Volume 4, Issue 12 (Winter 2018)                   Caspian.J.Neurol.Sci 2018, 4(12): 30-34 | Back to browse issues page

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Barzegar M, Shaygannejad V, Mirmosayyeb O, Valipour S, Akbari R, Vesal S, et al . Serum Bilirubin Level Changes in Multiple Sclerosis Patients. Caspian.J.Neurol.Sci. 2018; 4 (12) :30-34
URL: http://cjns.gums.ac.ir/article-1-217-en.html
1- Neurosciences Research Center, Alzahra Institute, Isfahan University of Medical Sciences, Isfahan, Iran
2- PhD Department of Immunology, School of Medicine, Isfahan University of Medical Sciences, Isfahan, Iran
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ultiple sclerosis (MS) is a demyelinating immune disease of Central Nervous System (CNS) and a cause of disability in middle-aged people [1, 2]. It is characterize by clinical attacks, inflammation, demyelination and plaques in the brain and spinal cord [3]. In addition to role of immune mediated mechanisms in neuroimmunological disorders such as MS, recent findings suggest that oxidation of brain phospholipids and proteins have an important role in pathogenesis of these disorders [3-5]. Over production of the Reactive Oxygen Species (ROS) or Reactive Nitrogen Species (RNS) that cause oxidative and nitrosative stress plays a main role in neuropathology of Multiple Sclerosis (MS) during the disease onset and its promotion [6, 7].
Macrophages and microglial cells generate ROS and RNS during CNS inflammation. These cells mediate demyelination and neurodegeneration through apoptosis of oligodendrocytes and also oxidize cardinal cellular components such as DNA molecules, lipids and proteins [8, 9]. This pathological condition appears as a result of impaired in the redox homeostatic mechanisms and reducing in the endogenous antioxidants such as bilirubin levels. Bilirubin as an endogenous antioxidant plays critical role in the control of oxidative stress and ROS production [10]. It is an endogenous product of heme breakdown. Senescent heme degrades by hemeoxygenase inside macrophages of the spleen and metabolize to biliverdin, carbon monoxide and iron. Afterward, biliverdin is converted to bilirubin by biliverdin reductase [11, 12]
Two decades, researchers have indicated bilirubin is an important physiological neuroprotective antioxidant. Experimental researches on rats have displayed bilirubin with a protective effect against traumatic brain injuries, cerebral ischemia damage and intracerebral hemorrhage [13, 14]. Bilirubin protects Blood Brain Barrier (BBB) from immune cell invasion and scavenges the ROS in spinal cord lesions and protects CNS from damage in MS patients. Additionally in animal model of MS, therapeutic effect of bilirubin in long term has been more effective than dexamethasone and other antioxidants [15]. Liu.et al have explained the immunomodulatory role of bilirubin in the Experimental Autoimmune Encephalomyelitis (EAE) as a model for multiple sclerosis. Bilirubin as an immunomodulator, suppresses both polyclonal and antigen-specific T cell responses and also induces anergy against reactive CD4+T cells via inhibition of costimulatory signaling [16]. Despite decisive antioxidative and immunmodulatory properties of bilirubin, there is inadequate research that surveys its role in MS patients. So in the present study, we assessed the levels of bilirubin (total, indirect and direct), in order to determine the changes of this natural endogenous antioxidant in the serum of MS patients compared with healthy control subjects We also looked for a possible association between bilirubin serum concentrations and clinical characteristics of disorder and its progression.
Materials and Methods
Serum samples were collected from 174 individuals comprising 80 MS patients who attended to Kashani hospital MS clinic, Isfahan, Iran and 94 healthy controls. Venous blood was collected in the morning after an overnight fast from patients and Healthy Controls (HCs) for measurement of serum bilirubin. Serum total bilirubin (normal range: 0.1-1.3 mg/dl), Direct bilirubin (normal range: .01-0.4 mg/dl) and inDirect bilirubin (normal range: 0.1-0.9 mg/dl) concentration were measured using dichloroaniline method. At the same time, Alanine Transaminase (ALT) (normal range: 1-31 mg/dl for female and 1-41 mg/dl for male) and Aspartate Transaminase (AST) concentrations (normal range: 1-38 mg/dl for female and 1-40 mg/dl for male) were also measured with International Federation of Clinical Chemistry (IFCC) method. Four subjects (One patient and 3 HCs) were excluded from the study because of abnormal liver enzymes. Patient’s data were compared with 94 HCs. Demographic and clinically information of subjects are presented and summarized in Table 1. All patients had definite MS according to the criteria of McDonald et al. 2010 [17]. The severity of disease in patients was evaluated by using the Expanded Disability Status Scale (EDSS) score [18].
Exclusion criteria were liver enzyme abnormality, other neurological and autoimmune diseases, receiving of immunosuppressive or interferon medication at least 6 months prior to study entry. The study was approved by Vice President of research of Isfahan University of Medical Sciences (project number: 293398) and written informed consents were obtained from all subjects before enrollment.
The comparisons of all types of bilirubin concentrations between MS patients and HC subjects were examined with covariance. The relation between sex and age with serum levels of bilirubin in MS patients and HCs was assessed with covariance analysis. The relation between bilirubin concentration in MS patients and EDSS was analyzed using regression analysis. All statistical calculations were done using the SPSS 17.0 for Windows (SPSS, Chicago, IL, USA). All data for both groups are presented as mean (SD). The P<0.05 was considered as significant.
