Metabolic dysfunction-associated steatotic liver disease (MASLD) is a clinical syndrome that is defined as hepatic macrovesicular steatosis (intracellular triglycerides of more than 5%)1. MASLD encompasses a spectrum from simple steatosis to- non-alchoholic steatohepatitis, fibrosis and cirrohsis2. As the prevalence of obesity, diabetes, and metabolic syndrome is growing among the population, MASLD has become the most common chronic liver disease all over the world3,4,5. The prevalence of MASLD is 38% globally6. South America has the most abundance, and Africa has the least abundance of this disease7. Its prevalence in Iran is 33.9%4. Diagnosis of MASLD is based on imaging methods like sonography, computed tomography (CT) scan, and magnetic resonance imaging (MRI), and rules out alcohol consumption; however, a biopsy is the gold standard of diagnosis8.

As most of the diseases that affect the liver are preventable, identifying fibrosis and its severity is essential to the management and follow-up of patients7,9. Zinc (Zn) is an important mineral that can eliminate oxygen radicals in the human body and necessary factor in lipid metabolism in liver cells10. Also, copper (Cu) is a necessary mineral for human and plant metabolism which can neutralize oxidative stress enzymes. In liver hepatocytes, Cu is incorporated into apo-ceruloplasmin using P-type ATPase enzymes11,12. Ceruloplasmin, a ferroxidase enzyme, is released into the bloodstream and is responsible for transporting over 95% of serum copper, oxidizing Fe2+ to Fe2+, regulating intracellular iron, and eliminating free radicals9,13,14,15,16,17,18. Recent studies found that serum levels of Zn and Cu are correlated with the hepatic necroinflammation19,20. This correlation is explained with these minerals important roles in oxidative stress, endoplasmic reticulum stress, apoptosis, and inflammation, which have a significant association with MASLD21,22. Despite known roles of zinc in liver health, its relationship with non-invasive fibrosis scores remains underexplored. Recently, liver fibrosis association with serum Zn in MASLD patients has been proved through biopsy; however, biopsy is an invasive and costly method, so it couldn’t be used to follow up and management of patients with chronic liver disease. To facilitate liver fibrosis assessment, some non-invasive indices like Fibrosis-4 (FIB-4), Nonalcoholic Fatty Liver Disease fibrosis score (NFS), and BARD have been introduced. Among these items, FIB-4 is the most proper index relative to the others23,24.

Because of the significance of non-invasive assessment of fibrosis in MASLD patients and the presence of nutritional metabolic role in the basis of MASLD, we are to examine the association between serum levels of Zn, Cu, and ceruloplasmin with non-invasive indices like FIB-4, NFS, and BARD.

Materials and methods

Study design and setting

This study was a prospective descriptive-analytic cross-sectional study that examined serum and liver lab tests of MASLD patients who had been referred to Imam Reza Research and Educational Hospital in Tabriz, Iran, during the year 2022. Abdominal sonography was used to diagnose and assess hepatic steatosis. To calculate the sample size, the PASS (version 15) application was used. According to a study by Ito Takanori, et al.23 which was done to assess serum Zn level and liver fibrosis association, with 80% power and 5% of type one error, the sample size was calculated to be 97 patients; Also for increasing study accuracy and the possibility of 20% sample loss, we considered 122 as sample size. And finally, 134 patients were included in this study.

