Cardiovascular comorbidities are risk factors for increased oxidative stress and DNA damage in migraine patients: a prospective cohort study

Abstract

Background

Migraine is a prevalent neurovascular disorder frequently linked with oxidative stress and an elevated risk of cardiovascular diseases (CVDs), particularly in patient with comorbidities. This study aimed to investigate the relationships between oxidative stress and DNA damage biomarkers, cardiovascular comorbidities, and the effects of six months of migraine prophylaxis.

Methods

A prospective cohort study was conducted between January and September 2024 at a tertiary neurology clinic, enrolling 75 women who were divided into three groups: migraine with cardiovascular comorbidities (MC, n = 25), migraine without comorbidities (M, n = 25), and age-matched healthy controls (C, n = 25). Migraine diagnosis was confirmed according to the International Classification of Headache Disorders, 3rd edition (ICHD-3), and patients with renal/hepatic dysfunction, active infections, migraine with aura, pregnancy, or other neurological/psychiatric disorders were excluded. Venous blood samples were obtained during the interictal period (≥ 72 h migraine-free) at baseline and after 6 months of standard acute migraine treatment. Biochemical analyses included total oxidant status (TOS), total antioxidant status (TAS), and calculation of oxidative stress index (OSI) using automated colorimetric assays. DNA damage was quantified by comet assay (single-cell gel electrophoresis), whereas ischemia-modified albumin (IMA) and hypoxia-inducible factor-1α (HIF-1α) were measured via ELISA. Statistical analyses were performed using ANOVA, paired and independent t tests, and Pearson correlation, with p < 0.05 considered significant.

Results

Migraine patients exhibited significantly higher oxidative stress and DNA damage levels compared to controls, with the highest levels in those with cardiovascular comorbidities. After six months of treatment, biomarker levels decreased but remained elevated relative to controls. Ischemic markers (IMA and HIF-1α) were consistently higher in migraine patients, especially in the MC group, and although reduced post-treatment, did not normalise to control values.

Conclusions

Cardiovascular comorbidities substantially increase oxidative stress and DNA damage in migraine patients, potentially heightening long-term cardiovascular risks. Monitoring these biomarkers may facilitate personalised risk stratification and management in clinical practice.

Supplementary Information

The online version contains supplementary material available at 10.1186/s12967-025-07055-4.

Highlights

• Previous studies have reported the role of oxidative stress and ischemia-related biomarkers (e.g. IMA, HIF-1α) in migraine, but none have comprehensively examined the additive impact of cardiovascular comorbidities on oxidative stress, DNA damage, and ischemic burden.

• In this prospective cohort study, we demonstrated that migraine patients with cardiovascular comorbidities exhibit markedly higher levels of oxidative stress, ischemia-modified albumin, hypoxia-inducible factor-1α, and DNA damage compared with migraine patients without comorbidities and healthy controls. Six months of treatment reduced these abnormalities but did not restore them to normal levels.

• Cardiovascular comorbidities exacerbate oxidative and ischemic processes in migraine, highlighting the need for integrated monitoring and management of vascular risk factors in this population. Incorporating oxidative stress and ischemic biomarkers into clinical practice may facilitate more effective risk stratification and long-term preventive strategies.

Supplementary Information

The online version contains supplementary material available at 10.1186/s12967-025-07055-4.

Introduction

Migraine Disorders are common, chronic neurovascular conditions that affect nearly 15% of the global population and carry considerable personal and societal burden [1–3]. Increasing evidence recognises migraine as an independent risk factor for Cardiovascular Diseases (CVDs), including ischaemic and haemorrhagic stroke, myocardial infarction, and atrial arrhythmias [4, 5]. Women under 50 years of age with migraine are particularly vulnerable to ischaemic stroke, and this risk is further amplified by smoking and the use of oral contraceptives [1]. Epidemiological studies have also demonstrated that individuals with migraine have a 1.5-fold higher risk of haemorrhagic stroke [5–8]. In addition to stroke, migraine has been associated with increased rates of myocardial infarction, coronary interventions, and cardiovascular mortality, especially in women and postpartum patients. This association is thought to be driven by several pathophysiological mechanisms, including blood vessel spasm (vasospasm), endothelial dysfunction, and enhanced platelet activation, all of which may increase the risk of vascular events in migraine patients [9].

