المقدمة: التصلب اللويحي (MS) مرض مناعي التهابي مزمن مزيل للميالين يؤثر على الجهاز العصبي المركزي (CNS). يعد التصلب اللويحي مرض متعدد العوامل حيث تتفاعل فيه العوامل الوراثية والبيئية لزيادة خطر تكوين الآفات الالتهابية التي تؤدي إلى تلف الخلايا الدبقية القليلة التغصن وإزالة الميالين بشكل تدريجي. تم استخدام مقياس اتساع الإعاقة (EDSS) لتقييم الإعاقة الحركيّة الناجمة عن التصلب اللويحي، ويتراوح من 0 إلى 10. يصاحب التصلب اللويحي زيادة في المركبات المؤكسدة في الدماغ وخاصة البيروكسينتريت ، واختلال في استقلاب الطاقة في الخلايا العصبية، بالإضافة إلى تسلل للخلايا المناعية التي تحفز الحالة الالتهابية مما يؤدي إلى تكوين لويحات مزيلة للميالين في الجهاز العصبي المركزي. لذلك، هدفت هذه الدراسة في المقام الأول إلى التحقيق في مستويات حمض البول واللاكتات ونسبة العدلات إلى اللمفاويات (NLR) بسبب ارتباطها المباشر بالآلية المرضية المرتبطة بالتصلب اللويحي ، وإمكانية اقتراحها كمعايير جديدة في منهج تحديد تطور وشدة التصلب اللويحي.
المواد والطرائق: كان البحث دراسة حالة – شاهد، شملت 80 مشاركاً سوريًا من كلا الجنسين مقسمين إلى مجموعتين: مجموعة مرضى التصلب اللويحي (40n=) ومجموعة الشّواهد (40n=) مطابقة في العمر والجنس مع مجموعة المرضى. تم قياس مستويات حمض البول واللاكتات بطريقة لونية إنزيمية باستخدام Roche Cobas Uric acid kit (Roche، ألمانيا) و Roche Cobas Lactate kit (Roche، ألمانيا)، في حين تم إجراء تعداد دموي كامل للعينات باستخدام جهاز ADVIA 2120i Siemens وتم حساب NLR. تم إجراء التحليل الإحصائي باستخدام SPSS الإصدار 25 (SPSS Inc.، شيكاغو، إلينوي، الولايات المتحدة الأمريكية). تم تقديم القيم على أنها متوسط ± انحراف معياري. تم اعتبار قيمة P < 0.05 قيمة مهمة إحصائياً.
النتائج: كانت مستويات حمض البول لدى مجموعة التصلب اللويحي أقل من مستوياتها في مجموعة الشواهد (P = 0.0002)، في حين كانت مستويات اللاكتات وNLR لدى مجموعة التصلب اللويحي أعلى من تلك الموجودة في مجموعة الشواهد (P = 0.0001). وجدت هذه الدراسة أيضًا أن مستويات حمض البول لدى المرضى الذين لديهم EDSS>3 كانت أقل من تلك الموجودة لدى المرضى الذين لديهم EDSS ≤3 (P = 0.0001)، في حين كانت NLR لدى المرضى الذين لديهم EDSS>3 أعلى من تلك الموجودة لدى المرضى الذين لديهم EDSS ≤3 (P = 0.0001). كانت مستويات حمض البول لدى مرضى التصلب اللويحي في حالة الهجمة أقل من تلك الموجودة لدى مرضى التصلب اللويحي في حالة الهدأة (P = 0.0001)، في حين كانت مستويات اللاكتات وNLR أعلى لدى مرضى التصلب اللويحي في حالة الهجمة من تلك الموجودة في مرضى التصلب المتعدد في حالة الهدأة (P = 0.0001).
الاستنتاجات: أشارت نتائجنا إلى أن مستويات حمض البول انخفضت لدى مرضى التصلب اللويحي، على النقيض من مستويات اللاكتات وNLR، والتي زادت لدى هؤلاء المرضى. وأظهرت هذه النتائج أيضًا وجود علاقة بين هذه المعايير وحالة التصلب اللويحي المعبر عنها بدرجة EDSS، ووجود أو غياب نوبة التصلب المتعدد. بناءً على نتائجنا، ترتبط مستويات حمض البول واللاكتات وNLR في الدم بالتصلب اللويحي ويمكن اقتراحها كمعايير جديدة في منهج تحديد تطور وشدة التصلب اللويحي.
