An investigation of oxidant/antioxidant balance in patients with migraine: a case-control study

Migraine has been identified as the leading cause of disability in people under 50 years of age and affects approximately 11% of the adult population of the world [1, 2]. Migraine is categorized into chronic migraine ((CM) defined as experiencing more than 15 days of headache per month with at least 8 days with migraine characteristics or response to Triptans, for at least 3 months) and episodic migraine ((EM) defined as having less than 15 days of migraine headache per month) [3, 4]. If EM patients do not receive proper treatment, their headaches can progress to CM over time [5]. In comparison with EM sufferers, subjects with CM experience greater headache-related disability [6].

Although much effort has been made to elucidate migraine headache pathogenesis, its exact underlying mechanism remains to be understood. To date, different mechanisms have been proposed, including vascular dysfunction, neurogenic inflammation, and activation of the trigeminovascular pathway [7].

Nociceptive information is conducted from the meninges to the brain’s central region and the cortex through the trigeminovascular pathway. In the other direction, trigeminal ganglion cells are responsible for pain sensation through nociceptive receptors in meningeal vasculatures [7]. Headache attacks initiate with stimulation of nociceptive neurons. During this process neuropeptides with vasoactive properties (e.g. calcitonin gene-related peptide (CGRP) and pituitary adenylate cyclase-activating peptide (PACAP)) are released and could result in vasodilatation of arteries, degranulation of mast cells and plasma leakage [7].

The disturbed balance between antioxidant and prooxidant indicators is established as oxidative stress. According to the literature, the important effect of oxidative stress in increasing the risk of a variety of chronic disorders such as cancer, diabetes, hepatic disorders, cardiovascular disease (CVD), and neurodegenerative conditions is well-known [8]. This complicated condition may be a consequence of the disturbances in cellular biochemical pathways that result in increased levels of reactive oxygen and nitrogen species (ROS and RNS), greater vulnerability to oxidative compounds in the environment and reduced antioxidative defense [8]. During oxidative stress, several parameters are involved such as disturbed metabolism of cellular energy phosphate, production of pro-inflammatory mediators and nitric oxide (NO) metabolites, and the presence of high levels of polyunsaturated fatty acids in cell membranes that could predispose the cell to a heightened risk of lipid peroxidation [9]. The antioxidative scavenging system, which is involved in protecting against production of oxidants (e.g. ROS and RNS), consists of enzymatic agents (including superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx)) and non-enzymatic agents (including glutathione and a number of dietary antioxidants such as vitamin A, C and E) [8]. On the other hand, augmented production of ROS and RNS could damage the intracellular molecules [8]. Oxidation of these molecules, including the proteins and DNA, could lead to dysfunction in cellular structures and organelles and subsequent disruption in receptor function and dysregulation in signaling pathways and the transport system. Emerging animal studies point to the contribution of these events in headache genesis [10, 11]. In this context, measuring levels of oxidative stress indicators can be a noteworthy tool for exploring the relation of oxidant/antioxidant status and pathophysiologic mechanisms of disease development and/or progression [8].

In recent years, the role of neuroinflammation and oxidative stress in migraine pathogenesis has received considerable attention; however, to date findings are equivocal [10, 12‐19]. It has been reported that compared to healthy subjects, migraineurs may have higher levels of NO metabolites and malondialdehyde (MDA) that may be produced following an increase in levels of ROS [8, 20]. Also, according to the findings of a meta-analysis of 19 studies on the association between oxidative/nitrosative pathways in migraine, there may be higher levels of oxidative stress indicators and conversely, lower activity of the antioxidant enzyme, SOD, in migraine patients compared to controls [21]. Similar findings have been shown in a number of studies [15, 22‐28]. However, there are some studies which have failed to find any differences in the MDA, GPx, SOD and CAT levels of migraine suffers compared to control subjects [9, 18, 25]. Hence, there seems to be a highlighted need to design a study in which the levels of these biomarkers are assessed simultaneously.

Thus, it could be hypothesized that oxidative and nitrosative stress—measured both by serum levels of MDA [8], the most studied biomarker of lipid oxidation end product, and NO [8, 29‐31], a well-known oxidant/vasodilator with a pivotal role in migraine pathogenesis—may be present to a greater extent in migraine patients compared with the healthy population while, at the same time, lower antioxidant defense assessed by total non-enzymatic antioxidant capacity and the most-known antioxidant enzymes (CAT, SOD, GPx-1) [8] might also be detected in this population. It could also be assumed that along with an increase in the number of headache days per month, levels of oxidative stress biomarkers may increase whereas the total antioxidant capacity and antioxidant enzymes might decrease. Therefore, we aimed to investigate the serum status of MDA and NO, on the one hand, and total antioxidant capacity, CAT, SOD, and GPx-1, on the other hand, in populations of EM and CM patients compared to controls. Moreover, this research aimed to explore the correlation between the mentioned biomarkers and the number of headache days per month.

