The possible role of oxidative stress marker glutathione in the assessment of cognitive impairment in multiple sclerosis

2.1 Study population

The study was conducted in the General Hospital Dubrovnik from September 2020 to October 2021.

The subjects were diagnosed based on the revised McDonald’s criteria with the inclusion of criteria (duration of the disease less than 10 years, clinical confirmation of at least one relapse in a year, expanded disability status scale [EDSS] number less than 5.0). According to the revised McDonald’s criteria from 2017, the diagnosis of MS is based on clinical and paraclinical criteria (MR, cerebrospinal fluid diagnostics) which are based on two basic postulates, dissemination in space and dissemination in time, which can be satisfied clinically and/or magnetic resonance imaging. The criterion of dissemination in space is met when we have clinical or MR signs of damage in at least two systems, and dissemination in time is satisfied if the patient has either clinical or neuroradiological evidence that two damages occurred at different times, or positive IgG oligoclonal bands. There were no relapses within 3 months before cognitive testing.

1. Ethical approval: The study had been approved by the Medical Ethics Committee of General Hospital Dubrovnik and School of Medicine Zagreb (380-59-10106-16-20/157).

2. Informed consent: The patients and their families understood the research content and methods and agreed to sign the corresponding informed consent.

2.2 Biological markers of antioxidative defense in serum

Blood samples were collected in the morning, and after 30 min, they were centrifuged for 15 min at 1,000×g. The absolute serum concentrations of oxidative stress markers (GSH, catalase) were determined by spectrophotometric reading. Oxidative stress marker samples were stored at −70°C, all according to the kit manufacturer’s instructions.

The determination of catalase (CAT) activity in samples relied on the H₂O₂ degradation rate in the reaction mix. The reaction was initiated by adding 100 µL of sample to 900 µL of the reaction mixture containing 33 mmol/L H₂O₂ in 50 mmol/L phosphate buffer (pH 7.0). The absorbance at 240 nm (Libro S22 Spectrophotometer Biochrom Ltd. Cambridge, UK) was recorded for 3 min, and CAT activity was calculated from mean absorbance change per minute and molar absorption coefficient for H₂O₂ (43.6 mol/L cm). The results are expressed as U/mg protein.

Reduced GSH concentrations were determined following Ellman’s method as described elsewhere [8,12,13]. Briefly, 20 µL of sample was incubated with 40 µL of 35 mmol/L HCL for 10 min. At the same time, we prepared the enzyme working solution by adding 20 µL of GSH reductase (0.2 U/mL) to 9.98 mL of NADPH (0.8 mmol/L). The reaction mixture was prepared in a 96-well plate by pipetting 40 µL of 10 mmol/L DTNB, pretreated sample, and 100 µL of enzyme working solution. The absorbance at 412 nm was monitored for 5 min (ELISA plate reader, BIORAD Laboratories, Hercules CA, USA) to obtain the mean change per minute. GSH concentration was calculated using the calibration curve, and the results are reported as nmol/L per mg of proteins.

2.3 Cognitive and parameters of CNS damage

All subjects (with RRMS and the control group) underwent an magnetic resonance imaging (MRI) of the CNS (brain and cervical spinal cord), within 1 week after blood sampling, on a 1.5 Tesla (Siemens Somatom Essenza). Images are taken in T1, T2, and FLAIR measurement times, in sagittal, coronal, and transverse sections and analyzed by neuroradiologist.

EDSS is the gold standard scale for assessing the disability of MS patients. This scale quantitatively assesses the degree of clinical damage for individual systems (pyramidal, sensory, brainstem system, cerebellum, visual system, cognitive functions, others). Based on the number in those systems, the total EDSS number is calculated, which gives neurologists an objective quantification of disability, and the total value can be from 0 to 10, with EDSS values of 0–4.5 referring to patients who are fully ambulatory, while values of 5.0–9.5 are defined with movement difficulties [14].

The Montreal Scale of Cognitive Assessment (MoCA) was designed as a quick test to screen patients with mild cognitive impairment, a function that includes different cognitive domains: attention, concentration, executive functions, memory, language, visual construction abilities, conceptual thinking, calculation, and orientation [15]. The time of application of the MoCA test is approximately 10 min. The total possible score is 30 points, a score of 26 points or more is considered normal.

The Beck Depression Inventory II (BDI-II) is a self-report scale consisting of 21 questions, each of which can be answered with four answers that are graded from 0 to 3. The minimum grade is 0, and the maximum is 63. The questions refer to mood disorder, loss hopes, feelings of rejection, inability to enjoy, feelings of guilt, need for punishment, self-hatred, self-judgment, tendency suicide, tearfulness, irritability, disturbance in relationship with other people, indecisiveness, negative self-image, inability of work, sleep disturbance, fatigue, lack of appetite, weight loss, hypochondria and loss of libido. The average results show that respondents who scored up to 11 points do not have depression, and respondents who scored between 12 and 28 points have mild or moderate depression [16]. A major depressive episode is diagnosed when a score greater than 28 is reached [17].

