Oxidative and Nitrosative Stress

<p>From: <a href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3964747/">Oxidative and Nitrosative Stress and Immune-Inflammatory Pathways in Patients with Myalgic Encephalomyelitis (ME)/Chronic Fatigue Syndrome (CFS)</a></p>

<p>Myalgic Encephalomyelitis (ME) / Chronic Fatigue Syndrome (CFS) has been classified as a disease of the central nervous system by the WHO since 1969. Many patients carrying this diagnosis do demonstrate an almost bewildering array of biological abnormalities particularly the presence of oxidative and nitrosative stress (O&NS) and a chronically activated innate immune system. The proposal made herein is that once generated chronically activated O&NS and immune-inflammatory pathways conspire to generate a multitude of self-sustaining and self-amplifying pathological processes which are associated with the onset of ME/CFS. Sources of continuous activation of O&NS and immune-inflammatory pathways in ME/CFS are chronic, intermittent and opportunistic infections, bacterial translocation, autoimmune responses, mitochondrial dysfunctions, activation of the Toll-Like Receptor Radical Cycle, and decreased antioxidant levels. Consequences of chronically activated O&NS and immune-inflammatory pathways in ME/CFS are brain disorders, including neuroinflammation and brain hypometabolism / hypoperfusion, toxic effects of nitric oxide and peroxynitrite, lipid peroxidation and oxidative damage to DNA, secondary autoimmune responses directed against disrupted lipid membrane components and proteins, mitochondrial dysfunctions with a disruption of energy metabolism (e.g. compromised ATP production) and dysfunctional intracellular signaling pathways. The interplay between all of these factors leads to self-amplifying feed forward loops causing a chronic state of activated O&NS, immune-inflammatory and autoimmune pathways which may sustain the disease.</p>

<p id="__p35" class="p p-first">Proposal made herein is that once generated chronically increased levels of O&NS and immune-inflammatory substances in ME/CFS conspire to generate a multitude of self-sustaining and self-amplifying pathological processes which lead to the development of a chronic illness. The pathology stemming from chronically elevated levels of NO and peroxinitrite cannot readily be overstated. At high levels NO becomes a neurotoxin and a facilitator of glutamate-generated exitotoxicity and other NDMA receptor related pathology. Its role in modifying the activity of vital cellular enzymes and signaling pathways coupled with its ability to inhibit the performance of mitochondrial electron transport chain and compromise the production of ATP merely serves to magnify its importance as a source of pathology.</p>

<p id="__p36">Peroxinitrite is a major regulator of cellular processes and elevated levels lead to chronically elevated inflammatory pathways and inactivation of vital regulatory enzymes responsible for the maintenance of other vital cell systems. The toxic effects of peroxinitrite on energy production are multiple, ranging from inhibition of enzymes within the mitochondrial respiratory chain, depletion or inactivation of vital players in the tricarboxylic acid cycle, disruption of the mitochondrial membrane potential and inactivation of p53. Loss of function of the latter molecule compromises the cells ability to respond to increasing energy demands and, <em>via </em>a range of processes, leads to loss of immune homeostasis. Compromised electron transfer chain function leads to increased production of O&NS species which in turn further compromise energy production both directly and indirectly and is a stark example of pathology which becomes self-sustaining and self-amplifying once initiated. It is held that the genesis of these self-sustaining pathological systems stems from prolonged and or intense pathogen invasion. Prolonged and or persistent infections, resulting in the development of autoimmune processes and bacterial translocation lead to chronic activation of the immune system and subsequent elevated levels of pro-inflammatory cytokines and O&NS leading in turn to O&NS damage to self epitopes and likely activation of the inflammasome which further acts to maintain chronic immune activation and increase the magnitude of inflammation.</p>

<p id="__p37">The flame of chronic immune activation and elevated O&NS can be fanned by TLR engagement in the periphery and the CNS, lowered antioxidant defenses and a deficiency in ω3PUFA levels. The processes underpinning neuro-inflammation involving conspirational interactions between astrocytes, microglia, O&NS, pro-inflammatory cytokines and other inflammatory mediators are also self-sustaining once initiated. The chronic activation of inflammatory pathways also makes a major contribution to disease persistence. It is also worthy of note that a pathogen invasion of sufficient intensity can leave microglia, and hence astrocytes, chronically active even once the initial invasion has been cleared. The interplay between all of these factors leads to the formation of self-sustaining, self-amplifying auto inflammatory feed forward loops leading to a state of elevated oxidative stress and chronic immune activation which sustains the disease.</p>

<p id="__p38" class="p p-last">Investigation into pathogens as causative agents need to be mindful of the experiences in Multiple Sclerosis where initial skepticism has now evaporated and the consensus view is that at least two pathogens are responsible for initiating and maintaining the disease in a significant proportion of patients. This consensus has developed following the realization that the blood compartment may reveal no association between viruses and disease causation while examination of other body compartments for the presence of virus reveal strong associations. Hence further research into virus causation in ME/CFS should focus on lymphoid and muscle tissue in the periphery and sampling CSF. The exploration of relationships between polymorphisms and the presence of actively replicating viruses in people with ME/CFS appears to be urgently required. There would also seem to be a great need for the investigation of intracellular signaling networks, such as NF-kappa b, MAPK, JAK-STAT, p53, the role of apoptosis and mitochondrial signaling cascades by means of high throughput methods analyzed by systems biology. Pharmaceuticals and nutraceuticals aimed at reducing O&NS levels are needed in order to treat individuals with ME/CFS. Endotherapia would seem to be a worthy candidate as its actions in ameliorating oxidative stress has provided objectively measureable improvements in patients with MS. Minocycline would be a candidate drug for reducing neuroinflammatory processes because of its nature as an inhibitor of microglia. Dietary supplementation with ω3 PUFAs, zinc and other antioxidants has already demonstrated promise and supplementation with IV glutathione or its precursors such as N-acetyl cysteine should be considered.</p>

<p class="p p-last">From: <a id="null" class="null" href="https://www.nature.com/articles/s41598-018-31270-3">https://www.nature.com/articles/s41598-018-31270-3</a></p>

<p>The linkage between oxidative stress and idiopathic chronic fatigue (ICF) has not been explored in detail. This study thoroughly compared the serum levels of biomarkers for oxidative stress and antioxidants from 103 subjects with ICF (20 men and 83 women) to those of 82 healthy volunteers (27 men and 55 women). Oxidative parameters, which included reactive oxygen species (ROS), malondialdehyde (MDA) and F2-isoprotan, and tumor necrosis factor-alpha (TNF-α) were significantly elevated, while antioxidant parameters, which included total antioxidant activity (TAC), catalase, superoxide dismutase, SOD and GSH activity, were decreased compared to those of healthy subjects (by approximately 1.2- to 2.3-fold, p < 0.05 or 0.01). Our results confirmed that oxidative stress is a key contributor in the pathophysiology of ICF, and firstly explored the features of oxidative stress parameters in ICF subjects compared to a healthy population.</p>

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<h2 id="Sec1" class="js-section-title section-title strong position-relative tighten-line-height background-gray-light pt20 pb6 pl0 pr20 standard-space-below small-space-above mq640-pt10 mq640-pb10 mq640-pl20 mq640-mt0 mq640-ml-20 mq640-mr-20 extend-left">Introduction</h2>

