Introduction

Multiple sclerosis (MS) is the most common autoimmune disease of the central nervous system (CNS) and one of the main causes of neurological disability in young adults. The prevalence of MS varies between 2 and 160 per 100,000 according to the geographical area [1]. The etiology of the disease is not fully understood, though all the proposed theories point to the interplay between genetic and environmental risk factors [2]. Major histocompatibility complex with HLA-DRB1 showed the strongest genetic predisposition to MS [3]. Many genes have been proposed as well, Zn-ion binding genes which are involved in interferon-γ (IFN-γ) expression can predict the clinical outcome in relapsing remitting multiple sclerosis (RRMS) patients with high accuracy [4].

Zinc is an essential trace element, required by a considerable number of enzymes, transcription factors, and proteins [5, 6]. Actually, Zn deficiency suppress several immune system functions including T-helper cell function, cytotoxic T cell activity, peripheral T cell count, natural killer (NK) cells, neutrophil, and macrophage functions [7].

The brain has an abundant amount of Zn which is mainly found in the presynaptic vesicles. Efficient Zn homeostasis in neural cells is of great importance; deficiency of Zn can induce apoptosis while high level of Zn is reported to be neurotoxic [8, 9].

To what extent zinc deficiency could play a critical role in the pathogenesis of multiple sclerosis and therefore affect disease progression, clinical outcome and disability status remain a matter of debate.

Aim of the work

The aim of this study was to assess serum Zinc level in patients with relapsing remittent multiple sclerosis at time of relapse and during remission and to correlate serum zinc with degree of disability, fatigue, response to disease modifying drugs, and magnetic resonance imaging (MRI) findings.

Subjects and methods

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.

Results

The mean age for patients in relapse was (30.88 ± 9.01) years, for patients in remission was (32.28 ± 8.07) years, and for controls was (28.28 ± 7.08) years. Regarding sex, 16% (n = 4) of patients in relapse were males and 84% (n = 21) were females, and 36% (n = 9) of patients in remission were males and 64% (n = 16) were females. As for controls, 44% (n = 11) were males and 56% (n = 14) were females (p = 0.47). There was no statistically significant difference between patients and controls in either age (P value = 0.214) or sex (P value = 0.092) (Table 1).

Table 1 Demographics of MS patients and control group
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Regarding clinical and radiological characteristics of MS patients, there was no statistically significant difference between patients in relapse and patients in remission in either disease duration, number of relapses in the last 2 years, total number of relapses, EDSS, MRS, MFIS, or MRI lesion load (Table 2).

Table 2 Clinical and radiological characteristics of MS patients
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Regarding serum zinc level, patients in relapse were found to have significantly lower mean values (65.29 ± 16.92) μg/dL than the control group (82.15 ± 15.496) μg/dL (P value = 0.001). Patients in remission were also found to have significantly lower mean values (67.97 ± 15.28) μg/dL than the control group (82.15 ± 15.496) μg/dL (P value < 0.001). There was no statistically significant difference between patients in relapse and patients in remission in serum zinc level (P value = 0.56) (Table 3).

Table 3 Serum zinc level in MS patients and control group
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There was a statistically significant negative correlation 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 (Table 4).

Table 4 Correlation between serum Zinc level and clinical and radiological characteristics of MS
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Discussion

In the last few years, serum zinc levels was thoroughly investigated in patients with multiple sclerosis to understand its potential role in pathogenesis of MS. Lower serum zinc levels were found to be associated with higher disease disability[14].

The aim of this work was to evaluate serum Zinc level in patients with relapsing remittent multiple sclerosis at time of relapse and during remission and to correlate serum zinc with degree of disability, fatigue, response to disease modifying drugs, and MRI findings.

In the present study, MS patients in relapse and remission were found to have significantly lower serum Zinc level compared to controls. There was a statistically significant negative correlation 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.

Similar to our findings, Pawlitzki and colleagues found that MS patients had a significantly lower concentrations of zinc compared to healthy controls but there was no significant relationship between serum zinc and either disease duration, median number of relapses, annual relapse rate, or EDSS [15].

Another study carried out by Ho and colleagues reported that serum levels of Zn significantly decrease during relapse and increase during remission [16].

On the other hand, Geeta and colleagues found no difference in serum zinc levels between MS patients and control groups [17]. Additionally, Sedighidid and colleagues did not find a significant difference between serum zinc levels in MS patients and controls [18]. The same findings were also obtained by Mellow and colleagues who compared zinc level in patients with MSto control group [19].

A recent meta-analysis carried out by Bredholt and Frederiksen included 13 studies evaluating plasma or serum Zn levels in MS patients. The investigators found that MS patients had a statistically significant reduction in overall plasma or serum Zn levels, with few studies reporting either increase or no difference in plasma or serum Zn levels [14].

Zinc plays an important role in suppression of potentially harmful immune reactions against T lymphocytes, and predisposing inflammatory responses of MS. Zn also has an antioxidant effect protecting myelin and cell membranes [20].

During active disease stages, upregulation of zinc-dependent matrix metalloproteinases occurs which in turn leads to lower zinc levels in MS patients [21]. Zinc deficiency is also reported to play an important role in inducing an imbalance between Th1 and Th2 functions [22], in addition to its role in prohibiting downregulation of Th17 lymphocytes [23]. These changes have been proposed to be major mechanisms in the pathogenesis of MS [24].

Also, zinc was reported to be involved in releasing tumor necrosis factor alpha (TNFα), which plays an important role in immune system activation. Consequently, even a mild Zn deficiency can impair the immune system functions [25, 26].

The results of studies which assessed the effect of Zn supplementation revealed a reduction in proliferation and activation of T cell following Zn supplementation thereby considered a potential therapeutic approach to MS [27]. On the other hand, a recent study revealed that neurological signs did not improve in patients with MS after Zn supplementation for 3 months compared to the placebo group [28]. An interesting study carried out by Choi and colleagues examined the effect of redistributing Zn in Experimental Autoimmune Encephalitis (EAE) in an animal model of MS using Clioquinol (a Zincor Copper chelator) [29]. The study revealed promising results regarding suppression of demyelination, inhibition of BBB disruption, reduced Matrix metalloproteinase-9 activation, reduced infiltration of encephalitogenic immune cells, and reduced clinical score of EAE following treatment with Clioquinol.

Conclusion

At the end of the study, we conclude that serum Zinc level is significantly lower in patients with multiple sclerosis than healthy controls. It is negatively correlated with disease duration, number of relapses, motor disability, fatigue, and MRI lesion load.

The limitation of this work is the relatively small number of patients due to financial issues and the limitation of resources.

Further studies should be conducted on a larger number of MS patients to estimate the effect of zinc supplementation on motor disability, fatigability, and number of relapses.