Saliva and Serum Acetylcholinesterase Activity in Multiple Sclerosis

Multiple sclerosis is mediated by the immune system that damages the myelin sheath. Most patients experience inflammation. Since one of the factors that have a role in reducing inflammation is acetylcholine, and according to the benefits of saliva, in this study, the level of salivary and serum cholinesterase activity in patients with multiple sclerosis and healthy were evaluated. Thirty women with multiple sclerosis who were hospitalized in the neurology ward of Imam Reza and Hazrat Rasoul Hospitals and 30 healthy females participated in the study. The severity of multiple sclerosis was calculated by expanded disability status scale (EDSS). Saliva and serum samples were collected in the morning. Cholinesterase activity was assessed by a photometric method. The mean cholinesterase activity in stimulated and unstimulated saliva and serum significantly reduced in the multiple sclerosis group. The cutoff for differentiation of multiple sclerosis patients from healthy individuals by assessing cholinesterase activity (IU/L) was 3577 in serum, 241 in unstimulated saliva, and 266 in stimulated saliva. It seems that cholinesterase activity decreases in patients with multiple sclerosis.


Introduction
Multiple sclerosis is a chronic autoimmune and inflammatory disease of the brain characterized by myelin destruction and neuronal damage in the spinal cord and brain [1,2]. Clinical signs of multiple sclerosis vary depending on the level and location of the lesion and include muscle weakness, blurred vision, dizziness, fatigue, and imbalance [3]. It is projected that more than two million persons toil from multiple sclerosis. The occurrence of multiple sclerosis is higher in females, especially young people [4]. The etiology and pathogenesis of multiple sclerosis have not been elucidated to date, but complex interplays between environmental influences and genetics appear to lead to multiple sclerosis [5]. T cells are believed to have a pivotal function in the pathogenesis of multiple sclerosis. T cells are probably stimulated by antigens in the peripheral tissue and form a colony, and by crossing the blood-brain barrier, they attach to the antigens in the brain and become more active, producing cytokines that lead to the demyelination of neurons [6]. Antibodies and B cells also have a fundamental function in the pathogenesis of multiple sclerosis [7].
To prove multiple sclerosis, magnetic resonance imaging (MRI) is used to display lesions and oligoclonal groups of IgG assays in CSF [8]. However, MRI is a time-consuming and expensive technique. CSF is an option for assessing multiple sclerosis and other CNS disorders [8]. However, CSF sampling is a very invasive method, and CSF tests to check for disease and drug response are usually impractical. To date, no specific diagnostic tests are available for multiple sclerosis [9]. Therefore, it is necessary to study new markers, which can be applied in conjunction with existing clinical findings for more precise and initial diagnosis and recognition of the effectiveness of treatment. The potential usage of saliva as an investigative fluid for oral and systemic diseases has been extensively studied [10][11][12][13][14][15][16]. However, there are few studies on saliva in multiple sclerosis [8].
Studies have shown that inflammation stimulates the vagus nerve and releases acetylcholine (Ach) from its end [17]. Acetylcholine can inhibit the production of proinflammatory cytokines and directly inhibit inflammation [18]. The amount of Ach depends on the balance between production and hydrolysis.
There have been reports of decreased cholinesterase activity in a variety of chronic and severe inflammatory diseases [19][20][21]. It has also been shown that serum cholinesterase concentrations increase in patients with multiple sclerosis [22]. Saliva, as compared to blood sampling, is easily and noninvasively available without stress and can be collected without causing discomfort to the patient [10]. Unfortunately, it has not yet been routinely used for diagnostic purposes like other biological samples (such as blood plasma). Evaluation of biomarkers in saliva can be considered in the fast diagnosis of diseases [10][11][12][13][14][15][16][17]. The level of salivary and serum cholinesterase activity in patients with multiple sclerosis and healthy individuals was evaluated for answers to the question of whether salivary and serum levels of cholinesterase activity are affected in multiple sclerosis.

Method
Thirty females with relapsing-remitting multiple sclerosis (RRMS) who were hospitalized in Imam Reza and Hazrat Rasoul Hospitals in the active phase of the disease and 30 healthy females were involved in a cross-sectional study. Multiple sclerosis was confirmed according to McDonald 2010 [9]. Clinical status in multiple sclerosis patients was assessed by EDSS (expanded disability status scale) by two neurologists. EDSS for the patients ranged between 1 and 7; the mean (± SD) EDSS was 3.5 ± 2.2. Individuals with ulcers and inflammation in the oral cavity and any systemic blood abnormalities were excluded from the study.
The participants refrain from eating or drinking for 2 h before sampling. For unstimulated saliva sampling, participants first swallow the saliva in the mouth and then pours any amount of saliva that collects in their mouth (without performing mouth movements such as sucking the wall of the mouth) into a calibrated Falcon tube. Then they chewed a piece of neutral natural chewing gum stimulating saliva sampling. The saliva that was collected during chewing gum was swallowed for 2 min, and then, while chewing, we asked the patient to pour any amount of saliva that collects in the mouth into another Falcon tube. For blood sampling, 2 mL of the volunteers' venous blood was taken and poured into a test tube. The samples were centrifuged at 5000 g for 5 min and the supernatant of saliva and serum were transferred into microtubes and kept in − 70 °C until the cholinesterase activity was measured by the photometric method according to the instructions of the manufacturer (Pars Azmoun, Karaj, Iran). Each sample was measured at least twice and if the coefficient of variation (CV) was more than 15%, the measurement was repeated until the CV reached less than 15%. Mean replications were considered for statistical analysis. Data are indicated as mean ± SEM and analyzed by Spearman, receiver operating characteristic (ROC), and unpaired Student's t-tests using SPSS software.
The ethics committee of the Iran University of Medical Sciences approved the study (IR.IUMS.FMD. REC.1400.482) and informed consent was obtained from all individuals.