Oxidative Stress is important to the pathophysiology of migraine and its link to CVD. An imbalance between Reactive Oxygen Species (ROS) production and antioxidant defences leads to oxidative damage to DNA, lipids, and proteins. This imbalance contributes to neurovascular inflammation, cortical spreading depression, and sensitisation of pain pathways [10–12]. Beyond trigeminal nerve activation, recent theories emphasise a multifactorial pathogenesis that includes neurogenic inflammation, cortical hyperexcitability, vascular dysregulation, and endothelial dysfunction, all of which may converge to exacerbate systemic vascular stress and increase cardiovascular vulnerability. Hypoxia-Inducible Factor 1, alpha Subunit (HIF-1α) is a key regulator of the cellular adaptive response to oxidative stress. It translocates to the nucleus and activates target genes, thereby preserving mitochondrial integrity and reducing cellular injury. [13–16]. Impairment of this pathway may further elevate cardiovascular risk in migraine patients [17]. Another relevant biomarker is Ischaemia-Modified Albumin (IMA), measured by the albumin–cobalt-binding assay, which provides an early marker of ischaemic stress [18].

Although HIF-1α and IMA do not interact directly, they are triggered by overlapping biological mechanisms, primarily hypoxia and oxidative stress. HIF-1α reflects intracellular adaptive processes, whereas IMA indicates systemic biochemical alterations caused by ischaemia. Clinical studies in myocardial infarction, stroke, malignancies, and critical illness consistently demonstrate parallel elevations of both markers. This pattern suggests that the same ROS and hypoxic conditions that stabilise HIF-1α also promote albumin modification, resulting in increased IMA levels [18–20]. Evaluating these markers together may provide a more comprehensive view of oxidative and ischaemic processes in migraine and its cardiovascular comorbidities. While no prior study has investigated their direct relationship in migraine, the literature supports their complementary roles. Furthermore, IMA has been consistently elevated in diverse oxidative and inflammatory conditions, highlighting its potential as a surrogate marker of cardiovascular risk in migraine [21, 22].

Migraine frequently coexists with psychiatric, neurological, and cardiovascular comorbidities [23, 24]. These include major depressive disorder, anxiety, hypertension, coronary artery disease, and stroke [23]. Understanding such comorbidities is essential for refining diagnostic strategies and improving therapeutic outcomes. The overlap of symptoms and complications across disorders underscores the need for targeted evaluation. Although previous studies have explored oxidative stress and ischaemia-related biomarkers in migraine, none has specifically assessed the contribution of cardiovascular comorbidities. This study addresses this gap by prospectively evaluating oxidative stress, ischaemic biomarkers, and DNA Damage in migraine patients with and without cardiovascular comorbidities. The findings provide novel insights into the additive impact of comorbid conditions on migraine pathophysiology.

Methods

This prospective study was conducted between January and September 2024 in the neurology clinic of a tertiary university hospital. The protocol was approved by the Clinical Research Ethics Committee (Decision No: 2024-KAEK-03). Written informed consent was obtained from all participants before enrolment. All procedures complied with the ethical standards of the University of Siena and the principles of the Declaration of Helsinki (1964) and its later amendments.

A total of 264 patients with Migraine were initially screened. Of these, 135 were excluded as they did not meet the eligibility criteria. Forty-seven were excluded because of incomplete data, and seven were excluded due to haemolysed blood samples. Finally, 75 female participants were enrolled (Fig. 1). This study population comprised three groups: 25 patients with migraine and cardiovascular comorbidities (MC group), 25 patients with migraine without comorbidities (M group), and 25 age-matched healthy controls (C group). The control group included healthcare professionals and medical students who fulfilled the eligibility criteria, provided consent, and were matched with the patient groups by age.

Fig. 1.

Flowchart illustrating the patient selection process for the study

The study's inclusion criteria included outpatients > 18 years of age who were admitted to the hospital with a confirmed diagnosis of migraine without aura with the ICD-10 codes G43.0 and G43.1 and who provided informed consent for participation in the study. Migraine without aura was diagnosed on the basis of the International Classification of Headache Disorders (ICHD-3) criteria [2] by neurologists in the clinic before the participants were enrolled. The exclusion criteria for the study included individuals in both patient groups (MC and M) who had renal or hepatic dysfunction, those with active infections, individuals who had migraine with aura, individuals who were pregnant or breastfeeding, and those who had other chronic neurological disorders. Also, participants in the MC group who had migraine along with two or more additional comorbidities were excluded from the study. In the C group, individuals with other types of headache (tension-type, cluster and secondary headache), severe psychiatric (e.g., major depressive disorder, Schizotypal personality disorder) or neurologic disorders (neurodegenerative or demyelinating diseases, stroke or tumors), vision and hearing problems, uncontrolled hypertension, alcohol, substance addiction, pregnancy, or breastfeeding were excluded. Individuals in the C group were screened to ensure that they had no personal or family history of migraine, as this could introduce confounding factors. Additionally, patients who did not provide consent, as well as individuals lacking cognitive abilities or decision-making capacity, were not included in the patient groups.