In the current study on populations of EM and CM patients and healthy controls, serum levels of MDA (a lipid oxidation end product) and NO (an oxidant vasodilator agent) as biomarkers of oxidative stress were investigated. Further, in order to evaluate antioxidant defense status, three well-known antioxidant enzymes were explored including SOD, catalyzing superoxide (O2−) radical dismutation into O₂ or H₂O₂; CAT, catalyzing H₂O₂ conversion to H₂O and O₂; and GPx, catalyzing H₂O₂ reduction to H₂O and alcohols via conversion of glutathione oxidation to oxidized glutathione (GSSG) [8]. Total non-enzymatic antioxidant capacity reflecting serum content of non-enzymatic agents with antioxidant features along with dietary antioxidants (such as carotenoids, ascorbic acid, tocopherols, and phenolics agents) [8]. was also determined through TEAC assay. Collectively, the results revealed an imbalance in oxidant/antioxidant status in favor of a higher level of oxidative and nitrosative stress factors in migraine patients than healthy controls. It was shown that, except for GPx-1, serum concentrations of antioxidant enzymes were significantly lower in CM and EM patients than in controls. In addition, it was indicated that chronic migraineurs had lower total non-enzymatic antioxidant capacity than EM and control individuals. While elevated serum levels of NO and MDA were detected among chronic and episodic migraineurs compared to controls.

In line with our results, higher platelet levels of NO metabolites and MDA were detected among migraineurs than in healthy subjects especially during the ictal period in a study by Yilmaz et al. [20]. On the contrary, Bernecker et al. [9] failed to detect significant differences in MDA serum levels for women with migraine compared to controls; it was, however, found that these patients had higher levels than controls of 4-hydroxy-2-nonenal (HNE), another biomarker of lipid peroxidation [9].

MDA is a lipid oxidation end product and could be an acceptable biomarker of oxidative stress and an indicator of increased production of ROS [8]. Based on the relevant literature of animal and human studies, the elevated level of this oxidative mediator has been linked to cortical spreading depression (CSD), one of the main proposed pathological mechanisms in migraine pain initiation particularly in migraine with aura [10, 22, 23, 25, 27, 33‐35]. As such, it has been suggested that MDA levels may be attenuated following administration of curcumin, a dietary agent with antioxidant features, in umbilical vein endothelial cells which were considered a model for exploring oxidative stress status in migraine [36].

Additionally, NO is an oxidant vasodilator which could provoke pathways of pain initiation in migraine [8, 30]. As a messenger molecule, NO may be involved in synaptic transmission in neurons and in some of the brain functions including learning, memory, and nociception [29‐31]. The present findings on increased levels of NO in both groups of migraineurs (CM and EM) compared to healthy controls is completely in accordance with the current empirical and human evidence that emphasizes the notable role played by NO in migraine pain genesis [29‐31]. The stimulating effect of this factor in causing migraine attacks has been studied in depth. This role is highlighted by the elevated production of NO and over-activation of the L-arginine-NO/cyclic GMP signaling pathway in migraine suffers [29‐31]. It has been noted that NO could provoke migraine attacks directly by vasodilation through the NO/cyclic GMP signaling pathway in the first phase of attacks and also via increasing CGRP production and activating the nociceptor neurons especially in the trigeminovascular system [29‐31]. On the other hand, it has been shown that pharmacological suppression of NO synthesis and/or the signaling pathway could result in decreasing the level of neurogenic vasodilation, neuro-inflammation and treat migraine headaches. NO is believed to be involved in causing neuroinflammation through increasing CGRP and substance P (SP) production in the perivascular nociceptors and stimulation of their activity in trigeminal neurons [29‐31].

Additionally, our findings regarding lower serum total non-enzymatic antioxidant capacity, CAT, and SOD levels in migraineurs than controls are partially in accordance with the findings of a meta-analysis on the association between oxidative/nitrosative pathways in migraine. This meta-analysis included 19 studies and reported an increase of about two times in the concentrations of thiobarbituric acid reactive substances (TBARS) in migraineurs compared to controls. While similar to our findings, the activity of the antioxidant enzyme SOD was lower in the studied subjects [21]. Further, in 2014 Aytac et al. conducted an analysis of serum MDA levels and SOD, GPx and CAT activities in 17 controls and 32 migraine suffers (18 patients with deep white matter hyperintensities (WMH)). Similar to the present findings, they showed that the patients had diminished antioxidant response indicated by decreased CAT levels and increased MDA concentrations. They also observed that patients with WMH had lower serum CAT than migraineurs without WMH and healthy subjects [22]. Like the current findings, the higher level of oxidative stress in migraine subjects in comparison with healthy individuals as revealed by augmented values of MDA, NO metabolites, TBARS, lipid peroxidation, total oxidant status (TOS), oxidative stress index (OSI), and plasma lymphocyte DNA damage has also been confirmed by other studies [15, 23‐27]. Our results regarding lower TEAC values and two of the antioxidant enzymes (SOD and CAT) are, at least in part, in line with those reported by Bolayir et al. [28], Tripathi et al. [26], and Yigit et al. [27].