Results
In this study, 30 women with multiple sclerosis with a mean age of 32.6 ± 1.9 years and 30 healthy women with a mean age of 34.8 ± 0.9 years were included (P > 0.05).
In the multiple sclerosis group, the mean cholinesterase activity in serum (P = 0.001), unstimulated saliva (P = 0.001), and stimulated saliva (P = 0.028) was significantly lower than in the control group (Fig. 1). There was a positive correlation in cholinesterase activity between serum and unstimulated saliva (r = 0.475; P = 0.001) as well as stimulated saliva (r = 0.300; P = 0.025).
The cutoff for differentiation of multiple sclerosis patients from healthy individuals by assessing cholinesterase activity was 3577 IU/L in serum, 241 IU/L in unstimulated saliva, and 266 IU/L in stimulated saliva (Table 1 and Fig. 2).

Discussion
Today, a large number of studies are underway to establish and standardize laboratory methods for using saliva as a diagnostic fluid. The main reason for these efforts is the simplicity and non-invasiveness of saliva sampling, which allows easy, low-cost, painless, and frequent sampling of patients. On the other hand, it is thought that saliva is a reflector of the body and will be a diagnostic fluid in the future and therefore will be included in the health maintenance program in the diagnosis, monitoring, and screening of diseases [23]. According to the benefits of saliva, in this study, serum and salivary levels of cholinesterase activity in healthy individuals and patients with multiple sclerosis were evaluated and the results showed that serum and salivary activity of cholinesterase in patients with MS is lower than in healthy people.
In multiple sclerosis, brain tissue becomes inflamed [24]. Inflammation activates the "cholinergic anti-inflammatory pathway" that stimulates the vagus nerve and releases Ach from its end [17]. Acetylcholine can inhibit inflammation [18] and affects macrophages via the nicotinic receptor α7, which consequences in the blocking of the NF-κB nuclear transport signaling pathway, leading to a reduction in the production of proinflammatory cytokines such as TNF-α, IL-1β, IL-18, and IL6. Thus, the cholinergic system can improve inflammation in patients by receiving information from different parts of the body, rapidly through the vagus nerve and through nicotinic alpha 7 receptors [17,20].
Cholinesterase activity decreases in a variety of chronic and severe inflammatory diseases [19,21]. Because cholinesterase is produced in the liver, it is also reduced in patients with liver disease and malnutrition [17,25]. Cholinesterase activity decreases in many inflammation diseases such as burns [25], gingivitis and periodontitis [26], cancer [27], heart failure [28], brain injury [29], and ischemic stroke [30] that are in agreement with the results of our study. There is a study that shows an increase in the concentration of cholinesterase in the blood and lymphocytes of patients with multiple sclerosis [22], which contradicts the results of our study. Perhaps the reason for the difference is that we examined the activity of the enzyme while Polachini et al. measured its concentration. It has been recommended that in future studies, the level and activity of this enzyme in MS patients be examined simultaneously.
The amount of cholinesterase is so strongly affected by inflammation that it decreases in the acute phase of inflammation and increases rapidly when the inflammation improves [25]. It has been shown that in severe systemic inflammation, the level of cholinesterase activity in the blood decreases [20]. Since serum cholinesterase levels show changes in the body's overall ability to hydrolyze Ach, an increase in serum cholinesterase levels is related to a decreasing regulation of cholinesterase activity as a compensatory response to increase acetylcholine anti-inflammatory activity [17] which can justify our results in reducing cholinesterase activity in saliva and serum.
In this study, the ROC test was applied to evaluate the diagnostic significance of cholinesterase activity in serum and saliva, to diagnose the multiple sclerosis group. The cutoff point of cholinesterase activity in serum and saliva was significant for separating patients from healthy individuals and also a positive correlation was detected between serum and salivary values. Therefore, it can be said that serum and salivary cholinesterase activity can have diagnostic value. Therefore, measuring cholinesterase activity can be helpful in diagnosing multiple sclerosis.

Conclusion
It seems that cholinesterase activity decreases in patients with multiple sclerosis.