Data collection and study performance

A total of 134 MASLD patients who had been referred to Imam Reza Hospital during the year 2022 who had the inclusion criteria were selected and included in this study. The patients were selected through simple randomization. Patients younger than 18 or older than 65, those with a history of alcohol consumption, a history of Zn supplements, a history of viral or autoimmune hepatitis, and a history of consuming drugs known to alter Zn or Cu levels, such as penicillamine, diuretics, and corticosteroids were excluded from the study. After getting informed consent, their demographic data, like sex, age, height, weight, and patient’s medical history, like diabetes and hypertension, were obtained and registered in the previously accepted checklist. Also, a fasting blood sample was gained to examine serum levels of Zn, Cu, ceruloplasmin, albumin, complete blood count (CBC), fasting blood sugar (FBS), and hepatic enzymes like Alanine aminotransferase (ALT) and aspartate aminotransferase (AST). Serum Zn, Cu, and ceruloplasmin levels were measured using atomic absorption spectrophotometry and colorimetric assays, and interpreted according to standard reference ranges. By using these data, non-invasive indices of liver fibrosis like FIB-4, NFS, and BARD were calculated. Age, platelet level, ALT, and AST are needed to calculate FIB-4. FIB-4 under 1.45 was considered a 0–1 fibrosis stage, a score between 1.45 and 3.25 was considered a 2–3 fibrosis stage, and a score of more than 3.25 was considered a 4–6 fibrosis stage25. The NFS index was calculated by using age, body mass index (BMI), platelet level, albumin, AST, ALT, and the existence of glucose tolerance disorder. In this index, a score under − 1.455 was equalized as fibrosis absence (F = 0), mild fibrosis (F = 1), or moderate fibrosis (F = 2). A score between − 1.455 and 0.675a was considered an unclear level of fibrosis. A score over 0.675 was considered severe fibrosis (F = 3) or cirrhosis (F = 4)4. BARD index was calculated by utilizing BMI, AST, ALT, and the existence of diabetes mellitus. A score between 0 and 1 was considered a low possibility of fibrosis; 2 to 4 is considered a severe stage of fibrosis26.

In this article, to simplify the comparison, we considered a FIB-4 below 1.45 as Category 1, between 1.45 and 3.25 as Category 2, and above 3.25 as Category 3. In NFS, we considered below − 1.455 as category 1, between − 1.455 and 0.675 as category 2, and above 0.675 as category 3, and in BARD, 0 was considered as category 0, 1 as category 1, 2 as category 2, 3 as category 3, and 4 as category 4.

After calculating these indices, their association with the level of serum Zn, Cu, and ceruloplasmin was examined.

Statistical analysis

All data were analyzed using IBM SPSS 26; Descriptive results were presented with mean (standard deviation (SD)) and frequency (percentage) for numerical and categorical variables, respectively. The normality of the variables was assessed and assured by the descriptive measures of distribution, the skewness (within ± 1.5), and the kurtosis (within ± 2). One-way ANOVA and independent t-test for normal variables were used to compare the significance. * Multivariable logistic regression analysis was conducted to identify potential cofounding factors influencing NFS and FIB-4. An odds ratio (OR) with a 95% confidence interval (CI) was presented. Factors with a P-value of less than 0.05 were considered statistically significant.

Ethical considerations

The current study was approved by the ethical committee of Tabriz University of Medical Sciences with code IR.TBZMED.REC.1401.292. All methods were carried out according to relevant guidelines and regulations. All experimental protocols were checked by the liver and gastrointestinal diseases research center and then approved by the scientific committee of the medicine faculty. All patients’ information was kept confidential, and their personal information was not mentioned or published anywhere. Furthermore, all needed tests were performed for patients, and no extra fees were charged to the patients. Informed consent was obtained from all patients.

Results

The demographic and laboratory data are presented in Table 1. The mean (SD) age was 50.05 ± 9.5 years. 78 patients (58.2%) were female. Serum markers of patients and their non-invasive liver fibrosis indices are demonstrated in Table 1.

Table 1 Demographic data and underlying disease of participants, and Table 2: blood markers and non-invasive indices of liver fibrosis in participants.
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In Table 2, the association between serum Zn, Cu, and ceruloplasmin levels with FIB-4, NFS and BARD scores was assessed. Lower serum Zn levels were found to have a significant correlation with higher FIB_4 and NFS scores (P-value < 0.05).

Table 2 Serum zinc, copper, and ceruloplasmin level association with FIB-4, NFS and BARD scores in participants.
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In Table 3, the results indicate that in the unadjusted (univariate) model, except for the variable Zn, none of neither variable Cu nor Ceruloplasmin showed a significant relationship with the variable FIB-4. Therefore, in both adjusted and unadjusted models, no significant difference in the probability of event occurrence was observed between FIB-4 and Cu and Ceruloplasmin. However, logistic regression analysis showed that after adjusting for age, BMI, and sex in the model, a significant relationship between the variable Zn and FIB-4 was still observed.