<p>Chronic fatigue is a widespread symptom, with a prevalence of approximately 10% to 15% in the general population worldwide<sup><a id="ref-link-section-d1349e411" title="Wong, W. S. & Fielding, R. Prevalence of chronic fatigue among Chinese adults in Hong Kong: a population-based study. J Affect Disord 127, 248–56 (2010)." href="https://www.nature.com/articles/s41598-018-31270-3#ref-CR1" data-track="click" data-track-action="reference anchor" data-track-label="link" data-test="citation-ref">1</a>,<a id="ref-link-section-d1349e414" title="Watanabe, N., Stewart, R., Jenkins, R., Bhugra, D. K. & Furukawa, T. A. The epidemiology of chronic fatigue, physical illness, and symptoms of common mental disorders: a cross-sectional survey from the second British National Survey of Psychiatric Morbidity. J Psychosom Res 64, 357–62 (2008)." href="https://www.nature.com/articles/s41598-018-31270-3#ref-CR2" data-track="click" data-track-action="reference anchor" data-track-label="link" data-test="citation-ref">2</a></sup>. This subjective illness generally resolves with rest or after treating the causative diseases, but uncontrolled chronic fatigue impairs quality of life in the physical, social and occupational well-being domains<sup><a id="ref-link-section-d1349e418" title="Jorgensen, R. Chronic fatigue: an evolutionary concept analysis. J Adv Nurs 63, 199–207 (2008)." href="https://www.nature.com/articles/s41598-018-31270-3#ref-CR3" data-track="click" data-track-action="reference anchor" data-track-label="link" data-test="citation-ref">3</a></sup>. In particular, medically unexplained chronic fatigue, such as chronic fatigue syndrome (CFS) and idiopathic chronic fatigue (ICF), are debilitating illnesses<sup><a id="ref-link-section-d1349e422" title="Morris, G., Anderson, G., Galecki, P., Berk, M. & Maes, M. A. Narrative review on the similarities and dissimilarities between myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS) and sickness behavior. BMC Med 11, 64 (2013)." href="https://www.nature.com/articles/s41598-018-31270-3#ref-CR4" data-track="click" data-track-action="reference anchor" data-track-label="link" data-test="citation-ref">4</a></sup>.</p>

<p>The etiologies of CFS and ICF are poorly understood, and currently there is a lack of effective therapeutic treatments for these conditions<sup><a id="ref-link-section-d1349e429" title="Norheim, K. B., Jonsson, G. & Omdal, R. Biological mechanisms of chronic fatigue. Rheumatology (Oxford). 50(6), 1009–18 (2011)." href="https://www.nature.com/articles/s41598-018-31270-3#ref-CR5" data-track="click" data-track-action="reference anchor" data-track-label="link" data-test="citation-ref">5</a></sup>. Accumulated data suggest a link between oxidative stress and chronic fatigue, especially in CFS<sup><a id="ref-link-section-d1349e433" title="Maes, M. Inflammatory and oxidative and nitrosative stress pathways underpinning chronic fatigue, somatization and psychosomatic symptoms. Curr Opin Psychiatry. 22(1), 75–83 (2009)." href="https://www.nature.com/articles/s41598-018-31270-3#ref-CR6" data-track="click" data-track-action="reference anchor" data-track-label="link" data-test="citation-ref">6</a></sup>. In one study, evidence indicated increased levels of two oxidative stress biomarkers, plasma peroxides and serum oxidized LDL antibodies, in CFS patients<sup><a id="ref-link-section-d1349e437" title="Maes, M., Kubera, M., Uytterhoeven, M., Vrydags, N. & Bosmans, E. Increased plasma peroxides as a marker of oxidative stress in myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS). Med SciMonit. 17(4), SC11–5 (2011)." href="https://www.nature.com/articles/s41598-018-31270-3#ref-CR7" data-track="click" data-track-action="reference anchor" data-track-label="link" data-test="citation-ref">7</a></sup>. Other studies reported that CFS symptoms were correlated with the blood levels of oxidative stress biomarkers, such as malondialdehyde and isoprostane<sup><a id="ref-link-section-d1349e441" title="Richards, R. S., Roberts, T. K., McGregor, N. R., Dunstan, R. H. & Butt, H. L. Blood parameters indicative of oxidative stress are associated with symptom expression in chronic fatigue syndrome. Redox Rep. 5(1), 35–41 (2000)." href="https://www.nature.com/articles/s41598-018-31270-3#ref-CR8" data-track="click" data-track-action="reference anchor" data-track-label="link" data-test="citation-ref">8</a>,<a id="ref-link-section-d1349e444" title="Kennedy, G. et al. Oxidative stress levels are raised in chronic fatigue syndrome and are associated with clinical symptoms. Free RadicBiol Med. 39(5), 584–9 (2005)." href="https://www.nature.com/articles/s41598-018-31270-3#ref-CR9" data-track="click" data-track-action="reference anchor" data-track-label="link" data-test="citation-ref">9</a></sup>.</p>

<p>ICF is defined as clinically sustained fatigue with no known underlying pathologies, and it does not meet the criteria of CFS<sup><a id="ref-link-section-d1349e451" title="Fukuda, K. et al. The chronic fatigue syndrome: a comprehensive approach to its definition and study. AnnIntern Med 121, 953–959 (1994)." href="https://www.nature.com/articles/s41598-018-31270-3#ref-CR10" data-track="click" data-track-action="reference anchor" data-track-label="link" data-test="citation-ref">10</a></sup>. ICF generally has a prevalence that is 10 times higher than that of CFS in the general population<sup><a id="ref-link-section-d1349e455" title="Jason, L. A., Porter, N., Herrington, J., Sorenson, M. & Kubow, S. Kindling and Oxidative Stress as Contributors to Myalgic Encephalomyelitis/Chronic Fatigue Syndrome. J Behav Neurosci Res. 7(2), 1–17 (2009)." href="https://www.nature.com/articles/s41598-018-31270-3#ref-CR11" data-track="click" data-track-action="reference anchor" data-track-label="link" data-test="citation-ref">11</a>,<a id="ref-link-section-d1349e458" title="Wessely, S., Chalder, T., Hirsch, S., Wallace, P. & Wright, D. The prevalence and morbidity of chronic fatigue and chronic fatigue syndrome: a prospective primary care study. Am J Public Health. 87(9), 1449–55 (1997)." href="https://www.nature.com/articles/s41598-018-31270-3#ref-CR12" data-track="click" data-track-action="reference anchor" data-track-label="link" data-test="citation-ref">12</a></sup>. One study suggested that ICF was linked to an alteration of the mitochondrial content rather than to oxidative damage in skeletal muscle of older adults<sup><a id="ref-link-section-d1349e462" title="Wawrzyniak, N. R. et al. Idiopathic chronic fatigue in older adults is linked to impaired mitochondrial content and biogenesis signaling in skeletal muscle. Oncotarget. 7(33), 52695–52709 (2016)." href="https://www.nature.com/articles/s41598-018-31270-3#ref-CR13" data-track="click" data-track-action="reference anchor" data-track-label="link" data-test="citation-ref">13</a></sup>. However, no comparative study analyzing the blood status of oxidative stress and symptom expression in ICF subjects and healthy controls has been performed to date.</p>

<p>In the present study, we aimed to explore the features of oxidative stress parameters in subjects with ICF compared to a healthy population.</p>

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<h3 class="h3 strong mb4" data-test="sub-heading">Subjects’ characteristics</h3>

