Effects of N‐acetylcysteine on oxidative stress biomarkers, depression, and anxiety symptoms in patients with multiple sclerosis

Abstract

Aim

N‐acetylcysteine (NAC), a thiol‐containing antioxidant and glutathione (GSH) precursor, attenuates oxidative stress, and possibly improves psychiatric disorders. This study aimed to evaluate the effects of oral NAC on oxidative stress, depression, and anxiety symptoms in patients with multiple sclerosis (MS).

Methods

This clinical trial was conducted on 42 MS patients randomly assigned to intervention (n = 21) and control (n = 21) groups. The intervention group received 600 mg of NAC twice daily for 8 weeks, and the control group received a placebo with the same prescription form. An analysis of serum malondialdehyde (MDA), serum nitric oxide (NO), and erythrocyte GSH was carried out on both groups, along with a complete blood count. The Hospital Anxiety and Depression Scale (HADS) was used to assess symptoms of depression (HADS‐D) and anxiety (HADS‐A).

Results

Compared to the control group, NAC consumption significantly decreased serum MDA concentrations (−0.33 [−5.85–2.50] vs. 2.75 [−0.25–5.22] μmol/L; p = 0.03) and HADS‐A scores (−1.6 ± 2.67 vs. 0.33 ± 2.83; p = 0.02). No significant changes were observed in serum NO concentrations, erythrocyte GSH levels, and HADS‐D scores (p > 0.05).

Conclusions

Based on the findings of the present study, NAC supplementation for 8 weeks decreased lipid peroxidation and improved anxiety symptoms in MS patients. The aforementioned results suggest that adjunctive therapy with NAC can be considered an effective strategy for MS management. Further randomized controlled studies are warranted.

N‐acetylcysteine (NAC) supplementation for 8 weeks decreased lipid peroxidation and improved anxiety symptoms in multiple sclerosis (MS) patients. The aforementioned results suggest that adjunctive therapy with NAC can be considered an effective strategy for MS management.

1. INTRODUCTION

Multiple sclerosis (MS) is a multifactorial disease of the central nervous system (CNS). ¹ , ² , ³ MS is one of the most common types of neurological disorders in young adults and is induced by some factors, such as genetics and environmental factors. ⁴ , ⁵ MS affects more than 2 million individuals worldwide, ⁶ and the disease's average global prevalence increased from about 29 per 100 000 individuals in 2013 to 44 per 100 000 individuals in 2020. ⁷ Currently, Iran is well‐known for having a high prevalence of MS in the world, and Khuzestan province (southwest Iran) has been one of the high‐risk areas for MS, with a prevalence of >25 per 100 000 individuals. ⁸

Multiple sclerosis affects both the white and gray matter of the brain, which causes clinical signs and symptoms that eventually cause physical disability and cognitive and psychological disorders in patients. ⁹ , ¹⁰ Some studies suggest that oxidative stress and reactive oxygen species (ROS) are significantly involved in chronic inflammation and neurodegeneration during MS. ¹¹ , ¹² , ¹³ Several studies have demonstrated that erythrocytes, serum, and cerebrospinal fluid (CSF) levels of glutathione (GSH), a vital antioxidant, are reduced in MS patients. Nevertheless, the serum and CSF levels of oxidative markers, such as nitric oxide (NO) and malondialdehyde (MDA), are increased in these individuals. ¹¹ , ¹⁴ , ¹⁵ , ¹⁶ , ¹⁷ Moreover, oxidative stress might play an important role in psychiatric disorders. ¹⁸ Serum total antioxidant potentials and brain GSH levels were shown to be decreased in patients with depressive disorder. ¹⁹ On the other hand, research on humans and animals showed a significant link between anxiety and oxidative stress. ²⁰

Bulut et al. ²¹ reported that an increase in MDA levels might be associated with some neuropsychiatric disorders. Additionally, Chakraborty et al. ²² showed that the increased severity of obsessive‐compulsive disorder (OCD) in the patients might be associated with elevated markers of serum lipid peroxidation. Another study also showed that MDA levels were significantly higher in patients with OCD. ²³

