Serum copper and ceruloplasmin levels as biomarkers reflecting liver fibrosis in children with autoimmune hepatitis

Article information

Clin Exp Pediatr. 2025;68(11):909-920

Publication date (electronic) : 2025 August 6

doi : https://doi.org/10.3345/cep.2025.01011

Salma Abdel Megeed Nagi, MD^,1,2, Mai Ibrahim Elashmawy, MD³, Amany E. Elashkar, MD³, Mohamed Zaeim Hafez, MD⁴, Ashraf A.E. Emara, MD⁵, Osama Mohammad Abdelhay, MD⁵, Albayoumi A.B. Fouda, MD⁵, Mohamed AbdelAziz Doma, MD⁶, Ahmad Mohamed Awad, MD⁶, Ahmed Mohammed Saba, MD⁶, Hesham Abdelrahman Ahmed, MD⁷, Ahmed Mohamed Gad Allah, MD⁸, Fatma Mahmoud Abdelraheem, MD⁹, Mohamed A. Gad, MD¹⁰, Mohamad A. Soliman, MD¹¹, Tamer I. Abdalrhman, MD¹¹, Khaled Hassaan Awad, MD¹², Ismael A.K.M. El-lebedy, MD¹², Mostafa M. Abdelnaser, MD¹³, Mohammed Z. Abdel Kareem, MD¹³, Marwa Fekry Hassan, MD¹⁴, Shymaa Sobhy Menshawy Khalifa, MD¹⁵

¹Faculty of Medicine, King Salman International University, South Sinai, Egypt

²Pediatric Hepatology, Gastroenterology, and Nutrition Department, National Liver Institute, Menoufia University, Shebin ElKom, Egypt

³Clinical Pathology Department, National Liver Institute, Menoufia University, Shebin ElKom, Egypt

⁴Medical Physiology Department, Faculty of Medicine, Al-Azhar University (Assiut), Assiut, Egypt

⁵Medical Physiology Department, Faculty of Medicine, Al-Azhar University, Cairo, Egypt

⁶Medical Biochemistry Department, Faculty of Medicine, Al-Azhar University, Cairo, Egypt

⁷Medical Biochemistry Department, Faculty of Medicine, Al-Azhar University (Assiut), Assiut, Egypt

⁸Forensic Medicine and Clinical Toxicology Department, Faculty of Medicine, Al-Azhar University (Assiut), Assiut, Egypt

⁹Histology and Cell Biology Department, Faculty of Medicine, Assiut University, Assiut, Egypt

¹⁰Medical Biochemistry Department, Faculty of Medicine, Al-Azhar University, Damietta, Egypt

¹¹Medical Histology and Cell Biology Department, Faculty of Medicine, Al-Azhar University (Assiut), Assiut, Egypt

¹²Pediatric Department, Faculty of Medicine, Al-Azhar University (Assiut), Assiut, Egypt

¹³Medical Microbiology and Immunology Departments, Faculty of Medicine, Al-Azhar University (Assiut), Assiut, Egypt

¹⁴Epidemiology and Preventive Medicine Department, National Liver Institute, Menoufia University, Menoufia, Egypt

¹⁵Pediatrics Department, Faculty of Medicine, Menoufia University, Shebin ElKom, Egypt

Corresponding author: Salma Abdel Megeed Nagi, MD. Faculty of Medicine, King Salman International University, El Tor Branch, Sahely Road, South Sinai, Shebin ElKom 32511, Egypt Email: salmanage@liver.menofia.edu.eg

Received 2025 May 4; Revised 2025 June 11; Accepted 2025 June 11.

Abstract

Introduction

According to Beretta-Piccoli et al. [1] and Yu et al. [2], autoimmune hepatitis (AIH) is an immune-mediated hepatocyte damage that results in inflammation, liver failure, and fibrosis by destroying liver cells. AIH in children is an autoimmune inflammatory illness that typically necessitates long-term immunosuppression. Current medications do not suppress intrahepatic immunological processes, as evidenced by the frequent relapses following therapy withdrawal [3,4].