<p>The subjects’ characteristics are summarized in Table <a href="https://www.nature.com/articles/s41598-018-31270-3#Tab1" data-track="click" data-track-label="link" data-track-action="table anchor">1</a>. The median age of 82 healthy subjects (27 men and 55 women) and 103 ICF subjects (20 men and 83 women) was 44 years (range 19 ~ 64) and 40 years (range 23₆₁₎, respectively. The median height and body weight were 162 cm and 61.0 kg for healthy subjects and 162 cm and 58.3 kg for ICF subjects. The median BMIs were 23.3 for healthy subjects and 22.8 for ICF subjects, and no statistical differences existed between the healthy and ICF group for any of these items.</p>

<figcaption><strong id="Tab1" class="block small-space-below icon icon-table tighten-line-height" data-test="table-caption">Table 1 Characteristics of subjects.</strong></figcaption>

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<h3 class="h3 strong mb4" data-test="sub-heading">Comparison of the NRS and VAS scores</h3>

<p>The NRS scores were approximately 3-fold higher in ICF subjects compared to healthy subjects; 61.1 ± 11.7 <em>vs</em>. 19.7 ± 9.6 for the total score, 40.6 ± 8.2 <em>vs</em>. 12.8 ± 6.8 for the physical score and 20.6 ± 6.3 <em>vs</em>. 7.1 ± 4.6 for the mental score, respectively. The VAS score of ICF subjects was higher by approximately 2.7-fold than that of healthy subjects. For all scores, the differences between ICF subjects and healthy controls were statistically significant (p < 0.01), and male and female subgroups showed an extremely similar pattern (Table <a href="https://www.nature.com/articles/s41598-018-31270-3#Tab2" data-track="click" data-track-label="link" data-track-action="table anchor">2</a>).</p>

<figcaption><strong id="Tab2" class="block small-space-below icon icon-table tighten-line-height" data-test="table-caption">Table 2 Comparison of fatigue severity in two groups.</strong></figcaption>

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<h3 class="h3 strong mb4" data-test="sub-heading">Comparison of the serum ROS, MDA and 8-iso-PGF2α levels</h3>

<p>The serum level of the total ROS in ICF subjects was significantly higher, at 184.2 ± 22.8 units, compared to 135.3 ± 35.3 units in the healthy controls (p < 0.01). The serum levels of MDA and 8-iso-PGF2α were also significantly higher in ICF subjects compared to healthy controls, at 9.0 ± 6.0 μM <em>vs</em>. 3.9 ± 2.7 μM (p < 0.01) and 230.2 ± 117.5 pg/mL <em>vs</em>. 276.5 ± 127.9 pg/mL (p < 0.05), respectively. These differences were significant in the subgroups of males (with the exception of 8-iso-PGF2α, p > 0.05) and females (Fig. <a href="https://www.nature.com/articles/s41598-018-31270-3#Fig1" data-track="click" data-track-label="link" data-track-action="figure anchor">1</a>).</p>

<figcaption><strong id="Fig1" class="block tiny-space-below" data-test="figure-caption-text">Figure 1</strong></figcaption>

<div class="inline-block max-width"><a href="https://spindoctor-staging.dev.cf.private.springer.com/m685/springer-static/image/art%3A10.1038%2Fs41598-018-31270-3/MediaObjects/41598_2018_31270_Fig1_HTML.png">https://spindoctor-staging.dev.cf.private.springer.com/m685/springer-static/image/art%3A10.1038%2Fs41598-018-31270-3/MediaObjects/41598_2018_31270_Fig1_HTML.png</a></div>

<p>Serum parameters of oxidative stress. Three blood parameters of oxidative stress were compared between 82 healthy controls and 103 ICF subjects, regarding ROS <strong>(a)</strong>, MDA <strong>(b)</strong>, 8-iso-PGF2α <strong>(c)</strong>, respectively. The correlation between NRS score and TAC <strong>(d)</strong> was conducted. Data are expressed as the mean ± SD. <em>p</em> < 0.05 and *<em>p</em> < 0.01 compared with the healthy control group.</p>

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<h3 class="h3 strong mb4" data-test="sub-heading">Comparison of the serum TAC level and activity of catalase and SOD</h3>

<p>The serum TAC level, expressed as GEAC, and the activities of catalase and SOD were significantly lower in ICF subjects compared to healthy controls: 232.8 ± 46.6 μM <em>vs</em>. 312.8 ± 55.6 μM (p < 0.01), 197.0 ± 72.6 units <em>vs</em>. 337.4 ± 193.8 units (p < 0.01) and 1.6 ± 0.7 units <em>vs</em>. 2.4 ± 0.9 units (p < 0.01), respectively. These differences were also significant in subgroup analysis for males and females (Table <a href="https://www.nature.com/articles/s41598-018-31270-3#Tab3" data-track="click" data-track-label="link" data-track-action="table anchor">3</a>).</p>

<figcaption><strong id="Tab3" class="block small-space-below icon icon-table tighten-line-height" data-test="table-caption">Table 3 Comparison of the serum parameters for oxidative stress and antioxidants.</strong></figcaption>

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<h3 class="h3 strong mb4" data-test="sub-heading">Comparison of the serum total GSH content, GSH/GSSG ratio, and activities of GSH-Px and GSH-Rd</h3>

<p>The serum GSH concentration, GSH/GSSG ratio, and activities of GSH-Px and GSH-Rd were significantly lower in ICF subjects compared to healthy controls: 28.8 ± 37.1 μM <em>vs</em>. 54.6 ± 48.5 μM (p < 0.01), 11.1 ± 2.5 <em>vs</em>. 14.6 ± 4.2 (p < 0.01), 0.36 ± 0.14 units <em>vs</em>. 0.81 ± 0.86 units (p < 0.05) and 0.23 ± 0.10 units <em>vs</em>. 0.52 ± 0.12 units (p < 0.01), respectively. In the subgroup analysis by sex, the GSH content (in females), GSH-Px (in males) and GSH/GSSG ratio & GSH-Rd (in both genders) were significant (Table <a href="https://www.nature.com/articles/s41598-018-31270-3#Tab3" data-track="click" data-track-label="link" data-track-action="table anchor">3</a>).</p>

<h3 class="h3 strong mb4" data-test="sub-heading">Comparison of the serum concentration of the HO-1 protein</h3>

<p>The serum protein concentration of HO-1 was 2.0 ± 0.9 ng/mL and 1.8 ± 0.8 ng/mL in ICF subjects and healthy controls, respectively, which were not different statistically between the two groups (p > 0.05; Table <a href="https://www.nature.com/articles/s41598-018-31270-3#Tab3" data-track="click" data-track-label="link" data-track-action="table anchor">3</a>).</p>

<h3 class="h3 strong mb4" data-test="sub-heading">Comparison of the serum concentration of TNF-α and IFN-γ</h3>

<p>The serum concentration of TNF-α was significantly higher in ICF subjects compared to healthy controls, at 57.2 ± 42.5 pg/mL and 23.9 ± 16.4 pg/mL, respectively (p < 0.01). The sub-group comparisons were very similar for both males and females (p < 0.01). The serum IFN-γ concentration in the two groups was not significantly different (p > 0.05), at 25.3 ± 39.3 pg/mL <em>vs</em>. 30.3 ± 28.0 pg/mL, respectively (Table <a href="https://www.nature.com/articles/s41598-018-31270-3#Tab3" data-track="click" data-track-label="link" data-track-action="table anchor">3</a>).</p>

