Effect of high dose N-acetyl cysteine supplementation on markers of oxidative stress and insulin resistance in non-diabetic patients with metabolic dysfunction associated steatotic liver disease: a randomized controlled trial

Abstract

Background

Metabolic dysfunction–associated steatotic liver disease (MASLD) is a major cause of chronic liver disease. N-acetylcysteine (NAC) has demonstrated antioxidant and hepatoprotective effects in non-alcoholic steatohepatitis. This study evaluated the impact of high-dose NAC on oxidative stress, insulin resistance, and liver-related outcomes in non-diabetic MASLD patients.

Methods

This prospective, randomized, open-label controlled trial included 60 non-diabetic adults with MASLD. Participants were randomly assigned to receive either oral NAC (2400 mg/day) combined with lifestyle intervention (n = 30) or lifestyle intervention alone (n = 30) for 12 weeks. The primary outcome was change in serum malondialdehyde (MDA). Secondary outcomes included markers of insulin resistance (leptin, fasting insulin, and HOMA-IR), lipid profile, liver steatosis and fibrosis assessed by FibroScan^® and non-invasive scores, and quality of life (QOL).

Results

No significant differences in serum MDA, leptin, fasting insulin, or HOMA-IR (all p > 0.05) between groups. Similarly, FibroScan^® parameters, non-invasive steatosis and fibrosis scores did not differ significantly between groups. A significant reduction in liver steatosis scores in the control group (p = 0.004) while the Hepatic steatosis index improved in both groups (p = 0.008).Triglyceride levels decreased significantly in the control group, whereas HDL-cholesterol increased significantly in the NAC group. QOL scores remained unchanged overall, except for a significant increase in the emotional domain scores in the NAC group.

Conclusions

High-dose NAC for three months did not significantly improve oxidative stress, insulin resistance, or hepatic steatosis or fibrosis in non-diabetic MASLD patients. NAC was safe and well tolerated during the study period.

Trial registration

This study was registered on clinicaltrials.gov under the identifier number NCT05589584 in October 2022.

Graphical Abstract

Supplementary Information

The online version contains supplementary material available at 10.1186/s12876-026-05157-x.

Background

Metabolic dysfunction-associated steatotic liver disease (MASLD) is the main cause of chronic hepatic disorders and liver-related mortality. It was previously termed as non-alcoholic fatty liver disease (NAFLD). Globally, MASLD prevalence has been increasing alongside obesity and T2DM from around 25% to 32% of the adult population with attributable mortality of 0.17% in 2023 [1]. MASLD encompasses a large spectrum of clinical disorders, from simple hepatic steatosis to hepatocellular inflammation and ballooning known as metabolic-associated steatohepatitis (MASH), which can advance to cirrhosis and hepatocellular carcinoma (HCC) [2].

MASLD is diagnosed by the existence of hepatic steatosis ≥ 5% of hepatic cells and one or more of the five cardiometabolic risk abnormalities [3]. Despite the complexity of the pathogenesis, both insulin resistance (IR) and oxidative stress play a pivotal role in steatosis initiation and progression [4, 5]. Due to the strong association between MASLD and cardiometabolic conditions, it is recommended to target all factors to enhance hepatic and extrahepatic outcomes [6]. Being a manifestation of metabolic disorder, the therapeutic lifestyle intervention aimed at weight reduction is the main cornerstone in MASLD management [6, 7]. Recently, some medications have been approved as additional treatments (Semaglutide and Resmetriom) for certain circumstances of MASH, if the weight loss goals are not met with lifestyle interventions alone [7, 8]. Semaglutide, a glucagon-like peptide- 1 receptor agonist (GLP-1RA), has recently been FDA-approved for stage 2–3 fibrosis (F2-F3) patients with MASH, especially in those who have other co-morbid conditions (obesity and diabetes) that would benefit from GLP-1-based therapy [8, 9]. Resmetirom, the first FDA-approved for non-cirrhotic MASH patients with stages 2–3 fibrosis (F2-F3), is a selective thyroid hormone receptor beta agonist [10], which is suggested as an adjunctive therapy for patients who can not tolerate GLP-1-based therapy.

The amino acid l-cysteine’s acetylated form, N-acetylcysteine (NAC), is a precursor of thiol (SH) groups that promote the synthesis of glutathione, which scavenges free radicals. The anti-inflammatory and antioxidant qualities of NAC are widely recognized and used to counteract the hepatotoxic effects of acetaminophen overdose [11].

Over time, reducing oxidative stress and inflammation in metabolic dysfunction has required the use of potent antioxidants like NAC. Earlier clinical studies reported the efficacy of NAC in the reduction of spleen size and alanine aminotransferase (ALT) levels [12], aspartate aminotransferase (AST) and gamma-glutamyl transferase (GGT) [13]. Similarly, 1.2 gm of oral NAC combined with 850–1000 mg of oral metformin significantly reduced not only ALT but also glucose, insulin, HOMA-IR, and degree of liver steatosis in a pilot study of 20 biopsy-proven non-alcoholic steatohepatitis patients [14].

Another study by Oliveria et al. (2019) showed that the combination of NAC and metformin improved the NAFLD Activity Score (NAS), hepatocyte ballooning, and degree of steatosis [15].

Treatment with 600 and 1200 mg of oral NAC improved oxidative stress indicators glutathione (GSH), IR (HOMA-IR index), inflammatory markers (interleukin-6 and high-sensitivity C-reactive protein), and decreased liver stiffness in a phase II double-blind trial of obese children with biopsy-proven MASLD [16]. Till now, all previous studies of NAC on steatotic patients used NAC doses ranging from 600 to 1200 mg, targeted diabetic patients with steatosis, and focused on its impact on elevated transaminases and changes in ultrasonography or liver biopsy.

N-Acetylcysteine (NAC) is considered safe when taken in doses ranging from 600 to 3000 mg per day [17]. The appropriate dosage of NAC depends on the specific treatment and the intended therapeutic outcome. Higher doses of NAC can act as a precursor to glutathione (GSH), an essential cellular antioxidant. By replenishing GSH levels in the liver, NAC aids in neutralizing reactive oxygen species (ROS) and mitigating oxidative stress, which is a significant contributor to liver damage. Additionally, high doses of NAC have been tested in other conditions, such as cystic fibrosis, and have been shown to be safe and well-tolerated [18]. Hence, this study aimed to investigate the efficacy and safety of high-dose NAC (2400 mg /day) on non-diabetic patients with MASLD.

Patients and methods

Study design and setting

A prospective, randomized, open-label, parallel-group, controlled clinical trial conducted from November 2022 to June 2024. Sixty non-diabetic MASLD patients were recruited from the National Hepatology and Tropical Medicine Research Institute (NHTMRI), Cairo, Egypt.