Background
Multiple sclerosis (MS) is a chronic inflammatory demyelinating disease that affects the central nervous system (CNS) and can lead to physical disability, cognitive impairment, and decreased quality of life (1). It is one of the most common non-painful diseases that affect young people aged 20-30 years, and it is more common in females than in males by approximate ratio of 3:1 (2). It is estimated that over 1.8 million people have MS worldwide according to world health organization (WHO) statistics in 2023 (3). In Syria in particular, 4,000 patients have been registered until 2022 in the treatment center at Ibn al-Nafis Hospital in Damascus. MS is a multifactorial disease in which genetic and environmental factors interact to increase the risk of developing MS. The combination of the Epstein–Barr virus, smoking, low levels of vitamin D, etc., along with the individual’s genetic background, increases the likelihood of developing MS (2). MS is characterized by perivenular inflammatory lesions that lead to the formation of demyelinating plaques, resulting in damage to oligodendrocytes and gradual demyelination of axons, as axons are preserved in the early stages of the disease, irreversible axonal damage occurs as they progress (4). MS can be considered as a disease with clinical subtypes beginning with relapsing/remitting MS (RRMS), which represents 85% of cases at onset and is characterized by discrete attacks lasting from days to weeks, the first attack of which is followed by significant recovery, which decreases over time. RRMS then develops into Secondary Progressive MS (SPMS), which includes increasing severity of symptoms and persistent deterioration that is unrelated with attacks (1)(5). About 15% of MS cases begin with Primary progressive MS (PPMS), which has a relatively lower frequency of attacks, while MS patients with progressive/relapsing condition are rare (less than 5% of MS patients). Clinically isolated syndrome (CIS) is another subtype of MS although the diagnostic criteria for MS are not fully verified in this subtype (1)(5). The clinical severity and functional disability resulting from MS are described by several methods, the most important of which is Kurtzke’s Expanded Disability Status Scale (EDSS). This scale is used to evaluate the functional systems of the central nervous system by describing the progression of MS and evaluating the effectiveness of therapeutic interventions. This scale consists of an ordinal rating system ranging from 0 (normal neurological status) to 10 (death caused by MS) in an interval of 0.5 (at EDSS=1) (6)(7). At the molecular level, MS is accompanied by an excessive production of reactive oxygen and nitrogen species in the brain and the resulting incitement of programmed cell death (8), as many studies have confirmed the presence of peroxynitrite in the brain, cerebrospinal fluid (CSF), and blood of MS patients (9)(10). At the immune level, T lymphocytes, especially CD+8, are the most abundant in the inflammatory infiltrates. B cells and plasma cells are also present, but in much lower numbers. These cells are the main causes of chronic inflammation. In contrast, neutrophils and macrophages migrate to the inflammation sites via chemokines and cytokines responsible for acute inflammation (1)(2)(4)(11). According to the pathogenesis of MS, several studies have investigated new parameters that can be used to detect the extent of disability resulting from MS, and their role in assessing the clinical condition and its severity, and uric acid, lactate, and NLR were suggested. It was found that uric acid has a protective role in MS by scavenging free radicals that are associated with MS, as uric acid – which is the end of the purine metabolism – acts as a free radical scavenger by preventing lipid oxidation and removing free radicals under hydrophilic conditions, especially peroxynitrite, by selectively binding and inactivating them (12)(13). On the other hand, there has been increasing evidence that multiple sclerosis is associated with mitochondrial dysfunction, which results in a disruption in energy metabolism, resulting in neurodegeneration that enhances the development of MS. Due to the lack of energy supply, brain tissue will resort to increasing the process of glycolysis until it exceeds the ability of mitochondria to metabolize pyruvate, leading to increased lactate production (14). Based on the above, this study aimed to investigate the levels of uric acid, lactate and NLR in MS patients, their alterations due to the extent of the disability resulting from it, and their role in assessing the clinical condition and its severity, as many studies indicated the association of these parameters with the pathogenic mechanisms involved in the development of MS, which may allow the addition of new parameters in the approach to determine the development and severity of MS.
Methods
– This case-control study, recruited 80 Syrian individuals of both sexes, divided into two groups: MS patients group (n=40) and control group (n=40) matched in age and sex to the patients group.
