2.1. Study design and population
In this cross-sectional study, 265 patients diagnosed with migraine were recruited. From August 2019 to June 2020, we consecutively evaluated patients with a suspect of migraine in the Imam Moosa Sadr neurology clinic and Khurshid neurology clinic, both affiliated with Isfahan University of Medical Sciences, Isfahan, Iran. Using a simple random sampling method, participants were selected. Patients were included if they met the following criteria: individuals (20–50 years old) with a diagnosis of migraine by a neurologist (F.K) based on the International Classification of Headache Disorders 3 (ICHD3) criteria [11]; visiting the neurology clinic for the first time, and; body mass index (BMI) of 18.5–30. Participants with a history of cardiovascular disease, hypertension, diabetes, cancer, hepatic, renal or thyroid disease, and other neurological disorders were excluded due to possible disease-related changes in diet, along with those who were taking nutritional and herbal supplements including riboflavin, magnesium, coenzyme Q10, and feverfew. Patients with reported daily energy intakes lower than 800 kcal/d (3347 kJ/d) or higher than 4200 kcal/d (17573 kJ/d), were also excluded [12]. All participants provided written informed consent forms. From 298 invited participants, 262 of them completed the study (response rate 87%). The study protocol was approved by the research ethics committee of Isfahan University of Medical Sciences (IR.MUI.RESEARCH.REC.1398.352) on 3 November 2019.
2.2. 24-h urine collection
24-h urine collection containers were distributed among patients, along with verbal and written instructions on how to complete the procedure. It was highlighted that no alteration in dietary habits during the day of collection was allowed. 24-h sampling was done over the weekends from Friday to Saturday during a 24-h period. Each individual was provided with a polypropylene container (2.5 liters) used for the collection of the 24-h urine samples. A single 24-h urine collection was obtained with the first voided urine on waking on Friday morning being discarded and participants then collecting all voided urine up to and including the first void of the Saturday morning. All patients were asked to keep the containers in dry and cool places and samples were transferred immediately to the laboratory on Saturday for analysis to prevent microbial degradation. Sodium and potassium were assessed using the ion-selective electrode method (ProLyte Electrolyte Analyzer) and urine creatinine (Cr) was measured by the Jaffe reaction method (BT 3000) [13]. Suspected inaccurate urine collections were defined as urinary creatinine < 6 mmol/day for men, or < 4 mmol/day for women, or a 24-h urine volume < 500 mL which were excluded [14]. For each individual, the 24-h sodium and potassium excretion value (mmol/day) was calculated as the concentration of sodium and potassium in the urine (mmol/L) multiplied by the urinary volume (L/day). The conversion from mmol to mg was made by multiplying by 23 for sodium and 39 for potassium, and the conversion from mg of sodium to salt by multiplying by 2.542 [14].
2.3. Dietary assessment
The dietary intake of participants over the past year was assessed using a semi-quantitative food frequency questionnaire (FFQ). This questionnaire included a list of 168 food items, along with a standard serving size for each, and its reliability and validity had been approved previously among the Iranian population [15–17]. The patients were asked to report the consumption frequency of a given serving of each food item on a daily, weekly, or monthly basis. All of the consumed foods portion sizes were transformed into grams using household measures [18]. FFQ was completed by a trained dietitian through face to face interview and data were analyzed by Nutritionist IV software (First Databank, Hearst Corp, San Bruno, CA, USA).
2.4. Anthropometric measurements
Bodyweight was measured to the nearest 100 g using a digital scale (Omron BF511 (Omron Corp, Kyoto, Japan)), while the subjects were clothed minimally (i.e. no belts, sweaters, or jackets) and not wearing shoes. Height was measured using an upstretched tape to the nearest 1 mm, while the subject was in a standing position without shoes, and the shoulders were relaxed. BMI was calculated from the height in m2 and weight in kg, using the “weight (kg)/height2 (m2)” equation.
2.5. Assessments of migraine clinical features
Headache diaries regarding the clinical features of migraine during the previous month were obtained from all of the participants. A 30-day headache diary was given to all participants, along with verbal and written instructions on how to complete the procedure. Clinical features of migraine including the time of migraine attack onset, duration, and severity scores (ranged from 0–10 based on a visual analog scale (VAS)) had to be recorded accurately after each migraine attack no matter what time of day. If the patients had any difficulties in filling their diaries, there was a contact person (A.A) who would answer their questions via phone calls or text messages. The participants were asked to complete their headache diaries during the upcoming month [19]. The VAS questionnaire was used to assess the severity of headache [20]. Based on this scale, the headache severity is ranked from 0 to 10, with “0” indicating no pain and “10” the worst imaginable pain. The participants denote on the point that they feel represents their perception of their current pain. The frequency was defined as the number of attacks per month. The mean duration of headache attacks in hour was considered as the duration. Furthermore, migraine headache index score (MHIS) calculated using “frequency × duration × severity” equation [21].
The headache impact test (HIT-6) was used to evaluate the impact of headaches on the patients’ quality of life. The HIT-6 is a validated questionnaire [22] containing 6 questions with 5 options for each including never (scored as 6), rarely (8), sometimes (10), very often (11), and always (13), with a total possible score of 36–78. The scores of 36–49, 50–55, 56–59, and ≥ 60 indicate that headache has no, moderate, substantial, and severe impact on the quality of life of the participants [23].
2.6. Assessment of other variables
Additional information was collected by researchers using a demographic questionnaire, which contains questions about age, gender, marital status, number of family members, family history of migraine, time since diagnosis of migraine, and drug consumption.
Physical activity (PA) status was assessed via International Physical Activity Questionnaire (IPAQ), a self-administered, 7-day recall instrument, which its validity and reliability had been approved previously among the Iranian population [24]. PA levels were stated as metabolic equivalent hours per day (METs h/day).
Fasting Blood samples (10 ml) were centrifuged (Avanti J-25, Beckman, Brea, CA, USA) at 3500 rpm for 10 min to separate serum immediately after collection and were then maintained at − 80 °C for further analyses. The serum nitric oxide (NO) values were assessed using the Griess method via commercial kits (Kiazist Life Sciences, Iran).
2.7. Statistical Analyses
A suggested formula for estimating mean was used to compute the sample size. Based on α = 0.05, = 0.9, and d = 0.1 based on sodium level in patients with migraine, we reached to 260 subjects [25]. Continuous and categorical variables were presented as mean ± standard error (SE) and number (percentage), respectively. The characteristics distribution of the study population was compared by gender using independent t-test or Chi-square test as appropriate. Analysis of variance (ANOVA) was used for comparing clinical features of migraine headache across quartiles (Q) of 24-hour urinary sodium, potassium, and Na/K ratio. To assess relationships between the 24-hour urinary sodium, potassium, and Na/K ratio with clinical features of migraine headache, multiple linear regression analysis was performed in different models. Adjusted Beta (β)s with 95% confidence interval (CI) are presented in 2 different models. First, we adjusted for age, sex, marital status, smoking status, migraine type, migraine characteristic, family history, mean arterial pressure, and physical activity. In the last model, further adjustments were made for BMI and energy intake. Data analyses were performed using Statistical Package for Social Sciences (SPSS) version 21 (IBM Corp, Armonk, NY, USA). P-values < 0.05 were considered statistically significant.