There is no discernible pattern of genetic susceptibility or recognizable environmental impacts for AIH, even though there appear to be some regional variations [5]. Female predilection raised aminotransferase levels, hypergammaglobulinemia or elevated immunoglobulin G (IgG) levels, positive results for autoimmune antibodies, and interface hepatitis on biopsy are the typical characteristics of AIH. An autoimmune attack on the liver may be caused by a combination of genetic predisposition, environmental stimuli, and immunological tolerance mechanisms failing [6].

Elevated liver enzymes, autoantibody positivity, IgG elevation, compatible or typical liver histology results, and ruling out other liver disease causes are the basis for the diagnosis [7]. The likelihood of developing AIH is increased by extrahepatic autoimmunity and/or a family history of autoimmune disorders. Interface hepatitis, plasma cell-rich infiltrates, emperipolesis, and rosette formation are the most common histological findings [8,9]. However, only 56% of infants with AIH had all these histological characteristics, and histological results are not always definitive, particularly when the disease is still in its early stages [10]. In addition to the serological characteristics, new criteria were recently proposed for the histological diagnosis of AIH; however, these are still awaiting validation [11]. Additionally, several illnesses could be mistakenly labeled as AIH since they appear in children [12-15].

One essential trace element is copper. It functions as a cofactor for enzymes that are involved in fibrogenesis, antioxidant defense, and cellular energy metabolism. Few studies have linked these processes to changes in copper levels, even though they are essential to the pathophysiology of many liver illnesses [6]. The primary protein in the blood that carries copper is called ceruloplasmin (CP). Non-copper-bound apo-CP degrades quickly due to the copper transportation problem [16]. Numerous liver illnesses are linked to abnormalities in CP function and metabolism [17]. The purpose of this study was to determine whether serum CP and copper play a part in determining liver fibrosis in child with AIH.

Methods

5. Liver biopsy

As seen in Fig. 1A–D, Masson’s trichrome and hematoxylin and eosin (H&E) were used to stain all liver biopsies from patients who had undergone treatment prior to treatment. To determine the stage of liver fibrosis and grade the level of inflammation, the Ishak score was employed. We took liver biopsies before starting treatment for AIH. The duration of AIH before biopsy was around 2 weeks till 1 month.

Fig. 1.

(A) Moderate portal fibrosis with porto-portal link development (black arrows) and (B) mild portal inflammation rich in plasma cells (blue arrow) are visible on a hematoxylin and eosin (H&E)-stained slide of a patient with autoimmune hepatitis (AIH). An instance of AIH with modest portal fibrosis (black arrow) and mild portal inflammation rich in plasma cells (blue arrow) is depicted on an H&E-stained slide (×200), (C) An H&E-stained slide of a sample from a patient with AIH showing moderate portal inflammation rich in plasma cells (blue arrow), notable portal fibrosis (green arrows), cirrhosis, and regenerative nodules (black arrows) (×200), (D) An H&E-stained slide of a sample from a patient with AIH revealing modest portal inflammation with periportal glycogenotic nuclei (yellow arrow) and plasma cells (blue arrow) as well as significant portal fibrosis with the creation of porto-portal connections (black arrows).

Results

A flowchart of the 109 children was enrolled in the current study who were first diagnosed with AIH at Menoufia University's National Liver Institute's Pediatric Hepatology Department. 100 people took part in the trial, and 9 patients were eliminated (3 patients refused consent, and 6 did not fit the inclusion criteria). Patients were divided into 3 categories based on their autoantibody profiles: seronegative AIH (n=16), AIH type 1 (n=75), and AIH type 2 (n=9). Additional classification was carried out according to the stage of fibrosis, with stages 3 and 4 being regarded as advanced fibrosis (Fig. 2).

Fig. 2.

Flowchart of enrolled children with autoimmune hepatitis (AIH).

