Kennedy G, Spence VA, McLaren M, Hill A, Underwood C, Belch JJF
Vascular Diseases Research Unit, The Institute of Cardiovascular Research, Ninewells Hospital and Medical School, Dundee, UK
The study was funded by ME Research UK, and further support was received from the Sir John Fisher Foundation (Educational Grant).
The aetiology of chronic fatigue syndrome (CFS) is unknown; however, recent evidence suggests that excessive free radical (FR) generation may be involved. This study investigated for the first time levels of 8-iso-prostaglandin-F2a-isoprostanes alongside other plasma markers of oxidative stress in CFS patients and control subjects.
Forty-seven patients (18 males, 29 females, mean age 48 [19–63] years) who fulfilled the Centres for Disease Control classification for CFS and 34 sex and age-matched healthy volunteers (13 males, 21 females, 46 [19–63] years) were enrolled in the study. The CFSpatients were divided into two groups: those with previously defined cardiovascular risk factors of obesity and hypertension (group 1) and those who were normotensive and non-obese (group 2). Blood samples were collected, from which red blood cell GSH levels were measured on a spectrophotometer, oxidised low-density lipoprotein levels were measured by ELISA, plasma isoprostanes were measured by gas chromatography–mass spectrometry, and high-density lipoprotein levels were measured on a Cobas Bio centrifugal analyser.
Patients with CFS had significantly increased levels of isoprostanes (group 1, p=0.007; group 2, p=0.03) and oxidised low-density lipoproteins (group 2, p=0.02), compared with controls, indicative of a FR attack on lipids. Patients also had significantly lower high-density lipoproteins (group 1, p=0.011; group 2, p=0.005), and lower levels of the antioxidant GSH (p=0.05). CFS symptoms correlated with isoprostane levels (total symptom score, p=0.005; joint pain, p=0.002; post-exertional malaise, p=0.027), but only in group 2 CFS patients with low cardiovascular risk.
This new data provides further evidence of dysfunction to oxidative pathways in CFS. The finding of high levels of isoprostanes in people with CFS is particularly important given this measure’s sensitivity, reliability and correlation with other measures of lipid peroxidation in vivo. Furthermore, isoprostanes may not only be markers of oxidative injury, but may in fact mediate the effects of free radicals and reactive oxygen species.
- International Society on Thrombosis and Haemostasis, Birmingham, July 2003
Kennedy G, Spence VA, McLaren M, Hill A, Belch JJF. Increased plasma isoprostanes and other markers of oxidative stress in chronic fatigue syndrome.Journal of Thrombosis and Haemostasis, 2003 Jul 12–18; 1(Suppl 1): P0182
- American Association of Chronic Fatigue Syndrome biennial meeting, Washington, January 2003
- Scottish Society for Experimental Medicine, Edinburgh, November 2002
The body contains highly reactive molecules called free radicals. They are normally kept under control by natural processes which remove them from the circulation, but when an imbalance occurs they can be left to cause damage unchecked. This damage is called oxidative stress. In particular, free radicals can change our normal ‘good’ cholesterol into something more harmful, leading to heart and circulation problems.
We found that patients with ME/CFS had higher than normal levels in their blood of chemical markers which indicate oxidative stress, as well as lower levels of their ‘good’ cholesterol.
This suggests that the normal processes controlling free radicals are not working properly in patients with ME/CFS.
Comment by ME Research UK
Circulating in the bloodstream are highly reactive molecules, known as free radicals, which can cause damage to the cells of the body; a process called oxidative stress. In healthy people, increases in oxidative free radicals are neutralised by antioxidant defences, and it is only when these defences are overwhelmed that oxidative stress and consequently cell injury results. Such damage is implicated in a number of conditions, including cardiovascular disease, most neurological diseases (including Alzheimer’s), and the ageing process. Evidence is now increasing that oxidative stress and, more specifically, lipid peroxidation contributes to the disease process in ME/CFS (1–6) and to some of the symptoms in the illness (1).
