We have shown that apart from oxLDL and HbA1c most other markers of mitochondrial functioning showed abnormalities in a significant proportion (>30%) of the patients examined.
To the best of our knowledge ALA levels have not previously been determined in migraine. Almost 90% of patients in this sample had abnormally low values of ALA. ALA, also known as thioctic acid, is an eight-carbon, sulfur-containing compound that functions as a water- and fat-soluble antioxidant (79,80). It can directly (by removing reactive species) and indirectly (by chelating transition metal ions) reduce oxidative stress (79,80). The human body can synthesize small amounts of ALA (79). ALA also plays an important role as co-enzyme in energy metabolism (79–81). Furthermore, it is able to regenerate other antioxidants, such as vitamin C and E, CoQ10, it increases intracellular glutathione and activates endogenous antioxidant systems (82–84). Apart from its anti-oxidant action, ALA seems to assist weight loss (85), increase insulin sensitivity and decrease blood lipids (86). All of these mechanisms are probably migraine relevant. Interestingly, ALA supplementation (300-600mg) per day has been shown to significantly reduce migraine attack frequency, severity and duration (37–39), which seems to align with our findings. Further research is needed to see, whether this finding is specific to our medium-high frequency episodic migraine population or a general characteristic of migraine or even a general characteristic of other (neurological) diseases with a mitochondrial / oxidative stress component. Should this finding be replicated and migraine specific, ALA might represent a potential biomarker.
Serum (or plasma) concentrations of different antioxidants can be measured separately, but since the measurement of different antioxidant molecules individually is impractical and costly and their antioxidant effects are additive, the total antioxidant capacity of a sample is typically measured, and this is typically referred to as total antioxidant capacity (TAC), total antioxidant status (TAS) or other synonyms, which will be used interchangeably.
Almost 40% of our patients had abnormally low TAC being in line with results of previous research. A study on 75 MO patients demonstrated that the levels of total antioxidants were decreased and the levels of total oxidants and the oxidative stress index were increased (56). Another study found TAC to be significantly reduced in migraineurs compared to controls (67). TAC levels increased after successful prophylactic treatment compared to the baseline, irrespective of treatment modality (rTMS versus amitriptyline) and the increase correlated with treatment success (67).We assume higher TAC with lower migraine severity, less recent oxidative stress exposure, and increased distance to previous and future migraine attack. These assumptions would have to be validated in future research.
Lipid peroxidation is the oxidative degradation of lipids via free radical damage of the lipids in cell membranes, polyunsaturated fatty acids in particular. The end products of lipid peroxidation are reactive aldehydes, such as 4-hydroxynonenal (HNE) and malondialdehyde (MDA). Free radicals cause increased accumulation of these lipid peroxidation by-products in the blood. About half of the patients had abnormally high total PerOx levels, being in line with previous research. Several studies have found serum levels of MDAs to be significantly elevated in migraine patients (57)(68), even in the interictal phase (87).
Oxidized low-density lipoprotein (LDL) is a harmful type of cholesterol that is produced when normal LDL cholesterol is damaged by chemical interactions with ROS. All but one patient had normal levels for oxLDL, which is in contrast to the study of Bernecker et al. that found highly significantly elevated levels oxLDL in female migraineurs (58). This result could be due to differences in study population, as the migraineurs of the Berecker et al. study tended to have metabolic syndrome and had generally higher BMIs as our migraine patient population.
The term “thiol” refers to organic compounds containing sulfur (in form of the functional group -SH, the thiol group). Thiol groups are able to destroy ROS and other free radicals by enzymatic as well as nonenzymatic mechanisms (88). Total thiol levels have previously been used to evaluate excess free radical generation, both in physiological and pathological conditions (89). Protein thiol levels in serum have been shown to be a direct measure of the in vivo reduction/oxidation (redox) status in humans, because thiols react readily with ROS to form disulfides (77). Thiol redox homeostasis plays an important role in neurogenerative diseases (90) and in nine other categories of human disorders serum protein thiols have been found to be significantly reduced compared to healthy controls (77).
Only about one third of patients had abnormally low serum thiol levels, but this seems to be in line with previous research. A larger study found significantly reduced thiol levels in 151 migraine patients (74 MO, 77MA) compared to 70 healthy controls and there was a negative correlation with migraine disability (62). A negative correlation between the levels of total thiols and the duration of the headaches has also been demonstrated (56). However, others studies found no significant difference in thiol groups between patients and controls, even during attacks (69) and one study even found higher total (-SH+-S-S-) & native thiol (-SH) levels in serum of migraineurs, but this did not correlated with disease severity or migraine type (64). Recent exposure to oxidative stress, migraine severity, time in the migraine cycle and similar aspects could explain the different results.
HbA1c (glycated hemoglobin) is an indication of the average blood glucose levels over the last two to three months. Just over 20% of patients had abnormally low HbA1c levels and none of them had HbA1c levels that were above 5.6%. To the best of our knowledge HbA1c has rarely been looked at in migraine. One study found no significant difference in HbA1c levels between CM, EM and healthy controls (20)
However, magnetic resonance spectroscopy (MRS) studies in migraine have consistently shown abnormalities of mitochondrial oxidative phosphorylation (OXPHOS), such as hypometabolism between (3–10) and during migraine attacks (11), in the resting brain and in the muscle following exercise (3,12,13). A 16% decrease of absolute ATP levels in migraine without aura patients was also demonstrated interictally using 31P-MRS (14). These findings are supported by early studies showing that metabolic changes induced by fasting, glucose or insulin administration can trigger migraine attacks; e.g. a 50g glucose tolerance test (GTT) after a 10-hour fast triggered a migraine in 6 out of 10 migraine patients reporting attacks associated with fasting (15). Abnormal metabolic responses were also reported in GTT studies (15,16) and interictal impaired glucose tolerance and insulin resistance has been reported in various other studies (17–21). While only 20% of our migraineurs had abnormally low HbA1c levels, all levels tended to be on the lower side, despite reported higher carbohydrate diets. As HbA1c levels correspond to an average blood sugar measurement, low average values despite probable highs after carbohydrate rich meals could be an indication that there might be lows as well. This would be in line with previous neuroimaging and GTT research results, but it is speculation only and these assumption need to be confirmed by future research.