– The study included MS patients of both sexes whose duration of illness did not exceed 3 years and who were diagnosed and reviewed by specialized neurologists at the Multiple Sclerosis committee at Ibn Al-Nafis Hospital in Damascus. Individuals with gout, epilepsy, pancreatitis, malignant tumors, alcohol consumption, active infections, or receiving medications that affect uric acid and lactate levels, or immunotherapy that affects lymphocyte counts, and those with a history of surgical intervention within the past three months and pregnant women, were all excluded.
– 5 ml of venous blood samples were drawn from each participant between April 2023 and January 2024 after obtaining their inform consent. Each blood sample was distributed into 3 tubes: a dry tube to measure serum uric acid levels, a heparin tube to measure lactate levels in plasma, and an EDTA tube to calculate the NLR ratio.
– Patients informations were obtained from their medical records archived in the Multiple Sclerosis Committee at Ibn Al-Nafis Hospital. A form that included personal information, EDSS score, MS subtype, and current MS status was filled out for each patient.
– Measurements of uric acid and lactate levels were conducted at Al-Assad Hospital at Damascus University using an enzymatic colorimetric method of Roche Cobas Uric acid kit and Roche Cobas Lactate kit, respectively, while NLR was calculated after complete blood count was performed using ADVIA 2120i Siemens device.
– Serum uric acid levels were determined using the Uricease test, while lactate levels were determined using the Lactate oxidase method.
– This study obtained the approval of the Biomedical Research Ethics Council at the University of Damascus (1/29/2022) (ID no. PH-290122-34).
– The statistical analysis was conducted using SPSS version 25 (SPSS Inc., Chicago, IL, USA). Values were presented as mean ± standard deviation. Mann-Whitney test was conducted to compare values between study groups, as all values were not normally distributed. A P-value < 0.05 was considered to indicate statistical significance.
Results
– A total of 80 individuals were included in this study, and divided into two groups, MS patients group (n=40) and control group (n=40) matched in terms of gender and age (Table 1). The MS patients were distributed according to MS subclinical type, EDSS score, MS current status, family history, and MS duration (Table 2).
Table 1. The general data of the study groups and their distribution according to gender and Age:
|
Age, years (mean ± Std.) |
Gender, no. (%) |
Study group |
|
|
6.98 ± 29.83 |
15 (37.5%) |
Male |
control group (n=40) |
|
25 (62.5%) |
Female |
||
|
6.98 ± 29.83 |
15 (37.5%) |
Male |
MS group (n=40) |
|
25 (62.5%) |
Female |
||
Table 2. The general data of the MS patients and their distribution according to subclinical type, EDSS score, MS current status, family history, and MS duration:
|
MS subclinical type, no. (%) |
|
| 37 (92.5%) | RRMS |
| 3 (7.5%) | CIS |
|
EDSS score, no. (%) |
|
| 19 (47.5%) | EDSS ≤ 3 |
| 21 (52.2%) | EDSS > 3 |
|
MS current status, no. (%) |
|
| 24 (60%) | remission |
| 16 (40%) | attack |
|
Family history of MS, no. (%) |
|
|
33 (82.5%) |
Negative |
|
7 (17.5%) |
Positive |
| Duration of MS, months (mean ± Std.) | |
|
21.83 ± 9.46 |
Duration of MS |
RRMS: relapsing/remitting MS, CIS: Clinically isolated syndrome, EDSS: Expanded Disability Status Scale, MS: Multiple sclerosis.
Comparison of uric acid and lactate levels and NLR between MS patients group and control group:
Our findings showed lower levels of uric acid in MS patients group (3.80 ± 0.72 mg/dl) compared to its levels in control group (5.57 ± 0.97 mg/dl) (P=0.0002), while lactate levels in MS patient group (3.36 ± 1.43 mmol/l) were higher than that of control group (1.80 ± 0.45 mmol/l) (P=0.0001). Also, NLR in the MS patient group (2.75 ± 1.12) was higher than that in control group (1.77 ± 0.21) (P=0.0001) (Table 3).
