Migraine is one of the most prevalent and disabling neurological disorders worldwide, imposing an immense personal and socioeconomic burden [1]. Characterized by recurrent throbbing headaches typically accompanied by autonomic symptoms, photophobia, and phonophobia, migraine pathophysiology involves complex interactions between central neuronal hyperexcitability, trigeminovascular system activation, and sterile neurogenic inflammation [2, 3]. Despite decades of pharmacological advancements, managing refractory migraine remains challenging, prompting intense clinical interest in identifying modifiable metabolic risk factors and biochemical determinants that influence disease severity and frequency [4, 5]. Among various biochemical candidates, micronutrient status—specifically vitamin D and magnesium—alongside systemic inflammatory markers such as high-sensitivity C-reactive protein (hs-CRP), have emerged as pivotal modulators of neurovascular homeostasis [6, 7]. Vitamin D, traditionally recognized for its endocrine role in calcium and bone metabolism, functions as a potent neurosteroid within the central nervous system [8]. Receptors for 1,25-dihydroxyvitamin D3 are widely distributed across pain-modulating brain regions, including the periaqueductal gray, thalamus, and trigeminal nucleus caudalis [9]. Adequate vitamin D exerts neuroprotective, anti-inflammatory, and immunomodulatory effects by suppressing pro-inflammatory cytokine transcription and upregulating neurotrophic factors [10]. Emerging clinical investigations indicate that vitamin D deficiency is disproportionately prevalent among individuals suffering from chronic pain syndromes, including migraine, where it correlates inversely with attack frequency and headache intensity [11, 12]. Nevertheless, regional data from South Asian cohorts remain critical given distinct dietary habits, skin pigmentation, and sunlight exposure patterns [13]. Similarly, magnesium plays a fundamental physiological role in neurological function, acting as a natural physiological antagonist of N-methyl-D-aspartate (NMDA) receptors and a critical cofactor for numerous enzymatic reactions governing vascular tone and energy metabolism [14, 15]. Intracellular magnesium depletion lowers the threshold for cortical spreading depression—the electrophysiological phenomenon underlying migraine aura—and promotes abnormal neuronal depolarization and trigeminal activation [16]. Clinical trials have demonstrated that magnesium supplementation can mitigate migraine frequency and severity, yet routine biochemical assessment of serum magnesium in migraineurs remains underutilized in clinical practice [17, 18]. Furthermore, magnesium homeostasis is intimately linked with inflammatory pathways, wherein depletion triggers oxidative stress and endothelial activation [19]. Systemic low-grade inflammation is increasingly recognized as a core pathophysiological component linking metabolic abnormalities to migraine pathogenesis [20]. High-sensitivity C-reactive protein (hs-CRP), a canonical acute-phase reactant synthesized by the liver in response to interleukin-6 and other pro-inflammatory stimuli, serves as a reliable systemic marker of vascular inflammation [21]. During migraine attacks, sterile neurogenic inflammation within the trigeminovascular system releases calcitonin gene-related peptide (CGRP) and substance P, which subsequently stimulate hepatic acute-phase responses and elevate circulating hs-CRP levels [22, 23]. Elevated hs-CRP has been consistently linked to higher headache frequency, chronification from episodic to chronic migraine, and greater functional disability measured via standardized instruments such as the Migraine Disability Assessment (MIDAS) scale [24, 25]. While individual studies have examined vitamin D, magnesium, or hs-CRP in isolation, comprehensive investigations evaluating their interconnected tripartite relationship with migraine severity, frequency, and functional disability within an Indian hospital setting are exceptionally scarce [26]. Understanding how these biochemical parameters interact could provide valuable prognostic insights and support targeted adjunctive nutritional and anti-inflammatory strategies [27, 28]. Therefore, the present prospective observational study was undertaken to evaluate the association of serum vitamin D, magnesium, and high-sensitivity C-reactive protein with migraine severity, attack frequency, and functional disability among patients attending Mallareddy Medical College for Women in Hyderabad, Telangana [29, 30].