The study consisted of two phases. In the first phase, on day 0, the demographic information of the patients was collected, and approximately 4–5 ml of venous blood was collected into gel tubes before migraine treatment. All analyses of oxidative stress and DNA damage were conducted on serum or whole blood samples, depending on the biomarker. The samples were subjected to centrifugation at 3000 × g for 10 min, after which the serum was collected, transferred to Eppendorf tubes, and stored at − 80 °C for future analysis. In the second phase (on day 90), the patients were re-evaluated in the neurology clinic, the same tests and questionnaires were administered, and blood samples were collected again. Blood samples were collected during scheduled outpatient appointments, and only if the patient had remained completely free of migraine symptoms for at least 72 h prior to sampling. No prophylactic therapy was permitted during follow-up; blood sampling was performed during the interictal phase (≥ 72 h migraine-free) to minimise attack- and treatment-related variability. This 72-h symptom-free interval was used as an operational definition of the interictal phase, in accordance with established criteria in migraine research. Patients experiencing aura, prodromal symptoms, or postdromal fatigue were excluded from blood sampling until the interictal window was confirmed.

The effects of treatment on biomarkers were subsequently examined, and the data from day 0 and day 180 were compared for evaluation. During the 6-month treatment period, patients did not use any prophylactic medication; instead, they used medications specifically for acute migraine attacks. These acute therapies typically included triptans, non-steroidal anti-inflammatory drugs (NSAIDs), and other supportive medications, prescribed in accordance with current clinical guidelines.”

Measurement of biochemical parameters

The total antioxidant status (TAS) and total oxidant status (TOS) were assessed via fully automated techniques established by Erel [25]. TAS measures the body's total antioxidant level against potent free radicals, whereas TOS quantifies the oxidants present in a sample. TAS is based on the reaction of antioxidants with reactive oxygen species (ROS), which prevents the formation of color by stopping oxidation reactions, as measured spectrophotometrically. TOS uses a similar spectrophotometric method in which oxidants generate a color reaction [26]. TOS and TAS were measured using commercially available colorimetric assay kits (Rel Assay Diagnostics; Cat. No: RL0024 for TAS and RL0023 for TOS). The oxidative stress index (OSI) is determined by the ratio of TOS to TAS, expressed in specific units (μmol H2O2 equivalent/L for TOS and μmol Trolox equivalent/L for TAS).

DNA damage was analyzed via the comet assay, which is a method based on alkaline single-cell gel electrophoresis [27] (Abcam, UK; Cat. No: ab201734). To achieve this goal, 6 µL of whole blood was combined with 0.7% low melting point agarose and spread onto slides that were precoated with 1% normal melting point agarose gel. A coverslip was then placed on top, and the gel was allowed to solidify under cold conditions. Once solidified, the coverslips were removed, and the cells were lysed for at least 4 h in lysis buffer. Next, electrophoresis was performed for 20 min in alkaline buffer (pH 13) at 300 mA. After electrophoresis, the cells were stained with ethidium bromide (5 mg/mL) and examined via fluorescence microscopy (absorbance: 546 nm; emission: 20 nm). Tail intensity (% tail) was analyzed as an indicator of DNA damage. The comet analyses were performed via Comet Assay IV software (Perceptive Instruments, Suffolk, UK), and 50 cells were counted on average. Serum IMA and HIF-1α levels were measured via commercially available ELISA kits (RL0060, RelAssay, TR for IMA and E-EL-H6066, Elabscience, USA for HIF-1α) following the manufacturer’s instructions, with absorbance readings for both assays taken via a Synergy-HTX microplate reader (Synergy-HTX, Biotek, USA).

Statistical analysis

The primary endpoint was the comparison of oxidative stress markers between the MC, M, and control groups, whereas the secondary endpoint was the change in these markers following a 6-month treatment. The required sample size was determined through a priori power analysis based on the OSI. Reference was made to the findings of Geyik et al. [28], and the expected mean difference between healthy controls and patient groups was assumed to be 0.05 arbitrary units (AU), with a standard deviation difference of 0.02 AU. Using these parameters, the calculated effect size (Cohen’s d) was 0.7936. With a significance level (α) of 0.05 and a desired power (1 − β) of 0.95, the minimum required sample size was calculated as 23 participants per group.

The Shapiro–Wilk test was applied to assess whether continuous variables followed a normal distribution, and the results confirmed that the data were normally distributed. One-way ANOVA was used to assess differences in demographic, clinical, and biochemical variables between the control (C), migraine (M), and migraine with comorbidity (MC) groups. Post hoc comparisons were performed for pairwise group comparisons (Tukey’s HSD test). Paired-sample t tests were used to evaluate differences in biochemical parameters within the same group before and after treatment. Independent-sample t tests were conducted to compare biochemical parameters before and after treatment between the groups. Pearson’s correlation coefficients (r) were used to assess the relationships between these parameters. A p value of < 0.05 was deemed statistically significant for all tests. The results are reported as the means ± standard deviations (SDs). Statistical analyses were performed via SPSS for Windows, version 22.0 (IBM Corp., USA).