Table 3. Uric acid and lactate levels and NLR in both MS patients group and control group.
| P value * | MS group (n=40)
(mean ± Std.) |
control group (n=40)
(mean ± Std.) |
Parameter |
| 0.0002 | 0.72 ± 3.80 | 0.97 ± 5.57 | Uric acid (mg/dl) |
| 0.0001 | 1.43 ± 3.36 | 0.45 ± 1.80 | Lactate (mmol/l) |
| 0.0001 | 1.12 ± 2.75 | 0.21 ± 1.77 | NLR |
* P-value > 0.05 is not significant
Relationship of uric acid and lactate levels and NLR with EDSS:
Our results gave an evidence that uric acid levels in patients with EDSS>3 (3.29 ± 0.41 mg/dl) were lower than in patients with EDSS≤3 (4.36 ± 0.55 mg/dl) (P=0.0001). A statistically significant difference between lactate levels in patients with (EDSS>3) (3.70 ± 1.39 mmol/l) and patients with (EDSS≤3) (2.98 ± 1.41 mmol/l) (P=0.065) was not observed. Nevertheless NLR in patients with EDSS>3 (3.01 ± 0.72) was higher than in patients with EDSS≤3 (2.47 ± 1.43) (P=0.0001) (Table 4).
Table 4. Uric acid and lactate levels and NLR in both EDSS≤3 group and EDSS>3 group.
| P value * | EDSS>3 (n=21) | EDSS≤3 (n=19) | Parameter |
| 0.0001 | 3.29 ± 0.41 | 4.36 ± 0.55 | Uric acid (mg/dl) |
| 0.065 | 3.70 ± 1.39 | 2.98 ± 1.41 | Lactate (mmol/l) |
| 0.0001 | 3.01 ± 0.72 | 2.47 ± 1.43 | NLR |
* P-value > 0.05 is not significant
EDSS: Expanded Disability Status Scale, EDSS≤3: mild motor disability, EDSS>3: severe motor disability.
Relationship of uric acid and lactate levels and NLR with MS current status:
When analysing the relationship of uric acid and lactate levels and NLR with the presence of an attack in MS patients, we found that uric acid levels in patients with an attack (3.21 ± 0.43 mg/dl) were lower than in patients in remission (4.19 ± 0.60 mg/dl) (P=0.0001), while lactate levels (4.76 ± 1.10 mmol/l) and NLR (3.57 ± 1.92) in patients with an attack were higher than in patients in remission (2.42 ± 0.63 mmol/l) (2.21 ± 0.52), respectively. ((P=0.0001) and (P=0.0001) respectively) (Table 5).
Table 5. Uric acid and lactate levels and NLR in both MS patients undergoing disease attack group and MS patients under remission group.
| P value * | Attack (n=16) | Remission (n=24) | Parameter |
| 0.0001 | 3.21 ± 0.43 | 4.19 ± 0.60 | Uric acid (mg/dl) |
| 0.0001 | 4.76 ± 1.10 | 2.42 ± 0.63 | Lactate (mmol/l) |
| 0.0001 | 3.57 ± 1.92 | 2.21 ± 0.52 | NLR |
* P-value > 0.05 is not significant
NLR: neutrophil to lymphocyte ratio.
Discussion
Our findings suggest that, uric acid and lactate levels and NLR may represent vital parameters that can be included in the evaluation approach of the clinical condition and severity of MS, as many studies have indicated that the levels of these parameters differ according to the clinical condition of MS patients (1) (8) (15) (16) (17). Uric acid levels decreased in MS patients compared to controls. Its levels also decreased further as EDSS score increased, and during MS attack compared to the remission state. These results may be explained by the fact that MS is accompanied by an increase in the production of reactive oxygen and nitrogen compounds that are toxic to cells, especially peroxynitrite, which causes demyelination and damage to the axons of nerve cells (9), leading to the consumption of uric acid and consequently a decrease in its levels, as it is a natural scavenger of peroxynitrite (18), that works to reduce the resulting demyelination caused by the inflammatory event. High uric acid levels play a protective role against neurodegeneration because they are associated with less disability in MS, which is indicated by the EDSS score (1)(18). This is consistent with a study performed in (Turkey, 2021) (1), which included 150 MS patients and 150 controls, where uric acid levels were lower in MS patients compared to controls (P<0.001), as well as in patients with high EDSS compared to low EDSS (P<0.001) (this study adopted EDSS=3 as the dividing value between mild motor disability and severe disability, which is the value we adopted in our study). In another study (Italy, 2015) (15) included 362 MS patients and 181 controls, it indicated that uric acid levels were lower in MS patients than in controls (P=0.014) as well as in patients with high EDSS compared to low EDSS (P=0.002) (this study adopted EDSS=5 as a dividing value between mild motor disability and severe disability). Also, another study (Egypt, 2015) (16), which included 55 MS patients and 34 controls, showed that uric acid levels were lower in MS patients compared to controls (P=0.001), while they did not find a correlation between EDSS score and uric acid levels in MS patients (r=0.181; P=0.203). In contrast to uric acid, our results indicated that lactate levels were higher in the patients group compared to the control group, and in MS patients undergoing disease attack compared to MS patients under