2.4 Statistical analysis

Statistical data processing was carried out using the SPSS statistical program. The quantitative analytical paradigm was applied in the data analysis. The results of the quantitative data analysis were interpreted with at least a 5% level of significance and analyzed with the help of the program support STATISTICS 11. Stat Soft. Inch. The distribution of qualitative data was presented with contingency tables, and the data were analyzed with the χ ² test or, if necessary, with Fisher’s exact test. Distributions of quantitative measurements (properties) are tested for normality with the Smirnov-Kolmogorov test and depending on the outcome in the description and analysis, some of the appropriate statistical-analytical procedures were applied (Student’s T test, Mann–Whitney U test, analysis of variance [ANOVA], Kruskal–Walis ANOVA test). Correlation and regression models are used in data analysis with regard to data distribution (e.g., Spearman’s correlation).

4.1 GSH in RRMS

GSH values are higher in healthy subjects compared to subjects with MS, as published in the study by Lubisavljević et al. [18]. They included 50 patients with clinically isolated syndrome (CIS) and 57 patients with RRMS. Significantly reduced GSH values were obtained in both examined groups in comparison with control values. Depletions were more pronounced in RRMS than in CIS patients (p = 0.009 for GSH). The results show that GSH can be important in the development of neuroinflammation and serve as a marker that is closely related to neurological and radiological signs of acute inflammation of the CNS. In the study by Choi et al. [19] lower GSH values were also found in 21 subjects with RRMS, 20 with SPMS, and 20 with PPMS compared to 28 healthy controls. The results have shown markedly lower GSH in progressive MS than in RRMS, indicating a more pronounced involvement of oxidative stress in the progressive stage of MS than in the inflammatory stage. The connection between GSH and brain atrophy indicates an important contributing role of oxidative stress neurodegeneration in progressive MS. Calabrese et al. [13] showed significantly lower GSH values in the cerebrospinal fluid of patients with MS, compared to controls, which probably reflects lower GSH in the CNS. A 2011 study by Van Horssen et al. [3] shows that oligodendrocytes have particularly low levels of GSH, making them more sensitive to oxidative stress, and in this way, they also produce reduced ability to restore the myelin sheath. Krotenko et al. [20] analyzed the peripheral blood of 79 MS patients and 75 healthy patients. They found lower GSH levels in RRMS patients compared to controls. The authors report a lower level of GSH in the phase of remission and in the phase of exacerbation, both in RRMS and secondary progressive MS. Di Giussepe et al. [21] measured GSH in very fluid plasma chromatography and concluded that there are no significant differences in GSH concentration between healthy and MS patients. In the studies of Tasset et al. [22] on 24 patients with RRMS and 15 healthy controls, GSH levels in the blood of patients with MS were increased, which may be partly explained by an increase in the activity of GSH reductase, which regenerates GSH from oxidation form of GSH. Given the variation in blood measurements of GSH and GSH-related enzymes and cerebrospinal fluid in different studies, imaging methods that allow non-invasive quantification of brain GSH are useful in providing accurate measurements in real time. One of the causes of increased GSH was shown by stimulation of extracellular glutamate, indicating that GSH may have neuroprotective role to reduce glutamate toxicity and thus affect neurodegeneration [23]. Also, the reduced level of GSH in the brain, primarily in the white matter, should be taken into account, as it can be used to identify patients who have a higher risk of progression disease, and should be investigated in terms of how this reduction affects functional and cognitive activity [19,23].

4.2 GSH and MoCA in RRMS

In this study, GSH was significantly negatively correlated with the degree of cognitive impairment (MoCA test, Table 4), although we did not find the statistically significant correlation in the multivariant model (Table 5). This is the first study of subjects with RRMS that did the aforementioned research. It is likely that changes in regional GSH levels in the brain may affect cognitive and sensorimotor function, although it is unclear whether this relationship was seen in normal aging or only in pathological conditions. In previous research, GSH was shown as a potential marker, therefore we conducted our research. In the similar research of Hupfeld et al. [24], who tested the association between brain GSH levels and cognitive performance in 37 young (mean age 21.8) and 37 elderly (mean age 72.8) healthy individuals, predicted regionally specific relationships in which frontal GSH levels would be related to cognitive performance, and sensorimotor GSH levels were related to motor performance. They concluded that there was no association between GSH levels and cognitive performance determined by the MoCA test, although MoCA test results are sensitive enough to identify associations between neurometabolites measured by magnetic resonance spectroscopy and cognitive status. Based on this research, it could be said that correlations between GSH and cognitive deficit appear only in cases of more serious cognitive decline, when brain resources (such as the availability of antioxidants) have significantly decreased, that is, in pathological conditions of the CNS. The negative correlation of GSH levels and MoCA test results in this study could indicate a compensatory and neuroprotective response of GSH to oxidative stress and associated tissue damage in the brain [24], suggesting a GSH response to increased oxidative stress in RRMS. The limitation of the study is a small number of patients. Future investigations should be conducted on a larger number of patients with possible distinctions with all MS types.