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<h2 id="Sec10" class="js-section-title section-title strong position-relative tighten-line-height background-gray-light pt20 pb6 pl0 pr20 standard-space-below small-space-above mq640-pt10 mq640-pb10 mq640-pl20 mq640-mt0 mq640-ml-20 mq640-mr-20 extend-left">Discussion</h2>

<p>Oxidative stress, a status of excessive ROS generation exceeding the capacity of antioxidant defense, is known to be involved in a wide range of pathophysiological processes, such as inflammation, vascular disorder, cancer, aging and chronic fatigue<sup><a id="ref-link-section-d1349e1547" title="Visconti, R. & Grieco, D. New insights on oxidative stress in cancer. Curr Opin Drug Discov Devel. 12(2), 240–5 (2009)." href="https://www.nature.com/articles/s41598-018-31270-3#ref-CR14" data-track="click" data-track-action="reference anchor" data-track-label="link" data-test="citation-ref">14</a></sup>. To gain the information necessary to establish the contribution of oxidative stress to ICF, we analyzed serum parameters between ICF subjects and healthy controls. Our present data clearly provided evidence of significant alterations of oxidative parameters as well as antioxidant parameters. The serum level of ROS, a typical oxidative stressor, was 1.4-fold higher in ICF subjects than healthy controls and was correlated with the serum levels of two typical oxidized lipid products, MDA (2.3-fold) and F2-isoprotane (1.2-fold) (Fig. <a href="https://www.nature.com/articles/s41598-018-31270-3#Fig1" data-track="click" data-track-label="link" data-track-action="figure anchor">1a–b</a>). An elevated serum level of MDA was observed in subjects with CFS<sup><a id="ref-link-section-d1349e1554" title="Brkic, S., Tomic, S., Maric, D., Novakov Mikic, A. & Turkulov, V. Lipid peroxidation is elevated in female patients with chronic fatigue syndrome. Med Sci Monit. 16(12), CR628–32 (2010)." href="https://www.nature.com/articles/s41598-018-31270-3#ref-CR15" data-track="click" data-track-action="reference anchor" data-track-label="link" data-test="citation-ref">15</a></sup>, while the F2-isoprotane level was also correlated with the presence of CFS and severity of CFS symptoms<sup><a id="ref-link-section-d1349e1558" title="Kennedy, G. et al. Oxidative stress levels are raised in chronic fatigue syndrome and are associated with clinical symptoms. Free RadicBiol Med. 39(5), 584–9 (2005)." href="https://www.nature.com/articles/s41598-018-31270-3#ref-CR9" data-track="click" data-track-action="reference anchor" data-track-label="link" data-test="citation-ref">9</a></sup>.</p>

<p>All aerobic organisms that derive energy from the reduction of oxygen are forced to produce ROS, so they have evolved highly efficient and adaptive antioxidant defense mechanisms: enzyme-based antioxidants and non-enzyme derived antioxidant molecules<sup><a id="ref-link-section-d1349e1565" title="Halliwell, B. Biochemistry of oxidative stress. Biochem Soc Trans. 35(Pt 5), 1147–50 (2007)." href="https://www.nature.com/articles/s41598-018-31270-3#ref-CR16" data-track="click" data-track-action="reference anchor" data-track-label="link" data-test="citation-ref">16</a></sup>. As was our expectation, most of the antioxidant parameters, including the TAC, catalase, SOD and GSH-related biomarkers, were significantly different in the healthy control group (Table <a href="https://www.nature.com/articles/s41598-018-31270-3#Tab3" data-track="click" data-track-label="link" data-track-action="table anchor">3</a>). In contrast with our results, one study showed no significant differences between non-fatigue and ICF groups in the 4HNE adducts, catalase and GSH-peroxidase activity<sup><a id="ref-link-section-d1349e1572" title="Wawrzyniak, N. R. et al. Idiopathic chronic fatigue in older adults is linked to impaired mitochondrial content and biogenesis signaling in skeletal muscle. Oncotarget. 7(33), 52695–52709 (2016)." href="https://www.nature.com/articles/s41598-018-31270-3#ref-CR13" data-track="click" data-track-action="reference anchor" data-track-label="link" data-test="citation-ref">13</a></sup>, however, their investigation was conducted in skeletal muscle, but not serum.</p>

<p>Regarding the measurement of the serum HO-1 change, HO-1 was significantly higher in only male ICF subjects (not total subjects) compared to the control group. Unlike “classic” antioxidant enzymes (e.g., catalase, SOD and glutathione peroxidase), HO-1 is an oxidative stress-response protein that has a variety of roles, and a high concentration of blood HO-1 is generally a risk factor in many pathogenesis<sup><a id="ref-link-section-d1349e1579" title="Bao, W. et al. Plasma heme oxygenase-1 concentration is elevated in individuals with type 2 diabetes mellitus. PLoS One. 5(8), e12371 (2010)." href="https://www.nature.com/articles/s41598-018-31270-3#ref-CR17" data-track="click" data-track-action="reference anchor" data-track-label="link" data-test="citation-ref">17</a>,<a id="ref-link-section-d1349e1582" title="Li, X. et al. Higher level of heme oxygenase-1 in patients with stroke than TIA. J Thorac Dis. 6(6), 772–7 (2014)." href="https://www.nature.com/articles/s41598-018-31270-3#ref-CR18" data-track="click" data-track-action="reference anchor" data-track-label="link" data-test="citation-ref">18</a></sup>. Above results are in accordance with the outcomes of antifatigue-targeted clinical studies. One clinical study reported that <em>ginseng</em>, a typical medicinal herb used for chronic fatigue, reduced the serum levels of ROS and MDA but increased the GSH contents and led to symptom improvement in ICF subjects<sup><a id="ref-link-section-d1349e1589" title="Kim, H. G. et al. Antifatigue effects of Panax ginseng C.A. Meyer: a randomised, double-blind, placebo-controlled trial. PLoS One. 8(4), e61271 (2013)." href="https://www.nature.com/articles/s41598-018-31270-3#ref-CR19" data-track="click" data-track-action="reference anchor" data-track-label="link" data-test="citation-ref">19</a></sup>; additionally, <em>ginsengdecreased</em> the serum F2-isoprotane level in healthy volunteers<sup><a id="ref-link-section-d1349e1596" title="Lee, L. S. et al. Possible differential induction of phase 2 enzyme and antioxidant pathways by american ginseng, Panax quinquefolius. J Clin Pharmacol. 48(5), 599–609 (2008)." href="https://www.nature.com/articles/s41598-018-31270-3#ref-CR20" data-track="click" data-track-action="reference anchor" data-track-label="link" data-test="citation-ref">20</a></sup>.</p>