Since ROS plays a key role in both the psychiatric and degenerative processes of MS, ²⁴ , ²⁵ antioxidant supplementation might be an attractive strategy for MS management. Considering the reduced levels of GSH and changes in the activity of GSH‐associated enzymes in MS, maintaining or retrieving GSH levels might be a promising therapeutic strategy to improve the health status of patients with MS. Among GSH precursors, N‐acetylcysteine (NAC), an antioxidant, was reported to have protective effects against several neurological diseases. ²⁶ , ²⁷

N‐acetylcysteine is an organosulfur compound that increases GSH S‐transferase activity. ²⁸ , ²⁹ , ³⁰ Animal studies demonstrated that NAC consumption reduces oxidative stress and inflammation in experimental autoimmune encephalomyelitis. ³¹ , ³² Clinical trials, including various psychiatric and neurological assessments, were performed on the effects of treatment with NAC on neurological disorders, such as schizophrenia, neuropathy, and traumatic brain injury. ³³ , ³⁴ , ³⁵ , ³⁶ To the best of our knowledge, there is currently no randomized controlled trial on the effect of NAC supplementation on lipid peroxidation, depression, or anxiety in MS patients. Therefore, this study was conducted to evaluate the effects of daily consumption of NAC on oxidative stress, depression, anxiety, and hematological parameters in MS patients.

2. METHODS

2.1. Participants

The MS patients (n = 54) were recruited from the MS Society of Khuzestan province, Ahvaz, Iran. A total of 100 patients with MS were interviewed, and 54 eligible patients were recruited. The inclusion criteria were the age range of 18–60 years with clinically definite relapsing–remitting MS (RRMS) diagnosed based on McDonald criteria and an Expanded Disability Status Scale (EDSS) score ≤4.5, a score of 8–15 (mild‐to‐moderate anxiety and depression) from the Hospital Anxiety and Depression Scale (HADS) questionnaire in depression (HADS‐D) and anxiety (HADS‐A) subscales, no history of severe depression, anxiety, suicide attempt, and any chronic diseases, lack of consuming medications including antidepressants, fatigue modulating drugs, or drugs that would interact with NAC or affect its metabolism, no history of drug or alcohol addiction, lack of consuming oral or systemic glucocorticoids and any regular intake of antioxidants or vitamin supplements within 30 days prior to the study, and lack of pregnancy or lactation. The exclusion criteria were starting to consume any of the above‐mentioned medications or supplements, pregnancy during the study, imperfect compliance (consuming fewer than 90% of tablets), other health issues (e.g., chronic or other autoimmune diseases), relapse attacks during the study, adverse events leading to patient intolerance or complications, and a lack of consent to complete the trial.

2.2. Study design

This study was conducted on RRMS patients in Ahvaz, Iran, from June 2018 to January 2019. The MS cases were randomly assigned to NAC (n = 27) or placebo (n = 27) groups using the blocked randomization method. The randomized allocation sequences, enrolling participants and allocating them to interventions, were carried out by a dietitian who was not a member of the main research team and was not aware of random sequences. Up until the conclusion of the study, the researchers and patients were not aware of the randomization or allocation. The participants in the intervention group received NAC effervescent tablets (acetylcysteine 600 mg effervescent tablets; Zambon Switzerland Ltd., Switzerland) orally twice a day for 8 weeks, and the control group received two matching placebo effervescent tablets per day for the same time. The NAC and placebo tablets were identical in shape, size, color, and packaging.

Since evidence‐based appropriate dosage for NAC in MS patients is scarce, the recommended dose was chosen based on the results of a previous study on patients with high oxidative stress conditions, which indicated that a higher dose than 1200 mg NAC per day might not be needed for GSH increments accompanied by gastrointestinal side effects. ³⁷ On the first visit, 60 tablets were given to the participants for 1‐month consumption. The rest of the tablets (60 tablets) were given to the patients on the second visit.

Using each participant's checklist, the investigators telephoned participants to gauge their compliance during the survey. At the end of the study, the participants were asked to return the empty supplement boxes to verify that they had consumed all the supplements. The patients were excluded from the study if they consumed fewer than 90% of the tablets. The primary outcomes included serum NO and MDA levels, erythrocyte GSH concentrations, and HADS‐D and HADS‐A scores. The changes in blood cell values were considered secondary outcomes.