Results

As shown in Table 1, there were no significant differences in demographic characteristics, such as age, height, weight, and BMI, among the control (C), migraine (M), and migraine with comorbidity (MC) groups (p > 0.05 for all). Smoking was reported in 40% (n = 10) of participants in both the M and MC groups, whereas no participants in the control group smoked (p = 0.001). Alcohol consumption was also more common in the M and MC groups (24% (n = 6) in both) than in the control group (0%, p = 0.027). The duration of migraine attacks was shorter with medication in both M (2.36 ± 1.38 h) and MC (2.72 ± 1.74 h) groups, whereas untreated attack durations were significantly longer (M: 28.80 ± 15.80 h; MC: 32.16 ± 17.56 h; p < 0.001). As expected, the MC group had multiple comorbidities, including hypertension (n = 9), hyperlipidemia (n = 7), heart failure (n = 4), arrhythmia (n = 3), dilated cardiomyopathy (n = 1), and pulmonary hypertension (n = 1), all of which were absent in the M and C groups (p < 0.001 for each). During the 6-month follow-up period, patients did not receive prophylactic therapy but were permitted to use medications for acute migraine attacks. The most frequently used drugs were non-steroidal anti-inflammatory drugs (NSAIDs), analgesics, and triptans. Specifically, dexketoprofen (n = 11, 22%), paracetamol + codeine (n = 7, 14%), flurbiprofen (n = 6, 12%), metamizole (n = 4, 8%), sumatriptan (n = 4, 8%), rizatriptan (n = 3, 6%), diclofenac (n = 3, 6%), naproxen (n = 3, 6%), paracetamol (n = 2, 4%), and eletriptan (n = 2, 4%) were used.

Table 1.

Comparison of demographic and clinical characteristics among the control, migraine, and migraine with comorbidity groups

		Control (C) (n = 25)	Migraine (M) (n = 25)	Migraine with Comorbidity (MC) (n = 25)	p valuex*
Age (years)		38.60 ± 9.28	39.20 ± 5.17	37.37 ± 5.62	ns
Height (cm)		164.08 ± 7.98	159.36 ± 7.81	160.36 ± 8.57	ns
Weight (kg)		62.68 ± 7.64	60.84 ± 8.32	62.76 ± 11.25	ns
BMI (kg m−1)		23.48 ± 3.88	24.16 ± 4.40	24.67 ± 5.62	ns
Headache history (years)		0 ± 0	10.64 ± 6.28	10.64 ± 3.75	< 0.001
Smoking ( +)		0	40% (10)	40% (10)	0.001
Alcohol ( +)		0	24% (6)	24% (6)	0.027
Monthly attack frequency		0 ± 0	5.80 ± 1.73	6.32 ± 1.93	< 0.001
Attack duration (h)	With medicine	0	2.36 ± 1.38	2.72 ± 1.74	< 0.001
	Without medicine	0	28.8 ± 15.8	32.16 ± 17.56	< 0.001
Comorbidities (n)
Hypertension		0	0	9	< 0.001
Hyperlipidemia		0	0	7	< 0.001
Heart failure		0	0	4	< 0.001
Arrhythmia		0	0	3	< 0.001
Dilated cardiomyopathy		0	0	1	< 0.001
Pulmonary hypertension		0	0	1	< 0.001

BMI, body mass index

^xOne-way ANOVA

*p < 0.05 was considered statistically significant

Biochemical analysis (Table 2) revealed significant differences in TOS, TAS, OSI, IMA, HIF-1α, and DNA damage among the C, M, and MC groups before and after treatment. TOS levels were significantly higher in the M (14.81 ± 1.48 µmol H₂O₂ Eq./L) and MC (16.51 ± 1.78 µmol H₂O₂ Eq./L) groups compared to the control group (9.31 ± 1.70 µmol H₂O₂ Eq./L) before treatment (p < 0.001). Although posttreatment TOS levels decreased in the M (12.82 ± 1.45) and MC (13.67 ± 1.61) groups, they remained significantly higher than in controls (9.59 ± 1.63; p < 0.001). TAS levels were significantly lower in the M (0.75 ± 0.12 mmol Trolox Eq./L) and MC (0.65 ± 0.08 mmol Trolox Eq./L) groups compared to the C group (1.07 ± 0.06 mmol Trolox Eq./L) at baseline (p < 0.001). Posttreatment TAS levels showed slight increases (M: 0.80 ± 0.13, MC: 0.73 ± 0.08), but remained significantly lower than controls (1.07 ± 0.04; p < 0.001). OSI, calculated as TOS/TAS × 100, was markedly elevated in the M (20.38 ± 5.03) and MC (25.52 ± 4.42) groups compared to the C group (8.69 ± 1.71) before treatment (p < 0.001). While OSI decreased after treatment (M: 16.53 ± 3.94; MC: 18.96 ± 3.03), values remained significantly higher than in controls (8.97 ± 1.67; p < 0.001).