<p>Our data indicated that oxidative stress plays a role as an important factor in ICF. Alterations of redox system parameters were well evidenced by the comparison between healthy subjects and patients with CFS in other study<sup><a id="ref-link-section-d1349e1603" title="Fukuda, S. et al. A potential biomarker for fatigue: Oxidative stress and anti-oxidative activity. Biol Psychol. 118, 88–93 (2016)." href="https://www.nature.com/articles/s41598-018-31270-3#ref-CR21" data-track="click" data-track-action="reference anchor" data-track-label="link" data-test="citation-ref">21</a></sup> and by the results from the fatigue-induced clinical model<sup><a id="ref-link-section-d1349e1607" title="Bouzid, M. A., Hammouda, O., Matran, R., Robin, S. & Fabre, C. Changes in oxidative stress markers and biological markers of muscle injury with aging at rest and in response to an exhaustive exercise. PLoS One. 9(3), e90420 (2014)." href="https://www.nature.com/articles/s41598-018-31270-3#ref-CR22" data-track="click" data-track-action="reference anchor" data-track-label="link" data-test="citation-ref">22</a></sup>. Thus, the linkage of redox imbalance with ICF is expected, but until now, no study has described its specific features. When we analyzed the fatigue severity and TAC levels, a representative parameter of antioxidant capacity, of total 185 individuals, they showed the inverse correlation as <em>R</em>-squared 0.3477 (p < 0.001; Fig. <a href="https://www.nature.com/articles/s41598-018-31270-3#Fig1" data-track="click" data-track-label="link" data-track-action="figure anchor">1d</a>).</p>

<p>Our study protocol distinguishes ICF patients because it excluded patients with CFS and any subjects complaining of chronic fatigue with medically explained disorders, including any abnormality in physical examinations or severe obesity (BMI > 30) due to the positive correlation between obesity and oxidative stress<sup><a id="ref-link-section-d1349e1621" title="Kelli, H. M. et al. Relation of Changes in Body Fat Distribution to Oxidative Stress. Am J Cardiol. 120(12), 2289–2293 (2017)." href="https://www.nature.com/articles/s41598-018-31270-3#ref-CR23" data-track="click" data-track-action="reference anchor" data-track-label="link" data-test="citation-ref">23</a></sup>. No difference in the physical characteristics of subjects between the ICF and healthy groups existed (Table <a href="https://www.nature.com/articles/s41598-018-31270-3#Tab1" data-track="click" data-track-label="link" data-track-action="table anchor">1</a>). When we analyzed the status of the redox imbalance between males and females, females had more altered parameters than male subjects, except for GSH-Px and HO-1; this difference was also observed in the fatigue score (Table <a href="https://www.nature.com/articles/s41598-018-31270-3#Tab2" data-track="click" data-track-label="link" data-track-action="table anchor">2</a>). Intriguingly alterations between healthy and ICF subjects were different in according to gender, especially results in HO-1, iso-PGF2α and GSH contents (Table <a href="https://www.nature.com/articles/s41598-018-31270-3#Tab3" data-track="click" data-track-label="link" data-track-action="table anchor">3</a>). These phenomenons are frequently shown as reported by the previous pathophysiological studies involving antioxidant activity<sup><a id="ref-link-section-d1349e1634" title="Liu, H., Harrell, L. E., Shenvi, S., Hagen, T. & Liu, R. M. Gender differences in glutathione metabolism in Alzheimer’s disease. J Neurosci Res. 79(6), 861–7 (2005)." href="https://www.nature.com/articles/s41598-018-31270-3#ref-CR24" data-track="click" data-track-action="reference anchor" data-track-label="link" data-test="citation-ref">24</a>,<a id="ref-link-section-d1349e1637" title="Chilelli, N. C., Cosma, C., Burlina, S., Plebani, M. & Lapolla, A. Antioxidant capacity in patients with type 2 diabetes: a preliminary investigation on gender-specific differences in an Italian population. Clin Chem Lab Med. 56(4), 101–104 (2018)." href="https://www.nature.com/articles/s41598-018-31270-3#ref-CR25" data-track="click" data-track-action="reference anchor" data-track-label="link" data-test="citation-ref">25</a></sup>. Chronic fatigue, including CFS, is known to be predominant in women, who also display a greater degree of fatigue than men<sup><a id="ref-link-section-d1349e1642" title="Faro, M. et al. Gender differences in chronic fatigue syndrome. Reumatol Clin. 12(2), 72–7 (2016)." href="https://www.nature.com/articles/s41598-018-31270-3#ref-CR26" data-track="click" data-track-action="reference anchor" data-track-label="link" data-test="citation-ref">26</a></sup>.</p>

<p>Immune shifting is also mutually linked to oxidative stress, inflammation and fatigue with or without a medically explained cause<sup><a id="ref-link-section-d1349e1649" title="Nagai, M. et al. Effects of fatigue on immune function in nurses performing shift work. J Occup Health. 53(5), 312–9 (2011)." href="https://www.nature.com/articles/s41598-018-31270-3#ref-CR27" data-track="click" data-track-action="reference anchor" data-track-label="link" data-test="citation-ref">27</a>,<a id="ref-link-section-d1349e1649_1" title="Russell, L. et al. Illness progression in chronic fatigue syndrome: a shifting immune baseline. BMC Immunol. 17, 3 (2016)." href="https://www.nature.com/articles/s41598-018-31270-3#ref-CR28" data-track="click" data-track-action="reference anchor" data-track-label="link" data-test="citation-ref">28</a>,<a id="ref-link-section-d1349e1652" title="Cristani, M. et al. Circulating Advanced Oxidation Protein Products as Oxidative Stress Biomarkers and Progression Mediators in Pathological Conditions Related to Inflammation and Immune Dysregulation. Curr Med Chem. 23(34), 3862–3882 (2016)." href="https://www.nature.com/articles/s41598-018-31270-3#ref-CR29" data-track="click" data-track-action="reference anchor" data-track-label="link" data-test="citation-ref">29</a></sup>. It is well-known that oxidative & nitrosative stress is chronically activated in myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS) patients and involved in the immune-inflammatory pathway<sup><a id="ref-link-section-d1349e1656" title="Morris, G. & Maes, M. Oxidative and Nitrosative Stress and Immune-Inflammatory Pathways in Patients with Myalgic Encephalomyelitis (ME)/Chronic Fatigue Syndrome (CFS). Curr Neuropharmacol. 12(2), 168–85 (2014)." href="https://www.nature.com/articles/s41598-018-31270-3#ref-CR30" data-track="click" data-track-action="reference anchor" data-track-label="link" data-test="citation-ref">30</a></sup>.</p>

