determine if the scenario described above occurs in CFS patients. High fatigue levels have been correlated with low succinic acid and asparagnine excretion and reduced tyrosine and phenylalanine serum levels. Not surprisingly CFS patients, in contrast to controls, exhibited a similar pattern. This data suggests that increased catabolism (breakdown) of asparagnine and phenylalanine and possibly tyrosine results in increased glycolysis in CFS patients. That serum glucose and succinate excretion are positively correlated suggests that glycolysis is upregulated (anerobic phosphorylation) and oxidative phosphorylation (aerobic phosphorylation) is inhibited in CFS. (If asparagnine, phenylalanine and tyrosine are reduced in CFS then the output of the last half of the citric acid cycle is reduced.)
(To make matters worse) the precursors of catecholamines appear to be broken down by this mechanism. (Catecholamines are neurotransmitters; epinephrine, norepinephrine and dopamine. They are all derived from tyrosine. The authors earlier noted that tyrosine levels were reduced in CFS patients relative to controls.) Reduced catecholamine levels may lead to many of the sympathetic nervous system associated symptoms found in CFS.
The authors suggest that the general CFS symptoms are the result of acute cytokine mediated responses (to infectious events). The muscoskeletal, neurocognitive, and mood change/psychiatric symptoms result from the depletion or accumulation of components that are altered by the continuing cytokine responses or by ‘co-morbid conditions’ that effect cellular homeostasis.
(Talk about a complex process. If I understand it (a big if!) it appears that high NO levels combined with increased levels of cytokine production as a result of bacterial or viral attack impairs mitochondrial functioning and causes increased catabolism of amino acids involved in the citric acid cycle. The impairment of aerobic ATP production results in increased anerobic activity (glycolysis) in CFS patients. Abnormalities of the sympathetic nervous system occur when a neurotransmitter precursor, tyrosine, is also broken down.)
*Update – (2003, Clin Sci, Apr. 23) - Amino acid modulators of serotonin and dopamine function were measured in CFS and controls in an investigation of the central neural system (CNS) in CFS. Levels of free tryptophan, the rate-limiting serotonin precursor were significantly higher and levels of tyrosine, the dopamine precursor and branch chain and large neutral amino acids were significantly lower in CFS patients. The authors state that these finding implicate the CNS in CFS pathology. The finding of low tyrosine in CFS patients is replicated.
*Update - Two 2005 studies by Jones and Chalmers suggested that McGregor and De Becker’s amino acid analyses were based on a faulty testing procedure and that their results cannot be trusted.
Data from over 1500 (!) patients indicates that increased pain and fatigue in CFS is correlated with reduced amino acid excretion. The authors believe that pro-inflammatory fatty acids and precursors increase and amino acids decrease in CFS patients over time. Each increase in the general CFS symptom factor scores is associated with increased cytokine driven amino acidaemia and diuresis and losses of sodium and amino acids.
A multivariate analysis indicated that n-6 fatty acids were positively correlated with all four symptom factor while saturated fatty acids were negatively correlated with increased symptomology. (As CFS patients experienced more and more negative symptoms the levels of their n-6 fatty acids rose and their saturated fatty acid levels declined.) This pattern is consistent with an increased inflammatory and/or ‘prohyperalgesia response’.
CO-MORBID DISEASE IN CFS
While the co-morbid disease/CFS interaction is complex, the evidence to date suggests a model based on AIDS may be useful. Two broadly defined ‘factors’ appear to result in the reduced energy that is the hallmark of CFS; those that increase cytokine production, and those that alter energy availability.
FACTORS INFLUENCING CYTOKINE PRODUCTION
Viruses - The authors believe that viral reactivation is an important factor for most CFS patients. That CFS patients evidence increased rates of viral reactivation yet do not display an increased prevalence of specific viruses, suggests, however, that viral reactivation is not causative in CFS. Instead viral reactivation is a co-morbid feature of the disease; that is, the underlying disease process in CFS contributes to the increased incidence of viral reactivations found in CFS. (Interestingly enough) different viruses trigger different parts on the interferon/2-5OAS/RNaseL enzyme system. The potential therefore exists for each to alter that system, and therefore the body chemistry of each CFS patient, and thus the symptoms that each CFS patient experiences in distinct ways.
Viral reactivation or infection in CFS is likely not only to cause different symptoms in CFS patients, but also to cause variations in lymphocyte activity and probably cytokine production and biochemistry. The variable results seen in many CFS studies may be the result of a heterogeneous co-morbid disease process that has yet to be accounted for.
Bacterial Toxins - Bacterial toxins can induce cytokine activity. Skin coagulase-negative staphylococci (CoNS) produces a membrane damaging toxin that, the authors have found, is implicated in myofascial pain syndrome (MFPS), a commonly occurring syndrome characterized by muscle tenderness. That CFS patients with more toxin producing strains of CoNS were found to have more severe core CFS symptoms, and that toxin levels were associated with increased RNase L activity, suggests that bacterial toxins are another co-morbid condition that exacerbates CFS symptoms.
Another study indicated that CFS patients exhibit significant disruptions of bowel microflora relative to controls. Gastrointestinal upset was predominantly associated with general CFS symptoms. CFS patients were significantly more likely than controls to harbor Enterococcusand Staphylococcus spp. (the ‘bad’ bacteria), and significantly less likely to haveBifidobacterium or Lactobaccillus spp. in their bowels.