The total sample size of 54 patients was calculated based on a previous study on the erythrocyte oxidized/reduced GSH ratios, ³⁸ α = 0.05, and β = 0.20 (power = 80%), considering the probability of sample loss. Six patients were excluded from the NAC group regarding shortness of breath (n = 1), corticosteroid therapy (n = 2), personal reasons (n = 2), and clinical relapses during the study (n = 1). Furthermore, six patients were excluded from the placebo group due to clinical relapses during the study (n = 3), taking other supplements (n = 1), and personal reasons (n = 2). Finally, 42 patients were included in the analysis. Figure 1 depicts the flowchart of the study design.

FIGURE 1.

Flow chart of the study.

At the beginning of the study, data on demographic factors, disease severity, disease duration, and age of disease onset were obtained. Subsequently, neuropsychiatric disorders and biochemical parameters were assessed as follows.

2.3. Assessment of neuropsychiatric disorders

Depression and anxiety were measured with a valid and reliable screening instrument for MS patients, namely the HADS, at baseline and at the end of the study. ³⁹ , ⁴⁰ The HADS quantitatively measures psychological distress and comprises 14 items with two subscales, HADS‐A and HADS‐D. Scores range from 0 to 21 on each subscale, with higher scores indicating more severe symptoms of depression/anxiety. Cut‐offs of 8 and higher on both subscales show optimal sensitivity and specificity in identifying disorders of anxiety and depression. ⁴⁰ Consequently, the patients with MS were divided into depression and anxiety states based on a threshold >8, and only patients with depression and anxiety were included in the study.

2.4. Biochemical assessment

At the beginning of the study and in the eighth week, the blood samples were collected by venipuncture from 07:00 to 08:30 a.m. after fasting from the previous midnight in two separate tubes, one of which was an ethylenediaminetetraacetic acid (EDTA) tube and the other without EDTA to determine serum NO and MDA levels. The non‐EDTA tubes were stored for 30 min at room temperature to clot and then centrifuged at 1160 x g for 10 min to separate the serum. The samples were then stored at −70°C until final analysis.

The tubes containing EDTA were used for the measurement of erythrocyte GSH levels after hematologic evaluation. A complete blood count (CBC) analysis was performed within 2 h after blood sample collection using a Mindray hematology analyzer (BC5800, China). Then, the blood was immediately centrifuged at 850 x g for 10 min, and the supernatant, corresponding to plasma, was aspirated. Four volumes of a 5% metaphosphoric acid solution were added, and the precipitate was mixed. The sample was centrifuged at 13680 x g for 10 min. Then, the supernatant was collected and stored at −70°C until analysis.

The chemical colorimetric method was performed for all biochemical assessments. Serum MDA and NO concentrations and erythrocyte GSH levels were measured using the ZellBio kit (Germany) with a sensitivity of 0.1 μmol/L, 1 μmol/L, and 0.01 mmol/L, respectively.

2.5. Statistical analysis

The Kolmogorov–Smirnov test was used to assess the distribution of quantitative data. Within‐group analyses were conducted using the paired samples t‐test or Wilcoxon paired rank test (when the data were not normally distributed) based on a change from the baseline. Moreover, the independent t‐test and Mann–Whitney U test (for non‐normally distributed data) were applied to compare the changes (endpoint minus baseline) after 8 weeks of intervention between the two groups. The data were reported as mean ± standard deviation (SD) or median (25th and 75th percentile) for parametric and nonparametric data, respectively. The Chi‐square test and Fisher's exact test were used for some baseline characteristic data. SPSS statistical software (version 16; SPSS Inc., Chicago, IL, USA) was used for all the statistical analyses. The main outcomes were presented at 95% confidence intervals (CI). For all tests, two‐sided p‐values less than 0.05 were considered statistically significant.

3. RESULTS

3.1. Baseline characteristics

Table 1 shows the demographic and clinical characteristics of the participants. The mean ages of the patients in the NAC and placebo groups were 36.9 ± 8.33 and 38.4 ± 8.31 years, respectively. No significant differences (p ≥ 0.05) were observed in demographic and anthropometric characteristics, EDSS, disease duration, age of MS onset, physical activity, and dietary intake between the two groups at baseline (Table 1). In addition, there were no differences between the placebo and NAC groups regarding HADS‐A and HADS‐D scores and levels of serum NO, serum MDA, and erythrocyte GSH levels (p ≥ 0.05; Table 1).

TABLE 1.

Baseline characteristics of participants in the NAC‐treated and placebo groups.