<p>We examined the changes of the serum levels of TNF-α, a proinflammatory cytokine, and IFN-γ, a key cytokine for both innate and adaptive immunity. Many studies reported an increased serum level of TNF-α and reduction of IFN-γ and NK cell activity in CFS patients compared to healthy individuals<sup><a id="ref-link-section-d1349e1663" title="Broderick, G. et al. A formal analysis of cytokine networks in chronic fatigue syndrome. Brain Behav Immun. 24(7), 1209–17 (2010)." href="https://www.nature.com/articles/s41598-018-31270-3#ref-CR31" data-track="click" data-track-action="reference anchor" data-track-label="link" data-test="citation-ref">31</a>,<a id="ref-link-section-d1349e1666" title="Brenu, E. W. et al. Role of adaptive and innate immune cells in chronic fatigue syndrome/myalgic encephalomyelitis. Int Immunol. 26(4), 233–42 (2014)." href="https://www.nature.com/articles/s41598-018-31270-3#ref-CR32" data-track="click" data-track-action="reference anchor" data-track-label="link" data-test="citation-ref">32</a></sup>. Our results showed an alteration in only TNF-α, but not in IFN-γ, which may distinguish ICF from CFS. An anti-TNF therapy improved fatigue in rheumatoid arthritis patients<sup><a id="ref-link-section-d1349e1670" title="van Hoogmoed, D. et al. The effect of anti-TNF-α vs. DMARDs on fatigue in rheumatoid arthritis patients. Scand J Rheumatol. 42(1), 15–9 (2013)." href="https://www.nature.com/articles/s41598-018-31270-3#ref-CR33" data-track="click" data-track-action="reference anchor" data-track-label="link" data-test="citation-ref">33</a></sup>. N-acetylcysteine (a well-known antioxidant) supplementation significantly attenuated exercise-induced fatigue and alterations of blood TNF-α and TAC levels<sup><a id="ref-link-section-d1349e1674" title="Leelarungrayub, D., Khansuwan, R., Pothongsunun, P. & Klaphajone, J. N-acetylcysteine supplementation controls total antioxidant capacity, creatine kinase, lactate, and tumor necrotic factor-alpha against oxidative stress induced by graded exercise in sedentary men. Oxid Med Cell Longev. 2011, 329643 (2011)." href="https://www.nature.com/articles/s41598-018-31270-3#ref-CR34" data-track="click" data-track-action="reference anchor" data-track-label="link" data-test="citation-ref">34</a></sup>. Accordingly, TNF-α is, however, not specific for CFS and ICF, which is attributed to the diverse forms of fatigue, such as acute and chronic as well as disease-associated or idiopathic conditions. The current findings have limitations, such as the absence of a long-term follow-up for changes of these parameters and use of a design that cannot distinguish ICF from CFS. To our knowledge, however, this study is the first clinical observation to provide systematic features of blood parameters of the redox system in subjects with ICF compared to healthy individuals.</p>

<p>This study firstly demonstrated that oxidative stress is intensively involved in the pathology of ICF, and explored the features of oxidative stress parameters in ICF subjects compared to a healthy population. These data will help us to further explore the pathophysiology of unexplained chronic fatigue.</p>

</div>

</section>

<h2 id="Sec11" class="js-section-title section-title strong position-relative tighten-line-height background-gray-light pt20 pb6 pl0 pr20 standard-space-below small-space-above mq640-pt10 mq640-pb10 mq640-pl20 mq640-mt0 mq640-ml-20 mq640-mr-20 extend-left">Subjects and Methods</h2>

<h3 class="h3 strong mb4" data-test="sub-heading">Subjects and study design</h3>

<p>This study enrolled two adult groups (aged 20 to 65 years) that were composed of healthy adults with no physical and psychological distress, including fatigue by self-judgment, and subjects who had experienced fatigue for longer than 6 months. A physician and radiologist examined potential candidates at Daejeon University Hospital, Republic of Korea, and thus excluded subjects who had any hematological or radiological test abnormalities. Subjects who worked at night, used alcohol, smoked, took medication or were severely obese or lean (body mass index, BMI >30 or <17) were excluded. Using the Korean version of the Beck Depression Inventory (BDI) and Korean translation of the State-Trait Anxiety Inventory (STAI), subjects with a history of psychological disorders or who were currently experiencing severe depression (BDI score >29) or anxiety (STAI score >70) were excluded<sup><a id="ref-link-section-d1349e1695" title="Jo, S. A., Park, M. H., Jo, I., Ryu, S. H. & Han, C. Usefulness of Beck Depression Inventory (BDI) in the Korean elderly population. Int J Geriatr Psychiatry 22, 218–23 (2007)." href="https://www.nature.com/articles/s41598-018-31270-3#ref-CR35" data-track="click" data-track-action="reference anchor" data-track-label="link" data-test="citation-ref">35</a>,<a id="ref-link-section-d1349e1698" title="Oei, T. P., Evans, L. & Crook, G. M. Utility and validity of the STAI with anxiety disorder patients. Br J ClinPsychol 29(Pt 4), 429–32 (1990)." href="https://www.nature.com/articles/s41598-018-31270-3#ref-CR36" data-track="click" data-track-action="reference anchor" data-track-label="link" data-test="citation-ref">36</a></sup>. For selection of the ICF group, only subjects who had chronic fatigue with at most three of the following 8-symptoms were included:</p>

<p>Post-exertion malaise lasting for more than 24 hours.</p>

</li>

<p>Unrefreshing sleep.</p>

</li>

<p>Significant impairment of short-term memory or concentration.</p>

</li>

<p>Muscle pain.</p>

</li>

<p>Pain in the joints without swelling or redness.</p>

</li>

<p>Headaches of a new pattern.</p>

</li>

<p>Tender lymph nodes in the neck or armpit.</p>

</li>

<p>Sore throat that is frequent or recurring.</p>

</li>

</ol>

<p>Patients who met the criteria for CFS (positive at least four of above 8-symptoms) were excluded<sup><a id="ref-link-section-d1349e1804" title="Fukuda, K. et al. The chronic fatigue syndrome: a comprehensive approach to its definition and study. AnnIntern Med 121, 953–959 (1994)." href="https://www.nature.com/articles/s41598-018-31270-3#ref-CR10" data-track="click" data-track-action="reference anchor" data-track-label="link" data-test="citation-ref">10</a></sup>. Ultimately, 82 healthy subjects (27 men and 55 women) and 103 ICF subjects (20 men and 83 women) were selected.</p>

<p>The study protocol was approved by the institutional ethics committee of Daejeon University Hospital (DJOMC-51), and all methods were performed in accordance with the relevant guidelines and regulations of the institution. The informed consent was obtained from all study participants.</p>

<h3 class="h3 strong mb4" data-test="sub-heading">Assessment of fatigue severity using a numerical rating scale and visual analogue scale</h3>

<p>Fatigue severity was accessed using the same self-rating instrument for the two groups, which included a numerical rating scale (NRS) and visual analogue scale (VAS). The NRS was used with the Korean-translated Chalder fatigue severity questionnaire<sup><a id="ref-link-section-d1349e1820" title="Morriss, R. K., Wearden, A. J. & Mullis, R. Exploring the validity of the Chalder Fatigue scale in chronic fatigue syndrome. J Psychosom Res. 45(5), 411–7 (1998)." href="https://www.nature.com/articles/s41598-018-31270-3#ref-CR37" data-track="click" data-track-action="reference anchor" data-track-label="link" data-test="citation-ref">37</a></sup>. The survey consisted of seven physical health-related questions (number 1 to 7) and four mental health-related questions (number 8 to 11) as follows: (1) How tired do you feel? (2) How strongly do you currently feel the need to rest? (3) How sleepy or drowsy do you feel? (4) Do you have problems starting things? (5) Are you lacking energy? (6) Do you have less strength in your muscles? (7) Do you feel weak? (8) Do you have difficulty concentrating? (9) Do you have difficulty thinking clearly? (10) Do you make slips of the tongue when speaking? (11) How is your memory? All subjects scored each item on a 10-point scale (0 = not at all to 9 = unbearably severe condition). Additionally, patients were asked to indicate their feeling of general fatigue by drawing a vertical line on a 10 cm visual analogue scale (VAS).</p>

<h3 class="h3 strong mb4" data-test="sub-heading">Determination of total reactive oxygen species</h3>