Patients

All patients presenting to the MASLD Outpatient Clinic at NHTMRI were evaluated for eligibility. Patients were included if they were aged from 18 to 60 years and non-diabetic, diagnosed with MASLD. The diagnosis of MASLD was based on the presence of hepatic steatosis on previous imaging confirmed by FibroScan CAP score > 248 (dB/m1) [19], plus one or more cardiometabolic risk factors and absence of any other etiologies of liver diseases [3, 6]. Exclusion criteria included ethanol consumption, drug addiction, history of schistosomiasis, active viral hepatitis C infection, viral hepatitis B infection, autoimmune liver disease, hemochromatosis, Wilson’s disease, decompensated liver disease (Hepatic encephalopathy, ascites and esophageal varices) or liver cirrhosis, diabetes Miletus, consumption of any antioxidant supplements, hepatotoxic drugs (amiodarone, valproic acid and methotrexate) or liver support medications (ursodeoxycholic acid and silymarin) or drugs known to affect MASLD (vitamin E, pioglitazone, dipeptidyl peptidase IV inhibitors, and GLP-1 analogs) within the 6 months prior to the study, pregnancy, breastfeeding, malignancy, hypersensitivity to N-acetylcysteine and current N-acetylcysteine consumption.

Randomization and treatment allocation

Using the free online program “Sealed Envelope” to perform block randomization with a block size of four by an independent researcher not involved in patient recruitment. To ensure allocation concealment within the randomization, treatment assignment was revealed only after participant enrollment and completion of baseline assessment. The 60 eligible participants were randomly assigned by at a 1:1 ratio into two groups; the NAC-treated group (30 patients) were assigned to 2400 mg/ day of N-acetyl cysteine oral therapy (N-acetyl cysteine, 600 mg effervescent sachets, Mucobrave^®, XEEDIA pharma -Egypt); two 600 mg sachets twice daily (1200 mg at the morning and 1200 mg at the evening) plus Mediterranean diet (MD) plan for 3 months. To lower the frequency of NAC-induced gastrointestinal tract (GIT) adverse events, proton pump inhibitors (PPIs) were added to the NAC group. The control group was only assigned to the same MD plan for 3 months. The MD plan was based on limiting ultra-processed and highly sugary foods and increasing vegetables, whole grains, nuts, olive oil, and fruit intake [20] aiming to achieve a gradual weight reduction (3–5% reduction in body weight) as recommended [6, 7]. It was provided by nutritionists who were available onsite while being blinded to the patient’s allocation. Prior to the study, none of the participants had received nutritional interventions. All participants did their own routine daily activities without any increased physical activities.

Methods

At baseline, all patients were subjected to a full physical examination and thorough data collection that included demographics, complete medical and medication history and other relevant clinical data collection.

Using routine laboratory techniques, laboratory investigations such as complete blood count (CBC), fasting blood glucose (FBG), glycated hemoglobin (HbA1c), serum albumin, liver enzymes like aspartate aminotransferase (AST) and alanine aminotransferase (ALT), as well as lipid profiles including total cholesterol (TC), high-density lipoprotein cholesterol (HDL-C), low-density lipoprotein cholesterol (LDL-C) and triglycerides (TG) were measured for all patients at baseline and after 3 months.

To quantify hepatic steatosis in dB/m and fibrosis in KPa, vibration-controlled transient elastography (FibroScan^®) with controlled attenuation parameter (CAP) was performed. Additionally, non-invasive tests (NITs), including hepatic steatosis index (HSI), NAFLD-fibrosis score (NFS) and fibrosis- 4 score (FIB-4) were calculated for all participants at the baseline and after 3 months.

At baseline and after 3 months, malondialdehyde (MDA) and leptin were assessed as markers of oxidative stress and insulin resistance (IR), respectively, for all participants. Also, fasting insulin and fasting blood glucose (FBG) were measured to estimate IR by calculating the homeostasis model assessment of insulin resistance (HOMA-IR) using the following formula [21] ; HOMA-IR = [fasting insulin (µIU/ml) × fasting glucose (mg/dl) /405].

All blood samples were obtained from patients after 10 h of fasting. Serum samples were stored at -80 °C till sample analysis. Leptin, MDA, and fasting insulin were measured using enzyme-linked immunosorbent assay (ELISA) commercial kits provided by BT LAB and DiaMetra companies, respectively. FibroScan^® was done at NHTMRI, using Transient Elastography, FibroScan^® 502, Echosense, US; with XL probe. All laboratory measurements were conducted at an external medical laboratory “My Lab for Hepatology and Oncology Laboratory”, Cairo, Egypt.

Assessment of quality of life (QOL) was performed using a validated Arabic form of chronic liver disease questionnaire-NAFLD version (CLDQ-NAFLD/NASH) that consists of six domains; abdominal, activity, worry, systemic, emotional and fatigue symptoms [22]. The assessment was done at the baseline and at the 3^rd month through face-to-face interviews. Patients were asked close-ended questions about each symptom and were given discrete choices ranging from 1 ‘all of the time’ to 7 ‘none of the time’ to pick one of them. The higher the score, the better the patient’s quality of life.

Patient education

All patients were educated regarding the following: disease definition, complications, the role of NAC, expected NAC side effects, and how to report them using side effect card.

Follow up-evaluations

All patients in the 2 groups were followed up by weekly phone calls and every other week visits and were re-assessed for their compliance to NAC, the MD plan and the occurrence of side effects. Patient’s compliance to NAC was measured by counting the remaining sachets during refill to ensure that the patients were taking the medication exactly as prescribed. While patients’ compliance with the MD plan was assessed by monitoring the change in measurements of body weight and waist circumference (WCr) at each visit.

Ethical considerations

Every procedure involving human subjects in this study complied with the Declaration of Helsinki as revised in 2013 and its updates in 2024 [23].

The study was registered on clinicaltrials.gov under the identifier number (NCT05589584) in October 2022. In addition, the study protocol was approved by the Research and Ethics Committee of the Faculty of Pharmacy, Ain Shams University, approval number (RHDIRB2020110301 REC # 114). Also, it was approved by the Research and Ethics Committee for Experimental and Clinical Studies, NHTMRI (Approval No. 2/22).

Patient consent

All recruited patients were asked to fill out a written informed consent before enrollment in the trial after a clear and complete clarification of the nature, purpose, and potential risks of the study.

Outcome measures

The primary outcomes of this study were to evaluate NAC’s antioxidant and insulin-sensitizing effects on MDA, leptin, and HOMA-IR in non-diabetic patients with MASLD. The secondary outcomes were the impact on liver steatosis and stiffness, lipid profile, safety and QOL.

Sample size calculations

Using G*Power© software (Institutfür Experimentelle Psychologie, Heinrich Heine Universität, Düsseldorf, Germany) version 3.1.9.2, the sample size was calculated by anticipating a large effect size of 0.8 for the NAC treatment group versus the control group. The large effect size 0.8 was used owing to lack of previous studies examining the impact of NAC on leptin and MDA in patients with MASLD. A minimum of 26 patients per group, or 52 patients overall, were needed to reach the target power of 0.80 and an alpha error of 0.05. The sample size was expanded by 15% to compensate for the loss of follow-up to include 30 patients in each group, for a total of 60 patients.

Statistical analysis

IBM SPSS^® Statistics version 23 (IBM^® Corp., Armonk, NY, USA) was used for statistical analysis. The mean and standard deviation or, if applicable, the median and range were used to represent numerical data. Frequencies and percentages were used to express qualitative data. The relation between qualitative variables was examined using either Fisher’s exact test or Pearson’s Chi-square test.