Variables	NAC group (n = 21)	Placebo group (n = 21)	p‐Value
Age (years)	36.9 ± 8.33	38.4 ± 8.31	0.56
Sex, n (%)
Male	6 (28.6%)	6 (28.6%)	0.999
Female	15 (71.4%)	15 (71.4%)
BMI (kg/m2)	25.06 ± 4.93	26.32 ± 5.31	0.43
EDSS	1.58 ± 1.003	1.7 ± 1.28	0.74
MS duration (months)	78.43 ± 54.5	72.05 ± 54.22	0.70
Age of onset (years)	30.14 ± 9.22	31.66 ± 6.71	0.54
Serum NO (μmol/L)	40.54 ± 10.90	36.77 ± 7.80	0.205
Serum MDA (μmol/L)	13.48 (8.33–34.30)	13.06 (9.42–33.08)	0.99
Erythrocyte GSH (mmol/L)	0.41 ± 0.14	0.43 ± 0.15	0.64
HADS‐A (Score)	10.05 ± 1.83	9.28 ± 1.70	0.17
HADS‐D (Score)	9.43 ± 1.75	8.76 ± 1.44	0.19
Physical activity (MET h/day)	36.57 ± 2.9	36.67 ± 4.18	0.931
Energy intake (Kcal/day)	1746.43 ± 354.91	1875.57 ± 355.71	0.25
Total fat intake (% of energy)	30.91 ± 7.46	29.89 ± 6.44	0.638
Protein intake (% of energy)	14.98 ± 4.69	14.40 ± 2.59	0.626
Carbohydrate intake (% of energy)	54.35 ± 9.14	55.94 ± 6.92	0.529

Note: Values are presented as mean ± standard deviation (SD) and median (IQR = Interquartile Range) or number and percent. Mann–Whitney U test and independent sample t‐test were used to compare nonparametric and parametric variables between the two groups, respectively.

Abbreviations: BMI, body mass index; EDSS, expanded disability status scale; GSH, reduced glutathione; HADS‐A, anxiety score from the Hospital Anxiety and Depression Scale questionnaire; HADS‐D, depression score from the Hospital Anxiety and Depression Scale questionnaire; MDA, malondialdehyde; MS, multiple sclerosis; NO, nitric oxide; NAC, N‐acetylcysteine.

3.2. Primary outcomes

The NAC administration for 8 weeks led to a significant decrease in serum MDA levels (−0.33 [−5.85–2.50] vs. 2.75 [−0.25–5.22] μmol/L; p = 0.03) and HADS‐A scores (−1.6 ± 2.67 vs. 0.33 ± 2.83; p = 0.02), compared to the control group (Table 2). Furthermore, the within‐group analysis identified that NAC supplementation was associated with a significant reduction in HADS‐A scores after 8 weeks (10.05 ± 1.83 to 8.38 ± 2.56; p = 0.01; Figure 2). There was no significant reduction in the HADS‐A scores of the placebo group (p ≥ 0.05). Regarding NO and GSH levels and HADS‐D scores, there was no significant difference between or within the two groups before and after the intervention (p ≥ 0.05; Table 2).

TABLE 2.

Results of primary outcomes at baseline and after 8 weeks of intervention in the NAC‐treated and placebo groups.

Variable	Placebo group (n = 21)				NAC group (n = 21)				p‐Value**
	Baseline	End of trial	Change	p‐Value*	Baseline	End of trial	Change	p‐Value*
Serum NO (μmol/L)	36.77 ± 7.80	39.59 ± 15.80	2.82 ± 17.1	0.46	40.54 ± 10.90	37.71 ± 13.23	−2.82 ± 15.5	0.41	0.27
Serum MDA (μmol/L)	13.06 (9.42–33.08)	13.21 (10.33–32.34)	2.75 (−0.25–5.22)	0.07	13.48 (8.33–34.30)	11.66 (8.72–27.60)	−0.33 (−5.85–2.50)	0.32	0.03
Erythrocyte GSH (mmol/L)	0.43 ± 0.15	0.38 ± 0.09	−0.05 ± 0.12	0.07	0.41 ± 0.14	0.43 ± 0.19	0.02 ± 0.24	0.67	0.21
HADS‐A	9.28 ± 1.70	9.62 ± 3.07	0.33 ± 2.83	0.6	10.05 ± 1.83	8.38 ± 2.56	−1.6 ± 2.67	0.01	0.02
HADS‐D	8.76 ± 1.44	8.43 ± 2.78	−0.33 ± 2.78	0.59	9.43 ± 1.75	9.09 ± 1.99	−0.33 ± 2.10	0. 48	1.0

Abbreviations: GSH, reduced glutathione; HADS‐A, anxiety score from Hospital Anxiety and Depression Scale questionnaire; HADS‐D, depression score from Hospital Anxiety and Depression Scale questionnaire; MDA, malondialdehyde; NO, nitric oxide; NAC, N‐acetylcysteine.