<p>The serum levels of the biomarkers associated with oxidative stress and antioxidants were measured after an 8 hour fast. The total amount of reactive oxygen species (ROS) in serum was determined according to Hayashi’s method<sup><a id="ref-link-section-d1349e1833" title="Hayashi, I. et al. High-throughput spectrophotometric assay of reactive oxygen species in serum. Mutat Res 631, 55–61(2007)." href="https://www.nature.com/articles/s41598-018-31270-3#ref-CR38" data-track="click" data-track-action="reference anchor" data-track-label="link" data-test="citation-ref">38</a></sup>. Briefly, H2O2 was used to generate the calibration curve as the standard. N, N-diethyl-para-phenylenediamine (DEPPD) and ferrous sulfate solutions were prepared beforehand. Five microliters of standard solution or serum was added to 140 μL of 0.1 M sodium acetate buffer (pH 4.8) in 96-well plates and incubated at 37 °C for 5 min. One-hundred microliters of DEPPD and a ferrous mixture solution was added to each well, and the amount of ROS was determined at 505 nm using a UV spectrophotometer (Molecular Devices; Sunnyvale, CA, USA).</p>

<h3 class="h3 strong mb4" data-test="sub-heading">Determination of lipid peroxide as malondialdehyde</h3>

<p>The serum lipid peroxide levels were determined using thiobarbituric acid reactive substances (TBARS) as described by Kamal<sup><a id="ref-link-section-d1349e1852" title="Kamal, A. A., Gomaa, A., el Khafif, M. & Hammad, A. S. Plasma lipid peroxides among workers exposed to silica or asbestos dusts. Environ Res 49, 173–80 (1989)." href="https://www.nature.com/articles/s41598-018-31270-3#ref-CR39" data-track="click" data-track-action="reference anchor" data-track-label="link" data-test="citation-ref">39</a></sup>. The TBARS concentration was expressed as μM malondialdehyde (MDA) in serum. Briefly, 250 μL of serum or standard solution was added to 2.5 mL of 20% trichloroacetic acid (TCA) and then mixed with 1 mL of 0.67% thiobarbituric acid (TBA), followed by heating at 100 °C for 30 min, cooling on ice and vigorously vortexing with 4 mL of <em>n-butanol.</em> After centrifugation at 3000 × g for 20 min, the absorbance of the upper organic layer was measured at 535 nm with a UV spectrophotometer (Molecular Devices) and compared with a 1, 1, 3, 3-tetraethoxypropane (TEP) standard curve.</p>

<h3 class="h3 strong mb4" data-test="sub-heading">Determination of F2‐isoprostane</h3>

<p>To prepare the serum concentration of <em>F2‐isoprostane</em>, 8-Iso-Prostaglandin F2α (8-iso-PGF2α) was measured with a direct 8-iso-PGF2α ELISA kit (Enzo Life Sciences, Inc. Farmingdale, NY 11735) according to the manufacturer’s instructions. The absorbance was read at 405 nm with a UV spectrophotometer (Molecular Devices).</p>

<h3 class="h3 strong mb4" data-test="sub-heading">Determination of the total antioxidant capacity</h3>

<p>The total antioxidant capacity (TAC) was determined according to Kambsyashi<sup><a id="ref-link-section-d1349e1884" title="Kambayashi, Y. et al. Efficient assay for total antioxidant capacity in human plasma using a 96-well microplate. J Clin Biochem Nutr 44, 46–51 (2009)." href="https://www.nature.com/articles/s41598-018-31270-3#ref-CR40" data-track="click" data-track-action="reference anchor" data-track-label="link" data-test="citation-ref">40</a></sup>. Ninety microliters of 10 mM phosphate-buffered saline (PBS; pH 7.2), 50 μL of 18 μM myoglobin solution and 20 μL of a 3 mM 2, 2′-azino-bis (3-ethylbenzthiazoline-6-sulfonic acid) diammonium salt (ABTS) solution were mixed with 20 μL of diluted serum sample or various concentrations of gallic acid in a 96-well microplate at 25 °C for 3 min. Then, 20 μL of H2O2 was added to each well and incubated for 5 min. The absorbance was measured using a plate reader (Molecular Device) at 600 nm. TAC was expressed as the gallic acid equivalent antioxidant capacity (GEAC).</p>

<h3 class="h3 strong mb4" data-test="sub-heading">Determination of the catalase and superoxide dismutase activities</h3>

<p>The serum catalase activity was assayed as described previously<sup><a id="ref-link-section-d1349e1903" title="Wheeler, C. R., Salzman, J. A., Elsayed, N. M., Omaye, S. T. & Korte, D. W. Jr. Automated assays for superoxide dismutase, catalase, glutathione peroxidase, and glutathione reductase activity. Anal Biochem. 184(2), 193–9 (1990)." href="https://www.nature.com/articles/s41598-018-31270-3#ref-CR41" data-track="click" data-track-action="reference anchor" data-track-label="link" data-test="citation-ref">41</a></sup>. Briefly, 150 μL of phosphatase buffer (250 mM, pH 7.0), 150 μL of 12 mM methanol and 30 μL of hydrogen peroxide were mixed with 300 μL of the serum sample or standard solutions in a 13 × 100 mm test tube. The reaction was allowed to proceed for 10 to 20 min and was stopped by the addition of 450 μL of Purpald solution (22.8 mM Purpald in 2 N potassium hydroxide). The mixture was left for 20 min at 25 °C, followed by the addition of 150 μL of potassium periodate (65.2 mM in 0.5 N potassium hydrate). The absorbance of the purple formaldehyde adduct was measured at 550 nm using a spectrophotometer (Molecular Devices)</p>

<p>The serum superoxide dismutase (SOD) activity was determined using an SOD assay kit (Dojindo Laboratories, Kumamoto, Japan) according to the manufacturer’s protocol. Absorbance was measured at 450 nm using UV spectrophotometer (Molecular Devices). Dilutions of bovine erythrocyte SOD ranging from 0.01–50 unit/mL were used as standards.</p>

<h3 class="h3 strong mb4" data-test="sub-heading">Determination of the total glutathione content, reduced & oxidized glutathione ratio, and activities of glutathione peroxidase and glutathione reductase</h3>

<p>The total glutathione (GSH) content was determined as described previously<sup><a id="ref-link-section-d1349e1919" title="Ellman, G. L. Tissue sulfhydryl groups. Arch Biochem Biophys 82, 70–7 (1959)." href="https://www.nature.com/articles/s41598-018-31270-3#ref-CR42" data-track="click" data-track-action="reference anchor" data-track-label="link" data-test="citation-ref">42</a></sup>. Briefly, 50 µL of diluted serum (in PBS 10 mM, pH 7.2) or total GSH standard was combined with 80 µL of a DTNB/NADPH mixture (10 μL of 4 mM DTNB and 70 μL of 0.3 mM NADPH) in a 96-well microplate. Next, 20 μL (0.06 U) of a GSH-reductase (GSH-Rd) solution was added to each well. The serum levels of reduced and oxidized GSH were determined by using the GSH & GSSG assay kit (DoGen Bio Co., Ltd., Seoul, Republic of Korea) according to the manufacturer’s instructions, and GSH/GSSG ratio was calculated based on their concentration. Absorbance was measured using a plate reader at 412 nm (Molecular Devices).</p>

<p>The GSH-peroxidase (GSH-Px) activity was determined according to the method of Paglia<sup><a id="ref-link-section-d1349e1926" title="Paglia, D. E. & Valentine, W. N. Studies on the quantitative and qualitative characterization of erythrocyte glutathione peroxidase. J Lab Clin Med 70, 158–69 (1967)." href="https://www.nature.com/articles/s41598-018-31270-3#ref-CR43" data-track="click" data-track-action="reference anchor" data-track-label="link" data-test="citation-ref">43</a></sup>. Briefly, 50 μL of NADPH reagent (5 mM NADPH, 42 mM GSH and 10 units/mL of GSH-Rd in 1.25 mL of distilled water) was added to 890 μL of GSH-Px buffer (50 mM Tris HCl, pH 8.0, with 0.5 mM EDTA). Then, 50 μL of serum and 10 μL of 30 mM tert-butyl hydroperoxide solution were added to the mixture. The final absorbance was measured at 340 nm using a UV-visible spectrophotometer (Molecular Devices).</p>