Data were tested for normality using the Shapiro-Wilk and Kolmogorov-Smirnov tests; quantitative variables were compared between two groups using either the Mann-Whitney (non-parametric t-test) or the Student t-test (for normally distributed data); two consecutive measurements of numerical variables were compared using either the paired t-test or the Wilcoxon Signed Ranks (non-parametric paired t-test); and categorical variables were compared at baseline and after the trial in each group using the McNamar-Bowker test. Using the Spearman-Rho correlation, the correlation between numerical variables was examined. The Bonferroni method was utilized to correct the p-value because of multiple comparisons. Analysis of covariance (ANCOVA) was performed to compare post-treatment readings between the two groups while controlling the effects of gender and intake of PPIs. The main analysis was performed based on the Per-Protocol principle. All tests were two-tailed. P-values less than 0.05 were regarded as statistically significant.

Results

Baseline assessment

Hepatitis B surface antigen (HBsAg), HCV antibodies and human immunodeficiency virus (HIV) antigen/antibody were tested in 140 individuals, ages 18 to 60, who had steatotic liver on ultrasonography. Just 60 patients consented to enroll in the trial after 112 patients were determined to be eligible. Nine patients were dropped after few weeks of enrollment (5 from the NAC group and 4 from the control group), Those patients were lost to follow-up after few weeks. Therefore, only 51 patients completed the study (control, n = 26; NAC, n = 25). The study flow chart is displayed in Fig. 1.

Fig. 1.

The consort flow diagram of the study

Patients’ demographics are presented in Table 1. At baseline, patients in the two groups were comparable (p-value > 0.05) with regards to their age, gender, height, weight, BMI, waist circumference, hepatomegaly, comorbidities, current medications, degree of steatosis, hepatic steatosis index (HSI), NAFLD fibrosis Score (NFS), and Fibrosis-4 (FIB-4) scores, except for the use of PPIs in NAC treated group was significantly different p-value (< 0.004). ). This unbalanced administration of PPI didn’t affect any of clinical outcomes in both groups as analyzed by ANCOVA. Females constituted a higher proportion of the study population than males; however, the difference between groups was not statistically significant. A univariate analysis of variance was conducted to assess the effect of sex on the study outcomes, and no statistically significant influence of female sex was observed on any of the reported clinical outcomes (p-value > 0.05), as represented in Supplementary Tables S1-S6.

Table 1.

Patients’ demographic data, medications used and clinical characteristics

Parameters		NAC group (n = 25)	Control group (n = 26)	p-value
Age (years); mean ± SD		42.9 ± 9.2	45.2 ± 10	1 a
Gender n (%)	Females	21 (84%)	24 (92.3%)	0.419 b
	Males	4 (16%)	2 (7.7%)
Weight (Kg); mean ± SD		98.6 ± 13.7	91.6 ± 16.2	0.424 a
Height (m); mean ± SD		1.596 ± 0.079	1.589 ± 0.06	1 a
BMI (Kg/m2); mean ± SD		38.9 ± 6.4	36.3 ± 6.2	0.588 a
Waist circumference (cm); mean ± SD		115.7 ± 11.6	109.4 ± 8.5	0.124 a
Comorbidities n (%)	Hepatomegaly	21 (84%)	19 (73%)	1 b
	Dyslipidemia	15 (60%)	16 (61.5%)	1 b
	Hypertension	8 (32%)	3 (11.5%)	0.304 b
	Hypothyroidism	1 (4%)	1 (3.8%)	NA c
	Anemia	8 (32%)	4 (15.4%)	0.648 b
	BPH	1 (4%)	1 (3.8%)	NA c
	IHD	1 (4%)	1 (3.8%)	NA c
	Rheumatic fever	0%	2 (7.7%)	NA c
	Asthma	0 (0%)	1 (3.8%)	NA c
Medications n (%)	Iron supplement	2 (8%)	2 (7.7%)	1d
	Inhaled corticosteroids	0%	2 (7.7%)	NA c
	Vitamin B 12 complex	12 (48%)	12 (46.2%)	1 b
	COC	6 (24%)	3 (11.5%)	1 d
	Analgesics	12 (48%)	16 (61.5%)	1 b
		13 (52%)	10 (38.5%)
	Statins	5 (20%)	9 (34.6%)	0.968 b
	Beta-blockers	7 (28%)	2 (7.7%)	0.3 d
	PPI	25 (100%)	10 (38.5%)	<0.001* b
	Levothyroxine	1 (4%)	1 (3.8%)	NA c

All p-values were corrected using Bonferroni method

Abbreviations: BPH Benign Prostatic Hyperplasia, BMI Body Mass Index, CAP Controlled Attenuation Parameter, COC Combined Oral Contraceptive Pills, IHD Ischemic Heart Disease, NAC N-Acetyl Cysteine, PPI Proton Pump Inhibitors

* Level of significance p ˂0.05

Statistical tests; ^a, Independent samples t-test; ^b, Pearson Chi-Square; ^c, no p-value due to small number of cases within subgroups; ^d, Fisher’s Exact Test

Regarding the biochemical data, the baseline biochemical profiles of patients in both groups were similar (p-value > 0.05).

Evaluation of NAC efficacy

After 12 weeks, the median MDA serum level showed a non-significant reduction in the NAC-treated group and a non-significant increase in the control group (both P- values = 1). Also, there was no significant difference between the study groups with respect to MDA serum level (p = 1), Table 2 (Fig. 2).

Table 2.

Changes in liver enzymes, oxidative stress, and insulin resistance after three months

Parameters	Time of assessment		NAC group (n = 25)	Control group (n = 26)	p-value
ALT (U/L) Median (IQR)		Baseline	22 (15–31)	22.5 (18–30)	1 a
		After 3 months	23 (18–26)	21 (17–30)	1 a
		p-value	1 c	1 c
AST (U/L) Median (IQR)		Baseline	25 (21–30)	25 (18–34)	1 a
		After 3 months	26 (22–28)	25.5 (23–29)	1 a
		p-value	1 c	1 c
MDA (nmol/ml) Median (IQR)	Baseline		7 (5.4–13)	6.9 (4.3–11.4)	1 a
	After 3 months		5.9 (5 -14.1)	8.9 (5 -12.7)	1 a
	p-value		1 c	1 c
	Percent change		0 ((-21.6) – 14.5)	0 ((-4.7)- 30.5)	0.249 a
Leptin (ng/ml) Median (IQR)	Baseline		1.6 (0.8 -2)	1.3 (0.8–1.8)	1 a
	After 3 months		0.9 (0.7–2.1)	1.2 (0.8-2)	1 a
	p-value		0.612 c	1 c
	Percent change		-12.5 ((-25) − 0)	-9 ((-20) -16.67)	0.406 a
Hb A1c (%) Mean ± SD	Baseline		5.6 ± 0.5	5.5 ± 0.5	1 b
	After 3 months		5.3 ± 0.7	5.3 ± 0.6	1 b
	p-value		0.148 d	0.116 d
Fasting insulin (µIU/ml) Median (IQR)	Baseline		9.9 (5.5–31.1)	5.3 (4.3–12.9)	0.288 a
	After 3 months		6.4 (4–24)	4.9 (4 -9.6)	0.812 a
	p-value		1 c	0.748 c
	Percent change		-18.5 ((-44.2) – 3.3)	-6.1((-47.8) -17.9)	0.792 a
FBG (mg/dl) Mean ± SD	Baseline		103.5 ± 17.9	113.3 ± 22.9	0.380 b
	After 3 months		109.4 ± 17.4	113 ± 15.7	1 b
	p-value		0.704 d	1 d
HOMA-IR index Median (IQR)	Baseline		2.1 (1.5–7.3)	1.4 (1.2–3.3)	0.620 a
	After 3 months		1.9 (1.1–6.8)	1.2 (1- 2.4)	1 a
	p-value		1 c	0.912 c
	Percent change		-17.4 ((-47.6) -33.6)	-9.1 ((-43.9) -28.6)	1 a