Table 2.

Biochemical parameters in the control, migraine, and migraine with comorbidity groups before and after treatment

		Control (C)	Migraine (M)	Migraine with comorbidity (MC)	p valuex*	Significant between
OSI (arbitrary unit)	BT	8.69 ± 1.71	20.38 ± 5.03	25.52 ± 4.42	< 0.001	C vs M, C vs MC, M vs MC
	AT	8.97 ± 1.67	16.53 ± 3.94	18.96 ± 3.03	< 0.001	C vs M, C vs MC, M vs MC
	ΔOSI	0.28 ± 0.43	− 3.84 ± 2.29	− 6.56 ± 3.90	< 0.001	C vs M, C vs MC, M vs MC
TOS (µmol H2O2 Eq./L)	BT	9.31 ± 1.70	14.81 ± 1.48	16.51 ± 1.78	< 0.001	C vs M, C vs MC, M vs MC
	AT	9.59 ± 1.63	12.82 ± 1.45	13.67 ± 1.61	< 0.001	C vs M, C vs MC, M vs MC
	ΔTOS	0.28 ± 0.43	− 1.98 ± 1.49	− 2.84 ± 2.32	< 0.001	C vs M, C vs MC, M vs MC
TAS (mmol Trolox Eq./L)	BT	1.07 ± 0.06	0.75 ± 0.12	0.65 ± 0.08	< 0.001	C vs M, C vs MC, M vs MC
	AT	1.07 ± 0.04	0.80 ± 0.13	0.73 ± 0.08	< 0.001	C vs M, C vs MC, M vs MC
	ΔTAS	− 0.00 ± 0.04	0.05 ± 0.05	0.07 ± 0.11	0.003	C vs M, C vs MC, M vs MC
IMA (ABSU)	BT	0.66 ± 0.10	1.03 ± 0.19	1.20 ± 0.13	< 0.001	C vs M, C vs MC, M vs MC
	AT	0.65 ± 0.10	0.98 ± 0.14	1.16 ± 0.10	< 0.001	C vs M, C vs MC, M vs MC
	ΔIMA	− 0.00 ± 0.03	− 0.04 ± 0.09	− 0.04 ± 0.08	0.156
HIF-1α (pg/mL)	BT	952.65 ± 104.83	1621.21 ± 170.37	1794.46 ± 209.60	< 0.001	C vs M, C vs MC, M vs MC
	AT	951.59 ± 92.53	1476.28 ± 167.54	1652.72 ± 202.49	< 0.001	C vs M, C vs MC, M vs MC
	ΔHIF − 1 α	− 1.06 ± 19.72	− 144.92 ± 146.60	− 141.74 ± 134.67	< 0.001	C vs M, C vs MC
DNA damage (%tail intensity)	BT	1.77 ± 0.95	8.43 ± 1.66	10.53 ± 3.19	< 0.001	C vs M, C vs MC
	AT	1.63 ± 0.93	7.79 ± 1.47	8.18 ± 1.61	< 0.001	C vs M, C vs MC, M vs MC
	ΔDNA Damage	− 0.13 ± 0.30	− 0.63 ± 0.69	− 2.34 ± 2.95	0.001	C vs M, C vs MC, M vs MC

TOS, total oxidant status; TAS, total antioxidant status; OSI, oxidative stress index; IMA, ischemic modified albumin; HIF-1α, hypoxia inducible factor-1α; BT, before treatment; AT, after treatment

Data are expressed as the mean ± standard deviation (SD)

The Δ row represents the difference between pre- and posttreatment values

^xOne-way ANOVA

*p < 0.05 was considered statistically significant

IMA levels were elevated in the M (1.03 ± 0.19 ABSU) and MC (1.20 ± 0.13 ABSU) groups relative to controls (0.66 ± 0.10 ABSU) at baseline (p < 0.001), and posttreatment levels (M: 0.98 ± 0.14; MC: 1.16 ± 0.10) remained significantly higher than in the C group (0.65 ± 0.10; p < 0.001).

HIF-1α levels were also significantly higher in the M (1621.21 ± 170.37 pg/mL) and MC (1794.46 ± 209.60 pg/mL) groups than in controls (952.65 ± 104.83 pg/mL) at baseline (p < 0.001), and although they decreased after treatment (M: 1476.28 ± 167.54; MC: 1652.72 ± 202.49), levels remained significantly elevated compared to the C group (951.59 ± 92.53; p < 0.001).