Lactate is typically measured to assess tissue oxygenation, arising from either decreased oxygen delivery or a disorder in oxygen use, both of which lead to increased anaerobic metabolism and increases in lactate levels. In certain types of migraine, especially migrainous stroke, elevated serum lactate and pyruvate levels have previously been reported (47,48). In contrast to this, only 2 patients had abnormally high serum lactate levels in our cohort and over 70% of patients serum lactate levels were abnormally low.
While there is little data on serum lactate levels in migraine, data on brain lactate analysed with 1H-MRS have also been shown to vary due to patient selection (see review by Reyngoudt et al (2012) for details (9)). Elevated brain lactate levels were found in some studies of MA (91,92), but not in MO (93–96). Occipital baseline lactate levels were increased in patients with visual auras, but not in those having complex neurological auras. By contrast, during photic stimulation lactate increased significantly in the latter, but not in the former (91). Stimulus-induced lactate increases are physiological (97) and can be explained by the neuron-astrocyte lactate shuttle (98). Hence, their absence in migraine patients, whose neuronal activation is energetically more demanding (99), could be considered pathological and might be contributing to an energetic crisis.
To the best of our knowledge, no recent studies have looked at baseline serum lactate levels in episodic migraine patients or subgroups thereof. More research is needed to replicate this finding; in particular a study combining lactate level quantification in the cortex with that of the periphery and with brain energetics seems warranted. We can only speculate as to why lactate levels were predominantly low in the majority of our patients. They all came rested, but fasted overnight to the trial site. Decreased baseline lactate levels might be a sign of increased cerebral lactate consumption and an indicator of an increased cerebral energy demand of the migraine brain, as in addition to ketone bodies, lactate constitutes the only other major alternative brain energy substrate from glucose and is used especially during times of high metabolic demands or hypoglycemia (100). A study using 13 C-L-lactate and magnetic resonance spectroscopy suggested that the contribution of plasma lactate to brain metabolism can be up to 60% (101), which is very similar to ketone bodies. It could also be a sign of decreased lactate synthesis as demonstrated with 1H-MRS (91).
In summary, we have shown that apart from oxLDL and HbA1c most other markers of mitochondrial functioning are abnormal in at least >30% of the patients examined. As oxidative stress is a complex mechanism including different sources of ROS and various pathways, differing results in previous research may at least be partially caused by different oxidative stress parameters examined, e.g. MDA versus HNE, as well as by different study groups investigated, e.g. adults versus children, MA versus MO, females versus males, and differences in migraine severity, recent oxidative stress exposure and the time within the individual migraine cycle, where measurements were taken. Genetic research examining oxidative stress related genes in larger homogenous migraine cohorts could be interesting future research that would hardly be influenced by these factors.
Our data provide no evidence for correlations between any of the seven mitochondrial function / oxidative stress markers and migraine severity. This could be due to our sample population being fairly homogenous or the sample size being too small. In addition, we found no evidence for an effect of migraine prophylaxis. This is not surprising, since patients were still suffering from a substantial number of migraine days / months despite the prophylactic treatment (5-14 days / months), suggesting that the critical migraine pathophysiological mechanisms remained active. Furthermore, no evidence for an effect of a preceding or subsequent migraine attack has been found. This might be due to only 5 patients being migraine attack free within 2 days before and after the venous puncture, making an analysis of the potential impact of an attack difficult. We also found no evidence for a difference between MA and MO patients. For a randomly selected migraine cohort mainly recruited via public advertisements, the number of MA patients was unusually high (62.5%) in our study population. We can only speculate as to why this might be the case. Since participants were part of the 9 months MigraKet intervention trial (71), it seems plausible that MA patients might have been more motivated to take place in such a lengthy trial and this led to the observed over-representation.
While we found no correlation between these mitochondrial function / oxidative stress markers and disease severity, differences in methodologies used and patient characteristics, recent oxidative stress exposure and also time in the respective migraine cycle is likely to play a role. Future research examining these markers at different time points during the migraine cycle and in different migraine types would be interesting.
The most important limitation of this study is the absence of a matched control group. While abnormally low levels in 90% of patients in the case of ALA are likely to be of importance, we cannot be sure that PerOx, TAC and thiol level findings would have been significantly different from controls. Future research is needed to replicate these findings in the presence of a control group. Secondly, the sample size was fairly small, in particular with regards to the correlation analyses. In addition, one third of patients was using a migraine prophylaxis. While our data provide no evidence for an effect of migraine prophylaxis, the inclusion of patients who are using a prophylaxis is not ideal.
In conclusion, this study provides further support for metabolic abnormalities in migraine, in particular the role of increase oxidative stress and decreased anti-oxidant capacity respectively in migraine pathophysiology. The peripheral markers assessed here could easily be examined in most doctor’s offices and might assist personalised migraine treatment that targets oxidative stress and mitochondrial functioning; however, further research is needed to replicate these findings, ideally in the presence of a control group.