All p-values were corrected using Bonferroni adjustment

AbbreviationsALT Alanine Aminotransferase, AST Aspartate Aminotransferase, FBG Fasting Blood Glucose, Hb A1c Glycated Hemoglobin, HOMA-IR Homeostatic Model Assessment for Insulin Resistance, MDA Malondialdehyde, NAC N-Acetyl Cysteine

* Level of significance p ˂0.05

Statistical tests; ^a, Mann-Whitney Test; ^b, Independent samples t-test; ^c, Wilcoxon Signed Ranks Test; ^d, Paired T-Test

Fig. 2.

Comparison of malondialdehyde MDA (nmol/ml) levels between groups and change over time in each group

Regarding the insulin resistance (IR) markers, there was a non-significant difference within groups and between groups regarding leptin (Fig. 3), FBG, HOMA-IR, and fasting insulin as presented in Table 2.

Fig. 3.

Comparison of leptin (ng/ml) levels between groups and change over time in each group

FibroScan^®was used as a non-invasive tool to measure liver steatosis and fibrosis expressed in controlled attenuation parameter (CAP) score and Kpa, respectively. However, there was no significant difference between the groups (p = 1), It was observed that within the NAC group, the CAP score decreased non-significantly (p = 0.460), while it decreased significantly within the control group (p = 0.004).

Regarding the change in Kpa, there was non-significant reduction within and between groups as described in Table 3. The change in the degree of liver steatosis and fibrosis over time is presented in Table 4.

Table 3.

Comparison of FibroScan and score-based steatosis and fibrosis grades over three months

Parameters		Time of assessment	NAC group (n = 25)		Control group (n = 26)	p-value
Steatosis/ CAP score (dB/m)		Baseline median (IQR)	297 (264–310)		289 (249–329)	1 a
		After median (IQR)	274 (246–298)		266 (206–296)	1 a
		p-value	0.460 b		0.004* b
		Percent change	-1.14 ((-18) – 4.6)		-12.8 ((-23) – (-0.34)	0.094 a
Fibrosis (KPa)		Baseline Median (IQR)_	5.4 (4.3–11)		4.6 (4 -5.7)	1 a
		After Median (IQR)	5.5 (2.6–7.1)		4.7 (3-10.4)	0.908 a
		p-value	0.940 b		1 b
		Percent change	-4.7 ((-15.9) – 5.5)		0.25 ((-13) – 27.5)	0.296 a
HSI Median (IQR)	Baseline		47.9 (41.4–51.9)	44.6 (40.3–48.2)		1 a
	After 3 months		46.9 (39.2–50.9)	42.9 (38.4–47.6)		0.828 a
	p-value		0.008* b	0.008* b
NFS Median (IQR)	Baseline		-2 ((-3.1) – (-1))	-1.6 ((-2.9)- (-0.9))		1 a
	After 3 months		-2.7 ((-3.3) – (-1.8))	-2.1 ((-2.6) – (-0.7))		0.800 a
	p-value		0.924 b	1 b
FIB-4 Median (IQR)	Baseline		0.72 (0.5–1)	0.84 (0.5–1.3)		1 a
	After 3 months		0.68 (0.5–0.9)	0.86 (0. 7–1.3)		0.158 a
	p-value		1 b	1 b

All p-values were corrected using Bonferroni adjustment

AbbreviationsCAP Controlled Attenuation Parameter, FIB-4 Fibrosis-4 Score, HSI Hepatic Steatosis Index, NAC N-Acetyl Cysteine, NFS NAFLD Fibrosis Score

* Level of significance p ˂0.05

Statistical tests; ^a, Mann-Whitney Test; ^b, Wilcoxon Signed Ranks Test

Table 4.

Change in steatosis and fibrosis within each group

All p-values were corrected using Bonferroni method

Abbreviations: CAP Controlled Attenuation Parameter; NAC N-Acetyl Cysteine

*Level of significance p ˂0.05

Progression or worsening,   Stable,  Improvement

Statistical tests; *, level of significance p <0.05, corrected p-value using Bonferroni method; a, McNemar-Bowker Test at p <0.05

Hepatic steatosis index (HSI), fibrosis-4 (FIB-4) score, and NAFLD fibrosis score (NFS) were calculated for each participant at the start and the end of the study. Despite the non-significant difference between the study groups regarding HSI (p = 0.828), HSI was significantly improved within both groups (both p-values = 0.008) (Fig. 4).

Fig. 4.

Comparison of hepatic steatosis index (HSI) between groups and change over time in each group. * p-value = 0.008

According to scores assessing liver fibrosis (NFS and FIB-4), there was a reduction in the median of FIB-4 within the NAC group versus an increase within the control group, but this change didn’t reach significance (both p-values = 1). Also, the intergroup analysis of FIB-4 showed no significance (p = 1). Regarding NFS, there was a non-significant difference within and between the study groups as presented in Table 3.

After 12 weeks, both groups were comparable regarding lipid profiles. However, there was a significant rise within the NAC group with respect to HDL-C (p = 0.028) (Fig. 5) while triglycerides serum level was significantly decreased within the control group (p = 0.008) Table 5.

Fig. 5.

Comparison of high-density lipoprotein cholesterol (HDL-C) levels in mg/dL between groups and change over time in each group. ^*P-value = 0.028

Table 5.

Change in routine laboratory parameters between and within both Groups

Parameters	Time of assessment	NAC group (n = 25)		Control group (n = 26)	p-value
TLC (109/L) Mean ± SD	Baseline		8.06 ± 2.57	6.66 ± 2.69	0.256 a
	After 3 months		7.42 ± 2.45	6.73 ± 2.51	1 a
	p-value		0.088 b	1 b
ANC (109/L) Mean ± SD	Baseline		4.51 ± 1.61	3.61 ± 1.85	0.276 a
	After 3 months		4.29 ± 1.82	3.92 ± 1.6	1 a
	p-value		1 b	1 b
NLR Mean ± SD	Baseline		0.56 ± 0.08	0.54 ± 0.12	1 a
	After 3 months		0.56 ± 0.7	0.58 ± 0.06	1 a
	p-value		1 b	0.268 b
Hb (g/dl) Mean ± SD	Baseline		12 ± 1.6	12.7 ± 1.3	0.520 a
	After 3 months		12.2 ± 1.8	12.3 ± 1.2	1 a
	p-value		1 b	0.240 b
Platelets (109/L) Median (IQR)	Baseline		311 (262–351)	268 (247–308)	0.344 c
	After 3 months		307 (257–369)	259.5 (220–310)	0.148 c
	p-value		1 d	0.972 d
Serum albumin (g/dl) Mean ± SD	Baseline		4.1 ± 0.5	4 ± 0.3	1 a
	After 3 months		4.1 ± 0.4	3.9 ± 0.3	1 a
	p-value		1 b	1 b
Total cholesterol (mg/dl) Mean ± SD	Baseline		193.2 ± 39.1	203.9 ± 50.8	1 a
	After 3 months		181.5 ± 37.4	176.8 ± 36.7	1 a
	p-value		1 b	0.128 b
Triglycerides (mg/dl) Mean ± SD	Baseline		137 ± 53.2	134.9 ± 53.6	1 a
	After 3 months		123.7 ± 46.7	99.5 ± 28.6	0.116 a
	p-value		0.384 b	0.008 * b
HDL-C (mg/dl) Mean ± SD	Baseline		48.6 ± 12.1	51 ± 6.9	1 a
	After 3 months		55 ± 10.2	52.7 ± 13.6	1 a
	p-value		0.028 * b	1 b
LDL-C (mg/dl) Mean ± SD	Baseline		119.7 ± 41.6	123.6 ± 43	1 a
	After 3 months		101.2 ± 31.6	106.5 ± 35.9	1 a
	p-value		0.284 b	0.496 b