<p>GSH-Rd activity was determined according to the method of Worthington with slight modifications<sup><a id="ref-link-section-d1349e1933" title="Worthington, D. J. & Rosemeyer, M. A. Human glutathione reductase: purification of the crystalline enzyme from erythrocytes. Eur J Biochem 48, 167–77 (1974)." href="https://www.nature.com/articles/s41598-018-31270-3#ref-CR44" data-track="click" data-track-action="reference anchor" data-track-label="link" data-test="citation-ref">44</a></sup>. Briefly, 150 μL of GSSG with 30 µL of GSH-Rd assay buffer (100 mM potassium phosphate buffer, pH 7.5, with 1 mM EDTA) was added to 30 µL of the serum sample and diluted with GSH-Rd dilution buffer (100 mM potassium phosphate buffer, pH 7.5, with 1 mM EDTA and 1 mg/mL bovine serum albumin). Then, 75 μL of DTNB and 2 mM NADPH were added, and the absorbance was read at 412 nm.</p>

<h3 class="h3 strong mb4" data-test="sub-heading">Determination of the heme oxygenase 1 activity</h3>

<p>The serum protein concentration of heme oxygenase 1 (HO-1) was measured by a Human Heme Oxygenase 1 ELISA Kit (Abcam, Inc., Cambridge, MA, USA) according to the manufacturer’s instructions. The absorbance was read at 450 nm with a spectrophotometer (Molecular Devices).</p>

<h3 class="h3 strong mb4" data-test="sub-heading">Determination of the serum levels of tumor necrosis factor-alpha (TNF-α) and interferon-gamma (IFN-γ)</h3>

<p>The serum levels of TNF-α and IFN-γ were determined using a commercial ELISA kit (R&D system, CA, USA). The absorbance was read at 450 and 570 nm with a spectrophotometer (Molecular Devices).</p>

<h3 class="h3 strong mb4" data-test="sub-heading">Statistical analysis</h3>

<p>The average of each item for fatigue severity and the biochemical parameters of oxidative stress and antioxidants between ICF patients and healthy subjects were analyzed by Student’s t-test using SPSS (SPSS® 18.0 KO. for Windows; SPSS, Inc., Chicago, IL, USA). A p-value < 0.05 was considered to indicate statistical significance. Regarding for the correlation between fatigue severity (NRS) and antioxidant capacity (TAC), Pearson’s correlation coefficient and Linear regression analysis were performed.</p>

</div>

</section>

<p class="p p-last">From: <a href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3847236/">Myalgic encephalomyelitis/chronic fatigue syndrome and encephalomyelitis disseminata/multiple sclerosis show remarkable levels of similarity in phenomenology and neuroimmune characteristics</a></p>

<p id="__p69" class="p p-first">Increased levels of O+NS occur in both illnesses and patients share an almost identical range of empirically determined abnormalities as evidenced by elevated levels of peroxinitrite, NO and iNOS, lipid peroxidation and nitration of amino acids. The consequences of O+NS damage to self-epitopes is evidenced by the almost bewildering and almost identical array of autoantibodies formed against damaged epitopes seen in both illnesses. Reduced levels of antioxidants, including vitamin E, zinc, glutathione are also found in both diseases. Evidence of chronic immune activation coupled with disordered T cell homeostasis is seen in both diseases. Similar abnormalities exist in levels of proinflammatory cytokines, serum neopterin and T cell antigens. Although reduced NKC function in ME/CFS has been emphasized over many years the identical abnormalities in MS has not received such widespread attention. The findings produced by neuroimaging using PET, SPECT and nuclear MRS are similar in both illnesses and in MS the severity of the abnormality in glucose metabolism correlates well with disease activity while MRI findings do not.</p>

<p id="__p70">There are however also differences in symptomatic and immune profiles between both diagnoses. Thus, patients with ME/CFS seem more sensitive to increases in physical or cognitive activity than patients with MS. ME/CFS patients may have a worse experience with regard to infections, while the number of infections is associated with increasing symptom severity. While MS is characterized by increased expression of CD69, a decreased CD69 expression is seen in ME/CFS. Accelerated glycolysis is reported in ME/CFS but not in MS. Neuronal damage to the respiratory chain has been found in MS but not in ME/CFS. T cell exhaustion seems to be more of an issue in ME/CFS than in MS. When taken together the range of surrogate markers for O+NS and the range of autoantibodies are wider in ME/CFS than in MS and this may be due to an increased severity of O+NS in ME/CFS. While coenzyme Q10 is related to fatigue in ME/CFS, the findings in MS are less evident. Finally, while both ME/CFS and MS are chronic immunoinflammatory diseases, inflammation of the central nervous system is clearly more prominent in MS than in ME/CFS.</p>

<p id="__p71">Nevertheless, the strong similarities between both disorders in terms of phenomenological, neurobehavior and neuroimmune characteristics further underscore that ME/CFS belongs to the spectrum of neuroimmune disorders. In addition, the data show that the comorbidity between both disorders and the high prevalence of ME/CFS symptoms in patients with MS may be explained by neuroimmune mechanisms.</p>

<p>From: <a id="null" class="null" href="https://www.fasebj.org/doi/abs/10.1096/fasebj.26.1_supplement.678.10">https://www.fasebj.org/doi/abs/10.1096/fasebj.26.1_supplement.678.10</a></p>

<p>Local cutaneous heating causes vasodilation as an initial peak, a nadir, and increase to plateau. We showed reactive oxygen species (ROS) produced by Ang-II via NADPH and xanthine oxidase, modulates this response in healthy controls. The initial peak, due to C-fibre nociceptor-mediated axon reflex and local release of calcitonin gene-related peptide, substance P and neuropeptide Y, is blunted with local anesthetics. To determine the role of ROS in this neurovascular response, we studied orthostatically-challenged, Chronic Fatigue Syndrome (CFS) subjects. We heated the skin of 9 CFS subjects (15.8–22.0 years) to 42°C and measured local blood flow. While CFS subjects had lower baseline blood flow (79% of control), there were no differences between groups to local heat. We then measured CFS subjects while perfusing apocynin (NADPH oxidase inhibitor), allopurinol (xanthine oxidase inhibitor), tempol (superoxide inhibitor), and ebselen to reduce H2O2 via microdialysis catheters. Allopurinol and ebselen only enhanced the first heat peak (117% and 126% of heat-alone, respectively). Heat with apocynin caused an increase in baseline (170% of non-drug) and of the first heat peak (114% of heat-alone). Tempol had no effect. Thus the response to heat, as indicated by the first peak, may be altered by local levels of ROS in CFS subjects, and may be related to hyperesthesia/hyperalgesia reported by many individuals with CFS. Supported by the National Heart, Lung, and Blood Institute Grants 1-RO1-HL-074873, 1-RO1-HL-087803 and 1-F30-HL-097380 and a grant from the Chronic Fatigue and Immune Dysfunction Syndrome Association.</p>