DNA damage, expressed as % tail intensity, was significantly increased in the M (8.43 ± 1.66%) and MC (10.53 ± 3.19%) groups compared to the C group (1.77 ± 0.95%) before treatment (p < 0.001). Posttreatment values (M: 7.79 ± 1.47%; MC: 8.18 ± 1.61%) remained significantly higher than in the C group (1.63 ± 0.93%; p < 0.001).

Correlation analysis (Table 3) revealed several significant relationships between the biochemical parameters before and after treatment in migraine patients. A strong positive correlation was found between TOS and OSI (r = 0.718, p < 0.001), as expected due to the mathematical derivation of OSI. TOS was also positively correlated with IMA (r = 0.314, p < 0.001) and HIF-1α (r = 0.265, p < 0.05). In contrast, TOS and TAS showed a significant negative correlation (r =  − 0.605, p < 0.001). TAS was negatively correlated with IMA (r =  − 0.285, p < 0.01) and HIF-1α (r =  − 0.265, p < 0.05), and showed a strong negative correlation with OSI (r =  − 0.605, p < 0.001). A positive correlation was observed between TAS and DNA damage (r =  − 0.347, p < 0.001). OSI showed significant positive correlations with IMA (r = 0.470, p < 0.001), HIF-1α (r = 0.463, p < 0.001), and DNA damage (r = 0.438, p < 0.001). Although IMA was not significantly correlated with HIF-1α (r = 0.053, ns) or DNA damage (r = 0.136, ns), HIF-1α was significantly correlated with DNA damage (r = 0.359, p < 0.001).

Table 3.

Correlation analysis between pre- and posttreatment biochemical parameters

	TOS	TAS	OSI	IMA	HIF-1α	DNA Damage
TOS		0.005 ns	0.718***	0.314***	0.265*	0.139 ns
TAS			− 0.605***	− 0.285**	0.265*	− 0.347***
OSI				0.470***	0.463***	0.438***
IMA					0.053 ns	0.136 ns
HIF-1α						0.359***
DNA damage

TOS, total oxidant status; TAS, total antioxidant status; OSI, oxidative stress index; IMA, ischemic modified albumin; HIF-1α, hypoxia inducible factor-1α, ^ns: Not significant

Pearson correlation analysis was performed

*p < 0.05, ** p < 0.01, *** p < 0.001 was considered statistically significant

Discussion

Compared with healthy controls, patients with migraine showed an imbalance in redox homeostasis, characterised by elevated TOS and reduced TAS. The increase in TOS reflects a heightened burden of ROS, which are implicated in triggering migraine attacks through the promotion of neurovascular inflammation and cortical spreading depression. Conversely, the reduction in TAS denotes an impaired antioxidant defence capacity, thereby perpetuating oxidative stress and contributing to the persistence of migraine symptoms. [29]. Tripathi et al. demonstrated that TAS levels were significantly lower in migraine patients with aura than in healthy individuals, further emphasizing the role of oxidative stress in migraine headaches [30]. Similarly, in another experimental study of patients with migraine without aura, TAS levels were found to decrease, whereas TOS and OSI levels increased in the patient group [31]. In line with these findings, another study reported elevated TOS and OSI levels in migraine patients compared with controls [32]. However, some studies have reported no significant differences in TAS, TOS, or OSI levels between migraine patients and control groups [28, 33]. The lack of sufficient subgrouping in these studies was noted as a potential limitation contributing to the inconsistent results. In one study, although no differences in the TAS, TOS, or OSI were observed between migraine patients and controls, the serum thiol level, an indicator of antioxidant capacity, was significantly lower in migraine patients than in controls [12]. Current study demonstrated that the presence of comorbidities in migraine patients further increases oxidative stress levels. While 6 months of migraine treatment significantly reduced oxidative stress in both migraine groups, the oxidative stress levels remained higher than those in the control group did, particularly in those with comorbidities, indicating that the treatment alleviated symptoms but did not fully resolve the underlying oxidative stress.

In the current study, IMA levels were significantly elevated in both migraine groups compared to the control group, with the highest levels observed in the migraine with MC group. Moreover, IMA showed a positive correlation with OSI, suggesting a mechanistic link between ischaemic stress and oxidative imbalance in migraine patients. Although IMA levels slightly decreased after six months of treatment, they remained significantly higher than those in healthy controls, indicating that ischaemic stress may persist despite symptomatic relief. This persistent elevation may reflect a chronic underlying endothelial dysfunction, particularly in patients with cardiovascular comorbidities. Indeed, IMA is increasingly recognised not only as an acute marker of ischaemia but also as an indicator of sustained endothelial alterations and vascular stress. Such dual functionality may explain why elevated IMA is more pronounced in patients with migraine who also carry cardiovascular risk factors, as comorbid conditions exacerbate redox imbalance and perpetuate vascular injury [4, 34–36]. These findings are consistent with the literature, which supports IMA as a sensitive biomarker for ischemia. IMA is currently the only ischemic marker approved by the U.S. Food and Drug Administration, and previous research has shown its elevation during acute ischemic and hemorrhagic strokes [34–36]. Although only a few studies have directly examined IMA in the context of migraine, Ersoy et al. demonstrated that increased serum IMA levels in migraine patients indicate ischemia/hypoxia and an oxidant–antioxidant imbalance. Similarly, Say et al. reported elevated IMA and prolidase levels in migraine, reinforcing the association between oxidative stress and migraine pathophysiology [4, 37]. The higher IMA levels observed in the current study MC group may reflect the additive effect of migraine and comorbid vascular conditions, aligning with studies that link migraine—especially with aura—to increased risk of ischemic stroke and myocardial infarction [4, 34–36].