While free radicals may generate tissue oxidative injury, it is also evident that other oxidative byproducts, especially peroxidised lipids such as 8-iso-prostaglandin F2a, may be even more pivotal in the pathological process. For this reason, Kennedy et al set out to measure levels of 8-iso-prostaglandin F2a alongside other markers of oxidative stress and antioxidant status in a group of 47 well defined ME/CFS patients and comparable control subjects, and to relate these levels to reported clinical symptoms of ME/CFS. Given the association of oxidative stress with obesity and high blood pressure — obesity and hypertension independently promote F2a isoprostanes levels (7) — the authors had to divide the patient group into two: those obese (body mass index > 30) and with high blood pressure (ME/CFS high cardiovascular risk factor group) and those with normal blood pressure and with a body mass index < 30 (low cardiovascular risk factor group).
As expected, the ME/CFS high cardiovascular-risk factor group had significantly increased 8-iso-prostaglandin F2a isoprostanes and significantly lower HDL compared with their control group. Importantly however, the ME/CFS low cardiovascular risk factor group also had significantly higher levels of 8-iso-prostaglandin F2a isoprostanes and significantly lower HDL levels than their matched control group. And in the low cardiovascular-risk factor group, 8-iso-prostaglandin F2a isoprostane levels were significantly and positively correlated with joint pain and post-exertional malaise. Indeed, it was found that in ME/CFS patients reporting the most severe joint pain, 8-iso-prostaglandin F2a isoprostane levels were significantly higher than in patients reporting milder joint pain (e.g., mean 318.9 vs 659.2 pg/mL for patients with no pain vs severe pain); and similar results were reported for post-exercise malaise.
The fact that the pattern of oxidative stress (increased 8-iso-prostaglandin F2a isoprostanes and oxLDL in combination with decreased HDL) exists in ME/CFS patients who are not hypertensive or obese suggests that the “pro-oxidant” state is a consequence of their illness and not a secondary effect to the presence of any known cardiovascular risk factors.
The source of excessive free radical generation in ME/CFS patients which involves oxidation of lipids and proteins (8) may be associated with a variety of altered biological processes. Exercising muscle is a prime contender for excessive free radical generation with recent evidence pointing to good correlations between muscle pain thresholds and fatigue with various blood markers of oxidative injury in CFS patients (5), and further evidence of viral persistence in muscle tissue in at least some patients with the illness (9). Fulle et al (10) demonstrated oxidative damage to DNA and lipids within muscle biopsies of ME/CFS patients consistent with metabolic abnormalities to both mitochondria and phospholipids. The samples in Kennedy et al‘s report were taken from well-rested subjects, and recent research has demonstrated that incremental exercise challenge induces a prolonged and accentuated oxidant stress that might well account for post-exercise symptoms in ME/CFS patients (11). ME/CFS is also associated with immune activation (12), and an equally compelling case can be made for excessive free radicals and reactive molecular intermediates being generated by activated white blood cells (13), either as a consequence of persistent infection (14) or environmental stressors (15). A further consideration is that viral infections are also associated with excessive free radical production (16, 17) and, in animal models at least, herpes simplex virus type 1 (HSV-1) infection is associated with significantly elevated levels of F2-isoprostanes (18). Of course, the fact that the “diagnosis” ME/CFS seems to catch a highly heterogeneous group of patients (‘ME/CFS: A research and clinical conundrum’) does not make investigation easy. However, Kennedy et al have previously reported raised concentrations of active transforming growth factor β1 and increased neutrophil apoptosis in chronic fatigue syndrome (19), and it could be suggested that many patients currently diagnosed with ME/CFS could have an inflammatory condition and be in a “pro-oxidant state”.
The novel findings of this study are that patients with ME/CFS have significantly elevated levels of F2-isoprostanes alongside other key markers of oxidative stress, and that these correlate with various ME/CFS symptoms. On balance, ME/CFS patients have a lipid profile and oxidant biology that is consistent with cardiovascular risk, and the presence of high levels of F2-isoprostanes may explain some of the symptoms of the disease. Importantly, obesity and hypertension represent a potentially additional burden to free radical formation and CFS pathology, an issue of which patients should be aware.
