This is a case-control study which included 75 subjects divided into three groups, 25 patients diagnosed with relapsing remitting multiple sclerosis (during relapse), 25 patients diagnosed with relapsing remitting multiple sclerosis (during remission), and 25 age- and sex-matched healthy controls. The study was approved by the ethical committee in Faculty of medicine, Beni-Suef University (FWA00015574in 9th of October 2018), and all participants in this study signed a written informed consent.
Patients were diagnosed as relapsing remitting multiple sclerosis (RRMS) according to the International Panel on Diagnosis of Multiple Sclerosis “McDonald’s criteria 2017” [10] (25 patients during relapse and 25 patients during remission). Relapse was defined by the appearance of new neurological symptoms or worsening of pre-existing neurological symptoms lasting at least 24 h in the absence of fever or infection in a patient who was neurologically stable or improving for the previous 30 days accompanied by objective changes on neurological examination (worsening on the Expanded Disability Status Scale (EDSS) of 0.5 point or a worsening by one point on the pyramidal, cerebellar, brainstem, or visual functional system scores). Age of the included patients ranged between 15 and 45 years.
The following patients were excluded from the study: patients with history of any other autoimmune disease, cerebrovascular disease, malignancy, hypertension, diabetes mellitus, any infectious or inflammatory diseases in the previous month, or pregnant females.
All patients included in the study were subjected to the following:
Neurological assessment
Expanded Disability Status Scale (EDSS): a scale used for assessment of the neurological disability status of MS patients. The functional systems included are pyramidal, brain stem, cerebellar, sensory, bowel and bladder, cerebral and visual. Its score ranged from 0 (normal) to 10 (death due to MS) [11].
Modified fatigue impact scale (MFIS): the modified version of the 40-item Fatigue Impact Scale (FIS), which was used to evaluate the effect of fatigue on quality of life in patients with MS and can be used in patients with other chronic diseases. The full-length MFIS consists of 21 items; 10 “cognitive” items, 9 “physical” items, and 2 “psychosocial” items. Higher scores indicate a greater impact on patient’s quality of life, and the maximum possible score is 84. The time needed to complete the scale is approximately 5–10 min [12].
Modified RIO score (MRS): a 0 to 3 scale which evaluates the response of MS patients to disease modifying therapy. It is obtained by a combination of MRI and relapse criteria. (i) MRI criterion 1 point for patients with > 4 new T2 lesions; (ii) relapse criterion 1 point for patients experiencing 1 relapse and 2 points for patients experiencing ≥ 2 relapses [13].
Laboratory assessment
Serum level of zinc was measured for all patients and controls included in this study using direct colorimetric test method (Reactivos GPL, Barcelona, Spain). This was applied at clinical pathology department, Beni-Suef University hospital on a semi-automated photometer (ELITechGroup VITAL, Dieren–The Netherlands). Five milliliters of whole blood was collected from both patients and controls into sterile plain tube and centrifuged for 10–15 min at 3000 r.p.m. Supernatant clear sera were collected, and Zinc was immediately measured at wave length 578 nm at 37 °C (range of normal value = 68–107 μg/dL)
Radiological assessment
Magnetic resonance imaging (MRI) on brain, cervical, and dorsal segments of the spinal cord were carried out for all patients included in this study to detect site, size, and number of MS plaques and to exclude other structural lesions. The scan was done by a 1.5 Tesla Siemens scanner, Germany. The following sequences were used: T1-weighted images (axial, sagittal), T2-weighted images (axial, coronal), fluid-attenuated inversion recovery (FLAIR), and gadolinium-enhanced T1-weighted axial and sagittal images for patients during relapse.
Statistical methods
The data were coded and entered using the statistical package for social science (SPSS) version 18 (SPSS Inc. Released 2009. PASW Statistics for Windows, Version 18.0. Chicago: SPSS Inc.). Student t test was used for comparison between means of quantitative variables in patients and control group. Chi-square test was used for comparison between categorical data in patients and control group. The one way analysis of variance (ANOVA) and post hoc analysis (Bonferonni test) were used to assess statistical differences between quantitative data inpatients in relapse, patients in remission, and control group. The Pearson correlation coefficient (r) was used to describe the degree of relationship between serum zinc level and disease duration, number of relapses in the last 2 years, total number of relapses, EDSS before and after pulse steroids, MRS, MFIS, and MRI lesion load. P value equal to or less than 0.05 was considered significant.