According to the results of liver biopsy analysis, the patients under study were divided into 2 groups: those with advanced fibrosis and those without. 84 of the 100 AIH patients (84%) had nonadvanced liver fibrosis, while 16 (16%) had advanced fibrosis. Females made up 87.5% of patients with advanced fibrosis and 61% of all AIH patients. However, only 39% of AIH patients and 12.5% of patients with severe fibrosis were males. Patients with advanced fibrosis were statistically significantly more likely to be female (P=0.018). At diagnosis, the average age was 8.03±4.27. In terms of laboratory markers, individuals with extensive fibrosis had significantly lower levels of Hb, ALT, AST, and CP (P=0.019, P=0.043, P=0.013, and P<0.001, respectively). Patients with advanced fibrosis had considerably higher copper levels (P<0.001)(Fig. 3). Regarding the kind of AIH and other laboratory characteristics, no notable variations were found, as shown in Table 1.

Fig. 3.

Serum ceruloplasmin (A) and copper (B) levels by liver fibrosis stage. *P<0.05, statistically significant differences.

Table 1.

Demographic data and laboratory values of patients with AIH by liver fibrosis stage

Patients were divided into 3 categories based on autoantibody results: seronegative AIH, AIH type 1, and AIH type 2. Nine of the 100 individuals (9%) with AIH had type 2 AIH, 16 (16%) had seronegative AIH, and 75 (75%) had type 1 AIH. Table 2 shows basic laboratory, clinical, and demographic data. Except for type 2 AIH having a considerably lower CP level than type 1 and seronegative groups (P=0.024 and P=0.011, respectively), there were no discernible differences between the various forms of AIH. According to Table 2, the levels of CP were 16.22±2.81 in type 2 AIH, 20.42±5.07 in type 1 AIH, and 21.81±6.63 in seronegative AIH.

Table 2.

Demographic data and laboratory values of patients with AIH by disease type

Regarding ultrasonography examination, there were no appreciable differences between the forms of AIH under study (P>0.05). Nonetheless, there were notable variations in the fibrosis stage when taking histological findings into account (P=0.026). Seronegative AIH had a substantially higher prevalence of mild fibrosis (n=8, 50%), but type 1 and type 2 AIH had higher prevalences of intermediate fibrosis (n=38, 50.6%; n=6, 66.6%, respectively). The prevalence of severe (advanced) fibrosis was higher in type 2 AIH (n=3, 33.3%), seronegative AIH (n=4, 25%), and type 1 AIH (n=9, 12%) (Table 3).

Table 3.

Ultrasound and histopathological findings of the study patients

The levels of total and direct bilirubin, AST, and PLTs were significantly positively correlated with CP (r=0.20, P=0.042, r=0.21, P=0.033, r=0.20, P=0.040, and r=0.32, P=0.001, respectively). Serum copper levels and both total and direct bilirubin showed a strong positive connection (r=0.20, P=0.042, and r=0.21, P=0.033, respectively). Serum copper levels and AST showed a strong negative connection (r=-0.22, P=0.023) (Table 4).

Table 4.

Correlations between ceruloplasmin and serum copper levels and study variables

The risk variables for progressive liver fibrosis in AIH patients were calculated using binary logistic regression analysis (Table 5). In the univariate logistic regression analysis, patients with advanced liver fibrosis were more likely to be female (P=0.03), have greater serum copper (P=0.004), lower CP levels (P=0.005), higher ALT (P=0.029), and higher AST (P=0.024). However, CP and copper levels were found to be independent risk factors for predicting advanced liver fibrosis in AIH patients following multivariate logistic regression analysis. The odds ratios (ORs) were 2.71 (95% confidence interval [CI], 1.263–5.796; P=0.010) and 0.30 (95% CI, 0.119–0.757; P=0.011), respectively (Table 5).

Table 5.

Uni- and multivariate analyses of factors associated with advanced liver fibrosis

In contrast to serum copper levels, which have an area under the curve of 0.939 at 95% CI (0.887–0.991, P<0.001), with a cutoff of >24.7 mg/dL with 90.8% sensitivity and 66.9% specificity, CP levels in our study had an area under the curve of 0.945 at 95% CI of (0.889–1.00, P<0.001) with a cutoff of <17.5 ng/mL with 97.3% sensitivity and 61.5% specificity (Fig. 4).