The basic demographic and clinical characteristics of the study subjects are given in brief version in Table 1. There was no difference between two groups in terms of gender and age (P>0.05). The mean EDSS of patients were 2.04±1.3. The serum concentrations of Direct bilirubin (Dbil) were lower in MS patients compared to healthy controls (P<0.05). Tbil and Ibil levels were higher in MS patients than those observed in healthy controls, but it was not statistically significant (Figure 1).
There was no statistical difference in Tbil, Dbil, and Ibil levels between female healthy controls and male healthy controls. But Tbil and Ibil levels in female MS patients were significantly lower compared to male MS patients. The comparison of Tbil, Dbil, and Ibil levels between the same genders in different groups showed that Tbil and Ibil levels were higher in male and female MS patients than those observed in healthy controls of the same gender, but it was not statistically significant (Table 2). There was a negative correlation between EDSS of MS patients and bilirubin levels, but it was not statistically significant (P>0.05). There was no correlation between disease duration and bilirubin levels in MS patients (P>0.05).
In the present study, we observed significantly lower serum concentration of Dbil in MS patients. But we found higher levels of Tbil and Ibili in male and female MS patients compared to healthy controls of the same gender. Some of previous studies have suggested patients with MS have a low level of serum bilirubin that our findings were inconsistent with these studies [19, 20].
To our knowledge, major limitations in previous studies have been a small number of patients included. Increasing of ROS activity by oxidative stress leads to nerve injury and may simultaneously cause an overuse of bilirubin. Although bilirubin concentrations in sera might be reduced as a result of an over consumption of bilirubin by produced ROS cleaning, but increasing of bilirubin in male and female MS patients in our study may be secondary for compensation of bilirubin over-consumption [21]. Liu et al. have suggested that increasing of bilirubin levels closely related to reduced neuroinflammation and better clinical manifestation of experimental disease [15]. Changes in activity of key enzymes which are involved in breakdown of porphyrin ring maybe effects on bilirubin production. Hemeoxygenase enzymes have key role in heme catabolism and yield equimolar amounts of biliverdin that is quickly converted by biliverdin reductase into bilirubin. Inducible Hemeoxygenase (HO-1) which induce by high levels of ROS in MS patients have antioxidative and anti-inflammatory properties, while constitutive Hemeoxygenase (HO-2) doesn’t have such properties [22, 23]
Many studies have shown enhanced HO-1 expression in residential CNS cells such as astrocytes, reactive microglia, and infiltrated macrophages in animals suffering from EAE. Residential cells in CNS play main role in neuroinflammation and increasing of HO-1 in these cells underline contribution of these cells in neuroinflammation which maybe mediated by bilirubin and its metabolism modulator enzymes [24, 25]. Also it should be noted that, in addition to antioxidant activity bilirubin has immunimodulatory activities such as inhibition of complement cascade and inhibition of antibody dependent cell-mediated cytotoxic activities of lymphocytes [26, 27]. Therefore, we should consider other activities of bilirubin might also be involved in MS pathogenesis and increasing of bilirubin in MS patients probably related to its other activities. It is so complicated to address when bilirubin concentration is being reduced or increased in MS patients. So it is either basically increased by virtue of its protective role against oxidants. 
Furthermore, in the present study we found a negative correlation between EDSS of MS patients and bilirubin levels (although it was not significant), which is in agreement with all facts that mentioned above and emphasizes the positive effects of antioxidants on improving of MS.
We found that bilirubin levels (Tbil and Ibil) were significantly lower in female than in male patients, which is in agreement with similar studies [10, 6]. Because the prevalence of MS is higher in women, whether lower endogenous antioxidants levels are related to higher incidence of this disease among females is still unknown. Further studies with larger samples are necessary to judge how bilirubin levels relate to MS disease and the gender of patients.
The study concludes that bilirubin levels in MS patients probably are related to its both antioxidant and immunomodulatory functions. However, more controlled studies are needed in order to investigate the role of bilirubin and its mode of action in MS disease. 
Isfahan University of Medical Sciences has financially supported the present paper. We thank our patients for their collaboration. Also the authors would like to thank the efforts of the staff at the MS clinic in Kashani hospital. The neurosciences research center, Alzahra hospital, Isfahan, Iran has supported this study financially and so we acknowledge this gesture.
Conflict of Interest
The authors have no conflicts of interest.

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Type of Study: Research | Subject: Special
Received: 2018/02/24 | Accepted: 2018/02/24 | Published: 2018/02/24

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