All p-values were corrected using Bonferroni adjustment

AbbreviationsANC Absolute Neutrophil Count, AST Aspartate Aminotransferase, Hb Hemoglobin, HDL High-Density Lipoprotein, LDL Low-Density Lipoprotein, NAC N-Acetyl Cysteine, NLR Neutrophil-Lymphocyte Ratio, TLC Total Leukocyte Count

* Level of significance p ˂0.05

Statistical tests; ^a, Independent samples t-test; ^b, Paired T-Test; ^c, Mann-Whitney Test; ^d, Wilcoxon Signed Ranks Test

There was also a non-significant change between both groups in relation to weight, BMI, and waist circumference (p = 0.524, p = 0.688, p = 0.144), respectively. While there was a significant decrease in body weight, BMI, and waist circumference measures within each group (p ˂ 0.001) (Table 6).

Table 6.

Change in weight, body mass index and waist circumference between and within both Groups

Parameters	Time of assessment	NAC group (n = 25)		Control group (n = 26)	p-value
Weight (Kg) Mean ± SD	Baseline		98.6 ± 13.7	91.6 ± 16.2	0.106 a
	After 3 months		94 ± 14.4	87.5 ± 15.7	0.131 a
	p-value		˂ 0.001* b	˂ 0.001* b
BMI (Kg/m2) Mean ± SD	Baseline		38.9 ± 6.4	36.3 ± 6.2	0.147 a
	After 3 months		37.1 ± 6.8	34.7 ± 5.9	0.172 a
	p-value		˂ 0.001* b	˂ 0.001* b
Waist circumference (cm) Mean ± SD	Baseline		115.7 ± 11.6	109.4 ± 8.5	0.124 a
	After 3 months		111.5 ± 12.1	105 ± 9.1	0.144 a
	p-value		˂ 0.001* b	˂ 0.001* b

All p-values were corrected using Bonferroni method

Abbreviations: BMI Body Mass Index, NAC N-acetyl cysteine

* Level of significance p ˂0.05

Statistical tests; ^a, Independent samples t-test; ^b, Paired T-Test

At baseline, the two groups were comparable with regards to the individual domains’ scores and the overall scores. After 3 months, it was revealed that the overall score and the domains’ scores were not significantly improved within and between groups. However, the emotional domain score was significantly improved within the NAC group (p = 0.044) (Table 7).

Table 7.

Change in patients’ answers to the CLDQ-NAFLD/NASH questionnaire between and within both Groups

Domain	Time of assessment	NAC group (n = 25)	Control group (n = 26)	p-value
Abdominal domain Median (IQR)	Baseline	3.7 (2.3–5.3)	4.3 (2.7–5.3)	1 a
	After 3 months	4.7 (3-5.7)	4.8 (3.3–6.7)	1 a
	p-value	1 b	0.068 b
Activity domain Median (IQR)	Baseline	5.6 (4.4–7)	5.6 (3.4–7.2)	1 a
	After 3 months	5.2 (4.6–6.8)	6.1 (3.8–7.4)	1 a
	p-value	1 b	1 b
Emotional domain Median (IQR)	Baseline	4.1 (3.1–5.2)	3.6 (3- 4.7)	1 a
	After 3 months	4.3 (3.3–5.9)	4 (2.8–5.1)	0.584 a
	p-value	0.044 * b	1 b
Fatigue domain Median (IQR)	Baseline	3.5 (2.5–4.7)	3.2 (1.8–4.3)	1 a
	After 3 months	3.8 (2.3–5.2)	3.5 (2.7–5.5)	1 a
	p-value	1 b	1 b
Systematic domain Median (IQR)	Baseline	4.5 (3.5–5.3)	3.5 (2.8- 5)	0.352 a
	After 3 months	4.5 (3.7–5.5)	3.9 (3–4.8)	0.480 a
	p-value	1 b	1 b
Worry domain Median (IQR)	Baseline	5.1 (3.7–6.1)	5.3 (3.1–6.1)	1 a
	After 3 months	6.3 (4.1–7)	6.3 (3.7- 7)	1 a
	p-value	0.112 b	0.416 b
Overall score Median (IQR)	Baseline	4.1 (3.2–5.5)	3.9 (3.3–4.6)	1 a
	After 3 months	4.4 (3.8–5.9)	4.6 (3.6–5.2)	1 a
	p-value	0.180 b	0.392 b

All p-values were corrected using Bonferroni method

Abbreviations:NAC N-acetyl cysteine

* Level of significance p ˂0.05

Statistical tests; ^a, Mann-Whitney test; ^b, Wilcoxon Signed Ranks Test

Correlations

In the correlation analysis of the whole study population, CAP score showed a significant fair positive correlation with BMI at baseline (r = 0.405, p-value = 0.003), HOMA-IR index at baseline (r = 0.377, p-value = 0.006), and Absolute neutrophil count at the end of study (r = 0.3, p-value = 0.003), while MDA was strongly positively correlated with leptin at baseline (r = 0.837, p-value = 0.00) and at the end of the study (r = 0.755, p-value = 0.00).

Regarding the correlation analysis of the NAC group, the end-of-study overall mean score of the CLDQ-NAFLD/NASH questionnaire was negatively correlated with the baseline fasting insulin levels (r = − 0.456, p-value = 0.022) and with end HOMA-IR index (r = − 0.419, p-value = 0.037), respectively. At the end of the study, the Absolute neutrophil count was positively correlated with CAP score (r = 0.905, p-value = 0.009) and HSI (r = 0.659, p-value < 0.001).

QOL evaluation

Table 7 compares the QOL between groups using the CLDQ-NAFLD/NASH questionnaire. There was no statistically significant difference between both groups in all domains except the emotional domain; there was a statistically significant improvement within the NAC group (p = 0.044).

Tolerability evaluations

Gastrointestinal symptoms (nausea, vomiting, fullness), skin itching, metallic taste, and mucus expulsion were the most frequently reported adverse effects in NAC treated group. There was no significant difference between both groups regarding gastrointestinal symptoms and skin itching. Mucus expulsion was resolved within one week. In the NAC group, two patients reported side effects like mild abdominal pain and mild difficulty with breathing.