Fig. 4.

Receiver operating characteristic (ROC) analysis of ceruloplasmin (A) and serum copper levels (B) for the detection of liver fibrosis in children with chronic liver diseases. AUC, area under the curve; CI, confidence interval. *P<0.05, statistically significant differences.

Except for sex (P=0.019), there was no significant correlation between the CP levels and the factors under study (P>0.05). Male patients had a substantially higher level of CP (median, 20; range, 12–34) than female patients (median, 19; range, 10–33). Serum copper levels did not substantially correlate with other factors under study (P>0.05) (Table 6).

Table 6.

Correlations between serum ceruloplasmin and copper levels and other study variables

Discussion

To stop the development of cirrhosis, AIH, a chronic liver disease, must be diagnosed early [15]. The majority of AIH patients appear with substantial fibrosis or even cirrhosis because there are no diagnostic markers for these patients [20]. When choosing a treatment plan and estimating the long-term prognosis for patients with AIH, it is crucial to assess the stages of liver fibrosis [21]. It is crucial to look for noninvasive indicators of liver fibrosis, and CP has been suggested as a fibrosis biomarker in several liver illnesses, including nonalcoholic fatty liver disease/steatohepatitis and chronic hepatitis B (CHB) [22]. To ascertain their clinical significance, more study is necessary because the involvement of serum CP and copper in fibrosis assessment in pediatric AIH is not well established. It is important to note that no research has examined the amounts of copper or CP in the serum of children with AIH. Therefore, the purpose of this study was to investigate whether serum copper and serum CP play a part in identifying liver fibrosis in children with AIH.

A H&E-stained slide of a patient with AIH showing significant portal fibrosis with the formation of portoportal connections (black arrows) and mild portal inflammation rich in plasma cells (blue arrow). (×200), An H&E-stained slide of a patient with AIH showing modest portal fibrosis (black arrow) and portal inflammation rich in plasma cells (blue arrow) (×200), An H&E-stained slide of a case of AIH showing considerable portal fibrosis (green arrows), cirrhosis and the development of regenerative nodules (black arrows), and mild portal inflammation rich in plasma cells (blue arrow). Zeng et al. [22] and Faddan et al. [23] reported a female-to-male ratio of 2.4:1 in a cohort of 34 children, while studies from Argentina showed an even higher ratio of 4:1 in adults. These findings are consistent with the female predominance in AIH identified in our study. However, Oettinger et al. [24] observed an equal male-to-female ratio (1:1) throughout the United States, indicating diversity in sex distribution. Notwithstanding these regional variations, most of the research indicates that women are more likely to have AIH, which supports the condition's status as an autoimmune disease. Additionally, seronegative AIH was found in only 16% of cases in our sample, whereas AIH-1 and AIH-2 accounted for 75% and 9% of cases, respectively [24-26], who also reported AIH-1 as the prevalent form, concur with our study's increased frequency of AIH-1 relative to AIH-2. According to Maggiore et al. [27], a strong index of suspicion is necessary for identifying seronegative AIH because it can be difficult to start corticosteroid treatment when there are no detectable autoantibodies. According to Faddan et al. [23], children with AIH-2 were considerably younger than those with AIH-1 in terms of age of diagnosis. Similarly, children with AIH-1 tend to be older than those with AIH-2, according to Bellomo-Brandão et al. [28] in the British cohort.