However, there are also some researches that have shown conflicting results in this field. For example, in a study by Gupta et al. it was indicated that patients suffering from migraines with aura had higher SOD activity than migraineurs without aura [25]. Furthermore, Tuncel et al. failed to show significant differences in SOD and CAT activities of migraine suffers compared to control subjects [25]. Shukla et al. evaluated the activity of CAT, SOD, and GPx in the blood polymorphonuclear neutrophils of participants but did not observe any significant differences in the activity of these enzymes between migraine cases and control individuals [18].

Surprisingly, present findings on the serum GPx-1 levels revealed that the mean serum GPx-1 levels of the CM group were slightly higher than EM and control subjects. Also, there was a significant medium positive correlation between the number of headache days and serum GPx-1 levels whereas in the research conducted by Bolayir et al. [28] it was demonstrated that migraine patients had lower GPx activities than the patients with tension-type headache and controls. Moreover, Tripathi et al. reported diminished glutathione and glutathione S-transferase (GST) in migraine subjects in comparison with the control group [26]. The possible cause of these results could have been related to the compensatory mechanism in which the levels of GPx-1 may decrease subsequent to the increase of other antioxidant enzymes [37]. Further, it is proposed that as a compensatory process the elevated formation of antioxidant enzymes might occur in response to higher levels of oxidative stress [8]. In this regard, the increased level of GPx in migraineurs observed in the current study may likely be due, at least in part, to reducing SOD and CAT levels in order to preserve balance in the state of oxidative stress [8]. Moreover, it has been pointed out that excess GPx-1 levels, which might be a result of pathological changes in the body or vascular mechanical stress, could lead to adverse effects including the elimination of essential oxidant agents that may cause impaired physiological responses, mitochondrial dysfunction and decreased apoptosis [37].

Thus, although our findings were consistent with most previous reports, there could be some explanations for controversial results obtained in a number of prior researches that make comparison difficult such as the use of different techniques to assess the oxidative/antioxidant balance (e.g. OSI, TOS, total antioxidant status (TAS), ferric reducing ability of plasma (FRAP), TBARS, activities of antioxidant enzymes in erythrocytes or their concentrations in serum).

The positive correlation between the number of headache days and oxidant levels found by Alp et al. in 2010 [16] was partly consistent with the present results. Likewise, Erol et al. showed lower erythrocyte CAT and GPx activities among migraineurs compared to the control group [19]. Our study also showed that increasing oxidative and nitrosative biomarkers and decreasing levels of antioxidant factors were correlated with more frequent headaches which may suggest an inflammatory mechanism in the pathophysiology of EM progression to CM.

Collectively, although the exact mechanisms by which oxidative stress may be involved in head pain pathophysiology has not been clearly defined, there might be some explanations. It is assumed that increased production of NO metabolites and ROS in addition to pro-inflammatory mediators may lead to peroxidation of cell membrane phospholipids, destruction of intracellular molecules (particularly proteins and DNA) and consequently impairment in the normal function of cells [8, 10, 11]. In this respect, emerging evidence has highlighted the contribution of protein and DNA oxidation and subsequent disruption in receptor function, dysregulation in signaling pathways and the transport system to hyperexcitability of cortical neurons, CSD, neurogenic inflammation, and trigeminovascular pathway activation, that ultimately could lead to headache [10, 11]. In addition, mitochondrial biogenesis of the trigeminal nerve may be disrupted in migraine, which could subsequently result in decreased ATP production and increased ROS synthesis [38]. Additionally, CSD, a well-known mechanism in aura generation in migraine, may contribute to increased production of ROS in different parts of CNS including the cortex, meninges and trigeminal ganglia. ROS might induce nociceptive responses directly or through stimulating the secretion of pain mediators in migraine such as CGRP [34, 39]. CGRP, a vasodilator neuropeptide which is known as the main mediator of migraine pain, may be associated with increased formation of pro-inflammatory cytokines and oxidative factors that all are believed to play a pivotal role in pain sensitization [34, 39]. On the other hand, the antioxidant defense consisting of enzymatic or non-enzymatic factors could eliminate ROS and free radicals, protect against oxidative stress induced in migraine [10] and thus alleviate head pain.

The present research revealed an imbalance in oxidant/antioxidant status in favor of higher levels of oxidative biomarkers in migraine patients than healthy controls. Also, it was highlighted that chronic migraineurs had lower total non-enzymatic antioxidant capacity than episodic migraine and control individuals. It was also demonstrated that as the frequency of headache days increased, the level of NO and MDA as biomarkers of oxidative stress went up and the antioxidant defense (as shown by TEAC values, SOD, CAT serum levels) went down. This changing state might be the basis of the progression of migraine from the episodic to the chronic stage. In sum, the current findings seem to further support the relationship of oxidative stress in migraine pathophysiology. Thus, novel prophylactic medications or dietary supplements with antioxidant properties might be promising in migraine treatment generally and also in the prevention of EM becoming CM. Well-designed randomized controlled trials should be conducted in order to assess the effects of the above-mentioned agents on migraine headaches.