Discussion

MASLD is a serious global health concern. The pathogenesis of MASLD is complicated and involves genetic, hormonal, and nutritional factors. Both oxidative stress and IR contribute to the pathophysiology of MASLD by enhancing the deposition of free fatty acids (FFAs), enhancing the hepatic de novo lipogenesis (DNL), and damaging the mitochondrial genome [24]. Thus, the reduction of oxidative stress status and increasing the sensitivity to insulin in patients with MASLD are the main goals in MASLD management.

Beyond being the most popular antidote for acetaminophen-induced hepatotoxicity and a well-known antioxidant, Experimental and clinical trials have demonstrated the significant usefulness of N-acetyl cysteine in a variety of clinical conditions [25] as well as in steatohepatitis [12–16].

The purpose of the current study was to investigate the possible impact of high dose of NAC as an antioxidant and insulin sensitizer on the clinical outcomes of patients with MASLD. To our knowledge, this is the first trial to investigate the impact of NAC at this high dose (2400 mg daily) on MDA and leptin, which are indicators of oxidative stress and IR in this patient population, respectively. According to the study’s findings, oral supplementation with 2400 mg of NAC did not affect either the MDA, leptin levels, or liver steatosis (p > 0.05).

MDA serum level was used as a surrogate measure of intrinsic oxidative stress. No significant effect was found in serum MDA in our study. Similarly, a study by Garicano et al. [26] observed no impact on MDA serum concentrations in patients with metabolic syndrome and at high risk for MASLD after three months of administration of 100 mg of NAC combined with thioctic acid (75 mg), S-adenosyl-L-methionine (200 mg) and vitamin B6 (0.65 mg) under the trade name of MetioNac^®. Simultaneously, an RCT trial found that taking 1800 mg of NAC for 3 months did not affect MDA levels in obese subjects with metabolic syndrome [27]. Another trial reported that following eight weeks of NAC administration in patients with MASLD, no significant changes were observed compared to the placebo on MDA levels(p > 0.05) [28].

This was supported by results reported by Thong-Ngam et al. [29], that NAC administration at 20 mg/kg/d for 6 weeks did not affect the level of MDA in rats subjected to high-fat diet-induced MASLD. In contrast to these results, some studies reported that NAC ameliorated MDA serum levels in preclinical models of MASLD [30, 31]. A meta-analysis revealed a significant reduction in MDA levels with N-acetylcysteine NAC treatment (p < 0.001) [32]. Variability in effect size between our study and others may be explained by heterogeneity of NAC effect in different patient populations, such as individuals with liver disease versus others treated for other diseases, such as burns, chronic obstructive pulmonary disease (COPD), community-acquired pneumonia (CAP), and chronic kidney disease (CKD).

Both insulin resistance (IR) and adipokines, such as leptin, are related to the pathogenesis and progression of MASLD [33]. Prospective studies observed elevated serum leptin concentrations among MASLD patients [34, 35]. Although NAC was found to reduce serum leptin levels in methionine–choline-deficient diet )MCDD( induced liver steatosis in rats [31], this study’s results didn’t show an effect of NAC on serum leptin levels. This finding was not consistent with the finding reported by Cheraghi et al., that NAC decreased follicular leptin concentration in PCOS women candidates for intracytoplasmic sperm injection [36]. This may be explained that leptin appears to be context-dependent and potentially related to changes in underlying metabolic and inflammatory conditions like PCOS or severe obesity-related inflammation rather than a universal effect in all human subjects.

According to the recent joint European guidelines for MASLD, HOMA-IR may be considered to describe insulin resistance in non-diabetic persons with suspicion of MASLD [6]. NAC showed a significant impact on HOMA-IR and fasting insulin index, not only in women with PCOS [37–39] but also in patients with metabolic syndrome [27]. Also, a year of oral NAC at 1.2 g coupled with 850–1000 mg of oral metformin significantly reduced the HOMA-IR index and serum insulin in 20 biopsy-proven non-alcoholic steatohepatitis (NASH) patients, according to Oliveira et al. [14]. Similarly, Babu Balagopal et al. reported decrease in HOMA-IR upon 4 months of NAC treatment in obese children with biopsy-proven MASLD [16]. In the present study, FBG, fasting insulin, and HOMA-IR index did not significantly differ between or within the study groups. This came in concordance with an open-label pilot study in subjects with type 2 diabetes that compared the effect of 1200 mg daily and 2400 mg daily of NAC. Both NAC dosages had no effect on oxidative indicators, insulin release, glucose tolerance, or glycemic control [40]. The discrepancy between the findings may be attributed to the longer duration used by Oliveira et al. and Babu Balagopal et al. (12 months and 4 months), respectively. Oliveira et al. evaluated the impact of NAC in combination with metformin, not independently as compared to the 12 weeks of the current study.

Regarding clinical outcomes, the FibroScan^® was one of the non-invasive diagnostic modalities that was recommended for hepatic stiffness assessment [7]. The controlled attenuation parameter (CAP) feature quantifies ultrasound attenuation during hepatic stiffness measurement by transient elastography. It provides a numerical value correlated with histological steatosis and fibrosis degree, expressed in dB/m and Kpa, respectively [41]. It was reported that NAC, individually or in combination with metformin, reduced hepatic steatosis in rats with MASLD [42]. Tsai et al. concluded that 12 months of administration of NAC decreased high-fat diet-induced steatosis in mice [43].

In our study, there were no significant differences between both groups regarding CAP and Kpa scores at baseline and the end of this trial. Only a significant reduction in CAP scores was observed within the control group. This finding was in agreement with previous studies that had reported that the Mediterranean Diet significantly improved the CAP scores and liver fibrosis among MASLD patients [44, 45]. On the other hand, Khoshbaten et al. [12] reported that NAC at a dose of 1.2 gm/day non-significantly improved liver steatosis grades when compared to 2gm of vitamin C.

On the contrary, another study by Oliveria et al. (2019) revealed that one-year administration of the combination of NAC and metformin ameliorated hepatocyte ballooning and the degree of steatosis as provided by the end of follow-up liver biopsy [15]. This may be attributable to the heterogeneous modalities used to assess liver steatosis and stiffness. Also, many factors can influence the FibroScan^® interpretation with CAP scores, as BMI, intrahepatic masses, the position of the probe, non-fasting state, and operator inexperience [46–49].

The primary diagnostic method for detecting hepatic steatosis is abdominal ultrasonography [50]. However, because of the cost and necessity of equipment and specialized staff, Steatosis indices like HSI have been used to diagnose MASLD [51]. In this study, the intragroup analysis of both groups displayed a significant reduction in the HSI scores, while the intergroup analysis of HSI showed no significance. This came in agreement with Sinaeinejad et al. [28] who showed that NAC administration in patients with MASLD does not significantly impact hepatic steatosis grade (p = 0.215). This can be explained by the fact that oxidative stress and inflammation are the NAC primary mechanisms targeted, not the underlying fat accumulation in the liver, which is estimated by the hepatic steatosis index (HSI).

On the other hand, one study has reported improvement of liver histopathology by NAC in non-alcoholic fatty steatosis (NASH) in a rat model [29]. Another study has reported a significant decrease in liver steatosis and fibrosis in patients with NASH receiving Metformin and NAC [14]. This may be attributed to the effect of combination therapy on the hepatic steatosis index.