Compared to AIH-1 and seronegative AIH, patients with AIH-2 were younger in our group. However, because the P value was 0.082, this difference did not approach statistical significance. According to our research, patients with severe fibrosis had a much lower serum CP level than those without the disease. However, compared to patients in nonadvanced stages, those with advanced fibrosis have much higher serum copper levels. Additionally, children with type 2 AIH had considerably lower serum CP levels than children with type 1 and seronegative AIH. However, there was no statistically significant difference in copper levels between seronegative AIH, type 1, and type 2. These results are consistent with those of Raouf [29], who found that children with chronic liver disease (CLD) had noticeably higher serum copper levels than controls. This discovery was also highlighted by Sayed et al. [30], who found comparable results in a rural community in Egypt, and Roberts and Schilsky [31], who discovered higher serum copper levels in cirrhotic patients. According to this research, elevated serum copper might not be a specific indicator of Wilson disease but rather a common characteristic of liver disorders. As first described by Sternlieb and Scheinberg [32], CP is an alpha-2 glycoprotein that is mostly produced in the liver and is known for its function in acute phase reactions, where serum levels usually increase after inflammation or tissue injury. Our results, however, are more in line with those of Walshe [33] and Perman et al. [34], who noted lower CP levels in diseases like decompensated cirrhosis, fulminant hepatitis, and severe hepatitis. This suggests that CP reduction may be linked to compromised hepatic synthetic function rather than just the inflammatory stimulus. In a similar vein, Zeng et al. [35] found a strong relationship between CP levels and the severity of liver inflammation, demonstrating that CP levels significantly decreased in moderate or severe inflammation (grades 3–4) when there was widespread hepatocyte necrosis. This confirms our finding that lower serum CP levels are a result of moderate to severe liver injury, which is in line with the liver's impaired capacity to produce CP in advanced fibrosis. Ebara et al. [36] found that HCV-positive patients with chronic hepatitis or cirrhosis had higher copper levels in their liver parenchyma, which increased CP's oxidation activity. This finding partially supports our findings. Similarly, Ranganathan et al. [37] postulated that hepatic copper overload may contribute to the development of hepatocellular carcinoma by increasing the metalation of the CP protein, which results in a higher fraction of its active holo form. Our findings were corroborated by Zeng et al. [35], who found that CP was a unique biomarker that was adversely connected with liver fibrosis. They hypothesized that lower CP levels might result from a selective decrease in hepatocyte production as the fibrosis progressed. Our results, however, contrast with those of Habeeb et al. [38], who discovered an unanticipated rise in CP activity among HCV patients relative to HBV patients and observed no significant correlation between CP concentration and CLD caused by HBV and HCV. These disparities could be the result of changes in study methodology, patient demographics, or stages of illness. With an AUC of 0.945 (95% CI, 0.889–1.00) at a threshold of <17.5 ng/mL, our ROC analysis showed that CP is a highly sensitive marker for diagnosing liver fibrosis in juvenile AIH, attaining 97.3% sensitivity and 61.5% specificity. With an AUC of 0.939 (95% CI, 0.887–0.991) at a cutoff of >24.7 mg/dL, serum copper likewise demonstrated good performance, exhibiting 66.9% specificity and 90.8% sensitivity. These results imply that copper levels and CP levels may both be useful noninvasive methods for determining fibrosis in juvenile AIH. Our findings are corroborated by Zeng et al. [35] who reported AUCs of 0.74 for substantial fibrosis (F≥4), 0.75 for moderate fibrosis (F≥2), and 0.71 for advanced fibrosis (F≥3), indicating the use of CP in assessing fibrosis in CHB patients. Their investigation demonstrated CP's ability to lessen reliance on liver biopsies, especially in male patients, using a grading methodology akin to that of Scheuer et al. [39] and Bedossa [40]. The work of Zeng et al. [35] in CHB further supports the importance of CP as a trustworthy biomarker for liver fibrosis across various liver disorders, even though our study concentrated on pediatric AIH. Nonetheless, variability in patient groups and illness etiology may be reflected in disparities in cutoff values and diagnostic performance, indicating that the best thresholds should be confirmed clinical settings.

It is important to note that no research has examined the levels of copper or CP in the serum of children with AIH. There are certain restrictions on this study, though. First, the low prevalence of AIH means that there aren't many cases. Therefore, additional research with a sizable sample of AIH patients from several institutions is required to validate our findings.

In conclusion, this study concludes by indicating that blood CP and copper levels may provide useful information for identifying liver fibrosis in children with AIH. In the end, serum copper and CP levels ought to be considered as a component of a more comprehensive evaluation strategy for pediatric AIH patients.

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