^** p‐value for comparing the changes in variables between the groups, independent t‐test and Mann–Whitney U test were used.

^* p‐value for comparing baseline, with endpoint values within each group, Wilcoxon and Paired t‐tests were used. Values are expressed as mean ± standard deviation (SD) for parametric data and median (25th, 75th percentiles) for nonparametric data.

FIGURE 2.

The effect of N‐acetylcysteine on anxiety symptoms in multiple sclerosis.

3.3. Secondary outcomes

Table 3 shows the mean and median of blood cell values during the study. Both between‐group and within‐group comparisons showed that NAC supplementation had no effect on blood cell values, including white blood cells (WBC), red blood cells (RBC), hemoglobin (Hgb), hematocrit (HCT), mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), and platelets (PLT; p ≥ 0.05).

TABLE 3.

Hematological parameters at baseline and after 8 weeks of intervention in the NAC‐treated and placebo groups.

Variable	Placebo group (n = 21)				NAC group (n = 21)				p‐Value**
	Baseline	End of trial	Change	p‐Value*	Baseline	End of trial	Change	p‐Value*
WBC × 103/μL	6.77 ± 1.83	6.35 ± 1.70	−0.42 ± 1.44	0.19	6.06 ± 1.71	5.90 ± 0.81	−0.15 ± 1.39	0.62	0.54
RBC × 106/μL	4.71 ± 0.6	4.58 ± 0.57	−0.12 ± 0.27	0.04	4.63 ± 0.5	4.59 ± 0.45	−0.04 ± 0.17	0.25	0.24
Hgb g/dL	13.20 ± 1.69	12.96 ± 1.57	−0.24 ± 0.98	0.28	13.49 ± 1.70	13.35 ± 1.56	−0.14 ± 0.72	0.37	0.72
HCT (%)	39.30 (37.20–43.05)	38.90 (35.80–40)	−0.4 (−2.1–0.05)	0.12	39.20 (37–43.45)	38.60 (37.15–42)	−0.2 (−2–0.4)	0.17	0.56
MCV (fL)	85.30 (83.55–91.90)	84.10 (80.50–88.50)	−0.6 (−2.25–0.6)	0.16	87.10 (81.05–90.3)	86 (81.9–88.75)	−0.7 (−1–0.9)	0.35	0.53
MCH (pg)	28.24 ± 3.53	28.49 ± 3.49	0.25 ± 1.51	0.45	29.11 ± 1.99	29.12 ± 1.67	0.01 ± 0.96	0.96	0.54
MCHC g/dL	32.66 ± 1.86	33.46 ± 2.93	0.79 ± 2.47	0.15	33.75 ± 1.63	34.17 ± 1.82	0.42 ± 1.48	0.20	0.56
PLT × 103/μL	246.71 ± 73.37	243.28 ± 63.82	−3.43 ± 24.12	0.52	264.86 ± 84.52	269.05 ± 79.47	4.19 ± 33.86	0.57	0.4

Abbreviations: Hgb, hemoglobin; HCT, hematocrit; MCV, mean corpuscular volume; MCH, mean corpuscular hemoglobin; MCHC, mean corpuscular hemoglobin concentration; NAC, N‐acetylcysteine; PLT, platelet; RBC, red blood cells; WBC, white blood cells.

^** p‐value for comparing the changes in variables between the groups, independent t‐test and Mann–Whitney U test were used.

^* p‐value for comparing baseline, with endpoint values within each group; Wilcoxon and Paired t‐test were used. Values are expressed as mean ± standard deviation (SD) for parametric and median (25th, 75th percentiles) for nonparametric data.

3.4. Side effects

Among the patients, one complained of shortness of breath, which was excluded from the study. However, this symptom disappeared when the supplement was discontinued. The dosage was very well tolerated, and none of the participants experienced gastrointestinal or any major side effects.