HIF-1α was found to be significantly elevated in the M and MC groups compared with the control group. Although HIF-1α did not correlate with IMA levels, it was positively correlated with OSI levels. Elevated HIF-1α levels are likely a compensatory response to chronic oxidative stress in migraine patients, as this factor promotes angiogenesis and adaptive cellular mechanisms to counteract hypoxia-induced damage [38]. The decrease in HIF-1α levels after treatment in both groups suggests that migraine therapies may reduce hypoxia-induced stress; however, the persistence of elevated levels compared with those in controls indicates that the underlying hypoxic conditions are not entirely resolved. The increase in HIF-1α observed in the current study may therefore reflect both tissue hypoxia and the accumulation of reactive oxygen species (ROS). In the presence of cardiovascular comorbidities, this hypoxia–ROS interplay is particularly pronounced, potentially accounting for the higher HIF-1α levels detected in the MC group. These findings are consistent with broader literature that positions HIF-1α as a central mediator of hypoxic and oxidative responses in cerebrovascular and systemic disease. Supporting this interpretation, a clinical study reported that HIF-1α levels were elevated in paediatric patients experiencing migraine attacks, with serum peptide levels associated with ROS showing a positive correlation with HIF-1α [39]. Another study demonstrated that HIF-1α levels remain increased in chronic migraine patients receiving long-term antimigraine treatment [40].

In this study, the MC group presented significantly greater levels of DNA damage than did the M group, suggesting that comorbid conditions further exacerbate oxidative DNA damage in migraine patients. In migraine, mitochondrial dysfunction results in inefficient ATP production and excessive ROS generation. This impaired energy metabolism contributes to oxidative DNA damage, neuroinflammation, and sensitisation of meningeal pain pathways. [41]. Current studys’ findings align with the broader literature, which indicates that chronic exposure to oxidative stress can lead to cumulative DNA damage over time, thereby increasing the risk of long-term complications, including cardiovascular diseases [28, 32, 41]. Supporting this interpretation, Geyik et al. reported that both migraine patients with and without aura demonstrated significantly elevated plasma levels of 8-hydroxy-2′-deoxyguanosine, a marker of oxidative DNA damage, compared with healthy individuals [28]. Similarly, Yigit et al. showed that plasma DNA damage, TOS, malondialdehyde (MDA) levels, and OSI values were higher in migraine patients than in controls [32]. Importantly, in this study, the greater reduction in DNA damage observed in the MC group compared with the M group after six months of treatment suggests that antimigraine therapy may be particularly effective in mitigating oxidative DNA damage in patients with comorbidities. Taken together, these results not only confirm the role of oxidative stress in migraine pathophysiology but also highlight the potential long-term vascular and systemic implications of sustained DNA damage, consistent with prior evidence linking oxidative DNA injury to vascular and neurodegenerative disorders [28, 32, 41].

Moreover, the positive correlation between HIF-1α and DNA damage observed in current studys’ suggests a close link between hypoxic stress and oxidative DNA damage in migraine patients. This association highlights the potential role of HIF-1α as a biomarker for oxidative stress-induced cellular damage, which may contribute to the increased cardiovascular risk observed in migraine patients, especially those with comorbidities. However, no significant correlation was found between IMA and HIF-1α or between IMA and DNA damage. This may reflect distinct temporal dynamics and compartmental responses of these biomarkers. HIF-1α is a transcription factor primarily activated within hypoxic tissues, regulating downstream gene expression, whereas IMA reflects a circulating biochemical response to ischemia and oxidative modification of albumin [15, 16]. The lack of correlation could suggest that while both respond to ischemia, they do so through different mechanisms or on different timelines. Similarly, the absence of a correlation between IMA and DNA damage may be due to the fact that DNA damage reflects cumulative oxidative stress at the nuclear level, whereas IMA is more indicative of acute extracellular oxidative changes [42–45].