Table 3 Results of univariate and multivariable binary logistic regression models of factors affecting FIB-4.
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In Table 4, the results show that in the unadjusted model, by comparing categories 1 and 2 with the reference category 3, no significant difference in the odds of outcome occurrence for Cu and ceruloplasmin variables was observed. By adjusting for age, BMI, and sex in the multivariate model, no significant difference was observed in the results. Although the Zn variable was a significant predictor for different levels of NFS in the unadjusted model. Thus, when comparing categories 1 and 2 with the reference category 3, a significant difference in the odds of outcome occurrence for the Zn variable was observed. Similar results were found in multivariate findings based on adjusted odds ratios, showing a significant relationship between NFS and Zn after adjusting for confounding variables, age, BMI, and sex. In other words, the odds of NFS outcome occurrence in categories 1 and 2 compared to category 4 were significantly influenced by changes in the Zn variable.

Table 4 Results of univariable and multivariable multinomial logistic regression models of factors affecting NFS.
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Discussion

This study was designed to investigate serum Cu, Zn, and ceruloplasmin levels with three top non-invasive hepatic fibrosis indices: FIB-4, NFS, and BARD. This study found that among the three variables (Cu, Zn, and ceruloplasmin), serum Zn level had a significant reverse association with the FIB-4 and NFS scores. In the study by Iwata et al. lower serum Zn levels was found to be associated with hepatic fibrosis in hepatitis B patients. They suggested that disturbance in Zn metabolism may predict hepatic fibrosis27. Also a cross-sectional study done by Kim et al. found that higher FIB-4 index is associated with lower serum Zn level and Lower serum Zn levels could be an independent risk factor for hepatic fibrosis in MASLD patients3. Moreover, the Ito et al. study also found a significant correlation between low serum Zn and the hepatic fibrosis and higher FIB-4 and NSF scores in MASLD patients23. In the Moriyama et al. study, Zn levels were found to be significantly associated with FIB-4, NFS, and hyaluronic acid28. Finally, in recent study published by Semeya et al. Zn deficiency was reported to have a strong correlation with the severity of liver cirrhosis and hepatic encephalopathy29. The results of this study were compatible with all these findings, demonstrating a possible significant association between serum Zn level and hepatic non-invasive indices. Zn being an important part of a constitution of metabolic, anti-inflammatory, and antioxidant enzymes which have huge impacts on distorted metabolism in chronic liver disease as well as its role in reduction of the degree of liver injury and normalization of lipid peroxidation are the possible explanations for these findings30,31. Furthermore, in this study, there was no significant association between serum ceruloplasmin and FIB-4, NFS, and BARD scores. Studies report conflicting results regarding this matter. For example, In the Nobili et al. study, on children with MASLD proved by biopsy, The results claim that although serum ceruloplasmin is associated with the possibility of Nonalcoholic Steatohepatitis, inflammation, and steatosis, but it is not associated with fibrosis32. Also, in the review study by Chen et al. the relationship between serum/hepatic Cu or ceruloplasmin concentration and MASLD was evaluated. Their meta-analysis demonstrated that serum Cu and ceruloplasmin were not associated with MASLD33. However, Corradini et al. reported that ceruloplasmin variants were independently associated with liver fibrosis34. Moreover, in the Issa et al. study, Ceruloplasmin level was significantly lower in patients with higher levels of fibrosis in comparison with patients with no fibrosis35. The results of this study, showed no association between ceruloplasmin level and non-invasive hepatic indices. This contrast may arise from the target population, study environment, inclusion and exclusion criteria, and underlying diseases. Moreover, the limited sample size and the single-border nature of this research can also be another explanation for this contrast.

To our knowledge this study is one of the first studies investigating three serum elements correlation with three scoring systems in MASLD patients. The findings of this study could be used for an easier risk stratification of liver fibrosis in MASLD patients. However, some limitations may affect our findings. One of them is the lack of long-term follow-up in the study. Also, hepatic steatosis was assessed using ultrasonography, which might underrepresent the prevalence of MASLD in the studied population specially in the early stages of MASLD. Moreover, the design of this study does not allow for confirming or negating causality between the assessed parameters. Finally lack of control group is another major limitation of this study. In addition, there is a need for future prospective multicenter studies that could use MRI/elastography for early diagnosis of MASLD to provide a higher level of evidence in this regard.

Conclusion

This study found a significant reverse correlation between serum Zn levels and FIB-4 and NFS scores in MASLD patients. Therefore, serum Zn may serve as a risk stratification factor in MASLD patients. Serum Cu and ceruloplasmin showed no significant association with the FIB-4 and NFS scores. Lastly, none of these three markers showed a significant association with the BARD scoring system. Future prospective studies are recommended for further evaluation of these findings.