4. DISCUSSION

This study's results showed that oral NAC supplements for MS patients had beneficial effects that might be associated with their antioxidant function. Lower MDA levels and improved HADS‐A scores, compared to the control group, indicated that NAC supplementation was associated with a significant reduction in oxidative stress in MS patients. As previously mentioned, oxidative stress and the depletion of endogenous antioxidants play important roles in the etiology of neurodegenerative disorders, including MS. Therefore, antioxidant therapy might be a helpful approach to limiting disease progression. ⁴¹

In this study, NAC was associated with a significant reduction in serum MDA levels without affecting the erythrocyte GSH concentration. This finding is in line with the findings of another study showing that supplementation with 1800 mg NAC did not significantly affect plasma GSH levels in patients with systemic lupus erythematosus; however, it significantly decreased plasma MDA levels. ⁴² Nevertheless, in a study by Nur et al., ³⁷ GSH increments were observed in patients with sickle cell disease treated with 1200 mg NAC for 6 weeks.

The use of NAC has also been associated with a significant reduction in lipid peroxidation and a significant increase in GSH levels in the liver and erythrocytes of mice. ⁴³ Moreover, a report showed that NAC is effective in reducing plasma lipid peroxidation in individuals undergoing chronic hemodialysis ⁴⁴ or patients with acute ischemic stroke. ⁴⁵ A possible explanation for this discrepancy could be the difference in disease and health outcomes in different studies.

Glutathione plays an important role in antioxidant and cellular redox regulation. ⁴⁶ Previous studies reported that GSH concentrations consistently decreased in the serum, erythrocytes, and CSF in patients with MS ¹⁴ , ¹⁷ and have stimulated numerous studies to develop new potential approaches to maintain or restore GSH levels in these patients. GSH is composed of amino acids, including glycine, glutamic acid, and cysteine. Additionally, under physiological conditions, the intracellular availability of cysteine is considered to be the rate‐determining factor in GSH synthesis. However, cysteine supplementation can cause toxicity due to the formation of free radicals during the autoxidation of cysteine. ²⁷ , ⁴⁷ Consequently, compounds that can be metabolized into cysteine can also be used as prodrugs to increase GSH levels in neurons. ⁴⁸ In the present study, supplementation with NAC could not significantly affect GSH levels; probably, higher doses or a longer supplementation duration were needed for significant GSH changes in the NAC group. Moreover, NAC supplementation depends on the body's ability to synthesize GSH from available substrates (e.g., glycine and glutamate amino acids), an ability that was reported to be diminished with age and in the presence of certain diseases, especially liver dysfunction. ⁴⁹ , ⁵⁰

This study's results indicated that NAC supplementation lowered MDA levels. However, the exact mechanism of the effect of NAC on MDA is not clear. MDA synthesis results from lipid peroxidation, which is inhibited by NAC antioxidant function. ⁴⁴ As previously mentioned, in some conditions where endogenous cysteine and GSH are significantly depleted, NAC might act as a direct antioxidant for some oxidant species. ⁵¹ Nevertheless, MDA induces the expression of the aldehyde reductase gene, which upregulates lipid peroxide production. Subsequently, NAC suppresses this MDA‐induced genetic stimulation. ⁴⁴

This study's results showed that NAC supplementation did not affect NO levels compared to the control group. NO is a free radical signaling molecule that is present at higher‐than‐normal levels in inflammatory MS lesions. These high levels are due to the presence of inducible NO synthase in cells such as macrophages and astrocytes. The CSF, blood, and urine of patients with MS have elevated levels of NO‐producing markers (e.g., nitrates and nitrites). The evidence points to the involvement of NO in several features of the disease, such as blood–brain barrier disruption, oligodendrocyte damage and demyelination, axonal degeneration, and loss of function as a result of reduced axonal conduction. There has even been some evidence that NO generation might have some immunomodulating effects on MS. ⁵² Several studies reported a significant decrease in NO levels using GSH ⁵³ or NAC. ⁵⁴ , ⁵⁵ , ⁵⁶ However, most of the studies on the effect of NAC on NO levels were animal studies. Sabetghadam et al. reported that high‐dose NAC administration in 68 patients with acute ischemic stroke resulted in significantly decreased serum levels of NO. ⁴⁵ It is possible that NAC is an effective NO inhibitor at high doses in humans; probably, higher doses could have a significant effect on the NO serum level.