The association between migraine and cardiovascular disease has been well documented, particularly for women with migraine with aura, who are at a greater risk for ischemic stroke and myocardial infarction [5, 46]. Current studys’ findings support this finding, as the MC group presented higher oxidative stress levels and more pronounced changes in the levels of ischemic markers, such as IMA and HIF-1α, than the non-MC group did. This condition may render them vulnerable to further cardiovascular events due to increased oxidative stress and increased ischemic burden. Thus, this population should be targeted for tight surveillance and the management of CVD risk factors [9].

Moreover, results obtained in this current study suggest that despite symptomatic treatment, migraine patients with cardiovascular comorbidities continue to exhibit elevated levels of oxidative stress, ischemia-related biomarkers (IMA and HIF-1α), and DNA damage. These biochemical abnormalities likely reflect a persistent state of vascular dysfunction that is not adequately addressed by conventional migraine therapies. Given the elevated burden of ischemic and oxidative markers, cardiovascular risk stratification and routine monitoring should be considered integral to the management of migraine patients with comorbidities. Additionally, incorporating adjunctive strategies—such as pharmacologic agents with vascular protective properties (e.g., statins, ACE inhibitors) and lifestyle modifications (e.g., smoking cessation, dietary interventions)—may help reduce long-term vascular risk and improve outcomes beyond migraine relief [30, 42, 47]. This integrative, risk-targeted approach could prove valuable in preventing cerebrovascular and cardiovascular complications in this vulnerable patient population. Taken together, these results highlight the translational relevance of integrating cardiovascular risk management into migraine care, especially in patients with comorbid conditions, in order to mitigate both neurological and vascular complications.

Limitations

While this study offers valuable insights, there are several limitations to consider. While conducting the study in a controlled, single-institution setting ensured methodological consistency and standardized data collection, we acknowledge that it may have limited the generalizability of current studys’ findings. The patient population may not fully represent broader demographic or clinical profiles, and thus, selection bias cannot be entirely excluded. Although lifestyle factors were comparable between migraine groups and sampling was standardised, we could not fully standardise cardiovascular medications in the MC group; residual confounding cannot be entirely excluded. In addition, the study recruited only females; thus, the generalizability of these findings may be limited to males. The relatively short follow-up period limits the assessment of long-term cardiovascular outcomes in migraine patients. Furthermore, only the serum levels of the peptides were measured; there has been no investigation of either their expression or activity in the brain, where migraine pathology takes place. Further studies with large and various populations with longer follow-up periods are needed to investigate the long-term effects of oxidative stress and ischemia. Additionally, the menstrual cycle status of females was not considered during current study. This may have influenced the evaluation of migraine-type headaches and the stratification of the results we obtained. Additional studies are needed to determine the effects of specific antioxidant and anti-ischemic therapies on migraine frequency and cardiovascular risk.

Conclusions

This study shows that cardiovascular comorbidities significantly raise oxidative stress and DNA damage in migraine patients, with those in the MC group showing higher levels than both non-comorbid migraine patients and healthy controls. Although some improvement was observed after six months of treatment, levels did not return to normal, indicating persistent underlying vascular and ischemic issues. These findings underscore the value of oxidative stress and DNA damage as clinical biomarkers, especially in patients with comorbidities, who should be included in long-term cardiovascular risk strategies. Future studies with larger, more diverse groups are needed to assess the effectiveness of targeted antioxidant and anti-ischemic treatments.

Supplementary Information

Below is the link to the electronic supplementary material.

Abbreviations

CVD

Cardiovascular disease

HIF-1α

Hypoxia-inducible factor-1α

ICD-10

International classification of diseases, 10th revision

ICHD-3

International classification of headache disorders, 3rd edition

IMA

Ischemia-modified albumin

MC

Migraine with comorbidity (group)

M

Migraine without comorbidity (group)

MDA

Malondialdehyde

OSI

Oxidative stress index

ROS

Reactive oxygen species

TAS

Total antioxidant status

TOS

Total oxidant status

Author contributions

MG: conceptualization, data curation, formal analysis, investigation, methodology, validation, and writing—original draft. MYB: investigation, funding, conceptualization, methodology, resources, supervision, validation, and writing—review and editing. MU: resources, and writing—review and editing. CU: conceptualization, resources, visualization. FU: conceptualization, resources, visualization. EMG: methodology, supervision, validation, and writing—review and editing.

Funding

This study was not funded by any organization.

Data availability

The datasets generated and/or analyzed during this study are not publicly available for ethical reasons but are available from the corresponding author upon reasonable request.

Declarations

Ethics approval and consent to participate

Study approval was obtained from the Ethics Committee of Clinical Research (2024-KAEK-03). Informed consent was obtained from all the subjects prior to the study.

Consent for publication

All participants involved in this study provided written informed consent for the publication of their anonymized data. The authors affirm that personal or identifiable information is not disclosed in the manuscript. The study strictly adhered to ethical guidelines, ensuring that the privacy and confidentiality of all participants were protected throughout the research process.

Competing interests

The authors declare that they have no competing interests.