The importance of these findings cannot be overstated. F2-isoprostanes are now recognised as one of the most reliable approaches to assessing in-vivo oxidative stress and, in conjunction with their central role in oxidation, isoprostanes also exert potent biological activities and are likely to participate as mediators of oxidative injury. It has also emerged that F2-isoprostanes have powerful vascular actions utilising a novel mechanism of inducing the formation of thromboxane in the endothelium, which in turn contracts the vascular smooth muscle and also causes endothelial cell death. Such mechanisms have been demonstrated in the brain vasculature and are also thought to be relevant to other vascular beds (20). It is also clear that the formation of isoprostanes is oxygen dependent; i.e., oxygen concentration differentially modulates the formation of isoprostanes and isofurans. These findings may therefore have relevance to the brain symptoms that characterise many ME/CFS patients, and might also help to explain some of the peripheral vascular consequences of being upright, as recently reviewed in the Biologist (‘Standing up for ME’).
Finally, the data of Kennedy et al are strengthened by the results of recent gene investigations in ME/CFS (‘Gene Research: A Scientific Signature for ME/CFS?’). The finding by Kaushik and colleagues (21) of upregulation of the genes ABCD4 and PEX16 (suggesting enhanced defence to oxidative stress in CFS) and Dr John Gow’s recent evidence (22) of alterations to genes controlling the metabolism of prostaglandin (prostaglandin endoperoxides are intermediates in the formation of isoprostanes), provide a tantalising new context for these novel results.
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- Pall ML, Scatterle JD. Elevated nitric oxide/peroxynitrite mechanism for the common etiology of multiple chemical sensitivity, chronic fatigue syndrome, and posttraumatic stress disorder. Ann N Y Acad Sci 2001; 933: 323–329.
- Manuel Y et al. Antioxidant status and lipoprotein peroxidation in chronic fatigue syndrome. Life Sci 2001; 68: 2037–2049.
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- Vecchiet J et al. Relationship between musculoskeletal symptoms and blood markers of oxidative stress in patients with chronic fatigue syndrome. Neurosci Lett 2003; 335: 151–154.
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- Keaney JF Jr et al. Obesity and systemic oxidative stress: clinical correlates of oxidative stress in the Framingham Study. Arterioscler Thromb Vasc Biol. 2003; 23: 434–439.
- Smirnova IV, Pall ML. Elevated levels of protein carbonyls in sera of chronic fatigue syndrome patients. Mol Cell Biochem 2003; 248: 93–95.
- Lane RJM et al. Enterovirus related metabolic myopathy: a post viral fatigue syndrome. J Neurol Neurosurg Psychiatry 2003; 74: 1382–1386.
- Fulle S et al. Specific oxidative alterations in vastus lateralis muscle of patients with the diagnosis of chronic fatigue syndrome. Free Radic Biol Med 2000; 29: 1252–1259.
- Jammes Y et al. Chronic fatigue syndrome: assessment of increased oxidative stress and altered muscle excitability in response to incremental exercise. J Intern Med 2005; 257: 299–310.
- Patarca-Montero R et al. Cytokine and other immunologic markers in chronic fatigue syndrome and their relation to neuropsychological factors. Appl Neuropsychol 2001; 8: 51–64.
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- Cai J et al. Inhibition of influenza infection by glutathione. Free Radic Biol Med 2003; 34: 928–936.
- Beck MA et al. The role of oxidative stress in viral infections. Ann N Y Acad Sci 2000; 917: 906–912.
- Milatovic D et al. Herpes simplex virus type 1 encephalitis is associated with elevated levels of F2-isoprostanes and F4-neuroprostanes. J Neurovirol 2002; 8: 295–305.
- Kennedy G et al. Increased neutrophil apoptosis in chronic fatigue syndrome. J Clin Path 2004; 57: 891–893.
- Jackson L et al. The Biochemistry of the Isoprostane, Neuroprostane, and Isofuran Pathways of Lipid Peroxidation. Brain Pathology 2005; 15: 143–148.
- Kaushik N et al. Gene expression in peripheral blood mononuclear cells from patients with chronic fatigue syndrome. J Clin Pathol 2005; 58; 826–832.
- Gow JL et al. Whole-Genome (33,000 genes) Affymetrix DNA Microarray Analysis of Gene Expression in Chronic Fatigue Syndrome. International Conference on Fatigue Science, Karuizawa, Japan. February 9–11, 2005. Abstract available here.
Read more (pdf 376 KB) in the Winter 2005 issue of Breakthrough.