The findings of the present study demonstrated a significant improvement in the HADS‐A score. In line with the results of this study, Strawn and Saldana reported that 2 months of adjunctive administration of NAC (1200–2400 mg/day) in a 17‐year‐old male case with a history of generalized anxiety disorder and generalized social phobia decreased anxiety, improved his anxiety‐related insomnia, sense of inner tension, and restlessness, and diminished somatic symptoms. ⁵⁷ Moreover, Ghanizadeh et al. studied the effect of 800 mg/day NAC consumption on chronic nail biting in children and adolescents and reported that NAC decreased nail biting behavior over the short term. ⁵⁸ In another study, 8 weeks of NAC (1200 mg/day) consumption caused a significant decrease in irritability in children and adolescents with autism spectrum disorders. ⁵⁹ Furthermore, a response to treatment was observed in Grant et al.'s ⁶⁰ and Rodrigues‐Barata et al.'s ⁶¹ studies on trichotillomania disorder.

In the current study, NAC supplementation had no significant effect on the patient's HADS‐D score. Despite the results of this study, some studies showed that NAC (1000–2000 mg/day) consumption has beneficial effects on the treatment of depression‐related neuropsychiatric disorders in bipolar individuals. ⁶² , ⁶³ , ⁶⁴ , ⁶⁵ Moreover, the changes in depression were related to changes in oxidative stress when using coenzyme Q10 in a previous study. ⁶⁶ The underlying mechanisms of action of NAC to improve neuropsychiatric disorders are not yet clear.

However, the present study has some limitations. First, only one dose of NAC was used. Second, the biochemical assessment of the CNS and brain markers was not applicable, and the inflammatory markers were not evaluated. Third, the short duration of the intervention and the small sample size from a single institution might affect the accuracy of the obtained results. Fourth, the effect of the confounding factors was not adjusted. However, given that all patients were selected from one center, they received the same treatment and probably did not differ significantly in socioeconomic status. Fifth, the plasma thiol concentrations were not assessed. Finally, data on the dietary intake of the participants was not available. The MS‐related biochemical and pathological parameters should be considered in future studies with larger sample sizes and higher doses of NAC.

5. CONCLUSION

To conclude, this study demonstrated that supplementation with 600 mg of NAC twice daily for 2 months improved lipid peroxidation and anxiety in patients with MS. Further studies with larger sample sizes and longer durations are needed to confirm the aforementioned findings and discover the underlying mechanisms of the possible effects of NAC on MS.

AUTHOR CONTRIBUTIONS

Golsa Khalatbari Mohseni and Seyed Ahmad Hosseini designed the study and carried out data collection. Seyed Ahmad Hosseini, Bahman Cheraghian, Nastaran Majdinasab, and Golsa Khalatbari Mohseni designed the study, analyzed the data, and critically reviewed the manuscript.

FUNDING INFORMATION

This study was financially supported by the Vice‐Chancellor for Research Affairs of Ahvaz Jundishapur University of Medical Sciences (NRC‐9625).

CONFLICT OF INTEREST STATEMENT

The authors declare no conflict of interest.

ETHICS STATEMENT

Approval of the Research Protocol by an Institutional Review Board: This study was approved by the Local Ethics Review Board of Ahvaz University of Medical Sciences (IR.AJUMS.REC.1396.888). The research protocol for this study was approved by the Iranian Registry of Clinical Trials (IRCT20180515039662N1).

Informed Consent: The present study followed the Declaration of Helsinki, and written informed consent was obtained from all the participants.

Registry and Registration Number of Study/Trial: Clinical trial registration number: IRCT20180515039662N1.

Animal Studies: Not applicable.

Khalatbari Mohseni G, Hosseini SA, Majdinasab N, Cheraghian B. Effects of N‐acetylcysteine on oxidative stress biomarkers, depression, and anxiety symptoms in patients with multiple sclerosis. Neuropsychopharmacol Rep. 2023;43:383–391. 10.1002/npr2.12360

DATA AVAILABILITY STATEMENT

Not all data are freely accessible because no informed consent was given by the participating agencies for open data sharing. However, the data are available from the corresponding author on reasonable request, following approval by the Iranian Registry of Clinical Trials (IRCT20180515039662N1).