remission. Amorini et al. (8) explained that MS patients suffer from mitochondrial dysfunction that results in a decrease in the supply of ATP, which prompts brain tissue to increase the process of glycolysis until it exceeds the ability of the mitochondria to metabolize pyruvate, leading to an increase in the production of lactate (8)(14), and most of the excess lactate moves from the cerebrospinal fluid (CSF) to the blood (8). Furthermore they concluded that with the occurrence of an MS attack, the inflammatory process increases, leading to increased demyelination, which is accompanied by an increase in the processes of glycolysis in nerve cells, thus increasing the source of lactate production. Intracellular acidification resulting from increased lactate production will, in turn, harm the functions of mitochondria and their ability to process pyruvate resulting from glycolysis, causing the cell to enter a vicious cycle of mitochondrial dysfunction and the resulting metabolites (8)(19). On the other hand, we did not find a statistically significant difference between lactate levels in patients with high EDSS score and patients with low EDSS score. These results were consistent with the study of Hassan A et al. (16) which found that lactate levels in patients group were higher than those in control group (P=0.005), while no correlation was found between EDSS score and lactate levels in patients (r= – 0.194; P=0.213). Amorini AM et al. (8) in a study that included 613 MS patients and 625 controls, found that lactate levels in MS patients were higher than in control group (P<0.0001), and they also found a positive correlation between high lactate levels and increased EDSS score (R2 = 0.419; P<0.001). This inconsistency between this study and our study can be due to the difference in the number of participants. When calculating NLR, it was found that this ratio has increased more in MS patients group compared to control group, as well as in MS patients with high EDSS compared to MS patients with low EDSS, and also in MS patients undergoing disease attack compared to patients in remission. NLR is more accurate than the neutrophil or lymphocyte count alone in detecting systemic inflammation (1)(4)(20). This ratio increases in patients who suffer from autoimmune diseases, including Sjogren’s syndrome, sarcoidosis, ulcerative colitis, rheumatoid arthritis, etc. (21). The increased NLR can be explained by a decreased T lymphocytes count due to their infiltration into the CNS tissues through their adhesion to the endothelial cells of the cerebral blood vessels (a decreased number of lymphocytes is a marker of immune and inflammatory diseases), as well as by an increased number of neutrophils associated with increased expression of Toll-like receptor 2 (TLR2), cluster of differentiation 43 (CD43) and related phenotypic changes of formyl peptide receptor 1 (FPFR1). Neutrophil migration to the site of inflammation is an important step in inflammation, and is manifested by increased neutrophil production and accelerated lymphocyte death, which together lead to an increased NLR (22). Bolayir A et al. study (1) found that NLR in MS patients was higher than in controls (P=0.02), as well as in patients with high EDSS compared to low EDSS (P<0.001). Fahmi RM et al. (17) conducted a study that included 140 MS patients and 140 controls and indicated that NLR was higher in MS patients compared to controls (P<0.001) as well as in patients with high EDSS compared to low EDSS (P<0.001). According to what was mentioned above, our finding concluded that uric acid and lactate levels and NLR can be proposed as supporting parameters for procedures used to evaluate the clinical status of MS patients. There are still several limitations of our study should be noted. First, it included only patients referring to medical committee of multiple sclerosis at Ibn Al-Nafis Hospital in Damascus. Second, the limited number of participants in this study.
Conclusions
In this study, we demonstrated that blood uric acid levels decreased in MS patients, in contrast to lactate levels and NLR, which increased in MS patients. Our study also investigated the relationship of these parameters with the MS status expressed by EDSS score and the presence or absence of MS attack. Based on our findings, uric acid and lactate levels and NLR in the blood are related to multiple sclerosis and its status and can be proposed as new parameters in the approach to determine the development and severity of multiple sclerosis. The use of these parameters as vital, easy-to-analyze parameters for indicating the progression of the disease and the degree of severity of disability requires further study and research on a larger group of patients.
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