Study Design and Setting This investigation was executed as a prospective, single-center observational clinical study at Mallareddy Medical College for Women, Hyderabad, Telangana, India. The study was conducted over a two-year period, spanning from July 2023 to December 2024. Participants were recruited from the inpatient neurology wards and outpatient neurology and general medicine clinics. The study protocol adhered strictly to the ethical principles outlined in the Declaration of Helsinki (2013 revision). Formal institutional ethical approval was granted by the Institutional Ethics Committee prior to commencement, and written informed consent was acquired from all enrolled participants. Participant Selection and Eligibility Criteria A total of 420 patients presenting with recurrent headaches were screened for eligibility during the study window. Inclusion criteria comprised adult patients aged 18 to 65 years who fulfilled the International Classification of Headache Disorders (ICHD-3) diagnostic criteria for episodic or chronic migraine. Exclusion criteria included secondary headache disorders, pregnancy or lactation, chronic renal or hepatic failure, active infectious diseases, autoimmune disorders, malignancy, current supplementation with vitamin D or magnesium within the preceding three months, and chronic use of anti-inflammatory medications or corticosteroids. Following rigorous application of these criteria, 350 eligible patients were successfully enrolled and included in the final analysis. Clinical and Biochemical Assessments Comprehensive baseline demographic data, medical history, headache characteristics, and medication use were systematically recorded using a standardized patient assessment form. Migraine severity was quantified using the Visual Analog Scale (VAS, ranging from 0 to 10). Attack frequency was determined prospectively via headache diaries over a three-month observation period. Functional disability was evaluated using the Migraine Disability Assessment (MIDAS) questionnaire, categorizing patients into minimal, mild, moderate, or severe disability tiers. Venous blood samples (10 mL) were collected from each participant under aseptic fasting conditions between 8:00 AM and 10:00 AM. Samples were centrifuged at 3000 rpm for 10 minutes to separate serum, which was immediately aliquoted and stored at -80°C until biochemical analysis. Serum 25-hydroxyvitamin D [25(OH)D] concentrations were quantified using chemiluminescent microparticle immunoassay (CMIA). Serum magnesium levels were measured using xylidyl blue colorimetric assay on an automated chemistry analyzer. Serum high-sensitivity C-reactive protein (hs-CRP) levels were determined via particle-enhanced immunoturbidimetric assay, with values expressed in mg/L. Statistical Analysis All statistical evaluations were performed using SPSS software (version 26.0, IBM Corp., Armonk, NY, USA). Quantitative variables were tested for normality using the Kolmogorov-Smirnov test and expressed as mean ± standard deviation (SD) or median with interquartile range. Categorical variables were presented as absolute frequencies and percentages. Pearson's or Spearman's correlation coefficients were calculated to assess relationships between biochemical parameters (vitamin D, magnesium, hs-CRP) and clinical metrics (VAS, attack frequency, MIDAS score). Multivariate linear and ordinal logistic regression models were constructed to identify independent predictors of migraine disability. A two-tailed probability value of p < 0.05 was established as statistically significant.