Regarding the progression of fibrosis, we compared the NFS and FIB-4 scores at baseline to the same scores at the end of the follow-up period. There was no significant difference between and within the study groups. This finding is in line with Garicano et al.‘s study that found no impact on NFS in patients with metabolic syndrome and high risk for MASLD after three months of MetioNac^® administration [26]. This was also consistent with the results of Shaker et al., that reported a non-significant impact of 200 mg of Phyllanthus niruri extract combined with 100 mg Milk Thistle under the name of Heptex^® on the FIB-4 score after 36 weeks of receiving both high (2 capsules) and low doses (1 capsule inserted into 2 capsules for blinding) in patients with apparent risk for NASH [52]. Both Phyllanthus niruri and Milk Thistle possess an antioxidant effect like NAC [53]. In contrast to our results, a recent RCT reported that NAC caused a significant reduction in the mean value of fibrosis (p = 0.001), and this may be explained that the underlying causes of advanced fibrosis are complex and multifactorial, and NAC’s primary effect is mainly on oxidative stress rather than the full spectrum of disease progression [54].

Dyslipidemia is one of the cardio-metabolic risk factors that are involved in MASLD progression to HCC [55]. In clinical practice, biochemical testing reveals that patients with MASLD have higher triglycerides, higher LDL-C, and lower HDL-C [56].

In our study, there was no significant difference between study groups regarding the lipid profile panel (TC, TG, LDL-C and HDL-C), but the NAC-treated group showed a significant elevation of HDL-C levels from 48.6 ± 12.1 to 55 ± 10.2 mg/dl (p = 0.028). This is supported by the results obtained from an RCT that compared the metabolic effect of NAC and metformin on PCOS, in which HDL-C level was increased in the NAC arm [38] Moreover, there was a significant reduction in TG levels within the control group, while the TG reduction within the NAC group did not reach statistical significance. Also, Younes et al. reported that NAC intake had substantially improved HDL-cholesterol concentrations but did not alter other lipid parameters [27].

Moreover, Sinaeinejad et al. documented that changes in serum lipid profile parameters were not statistically significant. TG levels decreased from 182.2 ± 59.8 to 162.6 ± 41.9 mg/ dL in the NAC group (p = 0.15) and from 183.2 ± 63.0 to 176.0 ± 57.3 mg/dL in the placebo group [28]. TC, LDL-C, and HDL-C levels showed similar non-significant trends in both groups. This may be explained that NAC does not directly alter lipid metabolism and so does not impact lipid profiles.

Also, Javanmanesh et al. [38] demonstrated that NAC at a dosage of 1800 mg/day, compared with metformin, for 6 months in PCOS women, considerably decreased serum triglycerides levels, but did not influence other lipid profile findings. Moreover, Oliveira et al. [14] reported that NAC and metformin administered to nonalcoholic steatohepatitis patients for 12 months improved serum HDL-cholesterol, and triglycerides levels, but did not affect serum concentrations of total cholesterol. Also, NAC (1200 mg/day) after 6 weeks improved HDL-cholesterol, and triglycerides levels in metabolic syndrome patients [57].

Regarding the significant reduction in TG shown in the control group, Abenavoli et al. [58, 59]found a significant reduction in TG level within the low-calorie diet (MD) group. On the contrary, Garicano et al. reported that MetioNac^® significantly decreased TG levels in patients with high risk for MASLD and metabolic syndrome [26].

Treatment for MASLD necessitates sustained lifestyle modifications, particularly weight loss and physical activity, which are associated with health benefits, as evidenced by existing literature and guidelines [6, 7]. In the current study, the Mediterranean diet (MD) was programmed for weight loss in both study groups. The statistical analysis within the control group and within the NAC group showed a significant reduction in body weight, BMI, and waist circumference but the intergroup analysis did not reach significance. Thus, we can conclude that the MD was the main cause of the reduction of body weight, BMI, and waist circumference which is in accordance with previous studies by Abenavoli et al. [58, 59], Babu Balagopal et al. [16] and Garicano et al. [26].

In MASLD, a modest reduction in body weight exceeding 5% has been associated with improvements in hepatic steatosis, liver enzyme levels, and extrahepatic metabolic abnormalities [60]. More substantial weight loss of over 10% leads to pronounced reductions in liver fat and is linked to significant improvement in NASH and regression of liver fibrosis [61–63]. Tapper et al. [64] also reported that weight reduction positively affects quality of life in MASLD patients across all domains of the Chronic Liver Disease Questionnaire (CLDQ) compared with patients who did not lose weight.

Moreover, a meta-analysis conducted by Fernández et al. showed that lifestyle modification is an effective therapeutic approach for MASLD. Interventions combining dietary changes and physical activity were more effective than either approach alone in improving liver enzyme concentrations and insulin resistance, as measured by HOMA-IR [65]. Similarly, a review by Maria Corina Plaz Torres highlighted that adherence to the Mediterranean diet improves lipid profiles and fibrosis scores in patients with MASLD [66], these findings are consistent with the results of the present study.

On the other hand, our findings are consistent with a meta-analysis that showed that NAC treatment had no significant impact on waist circumference, body weight, or BMI [67]. However, recently patients administered NAC at a dosage of 1200 mg twice daily, for six months, had experienced a decrease in lean body weight and BMI (p < 0.05). Waist circumference (WCr) and hip circumference (HC) demonstrated a significant reduction (p = 0.001) [54].

Body mass index and waist circumference are recognized as key risk factors for MASLD and its related cardiometabolic disorders, primarily reflecting the impact of visceral fat accumulation. Current clinical guidelines for MASLD management prioritize a 7–10% reduction in body weight achieved through dietary and exercise interventions, as this level of weight loss is associated with meaningful histological improvement. By contrast, N-acetylcysteine (NAC) exerts its effects mainly at the cellular level by attenuating oxidative stress and inflammatory pathways in the liver and adipose tissue, rather than by promoting substantial overall weight reduction.

MASLD is described as an asymptomatic disease in which patients often experience non-specific symptoms, such as abdominal upset, fatigue, somnolence, and anxiety [68]. By comparing the change in answers to the “Chronic Liver Disease Questionnaire (CLDQ -NAFLD/NASH)” of each participant in this trial at the beginning and after 3 months, it was revealed that NAC had no impact on overall score and domain scores except for the emotional domain score, which was increased within the NAC group. The increase in scores of the emotional domain of CLDQ-NAFLD/NASH may be attributed to the impact of NAC on interleukin-6 (IL-6) [69] and C-reactive proteins (CRP) [57] as it was reported that the emotional domain of CLDQ-NAFLD/NASH was negatively correlated to both [70]. However, this can’t be concluded as the study was an open-label trial.

On the other hand, Zakaria et al., reported that NAC showed a significant increase in physical functioning domains in SF36 HRQOL questionnaire by 22.22% (p = 0.001); in addition, the improvements in role limitations due to physical health, and role limitations due to emotional scale [54].

This study showed a statistically significant positive correlation between the baseline value of the CAP score (dB/m) and the baseline value of both BMI (kg/m²) and HOMA-IR index. This was in agreement with the results obtained from previous studies in which the CAP score was correlated with metabolic syndrome features, especially BMI and HOMA-IR index [71–73].