A total of 350 patients diagnosed with migraine completed the prospective evaluation protocol at Mallareddy Medical College for Women, Hyderabad, Telangana, between July 2023 to December 2024. The cohort comprised 274 females (78.3%) and 76 males (21.7%), with a mean age of 38.6 ± 10.4 years. The clinical profile revealed 242 patients (69.1%) with episodic migraine and 108 patients (30.9%) with chronic migraine. 3.1 Baseline Biochemical Profile and Migraine Characteristics Evaluation of micronutrient and inflammatory markers demonstrated widespread deficiencies across the cohort. The mean serum 25-hydroxyvitamin D level was 20.4 ± 7.8 ng/mL, with 64.3% of participants exhibiting vitamin D deficiency (<20 ng/mL). The mean serum magnesium concentration was 1.82 ± 0.31 mg/dL, with 48.6% classified as hypomagnesemic (<1.8 mg/dL). Serum hs-CRP concentrations showed a skewed distribution, with a median of 3.2 mg/L (IQR: 1.6 - 5.8 mg/L). Mean VAS pain severity score stood at 7.4 ± 1.5, mean monthly attack frequency was 8.6 ± 4.2 attacks per month, and mean MIDAS disability score was 31.4 ± 16.8 days, reflecting substantial clinical burden (Table 1; Figure 1). Table 1. Baseline Demographic, Clinical, and Biochemical Characteristics of Study Participants (N = 350) Parameter Mean ± SD / Frequency Percentage / Range Age (Years) 38.6 ± 10.4 19 - 62 years Gender (Female / Male) 274 / 76 78.3% / 21.7% Migraine Subtype (Episodic / Chronic) 242 / 108 69.1% / 30.9% Serum Vitamin D (ng/mL) 20.4 ± 7.8 8.2 - 42.5 ng/mL Serum Magnesium (mg/dL) 1.82 ± 0.31 1.12 - 2.55 mg/dL Serum hs-CRP (mg/L)* 3.2 (1.6 - 5.8) 0.4 - 14.2 mg/L *Values for serum hs-CRP are expressed as median and interquartile range (IQR) due to non-normal distribution. .2 Correlation Analysis Between Biochemical Biomarkers and Migraine Metrics Bivariate correlation analyses revealed highly significant relationships between biochemical markers and clinical severity parameters. Serum vitamin D demonstrated strong inverse correlations with VAS pain score (r = -0.542, p < 0.001), attack frequency (r = -0.612, p < 0.001), and MIDAS disability score (r = -0.658, p < 0.001). Similarly, serum magnesium exhibited robust inverse correlations with VAS score (r = -0.485, p < 0.001), attack frequency (r = -0.681, p < 0.001), and MIDAS score (r = -0.594, p < 0.001). In contrast, serum hs-CRP showed positive correlations with headache severity (r = 0.512, p < 0.001), attack frequency (r = 0.634, p < 0.001), and functional disability (r = 0.605, p < 0.001) (Figure 2). 3.3 Association of Micronutrient Quartiles with Functional Disability and Inflammation Stratification of the cohort into vitamin D quartiles demonstrated a stepwise gradient in functional disability. Patients in the lowest quartile of vitamin D (<15 ng/mL) suffered from a mean MIDAS disability score of 48.6 ± 12.4 days, compared to 14.1 ± 5.2 days for those in the highest quartile (>30 ng/mL, p < 0.001). Furthermore, serum hs-CRP levels were significantly elevated across migraine subtypes, peaking in chronic migraine (5.7 ± 2.1 mg/L) compared to episodic migraine without aura (1.9 ± 0.8 mg/L, p < 0.001) (Table 2; Figure 3, 4). Table 2: Correlation Coefficients (r) Between Biochemical Markers and Migraine Clinical Parameters Biochemical Marker VAS Severity Score Attack Frequency (attacks/mo) MIDAS Disability Score Serum Vitamin D (ng/mL) r = -0.542 (p < 0.001) r = -0.612 (p < 0.001) r = -0.658 (p < 0.001) Serum Magnesium (mg/dL) r = -0.485 (p < 0.001) r = -0.681 (p < 0.001) r = -0.594 (p < 0.001) Serum hs-CRP (mg/L) r = +0.512 (p < 0.001) r = +0.634 (p < 0.001) r = +0.605 (p < 0.001)

The present prospective observational study conducted at Tertiary care hospital, Hyderabad, Telangana, provides compelling evidence regarding the robust associations linking serum vitamin D, magnesium, and high-sensitivity C-reactive protein with migraine severity, attack frequency, and functional disability. Our findings confirm that micronutrient deficiencies and systemic low-grade inflammation are highly prevalent among Indian migraineurs and serve as critical determinants of disease burden [11, 19]. The strong inverse correlations observed between serum vitamin D levels and clinical severity parameters reinforce the concept that vitamin D plays a neuroprotective and anti-nociceptive role in migraine pathophysiology [8]. As a potent neurosteroid, vitamin D modulates pain perception pathways within the central nervous system, inhibits pro-inflammatory cytokine synthesis, and reduces inducible nitric oxide synthase expression [10]. Our observation that patients in the lowest vitamin D quartile experienced severe functional disability (MIDAS > 45 days) aligns with international literature emphasizing the clinical relevance of correcting hypovitaminosis D in chronic pain management [12]. Similarly, serum magnesium concentrations demonstrated a striking inverse relationship with monthly attack frequency and headache intensity. Magnesium acts as a physiological NMDA receptor blocker, preventing calcium influx and subsequent cortical spreading depression [15, 16]. Intracellular magnesium depletion lowers seizure and spreading depression thresholds, precipitating recurrent trigeminovascular activation [17]. The robust correlation (r = -0.681) between serum magnesium and attack frequency underscores the imperative for evaluating and replenishing magnesium stores in clinical practice [18]. Elevated serum hs-CRP concentrations in our cohort highlight the contribution of sterile neurogenic inflammation to migraine chronification [21, 22]. The significant elevation of hs-CRP in chronic migraine compared to episodic subtypes corroborates the hypothesis that recurrent trigeminal activation perpetuates a systemic acute-phase response [23]. By integrating vitamin D, magnesium, and hs-CRP assessments, clinicians can better stratify patients at high risk for disabling migraine trajectories and implement targeted multimodal therapeutic interventions

In conclusion, this prospective hospital-based study demonstrates that serum vitamin D and magnesium deficiencies, alongside elevated high-sensitivity C-reactive protein levels, are significantly and independently associated with increased migraine severity, attack frequency, and functional disability among Indian patients. Routine biochemical screening for micronutrient status and systemic inflammation can enhance prognostic accuracy and guide personalized adjunctive management strategies in clinical neurology.

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