Regarding the inflammatory role in the development and progression of MASLD [74], neutrophil to lymphocyte count ratio (NLR) is one of the inflammatory markers that combines two types of immune responses; neutrophils, which represent continuous inflammation, while the regulatory pathway is illustrated by lymphocytes [75]. NLR may be used for steatosis prediction [76, 77]. Lesmana et al. reported a strong correlation between the degree of steatosis measured by the CAP score and NLR [78]. However, our study demonstrated no significant correlation between CAP score and NLR. We found a significant correlation between the change in CAP score and the change in ANC for the whole study group (r = 0.3, p = 0.003), and the NAC-treated group had a strong positive correlation between both variables (r = 0.905, p = 0.009) which could be attributed to the inflammatory role of neutrophils in MASH progression Also, in line with a previous study on obese subjects [79]. The present study found a strong positive correlation between serum levels of MDA and leptin and a negative correlation between the end-of-study overall mean score of the CLDQ-NAFLD/NASH questionnaire and the baseline serum fasting insulin levels and end-of-study HOMA-IR index.

In the present study, the high dose of NAC (2400 mg/ day) was found to be tolerable with minimal side effects; 6 patients reported some GIT upset, 6 patients reported mucus expulsion, 2 patients suffered nausea and 2 patients experienced mild skin itching. The GIT side effects were managed using proton pump inhibitors (PPIs), while other side effects were self-limited, resolved within 1 to 2 weeks of treatment. The unbalanced administration of PPI didn’t affect our clinical outcomes as revealed by univariate analysis.

Only 2 patients stopped NAC; one patient suffered from difficulty breathing that didn’t require any medical interventions, while the other patient experienced mild GIT upset. This came in concordance with previous studies, NAC has a well-established safety profile with tolerable and self-controlled side effects [80–82]. NAC toxicity is not common and depends on the dose and route of administration. NAC’s side effects have been more apparent when given intravenously and at large dosages (> 3 g/day) [83]. The dose of 2400 mg of NAC was used before and showed a good safety profile [84–86]. GIT upsets were the most frequent adverse effects [80].

The strength points of the current study include being the first study to use such a high dose of NAC (2400 mg/day) in patients with MASLD. Also, no previous studies have investigated the effect of NAC on MDA, leptin, and HOMA-IR in non-diabetic MASLD patients.

Limitations

This trial had some limitations, including a small sample size, a single site, a short duration, a population in the early stages of the disease and an open-label design.

Other important limitations are the potential confounding effect of proton pump inhibitor (PPIs) use. In our study, all participants in the NAC group were on PPIs, compared to 38.5% in the control group. PPIs can alter hepatic drug metabolism [87], increase gastric pH leading to gut microbiota dysbiosis [88], and modulate systemic inflammatory markers [89]. These changes may have impaired NAC absorption, bioavailability, or efficacy, potentially blunting its antioxidant and insulin-sensitizing effects in MASLD patients. Therefore, the exclusive use of PPIs in the NAC group limits causal interpretation of our findings. On the other hand, the absence of GGT and ferritin as common clinical markers could be justified as they are indirect measures of oxidative stress and can be influenced by iron storage or general systemic inflammation. In this study, we measured malondialdehyde (MDA), a direct biomarker of lipid peroxidation, a hallmark of MASLD.

Conclusion

In conclusion, the consumption of high dose 2400 mg/day of NAC orally for 3 months in non-diabetic patients with MASLD had no effect on levels of MDA, leptin, and HOMA-IR index and did not impact hepatic steatosis or fibrosis. NAC administration improved the QOL in terms of emotional aspects, resulted in an elevation of HDL-C levels, and was tolerable with minimal side effects.

Recommendations

More clinical trials with larger sample sizes, longer durations, and varied outcomes on a wide spectrum of MASLD (diabetic MASLD and MASH), measuring other markers such as interleukin 1, interleukin 6, TNF-alpha, uric acid, and ferritin, are required to examine the NAC effect in the long term.

Abbreviations

ALT

Alanine aminotransferase

AST

Aspartate aminotransferase

BMI

Body mass index

BPH

Benign Prostatic Hyperplasia

CAP

Community acquired pneumonia

CAP

Controlled attenuation parameter

CBC

Complete blood count

CKD

Chronic kidney disease

COPD

Chronic obstructive pulmonary disease

CRP

C-reactive proteins

ELISA

Enzyme-linked immunosorbent assay

FBG

Fasting blood glucose

FIB-4

Fibrosis- 4 score

GGT

Gamma-glutamyl transferase

GLP-1RA

Glucagon-like peptide- 1 receptor agonist

GSH

Glutathione

HbA1c

Glycated hemoglobin

HCC

Hepatocellular carcinoma

HDL-C

High-density lipoprotein cholesterol

HOMA-IR

the homeostasis model assessment of insulin resistance

HSI

Hepatic steatosis index

IL-6

Interleukin-6

IR

Insulin resistance

LDL-C

Low-density lipoprotein cholesterol

MASLD

Metabolic dysfunction-associated steatotic liver disease

MASH

Metabolic-associated steatohepatitis

MCDD

Methionine–choline-deficient diet

MDA

Malondialdehyde

MD

Mediterranean diet

NAC

N-acetylcysteine

NAFLD

Non-alcoholic fatty liver disease

NAS

NAFLD Activity Score

NASH

Non-alcoholic steatohepatitis

NFS

NAFLD-fibrosis score

NITs

Non-invasive tests

NLR

Neutrophil to lymphocyte count ratio

PCOS

Polycystic ovary syndrome

PPIs

Proton pump inhibitors

QOL

Quality of life

ROS

Reactive oxygen species

TC

Total cholesterol

TG

Triglycerides

WCr

Waist circumference

Authors’ contributions

**Asmaa M. Ramadan** : Conceptualization; Data curation; Formal analysis; Investigation; Methodology; Visualization, and Writing – original draft.**Lamia M. El Wakeel** : Conceptualization; Formal analysis; Methodology; Supervision; Writing – reviewing and editing.**AbdulMoneim Adel** : Investigation; Methodology; Supervision. Reviewing and editing.**Mohamed Abdel-Wahab** : Conceptualization; Formal analysis; Methodology; Supervision; Writing – reviewing and editing.**Sarah Farid** : Conceptualization; Formal analysis; Methodology; Supervision; Writing – reviewing and editing.

Funding

All authors received no financial support for the research, authorship, and/or publication of this article.

Data availability

The datasets generated and analyzed during this study are available from the corresponding author upon reasonable request.

Declarations

Ethics approval and consent to participate

This study was approved by the Ain Shams University Ethical Review Board (Approval No. RHDIRB2020110301 REC # 114). Also, the Research and Ethics Committee approved it for Experimental and Clinical Studies, NHTMRI (Approval No. 2/22) and was conducted in accordance with the Declaration of Helsinki and Good Clinical Practice guidelines. In addition, it was registered at clinicaltrials.gov (https://clinicaltrials.gov/study/NCT05589584) on October 2022. Written informed consent was obtained from all participants prior to enrollment.

Consent for publication

Not applicable.

Competing interests

The authors declare no competing interests.